"Medicine, Faculty of"@en . "Medicine, Department of"@en . "Experimental Medicine, Division of"@en . "DSpace"@en . "UBCV"@en . "Hamilton, Melisa June"@en . "2011-12-13T18:54:45Z"@en . "2011"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Although the importance of immunomodulatory myeloid cells in both normal physiology and carcinogenesis is well established, many questions remain regarding the specific roles and regulation of these cells. In this thesis, we explore the immunosuppressive features of macrophages [M\u00CF\u0086s] and elucidate the mechanisms by which they suppress T cell proliferation/activation, the factors that regulate their suppressive properties, the relative potency of macrophage suppression compared to other myeloid cells, such as myeloid-derived suppressor cells (MDSCs), and the role these cells play in promoting tumor growth and metastasis. \nWe demonstrate herein that in response to interferon (IFN)-beta, which is secreted by activated T cells, resident macrophages from non-tumor-bearing mice acquire immunosuppressive properties that are mediated by nitric oxide (NO). Moreover, our data reveal a novel role for Toll-like receptor (TLR)-induced IFN-beta in regulating the immunosuppressive properties of macrophages. \n\nWe also demonstrate for the first time that in vitro culture conditions profoundly affect the immunosuppressive functions of MDSCs. Specifically, we show that serum antagonizes the suppressive abilities of MDSCs from 4T1 tumor-bearing mice and that the major serum protein albumin mediates these effects, in part by reducing reactive oxygen species (ROS) production from MDSCs. These findings have important implications, since the accurate detection and quantification of immunosuppression is critical for both the identification and functional analysis of tumor-induced MDSCs. \n\nWe also explore the phenotypic and functional heterogeneity of tumor-induced myeloid cells and compare the immunosuppressive functions of different populations isolated from normal and tumor-bearing mice. We show that tumors that induce the accumulation of myeloid cells also enhance the suppressive functions of these cells. In addition, we demonstrate that, in vitro, tumor-induced macrophages are significantly more potent immune suppressors than tumor-induced MDSCs on a per cell basis, and suppress T cell responses via distinct mechanisms. Finally, we present data showing that treating metastatic mammary tumor-bearing mice with all-trans-retinoic acid (ATRA) decreases MDSCs, increases macrophages, and enhances metastatic growth. \n\nTaken together, these findings advance our understanding of the factors that regulate myeloid cell functions in normal and neoplastic tissues and may lead to improved immunotherapies to treat human disease."@en . "https://circle.library.ubc.ca/rest/handle/2429/39661?expand=metadata"@en . "IMMUNOSUPPRESSIVE\u00C2\u00A0MYELOID\u00C2\u00A0CELLS\u00C2\u00A0 UNDER\u00C2\u00A0NORMAL\u00C2\u00A0AND\u00C2\u00A0NEOPLASTIC\u00C2\u00A0CONDITIONS\u00C2\u00A0 \u00C2\u00A0 by\u00C2\u00A0 \u00C2\u00A0 MELISA\u00C2\u00A0JUNE\u00C2\u00A0HAMILTON\u00C2\u00A0 B.Sc.,\u00C2\u00A0The\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia,\u00C2\u00A02005\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0THESIS\u00C2\u00A0SUBMITTED\u00C2\u00A0IN\u00C2\u00A0PARTIAL\u00C2\u00A0FULFILLMENT\u00C2\u00A0OF\u00C2\u00A0 THE\u00C2\u00A0REQUIREMENTS\u00C2\u00A0FOR\u00C2\u00A0THE\u00C2\u00A0DEGREE\u00C2\u00A0OF\u00C2\u00A0 \u00C2\u00A0 DOCTOR\u00C2\u00A0OF\u00C2\u00A0PHILOSOPHY\u00C2\u00A0 in\u00C2\u00A0 THE\u00C2\u00A0FACULTY\u00C2\u00A0OF\u00C2\u00A0GRADUATE\u00C2\u00A0STUDIES\u00C2\u00A0 (Experimental\u00C2\u00A0Medicine)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 THE\u00C2\u00A0UNIVERSITY\u00C2\u00A0OF\u00C2\u00A0BRITISH\u00C2\u00A0COLUMBIA\u00C2\u00A0 (Vancouver)\u00C2\u00A0 December\u00C2\u00A02011\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A9\u00C2\u00A0Melisa\u00C2\u00A0June\u00C2\u00A0Hamilton,\u00C2\u00A02011\u00C2\u00A0 ii ABSTRACT\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0 the\u00C2\u00A0 importance\u00C2\u00A0 of\u00C2\u00A0 immunomodulatory\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 normal\u00C2\u00A0 physiology\u00C2\u00A0 and\u00C2\u00A0 carcinogenesis\u00C2\u00A0 is\u00C2\u00A0 well\u00C2\u00A0 established,\u00C2\u00A0 many\u00C2\u00A0 questions\u00C2\u00A0 remain\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 roles\u00C2\u00A0 and\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 cells.\u00C2\u00A0 In\u00C2\u00A0 this\u00C2\u00A0 thesis,\u00C2\u00A0 we\u00C2\u00A0 explore\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 features\u00C2\u00A0of\u00C2\u00A0macrophages\u00C2\u00A0 (M\u00CF\u0086s)\u00C2\u00A0and\u00C2\u00A0elucidate\u00C2\u00A0 the\u00C2\u00A0mechanisms\u00C2\u00A0by\u00C2\u00A0 which\u00C2\u00A0 they\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation/activation,\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 their\u00C2\u00A0 suppressive\u00C2\u00A0 properties,\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 potency\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 suppression\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 other\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 (MDSCs),\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0play\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0demonstrate\u00C2\u00A0herein\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0interferon\u00C2\u00A0(IFN)\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0secreted\u00C2\u00A0 by\u00C2\u00A0 activated\u00C2\u00A0 T\u00C2\u00A0 cells,\u00C2\u00A0 resident\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 non\u00E2\u0080\u0090tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 acquire\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0 that\u00C2\u00A0are\u00C2\u00A0mediated\u00C2\u00A0by\u00C2\u00A0nitric\u00C2\u00A0oxide\u00C2\u00A0(NO).\u00C2\u00A0Moreover,\u00C2\u00A0our\u00C2\u00A0 data\u00C2\u00A0 reveal\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0 Toll\u00E2\u0080\u0090like\u00C2\u00A0 receptor\u00C2\u00A0 (TLR)\u00E2\u0080\u0090induced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 in\u00C2\u00A0 regulating\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0also\u00C2\u00A0demonstrate\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0time\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0profoundly\u00C2\u00A0 affect\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 MDSCs.\u00C2\u00A0 Specifically,\u00C2\u00A0 we\u00C2\u00A0 show\u00C2\u00A0 that\u00C2\u00A0 serum\u00C2\u00A0 antagonizes\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0 the\u00C2\u00A0major\u00C2\u00A0 serum\u00C2\u00A0protein\u00C2\u00A0albumin\u00C2\u00A0mediates\u00C2\u00A0 these\u00C2\u00A0effects,\u00C2\u00A0 in\u00C2\u00A0part\u00C2\u00A0by\u00C2\u00A0 reducing\u00C2\u00A0 reactive\u00C2\u00A0 oxygen\u00C2\u00A0 species\u00C2\u00A0 (ROS)\u00C2\u00A0 production\u00C2\u00A0 from\u00C2\u00A0 MDSCs.\u00C2\u00A0 These\u00C2\u00A0 findings\u00C2\u00A0 have\u00C2\u00A0 important\u00C2\u00A0 implications,\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0accurate\u00C2\u00A0detection\u00C2\u00A0and\u00C2\u00A0quantification\u00C2\u00A0of\u00C2\u00A0 immunosuppression\u00C2\u00A0is\u00C2\u00A0 critical\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0identification\u00C2\u00A0and\u00C2\u00A0functional\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0tumor\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0also\u00C2\u00A0explore\u00C2\u00A0the\u00C2\u00A0phenotypic\u00C2\u00A0and\u00C2\u00A0functional\u00C2\u00A0heterogeneity\u00C2\u00A0of\u00C2\u00A0tumor\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0compare\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0populations\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0normal\u00C2\u00A0and\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice.\u00C2\u00A0 \u00C2\u00A0We\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0 tumors\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 the\u00C2\u00A0 accumulation\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0also\u00C2\u00A0enhance\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells.\u00C2\u00A0In\u00C2\u00A0 addition,\u00C2\u00A0 we\u00C2\u00A0 demonstrate\u00C2\u00A0 that,\u00C2\u00A0 in\u00C2\u00A0 vitro,\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 significantly\u00C2\u00A0 more\u00C2\u00A0 iii potent\u00C2\u00A0 immune\u00C2\u00A0 suppressors\u00C2\u00A0 than\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 per\u00C2\u00A0 cell\u00C2\u00A0 basis,\u00C2\u00A0 and\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 distinct\u00C2\u00A0mechanisms.\u00C2\u00A0 Finally,\u00C2\u00A0we\u00C2\u00A0 present\u00C2\u00A0 data\u00C2\u00A0 showing\u00C2\u00A0 that\u00C2\u00A0 treating\u00C2\u00A0 metastatic\u00C2\u00A0 mammary\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 with\u00C2\u00A0 all\u00E2\u0080\u0090trans\u00E2\u0080\u0090retinoic\u00C2\u00A0 acid\u00C2\u00A0 (ATRA)\u00C2\u00A0decreases\u00C2\u00A0MDSCs,\u00C2\u00A0increases\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0and\u00C2\u00A0enhances\u00C2\u00A0metastatic\u00C2\u00A0growth.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 these\u00C2\u00A0 findings\u00C2\u00A0 advance\u00C2\u00A0 our\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 functions\u00C2\u00A0 in\u00C2\u00A0 normal\u00C2\u00A0 and\u00C2\u00A0 neoplastic\u00C2\u00A0 tissues\u00C2\u00A0 and\u00C2\u00A0 may\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 improved\u00C2\u00A0immunotherapies\u00C2\u00A0to\u00C2\u00A0treat\u00C2\u00A0human\u00C2\u00A0disease.\u00C2\u00A0\u00C2\u00A0 iv PREFACE\u00C2\u00A0 \u00C2\u00A0 All\u00C2\u00A0 experiments\u00C2\u00A0 were\u00C2\u00A0 conducted\u00C2\u00A0 by\u00C2\u00A0 Melisa\u00C2\u00A0 J.\u00C2\u00A0 Hamilton,\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 exception\u00C2\u00A0 of\u00C2\u00A0 those\u00C2\u00A0described\u00C2\u00A0below.\u00C2\u00A0 I\u00C2\u00A0designed\u00C2\u00A0 the\u00C2\u00A0studies\u00C2\u00A0presented\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0dissertation,\u00C2\u00A0analyzed\u00C2\u00A0 and\u00C2\u00A0interpreted\u00C2\u00A0all\u00C2\u00A0the\u00C2\u00A0data,\u00C2\u00A0and\u00C2\u00A0composed\u00C2\u00A0and\u00C2\u00A0edited\u00C2\u00A0the\u00C2\u00A0thesis.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0version\u00C2\u00A0of\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0was\u00C2\u00A0previously\u00C2\u00A0published.\u00C2\u00A0Hamilton\u00C2\u00A0MJ,\u00C2\u00A0Antignano\u00C2\u00A0F,\u00C2\u00A0von\u00C2\u00A0 Rossum\u00C2\u00A0 A,\u00C2\u00A0 Boucher\u00C2\u00A0 JL,\u00C2\u00A0 Bennewith\u00C2\u00A0 KL,\u00C2\u00A0 Krystal\u00C2\u00A0 G.\u00C2\u00A0 \u00E2\u0080\u009CTLR\u00C2\u00A0 agonists\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 abrogate\u00C2\u00A0 resident\u00C2\u00A0 macrophage\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cells\u00E2\u0080\u009D.\u00C2\u00A0 The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 Immunology,\u00C2\u00A0 185(8):4545\u00E2\u0080\u00904553,\u00C2\u00A02010.\u00C2\u00A0Copyright\u00C2\u00A02010.\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0 Inc.\u00C2\u00A0 Several\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 experiments\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 were\u00C2\u00A0 conducted\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 technical\u00C2\u00A0 assistance\u00C2\u00A0 of\u00C2\u00A0 co\u00E2\u0080\u0090operative\u00C2\u00A0 education\u00C2\u00A0 students\u00C2\u00A0 Anna\u00C2\u00A0 von\u00C2\u00A0 Rossum\u00C2\u00A0 (Terry\u00C2\u00A0 Fox\u00C2\u00A0 Laboratory),\u00C2\u00A0 who\u00C2\u00A0 worked\u00C2\u00A0 under\u00C2\u00A0 my\u00C2\u00A0 supervision,\u00C2\u00A0 and\u00C2\u00A0 Kim\u00C2\u00A0 Snyder\u00C2\u00A0 (Terry\u00C2\u00A0 Fox\u00C2\u00A0 Laboratory),\u00C2\u00A0who\u00C2\u00A0worked\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0supervision\u00C2\u00A0of\u00C2\u00A0Vivian\u00C2\u00A0Lam\u00C2\u00A0(Terry\u00C2\u00A0Fox\u00C2\u00A0Laboratory).\u00C2\u00A0 In\u00C2\u00A0addition,\u00C2\u00A0Dr.\u00C2\u00A0Frann\u00C2\u00A0Antignano\u00C2\u00A0(Terry\u00C2\u00A0Fox\u00C2\u00A0Laboratory)\u00C2\u00A0developed\u00C2\u00A0the\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0ELISA\u00C2\u00A0in\u00C2\u00A0 collaboration\u00C2\u00A0 with\u00C2\u00A0 Victor\u00C2\u00A0 Ho\u00C2\u00A0 (Terry\u00C2\u00A0 Fox\u00C2\u00A0 Laboratory),\u00C2\u00A0 and\u00C2\u00A0 participated\u00C2\u00A0 in\u00C2\u00A0 helpful\u00C2\u00A0 discussions\u00C2\u00A0 that\u00C2\u00A0 led\u00C2\u00A0 to\u00C2\u00A0 hypothesis\u00C2\u00A0 generation\u00C2\u00A0 and\u00C2\u00A0 experimental\u00C2\u00A0 ideas.\u00C2\u00A0 Dr.\u00C2\u00A0 Scott\u00C2\u00A0 Patterson\u00C2\u00A0(CFRI)\u00C2\u00A0assisted\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Foxp3\u00C2\u00A0staining\u00C2\u00A0protocol.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 version\u00C2\u00A0 of\u00C2\u00A0 Chapter\u00C2\u00A0 4\u00C2\u00A0 is\u00C2\u00A0 currently\u00C2\u00A0 in\u00C2\u00A0 press.\u00C2\u00A0 Hamilton\u00C2\u00A0 MJ,\u00C2\u00A0 Ban\u00C3\u00A1th\u00C2\u00A0 JP,\u00C2\u00A0 Lam\u00C2\u00A0 V,\u00C2\u00A0 Lepard\u00C2\u00A0NE,\u00C2\u00A0Krystal\u00C2\u00A0G,\u00C2\u00A0Bennewith\u00C2\u00A0KL.\u00C2\u00A0\u00E2\u0080\u009CSerum\u00C2\u00A0inhibits\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0function\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00E2\u0080\u009D\u00C2\u00A0 Cancer\u00C2\u00A0 Immunology\u00C2\u00A0Immunotherapy.\u00C2\u00A0Studies\u00C2\u00A0in\u00C2\u00A0Chapters\u00C2\u00A04\u00C2\u00A0and\u00C2\u00A05\u00C2\u00A0involving\u00C2\u00A0tumor\u00C2\u00A0models\u00C2\u00A0were\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 collaboration\u00C2\u00A0with\u00C2\u00A0Dr.\u00C2\u00A0Kevin\u00C2\u00A0Bennewith\u00E2\u0080\u0099s\u00C2\u00A0 laboratory,\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 technical\u00C2\u00A0 assistance\u00C2\u00A0of\u00C2\u00A0Dr.\u00C2\u00A0Judit\u00C2\u00A0Ban\u00C3\u00A1th,\u00C2\u00A0Nancy\u00C2\u00A0LePard,\u00C2\u00A0Momir\u00C2\u00A0Bosiljcic,\u00C2\u00A0Denise\u00C2\u00A0McDougal,\u00C2\u00A0Jessica\u00C2\u00A0 Jia,\u00C2\u00A0and\u00C2\u00A0Dr.\u00C2\u00A0Kevin\u00C2\u00A0Bennewith,\u00C2\u00A0(Department\u00C2\u00A0of\u00C2\u00A0Integrative\u00C2\u00A0Oncology,\u00C2\u00A0BC\u00C2\u00A0Cancer\u00C2\u00A0Research\u00C2\u00A0 Centre).\u00C2\u00A0 Specifically,\u00C2\u00A0 lab\u00C2\u00A0 members\u00C2\u00A0 assisted\u00C2\u00A0 with\u00C2\u00A0 tumor\u00C2\u00A0 injections,\u00C2\u00A0 tissue\u00C2\u00A0 processing,\u00C2\u00A0 immunofluorescence\u00C2\u00A0 analyses,\u00C2\u00A0 clonogenic\u00C2\u00A0 assays,\u00C2\u00A0 and\u00C2\u00A0 some\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0 analyses.\u00C2\u00A0 Victor\u00C2\u00A0Ho\u00C2\u00A0also\u00C2\u00A0assisted\u00C2\u00A0with\u00C2\u00A0tissue\u00C2\u00A0collection,\u00C2\u00A0though\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0contribute\u00C2\u00A0directly\u00C2\u00A0to\u00C2\u00A0any\u00C2\u00A0 specific\u00C2\u00A0 figure.\u00C2\u00A0 In\u00C2\u00A0addition,\u00C2\u00A0co\u00E2\u0080\u0090operative\u00C2\u00A0education\u00C2\u00A0students\u00C2\u00A0Kim\u00C2\u00A0Snyder\u00C2\u00A0and\u00C2\u00A0Ann\u00C2\u00A0Hsu\u00E2\u0080\u0090 v An\u00C2\u00A0Lin\u00C2\u00A0(Terry\u00C2\u00A0Fox\u00C2\u00A0Laboratory),\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0supervision\u00C2\u00A0of\u00C2\u00A0me,\u00C2\u00A0Vivian\u00C2\u00A0Lam,\u00C2\u00A0and/or\u00C2\u00A0Victor\u00C2\u00A0 Ho,\u00C2\u00A0 assisted\u00C2\u00A0with\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0 analyses.\u00C2\u00A0 In\u00C2\u00A0Chapter\u00C2\u00A04,\u00C2\u00A0 the\u00C2\u00A0microscopy\u00C2\u00A0 in\u00C2\u00A0Fig.\u00C2\u00A04.3\u00C2\u00A0was\u00C2\u00A0 performed\u00C2\u00A0by\u00C2\u00A0Dr.\u00C2\u00A0Judit\u00C2\u00A0Ban\u00C3\u00A1th\u00C2\u00A0and\u00C2\u00A0Dr.\u00C2\u00A0Kevin\u00C2\u00A0Bennewith,\u00C2\u00A0and\u00C2\u00A0Vivian\u00C2\u00A0Lam\u00C2\u00A0prepared\u00C2\u00A0the\u00C2\u00A0 fetal\u00C2\u00A0calf\u00C2\u00A0serum\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A04.5\u00C2\u00A0and\u00C2\u00A0Fig.\u00C2\u00A04.7.\u00C2\u00A0In\u00C2\u00A0Chapter\u00C2\u00A05,\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0Dr.\u00C2\u00A0Kevin\u00C2\u00A0 Bennewith\u00E2\u0080\u0099s\u00C2\u00A0laboratory\u00C2\u00A0collected\u00C2\u00A0the\u00C2\u00A0raw\u00C2\u00A0data\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A05.1,\u00C2\u00A0Fig.\u00C2\u00A05.2,\u00C2\u00A0Fig.\u00C2\u00A05.3,\u00C2\u00A0Fig.\u00C2\u00A0 5.28,\u00C2\u00A0and\u00C2\u00A0Fig.\u00C2\u00A05.30A.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Mice\u00C2\u00A0were\u00C2\u00A0housed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0Animal\u00C2\u00A0Resource\u00C2\u00A0Centre\u00C2\u00A0 (ARC)\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0assistance\u00C2\u00A0of\u00C2\u00A0 ARC\u00C2\u00A0staff.\u00C2\u00A0Mouse\u00C2\u00A0studies\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0under\u00C2\u00A0UBC\u00C2\u00A0Animal\u00C2\u00A0Care\u00C2\u00A0protocols\u00C2\u00A0A07\u00E2\u0080\u00900221\u00C2\u00A0 (Dr.\u00C2\u00A0Gerald\u00C2\u00A0Krystal)\u00C2\u00A0and\u00C2\u00A0A09\u00E2\u0080\u00900251\u00C2\u00A0(Dr.\u00C2\u00A0Kevin\u00C2\u00A0Bennewith).\u00C2\u00A0 \u00C2\u00A0 Drs.\u00C2\u00A0 Gerald\u00C2\u00A0 Krystal\u00C2\u00A0 and\u00C2\u00A0 Kevin\u00C2\u00A0 Bennewith\u00C2\u00A0 contributed\u00C2\u00A0 to\u00C2\u00A0 experimental\u00C2\u00A0 design,\u00C2\u00A0 data\u00C2\u00A0interpretation,\u00C2\u00A0and\u00C2\u00A0editing\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0dissertation.\u00C2\u00A0 \u00C2\u00A0 vi TABLE\u00C2\u00A0OF\u00C2\u00A0CONTENTS\u00C2\u00A0 ABSTRACT\u00C2\u00A0.........................................................................................................................................\u00C2\u00A0ii\u00C2\u00A0 PREFACE\u00C2\u00A0............................................................................................................................................\u00C2\u00A0iv\u00C2\u00A0 TABLE\u00C2\u00A0OF\u00C2\u00A0CONTENTS\u00C2\u00A0...................................................................................................................\u00C2\u00A0vi\u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0TABLES\u00C2\u00A0................................................................................................................................\u00C2\u00A0x\u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0FIGURES\u00C2\u00A0.............................................................................................................................xi\u00C2\u00A0 LIST\u00C2\u00A0OF\u00C2\u00A0ABBREVIATIONS\u00C2\u00A0..........................................................................................................\u00C2\u00A0xiv\u00C2\u00A0 ACKNOWLEDGEMENTS\u00C2\u00A0..............................................................................................................\u00C2\u00A0xix\u00C2\u00A0 DEDICATION\u00C2\u00A0....................................................................................................................................\u00C2\u00A0xx\u00C2\u00A0 CHAPTER\u00C2\u00A01\u00C2\u00A0:\u00C2\u00A0INTRODUCTION\u00C2\u00A0......................................................................................................\u00C2\u00A01\u00C2\u00A0 1.1\u00C2\u00A0 History\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0immunology\u00C2\u00A0....................................................................................................................\u00C2\u00A01\u00C2\u00A0 1.2\u00C2\u00A0 Cancer\u00C2\u00A0immunoediting\u00C2\u00A0versus\u00C2\u00A0tumor\u00E2\u0080\u0090induced\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0.......................................\u00C2\u00A02\u00C2\u00A0 1.3\u00C2\u00A0 Brief\u00C2\u00A0overview\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0....................................................................................................\u00C2\u00A03\u00C2\u00A0 1.4\u00C2\u00A0 Tolerance\u00C2\u00A0versus\u00C2\u00A0autoimmunity\u00C2\u00A0................................................................................................................\u00C2\u00A05\u00C2\u00A0 1.5\u00C2\u00A0 TLR\u00C2\u00A0signaling\u00C2\u00A0.....................................................................................................................................................\u00C2\u00A06\u00C2\u00A0 1.6\u00C2\u00A0 Immune\u00C2\u00A0escape\u00C2\u00A0...............................................................................................................................................\u00C2\u00A011\u00C2\u00A0 1.7\u00C2\u00A0 Pro\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0.........................................................................\u00C2\u00A012\u00C2\u00A0 1.7.1\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0.......................................................................................................................................................\u00C2\u00A016\u00C2\u00A0 1.7.1.1\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0activation\u00C2\u00A0.................................................................................................................\u00C2\u00A019\u00C2\u00A0 1.7.1.2\u00C2\u00A0 TAMs\u00C2\u00A0.................................................................................................................................\u00C2\u00A023\u00C2\u00A0 1.7.2\u00C2\u00A0 Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0...........................................................................................\u00C2\u00A026\u00C2\u00A0 1.8\u00C2\u00A0 Tumor\u00C2\u00A0metastasis\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0pre\u00E2\u0080\u0090metastatic\u00C2\u00A0niche\u00C2\u00A0..............................................................................\u00C2\u00A029\u00C2\u00A0 1.9\u00C2\u00A0 4T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0murine\u00C2\u00A0mammary\u00C2\u00A0tumor\u00C2\u00A0models\u00C2\u00A0.............................................................................\u00C2\u00A031\u00C2\u00A0 1.10\u00C2\u00A0 Aims\u00C2\u00A0of\u00C2\u00A0study\u00C2\u00A0...................................................................................................................................................\u00C2\u00A033\u00C2\u00A0 CHAPTER\u00C2\u00A02\u00C2\u00A0:\u00C2\u00A0MATERIALS\u00C2\u00A0AND\u00C2\u00A0METHODS\u00C2\u00A0............................................................................\u00C2\u00A035\u00C2\u00A0 2.1\u00C2\u00A0 Mice\u00C2\u00A0.....................................................................................................................................................................\u00C2\u00A035\u00C2\u00A0 2.2\u00C2\u00A0 Media\u00C2\u00A0...................................................................................................................................................................\u00C2\u00A035\u00C2\u00A0 2.3\u00C2\u00A0 Reagents\u00C2\u00A0and\u00C2\u00A0cytokines\u00C2\u00A0...............................................................................................................................\u00C2\u00A036\u00C2\u00A0 2.4\u00C2\u00A0 Isolation\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0............................................................................................................................\u00C2\u00A037\u00C2\u00A0 2.4.1\u00C2\u00A0 Peritoneal\u00C2\u00A0macrophages\u00C2\u00A0.............................................................................................................\u00C2\u00A037\u00C2\u00A0 2.4.2\u00C2\u00A0 Splenic,\u00C2\u00A0pulmonary,\u00C2\u00A0or\u00C2\u00A0tumor\u00E2\u0080\u0090associated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0..........................................\u00C2\u00A037\u00C2\u00A0 2.5\u00C2\u00A0 Flow\u00C2\u00A0cytometry\u00C2\u00A0...............................................................................................................................................\u00C2\u00A038\u00C2\u00A0 2.6\u00C2\u00A0 T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0assay\u00C2\u00A0and\u00C2\u00A0cytokine\u00C2\u00A0assays\u00C2\u00A0...................................................................................\u00C2\u00A040\u00C2\u00A0 vii 2.7\u00C2\u00A0 In\u00C2\u00A0vitro\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0skewing\u00C2\u00A0.......................................................................................................................................\u00C2\u00A041\u00C2\u00A0 2.8\u00C2\u00A0 SDS\u00E2\u0080\u0090PAGE\u00C2\u00A0and\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0....................................................................................................\u00C2\u00A041\u00C2\u00A0 2.9\u00C2\u00A0 Viability\u00C2\u00A0and\u00C2\u00A0morphology\u00C2\u00A0assays\u00C2\u00A0.............................................................................................................\u00C2\u00A042\u00C2\u00A0 2.10\u00C2\u00A0 Nitric\u00C2\u00A0oxide\u00C2\u00A0assay\u00C2\u00A0...........................................................................................................................................\u00C2\u00A043\u00C2\u00A0 2.11\u00C2\u00A0 In\u00C2\u00A0vitro\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0and\u00C2\u00A0stimulation\u00C2\u00A0.........................................................................................\u00C2\u00A043\u00C2\u00A0 2.12\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0ELISA\u00C2\u00A0......................................................................................................................................................\u00C2\u00A044\u00C2\u00A0 2.13\u00C2\u00A0 Tumor\u00C2\u00A0models\u00C2\u00A0.................................................................................................................................................\u00C2\u00A044\u00C2\u00A0 2.14\u00C2\u00A0 Serum\u00C2\u00A0treatment\u00C2\u00A0for\u00C2\u00A0MDSC\u00C2\u00A0assay\u00C2\u00A0studies............................................................................................\u00C2\u00A045\u00C2\u00A0 2.15\u00C2\u00A0 ROS\u00C2\u00A0Detection\u00C2\u00A0..................................................................................................................................................\u00C2\u00A045\u00C2\u00A0 2.16\u00C2\u00A0 Clonogenic\u00C2\u00A0assays\u00C2\u00A0..........................................................................................................................................\u00C2\u00A046\u00C2\u00A0 2.17\u00C2\u00A0 Immunofluorescence\u00C2\u00A0....................................................................................................................................\u00C2\u00A046\u00C2\u00A0 2.18\u00C2\u00A0 Statistical\u00C2\u00A0analysis\u00C2\u00A0.........................................................................................................................................\u00C2\u00A047\u00C2\u00A0 CHAPTER\u00C2\u00A03\u00C2\u00A0:\u00C2\u00A0TOLL\u00C2\u00ADLIKE\u00C2\u00A0RECEPTOR\u00C2\u00A0AGONISTS\u00C2\u00A0THAT\u00C2\u00A0INDUCE\u00C2\u00A0IFN\u00C2\u00AD\u00CE\u00B2\u00C2\u00A0ABROGATE\u00C2\u00A0 RESIDENT\u00C2\u00A0MACROPHAGE\u00C2\u00A0SUPPRESSION\u00C2\u00A0OF\u00C2\u00A0T\u00C2\u00A0CELLS\u00C2\u00A0.......................................................\u00C2\u00A048\u00C2\u00A0 3.1\u00C2\u00A0 Introduction\u00C2\u00A0.....................................................................................................................................................\u00C2\u00A048\u00C2\u00A0 3.2\u00C2\u00A0 Results\u00C2\u00A0................................................................................................................................................................\u00C2\u00A049\u00C2\u00A0 3.2.1\u00C2\u00A0 Characterization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0PC\u00C2\u00A0cells\u00C2\u00A0.........................\u00C2\u00A049\u00C2\u00A0 3.2.2\u00C2\u00A0 Resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0phenotype\u00C2\u00A0...........................................................................\u00C2\u00A050\u00C2\u00A0 3.2.3\u00C2\u00A0 Resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0and\u00C2\u00A0cytokine\u00C2\u00A0production\u00C2\u00A0.....................\u00C2\u00A051\u00C2\u00A0 3.2.4\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0contact\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0.................\u00C2\u00A054\u00C2\u00A0 3.2.5\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090induced\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0.......................\u00C2\u00A057\u00C2\u00A0 3.2.6\u00C2\u00A0 Pre\u00E2\u0080\u0090treatment\u00C2\u00A0 with\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 CpG\u00C2\u00A0 or\u00C2\u00A0 PGN,\u00C2\u00A0 decreases\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0NO\u00C2\u00A0and\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0..........................................................\u00C2\u00A059\u00C2\u00A0 3.2.7\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 contributes\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 reduced\u00C2\u00A0 suppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0..............................................................................................................................\u00C2\u00A062\u00C2\u00A0 3.2.8\u00C2\u00A0 Inhibition\u00C2\u00A0of\u00C2\u00A0Arg1\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0...\u00C2\u00A065\u00C2\u00A0 3.3\u00C2\u00A0 Discussion\u00C2\u00A0.........................................................................................................................................................\u00C2\u00A066\u00C2\u00A0 CHAPTER\u00C2\u00A0 4\u00C2\u00A0 :\u00C2\u00A0 SERUM\u00C2\u00A0 INHIBITS\u00C2\u00A0 THE\u00C2\u00A0 IMMUNOSUPPRESSIVE\u00C2\u00A0 FUNCTION\u00C2\u00A0 OF\u00C2\u00A0 MYELOID\u00C2\u00ADDERIVED\u00C2\u00A0 SUPPRESSOR\u00C2\u00A0 CELLS\u00C2\u00A0 ISOLATED\u00C2\u00A0 FROM\u00C2\u00A0 4T1\u00C2\u00A0 TUMOR\u00C2\u00AD BEARING\u00C2\u00A0MICE\u00C2\u00A0...............................................................................................................................\u00C2\u00A071\u00C2\u00A0 4.1\u00C2\u00A0 Introduction\u00C2\u00A0.....................................................................................................................................................\u00C2\u00A071\u00C2\u00A0 4.2\u00C2\u00A0 Results\u00C2\u00A0................................................................................................................................................................\u00C2\u00A072\u00C2\u00A0 4.2.1\u00C2\u00A0 Validation\u00C2\u00A0of\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0assay\u00C2\u00A0systems\u00C2\u00A0................................................\u00C2\u00A072\u00C2\u00A0 viii 4.2.2\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 only\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 under\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 conditions\u00C2\u00A0..........................................................................................................................................\u00C2\u00A073\u00C2\u00A0 4.2.3\u00C2\u00A0 Serum\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0increase\u00C2\u00A0the\u00C2\u00A0viability\u00C2\u00A0of\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0....................................\u00C2\u00A075\u00C2\u00A0 4.2.4\u00C2\u00A0 Serum\u00C2\u00A0directly\u00C2\u00A0inhibits\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0............................\u00C2\u00A076\u00C2\u00A0 4.2.5\u00C2\u00A0 FCS\u00C2\u00A0inhibits\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0dose\u00E2\u0080\u0090dependent\u00C2\u00A0manner\u00C2\u00A0..................\u00C2\u00A076\u00C2\u00A0 4.2.6\u00C2\u00A0 Effect\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0serum\u00C2\u00A0treatments\u00C2\u00A0on\u00C2\u00A0MDSC\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0......................\u00C2\u00A078\u00C2\u00A0 4.2.7\u00C2\u00A0 BSA\u00C2\u00A0blunts\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0.....................................................\u00C2\u00A080\u00C2\u00A0 4.2.8\u00C2\u00A0 BSA\u00C2\u00A0antagonizes\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0by\u00C2\u00A0inhibiting\u00C2\u00A0ROS\u00C2\u00A0 production\u00C2\u00A0.........................................................................................................................................\u00C2\u00A082\u00C2\u00A0 4.3\u00C2\u00A0 Discussion\u00C2\u00A0.........................................................................................................................................................\u00C2\u00A084\u00C2\u00A0 CHAPTER\u00C2\u00A0 5\u00C2\u00A0 :\u00C2\u00A0 MACROPHAGES\u00C2\u00A0 ARE\u00C2\u00A0 MORE\u00C2\u00A0 POTENT\u00C2\u00A0 IMMUNE\u00C2\u00A0 SUPPRESSORS\u00C2\u00A0 THAN\u00C2\u00A0 MYELOID\u00C2\u00ADDERIVED\u00C2\u00A0 SUPPRESSOR\u00C2\u00A0 CELLS\u00C2\u00A0 IN\u00C2\u00A0 MURINE\u00C2\u00A0 METASTATIC\u00C2\u00A0 MAMMARY\u00C2\u00A0CARCINOMA\u00C2\u00A0.............................................................................................................\u00C2\u00A089\u00C2\u00A0 5.1\u00C2\u00A0 Introduction\u00C2\u00A0.....................................................................................................................................................\u00C2\u00A089\u00C2\u00A0 5.2\u00C2\u00A0 Results\u00C2\u00A0................................................................................................................................................................\u00C2\u00A091\u00C2\u00A0 5.2.1\u00C2\u00A0 Characterization\u00C2\u00A0of\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0 tumor\u00C2\u00A0models\u00C2\u00A0...................................................................................................................................\u00C2\u00A091\u00C2\u00A0 5.2.2\u00C2\u00A0 4T1\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0induce\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0..........................................................\u00C2\u00A093\u00C2\u00A0 5.2.3\u00C2\u00A0 Phenotypic\u00C2\u00A0 characterization\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 versus\u00C2\u00A0 tumor\u00E2\u0080\u0090 bearing\u00C2\u00A0mice\u00C2\u00A0.....................................................................................................................................\u00C2\u00A095\u00C2\u00A0 5.2.3.1\u00C2\u00A0 TAMs\u00C2\u00A0.................................................................................................................................\u00C2\u00A095\u00C2\u00A0 5.2.3.2\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0..................................................................................................................................\u00C2\u00A097\u00C2\u00A0 5.2.3.3\u00C2\u00A0 Splenic\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0.....................................................................................................................\u00C2\u00A097\u00C2\u00A0 5.2.3.4\u00C2\u00A0 Splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0.......................................................................................................\u00C2\u00A0100\u00C2\u00A0 5.2.4\u00C2\u00A0 Summary\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0tumors\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0phenotypes\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0.........\u00C2\u00A0100\u00C2\u00A0 5.2.5\u00C2\u00A0 Comparison\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 versus\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0.....................................................................................................\u00C2\u00A0102\u00C2\u00A0 5.2.5.1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0...............................................................................................................................\u00C2\u00A0102\u00C2\u00A0 5.2.5.2\u00C2\u00A0 TAMs\u00C2\u00A0..............................................................................................................................\u00C2\u00A0103\u00C2\u00A0 5.2.5.3\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0.............................................................................................................................\u00C2\u00A0103\u00C2\u00A0 5.2.5.4\u00C2\u00A0 Splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0.......................................................................................................\u00C2\u00A0107\u00C2\u00A0 5.2.5.5\u00C2\u00A0 Gr1+\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0different\u00C2\u00A0tissues\u00C2\u00A0.........................................................................\u00C2\u00A0109\u00C2\u00A0 5.2.6\u00C2\u00A0 Summary\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0populations\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0.............................................................\u00C2\u00A0111\u00C2\u00A0 ix 5.2.7\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0TAMs,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0decrease\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0.............\u00C2\u00A0119\u00C2\u00A0 5.2.8\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0contact\u00E2\u0080\u0090independent\u00C2\u00A0mechanism\u00C2\u00A0..\u00C2\u00A0121\u00C2\u00A0 5.2.9\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 suppress\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 NO\u00E2\u0080\u0090 dependent\u00C2\u00A0mechanism\u00C2\u00A0..............................................................................................................\u00C2\u00A0123\u00C2\u00A0 5.2.10\u00C2\u00A0 The\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0abrogated\u00C2\u00A0by\u00C2\u00A0TLR\u00C2\u00A0 stimulation\u00C2\u00A0.....................................................................................................................................\u00C2\u00A0125\u00C2\u00A0 5.2.11\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086\u00C2\u00A0suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0reversed\u00C2\u00A0by\u00C2\u00A0N\u00E2\u0080\u0090acetyl\u00E2\u0080\u0090cysteine\u00C2\u00A0..........................\u00C2\u00A0126\u00C2\u00A0 5.2.12\u00C2\u00A0 The\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0pulmonary\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0inhibited\u00C2\u00A0 by\u00C2\u00A0catalase\u00C2\u00A0......................................................................................................................................\u00C2\u00A0128\u00C2\u00A0 5.2.13\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 different\u00C2\u00A0 ROS\u00E2\u0080\u0090mediated\u00C2\u00A0 mechanisms\u00C2\u00A0...................................................................................................................................\u00C2\u00A0130\u00C2\u00A0 5.2.14\u00C2\u00A0 ATRA\u00C2\u00A0increases\u00C2\u00A0lung\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A04T1\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0...................................\u00C2\u00A0133\u00C2\u00A0 5.2.15\u00C2\u00A0 ATRA\u00C2\u00A0 increases\u00C2\u00A0 the\u00C2\u00A0 proportion\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 much\u00C2\u00A0 more\u00C2\u00A0 immunosuppressive\u00C2\u00A0than\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0lungs\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0.......\u00C2\u00A0135\u00C2\u00A0 5.3\u00C2\u00A0 Discussion\u00C2\u00A0......................................................................................................................................................\u00C2\u00A0139\u00C2\u00A0 CHAPTER\u00C2\u00A06\u00C2\u00A0:\u00C2\u00A0SUMMARY\u00C2\u00A0AND\u00C2\u00A0FUTURE\u00C2\u00A0DIRECTIONS\u00C2\u00A0......................................................\u00C2\u00A0149\u00C2\u00A0 REFERENCES\u00C2\u00A0................................................................................................................................\u00C2\u00A0160\u00C2\u00A0 x LIST\u00C2\u00A0OF\u00C2\u00A0TABLES\u00C2\u00A0 \u00C2\u00A0 CHAPTER\u00C2\u00A01\u00C2\u00A0 Table\u00C2\u00A01.1\u00C2\u00A0 Summary\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0known\u00C2\u00A0TLRs\u00C2\u00A0..........................................................................\u00C2\u00A08\u00C2\u00A0 Table\u00C2\u00A01.2\u00C2\u00A0 Diversity\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0tissues\u00C2\u00A0.......................................................................................\u00C2\u00A019\u00C2\u00A0 \u00C2\u00A0 CHAPTER\u00C2\u00A02\u00C2\u00A0 Table\u00C2\u00A02.1\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0antibodies\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0thesis\u00C2\u00A0......................................................................................\u00C2\u00A039\u00C2\u00A0 \u00C2\u00A0 xi LIST\u00C2\u00A0OF\u00C2\u00A0FIGURES\u00C2\u00A0 \u00C2\u00A0 CHAPTER\u00C2\u00A01\u00C2\u00A0 Figure\u00C2\u00A01.1\u00C2\u00A0 Immune\u00C2\u00A0 cells\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 promotion\u00C2\u00A0 and\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0................................................................................................................................................\u00C2\u00A013\u00C2\u00A0 Figure\u00C2\u00A01.2\u00C2\u00A0 Development\u00C2\u00A0and\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mononuclear\u00C2\u00A0phagocytic\u00C2\u00A0lineage\u00C2\u00A0......................\u00C2\u00A017\u00C2\u00A0 Figure\u00C2\u00A01.3\u00C2\u00A0 Polarization\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0phenotypes\u00C2\u00A0.................................................................................................\u00C2\u00A021\u00C2\u00A0 Figure\u00C2\u00A01.4\u00C2\u00A0 TAMs\u00C2\u00A0promote\u00C2\u00A0tumorigenesis\u00C2\u00A0via\u00C2\u00A0multiple\u00C2\u00A0mechanisms\u00C2\u00A0..............................................\u00C2\u00A025\u00C2\u00A0 \u00C2\u00A0 CHAPTER\u00C2\u00A03\u00C2\u00A0 Figure\u00C2\u00A03.1\u00C2\u00A0 Resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0possess\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0..............................................\u00C2\u00A050\u00C2\u00A0 Figure\u00C2\u00A03.2\u00C2\u00A0 Resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0phenotype\u00C2\u00A0and\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0skewed\u00C2\u00A0to\u00C2\u00A0either\u00C2\u00A0M1\u00C2\u00A0or\u00C2\u00A0 M2\u00C2\u00A0with\u00C2\u00A0appropriate\u00C2\u00A0stimulation\u00C2\u00A0............................................................................................\u00C2\u00A051\u00C2\u00A0 Figure\u00C2\u00A03.3\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 and\u00C2\u00A0 cytokine\u00C2\u00A0 production\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 dose\u00E2\u0080\u0090 dependent\u00C2\u00A0manner\u00C2\u00A0........................................................................................................................\u00C2\u00A053\u00C2\u00A0 Figure\u00C2\u00A03.4\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0increase\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0dead\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0.........................................................\u00C2\u00A054\u00C2\u00A0 Figure\u00C2\u00A03.5\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0contact\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0.................\u00C2\u00A056\u00C2\u00A0 Figure\u00C2\u00A03.6\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0through\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0...........................................\u00C2\u00A058\u00C2\u00A0 Figure\u00C2\u00A03.7\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0...............................................................................\u00C2\u00A059\u00C2\u00A0 Figure\u00C2\u00A03.8\u00C2\u00A0 Pre\u00E2\u0080\u0090treatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0agonists\u00C2\u00A0that\u00C2\u00A0signal\u00C2\u00A0through\u00C2\u00A0TRIF\u00C2\u00A0desensitizes\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0subsequent\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0stimulation,\u00C2\u00A0decreasing\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0NO\u00C2\u00A0 and\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0......................................................................................................................\u00C2\u00A061\u00C2\u00A0 Figure\u00C2\u00A03.9\u00C2\u00A0 Pre\u00E2\u0080\u0090treatment\u00C2\u00A0with\u00C2\u00A0LPS,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CpG,\u00C2\u00A0reduces\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0sensitivity\u00C2\u00A0to\u00C2\u00A0subsequent\u00C2\u00A0 stimulation\u00C2\u00A0........................................................................................................................................\u00C2\u00A062\u00C2\u00A0 Figure\u00C2\u00A03.10\u00C2\u00A0 LPS\u00C2\u00A0stimulation\u00C2\u00A0induces\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0secondary\u00C2\u00A0factor\u00C2\u00A0that\u00C2\u00A0inhibits\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090 induced\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0................................................................................................\u00C2\u00A063\u00C2\u00A0 Figure\u00C2\u00A03.11\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 contributes\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 reduced\u00C2\u00A0 suppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0..............................................................................................................................\u00C2\u00A064\u00C2\u00A0 Figure\u00C2\u00A03.12\u00C2\u00A0 The\u00C2\u00A0Arg1\u00C2\u00A0inhibitor\u00C2\u00A0BEC\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0....\u00C2\u00A065\u00C2\u00A0 Figure\u00C2\u00A03.13\u00C2\u00A0 The\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 resident\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 is\u00C2\u00A0 abrogated\u00C2\u00A0 when\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0PGN\u00C2\u00A0........................\u00C2\u00A067\u00C2\u00A0 \u00C2\u00A0 xii CHAPTER\u00C2\u00A04\u00C2\u00A0 Figure\u00C2\u00A04.1\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090stimulated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0proliferate\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0serum\u00E2\u0080\u0090free\u00C2\u00A0 and\u00C2\u00A0serum\u00E2\u0080\u0090containing\u00C2\u00A0conditions\u00C2\u00A0...........................................................................................\u00C2\u00A073\u00C2\u00A0 Figure\u00C2\u00A04.2\u00C2\u00A0 MDSCs\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 only\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 under\u00C2\u00A0serum\u00E2\u0080\u0090free\u00C2\u00A0conditions\u00C2\u00A0....................................................................................................\u00C2\u00A074\u00C2\u00A0 Figure\u00C2\u00A04.3\u00C2\u00A0 Serum\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0alter\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0MDSC\u00C2\u00A0subtypes\u00C2\u00A0............................\u00C2\u00A075\u00C2\u00A0 Figure\u00C2\u00A04.4\u00C2\u00A0 Serum\u00C2\u00A0directly\u00C2\u00A0inhibits\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0........................\u00C2\u00A077\u00C2\u00A0 Figure\u00C2\u00A04.5\u00C2\u00A0 The\u00C2\u00A0 inhibitory\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0on\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 are\u00C2\u00A0dose\u00E2\u0080\u0090dependent\u00C2\u00A0......................................................................................................................\u00C2\u00A078\u00C2\u00A0 Figure\u00C2\u00A04.6\u00C2\u00A0 The\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0on\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0cannot\u00C2\u00A0be\u00C2\u00A0reversed\u00C2\u00A0by\u00C2\u00A0filtration,\u00C2\u00A0 dialyzation,\u00C2\u00A0or\u00C2\u00A0heat\u00C2\u00A0inactivation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0serum\u00C2\u00A0...................................................................\u00C2\u00A079\u00C2\u00A0 Figure\u00C2\u00A04.7\u00C2\u00A0 BSA\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0..................\u00C2\u00A081\u00C2\u00A0 Figure\u00C2\u00A04.8\u00C2\u00A0 Serum\u00C2\u00A0albumin\u00C2\u00A0restricts\u00C2\u00A0ROS\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0...........................\u00C2\u00A083\u00C2\u00A0 \u00C2\u00A0 CHAPTER\u00C2\u00A05\u00C2\u00A0 Figure\u00C2\u00A05.1\u00C2\u00A0 Characterization\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0...........................................................\u00C2\u00A092\u00C2\u00A0 Figure\u00C2\u00A05.2\u00C2\u00A0 Visualization\u00C2\u00A0 of\u00C2\u00A0 hypoxia\u00C2\u00A0 and\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 infiltration\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 tumors.................................................................................................................................................\u00C2\u00A093\u00C2\u00A0 Figure\u00C2\u00A05.3\u00C2\u00A0 Induction\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0......................................................\u00C2\u00A094\u00C2\u00A0 Figure\u00C2\u00A05.4\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0TAMs\u00C2\u00A0...................................................................................\u00C2\u00A096\u00C2\u00A0 Figure\u00C2\u00A05.5\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0....................................................................................\u00C2\u00A098\u00C2\u00A0 Figure\u00C2\u00A05.6\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0..................................................................................\u00C2\u00A099\u00C2\u00A0 Figure\u00C2\u00A05.7\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0.........................................................\u00C2\u00A0101\u00C2\u00A0 Figure\u00C2\u00A05.8\u00C2\u00A0 Analysis\u00C2\u00A0of\u00C2\u00A0protein\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0...................\u00C2\u00A0102\u00C2\u00A0 Figure\u00C2\u00A05.9\u00C2\u00A0 Tumors\u00C2\u00A0augment\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0...................................\u00C2\u00A0104\u00C2\u00A0 Figure\u00C2\u00A05.10\u00C2\u00A0 67NR\u00C2\u00A0TAMs\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0immunosuppressive\u00C2\u00A0than\u00C2\u00A04T1\u00C2\u00A0TAMs\u00C2\u00A0....................................\u00C2\u00A0105\u00C2\u00A0 Figure\u00C2\u00A05.11\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0only\u00C2\u00A0immunosuppressive\u00C2\u00A0at\u00C2\u00A0high\u00C2\u00A0concentrations\u00C2\u00A0...................................\u00C2\u00A0106\u00C2\u00A0 Figure\u00C2\u00A05.12\u00C2\u00A0 4T1\u00C2\u00A0tumors\u00C2\u00A0enhance\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0....\u00C2\u00A0108\u00C2\u00A0 Figure\u00C2\u00A05.13\u00C2\u00A0 4T1\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 different\u00C2\u00A0 tissues\u00C2\u00A0 are\u00C2\u00A0 equally\u00C2\u00A0 immunosuppressive\u00C2\u00A0..................................................................................................................\u00C2\u00A0110\u00C2\u00A0 Figure\u00C2\u00A05.14\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 more\u00C2\u00A0 potent\u00C2\u00A0 suppressors\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 than\u00C2\u00A0 4T1\u00C2\u00A0 MDSCs\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis\u00C2\u00A0.........................................................................................................\u00C2\u00A0112\u00C2\u00A0 xiii Figure\u00C2\u00A05.15\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0 suppressors\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 cytokine\u00C2\u00A0production\u00C2\u00A0 than\u00C2\u00A0 4T1\u00C2\u00A0MDSCs\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis\u00C2\u00A0...............................................................................................\u00C2\u00A0113\u00C2\u00A0 Figure\u00C2\u00A05.16\u00C2\u00A0 Visualization\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 populations\u00C2\u00A0....................................................................................................................................\u00C2\u00A0115\u00C2\u00A0 Figure\u00C2\u00A05.17\u00C2\u00A0 Summary\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0to\u00C2\u00A0inhibit\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 division\u00C2\u00A0as\u00C2\u00A0determined\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometric\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0CFSE\u00C2\u00A0intensity\u00C2\u00A0................\u00C2\u00A0117\u00C2\u00A0 Figure\u00C2\u00A05.18\u00C2\u00A0 Comparison\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0the\u00C2\u00A0 proportion\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0...................................................................................................................\u00C2\u00A0118\u00C2\u00A0 Figure\u00C2\u00A05.19\u00C2\u00A0 Effect\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0on\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0..................................................\u00C2\u00A0120\u00C2\u00A0 Figure\u00C2\u00A05.20\u00C2\u00A0 At\u00C2\u00A0high\u00C2\u00A0concentrations,\u00C2\u00A0both\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0contact\u00E2\u0080\u0090 independent\u00C2\u00A0mechanisms\u00C2\u00A0........................................................................................................\u00C2\u00A0122\u00C2\u00A0 Figure\u00C2\u00A05.21\u00C2\u00A0 Control\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0but\u00C2\u00A0no\u00C2\u00A0other\u00C2\u00A0cell\u00C2\u00A0 types\u00C2\u00A0 tested,\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 responses\u00C2\u00A0via\u00C2\u00A0 NO\u00C2\u00A0production\u00C2\u00A0..............................................................................................................................\u00C2\u00A0124\u00C2\u00A0 Figure\u00C2\u00A05.22\u00C2\u00A0 The\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 reversed\u00C2\u00A0 by\u00C2\u00A0 pre\u00E2\u0080\u0090 treatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0ligands\u00C2\u00A0....................................................................................................\u00C2\u00A0125\u00C2\u00A0 Figure\u00C2\u00A05.23\u00C2\u00A0 NAC\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0...................................\u00C2\u00A0127\u00C2\u00A0 Figure\u00C2\u00A05.24\u00C2\u00A0 The\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0reversed\u00C2\u00A0by\u00C2\u00A0pre\u00E2\u0080\u0090 treatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0ligands\u00C2\u00A0....................................................................................................\u00C2\u00A0128\u00C2\u00A0 Figure\u00C2\u00A05.25\u00C2\u00A0 Catalase\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0.........................................\u00C2\u00A0129\u00C2\u00A0 Figure\u00C2\u00A05.26\u00C2\u00A0 4T1\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0ROS\u00E2\u0080\u0090mediated\u00C2\u00A0 mechanisms\u00C2\u00A0...................................................................................................................................\u00C2\u00A0131\u00C2\u00A0 Figure\u00C2\u00A05.27\u00C2\u00A0 Myeloid\u00C2\u00A0cells\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0mechanisms\u00C2\u00A0and\u00C2\u00A0to\u00C2\u00A0 different\u00C2\u00A0degrees\u00C2\u00A0.........................................................................................................................\u00C2\u00A0132\u00C2\u00A0 Figure\u00C2\u00A05.28\u00C2\u00A0 ATRA\u00C2\u00A0 treatment\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 alter\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 but\u00C2\u00A0 increases\u00C2\u00A0 lung\u00C2\u00A0 metastasis\u00C2\u00A0.......................................................................................................................................\u00C2\u00A0134\u00C2\u00A0 Figure\u00C2\u00A05.29\u00C2\u00A0 ATRA\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0change\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0or\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0.\u00C2\u00A0136\u00C2\u00A0 Figure\u00C2\u00A05.30\u00C2\u00A0 ATRA\u00C2\u00A0 increases\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 reduces\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0.........................................................................................................................................\u00C2\u00A0137\u00C2\u00A0 Figure\u00C2\u00A05.31\u00C2\u00A0 ATRA\u00C2\u00A0 induces\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 into\u00C2\u00A0more\u00C2\u00A0 immunosuppressive\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0promotes\u00C2\u00A0lung\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A04TO7\u00C2\u00A0tumor\u00C2\u00A0models\u00C2\u00A0............\u00C2\u00A0138\u00C2\u00A0 \u00C2\u00A0 xiv LIST\u00C2\u00A0OF\u00C2\u00A0ABBREVIATIONS\u00C2\u00A0 \u00C2\u00A0 Ab\u00C2\u00A0 \u00C2\u00A0 antibody\u00C2\u00A0 Ag\u00C2\u00A0 \u00C2\u00A0 antigen\u00C2\u00A0 AP\u00E2\u0080\u00901\u00C2\u00A0 \u00C2\u00A0 activator\u00C2\u00A0protein\u00C2\u00A01\u00C2\u00A0 APC\u00C2\u00A0 \u00C2\u00A0 antigen\u00C2\u00A0presenting\u00C2\u00A0cell\u00C2\u00A0 APCy\u00C2\u00A0 \u00C2\u00A0 allophycocyanin\u00C2\u00A0 AML\u00C2\u00A0 \u00C2\u00A0 acute\u00C2\u00A0myeloid\u00C2\u00A0leukemia\u00C2\u00A0 APL\u00C2\u00A0 \u00C2\u00A0 acute\u00C2\u00A0promyelocytic\u00C2\u00A0leukemia\u00C2\u00A0 Arg1\u00C2\u00A0 \u00C2\u00A0 arginase\u00C2\u00A01\u00C2\u00A0 ATRA\u00C2\u00A0 \u00C2\u00A0 all\u00E2\u0080\u0090trans\u00C2\u00A0retinoic\u00C2\u00A0acid\u00C2\u00A0 \u00CE\u00B2\u00E2\u0080\u0090ME\u00C2\u00A0 \u00C2\u00A0 beta\u00E2\u0080\u0090mercaptoethanol\u00C2\u00A0 Bcl\u00C2\u00A0 \u00C2\u00A0 B\u00C2\u00A0cell\u00C2\u00A0lymphoma\u00C2\u00A0 BEC\u00C2\u00A0 \u00C2\u00A0 [S]\u00E2\u0080\u0090[2\u00E2\u0080\u0090boronoethyl]\u00E2\u0080\u0090L\u00E2\u0080\u0090cysteine\u00E2\u0080\u0090HCl\u00C2\u00A0 BM\u00C2\u00A0 \u00C2\u00A0 bone\u00C2\u00A0marrow\u00C2\u00A0 BSA\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 bovine\u00C2\u00A0serum\u00C2\u00A0albumin\u00C2\u00A0 C/EBP\u00C2\u00A0\u00C2\u00A0 CCAAT/enhancer\u00C2\u00A0binding\u00C2\u00A0protein\u00C2\u00A0 CCL\u00C2\u00A0 \u00C2\u00A0 CC\u00C2\u00A0chemokine\u00C2\u00A0ligand\u00C2\u00A0 CCR\u00C2\u00A0 \u00C2\u00A0 CC\u00C2\u00A0chemokine\u00C2\u00A0receptor\u00C2\u00A0 CD\u00C2\u00A0 \u00C2\u00A0 cluster\u00C2\u00A0of\u00C2\u00A0differentiation\u00C2\u00A0 CFSE\u00C2\u00A0 \u00C2\u00A0 carboxyfluorescein\u00C2\u00A0succinimidyl\u00C2\u00A0ester\u00C2\u00A0 CMP\u00C2\u00A0 \u00C2\u00A0 common\u00C2\u00A0myeloid\u00C2\u00A0progenitor\u00C2\u00A0 COX\u00C2\u00A0 \u00C2\u00A0 cyclooxygenase\u00C2\u00A0 CpG\u00C2\u00A0 \u00C2\u00A0 CpG\u00E2\u0080\u0090dinucleotides\u00C2\u00A0 cpm\u00C2\u00A0 \u00C2\u00A0 counts\u00C2\u00A0per\u00C2\u00A0minute\u00C2\u00A0 CSF\u00E2\u0080\u00901\u00C2\u00A0 \u00C2\u00A0 colony\u00E2\u0080\u0090stimulating\u00C2\u00A0factor\u00C2\u00A01\u00C2\u00A0 CTL\u00C2\u00A0 \u00C2\u00A0 cytotoxic\u00C2\u00A0T\u00C2\u00A0lymphocyte\u00C2\u00A0 CTLA\u00E2\u0080\u00904\u00C2\u00A0 cytotoxic\u00C2\u00A0T\u00E2\u0080\u0090lymphocyte\u00C2\u00A0antigen\u00C2\u00A04\u00C2\u00A0(or\u00C2\u00A0CD152)\u00C2\u00A0 CXCL\u00C2\u00A0 \u00C2\u00A0 CXC\u00C2\u00A0chemokine\u00C2\u00A0ligand\u00C2\u00A0 Cy\u00C2\u00A0 \u00C2\u00A0 cyanine\u00C2\u00A0 xv DAMP\u00C2\u00A0 \u00C2\u00A0 damage\u00E2\u0080\u0090associated\u00C2\u00A0molecular\u00C2\u00A0pattern\u00C2\u00A0molecule\u00C2\u00A0 DAPI\u00C2\u00A0 \u00C2\u00A0 4',6\u00E2\u0080\u0090diamidino\u00E2\u0080\u00902\u00E2\u0080\u0090phenylindole\u00C2\u00A0 DC\u00C2\u00A0 \u00C2\u00A0 dendritic\u00C2\u00A0cell\u00C2\u00A0 dsRNA\u00C2\u00A0\u00C2\u00A0 double\u00C2\u00A0stranded\u00C2\u00A0ribonucleic\u00C2\u00A0acid\u00C2\u00A0 ECM\u00C2\u00A0 \u00C2\u00A0 extracellular\u00C2\u00A0matrix\u00C2\u00A0 EDTA\u00C2\u00A0 \u00C2\u00A0 ethylenediaminetetraacetic\u00C2\u00A0acid\u00C2\u00A0 ELISA\u00C2\u00A0 \u00C2\u00A0 enzyme\u00E2\u0080\u0090linked\u00C2\u00A0immunosorbent\u00C2\u00A0assay\u00C2\u00A0 FA\u00C2\u00A0 \u00C2\u00A0 fatty\u00C2\u00A0acid\u00C2\u00A0 FCS\u00C2\u00A0 \u00C2\u00A0 fetal\u00C2\u00A0calf\u00C2\u00A0serum\u00C2\u00A0 FITC\u00C2\u00A0 \u00C2\u00A0 fluorescein\u00C2\u00A0isothiocyanate\u00C2\u00A0 Flt3L\u00C2\u00A0 \u00C2\u00A0 Flt3\u00C2\u00A0ligand\u00C2\u00A0 G\u00E2\u0080\u0090MDSC\u00C2\u00A0 granulocytic\u00E2\u0080\u0090myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0 GAPDH\u00C2\u00A0 glyceraldehyde\u00C2\u00A03\u00E2\u0080\u0090phosphate\u00C2\u00A0dehydrogenase\u00C2\u00A0 GM\u00E2\u0080\u0090CFU\u00C2\u00A0 granulocyte\u00C2\u00A0macrophage\u00C2\u00A0colony\u00E2\u0080\u0090forming\u00C2\u00A0unit\u00C2\u00A0 GM\u00E2\u0080\u0090CSF\u00C2\u00A0 granulocyte\u00C2\u00A0macrophage\u00C2\u00A0colony\u00E2\u0080\u0090stimulating\u00C2\u00A0factor\u00C2\u00A0 h\u00C2\u00A0 \u00C2\u00A0 hour\u00C2\u00A0 H2O2\u00C2\u00A0 \u00C2\u00A0 hydrogen\u00C2\u00A0peroxide\u00C2\u00A0 HBSS\u00C2\u00A0 \u00C2\u00A0 Hank\u00E2\u0080\u0099s\u00C2\u00A0balanced\u00C2\u00A0salt\u00C2\u00A0solution\u00C2\u00A0 HEPES\u00C2\u00A0\u00C2\u00A0 4\u00E2\u0080\u0090(2\u00E2\u0080\u0090hydroxyethyl)\u00E2\u0080\u00901\u00E2\u0080\u0090piperazineethanesulfonic\u00C2\u00A0acid\u00C2\u00A0 HFN\u00C2\u00A0 \u00C2\u00A0 Hank\u00E2\u0080\u0099s\u00C2\u00A0balanced\u00C2\u00A0salt\u00C2\u00A0solution\u00C2\u00A0containing\u00C2\u00A02%\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A00.05%\u00C2\u00A0azide\u00C2\u00A0 HIF\u00E2\u0080\u00901\u00CE\u00B1\u00C2\u00A0 hypoxia\u00E2\u0080\u0090inducible\u00C2\u00A0factor\u00E2\u0080\u00901\u00C2\u00A0alpha\u00C2\u00A0 HMGB1\u00C2\u00A0 high\u00E2\u0080\u0090mobility\u00C2\u00A0group\u00C2\u00A0protein\u00C2\u00A0B1\u00C2\u00A0 HPC\u00C2\u00A0 \u00C2\u00A0 hematopoietic\u00C2\u00A0progenitor\u00C2\u00A0cell\u00C2\u00A0 HPLC\u00C2\u00A0 \u00C2\u00A0 high\u00E2\u0080\u0090performance\u00C2\u00A0liquid\u00C2\u00A0chromatography\u00C2\u00A0 IDO\u00C2\u00A0 \u00C2\u00A0 indoleamine\u00C2\u00A02,3\u00E2\u0080\u0090dioxygenase\u00C2\u00A0 IFN\u00C2\u00A0 \u00C2\u00A0 interferon\u00C2\u00A0 Ig\u00C2\u00A0 \u00C2\u00A0 immunoglobulin\u00C2\u00A0 I\u00CE\u00BAKi\u00C2\u00A0 \u00C2\u00A0 inducible\u00C2\u00A0I\u00CE\u00BAB\u00C2\u00A0kinase\u00C2\u00A0 IL\u00C2\u00A0 \u00C2\u00A0 interleukin\u00C2\u00A0 IMC\u00C2\u00A0 \u00C2\u00A0 immature\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 xvi IMDM\u00C2\u00A0 \u00C2\u00A0 Iscove\u00E2\u0080\u0099s\u00C2\u00A0modified\u00C2\u00A0Dulbecco\u00E2\u0080\u0099s\u00C2\u00A0medium\u00C2\u00A0 iNOS\u00C2\u00A0 \u00C2\u00A0 inducible\u00C2\u00A0nitric\u00C2\u00A0oxide\u00C2\u00A0synthase\u00C2\u00A0 IP\u00C2\u00A0 \u00C2\u00A0 intraperitoneal\u00C2\u00A0 IRAK1\u00C2\u00A0\u00C2\u00A0 interleukin\u00E2\u0080\u00901\u00C2\u00A0receptor\u00E2\u0080\u0090associated\u00C2\u00A0kinase\u00C2\u00A01\u00C2\u00A0 IV\u00C2\u00A0 \u00C2\u00A0 intravenous\u00C2\u00A0 JAK\u00C2\u00A0 \u00C2\u00A0 Janus\u00C2\u00A0kinase\u00C2\u00A0 kDa\u00C2\u00A0 \u00C2\u00A0 kilodalton\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 \u00C2\u00A0 L\u00E2\u0080\u0090arginine\u00C2\u00A0 LFA\u00E2\u0080\u00901\u00C2\u00A0 \u00C2\u00A0 lymphocyte\u00C2\u00A0function\u00E2\u0080\u0090associated\u00C2\u00A0antigen\u00C2\u00A01\u00C2\u00A0 L\u00E2\u0080\u0090NMMA\u00C2\u00A0 NG\u00E2\u0080\u0090monomethyl\u00E2\u0080\u0090L\u00E2\u0080\u0090arginine\u00C2\u00A0 LAP\u00C2\u00A0 \u00C2\u00A0 latency\u00C2\u00A0associated\u00C2\u00A0peptide\u00C2\u00A0 LPS\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 lipopolysaccharide\u00C2\u00A0 LRR\u00C2\u00A0 \u00C2\u00A0 leucine\u00E2\u0080\u0090rich\u00C2\u00A0repeat\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 \u00C2\u00A0 macrophage\u00C2\u00A0 M1\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0\u00C2\u00A0 classically\u00C2\u00A0activated\u00C2\u00A0macrophage\u00C2\u00A0 M2\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0\u00C2\u00A0 alternatively\u00C2\u00A0activated\u00C2\u00A0macrophage\u00C2\u00A0 mAb\u00C2\u00A0 \u00C2\u00A0 monoclonal\u00C2\u00A0antibody\u00C2\u00A0 M\u00E2\u0080\u0090CFU\u00C2\u00A0\u00C2\u00A0 macrophage\u00C2\u00A0colony\u00E2\u0080\u0090forming\u00C2\u00A0unit\u00C2\u00A0 M\u00E2\u0080\u0090CSF\u00C2\u00A0\u00C2\u00A0 macrophage\u00C2\u00A0colony\u00E2\u0080\u0090stimulating\u00C2\u00A0factor\u00C2\u00A0 M\u00E2\u0080\u0090CSFR\u00C2\u00A0 macrophage\u00C2\u00A0colony\u00E2\u0080\u0090stimulating\u00C2\u00A0factor\u00C2\u00A0receptor\u00C2\u00A0 M\u00E2\u0080\u0090MDSC\u00C2\u00A0 monocytic\u00E2\u0080\u0090myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0 MAL\u00C2\u00A0 \u00C2\u00A0 MyD88\u00C2\u00A0adaptor\u00E2\u0080\u0090like\u00C2\u00A0 MCP\u00E2\u0080\u00901\u00C2\u00A0\u00C2\u00A0 monocyte\u00C2\u00A0chemotactic\u00C2\u00A0protein\u00E2\u0080\u00901\u00C2\u00A0 mDC\u00C2\u00A0 \u00C2\u00A0 myeloid\u00C2\u00A0dendritic\u00C2\u00A0cell\u00C2\u00A0 MDSC\u00C2\u00A0 \u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0 MFI\u00C2\u00A0 \u00C2\u00A0 mean\u00C2\u00A0fluorescence\u00C2\u00A0intensity\u00C2\u00A0 MHC\u00C2\u00A0 \u00C2\u00A0 major\u00C2\u00A0histocompatibility\u00C2\u00A0complex\u00C2\u00A0 MHCII\u00C2\u00A0\u00C2\u00A0 major\u00C2\u00A0histocompatibility\u00C2\u00A0complex\u00C2\u00A0class\u00C2\u00A0II\u00C2\u00A0 min\u00C2\u00A0 \u00C2\u00A0 minute\u00C2\u00A0 MMP\u00C2\u00A0 \u00C2\u00A0 matrix\u00C2\u00A0metalloproteinase\u00C2\u00A0 xvii MPS\u00C2\u00A0 \u00C2\u00A0 mononuclear\u00C2\u00A0phagocyte\u00C2\u00A0system\u00C2\u00A0 MSF\u00C2\u00A0 \u00C2\u00A0 migration\u00C2\u00A0stimulating\u00C2\u00A0factor\u00C2\u00A0 MTG\u00C2\u00A0 \u00C2\u00A0 monothioglycerate\u00C2\u00A0 MyD88\u00C2\u00A0 myeloid\u00C2\u00A0differentiation\u00C2\u00A0factor\u00C2\u00A088\u00C2\u00A0 NAC\u00C2\u00A0 \u00C2\u00A0 N\u00E2\u0080\u0090acetyl\u00C2\u00A0cysteine\u00C2\u00A0 NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0\u00C2\u00A0 nuclear factor kappa-light-chain-enhancer of activated B cells\u00C2\u00A0 NK\u00C2\u00A0 \u00C2\u00A0 natural\u00C2\u00A0killer\u00C2\u00A0cell\u00C2\u00A0 NKT\u00C2\u00A0 \u00C2\u00A0 natural\u00C2\u00A0killer\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 NO\u00C2\u00A0 \u00C2\u00A0 nitric\u00C2\u00A0oxide\u00C2\u00A0 NO2\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 nitrite\u00C2\u00A0 O2\u00E2\u0080\u0090\u00C2\u00A0 \u00C2\u00A0 superoxide\u00C2\u00A0 O/N\u00C2\u00A0 \u00C2\u00A0 overnight\u00C2\u00A0 OCT\u00C2\u00A0 \u00C2\u00A0 optimal\u00C2\u00A0cutting\u00C2\u00A0temperature\u00C2\u00A0 ONOO\u00E2\u0080\u0090\u00C2\u00A0\u00C2\u00A0 peroxynitrite\u00C2\u00A0 OVA\u00C2\u00A0 \u00C2\u00A0 ovalbumin\u00C2\u00A0 PAGE\u00C2\u00A0 \u00C2\u00A0 polyacrylamide\u00C2\u00A0gel\u00C2\u00A0electrophoresis\u00C2\u00A0 PAMP\u00C2\u00A0 \u00C2\u00A0 pathogen\u00E2\u0080\u0090associated\u00C2\u00A0molecular\u00C2\u00A0pattern\u00C2\u00A0 PBS\u00C2\u00A0 \u00C2\u00A0 phosphate\u00C2\u00A0buffered\u00C2\u00A0saline\u00C2\u00A0 PC\u00C2\u00A0 \u00C2\u00A0 peritoneal\u00C2\u00A0cavity\u00C2\u00A0 PD\u00E2\u0080\u0090L1\u00C2\u00A0 \u00C2\u00A0 programmed\u00C2\u00A0death\u00C2\u00A0ligand\u00C2\u00A01\u00C2\u00A0 pDC\u00C2\u00A0 \u00C2\u00A0 plasmacytoid\u00C2\u00A0dendritic\u00C2\u00A0cell\u00C2\u00A0 PE\u00C2\u00A0 \u00C2\u00A0 phycoerythrin\u00C2\u00A0 PFA\u00C2\u00A0 \u00C2\u00A0 paraformaldehyde\u00C2\u00A0 PG\u00C2\u00A0 \u00C2\u00A0 prostaglandin\u00C2\u00A0 PGN\u00C2\u00A0 \u00C2\u00A0 peptidoglycan\u00C2\u00A0 PI\u00C2\u00A0 \u00C2\u00A0 propidium\u00C2\u00A0iodide\u00C2\u00A0 PM\u00CF\u0086\u00C2\u00A0 \u00C2\u00A0 peritoneal\u00C2\u00A0macrophage\u00C2\u00A0 PMN\u00C2\u00A0 \u00C2\u00A0 polymorphonuclear\u00C2\u00A0neutrophil\u00C2\u00A0 Poly\u00C2\u00A0I:C\u00C2\u00A0 polyinosinic:polycytidylic\u00C2\u00A0 PRR\u00C2\u00A0 \u00C2\u00A0 pattern\u00C2\u00A0recognition\u00C2\u00A0receptor\u00C2\u00A0 xviii PTIO\u00C2\u00A0 \u00C2\u00A0 carboxy\u00E2\u0080\u00902\u00E2\u0080\u0090phenyl\u00E2\u0080\u00904,4,5,5\u00E2\u0080\u0090tetramethylimidazoline\u00E2\u0080\u00901\u00E2\u0080\u0090oxyl\u00E2\u0080\u00903\u00E2\u0080\u0090oxide\u00C2\u00A0 RC\u00C2\u00A0 \u00C2\u00A0 responder\u00C2\u00A0control\u00C2\u00A0 ROS\u00C2\u00A0 \u00C2\u00A0 reactive\u00C2\u00A0oxygen\u00C2\u00A0species\u00C2\u00A0 rm\u00C2\u00A0 \u00C2\u00A0 recombinant\u00C2\u00A0mouse\u00C2\u00A0 RNS\u00C2\u00A0 \u00C2\u00A0 reactive\u00C2\u00A0nitrogen\u00C2\u00A0species\u00C2\u00A0 RPMI\u00C2\u00A0 \u00C2\u00A0 Roswell\u00C2\u00A0Park\u00C2\u00A0Memorial\u00C2\u00A0Institute\u00C2\u00A0 SCCVII\u00C2\u00A0\u00C2\u00A0 squamous\u00C2\u00A0cell\u00C2\u00A0carcinoma\u00C2\u00A0VII\u00C2\u00A0 SDS\u00C2\u00A0 \u00C2\u00A0 sodium\u00C2\u00A0dodecyl\u00C2\u00A0sulfate\u00C2\u00A0 SEM\u00C2\u00A0 \u00C2\u00A0 standard\u00C2\u00A0error\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0 SOD\u00C2\u00A0 \u00C2\u00A0 superoxide\u00C2\u00A0dismutase\u00C2\u00A0 SpM\u00CF\u0086\u00C2\u00A0 \u00C2\u00A0 splenic\u00C2\u00A0macrophage\u00C2\u00A0 STAT\u00C2\u00A0 \u00C2\u00A0 signal\u00C2\u00A0transducer\u00C2\u00A0and\u00C2\u00A0activator\u00C2\u00A0of\u00C2\u00A0transcription\u00C2\u00A0 TAA\u00C2\u00A0 \u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0antigen\u00C2\u00A0 TAM\u00C2\u00A0 \u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0macrophage\u00C2\u00A0 TBK1\u00C2\u00A0 \u00C2\u00A0 TANK\u00E2\u0080\u0090binding\u00C2\u00A0kinase\u00C2\u00A01\u00C2\u00A0 TCR\u00C2\u00A0 \u00C2\u00A0 T\u00C2\u00A0cell\u00C2\u00A0receptor\u00C2\u00A0 TF\u00C2\u00A0 \u00C2\u00A0 transcription\u00C2\u00A0factor\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 \u00C2\u00A0 transforming\u00C2\u00A0growth\u00C2\u00A0factor\u00C2\u00A0beta\u00C2\u00A0 Th\u00C2\u00A0 \u00C2\u00A0 T\u00C2\u00A0helper\u00C2\u00A0cell\u00C2\u00A0 thy\u00C2\u00A0 \u00C2\u00A0 thymidine\u00C2\u00A0 TIL\u00C2\u00A0 \u00C2\u00A0 tumor\u00E2\u0080\u0090infiltrating\u00C2\u00A0lymphocyte\u00C2\u00A0 TIR\u00C2\u00A0 \u00C2\u00A0 Toll\u00E2\u0080\u0090interleukin\u00E2\u0080\u00901\u00C2\u00A0receptor\u00C2\u00A0 TIRAP\u00C2\u00A0\u00C2\u00A0 TIR\u00E2\u0080\u0090domain\u00C2\u00A0containing\u00C2\u00A0adaptor\u00C2\u00A0protein\u00C2\u00A0 TLR\u00C2\u00A0 \u00C2\u00A0 Toll\u00E2\u0080\u0090like\u00C2\u00A0receptor\u00C2\u00A0 TNF\u00C2\u00A0 \u00C2\u00A0 tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0 TRAM\u00C2\u00A0 \u00C2\u00A0 TRIF\u00E2\u0080\u0090related\u00C2\u00A0adaptor\u00C2\u00A0molecule\u00C2\u00A0 TRIF\u00C2\u00A0 \u00C2\u00A0 TIR\u00E2\u0080\u0090domain\u00E2\u0080\u0090containing\u00C2\u00A0adapter\u00E2\u0080\u0090inducing\u00C2\u00A0interferon\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 Treg\u00C2\u00A0 \u00C2\u00A0 regulatory\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 VEGF\u00C2\u00A0 \u00C2\u00A0 vascular\u00C2\u00A0endothelial\u00C2\u00A0growth\u00C2\u00A0factor\u00C2\u00A0 \u00C2\u00A0 xix ACKNOWLEDGEMENTS\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0am\u00C2\u00A0extremely\u00C2\u00A0appreciative\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0many\u00C2\u00A0people\u00C2\u00A0in\u00C2\u00A0my\u00C2\u00A0life\u00C2\u00A0that\u00C2\u00A0supported\u00C2\u00A0me\u00C2\u00A0in\u00C2\u00A0 each\u00C2\u00A0 step\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 journey.\u00C2\u00A0 I\u00C2\u00A0 thank\u00C2\u00A0 my\u00C2\u00A0 family\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 constant\u00C2\u00A0 love,\u00C2\u00A0 support,\u00C2\u00A0 and\u00C2\u00A0 encouragement.\u00C2\u00A0I\u00C2\u00A0also\u00C2\u00A0thank\u00C2\u00A0my\u00C2\u00A0husband\u00C2\u00A0for\u00C2\u00A0his\u00C2\u00A0never\u00E2\u0080\u0090ending\u00C2\u00A0support,\u00C2\u00A0understanding,\u00C2\u00A0 advice,\u00C2\u00A0and\u00C2\u00A0(sometimes\u00C2\u00A0tough)\u00C2\u00A0love.\u00C2\u00A0Thank\u00C2\u00A0you,\u00C2\u00A0Evan,\u00C2\u00A0for\u00C2\u00A0being\u00C2\u00A0my\u00C2\u00A0partner,\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0and\u00C2\u00A0 all\u00C2\u00A0aspects\u00C2\u00A0of\u00C2\u00A0life.\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0 sincerely\u00C2\u00A0 thank\u00C2\u00A0my\u00C2\u00A0supervisor,\u00C2\u00A0Dr.\u00C2\u00A0Gerald\u00C2\u00A0Krystal,\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0opportunity\u00C2\u00A0 to\u00C2\u00A0 join\u00C2\u00A0 his\u00C2\u00A0laboratory\u00C2\u00A0and\u00C2\u00A0conduct\u00C2\u00A0these\u00C2\u00A0studies\u00C2\u00A0under\u00C2\u00A0his\u00C2\u00A0guidance.\u00C2\u00A0Thank\u00C2\u00A0you,\u00C2\u00A0Gerry,\u00C2\u00A0for\u00C2\u00A0your\u00C2\u00A0 support\u00C2\u00A0and\u00C2\u00A0encouragement\u00C2\u00A0these\u00C2\u00A0past\u00C2\u00A0six\u00C2\u00A0years.\u00C2\u00A0I\u00C2\u00A0appreciate\u00C2\u00A0your\u00C2\u00A0kind\u00C2\u00A0spirit,\u00C2\u00A0scientific\u00C2\u00A0 curiosity,\u00C2\u00A0creativity,\u00C2\u00A0and\u00C2\u00A0passion\u00C2\u00A0for\u00C2\u00A0clinically\u00E2\u0080\u0090relevant\u00C2\u00A0research.\u00C2\u00A0I\u00C2\u00A0am\u00C2\u00A0also\u00C2\u00A0grateful\u00C2\u00A0to\u00C2\u00A0Dr.\u00C2\u00A0 Kevin\u00C2\u00A0Bennewith\u00C2\u00A0for\u00C2\u00A0his\u00C2\u00A0collaboration\u00C2\u00A0and\u00C2\u00A0mentorship.\u00C2\u00A0Kevin,\u00C2\u00A0thank\u00C2\u00A0you\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0the\u00C2\u00A0time\u00C2\u00A0 and\u00C2\u00A0 effort\u00C2\u00A0 you\u00C2\u00A0 invested\u00C2\u00A0 in\u00C2\u00A0 these\u00C2\u00A0 studies\u00C2\u00A0 and\u00C2\u00A0 your\u00C2\u00A0 continual\u00C2\u00A0 guidance\u00C2\u00A0 and\u00C2\u00A0 advice.\u00C2\u00A0 I\u00C2\u00A0 would\u00C2\u00A0 also\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0 recognize\u00C2\u00A0 the\u00C2\u00A0members\u00C2\u00A0 of\u00C2\u00A0my\u00C2\u00A0 supervisory\u00C2\u00A0 committee,\u00C2\u00A0 Drs.\u00C2\u00A0 Fumio\u00C2\u00A0 Takei,\u00C2\u00A0Megan\u00C2\u00A0Levings,\u00C2\u00A0and\u00C2\u00A0Kevin\u00C2\u00A0Bennewith,\u00C2\u00A0for\u00C2\u00A0their\u00C2\u00A0input,\u00C2\u00A0suggestions,\u00C2\u00A0and\u00C2\u00A0direction\u00C2\u00A0 throughout\u00C2\u00A0my\u00C2\u00A0PhD\u00C2\u00A0 studies.\u00C2\u00A0 I\u00C2\u00A0 thank\u00C2\u00A0 all\u00C2\u00A0 of\u00C2\u00A0my\u00C2\u00A0 lab\u00C2\u00A0mates,\u00C2\u00A0 past\u00C2\u00A0 and\u00C2\u00A0 present,\u00C2\u00A0 especially\u00C2\u00A0 Victor\u00C2\u00A0 Ho,\u00C2\u00A0 Vivian\u00C2\u00A0 Lam,\u00C2\u00A0 Frann\u00C2\u00A0 Antignano,\u00C2\u00A0 and\u00C2\u00A0 Christina\u00C2\u00A0 Thomas,\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 help\u00C2\u00A0 and\u00C2\u00A0 friendship.\u00C2\u00A0 I\u00C2\u00A0 would\u00C2\u00A0 also\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0 thank\u00C2\u00A0 the\u00C2\u00A0 members\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Terry\u00C2\u00A0 Fox\u00C2\u00A0 Laboratory\u00C2\u00A0 for\u00C2\u00A0 helpful\u00C2\u00A0 discussions\u00C2\u00A0 and\u00C2\u00A0 collaborations,\u00C2\u00A0 and\u00C2\u00A0 express\u00C2\u00A0 my\u00C2\u00A0 gratitude\u00C2\u00A0 to\u00C2\u00A0 Drs.\u00C2\u00A0 Vincent\u00C2\u00A0 Duronio\u00C2\u00A0 and\u00C2\u00A0 Connie\u00C2\u00A0 Eaves\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 support\u00C2\u00A0 of\u00C2\u00A0 funding\u00C2\u00A0 applications;\u00C2\u00A0 the\u00C2\u00A0 research\u00C2\u00A0 described\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0thesis\u00C2\u00A0was\u00C2\u00A0supported\u00C2\u00A0by\u00C2\u00A0personal\u00C2\u00A0fellowships\u00C2\u00A0awarded\u00C2\u00A0to\u00C2\u00A0me\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 Canadian\u00C2\u00A0Institutes\u00C2\u00A0of\u00C2\u00A0Health\u00C2\u00A0Research,\u00C2\u00A0Michael\u00C2\u00A0Smith\u00C2\u00A0Foundation\u00C2\u00A0for\u00C2\u00A0Health\u00C2\u00A0Research,\u00C2\u00A0 and\u00C2\u00A0Canadian\u00C2\u00A0Federation\u00C2\u00A0of\u00C2\u00A0University\u00C2\u00A0Women.\u00C2\u00A0 \u00C2\u00A0 xx DEDICATION\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0dedicate\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0to\u00C2\u00A0my\u00C2\u00A0family.\u00C2\u00A0 1 CHAPTER\u00C2\u00A01\u00C2\u00A0: INTRODUCTION\u00C2\u00A0 \u00C2\u00A0 1.1 History\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0immunology\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0relationship\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0and\u00C2\u00A0cancer\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0the\u00C2\u00A0subject\u00C2\u00A0of\u00C2\u00A0 much\u00C2\u00A0 debate\u00C2\u00A0 throughout\u00C2\u00A0 the\u00C2\u00A0 past\u00C2\u00A0 century\u00C2\u00A0 and\u00C2\u00A0 remains\u00C2\u00A0 one\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 challenging\u00C2\u00A0 issues\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 field\u00C2\u00A0 of\u00C2\u00A0 immunology.\u00C2\u00A0 The\u00C2\u00A0 concept\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 provides\u00C2\u00A0 protection\u00C2\u00A0from\u00C2\u00A0cancer\u00C2\u00A0was\u00C2\u00A0first\u00C2\u00A0suggested\u00C2\u00A0by\u00C2\u00A0Paul\u00C2\u00A0Ehrlich\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0early\u00C2\u00A01900s\u00C2\u00A0(Ehrlich,\u00C2\u00A0 1909),\u00C2\u00A0 but\u00C2\u00A0 it\u00C2\u00A0 was\u00C2\u00A0 not\u00C2\u00A0 until\u00C2\u00A0 after\u00C2\u00A0 major\u00C2\u00A0 advances\u00C2\u00A0 in\u00C2\u00A0 immune\u00C2\u00A0 cell\u00C2\u00A0 identification\u00C2\u00A0 and\u00C2\u00A0 function\u00C2\u00A0that\u00C2\u00A0experiments\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0performed\u00C2\u00A0to\u00C2\u00A0test\u00C2\u00A0this\u00C2\u00A0hypothesis.\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0mid\u00C2\u00A01950s,\u00C2\u00A0Burnet\u00C2\u00A0and\u00C2\u00A0Thomas\u00C2\u00A0proposed\u00C2\u00A0 the\u00C2\u00A0 idea\u00C2\u00A0of\u00C2\u00A0 cancer\u00C2\u00A0 immunosurveillance,\u00C2\u00A0 i.e.,\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 can\u00C2\u00A0 recognize\u00C2\u00A0 and\u00C2\u00A0 destroy\u00C2\u00A0 emerging\u00C2\u00A0 transformed\u00C2\u00A0 cells\u00C2\u00A0 (Burnet,\u00C2\u00A0 1957).\u00C2\u00A0 However,\u00C2\u00A0 initial\u00C2\u00A0 studies\u00C2\u00A0 produced\u00C2\u00A0 mixed\u00C2\u00A0 results\u00C2\u00A0 and\u00C2\u00A0 this\u00C2\u00A0 idea\u00C2\u00A0 was\u00C2\u00A0 largely\u00C2\u00A0 abandoned\u00C2\u00A0 (Prehn\u00C2\u00A0 and\u00C2\u00A0 Main,\u00C2\u00A0 1957;\u00C2\u00A0 Schreiber\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011;\u00C2\u00A0 Stutman,\u00C2\u00A0 1974;\u00C2\u00A0 Stutman,\u00C2\u00A0 1975).\u00C2\u00A0 Nevertheless,\u00C2\u00A0 interest\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 immunology\u00C2\u00A0 was\u00C2\u00A0 revived\u00C2\u00A0 in\u00C2\u00A0 1982\u00C2\u00A0 when\u00C2\u00A0 van\u00C2\u00A0 Pel\u00C2\u00A0 and\u00C2\u00A0 Boon\u00C2\u00A0 made\u00C2\u00A0 the\u00C2\u00A0 seminal\u00C2\u00A0 discovery\u00C2\u00A0 that\u00C2\u00A0 vaccinating\u00C2\u00A0 mice\u00C2\u00A0 with\u00C2\u00A0 mutagenized\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0provided\u00C2\u00A0protection\u00C2\u00A0from\u00C2\u00A0spontaneous\u00C2\u00A0tumors,\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0 spontaneous\u00C2\u00A0 tumors\u00C2\u00A0 possessed\u00C2\u00A0 tumor\u00C2\u00A0 antigens\u00C2\u00A0 (Ags)\u00C2\u00A0 (Van\u00C2\u00A0 Pel\u00C2\u00A0 and\u00C2\u00A0 Boon,\u00C2\u00A0 1982).\u00C2\u00A0 Advances\u00C2\u00A0 in\u00C2\u00A0 technology,\u00C2\u00A0 including\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 improved\u00C2\u00A0 mouse\u00C2\u00A0 models,\u00C2\u00A0 combined\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0 better\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 has\u00C2\u00A0 allowed\u00C2\u00A0 major\u00C2\u00A0 advances\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0made\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 immunology\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0past\u00C2\u00A0 two\u00C2\u00A0decades\u00C2\u00A0 (Schreiber\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 Immune\u00C2\u00A0 surveillance\u00C2\u00A0 has\u00C2\u00A0 now\u00C2\u00A0 been\u00C2\u00A0 clearly\u00C2\u00A0 demonstrated,\u00C2\u00A0 since\u00C2\u00A0 mice\u00C2\u00A0 with\u00C2\u00A0 genetic\u00C2\u00A0 immune\u00C2\u00A0 deficiencies\u00C2\u00A0 are\u00C2\u00A0 more\u00C2\u00A0 susceptible\u00C2\u00A0 to\u00C2\u00A0 chemically\u00E2\u0080\u0090induced,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 spontaneous,\u00C2\u00A0 tumors\u00C2\u00A0 (Laoui\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011;\u00C2\u00A0 Shankaran\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2001),\u00C2\u00A0 and\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 now\u00C2\u00A0 a\u00C2\u00A0 large\u00C2\u00A0body\u00C2\u00A0of\u00C2\u00A0evidence\u00C2\u00A0supporting\u00C2\u00A0the\u00C2\u00A0general\u00C2\u00A0hypothesis\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0can\u00C2\u00A0 act\u00C2\u00A0to\u00C2\u00A0restrict\u00C2\u00A0cancer\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0progression\u00C2\u00A0(Vesely\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0However,\u00C2\u00A0these\u00C2\u00A0 advances\u00C2\u00A0 have\u00C2\u00A0 also\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 relationship\u00C2\u00A0 between\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 and\u00C2\u00A0 cancer\u00C2\u00A0is\u00C2\u00A0extremely\u00C2\u00A0complex.\u00C2\u00A0In\u00C2\u00A0fact,\u00C2\u00A0we\u00C2\u00A0now\u00C2\u00A0appreciate\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0 dual\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 cancer;\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0one\u00C2\u00A0hand,\u00C2\u00A0 it\u00C2\u00A0 can\u00C2\u00A0 inhibit\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0by\u00C2\u00A0 recognizing\u00C2\u00A0 and\u00C2\u00A0 destroying\u00C2\u00A0 cancer\u00C2\u00A0 cells,\u00C2\u00A0 but\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 other,\u00C2\u00A0 it\u00C2\u00A0 can\u00C2\u00A0 encourage\u00C2\u00A0 tumor\u00C2\u00A0 progression\u00C2\u00A0 by\u00C2\u00A0 2 selecting\u00C2\u00A0 for\u00C2\u00A0 less\u00C2\u00A0 immunogenic\u00C2\u00A0 tumors\u00C2\u00A0 and\u00C2\u00A0by\u00C2\u00A0providing\u00C2\u00A0 resources\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 that\u00C2\u00A0 support\u00C2\u00A0its\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0(Schreiber\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.2 Cancer\u00C2\u00A0immunoediting\u00C2\u00A0versus\u00C2\u00A0tumor\u00C2\u00ADinduced\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 2001,\u00C2\u00A0 experimental\u00C2\u00A0 evidence\u00C2\u00A0 from\u00C2\u00A0 Schreiber\u00C2\u00A0 and\u00C2\u00A0 colleagues\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 demonstrated\u00C2\u00A0the\u00C2\u00A0existence\u00C2\u00A0of\u00C2\u00A0immune\u00C2\u00A0surveillance,\u00C2\u00A0but\u00C2\u00A0also\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0 system\u00C2\u00A0can\u00C2\u00A0influence\u00C2\u00A0tumor\u00C2\u00A0immunogenicity\u00C2\u00A0(Dunn\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002;\u00C2\u00A0Shankaran\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02001).\u00C2\u00A0 Based\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0findings,\u00C2\u00A0they\u00C2\u00A0proposed\u00C2\u00A0what\u00C2\u00A0they\u00C2\u00A0called\u00C2\u00A0cancer\u00C2\u00A0immunoediting\u00C2\u00A0(Dunn\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02002),\u00C2\u00A0which\u00C2\u00A0suggests\u00C2\u00A0 three\u00C2\u00A0sequential\u00C2\u00A0steps:\u00C2\u00A0elimination,\u00C2\u00A0equilibrium,\u00C2\u00A0and\u00C2\u00A0escape\u00C2\u00A0 (Schreiber\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0During\u00C2\u00A0the\u00C2\u00A0elimination\u00C2\u00A0phase,\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0detects\u00C2\u00A0and\u00C2\u00A0 destroys\u00C2\u00A0developing\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0(Dunn\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002).\u00C2\u00A0Although\u00C2\u00A0the\u00C2\u00A0mechanisms\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 becomes\u00C2\u00A0 aware\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 nascent\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 fully\u00C2\u00A0 understood,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 a\u00C2\u00A0 combination\u00C2\u00A0 of\u00C2\u00A0 danger\u00C2\u00A0 signals\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 involved,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 Type\u00C2\u00A0 I\u00C2\u00A0 interferons\u00C2\u00A0 (IFNs),\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 induced\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 early\u00C2\u00A0 stages\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 development\u00C2\u00A0 (Matzinger,\u00C2\u00A0 1994),\u00C2\u00A0 damage\u00E2\u0080\u0090associated\u00C2\u00A0 molecular\u00C2\u00A0 pattern\u00C2\u00A0 molecules\u00C2\u00A0 (DAMPs)\u00C2\u00A0(e.g.\u00C2\u00A0high\u00C2\u00A0mobility\u00C2\u00A0group\u00C2\u00A0box\u00C2\u00A01;\u00C2\u00A0HMGB1),\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0released\u00C2\u00A0from\u00C2\u00A0dying\u00C2\u00A0tumor\u00C2\u00A0 cells\u00C2\u00A0or\u00C2\u00A0 injured\u00C2\u00A0tissues\u00C2\u00A0(Sims\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010),\u00C2\u00A0or\u00C2\u00A0stress\u00C2\u00A0 ligands\u00C2\u00A0(i.e.\u00C2\u00A0RAE\u00E2\u0080\u00901\u00C2\u00A0or\u00C2\u00A0H60\u00C2\u00A0in\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0MICA/B\u00C2\u00A0in\u00C2\u00A0humans),\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0expressed\u00C2\u00A0on\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0(Guerra\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0 This\u00C2\u00A0 elimination\u00C2\u00A0phase\u00C2\u00A0 appears\u00C2\u00A0 to\u00C2\u00A0 require\u00C2\u00A0players\u00C2\u00A0 from\u00C2\u00A0both\u00C2\u00A0 the\u00C2\u00A0 innate\u00C2\u00A0 and\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 system,\u00C2\u00A0 and\u00C2\u00A0 if\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 fully\u00C2\u00A0 successful,\u00C2\u00A0 elimination\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 possible\u00C2\u00A0 endpoint\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 cancer\u00C2\u00A0 immunoediting\u00C2\u00A0 process\u00C2\u00A0 (Schreiber\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 However,\u00C2\u00A0 if\u00C2\u00A0 tumor\u00C2\u00A0 variants\u00C2\u00A0 survive,\u00C2\u00A0 the\u00C2\u00A0 equilibrium\u00C2\u00A0 phase\u00C2\u00A0 begins,\u00C2\u00A0 whereby\u00C2\u00A0 the\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 holds\u00C2\u00A0 tumors\u00C2\u00A0 in\u00C2\u00A0 check\u00C2\u00A0 by\u00C2\u00A0 preventing\u00C2\u00A0 the\u00C2\u00A0 outgrowth\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cells,\u00C2\u00A0 and\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 immune\u00C2\u00A0 surveillance\u00C2\u00A0 enter\u00C2\u00A0 a\u00C2\u00A0 dynamic\u00C2\u00A0 balance\u00C2\u00A0 (Schreiber\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 Since\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 is\u00C2\u00A0 best\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 recognize\u00C2\u00A0 and\u00C2\u00A0 control\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 immunogenic\u00C2\u00A0 tumor\u00C2\u00A0 variants,\u00C2\u00A0it\u00C2\u00A0exerts\u00C2\u00A0a\u00C2\u00A0constant\u00C2\u00A0selective\u00C2\u00A0pressure\u00C2\u00A0on\u00C2\u00A0genetically\u00C2\u00A0unstable\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0 promotes\u00C2\u00A0the\u00C2\u00A0outgrowth\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0variants\u00C2\u00A0that\u00C2\u00A0have\u00C2\u00A0acquired\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0immunoevasive\u00C2\u00A0 mutations\u00C2\u00A0(Schreiber\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0The\u00C2\u00A0final\u00C2\u00A0stage\u00C2\u00A0described\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0surveillance\u00C2\u00A0 hypothesis\u00C2\u00A0 is\u00C2\u00A0 escape,\u00C2\u00A0 during\u00C2\u00A0 which\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 have\u00C2\u00A0 evaded\u00C2\u00A0 immune\u00C2\u00A0 detection\u00C2\u00A0 emerge\u00C2\u00A0and\u00C2\u00A0flourish\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0clinically\u00E2\u0080\u0090relevant\u00C2\u00A0tumors\u00C2\u00A0(Schreiber\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0\u00C2\u00A0 3 \u00C2\u00A0 Although\u00C2\u00A0the\u00C2\u00A0reciprocal\u00C2\u00A0relationship\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0and\u00C2\u00A0tumors\u00C2\u00A0is\u00C2\u00A0 widely\u00C2\u00A0 accepted,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 some\u00C2\u00A0 investigators\u00C2\u00A0 who\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 subscribe\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 immunoediting\u00C2\u00A0 hypothesis.\u00C2\u00A0 Instead,\u00C2\u00A0 these\u00C2\u00A0 scientists\u00C2\u00A0 propose\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0most\u00C2\u00A0 important\u00C2\u00A0 determinants\u00C2\u00A0of\u00C2\u00A0successful\u00C2\u00A0tumorigenesis\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0mechanisms\u00C2\u00A0that\u00C2\u00A0 are\u00C2\u00A0 acquired\u00C2\u00A0by\u00C2\u00A0 tumors\u00C2\u00A0 to\u00C2\u00A0 block\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 and\u00C2\u00A0 induce\u00C2\u00A0 a\u00C2\u00A0 state\u00C2\u00A0 of\u00C2\u00A0 tumor\u00E2\u0080\u0090specific\u00C2\u00A0tolerance.\u00C2\u00A0Although\u00C2\u00A0these\u00C2\u00A0two\u00C2\u00A0models\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0proposed\u00C2\u00A0as\u00C2\u00A0conflicting\u00C2\u00A0 hypotheses,\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 likely\u00C2\u00A0not\u00C2\u00A0mutually\u00C2\u00A0 exclusive\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2010).\u00C2\u00A0Rather,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 evidence\u00C2\u00A0 to\u00C2\u00A0 support\u00C2\u00A0 both\u00C2\u00A0models,\u00C2\u00A0 suggesting\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 selection\u00C2\u00A0 of\u00C2\u00A0 less\u00C2\u00A0 immunogenic\u00C2\u00A0 tumors\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 carcinogenesis\u00C2\u00A0 (Whiteside,\u00C2\u00A02010).\u00C2\u00A0In\u00C2\u00A0this\u00C2\u00A0thesis,\u00C2\u00A0the\u00C2\u00A0pro\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0 on\u00C2\u00A0 tumorigenesis,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 tumors\u00C2\u00A0 suppress\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0discussed.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.3 Brief\u00C2\u00A0overview\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 is\u00C2\u00A0 typically\u00C2\u00A0 divided\u00C2\u00A0 into\u00C2\u00A0 two\u00C2\u00A0 arms,\u00C2\u00A0 innate\u00C2\u00A0 and\u00C2\u00A0 adaptive.\u00C2\u00A0 Innate\u00C2\u00A0 immune\u00C2\u00A0 responses,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 carried\u00C2\u00A0 out\u00C2\u00A0 by\u00C2\u00A0monocytes,\u00C2\u00A0macrophages\u00C2\u00A0 (M\u00CF\u0086s),\u00C2\u00A0 dendritic\u00C2\u00A0 cells\u00C2\u00A0 (DCs),\u00C2\u00A0 polymorphonuclear\u00C2\u00A0 leukocytes\u00C2\u00A0 (PMNs,\u00C2\u00A0 a.k.a.\u00C2\u00A0 granulocytes),\u00C2\u00A0 natural\u00C2\u00A0 killer\u00C2\u00A0 (NK)\u00C2\u00A0 cells,\u00C2\u00A0 platelets,\u00C2\u00A0mast\u00C2\u00A0 cells,\u00C2\u00A0 epithelial,\u00C2\u00A0 and\u00C2\u00A0endothelial\u00C2\u00A0 cells,\u00C2\u00A0 provide\u00C2\u00A0 rapid\u00C2\u00A0host\u00C2\u00A0protection\u00C2\u00A0against\u00C2\u00A0pathogens\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0Innate\u00C2\u00A0immune\u00C2\u00A0cells\u00C2\u00A0 recognize\u00C2\u00A0 foreign\u00C2\u00A0 organisms,\u00C2\u00A0 including\u00C2\u00A0 nascent\u00C2\u00A0 tumor\u00C2\u00A0 cells,\u00C2\u00A0 via\u00C2\u00A0 sets\u00C2\u00A0 of\u00C2\u00A0 germ\u00E2\u0080\u0090line\u00C2\u00A0 encoded\u00C2\u00A0 pattern\u00C2\u00A0 recognition\u00C2\u00A0 receptors\u00C2\u00A0 (PRRs),\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 Toll\u00E2\u0080\u0090like\u00C2\u00A0 receptors\u00C2\u00A0 (TLRs)\u00C2\u00A0 (Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0finite\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0receptor\u00C2\u00A0structure\u00C2\u00A0and\u00C2\u00A0expression,\u00C2\u00A0the\u00C2\u00A0 innate\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 is\u00C2\u00A0 limited\u00C2\u00A0 in\u00C2\u00A0 its\u00C2\u00A0 breadth\u00C2\u00A0 and\u00C2\u00A0 is\u00C2\u00A0 only\u00C2\u00A0 activated\u00C2\u00A0 by\u00C2\u00A0 certain\u00C2\u00A0 evolutionarily\u00C2\u00A0conserved\u00C2\u00A0pathogen\u00E2\u0080\u0090associated\u00C2\u00A0molecular\u00C2\u00A0patterns\u00C2\u00A0(PAMPs)\u00C2\u00A0(Moresco\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02011).\u00C2\u00A0Receptor\u00C2\u00A0activation\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0maturation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0innate\u00C2\u00A0immune\u00C2\u00A0 cells,\u00C2\u00A0 which\u00C2\u00A0 in\u00C2\u00A0 turn\u00C2\u00A0 triggers\u00C2\u00A0 cytokine\u00C2\u00A0 production,\u00C2\u00A0 recruitment\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 cells,\u00C2\u00A0 complement\u00C2\u00A0 activation,\u00C2\u00A0 and\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 via\u00C2\u00A0 Ag\u00C2\u00A0 presentation\u00C2\u00A0 (Parkin\u00C2\u00A0 and\u00C2\u00A0 Cohen,\u00C2\u00A0 2001).\u00C2\u00A0 Myeloid\u00C2\u00A0 cell\u00C2\u00A0 populations,\u00C2\u00A0 including\u00C2\u00A0 PMNs,\u00C2\u00A0 4 monocytes,\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0and\u00C2\u00A0DCs\u00C2\u00A0are\u00C2\u00A0critical\u00C2\u00A0mediators\u00C2\u00A0of\u00C2\u00A0immune\u00C2\u00A0responses\u00C2\u00A0(Mantovani\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2011;\u00C2\u00A0 Soehnlein\u00C2\u00A0 and\u00C2\u00A0 Lindbom,\u00C2\u00A0 2010).\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 PMNs\u00C2\u00A0 both\u00C2\u00A0 eliminate\u00C2\u00A0 pathogens\u00C2\u00A0 and\u00C2\u00A0 damaged\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0phagocytosis\u00C2\u00A0but\u00C2\u00A0also\u00C2\u00A0have\u00C2\u00A0distinct\u00C2\u00A0 roles\u00C2\u00A0 (Ueha\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2011);\u00C2\u00A0PMNs\u00C2\u00A0 amplify\u00C2\u00A0inflammation\u00C2\u00A0by\u00C2\u00A0releasing\u00C2\u00A0cytotoxic\u00C2\u00A0granules,\u00C2\u00A0while\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0resolve\u00C2\u00A0inflammation\u00C2\u00A0 and\u00C2\u00A0restore\u00C2\u00A0tissue\u00C2\u00A0integrity\u00C2\u00A0after\u00C2\u00A0removal\u00C2\u00A0of\u00C2\u00A0inflammatory\u00C2\u00A0stimuli\u00C2\u00A0(Kennedy\u00C2\u00A0and\u00C2\u00A0DeLeo,\u00C2\u00A0 2009).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0key\u00C2\u00A0players\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0more\u00C2\u00A0recently\u00C2\u00A0evolved\u00C2\u00A0adaptive\u00C2\u00A0immune\u00C2\u00A0response\u00C2\u00A0are\u00C2\u00A0T\u00C2\u00A0 and\u00C2\u00A0B\u00C2\u00A0lymphocytes.\u00C2\u00A0Unlike\u00C2\u00A0innate\u00C2\u00A0immune\u00C2\u00A0cells,\u00C2\u00A0T\u00C2\u00A0and\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0express\u00C2\u00A0a\u00C2\u00A0large\u00C2\u00A0repertoire\u00C2\u00A0 of\u00C2\u00A0Ag\u00C2\u00A0 receptors\u00C2\u00A0 that\u00C2\u00A0are\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0site\u00E2\u0080\u0090specific\u00C2\u00A0 somatic\u00C2\u00A0 recombination\u00C2\u00A0 (Parkin\u00C2\u00A0and\u00C2\u00A0 Cohen,\u00C2\u00A0 2001).\u00C2\u00A0 This\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 receptor\u00C2\u00A0 generation\u00C2\u00A0 allows\u00C2\u00A0 for\u00C2\u00A0 a\u00C2\u00A0 specific\u00C2\u00A0 yet\u00C2\u00A0wide\u00E2\u0080\u0090 ranging\u00C2\u00A0 response,\u00C2\u00A0 allowing\u00C2\u00A0 the\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 to\u00C2\u00A0 distinguish\u00C2\u00A0 between\u00C2\u00A0 vast\u00C2\u00A0 numbers\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0Ags\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0Although\u00C2\u00A0the\u00C2\u00A0adaptive\u00C2\u00A0response\u00C2\u00A0is\u00C2\u00A0 not\u00C2\u00A0 as\u00C2\u00A0 fast\u00E2\u0080\u0090acting\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 innate\u00C2\u00A0 immune\u00C2\u00A0 system,\u00C2\u00A0 it\u00C2\u00A0 imparts\u00C2\u00A0 long\u00E2\u0080\u0090lasting\u00C2\u00A0 specific\u00C2\u00A0 protection\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0host\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0There\u00C2\u00A0are\u00C2\u00A0two\u00C2\u00A0main\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0effector\u00C2\u00A0T\u00C2\u00A0 cells,\u00C2\u00A0 cluster\u00C2\u00A0 of\u00C2\u00A0 differentiation\u00C2\u00A0 (CD)4+\u00C2\u00A0 T\u00C2\u00A0 helper\u00C2\u00A0 (Th)\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 CD8+\u00C2\u00A0 cytotoxic\u00C2\u00A0 T\u00C2\u00A0 lymphocytes\u00C2\u00A0 (CTLs)\u00C2\u00A0 (Parkin\u00C2\u00A0 and\u00C2\u00A0 Cohen,\u00C2\u00A0 2001).\u00C2\u00A0 Upon\u00C2\u00A0 Ag\u00C2\u00A0 recognition\u00C2\u00A0 Th\u00C2\u00A0 cells\u00C2\u00A0mature\u00C2\u00A0 into\u00C2\u00A0Th1\u00C2\u00A0or\u00C2\u00A0Th2\u00C2\u00A0cells,\u00C2\u00A0which\u00C2\u00A0produce\u00C2\u00A0cytokines\u00C2\u00A0that\u00C2\u00A0activate\u00C2\u00A0players\u00C2\u00A0involved\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0cell\u00E2\u0080\u0090 mediated\u00C2\u00A0 (i.e.\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 NK\u00C2\u00A0 cells,\u00C2\u00A0 CTLs)\u00C2\u00A0 or\u00C2\u00A0 antibody\u00C2\u00A0 (Ab)\u00E2\u0080\u0090mediated\u00C2\u00A0 (i.e.\u00C2\u00A0 B\u00C2\u00A0 cell)\u00C2\u00A0 response,\u00C2\u00A0 respectively\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0CTLs,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0are\u00C2\u00A0directly\u00C2\u00A0cytotoxic\u00C2\u00A0to\u00C2\u00A0 their\u00C2\u00A0 target\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 thus\u00C2\u00A0 have\u00C2\u00A0 important\u00C2\u00A0 functions\u00C2\u00A0 in\u00C2\u00A0 anti\u00E2\u0080\u0090viral\u00C2\u00A0 and\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immunity\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0Natural\u00C2\u00A0killer\u00C2\u00A0T\u00C2\u00A0(NKT)\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0lymphocytes\u00C2\u00A0that\u00C2\u00A0 share\u00C2\u00A0characteristics\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0NK\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0(Godfrey\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0NKT\u00C2\u00A0cells\u00C2\u00A0recognize\u00C2\u00A0 glycolipid\u00C2\u00A0Ags\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0context\u00C2\u00A0of\u00C2\u00A0CD1d,\u00C2\u00A0and\u00C2\u00A0express\u00C2\u00A0a\u00C2\u00A0semi\u00E2\u0080\u0090invariant\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0receptor\u00C2\u00A0(TCR)\u00C2\u00A0 and\u00C2\u00A0NK\u00C2\u00A0 cell\u00C2\u00A0markers\u00C2\u00A0 including\u00C2\u00A0NK1.1\u00C2\u00A0 (a.k.a.\u00C2\u00A0 CD161)\u00C2\u00A0 (Godfrey\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 NKT\u00C2\u00A0 cells\u00C2\u00A0 produce\u00C2\u00A0high\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0Th1\u00C2\u00A0and\u00C2\u00A0Th2\u00C2\u00A0cytokines\u00C2\u00A0and\u00C2\u00A0can\u00C2\u00A0 therefore\u00C2\u00A0activate\u00C2\u00A0or\u00C2\u00A0 suppress\u00C2\u00A0 immune\u00C2\u00A0responses\u00C2\u00A0by\u00C2\u00A0modulating\u00C2\u00A0 the\u00C2\u00A0 functions\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0 immune\u00C2\u00A0cells\u00C2\u00A0 (Godfrey\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2010).\u00C2\u00A0The\u00C2\u00A0 final\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0adaptive\u00C2\u00A0 immune\u00C2\u00A0system\u00C2\u00A0are\u00C2\u00A0B\u00C2\u00A0 cells,\u00C2\u00A0which\u00C2\u00A0produce\u00C2\u00A0 Abs\u00C2\u00A0upon\u00C2\u00A0activation.\u00C2\u00A0They\u00C2\u00A0also\u00C2\u00A0act\u00C2\u00A0as\u00C2\u00A0Ag\u00E2\u0080\u0090presenting\u00C2\u00A0cells\u00C2\u00A0(APCs),\u00C2\u00A0presenting\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 context\u00C2\u00A0 of\u00C2\u00A0 major\u00C2\u00A0 histocompatibility\u00C2\u00A0 complex\u00C2\u00A0 (MHC)\u00C2\u00A0 to\u00C2\u00A0 activate\u00C2\u00A0 Th\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 help\u00C2\u00A0 5 amplify\u00C2\u00A0the\u00C2\u00A0response\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0Abs\u00C2\u00A0function\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0 mechanisms\u00C2\u00A0 to\u00C2\u00A0 improve\u00C2\u00A0 the\u00C2\u00A0 effectiveness\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 innate\u00C2\u00A0 and\u00C2\u00A0 adaptive\u00E2\u0080\u0090mediated\u00C2\u00A0 responses\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0 \u00C2\u00A0 There\u00C2\u00A0is\u00C2\u00A0overlap\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0two\u00C2\u00A0arms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0since\u00C2\u00A0innate\u00C2\u00A0APCs\u00C2\u00A0 (i.e.\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 DCs)\u00C2\u00A0 are\u00C2\u00A0 critical\u00C2\u00A0 modulators\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 adaptive\u00C2\u00A0 response.\u00C2\u00A0 They\u00C2\u00A0 reside\u00C2\u00A0 in\u00C2\u00A0 tissues\u00C2\u00A0where\u00C2\u00A0 they\u00C2\u00A0 continually\u00C2\u00A0 survey\u00C2\u00A0 the\u00C2\u00A0environment,\u00C2\u00A0 taking\u00C2\u00A0up\u00C2\u00A0Ags\u00C2\u00A0 and\u00C2\u00A0displaying\u00C2\u00A0 them\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0surface\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0context\u00C2\u00A0of\u00C2\u00A0MHC\u00C2\u00A0molecules\u00C2\u00A0(Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0Immature\u00C2\u00A0 APCs,\u00C2\u00A0which\u00C2\u00A0have\u00C2\u00A0not\u00C2\u00A0 encountered\u00C2\u00A0a\u00C2\u00A0pathogen\u00C2\u00A0or\u00C2\u00A0harmful\u00C2\u00A0 foreign\u00C2\u00A0entity,\u00C2\u00A0 express\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 MHC\u00C2\u00A0 class\u00C2\u00A0 II\u00C2\u00A0 (MHCII)\u00C2\u00A0 and\u00C2\u00A0 lack\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 co\u00E2\u0080\u0090stimulatory\u00C2\u00A0 molecules\u00C2\u00A0 (e.g.\u00C2\u00A0 CD80/86,\u00C2\u00A0which\u00C2\u00A0bind\u00C2\u00A0to\u00C2\u00A0CD28\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0cells)\u00C2\u00A0(Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0However,\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0 to\u00C2\u00A0PRR\u00C2\u00A0activation\u00C2\u00A0or\u00C2\u00A0inflammatory\u00C2\u00A0cytokine\u00C2\u00A0production,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0DC\u00C2\u00A0mature,\u00C2\u00A0increasing\u00C2\u00A0 their\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0MHCII\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090stimulatory\u00C2\u00A0molecules\u00C2\u00A0(Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0These\u00C2\u00A0 APCs\u00C2\u00A0 then\u00C2\u00A0 migrate\u00C2\u00A0 to\u00C2\u00A0 specialized\u00C2\u00A0 peripheral\u00C2\u00A0 lymphoid\u00C2\u00A0 organs,\u00C2\u00A0 including\u00C2\u00A0 the\u00C2\u00A0 lymph\u00C2\u00A0 nodes\u00C2\u00A0 and\u00C2\u00A0 spleen,\u00C2\u00A0 where\u00C2\u00A0 they\u00C2\u00A0 interact\u00C2\u00A0 with\u00C2\u00A0 lymphocytes\u00C2\u00A0 (Martinez\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 Depending\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 activation\u00C2\u00A0 state,\u00C2\u00A0 APCs\u00C2\u00A0 can\u00C2\u00A0 either\u00C2\u00A0 tolerize\u00C2\u00A0 or\u00C2\u00A0 activate\u00C2\u00A0 na\u00C3\u00AFve\u00C2\u00A0 lymphocytes\u00C2\u00A0 (Parkin\u00C2\u00A0 and\u00C2\u00A0 Cohen,\u00C2\u00A0 2001).\u00C2\u00A0 When\u00C2\u00A0 a\u00C2\u00A0 na\u00C3\u00AFve\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 recognizes\u00C2\u00A0 a\u00C2\u00A0 peptide\u00C2\u00A0 displayed\u00C2\u00A0on\u00C2\u00A0an\u00C2\u00A0immature\u00C2\u00A0APC\u00C2\u00A0the\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0becomes\u00C2\u00A0anergic,\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 peripheral\u00C2\u00A0tolerance\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0On\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0Ag\u00C2\u00A0presented\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0mature\u00C2\u00A0APC\u00C2\u00A0induces\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0and\u00C2\u00A0clonal\u00C2\u00A0expansion\u00C2\u00A0 (Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.4 Tolerance\u00C2\u00A0versus\u00C2\u00A0autoimmunity\u00C2\u00A0 \u00C2\u00A0 Because\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0process\u00C2\u00A0of\u00C2\u00A0somatic\u00C2\u00A0hypermutation\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0generated,\u00C2\u00A0T\u00C2\u00A0 and\u00C2\u00A0B\u00C2\u00A0cell\u00C2\u00A0receptors\u00C2\u00A0have\u00C2\u00A0the\u00C2\u00A0potential\u00C2\u00A0to\u00C2\u00A0recognize\u00C2\u00A0an\u00C2\u00A0almost\u00C2\u00A0unlimited\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Ags,\u00C2\u00A0 including\u00C2\u00A0 self\u00E2\u0080\u0090Ags\u00C2\u00A0 (Munn\u00C2\u00A0 and\u00C2\u00A0 Mellor,\u00C2\u00A0 2003).\u00C2\u00A0 If\u00C2\u00A0 left\u00C2\u00A0 unchecked,\u00C2\u00A0 these\u00C2\u00A0 self\u00E2\u0080\u0090reactive\u00C2\u00A0 lymphocytes\u00C2\u00A0could\u00C2\u00A0cause\u00C2\u00A0tremendous\u00C2\u00A0damage\u00C2\u00A0to\u00C2\u00A0host\u00C2\u00A0tissues\u00C2\u00A0and\u00C2\u00A0lead\u00C2\u00A0to\u00C2\u00A0autoimmune\u00C2\u00A0 diseases.\u00C2\u00A0Many\u00C2\u00A0self\u00E2\u0080\u0090reactive\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0deleted\u00C2\u00A0during\u00C2\u00A0development\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0thymus\u00C2\u00A0via\u00C2\u00A0 central\u00C2\u00A0tolerance\u00C2\u00A0(Munn\u00C2\u00A0and\u00C2\u00A0Mellor,\u00C2\u00A02003).\u00C2\u00A0However,\u00C2\u00A0since\u00C2\u00A0some\u00C2\u00A0self\u00E2\u0080\u0090reactive\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0 6 able\u00C2\u00A0 to\u00C2\u00A0 mature\u00C2\u00A0 and\u00C2\u00A0 enter\u00C2\u00A0 the\u00C2\u00A0 periphery,\u00C2\u00A0 peripheral\u00C2\u00A0 tolerance\u00C2\u00A0 strategies\u00C2\u00A0 are\u00C2\u00A0 also\u00C2\u00A0 necessary\u00C2\u00A0 to\u00C2\u00A0 prevent\u00C2\u00A0 autoimmunity\u00C2\u00A0 (Munn\u00C2\u00A0 and\u00C2\u00A0 Mellor,\u00C2\u00A0 2003).\u00C2\u00A0 These\u00C2\u00A0 mechanisms\u00C2\u00A0 include\u00C2\u00A0 immune\u00C2\u00A0 ignorance,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0 in\u00C2\u00A0 immune\u00C2\u00A0privileged\u00C2\u00A0sites,\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0anergy\u00C2\u00A0 in\u00C2\u00A0 lymphocytes\u00C2\u00A0that\u00C2\u00A0encounter\u00C2\u00A0their\u00C2\u00A0Ag\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0co\u00E2\u0080\u0090stimulatory\u00C2\u00A0or\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0 of\u00C2\u00A0 co\u00E2\u0080\u0090inhibitory\u00C2\u00A0 signals,\u00C2\u00A0 and\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 by\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 actively\u00C2\u00A0 suppress\u00C2\u00A0 lymphocyte\u00C2\u00A0activation\u00C2\u00A0and\u00C2\u00A0proliferation\u00C2\u00A0(Nurieva\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Although\u00C2\u00A0these\u00C2\u00A0tolerance\u00C2\u00A0 mechanisms\u00C2\u00A0evolved\u00C2\u00A0to\u00C2\u00A0prevent\u00C2\u00A0autoimmune\u00C2\u00A0reactions,\u00C2\u00A0tumors\u00C2\u00A0have\u00C2\u00A0co\u00E2\u0080\u0090opted\u00C2\u00A0them\u00C2\u00A0to\u00C2\u00A0 hide\u00C2\u00A0 from\u00C2\u00A0 or\u00C2\u00A0 disable\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system.\u00C2\u00A0 Therefore,\u00C2\u00A0maintaining\u00C2\u00A0 the\u00C2\u00A0 proper\u00C2\u00A0 balance\u00C2\u00A0 between\u00C2\u00A0immune\u00C2\u00A0tolerance\u00C2\u00A0and\u00C2\u00A0response\u00C2\u00A0is\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0utmost\u00C2\u00A0importance;\u00C2\u00A0if\u00C2\u00A0the\u00C2\u00A0equilibrium\u00C2\u00A0 shifts\u00C2\u00A0 too\u00C2\u00A0 strongly\u00C2\u00A0 in\u00C2\u00A0 favour\u00C2\u00A0of\u00C2\u00A0 immune\u00C2\u00A0 tolerance\u00C2\u00A0 the\u00C2\u00A0organism\u00C2\u00A0 forgoes\u00C2\u00A0 its\u00C2\u00A0protection\u00C2\u00A0 against\u00C2\u00A0 harmful\u00C2\u00A0 pathogens\u00C2\u00A0 or\u00C2\u00A0 tumors,\u00C2\u00A0 yet\u00C2\u00A0 if\u00C2\u00A0 tolerance\u00C2\u00A0 is\u00C2\u00A0 diminished\u00C2\u00A0 too\u00C2\u00A0 much\u00C2\u00A0 the\u00C2\u00A0 organism\u00C2\u00A0is\u00C2\u00A0no\u00C2\u00A0longer\u00C2\u00A0protected\u00C2\u00A0from\u00C2\u00A0autoimmunity\u00C2\u00A0(Munn\u00C2\u00A0and\u00C2\u00A0Mellor,\u00C2\u00A02003).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.5 TLR\u00C2\u00A0signaling\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0important\u00C2\u00A0factor\u00C2\u00A0that\u00C2\u00A0regulates\u00C2\u00A0the\u00C2\u00A0balance\u00C2\u00A0between\u00C2\u00A0immune\u00C2\u00A0tolerance\u00C2\u00A0and\u00C2\u00A0 response\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0system\u00C2\u00A0 to\u00C2\u00A0distinguish\u00C2\u00A0 self\u00C2\u00A0 from\u00C2\u00A0non\u00E2\u0080\u0090self.\u00C2\u00A0 Innate\u00C2\u00A0 immune\u00C2\u00A0 cells\u00C2\u00A0 possess\u00C2\u00A0 PRRs\u00C2\u00A0 that\u00C2\u00A0 directly\u00C2\u00A0 recognize\u00C2\u00A0 pathogens\u00C2\u00A0 and\u00C2\u00A0 initiate\u00C2\u00A0 an\u00C2\u00A0 inflammatory\u00C2\u00A0response\u00C2\u00A0(Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0These\u00C2\u00A0receptors\u00C2\u00A0are\u00C2\u00A0located\u00C2\u00A0either\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0or\u00C2\u00A0within\u00C2\u00A0internal\u00C2\u00A0cell\u00C2\u00A0compartments\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0PMNs,\u00C2\u00A0DCs,\u00C2\u00A0epithelial,\u00C2\u00A0and\u00C2\u00A0 endothelial\u00C2\u00A0cells\u00C2\u00A0 (Fitzner\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Parkin\u00C2\u00A0and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0These\u00C2\u00A0 innate\u00C2\u00A0 immune\u00C2\u00A0 receptors\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 clonally\u00C2\u00A0 distributed\u00C2\u00A0 like\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 receptors;\u00C2\u00A0 all\u00C2\u00A0 cells\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 particular\u00C2\u00A0 subtype\u00C2\u00A0 express\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 set\u00C2\u00A0 of\u00C2\u00A0 PRRs\u00C2\u00A0 (Parkin\u00C2\u00A0 and\u00C2\u00A0 Cohen,\u00C2\u00A0 2001).\u00C2\u00A0 The\u00C2\u00A0 TLR\u00C2\u00A0 family\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0class\u00C2\u00A0of\u00C2\u00A0PRRs\u00C2\u00A0that\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0innate\u00C2\u00A0immune\u00C2\u00A0response.\u00C2\u00A0To\u00C2\u00A0date,\u00C2\u00A0 thirteen\u00C2\u00A0 TLRs,\u00C2\u00A0 named\u00C2\u00A0 TLR1\u00E2\u0080\u009013,\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 identified;\u00C2\u00A0 10\u00C2\u00A0 in\u00C2\u00A0 humans\u00C2\u00A0 and\u00C2\u00A0 12\u00C2\u00A0 in\u00C2\u00A0 mice\u00C2\u00A0 (Moresco\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 All\u00C2\u00A0 family\u00C2\u00A0 members\u00C2\u00A0 share\u00C2\u00A0 a\u00C2\u00A0 common\u00C2\u00A0 intracellular\u00C2\u00A0 Toll\u00E2\u0080\u0090 interleukin\u00E2\u0080\u00901\u00C2\u00A0 receptor\u00C2\u00A0 (TIR)\u00C2\u00A0 domain\u00C2\u00A0 and\u00C2\u00A0 have\u00C2\u00A0 multiple\u00C2\u00A0 extracellular\u00C2\u00A0 leucine\u00E2\u0080\u0090rich\u00C2\u00A0 repeats\u00C2\u00A0(LRRs)\u00C2\u00A0(Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0TLRs,\u00C2\u00A0named\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0homology\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0protein\u00C2\u00A0 coded\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 Toll\u00C2\u00A0 gene\u00C2\u00A0 in\u00C2\u00A0 Drosophila,\u00C2\u00A0 are\u00C2\u00A0 single\u00C2\u00A0 membrane\u00E2\u0080\u0090spanning\u00C2\u00A0 non\u00E2\u0080\u0090catalytic\u00C2\u00A0 receptors\u00C2\u00A0that\u00C2\u00A0function\u00C2\u00A0as\u00C2\u00A0homo\u00E2\u0080\u0090\u00C2\u00A0or\u00C2\u00A0hetero\u00E2\u0080\u0090dimers\u00C2\u00A0(Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Each\u00C2\u00A0receptor\u00C2\u00A0 7 or\u00C2\u00A0 receptor\u00C2\u00A0 dimer\u00C2\u00A0 is\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 recognize\u00C2\u00A0 a\u00C2\u00A0 distinct\u00C2\u00A0 set\u00C2\u00A0 of\u00C2\u00A0 PAMPs,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0molecular\u00C2\u00A0 patterns\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 broadly\u00C2\u00A0 shared\u00C2\u00A0by\u00C2\u00A0 a\u00C2\u00A0 class\u00C2\u00A0 of\u00C2\u00A0 pathogens\u00C2\u00A0 but\u00C2\u00A0 are\u00C2\u00A0 also\u00C2\u00A0 distinguishable\u00C2\u00A0 from\u00C2\u00A0normal\u00C2\u00A0host\u00C2\u00A0molecules\u00C2\u00A0(Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Since\u00C2\u00A0TLRs\u00C2\u00A0are\u00C2\u00A0germ\u00E2\u0080\u0090line\u00C2\u00A0encoded\u00C2\u00A0 and\u00C2\u00A0 their\u00C2\u00A0 specificity\u00C2\u00A0 cannot\u00C2\u00A0 be\u00C2\u00A0 easily\u00C2\u00A0 altered\u00C2\u00A0 by\u00C2\u00A0 evolution,\u00C2\u00A0 these\u00C2\u00A0 receptors\u00C2\u00A0 recognize\u00C2\u00A0 molecules\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 consistently\u00C2\u00A0 and\u00C2\u00A0 specifically\u00C2\u00A0 associated\u00C2\u00A0 with\u00C2\u00A0 threats,\u00C2\u00A0 including\u00C2\u00A0 pathogens\u00C2\u00A0or\u00C2\u00A0cellular\u00C2\u00A0stress\u00C2\u00A0(Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Stimulation\u00C2\u00A0 of\u00C2\u00A0 TLRs\u00C2\u00A0 activates\u00C2\u00A0 signaling\u00C2\u00A0 cascades\u00C2\u00A0 that\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 genes\u00C2\u00A0 essential\u00C2\u00A0 for\u00C2\u00A0 immune\u00C2\u00A0 responses,\u00C2\u00A0 including\u00C2\u00A0 those\u00C2\u00A0 that\u00C2\u00A0 encode\u00C2\u00A0 chemokines,\u00C2\u00A0 cytokines,\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090stimulatory\u00C2\u00A0 molecules\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004).\u00C2\u00A0 Since\u00C2\u00A0 different\u00C2\u00A0 classes\u00C2\u00A0 of\u00C2\u00A0microorganisms\u00C2\u00A0 possess\u00C2\u00A0 different\u00C2\u00A0 components,\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 thus\u00C2\u00A0 recognized\u00C2\u00A0 by\u00C2\u00A0 different\u00C2\u00A0 TLRs,\u00C2\u00A0 the\u00C2\u00A0 innate\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 is\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 identify\u00C2\u00A0 a\u00C2\u00A0 pathogen\u00C2\u00A0 as\u00C2\u00A0 foreign,\u00C2\u00A0but\u00C2\u00A0also\u00C2\u00A0initiate\u00C2\u00A0an\u00C2\u00A0appropriately\u00C2\u00A0targeted\u00C2\u00A0response.\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0extracellular\u00C2\u00A0 bacterial\u00C2\u00A0 PAMPs\u00C2\u00A0 induce\u00C2\u00A0 increased\u00C2\u00A0 phagocytosis,\u00C2\u00A0 Ag\u00C2\u00A0 processing\u00C2\u00A0 and\u00C2\u00A0 presentation,\u00C2\u00A0 and\u00C2\u00A0 secretion\u00C2\u00A0 of\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 cytokines,\u00C2\u00A0 which\u00C2\u00A0 stimulate\u00C2\u00A0 a\u00C2\u00A0 Th2\u00E2\u0080\u0090skewed\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0Takeda,\u00C2\u00A0 2004).\u00C2\u00A0 Viral\u00C2\u00A0 PAMPs,\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 trigger\u00C2\u00A0 decreased\u00C2\u00A0 protein\u00C2\u00A0 synthesis,\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis,\u00C2\u00A0 and\u00C2\u00A0 type\u00C2\u00A0 1\u00C2\u00A0 interferon\u00C2\u00A0 (IFN)\u00C2\u00A0 production\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004).\u00C2\u00A0 After\u00C2\u00A0 ligand\u00C2\u00A0 binding,\u00C2\u00A0 TLRs\u00C2\u00A0 dimerize,\u00C2\u00A0 undergo\u00C2\u00A0 conformational\u00C2\u00A0changes,\u00C2\u00A0and\u00C2\u00A0recruit\u00C2\u00A0different\u00C2\u00A0TIR\u00E2\u0080\u0090domain\u00E2\u0080\u0090containing\u00C2\u00A0adaptor\u00C2\u00A0proteins\u00C2\u00A0 including\u00C2\u00A0myeloid\u00C2\u00A0 differentiation\u00C2\u00A0 factor\u00C2\u00A0 88\u00C2\u00A0 (MyD88),\u00C2\u00A0 TIR\u00E2\u0080\u0090domain\u00C2\u00A0 containing\u00C2\u00A0 adaptor\u00C2\u00A0 protein\u00C2\u00A0 (TIRAP,\u00C2\u00A0 a.k.a.\u00C2\u00A0 MyD88\u00C2\u00A0 adaptor\u00E2\u0080\u0090like,\u00C2\u00A0 or\u00C2\u00A0 MAL),\u00C2\u00A0 TIR\u00E2\u0080\u0090domain\u00E2\u0080\u0090containing\u00C2\u00A0 adaptor\u00E2\u0080\u0090 inducing\u00C2\u00A0 interferon\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 (TRIF),\u00C2\u00A0and\u00C2\u00A0TRIF\u00E2\u0080\u0090related\u00C2\u00A0adaptor\u00C2\u00A0molecule\u00C2\u00A0(TRAM)\u00C2\u00A0(Moresco\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0These\u00C2\u00A0 adaptors,\u00C2\u00A0 in\u00C2\u00A0 turn,\u00C2\u00A0 activate\u00C2\u00A0 other\u00C2\u00A0molecules\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0 cell,\u00C2\u00A0 including\u00C2\u00A0 specific\u00C2\u00A0protein\u00C2\u00A0kinases\u00C2\u00A0(i.e.\u00C2\u00A0interleukin\u00E2\u0080\u00901\u00C2\u00A0receptor\u00E2\u0080\u0090associated\u00C2\u00A0kinase\u00C2\u00A01\u00C2\u00A0(IRAK1),\u00C2\u00A0TANK\u00E2\u0080\u0090 binding\u00C2\u00A0kinase\u00C2\u00A01\u00C2\u00A0 (TBK1),\u00C2\u00A0and\u00C2\u00A0 inducible\u00C2\u00A0 I\u00CE\u00BAB\u00C2\u00A0kinase\u00C2\u00A0 (IKKi))\u00C2\u00A0 that\u00C2\u00A0amplify\u00C2\u00A0 the\u00C2\u00A0signal\u00C2\u00A0and\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 or\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 genes\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 inflammatory\u00C2\u00A0 response\u00C2\u00A0 (Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Waltenbaugh\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0In\u00C2\u00A0general,\u00C2\u00A0TLR\u00C2\u00A0signaling\u00C2\u00A0pathways\u00C2\u00A0can\u00C2\u00A0 be\u00C2\u00A0 classified\u00C2\u00A0 as\u00C2\u00A0 MyD88\u00E2\u0080\u0090dependent\u00C2\u00A0 or\u00C2\u00A0 MyD88\u00E2\u0080\u0090independent,\u00C2\u00A0 each\u00C2\u00A0 of\u00C2\u00A0 which\u00C2\u00A0 activates\u00C2\u00A0 distinct\u00C2\u00A0 signaling\u00C2\u00A0 pathways\u00C2\u00A0 and\u00C2\u00A0 ultimate\u00C2\u00A0 end\u00E2\u0080\u0090points\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004).\u00C2\u00A0 Signaling\u00C2\u00A0 through\u00C2\u00A0 MyD88\u00C2\u00A0 activates\u00C2\u00A0 nuclear\u00C2\u00A0 factor\u00C2\u00A0 kappa\u00E2\u0080\u0090light\u00E2\u0080\u0090chain\u00E2\u0080\u0090enhancer\u00C2\u00A0 of\u00C2\u00A0 8 activated\u00C2\u00A0 B\u00C2\u00A0 cells\u00C2\u00A0 (NF\u00E2\u0080\u0090\u00CE\u00BAB)\u00C2\u00A0 and\u00C2\u00A0 activator\u00C2\u00A0 protein\u00C2\u00A0 1\u00C2\u00A0 (AP\u00E2\u0080\u00901)\u00C2\u00A0 transcription\u00C2\u00A0 factors\u00C2\u00A0 (TFs),\u00C2\u00A0 resulting\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0genes\u00C2\u00A0(Akira\u00C2\u00A0and\u00C2\u00A0Takeda,\u00C2\u00A02004).\u00C2\u00A0On\u00C2\u00A0the\u00C2\u00A0 other\u00C2\u00A0hand,\u00C2\u00A0in\u00C2\u00A0addition\u00C2\u00A0to\u00C2\u00A0activating\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB,\u00C2\u00A0signaling\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0MyD88\u00E2\u0080\u0090independent\u00C2\u00A0 pathway\u00C2\u00A0 also\u00C2\u00A0 triggers\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 Type\u00C2\u00A0 I\u00C2\u00A0 IFNs\u00C2\u00A0 (IFN\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 and\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2),\u00C2\u00A0 which\u00C2\u00A0 have\u00C2\u00A0 potent\u00C2\u00A0 anti\u00E2\u0080\u0090viral\u00C2\u00A0 effects\u00C2\u00A0 via\u00C2\u00A0 their\u00C2\u00A0 subsequent\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 IFN\u00E2\u0080\u0090inducible\u00C2\u00A0 genes\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0Takeda,\u00C2\u00A02004;\u00C2\u00A0Schroder\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004).\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0summary\u00C2\u00A0of\u00C2\u00A0known\u00C2\u00A0mammalian\u00C2\u00A0TLRs,\u00C2\u00A0their\u00C2\u00A0 PAMP\u00C2\u00A0ligands,\u00C2\u00A0signaling\u00C2\u00A0molecules,\u00C2\u00A0and\u00C2\u00A0expression\u00C2\u00A0patterns\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0found\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A01.1.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A01.1\u00C2\u00A0 Summary\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0known\u00C2\u00A0TLRs.\u00C2\u00A0 TLRs\u00C2\u00A0 recognize,\u00C2\u00A0 bind,\u00C2\u00A0 and\u00C2\u00A0 become\u00C2\u00A0 activated\u00C2\u00A0 by\u00C2\u00A0 different\u00C2\u00A0 ligands,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 derived\u00C2\u00A0 from\u00C2\u00A0 different\u00C2\u00A0 microbes\u00C2\u00A0 including\u00C2\u00A0 viruses,\u00C2\u00A0 fungi,\u00C2\u00A0 bacteria,\u00C2\u00A0 and\u00C2\u00A0 protozoa\u00C2\u00A0 or\u00C2\u00A0 from\u00C2\u00A0 damaged\u00C2\u00A0host\u00C2\u00A0cells.\u00C2\u00A0TLRs\u00C2\u00A0signal\u00C2\u00A0through\u00C2\u00A0different\u00C2\u00A0adaptor\u00C2\u00A0proteins\u00C2\u00A0that\u00C2\u00A0directly\u00C2\u00A0bind\u00C2\u00A0to\u00C2\u00A0 activated\u00C2\u00A0 TLRs\u00C2\u00A0 and\u00C2\u00A0 recruit\u00C2\u00A0 different\u00C2\u00A0 downstream\u00C2\u00A0 signaling\u00C2\u00A0 components.\u00C2\u00A0 While\u00C2\u00A0 most\u00C2\u00A0 receptors\u00C2\u00A0are\u00C2\u00A0displayed\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0TLRs\u00C2\u00A0that\u00C2\u00A0recognize\u00C2\u00A0nucleic\u00C2\u00A0acids\u00C2\u00A0(TLR3,\u00C2\u00A07,\u00C2\u00A0 8,\u00C2\u00A09)\u00C2\u00A0are\u00C2\u00A0located\u00C2\u00A0in\u00C2\u00A0intracellular\u00C2\u00A0endosomes.\u00C2\u00A0TLR10\u00C2\u00A0is\u00C2\u00A0only\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0humans\u00C2\u00A0and\u00C2\u00A0TLRs\u00C2\u00A0 11\u00E2\u0080\u009013\u00C2\u00A0are\u00C2\u00A0only\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0mice.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Moresco\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Fitzner\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0 and\u00C2\u00A0Waltenbaugh\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008.\u00C2\u00A0 \u00C2\u00A0 Receptor\u00C2\u00A0 Ligand(s)\u00C2\u00A0 Ligand\u00C2\u00A0 Location\u00C2\u00A0 Adaptor(s)\u00C2\u00A0 Cell\u00C2\u00A0Types\u00C2\u00A0 Location\u00C2\u00A0 TLR1\u00C2\u00A0 Triacyl\u00C2\u00A0 lipopeptides\u00C2\u00A0 Bacteria MyD88/MAL Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0\u00C2\u00A0 DC\u00C2\u00A0subsets,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0surface TLR2\u00C2\u00A0 Glycolipids,\u00C2\u00A0 lipopeptides,\u00C2\u00A0 lipoproteins,\u00C2\u00A0 lipoteichoic\u00C2\u00A0 acid\u00C2\u00A0 Bacteria MyD88/MAL Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 myeloid\u00C2\u00A0 (m)DCs\u00C2\u00A0,\u00C2\u00A0 mast\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0surface HSP70\u00C2\u00A0 Host\u00C2\u00A0cells Zymosan\u00C2\u00A0 Fungi TLR3\u00C2\u00A0 Double\u00E2\u0080\u0090 stranded\u00C2\u00A0RNA\u00C2\u00A0 (dsRNA),\u00C2\u00A0poly\u00C2\u00A0 I:C\u00C2\u00A0 Viruses TRIF DCs,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0 compartment\u00C2\u00A0 9 \u00C2\u00A0 Receptor\u00C2\u00A0 Ligand(s)\u00C2\u00A0 Ligand\u00C2\u00A0 Location\u00C2\u00A0 Adaptor(s)\u00C2\u00A0 Cell\u00C2\u00A0Types\u00C2\u00A0 Location\u00C2\u00A0 TLR4\u00C2\u00A0 Lipopolysacch\u00E2\u0080\u0090 aride\u00C2\u00A0(LPS)\u00C2\u00A0 Gram\u00E2\u0080\u0090 negative\u00C2\u00A0 bacteria\u00C2\u00A0 MyD88/MAL/ TRIF/TRAM\u00C2\u00A0 Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 mDCs,\u00C2\u00A0 mast\u00C2\u00A0cells,\u00C2\u00A0 intestinal\u00C2\u00A0 epithelium,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0surface \u00C2\u00A0 TLR5\u00C2\u00A0 Heat\u00C2\u00A0shock\u00C2\u00A0 proteins\u00C2\u00A0 Bacteria\u00C2\u00A0and\u00C2\u00A0 host\u00C2\u00A0cells\u00C2\u00A0 MyD88\u00C2\u00A0 Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 DC\u00C2\u00A0subsets,\u00C2\u00A0 intestinal\u00C2\u00A0 epithelium,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 \u00C2\u00A0 Cell\u00C2\u00A0surface\u00C2\u00A0 Fibrinogen,\u00C2\u00A0 heparin\u00C2\u00A0sulfate\u00C2\u00A0 fragments,\u00C2\u00A0 hyaluronic\u00C2\u00A0acid\u00C2\u00A0 fragments\u00C2\u00A0 Host\u00C2\u00A0Cells Flagellin\u00C2\u00A0 Bacteria TLR6\u00C2\u00A0 Diacyl\u00C2\u00A0 lipopeptides\u00C2\u00A0 Mycoplasma MyD88/MAL Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 mast\u00C2\u00A0cells,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0surface TLR7\u00C2\u00A0 Single\u00E2\u0080\u0090 stranded\u00C2\u00A0RNA\u00C2\u00A0 and\u00C2\u00A0synthetic\u00C2\u00A0 analogs\u00C2\u00A0 (imidazoguino\u00E2\u0080\u0090 line,\u00C2\u00A0loxoribine,\u00C2\u00A0 bropirimine)\u00C2\u00A0 Synthetic\u00C2\u00A0 compounds\u00C2\u00A0 MyD88 Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 plasma\u00E2\u0080\u0090 cytoid\u00C2\u00A0 (p)DCs,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0 compartment\u00C2\u00A0 TLR8\u00C2\u00A0 Single\u00E2\u0080\u0090 stranded\u00C2\u00A0RNA,\u00C2\u00A0 small\u00C2\u00A0synthetic\u00C2\u00A0 compounds\u00C2\u00A0 Synthetic\u00C2\u00A0 compounds\u00C2\u00A0 MyD88 Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0DC\u00C2\u00A0 subsets,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0 compartment\u00C2\u00A0 TLR9\u00C2\u00A0 Unmethylated\u00C2\u00A0 CpG\u00C2\u00A0DNA\u00C2\u00A0 Bacteria MyD88 Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0pDCs,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0 compartment\u00C2\u00A0 10 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Receptor\u00C2\u00A0 Ligand(s)\u00C2\u00A0 Ligand\u00C2\u00A0 Location\u00C2\u00A0 Adaptor(s)\u00C2\u00A0 Cell\u00C2\u00A0Types\u00C2\u00A0 Location\u00C2\u00A0 TLR10\u00C2\u00A0 Unknown\u00C2\u00A0 Unknown Unknown Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 B\u00C2\u00A0cells,\u00C2\u00A0\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 Cell\u00C2\u00A0surface TLR11\u00C2\u00A0 Profilin\u00C2\u00A0 Toxoplasma\u00C2\u00A0 gondii\u00C2\u00A0 MyD88 Monocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 liver\u00C2\u00A0cells,\u00C2\u00A0 kidney\u00C2\u00A0 cells,\u00C2\u00A0 bladder\u00C2\u00A0 epithelium\u00C2\u00A0 Cell\u00C2\u00A0 compartment\u00C2\u00A0 TLR12\u00C2\u00A0 Unknown\u00C2\u00A0 Unknown Unknown Neurons\u00C2\u00A0 Unknown TLR13\u00C2\u00A0 Unknown\u00C2\u00A0 Virus MyD88,\u00C2\u00A0TAK\u00E2\u0080\u00901 Unknown\u00C2\u00A0 Unknown 11 1.6 Immune\u00C2\u00A0escape\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 tumors\u00C2\u00A0 are\u00C2\u00A0 frequently\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 develop\u00C2\u00A0 and\u00C2\u00A0 thrive\u00C2\u00A0 in\u00C2\u00A0 immune\u00C2\u00A0 competent\u00C2\u00A0hosts\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 is\u00C2\u00A0 often\u00C2\u00A0unable\u00C2\u00A0 to\u00C2\u00A0 prevent\u00C2\u00A0 cancer.\u00C2\u00A0 There\u00C2\u00A0 are\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 this\u00C2\u00A0 failure\u00C2\u00A0 of\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immunity\u00C2\u00A0 including\u00C2\u00A0low\u00C2\u00A0immunogenicity\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0cells,\u00C2\u00A0tolerance,\u00C2\u00A0and\u00C2\u00A0immune\u00C2\u00A0evasion\u00C2\u00A0(Dunn\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02002).\u00C2\u00A0In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0successful,\u00C2\u00A0tumors\u00C2\u00A0must\u00C2\u00A0evolve\u00C2\u00A0mechanisms\u00C2\u00A0to\u00C2\u00A0escape\u00C2\u00A0from\u00C2\u00A0or\u00C2\u00A0 suppress\u00C2\u00A0 host\u00C2\u00A0 immune\u00C2\u00A0 responses,\u00C2\u00A0 and\u00C2\u00A0 this\u00C2\u00A0 immune\u00C2\u00A0 evasion\u00C2\u00A0 is\u00C2\u00A0 increasingly\u00C2\u00A0 being\u00C2\u00A0 recognized\u00C2\u00A0 as\u00C2\u00A0 an\u00C2\u00A0 important\u00C2\u00A0 component\u00C2\u00A0 of\u00C2\u00A0 cancer\u00C2\u00A0 development\u00C2\u00A0 (Hanahan\u00C2\u00A0 and\u00C2\u00A0 Weinberg,\u00C2\u00A0 2011).\u00C2\u00A0 Immune\u00C2\u00A0 evasion\u00C2\u00A0 is\u00C2\u00A0 mediated\u00C2\u00A0 by\u00C2\u00A0 several\u00C2\u00A0 distinct\u00C2\u00A0 molecular\u00C2\u00A0 mechanisms\u00C2\u00A0that\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0divided\u00C2\u00A0into\u00C2\u00A0two\u00C2\u00A0general\u00C2\u00A0categories;\u00C2\u00A0those\u00C2\u00A0that\u00C2\u00A0allow\u00C2\u00A0tumors\u00C2\u00A0to\u00C2\u00A0 hide\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 and\u00C2\u00A0 those\u00C2\u00A0 that\u00C2\u00A0 enable\u00C2\u00A0 tumors\u00C2\u00A0 to\u00C2\u00A0 disable\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system.\u00C2\u00A0 \u00C2\u00A0 Transformed\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 hide\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 by\u00C2\u00A0 down\u00E2\u0080\u0090regulating\u00C2\u00A0 Ag\u00C2\u00A0 presentation\u00C2\u00A0 (either\u00C2\u00A0 by\u00C2\u00A0 decreasing\u00C2\u00A0 Ag\u00C2\u00A0 or\u00C2\u00A0 co\u00E2\u0080\u0090stimulatory\u00C2\u00A0 molecule\u00C2\u00A0 expression),\u00C2\u00A0 by\u00C2\u00A0 antigenic\u00C2\u00A0 drift,\u00C2\u00A0 or\u00C2\u00A0 by\u00C2\u00A0 acquiring\u00C2\u00A0 defects\u00C2\u00A0 in\u00C2\u00A0 Ag\u00C2\u00A0 processing\u00C2\u00A0 or\u00C2\u00A0 presentation\u00C2\u00A0 machinery\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2006).\u00C2\u00A0 Another\u00C2\u00A0way\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 hide\u00C2\u00A0 is\u00C2\u00A0 by\u00C2\u00A0 decreasing\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 recruitment\u00C2\u00A0 into\u00C2\u00A0tumor\u00C2\u00A0sites\u00C2\u00A0via\u00C2\u00A0reducing\u00C2\u00A0chemokine\u00C2\u00A0and\u00C2\u00A0adhesion\u00C2\u00A0molecule\u00C2\u00A0expression\u00C2\u00A0(Whiteside,\u00C2\u00A0 2006).\u00C2\u00A0 Tumor\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 also\u00C2\u00A0 surmount\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immunity\u00C2\u00A0 by\u00C2\u00A0 increasing\u00C2\u00A0 their\u00C2\u00A0 resistance\u00C2\u00A0 to\u00C2\u00A0 immune\u00C2\u00A0 effector\u00C2\u00A0 mechanisms.\u00C2\u00A0 For\u00C2\u00A0 example,\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 acquire\u00C2\u00A0 mutations\u00C2\u00A0that\u00C2\u00A0make\u00C2\u00A0them\u00C2\u00A0less\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0apoptosis,\u00C2\u00A0including\u00C2\u00A0over\u00E2\u0080\u0090expression\u00C2\u00A0of\u00C2\u00A0anti\u00E2\u0080\u0090 apoptotic\u00C2\u00A0 molecules\u00C2\u00A0 (e.g.\u00C2\u00A0 B\u00C2\u00A0 cell\u00C2\u00A0 lymphoma\u00C2\u00A0 (Bcl)\u00E2\u0080\u00902,\u00C2\u00A0 Bcl\u00E2\u0080\u0090XL,\u00C2\u00A0 survivin),\u00C2\u00A0 up\u00E2\u0080\u0090regulation\u00C2\u00A0 of\u00C2\u00A0 molecules\u00C2\u00A0 that\u00C2\u00A0 interfere\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 perforin/granzyme\u00C2\u00A0 pathway\u00C2\u00A0 (e.g.\u00C2\u00A0 serine\u00C2\u00A0 protease\u00C2\u00A0 inhibitors),\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 soluble\u00C2\u00A0 death\u00C2\u00A0 receptors\u00C2\u00A0 that\u00C2\u00A0 act\u00C2\u00A0 as\u00C2\u00A0 decoys,\u00C2\u00A0 and\u00C2\u00A0 shedding\u00C2\u00A0 surface\u00C2\u00A0molecules\u00C2\u00A0that\u00C2\u00A0activate\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0(Whiteside,\u00C2\u00A02006).\u00C2\u00A0 \u00C2\u00A0 Alternatively,\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0can\u00C2\u00A0escape\u00C2\u00A0from\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0immunity\u00C2\u00A0by\u00C2\u00A0disabling\u00C2\u00A0the\u00C2\u00A0 immune\u00C2\u00A0 system.\u00C2\u00A0 One\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 key\u00C2\u00A0mechanisms\u00C2\u00A0 tumors\u00C2\u00A0 use\u00C2\u00A0 to\u00C2\u00A0 do\u00C2\u00A0 this\u00C2\u00A0 is\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 either\u00C2\u00A0 disable\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 directly\u00C2\u00A0 or\u00C2\u00A0 that\u00C2\u00A0 promote\u00C2\u00A0 recruitment\u00C2\u00A0 and\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0 cells\u00C2\u00A0 (Whiteside,\u00C2\u00A0 12 2006).\u00C2\u00A0Tumors\u00C2\u00A0secrete\u00C2\u00A0a\u00C2\u00A0plethora\u00C2\u00A0of\u00C2\u00A0molecules\u00C2\u00A0that\u00C2\u00A0induce\u00C2\u00A0an\u00C2\u00A0immunosuppressive\u00C2\u00A0state\u00C2\u00A0 either\u00C2\u00A0 locally,\u00C2\u00A0 i.e.,\u00C2\u00A0 within\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 microenvironment,\u00C2\u00A0 or\u00C2\u00A0 systemically.\u00C2\u00A0 These\u00C2\u00A0 can\u00C2\u00A0 include\u00C2\u00A0 immunosuppressive\u00C2\u00A0 small\u00C2\u00A0 molecules\u00C2\u00A0 (i.e.\u00C2\u00A0 prostaglandin\u00C2\u00A0 (PG)E2,\u00C2\u00A0 histamine,\u00C2\u00A0 reactive\u00C2\u00A0 oxygen\u00C2\u00A0 species\u00C2\u00A0 (ROS)\u00C2\u00A0 and\u00C2\u00A0 reactive\u00C2\u00A0 nitrogen\u00C2\u00A0 species\u00C2\u00A0 (RNS)),\u00C2\u00A0 cytokines\u00C2\u00A0 (i.e.\u00C2\u00A0 vascular\u00C2\u00A0endothelial\u00C2\u00A0growth\u00C2\u00A0factor\u00C2\u00A0(VEGF),\u00C2\u00A0granulocyte\u00C2\u00A0macrophage\u00C2\u00A0colony\u00E2\u0080\u0090stimulating\u00C2\u00A0 factor\u00C2\u00A0 (GM\u00E2\u0080\u0090CSF),\u00C2\u00A0 interleukin\u00C2\u00A0 (IL)\u00E2\u0080\u00904,\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 transforming\u00C2\u00A0 growth\u00C2\u00A0 factor\u00C2\u00A0 beta\u00C2\u00A0 (TGF\u00E2\u0080\u0090\u00CE\u00B2)),\u00C2\u00A0 and\u00C2\u00A0 enzymes\u00C2\u00A0 (i.e.\u00C2\u00A0 indoleamine\u00C2\u00A0 2,3\u00C2\u00A0 dioxygenase\u00C2\u00A0 (IDO),\u00C2\u00A0 arginase\u00C2\u00A0 1(Arg1))\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2010).\u00C2\u00A0 \u00C2\u00A0 Furthermore,\u00C2\u00A0 tumors\u00C2\u00A0 secrete\u00C2\u00A0 growth\u00C2\u00A0 factors\u00C2\u00A0 and\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 cytokines\u00C2\u00A0 that\u00C2\u00A0promote\u00C2\u00A0the\u00C2\u00A0recruitment\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0regulatory\u00C2\u00A0cells\u00C2\u00A0(i.e.\u00C2\u00A0 \u00C2\u00A0regulatory\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 (Tregs),\u00C2\u00A0 tolerogenic\u00C2\u00A0 DCs,\u00C2\u00A0 mast\u00C2\u00A0 cells,\u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0 macrophages\u00C2\u00A0 (TAMs),\u00C2\u00A0 and\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0(MDSCs))\u00C2\u00A0(Whiteside,\u00C2\u00A02006),\u00C2\u00A0whose\u00C2\u00A0functions\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0 discussed\u00C2\u00A0in\u00C2\u00A0detail\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A01.7.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.7 Pro\u00C2\u00AD\u00C2\u00A0and\u00C2\u00A0anti\u00C2\u00ADtumor\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0is\u00C2\u00A0well\u00C2\u00A0established\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0promotion\u00C2\u00A0 and\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0metastasis\u00C2\u00A0 (Schreiber\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011)\u00C2\u00A0 (Fig.\u00C2\u00A0 1.1).\u00C2\u00A0 In\u00C2\u00A0 terms\u00C2\u00A0of\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0immunity,\u00C2\u00A0members\u00C2\u00A0from\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0innate\u00C2\u00A0and\u00C2\u00A0adaptive\u00C2\u00A0arms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 immune\u00C2\u00A0 system,\u00C2\u00A0 including\u00C2\u00A0 CD8+\u00C2\u00A0 CTLs,\u00C2\u00A0 CD4+\u00C2\u00A0 Th\u00C2\u00A0 cells,\u00C2\u00A0 NK\u00C2\u00A0 cells,\u00C2\u00A0 DCs,\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0 and\u00C2\u00A0 B\u00C2\u00A0 cells,\u00C2\u00A0 work\u00C2\u00A0together\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0and\u00C2\u00A0kill\u00C2\u00A0foreign\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0mechanisms\u00C2\u00A0used\u00C2\u00A0 to\u00C2\u00A0eradicate\u00C2\u00A0microorganisms\u00C2\u00A0(Bhardwaj,\u00C2\u00A02007).\u00C2\u00A0As\u00C2\u00A0mentioned\u00C2\u00A0earlier,\u00C2\u00A0while\u00C2\u00A0there\u00C2\u00A0was\u00C2\u00A0 initially\u00C2\u00A0some\u00C2\u00A0doubt\u00C2\u00A0about\u00C2\u00A0whether\u00C2\u00A0the\u00C2\u00A0 immune\u00C2\u00A0system\u00C2\u00A0could\u00C2\u00A0successfully\u00C2\u00A0distinguish\u00C2\u00A0 transformed\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0normal\u00C2\u00A0cells,\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0now\u00C2\u00A0convincing\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0cancer\u00C2\u00A0cells\u00C2\u00A0 express\u00C2\u00A0 specific\u00C2\u00A0Ags,\u00C2\u00A0 termed\u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0Ags\u00C2\u00A0 (TAAs)\u00C2\u00A0 that\u00C2\u00A0distinguish\u00C2\u00A0 them\u00C2\u00A0 from\u00C2\u00A0 their\u00C2\u00A0non\u00E2\u0080\u0090transformed\u00C2\u00A0counterparts\u00C2\u00A0(Whiteside,\u00C2\u00A02010).\u00C2\u00A0These\u00C2\u00A0Ags\u00C2\u00A0are\u00C2\u00A0distinctive\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0 a\u00C2\u00A0combination\u00C2\u00A0of\u00C2\u00A0mutagenic\u00C2\u00A0and\u00C2\u00A0epigenetic\u00C2\u00A0alterations\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011)\u00C2\u00A0and\u00C2\u00A0include\u00C2\u00A0 products\u00C2\u00A0 of\u00C2\u00A0 viral,\u00C2\u00A0 aberrantly\u00C2\u00A0 expressed,\u00C2\u00A0 or\u00C2\u00A0mutated\u00C2\u00A0 genes\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2010).\u00C2\u00A0 Tumor\u00E2\u0080\u0090 specific\u00C2\u00A0 T\u00C2\u00A0 cells,\u00C2\u00A0 i.e.,\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 with\u00C2\u00A0 TCRs\u00C2\u00A0 that\u00C2\u00A0 recognize\u00C2\u00A0 TAAs,\u00C2\u00A0 are\u00C2\u00A0 theoretically\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 eradicate\u00C2\u00A0 even\u00C2\u00A0metastatic\u00C2\u00A0 tumors\u00C2\u00A0 (Vesely\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 CTLs\u00C2\u00A0 are\u00C2\u00A0 critical\u00C2\u00A0mediators\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 killing\u00C2\u00A0 and\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 effects\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 mechanisms,\u00C2\u00A0 13 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A01.1\u00C2\u00A0 Immune\u00C2\u00A0cells\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0promotion\u00C2\u00A0and\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0 growth.\u00C2\u00A0\u00C2\u00A0 Both\u00C2\u00A0innate\u00C2\u00A0and\u00C2\u00A0adaptive\u00C2\u00A0immune\u00C2\u00A0cells\u00C2\u00A0mediate\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0immunity,\u00C2\u00A0including\u00C2\u00A0CD8+\u00C2\u00A0CTLs,\u00C2\u00A0 CD4+\u00C2\u00A0Th\u00C2\u00A0cells,\u00C2\u00A0NK\u00C2\u00A0cells,\u00C2\u00A0M1\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0DCs,\u00C2\u00A0and\u00C2\u00A0B\u00C2\u00A0cells.\u00C2\u00A0Together\u00C2\u00A0 these\u00C2\u00A0cell\u00C2\u00A0 types\u00C2\u00A0can\u00C2\u00A0detect\u00C2\u00A0and\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 cells,\u00C2\u00A0which\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0decreased\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0metastasis.\u00C2\u00A0 However,\u00C2\u00A0 successful\u00C2\u00A0 tumors\u00C2\u00A0 develop\u00C2\u00A0 strategies\u00C2\u00A0 to\u00C2\u00A0 evade\u00C2\u00A0 immune\u00C2\u00A0 detection,\u00C2\u00A0 including\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 cytokines,\u00C2\u00A0 growth\u00C2\u00A0 factors,\u00C2\u00A0 and\u00C2\u00A0 chemokines\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0cells,\u00C2\u00A0including\u00C2\u00A0Tregs,\u00C2\u00A0mast\u00C2\u00A0cells,\u00C2\u00A0MDSCs,\u00C2\u00A0tolerogenic\u00C2\u00A0DCs,\u00C2\u00A0and\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 These\u00C2\u00A0 suppressive\u00C2\u00A0 immune\u00C2\u00A0 cells\u00C2\u00A0 actively\u00C2\u00A0 inhibit\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immunity\u00C2\u00A0 and\u00C2\u00A0produce\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 promote\u00C2\u00A0 angiogenesis,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 invasion,\u00C2\u00A0 and\u00C2\u00A0 thereby\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 progression\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 primary\u00C2\u00A0 and\u00C2\u00A0 metastatic\u00C2\u00A0 tumors.\u00C2\u00A0 Adapted\u00C2\u00A0 from\u00C2\u00A0 references\u00C2\u00A0in\u00C2\u00A0section\u00C2\u00A01.7.\u00C2\u00A0\u00C2\u00A0 14 including\u00C2\u00A0 indirect\u00C2\u00A0 killing\u00C2\u00A0 through\u00C2\u00A0 release\u00C2\u00A0of\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0 tumor\u00C2\u00A0necrosis\u00C2\u00A0 factor\u00C2\u00A0 (TNF)\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 and\u00C2\u00A0 soluble\u00C2\u00A0Fas\u00C2\u00A0ligand,\u00C2\u00A0and\u00C2\u00A0direct\u00C2\u00A0killing\u00C2\u00A0by\u00C2\u00A0engagement\u00C2\u00A0of\u00C2\u00A0membrane\u00E2\u0080\u0090bound\u00C2\u00A0Fas\u00C2\u00A0ligand\u00C2\u00A0to\u00C2\u00A0 Fas\u00C2\u00A0on\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0or\u00C2\u00A0by\u00C2\u00A0secretion\u00C2\u00A0of\u00C2\u00A0perforin\u00C2\u00A0and\u00C2\u00A0granzymes\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0cell\u00C2\u00A0(Vesely\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0addition,\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0is\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0indirectly\u00C2\u00A0protect\u00C2\u00A0the\u00C2\u00A0host\u00C2\u00A0from\u00C2\u00A0tumors\u00C2\u00A0 by\u00C2\u00A0 both\u00C2\u00A0 eliminating\u00C2\u00A0 pathogens\u00C2\u00A0 and\u00C2\u00A0 limiting\u00C2\u00A0 inflammation.\u00C2\u00A0 Specifically,\u00C2\u00A0 since\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0system\u00C2\u00A0evolved\u00C2\u00A0in\u00C2\u00A0part\u00C2\u00A0to\u00C2\u00A0protect\u00C2\u00A0the\u00C2\u00A0host\u00C2\u00A0from\u00C2\u00A0viral\u00C2\u00A0infection\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0quite\u00C2\u00A0adept\u00C2\u00A0 at\u00C2\u00A0 preventing\u00C2\u00A0 virus\u00E2\u0080\u0090induced\u00C2\u00A0 tumors\u00C2\u00A0 (Schreiber\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 Similarly,\u00C2\u00A0 by\u00C2\u00A0 efficiently\u00C2\u00A0 resolving\u00C2\u00A0 infections,\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 reduces\u00C2\u00A0 the\u00C2\u00A0 duration\u00C2\u00A0 and\u00C2\u00A0 extent\u00C2\u00A0 of\u00C2\u00A0 inflammation\u00C2\u00A0(Schreiber\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Inflammation\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0multifaceted\u00C2\u00A0process\u00C2\u00A0that\u00C2\u00A0acts\u00C2\u00A0 to\u00C2\u00A0 protect\u00C2\u00A0 the\u00C2\u00A0 host\u00C2\u00A0 and\u00C2\u00A0 maintain\u00C2\u00A0 tissue\u00C2\u00A0 homeostasis.\u00C2\u00A0 Inflammation\u00C2\u00A0 is\u00C2\u00A0 initiated\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0to\u00C2\u00A0stresses\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0infection,\u00C2\u00A0physical\u00C2\u00A0 injury,\u00C2\u00A0or\u00C2\u00A0 local\u00C2\u00A0 immune\u00C2\u00A0responses\u00C2\u00A0and\u00C2\u00A0 functions\u00C2\u00A0to\u00C2\u00A0eliminate\u00C2\u00A0the\u00C2\u00A0threat\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0organism\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0trigger\u00C2\u00A0the\u00C2\u00A0healing\u00C2\u00A0process\u00C2\u00A0 (Ostrand\u00E2\u0080\u0090Rosenberg\u00C2\u00A0 and\u00C2\u00A0 Sinha,\u00C2\u00A0 2009).\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 involves\u00C2\u00A0 the\u00C2\u00A0 local\u00C2\u00A0 accumulation\u00C2\u00A0 of\u00C2\u00A0 fluid,\u00C2\u00A0 plasma\u00C2\u00A0proteins,\u00C2\u00A0and\u00C2\u00A0immune\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0generally\u00C2\u00A0classified\u00C2\u00A0as\u00C2\u00A0either\u00C2\u00A0acute\u00C2\u00A0or\u00C2\u00A0chronic;\u00C2\u00A0 acute\u00C2\u00A0inflammation\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0response\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0organism\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0threat\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0normally\u00C2\u00A0a\u00C2\u00A0 short\u00E2\u0080\u0090lived\u00C2\u00A0 process,\u00C2\u00A0 while\u00C2\u00A0 chronic\u00C2\u00A0 inflammation\u00C2\u00A0 occurs\u00C2\u00A0 when\u00C2\u00A0 inflammation\u00C2\u00A0 is\u00C2\u00A0 prolonged\u00C2\u00A0(e.g.\u00C2\u00A0autoimmune\u00C2\u00A0disease,\u00C2\u00A0persistent\u00C2\u00A0foreign\u00C2\u00A0body,\u00C2\u00A0or\u00C2\u00A0cancer)\u00C2\u00A0(Sansone\u00C2\u00A0and\u00C2\u00A0 Bromberg,\u00C2\u00A02011).\u00C2\u00A0Whereas\u00C2\u00A0acute\u00C2\u00A0inflammation\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0necessary\u00C2\u00A0aspect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0 response,\u00C2\u00A0 chronic\u00C2\u00A0 inflammation\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 all\u00C2\u00A0 stages\u00C2\u00A0 of\u00C2\u00A0 tumorigenesis,\u00C2\u00A0 including\u00C2\u00A0 cancer\u00C2\u00A0 development,\u00C2\u00A0 progression,\u00C2\u00A0 and\u00C2\u00A0 metastasis\u00C2\u00A0 (Sansone\u00C2\u00A0 and\u00C2\u00A0Bromberg,\u00C2\u00A02011).\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0Mantovani\u00C2\u00A0 and\u00C2\u00A0 colleagues\u00C2\u00A0have\u00C2\u00A0 recently\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 inflammation\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0considered\u00C2\u00A0the\u00C2\u00A0seventh\u00C2\u00A0hallmark\u00C2\u00A0of\u00C2\u00A0cancer\u00C2\u00A0(Mantovani,\u00C2\u00A02009;\u00C2\u00A0 Mantovani\u00C2\u00A0 and\u00C2\u00A0 Sica,\u00C2\u00A0 2010)\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0 recent\u00C2\u00A0 review\u00C2\u00A0 Hanahan\u00C2\u00A0 and\u00C2\u00A0 Weinberg\u00C2\u00A0 highlighted\u00C2\u00A0 tumor\u00E2\u0080\u0090promoting\u00C2\u00A0 inflammation\u00C2\u00A0 as\u00C2\u00A0 an\u00C2\u00A0 enabling\u00C2\u00A0 characteristic\u00C2\u00A0 of\u00C2\u00A0 cancer\u00C2\u00A0 (Hanahan\u00C2\u00A0and\u00C2\u00A0Weinberg,\u00C2\u00A02011).\u00C2\u00A0Inflammation\u00C2\u00A0and\u00C2\u00A0cancer\u00C2\u00A0are\u00C2\u00A0linked\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0major\u00C2\u00A0ways;\u00C2\u00A0 on\u00C2\u00A0one\u00C2\u00A0hand,\u00C2\u00A0oncogene\u00C2\u00A0activation\u00C2\u00A0drives\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0inflammatory\u00C2\u00A0mediators\u00C2\u00A0and\u00C2\u00A0 leads\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0microenvironment\u00C2\u00A0and,\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0 inflammatory\u00C2\u00A0 conditions\u00C2\u00A0 promote\u00C2\u00A0 cancer\u00C2\u00A0 development\u00C2\u00A0 (Mantovani\u00C2\u00A0 and\u00C2\u00A0 Sica,\u00C2\u00A0 2010).\u00C2\u00A0 Therefore,\u00C2\u00A0 15 prompt\u00C2\u00A0 resolution\u00C2\u00A0 of\u00C2\u00A0 inflammation\u00C2\u00A0 by\u00C2\u00A0 immune\u00C2\u00A0 cells\u00C2\u00A0 is\u00C2\u00A0 critical\u00C2\u00A0 to\u00C2\u00A0 reducing\u00C2\u00A0 cancer\u00C2\u00A0 incidence\u00C2\u00A0and\u00C2\u00A0development.\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0these\u00C2\u00A0protective\u00C2\u00A0mechanisms\u00C2\u00A0are\u00C2\u00A0effective\u00C2\u00A0in\u00C2\u00A0some\u00C2\u00A0cases,\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0 tumors\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0develop\u00C2\u00A0and\u00C2\u00A0thrive\u00C2\u00A0in\u00C2\u00A0immune\u00C2\u00A0competent\u00C2\u00A0hosts\u00C2\u00A0indicates\u00C2\u00A0that\u00C2\u00A0 the\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0are\u00C2\u00A0frequently\u00C2\u00A0inadequate\u00C2\u00A0to\u00C2\u00A0protect\u00C2\u00A0the\u00C2\u00A0 host\u00C2\u00A0 from\u00C2\u00A0 developing\u00C2\u00A0 cancer.\u00C2\u00A0 Related\u00C2\u00A0 to\u00C2\u00A0 this,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 numerous\u00C2\u00A0 lines\u00C2\u00A0 of\u00C2\u00A0 evidence\u00C2\u00A0 suggesting\u00C2\u00A0 both\u00C2\u00A0 early\u00C2\u00A0 and\u00C2\u00A0 late\u00C2\u00A0 involvement\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 in\u00C2\u00A0 promoting\u00C2\u00A0 tumorigenesis\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2010).\u00C2\u00A0 Early\u00C2\u00A0 tumors\u00C2\u00A0 and\u00C2\u00A0 even\u00C2\u00A0 premalignant\u00C2\u00A0 foci\u00C2\u00A0 are\u00C2\u00A0 frequently\u00C2\u00A0 infiltrated\u00C2\u00A0 with\u00C2\u00A0 hematopoietic\u00C2\u00A0 cells,\u00C2\u00A0 including\u00C2\u00A0 lymphocytes,\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 and\u00C2\u00A0 occasionally\u00C2\u00A0PMNs\u00C2\u00A0(Kornstein\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01983;\u00C2\u00A0von\u00C2\u00A0Kleist\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01987).\u00C2\u00A0Although\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0 of\u00C2\u00A0 tumor\u00E2\u0080\u0090infiltrating\u00C2\u00A0 lymphocytes\u00C2\u00A0 (TILs),\u00C2\u00A0 especially\u00C2\u00A0 CD8+\u00C2\u00A0 CTLs,\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 associated\u00C2\u00A0 with\u00C2\u00A0 improved\u00C2\u00A0 patient\u00C2\u00A0 survival\u00C2\u00A0 in\u00C2\u00A0 some\u00C2\u00A0 studies,\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 largely\u00C2\u00A0 unsuccessful\u00C2\u00A0 in\u00C2\u00A0 inhibiting\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0(Whiteside,\u00C2\u00A02010).\u00C2\u00A0Moreover,\u00C2\u00A0TILs\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0advanced\u00C2\u00A0or\u00C2\u00A0 metastatic\u00C2\u00A0tumors\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0more\u00C2\u00A0functionally\u00C2\u00A0impaired\u00C2\u00A0than\u00C2\u00A0those\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0early\u00C2\u00A0 lesions\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2010).\u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 in\u00C2\u00A0 large\u00C2\u00A0 part\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 bone\u00C2\u00A0 marrow\u00C2\u00A0 (BM)\u00C2\u00A0 derived\u00C2\u00A0 immunosuppressive\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 develop\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 recruited\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 site\u00C2\u00A0 by\u00C2\u00A0 tumor\u00E2\u0080\u0090 produced\u00C2\u00A0 factors\u00C2\u00A0 (Whiteside,\u00C2\u00A0 2008)\u00C2\u00A0 including\u00C2\u00A0 Tregs\u00C2\u00A0 (Bettini\u00C2\u00A0 and\u00C2\u00A0 Vignali,\u00C2\u00A0 2009),\u00C2\u00A0 alternatively\u00C2\u00A0 activated\u00C2\u00A0 (M2)\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 (Gordon\u00C2\u00A0 and\u00C2\u00A0 Martinez,\u00C2\u00A0 2010),\u00C2\u00A0 tolerogenic\u00C2\u00A0 DCs\u00C2\u00A0 (Whiteside,\u00C2\u00A02010),\u00C2\u00A0mast\u00C2\u00A0cells\u00C2\u00A0(Maltby\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009),\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0(Ostrand\u00E2\u0080\u0090Rosenberg\u00C2\u00A0and\u00C2\u00A0 Sinha,\u00C2\u00A0 2009;\u00C2\u00A0 Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 These\u00C2\u00A0 immunosuppressive\u00C2\u00A0 cells,\u00C2\u00A0 which\u00C2\u00A0 function\u00C2\u00A0 physiologically\u00C2\u00A0 to\u00C2\u00A0 prevent\u00C2\u00A0 auto\u00E2\u0080\u0090immunity\u00C2\u00A0 and\u00C2\u00A0 resolve\u00C2\u00A0 inflammation,\u00C2\u00A0 directly\u00C2\u00A0 oppose\u00C2\u00A0 the\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 by\u00C2\u00A0 actively\u00C2\u00A0 suppressing\u00C2\u00A0 cytotoxic\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0critical\u00C2\u00A0mediators\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0progression\u00C2\u00A0via\u00C2\u00A0 production\u00C2\u00A0of\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0foster\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0(Hanahan\u00C2\u00A0and\u00C2\u00A0Weinberg,\u00C2\u00A0 2011;\u00C2\u00A0 Whiteside,\u00C2\u00A0 2006).\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 two\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 integral\u00C2\u00A0 pro\u00E2\u0080\u0090tumor\u00C2\u00A0 immune\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 focus\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 thesis.\u00C2\u00A0 The\u00C2\u00A0 roles\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 in\u00C2\u00A0 suppressing\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0immunity\u00C2\u00A0and\u00C2\u00A0fostering\u00C2\u00A0tumorigenesis\u00C2\u00A0will\u00C2\u00A0now\u00C2\u00A0be\u00C2\u00A0discussed\u00C2\u00A0in\u00C2\u00A0 greater\u00C2\u00A0detail.\u00C2\u00A0 \u00C2\u00A0 16 1.7.1 M\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 terminally\u00C2\u00A0 differentiated\u00C2\u00A0 cells\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 mononuclear\u00C2\u00A0 phagocyte\u00C2\u00A0 system\u00C2\u00A0 (MPS),\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 comprised\u00C2\u00A0 of\u00C2\u00A0 mononuclear\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 possess\u00C2\u00A0 phagocytic\u00C2\u00A0 abilities,\u00C2\u00A0 including\u00C2\u00A0lineage\u00C2\u00A0committed\u00C2\u00A0BM\u00C2\u00A0precursors,\u00C2\u00A0MDSCs,\u00C2\u00A0monocytes,\u00C2\u00A0DCs,\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(Pollard,\u00C2\u00A0 2009).\u00C2\u00A0Terminally\u00E2\u0080\u0090differentiated\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0MPS\u00C2\u00A0arise\u00C2\u00A0as\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0normal\u00C2\u00A0myelopoiesis\u00C2\u00A0 from\u00C2\u00A0 pluripotent\u00C2\u00A0 hematopoietic\u00C2\u00A0 progenitor\u00C2\u00A0 cells\u00C2\u00A0 (HPCs)\u00C2\u00A0 that\u00C2\u00A0 give\u00C2\u00A0 rise\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 lineage\u00E2\u0080\u0090restricted\u00C2\u00A0progenitor\u00C2\u00A0 cells,\u00C2\u00A0 or\u00C2\u00A0 immature\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 (IMCs)\u00C2\u00A0 (Fig.\u00C2\u00A0 1.2).\u00C2\u00A0These\u00C2\u00A0 IMCs,\u00C2\u00A0which\u00C2\u00A0 lack\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0 in\u00C2\u00A0healthy\u00C2\u00A0 individuals\u00C2\u00A0 (Delano\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007),\u00C2\u00A0generate\u00C2\u00A0pro\u00E2\u0080\u0090monocytes\u00C2\u00A0and\u00C2\u00A0monocytes\u00C2\u00A0that\u00C2\u00A0circulate\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0bloodstream\u00C2\u00A0(Fig.\u00C2\u00A0 1.2)\u00C2\u00A0 (Pollard,\u00C2\u00A0 2009).\u00C2\u00A0 Murine\u00C2\u00A0 monocytes\u00C2\u00A0 express\u00C2\u00A0 both\u00C2\u00A0 CD11b\u00C2\u00A0 and\u00C2\u00A0 CD115\u00C2\u00A0 and\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 further\u00C2\u00A0 classified\u00C2\u00A0 into\u00C2\u00A0 functional\u00C2\u00A0 subpopulations\u00C2\u00A0 of\u00C2\u00A0 resident\u00C2\u00A0 (Gr1\u00E2\u0080\u0090Ly6C\u00E2\u0080\u0090)\u00C2\u00A0 and\u00C2\u00A0 inflammatory\u00C2\u00A0(Gr1+Ly6C+)\u00C2\u00A0monocytes\u00C2\u00A0(Geissmann\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Geissmann\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0 In\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 inflammatory\u00C2\u00A0 cytokines,\u00C2\u00A0 chemokines,\u00C2\u00A0 and\u00C2\u00A0 other\u00C2\u00A0 factors,\u00C2\u00A0 monocytes\u00C2\u00A0 migrate\u00C2\u00A0into\u00C2\u00A0tissues\u00C2\u00A0and\u00C2\u00A0undergo\u00C2\u00A0terminal\u00C2\u00A0differentiation\u00C2\u00A0into\u00C2\u00A0resident\u00C2\u00A0DCs\u00C2\u00A0or\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A0 1.2)\u00C2\u00A0(Siveen\u00C2\u00A0and\u00C2\u00A0Kuttan,\u00C2\u00A02009).\u00C2\u00A0\u00C2\u00A0Recruitment\u00C2\u00A0of\u00C2\u00A0monocytes\u00C2\u00A0to\u00C2\u00A0these\u00C2\u00A0inflammatory\u00C2\u00A0sites\u00C2\u00A0 is\u00C2\u00A0 mediated\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 large\u00C2\u00A0 extent\u00C2\u00A0 by\u00C2\u00A0 CC\u00C2\u00A0 chemokine\u00C2\u00A0 ligand\u00C2\u00A0 2\u00C2\u00A0 (CCL2,\u00C2\u00A0 a.k.a.\u00C2\u00A0 monocyte\u00C2\u00A0 chemotactic\u00C2\u00A0 protein\u00E2\u0080\u00901,\u00C2\u00A0 MCP\u00E2\u0080\u00901),\u00C2\u00A0 which\u00C2\u00A0 binds\u00C2\u00A0 to\u00C2\u00A0 CC\u00C2\u00A0 chemokine\u00C2\u00A0 receptor\u00C2\u00A0 2\u00C2\u00A0 (CCR2)\u00C2\u00A0 on\u00C2\u00A0 monocytes\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Overall,\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0MPS\u00C2\u00A0are\u00C2\u00A0critical\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0 physiological\u00C2\u00A0processes,\u00C2\u00A0including\u00C2\u00A0tissue\u00C2\u00A0development,\u00C2\u00A0homeostasis,\u00C2\u00A0and\u00C2\u00A0regulating\u00C2\u00A0the\u00C2\u00A0 balance\u00C2\u00A0 between\u00C2\u00A0 pro\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 anti\u00E2\u0080\u0090inflammatory\u00C2\u00A0 responses,\u00C2\u00A0 and\u00C2\u00A0 accordingly,\u00C2\u00A0 exhibit\u00C2\u00A0 enormous\u00C2\u00A0heterogeneity\u00C2\u00A0and\u00C2\u00A0versatility\u00C2\u00A0(Qian\u00C2\u00A0and\u00C2\u00A0Pollard,\u00C2\u00A02010).\u00C2\u00A0 17 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A01.2\u00C2\u00A0 Development\u00C2\u00A0and\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mononuclear\u00C2\u00A0phagocytic\u00C2\u00A0lineage.\u00C2\u00A0 Mononuclear\u00C2\u00A0phagocyte\u00C2\u00A0development\u00C2\u00A0begins\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0BM\u00C2\u00A0where\u00C2\u00A0pluripotent\u00C2\u00A0HPCs\u00C2\u00A0give\u00C2\u00A0rise\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 progenitor\u00C2\u00A0 cells,\u00C2\u00A0 i.e.,\u00C2\u00A0 common\u00C2\u00A0 myeloid\u00C2\u00A0 progenitors\u00C2\u00A0 (CMPs),\u00C2\u00A0 granulocyte/macrophage\u00C2\u00A0 colony\u00E2\u0080\u0090forming\u00C2\u00A0 units\u00C2\u00A0 (GM\u00E2\u0080\u0090CFUs),\u00C2\u00A0 macrophage\u00C2\u00A0 colony\u00E2\u0080\u0090forming\u00C2\u00A0 units\u00C2\u00A0 (M\u00E2\u0080\u0090CFUs),\u00C2\u00A0monoblasts,\u00C2\u00A0 and\u00C2\u00A0pro\u00E2\u0080\u0090monocytes.\u00C2\u00A0Terminally\u00C2\u00A0differentiated\u00C2\u00A0osteoclasts\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 arise\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 BM,\u00C2\u00A0 while\u00C2\u00A0 all\u00C2\u00A0 other\u00C2\u00A0 mature\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 arise\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 tissues\u00C2\u00A0 from\u00C2\u00A0 blood\u00C2\u00A0 monocytes.\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0plays\u00C2\u00A0 an\u00C2\u00A0 important\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0promoting\u00C2\u00A0 the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0mononuclear\u00C2\u00A0 phagocytes\u00C2\u00A0at\u00C2\u00A0multiple\u00C2\u00A0stages.\u00C2\u00A0The\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0factors\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0tissues,\u00C2\u00A0including\u00C2\u00A0GM\u00E2\u0080\u0090CSF,\u00C2\u00A0 IL\u00E2\u0080\u00904,\u00C2\u00A0IL\u00E2\u0080\u009013,\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1,\u00C2\u00A0promote\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0and\u00C2\u00A0determine\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0phenotype\u00C2\u00A0 and\u00C2\u00A0 physiological\u00C2\u00A0 roles\u00C2\u00A0 of\u00C2\u00A0 mature\u00C2\u00A0 DCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 *\u00E2\u0080\u0099s\u00C2\u00A0 indicate\u00C2\u00A0 IMCs,\u00C2\u00A0 which\u00C2\u00A0 lack\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0 in\u00C2\u00A0healthy\u00C2\u00A0individuals.\u00C2\u00A0However,\u00C2\u00A0 in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0 inflammatory\u00C2\u00A0factors,\u00C2\u00A0or\u00C2\u00A0disease\u00C2\u00A0states,\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0can\u00C2\u00A0acquire\u00C2\u00A0immunosuppressive\u00C2\u00A0functions\u00C2\u00A0 and\u00C2\u00A0are\u00C2\u00A0termed\u00C2\u00A0MDSCs.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0Pollard,\u00C2\u00A02009\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0references\u00C2\u00A0in\u00C2\u00A0section\u00C2\u00A01.7.1.\u00C2\u00A0\u00C2\u00A0 18 Mature\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0found\u00C2\u00A0in\u00C2\u00A0almost\u00C2\u00A0every\u00C2\u00A0tissue\u00C2\u00A0where\u00C2\u00A0they\u00C2\u00A0perform\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0variety\u00C2\u00A0 of\u00C2\u00A0critical\u00C2\u00A0functions\u00C2\u00A0(Table\u00C2\u00A01.2).\u00C2\u00A0Under\u00C2\u00A0steady\u00E2\u0080\u0090state\u00C2\u00A0conditions,\u00C2\u00A0terminally\u00C2\u00A0differentiated\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0very\u00C2\u00A0 stable\u00C2\u00A0 and\u00C2\u00A0 can\u00C2\u00A0exist\u00C2\u00A0 in\u00C2\u00A0 tissues\u00C2\u00A0 for\u00C2\u00A0months,\u00C2\u00A0 or\u00C2\u00A0 even\u00C2\u00A0years\u00C2\u00A0 (Cuenca\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0defined\u00C2\u00A0by\u00C2\u00A0specific\u00C2\u00A0phenotypic\u00C2\u00A0characteristics\u00C2\u00A0and\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0 certain\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0markers,\u00C2\u00A0none\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0specifically\u00C2\u00A0limited\u00C2\u00A0to\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(Gordon\u00C2\u00A0and\u00C2\u00A0 Taylor,\u00C2\u00A02005).\u00C2\u00A0With\u00C2\u00A0the\u00C2\u00A0exception\u00C2\u00A0of\u00C2\u00A0alveolar\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0which\u00C2\u00A0express\u00C2\u00A0CD11c\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CD11b\u00C2\u00A0 due\u00C2\u00A0to\u00C2\u00A0their\u00C2\u00A0unique\u00C2\u00A0 lung\u00C2\u00A0environment\u00C2\u00A0(Guth\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009),\u00C2\u00A0murine\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0express\u00C2\u00A0CD11b,\u00C2\u00A0 F4/80,\u00C2\u00A0and\u00C2\u00A0macrophage\u00C2\u00A0colony\u00E2\u0080\u0090stimulating\u00C2\u00A0 factor\u00C2\u00A0receptor\u00C2\u00A0(M\u00E2\u0080\u0090CSFR;\u00C2\u00A0CD115)\u00C2\u00A0and\u00C2\u00A0 lack\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 Gr1\u00C2\u00A0 (Qian\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A0 2010).\u00C2\u00A0 In\u00C2\u00A0 combination,\u00C2\u00A0 these\u00C2\u00A0 characteristics\u00C2\u00A0 distinguish\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0other\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0myeloid\u00C2\u00A0lineage\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0PMNs,\u00C2\u00A0monocytes,\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0 (Joyce\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A0 2009).\u00C2\u00A0 The\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 is\u00C2\u00A0 regulated\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0growth\u00C2\u00A0factors,\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0important\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0macrophage\u00C2\u00A0colony\u00E2\u0080\u0090 stimulating\u00C2\u00A0factor\u00C2\u00A0(M\u00E2\u0080\u0090CSF,\u00C2\u00A0a.k.a.\u00C2\u00A0colony\u00E2\u0080\u0090stimulating\u00C2\u00A0factor\u00E2\u0080\u00901\u00C2\u00A0or\u00C2\u00A0CSF\u00E2\u0080\u00901),\u00C2\u00A0as\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0 at\u00C2\u00A0multiple\u00C2\u00A0stages\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0process\u00C2\u00A0(Fig.\u00C2\u00A01.2)\u00C2\u00A0(Pollard,\u00C2\u00A02009).\u00C2\u00A0In\u00C2\u00A0fact,\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 essential\u00C2\u00A0 for\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 development,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 also\u00C2\u00A0 necessary\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 survival\u00C2\u00A0 and\u00C2\u00A0 proliferation\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0(Chitu\u00C2\u00A0and\u00C2\u00A0Stanley,\u00C2\u00A02006).\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0phenotypes\u00C2\u00A0and\u00C2\u00A0 perform\u00C2\u00A0 many\u00C2\u00A0 diverse\u00C2\u00A0 functions\u00C2\u00A0 including\u00C2\u00A0 phagocytosis\u00C2\u00A0 of\u00C2\u00A0 apoptotic\u00C2\u00A0 cells,\u00C2\u00A0 debris\u00C2\u00A0 removal,\u00C2\u00A0destruction\u00C2\u00A0of\u00C2\u00A0pathogens,\u00C2\u00A0coordination\u00C2\u00A0of\u00C2\u00A0wound\u00C2\u00A0healing,\u00C2\u00A0tissue\u00C2\u00A0development\u00C2\u00A0 and\u00C2\u00A0 remodeling,\u00C2\u00A0 Ag\u00C2\u00A0 presentation,\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 responses,\u00C2\u00A0 and\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0innate\u00C2\u00A0and\u00C2\u00A0adaptive\u00C2\u00A0immunity\u00C2\u00A0(Fig.\u00C2\u00A01.2)\u00C2\u00A0(Cesta,\u00C2\u00A02006;\u00C2\u00A0Gordon\u00C2\u00A0and\u00C2\u00A0Martinez,\u00C2\u00A02010;\u00C2\u00A0 Martinez\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008;\u00C2\u00A0Munn\u00C2\u00A0 and\u00C2\u00A0Mellor,\u00C2\u00A0 2003;\u00C2\u00A0 Pollard,\u00C2\u00A0 2009).\u00C2\u00A0 These\u00C2\u00A0 roles\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 normal\u00C2\u00A0 tissues\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 exploited\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 developing\u00C2\u00A0 tumor\u00C2\u00A0 and,\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 result,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 can\u00C2\u00A0 contribute\u00C2\u00A0to\u00C2\u00A0all\u00C2\u00A0phases\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cancer\u00C2\u00A0process\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 19 Table\u00C2\u00A01.2\u00C2\u00A0 Diversity\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0tissues.\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 found\u00C2\u00A0 in\u00C2\u00A0 almost\u00C2\u00A0 every\u00C2\u00A0 tissue\u00C2\u00A0 in\u00C2\u00A0 mammals\u00C2\u00A0 and\u00C2\u00A0 perform\u00C2\u00A0 a\u00C2\u00A0 wide\u00C2\u00A0 variety\u00C2\u00A0 of\u00C2\u00A0 immunogenic\u00C2\u00A0 and\u00C2\u00A0 non\u00E2\u0080\u0090immunogenic\u00C2\u00A0 roles.\u00C2\u00A0 Adapted\u00C2\u00A0 from\u00C2\u00A0 Pollard,\u00C2\u00A0 2009;\u00C2\u00A0 Guth\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009;\u00C2\u00A0and\u00C2\u00A0Cesta,\u00C2\u00A02006.\u00C2\u00A0 \u00C2\u00A0 Tissue\u00C2\u00A0\u00C2\u00A0 Specific\u00C2\u00A0Name\u00C2\u00A0 Function\u00C2\u00A0 Bone\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Osteoclast\u00C2\u00A0 \u00C2\u00A0 Bone\u00C2\u00A0remodelling;\u00C2\u00A0provide\u00C2\u00A0stem\u00C2\u00A0cell\u00C2\u00A0niches Bone\u00C2\u00A0Marrow\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 Erythropoiesis\u00C2\u00A0(identify\u00C2\u00A0and\u00C2\u00A0degrade\u00C2\u00A0ejected\u00C2\u00A0 nuclei)\u00C2\u00A0 Brain\u00C2\u00A0 Microglial\u00C2\u00A0cell Neuronal\u00C2\u00A0survival\u00C2\u00A0and\u00C2\u00A0connectivity;\u00C2\u00A0repair Epidermis\u00C2\u00A0 Langerhans\u00C2\u00A0cell Immune\u00C2\u00A0surveillance Eye\u00C2\u00A0 N/A\u00C2\u00A0 Vascular\u00C2\u00A0remodelling Intestine\u00C2\u00A0 Crypt\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 Immune\u00C2\u00A0surveillance Kidney\u00C2\u00A0 N/A\u00C2\u00A0 Ductal\u00C2\u00A0development Liver\u00C2\u00A0 Kupffer\u00C2\u00A0cell\u00C2\u00A0 Clearance\u00C2\u00A0of\u00C2\u00A0debris\u00C2\u00A0from\u00C2\u00A0blood;\u00C2\u00A0tissue\u00C2\u00A0 regeneration\u00C2\u00A0 Lung\u00C2\u00A0 Alveolar\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 Regulation\u00C2\u00A0of immune\u00C2\u00A0responses\u00C2\u00A0to\u00C2\u00A0inhaled\u00C2\u00A0 pathogens\u00C2\u00A0and\u00C2\u00A0allergens\u00C2\u00A0 Mammary\u00C2\u00A0gland\u00C2\u00A0 N/A\u00C2\u00A0 Branching\u00C2\u00A0morphogenesis;\u00C2\u00A0ductal\u00C2\u00A0development Ovary\u00C2\u00A0 N/A\u00C2\u00A0 Steroid\u00C2\u00A0hormone\u00C2\u00A0production;\u00C2\u00A0ovulation\u00C2\u00A0 Pancreas\u00C2\u00A0 N/A\u00C2\u00A0 Islet\u00C2\u00A0development Peritoneal\u00C2\u00A0Cavity\u00C2\u00A0 Peritoneal\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 Immune\u00C2\u00A0surveillance Spleen\u00C2\u00A0 Splenic\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 Clearance\u00C2\u00A0of\u00C2\u00A0debris\u00C2\u00A0and\u00C2\u00A0damaged\u00C2\u00A0erythrocytes Testis\u00C2\u00A0 N/A\u00C2\u00A0 Steroid\u00C2\u00A0hormone\u00C2\u00A0production\u00C2\u00A0 Uterus\u00C2\u00A0 Uterine\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 Cervical\u00C2\u00A0ripening N/A,\u00C2\u00A0not\u00C2\u00A0applicable\u00C2\u00A0 \u00C2\u00A0 1.7.1.1 M\u00CF\u0086\u00C2\u00A0activation\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 hallmark\u00C2\u00A0 characteristics\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 is\u00C2\u00A0 their\u00C2\u00A0 functional\u00C2\u00A0 plasticity\u00C2\u00A0 (Mantovani\u00C2\u00A0and\u00C2\u00A0Sica,\u00C2\u00A02010).\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0range\u00C2\u00A0of\u00C2\u00A0phenotypes\u00C2\u00A0and\u00C2\u00A0perform\u00C2\u00A0many\u00C2\u00A0 different\u00C2\u00A0functions\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0environmental\u00C2\u00A0stimuli\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0exposed\u00C2\u00A0\u00C2\u00A0 (Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008)\u00C2\u00A0(Fig.\u00C2\u00A01.3).\u00C2\u00A0This\u00C2\u00A0diversity\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0has\u00C2\u00A0led\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0proposed\u00C2\u00A0 classification\u00C2\u00A0 systems\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 activating\u00C2\u00A0 stimuli\u00C2\u00A0 (Gordon,\u00C2\u00A0 2003)\u00C2\u00A0 or\u00C2\u00A0 different\u00C2\u00A0 homeostatic\u00C2\u00A0activities\u00C2\u00A0(i.e.\u00C2\u00A0host\u00C2\u00A0defense,\u00C2\u00A0wound\u00C2\u00A0healing,\u00C2\u00A0or\u00C2\u00A0immune\u00C2\u00A0regulation)\u00C2\u00A0(Mosser\u00C2\u00A0 and\u00C2\u00A0 Edwards,\u00C2\u00A0 2008).\u00C2\u00A0 The\u00C2\u00A0 M1/M2\u00C2\u00A0 classification\u00C2\u00A0 scheme\u00C2\u00A0 described\u00C2\u00A0 below\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 widely\u00C2\u00A0 used\u00C2\u00A0 model\u00C2\u00A0 and\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 elaborated\u00C2\u00A0 upon\u00C2\u00A0 to\u00C2\u00A0 include\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 subclasses\u00C2\u00A0 (i.e.\u00C2\u00A0M2a,\u00C2\u00A0M2b,\u00C2\u00A0M2c)\u00C2\u00A0 (Mantovani\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2004).\u00C2\u00A0However,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 important\u00C2\u00A0 to\u00C2\u00A0 bear\u00C2\u00A0 in\u00C2\u00A0mind\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 activation\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0 a\u00C2\u00A0 cyclic\u00C2\u00A0 process\u00C2\u00A0 that\u00C2\u00A0 balances\u00C2\u00A0 classical\u00C2\u00A0 and\u00C2\u00A0 20 alternative\u00C2\u00A0 activation\u00C2\u00A0 to\u00C2\u00A0 achieve\u00C2\u00A0 proper\u00C2\u00A0 immunologic\u00C2\u00A0 function\u00C2\u00A0 (McGrath\u00C2\u00A0 and\u00C2\u00A0Kodelja,\u00C2\u00A0 1999).\u00C2\u00A0 Thus,\u00C2\u00A0 polarization\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 useful,\u00C2\u00A0 simplified\u00C2\u00A0 conceptual\u00C2\u00A0 framework\u00C2\u00A0 for\u00C2\u00A0 describing\u00C2\u00A0what\u00C2\u00A0 is\u00C2\u00A0most\u00C2\u00A0 likely\u00C2\u00A0a\u00C2\u00A0continuum\u00C2\u00A0of\u00C2\u00A0diverse\u00C2\u00A0 functional\u00C2\u00A0 states\u00C2\u00A0 (Mantovani\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02004).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Classically\u00C2\u00A0 activated,\u00C2\u00A0 or\u00C2\u00A0 M1\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 are\u00C2\u00A0 induced\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 type\u00C2\u00A0 I,\u00C2\u00A0 or\u00C2\u00A0 pro\u00E2\u0080\u0090 inflammatory\u00C2\u00A0 cytokines,\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 and\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 (Martinez\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2006).\u00C2\u00A0 In\u00C2\u00A0 turn,\u00C2\u00A0M1\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 produce\u00C2\u00A0 high\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 cytokines\u00C2\u00A0 (i.e.\u00C2\u00A0 IL\u00E2\u0080\u00901\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u00906,\u00C2\u00A0 IL\u00E2\u0080\u009012,\u00C2\u00A0 IL\u00E2\u0080\u009023,\u00C2\u00A0 TNF\u00E2\u0080\u0090\u00CE\u00B1)\u00C2\u00A0and\u00C2\u00A0low\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0anti\u00E2\u0080\u0090inflammatory\u00C2\u00A0cytokines\u00C2\u00A0(i.e.\u00C2\u00A0IL\u00E2\u0080\u009010)\u00C2\u00A0(Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006).\u00C2\u00A0\u00C2\u00A0 Upon\u00C2\u00A0activation\u00C2\u00A0by\u00C2\u00A0Th1\u00C2\u00A0or\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0produced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0M1\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0secrete\u00C2\u00A0ROS\u00C2\u00A0and\u00C2\u00A0RNS\u00C2\u00A0and\u00C2\u00A0 thereby\u00C2\u00A0become\u00C2\u00A0potent\u00C2\u00A0killers\u00C2\u00A0of\u00C2\u00A0intracellular\u00C2\u00A0microorganisms\u00C2\u00A0and\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0(Parkin\u00C2\u00A0 and\u00C2\u00A0Cohen,\u00C2\u00A02001).\u00C2\u00A0These\u00C2\u00A0cytotoxic\u00C2\u00A0functions,\u00C2\u00A0combined\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0 process\u00C2\u00A0 and\u00C2\u00A0 present\u00C2\u00A0 Ags,\u00C2\u00A0 makes\u00C2\u00A0 them\u00C2\u00A0 important\u00C2\u00A0 inducers\u00C2\u00A0 and\u00C2\u00A0 effectors\u00C2\u00A0 of\u00C2\u00A0 Th1\u00C2\u00A0 cell\u00E2\u0080\u0090 mediated\u00C2\u00A0immune\u00C2\u00A0responses\u00C2\u00A0(Gordon\u00C2\u00A0and\u00C2\u00A0Martinez,\u00C2\u00A02010;\u00C2\u00A0Munn\u00C2\u00A0and\u00C2\u00A0Mellor,\u00C2\u00A02003).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Alternatively\u00C2\u00A0 activated,\u00C2\u00A0 or\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reported\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 both\u00C2\u00A0 specific\u00C2\u00A0 and\u00C2\u00A0 non\u00E2\u0080\u0090specific\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 activation.\u00C2\u00A0 In\u00C2\u00A0 general,\u00C2\u00A0 they\u00C2\u00A0 arise\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0to\u00C2\u00A0stimulation\u00C2\u00A0by\u00C2\u00A0type\u00C2\u00A0II\u00C2\u00A0cytokines,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0IL\u00E2\u0080\u00904,\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0IL\u00E2\u0080\u009013,\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0and\u00C2\u00A0PGE2\u00C2\u00A0 and\u00C2\u00A0 produce\u00C2\u00A0 anti\u00E2\u0080\u0090inflammatory\u00C2\u00A0 cytokines\u00C2\u00A0 (i.e.\u00C2\u00A0 IL\u00E2\u0080\u009010)\u00C2\u00A0 (Martinez\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 Interestingly,\u00C2\u00A0M2\u00C2\u00A0activation\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0regulated\u00C2\u00A0in\u00C2\u00A0part\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0p50\u00C2\u00A0subunit\u00C2\u00A0of\u00C2\u00A0NF\u00E2\u0080\u0090 \u00CE\u00BAB,\u00C2\u00A0 which\u00C2\u00A0 inhibits\u00C2\u00A0 NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0 induced\u00C2\u00A0 M1\u00E2\u0080\u0090polarizing\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 production\u00C2\u00A0 and\u00C2\u00A0 consequently\u00C2\u00A0 promotes\u00C2\u00A0 M2\u00C2\u00A0 activation\u00C2\u00A0 (Porta\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009).\u00C2\u00A0 M2\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 important\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 killing\u00C2\u00A0 extracellular\u00C2\u00A0 microorganisms\u00C2\u00A0 and\u00C2\u00A0 parasites\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 promoting\u00C2\u00A0 wound\u00C2\u00A0 healing\u00C2\u00A0 by\u00C2\u00A0 initiating\u00C2\u00A0 tissue\u00C2\u00A0 remodeling\u00C2\u00A0 and\u00C2\u00A0 angiogenesis\u00C2\u00A0 (Martinez\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 A\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 murine\u00C2\u00A0 M2\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 markers\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 described\u00C2\u00A0 including\u00C2\u00A0 scavenger,\u00C2\u00A0 mannose,\u00C2\u00A0 and\u00C2\u00A0 galactose\u00E2\u0080\u0090type\u00C2\u00A0receptors\u00C2\u00A0(Gordon,\u00C2\u00A02003;\u00C2\u00A0Stein\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01992),\u00C2\u00A0FIZZ1\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0mammalian\u00C2\u00A0 chitinase\u00C2\u00A0Ym1\u00C2\u00A0(Ho\u00C2\u00A0and\u00C2\u00A0Sly,\u00C2\u00A02009;\u00C2\u00A0Holcomb\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02000;\u00C2\u00A0Raes\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002),\u00C2\u00A0and\u00C2\u00A0recently,\u00C2\u00A0 migration\u00E2\u0080\u0090stimulating\u00C2\u00A0factor\u00C2\u00A0(MSF)\u00C2\u00A0(Solinas\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0to\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation/proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0mechanisms\u00C2\u00A0including\u00C2\u00A0secretion\u00C2\u00A0 21 \u00C2\u00A0 Figure\u00C2\u00A01.3\u00C2\u00A0 Polarization\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0phenotypes.\u00C2\u00A0\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 activation\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 cyclical\u00C2\u00A0 process\u00C2\u00A0 that\u00C2\u00A0 balances\u00C2\u00A0 classical\u00C2\u00A0 (M1)\u00C2\u00A0 and\u00C2\u00A0 alternative\u00C2\u00A0 (M2)\u00C2\u00A0 activation\u00C2\u00A0 to\u00C2\u00A0 eradicate\u00C2\u00A0 invading\u00C2\u00A0 pathogens\u00C2\u00A0 and\u00C2\u00A0 promote\u00C2\u00A0 subsequent\u00C2\u00A0 healing.\u00C2\u00A0 Blood\u00C2\u00A0 monocytes\u00C2\u00A0 that\u00C2\u00A0 differentiate\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 LPS/IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 mature\u00C2\u00A0 into\u00C2\u00A0 M1\u00C2\u00A0 or\u00C2\u00A0 \u00E2\u0080\u009Cclassically\u00C2\u00A0 activated\u00E2\u0080\u009D\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0These\u00C2\u00A0M1\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0effective\u00C2\u00A0APCs\u00C2\u00A0and\u00C2\u00A0 thus\u00C2\u00A0promote\u00C2\u00A0 immune\u00C2\u00A0responses.\u00C2\u00A0 In\u00C2\u00A0 addition,\u00C2\u00A0 they\u00C2\u00A0 produce\u00C2\u00A0 high\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 cytokines,\u00C2\u00A0 ROS,\u00C2\u00A0 and\u00C2\u00A0 RNS,\u00C2\u00A0 which\u00C2\u00A0 enable\u00C2\u00A0 them\u00C2\u00A0 to\u00C2\u00A0 kill\u00C2\u00A0 pathogens\u00C2\u00A0 and\u00C2\u00A0 transformed\u00C2\u00A0 cells.\u00C2\u00A0 On\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 monocytes\u00C2\u00A0 that\u00C2\u00A0 differentiate\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 IL\u00E2\u0080\u00904,\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 IL\u00E2\u0080\u009013,\u00C2\u00A0 or\u00C2\u00A0 corticosteroids\u00C2\u00A0 mature\u00C2\u00A0 into\u00C2\u00A0 M2\u00C2\u00A0 or\u00C2\u00A0 \u00E2\u0080\u009Calternatively\u00C2\u00A0 activated\u00E2\u0080\u009D\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 which\u00C2\u00A0 secrete\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u00901ra,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 IL\u00E2\u0080\u00901R\u00C2\u00A0 decoy\u00C2\u00A0 protein.\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0scavenge\u00C2\u00A0dead\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0debris,\u00C2\u00A0 remodel\u00C2\u00A0and\u00C2\u00A0repair\u00C2\u00A0damaged\u00C2\u00A0 tissues,\u00C2\u00A0and\u00C2\u00A0 promote\u00C2\u00A0angiogenesis.\u00C2\u00A0They\u00C2\u00A0also\u00C2\u00A0 function\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0M1\u00C2\u00A0M\u00CF\u0086\u00E2\u0080\u0090mediated\u00C2\u00A0immune\u00C2\u00A0responses\u00C2\u00A0 as\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0resolving\u00C2\u00A0inflammation.\u00C2\u00A0Another\u00C2\u00A0key\u00C2\u00A0difference\u00C2\u00A0between\u00C2\u00A0M1\u00C2\u00A0and\u00C2\u00A0M2\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0is\u00C2\u00A0how\u00C2\u00A0they\u00C2\u00A0metabolize\u00C2\u00A0the\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0L\u00E2\u0080\u0090Arg;\u00C2\u00A0M1\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0express\u00C2\u00A0iNOS\u00C2\u00A0and\u00C2\u00A0convert\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0 into\u00C2\u00A0 NO\u00C2\u00A0 while\u00C2\u00A0 M2\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 express\u00C2\u00A0 the\u00C2\u00A0 enzyme\u00C2\u00A0 Arg1,\u00C2\u00A0 which\u00C2\u00A0 converts\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 into\u00C2\u00A0 L\u00E2\u0080\u0090ornithine,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 then\u00C2\u00A0 converted\u00C2\u00A0 into\u00C2\u00A0 polyamines\u00C2\u00A0 (i.e.\u00C2\u00A0 putrescine,\u00C2\u00A0 spermidine,\u00C2\u00A0 spermine)\u00C2\u00A0 that\u00C2\u00A0 promote\u00C2\u00A0 cell\u00C2\u00A0 division\u00C2\u00A0 and\u00C2\u00A0 L\u00E2\u0080\u0090proline,\u00C2\u00A0 which\u00C2\u00A0 supports\u00C2\u00A0 collagen\u00C2\u00A0 synthesis.\u00C2\u00A0 Adapted\u00C2\u00A0 from\u00C2\u00A0 references\u00C2\u00A0in\u00C2\u00A0section\u00C2\u00A01.7.1.1.\u00C2\u00A0\u00C2\u00A0 22 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0 cytokines\u00C2\u00A0 (i.e.\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u009010)\u00C2\u00A0 (Munn\u00C2\u00A0 and\u00C2\u00A0 Mellor,\u00C2\u00A0 2003),\u00C2\u00A0 Treg\u00C2\u00A0 induction\u00C2\u00A0(Brem\u00E2\u0080\u0090Exner\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Hoves\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006),\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0PGs,\u00C2\u00A0ROS,\u00C2\u00A0and\u00C2\u00A0RNS\u00C2\u00A0 (Albina\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01991;\u00C2\u00A0Allison,\u00C2\u00A01978;\u00C2\u00A0Metzger\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01980;\u00C2\u00A0Strickland\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01996),\u00C2\u00A0and\u00C2\u00A0by\u00C2\u00A0 depletion\u00C2\u00A0of\u00C2\u00A0amino\u00C2\u00A0acids\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0tryptophan\u00C2\u00A0(Munn\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01999)\u00C2\u00A0and\u00C2\u00A0arginine\u00C2\u00A0(Kung\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A01977;\u00C2\u00A0Rodriguez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003)\u00C2\u00A0via\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0IDO\u00C2\u00A0and\u00C2\u00A0Arg1,\u00C2\u00A0respectively.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Based\u00C2\u00A0 on\u00C2\u00A0 in\u00C2\u00A0 depth\u00C2\u00A0 studies\u00C2\u00A0 exploring\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 polarization,\u00C2\u00A0 Mantovani\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 have\u00C2\u00A0 proposed\u00C2\u00A0that\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0further\u00C2\u00A0subdivided\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0induce\u00C2\u00A0their\u00C2\u00A0 activation\u00C2\u00A0(Mantovani\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004).\u00C2\u00A0Specifically,\u00C2\u00A0M2a\u00C2\u00A0are\u00C2\u00A0activated\u00C2\u00A0by\u00C2\u00A0IL\u00E2\u0080\u00904\u00C2\u00A0or\u00C2\u00A0IL\u00E2\u0080\u009013,\u00C2\u00A0M2b\u00C2\u00A0 by\u00C2\u00A0immune\u00C2\u00A0complexes\u00C2\u00A0in\u00C2\u00A0combination\u00C2\u00A0with\u00C2\u00A0IL\u00E2\u0080\u00901\u00CE\u00B2\u00C2\u00A0or\u00C2\u00A0lipopolysaccharide\u00C2\u00A0(LPS),\u00C2\u00A0and\u00C2\u00A0M2c\u00C2\u00A0 by\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0or\u00C2\u00A0glucocortocoids\u00C2\u00A0(Mantovani\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004;\u00C2\u00A0Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0The\u00C2\u00A0 phenotype\u00C2\u00A0 and\u00C2\u00A0 function\u00C2\u00A0 of\u00C2\u00A0 each\u00C2\u00A0 subtype,\u00C2\u00A0 including\u00C2\u00A0 chemokine,\u00C2\u00A0 cytokine,\u00C2\u00A0 effector\u00C2\u00A0 molecule,\u00C2\u00A0and\u00C2\u00A0receptor\u00C2\u00A0expression,\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0thoroughly\u00C2\u00A0elucidated\u00C2\u00A0by\u00C2\u00A0genetic\u00C2\u00A0profiling,\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0functional\u00C2\u00A0assays\u00C2\u00A0(Mantovani\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004;\u00C2\u00A0Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006).\u00C2\u00A0Among\u00C2\u00A0their\u00C2\u00A0 many\u00C2\u00A0intriguing\u00C2\u00A0findings,\u00C2\u00A0Martinez\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0report\u00C2\u00A0that\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0driven\u00C2\u00A0human\u00C2\u00A0monocyte\u00C2\u00A0to\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0differentiation\u00C2\u00A0leads\u00C2\u00A0to\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0M2\u00C2\u00A0transcripts,\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0M2\u00C2\u00A0activation\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 default\u00C2\u00A0 pathway\u00C2\u00A0 under\u00C2\u00A0 steady\u00E2\u0080\u0090state\u00C2\u00A0 conditions\u00C2\u00A0 (Martinez\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006).\u00C2\u00A0 Furthermore,\u00C2\u00A0they\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0upregulated\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0cell\u00C2\u00A0cycle\u00E2\u0080\u0090 associated\u00C2\u00A0 genes,\u00C2\u00A0 revealing\u00C2\u00A0 the\u00C2\u00A0 previously\u00C2\u00A0 unappreciated\u00C2\u00A0 proliferation\u00C2\u00A0 potential\u00C2\u00A0 of\u00C2\u00A0 monocytes\u00C2\u00A0(Martinez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006).\u00C2\u00A0These\u00C2\u00A0 important\u00C2\u00A0 findings\u00C2\u00A0regarding\u00C2\u00A0the\u00C2\u00A0phenotypic\u00C2\u00A0 profiles\u00C2\u00A0 of\u00C2\u00A0 M1\u00C2\u00A0 versus\u00C2\u00A0 M2\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 recently\u00C2\u00A0 and\u00C2\u00A0 comprehensively\u00C2\u00A0 reviewed\u00C2\u00A0 (Gordon\u00C2\u00A0and\u00C2\u00A0Martinez,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Contributing\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 distinct\u00C2\u00A0 responses\u00C2\u00A0 of\u00C2\u00A0 M1\u00C2\u00A0 and\u00C2\u00A0 M2\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 way\u00C2\u00A0 they\u00C2\u00A0 metabolize\u00C2\u00A0 the\u00C2\u00A0 amino\u00C2\u00A0 acid\u00C2\u00A0 L\u00E2\u0080\u0090arginine\u00C2\u00A0 (L\u00E2\u0080\u0090Arg).\u00C2\u00A0 M1\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 rapidly\u00C2\u00A0 upregulate\u00C2\u00A0 inducible\u00C2\u00A0 nitric\u00C2\u00A0oxide\u00C2\u00A0synthase\u00C2\u00A0(iNOS,\u00C2\u00A0or\u00C2\u00A0NOS2)\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0danger\u00C2\u00A0signals\u00C2\u00A0(Rauh\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005;\u00C2\u00A0 Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005a)\u00C2\u00A0to\u00C2\u00A0convert\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0into\u00C2\u00A0nitric\u00C2\u00A0oxide\u00C2\u00A0(NO)\u00C2\u00A0to\u00C2\u00A0kill\u00C2\u00A0bacteria,\u00C2\u00A0viruses,\u00C2\u00A0and\u00C2\u00A0 tumor\u00C2\u00A0cells\u00C2\u00A0(Gordon,\u00C2\u00A02003;\u00C2\u00A0Mantovani\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002).\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0which\u00C2\u00A0 typically\u00C2\u00A0 function\u00C2\u00A0 in\u00C2\u00A0 wound\u00C2\u00A0 healing\u00C2\u00A0 after\u00C2\u00A0 an\u00C2\u00A0 infectious\u00C2\u00A0 agent\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 destroyed,\u00C2\u00A0 possess\u00C2\u00A0constitutively\u00C2\u00A0high\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0Arg1,\u00C2\u00A0which\u00C2\u00A0sequesters\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0away\u00C2\u00A0 from\u00C2\u00A0 iNOS\u00C2\u00A0 to\u00C2\u00A0 23 generate\u00C2\u00A0ornithine.\u00C2\u00A0Ornithine,\u00C2\u00A0 in\u00C2\u00A0 turn,\u00C2\u00A0 stimulates\u00C2\u00A0host\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 (via\u00C2\u00A0ornithine\u00C2\u00A0 decarboxylase\u00E2\u0080\u0090mediated\u00C2\u00A0 conversion\u00C2\u00A0 into\u00C2\u00A0 polyamines)\u00C2\u00A0 and\u00C2\u00A0 collagen\u00C2\u00A0 synthesis\u00C2\u00A0 (via\u00C2\u00A0 ornithine\u00E2\u0080\u0090derived\u00C2\u00A0proline)\u00C2\u00A0(Gordon\u00C2\u00A0and\u00C2\u00A0Martinez,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.7.1.2 TAMs\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0is\u00C2\u00A0well\u00C2\u00A0established\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0critical\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0biology.\u00C2\u00A0Solid\u00C2\u00A0tumors,\u00C2\u00A0 including\u00C2\u00A0 both\u00C2\u00A0 primary\u00C2\u00A0 tumors\u00C2\u00A0 and\u00C2\u00A0 metastatic\u00C2\u00A0 lesions,\u00C2\u00A0 are\u00C2\u00A0 infiltrated\u00C2\u00A0 by\u00C2\u00A0 significant\u00C2\u00A0 numbers\u00C2\u00A0of\u00C2\u00A0TAMs\u00C2\u00A0(Siveen\u00C2\u00A0and\u00C2\u00A0Kuttan,\u00C2\u00A02009).\u00C2\u00A0In\u00C2\u00A0fact,\u00C2\u00A0there\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reports\u00C2\u00A0that\u00C2\u00A0TAMs\u00C2\u00A0 can\u00C2\u00A0 comprise\u00C2\u00A0 up\u00C2\u00A0 to\u00C2\u00A0 80%\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0mass\u00C2\u00A0 in\u00C2\u00A0 breast\u00C2\u00A0 tumors\u00C2\u00A0 (Bingle\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2002).\u00C2\u00A0 The\u00C2\u00A0 presence\u00C2\u00A0of\u00C2\u00A0extensive\u00C2\u00A0TAM\u00C2\u00A0infiltration\u00C2\u00A0correlates\u00C2\u00A0with\u00C2\u00A0poor\u00C2\u00A0prognosis\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A0 a\u00C2\u00A0 variety\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0 cancers,\u00C2\u00A0 including\u00C2\u00A0 breast,\u00C2\u00A0 cervix,\u00C2\u00A0 and\u00C2\u00A0 bladder\u00C2\u00A0 (Leek\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1996;\u00C2\u00A0 Siveen\u00C2\u00A0 and\u00C2\u00A0 Kuttan,\u00C2\u00A0 2009),\u00C2\u00A0 a\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0 underscores\u00C2\u00A0 the\u00C2\u00A0 vital\u00C2\u00A0 role\u00C2\u00A0 TAMs\u00C2\u00A0 play\u00C2\u00A0 in\u00C2\u00A0 promoting\u00C2\u00A0carcinogenesis.\u00C2\u00A0Monocytes\u00C2\u00A0are\u00C2\u00A0recruited\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0site\u00C2\u00A0by\u00C2\u00A0tumor\u00E2\u0080\u0090derived\u00C2\u00A0 chemokines\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0CCL2\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0mature\u00C2\u00A0and\u00C2\u00A0become\u00C2\u00A0activated\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0tumor\u00E2\u0080\u0090 derived\u00C2\u00A0cytokines,\u00C2\u00A0growth\u00C2\u00A0factors\u00C2\u00A0(i.e.\u00C2\u00A0M\u00E2\u0080\u0090CSF,\u00C2\u00A0VEGF,\u00C2\u00A0IL\u00E2\u0080\u00904,\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2),\u00C2\u00A0and\u00C2\u00A0PGs\u00C2\u00A0(Siveen\u00C2\u00A0 and\u00C2\u00A0Kuttan,\u00C2\u00A02009).\u00C2\u00A0Once\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 site,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 tend\u00C2\u00A0 to\u00C2\u00A0 cluster\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 leading\u00C2\u00A0edge\u00C2\u00A0of\u00C2\u00A0 tumors,\u00C2\u00A0 i.e.,\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 tumor\u00E2\u0080\u0090host\u00C2\u00A0 tissue\u00C2\u00A0 interface\u00C2\u00A0 (Siveen\u00C2\u00A0and\u00C2\u00A0Kuttan,\u00C2\u00A02009).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0general,\u00C2\u00A0 TAMs\u00C2\u00A0are\u00C2\u00A0thought\u00C2\u00A0to\u00C2\u00A0display\u00C2\u00A0a\u00C2\u00A0molecular\u00C2\u00A0and\u00C2\u00A0functional\u00C2\u00A0phenotype\u00C2\u00A0similar\u00C2\u00A0to\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (Mantovani\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002).\u00C2\u00A0However,\u00C2\u00A0there\u00C2\u00A0are\u00C2\u00A0reports\u00C2\u00A0that\u00C2\u00A0some\u00C2\u00A0TAMs\u00C2\u00A0can\u00C2\u00A0express\u00C2\u00A0both\u00C2\u00A0 iNOS\u00C2\u00A0 and\u00C2\u00A0Arg1\u00C2\u00A0 and\u00C2\u00A0 thus\u00C2\u00A0possess\u00C2\u00A0 a\u00C2\u00A0 combination\u00C2\u00A0 of\u00C2\u00A0M1\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0M2\u00E2\u0080\u0090like\u00C2\u00A0 features\u00C2\u00A0 (Gordon\u00C2\u00A0 and\u00C2\u00A0 Martinez,\u00C2\u00A0 2010).\u00C2\u00A0 Interestingly,\u00C2\u00A0 although\u00C2\u00A0 these\u00C2\u00A0 M1/M2\u00C2\u00A0 TAMs\u00C2\u00A0 produce\u00C2\u00A0 pro\u00E2\u0080\u0090 inflammatory\u00C2\u00A0 cytokines\u00C2\u00A0 and\u00C2\u00A0 ROS,\u00C2\u00A0 they\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 seem\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 cytotoxic\u00C2\u00A0 to\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 (Gordon\u00C2\u00A0 and\u00C2\u00A0 Martinez,\u00C2\u00A0 2010).\u00C2\u00A0 A\u00C2\u00A0 recent\u00C2\u00A0 study\u00C2\u00A0 by\u00C2\u00A0 Movahedi\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 highlighted\u00C2\u00A0 the\u00C2\u00A0 heterogeneity\u00C2\u00A0 of\u00C2\u00A0 TAMs,\u00C2\u00A0 demonstrating\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 multiple\u00C2\u00A0 phenotypically\u00C2\u00A0 and\u00C2\u00A0 functionally\u00C2\u00A0distinct\u00C2\u00A0TAM\u00C2\u00A0subsets,\u00C2\u00A0which\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0 characterized\u00C2\u00A0as\u00C2\u00A0either\u00C2\u00A0M1\u00E2\u0080\u0090\u00C2\u00A0or\u00C2\u00A0M2\u00E2\u0080\u0090 like\u00C2\u00A0(Movahedi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Consistent\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 in\u00C2\u00A0 normal\u00C2\u00A0 wound\u00C2\u00A0 healing,\u00C2\u00A0 TAMs\u00C2\u00A0 promote\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 metastasis\u00C2\u00A0 via\u00C2\u00A0 promotion\u00C2\u00A0 of\u00C2\u00A0 angiogenesis\u00C2\u00A0 and\u00C2\u00A0 vascularization,\u00C2\u00A0 24 stroma\u00C2\u00A0 formation\u00C2\u00A0 and\u00C2\u00A0 remodeling,\u00C2\u00A0 enhancement\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 invasion,\u00C2\u00A0 recruitment\u00C2\u00A0 of\u00C2\u00A0 additional\u00C2\u00A0 inflammatory\u00C2\u00A0 cells,\u00C2\u00A0 and\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 (Mantovani\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2002;\u00C2\u00A0 Siveen\u00C2\u00A0 and\u00C2\u00A0Kuttan,\u00C2\u00A0 2009)\u00C2\u00A0 (Fig.\u00C2\u00A0 1.4).\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 it\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0suggested\u00C2\u00A0that\u00C2\u00A0TAMs\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0critical\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0facilitating\u00C2\u00A0tumor\u00C2\u00A0cell\u00C2\u00A0migration\u00C2\u00A0out\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0primary\u00C2\u00A0tumor,\u00C2\u00A0entry\u00C2\u00A0into\u00C2\u00A0blood\u00C2\u00A0or\u00C2\u00A0lymph\u00C2\u00A0vessels,\u00C2\u00A0and\u00C2\u00A0seeding\u00C2\u00A0in\u00C2\u00A0distant\u00C2\u00A0sites\u00C2\u00A0(Qian\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0TAMs\u00C2\u00A0carry\u00C2\u00A0out\u00C2\u00A0these\u00C2\u00A0functions\u00C2\u00A0by\u00C2\u00A0producing\u00C2\u00A0a\u00C2\u00A0plethora\u00C2\u00A0of\u00C2\u00A0growth\u00C2\u00A0factors,\u00C2\u00A0 proteolytic\u00C2\u00A0enzymes,\u00C2\u00A0cytokines,\u00C2\u00A0and\u00C2\u00A0inflammatory\u00C2\u00A0mediators\u00C2\u00A0(Bingle\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002;\u00C2\u00A0Siveen\u00C2\u00A0 and\u00C2\u00A0Kuttan,\u00C2\u00A02009)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0mechanisms\u00C2\u00A0involved\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0detailed\u00C2\u00A0in\u00C2\u00A0several\u00C2\u00A0 reviews\u00C2\u00A0(Bingle\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002;\u00C2\u00A0Condeelis\u00C2\u00A0and\u00C2\u00A0Pollard,\u00C2\u00A02006;\u00C2\u00A0Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Martinez\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02008;\u00C2\u00A0Qian\u00C2\u00A0and\u00C2\u00A0Pollard,\u00C2\u00A02010).\u00C2\u00A0 25 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A01.4\u00C2\u00A0 TAMs\u00C2\u00A0promote\u00C2\u00A0tumorigenesis\u00C2\u00A0via\u00C2\u00A0multiple\u00C2\u00A0mechanisms.\u00C2\u00A0\u00C2\u00A0 TAMs\u00C2\u00A0 resemble\u00C2\u00A0 M2\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 play\u00C2\u00A0 a\u00C2\u00A0 critical\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 metastasis.\u00C2\u00A0 TAMs\u00C2\u00A0 produce\u00C2\u00A0 a\u00C2\u00A0 plethora\u00C2\u00A0 of\u00C2\u00A0 cytokines,\u00C2\u00A0 growth\u00C2\u00A0 factors,\u00C2\u00A0 chemokines,\u00C2\u00A0 and\u00C2\u00A0 other\u00C2\u00A0 molecules\u00C2\u00A0 that\u00C2\u00A0 support\u00C2\u00A0 tumor\u00C2\u00A0 growth,\u00C2\u00A0 metastasis,\u00C2\u00A0 matrix\u00C2\u00A0 remodeling,\u00C2\u00A0 inflammation,\u00C2\u00A0 angiogenesis,\u00C2\u00A0 and\u00C2\u00A0 immune\u00C2\u00A0suppression.\u00C2\u00A0Adapted\u00C2\u00A0from\u00C2\u00A0references\u00C2\u00A0in\u00C2\u00A0section\u00C2\u00A01.7.1.2.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 26 1.7.2 Myeloid\u00C2\u00ADderived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 MDSCs\u00C2\u00A0are\u00C2\u00A0a\u00C2\u00A0heterogeneous\u00C2\u00A0population\u00C2\u00A0of\u00C2\u00A0relatively\u00C2\u00A0immature\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0 stages\u00C2\u00A0 of\u00C2\u00A0 differentiation\u00C2\u00A0 including\u00C2\u00A0 early\u00C2\u00A0 myeloid\u00C2\u00A0 progenitors\u00C2\u00A0 and\u00C2\u00A0 immature\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0\u00C2\u00A0 monocytes,\u00C2\u00A0PMNs,\u00C2\u00A0and\u00C2\u00A0DCs\u00C2\u00A0(Pastula\u00C2\u00A0and\u00C2\u00A0Marcinkiewicz,\u00C2\u00A02011).\u00C2\u00A0Murine\u00C2\u00A0MDSCs\u00C2\u00A0express\u00C2\u00A0 both\u00C2\u00A0the\u00C2\u00A0\u00CE\u00B1M\u00C2\u00A0 integrin\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0myeloid\u00C2\u00A0 lineage\u00C2\u00A0differentiation\u00C2\u00A0Ag\u00C2\u00A0Gr1\u00C2\u00A0(Ly6G\u00C2\u00A0and\u00C2\u00A0 Ly6C).\u00C2\u00A0 Two\u00C2\u00A0 subsets\u00C2\u00A0 of\u00C2\u00A0 murine\u00C2\u00A0 MDSCs\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 identified;\u00C2\u00A0 CD11b+Ly6G\u00E2\u0080\u0090Ly6Chigh\u00C2\u00A0 mononuclear\u00C2\u00A0 cells,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 termed\u00C2\u00A0 monocytic\u00C2\u00A0 MDSCs\u00C2\u00A0 (M\u00E2\u0080\u0090MDSCs)\u00C2\u00A0 and\u00C2\u00A0 CD11b+Ly6G+Ly6Clow\u00C2\u00A0cells,\u00C2\u00A0which\u00C2\u00A0have\u00C2\u00A0multi\u00E2\u0080\u0090lobed\u00C2\u00A0nuclei\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0termed\u00C2\u00A0granulocytic\u00E2\u0080\u0090 MDSCs\u00C2\u00A0 (G\u00E2\u0080\u0090MDSCs)\u00C2\u00A0 (Movahedi\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008;\u00C2\u00A0 Youn\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 Human\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 generally\u00C2\u00A0defined\u00C2\u00A0as\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0express\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0common\u00C2\u00A0myeloid\u00C2\u00A0marker\u00C2\u00A0CD33,\u00C2\u00A0 but\u00C2\u00A0lack\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0mature\u00C2\u00A0myeloid\u00C2\u00A0and\u00C2\u00A0lymphoid\u00C2\u00A0markers\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0MHCII\u00C2\u00A0molecule\u00C2\u00A0 HLA\u00E2\u0080\u0090DR\u00C2\u00A0(Almand\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02001;\u00C2\u00A0Diaz\u00E2\u0080\u0090Montero\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Nagaraj\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010;\u00C2\u00A0 Zea\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005).\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 greatly\u00C2\u00A0 expanded\u00C2\u00A0 in\u00C2\u00A0 practically\u00C2\u00A0 all\u00C2\u00A0 tested\u00C2\u00A0 mouse\u00C2\u00A0 tumor\u00C2\u00A0 models\u00C2\u00A0(Gallina\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006;\u00C2\u00A0Habibi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Rodriguez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005;\u00C2\u00A0Youn\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008)\u00C2\u00A0 and\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0patients\u00C2\u00A0(Almand\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02001;\u00C2\u00A0Diaz\u00E2\u0080\u0090Montero\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2008;\u00C2\u00A0 Mandruzzato\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009;\u00C2\u00A0 Ochoa\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007;\u00C2\u00A0 Valenti\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006;\u00C2\u00A0 Young\u00C2\u00A0 and\u00C2\u00A0 Lathers,\u00C2\u00A0 1999;\u00C2\u00A0 Zea\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005)\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 considered\u00C2\u00A0 one\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0main\u00C2\u00A0 contributors\u00C2\u00A0 to\u00C2\u00A0 immune\u00C2\u00A0suppression\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0(Nagaraj\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0In\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0tumor\u00E2\u0080\u0090 derived\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0promote\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 recruitment\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0 (i.e.\u00C2\u00A0M\u00E2\u0080\u0090CSF,\u00C2\u00A0GM\u00E2\u0080\u0090 CSF,\u00C2\u00A0VEGF,\u00C2\u00A0IL\u00E2\u0080\u00901\u00CE\u00B2,\u00C2\u00A0IL\u00E2\u0080\u00903,\u00C2\u00A0IL\u00E2\u0080\u00906,\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0Flt3\u00C2\u00A0ligand\u00C2\u00A0(Flt3L))\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0recruited\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0BM\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 site\u00C2\u00A0 and\u00C2\u00A0 secondary\u00C2\u00A0 lymphoid\u00C2\u00A0 organs\u00C2\u00A0 (i.e.\u00C2\u00A0 spleen,\u00C2\u00A0 lymph\u00C2\u00A0 nodes)\u00C2\u00A0 via\u00C2\u00A0 the\u00C2\u00A0 bloodstream\u00C2\u00A0 (Youn\u00C2\u00A0 and\u00C2\u00A0Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 In\u00C2\u00A0 addition\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0 can\u00C2\u00A0 also\u00C2\u00A0 home\u00C2\u00A0 to\u00C2\u00A0 and\u00C2\u00A0 accumulate\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 liver\u00C2\u00A0 (Ilkovitch\u00C2\u00A0 and\u00C2\u00A0 Lopez,\u00C2\u00A0 2009),\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 other\u00C2\u00A0 sites\u00C2\u00A0 including\u00C2\u00A0 the\u00C2\u00A0 kidneys,\u00C2\u00A0 bones,\u00C2\u00A0 and\u00C2\u00A0 lungs\u00C2\u00A0 (Pulaski\u00C2\u00A0 and\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A0 2001).\u00C2\u00A0 A\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 combinations\u00C2\u00A0 of\u00C2\u00A0markers\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 suggested\u00C2\u00A0 to\u00C2\u00A0more\u00C2\u00A0 specifically\u00C2\u00A0identify\u00C2\u00A0murine\u00C2\u00A0and\u00C2\u00A0human\u00C2\u00A0MDSCs,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0heterogeneity\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0makes\u00C2\u00A0 this\u00C2\u00A0difficult\u00C2\u00A0(Gallina\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006;\u00C2\u00A0Greenwald\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005;\u00C2\u00A0Huang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006;\u00C2\u00A0Kryczek\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02006;\u00C2\u00A0Nagaraj\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0Moreover,\u00C2\u00A0although\u00C2\u00A0 immature\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0 from\u00C2\u00A0 tumor\u00E2\u0080\u0090free\u00C2\u00A0 mice\u00C2\u00A0 lack\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 they\u00C2\u00A0 express\u00C2\u00A0 27 similar\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0proposed\u00C2\u00A0markers\u00C2\u00A0(Youn\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0Consequently,\u00C2\u00A0both\u00C2\u00A0murine\u00C2\u00A0 and\u00C2\u00A0human\u00C2\u00A0MDSCs\u00C2\u00A0must\u00C2\u00A0be\u00C2\u00A0identified\u00C2\u00A0functionally,\u00C2\u00A0which\u00C2\u00A0requires\u00C2\u00A0assays\u00C2\u00A0to\u00C2\u00A0accurately\u00C2\u00A0 assess\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0ex\u00C2\u00A0vivo.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 distinguished\u00C2\u00A0 by\u00C2\u00A0 their\u00C2\u00A0 myeloid\u00C2\u00A0 origin,\u00C2\u00A0 immature\u00C2\u00A0 state,\u00C2\u00A0 and,\u00C2\u00A0 most\u00C2\u00A0 notably,\u00C2\u00A0 by\u00C2\u00A0 their\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00E2\u0080\u0090mediated\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 (Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 They\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reported\u00C2\u00A0 to\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0mechanisms,\u00C2\u00A0which\u00C2\u00A0 are\u00C2\u00A0 indicative\u00C2\u00A0 of\u00C2\u00A0 their\u00C2\u00A0 heterogeneity.\u00C2\u00A0MDSCs\u00C2\u00A0 can\u00C2\u00A0express\u00C2\u00A0both\u00C2\u00A0iNOS\u00C2\u00A0(induced\u00C2\u00A0by\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3)\u00C2\u00A0and\u00C2\u00A0Arg1\u00C2\u00A0(induced\u00C2\u00A0by\u00C2\u00A0IL\u00E2\u0080\u00904,\u00C2\u00A0IL\u00E2\u0080\u009013,\u00C2\u00A0and\u00C2\u00A0PGE2),\u00C2\u00A0 which\u00C2\u00A0contribute\u00C2\u00A0 to\u00C2\u00A0MDSC\u00E2\u0080\u0090mediated\u00C2\u00A0suppression\u00C2\u00A0both\u00C2\u00A0 individually\u00C2\u00A0and\u00C2\u00A0synergistically\u00C2\u00A0 (Bronte\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003;\u00C2\u00A0Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0As\u00C2\u00A0mentioned\u00C2\u00A0previously,\u00C2\u00A0iNOS\u00C2\u00A0converts\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0 into\u00C2\u00A0 NO,\u00C2\u00A0 which\u00C2\u00A0 inhibits\u00C2\u00A0 IL\u00E2\u0080\u00902R\u00C2\u00A0 signaling\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 activation\u00C2\u00A0 while\u00C2\u00A0 Arg1\u00C2\u00A0 expression\u00C2\u00A0 depletes\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0microenvironment,\u00C2\u00A0which\u00C2\u00A0prevents\u00C2\u00A0re\u00E2\u0080\u0090expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0TCR\u00CE\u00B6\u00C2\u00A0 chain\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0signaling\u00C2\u00A0(Bronte\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003).\u00C2\u00A0However,\u00C2\u00A0when\u00C2\u00A0both\u00C2\u00A0iNOS\u00C2\u00A0and\u00C2\u00A0Arg1\u00C2\u00A0are\u00C2\u00A0 expressed\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0cell\u00C2\u00A0these\u00C2\u00A0two\u00C2\u00A0enzymes\u00C2\u00A0work\u00C2\u00A0together\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0synergistic\u00C2\u00A0fashion\u00C2\u00A0to\u00C2\u00A0 produce\u00C2\u00A0 ROS.\u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 (due\u00C2\u00A0 to\u00C2\u00A0 Arg1\u00C2\u00A0 expression),\u00C2\u00A0 iNOS\u00C2\u00A0 can\u00C2\u00A0 catalyze\u00C2\u00A0 superoxide\u00C2\u00A0 (O2\u00E2\u0080\u0090)\u00C2\u00A0 formation\u00C2\u00A0 via\u00C2\u00A0 its\u00C2\u00A0 reductase\u00C2\u00A0 domain,\u00C2\u00A0 which\u00C2\u00A0 combines\u00C2\u00A0 with\u00C2\u00A0 water\u00C2\u00A0 to\u00C2\u00A0 generate\u00C2\u00A0 hydrogen\u00C2\u00A0 peroxide\u00C2\u00A0 (H2O2)\u00C2\u00A0 or\u00C2\u00A0 with\u00C2\u00A0 NO\u00C2\u00A0 to\u00C2\u00A0 form\u00C2\u00A0 peroxynitrite\u00C2\u00A0 (ONOO\u00E2\u0080\u0090),\u00C2\u00A0 a\u00C2\u00A0 powerful\u00C2\u00A0oxidant\u00C2\u00A0that\u00C2\u00A0potently\u00C2\u00A0induces\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0dysfunction\u00C2\u00A0and\u00C2\u00A0apoptosis\u00C2\u00A0(Bronte\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2003;\u00C2\u00A0Xia\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01998).\u00C2\u00A0Consequently,\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0high\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0ROS\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0H2O2\u00C2\u00A0and\u00C2\u00A0ONOO\u00E2\u0080\u0090\u00C2\u00A0(Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0 reported\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0immunosuppressive\u00C2\u00A0factors\u00C2\u00A0including\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 and\u00C2\u00A0 PGE2\u00C2\u00A0 (Ostrand\u00E2\u0080\u0090Rosenberg\u00C2\u00A0 and\u00C2\u00A0 Sinha,\u00C2\u00A0 2009),\u00C2\u00A0 by\u00C2\u00A0 sequestering\u00C2\u00A0 the\u00C2\u00A0 amino\u00C2\u00A0acid\u00C2\u00A0L\u00E2\u0080\u0090cysteine,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0(Srivastava\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010),\u00C2\u00A0by\u00C2\u00A0 down\u00E2\u0080\u0090regulating\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 L\u00E2\u0080\u0090selectin,\u00C2\u00A0 which\u00C2\u00A0 prevents\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 trafficking\u00C2\u00A0 (Hanson\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009),\u00C2\u00A0and\u00C2\u00A0by\u00C2\u00A0 inducing\u00C2\u00A0the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0 functional\u00C2\u00A0Tregs\u00C2\u00A0(Huang\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02006;\u00C2\u00A0Serafini\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 There\u00C2\u00A0 are\u00C2\u00A0 reports\u00C2\u00A0 that\u00C2\u00A0 M\u00E2\u0080\u0090MDSCs\u00C2\u00A0 and\u00C2\u00A0 G\u00E2\u0080\u0090MDSCs\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 different\u00C2\u00A0mechanisms\u00C2\u00A0 (Movahedi\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008;\u00C2\u00A0 Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 G\u00E2\u0080\u0090MDSCs\u00C2\u00A0 28 suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0ROS\u00C2\u00A0production,\u00C2\u00A0which\u00C2\u00A0requires\u00C2\u00A0contact\u00C2\u00A0between\u00C2\u00A0G\u00E2\u0080\u0090MDSCs\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0for\u00C2\u00A0effective\u00C2\u00A0suppression.\u00C2\u00A0\u00C2\u00A0(Movahedi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0M\u00E2\u0080\u0090 MDSCs,\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reported\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 contact\u00C2\u00A0 independent\u00C2\u00A0mechanisms\u00C2\u00A0 including\u00C2\u00A0 upregulation\u00C2\u00A0 of\u00C2\u00A0 iNOS\u00C2\u00A0 and\u00C2\u00A0 consequent\u00C2\u00A0NO\u00C2\u00A0 production,\u00C2\u00A0 upregulation\u00C2\u00A0 of\u00C2\u00A0 Arg1\u00C2\u00A0 and\u00C2\u00A0 subsequent\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 depletion,\u00C2\u00A0 and\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0cytokines\u00C2\u00A0(Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0In\u00C2\u00A0addition,\u00C2\u00A0some\u00C2\u00A0groups\u00C2\u00A0 have\u00C2\u00A0 reported\u00C2\u00A0 that\u00C2\u00A0 M\u00E2\u0080\u0090MDSCs\u00C2\u00A0 are\u00C2\u00A0 more\u00C2\u00A0 potent\u00C2\u00A0 than\u00C2\u00A0 G\u00E2\u0080\u0090MDSCs\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 per\u00C2\u00A0 cell\u00C2\u00A0 basis;\u00C2\u00A0 however,\u00C2\u00A0 in\u00C2\u00A0most\u00C2\u00A0 tumor\u00C2\u00A0models\u00C2\u00A0 the\u00C2\u00A0 vast\u00C2\u00A0majority\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 are\u00C2\u00A0 comprised\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 G\u00E2\u0080\u0090 MDSC\u00C2\u00A0subtype\u00C2\u00A0(Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0addition\u00C2\u00A0 to\u00C2\u00A0suppressing\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 responses,\u00C2\u00A0MDSCs\u00C2\u00A0can\u00C2\u00A0also\u00C2\u00A0 impact\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immunity\u00C2\u00A0 via\u00C2\u00A0 interactions\u00C2\u00A0 with\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 DCs,\u00C2\u00A0 B\u00C2\u00A0 cells,\u00C2\u00A0 NK\u00C2\u00A0 cells,\u00C2\u00A0 and\u00C2\u00A0 NKT\u00C2\u00A0 cells\u00C2\u00A0 (Ostrand\u00E2\u0080\u0090 Rosenberg,\u00C2\u00A02010).\u00C2\u00A0Briefly,\u00C2\u00A0MDSCs\u00C2\u00A0can\u00C2\u00A0inhibit\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0IL\u00E2\u0080\u009012\u00C2\u00A0and\u00C2\u00A0polarize\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 towards\u00C2\u00A0an\u00C2\u00A0M2,\u00C2\u00A0 tumor\u00E2\u0080\u0090promoting\u00C2\u00A0phenotype\u00C2\u00A0(Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005b).\u00C2\u00A0 In\u00C2\u00A0addition,\u00C2\u00A0MDSCs\u00C2\u00A0 increase\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 programmed\u00C2\u00A0 death\u00C2\u00A0 ligand\u00C2\u00A0 1\u00C2\u00A0 (PD\u00E2\u0080\u0090L1),\u00C2\u00A0 a\u00C2\u00A0 negative\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 co\u00E2\u0080\u0090 stimulatory\u00C2\u00A0molecule\u00C2\u00A0 (Ilkovitch\u00C2\u00A0 and\u00C2\u00A0 Lopez,\u00C2\u00A0 2009).\u00C2\u00A0 Moreover,\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 MDSC\u00C2\u00A0 activation\u00C2\u00A0 (i.e.\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 stimulation)\u00C2\u00A0 inhibit\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0of\u00C2\u00A0DCs\u00C2\u00A0(Greifenberg\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009)\u00C2\u00A0and\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0some\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0 can\u00C2\u00A0also\u00C2\u00A0restrict\u00C2\u00A0B\u00C2\u00A0cell\u00C2\u00A0development\u00C2\u00A0(Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A02010).\u00C2\u00A0However,\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0 MDSCs\u00C2\u00A0on\u00C2\u00A0NK\u00C2\u00A0and\u00C2\u00A0NKT\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0less\u00C2\u00A0clear;\u00C2\u00A0there\u00C2\u00A0are\u00C2\u00A0conflicting\u00C2\u00A0reports\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0literature\u00C2\u00A0 regarding\u00C2\u00A0whether\u00C2\u00A0MDSCs\u00C2\u00A0suppress\u00C2\u00A0or\u00C2\u00A0activate\u00C2\u00A0 these\u00C2\u00A0cell\u00C2\u00A0 types\u00C2\u00A0 (Elkabets\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0 Nausch\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A02010;\u00C2\u00A0Pastula\u00C2\u00A0and\u00C2\u00A0Marcinkiewicz,\u00C2\u00A02011).\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0that\u00C2\u00A0different\u00C2\u00A0MDSC\u00C2\u00A0subsets\u00C2\u00A0have\u00C2\u00A0distinct\u00C2\u00A0effects\u00C2\u00A0on\u00C2\u00A0NK\u00C2\u00A0and/or\u00C2\u00A0NKT\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0 this\u00C2\u00A0may\u00C2\u00A0provide\u00C2\u00A0a\u00C2\u00A0rationale\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0apparently\u00C2\u00A0dissimilar\u00C2\u00A0results.\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0 implicated\u00C2\u00A0 in\u00C2\u00A0 fostering\u00C2\u00A0 cancer\u00C2\u00A0 progression\u00C2\u00A0 via\u00C2\u00A0 promotion\u00C2\u00A0 of\u00C2\u00A0 angiogenesis,\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 invasion,\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0 (Priceman\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005b;\u00C2\u00A0Yang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004;\u00C2\u00A0 Yang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0considered\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0main\u00C2\u00A0 factors\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 failure\u00C2\u00A0 of\u00C2\u00A0 immunotherapy,\u00C2\u00A0 both\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 patients\u00C2\u00A0 and\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice,\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0an\u00C2\u00A0attractive\u00C2\u00A0therapeutic\u00C2\u00A0target\u00C2\u00A0(Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A02010;\u00C2\u00A0Vieweg\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02007).\u00C2\u00A0Consequently,\u00C2\u00A0a\u00C2\u00A0better\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0 29 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 will\u00C2\u00A0 be\u00C2\u00A0 vital\u00C2\u00A0 to\u00C2\u00A0 developing\u00C2\u00A0 effective\u00C2\u00A0 immune\u00E2\u0080\u0090 mediated\u00C2\u00A0cancer\u00C2\u00A0therapies.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0is\u00C2\u00A0worth\u00C2\u00A0noting\u00C2\u00A0that\u00C2\u00A0although\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0most\u00C2\u00A0commonly\u00C2\u00A0studied\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 context\u00C2\u00A0 of\u00C2\u00A0 cancer,\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 induced\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 chronic\u00C2\u00A0 and\u00C2\u00A0 acute\u00C2\u00A0 inflammatory\u00C2\u00A0 conditions,\u00C2\u00A0 including\u00C2\u00A0 autoimmune\u00C2\u00A0 disease,\u00C2\u00A0 trauma,\u00C2\u00A0 burns,\u00C2\u00A0 parasitic\u00C2\u00A0 infections,\u00C2\u00A0 and\u00C2\u00A0 sepsis\u00C2\u00A0 (Cuenca\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011;\u00C2\u00A0 Van\u00C2\u00A0 Ginderachter\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 Indeed,\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 generate\u00C2\u00A0 a\u00C2\u00A0 normal\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 and\u00C2\u00A0 mobilization\u00C2\u00A0 of\u00C2\u00A0 mature\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 PMNs,\u00C2\u00A0 and\u00C2\u00A0 immature\u00C2\u00A0 populations\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 BM\u00C2\u00A0 and\u00C2\u00A0 blood\u00C2\u00A0 to\u00C2\u00A0 inflammatory\u00C2\u00A0 sites,\u00C2\u00A0 also\u00C2\u00A0 induce\u00C2\u00A0 accelerated\u00C2\u00A0myelopoiesis\u00C2\u00A0and\u00C2\u00A0expansion\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0(Ueda\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0Related\u00C2\u00A0 to\u00C2\u00A0 this,\u00C2\u00A0 the\u00C2\u00A0expansion,\u00C2\u00A0accumulation\u00C2\u00A0(i.e.\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0terminal\u00C2\u00A0differentiation),\u00C2\u00A0and\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0 MDSCs\u00C2\u00A0are\u00C2\u00A0driven\u00C2\u00A0by\u00C2\u00A0pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0factors\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0tumor,\u00C2\u00A0tumor\u00C2\u00A0stroma,\u00C2\u00A0 and\u00C2\u00A0 infiltrating\u00C2\u00A0 T\u00C2\u00A0 cells.\u00C2\u00A0 These\u00C2\u00A0 include\u00C2\u00A0 cytokines\u00C2\u00A0 (i.e.\u00C2\u00A0 IL\u00E2\u0080\u00901\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u00906)\u00C2\u00A0 (Bunt\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007;\u00C2\u00A0 Elkabets\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010),\u00C2\u00A0chemokines,\u00C2\u00A0PGE2\u00C2\u00A0(Eruslanov\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Serafini,\u00C2\u00A02010),\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 S100A8/A9\u00C2\u00A0 proteins\u00C2\u00A0 (Ostrand\u00E2\u0080\u0090Rosenberg\u00C2\u00A0 and\u00C2\u00A0 Sinha,\u00C2\u00A0 2009).\u00C2\u00A0 In\u00C2\u00A0 addition,\u00C2\u00A0 NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0 signaling\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0complete\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0(Delano\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2007).\u00C2\u00A0Exposure\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0these\u00C2\u00A0factors\u00C2\u00A0increases\u00C2\u00A0the\u00C2\u00A0pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0 MDSCs,\u00C2\u00A0 including\u00C2\u00A0 increased\u00C2\u00A0 ROS,\u00C2\u00A0 cytokine\u00C2\u00A0 (i.e.\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 TNF\u00E2\u0080\u0090\u00CE\u00B1),\u00C2\u00A0 and\u00C2\u00A0 chemokine\u00C2\u00A0 production\u00C2\u00A0 (i.e.\u00C2\u00A0 CCL3,\u00C2\u00A0 CCL4,\u00C2\u00A0 CCL5,\u00C2\u00A0 CXC\u00C2\u00A0 chemokine\u00C2\u00A0 ligand\u00C2\u00A0 12;\u00C2\u00A0 CXCL12)\u00C2\u00A0 (Delano\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007).\u00C2\u00A0The\u00C2\u00A0mechanisms\u00C2\u00A0regulating\u00C2\u00A0these\u00C2\u00A0processes\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0elucidated\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0 of\u00C2\u00A0 groups\u00C2\u00A0 (Corzo\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009;\u00C2\u00A0 Nagaraj\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007)\u00C2\u00A0 and\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 recently\u00C2\u00A0 reviewed\u00C2\u00A0 (Cuenca\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0 \u00C2\u00A0 1.8 Tumor\u00C2\u00A0metastasis\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0pre\u00C2\u00ADmetastatic\u00C2\u00A0niche\u00C2\u00A0 \u00C2\u00A0 Historically,\u00C2\u00A0 the\u00C2\u00A0 major\u00C2\u00A0 focus\u00C2\u00A0 of\u00C2\u00A0 cancer\u00C2\u00A0 research\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 development\u00C2\u00A0and\u00C2\u00A0progression.\u00C2\u00A0However,\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0vast\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0cancer\u00C2\u00A0patients\u00C2\u00A0die\u00C2\u00A0 from\u00C2\u00A0metastasis\u00C2\u00A0there\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0a\u00C2\u00A0recent\u00C2\u00A0shift\u00C2\u00A0in\u00C2\u00A0focus\u00C2\u00A0towards\u00C2\u00A0investigating\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0 that\u00C2\u00A0 control\u00C2\u00A0 tumor\u00C2\u00A0 metastasis\u00C2\u00A0 and\u00C2\u00A0 this\u00C2\u00A0 has\u00C2\u00A0 led\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 emergence\u00C2\u00A0 of\u00C2\u00A0 several\u00C2\u00A0 novel\u00C2\u00A0 hypotheses\u00C2\u00A0and\u00C2\u00A0models\u00C2\u00A0 that\u00C2\u00A0 shed\u00C2\u00A0 light\u00C2\u00A0on\u00C2\u00A0 this\u00C2\u00A0 intricate\u00C2\u00A0process\u00C2\u00A0 (Coghlin\u00C2\u00A0and\u00C2\u00A0Murray,\u00C2\u00A0 30 2010).\u00C2\u00A0In\u00C2\u00A0the\u00C2\u00A0past,\u00C2\u00A0metastasis\u00C2\u00A0was\u00C2\u00A0considered\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0a\u00C2\u00A0stepwise\u00C2\u00A0accumulation\u00C2\u00A0of\u00C2\u00A0genetic\u00C2\u00A0 events\u00C2\u00A0driven\u00C2\u00A0by\u00C2\u00A0clonal\u00C2\u00A0evolution\u00C2\u00A0(Fidler,\u00C2\u00A02003).\u00C2\u00A0However,\u00C2\u00A0new\u00C2\u00A0evidence\u00C2\u00A0from\u00C2\u00A0genetic\u00C2\u00A0 studies\u00C2\u00A0suggests\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0vast\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0 tumor\u00C2\u00A0cells\u00C2\u00A0 inherently\u00C2\u00A0possess\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0 to\u00C2\u00A0 metastasize\u00C2\u00A0 (Singh\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2002;\u00C2\u00A0 van\u00C2\u00A0 't\u00C2\u00A0 Veer\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2002)\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 may\u00C2\u00A0 separate\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0much\u00C2\u00A0earlier\u00C2\u00A0than\u00C2\u00A0previously\u00C2\u00A0appreciated\u00C2\u00A0(Husemann\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Schmidt\u00E2\u0080\u0090Kittler\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003).\u00C2\u00A0Furthermore,\u00C2\u00A0the\u00C2\u00A0critical\u00C2\u00A0role\u00C2\u00A0that\u00C2\u00A0host\u00E2\u0080\u0090derived\u00C2\u00A0 factors\u00C2\u00A0play\u00C2\u00A0in\u00C2\u00A0driving\u00C2\u00A0tumor\u00C2\u00A0metastasis\u00C2\u00A0is\u00C2\u00A0now\u00C2\u00A0becoming\u00C2\u00A0clear.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0metastasis\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0greatly\u00C2\u00A0influenced\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0idea\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0 behaviour\u00C2\u00A0of\u00C2\u00A0individual\u00C2\u00A0tumor\u00C2\u00A0metastases\u00C2\u00A0is\u00C2\u00A0highly\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0interactions\u00C2\u00A0between\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 (the\u00C2\u00A0 \u00E2\u0080\u0098seeds\u00E2\u0080\u0099)\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 host\u00C2\u00A0 microenvironment\u00C2\u00A0 (the\u00C2\u00A0 \u00E2\u0080\u0098soil\u00E2\u0080\u0099)\u00C2\u00A0 (Coghlin\u00C2\u00A0 and\u00C2\u00A0 Murray,\u00C2\u00A0 2010).\u00C2\u00A0 This\u00C2\u00A0 idea\u00C2\u00A0was\u00C2\u00A0 first\u00C2\u00A0 proposed\u00C2\u00A0 by\u00C2\u00A0 Stephen\u00C2\u00A0 Paget\u00C2\u00A0 in\u00C2\u00A0 1889\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 his\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 metastatic\u00C2\u00A0 spread\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 patient\u00C2\u00A0 autopsies\u00C2\u00A0 (Paget,\u00C2\u00A0 1889).\u00C2\u00A0 Today\u00C2\u00A0 we\u00C2\u00A0 acknowledge\u00C2\u00A0 that\u00C2\u00A0 multiple\u00C2\u00A0 layers\u00C2\u00A0 of\u00C2\u00A0 cross\u00E2\u0080\u0090talk\u00C2\u00A0 occur\u00C2\u00A0 between\u00C2\u00A0 malignant\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 normal\u00C2\u00A0 physiological\u00C2\u00A0 cells,\u00C2\u00A0 including\u00C2\u00A0 stromal,\u00C2\u00A0 endothelial,\u00C2\u00A0 inflammatory,\u00C2\u00A0 and/or\u00C2\u00A0 BM\u00E2\u0080\u0090 derived\u00C2\u00A0cells,\u00C2\u00A0which\u00C2\u00A0 together\u00C2\u00A0comprise\u00C2\u00A0 the\u00C2\u00A0metastatic\u00C2\u00A0microenvironment\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2011;\u00C2\u00A0 Psaila\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006).\u00C2\u00A0 There\u00C2\u00A0 is\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 although\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 continually\u00C2\u00A0 released\u00C2\u00A0 from\u00C2\u00A0a\u00C2\u00A0primary\u00C2\u00A0site,\u00C2\u00A0 relatively\u00C2\u00A0 few\u00C2\u00A0of\u00C2\u00A0 them\u00C2\u00A0are\u00C2\u00A0able\u00C2\u00A0 to\u00C2\u00A0efficiently\u00C2\u00A0 form\u00C2\u00A0macrometastases\u00C2\u00A0(Luzzi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01998).\u00C2\u00A0In\u00C2\u00A0fact,\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0probable\u00C2\u00A0outcome\u00C2\u00A0for\u00C2\u00A0these\u00C2\u00A0 cells\u00C2\u00A0is\u00C2\u00A0death,\u00C2\u00A0with\u00C2\u00A0extravasation\u00C2\u00A0and\u00C2\u00A0establishment\u00C2\u00A0of\u00C2\u00A0micrometastases\u00C2\u00A0serving\u00C2\u00A0as\u00C2\u00A0rate\u00E2\u0080\u0090 limiting\u00C2\u00A0 steps\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 process\u00C2\u00A0 (Joyce\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A0 2009).\u00C2\u00A0 Perhaps\u00C2\u00A0 one\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 important\u00C2\u00A0concepts\u00C2\u00A0that\u00C2\u00A0has\u00C2\u00A0transformed\u00C2\u00A0the\u00C2\u00A0way\u00C2\u00A0we\u00C2\u00A0conceptualize\u00C2\u00A0metastasis\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u0098pre\u00E2\u0080\u0090metastatic\u00C2\u00A0 niche\u00E2\u0080\u0099\u00C2\u00A0 (Coghlin\u00C2\u00A0 and\u00C2\u00A0Murray,\u00C2\u00A0 2010;\u00C2\u00A0 Kaplan\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006),\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 landmark\u00C2\u00A0findings\u00C2\u00A0by\u00C2\u00A0Kaplan\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0(Kaplan\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005).\u00C2\u00A0These\u00C2\u00A0studies\u00C2\u00A0demonstrate\u00C2\u00A0that\u00C2\u00A0 recruitment\u00C2\u00A0of\u00C2\u00A0VEGFR\u00E2\u0080\u00901\u00C2\u00A0expressing\u00C2\u00A0HPCs\u00C2\u00A0represents\u00C2\u00A0an\u00C2\u00A0essential\u00C2\u00A0 cellular\u00C2\u00A0event\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 process\u00C2\u00A0of\u00C2\u00A0metastasis\u00C2\u00A0(Kaplan\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005).\u00C2\u00A0Prior\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0arrival\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0tumor\u00C2\u00A0cells,\u00C2\u00A0 HPCs\u00C2\u00A0 home\u00C2\u00A0 to\u00C2\u00A0 specific\u00C2\u00A0 sites\u00C2\u00A0 and\u00C2\u00A0 form\u00C2\u00A0 clusters,\u00C2\u00A0 comprising\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u0098pre\u00E2\u0080\u0090metastatic\u00C2\u00A0 niche\u00E2\u0080\u0099,\u00C2\u00A0 that\u00C2\u00A0 identify\u00C2\u00A0 sites\u00C2\u00A0 of\u00C2\u00A0 future\u00C2\u00A0metastases\u00C2\u00A0 (Kaplan\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005).\u00C2\u00A0 Their\u00C2\u00A0 results\u00C2\u00A0 suggest\u00C2\u00A0 a\u00C2\u00A0 functional\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0 VEGFR\u00E2\u0080\u00901,\u00C2\u00A0 since\u00C2\u00A0 anti\u00E2\u0080\u0090VEGFR\u00E2\u0080\u00901\u00C2\u00A0 blocking\u00C2\u00A0 Abs\u00C2\u00A0 specifically\u00C2\u00A0 prevented\u00C2\u00A0 tumor\u00C2\u00A0metastases\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0model\u00C2\u00A0 (Kaplan\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005).\u00C2\u00A0 Furthermore,\u00C2\u00A0 they\u00C2\u00A0 report\u00C2\u00A0 that\u00C2\u00A0 soluble\u00C2\u00A0factors\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0can\u00C2\u00A0induce\u00C2\u00A0VEGFR\u00E2\u0080\u00901\u00C2\u00A0progenitor\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0 31 migrate\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 BM\u00C2\u00A0 to\u00C2\u00A0 specific\u00C2\u00A0 metastatic\u00C2\u00A0 sites,\u00C2\u00A0 where\u00C2\u00A0 they\u00C2\u00A0 direct\u00C2\u00A0 the\u00C2\u00A0 seeding\u00C2\u00A0 and\u00C2\u00A0 proliferation\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0lesions\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0specific\u00C2\u00A0manner\u00C2\u00A0(Kaplan\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005).\u00C2\u00A0However,\u00C2\u00A0 there\u00C2\u00A0are\u00C2\u00A0conflicting\u00C2\u00A0reports\u00C2\u00A0regarding\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0that\u00C2\u00A0comprise\u00C2\u00A0the\u00C2\u00A0niche\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0manner\u00C2\u00A0in\u00C2\u00A0which\u00C2\u00A0the\u00C2\u00A0niche\u00C2\u00A0functions\u00C2\u00A0(Qian\u00C2\u00A0and\u00C2\u00A0Pollard,\u00C2\u00A02010).\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0some\u00C2\u00A0 groups\u00C2\u00A0 have\u00C2\u00A0 challenged\u00C2\u00A0 whether\u00C2\u00A0 VEGFR\u00E2\u0080\u00901\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 metastasis\u00C2\u00A0 (Dawson\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009),\u00C2\u00A0 while\u00C2\u00A0 others\u00C2\u00A0 have\u00C2\u00A0 proposed\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 niche\u00C2\u00A0 provides\u00C2\u00A0 sites\u00C2\u00A0 for\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 adhere\u00C2\u00A0 and\u00C2\u00A0 grow\u00C2\u00A0 (Psaila\u00C2\u00A0 and\u00C2\u00A0 Lyden,\u00C2\u00A0 2009)\u00C2\u00A0 or\u00C2\u00A0 acts\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 reservoir\u00C2\u00A0 of\u00C2\u00A0 monocytes\u00C2\u00A0 that\u00C2\u00A0 differentiate\u00C2\u00A0into\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0upon\u00C2\u00A0tumor\u00C2\u00A0cell\u00C2\u00A0arrival\u00C2\u00A0(Qian\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0There\u00C2\u00A0is\u00C2\u00A0also\u00C2\u00A0evidence\u00C2\u00A0 that\u00C2\u00A0 hypoxia\u00C2\u00A0 mediates\u00C2\u00A0 metastatic\u00C2\u00A0 niche\u00C2\u00A0 development.\u00C2\u00A0 Specifically,\u00C2\u00A0 hypoxia\u00E2\u0080\u0090induced\u00C2\u00A0 lysyl\u00C2\u00A0oxidase\u00C2\u00A0(LOX)\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0to\u00C2\u00A0accumulate\u00C2\u00A0at\u00C2\u00A0pre\u00E2\u0080\u0090metastatic\u00C2\u00A0sites,\u00C2\u00A0where\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 recruitment\u00C2\u00A0 of\u00C2\u00A0 CD11b+\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0 which\u00C2\u00A0 in\u00C2\u00A0 turn\u00C2\u00A0 promote\u00C2\u00A0 the\u00C2\u00A0 invasion\u00C2\u00A0and\u00C2\u00A0 recruitment\u00C2\u00A0of\u00C2\u00A0 other\u00C2\u00A0BM\u00E2\u0080\u0090derived\u00C2\u00A0 cells,\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 (Erler\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0it\u00C2\u00A0 is\u00C2\u00A0now\u00C2\u00A0evident\u00C2\u00A0that\u00C2\u00A0BM\u00E2\u0080\u0090derived\u00C2\u00A0progenitors\u00C2\u00A0and\u00C2\u00A0inflammatory\u00C2\u00A0cells\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0metastasis\u00C2\u00A0 (Psaila\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006),\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 and\u00C2\u00A0mechanisms\u00C2\u00A0 involved\u00C2\u00A0 are\u00C2\u00A0 still\u00C2\u00A0 being\u00C2\u00A0 explored.\u00C2\u00A0 Substantial\u00C2\u00A0 evidence\u00C2\u00A0 suggests\u00C2\u00A0 both\u00C2\u00A0MDSCs\u00C2\u00A0 and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0roles\u00C2\u00A0in\u00C2\u00A0metastasis\u00C2\u00A0through\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0soluble\u00C2\u00A0factors\u00C2\u00A0(including\u00C2\u00A0growth\u00C2\u00A0factors,\u00C2\u00A0 proteolytic\u00C2\u00A0 enzymes,\u00C2\u00A0 cytokines,\u00C2\u00A0 chemokines,\u00C2\u00A0 and\u00C2\u00A0 inflammatory\u00C2\u00A0 mediators),\u00C2\u00A0 modification\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0extracellular\u00C2\u00A0matrix\u00C2\u00A0(ECM),\u00C2\u00A0and\u00C2\u00A0interactions\u00C2\u00A0with\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0(Qian\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A0 2010;\u00C2\u00A0 Siveen\u00C2\u00A0 and\u00C2\u00A0 Kuttan,\u00C2\u00A0 2009;\u00C2\u00A0 Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 Consequently,\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 function\u00C2\u00A0 to\u00C2\u00A0 promote\u00C2\u00A0 metastasis\u00C2\u00A0 through\u00C2\u00A0 several\u00C2\u00A0 mechanisms\u00C2\u00A0 including\u00C2\u00A0 encouragement\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 and\u00C2\u00A0 proliferation,\u00C2\u00A0 promotion\u00C2\u00A0 of\u00C2\u00A0 angiogenesis,\u00C2\u00A0 and\u00C2\u00A0 enhancement\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 migration\u00C2\u00A0 and\u00C2\u00A0 invasion\u00C2\u00A0 (Condeelis\u00C2\u00A0and\u00C2\u00A0Pollard,\u00C2\u00A02006;\u00C2\u00A0Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 1.9 4T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0murine\u00C2\u00A0mammary\u00C2\u00A0tumor\u00C2\u00A0models\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0mentioned\u00C2\u00A0above,\u00C2\u00A0metastatic\u00C2\u00A0disease\u00C2\u00A0is\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0deaths\u00C2\u00A0 in\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0patients\u00C2\u00A0(Psaila\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006)\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0animal\u00C2\u00A0models\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 32 developed\u00C2\u00A0over\u00C2\u00A0the\u00C2\u00A0years\u00C2\u00A0to\u00C2\u00A0investigate\u00C2\u00A0key\u00C2\u00A0aspects\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0metastatic\u00C2\u00A0process\u00C2\u00A0(Heppner\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2000).\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0most\u00C2\u00A0 useful\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 4T1\u00C2\u00A0murine\u00C2\u00A0mammary\u00C2\u00A0 carcinoma\u00C2\u00A0model\u00C2\u00A0 because\u00C2\u00A0 it\u00C2\u00A0 closely\u00C2\u00A0 resembles\u00C2\u00A0 the\u00C2\u00A0 natural\u00C2\u00A0 history\u00C2\u00A0 and\u00C2\u00A0 histopathology\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0 breast\u00C2\u00A0 cancer\u00C2\u00A0and\u00C2\u00A0because\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0sister\u00C2\u00A0models\u00C2\u00A0that\u00C2\u00A0represent\u00C2\u00A0the\u00C2\u00A0spectrum\u00C2\u00A0of\u00C2\u00A0 metastatic\u00C2\u00A0disease\u00C2\u00A0 (Heppner\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2000).\u00C2\u00A0 Specifically,\u00C2\u00A0 a\u00C2\u00A0 comparison\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0metastatic\u00C2\u00A0 4T1\u00C2\u00A0to\u00C2\u00A0its\u00C2\u00A0non\u00E2\u0080\u0090metastatic\u00C2\u00A0sister\u00C2\u00A067NR\u00C2\u00A0facilitates\u00C2\u00A0studies\u00C2\u00A0aimed\u00C2\u00A0at\u00C2\u00A0elucidating\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0 responsible\u00C2\u00A0for\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0versus\u00C2\u00A0metastasis.\u00C2\u00A0Both\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 were\u00C2\u00A0derived\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0single,\u00C2\u00A0spontaneous\u00C2\u00A0mouse\u00C2\u00A0mammary\u00C2\u00A0tumor\u00C2\u00A0(Aslakson\u00C2\u00A0and\u00C2\u00A0Miller,\u00C2\u00A0 1992;\u00C2\u00A0Miller\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01983).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0thioguanine\u00E2\u0080\u0090resistant\u00C2\u00A04T1\u00C2\u00A0subline\u00C2\u00A0was\u00C2\u00A0derived\u00C2\u00A0from\u00C2\u00A0this\u00C2\u00A0 parental\u00C2\u00A0 population\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 geneticin\u00E2\u0080\u0090resistant\u00C2\u00A0 67NR\u00C2\u00A0 subline\u00C2\u00A0 was\u00C2\u00A0 obtained\u00C2\u00A0 by\u00C2\u00A0 transfection\u00C2\u00A0 of\u00C2\u00A0 line\u00C2\u00A0 67\u00C2\u00A0 (Aslakson\u00C2\u00A0 and\u00C2\u00A0 Miller,\u00C2\u00A0 1992).\u00C2\u00A0 While\u00C2\u00A0 both\u00C2\u00A0 lines\u00C2\u00A0 are\u00C2\u00A0 highly\u00C2\u00A0 tumorigenic\u00C2\u00A0 and\u00C2\u00A0 form\u00C2\u00A0 primary\u00C2\u00A0 tumors\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 mammary\u00C2\u00A0 fat\u00C2\u00A0 pad,\u00C2\u00A0 the\u00C2\u00A0 4T1\u00C2\u00A0 cell\u00C2\u00A0 line\u00C2\u00A0 is\u00C2\u00A0 highly\u00C2\u00A0 metastatic\u00C2\u00A0 while\u00C2\u00A0 67NR\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 non\u00E2\u0080\u0090metastatic\u00C2\u00A0 (Aslakson\u00C2\u00A0 and\u00C2\u00A0 Miller,\u00C2\u00A0 1992).\u00C2\u00A0 Another\u00C2\u00A0 key\u00C2\u00A0 difference\u00C2\u00A0 between\u00C2\u00A0 these\u00C2\u00A0 two\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 vascularization\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 primary\u00C2\u00A0tumor;\u00C2\u00A0primary\u00C2\u00A04T1\u00C2\u00A0tumors\u00C2\u00A0are\u00C2\u00A0hypoxic\u00C2\u00A0while\u00C2\u00A067NR\u00C2\u00A0primary\u00C2\u00A0tumors\u00C2\u00A0are\u00C2\u00A0well\u00C2\u00A0 vascularized\u00C2\u00A0and\u00C2\u00A0normoxic\u00C2\u00A0(Aslakson\u00C2\u00A0and\u00C2\u00A0Miller,\u00C2\u00A01992).\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 studies\u00C2\u00A0have\u00C2\u00A0used\u00C2\u00A0 the\u00C2\u00A04T1\u00C2\u00A0 tumor\u00C2\u00A0model\u00C2\u00A0 to\u00C2\u00A0 investigate\u00C2\u00A0 the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 metastasis.\u00C2\u00A0 When\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 injected\u00C2\u00A0 orthotopically\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 mammary\u00C2\u00A0 fat\u00C2\u00A0 pad\u00C2\u00A0 of\u00C2\u00A0 syngeneic\u00C2\u00A0 BALB/c\u00C2\u00A0 mice,\u00C2\u00A0 they\u00C2\u00A0 share\u00C2\u00A0 several\u00C2\u00A0 important\u00C2\u00A0 characteristics\u00C2\u00A0 with\u00C2\u00A0 human\u00C2\u00A0 breast\u00C2\u00A0 carcinomas\u00C2\u00A0 (Heppner\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2000;\u00C2\u00A0 Pulaski\u00C2\u00A0 and\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A02001).\u00C2\u00A0In\u00C2\u00A0particular,\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0spontaneously\u00C2\u00A0metastasize\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0 lung,\u00C2\u00A0 liver,\u00C2\u00A0 bone,\u00C2\u00A0 and\u00C2\u00A0 brain\u00C2\u00A0 via\u00C2\u00A0 the\u00C2\u00A0 bloodstream\u00C2\u00A0 (Aslakson\u00C2\u00A0 and\u00C2\u00A0 Miller,\u00C2\u00A0 1992;\u00C2\u00A0 Heppner\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2000),\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 late\u00E2\u0080\u0090stage\u00C2\u00A0 human\u00C2\u00A0 breast\u00C2\u00A0 cancer.\u00C2\u00A0 As\u00C2\u00A0well,\u00C2\u00A0 mice\u00C2\u00A0 bearing\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 exhibit\u00C2\u00A0 significant\u00C2\u00A0 expansion\u00C2\u00A0 and\u00C2\u00A0 accumulation\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs,\u00C2\u00A0spleen,\u00C2\u00A0blood,\u00C2\u00A0and\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0(Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A02010;\u00C2\u00A0Youn\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0 These\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 accumulate\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 pro\u00E2\u0080\u0090 inflammatory\u00C2\u00A0mediators\u00C2\u00A0(i.e.\u00C2\u00A0IL\u00E2\u0080\u00901\u00CE\u00B2,\u00C2\u00A0IL\u00E2\u0080\u00906,\u00C2\u00A0PGE2,\u00C2\u00A0S100A8/A9)\u00C2\u00A0(Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A02010),\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 decreased\u00C2\u00A0 immune\u00C2\u00A0 surveillance\u00C2\u00A0 (Sinha\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005c),\u00C2\u00A0 and\u00C2\u00A0 block\u00C2\u00A0 both\u00C2\u00A0 innate\u00C2\u00A0 and\u00C2\u00A0 adaptive\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immunity\u00C2\u00A0 (Sinha\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005b).\u00C2\u00A0 They\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 33 reported\u00C2\u00A0 to\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 mechanisms\u00C2\u00A0 including\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0Arg1\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0resulting\u00C2\u00A0depletion\u00C2\u00A0of\u00C2\u00A0L\u00E2\u0080\u0090Arg,\u00C2\u00A0cysteine\u00C2\u00A0 sequestration,\u00C2\u00A0 and\u00C2\u00A0 down\u00E2\u0080\u0090regulation\u00C2\u00A0 of\u00C2\u00A0 L\u00E2\u0080\u0090selectin\u00C2\u00A0 expression\u00C2\u00A0 on\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 (Ostrand\u00E2\u0080\u0090 Rosenberg,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Less\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0about\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0play\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00A0model.\u00C2\u00A0However,\u00C2\u00A0it\u00C2\u00A0has\u00C2\u00A0 been\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0skew\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0into\u00C2\u00A0M2\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0 an\u00C2\u00A0IL\u00E2\u0080\u00906\u00E2\u0080\u0090dependent\u00C2\u00A0manner\u00C2\u00A0(Wang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0Furthermore,\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reported\u00C2\u00A0 to\u00C2\u00A0 work\u00C2\u00A0 together\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 immune\u00C2\u00A0 surveillance\u00C2\u00A0 against\u00C2\u00A0 metastasis;\u00C2\u00A0specifically,\u00C2\u00A0contact\u00E2\u0080\u0090dependent\u00C2\u00A0cross\u00E2\u0080\u0090talk\u00C2\u00A0between\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0leads\u00C2\u00A0to\u00C2\u00A0 increased\u00C2\u00A0IL\u00E2\u0080\u009010\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0decreased\u00C2\u00A0IL\u00E2\u0080\u009012\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(Sinha\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02007a;\u00C2\u00A0Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005b).\u00C2\u00A0On\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0relatively\u00C2\u00A0 little\u00C2\u00A0 is\u00C2\u00A0known\u00C2\u00A0about\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 or\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 67NR\u00C2\u00A0 tumor\u00C2\u00A0model.\u00C2\u00A0 Additional\u00C2\u00A0 studies\u00C2\u00A0will\u00C2\u00A0 likely\u00C2\u00A0 reveal\u00C2\u00A0 whether\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 non\u00E2\u0080\u0090metastatic\u00C2\u00A0model\u00C2\u00A0 of\u00C2\u00A0 mammary\u00C2\u00A0carcinoma.\u00C2\u00A0 \u00C2\u00A0 1.10 Aims\u00C2\u00A0of\u00C2\u00A0study\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0well\u00C2\u00A0established\u00C2\u00A0that\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0contributes\u00C2\u00A0to\u00C2\u00A0all\u00C2\u00A0stages\u00C2\u00A0 of\u00C2\u00A0 cancer,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 many\u00C2\u00A0 aspects\u00C2\u00A0 that\u00C2\u00A0 remain\u00C2\u00A0 unclear.\u00C2\u00A0 First,\u00C2\u00A0 multiple\u00C2\u00A0 myeloid\u00C2\u00A0 and\u00C2\u00A0 lymphoid\u00C2\u00A0 cell\u00C2\u00A0 populations\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 proposed\u00C2\u00A0 to\u00C2\u00A0 possess\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties.\u00C2\u00A0However,\u00C2\u00A0little\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0about\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0roles\u00C2\u00A0each\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0play\u00C2\u00A0 in\u00C2\u00A0different\u00C2\u00A0situations.\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0do\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0that\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0peripheral\u00C2\u00A0 tolerance\u00C2\u00A0 under\u00C2\u00A0 physiological\u00C2\u00A0 circumstances\u00C2\u00A0 also\u00C2\u00A0 promote\u00C2\u00A0 tumor\u00C2\u00A0 growth?\u00C2\u00A0 Moreover,\u00C2\u00A0 the\u00C2\u00A0 signaling\u00C2\u00A0 pathways\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 cells\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0yet\u00C2\u00A0well\u00C2\u00A0defined.\u00C2\u00A0Given\u00C2\u00A0the\u00C2\u00A0dual\u00C2\u00A0roles\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0play\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0promotion\u00C2\u00A0 and\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 responses,\u00C2\u00A0 understanding\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 their\u00C2\u00A0function\u00C2\u00A0is\u00C2\u00A0of\u00C2\u00A0particular\u00C2\u00A0importance.\u00C2\u00A0\u00C2\u00A0Furthermore,\u00C2\u00A0very\u00C2\u00A0few\u00C2\u00A0studies\u00C2\u00A0have\u00C2\u00A0directly\u00C2\u00A0 compared\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 potencies\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 cell\u00C2\u00A0 types,\u00C2\u00A0 or\u00C2\u00A0 the\u00C2\u00A0 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of\u00C2\u00A0therapies\u00C2\u00A0that\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0treat\u00C2\u00A0diseases\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0cancer\u00C2\u00A0and\u00C2\u00A0autoimmunity\u00C2\u00A0via\u00C2\u00A0 the\u00C2\u00A0modulation\u00C2\u00A0of\u00C2\u00A0immune\u00C2\u00A0suppression.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0work\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0thesis\u00C2\u00A0addresses\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0issues,\u00C2\u00A0especially\u00C2\u00A0 the\u00C2\u00A0different\u00C2\u00A0 roles\u00C2\u00A0 immunosuppressive\u00C2\u00A0 cells\u00C2\u00A0play\u00C2\u00A0 in\u00C2\u00A0normal\u00C2\u00A0physiology\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0 cancer.\u00C2\u00A0 We\u00C2\u00A0 chose\u00C2\u00A0 to\u00C2\u00A0 focus\u00C2\u00A0 on\u00C2\u00A0 elucidating\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 features\u00C2\u00A0 of\u00C2\u00A0 mononuclear\u00C2\u00A0 phagocytes,\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0particular,\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0regulatory\u00C2\u00A0lymphoid\u00C2\u00A0cells\u00C2\u00A0(i.e.\u00C2\u00A0Tregs)\u00C2\u00A0 was\u00C2\u00A0 being\u00C2\u00A0 carried\u00C2\u00A0 out\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0 collaborator\u00C2\u00A0 (Dr.\u00C2\u00A0 Megan\u00C2\u00A0 Levings,\u00C2\u00A0 CFRI)\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 DC\u00C2\u00A0 subsets\u00C2\u00A0 was\u00C2\u00A0 being\u00C2\u00A0 investigated\u00C2\u00A0 by\u00C2\u00A0 another\u00C2\u00A0 graduate\u00C2\u00A0student\u00C2\u00A0 in\u00C2\u00A0our\u00C2\u00A0 laboratory\u00C2\u00A0 (Antignano\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Moreover,\u00C2\u00A0 the\u00C2\u00A0 regulatory\u00C2\u00A0 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Jackson\u00C2\u00A0Laboratory;\u00C2\u00A0Bar\u00C2\u00A0Harbor,\u00C2\u00A0ME)\u00C2\u00A0were\u00C2\u00A0bred\u00C2\u00A0in\u00C2\u00A0house.\u00C2\u00A0BALB/c\u00C2\u00A0DO11.10\u00C2\u00A0transgenic\u00C2\u00A0 mice,\u00C2\u00A0 which\u00C2\u00A0 express\u00C2\u00A0 a\u00C2\u00A0 TCR\u00C2\u00A0 specific\u00C2\u00A0 for\u00C2\u00A0 chicken\u00C2\u00A0 ovalbumin\u00C2\u00A0 (OVA)\u00C2\u00A0 peptide\u00C2\u00A0 (323\u00E2\u0080\u0090339)\u00C2\u00A0 restricted\u00C2\u00A0to\u00C2\u00A0I\u00E2\u0080\u0090Ad\u00C2\u00A0 \u00C2\u00A0(Murphy\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01990),\u00C2\u00A0were\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0The\u00C2\u00A0Jackson\u00C2\u00A0Laboratory\u00C2\u00A0 and\u00C2\u00A0spleens\u00C2\u00A0from\u00C2\u00A0OTI\u00C2\u00A0transgenic\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0kindly\u00C2\u00A0provided\u00C2\u00A0by\u00C2\u00A0Dr.\u00C2\u00A0Hung\u00E2\u0080\u0090Sia\u00C2\u00A0Teh\u00C2\u00A0(The\u00C2\u00A0 University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia,\u00C2\u00A0Vancouver,\u00C2\u00A0BC,\u00C2\u00A0Canada).\u00C2\u00A0\u00C2\u00A0Female\u00C2\u00A0mice,\u00C2\u00A06\u00C2\u00A0to\u00C2\u00A012\u00C2\u00A0weeks\u00C2\u00A0of\u00C2\u00A0 age,\u00C2\u00A0 were\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0 all\u00C2\u00A0 experiments.\u00C2\u00A0 Mice\u00C2\u00A0 were\u00C2\u00A0 maintained\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 Animal\u00C2\u00A0 Resource\u00C2\u00A0 Centre\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0BC\u00C2\u00A0Cancer\u00C2\u00A0Research\u00C2\u00A0Centre\u00C2\u00A0under\u00C2\u00A0specific\u00E2\u0080\u0090pathogen\u00C2\u00A0free\u00C2\u00A0conditions.\u00C2\u00A0 \u00C2\u00A0All\u00C2\u00A0 animal\u00C2\u00A0 experiments\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 accordance\u00C2\u00A0 with\u00C2\u00A0 institutional\u00C2\u00A0 and\u00C2\u00A0 Canadian\u00C2\u00A0 Council\u00C2\u00A0on\u00C2\u00A0Animal\u00C2\u00A0Care\u00C2\u00A0guidelines.\u00C2\u00A0 \u00C2\u00A0 2.2 Media\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0the\u00C2\u00A0studies\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A03,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0in\u00C2\u00A0Iscove\u00E2\u0080\u0099s\u00C2\u00A0modified\u00C2\u00A0 Dulbecco\u00E2\u0080\u0099s\u00C2\u00A0medium\u00C2\u00A0(IMDM)\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0fetal\u00C2\u00A0calf\u00C2\u00A0serum\u00C2\u00A0(FCS)\u00C2\u00A0medium\u00C2\u00A0(i.e.\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00E2\u0080\u0090free\u00C2\u00A0IMDM\u00C2\u00A0 (HyClone,\u00C2\u00A0Logan,\u00C2\u00A0UT)\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0FCS,\u00C2\u00A0150\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0monothioglycerate\u00C2\u00A0(MTG),\u00C2\u00A050\u00C2\u00A0U/ml\u00C2\u00A0penicillin,\u00C2\u00A0 50\u00C2\u00A0\u00C2\u00B5g/ml\u00C2\u00A0streptomycin,\u00C2\u00A0and\u00C2\u00A0100\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0L\u00E2\u0080\u0090Arg).\u00C2\u00A0For\u00C2\u00A0the\u00C2\u00A0studies\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A04,\u00C2\u00A0cells\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 in\u00C2\u00A0 HL1\u00C2\u00A0 medium\u00C2\u00A0 (i.e.\u00C2\u00A0 HL\u00E2\u0080\u00901\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 medium\u00C2\u00A0 (BioWhittaker;\u00C2\u00A0 Basel,\u00C2\u00A0 Switzerland)\u00C2\u00A0 +\u00C2\u00A0 1%\u00C2\u00A0 penicillin,\u00C2\u00A0 1%\u00C2\u00A0 streptomycin,\u00C2\u00A0 1%\u00C2\u00A0 Glutamax,\u00C2\u00A0 5\u00C2\u00A0 x\u00C2\u00A0 10\u00E2\u0080\u00905\u00C2\u00A0 M\u00C2\u00A0 beta\u00E2\u0080\u0090 mercaptoethanol\u00C2\u00A0(\u00CE\u00B2\u00E2\u0080\u0090ME)),\u00C2\u00A0or\u00C2\u00A0Roswell\u00C2\u00A0Park\u00C2\u00A0Memorial\u00C2\u00A0Institute\u00C2\u00A0(RPMI)\u00C2\u00A01640\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0 medium\u00C2\u00A0 (i.e.\u00C2\u00A0RPMI\u00C2\u00A01640\u00C2\u00A0 (StemCell\u00C2\u00A0Technologies;\u00C2\u00A0Vancouver,\u00C2\u00A0BC,\u00C2\u00A0Canada)\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0FCS,\u00C2\u00A0 150\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0MTG,\u00C2\u00A050\u00C2\u00A0U/ml\u00C2\u00A0penicillin\u00C2\u00A0and\u00C2\u00A050\u00C2\u00A0\u00C2\u00B5g/ml\u00C2\u00A0streptomycin).\u00C2\u00A0For\u00C2\u00A0the\u00C2\u00A0studies\u00C2\u00A0presented\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A05,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0in\u00C2\u00A0HL1\u00C2\u00A0medium.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0the\u00C2\u00A0MDSC\u00C2\u00A0serum\u00C2\u00A0studies,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0four\u00C2\u00A0different\u00C2\u00A0sources\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0purchased\u00C2\u00A0 from\u00C2\u00A0 HyClone,\u00C2\u00A0 StemCell\u00C2\u00A0 Technologies,\u00C2\u00A0 Invitrogen\u00C2\u00A0 (Burlington,\u00C2\u00A0 ON,\u00C2\u00A0 Canada),\u00C2\u00A0 and\u00C2\u00A0 PAA\u00C2\u00A0 36 Laboratories\u00C2\u00A0(Pasching,\u00C2\u00A0Austria)\u00C2\u00A0and\u00C2\u00A0two\u00C2\u00A0sources\u00C2\u00A0of\u00C2\u00A0bovine\u00C2\u00A0serum\u00C2\u00A0albumin\u00C2\u00A0(BSA)\u00C2\u00A0from\u00C2\u00A0 Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0 (St.\u00C2\u00A0 Louis,\u00C2\u00A0 MO)\u00C2\u00A0 and\u00C2\u00A0 Roche\u00C2\u00A0 Diagnostics\u00C2\u00A0 (Laval,\u00C2\u00A0 QC,\u00C2\u00A0 Canada).\u00C2\u00A0 BALB/c\u00C2\u00A0 mouse\u00C2\u00A0serum\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0Innovative\u00C2\u00A0Research\u00C2\u00A0(Novi,\u00C2\u00A0Michigan)\u00C2\u00A0or\u00C2\u00A0harvested\u00C2\u00A0 by\u00C2\u00A0cardiac\u00C2\u00A0puncture\u00C2\u00A0from\u00C2\u00A0BALB/c\u00C2\u00A0mice.\u00C2\u00A0 \u00C2\u00A0 2.3 Reagents\u00C2\u00A0and\u00C2\u00A0cytokines\u00C2\u00A0 \u00C2\u00A0 Neutralizing\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0cytokines\u00C2\u00A0were\u00C2\u00A0purchased\u00C2\u00A0as\u00C2\u00A0follows:\u00C2\u00A0IL\u00E2\u0080\u00904\u00C2\u00A0(eBioscience;\u00C2\u00A0San\u00C2\u00A0 Diego,\u00C2\u00A0 CA);\u00C2\u00A0 IL\u00E2\u0080\u009010\u00C2\u00A0 and\u00C2\u00A0 cytotoxic\u00C2\u00A0 T\u00E2\u0080\u0090lymphocyte\u00C2\u00A0 antigen\u00C2\u00A0 4\u00C2\u00A0 (CTLA\u00E2\u0080\u00904)\u00C2\u00A0 (BD\u00C2\u00A0 Biosciences;\u00C2\u00A0 Mississauga,\u00C2\u00A0 ON,\u00C2\u00A0 Canada);\u00C2\u00A0 IL\u00E2\u0080\u009013,\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0 and\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 (R&D\u00C2\u00A0 Systems;\u00C2\u00A0 Minneapolis,\u00C2\u00A0 MN);\u00C2\u00A0 and\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0(Pestka\u00C2\u00A0Biomedical\u00C2\u00A0Laboratories;\u00C2\u00A0Piscataway,\u00C2\u00A0NJ).\u00C2\u00A0Reagents\u00C2\u00A0were\u00C2\u00A0purchased\u00C2\u00A0 as\u00C2\u00A0 follows:\u00C2\u00A0 recombinant\u00C2\u00A0human\u00C2\u00A0 latency\u00E2\u0080\u0090associated\u00C2\u00A0peptide\u00C2\u00A0 (LAP)\u00C2\u00A0 (R&D\u00C2\u00A0Systems);\u00C2\u00A0N6\u00E2\u0080\u0090 (1\u00E2\u0080\u0090iminoethyl)\u00E2\u0080\u0090L\u00E2\u0080\u0090lysine\u00C2\u00A0 (L\u00E2\u0080\u0090NIL),\u00C2\u00A0 a\u00C2\u00A0 specific\u00C2\u00A0 inhibitor\u00C2\u00A0 of\u00C2\u00A0 iNOS\u00C2\u00A0 (Calbiochem;\u00C2\u00A0 San\u00C2\u00A0 Diego,\u00C2\u00A0 CA);\u00C2\u00A0 carboxy\u00E2\u0080\u00902\u00E2\u0080\u0090phenyl\u00E2\u0080\u00904,4,5,5\u00E2\u0080\u0090tetramethylimidazoline\u00E2\u0080\u00901\u00E2\u0080\u0090oxyl\u00E2\u0080\u00903\u00E2\u0080\u0090oxide\u00C2\u00A0 (PTIO),\u00C2\u00A0 an\u00C2\u00A0 NO\u00C2\u00A0 scavenger\u00C2\u00A0 (Cayman\u00C2\u00A0 Chemicals;\u00C2\u00A0 Ann\u00C2\u00A0 Arbor,\u00C2\u00A0 MI);\u00C2\u00A0 soluble\u00C2\u00A0 anti\u00E2\u0080\u0090CD3\u00C2\u00A0 and\u00C2\u00A0 anti\u00E2\u0080\u0090CD28\u00C2\u00A0 (eBioscience);\u00C2\u00A0OVA\u00C2\u00A0peptides\u00C2\u00A0 (257\u00E2\u0080\u0090264\u00C2\u00A0and\u00C2\u00A0323\u00E2\u0080\u0090339)\u00C2\u00A0 (GenScript;\u00C2\u00A0 Piscataway,\u00C2\u00A0NJ);\u00C2\u00A0 and\u00C2\u00A0 Celebrex,\u00C2\u00A0 a\u00C2\u00A0 cyclooxygenase\u00C2\u00A0 (COX)\u00E2\u0080\u00902\u00C2\u00A0 inhibitor\u00C2\u00A0 (Pfizer;\u00C2\u00A0 St.\u00C2\u00A0 Louis,\u00C2\u00A0 MO).\u00C2\u00A0 [(S)\u00E2\u0080\u0090(2\u00E2\u0080\u0090 Boronoethyl)\u00E2\u0080\u0090L\u00E2\u0080\u0090cysteine]\u00C2\u00A0 (BEC),\u00C2\u00A0 a\u00C2\u00A0 competitive\u00C2\u00A0 inhibitor\u00C2\u00A0 of\u00C2\u00A0 Arg1\u00C2\u00A0 and\u00C2\u00A0 2\u00C2\u00A0 that\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 inhibit\u00C2\u00A0iNOS,\u00C2\u00A0was\u00C2\u00A0from\u00C2\u00A0Dr.\u00C2\u00A0Jean\u00E2\u0080\u0090Luc\u00C2\u00A0Boucher\u00C2\u00A0(Universit\u00C3\u00A9\u00C2\u00A0Paris\u00C2\u00A0Descartes,\u00C2\u00A0Paris,\u00C2\u00A0France).\u00C2\u00A0 TIB\u00E2\u0080\u0090218,\u00C2\u00A0 a\u00C2\u00A0 rat\u00C2\u00A0 IgG2a\u00CE\u00BA\u00C2\u00A0 Ab\u00C2\u00A0 selective\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 \u00CE\u00B2\u00C2\u00A0 subunit\u00C2\u00A0 of\u00C2\u00A0 mouse\u00C2\u00A0 lymphocyte\u00C2\u00A0 function\u00E2\u0080\u0090 associated\u00C2\u00A0 antigen\u00C2\u00A0 1\u00C2\u00A0 (LFA\u00E2\u0080\u00901)\u00C2\u00A0 and\u00C2\u00A0 CD11b\u00C2\u00A0 (CD18),\u00C2\u00A0 was\u00C2\u00A0 purified\u00C2\u00A0 from\u00C2\u00A0 hybridoma\u00C2\u00A0 supernatants\u00C2\u00A0 in\u00C2\u00A0 house.\u00C2\u00A0 E.coli\u00C2\u00A0 LPS\u00C2\u00A0 serotype\u00C2\u00A0 O127:B8\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 double\u00C2\u00A0 stranded\u00C2\u00A0 ribonucleic\u00C2\u00A0 acid\u00C2\u00A0 (dsRNA)\u00C2\u00A0 analog\u00C2\u00A0 polyinosinic:polycytidylic\u00C2\u00A0 (Poly\u00C2\u00A0 I:C)\u00C2\u00A0 acid\u00C2\u00A0 were\u00C2\u00A0 from\u00C2\u00A0 Sigma\u00E2\u0080\u0090Aldrich.\u00C2\u00A0 The\u00C2\u00A0 high\u00E2\u0080\u0090performance\u00C2\u00A0 liquid\u00C2\u00A0 chromatography\u00C2\u00A0 (HPLC)\u00E2\u0080\u0090purified\u00C2\u00A0 phosphorothioate\u00E2\u0080\u0090modified\u00C2\u00A0CpG\u00E2\u0080\u0090dinuclotide\u00C2\u00A0(CpG),\u00C2\u00A05'\u00E2\u0080\u0090tccatgacgttcctgacgtt\u00E2\u0080\u00903'\u00C2\u00A0was\u00C2\u00A0 from\u00C2\u00A0 Invitrogen\u00C2\u00A0 and\u00C2\u00A0 peptidoglycan\u00C2\u00A0 (PGN)\u00C2\u00A0 from\u00C2\u00A0 Staphylococcus\u00C2\u00A0 aureus\u00C2\u00A0 was\u00C2\u00A0 from\u00C2\u00A0 Fluka\u00C2\u00A0 (Buchs,\u00C2\u00A0 Switzerland).\u00C2\u00A0 Recombinant\u00C2\u00A0 mouse\u00C2\u00A0 (rm)\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 and\u00C2\u00A0 IL\u00E2\u0080\u00902\u00C2\u00A0 were\u00C2\u00A0 from\u00C2\u00A0 StemCell\u00C2\u00A0 Technologies\u00C2\u00A0 and\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 was\u00C2\u00A0 from\u00C2\u00A0 Sigma\u00E2\u0080\u0090Aldrich.\u00C2\u00A0 Unless\u00C2\u00A0 otherwise\u00C2\u00A0 stated,\u00C2\u00A0 all\u00C2\u00A0 tissue\u00C2\u00A0 culture\u00C2\u00A0 reagents\u00C2\u00A0were\u00C2\u00A0 from\u00C2\u00A0 StemCell\u00C2\u00A0 Technologies\u00C2\u00A0 and\u00C2\u00A0 all\u00C2\u00A0 other\u00C2\u00A0 reagents\u00C2\u00A0were\u00C2\u00A0 from\u00C2\u00A0 Sigma\u00E2\u0080\u0090Aldrich.\u00C2\u00A0 37 2.4 Isolation\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 2.4.1 Peritoneal\u00C2\u00A0macrophages\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0peritoneal\u00C2\u00A0cavities\u00C2\u00A0(PCs)\u00C2\u00A0of\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0 lavaged\u00C2\u00A0with\u00C2\u00A03\u00C2\u00A0x\u00C2\u00A05\u00C2\u00A0ml\u00C2\u00A0IMDM\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0 FCS\u00C2\u00A0medium\u00C2\u00A0or\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A01\u00C2\u00A0mM\u00C2\u00A0ethylenediaminetetraacetic\u00C2\u00A0acid\u00C2\u00A0(EDTA)\u00C2\u00A0 (USB\u00C2\u00A0Corporation,\u00C2\u00A0Cleveland,\u00C2\u00A0OH).\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0peritoneal\u00C2\u00A0macrophages\u00C2\u00A0(PM\u00CF\u0086s)\u00C2\u00A0was\u00C2\u00A0 visually\u00C2\u00A0 enumerated\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 hemocytometer\u00C2\u00A0 and\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0 fresh\u00C2\u00A0 medium\u00C2\u00A0without\u00C2\u00A0EDTA.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0plated\u00C2\u00A0and\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0adhere\u00C2\u00A0for\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A03\u00C2\u00A0hours\u00C2\u00A0(h)\u00C2\u00A0 before\u00C2\u00A0the\u00C2\u00A0non\u00E2\u0080\u0090adherent\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0away.\u00C2\u00A0When\u00C2\u00A0required,\u00C2\u00A0adherent\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 harvested\u00C2\u00A0 using\u00C2\u00A0 cell\u00C2\u00A0 dissociation\u00C2\u00A0 buffer\u00C2\u00A0 (Invitrogen/Gibco)\u00C2\u00A0 and\u00C2\u00A0 gentle\u00C2\u00A0 scraping.\u00C2\u00A0 Analysis\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0adherent\u00C2\u00A0 cells\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0>95%\u00C2\u00A0co\u00E2\u0080\u0090expressed\u00C2\u00A0F4/80\u00C2\u00A0and\u00C2\u00A0CD11b.\u00C2\u00A0 In\u00C2\u00A0 experiments\u00C2\u00A0 where\u00C2\u00A0 the\u00C2\u00A0 ratio\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 splenocytes\u00C2\u00A0 was\u00C2\u00A0 varied,\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 plated\u00C2\u00A0per\u00C2\u00A0well\u00C2\u00A0was\u00C2\u00A0adjusted\u00C2\u00A0to\u00C2\u00A0give\u00C2\u00A0the\u00C2\u00A0desired\u00C2\u00A0ratio,\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0splenocytes\u00C2\u00A0 remained\u00C2\u00A0 constant\u00C2\u00A0 (2\u00C2\u00A0 x\u00C2\u00A0 105\u00C2\u00A0 cells/well\u00C2\u00A0 [96\u00E2\u0080\u0090well\u00C2\u00A0 plate]\u00C2\u00A0 or\u00C2\u00A0 1\u00C2\u00A0 x\u00C2\u00A0 106\u00C2\u00A0 cells/well\u00C2\u00A0 [48\u00E2\u0080\u0090well\u00C2\u00A0 plate]).\u00C2\u00A0 \u00C2\u00A0 2.4.2 Splenic,\u00C2\u00A0pulmonary,\u00C2\u00A0or\u00C2\u00A0tumor\u00C2\u00ADassociated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 isolate\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 or\u00C2\u00A0 MDSCs,\u00C2\u00A0 single\u00E2\u0080\u0090cell\u00C2\u00A0 suspensions\u00C2\u00A0 were\u00C2\u00A0 prepared\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 spleens,\u00C2\u00A0kidneys,\u00C2\u00A0livers,\u00C2\u00A0lungs,\u00C2\u00A0and/or\u00C2\u00A0tumors\u00C2\u00A0of\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0or\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0female\u00C2\u00A0BALB/c\u00C2\u00A0 mice.\u00C2\u00A0 Spleens\u00C2\u00A0 and\u00C2\u00A0 livers\u00C2\u00A0 were\u00C2\u00A0 passed\u00C2\u00A0 through\u00C2\u00A0 a\u00C2\u00A0 70\u00C2\u00A0 \u00C2\u00B5m\u00C2\u00A0 filter\u00C2\u00A0 to\u00C2\u00A0 create\u00C2\u00A0 a\u00C2\u00A0 single\u00E2\u0080\u0090cell\u00C2\u00A0 suspension.\u00C2\u00A0Lungs,\u00C2\u00A0kidneys,\u00C2\u00A0and\u00C2\u00A0tumors\u00C2\u00A0were\u00C2\u00A0finely\u00C2\u00A0minced\u00C2\u00A0with\u00C2\u00A0crossed\u00C2\u00A0scalpels\u00C2\u00A0prior\u00C2\u00A0 to\u00C2\u00A0agitation\u00C2\u00A0for\u00C2\u00A040\u00C2\u00A0minutes\u00C2\u00A0(min)\u00C2\u00A0at\u00C2\u00A037\u00C2\u00B0C\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0enzyme\u00C2\u00A0suspension\u00C2\u00A0containing\u00C2\u00A00.5%\u00C2\u00A0 trypsin\u00C2\u00A0(BD\u00C2\u00A0Biosciences)\u00C2\u00A0and\u00C2\u00A00.08%\u00C2\u00A0collagenase\u00C2\u00A0(Sigma\u00E2\u0080\u0090Aldrich)\u00C2\u00A0in\u00C2\u00A0phosphate\u00C2\u00A0buffered\u00C2\u00A0 saline\u00C2\u00A0 (PBS)\u00C2\u00A0 (lungs\u00C2\u00A0 and\u00C2\u00A0 kidneys)\u00C2\u00A0 or\u00C2\u00A0 30\u00C2\u00A0 min\u00C2\u00A0 at\u00C2\u00A0 37\u00C2\u00B0C\u00C2\u00A0 with\u00C2\u00A0 125\u00C2\u00A0 \u00CE\u00BCg\u00C2\u00A0 Liberase\u00C2\u00A0 (Roche\u00C2\u00A0 Diagnostics)\u00C2\u00A0 in\u00C2\u00A0 IMDM\u00C2\u00A0 (tumors).\u00C2\u00A0 \u00C2\u00A0 After\u00C2\u00A0 incubation,\u00C2\u00A0 0.06%\u00C2\u00A0DNase\u00C2\u00A0 (Sigma\u00E2\u0080\u0090Aldrich)\u00C2\u00A0was\u00C2\u00A0 added,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0suspension\u00C2\u00A0was\u00C2\u00A0gently\u00C2\u00A0vortexed\u00C2\u00A0and\u00C2\u00A0filtered\u00C2\u00A0through\u00C2\u00A030\u00C2\u00A0\u00C2\u00B5m\u00C2\u00A0nylon\u00C2\u00A0 mesh\u00C2\u00A0 to\u00C2\u00A0 remove\u00C2\u00A0 clumps.\u00C2\u00A0 Samples\u00C2\u00A0 for\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0 were\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 ammonium\u00C2\u00A0 chloride\u00C2\u00A0 solution\u00C2\u00A0 (0.8%\u00C2\u00A0with\u00C2\u00A0 0.1\u00C2\u00A0 mM\u00C2\u00A0 EDTA;\u00C2\u00A0 7\u00C2\u00A0 min\u00C2\u00A0 on\u00C2\u00A0 ice)\u00C2\u00A0 for\u00C2\u00A0 erythrocyte\u00C2\u00A0 lysis\u00C2\u00A0 and\u00C2\u00A0 38 either\u00C2\u00A0fixed\u00C2\u00A0in\u00C2\u00A070%\u00C2\u00A0EtOH\u00C2\u00A0and\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A0\u00E2\u0080\u009020\u00C2\u00B0C\u00C2\u00A0for\u00C2\u00A0subsequent\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0analysis\u00C2\u00A0or\u00C2\u00A0 analyzed\u00C2\u00A0immediately.\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0 for\u00C2\u00A0magnetic\u00C2\u00A0 separation\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0by\u00C2\u00A0 centrifugation\u00C2\u00A0 (1200\u00C2\u00A0 rpm,\u00C2\u00A05\u00C2\u00A0 min)\u00C2\u00A0and\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A0PBS\u00C2\u00A0+\u00C2\u00A02%\u00C2\u00A0FCS\u00C2\u00A0+\u00C2\u00A01mM\u00C2\u00A0EDTA.\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0using\u00C2\u00A0F4/80\u00E2\u0080\u0090 PE\u00C2\u00A0positive\u00C2\u00A0selection\u00C2\u00A0(EasySep;\u00C2\u00A0StemCell\u00C2\u00A0Technologies)\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0using\u00C2\u00A0 Gr1\u00E2\u0080\u0090PE\u00C2\u00A0 positive\u00C2\u00A0 selection\u00C2\u00A0 (EasySep;\u00C2\u00A0 StemCell\u00C2\u00A0 Technologies),\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions\u00C2\u00A0 (Table\u00C2\u00A0 2.1).\u00C2\u00A0 Cell\u00C2\u00A0 numbers\u00C2\u00A0 were\u00C2\u00A0 quantified\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 hemocytometer\u00C2\u00A0and\u00C2\u00A0purity\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0>90%\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.5 Flow\u00C2\u00A0cytometry\u00C2\u00A0 \u00C2\u00A0 All\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0samples\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0in\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0V\u00E2\u0080\u0090bottom\u00C2\u00A0plates.\u00C2\u00A0To\u00C2\u00A0label\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A0 surface\u00C2\u00A0marker\u00C2\u00A0Abs,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 suspended\u00C2\u00A0 in\u00C2\u00A0Hank\u00E2\u0080\u0099s\u00C2\u00A0 balanced\u00C2\u00A0 salt\u00C2\u00A0 solution\u00C2\u00A0 (HBSS)\u00C2\u00A0 +\u00C2\u00A0 2%\u00C2\u00A0 FCS\u00C2\u00A0 +\u00C2\u00A0 0.05%\u00C2\u00A0NaN3\u00C2\u00A0 (HFN)\u00C2\u00A0 and\u00C2\u00A0 incubated\u00C2\u00A0with\u00C2\u00A0 anti\u00E2\u0080\u0090mouse\u00C2\u00A0 CD16/CD32\u00C2\u00A0 (2.4G2)\u00C2\u00A0 (BD\u00C2\u00A0 Pharmigen,\u00C2\u00A0 Mississauga,\u00C2\u00A0 ON,\u00C2\u00A0 Canada)\u00C2\u00A0 for\u00C2\u00A0 10\u00C2\u00A0 min\u00C2\u00A0 at\u00C2\u00A0 4\u00C2\u00B0C\u00C2\u00A0 to\u00C2\u00A0 block\u00C2\u00A0 Fc\u00C2\u00A0 receptors\u00C2\u00A0before\u00C2\u00A0labeling.\u00C2\u00A0Flurochrome\u00C2\u00A0conjugated\u00C2\u00A0Abs\u00C2\u00A0(Table\u00C2\u00A02.1)\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0at\u00C2\u00A0pre\u00E2\u0080\u0090 determined\u00C2\u00A0optimal\u00C2\u00A0 concentrations\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0 at\u00C2\u00A0 4\u00C2\u00B0C.\u00C2\u00A0 \u00C2\u00A0 Cells\u00C2\u00A0were\u00C2\u00A0 then\u00C2\u00A0washed\u00C2\u00A0 twice\u00C2\u00A0 and\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0 HFN\u00C2\u00A0 for\u00C2\u00A0 analysis.\u00C2\u00A0 Data\u00C2\u00A0 were\u00C2\u00A0 acquired\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 FACSCalibur\u00C2\u00A0 flow\u00C2\u00A0 cytometer\u00C2\u00A0 (BD\u00C2\u00A0 Biosciences)\u00C2\u00A0 and\u00C2\u00A0 analyzed\u00C2\u00A0 using\u00C2\u00A0 FlowJo\u00C2\u00A0 software\u00C2\u00A0 (Tree\u00C2\u00A0 Star,\u00C2\u00A0 Inc.,\u00C2\u00A0 Ashland,\u00C2\u00A0 OR).\u00C2\u00A0 Where\u00C2\u00A0 reported,\u00C2\u00A0 absolute\u00C2\u00A0 numbers\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 calculated\u00C2\u00A0 by\u00C2\u00A0 multiplying\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 (enumerated\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 hemocytometer)\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 proportion\u00C2\u00A0of\u00C2\u00A0that\u00C2\u00A0specific\u00C2\u00A0cell\u00C2\u00A0type,\u00C2\u00A0as\u00C2\u00A0determined\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0stain\u00C2\u00A0cells\u00C2\u00A0for\u00C2\u00A0intracellular\u00C2\u00A0Foxp3\u00C2\u00A0expression,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0 HFN\u00C2\u00A0+\u00C2\u00A02.4G2\u00C2\u00A0Ab\u00C2\u00A0and\u00C2\u00A0100\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0Fixation/Permeabilization\u00C2\u00A0working\u00C2\u00A0solution\u00C2\u00A0(eBioscience)\u00C2\u00A0 was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0sample.\u00C2\u00A0Following\u00C2\u00A0incubation\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A04\u00C2\u00B0C,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0 twice\u00C2\u00A0with\u00C2\u00A0100\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A01x\u00C2\u00A0Permeabilization\u00C2\u00A0Buffer\u00C2\u00A0(eBioscience)\u00C2\u00A0and\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A01x\u00C2\u00A0 Permeabilization\u00C2\u00A0Buffer\u00C2\u00A0 containing\u00C2\u00A02%\u00C2\u00A0rat\u00C2\u00A0 serum\u00C2\u00A0 (Stem\u00C2\u00A0Cell\u00C2\u00A0Technologies).\u00C2\u00A0Abs\u00C2\u00A0were\u00C2\u00A0 39 added\u00C2\u00A0as\u00C2\u00A0 indicated\u00C2\u00A0 in\u00C2\u00A0Table\u00C2\u00A02.1\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A04\u00C2\u00B0C.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0twice\u00C2\u00A0and\u00C2\u00A0 resuspended\u00C2\u00A0in\u00C2\u00A0HFN\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A02.1\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0antibodies\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0thesis.\u00C2\u00A0 \u00C2\u00A0 FC\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Flow\u00C2\u00A0Cytometry,\u00C2\u00A0WB\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Western\u00C2\u00A0Blotting,\u00C2\u00A0IF\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Immunofluorescence,\u00C2\u00A0\u00C2\u00A0 PS\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Positive\u00C2\u00A0Selection\u00C2\u00A0 Ms\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0mouse,\u00C2\u00A0Rb\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0rabbit,\u00C2\u00A0c\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0cells\u00C2\u00A0or\u00C2\u00A0cell\u00C2\u00A0equivalents\u00C2\u00A0 pAb\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0polyclonal\u00C2\u00A0Ab,\u00C2\u00A0mAb\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0monoclonal\u00C2\u00A0Ab,\u00C2\u00A0Ig\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0immunoglobulin\u00C2\u00A0\u00C2\u00A0 APCy\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0allophycocyanin,\u00C2\u00A0Cy\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0cyanine,\u00C2\u00A0FITC\u00C2\u00A0\u00E2\u0080\u0090\u00C2\u00A0fluorescein\u00C2\u00A0isothiocyanate,\u00C2\u00A0\u00C2\u00A0 PE\u00C2\u00A0\u00E2\u0080\u0090\u00C2\u00A0phycoerythrin\u00C2\u00A0 \u00C2\u00A0 Antibody\u00C2\u00A0(Ab)\u00C2\u00A0 Type\u00C2\u00A0 Concentration/\u00C2\u00A0 Dilution\u00C2\u00A0used\u00C2\u00A0 Procedure\u00C2\u00A0 Source\u00C2\u00A0 Annexin\u00C2\u00A0V\u00E2\u0080\u0090PE\u00C2\u00A0 n/a\u00C2\u00A0 5\u00C2\u00A0\u00CE\u00BCl/106\u00C2\u00A0c\u00C2\u00A0 FC BD Pharmingen\u00C2\u00A0 Arg\u00C2\u00A01\u00C2\u00A0 mAb\u00C2\u00A0(Ms)\u00C2\u00A0 1:2000 WB BD Bioscience\u00C2\u00A0 B220\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 0.2\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC BD Pharmingen\u00C2\u00A0 CD3\u00E2\u0080\u0090APCy\u00C2\u00A0 Armenian\u00C2\u00A0 Hamster\u00C2\u00A0 IgG1\u00C2\u00A0 0.2\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC BD Pharmingen\u00C2\u00A0 CD4\u00E2\u0080\u0090APCy\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 0.125\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 CD8\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC StemCell\u00C2\u00A0Technologies CD8\u00E2\u0080\u0090FITC\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 0.5\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 CD11b\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 1:100 IF eBioscience\u00C2\u00A0 CD11b\u00E2\u0080\u0090APCy\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 0.125\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 CD11b\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC StemCell\u00C2\u00A0Technologies CD11b\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0 PS StemCell\u00C2\u00A0Technologies CD11c\u00E2\u0080\u0090APCy\u00C2\u00A0 Armenian\u00C2\u00A0 Hamster\u00C2\u00A0 IgG\u00C2\u00A0 0.125\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 CD40\u00E2\u0080\u0090FITC\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC BD Pharmingen\u00C2\u00A0 CD86\u00E2\u0080\u0090FITC\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC BD Pharmingen\u00C2\u00A0 Fc\u00CE\u00B5R1\u00CE\u00B1\u00E2\u0080\u0090FITC\u00C2\u00A0 Armenian\u00C2\u00A0 Hamster\u00C2\u00A0 IgG1\u00C2\u00A0 0.125\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 F4/80\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 1:100 IF eBioscience\u00C2\u00A0 F4/80\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 0.1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC Invitrogen\u00C2\u00A0 F4/80\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0 PS Invitrogen\u00C2\u00A0 GAPDH\u00C2\u00A0 (glyceraldehyde\u00C2\u00A03\u00E2\u0080\u0090 phosphate\u00C2\u00A0 dehydrogenase)\u00C2\u00A0 mAb\u00C2\u00A0(Ms)\u00C2\u00A0 1:40,000 WB Research\u00C2\u00A0Diagnostics\u00C2\u00A0 Inc.\u00C2\u00A0(Flanders,\u00C2\u00A0NJ)\u00C2\u00A0 Gr1\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 1:100 IF eBioscience\u00C2\u00A0 Gr1\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 40 Antibody\u00C2\u00A0(Ab)\u00C2\u00A0 Type\u00C2\u00A0 Concentration/\u00C2\u00A0 Dilution\u00C2\u00A0used\u00C2\u00A0 Procedure\u00C2\u00A0 Source\u00C2\u00A0 Gr1\u00E2\u0080\u0090PE\u00C2\u00A0 Rat\u00C2\u00A0IgG2b\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 PS eBioscience\u00C2\u00A0 Foxp3\u00E2\u0080\u0090PE\u00E2\u0080\u0090Cy7\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 0.25\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC eBioscience\u00C2\u00A0 iNOS\u00C2\u00A0 pAb\u00C2\u00A0(Rb)\u00C2\u00A0 1:750 WB Santa\u00C2\u00A0Cruz\u00C2\u00A0 Biotechnology,\u00C2\u00A0(Santa\u00C2\u00A0 Cruz,\u00C2\u00A0CA)\u00C2\u00A0 MHCII\u00E2\u0080\u0090FITC\u00C2\u00A0 Rat\u00C2\u00A0IgG2a\u00C2\u00A0 1\u00C2\u00A0\u00CE\u00BCg/106\u00C2\u00A0c\u00C2\u00A0 FC BD Pharmingen\u00C2\u00A0 Pimonidazole\u00E2\u0080\u0090FITC\u00C2\u00A0 Mouse\u00C2\u00A0IgG\u00C2\u00A0 1:100 IF Hydroxyprobe,\u00C2\u00A0Inc.\u00C2\u00A0 (Burlington,\u00C2\u00A0MA)\u00C2\u00A0 pSTAT1\u00C2\u00A0Y701\u00C2\u00A0 pAb\u00C2\u00A0(Rb)\u00C2\u00A0 1:1000 WB Cell\u00C2\u00A0Signalling\u00C2\u00A0(Beverly,\u00C2\u00A0 MA)\u00C2\u00A0 Ym1\u00C2\u00A0 pAb\u00C2\u00A0(Rb)\u00C2\u00A0 1:2000 WB StemCell\u00C2\u00A0Technologies Goat\u00C2\u00A0anti\u00E2\u0080\u0090rat\u00C2\u00A0\u00C2\u00A0 Alexa\u00C2\u00A0488\u00C2\u00A0 pAb\u00C2\u00A0(Goat) 1:100 IF Invitrogen\u00C2\u00A0 Goat\u00C2\u00A0anti\u00E2\u0080\u0090rat\u00C2\u00A0\u00C2\u00A0 Alexa\u00C2\u00A0594\u00C2\u00A0 pAb\u00C2\u00A0(Goat) 1:100 IF Invitrogen\u00C2\u00A0 \u00C2\u00A0 2.6 T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0assay\u00C2\u00A0and\u00C2\u00A0cytokine\u00C2\u00A0assays\u00C2\u00A0 \u00C2\u00A0 Erythrocyte\u00E2\u0080\u0090depleted\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 in\u00C2\u00A0 IMDM\u00C2\u00A0 medium\u00C2\u00A0 or\u00C2\u00A0 RPMI\u00C2\u00A0 medium\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0FCS,\u00C2\u00A0or\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0at\u00C2\u00A02\u00C2\u00A0x\u00C2\u00A0105\u00C2\u00A0cells/well\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0irradiated\u00C2\u00A0(2000\u00C2\u00A0 rads)\u00C2\u00A0test\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0total\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0150\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0in\u00C2\u00A096\u00E2\u0080\u0090well,\u00C2\u00A0flat\u00E2\u0080\u0090bottom\u00C2\u00A0tissue\u00C2\u00A0culture\u00C2\u00A0plates.\u00C2\u00A0 These\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0stimulated\u00C2\u00A0as\u00C2\u00A0follows:\u00C2\u00A0C57BL/6\u00C2\u00A0or\u00C2\u00A0BALB/c\u00C2\u00A0splenocytes\u00C2\u00A0with\u00C2\u00A01\u00C2\u00A0 \u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090CD3\u00C2\u00A0+\u00C2\u00A05\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090CD28\u00C2\u00A0(polyclonal),\u00C2\u00A0OTII\u00C2\u00A0or\u00C2\u00A0DO11.10\u00C2\u00A0splenocytes\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0 \u00CE\u00BCg/ml\u00C2\u00A0OVA\u00C2\u00A0peptide\u00C2\u00A0(323\u00E2\u0080\u0090339);\u00C2\u00A0and\u00C2\u00A0OTI\u00C2\u00A0splenocytes\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0OVA\u00C2\u00A0peptide\u00C2\u00A0(257\u00E2\u0080\u0090 264).\u00C2\u00A0Inhibitors\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0as\u00C2\u00A0indicated.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A037\u00C2\u00B0C,\u00C2\u00A05%\u00C2\u00A0CO2\u00C2\u00A0for\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0 with\u00C2\u00A01\u00C2\u00A0\u00CE\u00BCCi/well\u00C2\u00A03H\u00E2\u0080\u0090thymidine\u00C2\u00A0(thy)\u00C2\u00A0(2\u00C2\u00A0Ci/mM;\u00C2\u00A0PerkinElmer,\u00C2\u00A0Woodbridge,\u00C2\u00A0ON,\u00C2\u00A0Canada)\u00C2\u00A0 added\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 last\u00C2\u00A0 18\u00C2\u00A0 h.\u00C2\u00A0 For\u00C2\u00A0 cell\u00C2\u00A0 contact\u00C2\u00A0 studies,\u00C2\u00A0 test\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 added\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 lower\u00C2\u00A0 chamber\u00C2\u00A0 of\u00C2\u00A0 Transwell\u00C2\u00A0 plates\u00C2\u00A0 (0.4\u00C2\u00A0 \u00CE\u00BCm\u00C2\u00A0 pores,\u00C2\u00A0 polycarbonate\u00C2\u00A0 membrane;\u00C2\u00A0 Corning\u00C2\u00A0 Life\u00C2\u00A0 Sciences,\u00C2\u00A0Corning,\u00C2\u00A0NY)\u00C2\u00A0and\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0upper\u00C2\u00A0chamber.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0 harvested\u00C2\u00A0 onto\u00C2\u00A0 glass\u00C2\u00A0 fiber\u00C2\u00A0 filter\u00C2\u00A0 mats\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 96\u00E2\u0080\u0090well\u00C2\u00A0 harvester\u00C2\u00A0 (Molecular\u00C2\u00A0 Devices,\u00C2\u00A0 Sunnyvale,\u00C2\u00A0CA).\u00C2\u00A0Filter\u00C2\u00A0mats\u00C2\u00A0were\u00C2\u00A0sealed\u00C2\u00A0in\u00C2\u00A0plastic\u00C2\u00A0bags\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0ml\u00C2\u00A0of\u00C2\u00A0scintillation\u00C2\u00A0fluid\u00C2\u00A0 and\u00C2\u00A0 3H\u00E2\u0080\u0090thy\u00C2\u00A0 incorporation\u00C2\u00A0 measured\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 Betaplate\u00C2\u00A0 liquid\u00C2\u00A0 scintillation\u00C2\u00A0 counter\u00C2\u00A0 (Wallac,\u00C2\u00A0Waltham,\u00C2\u00A0MA).\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0expressed\u00C2\u00A0as\u00C2\u00A0counts\u00C2\u00A0per\u00C2\u00A0minute\u00C2\u00A0(cpm)\u00C2\u00A0(mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM)\u00C2\u00A0 of\u00C2\u00A0triplicate\u00C2\u00A0cultures.\u00C2\u00A0Responder\u00C2\u00A0control\u00C2\u00A0(RC)\u00C2\u00A0indicates\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0alone.\u00C2\u00A0 \u00C2\u00A0 41 The\u00C2\u00A0relative\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0splenocyte\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0calculated\u00C2\u00A0as:\u00C2\u00A0 (Proliferation\u00C2\u00A0of\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0with\u00C2\u00A0test\u00C2\u00A0cells)\u00C2\u00A0x\u00C2\u00A0100%\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0(Proliferation\u00C2\u00A0of\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(RC)\u00C2\u00A0alone)\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 measure\u00C2\u00A0 cytokine\u00C2\u00A0 production,\u00C2\u00A0 the\u00C2\u00A0 above\u00C2\u00A0 assay\u00C2\u00A0 was\u00C2\u00A0 performed\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 following\u00C2\u00A0 changes:\u00C2\u00A0 5\u00C2\u00A0 x\u00C2\u00A0 105\u00C2\u00A0 splenocytes/well\u00C2\u00A0were\u00C2\u00A0 added\u00C2\u00A0 to\u00C2\u00A0 48\u00E2\u0080\u0090well\u00C2\u00A0 plates,\u00C2\u00A0 the\u00C2\u00A0 assay\u00C2\u00A0 was\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 total\u00C2\u00A0 volume\u00C2\u00A0 of\u00C2\u00A0 375\u00C2\u00A0 \u00CE\u00BCl,\u00C2\u00A0 and\u00C2\u00A0 no\u00C2\u00A0 3H\u00E2\u0080\u0090thy\u00C2\u00A0 was\u00C2\u00A0 added.\u00C2\u00A0 Cell\u00E2\u0080\u0090free\u00C2\u00A0 supernatants\u00C2\u00A0 were\u00C2\u00A0 collected\u00C2\u00A0 after\u00C2\u00A0 72\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 IL\u00E2\u0080\u00902,\u00C2\u00A0 IL\u00E2\u0080\u00904,\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 and\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 production\u00C2\u00A0 assayed\u00C2\u00A0 using\u00C2\u00A0 cytokine\u00C2\u00A0 enzyme\u00E2\u0080\u0090linked\u00C2\u00A0 immunosorbent\u00C2\u00A0 assay\u00C2\u00A0 (ELISA)\u00C2\u00A0 kits\u00C2\u00A0 (BD\u00C2\u00A0 Biosciences),\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0instructions.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0assay\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry,\u00C2\u00A0erythrocyte\u00E2\u0080\u0090depleted\u00C2\u00A0splenocytes\u00C2\u00A0 were\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A037\u00C2\u00B0C\u00C2\u00A0PBS\u00C2\u00A0and\u00C2\u00A05\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0carboxyfluorescein\u00C2\u00A0succinimidyl\u00C2\u00A0ester\u00C2\u00A0(CFSE)\u00C2\u00A0 (Sigma\u00E2\u0080\u0090Aldrich)\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0sample.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A037\u00C2\u00B0C\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0washed\u00C2\u00A0twice\u00C2\u00A0in\u00C2\u00A04\u00C2\u00B0C\u00C2\u00A0PBS\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0stimulation\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0test\u00C2\u00A0cells.\u00C2\u00A0After\u00C2\u00A0 72\u00C2\u00A0h,\u00C2\u00A0non\u00E2\u0080\u0090adherent\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0harvested,\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0fluorescent\u00C2\u00A0Abs\u00C2\u00A0against\u00C2\u00A0CD3,\u00C2\u00A0CD4,\u00C2\u00A0 and/or\u00C2\u00A0CD8,\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0Histogram\u00C2\u00A0peaks\u00C2\u00A0represent\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 T\u00C2\u00A0cell\u00C2\u00A0divisions\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0divided\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0indicated.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.7 In\u00C2\u00A0vitro\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0skewing\u00C2\u00A0 \u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0plated\u00C2\u00A0at\u00C2\u00A01.25\u00C2\u00A0x\u00C2\u00A0105\u00C2\u00A0cells/well\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0IMDM\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A0treated\u00C2\u00A0 with\u00C2\u00A0this\u00C2\u00A0medium\u00C2\u00A0alone,\u00C2\u00A0with\u00C2\u00A0100\u00C2\u00A0ng/ml\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0+\u00C2\u00A0100\u00C2\u00A0ng/ml\u00C2\u00A0LPS,\u00C2\u00A0or\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0ng/ml\u00C2\u00A0IL\u00E2\u0080\u00904\u00C2\u00A0 +\u00C2\u00A010\u00C2\u00A0ng/ml\u00C2\u00A0IL\u00E2\u0080\u009013.\u00C2\u00A0After\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0subjected\u00C2\u00A0to\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0analysis.\u00C2\u00A0 \u00C2\u00A0 2.8 SDS\u00C2\u00ADPAGE\u00C2\u00A0and\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0 \u00C2\u00A0 Cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0PBS\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0pellets\u00C2\u00A0lysed\u00C2\u00A0with\u00C2\u00A01x\u00C2\u00A0sodium\u00C2\u00A0dodecyl\u00C2\u00A0sulfate\u00C2\u00A0 (SDS)\u00C2\u00A0 sample\u00C2\u00A0 buffer\u00C2\u00A0 (i.e.\u00C2\u00A0 8.5%\u00C2\u00A0 (v/v)\u00C2\u00A0 glycerol,\u00C2\u00A0 0.5%\u00C2\u00A0 (w/v)\u00C2\u00A0 SDS\u00C2\u00A0 and\u00C2\u00A0 0.71\u00C2\u00A0 M\u00C2\u00A0 \u00CE\u00B2\u00E2\u0080\u0090ME).\u00C2\u00A0 Samples\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0boiled\u00C2\u00A0for\u00C2\u00A02\u00C2\u00A0min,\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0cool,\u00C2\u00A0and\u00C2\u00A0DNA\u00C2\u00A0sheared\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A026\u00C2\u00A0gauge\u00C2\u00A0 needle.\u00C2\u00A0 Equal\u00C2\u00A0 numbers\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 (total\u00C2\u00A0 cell\u00C2\u00A0 lysates)\u00C2\u00A0 were\u00C2\u00A0 then\u00C2\u00A0 loaded\u00C2\u00A0 onto\u00C2\u00A0 10%\u00C2\u00A0 42 polyacrylamide\u00C2\u00A0 gels\u00C2\u00A0 and\u00C2\u00A0 subjected\u00C2\u00A0 to\u00C2\u00A0 SDS\u00E2\u0080\u0090polyacrylamide\u00C2\u00A0 gel\u00C2\u00A0 electrophoresis\u00C2\u00A0 (PAGE)\u00C2\u00A0 and\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0previously\u00C2\u00A0(Damen\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01995).\u00C2\u00A0 \u00C2\u00A0Abs\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0 Western\u00C2\u00A0blotting\u00C2\u00A0are\u00C2\u00A0indicated\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A02.1.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.9 Viability\u00C2\u00A0and\u00C2\u00A0morphology\u00C2\u00A0assays\u00C2\u00A0 \u00C2\u00A0 Following\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0of\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0with\u00C2\u00A0myeloid\u00C2\u00A0test\u00C2\u00A0cells,\u00C2\u00A0non\u00E2\u0080\u0090 adherent\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0 flow\u00C2\u00A0cytometry\u00C2\u00A0 for\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0CD3,\u00C2\u00A0 CD4,\u00C2\u00A0CD8,\u00C2\u00A0Annexin\u00C2\u00A0V\u00E2\u0080\u0090PE,\u00C2\u00A0and/or\u00C2\u00A0propidium\u00C2\u00A0iodide\u00C2\u00A0(PI)\u00C2\u00A0(Table\u00C2\u00A02.1).\u00C2\u00A0Annexin\u00C2\u00A0V\u00C2\u00A0staining\u00C2\u00A0 was\u00C2\u00A0 performed\u00C2\u00A0 by\u00C2\u00A0 washing\u00C2\u00A0 cells\u00C2\u00A0 (that\u00C2\u00A0 had\u00C2\u00A0 been\u00C2\u00A0 previously\u00C2\u00A0 stained\u00C2\u00A0 with\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 Abs)\u00C2\u00A0twice\u00C2\u00A0with\u00C2\u00A0cold\u00C2\u00A0PBS\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0resuspending\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0100\u00CE\u00BCl\u00C2\u00A01x\u00C2\u00A0Binding\u00C2\u00A0Buffer\u00C2\u00A0(100\u00C2\u00A0 mM\u00C2\u00A0 4\u00E2\u0080\u0090(2\u00E2\u0080\u0090hydroxyethyl)\u00E2\u0080\u00901\u00E2\u0080\u0090piperazineethanesulfonic\u00C2\u00A0 acid\u00C2\u00A0 (HEPES),\u00C2\u00A0 pH\u00C2\u00A0 7.4;\u00C2\u00A0 140\u00C2\u00A0 mM\u00C2\u00A0 NaCl;\u00C2\u00A02.5\u00C2\u00A0mM\u00C2\u00A0CaCl2).\u00C2\u00A05\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0Annexin\u00C2\u00A0V\u00E2\u0080\u0090PE\u00C2\u00A0and\u00C2\u00A0100\u00C2\u00A0ng\u00C2\u00A0PI\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0sample,\u00C2\u00A0cells\u00C2\u00A0 were\u00C2\u00A0gently\u00C2\u00A0mixed,\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A015\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A023\u00C2\u00B0C\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0dark.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0diluted\u00C2\u00A0 with\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A01x\u00C2\u00A0Binding\u00C2\u00A0Buffer\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0 flow\u00C2\u00A0cytometry.\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0classified\u00C2\u00A0as\u00C2\u00A0 viable\u00C2\u00A0(Annexin\u00C2\u00A0V\u00E2\u0080\u0090PE\u00C2\u00A0and\u00C2\u00A0PI\u00C2\u00A0negative),\u00C2\u00A0undergoing\u00C2\u00A0apoptosis\u00C2\u00A0(Annexin\u00C2\u00A0V\u00E2\u0080\u0090PE\u00C2\u00A0positive,\u00C2\u00A0PI\u00C2\u00A0 negative),\u00C2\u00A0 or\u00C2\u00A0 dead\u00C2\u00A0 (Annexin\u00C2\u00A0 V\u00E2\u0080\u0090PE\u00C2\u00A0 and\u00C2\u00A0 PI\u00C2\u00A0 positive).\u00C2\u00A0 For\u00C2\u00A0 some\u00C2\u00A0 experiments\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 stained\u00C2\u00A0for\u00C2\u00A0CD3,\u00C2\u00A0CD4,\u00C2\u00A0and/or\u00C2\u00A0CD8,\u00C2\u00A0and\u00C2\u00A0PI\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0flow\u00C2\u00A0cytometric\u00C2\u00A0analysis\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0 bulk\u00C2\u00A0 (total\u00C2\u00A0 cells),\u00C2\u00A0 total\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 (CD4+\u00C2\u00A0 or\u00C2\u00A0 CD8+),\u00C2\u00A0 or\u00C2\u00A0 CD4+\u00C2\u00A0 or\u00C2\u00A0 CD8+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 viability.\u00C2\u00A0 The\u00C2\u00A0 increase\u00C2\u00A0in\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0was\u00C2\u00A0calculated\u00C2\u00A0as:\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 (Percent\u00C2\u00A0viability\u00C2\u00A0of\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0with\u00C2\u00A0test\u00C2\u00A0cells)\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0x\u00C2\u00A0100%\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0(Percent\u00C2\u00A0viability\u00C2\u00A0of\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(RC)\u00C2\u00A0alone)\u00C2\u00A0 \u00C2\u00A0 Cell\u00C2\u00A0 viability\u00C2\u00A0was\u00C2\u00A0 also\u00C2\u00A0 determined\u00C2\u00A0 via\u00C2\u00A0 trypan\u00C2\u00A0 blue\u00C2\u00A0 exclusion.\u00C2\u00A0 Trypan\u00C2\u00A0 blue\u00C2\u00A0was\u00C2\u00A0 added\u00C2\u00A0to\u00C2\u00A0either\u00C2\u00A0adherent\u00C2\u00A0or\u00C2\u00A0non\u00E2\u0080\u0090adherent\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0excluded\u00C2\u00A0 trypan\u00C2\u00A0blue\u00C2\u00A0(viable\u00C2\u00A0cells)\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0hemocytometer.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 assess\u00C2\u00A0 MDSC\u00C2\u00A0 morphology,\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 plated\u00C2\u00A0 at\u00C2\u00A0 1\u00C2\u00A0 x\u00C2\u00A0 106\u00C2\u00A0 cells/ml\u00C2\u00A0 in\u00C2\u00A0 HL1\u00C2\u00A0 medium\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0overnight\u00C2\u00A0(O/N)\u00C2\u00A0at\u00C2\u00A037\u00C2\u00B0C.\u00C2\u00A0The\u00C2\u00A0next\u00C2\u00A0day,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0 and\u00C2\u00A0 cytospin\u00C2\u00A0preparations\u00C2\u00A0were\u00C2\u00A0 stained\u00C2\u00A0with\u00C2\u00A0Giemsa\u00E2\u0080\u0090Eosin.\u00C2\u00A0The\u00C2\u00A0 43 morphology\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 was\u00C2\u00A0 assessed\u00C2\u00A0 by\u00C2\u00A0 light\u00C2\u00A0 microscopy\u00C2\u00A0 using\u00C2\u00A0 an\u00C2\u00A0 Axiovert\u00C2\u00A0 S100\u00C2\u00A0 microscope\u00C2\u00A0(Carl\u00C2\u00A0Zeiss\u00C2\u00A0Canada\u00C2\u00A0Ltd.,\u00C2\u00A0Toronto,\u00C2\u00A0ON,\u00C2\u00A0Canada)\u00C2\u00A0and\u00C2\u00A0images\u00C2\u00A0captured\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0 Retiga\u00C2\u00A0EXi\u00C2\u00A0camera\u00C2\u00A0(QImaging,\u00C2\u00A0Surrey,\u00C2\u00A0BC,\u00C2\u00A0Canada).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 2.10 Nitric\u00C2\u00A0oxide\u00C2\u00A0assay\u00C2\u00A0 \u00C2\u00A0 NO\u00C2\u00A0production\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0indirectly\u00C2\u00A0by\u00C2\u00A0measuring\u00C2\u00A0the\u00C2\u00A0accumulation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 stable\u00C2\u00A0 end\u00C2\u00A0 product,\u00C2\u00A0 nitrite\u00C2\u00A0 (NO2\u00E2\u0080\u0090),\u00C2\u00A0 in\u00C2\u00A0 cell\u00E2\u0080\u0090free\u00C2\u00A0 culture\u00C2\u00A0 supernatants\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 Griess\u00C2\u00A0 assay\u00C2\u00A0(Kleinbongard\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02002).\u00C2\u00A0Briefly,\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0of\u00C2\u00A0supernatant\u00C2\u00A0was\u00C2\u00A0aliquoted\u00C2\u00A0into\u00C2\u00A0wells\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 96\u00E2\u0080\u0090well\u00C2\u00A0 plate\u00C2\u00A0 at\u00C2\u00A0 23\u00C2\u00B0C.\u00C2\u00A0 Then\u00C2\u00A0 50\u00C2\u00A0 \u00CE\u00BCl\u00C2\u00A0 of\u00C2\u00A0 solution\u00C2\u00A0 A\u00C2\u00A0 (1%\u00C2\u00A0 sulfanilamide\u00C2\u00A0 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In\u00C2\u00A0vitro\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0pre\u00C2\u00ADtreatment\u00C2\u00A0and\u00C2\u00A0stimulation\u00C2\u00A0 \u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0plated\u00C2\u00A0at\u00C2\u00A01.25\u00C2\u00A0x\u00C2\u00A0105\u00C2\u00A0cells/well\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0IMDM\u00C2\u00A0medium\u00C2\u00A0+\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0or\u00C2\u00A0 HL1\u00C2\u00A0medium\u00C2\u00A0in\u00C2\u00A048\u00E2\u0080\u0090well\u00C2\u00A0plates\u00C2\u00A0or\u00C2\u00A02.5\u00C2\u00A0x\u00C2\u00A0104\u00C2\u00A0cells/well\u00C2\u00A0 in\u00C2\u00A0100\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0 in\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plates.\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 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three\u00C2\u00A0times\u00C2\u00A0with\u00C2\u00A0wash\u00C2\u00A0buffer.\u00C2\u00A0100\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0sample\u00C2\u00A0or\u00C2\u00A0standard\u00C2\u00A0(rmIFN\u00E2\u0080\u0090\u00CE\u00B2)\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0 to\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0O/N\u00C2\u00A0at\u00C2\u00A04\u00C2\u00B0C.\u00C2\u00A0The\u00C2\u00A0wells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A07x\u00C2\u00A0with\u00C2\u00A0wash\u00C2\u00A0buffer\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0100\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0of\u00C2\u00A0detection\u00C2\u00A0Ab\u00C2\u00A0 (25\u00C2\u00A0U/ml\u00C2\u00A0 rabbit\u00C2\u00A0 anti\u00E2\u0080\u0090mouse\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0polyclonal\u00C2\u00A0Ab\u00C2\u00A0 in\u00C2\u00A0assay\u00C2\u00A0 diluent;\u00C2\u00A0R&D\u00C2\u00A0Systems,\u00C2\u00A0Minneapolis,\u00C2\u00A0MN)\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A02\u00C2\u00A0hrs\u00C2\u00A0 at\u00C2\u00A023\u00C2\u00B0C.\u00C2\u00A0The\u00C2\u00A0wells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A07x\u00C2\u00A0with\u00C2\u00A0wash\u00C2\u00A0buffer\u00C2\u00A0with\u00C2\u00A01\u00C2\u00A0min\u00C2\u00A0soaks\u00C2\u00A0between\u00C2\u00A0each\u00C2\u00A0 wash.\u00C2\u00A0ELISA\u00C2\u00A0substrate\u00C2\u00A0(100\u00C2\u00A0\u00CE\u00BCl)\u00C2\u00A0(BD\u00C2\u00A0OptEIA\u00C2\u00A0TMB\u00C2\u00A0Substrate\u00C2\u00A0Reagent\u00C2\u00A0Set;\u00C2\u00A0BD\u00C2\u00A0Biosciences,\u00C2\u00A0 Mississauga,\u00C2\u00A0ON,\u00C2\u00A0Canada)\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0plate\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A020\u00E2\u0080\u009030\u00C2\u00A0min\u00C2\u00A0 at\u00C2\u00A023\u00C2\u00B0C\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0dark.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0reaction\u00C2\u00A0was\u00C2\u00A0stopped\u00C2\u00A0with\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A02N\u00C2\u00A0H2SO4,\u00C2\u00A0the\u00C2\u00A0absorbance\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0samples\u00C2\u00A0at\u00C2\u00A0450\u00C2\u00A0nm\u00C2\u00A0determined,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0calculated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 standard\u00C2\u00A0curve.\u00C2\u00A0 \u00C2\u00A0 2.13 Tumor\u00C2\u00A0models\u00C2\u00A0 \u00C2\u00A0 4T1,\u00C2\u00A04TO7,\u00C2\u00A0 and\u00C2\u00A067NR\u00C2\u00A0murine\u00C2\u00A0mammary\u00C2\u00A0carcinoma\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0a\u00C2\u00A0kind\u00C2\u00A0gift\u00C2\u00A0 from\u00C2\u00A0 Dr.\u00C2\u00A0Fred\u00C2\u00A0Miller\u00C2\u00A0(Karmanos\u00C2\u00A0Cancer\u00C2\u00A0Institutes,\u00C2\u00A0Detroit,\u00C2\u00A0MI).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0were\u00C2\u00A0derived\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 spontaneous\u00C2\u00A0 mammary\u00C2\u00A0 tumor\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 BALB/cfC3H\u00C2\u00A0 mouse\u00C2\u00A0 (Dexter\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1978;\u00C2\u00A0 Miller\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1983)\u00C2\u00A0 and\u00C2\u00A0 represent\u00C2\u00A0 different\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0metastatic\u00C2\u00A0 propensity\u00C2\u00A0 (Aslakson\u00C2\u00A0 and\u00C2\u00A0Miller,\u00C2\u00A01992).\u00C2\u00A0\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0metastasize\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0lung,\u00C2\u00A0liver,\u00C2\u00A0bone,\u00C2\u00A0and\u00C2\u00A0brain;\u00C2\u00A04TO7\u00C2\u00A0 cells\u00C2\u00A0metastasize\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0lungs,\u00C2\u00A0but\u00C2\u00A0fail\u00C2\u00A0to\u00C2\u00A0grow\u00C2\u00A0into\u00C2\u00A0macroscopic\u00C2\u00A0metastases;\u00C2\u00A067NR\u00C2\u00A0cells\u00C2\u00A0 do\u00C2\u00A0not\u00C2\u00A0metastasize.\u00C2\u00A0\u00C2\u00A0BALB/c\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0anesthetized\u00C2\u00A0with\u00C2\u00A02%\u00C2\u00A0isoflurane\u00C2\u00A0in\u00C2\u00A0O2\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0 orthotopically\u00C2\u00A0inoculated\u00C2\u00A0with\u00C2\u00A0105\u00C2\u00A04T1\u00C2\u00A0cells,\u00C2\u00A0106\u00C2\u00A04TO7\u00C2\u00A0cells,\u00C2\u00A0or\u00C2\u00A02\u00C2\u00A0x\u00C2\u00A0105\u00C2\u00A067NR\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A050\u00C2\u00A0 45 \u00CE\u00BCl\u00C2\u00A0 PBS\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 fourth\u00C2\u00A0mammary\u00C2\u00A0 fat\u00C2\u00A0 pad.\u00C2\u00A0We\u00C2\u00A0have\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 cell\u00C2\u00A0 concentrations\u00C2\u00A0 produce\u00C2\u00A0 consistent\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 rates\u00C2\u00A0 with\u00C2\u00A0 tumor\u00C2\u00A0 volumes\u00C2\u00A0 that\u00C2\u00A0 approach\u00C2\u00A0 ethical\u00C2\u00A0 restrictions\u00C2\u00A0four\u00C2\u00A0weeks\u00C2\u00A0after\u00C2\u00A0implantation.\u00C2\u00A0\u00C2\u00A0Where\u00C2\u00A0indicated,\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0subcutaneously\u00C2\u00A0 implanted\u00C2\u00A0 with\u00C2\u00A0 5\u00C2\u00A0 mg\u00C2\u00A0 or\u00C2\u00A0 10\u00C2\u00A0 mg\u00C2\u00A0 slow\u00E2\u0080\u0090release\u00C2\u00A0 all\u00E2\u0080\u0090trans\u00C2\u00A0 retinoic\u00C2\u00A0 acid\u00C2\u00A0 (ATRA)\u00C2\u00A0 pellets\u00C2\u00A0 (Innovative\u00C2\u00A0Research\u00C2\u00A0of\u00C2\u00A0America,\u00C2\u00A0Sarasota,\u00C2\u00A0FL)\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0base\u00C2\u00A0of\u00C2\u00A0neck.\u00C2\u00A0Mice\u00C2\u00A0were\u00C2\u00A0sacrificed\u00C2\u00A0 at\u00C2\u00A0 various\u00C2\u00A0 time\u00C2\u00A0 points\u00C2\u00A0 and\u00C2\u00A0 weights\u00C2\u00A0 of\u00C2\u00A0 spleens\u00C2\u00A0 and\u00C2\u00A0 primary\u00C2\u00A0 tumors\u00C2\u00A0 were\u00C2\u00A0 measured.\u00C2\u00A0 Unless\u00C2\u00A0 otherwise\u00C2\u00A0 indicated,\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0 sacrificed\u00C2\u00A0 three\u00C2\u00A0weeks\u00C2\u00A0 post\u00E2\u0080\u0090tumor\u00C2\u00A0 implant\u00C2\u00A0 or\u00C2\u00A0 three\u00C2\u00A0weeks\u00C2\u00A0post\u00E2\u0080\u0090ATRA\u00C2\u00A0pellet\u00C2\u00A0implant.\u00C2\u00A0 \u00C2\u00A0 2.14 Serum\u00C2\u00A0treatment\u00C2\u00A0for\u00C2\u00A0MDSC\u00C2\u00A0assay\u00C2\u00A0studies\u00C2\u00A0 \u00C2\u00A0 FCS\u00C2\u00A0 was\u00C2\u00A0 untreated,\u00C2\u00A0 filtered\u00C2\u00A0 (0.22\u00C2\u00A0 \u00C2\u00B5m),\u00C2\u00A0 dialyzed\u00C2\u00A0 (3.5\u00C2\u00A0 kDa\u00C2\u00A0 (kilodalton)\u00C2\u00A0MW\u00C2\u00A0 cut\u00C2\u00A0 off),\u00C2\u00A0or\u00C2\u00A0heat\u00C2\u00A0inactivated\u00C2\u00A0(55\u00C2\u00B0C,\u00C2\u00A045\u00C2\u00A0min)\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0final\u00C2\u00A0serum\u00C2\u00A0 concentration\u00C2\u00A0of\u00C2\u00A010%.\u00C2\u00A0Albumin\u00C2\u00A0was\u00C2\u00A0removed\u00C2\u00A0from\u00C2\u00A0FCS\u00C2\u00A0using\u00C2\u00A0Affi\u00E2\u0080\u0090Gel\u00C2\u00A0Blue\u00C2\u00A0Gel\u00C2\u00A0(Bio\u00E2\u0080\u0090Rad;\u00C2\u00A0 Mississauga,\u00C2\u00A0 ON,\u00C2\u00A0 Canada)\u00C2\u00A0 affinity\u00C2\u00A0 chromatography\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions.\u00C2\u00A0 \u00C2\u00A0 2.15 ROS\u00C2\u00A0Detection\u00C2\u00A0 \u00C2\u00A0 Pulmonary\u00C2\u00A0 and\u00C2\u00A0 splenic\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 plated\u00C2\u00A0 O/N\u00C2\u00A0 in\u00C2\u00A0 6\u00E2\u0080\u0090well\u00C2\u00A0 plates\u00C2\u00A0 at\u00C2\u00A0 1\u00C2\u00A0 x\u00C2\u00A0 106\u00C2\u00A0 cells/ml\u00C2\u00A0 in\u00C2\u00A0 HL1\u00C2\u00A0medium\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 10%\u00C2\u00A0 FCS\u00C2\u00A0 or\u00C2\u00A0 3%\u00C2\u00A0 BSA.\u00C2\u00A0 The\u00C2\u00A0 next\u00C2\u00A0 day,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 harvested\u00C2\u00A0 using\u00C2\u00A01\u00C2\u00A0ml/well\u00C2\u00A0of\u00C2\u00A0cell\u00C2\u00A0dissociation\u00C2\u00A0buffer\u00C2\u00A0(Invitrogen/Gibco)\u00C2\u00A0and\u00C2\u00A0gentle\u00C2\u00A0scraping.\u00C2\u00A0Cells\u00C2\u00A0 were\u00C2\u00A0 washed\u00C2\u00A0 twice\u00C2\u00A0 with\u00C2\u00A0 PBS,\u00C2\u00A0 and\u00C2\u00A0 stained\u00C2\u00A0 with\u00C2\u00A0 20\u00C2\u00A0 \u00C2\u00B5M\u00C2\u00A0 DCFDA\u00C2\u00A0 dye\u00C2\u00A0 (Invitrogen/Molecular\u00C2\u00A0Probes)\u00C2\u00A0 in\u00C2\u00A0PBS\u00C2\u00A0 for\u00C2\u00A045\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A037\u00C2\u00B0C,\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0gently\u00C2\u00A0mixed\u00C2\u00A0 every\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0during\u00C2\u00A0this\u00C2\u00A0incubation.\u00C2\u00A0MDSCs\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0twice\u00C2\u00A0with\u00C2\u00A04\u00C2\u00B0C\u00C2\u00A0PBS\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0plated\u00C2\u00A0in\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plates\u00C2\u00A0for\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0staining,\u00C2\u00A0as\u00C2\u00A0previously\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0section\u00C2\u00A0 2.5.\u00C2\u00A0MDSCs\u00C2\u00A0were\u00C2\u00A0 stained\u00C2\u00A0 for\u00C2\u00A0 CD11b,\u00C2\u00A0 Gr1,\u00C2\u00A0 and\u00C2\u00A0PI,\u00C2\u00A0 and\u00C2\u00A0 then\u00C2\u00A0 suspended\u00C2\u00A0 for\u00C2\u00A0 analysis\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0cytometry.\u00C2\u00A0The\u00C2\u00A0fluorescent\u00C2\u00A0intensity\u00C2\u00A0of\u00C2\u00A0DCFDA\u00C2\u00A0(measured\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0FL\u00E2\u0080\u00901\u00C2\u00A0channel)\u00C2\u00A0of\u00C2\u00A0 live\u00C2\u00A0 MDSCs\u00C2\u00A0 (CD11b+Gr1+PI\u00E2\u0080\u0090)\u00C2\u00A0 was\u00C2\u00A0 determined\u00C2\u00A0 and\u00C2\u00A0 mean\u00C2\u00A0 fluorescence\u00C2\u00A0 intensity\u00C2\u00A0 (MFI)\u00C2\u00A0 recorded.\u00C2\u00A0\u00C2\u00A0 46 2.16 Clonogenic\u00C2\u00A0assays\u00C2\u00A0 \u00C2\u00A0 Monodispersed\u00C2\u00A0lung\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 ammonium\u00C2\u00A0chloride.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0in\u00C2\u00A0PBS,\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A0medium,\u00C2\u00A0and\u00C2\u00A0aliquots\u00C2\u00A0 of\u00C2\u00A0 3x103\u00C2\u00A0 to\u00C2\u00A0 106\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 plated\u00C2\u00A0 in\u00C2\u00A0 clonogenic\u00C2\u00A0 assays\u00C2\u00A0 containing\u00C2\u00A0 60\u00C2\u00B5M\u00C2\u00A0 or\u00C2\u00A0 30\u00C2\u00B5M\u00C2\u00A0 6\u00E2\u0080\u0090 thioguanine\u00C2\u00A0(for\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A04TO7\u00C2\u00A0respectively)\u00C2\u00A0or\u00C2\u00A0250\u00C2\u00B5g/ml\u00C2\u00A0geneticin\u00C2\u00A0(for\u00C2\u00A067NR).\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0 were\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A09\u00E2\u0080\u009012\u00C2\u00A0days\u00C2\u00A0(37\u00C2\u00B0C,\u00C2\u00A05%\u00C2\u00A0CO2)\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0colonies\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0malachite\u00C2\u00A0 green\u00C2\u00A0 and\u00C2\u00A0 manually\u00C2\u00A0 counted.\u00C2\u00A0 The\u00C2\u00A0 total\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0 was\u00C2\u00A0 calculated\u00C2\u00A0as\u00C2\u00A0follows:\u00C2\u00A0 \u00C2\u00A0 (Proportion\u00C2\u00A0of\u00C2\u00A0colony\u00C2\u00A0forming\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0x\u00C2\u00A0Total\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0lung\u00C2\u00A0cells)\u00C2\u00A0 \u00C2\u00A0 2.17 Immunofluorescence\u00C2\u00A0 \u00C2\u00A0 Pimonidazole\u00C2\u00A0 (100\u00C2\u00A0 mg/kg;\u00C2\u00A0 Hydroxyprobe,\u00C2\u00A0 Inc.;\u00C2\u00A0 Burlington,\u00C2\u00A0 MA)\u00C2\u00A0 was\u00C2\u00A0 injected\u00C2\u00A0 intraperitoneally\u00C2\u00A0 (IP)\u00C2\u00A0 90\u00C2\u00A0 min\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 tumor\u00C2\u00A0 excision\u00C2\u00A0 and\u00C2\u00A0 Hoecsht\u00C2\u00A0 33342\u00C2\u00A0 (500\u00C2\u00A0 \u00CE\u00BCg/mouse;\u00C2\u00A0 Sigma\u00E2\u0080\u0090Aldrich)\u00C2\u00A0 was\u00C2\u00A0 injected\u00C2\u00A0 intravenously\u00C2\u00A0 (IV)\u00C2\u00A0 20\u00C2\u00A0 min\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 tumor\u00C2\u00A0 excision.\u00C2\u00A0 Tumors\u00C2\u00A0 were\u00C2\u00A0 rinsed\u00C2\u00A0 in\u00C2\u00A0 4\u00C2\u00B0C\u00C2\u00A0 PBS\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 freezing\u00C2\u00A0 in\u00C2\u00A0 Optimal\u00C2\u00A0 Cutting\u00C2\u00A0 Temperature\u00C2\u00A0(OCT)\u00C2\u00A0medium\u00C2\u00A0(Sakura\u00C2\u00A0Finetek;\u00C2\u00A0Torrance,\u00C2\u00A0CA).\u00C2\u00A0Lungs\u00C2\u00A0were\u00C2\u00A0inflated\u00C2\u00A0with\u00C2\u00A0 300\u00C2\u00A0\u00CE\u00BCl\u00C2\u00A0 50:50\u00C2\u00A0OCT:PBS\u00C2\u00A0mixture\u00C2\u00A0 and\u00C2\u00A0 rinsed\u00C2\u00A0 in\u00C2\u00A04\u00C2\u00B0C\u00C2\u00A0PBS\u00C2\u00A0prior\u00C2\u00A0 to\u00C2\u00A0 freezing\u00C2\u00A0 in\u00C2\u00A0OCT.\u00C2\u00A0 Serial\u00C2\u00A0 sections\u00C2\u00A0 (8\u00E2\u0080\u009010\u00C2\u00A0 \u00CE\u00BCm)\u00C2\u00A0 were\u00C2\u00A0 cut\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0 cryostat\u00C2\u00A0 and\u00C2\u00A0 stained\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 appropriate\u00C2\u00A0 Abs\u00C2\u00A0 (Table\u00C2\u00A02.1)\u00C2\u00A0 in\u00C2\u00A0PBS\u00C2\u00A0 containing\u00C2\u00A04%\u00C2\u00A0calf\u00C2\u00A0 serum\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A00.1%\u00C2\u00A0Triton\u00C2\u00A0X,\u00C2\u00A0 for\u00C2\u00A0 anti\u00E2\u0080\u0090 pimonidazole\u00C2\u00A0or\u00C2\u00A0surface\u00C2\u00A0marker\u00C2\u00A0Abs,\u00C2\u00A0respectively.\u00C2\u00A0Where\u00C2\u00A0indicated,\u00C2\u00A0samples\u00C2\u00A0were\u00C2\u00A0fixed\u00C2\u00A0 with\u00C2\u00A0methanol\u00C2\u00A0and\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A04',6\u00E2\u0080\u0090diamidino\u00E2\u0080\u00902\u00E2\u0080\u0090phenylindole\u00C2\u00A0(DAPI).\u00C2\u00A0 47 2.18 Statistical\u00C2\u00A0analysis\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0are\u00C2\u00A0 represented\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 standard\u00C2\u00A0error\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0mean\u00C2\u00A0 (SEM)\u00C2\u00A0and\u00C2\u00A0all\u00C2\u00A0 experiments\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 a\u00C2\u00A0 minimum\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 times.\u00C2\u00A0 Two\u00E2\u0080\u0090tailed\u00C2\u00A0 Student\u00C2\u00A0 t\u00C2\u00A0 tests\u00C2\u00A0 were\u00C2\u00A0performed\u00C2\u00A0using\u00C2\u00A0Microsoft\u00C2\u00A0Office\u00C2\u00A0Excel\u00C2\u00A02007.\u00C2\u00A0Values\u00C2\u00A0of\u00C2\u00A0p\u00E2\u0089\u00A50.05\u00C2\u00A0were\u00C2\u00A0considered\u00C2\u00A0 not\u00C2\u00A0significant\u00C2\u00A0(NS)\u00C2\u00A0*,p<0.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<0.001\u00C2\u00A0 48 CHAPTER\u00C2\u00A03\u00C2\u00A0: TOLL\u00C2\u00ADLIKE\u00C2\u00A0RECEPTOR\u00C2\u00A0AGONISTS\u00C2\u00A0THAT\u00C2\u00A0INDUCE\u00C2\u00A0IFN\u00C2\u00AD\u00CE\u00B2\u00C2\u00A0 ABROGATE\u00C2\u00A0RESIDENT\u00C2\u00A0MACROPHAGE\u00C2\u00A0SUPPRESSION\u00C2\u00A0OF\u00C2\u00A0T\u00C2\u00A0CELLS\u00C2\u00A0 \u00C2\u00A0 3.1 Introduction\u00C2\u00A0 \u00C2\u00A0 Immune\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 function\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 are\u00C2\u00A0 critical\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 maintenance\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 homeostasis\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 prevention\u00C2\u00A0 of\u00C2\u00A0 auto\u00E2\u0080\u0090immune\u00C2\u00A0 disease.\u00C2\u00A0 Negative\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0occurs,\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0during\u00C2\u00A0development\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0thymus,\u00C2\u00A0but\u00C2\u00A0 also\u00C2\u00A0 in\u00C2\u00A0 peripheral\u00C2\u00A0 tissues\u00C2\u00A0 (Bettini\u00C2\u00A0 and\u00C2\u00A0Vignali,\u00C2\u00A0 2009),\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 lung\u00C2\u00A0 and\u00C2\u00A0PC.\u00C2\u00A0 Tregs,\u00C2\u00A0 myeloid\u00C2\u00A0DCs,\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0have\u00C2\u00A0all\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0peripheral\u00C2\u00A0tolerance\u00C2\u00A0(Bettini\u00C2\u00A0 and\u00C2\u00A0Vignali,\u00C2\u00A02009;\u00C2\u00A0Munn\u00C2\u00A0and\u00C2\u00A0Mellor,\u00C2\u00A02003);\u00C2\u00A0however,\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0as\u00C2\u00A0regulatory\u00C2\u00A0cells\u00C2\u00A0 has\u00C2\u00A0received\u00C2\u00A0little\u00C2\u00A0attention\u00C2\u00A0(Brem\u00E2\u0080\u0090Exner\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Munn\u00C2\u00A0and\u00C2\u00A0Mellor,\u00C2\u00A02003).\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 well\u00C2\u00A0 established\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 can\u00C2\u00A0 perform\u00C2\u00A0 both\u00C2\u00A0 immunogenic\u00C2\u00A0 and\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions,\u00C2\u00A0 the\u00C2\u00A0mechanisms\u00C2\u00A0 that\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0promote\u00C2\u00A0 or\u00C2\u00A0 inhibit\u00C2\u00A0 a\u00C2\u00A0 particular\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 well\u00C2\u00A0 understood.\u00C2\u00A0 Resting,\u00C2\u00A0 or\u00C2\u00A0 non\u00E2\u0080\u0090 activated,\u00C2\u00A0tissue\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0continually\u00C2\u00A0survey\u00C2\u00A0the\u00C2\u00A0microenvironment,\u00C2\u00A0ingesting\u00C2\u00A0large\u00C2\u00A0amounts\u00C2\u00A0 of\u00C2\u00A0extracellular\u00C2\u00A0 fluid,\u00C2\u00A0apoptotic\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0cellular\u00C2\u00A0debris,\u00C2\u00A0which\u00C2\u00A0they\u00C2\u00A0display\u00C2\u00A0as\u00C2\u00A0Ags\u00C2\u00A0on\u00C2\u00A0 their\u00C2\u00A0 surface\u00C2\u00A0 for\u00C2\u00A0 recognition\u00C2\u00A0by\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0 (Gordon\u00C2\u00A0and\u00C2\u00A0Martinez,\u00C2\u00A02010).\u00C2\u00A0 Since\u00C2\u00A0 these\u00C2\u00A0Ags\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0derived\u00C2\u00A0from\u00C2\u00A0either\u00C2\u00A0host\u00C2\u00A0cells\u00C2\u00A0(i.e.\u00C2\u00A0self\u00E2\u0080\u0090Ags)\u00C2\u00A0or\u00C2\u00A0pathogens,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0must\u00C2\u00A0be\u00C2\u00A0able\u00C2\u00A0 to\u00C2\u00A0 distinguish\u00C2\u00A0 whether\u00C2\u00A0 a\u00C2\u00A0 particular\u00C2\u00A0 Ag\u00C2\u00A0 should\u00C2\u00A0 provoke\u00C2\u00A0 an\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 or\u00C2\u00A0 be\u00C2\u00A0 tolerated.\u00C2\u00A0Therefore,\u00C2\u00A0we\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 activation;\u00C2\u00A0we\u00C2\u00A0determined\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0proliferation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0pathogens,\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0explored\u00C2\u00A0the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0 pathogen\u00E2\u0080\u0090derived\u00C2\u00A0 molecules\u00C2\u00A0 on\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression.\u00C2\u00A0 In\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0 we\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 secreted\u00C2\u00A0 by\u00C2\u00A0 TCR\u00E2\u0080\u0090activated\u00C2\u00A0 T\u00C2\u00A0 cells,\u00C2\u00A0 resident\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 acquire\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 mediated\u00C2\u00A0 by\u00C2\u00A0 NO.\u00C2\u00A0 Furthermore,\u00C2\u00A0we\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0agonists\u00C2\u00A0that\u00C2\u00A0activate\u00C2\u00A0TRIF\u00C2\u00A0(i.e.\u00C2\u00A0LPS\u00C2\u00A0 and\u00C2\u00A0dsRNA),\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0 those\u00C2\u00A0 that\u00C2\u00A0exclusively\u00C2\u00A0 signal\u00C2\u00A0 through\u00C2\u00A0MyD88\u00C2\u00A0(i.e.\u00C2\u00A0CpG\u00C2\u00A0and\u00C2\u00A0PGN),\u00C2\u00A0 eliminates\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0in\u00C2\u00A0part\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2.\u00C2\u00A0These\u00C2\u00A0 49 data\u00C2\u00A0further\u00C2\u00A0our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0how\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0tolerance\u00C2\u00A0and\u00C2\u00A0suggest\u00C2\u00A0a\u00C2\u00A0 novel\u00C2\u00A0role\u00C2\u00A0for\u00C2\u00A0TLR\u00C2\u00A0signaling\u00C2\u00A0and\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 in\u00C2\u00A0regulating\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0functions\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 This\u00C2\u00A0 thesis\u00C2\u00A0 chapter\u00C2\u00A0 is\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 previously\u00C2\u00A0 published\u00C2\u00A0 work\u00C2\u00A0 (Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010)\u00C2\u00A0 and\u00C2\u00A0 is\u00C2\u00A0 reprinted\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 permission\u00C2\u00A0 of\u00C2\u00A0 The\u00C2\u00A0 American\u00C2\u00A0 Association\u00C2\u00A0 of\u00C2\u00A0 Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 \u00C2\u00A0 3.2 Results\u00C2\u00A0 \u00C2\u00A0 3.2.1 Characterization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0PC\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0it\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0that\u00C2\u00A0negative\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0peripheral\u00C2\u00A0 tissues\u00C2\u00A0(Bettini\u00C2\u00A0and\u00C2\u00A0Vignali,\u00C2\u00A02009)\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0reside\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0PC\u00C2\u00A0(Shiku\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 1975),\u00C2\u00A0we\u00C2\u00A0hypothesized\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0PC\u00C2\u00A0may\u00C2\u00A0contain\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0possess\u00C2\u00A0immunosuppressive\u00C2\u00A0 properties.\u00C2\u00A0To\u00C2\u00A0test\u00C2\u00A0this,\u00C2\u00A0we\u00C2\u00A0isolated\u00C2\u00A0the\u00C2\u00A0PC\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0 present\u00C2\u00A0 and\u00C2\u00A0 their\u00C2\u00A0 relative\u00C2\u00A0proportions\u00C2\u00A0by\u00C2\u00A0 flow\u00C2\u00A0cytometry.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Fig.\u00C2\u00A0 3.1A,\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0abundant\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0CD11b+\u00C2\u00A0cells,\u00C2\u00A0 followed\u00C2\u00A0by\u00C2\u00A0DCs,\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0mast\u00C2\u00A0 cells.\u00C2\u00A0To\u00C2\u00A0 see\u00C2\u00A0 if\u00C2\u00A0 any\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0 could\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0proliferation,\u00C2\u00A0we\u00C2\u00A0 separated\u00C2\u00A0PC\u00C2\u00A0 cells\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 adherent\u00C2\u00A0 properties\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 adherent\u00C2\u00A0 cells\u00C2\u00A0 significantly\u00C2\u00A0 suppressed\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0while\u00C2\u00A0 non\u00E2\u0080\u0090adherent\u00C2\u00A0 cells\u00C2\u00A0 activated\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 (Fig.\u00C2\u00A0 3.1B).\u00C2\u00A0 Analysis\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 adherent\u00C2\u00A0 PC\u00C2\u00A0 cells\u00C2\u00A0 revealed\u00C2\u00A0 they\u00C2\u00A0were\u00C2\u00A0 exclusively\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0 co\u00E2\u0080\u0090 expressing\u00C2\u00A0 F4/80\u00C2\u00A0 and\u00C2\u00A0 CD11b\u00C2\u00A0 (Fig.\u00C2\u00A0 3.1C,\u00C2\u00A0 left)\u00C2\u00A0while\u00C2\u00A0 lacking\u00C2\u00A0 Gr1\u00C2\u00A0 (Fig.\u00C2\u00A0 3.1C,\u00C2\u00A0 right)\u00C2\u00A0 and\u00C2\u00A0 other\u00C2\u00A0non\u00E2\u0080\u0090M\u00CF\u0086\u00C2\u00A0lineage\u00C2\u00A0markers\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0To\u00C2\u00A0verify\u00C2\u00A0that\u00C2\u00A0these\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0indeed\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 suppression,\u00C2\u00A0 we\u00C2\u00A0 isolated\u00C2\u00A0 F4/80+\u00C2\u00A0 or\u00C2\u00A0 CD11b+\u00C2\u00A0 cells\u00C2\u00A0 by\u00C2\u00A0 positive\u00C2\u00A0 selection\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0effectively\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 to\u00C2\u00A0an\u00C2\u00A0equal\u00C2\u00A0extent\u00C2\u00A0(Fig.\u00C2\u00A03.1D).\u00C2\u00A0 50 3.2.2 Resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0phenotype\u00C2\u00A0 \u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0can\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0phenotypes\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0activation\u00C2\u00A0state,\u00C2\u00A0from\u00C2\u00A0 classically\u00C2\u00A0 activated,\u00C2\u00A0 M1,\u00C2\u00A0 to\u00C2\u00A0 alternatively\u00C2\u00A0 activated\u00C2\u00A0 M2a,\u00C2\u00A0 b,\u00C2\u00A0 or\u00C2\u00A0 c\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 phenotypes\u00C2\u00A0 (Martinez\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0We\u00C2\u00A0performed\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0 analysis\u00C2\u00A0on\u00C2\u00A0 lysates\u00C2\u00A0 from\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 their\u00C2\u00A0 activation\u00C2\u00A0 state\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 lacked\u00C2\u00A0 features\u00C2\u00A0of\u00C2\u00A0 either\u00C2\u00A0M1,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0iNOS\u00C2\u00A0expression,\u00C2\u00A0or\u00C2\u00A0M2\u00C2\u00A0activation,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0Arg1\u00C2\u00A0or\u00C2\u00A0Ym1\u00C2\u00A0expression\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.1\u00C2\u00A0 Resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0possess\u00C2\u00A0immunosuppressive\u00C2\u00A0properties.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0Bulk\u00C2\u00A0PC\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0fluorescent\u00C2\u00A0Abs\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 proportions\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 expressing\u00C2\u00A0 the\u00C2\u00A0 lineage\u00C2\u00A0 markers\u00C2\u00A0 B220\u00C2\u00A0 (B\u00C2\u00A0 cells),\u00C2\u00A0 CD11b\u00C2\u00A0 (myeloid\u00C2\u00A0cells),\u00C2\u00A0CD11c\u00C2\u00A0(DCs),\u00C2\u00A0CD3\u00C2\u00A0(T\u00C2\u00A0cells),\u00C2\u00A0and\u00C2\u00A0Fc\u00CE\u00B5R\u00C2\u00A0(mast\u00C2\u00A0cells).\u00C2\u00A0B,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 (white\u00C2\u00A0 bar)\u00C2\u00A0 adherent\u00C2\u00A0 or\u00C2\u00A0 non\u00E2\u0080\u0090 adherent\u00C2\u00A0PC\u00C2\u00A0cells\u00C2\u00A0(1\u00C2\u00A0PC\u00C2\u00A0cell:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0C,\u00C2\u00A0Adherent\u00C2\u00A0PC\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0fluorescent\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0F4/80\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0Gr1\u00C2\u00A0(right)\u00C2\u00A0 and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0The\u00C2\u00A0proportions\u00C2\u00A0of\u00C2\u00A0CD11b+F4/80+\u00C2\u00A0and\u00C2\u00A0CD11b+Gr1\u00E2\u0080\u0090\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0 indicated.\u00C2\u00A0D,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0 (white\u00C2\u00A0 bar)\u00C2\u00A0 F4/80+\u00C2\u00A0 or\u00C2\u00A0 CD11b+\u00C2\u00A0 PC\u00C2\u00A0 cells\u00C2\u00A0 (1\u00C2\u00A0 PC\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes)\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0\u00C2\u00A0Assays\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate\u00C2\u00A0(A,\u00C2\u00A0C)\u00C2\u00A0or\u00C2\u00A0triplicate\u00C2\u00A0(B,\u00C2\u00A0D).\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 (A)\u00C2\u00A0 or\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 (B,\u00C2\u00A0 C,\u00C2\u00A0 D).\u00C2\u00A0 *,p<\u00C2\u00A0 0.05;\u00C2\u00A0 **,p<0.01;\u00C2\u00A0 ***,p<0.001\u00C2\u00A0 relative\u00C2\u00A0 to\u00C2\u00A0 responder\u00C2\u00A0 control\u00C2\u00A0 (RC;\u00C2\u00A0 stimulated\u00C2\u00A0splenocytes\u00C2\u00A0alone).\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0 American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 \u00C2\u00A0 51 (Fig.\u00C2\u00A0 3.2,\u00C2\u00A0 left).\u00C2\u00A0 However,\u00C2\u00A0 these\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 could\u00C2\u00A0 become\u00C2\u00A0 either\u00C2\u00A0 M1\u00C2\u00A0 or\u00C2\u00A0 M2\u00C2\u00A0 polarized\u00C2\u00A0 with\u00C2\u00A0 appropriate\u00C2\u00A0 stimulation\u00C2\u00A0 (IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 +\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 IL\u00E2\u0080\u00904\u00C2\u00A0 +\u00C2\u00A0 IL\u00E2\u0080\u009013,\u00C2\u00A0 respectively)\u00C2\u00A0 (Fig.\u00C2\u00A0 3.2,\u00C2\u00A0 right),\u00C2\u00A0 confirming\u00C2\u00A0the\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0(non\u00E2\u0080\u0090activated)\u00C2\u00A0state\u00C2\u00A0of\u00C2\u00A0resident\u00C2\u00A0PM\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.3 Resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0and\u00C2\u00A0cytokine\u00C2\u00A0production\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 further\u00C2\u00A0 evaluate\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0we\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 resident\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 C57/Bl6\u00C2\u00A0 mice\u00C2\u00A0 with\u00C2\u00A0 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 potently\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0either\u00C2\u00A0polyclonal\u00C2\u00A0or\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00C2\u00A0 stimulation\u00C2\u00A0of\u00C2\u00A0 CD4+\u00C2\u00A0or\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0dose\u00E2\u0080\u0090dependent\u00C2\u00A0manner\u00C2\u00A0 (Fig.\u00C2\u00A0 3.3A).\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0 conducted\u00C2\u00A0these\u00C2\u00A0studies\u00C2\u00A0using\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0BALB/c\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0obtained\u00C2\u00A0similar\u00C2\u00A0results\u00C2\u00A0(Fig.\u00C2\u00A0 3.3B).\u00C2\u00A0Next,\u00C2\u00A0we\u00C2\u00A0 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Figure\u00C2\u00A03.3\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 and\u00C2\u00A0 cytokine\u00C2\u00A0 production\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 dose\u00C2\u00AD dependent\u00C2\u00A0manner.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 anti\u00E2\u0080\u0090CD3/anti\u00E2\u0080\u0090CD28\u00E2\u0080\u0090stimulated\u00C2\u00A0 C57BL/6\u00C2\u00A0 (Polyclonal)\u00C2\u00A0 or\u00C2\u00A0 OVA\u00E2\u0080\u0090peptide\u00E2\u0080\u0090stimulated\u00C2\u00A0 OTII\u00C2\u00A0 (CD4+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 Ag\u00E2\u0080\u0090specific)\u00C2\u00A0 or\u00C2\u00A0 OTI\u00C2\u00A0 (CD8+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 Ag\u00E2\u0080\u0090specific)\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0ratios\u00C2\u00A0(M\u00CF\u0086s:splenocytes;\u00C2\u00A01:2,\u00C2\u00A01:8,\u00C2\u00A01:32)\u00C2\u00A0 and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0\u00C2\u00A0Significance\u00C2\u00A0is\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0RC.\u00C2\u00A0B,\u00C2\u00A0Responder\u00C2\u00A0splenocytes\u00C2\u00A0 (polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0BALB/c\u00C2\u00A0 (Polyclonal)\u00C2\u00A0or\u00C2\u00A0OVA\u00E2\u0080\u0090peptide\u00E2\u0080\u0090stimulated\u00C2\u00A0DO11.10\u00C2\u00A0 (CD4+\u00C2\u00A0Ag\u00E2\u0080\u0090 specific))\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0without\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 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concentrations\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0(left),\u00C2\u00A0IL\u00E2\u0080\u009010\u00C2\u00A0(middle),\u00C2\u00A0and\u00C2\u00A0IL\u00E2\u0080\u00902\u00C2\u00A0(right)\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0of\u00C2\u00A0 2\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 assayed\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 (A,\u00C2\u00A0 B)\u00C2\u00A0 or\u00C2\u00A0 duplicate\u00C2\u00A0 (C).\u00C2\u00A0 *,p<\u00C2\u00A00.05;\u00C2\u00A0 **,p<0.01;\u00C2\u00A0 ***,p<0.001.\u00C2\u00A0 Reprinted\u00C2\u00A0 from\u00C2\u00A0 Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010\u00C2\u00A0 with\u00C2\u00A0 permission\u00C2\u00A0 of\u00C2\u00A0 The\u00C2\u00A0 American Association of Immunologists, Inc. 54 \u00C2\u00A0 \u00C2\u00A0 3.2.4 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0contact\u00C2\u00ADdependent\u00C2\u00A0mechanism\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 elucidate\u00C2\u00A0 the\u00C2\u00A0 mechanism(s)\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 suppressing\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 we\u00C2\u00A0 utilized\u00C2\u00A0 a\u00C2\u00A0 panel\u00C2\u00A0 of\u00C2\u00A0 blocking\u00C2\u00A0 Abs,\u00C2\u00A0 inhibitors,\u00C2\u00A0 and\u00C2\u00A0 exogenous\u00C2\u00A0 cytokines.\u00C2\u00A0 Each\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 reagents\u00C2\u00A0 tested\u00C2\u00A0were\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0biologically\u00C2\u00A0active\u00C2\u00A0 in\u00C2\u00A0our\u00C2\u00A0hands\u00C2\u00A0 in\u00C2\u00A0several\u00C2\u00A0different\u00C2\u00A0 systems\u00C2\u00A0(Alcon\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Kuroda\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Mace\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0 Rauh\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005;\u00C2\u00A0 Sly\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009;\u00C2\u00A0 Sly\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008,\u00C2\u00A0 and\u00C2\u00A0unpublished\u00C2\u00A0 results).\u00C2\u00A0We\u00C2\u00A0 first\u00C2\u00A0 determined\u00C2\u00A0whether\u00C2\u00A0cytokines\u00C2\u00A0that\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0to\u00C2\u00A0alter\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0phenotype\u00C2\u00A0(i.e.\u00C2\u00A0IL\u00E2\u0080\u00904,\u00C2\u00A0 IL\u00E2\u0080\u009013)\u00C2\u00A0(Rodriguez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003)\u00C2\u00A0or\u00C2\u00A0restrict\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0(i.e.\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2)\u00C2\u00A0(Levings\u00C2\u00A0 and\u00C2\u00A0Roncarolo,\u00C2\u00A02000)\u00C2\u00A0were\u00C2\u00A0 involved.\u00C2\u00A0Adding\u00C2\u00A0neutralizing\u00C2\u00A0Abs\u00C2\u00A0 to\u00C2\u00A0 IL\u00E2\u0080\u00904,\u00C2\u00A0 IL\u00E2\u0080\u009010,\u00C2\u00A0 IL\u00E2\u0080\u009013,\u00C2\u00A0or\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 reverse\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 cytokines\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0 involved\u00C2\u00A0 (Fig.\u00C2\u00A0 3.5A).\u00C2\u00A0 To\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 acting\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 sink\u00C2\u00A0for\u00C2\u00A0IL\u00E2\u0080\u00902\u00C2\u00A0(von\u00C2\u00A0Bergwelt\u00E2\u0080\u0090Baildon\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006)\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0restricting\u00C2\u00A0the\u00C2\u00A0availability\u00C2\u00A0of\u00C2\u00A0 IL\u00E2\u0080\u00902\u00C2\u00A0 for\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation,\u00C2\u00A0we\u00C2\u00A0added\u00C2\u00A0exogenous\u00C2\u00A0IL\u00E2\u0080\u00902\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0cultures,\u00C2\u00A0but\u00C2\u00A0 this\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 reverse\u00C2\u00A0 suppression\u00C2\u00A0 (Fig.\u00C2\u00A0 3.5A),\u00C2\u00A0 which\u00C2\u00A0 was\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 our\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 increased\u00C2\u00A0 IL\u00E2\u0080\u00902\u00C2\u00A0 levels\u00C2\u00A0 (Fig.\u00C2\u00A0 3.3C,\u00C2\u00A0 right).\u00C2\u00A0 Since\u00C2\u00A0 PGs\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 PGE2\u00C2\u00A0 have\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.4\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0increase\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0dead\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090\u00C2\u00A0 (Poly)\u00C2\u00A0or\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0 (OTI\u00C2\u00A0or\u00C2\u00A0OTII)\u00C2\u00A0 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0with\u00C2\u00A0 (black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0and\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0 of\u00C2\u00A0dead\u00C2\u00A0(PI+)\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0apoptotic\u00C2\u00A0(Annexin\u00C2\u00A0V+\u00C2\u00A0PI\u00E2\u0080\u0090)\u00C2\u00A0 (right)\u00C2\u00A0CD4+\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0assessed\u00C2\u00A0by\u00C2\u00A0 flow\u00C2\u00A0cytometry.\u00C2\u00A0Data\u00C2\u00A0shown\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A02\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0assayed\u00C2\u00A0in\u00C2\u00A0 duplicate.\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 55 been\u00C2\u00A0 implicated\u00C2\u00A0 in\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 (Allison,\u00C2\u00A01978;\u00C2\u00A0Metzger\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01980),\u00C2\u00A0we\u00C2\u00A0 tested\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 blocking\u00C2\u00A0 PGE2\u00C2\u00A0 synthesis.\u00C2\u00A0 However,\u00C2\u00A0 adding\u00C2\u00A0 the\u00C2\u00A0 COX\u00E2\u0080\u00902\u00C2\u00A0 inhibitor\u00C2\u00A0 Celebrex\u00C2\u00A0to\u00C2\u00A0co\u00E2\u0080\u0090cultures\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0alter\u00C2\u00A0suppression\u00C2\u00A0(Fig.\u00C2\u00A03.5A),\u00C2\u00A0which\u00C2\u00A0suggested\u00C2\u00A0PGE2\u00C2\u00A0was\u00C2\u00A0 not\u00C2\u00A0involved.\u00C2\u00A0Next,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0whether\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0suppressing\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0by\u00C2\u00A0an\u00C2\u00A0 arginine\u00C2\u00A0dependent\u00C2\u00A0mechanism.\u00C2\u00A0The\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0is\u00C2\u00A0necessary\u00C2\u00A0for\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0functions\u00C2\u00A0 and\u00C2\u00A0 arginine\u00C2\u00A0 depletion\u00C2\u00A0 inhibits\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 through\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 mechanisms,\u00C2\u00A0 including\u00C2\u00A0 preventing\u00C2\u00A0 re\u00E2\u0080\u0090expression\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 CD3\u00CE\u00B6\u00E2\u0080\u0090chain\u00C2\u00A0 (Rodriguez\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2002;\u00C2\u00A0Rodriguez\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003).\u00C2\u00A0However,\u00C2\u00A0adding\u00C2\u00A0exogenous\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0abrogate\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 (Fig.\u00C2\u00A0 3.5A).\u00C2\u00A0 Consistent\u00C2\u00A0 with\u00C2\u00A0 this\u00C2\u00A0 result,\u00C2\u00A0 inhibiting\u00C2\u00A0 Arg1,\u00C2\u00A0 which\u00C2\u00A0 converts\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 to\u00C2\u00A0 urea\u00C2\u00A0 and\u00C2\u00A0 L\u00E2\u0080\u0090ornithine,\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 inhibitor\u00C2\u00A0 BEC\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 reverse\u00C2\u00A0 suppression;\u00C2\u00A0 in\u00C2\u00A0 fact,\u00C2\u00A0 BEC\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 3.5A).\u00C2\u00A0 In\u00C2\u00A0 addition,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0whether\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0suppressing\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0indirectly\u00C2\u00A0via\u00C2\u00A0Treg\u00C2\u00A0 induction.\u00C2\u00A0However,\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0significantly\u00C2\u00A0 increase\u00C2\u00A0 the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0Tregs\u00C2\u00A0(Fig.\u00C2\u00A03.5B).\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 none\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 soluble\u00E2\u0080\u0090mediated\u00C2\u00A0 mechanisms\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 appeared\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 involved\u00C2\u00A0in\u00C2\u00A0M\u00CF\u0086\u00E2\u0080\u0090mediated\u00C2\u00A0immunosuppression,\u00C2\u00A0we\u00C2\u00A0asked\u00C2\u00A0whether\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0might\u00C2\u00A0 be\u00C2\u00A0contact\u00E2\u0080\u0090dependent.\u00C2\u00A0 \u00C2\u00A0To\u00C2\u00A0test\u00C2\u00A0this,\u00C2\u00A0we\u00C2\u00A0assessed\u00C2\u00A0proliferation\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0splenocytes\u00C2\u00A0 using\u00C2\u00A0a\u00C2\u00A0Transwell\u00C2\u00A0system.\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A03.5C,\u00C2\u00A0when\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0responder\u00C2\u00A0splenocytes\u00C2\u00A0 were\u00C2\u00A0 separated\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 semi\u00E2\u0080\u0090permeable\u00C2\u00A0 membrane,\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 suppression\u00C2\u00A0 was\u00C2\u00A0 abrogated,\u00C2\u00A0 suggesting\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0contact\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism.\u00C2\u00A0 \u00C2\u00A0 56 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.5\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0contact\u00C2\u00ADdependent\u00C2\u00A0mechanism.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010\u00C2\u00A0 \u00CE\u00BCg/ml\u00C2\u00A0rat\u00C2\u00A0 IgG,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u00904,\u00C2\u00A02\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u009010,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u009013,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090 TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0100\u00C2\u00A0U/well\u00C2\u00A0mIL\u00E2\u0080\u00902,\u00C2\u00A020\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0Celebrex,\u00C2\u00A02\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090arg,\u00C2\u00A0or\u00C2\u00A0200\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0BEC.\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0Significance\u00C2\u00A0is\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0 (\u00E2\u0080\u0090)\u00C2\u00A0co\u00E2\u0080\u0090cultures,\u00C2\u00A0where\u00C2\u00A0no\u00C2\u00A0inhibitors\u00C2\u00A0were\u00C2\u00A0added.\u00C2\u00A0B,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bar)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bar)\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes).\u00C2\u00A0After\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0the\u00C2\u00A0 proportion\u00C2\u00A0 of\u00C2\u00A0 Tregs\u00C2\u00A0 (CD4+Foxp3+\u00C2\u00A0 cells)\u00C2\u00A0was\u00C2\u00A0 analyzed\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0C,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 co\u00E2\u0080\u0090 cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A02\u00C2\u00A0splenocytes)\u00C2\u00A0 in\u00C2\u00A0control\u00C2\u00A0 (M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0 splenocytes\u00C2\u00A0 in\u00C2\u00A0contact)\u00C2\u00A0or\u00C2\u00A0Transwell\u00C2\u00A0 (M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0splenocytes\u00C2\u00A0separated\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0semi\u00E2\u0080\u0090permeable\u00C2\u00A0 membrane)\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plates.\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0 calculated.\u00C2\u00A0Data\u00C2\u00A0is\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate\u00C2\u00A0 (A,\u00C2\u00A0C)\u00C2\u00A0or\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0 ***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0 American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 57 3.2.5 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0IFN\u00C2\u00AD\u00CE\u00B3\u00C2\u00ADinduced\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 direct\u00C2\u00A0 contact\u00C2\u00A0 between\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 was\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 effective\u00C2\u00A0 suppression,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0whether\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0either\u00C2\u00A0by\u00C2\u00A0activating\u00C2\u00A0CTLA\u00E2\u0080\u00904,\u00C2\u00A0a\u00C2\u00A0 negative\u00C2\u00A0 co\u00E2\u0080\u0090stimulatory\u00C2\u00A0 molecule\u00C2\u00A0 found\u00C2\u00A0 on\u00C2\u00A0 T\u00C2\u00A0 cells,\u00C2\u00A0 by\u00C2\u00A0 producing\u00C2\u00A0 membrane\u00E2\u0080\u0090bound\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 or\u00C2\u00A0 via\u00C2\u00A0 integrin\u00E2\u0080\u0090mediated\u00C2\u00A0 cell\u00E2\u0080\u0090cell\u00C2\u00A0 interactions.\u00C2\u00A0 However,\u00C2\u00A0 suppression\u00C2\u00A0 was\u00C2\u00A0 not\u00C2\u00A0 reversed\u00C2\u00A0by\u00C2\u00A0adding\u00C2\u00A0a\u00C2\u00A0neutralizing\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0CTLA\u00E2\u0080\u00904,\u00C2\u00A0LAP,\u00C2\u00A0to\u00C2\u00A0bind\u00C2\u00A0and\u00C2\u00A0neutralize\u00C2\u00A0bioactive\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0membrane\u00C2\u00A0 (Gandhi\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007),\u00C2\u00A0 or\u00C2\u00A0 TIB218,\u00C2\u00A0 to\u00C2\u00A0 block\u00C2\u00A0 CD11b/LFA\u00E2\u0080\u00901\u00C2\u00A0 (Fig.\u00C2\u00A03.6A,\u00C2\u00A0left).\u00C2\u00A0Next,\u00C2\u00A0we\u00C2\u00A0investigated\u00C2\u00A0whether\u00C2\u00A0ROS\u00C2\u00A0or\u00C2\u00A0RNS\u00C2\u00A0were\u00C2\u00A0involved.\u00C2\u00A0Although\u00C2\u00A0ROS\u00C2\u00A0 and\u00C2\u00A0RNS\u00C2\u00A0are\u00C2\u00A0soluble\u00C2\u00A0mediators,\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0extremely\u00C2\u00A0short\u00E2\u0080\u0090lived\u00C2\u00A0and\u00C2\u00A0require\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0in\u00C2\u00A0 close\u00C2\u00A0 proximity\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 effects\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 transmitted.\u00C2\u00A0 Addition\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 ROS\u00C2\u00A0 inhibitors\u00C2\u00A0 catalase,\u00C2\u00A0 N\u00E2\u0080\u0090acetyl\u00E2\u0080\u0090L\u00E2\u0080\u0090cysteine\u00C2\u00A0 (NAC),\u00C2\u00A0 and\u00C2\u00A0 superoxide\u00C2\u00A0 dismutase\u00C2\u00A0 (SOD)\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 reverse\u00C2\u00A0 suppression;\u00C2\u00A0 in\u00C2\u00A0 fact,\u00C2\u00A0 catalase\u00C2\u00A0 and\u00C2\u00A0 NAC\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 3.6A,\u00C2\u00A0 right).\u00C2\u00A0 To\u00C2\u00A0 elucidate\u00C2\u00A0 whether\u00C2\u00A0 RNS\u00C2\u00A0 were\u00C2\u00A0 playing\u00C2\u00A0 a\u00C2\u00A0 role,\u00C2\u00A0 we\u00C2\u00A0 first\u00C2\u00A0 assayed\u00C2\u00A0 for\u00C2\u00A0 production\u00C2\u00A0of\u00C2\u00A0NO,\u00C2\u00A0a\u00C2\u00A0free\u00C2\u00A0radical\u00C2\u00A0gas\u00C2\u00A0that\u00C2\u00A0has\u00C2\u00A0cytostatic/cytotoxic\u00C2\u00A0effects\u00C2\u00A0(Bogdan,\u00C2\u00A02001).\u00C2\u00A0 We\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 produced\u00C2\u00A0 NO\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 dose\u00E2\u0080\u0090dependent\u00C2\u00A0 manner\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 activated\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0 (Fig.\u00C2\u00A03.6B\u00C2\u00A0and\u00C2\u00A0C).\u00C2\u00A0The\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0 iNOS\u00C2\u00A0 inhibitors\u00C2\u00A0NG\u00E2\u0080\u0090monomethyl\u00E2\u0080\u0090L\u00E2\u0080\u0090 arginine\u00C2\u00A0 (L\u00E2\u0080\u0090NMMA)\u00C2\u00A0 and\u00C2\u00A0 L\u00E2\u0080\u0090NIL,\u00C2\u00A0 or\u00C2\u00A0 the\u00C2\u00A0 NO\u00C2\u00A0 scavenger\u00C2\u00A0 PTIO,\u00C2\u00A0 to\u00C2\u00A0 co\u00E2\u0080\u0090cultures\u00C2\u00A0 effectively\u00C2\u00A0 reduced\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 (Fig.\u00C2\u00A0 3.6D,\u00C2\u00A0 left)\u00C2\u00A0 and\u00C2\u00A0 completely\u00C2\u00A0 abrogated\u00C2\u00A0 the\u00C2\u00A0 inhibitory\u00C2\u00A0 effect\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0(Fig.\u00C2\u00A03.6D,\u00C2\u00A0right),\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0 in\u00C2\u00A0large\u00C2\u00A0part\u00C2\u00A0via\u00C2\u00A0NO\u00C2\u00A0production.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 58 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.6\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0through\u00C2\u00A0NO\u00C2\u00A0production.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010\u00C2\u00A0 \u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090CTLA4,\u00C2\u00A0250\u00C2\u00A0ng/ml\u00C2\u00A0LAP,\u00C2\u00A0or\u00C2\u00A05\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0TIB218\u00C2\u00A0(left),\u00C2\u00A0or\u00C2\u00A0\u00C2\u00B1\u00C2\u00A01\u00C2\u00A0mg/ml\u00C2\u00A0catalase,\u00C2\u00A010\u00C2\u00A0mM\u00C2\u00A0 NAC,\u00C2\u00A0 or\u00C2\u00A0 200\u00C2\u00A0 U/ml\u00C2\u00A0 SOD\u00C2\u00A0 (right).\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Significance\u00C2\u00A0 is\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 control\u00C2\u00A0 (\u00E2\u0080\u0090)\u00C2\u00A0 co\u00E2\u0080\u0090cultures,\u00C2\u00A0 where\u00C2\u00A0 no\u00C2\u00A0 inhibitors\u00C2\u00A0 were\u00C2\u00A0 added.\u00C2\u00A0 B,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 alone\u00C2\u00A0 or\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0ratios\u00C2\u00A0(M\u00CF\u0086s:splenocytes;\u00C2\u00A01:4,\u00C2\u00A01:8,\u00C2\u00A01:16)\u00C2\u00A0for\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0 measured.\u00C2\u00A0 C,\u00C2\u00A0 Responder\u00C2\u00A0 splenocytes\u00C2\u00A0 (polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 C57BL/6\u00C2\u00A0 or\u00C2\u00A0 OVA\u00E2\u0080\u0090peptide\u00E2\u0080\u0090 stimulated\u00C2\u00A0 OTI\u00C2\u00A0 or\u00C2\u00A0 OTII)\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 ratios\u00C2\u00A0 (M\u00CF\u0086s:splenocytes;\u00C2\u00A0 1:4,\u00C2\u00A0 1:8,\u00C2\u00A0 1:16,\u00C2\u00A0 1:32).\u00C2\u00A0 After\u00C2\u00A0 72\u00C2\u00A0 h,\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 was\u00C2\u00A0 measured.\u00C2\u00A0 D,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 M\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0 splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.5\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090NMMA,\u00C2\u00A01\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090NIL,\u00C2\u00A0or\u00C2\u00A025\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0PTIO.\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0(left)\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 were\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated\u00C2\u00A0 (right).\u00C2\u00A0 Significance\u00C2\u00A0is\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0(\u00E2\u0080\u0090)\u00C2\u00A0co\u00E2\u0080\u0090cultures,\u00C2\u00A0where\u00C2\u00A0no\u00C2\u00A0inhibitors\u00C2\u00A0were\u00C2\u00A0added.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0 representative\u00C2\u00A0of\u00C2\u00A0 three\u00C2\u00A0 to\u00C2\u00A0 five\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 *,p<\u00C2\u00A00.05;\u00C2\u00A0 **,p<0.01;\u00C2\u00A0 ***,p<0.001;\u00C2\u00A0 NS,\u00C2\u00A0 not\u00C2\u00A0 significant.\u00C2\u00A0 Reprinted\u00C2\u00A0 from\u00C2\u00A0 Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010\u00C2\u00A0 with\u00C2\u00A0 permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0\u00C2\u00A0 59 Since\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 express\u00C2\u00A0 iNOS\u00C2\u00A0 (Fig.\u00C2\u00A0 3.2)\u00C2\u00A0 or\u00C2\u00A0 produce\u00C2\u00A0 NO\u00C2\u00A0 (Fig.\u00C2\u00A0 3.6B)\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0hypothesized\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0factor\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 was\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0NO\u00C2\u00A0production.\u00C2\u00A0Since\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 is\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0iNOS\u00C2\u00A0in\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 via\u00C2\u00A0Janus\u00C2\u00A0kinase\u00C2\u00A0(JAK)/signal\u00C2\u00A0transducer\u00C2\u00A0and\u00C2\u00A0activator\u00C2\u00A0of\u00C2\u00A0transcription\u00C2\u00A0(STAT)\u00C2\u00A0signaling\u00C2\u00A0 (Schroder\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004),\u00C2\u00A0we\u00C2\u00A0added\u00C2\u00A0an\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090neutralizing\u00C2\u00A0Ab\u00C2\u00A0during\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0 stimulated\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 this\u00C2\u00A0 eliminated\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 (Fig.\u00C2\u00A0 3.7,\u00C2\u00A0 left)\u00C2\u00A0 and\u00C2\u00A0 restored\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferative\u00C2\u00A0ability\u00C2\u00A0(Fig.\u00C2\u00A03.7,\u00C2\u00A0right).\u00C2\u00A0Taken\u00C2\u00A0together,\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0indicate\u00C2\u00A0 that\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0 induces\u00C2\u00A0na\u00C3\u00AFve,\u00C2\u00A0resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0express\u00C2\u00A0 iNOS\u00C2\u00A0 and\u00C2\u00A0produce\u00C2\u00A0NO,\u00C2\u00A0which\u00C2\u00A0negatively\u00C2\u00A0regulates\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.6 Pre\u00C2\u00ADtreatment\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0PGN,\u00C2\u00A0decreases\u00C2\u00A0the\u00C2\u00A0 ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0NO\u00C2\u00A0and\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 Our\u00C2\u00A0 results\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 pathogen\u00C2\u00A0 signal,\u00C2\u00A0 resident\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 become\u00C2\u00A0potent\u00C2\u00A0 suppressors\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 upon\u00C2\u00A0 stimulation\u00C2\u00A0 by\u00C2\u00A0T\u00C2\u00A0 cell\u00E2\u0080\u0090derived\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3.\u00C2\u00A0Consequently,\u00C2\u00A0we\u00C2\u00A0wanted\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0matured\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0ligands\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.7\u00C2\u00A0 IFN\u00C2\u00AD\u00CE\u00B3\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0NO\u00C2\u00A0production.\u00C2\u00A0\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0 splenocytes)\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 10\u00C2\u00A0 \u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0(left)\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated\u00C2\u00A0 (right).\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 to\u00C2\u00A0 five\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0***,p<0.001.\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0 permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0\u00C2\u00A0 60 would\u00C2\u00A0exhibit\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0suppressive\u00C2\u00A0phenotype.\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0although\u00C2\u00A0 LPS,\u00C2\u00A0CpG,\u00C2\u00A0dsRNA,\u00C2\u00A0and\u00C2\u00A0PGN\u00C2\u00A0were\u00C2\u00A0equally\u00C2\u00A0potent\u00C2\u00A0at\u00C2\u00A0 stimulating\u00C2\u00A0 the\u00C2\u00A0maturation\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0 i.e.,\u00C2\u00A0 increasing\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0MHCII,\u00C2\u00A0CD40,\u00C2\u00A0and\u00C2\u00A0CD86\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0similar\u00C2\u00A0extent\u00C2\u00A0(Fig.\u00C2\u00A03.8A;\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 CpG,\u00C2\u00A0 and\u00C2\u00A0 data\u00C2\u00A0 not\u00C2\u00A0 shown),\u00C2\u00A0 they\u00C2\u00A0 had\u00C2\u00A0 different\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression.\u00C2\u00A0 Pre\u00E2\u0080\u0090treatment\u00C2\u00A0 with\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 dsRNA,\u00C2\u00A0 which\u00C2\u00A0 signal\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 adaptor\u00C2\u00A0 TRIF\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004),\u00C2\u00A0 reduced\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0 activated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 to\u00C2\u00A0upregulate\u00C2\u00A0 iNOS\u00C2\u00A0expression\u00C2\u00A0(Fig.\u00C2\u00A03.8B),\u00C2\u00A0produce\u00C2\u00A0NO\u00C2\u00A0(Fig.\u00C2\u00A03.8C),\u00C2\u00A0or\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0(Fig.\u00C2\u00A03.8D).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0PGN,\u00C2\u00A0 which\u00C2\u00A0 signal\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 adaptor\u00C2\u00A0 MyD88\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004),\u00C2\u00A0 behaved\u00C2\u00A0 like\u00C2\u00A0 control\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 expressing\u00C2\u00A0 iNOS\u00C2\u00A0 (Fig.\u00C2\u00A0 3.8B),\u00C2\u00A0 producing\u00C2\u00A0 NO\u00C2\u00A0 (Fig.\u00C2\u00A0 3.8C),\u00C2\u00A0 and\u00C2\u00A0 significantly\u00C2\u00A0 inhibiting\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 (Fig.\u00C2\u00A0 3.8D).\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 worth\u00C2\u00A0 noting\u00C2\u00A0 that\u00C2\u00A0 although\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0reduced\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 iNOS\u00C2\u00A0expression\u00C2\u00A0upon\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0 the\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0STAT1\u00C2\u00A0phosphorylation\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0change\u00C2\u00A0(Fig.\u00C2\u00A03.8B),\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0JAK/STAT\u00C2\u00A0 signaling\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0reduced.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 previous\u00C2\u00A0 reports\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 pre\u00E2\u0080\u0090treatment\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 with\u00C2\u00A0 sub\u00E2\u0080\u0090lethal\u00C2\u00A0 doses\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0can\u00C2\u00A0cause\u00C2\u00A0a\u00C2\u00A0transient\u00C2\u00A0state\u00C2\u00A0of\u00C2\u00A0hypo\u00E2\u0080\u0090responsiveness\u00C2\u00A0to\u00C2\u00A0subsequent\u00C2\u00A0exposure\u00C2\u00A0 to\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 other\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 mediators\u00C2\u00A0 (Lorsbach\u00C2\u00A0 and\u00C2\u00A0 Russell,\u00C2\u00A0 1992),\u00C2\u00A0 we\u00C2\u00A0 hypothesized\u00C2\u00A0 that\u00C2\u00A0 pre\u00E2\u0080\u0090treatment\u00C2\u00A0 with\u00C2\u00A0 LPS\u00C2\u00A0 might\u00C2\u00A0 render\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 less\u00C2\u00A0 responsive\u00C2\u00A0 to\u00C2\u00A0 subsequent\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0stimulation.\u00C2\u00A0To\u00C2\u00A0test\u00C2\u00A0this,\u00C2\u00A0we\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0 CpG\u00C2\u00A0and\u00C2\u00A0 then\u00C2\u00A0stimulated\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0+\u00C2\u00A0LPS.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Fig.\u00C2\u00A03.9,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 that\u00C2\u00A0 had\u00C2\u00A0 been\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0 LPS\u00C2\u00A0 produced\u00C2\u00A0 significantly\u00C2\u00A0 less\u00C2\u00A0 NO\u00C2\u00A0 than\u00C2\u00A0 CpG\u00C2\u00A0 or\u00C2\u00A0 non\u00E2\u0080\u0090pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0with\u00C2\u00A0LPS,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CpG,\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0 ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0respond\u00C2\u00A0to\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 61 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.8\u00C2\u00A0 Pre\u00C2\u00ADtreatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0agonists\u00C2\u00A0that\u00C2\u00A0signal\u00C2\u00A0through\u00C2\u00A0TRIF\u00C2\u00A0desensitizes\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0subsequent\u00C2\u00A0IFN\u00C2\u00AD\u00CE\u00B3\u00C2\u00A0stimulation,\u00C2\u00A0decreasing\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0NO\u00C2\u00A0and\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0medium\u00C2\u00A0alone\u00C2\u00A0(grey\u00C2\u00A0fill),\u00C2\u00A0LPS\u00C2\u00A0(black\u00C2\u00A0line),\u00C2\u00A0or\u00C2\u00A0CpG\u00C2\u00A0(grey\u00C2\u00A0line).\u00C2\u00A0After\u00C2\u00A0 24\u00C2\u00A0h,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0for\u00C2\u00A0surface\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0MHC\u00C2\u00A0 class\u00C2\u00A0II,\u00C2\u00A0CD40,\u00C2\u00A0and\u00C2\u00A0CD86.\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0 or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0been\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0LPS,\u00C2\u00A0CpG,\u00C2\u00A0dsRNA,\u00C2\u00A0or\u00C2\u00A0PGN\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0 and\u00C2\u00A0washed\u00C2\u00A0prior\u00C2\u00A0to\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes).\u00C2\u00A0After\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0subjected\u00C2\u00A0to\u00C2\u00A0B,\u00C2\u00A0 Western\u00C2\u00A0blot\u00C2\u00A0analysis,\u00C2\u00A0and\u00C2\u00A0C,\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0and\u00C2\u00A0D,\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0 shown\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A04\u00C2\u00A0independent\u00C2\u00A0experiments,\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate\u00C2\u00A0(A)\u00C2\u00A0or\u00C2\u00A0the\u00C2\u00A0 mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A03\u00C2\u00A0independent\u00C2\u00A0experiments,\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate\u00C2\u00A0(B,\u00C2\u00A0C,\u00C2\u00A0D).\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0 **,p<0.01;\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0 permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 62 \u00C2\u00A0 \u00C2\u00A0 3.2.7 IFN\u00C2\u00AD\u00CE\u00B2\u00C2\u00A0contributes\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0reduced\u00C2\u00A0suppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0 pre\u00C2\u00ADtreated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0gain\u00C2\u00A0insight\u00C2\u00A0into\u00C2\u00A0how\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0pre\u00E2\u0080\u0090treatment\u00C2\u00A0reduced\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0respond\u00C2\u00A0to\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0whether\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0 was\u00C2\u00A0dependent\u00C2\u00A0 on\u00C2\u00A0a\u00C2\u00A0 secondary,\u00C2\u00A0 secreted\u00C2\u00A0 factor.\u00C2\u00A0 To\u00C2\u00A0perform\u00C2\u00A0 these\u00C2\u00A0 studies\u00C2\u00A0we\u00C2\u00A0utilized\u00C2\u00A0 polymyxin\u00C2\u00A0 B,\u00C2\u00A0 a\u00C2\u00A0 compound\u00C2\u00A0 that\u00C2\u00A0 binds\u00C2\u00A0 to\u00C2\u00A0 and\u00C2\u00A0 inactivates\u00C2\u00A0 LPS\u00C2\u00A0 (Morrison\u00C2\u00A0 and\u00C2\u00A0 Jacobs,\u00C2\u00A0 1976)\u00C2\u00A0 and\u00C2\u00A0 effectively\u00C2\u00A0 blocks\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 LPS\u00C2\u00A0 on\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 (Fig.\u00C2\u00A0 3.10A).\u00C2\u00A0We\u00C2\u00A0 treated\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 with\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 LPS\u00C2\u00A0 for\u00C2\u00A0 24\u00C2\u00A0 h,\u00C2\u00A0 and\u00C2\u00A0 then\u00C2\u00A0 added\u00C2\u00A0 polymyxin\u00C2\u00A0 B\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 supernatants\u00C2\u00A0before\u00C2\u00A0transferring\u00C2\u00A0them\u00C2\u00A0onto\u00C2\u00A0unstimulated\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0After\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0the\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 washed\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A03.10B,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0exposed\u00C2\u00A0to\u00C2\u00A0 supernatants\u00C2\u00A0 from\u00C2\u00A0LPS\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0had\u00C2\u00A0decreased\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0unstimulated\u00C2\u00A0supernatants.\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0 that\u00C2\u00A0LPS\u00C2\u00A0stimulation\u00C2\u00A0 induces\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0secrete\u00C2\u00A0a\u00C2\u00A0secondary\u00C2\u00A0 factor\u00C2\u00A0 that\u00C2\u00A0acts\u00C2\u00A0 in\u00C2\u00A0an\u00C2\u00A0autocrine\u00C2\u00A0manner\u00C2\u00A0 to\u00C2\u00A0reduce\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0from\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.9\u00C2\u00A0 Pre\u00C2\u00ADtreatment\u00C2\u00A0 with\u00C2\u00A0 LPS,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 CpG,\u00C2\u00A0 reduces\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 sensitivity\u00C2\u00A0 to\u00C2\u00A0 subsequent\u00C2\u00A0stimulation.\u00C2\u00A0\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0CpG\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0washed,\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0stimulated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3+LPS.\u00C2\u00A0After\u00C2\u00A0 72\u00C2\u00A0h,\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0was\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A03\u00C2\u00A0independent\u00C2\u00A0experiments,\u00C2\u00A0 performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0 with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 63 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 then\u00C2\u00A0 asked\u00C2\u00A0 if\u00C2\u00A0 this\u00C2\u00A0 secondary\u00C2\u00A0 factor\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 since\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 TRIF\u00C2\u00A0 pathway\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 produce\u00C2\u00A0 this\u00C2\u00A0 cytokine\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004).\u00C2\u00A0 Indeed,\u00C2\u00A0we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0stimulation\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0PGN,\u00C2\u00A0 induced\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0(Fig.\u00C2\u00A03.11A).\u00C2\u00A0Therefore,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0whether\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0was\u00C2\u00A0involved\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 abrogation\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 suppression\u00C2\u00A0 by\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 pre\u00E2\u0080\u0090 treatment\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 dose\u00E2\u0080\u0090dependently\u00C2\u00A0 reduced\u00C2\u00A0 iNOS\u00C2\u00A0 expression\u00C2\u00A0 (Fig.\u00C2\u00A0 3.11B,\u00C2\u00A0 left),\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0(Fig.\u00C2\u00A03.11B,\u00C2\u00A0middle)\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0immunosuppression\u00C2\u00A0(Fig.\u00C2\u00A03.11B,\u00C2\u00A0right).\u00C2\u00A0Adding\u00C2\u00A0 a\u00C2\u00A0neutralizing\u00C2\u00A0Ab\u00C2\u00A0 to\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0significantly\u00C2\u00A0 increased\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0 (Fig.\u00C2\u00A03.11C,\u00C2\u00A0 left)\u00C2\u00A0and\u00C2\u00A0 suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 (Fig.\u00C2\u00A03.11C,\u00C2\u00A0 right)\u00C2\u00A0by\u00C2\u00A0LPS\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0dsRNA\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 Moreover,\u00C2\u00A0 neutralizing\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 also\u00C2\u00A0 increased\u00C2\u00A0 iNOS\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 exposed\u00C2\u00A0 to\u00C2\u00A0 supernatants\u00C2\u00A0 from\u00C2\u00A0LPS\u00E2\u0080\u0090stimulated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 polymyxin\u00C2\u00A0 B\u00C2\u00A0 (Fig.\u00C2\u00A0 3.11D).\u00C2\u00A0 Taken\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.10\u00C2\u00A0 LPS\u00C2\u00A0stimulation\u00C2\u00A0 induces\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0secondary\u00C2\u00A0 factor\u00C2\u00A0 that\u00C2\u00A0 inhibits\u00C2\u00A0 IFN\u00C2\u00AD\u00CE\u00B3\u00C2\u00ADinduced\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0medium\u00C2\u00A0alone\u00C2\u00A0 (\u00E2\u0080\u0090),\u00C2\u00A0 LPS,\u00C2\u00A0or\u00C2\u00A0LPS\u00C2\u00A0+\u00C2\u00A0polymyxin\u00C2\u00A0B\u00C2\u00A0 (PB)\u00C2\u00A0 for\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0 washed,\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes).\u00C2\u00A0After\u00C2\u00A0 72\u00C2\u00A0h,\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0was\u00C2\u00A0measured.\u00C2\u00A0Significance\u00C2\u00A0is\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0non\u00E2\u0080\u0090pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(\u00E2\u0080\u0090).\u00C2\u00A0B,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 stimulated\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 LPS\u00C2\u00A0 for\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 supernatants\u00C2\u00A0 were\u00C2\u00A0 supplemented\u00C2\u00A0 with\u00C2\u00A0 polymyxin\u00C2\u00A0B\u00C2\u00A0(PB),\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0unstimulated\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0After\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0 splenocytes)\u00C2\u00A0 for\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A0NO\u00C2\u00A0 production\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0independent\u00C2\u00A0experiments.\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0 NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0 from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0 Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 64 together,\u00C2\u00A0 these\u00C2\u00A0 results\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 autocrine\u00E2\u0080\u0090acting\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 plays\u00C2\u00A0 an\u00C2\u00A0 important\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0abrogating\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.11\u00C2\u00A0 IFN\u00C2\u00AD\u00CE\u00B2\u00C2\u00A0contributes\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0reduced\u00C2\u00A0suppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0 pre\u00C2\u00ADtreated\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0stimulated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0LPS,\u00C2\u00A0CpG,\u00C2\u00A0dsRNA,\u00C2\u00A0or\u00C2\u00A0PGN\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0Supernatants\u00C2\u00A0were\u00C2\u00A0collected\u00C2\u00A0and\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 levels\u00C2\u00A0 measured.\u00C2\u00A0 Significance\u00C2\u00A0 is\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 non\u00E2\u0080\u0090pre\u00E2\u0080\u0090treated\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 (\u00E2\u0080\u0090).\u00C2\u00A0 B,\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090 stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0 been\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 (0,\u00C2\u00A0100,\u00C2\u00A01000,\u00C2\u00A05000\u00C2\u00A0U/ml),\u00C2\u00A0LPS,\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0 for\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A0washed\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 co\u00E2\u0080\u0090culture\u00C2\u00A0 (1\u00C2\u00A0 M\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0 splenocytes).\u00C2\u00A0 After\u00C2\u00A0 72\u00C2\u00A0 h,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 subjected\u00C2\u00A0 to\u00C2\u00A0 Western\u00C2\u00A0 blot\u00C2\u00A0 analysis\u00C2\u00A0 (left),\u00C2\u00A0 and\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 (middle)\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 (right)\u00C2\u00A0 measured.\u00C2\u00A0 Significance\u00C2\u00A0 is\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 untreated\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0C,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0medium\u00C2\u00A0 alone\u00C2\u00A0 (\u00E2\u0080\u0090),\u00C2\u00A0 LPS,\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0absence\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0of\u00C2\u00A0neutralizing\u00C2\u00A0anti\u00E2\u0080\u0090IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 (1000\u00C2\u00A0U/ml)\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0washed,\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0splenocytes).\u00C2\u00A0After\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0(left)\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0(right)\u00C2\u00A0were\u00C2\u00A0measured.\u00C2\u00A0 D,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0stimulated\u00C2\u00A0with\u00C2\u00A0medium\u00C2\u00A0alone\u00C2\u00A0(\u00E2\u0080\u0090),\u00C2\u00A0LPS,\u00C2\u00A0or\u00C2\u00A0LPS\u00C2\u00A0+\u00C2\u00A0anti\u00E2\u0080\u0090IFN\u00CE\u00B2\u00C2\u00A0 (500\u00C2\u00A0U/ml)\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0 Supernatants\u00C2\u00A0were\u00C2\u00A0collected,\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0polymyxin\u00C2\u00A0B,\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0unstimulated\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 After\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0 splenocytes)\u00C2\u00A0for\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0subjected\u00C2\u00A0to\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0analysis.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0 three\u00C2\u00A0to\u00C2\u00A0five\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate\u00C2\u00A0(A,\u00C2\u00A0D)\u00C2\u00A0or\u00C2\u00A0triplicate\u00C2\u00A0(B,\u00C2\u00A0C).\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0 ***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0 American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 65 3.2.8 Inhibition\u00C2\u00A0of\u00C2\u00A0Arg1\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0pre\u00C2\u00ADtreatment\u00C2\u00A0of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 observed\u00C2\u00A0 that,\u00C2\u00A0 in\u00C2\u00A0 addition\u00C2\u00A0 to\u00C2\u00A0 inhibiting\u00C2\u00A0 iNOS\u00C2\u00A0 expression\u00C2\u00A0 (Fig.\u00C2\u00A0 3.11B),\u00C2\u00A0 LPS\u00C2\u00A0 and,\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0lesser\u00C2\u00A0extent,\u00C2\u00A0dsRNA\u00C2\u00A0increased\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0Arg1\u00C2\u00A0in\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A03.11D\u00C2\u00A0and\u00C2\u00A0 data\u00C2\u00A0not\u00C2\u00A0 shown).\u00C2\u00A0Thus,\u00C2\u00A0we\u00C2\u00A0 investigated\u00C2\u00A0whether\u00C2\u00A0Arg1\u00C2\u00A0was\u00C2\u00A0playing\u00C2\u00A0a\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0LPS\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00E2\u0080\u0090induced\u00C2\u00A0abrogation\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0immunosuppression.\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A03.12,\u00C2\u00A0inhibiting\u00C2\u00A0 Arg1\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 Arg\u00E2\u0080\u0090specific\u00C2\u00A0 inhibitor\u00C2\u00A0 BEC\u00C2\u00A0 increased\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 (left)\u00C2\u00A0 and\u00C2\u00A0 suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0(right)\u00C2\u00A0by\u00C2\u00A0LPS\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0dsRNA\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0but,\u00C2\u00A0as\u00C2\u00A0expected,\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 alter\u00C2\u00A0 iNOS\u00C2\u00A0 expression\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown).\u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 observation\u00C2\u00A0 that\u00C2\u00A0 increasing\u00C2\u00A0Arg1\u00C2\u00A0expression\u00C2\u00A0or\u00C2\u00A0activity\u00C2\u00A0can\u00C2\u00A0decrease\u00C2\u00A0NO\u00C2\u00A0production,\u00C2\u00A0given\u00C2\u00A0that\u00C2\u00A0iNOS\u00C2\u00A0and\u00C2\u00A0 Arg1\u00C2\u00A0 compete\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 common\u00C2\u00A0 substrate,\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 (Gordon,\u00C2\u00A0 2003).\u00C2\u00A0 Therefore,\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00C2\u00A0decrease\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0NO\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0T\u00C2\u00A0cell\u00E2\u0080\u0090derived\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0by\u00C2\u00A0 both\u00C2\u00A0decreasing\u00C2\u00A0iNOS\u00C2\u00A0and\u00C2\u00A0increasing\u00C2\u00A0Arg1\u00C2\u00A0expression.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.12\u00C2\u00A0 The\u00C2\u00A0 Arg1\u00C2\u00A0 inhibitor\u00C2\u00A0 BEC\u00C2\u00A0 reduces\u00C2\u00A0 the\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 LPS\u00C2\u00A0 and\u00C2\u00A0 dsRNA\u00C2\u00A0 pre\u00C2\u00AD treatment.\u00C2\u00A0\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0medium\u00C2\u00A0alone\u00C2\u00A0(\u00E2\u0080\u0090),\u00C2\u00A0LPS,\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0 absence\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0 of\u00C2\u00A0 200\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0 BEC\u00C2\u00A0 for\u00C2\u00A0 24\u00C2\u00A0 h,\u00C2\u00A0 washed,\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0 splenocytes).\u00C2\u00A0 After\u00C2\u00A0 72\u00C2\u00A0 h,\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 (left)\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0(right)\u00C2\u00A0were\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A03\u00C2\u00A0independent\u00C2\u00A0experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant.\u00C2\u00A0Reprinted\u00C2\u00A0 from\u00C2\u00A0Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010\u00C2\u00A0 with\u00C2\u00A0permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0 66 3.3 Discussion\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 important\u00C2\u00A0 features\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 is\u00C2\u00A0 its\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 discriminate\u00C2\u00A0 between\u00C2\u00A0 Ags\u00C2\u00A0 that\u00C2\u00A0 should\u00C2\u00A0 provoke\u00C2\u00A0 an\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 and\u00C2\u00A0 those\u00C2\u00A0 that\u00C2\u00A0 should\u00C2\u00A0 be\u00C2\u00A0 tolerated.\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 well\u00C2\u00A0 known\u00C2\u00A0 that\u00C2\u00A0 APCs,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 DCs,\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 adaptive\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 to\u00C2\u00A0 respond\u00C2\u00A0 to\u00C2\u00A0 Ags.\u00C2\u00A0 However,\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 this\u00C2\u00A0 occurs\u00C2\u00A0 have\u00C2\u00A0 not\u00C2\u00A0 been\u00C2\u00A0 fully\u00C2\u00A0 elucidated\u00C2\u00A0 (Munn\u00C2\u00A0 and\u00C2\u00A0Mellor,\u00C2\u00A0 2003).\u00C2\u00A0In\u00C2\u00A0this\u00C2\u00A0chapter,\u00C2\u00A0we\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0process\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0resident\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 and\u00C2\u00A0 determined\u00C2\u00A0 how\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 pathogen\u00E2\u0080\u0090derived\u00C2\u00A0 molecules\u00C2\u00A0 influences\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0immunosuppression.\u00C2\u00A0Our\u00C2\u00A0results\u00C2\u00A0more\u00C2\u00A0clearly\u00C2\u00A0elucidate\u00C2\u00A0the\u00C2\u00A0mechanisms\u00C2\u00A0 by\u00C2\u00A0which\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects\u00C2\u00A0 and\u00C2\u00A0 suggest\u00C2\u00A0 a\u00C2\u00A0 key\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0 TLR\u00C2\u00A0 signaling\u00C2\u00A0and\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0in\u00C2\u00A0regulating\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0phenotype.\u00C2\u00A0 \u00C2\u00A0 Resident\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 other\u00C2\u00A0 cells\u00C2\u00A0 within\u00C2\u00A0 the\u00C2\u00A0 PC,\u00C2\u00A0 potently\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0via\u00C2\u00A0an\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0NO\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism.\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0model\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A0 3.13,\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 produced\u00C2\u00A0 by\u00C2\u00A0 TCR\u00E2\u0080\u0090activated\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 induces\u00C2\u00A0 na\u00C3\u00AFve\u00C2\u00A0 resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 express\u00C2\u00A0 iNOS\u00C2\u00A0and\u00C2\u00A0produce\u00C2\u00A0NO,\u00C2\u00A0which\u00C2\u00A0subsequently\u00C2\u00A0suppresses\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation.\u00C2\u00A0\u00C2\u00A0However,\u00C2\u00A0this\u00C2\u00A0 negative\u00C2\u00A0 feedback\u00C2\u00A0 loop\u00C2\u00A0 is\u00C2\u00A0 eliminated\u00C2\u00A0 by\u00C2\u00A0 TLR\u00C2\u00A0 agonists\u00C2\u00A0 that\u00C2\u00A0 signal\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 TRIF\u00C2\u00A0 cascade\u00C2\u00A0(i.e.\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA),\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0by\u00C2\u00A0those\u00C2\u00A0that\u00C2\u00A0signal\u00C2\u00A0exclusively\u00C2\u00A0through\u00C2\u00A0MyD88\u00C2\u00A0(i.e.\u00C2\u00A0 CpG\u00C2\u00A0or\u00C2\u00A0PGN).\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0stimulated\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0exhibit\u00C2\u00A0 low\u00C2\u00A0responsiveness\u00C2\u00A0to\u00C2\u00A0T\u00C2\u00A0cell\u00E2\u0080\u0090 produced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 and,\u00C2\u00A0 consequently,\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 upregulate\u00C2\u00A0 iNOS,\u00C2\u00A0 produce\u00C2\u00A0 NO,\u00C2\u00A0 or\u00C2\u00A0 exhibit\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties.\u00C2\u00A0The\u00C2\u00A0reduced\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0 respond\u00C2\u00A0 to\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 is\u00C2\u00A0 due,\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0 part,\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 autocrine\u00E2\u0080\u0090acting\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 which\u00C2\u00A0 occurs\u00C2\u00A0 following\u00C2\u00A0 TRIF\u00C2\u00A0 recruitment\u00C2\u00A0 and\u00C2\u00A0 IRF\u00E2\u0080\u00903\u00C2\u00A0 activation\u00C2\u00A0 (Akira\u00C2\u00A0 and\u00C2\u00A0 Takeda,\u00C2\u00A0 2004).\u00C2\u00A0This,\u00C2\u00A0together\u00C2\u00A0with\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0Arg1,\u00C2\u00A0reduces\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0allowing\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0proliferate\u00C2\u00A0and\u00C2\u00A0mount\u00C2\u00A0an\u00C2\u00A0 immune\u00C2\u00A0response.\u00C2\u00A0Although\u00C2\u00A0the\u00C2\u00A0 inhibitory\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0 LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0on\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 responsiveness\u00C2\u00A0and\u00C2\u00A0 iNOS\u00C2\u00A0expression\u00C2\u00A0are\u00C2\u00A0mediated\u00C2\u00A0by\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0dsRNA\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0Arg1\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0appear\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00E2\u0080\u0090dependent,\u00C2\u00A0 since\u00C2\u00A0blocking\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0reduce\u00C2\u00A0Arg1\u00C2\u00A0expression\u00C2\u00A0(Fig.\u00C2\u00A03.11D).\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0there\u00C2\u00A0 is\u00C2\u00A0 some\u00C2\u00A0evidence\u00C2\u00A0 that\u00C2\u00A0Arg1\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 induced\u00C2\u00A0via\u00C2\u00A0 the\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0pathway\u00C2\u00A0(Hagemann\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 67 2008),\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0activated\u00C2\u00A0by\u00C2\u00A0all\u00C2\u00A0TLR\u00C2\u00A0signaling\u00C2\u00A0pathways,\u00C2\u00A0although\u00C2\u00A0more\u00C2\u00A0rapidly\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0 MyD88\u00E2\u0080\u0090dependent\u00C2\u00A0pathway\u00C2\u00A0(Akira\u00C2\u00A0and\u00C2\u00A0Takeda,\u00C2\u00A02004).\u00C2\u00A0However,\u00C2\u00A0our\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0 increasing\u00C2\u00A0 Arg1\u00C2\u00A0 alone\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 sufficient\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression,\u00C2\u00A0but\u00C2\u00A0that\u00C2\u00A0 increased\u00C2\u00A0Arg1\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0only\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0decreasing\u00C2\u00A0 NO\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0combination\u00C2\u00A0with\u00C2\u00A0decreased\u00C2\u00A0iNOS\u00C2\u00A0expression.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.13\u00C2\u00A0 The\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0is\u00C2\u00A0abrogated\u00C2\u00A0 when\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0pre\u00C2\u00ADtreated\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0PGN.\u00C2\u00A0\u00C2\u00A0 Resident\u00C2\u00A0peritoneal\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0or\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0with\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0PGN,\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 via\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090induced\u00C2\u00A0NO\u00C2\u00A0production.\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0following\u00C2\u00A0TCR\u00C2\u00A0stimulation\u00C2\u00A0binds\u00C2\u00A0 to\u00C2\u00A0 its\u00C2\u00A0receptor\u00C2\u00A0on\u00C2\u00A0resident\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0recruits\u00C2\u00A0two\u00C2\u00A0JAKs\u00C2\u00A0(JAK\u00E2\u0080\u00901\u00C2\u00A0and\u00C2\u00A0JAK\u00E2\u0080\u00902),\u00C2\u00A0which\u00C2\u00A0trigger\u00C2\u00A0the\u00C2\u00A0 recruitment,\u00C2\u00A0 phosphorylation,\u00C2\u00A0 and\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 transcription\u00C2\u00A0 factor\u00C2\u00A0 STAT1.\u00C2\u00A0 The\u00C2\u00A0 phosphorylated\u00C2\u00A0 STAT1\u00C2\u00A0 homodimer\u00C2\u00A0 translocates\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 nucleus\u00C2\u00A0 and\u00C2\u00A0 binds\u00C2\u00A0 to\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090 activation\u00C2\u00A0sites\u00C2\u00A0(GAS)\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0iNOS\u00C2\u00A0promoter,\u00C2\u00A0 inducing\u00C2\u00A0iNOS\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0NO\u00C2\u00A0production,\u00C2\u00A0 which\u00C2\u00A0 in\u00C2\u00A0 turn\u00C2\u00A0suppresses\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation.\u00C2\u00A0 \u00C2\u00A0However,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA\u00C2\u00A0 lack\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties.\u00C2\u00A0 Exposure\u00C2\u00A0 of\u00C2\u00A0 resident\u00C2\u00A0 peritoneal\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 dsRNA\u00C2\u00A0 triggers\u00C2\u00A0TRIF\u00C2\u00A0recruitment,\u00C2\u00A0IRF\u00E2\u0080\u00903\u00C2\u00A0activation,\u00C2\u00A0and\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0and\u00C2\u00A0later,\u00C2\u00A0IFN\u00E2\u0080\u0090inducible\u00C2\u00A0 genes.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0these\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0subsequently\u00C2\u00A0detect\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 they\u00C2\u00A0fail\u00C2\u00A0 to\u00C2\u00A0upregulate\u00C2\u00A0 iNOS\u00C2\u00A0or\u00C2\u00A0produce\u00C2\u00A0NO,\u00C2\u00A0and\u00C2\u00A0consequently\u00C2\u00A0exhibit\u00C2\u00A0significantly\u00C2\u00A0reduced\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 as\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 na\u00C3\u00AFve,\u00C2\u00A0 or\u00C2\u00A0 CpG\u00C2\u00A0 or\u00C2\u00A0 PGN\u00C2\u00A0 pre\u00E2\u0080\u0090treated,\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 This\u00C2\u00A0 failure\u00C2\u00A0 of\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 dsRNA\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 respond\u00C2\u00A0 to\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 is,\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0 part,\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 autocrine\u00E2\u0080\u0090acting\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2.\u00C2\u00A0This,\u00C2\u00A0together\u00C2\u00A0with\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0Arg1,\u00C2\u00A0reduces\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0by\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 eliminates\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression.\u00C2\u00A0 Reprinted\u00C2\u00A0 from\u00C2\u00A0 Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010\u00C2\u00A0 with\u00C2\u00A0 permission\u00C2\u00A0of\u00C2\u00A0The\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0Immunologists,\u00C2\u00A0Inc.\u00C2\u00A0\u00C2\u00A0 68 There\u00C2\u00A0is\u00C2\u00A0some\u00C2\u00A0controversy\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0literature\u00C2\u00A0regarding\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0on\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 signaling\u00C2\u00A0(Gough\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Inaba\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01986;\u00C2\u00A0Ling\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01985;\u00C2\u00A0Schroder\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004;\u00C2\u00A0 Yoshida\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01988).\u00C2\u00A0The\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B1/\u00CE\u00B2\u00C2\u00A0signal\u00C2\u00A0pathways\u00C2\u00A0are\u00C2\u00A0partially\u00C2\u00A0overlapping,\u00C2\u00A0 which\u00C2\u00A0allows\u00C2\u00A0for\u00C2\u00A0cross\u00E2\u0080\u0090talk\u00C2\u00A0at\u00C2\u00A0multiple\u00C2\u00A0levels\u00C2\u00A0(Schroder\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004).\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0binds\u00C2\u00A0to\u00C2\u00A0its\u00C2\u00A0 receptor,\u00C2\u00A0 which\u00C2\u00A0 triggers\u00C2\u00A0 receptor\u00C2\u00A0 clustering\u00C2\u00A0 and\u00C2\u00A0 activates\u00C2\u00A0 a\u00C2\u00A0 JAK\u00E2\u0080\u0090STAT\u00C2\u00A0 signaling\u00C2\u00A0 pathway\u00C2\u00A0 that\u00C2\u00A0 culminates\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 binding\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 phosphorylated\u00C2\u00A0 STAT1\u00C2\u00A0 homodimer\u00C2\u00A0 to\u00C2\u00A0 promoter\u00C2\u00A0 elements.\u00C2\u00A0 This,\u00C2\u00A0 in\u00C2\u00A0 turn,\u00C2\u00A0 activates\u00C2\u00A0 or\u00C2\u00A0 suppresses\u00C2\u00A0 transcription\u00C2\u00A0 of\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090 regulated\u00C2\u00A0genes\u00C2\u00A0 (Schroder\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2004).\u00C2\u00A0There\u00C2\u00A0 is\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 type\u00C2\u00A0 I\u00C2\u00A0 IFNs\u00C2\u00A0 can\u00C2\u00A0prime\u00C2\u00A0 cells\u00C2\u00A0and\u00C2\u00A0increase\u00C2\u00A0responsiveness\u00C2\u00A0to\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0by\u00C2\u00A0inducing\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0signaling\u00C2\u00A0molecules,\u00C2\u00A0such\u00C2\u00A0 as\u00C2\u00A0STAT1\u00C2\u00A0(Gough\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Schroder\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004).\u00C2\u00A0On\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0our\u00C2\u00A0finding\u00C2\u00A0that\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0antagonizes\u00C2\u00A0the\u00C2\u00A0response\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0subsequent\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0 with\u00C2\u00A0several\u00C2\u00A0reports\u00C2\u00A0(Inaba\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01986;\u00C2\u00A0Ling\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01985;\u00C2\u00A0Yoshida\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01988)\u00C2\u00A0including\u00C2\u00A0 a\u00C2\u00A0 recent\u00C2\u00A0 study\u00C2\u00A0 suggesting\u00C2\u00A0 the\u00C2\u00A0 mechanism\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 down\u00E2\u0080\u0090regulation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3R\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0to\u00C2\u00A0IFN\u00E2\u0080\u0090\u00C2\u00A0\u00CE\u00B1/\u00CE\u00B2\u00C2\u00A0(Rayamajhi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0However,\u00C2\u00A0since\u00C2\u00A0we\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0see\u00C2\u00A0a\u00C2\u00A0decrease\u00C2\u00A0 in\u00C2\u00A0 STAT1\u00C2\u00A0 phosphorylation\u00C2\u00A0 following\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 treatment\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown),\u00C2\u00A0 down\u00E2\u0080\u0090 regulation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3R\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0findings.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0critical\u00C2\u00A0regulator\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0phenotype\u00C2\u00A0and\u00C2\u00A0function.\u00C2\u00A0It\u00C2\u00A0is\u00C2\u00A0well\u00C2\u00A0known\u00C2\u00A0that\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 stimulation\u00C2\u00A0 triggers\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 acquire\u00C2\u00A0 anti\u00E2\u0080\u0090microbial\u00C2\u00A0 and\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 properties,\u00C2\u00A0including\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0promote\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0(Schroder\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004).\u00C2\u00A0 However,\u00C2\u00A0 as\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 demonstrate,\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 can\u00C2\u00A0 also\u00C2\u00A0 trigger\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 become\u00C2\u00A0 immunosuppressive.\u00C2\u00A0Thus,\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090induced\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0have\u00C2\u00A0the\u00C2\u00A0potential\u00C2\u00A0to\u00C2\u00A0both\u00C2\u00A0stimulate\u00C2\u00A0and\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0dominating\u00C2\u00A0effect\u00C2\u00A0likely\u00C2\u00A0depends\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 resulting\u00C2\u00A0 local\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 NO.\u00C2\u00A0When\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0 low\u00C2\u00A0 numbers,\u00C2\u00A0little\u00C2\u00A0NO\u00C2\u00A0is\u00C2\u00A0produced\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0immunostimulatory\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0predominate\u00C2\u00A0 (Fig.\u00C2\u00A03.3A,\u00C2\u00A0 left).\u00C2\u00A0However,\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 is\u00C2\u00A0high,\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090induced\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 exhibit\u00C2\u00A0 anti\u00E2\u0080\u0090inflammatory\u00C2\u00A0 properties,\u00C2\u00A0 since\u00C2\u00A0 the\u00C2\u00A0 resulting\u00C2\u00A0 NO\u00C2\u00A0 is\u00C2\u00A0 sufficient\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 local\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation.\u00C2\u00A0This\u00C2\u00A0situation\u00C2\u00A0is\u00C2\u00A0physiologically\u00C2\u00A0relevant\u00C2\u00A0both\u00C2\u00A0in\u00C2\u00A0sites\u00C2\u00A0of\u00C2\u00A0acute\u00C2\u00A0 infection\u00C2\u00A0(e.g.\u00C2\u00A0wounds)\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0non\u00E2\u0080\u0090infected\u00C2\u00A0tissues,\u00C2\u00A0where\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0abundant\u00C2\u00A0than\u00C2\u00A0 lymphocytes\u00C2\u00A0 (Gordon,\u00C2\u00A0 2003;\u00C2\u00A0Martin\u00C2\u00A0 and\u00C2\u00A0Muir,\u00C2\u00A0 1990).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 supported\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 recent\u00C2\u00A0 69 report\u00C2\u00A0(Composto\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011)\u00C2\u00A0demonstrating\u00C2\u00A0that\u00C2\u00A0although\u00C2\u00A0resident\u00C2\u00A0PC\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0 phenotype\u00C2\u00A0 indicative\u00C2\u00A0 of\u00C2\u00A0 activation,\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 suppressed\u00C2\u00A0 by\u00C2\u00A0 resident\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0 which\u00C2\u00A0 outnumber\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0suppress\u00C2\u00A0via\u00C2\u00A0an\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0iNOS\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0co\u00E2\u0080\u0090existence\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0tissues\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0PC\u00C2\u00A0(Composto\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02011)\u00C2\u00A0suggests\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0play\u00C2\u00A0an\u00C2\u00A0important\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0maintaining\u00C2\u00A0immune\u00C2\u00A0homeostasis.\u00C2\u00A0\u00C2\u00A0 Our\u00C2\u00A0 results,\u00C2\u00A0 which\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 TLR\u00C2\u00A0 signaling\u00C2\u00A0 can\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0add\u00C2\u00A0to\u00C2\u00A0our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0how\u00C2\u00A0this\u00C2\u00A0homeostasis\u00C2\u00A0is\u00C2\u00A0maintained\u00C2\u00A0and\u00C2\u00A0 are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0TLRs\u00C2\u00A0in\u00C2\u00A0pathogen\u00C2\u00A0detection.\u00C2\u00A0In\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0PAMPs,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0 likely\u00C2\u00A0 that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 that\u00C2\u00A0detect\u00C2\u00A0T\u00C2\u00A0cell\u00E2\u0080\u0090produced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 sense\u00C2\u00A0 the\u00C2\u00A0 response\u00C2\u00A0as\u00C2\u00A0 inappropriate\u00C2\u00A0 and\u00C2\u00A0 exert\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects.\u00C2\u00A0 On\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 if\u00C2\u00A0 a\u00C2\u00A0 pathogen\u00C2\u00A0 is\u00C2\u00A0 present,\u00C2\u00A0 predisposition\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 allow\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 mount\u00C2\u00A0 an\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 advantageous.\u00C2\u00A0 However,\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 not\u00C2\u00A0 all\u00C2\u00A0 PAMPs\u00C2\u00A0 negate\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 suppressive\u00C2\u00A0 abilities.\u00C2\u00A0 Specifically,\u00C2\u00A0 while\u00C2\u00A0 TLR4\u00C2\u00A0 (LPS)\u00C2\u00A0 and\u00C2\u00A0 TLR3\u00C2\u00A0 (dsRNA)\u00C2\u00A0 agonists\u00C2\u00A0 abrogated\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression,\u00C2\u00A0TLR9\u00C2\u00A0(CpG)\u00C2\u00A0and\u00C2\u00A0TLR2\u00C2\u00A0(PGN)\u00C2\u00A0agonists\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0(Fig.\u00C2\u00A03.8).\u00C2\u00A0Although\u00C2\u00A0 the\u00C2\u00A0 physiological\u00C2\u00A0 rationale\u00C2\u00A0 for\u00C2\u00A0 these\u00C2\u00A0 differences\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 immediately\u00C2\u00A0 apparent,\u00C2\u00A0 we\u00C2\u00A0 hypothesize\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 abrogation\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 specific\u00C2\u00A0 to\u00C2\u00A0 viral\u00C2\u00A0 stimuli,\u00C2\u00A0given\u00C2\u00A0that\u00C2\u00A0only\u00C2\u00A0PAMPs\u00C2\u00A0that\u00C2\u00A0trigger\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0(Fig.\u00C2\u00A03.11A),\u00C2\u00A0a\u00C2\u00A0critical\u00C2\u00A0 mediator\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 anti\u00E2\u0080\u0090viral\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 (Schroder\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2004),\u00C2\u00A0 eliminated\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression\u00C2\u00A0(Fig.\u00C2\u00A03.8).\u00C2\u00A0Bacterial\u00C2\u00A0stimuli,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0reduce\u00C2\u00A0NO\u00C2\u00A0 production\u00C2\u00A0 by\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 which\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 advantageous\u00C2\u00A0 given\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 NO\u00C2\u00A0 to\u00C2\u00A0 kill\u00C2\u00A0 extracellular\u00C2\u00A0 pathogens,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 certain\u00C2\u00A0 classes\u00C2\u00A0 of\u00C2\u00A0 bacteria\u00C2\u00A0 (Bogdan,\u00C2\u00A0 2001).\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0well,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 indirect\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 (Taylor\u00E2\u0080\u0090Robinson\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1994)\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 (Wei\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1995)\u00C2\u00A0 evidence\u00C2\u00A0that\u00C2\u00A0Th1\u00C2\u00A0cells,\u00C2\u00A0which\u00C2\u00A0target\u00C2\u00A0intracellular\u00C2\u00A0pathogens\u00C2\u00A0(i.e.\u00C2\u00A0viruses)\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0 susceptible\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 inhibitory\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 NO\u00C2\u00A0 than\u00C2\u00A0 Th2\u00C2\u00A0 cells,\u00C2\u00A0 which\u00C2\u00A0 promote\u00C2\u00A0 attacks\u00C2\u00A0 on\u00C2\u00A0 extracellular\u00C2\u00A0 pathogens\u00C2\u00A0 (e.g.\u00C2\u00A0 extracellular\u00C2\u00A0 bacteria),\u00C2\u00A0 and\u00C2\u00A0 this\u00C2\u00A0may\u00C2\u00A0 suggest\u00C2\u00A0 a\u00C2\u00A0 potential\u00C2\u00A0 rationale\u00C2\u00A0 for\u00C2\u00A0 our\u00C2\u00A0 findings.\u00C2\u00A0 Further\u00C2\u00A0 studies\u00C2\u00A0 to\u00C2\u00A0 address\u00C2\u00A0 the\u00C2\u00A0 physiological\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0pathogens\u00C2\u00A0will\u00C2\u00A0provide\u00C2\u00A0additional\u00C2\u00A0insights\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0phenotype\u00C2\u00A0 by\u00C2\u00A0TLR\u00C2\u00A0agonists.\u00C2\u00A0 \u00C2\u00A0 70 The\u00C2\u00A0 central\u00C2\u00A0 role\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 in\u00C2\u00A0 regulating\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 activation\u00C2\u00A0 makes\u00C2\u00A0 them\u00C2\u00A0 attractive\u00C2\u00A0 therapeutic\u00C2\u00A0 targets.\u00C2\u00A0 Strategies\u00C2\u00A0 that\u00C2\u00A0promote\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 could\u00C2\u00A0 reduce\u00C2\u00A0 autoimmune\u00C2\u00A0 disease\u00C2\u00A0 and\u00C2\u00A0 enhance\u00C2\u00A0 transplantation\u00C2\u00A0 tolerance.\u00C2\u00A0 Alternatively,\u00C2\u00A0 therapies\u00C2\u00A0 that\u00C2\u00A0 reduce\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 iNOS\u00C2\u00A0 inhibitors,\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 LPS,\u00C2\u00A0 or\u00C2\u00A0 dsRNA\u00C2\u00A0 could\u00C2\u00A0 boost\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 and\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 more\u00C2\u00A0 effective\u00C2\u00A0 treatments\u00C2\u00A0 for\u00C2\u00A0 infection\u00C2\u00A0 and\u00C2\u00A0 cancer.\u00C2\u00A0 Furthermore,\u00C2\u00A0 given\u00C2\u00A0 the\u00C2\u00A0 current\u00C2\u00A0 debate\u00C2\u00A0 concerning\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 effective\u00C2\u00A0 vaccine\u00C2\u00A0 adjuvants\u00C2\u00A0 (McKee\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007)\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 contrasting\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 TLR\u00C2\u00A0 agonists\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 and\u00C2\u00A0 potentially\u00C2\u00A0 DCs,\u00C2\u00A0 agonists\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 dsRNA,\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 more\u00C2\u00A0effective\u00C2\u00A0 than\u00C2\u00A0CpG\u00C2\u00A0at\u00C2\u00A0 stimulating\u00C2\u00A0 immune\u00C2\u00A0 responses.\u00C2\u00A0 In\u00C2\u00A0 summary,\u00C2\u00A0 the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0both\u00C2\u00A0immune\u00C2\u00A0response\u00C2\u00A0and\u00C2\u00A0tolerance\u00C2\u00A0highlights\u00C2\u00A0their\u00C2\u00A0considerable\u00C2\u00A0 therapeutic\u00C2\u00A0potential,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0importance\u00C2\u00A0of\u00C2\u00A0increasing\u00C2\u00A0our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0function.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 71 CHAPTER\u00C2\u00A04\u00C2\u00A0: SERUM\u00C2\u00A0INHIBITS\u00C2\u00A0THE\u00C2\u00A0IMMUNOSUPPRESSIVE\u00C2\u00A0FUNCTION\u00C2\u00A0 OF\u00C2\u00A0MYELOID\u00C2\u00ADDERIVED\u00C2\u00A0SUPPRESSOR\u00C2\u00A0CELLS\u00C2\u00A0ISOLATED\u00C2\u00A0FROM\u00C2\u00A04T1\u00C2\u00A0 TUMOR\u00C2\u00ADBEARING\u00C2\u00A0MICE\u00C2\u00A0 \u00C2\u00A0 4.1 Introduction\u00C2\u00A0 \u00C2\u00A0 After\u00C2\u00A0 investigating\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 in\u00C2\u00A0 regulating\u00C2\u00A0 peripheral\u00C2\u00A0 tolerance\u00C2\u00A0 under\u00C2\u00A0physiological\u00C2\u00A0conditions,\u00C2\u00A0we\u00C2\u00A0next\u00C2\u00A0wanted\u00C2\u00A0to\u00C2\u00A0compare\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 types,\u00C2\u00A0 especially\u00C2\u00A0 MDSCs,\u00C2\u00A0 under\u00C2\u00A0 neoplastic\u00C2\u00A0 conditions.\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 a\u00C2\u00A0 heterogeneous\u00C2\u00A0 population\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 expand\u00C2\u00A0 and\u00C2\u00A0 become\u00C2\u00A0 activated\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 tumor\u00E2\u0080\u0090derived\u00C2\u00A0 factors\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 cancer\u00C2\u00A0 patients\u00C2\u00A0 and\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 (Nagaraj\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 MDSCs\u00C2\u00A0 can\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 cancer\u00C2\u00A0 progression\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 mechanisms\u00C2\u00A0 including\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00E2\u0080\u0090mediated\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0(Priceman\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005b;\u00C2\u00A0Yang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02004;\u00C2\u00A0Yang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0 Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 The\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 clearly\u00C2\u00A0 elucidated\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 heterogeneity\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 has\u00C2\u00A0 made\u00C2\u00A0 both\u00C2\u00A0 their\u00C2\u00A0 identification\u00C2\u00A0 and\u00C2\u00A0 isolation\u00C2\u00A0 difficult\u00C2\u00A0 (Nagaraj\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 \u00C2\u00A0 Consequently,\u00C2\u00A0 both\u00C2\u00A0 human\u00C2\u00A0 and\u00C2\u00A0 murine\u00C2\u00A0 MDSCs\u00C2\u00A0 must\u00C2\u00A0 be\u00C2\u00A0 identified\u00C2\u00A0 functionally,\u00C2\u00A0 which\u00C2\u00A0 requires\u00C2\u00A0 assays\u00C2\u00A0 to\u00C2\u00A0 accurately\u00C2\u00A0 assess\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0ex\u00C2\u00A0vivo.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 culture\u00C2\u00A0 systems\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 investigate\u00C2\u00A0 the\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 cancer\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs.\u00C2\u00A0 However,\u00C2\u00A0 the\u00C2\u00A0 influence\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 culture\u00C2\u00A0 conditions\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 ex\u00C2\u00A0 vivo\u00C2\u00A0 remains\u00C2\u00A0 unknown.\u00C2\u00A0 Given\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 functionally\u00C2\u00A0 defined,\u00C2\u00A0 accurate\u00C2\u00A0 assessment\u00C2\u00A0 of\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 is\u00C2\u00A0 extremely\u00C2\u00A0 important\u00C2\u00A0 for\u00C2\u00A0 studying\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 progression.\u00C2\u00A0 One\u00C2\u00A0 murine\u00C2\u00A0 cancer\u00C2\u00A0 model\u00C2\u00A0 often\u00C2\u00A0 employed\u00C2\u00A0 to\u00C2\u00A0 study\u00C2\u00A0 MDSC\u00E2\u0080\u0090induced\u00C2\u00A0 suppression\u00C2\u00A0 involves\u00C2\u00A0 orthotopic\u00C2\u00A0 injection\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 mammary\u00C2\u00A0 carcinoma\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 metastasize\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0 tissues\u00C2\u00A0(Aslakson\u00C2\u00A0and\u00C2\u00A0Miller,\u00C2\u00A01992;\u00C2\u00A0Heppner\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2000).\u00C2\u00A0One\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0primary\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0used\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0groups\u00C2\u00A0 studying\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0(Ko\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010)\u00C2\u00A0or\u00C2\u00A0absence\u00C2\u00A0(Srivastava\u00C2\u00A0et\u00C2\u00A0 72 al.,\u00C2\u00A0 2010)\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 in\u00C2\u00A0 ex\u00C2\u00A0 vivo\u00C2\u00A0 immunosuppression\u00C2\u00A0 assays.\u00C2\u00A0 In\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0 we\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 effectively\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 under\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 conditions,\u00C2\u00A0 but\u00C2\u00A0 fail\u00C2\u00A0 to\u00C2\u00A0 do\u00C2\u00A0 so\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 FCS.\u00C2\u00A0 Furthermore,\u00C2\u00A0we\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0major\u00C2\u00A0serum\u00C2\u00A0protein\u00C2\u00A0albumin\u00C2\u00A0mediates\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0 on\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0by\u00C2\u00A0inhibiting\u00C2\u00A0their\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0ROS.\u00C2\u00A0Our\u00C2\u00A0data\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0 different\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0can\u00C2\u00A0profoundly\u00C2\u00A0alter\u00C2\u00A0MDSC\u00C2\u00A0phenotype.\u00C2\u00A0These\u00C2\u00A0findings\u00C2\u00A0have\u00C2\u00A0 important\u00C2\u00A0 implications\u00C2\u00A0 for\u00C2\u00A0 investigating\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 in\u00C2\u00A0 cancer,\u00C2\u00A0 since\u00C2\u00A0 accurate\u00C2\u00A0 assessment\u00C2\u00A0of\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0is\u00C2\u00A0essential\u00C2\u00A0for\u00C2\u00A0identifying\u00C2\u00A0tumor\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 and\u00C2\u00A0for\u00C2\u00A0studying\u00C2\u00A0the\u00C2\u00A0functional\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0primary\u00C2\u00A0and\u00C2\u00A0metastatic\u00C2\u00A0tumor\u00C2\u00A0growth.\u00C2\u00A0 This\u00C2\u00A0 thesis\u00C2\u00A0 chapter\u00C2\u00A0 is\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 work\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 manuscript\u00C2\u00A0 currently\u00C2\u00A0 in\u00C2\u00A0 press\u00C2\u00A0 (Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0 \u00C2\u00A0 4.2 Results\u00C2\u00A0 \u00C2\u00A0 4.2.1 Validation\u00C2\u00A0of\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0assay\u00C2\u00A0systems\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 test\u00C2\u00A0our\u00C2\u00A0hypothesis\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0 are\u00C2\u00A0influenced\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0culture\u00C2\u00A0conditions,\u00C2\u00A0we\u00C2\u00A0designed\u00C2\u00A0assay\u00C2\u00A0systems\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0 ability\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0polyclonal\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090stimulated\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation.\u00C2\u00A0 As\u00C2\u00A0expected,\u00C2\u00A0splenic\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0BALB/c\u00C2\u00A0mice\u00C2\u00A0proliferated\u00C2\u00A0only\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulation,\u00C2\u00A0while\u00C2\u00A0splenic\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0BALB/c\u00C2\u00A0DO11.10\u00C2\u00A0mice\u00C2\u00A0proliferated\u00C2\u00A0in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 both\u00C2\u00A0 polyclonal\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090stimulation\u00C2\u00A0 (Fig.\u00C2\u00A0 4.1A).\u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 latter\u00C2\u00A0 system,\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulation\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0proliferation\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0therefore\u00C2\u00A0a\u00C2\u00A0 more\u00C2\u00A0 potent\u00C2\u00A0 stimulus\u00C2\u00A0 than\u00C2\u00A0Ag\u00E2\u0080\u0090driven\u00E2\u0080\u0090stimulation,\u00C2\u00A0which\u00C2\u00A0 specifically\u00C2\u00A0 activates\u00C2\u00A0 CD4+\u00C2\u00A0T\u00C2\u00A0 cells.\u00C2\u00A0Initial\u00C2\u00A0tests\u00C2\u00A0also\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0could\u00C2\u00A0proliferate\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0 equal\u00C2\u00A0extent\u00C2\u00A0whether\u00C2\u00A0or\u00C2\u00A0not\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0was\u00C2\u00A0present,\u00C2\u00A0while\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090stimulated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 proliferated\u00C2\u00A0more\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0(Fig.\u00C2\u00A04.1B).\u00C2\u00A0\u00C2\u00A0 73 \u00C2\u00A0 \u00C2\u00A0 4.2.2 4T1\u00C2\u00ADinduced\u00C2\u00A0MDSCs\u00C2\u00A0 only\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 under\u00C2\u00A0 serum\u00C2\u00AD free\u00C2\u00A0conditions\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0isolated\u00C2\u00A0MDSCs\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0spleens\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0Gr1\u00C2\u00A0positive\u00C2\u00A0selection\u00C2\u00A0 and\u00C2\u00A0 established\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 recovered\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 >95%\u00C2\u00A0 CD11b+Gr1+\u00C2\u00A0 and\u00C2\u00A0 contained\u00C2\u00A0 few\u00C2\u00A0 F4/80+\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 4.2A).\u00C2\u00A0 We\u00C2\u00A0 then\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 significantly\u00C2\u00A0 suppressed\u00C2\u00A0 both\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.1\u00C2\u00A0 Polyclonal\u00C2\u00AD\u00C2\u00A0and\u00C2\u00A0Ag\u00C2\u00ADspecific\u00C2\u00ADstimulated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0proliferate\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0serum\u00C2\u00AD free\u00C2\u00A0and\u00C2\u00A0serum\u00C2\u00ADcontaining\u00C2\u00A0conditions.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0BALB/c\u00C2\u00A0 (left)\u00C2\u00A0or\u00C2\u00A0DO11.10\u00C2\u00A0 (right)\u00C2\u00A0 splenocytes\u00C2\u00A0were\u00C2\u00A0 stimulated\u00C2\u00A0with\u00C2\u00A0anti\u00E2\u0080\u0090CD3/anti\u00E2\u0080\u0090CD28\u00C2\u00A0 (polyclonal)\u00C2\u00A0or\u00C2\u00A0OVA\u00C2\u00A0peptide\u00C2\u00A0(Ag\u00E2\u0080\u0090specific)\u00C2\u00A0in\u00C2\u00A0serum\u00E2\u0080\u0090free\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 measured.\u00C2\u00A0No\u00C2\u00A0stim\u00C2\u00A0=\u00C2\u00A0unstimulated\u00C2\u00A0splenocytes.\u00C2\u00A0B,\u00C2\u00A0Splenocytes\u00C2\u00A0were\u00C2\u00A0polyclonal\u00E2\u0080\u0090\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0Ag\u00E2\u0080\u0090 specific\u00E2\u0080\u0090\u00C2\u00A0 (right)\u00C2\u00A0 stimulated\u00C2\u00A0 in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 10%\u00C2\u00A0FCS\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0measured.\u00C2\u00A0 Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0(a)\u00C2\u00A0or\u00C2\u00A0five\u00C2\u00A0(b)\u00C2\u00A0independent\u00C2\u00A0experiments.\u00C2\u00A0***,p<0.001,\u00C2\u00A0NS,\u00C2\u00A0 not\u00C2\u00A0significant\u00C2\u00A0 74 polyclonal\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090stimulated\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0medium,\u00C2\u00A0 but\u00C2\u00A0 were\u00C2\u00A0no\u00C2\u00A0longer\u00C2\u00A0immunosuppressive\u00C2\u00A0when\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0was\u00C2\u00A0present\u00C2\u00A0(Fig.\u00C2\u00A04.2B).\u00C2\u00A0To\u00C2\u00A0ensure\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 results\u00C2\u00A0 were\u00C2\u00A0 not\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 specific\u00C2\u00A0 source\u00C2\u00A0 of\u00C2\u00A0 FCS\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 four\u00C2\u00A0 different\u00C2\u00A0 sources\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0they\u00C2\u00A0all\u00C2\u00A0completely\u00C2\u00A0prevented\u00C2\u00A0MDSC\u00E2\u0080\u0090induced\u00C2\u00A0suppression\u00C2\u00A0 of\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0did\u00C2\u00A0so\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0equal\u00C2\u00A0extent\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0tested\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0 mouse\u00C2\u00A0serum\u00C2\u00A0in\u00C2\u00A0our\u00C2\u00A0assay,\u00C2\u00A0but\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0 proliferate\u00C2\u00A0in\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A010%\u00C2\u00A0mouse\u00C2\u00A0serum\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.2\u00C2\u00A0 MDSCs\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00ADbearing\u00C2\u00A0mice\u00C2\u00A0only\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 under\u00C2\u00A0serum\u00C2\u00ADfree\u00C2\u00A0conditions.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 spleens\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 by\u00C2\u00A0 Gr1\u00C2\u00A0 positive\u00C2\u00A0 selection\u00C2\u00A0and\u00C2\u00A0surface\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0Gr1,\u00C2\u00A0CD11b,\u00C2\u00A0and\u00C2\u00A0F4/80\u00C2\u00A0assayed.\u00C2\u00A0B,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0 (right)\u00C2\u00A0 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 (white\u00C2\u00A0bars)\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A01\u00C2\u00A0splenocyte)\u00C2\u00A0in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 five\u00C2\u00A0 independent\u00C2\u00A0 experiments.\u00C2\u00A0 ***,p<0.001\u00C2\u00A0 75 4.2.3 Serum\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0increase\u00C2\u00A0the\u00C2\u00A0viability\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00ADinduced\u00C2\u00A0MDSCs\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 next\u00C2\u00A0 addressed\u00C2\u00A0 whether\u00C2\u00A0 the\u00C2\u00A0 inability\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0was\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0MDSCs\u00C2\u00A0or\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0First,\u00C2\u00A0we\u00C2\u00A0pre\u00E2\u0080\u0090incubated\u00C2\u00A0MDSCs\u00C2\u00A0O/N\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A010%\u00C2\u00A0FCS,\u00C2\u00A0washed\u00C2\u00A0the\u00C2\u00A0 cells,\u00C2\u00A0and\u00C2\u00A0performed\u00C2\u00A0morphological\u00C2\u00A0analysis\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0M\u00E2\u0080\u0090\u00C2\u00A0versus\u00C2\u00A0 G\u00E2\u0080\u0090MDSC\u00C2\u00A0 subsets\u00C2\u00A0 (Movahedi\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 We\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 although\u00C2\u00A0 M\u00E2\u0080\u0090MDSCs\u00C2\u00A0 were\u00C2\u00A0 present\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0serum\u00E2\u0080\u0090free\u00C2\u00A0and\u00C2\u00A0serum\u00E2\u0080\u0090containing\u00C2\u00A0conditions,\u00C2\u00A0the\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 G\u00E2\u0080\u0090MDSCs\u00C2\u00A0 under\u00C2\u00A0 both\u00C2\u00A0 conditions,\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 or\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 alter\u00C2\u00A0this\u00C2\u00A0ratio\u00C2\u00A0(Fig.\u00C2\u00A04.3A).\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0assayed\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0culturing\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 medium\u00C2\u00A0 containing\u00C2\u00A0 10%\u00C2\u00A0 FCS\u00C2\u00A0 slightly\u00C2\u00A0 reduced\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 and\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A0 proportion\u00C2\u00A0of\u00C2\u00A0non\u00E2\u0080\u0090adherent\u00C2\u00A0cells\u00C2\u00A0(Fig.\u00C2\u00A04.3B).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.3\u00C2\u00A0 Serum\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0alter\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0MDSC\u00C2\u00A0subtypes.\u00C2\u00A0\u00C2\u00A0 MDSCs\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0overnight\u00C2\u00A0 (O/N)\u00C2\u00A0 in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A0A,\u00C2\u00A0 cell\u00C2\u00A0morphology\u00C2\u00A0 (20x\u00C2\u00A0 magnification),\u00C2\u00A0 and\u00C2\u00A0 B,\u00C2\u00A0 viability\u00C2\u00A0 and\u00C2\u00A0 adherence\u00C2\u00A0 were\u00C2\u00A0 determined.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0independent\u00C2\u00A0experiments. 76 4.2.4 Serum\u00C2\u00A0directly\u00C2\u00A0inhibits\u00C2\u00A04T1\u00C2\u00ADinduced\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 pre\u00E2\u0080\u0090incubated\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 under\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 conditions.\u00C2\u00A0 MDSCs\u00C2\u00A0 pre\u00E2\u0080\u0090incubated\u00C2\u00A0 with\u00C2\u00A0 10%\u00C2\u00A0 FCS\u00C2\u00A0 displayed\u00C2\u00A0significantly\u00C2\u00A0reduced\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0under\u00C2\u00A0both\u00C2\u00A0polyclonal\u00C2\u00A0and\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00C2\u00A0conditions\u00C2\u00A0(Fig.\u00C2\u00A04.4A).\u00C2\u00A0Importantly,\u00C2\u00A0serum\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0compromise\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 T\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0suppressed\u00C2\u00A0since\u00C2\u00A0TAMs\u00C2\u00A0(Fig.\u00C2\u00A04.4B)\u00C2\u00A0and\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A04.4C)\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0tumor\u00E2\u0080\u0090 bearing\u00C2\u00A0mice\u00C2\u00A0potently\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0both\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0and\u00C2\u00A0absence\u00C2\u00A0 of\u00C2\u00A0 serum.\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 these\u00C2\u00A0 results\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 FCS\u00C2\u00A0 directly\u00C2\u00A0 ameliorates\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs.\u00C2\u00A0 \u00C2\u00A0 4.2.5 FCS\u00C2\u00A0inhibits\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0dose\u00C2\u00ADdependent\u00C2\u00A0manner\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 understand\u00C2\u00A0 how\u00C2\u00A0 serum\u00C2\u00A0 influences\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 MDSCs,\u00C2\u00A0 we\u00C2\u00A0 first\u00C2\u00A0 assayed\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 concentrations\u00C2\u00A0 of\u00C2\u00A0 FCS.\u00C2\u00A0As\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Fig.\u00C2\u00A0 4.5,\u00C2\u00A0 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Serum\u00C2\u00A0directly\u00C2\u00A0inhibits\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 Splenic\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 O/N\u00C2\u00A0 in\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 10%\u00C2\u00A0 FCS\u00E2\u0080\u0090containing\u00C2\u00A0 (black\u00C2\u00A0bars)\u00C2\u00A0HL1\u00C2\u00A0medium,\u00C2\u00A0washed,\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0ratios\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090\u00C2\u00A0(left)\u00C2\u00A0 or\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0(right)\u00C2\u00A0stimulated\u00C2\u00A0splenocytes\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0B,\u00C2\u00A0TAMs\u00C2\u00A0or\u00C2\u00A0 C,\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A02\u00C2\u00A0splenocytes)\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0(right)\u00C2\u00A0 stimulated\u00C2\u00A0splenocytes\u00C2\u00A0 in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0 are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0independent\u00C2\u00A0experiments.\u00C2\u00A0*,p<0.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<0.001\u00C2\u00A0\u00C2\u00A0 78 \u00C2\u00A0 \u00C2\u00A0 4.2.6 Effect\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0serum\u00C2\u00A0treatments\u00C2\u00A0on\u00C2\u00A0MDSC\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0 that\u00C2\u00A0 had\u00C2\u00A0 been\u00C2\u00A0 filtered\u00C2\u00A0 to\u00C2\u00A0 remove\u00C2\u00A0 components\u00C2\u00A0 >0.22\u00C2\u00A0 \u00C2\u00B5m\u00C2\u00A0 or\u00C2\u00A0 dialyzed\u00C2\u00A0 to\u00C2\u00A0 remove\u00C2\u00A0 components\u00C2\u00A0<3.5\u00C2\u00A0kDa.\u00C2\u00A0Neither\u00C2\u00A0 treatment\u00C2\u00A0restored\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0 pulmonary\u00C2\u00A0 or\u00C2\u00A0 splenic\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs,\u00C2\u00A0 although\u00C2\u00A0 dialysis\u00C2\u00A0 may\u00C2\u00A0 have\u00C2\u00A0 slightly\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0pulmonary\u00C2\u00A0MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 (Fig.\u00C2\u00A0 4.6A).\u00C2\u00A0 Similarly,\u00C2\u00A0 heat\u00E2\u0080\u0090inactivating\u00C2\u00A0 the\u00C2\u00A0 serum\u00C2\u00A0 to\u00C2\u00A0 destroy\u00C2\u00A0 complement\u00C2\u00A0 activity\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 restore\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 (Fig.\u00C2\u00A0 4.6B).\u00C2\u00A0 Our\u00C2\u00A0 experiments\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 using\u00C2\u00A0 HL1\u00C2\u00A0 medium\u00C2\u00A0 and\u00C2\u00A0 clearly\u00C2\u00A0 demonstrate\u00C2\u00A0 the\u00C2\u00A0 inhibitory\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 on\u00C2\u00A0 MDSC\u00C2\u00A0 immune\u00C2\u00A0 suppression.\u00C2\u00A0There\u00C2\u00A0are\u00C2\u00A0some\u00C2\u00A0reports\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0literature\u00C2\u00A0that\u00C2\u00A0splenic\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 are\u00C2\u00A0immunosuppressive\u00C2\u00A0when\u00C2\u00A0assayed\u00C2\u00A0in\u00C2\u00A0RPMI\u00C2\u00A01640\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0(Ko\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Kodumudi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Le\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0However,\u00C2\u00A0in\u00C2\u00A0our\u00C2\u00A0hands,\u00C2\u00A0both\u00C2\u00A0splenic\u00C2\u00A0 and\u00C2\u00A0 pulmonary\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 unable\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 RPMI\u00C2\u00A0 1640\u00C2\u00A0 medium\u00C2\u00A0containing\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0and,\u00C2\u00A0 in\u00C2\u00A0fact,\u00C2\u00A0pulmonary\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0appeared\u00C2\u00A0to\u00C2\u00A0 activate\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0proliferation\u00C2\u00A0under\u00C2\u00A0 these\u00C2\u00A0 conditions\u00C2\u00A0 (Fig.\u00C2\u00A04.6C).\u00C2\u00A0 Similarly,\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.5\u00C2\u00A0 The\u00C2\u00A0 inhibitory\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 on\u00C2\u00A0 4T1\u00C2\u00ADinduced\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0are\u00C2\u00A0dose\u00C2\u00ADdependent.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0pulmonary\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0splenic\u00C2\u00A0(right)\u00C2\u00A0MDSCs\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A01\u00C2\u00A0splenocyte)\u00C2\u00A0in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A01,\u00C2\u00A0 3,\u00C2\u00A0 or\u00C2\u00A0 10%\u00C2\u00A0 FCS\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments.\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0 79 MDSCs\u00C2\u00A0 failed\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 when\u00C2\u00A0 cultured\u00C2\u00A0 in\u00C2\u00A0 IMDM\u00C2\u00A0 medium\u00C2\u00A0 containing\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.6\u00C2\u00A0 The\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 on\u00C2\u00A0 4T1\u00C2\u00ADinduced\u00C2\u00A0 MDSCs\u00C2\u00A0 cannot\u00C2\u00A0 be\u00C2\u00A0 reversed\u00C2\u00A0 by\u00C2\u00A0 filtration,\u00C2\u00A0dialyzation,\u00C2\u00A0or\u00C2\u00A0heat\u00C2\u00A0inactivation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0serum.\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0pulmonary\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0splenic\u00C2\u00A0(right)\u00C2\u00A0MDSCs\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A01\u00C2\u00A0splenocyte)\u00C2\u00A0in\u00C2\u00A0A,\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 10%\u00C2\u00A0untreated,\u00C2\u00A0filtered,\u00C2\u00A0or\u00C2\u00A0dialyzed\u00C2\u00A0FCS,\u00C2\u00A0or\u00C2\u00A0B,\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010%\u00C2\u00A0heat\u00E2\u0080\u0090inactivated\u00C2\u00A0FCS,\u00C2\u00A0or\u00C2\u00A0C,\u00C2\u00A0 RPMI\u00C2\u00A01640\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010%\u00C2\u00A0untreated\u00C2\u00A0FCS,\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0independent\u00C2\u00A0experiments.\u00C2\u00A0*,p<0.05;\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0 80 4.2.7 BSA\u00C2\u00A0blunts\u00C2\u00A04T1\u00C2\u00ADinduced\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0the\u00C2\u00A0antagonism\u00C2\u00A0by\u00C2\u00A0serum\u00C2\u00A0of\u00C2\u00A0MDSC\u00E2\u0080\u0090induced\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0could\u00C2\u00A0not\u00C2\u00A0 be\u00C2\u00A0ameliorated\u00C2\u00A0by\u00C2\u00A0filtration,\u00C2\u00A0dialysis,\u00C2\u00A0or\u00C2\u00A0heat\u00E2\u0080\u0090inactivation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0serum,\u00C2\u00A0we\u00C2\u00A0considered\u00C2\u00A0 whether\u00C2\u00A0any\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0key\u00C2\u00A0protein\u00C2\u00A0components\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0playing\u00C2\u00A0a\u00C2\u00A0role.\u00C2\u00A0BSA\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0 most\u00C2\u00A0 abundant\u00C2\u00A0 protein\u00C2\u00A0 in\u00C2\u00A0 FCS\u00C2\u00A0 (Seifert\u00C2\u00A0 and\u00C2\u00A0 Resman\u00E2\u0080\u0090Targoff,\u00C2\u00A0 2006)\u00C2\u00A0 and\u00C2\u00A0 is\u00C2\u00A0 commonly\u00C2\u00A0 added\u00C2\u00A0to\u00C2\u00A0serum\u00E2\u0080\u0090deprived\u00C2\u00A0cultures\u00C2\u00A0to\u00C2\u00A0support\u00C2\u00A0cell\u00C2\u00A0growth\u00C2\u00A0(Monette\u00C2\u00A0and\u00C2\u00A0Sigounas,\u00C2\u00A01990).\u00C2\u00A0 We\u00C2\u00A0tested\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0BSA\u00C2\u00A0on\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0 BSA\u00C2\u00A0 decreased\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 pulmonary\u00C2\u00A0 and\u00C2\u00A0 splenic\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0dose\u00E2\u0080\u0090dependent\u00C2\u00A0manner\u00C2\u00A0(Fig.\u00C2\u00A04.7A).\u00C2\u00A0In\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0BSA,\u00C2\u00A0pulmonary\u00C2\u00A0 and\u00C2\u00A0 splenic\u00C2\u00A0MDSCs\u00C2\u00A0 suppressed\u00C2\u00A0 96.9%\u00C2\u00A0and\u00C2\u00A0 98.3%\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation,\u00C2\u00A0 respectively.\u00C2\u00A0 This\u00C2\u00A0 suppression\u00C2\u00A0was\u00C2\u00A0 reduced\u00C2\u00A0 to\u00C2\u00A0 51.4%\u00C2\u00A0 and\u00C2\u00A0 43.6%\u00C2\u00A0with\u00C2\u00A0 1%\u00C2\u00A0BSA,\u00C2\u00A0 and\u00C2\u00A0 to\u00C2\u00A0 29.8%\u00C2\u00A0 and\u00C2\u00A0 21.6%\u00C2\u00A0when\u00C2\u00A03%\u00C2\u00A0BSA\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0assay.\u00C2\u00A0Soluble\u00C2\u00A0BSA\u00C2\u00A0is\u00C2\u00A0400\u00C2\u00A0x\u00C2\u00A01400\u00C2\u00A0nm\u00C2\u00A0in\u00C2\u00A0size\u00C2\u00A0with\u00C2\u00A0 a\u00C2\u00A0molecular\u00C2\u00A0mass\u00C2\u00A0of\u00C2\u00A066.4\u00C2\u00A0kDa\u00C2\u00A0and,\u00C2\u00A0 therefore,\u00C2\u00A0would\u00C2\u00A0not\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0removed\u00C2\u00A0by\u00C2\u00A0either\u00C2\u00A0 filtration\u00C2\u00A0(>0.22\u00C2\u00A0\u00C2\u00B5m)\u00C2\u00A0or\u00C2\u00A0dialysis\u00C2\u00A0(<3.5\u00C2\u00A0kDa)\u00C2\u00A0of\u00C2\u00A0FCS,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0 (Fig.4.6A).\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0tested\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0human\u00C2\u00A0albumin\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0second\u00C2\u00A0source\u00C2\u00A0of\u00C2\u00A0BSA\u00C2\u00A0and\u00C2\u00A0 found\u00C2\u00A0that\u00C2\u00A0both\u00C2\u00A0significantly\u00C2\u00A0reduced\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0To\u00C2\u00A0verify\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0on\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0was\u00C2\u00A0 due\u00C2\u00A0to\u00C2\u00A0BSA,\u00C2\u00A0we\u00C2\u00A0removed\u00C2\u00A0BSA\u00C2\u00A0from\u00C2\u00A0FCS\u00C2\u00A0using\u00C2\u00A0Affi\u00E2\u0080\u0090Gel\u00C2\u00A0Blue\u00C2\u00A0bead\u00C2\u00A0affinity\u00C2\u00A0chromatography\u00C2\u00A0 and\u00C2\u00A0 compared\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 serum\u00C2\u00A0with\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0BSA.\u00C2\u00A0At\u00C2\u00A0all\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0tested,\u00C2\u00A0removing\u00C2\u00A0BSA\u00C2\u00A0restored\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 (Fig.\u00C2\u00A04.7B),\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0BSA\u00C2\u00A0 inhibits\u00C2\u00A0MDSC\u00C2\u00A0 immunosuppression.\u00C2\u00A0 Since\u00C2\u00A0 BSA\u00C2\u00A0 contains\u00C2\u00A0 both\u00C2\u00A0 high\u00C2\u00A0 and\u00C2\u00A0 low\u00C2\u00A0 affinity\u00C2\u00A0 binding\u00C2\u00A0 sites\u00C2\u00A0 for\u00C2\u00A0 fatty\u00C2\u00A0acids\u00C2\u00A0(FAs),\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0is\u00C2\u00A0an\u00C2\u00A0efficient\u00C2\u00A0source\u00C2\u00A0of\u00C2\u00A0lipids\u00C2\u00A0(Fasano\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005;\u00C2\u00A0Roche\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2008),\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 whether\u00C2\u00A0 BSA\u00E2\u0080\u0090associated\u00C2\u00A0 FAs\u00C2\u00A0 were\u00C2\u00A0 affecting\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression.\u00C2\u00A0 Interestingly,\u00C2\u00A0 we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 MDSC\u00C2\u00A0 suppression\u00C2\u00A0 was\u00C2\u00A0 restored\u00C2\u00A0 when\u00C2\u00A0 the\u00C2\u00A0 assay\u00C2\u00A0 was\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 medium\u00C2\u00A0 containing\u00C2\u00A0 FA\u00E2\u0080\u0090free\u00C2\u00A0 BSA\u00C2\u00A0 (Fig.\u00C2\u00A0 4.7C),\u00C2\u00A0 suggesting\u00C2\u00A0 that\u00C2\u00A0 BSA\u00E2\u0080\u0090associated\u00C2\u00A0 FAs\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 inhibiting\u00C2\u00A0 MDSC\u00C2\u00A0 immune\u00C2\u00A0 suppression.\u00C2\u00A0 81 \u00C2\u00A0 Figure\u00C2\u00A04.7\u00C2\u00A0 BSA\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00ADinduced\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0pulmonary\u00C2\u00A0 (left)\u00C2\u00A0or\u00C2\u00A0 splenic\u00C2\u00A0 (right)\u00C2\u00A0MDSCs\u00C2\u00A0 (1\u00C2\u00A0MDSC:\u00C2\u00A01\u00C2\u00A0 splenocyte)\u00C2\u00A0 in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 0.3,\u00C2\u00A0 1,\u00C2\u00A0 or\u00C2\u00A0 3%\u00C2\u00A0BSA\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0measured.\u00C2\u00A0B,\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0 pulmonary\u00C2\u00A0 (left)\u00C2\u00A0 or\u00C2\u00A0 splenic\u00C2\u00A0 (right)\u00C2\u00A0MDSCs\u00C2\u00A0(1\u00C2\u00A0MDSC:1\u00C2\u00A0splenocyte)\u00C2\u00A0in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0\u00C2\u00B1\u00C2\u00A01,\u00C2\u00A03,\u00C2\u00A0or\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0untreated\u00C2\u00A0 or\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0been\u00C2\u00A0depleted\u00C2\u00A0of\u00C2\u00A0BSA\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0C,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0pulmonary\u00C2\u00A0(left)\u00C2\u00A0or\u00C2\u00A0 splenic\u00C2\u00A0 (right)\u00C2\u00A0MDSCs\u00C2\u00A0 (1\u00C2\u00A0MDSC:1\u00C2\u00A0 splenocyte)\u00C2\u00A0 in\u00C2\u00A0HL1\u00C2\u00A0medium\u00C2\u00A0 containing\u00C2\u00A0 3%\u00C2\u00A0BSA\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 fatty\u00E2\u0080\u0090 acids\u00C2\u00A0(FAs)\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0measured.\u00C2\u00A0\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0independent\u00C2\u00A0 experiments.\u00C2\u00A0*,p<0.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 82 4.2.8 BSA\u00C2\u00A0antagonizes\u00C2\u00A04T1\u00C2\u00ADinduced\u00C2\u00A0MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0by\u00C2\u00A0 inhibiting\u00C2\u00A0 ROS\u00C2\u00A0production\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0 we\u00C2\u00A0 embarked\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 studies\u00C2\u00A0 to\u00C2\u00A0 investigate\u00C2\u00A0 the\u00C2\u00A0mechanism(s)\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0BSA\u00C2\u00A0inhibits\u00C2\u00A0MDSC\u00E2\u0080\u0090induced\u00C2\u00A0immune\u00C2\u00A0suppression.\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0to\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0mechanisms\u00C2\u00A0including\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0 iNOS\u00C2\u00A0 and/or\u00C2\u00A0 Arg1,\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 inhibitory\u00C2\u00A0 cytokines\u00C2\u00A0 (i.e.\u00C2\u00A0 TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u009010),\u00C2\u00A0 and\u00C2\u00A0 generation\u00C2\u00A0 of\u00C2\u00A0 ROS\u00C2\u00A0 species\u00C2\u00A0 (Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 Therefore,\u00C2\u00A0 we\u00C2\u00A0 investigated\u00C2\u00A0 whether\u00C2\u00A0BSA\u00C2\u00A0was\u00C2\u00A0 interfering\u00C2\u00A0with\u00C2\u00A0any\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0processes.\u00C2\u00A0We\u00C2\u00A0cultured\u00C2\u00A0MDSCs\u00C2\u00A0O/N\u00C2\u00A0in\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 or\u00C2\u00A0 10%\u00C2\u00A0 FCS\u00C2\u00A0 or\u00C2\u00A0 3%\u00C2\u00A0BSA\u00C2\u00A0 containing\u00C2\u00A0medium\u00C2\u00A0 (as\u00C2\u00A0 our\u00C2\u00A0 previous\u00C2\u00A0 studies\u00C2\u00A0 had\u00C2\u00A0 demonstrated\u00C2\u00A0 this\u00C2\u00A0 was\u00C2\u00A0 sufficient\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 function\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 (Fig.\u00C2\u00A0 4.4A)).\u00C2\u00A0Western\u00C2\u00A0blot\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0cell\u00C2\u00A0 lysates\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0both\u00C2\u00A0pulmonary\u00C2\u00A0and\u00C2\u00A0splenic\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 failed\u00C2\u00A0 to\u00C2\u00A0 express\u00C2\u00A0 either\u00C2\u00A0 iNOS\u00C2\u00A0or\u00C2\u00A0Arg1\u00C2\u00A0under\u00C2\u00A0 any\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0 culture\u00C2\u00A0 conditions\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0Furthermore,\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0or\u00C2\u00A0BSA\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0influence\u00C2\u00A0 the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0below\u00C2\u00A0the\u00C2\u00A0detection\u00C2\u00A0 limit\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0samples,\u00C2\u00A0or\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0 which\u00C2\u00A0was\u00C2\u00A0produced\u00C2\u00A0at\u00C2\u00A0low,\u00C2\u00A0but\u00C2\u00A0similar,\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0samples\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0Next,\u00C2\u00A0we\u00C2\u00A0 tested\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0or\u00C2\u00A0BSA\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0generate\u00C2\u00A0ROS\u00C2\u00A0and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 culturing\u00C2\u00A0 MDSCs\u00C2\u00A0 O/N\u00C2\u00A0 in\u00C2\u00A0 FCS\u00C2\u00A0 or\u00C2\u00A0 BSA\u00C2\u00A0 reduced\u00C2\u00A0 ROS\u00C2\u00A0 production\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 large\u00C2\u00A0 extent\u00C2\u00A0(Figure\u00C2\u00A04.8).\u00C2\u00A0Specifically,\u00C2\u00A0pulmonary\u00C2\u00A0MDSCs\u00C2\u00A0cultured\u00C2\u00A0in\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A0FCS\u00C2\u00A0 or\u00C2\u00A0BSA\u00C2\u00A0produced\u00C2\u00A03.6\u00C2\u00A0 fold\u00C2\u00A0and\u00C2\u00A06.5\u00C2\u00A0 fold\u00C2\u00A0 less\u00C2\u00A0ROS,\u00C2\u00A0respectively.\u00C2\u00A0Similarly,\u00C2\u00A0splenic\u00C2\u00A0MDSCs\u00C2\u00A0 produced\u00C2\u00A0 5.2\u00C2\u00A0 and\u00C2\u00A0 6.5\u00C2\u00A0 fold\u00C2\u00A0 less\u00C2\u00A0 ROS\u00C2\u00A0 when\u00C2\u00A0 cultured\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 FCS\u00C2\u00A0 or\u00C2\u00A0 BSA,\u00C2\u00A0 respectively.\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 our\u00C2\u00A0 results\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0greatly\u00C2\u00A0influenced\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0BSA\u00C2\u00A0 contained\u00C2\u00A0 in\u00C2\u00A0 FCS\u00C2\u00A0 blunts\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090derived\u00C2\u00A0 MDSCs,\u00C2\u00A0at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0part,\u00C2\u00A0by\u00C2\u00A0restricting\u00C2\u00A0ROS\u00C2\u00A0production.\u00C2\u00A0These\u00C2\u00A0 findings\u00C2\u00A0are\u00C2\u00A0vital\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 appropriate\u00C2\u00A0 design\u00C2\u00A0 and\u00C2\u00A0 interpretation\u00C2\u00A0 of\u00C2\u00A0 immunosuppression\u00C2\u00A0 assays\u00C2\u00A0 and\u00C2\u00A0 may\u00C2\u00A0 shed\u00C2\u00A0 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measured\u00C2\u00A0using\u00C2\u00A0DCFDA\u00C2\u00A0dye.\u00C2\u00A0B,\u00C2\u00A0Mean\u00C2\u00A0fluorescent\u00C2\u00A0intensity\u00C2\u00A0(MFI)\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0cultured\u00C2\u00A0O/N\u00C2\u00A0in\u00C2\u00A0 HL1\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A0no\u00C2\u00A0serum\u00C2\u00A0(grey\u00C2\u00A0fill),\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0(black\u00C2\u00A0line)\u00C2\u00A0or\u00C2\u00A03%\u00C2\u00A0BSA\u00C2\u00A0(grey\u00C2\u00A0line)\u00C2\u00A0and\u00C2\u00A0 labeled\u00C2\u00A0 with\u00C2\u00A0 DCFDA\u00C2\u00A0 dye.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 (A)\u00C2\u00A0 representative\u00C2\u00A0 or\u00C2\u00A0 (B)\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments.\u00C2\u00A0\u00C2\u00A0 84 4.3 Discussion\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0defined\u00C2\u00A0by\u00C2\u00A0their\u00C2\u00A0immunosuppressive\u00C2\u00A0properties,\u00C2\u00A0our\u00C2\u00A0work\u00C2\u00A0 demonstrates\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0influenced\u00C2\u00A0 by\u00C2\u00A0different\u00C2\u00A0culture\u00C2\u00A0conditions,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0or\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0serum.\u00C2\u00A0Specifically,\u00C2\u00A0 while\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 potently\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 under\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0 conditions,\u00C2\u00A0 they\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0 suppressive\u00C2\u00A0when\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0was\u00C2\u00A0 included\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 assay.\u00C2\u00A0Our\u00C2\u00A0 data\u00C2\u00A0 identify\u00C2\u00A0 BSA\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 principal\u00C2\u00A0 factor\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 on\u00C2\u00A0 4T1\u00E2\u0080\u0090 induced\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0model\u00C2\u00A0in\u00C2\u00A0which\u00C2\u00A0serum\u00C2\u00A0albumin\u00C2\u00A0acts\u00C2\u00A0directly\u00C2\u00A0 on\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 decrease\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 by\u00C2\u00A0 decreasing\u00C2\u00A0 ROS\u00C2\u00A0 production.\u00C2\u00A0Moreover,\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0some\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0BSA\u00E2\u0080\u0090associated\u00C2\u00A0FAs\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0 in\u00C2\u00A0 regulating\u00C2\u00A0 MDSC\u00C2\u00A0 suppression.\u00C2\u00A0 These\u00C2\u00A0 findings\u00C2\u00A0 have\u00C2\u00A0 important\u00C2\u00A0 implications\u00C2\u00A0 for\u00C2\u00A0 researchers\u00C2\u00A0 investigating\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 cancer,\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 accurate\u00C2\u00A0 detection\u00C2\u00A0 and\u00C2\u00A0 quantification\u00C2\u00A0 of\u00C2\u00A0 immunosuppression\u00C2\u00A0 is\u00C2\u00A0 critical\u00C2\u00A0 for\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 identification\u00C2\u00A0 and\u00C2\u00A0 functional\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 our\u00C2\u00A0 studies\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 serum\u00C2\u00A0 interferes\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0of\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs,\u00C2\u00A0we\u00C2\u00A0performed\u00C2\u00A0a\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0experiments\u00C2\u00A0to\u00C2\u00A0elucidate\u00C2\u00A0the\u00C2\u00A0 mechanism(s)\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 this\u00C2\u00A0 occurs.\u00C2\u00A0 Although\u00C2\u00A0 an\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 individual\u00C2\u00A0 components\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 most\u00C2\u00A0 helpful\u00C2\u00A0 to\u00C2\u00A0 address\u00C2\u00A0 this,\u00C2\u00A0 serum\u00C2\u00A0 represents\u00C2\u00A0 a\u00C2\u00A0 complex\u00C2\u00A0mixture\u00C2\u00A0 of\u00C2\u00A0 proteins\u00C2\u00A0 and\u00C2\u00A0other\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0well\u00C2\u00A0 defined\u00C2\u00A0 (Barnes\u00C2\u00A0 and\u00C2\u00A0 Sato,\u00C2\u00A0 1980;\u00C2\u00A0 Dainiak,\u00C2\u00A0 1985).\u00C2\u00A0 Nevertheless,\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 albumin,\u00C2\u00A0 a\u00C2\u00A0 soluble\u00C2\u00A0 protein\u00C2\u00A0 that\u00C2\u00A0 comprises\u00C2\u00A0 approximately\u00C2\u00A0 50\u00C2\u00A0 to\u00C2\u00A0 60%\u00C2\u00A0 of\u00C2\u00A0 total\u00C2\u00A0 protein\u00C2\u00A0 in\u00C2\u00A0 blood\u00C2\u00A0 plasma\u00C2\u00A0 (Seifert\u00C2\u00A0 and\u00C2\u00A0Resman\u00E2\u0080\u0090Targoff,\u00C2\u00A0 2006),\u00C2\u00A0 strongly\u00C2\u00A0 inhibits\u00C2\u00A0MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 (Fig.\u00C2\u00A0 4.7).\u00C2\u00A0Albumin,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 synthesized\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 liver,\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 important\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 vivo:\u00C2\u00A0 it\u00C2\u00A0controls\u00C2\u00A0oncotic\u00C2\u00A0pressure\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma,\u00C2\u00A0transports\u00C2\u00A0amino\u00C2\u00A0acids\u00C2\u00A0synthesized\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 liver\u00C2\u00A0 to\u00C2\u00A0 other\u00C2\u00A0 tissues,\u00C2\u00A0 and\u00C2\u00A0 acts\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 carrier\u00C2\u00A0 protein\u00C2\u00A0 for\u00C2\u00A0 ligands\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 relatively\u00C2\u00A0 insoluble\u00C2\u00A0 in\u00C2\u00A0 plasma,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 fatty\u00C2\u00A0 acids,\u00C2\u00A0metals,\u00C2\u00A0 cholesterol,\u00C2\u00A0 bile\u00C2\u00A0 pigments,\u00C2\u00A0 hormones,\u00C2\u00A0 and\u00C2\u00A0drugs\u00C2\u00A0(Fasano\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005;\u00C2\u00A0Seifert\u00C2\u00A0and\u00C2\u00A0Resman\u00E2\u0080\u0090Targoff,\u00C2\u00A02006).\u00C2\u00A0In\u00C2\u00A0addition,\u00C2\u00A0albumin\u00C2\u00A0 serves\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0important\u00C2\u00A0antioxidant,\u00C2\u00A0both\u00C2\u00A0directly\u00C2\u00A0via\u00C2\u00A0its\u00C2\u00A0thiol\u00C2\u00A0group\u00C2\u00A0and\u00C2\u00A0indirectly\u00C2\u00A0via\u00C2\u00A0its\u00C2\u00A0 ligand\u00E2\u0080\u0090binding\u00C2\u00A0 capabilities\u00C2\u00A0 (Fasano\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005;\u00C2\u00A0 Roche\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 Albumin\u00C2\u00A0 likely\u00C2\u00A0 85 carries\u00C2\u00A0out\u00C2\u00A0similar\u00C2\u00A0functions\u00C2\u00A0in\u00C2\u00A0vitro.\u00C2\u00A0In\u00C2\u00A0addition\u00C2\u00A0to\u00C2\u00A0serving\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0carrier\u00C2\u00A0for\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0 support\u00C2\u00A0cell\u00C2\u00A0growth,\u00C2\u00A0including\u00C2\u00A0FAs,\u00C2\u00A0trace\u00C2\u00A0minerals,\u00C2\u00A0hormones,\u00C2\u00A0and\u00C2\u00A0growth\u00C2\u00A0factors,\u00C2\u00A0there\u00C2\u00A0 are\u00C2\u00A0also\u00C2\u00A0reports\u00C2\u00A0that\u00C2\u00A0albumin\u00C2\u00A0can\u00C2\u00A0protect\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0mechanical\u00C2\u00A0shear\u00C2\u00A0damage\u00C2\u00A0and\u00C2\u00A0act\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 detoxifying\u00C2\u00A0 agent\u00C2\u00A0 for\u00C2\u00A0 H2O2\u00C2\u00A0 (Monette\u00C2\u00A0 and\u00C2\u00A0 Sigounas,\u00C2\u00A0 1990;\u00C2\u00A0 Roche\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 Interestingly,\u00C2\u00A0 we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 albumin\u00C2\u00A0 stripped\u00C2\u00A0 of\u00C2\u00A0 FAs\u00C2\u00A0 blocked\u00C2\u00A0 MDSC\u00E2\u0080\u0090induced\u00C2\u00A0 immunosuppression\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0lesser\u00C2\u00A0extent\u00C2\u00A0than\u00C2\u00A0control\u00C2\u00A0albumin\u00C2\u00A0(Fig.\u00C2\u00A04.7c),\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0 FAs\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0albumin\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0inhibiting\u00C2\u00A0MDSC\u00E2\u0080\u0090induced\u00C2\u00A0immunosuppression.\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0 addition,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0 that\u00C2\u00A0 albumin\u00C2\u00A0may\u00C2\u00A0 act\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 carrier\u00C2\u00A0 for\u00C2\u00A0 other\u00C2\u00A0molecules\u00C2\u00A0 that\u00C2\u00A0 restrict\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0 \u00C2\u00A0 Our\u00C2\u00A0 data\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 albumin\u00C2\u00A0 restricts\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0MDSCs,\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0 part,\u00C2\u00A0 by\u00C2\u00A0 decreasing\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 ROS.\u00C2\u00A0 MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 variety\u00C2\u00A0 of\u00C2\u00A0 murine\u00C2\u00A0 and\u00C2\u00A0 human\u00C2\u00A0 tumors\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reported\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 ROS,\u00C2\u00A0 including\u00C2\u00A0 H2O2,\u00C2\u00A0 O2\u00E2\u0080\u0090,\u00C2\u00A0 and\u00C2\u00A0 peroxynitrites\u00C2\u00A0 (Nagaraj\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010;\u00C2\u00A0 Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010).\u00C2\u00A0 Although\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 clearly\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 exposing\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0serum\u00C2\u00A0or\u00C2\u00A0albumin\u00C2\u00A0decreases\u00C2\u00A0ROS\u00C2\u00A0production\u00C2\u00A0(Fig.\u00C2\u00A04.8),\u00C2\u00A0 the\u00C2\u00A0flow\u00C2\u00A0cytometric\u00C2\u00A0approach\u00C2\u00A0we\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0assay\u00C2\u00A0ROS\u00C2\u00A0detects\u00C2\u00A0generalized\u00C2\u00A0oxidative\u00C2\u00A0stress\u00C2\u00A0 rather\u00C2\u00A0than\u00C2\u00A0any\u00C2\u00A0specific\u00C2\u00A0ROS\u00C2\u00A0(Eruslanov\u00C2\u00A0and\u00C2\u00A0Kusmartsev,\u00C2\u00A02010);\u00C2\u00A0therefore,\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0 ROS\u00C2\u00A0 involved\u00C2\u00A0 remain\u00C2\u00A0 unknown\u00C2\u00A0 and\u00C2\u00A0 their\u00C2\u00A0 identification\u00C2\u00A0 will\u00C2\u00A0 be\u00C2\u00A0 the\u00C2\u00A0 subject\u00C2\u00A0 of\u00C2\u00A0 future\u00C2\u00A0 studies.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0demonstrated\u00C2\u00A0that\u00C2\u00A0serum\u00C2\u00A0albumin\u00C2\u00A0inhibits\u00C2\u00A0the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0 ROS,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0possible\u00C2\u00A0that\u00C2\u00A0other\u00C2\u00A0components\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0and/or\u00C2\u00A0other\u00C2\u00A0mechanisms\u00C2\u00A0could\u00C2\u00A0also\u00C2\u00A0 be\u00C2\u00A0involved.\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0a\u00C2\u00A0recent\u00C2\u00A0report\u00C2\u00A0has\u00C2\u00A0suggested\u00C2\u00A0that\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0can\u00C2\u00A0 suppress\u00C2\u00A0 Ag\u00E2\u0080\u0090stimulated\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 by\u00C2\u00A0 restricting\u00C2\u00A0 the\u00C2\u00A0 availability\u00C2\u00A0 of\u00C2\u00A0 cysteine,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 an\u00C2\u00A0essential\u00C2\u00A0 amino\u00C2\u00A0acid\u00C2\u00A0 for\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 (Srivastava\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 therefore\u00C2\u00A0 possible\u00C2\u00A0 that\u00C2\u00A0 serum\u00C2\u00A0 contains\u00C2\u00A0 additional\u00C2\u00A0 cysteine/cystine\u00C2\u00A0 and\u00C2\u00A0 thus\u00C2\u00A0 prevents\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 from\u00C2\u00A0 suppressing\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation.\u00C2\u00A0 \u00C2\u00A0 However,\u00C2\u00A0 this\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 appear\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0the\u00C2\u00A0case\u00C2\u00A0since\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0were\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0potently\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0proliferation\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0dialyzed\u00C2\u00A0serum\u00C2\u00A0(Fig.\u00C2\u00A04.6A),\u00C2\u00A0which\u00C2\u00A0 lacks\u00C2\u00A0molecules\u00C2\u00A0 smaller\u00C2\u00A0 than\u00C2\u00A0 3.5\u00C2\u00A0 kDa,\u00C2\u00A0 including\u00C2\u00A0 amino\u00C2\u00A0 acids\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 cysteine\u00C2\u00A0 (121.15\u00C2\u00A0 Da).\u00C2\u00A0 Previous\u00C2\u00A0 86 studies\u00C2\u00A0 have\u00C2\u00A0 also\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 via\u00C2\u00A0 an\u00C2\u00A0 Arg1\u00E2\u0080\u0090 dependent\u00C2\u00A0mechanism\u00C2\u00A0(Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005c).\u00C2\u00A0Conceivably,\u00C2\u00A0proteins\u00C2\u00A0contained\u00C2\u00A0 in\u00C2\u00A0serum\u00C2\u00A0 may\u00C2\u00A0 inhibit\u00C2\u00A0MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 by\u00C2\u00A0 interfering\u00C2\u00A0with\u00C2\u00A0 Arg1\u00C2\u00A0 expression\u00C2\u00A0 or\u00C2\u00A0 L\u00E2\u0080\u0090Arg\u00C2\u00A0 metabolism,\u00C2\u00A0 although\u00C2\u00A0 our\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0 neither\u00C2\u00A0 pulmonary\u00C2\u00A0 nor\u00C2\u00A0 splenic\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0express\u00C2\u00A0Arg1\u00C2\u00A0directly\u00C2\u00A0ex\u00C2\u00A0vivo\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown)\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0support\u00C2\u00A0this\u00C2\u00A0hypothesis.\u00C2\u00A0 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to\u00C2\u00A0serum\u00C2\u00A0factors.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 While\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 some\u00C2\u00A0 reports\u00C2\u00A0 of\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 suppressing\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0under\u00C2\u00A0serum\u00E2\u0080\u0090containing\u00C2\u00A0culture\u00C2\u00A0conditions,\u00C2\u00A0i.e.,\u00C2\u00A0in\u00C2\u00A0RPMI\u00C2\u00A01640\u00C2\u00A0supplemented\u00C2\u00A0 with\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0(Ko\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Kodumudi\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Le\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009),\u00C2\u00A0the\u00C2\u00A0magnitude\u00C2\u00A0of\u00C2\u00A0 suppression\u00C2\u00A0is\u00C2\u00A0difficult\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0ratios\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0splenocytes\u00C2\u00A0have\u00C2\u00A0not\u00C2\u00A0 always\u00C2\u00A0been\u00C2\u00A0reported.\u00C2\u00A0However,\u00C2\u00A0in\u00C2\u00A0our\u00C2\u00A0hands,\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0could\u00C2\u00A0not\u00C2\u00A0suppress\u00C2\u00A0 T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0in\u00C2\u00A0RPMI\u00C2\u00A01640\u00C2\u00A0or\u00C2\u00A0IMDM\u00C2\u00A0media\u00C2\u00A0containing\u00C2\u00A010%\u00C2\u00A0FCS,\u00C2\u00A0even\u00C2\u00A0at\u00C2\u00A0very\u00C2\u00A0high\u00C2\u00A0 ratios\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A01\u00C2\u00A0splenocyte).\u00C2\u00A0In\u00C2\u00A0fact,\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0FCS,\u00C2\u00A0MDSCs\u00C2\u00A0stimulated\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 most\u00C2\u00A0 experiments\u00C2\u00A0 (Fig.\u00C2\u00A0 4.6C).\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 type\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 stimulation\u00C2\u00A0may\u00C2\u00A0affect\u00C2\u00A0MDSC\u00C2\u00A0immunosuppression\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0serum,\u00C2\u00A0given\u00C2\u00A0that\u00C2\u00A0 one\u00C2\u00A0 group\u00C2\u00A0 reported\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSC\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 stimulated\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 combination\u00C2\u00A0of\u00C2\u00A0bryostatin,\u00C2\u00A0 ionomycin,\u00C2\u00A0 IL\u00E2\u0080\u00902,\u00C2\u00A0and\u00C2\u00A0cultured\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 IL\u00E2\u0080\u00905,\u00C2\u00A0 IL\u00E2\u0080\u00907,\u00C2\u00A0 and\u00C2\u00A010%\u00C2\u00A0FCS\u00C2\u00A0(Le\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0In\u00C2\u00A0light\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0studies,\u00C2\u00A0we\u00C2\u00A0propose\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 capable\u00C2\u00A0of\u00C2\u00A0suppressing\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0in\u00C2\u00A0serum\u00E2\u0080\u0090containing\u00C2\u00A0RPMI\u00C2\u00A01640\u00C2\u00A0medium\u00C2\u00A0under\u00C2\u00A0 certain\u00C2\u00A0conditions,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0in\u00C2\u00A0immunosuppression\u00C2\u00A0assays\u00C2\u00A0can\u00C2\u00A0mask\u00C2\u00A0the\u00C2\u00A0full\u00C2\u00A0 suppressive\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Taken\u00C2\u00A0together,\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0demonstrate\u00C2\u00A0that\u00C2\u00A0different\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0 considerable\u00C2\u00A0 influence\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00C2\u00A0 Since\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 defined\u00C2\u00A0 functionally\u00C2\u00A0 via\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 assays,\u00C2\u00A0 these\u00C2\u00A0 findings\u00C2\u00A0 are\u00C2\u00A0 of\u00C2\u00A0 87 critical\u00C2\u00A0importance\u00C2\u00A0to\u00C2\u00A0accurately\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0MDSC\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 tumor\u00C2\u00A0 metastasis.\u00C2\u00A0 The\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 serum,\u00C2\u00A0 and\u00C2\u00A0 specifically\u00C2\u00A0 albumin,\u00C2\u00A0 conceals\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 MDSCs,\u00C2\u00A0 which\u00C2\u00A0 can\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 erroneous\u00C2\u00A0 conclusions\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 importance\u00C2\u00A0 of\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 immunosuppression.\u00C2\u00A0 Furthermore,\u00C2\u00A0these\u00C2\u00A0findings\u00C2\u00A0may\u00C2\u00A0provide\u00C2\u00A0insight\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0It\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0 that\u00C2\u00A0 high\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 albumin\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0 may\u00C2\u00A0 restrict\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0until\u00C2\u00A0 they\u00C2\u00A0reach\u00C2\u00A0the\u00C2\u00A0 tissues.\u00C2\u00A0This\u00C2\u00A0hypothesis\u00C2\u00A0 is\u00C2\u00A0 supported\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0study\u00C2\u00A0by\u00C2\u00A0Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 in\u00C2\u00A0which\u00C2\u00A0they\u00C2\u00A0demonstrate\u00C2\u00A0that,\u00C2\u00A0at\u00C2\u00A0higher\u00C2\u00A0ratios,\u00C2\u00A0 blood\u00C2\u00A0MDSCs\u00C2\u00A0were\u00C2\u00A0less\u00C2\u00A0suppressive\u00C2\u00A0than\u00C2\u00A0MDSCs\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0spleen,\u00C2\u00A0BM,\u00C2\u00A0and\u00C2\u00A0lung\u00C2\u00A0 of\u00C2\u00A04T1\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0(Sinha\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008).\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0stimulated\u00C2\u00A0 T\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0unable\u00C2\u00A0 to\u00C2\u00A0proliferate\u00C2\u00A0 in\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A010%\u00C2\u00A0mouse\u00C2\u00A0serum\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0 shown).\u00C2\u00A0 Although\u00C2\u00A0 the\u00C2\u00A0 basis\u00C2\u00A0 for\u00C2\u00A0 this\u00C2\u00A0 is\u00C2\u00A0 unclear,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0 that\u00C2\u00A0mouse\u00C2\u00A0 serum\u00C2\u00A0 lacks\u00C2\u00A0 some\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 required\u00C2\u00A0 to\u00C2\u00A0 support\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 and/or\u00C2\u00A0proliferation\u00C2\u00A0or\u00C2\u00A0 that\u00C2\u00A0 it\u00C2\u00A0 contains\u00C2\u00A0 inhibitory\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 anergy\u00C2\u00A0 or\u00C2\u00A0 death.\u00C2\u00A0 This\u00C2\u00A0 effect\u00C2\u00A0 may\u00C2\u00A0 have\u00C2\u00A0 physiological\u00C2\u00A0 importance,\u00C2\u00A0 since\u00C2\u00A0 proliferation\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 peripheral\u00C2\u00A0 circulation\u00C2\u00A0 could\u00C2\u00A0 have\u00C2\u00A0 deleterious\u00C2\u00A0 effects.\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 this\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 an\u00C2\u00A0 example\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 more\u00C2\u00A0 general\u00C2\u00A0 phenomenon,\u00C2\u00A0whereby\u00C2\u00A0serum\u00C2\u00A0constrains\u00C2\u00A0 the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0certain\u00C2\u00A0cell\u00C2\u00A0 types,\u00C2\u00A0 including\u00C2\u00A0 MDSCs\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0thereby\u00C2\u00A0maintains\u00C2\u00A0appropriate\u00C2\u00A0 immune\u00C2\u00A0cell\u00C2\u00A0 function.\u00C2\u00A0A\u00C2\u00A0recent\u00C2\u00A0 report\u00C2\u00A0 by\u00C2\u00A0 Haverkamp\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 also\u00C2\u00A0 indicates\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0 from\u00C2\u00A0 different\u00C2\u00A0 tissues\u00C2\u00A0 possess\u00C2\u00A0 different\u00C2\u00A0capacities\u00C2\u00A0to\u00C2\u00A0regulate\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0(Haverkamp\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Using\u00C2\u00A0the\u00C2\u00A0RM\u00E2\u0080\u0090 1\u00C2\u00A0prostate\u00C2\u00A0tumor\u00C2\u00A0model,\u00C2\u00A0they\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0while\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0peripheral\u00C2\u00A0tissues\u00C2\u00A0(i.e.\u00C2\u00A0spleen\u00C2\u00A0 and\u00C2\u00A0liver)\u00C2\u00A0could\u00C2\u00A0acquire\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0upon\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A0T\u00C2\u00A0cell\u00E2\u0080\u0090produced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0in\u00C2\u00A0vitro,\u00C2\u00A0only\u00C2\u00A0MDSCs\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0site\u00C2\u00A0revealed\u00C2\u00A0immediate\u00C2\u00A0suppressive\u00C2\u00A0 abilities\u00C2\u00A0 (Haverkamp\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 Our\u00C2\u00A0 findings,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 those\u00C2\u00A0 of\u00C2\u00A0 Haverkamp\u00C2\u00A0 et\u00C2\u00A0al.\u00C2\u00A0and\u00C2\u00A0others\u00C2\u00A0 (Nausch\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008),\u00C2\u00A0 support\u00C2\u00A0 the\u00C2\u00A0 idea\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0may\u00C2\u00A0 exhibit\u00C2\u00A0diverse\u00C2\u00A0properties\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0contexts\u00C2\u00A0or\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0certain\u00C2\u00A0treatments\u00C2\u00A0(i.e.\u00C2\u00A0 docetaxel)\u00C2\u00A0 (Kodumudi\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010),\u00C2\u00A0 a\u00C2\u00A0 feature\u00C2\u00A0 that\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 exploited\u00C2\u00A0 therapeutically.\u00C2\u00A0 Overall,\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 clearly\u00C2\u00A0 highlight\u00C2\u00A0 the\u00C2\u00A0 importance\u00C2\u00A0 of\u00C2\u00A0 testing\u00C2\u00A0 different\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 culture\u00C2\u00A0 conditions\u00C2\u00A0on\u00C2\u00A0MDSC\u00C2\u00A0phenotype\u00C2\u00A0to\u00C2\u00A0ensure\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0masking\u00C2\u00A0the\u00C2\u00A0 full\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0will\u00C2\u00A0enable\u00C2\u00A0more\u00C2\u00A0accurate\u00C2\u00A0 88 identification\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function\u00C2\u00A0 and\u00C2\u00A0 will\u00C2\u00A0 consequently\u00C2\u00A0advance\u00C2\u00A0our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0perform\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0 primary\u00C2\u00A0and\u00C2\u00A0metastatic\u00C2\u00A0tumor\u00C2\u00A0growth.\u00C2\u00A0 89 CHAPTER\u00C2\u00A05\u00C2\u00A0: MACROPHAGES\u00C2\u00A0ARE\u00C2\u00A0MORE\u00C2\u00A0POTENT\u00C2\u00A0IMMUNE\u00C2\u00A0 SUPPRESSORS\u00C2\u00A0THAN\u00C2\u00A0MYELOID\u00C2\u00ADDERIVED\u00C2\u00A0SUPPRESSOR\u00C2\u00A0CELLS\u00C2\u00A0IN\u00C2\u00A0 MURINE\u00C2\u00A0METASTATIC\u00C2\u00A0MAMMARY\u00C2\u00A0CARCINOMA\u00C2\u00A0 \u00C2\u00A0 5.1 Introduction\u00C2\u00A0 \u00C2\u00A0 Having\u00C2\u00A0 established\u00C2\u00A0 appropriate\u00C2\u00A0 culture\u00C2\u00A0 conditions\u00C2\u00A0 to\u00C2\u00A0 accurately\u00C2\u00A0 assay\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0embarked\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0experiments\u00C2\u00A0 to\u00C2\u00A0investigate\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0tumorigenesis.\u00C2\u00A0Immunosuppressive\u00C2\u00A0 myeloid\u00C2\u00A0cells,\u00C2\u00A0including\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0mature\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0play\u00C2\u00A0pivotal\u00C2\u00A0roles\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0promotion\u00C2\u00A0of\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 emerging\u00C2\u00A0 as\u00C2\u00A0 possible\u00C2\u00A0 players\u00C2\u00A0 in\u00C2\u00A0metastasis\u00C2\u00A0 (Qian\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A02010;\u00C2\u00A0Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010).\u00C2\u00A0However,\u00C2\u00A0little\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0about\u00C2\u00A0the\u00C2\u00A0relative\u00C2\u00A0 immunosuppressive\u00C2\u00A0potencies\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0types,\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 they\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 suppressive\u00C2\u00A0 effects,\u00C2\u00A0 or\u00C2\u00A0 how\u00C2\u00A0 the\u00C2\u00A0 strength\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0relates\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0or\u00C2\u00A0metastasis.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Characterization\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 phenotypes\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0roles\u00C2\u00A0each\u00C2\u00A0play\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0is\u00C2\u00A0difficult\u00C2\u00A0because\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0extreme\u00C2\u00A0heterogeneity\u00C2\u00A0exhibited\u00C2\u00A0by\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0myeloid\u00C2\u00A0cells.\u00C2\u00A0Different\u00C2\u00A0subtypes\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0classified\u00C2\u00A0 based\u00C2\u00A0on\u00C2\u00A0characteristics\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0morphology\u00C2\u00A0(i.e.\u00C2\u00A0M\u00E2\u0080\u0090MDSCs\u00C2\u00A0versus\u00C2\u00A0G\u00E2\u0080\u0090MDSCs),\u00C2\u00A0activation\u00C2\u00A0 state\u00C2\u00A0 (i.e.\u00C2\u00A0M1\u00C2\u00A0versus\u00C2\u00A0M2a,\u00C2\u00A0M2b,\u00C2\u00A0 or\u00C2\u00A0M2c\u00C2\u00A0M\u00CF\u0086s),\u00C2\u00A0 or\u00C2\u00A0 tissue\u00E2\u0080\u0090specificity\u00C2\u00A0 (Corzo\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010;\u00C2\u00A0 Haverkamp\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0However,\u00C2\u00A0these\u00C2\u00A0classifications\u00C2\u00A0only\u00C2\u00A0begin\u00C2\u00A0to\u00C2\u00A0describe\u00C2\u00A0the\u00C2\u00A0true\u00C2\u00A0 heterogeneity\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0 since\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 an\u00C2\u00A0 individual\u00C2\u00A0 cell\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 influenced\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0key\u00C2\u00A0 factors\u00C2\u00A0 including\u00C2\u00A0cell\u00C2\u00A0 lineage\u00C2\u00A0and\u00C2\u00A0molecules\u00C2\u00A0present\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 microenvironment\u00C2\u00A0 both\u00C2\u00A0 during\u00C2\u00A0 and\u00C2\u00A0 subsequent\u00C2\u00A0 to\u00C2\u00A0 differentiation\u00C2\u00A0 (Laoui\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0different\u00C2\u00A0populations\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0thought\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0suppressive\u00C2\u00A0effects\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0mechanisms,\u00C2\u00A0 including,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0 limited\u00C2\u00A0 to,\u00C2\u00A0 production\u00C2\u00A0of\u00C2\u00A0 inhibitory\u00C2\u00A0cytokines,\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0 inhibitory\u00C2\u00A0 receptors,\u00C2\u00A0 and\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0Arg1\u00C2\u00A0and\u00C2\u00A0iNOS,\u00C2\u00A0which\u00C2\u00A0lead\u00C2\u00A0to\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0depletion\u00C2\u00A0and\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0ROS\u00C2\u00A0and\u00C2\u00A0RNS\u00C2\u00A0(Cuenca\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0 90 \u00C2\u00A0 Although\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0TAMs\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0metastasis,\u00C2\u00A0many\u00C2\u00A0 questions\u00C2\u00A0still\u00C2\u00A0remain.\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0well\u00C2\u00A0established\u00C2\u00A0if\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0subtypes\u00C2\u00A0other\u00C2\u00A0than\u00C2\u00A0 TAMs\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0metastasis.\u00C2\u00A0Specifically,\u00C2\u00A0the\u00C2\u00A0contribution\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0extra\u00E2\u0080\u0090tumoral\u00C2\u00A0 (i.e.\u00C2\u00A0peripheral\u00C2\u00A0tissue)\u00C2\u00A0sites\u00C2\u00A0in\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0hosts\u00C2\u00A0remains\u00C2\u00A0unclear\u00C2\u00A0(Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A02009).\u00C2\u00A0Also,\u00C2\u00A0given\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0TAMs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0to\u00C2\u00A0promote\u00C2\u00A0metastasis\u00C2\u00A0 via\u00C2\u00A0similar\u00C2\u00A0mechanisms\u00C2\u00A0(Siveen\u00C2\u00A0and\u00C2\u00A0Kuttan,\u00C2\u00A02009;\u00C2\u00A0Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010)\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0 known\u00C2\u00A0whether\u00C2\u00A0MDSCs\u00C2\u00A0 and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 distinct\u00C2\u00A0 roles.\u00C2\u00A0 Finally,\u00C2\u00A0 little\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 about\u00C2\u00A0 the\u00C2\u00A0 relationship\u00C2\u00A0 between\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 and\u00C2\u00A0 metastasis.\u00C2\u00A0 The\u00C2\u00A0 concept\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 pre\u00E2\u0080\u0090 metastatic\u00C2\u00A0 niche\u00C2\u00A0 proposes\u00C2\u00A0 that\u00C2\u00A0 BM\u00E2\u0080\u0090derived\u00C2\u00A0 cells\u00C2\u00A0migrate\u00C2\u00A0 to\u00C2\u00A0 future\u00C2\u00A0 sites\u00C2\u00A0 of\u00C2\u00A0metastasis\u00C2\u00A0 ahead\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 promote\u00C2\u00A0 the\u00C2\u00A0 homing\u00C2\u00A0 and\u00C2\u00A0 growth\u00C2\u00A0 of\u00C2\u00A0 metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 (Kaplan\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005).\u00C2\u00A0 However,\u00C2\u00A0 whether\u00C2\u00A0 these\u00C2\u00A0 BM\u00E2\u0080\u0090derived\u00C2\u00A0 cells\u00C2\u00A0 possess\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0or\u00C2\u00A0whether\u00C2\u00A0 immune\u00C2\u00A0suppression\u00C2\u00A0contributes\u00C2\u00A0to\u00C2\u00A0tumor\u00C2\u00A0 metastasis\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0well\u00C2\u00A0established.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0investigate\u00C2\u00A0some\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0questions,\u00C2\u00A0including\u00C2\u00A0whether\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0play\u00C2\u00A0 distinct\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 vivo,\u00C2\u00A0 we\u00C2\u00A0 implanted\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 with\u00C2\u00A0 ATRA\u00C2\u00A0 pellets\u00C2\u00A0 to\u00C2\u00A0 promote\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0DCs.\u00C2\u00A0ATRA,\u00C2\u00A0a\u00C2\u00A0member\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0retinoid\u00C2\u00A0 family\u00C2\u00A0 and\u00C2\u00A0 structurally\u00C2\u00A0 related\u00C2\u00A0 to\u00C2\u00A0 vitamin\u00C2\u00A0 A,\u00C2\u00A0 exerts\u00C2\u00A0 strong\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 proliferation,\u00C2\u00A0 differentiation,\u00C2\u00A0 and\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 normal\u00C2\u00A0 and\u00C2\u00A0 cancer\u00C2\u00A0 cells\u00C2\u00A0 (Bastien\u00C2\u00A0 and\u00C2\u00A0Rochette\u00E2\u0080\u0090Egly,\u00C2\u00A02004).\u00C2\u00A0ATRA\u00C2\u00A0induces\u00C2\u00A0immature\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0terminally\u00C2\u00A0differentiate\u00C2\u00A0and\u00C2\u00A0 is\u00C2\u00A0used\u00C2\u00A0clinically\u00C2\u00A0to\u00C2\u00A0treat\u00C2\u00A0acute\u00C2\u00A0promyelocytic\u00C2\u00A0leukemia\u00C2\u00A0(APL)\u00C2\u00A0(a\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0acute\u00C2\u00A0myeloid\u00C2\u00A0 leukemia\u00C2\u00A0(AML)),\u00C2\u00A0in\u00C2\u00A0combination\u00C2\u00A0with\u00C2\u00A0chemotherapy\u00C2\u00A0(Sanz\u00C2\u00A0and\u00C2\u00A0Lo\u00E2\u0080\u0090Coco,\u00C2\u00A02011).\u00C2\u00A0Based\u00C2\u00A0 on\u00C2\u00A0its\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0MDSC\u00C2\u00A0levels\u00C2\u00A0and\u00C2\u00A0abrogate\u00C2\u00A0MDSC\u00E2\u0080\u0090mediated\u00C2\u00A0immune\u00C2\u00A0suppression,\u00C2\u00A0 the\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 ATRA\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 possible\u00C2\u00A0 cancer\u00C2\u00A0 therapy\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 proposed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 literature\u00C2\u00A0 (Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003;\u00C2\u00A0Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02008;\u00C2\u00A0Mirza\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006).\u00C2\u00A0In\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0we\u00C2\u00A0 report\u00C2\u00A0that,\u00C2\u00A0as\u00C2\u00A0expected,\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0ATRA\u00C2\u00A0had\u00C2\u00A0decreased\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0increased\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0their\u00C2\u00A0lungs.\u00C2\u00A0However,\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0ATRA\u00C2\u00A0displayed\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0lung\u00C2\u00A0 metastases\u00C2\u00A0 than\u00C2\u00A0 untreated\u00C2\u00A0 or\u00C2\u00A0 placebo\u00E2\u0080\u0090treated\u00C2\u00A0 mice.\u00C2\u00A0 Furthermore,\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0experiments,\u00C2\u00A0which\u00C2\u00A0reveal\u00C2\u00A0 that\u00C2\u00A0 lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0 91 immune\u00C2\u00A0suppressors\u00C2\u00A0than\u00C2\u00A0lung\u00C2\u00A0MDSCs\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis.\u00C2\u00A0These\u00C2\u00A0findings\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0 our\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 processes\u00C2\u00A0 and\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0metastasis,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0 lead\u00C2\u00A0to\u00C2\u00A0novel\u00C2\u00A0and\u00C2\u00A0more\u00C2\u00A0effective\u00C2\u00A0treatment\u00C2\u00A0strategies\u00C2\u00A0for\u00C2\u00A0cancer\u00C2\u00A0patients.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 5.2 Results\u00C2\u00A0 \u00C2\u00A0 5.2.1 Characterization\u00C2\u00A0of\u00C2\u00A0primary\u00C2\u00A0 tumor\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0 in\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A0 67NR\u00C2\u00A0tumor\u00C2\u00A0models\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0address\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis\u00C2\u00A0we\u00C2\u00A0used\u00C2\u00A0 two\u00C2\u00A0 murine\u00C2\u00A0 mammary\u00C2\u00A0 carcinoma\u00C2\u00A0 models.\u00C2\u00A0 After\u00C2\u00A0 three\u00C2\u00A0 weeks,\u00C2\u00A0 mice\u00C2\u00A0 orthotopically\u00C2\u00A0 implanted\u00C2\u00A0 with\u00C2\u00A0 metastatic\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 (i.e.\u00C2\u00A0 4T1\u00C2\u00A0 mice)\u00C2\u00A0 exhibited\u00C2\u00A0 large\u00C2\u00A0 primary\u00C2\u00A0 tumors\u00C2\u00A0 (Fig.\u00C2\u00A0 5.1A),\u00C2\u00A0 considerable\u00C2\u00A0 splenomegaly\u00C2\u00A0 (Fig.\u00C2\u00A0 5.1B)\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 extramedullary\u00C2\u00A0 myelopoiesis\u00C2\u00A0 (Ueha\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 2011),\u00C2\u00A0 and\u00C2\u00A0high\u00C2\u00A0numbers\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0 lungs\u00C2\u00A0 (Fig.\u00C2\u00A0 5.1C).\u00C2\u00A0 In\u00C2\u00A0 contrast,\u00C2\u00A0 although\u00C2\u00A0non\u00E2\u0080\u0090metastatic\u00C2\u00A0 67NR\u00C2\u00A0primary\u00C2\u00A0 tumors\u00C2\u00A0 reached\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0size\u00C2\u00A0as\u00C2\u00A04T1\u00C2\u00A0tumors,\u00C2\u00A0mice\u00C2\u00A0bearing\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0(i.e.\u00C2\u00A067NR\u00C2\u00A0mice)\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0exhibit\u00C2\u00A0 splenomegaly\u00C2\u00A0 (Fig.\u00C2\u00A0 5.1D),\u00C2\u00A0 and,\u00C2\u00A0 as\u00C2\u00A0 expected,\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 exhibit\u00C2\u00A0 any\u00C2\u00A0 lung\u00C2\u00A0metastases\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0 we\u00C2\u00A0 performed\u00C2\u00A0 immunofluorescence\u00C2\u00A0 on\u00C2\u00A0 4T1\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 sections\u00C2\u00A0to\u00C2\u00A0examine\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0perfusion\u00C2\u00A0and\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 infiltration.\u00C2\u00A0 \u00C2\u00A04T1\u00C2\u00A0tumors\u00C2\u00A0 were\u00C2\u00A0poorly\u00C2\u00A0vascularised\u00C2\u00A0and\u00C2\u00A0contained\u00C2\u00A0a\u00C2\u00A0high\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0hypoxic\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0while\u00C2\u00A0 67NR\u00C2\u00A0tumors\u00C2\u00A0were\u00C2\u00A0well\u00C2\u00A0vascularised\u00C2\u00A0and\u00C2\u00A0hypoxic\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0detectable\u00C2\u00A0(Fig.\u00C2\u00A05.2A).\u00C2\u00A0 Moreover,\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 CD11b+\u00C2\u00A0 cells\u00C2\u00A0 was\u00C2\u00A0 much\u00C2\u00A0 higher\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 (Fig.\u00C2\u00A0 5.2A),\u00C2\u00A0 indicating\u00C2\u00A0greater\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0infiltration.\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0examined\u00C2\u00A0the\u00C2\u00A0lungs\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0 found\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0metastatic\u00C2\u00A0tumor\u00C2\u00A0nodules\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0lung\u00C2\u00A0tissue\u00C2\u00A0were\u00C2\u00A0highly\u00C2\u00A0infiltrated\u00C2\u00A0with\u00C2\u00A0 myeloid\u00C2\u00A0 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Mice\u00C2\u00A0were\u00C2\u00A0implanted\u00C2\u00A0with\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0A,\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0weight,\u00C2\u00A0B,\u00C2\u00A0spleen\u00C2\u00A0weight,\u00C2\u00A0 and\u00C2\u00A0C,\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0 over\u00C2\u00A0 time.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0with\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A015\u00C2\u00A0mice\u00C2\u00A0per\u00C2\u00A0point.\u00C2\u00A0D,\u00C2\u00A0Mice\u00C2\u00A0were\u00C2\u00A0implanted\u00C2\u00A0with\u00C2\u00A04T1\u00C2\u00A0(\u00E2\u0080\u00A2)\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0 (\u00E2\u0096\u00A1)\u00C2\u00A0tumor\u00C2\u00A0cells,\u00C2\u00A0harvested\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0times,\u00C2\u00A0and\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0weights\u00C2\u00A0and\u00C2\u00A0spleen\u00C2\u00A0weights\u00C2\u00A0 measured.\u00C2\u00A0Each\u00C2\u00A0point\u00C2\u00A0represents\u00C2\u00A0an\u00C2\u00A0individual\u00C2\u00A0mouse.\u00C2\u00A0\u00C2\u00A0 93 \u00C2\u00A0 \u00C2\u00A0 5.2.2 4T1\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0induce\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 further\u00C2\u00A0 characterize\u00C2\u00A0 the\u00C2\u00A0 involvement\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0 tumor\u00C2\u00A0 models,\u00C2\u00A0 we\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 each\u00C2\u00A0 tumor\u00C2\u00A0 type\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 the\u00C2\u00A0 accumulation\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0 (CD11b+Gr1+\u00C2\u00A0 cells)\u00C2\u00A0 and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (F4/80+CD11b+\u00C2\u00A0 cells)\u00C2\u00A0 using\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0 Mice\u00C2\u00A0 bearing\u00C2\u00A0 4T1,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 67NR\u00C2\u00A0 tumors,\u00C2\u00A0 exhibited\u00C2\u00A0 significantly\u00C2\u00A0 higher\u00C2\u00A0 proportions\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0their\u00C2\u00A0lungs\u00C2\u00A0and\u00C2\u00A0spleens,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0(i.e.\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.2\u00C2\u00A0 Visualization\u00C2\u00A0of\u00C2\u00A0hypoxia\u00C2\u00A0and\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 infiltration\u00C2\u00A0 in\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0 tumors.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 4T1\u00C2\u00A0 (left)\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 (right)\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 sections\u00C2\u00A0 analyzed\u00C2\u00A0 for\u00C2\u00A0 anti\u00E2\u0080\u0090CD11b\u00C2\u00A0 (red),\u00C2\u00A0 pimonidazole\u00C2\u00A0(hypoxia;\u00C2\u00A0green),\u00C2\u00A0and\u00C2\u00A0Hoechst\u00C2\u00A033342\u00C2\u00A0(perfusion;\u00C2\u00A0blue).\u00C2\u00A0B,\u00C2\u00A0Lung\u00C2\u00A0sections\u00C2\u00A0from\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0analyzed\u00C2\u00A0for\u00C2\u00A0DAPI\u00C2\u00A0(nuclei;\u00C2\u00A0blue)\u00C2\u00A0and\u00C2\u00A0anti\u00E2\u0080\u0090Gr1\u00C2\u00A0(red;\u00C2\u00A0left)\u00C2\u00A0or\u00C2\u00A0anti\u00E2\u0080\u0090F4/80\u00C2\u00A0(red;\u00C2\u00A0right).\u00C2\u00A0 Areas\u00C2\u00A0 in\u00C2\u00A0dotted\u00C2\u00A0 lines\u00C2\u00A0are\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0nodules.\u00C2\u00A0All\u00C2\u00A0 images\u00C2\u00A0are\u00C2\u00A010x\u00C2\u00A0magnification.\u00C2\u00A0Data\u00C2\u00A0 are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0two\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0\u00C2\u00A0 94 non\u00E2\u0080\u0090tumor\u00E2\u0080\u0090bearing)\u00C2\u00A0mice\u00C2\u00A0 (Fig.\u00C2\u00A0 5.3A).\u00C2\u00A0 Similarly,\u00C2\u00A0 the\u00C2\u00A0 absolute\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0was\u00C2\u00A0 considerably\u00C2\u00A0 higher\u00C2\u00A0 in\u00C2\u00A0 4T1,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 67NR,\u00C2\u00A0mice,\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 control\u00C2\u00A0 mice\u00C2\u00A0(Fig.\u00C2\u00A05.3B).\u00C2\u00A0These\u00C2\u00A0contrasting\u00C2\u00A0effects\u00C2\u00A0were\u00C2\u00A0underscored\u00C2\u00A0by\u00C2\u00A0our\u00C2\u00A0observation\u00C2\u00A0that,\u00C2\u00A0in\u00C2\u00A0 some\u00C2\u00A0 cases,\u00C2\u00A0 67NR\u00C2\u00A0 mice\u00C2\u00A0 possessed\u00C2\u00A0 significantly\u00C2\u00A0 lower\u00C2\u00A0 proportions\u00C2\u00A0 and\u00C2\u00A0 numbers\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 than\u00C2\u00A0 control\u00C2\u00A0 animals,\u00C2\u00A0 while\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0 displayed\u00C2\u00A0 up\u00C2\u00A0 to\u00C2\u00A0 230\u00C2\u00A0 or\u00C2\u00A0 600\u00C2\u00A0 fold\u00C2\u00A0 more\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0or\u00C2\u00A0MDSCs,\u00C2\u00A0 respectively\u00C2\u00A0 (Fig.\u00C2\u00A05.3A,B).\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0ex\u00C2\u00A0 vivo\u00C2\u00A0 imaging\u00C2\u00A0 results\u00C2\u00A0 (Fig.\u00C2\u00A0 5.2)\u00C2\u00A0 and\u00C2\u00A0 reveal\u00C2\u00A0 clear\u00C2\u00A0 and\u00C2\u00A0 intriguing\u00C2\u00A0 differences\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0tumors.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.3\u00C2\u00A0 Induction\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0tumors.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0Proportion\u00C2\u00A0and\u00C2\u00A0\u00C2\u00A0B,\u00C2\u00A0total\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0(CD11b+Gr1+)\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(F4/80+CD11b+)\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 lungs\u00C2\u00A0and\u00C2\u00A0spleens\u00C2\u00A0of\u00C2\u00A0control\u00C2\u00A0(i.e.\u00C2\u00A0non\u00E2\u0080\u0090tumor\u00C2\u00A0bearing)\u00C2\u00A0(white\u00C2\u00A0bars),\u00C2\u00A04T1\u00C2\u00A0(black\u00C2\u00A0bars),\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0 (grey\u00C2\u00A0 bars)\u00C2\u00A0 mice\u00C2\u00A0 three\u00C2\u00A0 weeks\u00C2\u00A0 post\u00E2\u0080\u0090implant.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0 significant\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0mice;\u00C2\u00A0nd,\u00C2\u00A0not\u00C2\u00A0determined\u00C2\u00A0 95 5.2.3 Phenotypic\u00C2\u00A0 characterization\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 versus\u00C2\u00A0 tumor\u00C2\u00ADbearing\u00C2\u00A0mice\u00C2\u00A0 \u00C2\u00A0 5.2.3.1 TAMs\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 suggested\u00C2\u00A0 that\u00C2\u00A0 4T1,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 67NR,\u00C2\u00A0 tumors\u00C2\u00A0 induce\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0commenced\u00C2\u00A0a\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0studies\u00C2\u00A0to\u00C2\u00A0investigate\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0 of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0subtypes\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0and\u00C2\u00A0metastasis.\u00C2\u00A0We\u00C2\u00A0began\u00C2\u00A0 by\u00C2\u00A0 comparing\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 phenotype\u00C2\u00A0 and\u00C2\u00A0 protein\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 TAMs.\u00C2\u00A0 Flow\u00C2\u00A0 cytometric\u00C2\u00A0 analysis\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 TAMs\u00C2\u00A0 from\u00C2\u00A0 both\u00C2\u00A0 tumor\u00C2\u00A0 models\u00C2\u00A0 represented\u00C2\u00A0a\u00C2\u00A0heterogeneous\u00C2\u00A0population.\u00C2\u00A0Although\u00C2\u00A0almost\u00C2\u00A0all\u00C2\u00A0cells\u00C2\u00A0lacked\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0 Gr1,\u00C2\u00A0subsets\u00C2\u00A0of\u00C2\u00A0TAMs\u00C2\u00A0exhibited\u00C2\u00A0different\u00C2\u00A0degrees\u00C2\u00A0of\u00C2\u00A0F4/80\u00C2\u00A0and\u00C2\u00A0CD11b\u00C2\u00A0expression\u00C2\u00A0(Fig.\u00C2\u00A0 5.4A).\u00C2\u00A0In\u00C2\u00A0general,\u00C2\u00A0a\u00C2\u00A0larger\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0TAMs\u00C2\u00A0expressed\u00C2\u00A0high\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0F4/80\u00C2\u00A0 and\u00C2\u00A0 CD11b\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 67NR\u00C2\u00A0 TAMs\u00C2\u00A0 (Fig.\u00C2\u00A0 5.4A).\u00C2\u00A0 We\u00C2\u00A0 also\u00C2\u00A0 analyzed\u00C2\u00A0 cell\u00C2\u00A0 lysates\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0if\u00C2\u00A0TAMs\u00C2\u00A0were\u00C2\u00A0M1\u00C2\u00A0or\u00C2\u00A0M2\u00C2\u00A0skewed.\u00C2\u00A0As\u00C2\u00A0Figure\u00C2\u00A05.4B\u00C2\u00A0shows,\u00C2\u00A04T1\u00C2\u00A0TAMs\u00C2\u00A0expressed\u00C2\u00A0 Arg1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0TAMs\u00C2\u00A0expressed\u00C2\u00A0iNOS,\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0TAMs\u00C2\u00A0are\u00C2\u00A0M2\u00C2\u00A0skewed\u00C2\u00A0while\u00C2\u00A0 67NR\u00C2\u00A0TAMs\u00C2\u00A0exhibit\u00C2\u00A0features\u00C2\u00A0of\u00C2\u00A0M1\u00C2\u00A0activation.\u00C2\u00A0\u00C2\u00A0 96 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.4\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0TAMs.\u00C2\u00A0\u00C2\u00A0 TAMs\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0A,\u00C2\u00A0surface\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0F4/80,\u00C2\u00A0CD11b,\u00C2\u00A0and\u00C2\u00A0 Gr1\u00C2\u00A0 was\u00C2\u00A0 analyzed\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0 and\u00C2\u00A0 B,\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 Ym1,\u00C2\u00A0 Arg1,\u00C2\u00A0 and\u00C2\u00A0 GAPDH\u00C2\u00A0 was\u00C2\u00A0 analyzed\u00C2\u00A0by\u00C2\u00A0Western\u00C2\u00A0blot.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0at\u00C2\u00A0 least\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0 in\u00C2\u00A0duplicate.\u00C2\u00A0\u00C2\u00A0 97 5.2.3.2 PM\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0we\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 and\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0both\u00C2\u00A0control\u00C2\u00A0and\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0expressed\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0F4/80,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0Gr1\u00C2\u00A0 (Fig.\u00C2\u00A0 5.5A).\u00C2\u00A0 Interestingly,\u00C2\u00A0 control\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 a\u00C2\u00A0 single,\u00C2\u00A0 relatively\u00C2\u00A0 uniform\u00C2\u00A0 population,\u00C2\u00A0 while\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 appeared\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 composed\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 distinct\u00C2\u00A0 populations,\u00C2\u00A0 one\u00C2\u00A0 that\u00C2\u00A0 expressed\u00C2\u00A0moderate\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0F4/80\u00C2\u00A0and\u00C2\u00A0one\u00C2\u00A0that\u00C2\u00A0expressed\u00C2\u00A0high\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 two\u00C2\u00A0markers\u00C2\u00A0(Fig.\u00C2\u00A05.5A).\u00C2\u00A0Next,\u00C2\u00A0we\u00C2\u00A0 investigated\u00C2\u00A0whether\u00C2\u00A0 these\u00C2\u00A0cells\u00C2\u00A0expressed\u00C2\u00A0 features\u00C2\u00A0 of\u00C2\u00A0 M1\u00C2\u00A0 or\u00C2\u00A0 M2\u00C2\u00A0 activation.\u00C2\u00A0 Western\u00C2\u00A0 blot\u00C2\u00A0 analysis\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 expressed\u00C2\u00A0high\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0Arg1\u00C2\u00A0and\u00C2\u00A0Ym1,\u00C2\u00A067NR\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0expressed\u00C2\u00A0very\u00C2\u00A0low\u00C2\u00A0Arg1\u00C2\u00A0and\u00C2\u00A0 no\u00C2\u00A0Ym1,\u00C2\u00A0and\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0lacked\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0proteins\u00C2\u00A0(Fig.\u00C2\u00A05.5B).\u00C2\u00A0Furthermore,\u00C2\u00A0 none\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0expressed\u00C2\u00A0iNOS\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0Overall,\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0M2\u00C2\u00A0skewed,\u00C2\u00A0while\u00C2\u00A067NR\u00C2\u00A0and\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0a\u00C2\u00A0na\u00C3\u00AFve\u00C2\u00A0phenotype.\u00C2\u00A0 \u00C2\u00A0 5.2.3.3 Splenic\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 Analysis\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 surface\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 splenic\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 (SpM\u00CF\u0086s)\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 although,\u00C2\u00A0as\u00C2\u00A0expected,\u00C2\u00A0a\u00C2\u00A0large\u00C2\u00A0proportion\u00C2\u00A0expressed\u00C2\u00A0F4/80,\u00C2\u00A0few\u00C2\u00A0expressed\u00C2\u00A0CD11b\u00C2\u00A0(Fig.\u00C2\u00A0 5.6A).\u00C2\u00A0Moreover,\u00C2\u00A0SpM\u00CF\u0086\u00C2\u00A0samples\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0appeared\u00C2\u00A0to\u00C2\u00A0contain\u00C2\u00A0some\u00C2\u00A0Gr1+\u00C2\u00A0 cells\u00C2\u00A0 (Fig.\u00C2\u00A0 5.6B).\u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 perhaps\u00C2\u00A0 not\u00C2\u00A0 surprising\u00C2\u00A0 given\u00C2\u00A0 the\u00C2\u00A0 large\u00C2\u00A0 proportion\u00C2\u00A0 of\u00C2\u00A0 CD11b+/Gr1+\u00C2\u00A0 MDSCs\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0 spleens\u00C2\u00A0 (Fig.\u00C2\u00A0 5.3A).\u00C2\u00A0 However,\u00C2\u00A0 since\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 reports\u00C2\u00A0 that\u00C2\u00A0 some\u00C2\u00A0 MDSCs\u00C2\u00A0 express\u00C2\u00A0 F4/80,\u00C2\u00A0 CD11b,\u00C2\u00A0 and\u00C2\u00A0 Gr1\u00C2\u00A0 (Ueha\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011),\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 difficult\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0whether\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0 should\u00C2\u00A0 be\u00C2\u00A0 classified\u00C2\u00A0 as\u00C2\u00A0 SpM\u00CF\u0086s,\u00C2\u00A0MDSCs,\u00C2\u00A0 or\u00C2\u00A0 a\u00C2\u00A0 mixed\u00C2\u00A0population.\u00C2\u00A0Interestingly\u00C2\u00A0very\u00C2\u00A0few\u00C2\u00A0F4/80+\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0spleens\u00C2\u00A0of\u00C2\u00A0control\u00C2\u00A0(Fig.\u00C2\u00A0 5.6A)\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0(Fig.\u00C2\u00A05.6C)\u00C2\u00A0mice\u00C2\u00A0co\u00E2\u0080\u0090expressed\u00C2\u00A0Gr1+\u00C2\u00A0.\u00C2\u00A0\u00C2\u00A0 98 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.5\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control,\u00C2\u00A0 4T1,\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0A,\u00C2\u00A0 surface\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 F4/80,\u00C2\u00A0 CD11b,\u00C2\u00A0and\u00C2\u00A0Gr1\u00C2\u00A0was\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0and\u00C2\u00A0B,\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0Ym1,\u00C2\u00A0Arg1,\u00C2\u00A0and\u00C2\u00A0GAPDH\u00C2\u00A0 was\u00C2\u00A0 analyzed\u00C2\u00A0 by\u00C2\u00A0 Western\u00C2\u00A0 blot.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 three\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0 \u00C2\u00A0 99 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.6\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0SpM\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0A,\u00C2\u00A0 control,\u00C2\u00A0B,\u00C2\u00A0 4T1,\u00C2\u00A0 or\u00C2\u00A0C,\u00C2\u00A0 67NR\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 surface\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0\u00C2\u00A0 F4/80,\u00C2\u00A0CD11b,\u00C2\u00A0and\u00C2\u00A0Gr1\u00C2\u00A0was\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0 three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0\u00C2\u00A0 100 5.2.3.4 Splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 Lastly,\u00C2\u00A0we\u00C2\u00A0considered\u00C2\u00A0the\u00C2\u00A0phenotypic\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0 spleens\u00C2\u00A0of\u00C2\u00A0control,\u00C2\u00A04T1,\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0mice.\u00C2\u00A0Intriguingly,\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0phenotypes\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0 three\u00C2\u00A0populations\u00C2\u00A0varied\u00C2\u00A0significantly.\u00C2\u00A0Control\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0appeared\u00C2\u00A0to\u00C2\u00A0 be\u00C2\u00A0 comprised\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 distinct\u00C2\u00A0 populations\u00C2\u00A0 that\u00C2\u00A0 differentially\u00C2\u00A0 expressed\u00C2\u00A0 Gr1:\u00C2\u00A0 Gr1loCD11b\u00E2\u0080\u0090,\u00C2\u00A0 Gr1midCD11b+,\u00C2\u00A0 and\u00C2\u00A0Gr1brightCD11b+\u00C2\u00A0 (Fig.\u00C2\u00A0 5.7).\u00C2\u00A0 In\u00C2\u00A0 contrast,\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A04T1\u00C2\u00A0 mice\u00C2\u00A0were\u00C2\u00A0 a\u00C2\u00A0 uniform\u00C2\u00A0Gr1highCD11b+\u00C2\u00A0 population\u00C2\u00A0 (Fig.\u00C2\u00A0 5.7),\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 general\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 MDSCs.\u00C2\u00A0 In\u00C2\u00A0 addition,\u00C2\u00A0 we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 none\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Gr1+\u00C2\u00A0 cell\u00C2\u00A0 populations\u00C2\u00A0 isolated\u00C2\u00A0from\u00C2\u00A0any\u00C2\u00A0group\u00C2\u00A0of\u00C2\u00A0mice\u00C2\u00A0expressed\u00C2\u00A0F4/80\u00C2\u00A0(Fig.\u00C2\u00A05.7).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 5.2.4 Summary\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0tumors\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0phenotypes\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 Taken\u00C2\u00A0together,\u00C2\u00A0our\u00C2\u00A0data\u00C2\u00A0demonstrate\u00C2\u00A0that,\u00C2\u00A0unlike\u00C2\u00A0non\u00E2\u0080\u0090metastatic\u00C2\u00A067NR\u00C2\u00A0tumors,\u00C2\u00A0 metastatic\u00C2\u00A04T1\u00C2\u00A0tumors\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0profound\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0phenotypes\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 populations.\u00C2\u00A0 Whereas\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 67NR\u00C2\u00A0 mice\u00C2\u00A0 closely\u00C2\u00A0 resembled\u00C2\u00A0 that\u00C2\u00A0of\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0control\u00C2\u00A0mice,\u00C2\u00A04T1\u00C2\u00A0 tumors\u00C2\u00A0modified\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0 surface\u00C2\u00A0and\u00C2\u00A0 intracellular\u00C2\u00A0 protein\u00C2\u00A0 expression.\u00C2\u00A0 In\u00C2\u00A0 general,\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 increased\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 proteins\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0M2\u00C2\u00A0activation\u00C2\u00A0(i.e.\u00C2\u00A0 inducing\u00C2\u00A0Arg1,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0 iNOS)\u00C2\u00A0 in\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0 TAMs\u00C2\u00A0 (Fig.\u00C2\u00A05.8A).\u00C2\u00A0 Furthermore,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0noteworthy\u00C2\u00A0 that\u00C2\u00A0neither\u00C2\u00A0Gr1+\u00C2\u00A0nor\u00C2\u00A0F4/80+\u00C2\u00A0 splenic\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 or\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 exhibited\u00C2\u00A0 Arg1\u00C2\u00A0 expression,\u00C2\u00A0 but\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0tumors\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0did\u00C2\u00A0express\u00C2\u00A0both\u00C2\u00A0Arg1\u00C2\u00A0and\u00C2\u00A0Ym1\u00C2\u00A0(Fig.\u00C2\u00A05.8B).\u00C2\u00A0 101 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.7\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 spleens\u00C2\u00A0 of\u00C2\u00A0 control,\u00C2\u00A0 4T1,\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 surface\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 Gr1,\u00C2\u00A0 F4/80,\u00C2\u00A0 and\u00C2\u00A0 CD11b\u00C2\u00A0 was\u00C2\u00A0 analyzed\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0of\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0 \u00C2\u00A0 102 \u00C2\u00A0 \u00C2\u00A0 5.2.5 Comparison\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0versus\u00C2\u00A0tumor\u00C2\u00ADbearing\u00C2\u00A0mice\u00C2\u00A0 \u00C2\u00A0 5.2.5.1 PM\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0we\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0tumors\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0 of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0populations.\u00C2\u00A0Our\u00C2\u00A0previous\u00C2\u00A0results\u00C2\u00A0revealed\u00C2\u00A0 that\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 could\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0and\u00C2\u00A0we\u00C2\u00A0were\u00C2\u00A0interested\u00C2\u00A0to\u00C2\u00A0discover\u00C2\u00A0whether\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 also\u00C2\u00A0 possessed\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties.\u00C2\u00A0 We\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0control,\u00C2\u00A04T1,\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0mice\u00C2\u00A0all\u00C2\u00A0potently\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0(Fig.\u00C2\u00A0 5.9A).\u00C2\u00A0 However,\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 our\u00C2\u00A0 phenotypic\u00C2\u00A0 data,\u00C2\u00A0 different\u00C2\u00A0 tumor\u00C2\u00A0 models\u00C2\u00A0 had\u00C2\u00A0 distinct\u00C2\u00A0 effects\u00C2\u00A0on\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function.\u00C2\u00A0 Specifically,\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A067NR\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0immunosuppressive\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis\u00C2\u00A0than\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A05.9A)\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0trend\u00C2\u00A0held\u00C2\u00A0true\u00C2\u00A0over\u00C2\u00A0multiple\u00C2\u00A0PM\u00CF\u0086:splenocyte\u00C2\u00A0ratios\u00C2\u00A0(Fig.\u00C2\u00A05.9B).\u00C2\u00A0We\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.8\u00C2\u00A0 Analysis\u00C2\u00A0of\u00C2\u00A0protein\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0populations.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells,\u00C2\u00A0 SpM\u00CF\u0086s,\u00C2\u00A0 or\u00C2\u00A0 TAMs\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 or\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0iNOS,\u00C2\u00A0Arg1,\u00C2\u00A0and\u00C2\u00A0GAPDH\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0Western\u00C2\u00A0blot.\u00C2\u00A0B,\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0tumors\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0Ym1,\u00C2\u00A0Arg1,\u00C2\u00A0and\u00C2\u00A0GAPDH\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0Western\u00C2\u00A0 blot.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0\u00C2\u00A0 103 also\u00C2\u00A0 performed\u00C2\u00A0 these\u00C2\u00A0 studies\u00C2\u00A0 using\u00C2\u00A0 C3H/HeN\u00C2\u00A0mice\u00C2\u00A0 bearing\u00C2\u00A0metastatic\u00C2\u00A0 squamous\u00C2\u00A0 cell\u00C2\u00A0 carcinoma\u00C2\u00A0(SCCVII)\u00C2\u00A0tumors\u00C2\u00A0(Suit\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01985)\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0suppressive\u00C2\u00A0 than\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0at\u00C2\u00A0higher\u00C2\u00A0concentrations\u00C2\u00A0 (i.e.\u00C2\u00A0 lower\u00C2\u00A0 ratios)\u00C2\u00A0 (Fig.\u00C2\u00A0 5.9C).\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 certain\u00C2\u00A0 tumors,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 metastatic\u00C2\u00A0 4T1\u00C2\u00A0 and\u00C2\u00A0 SCCVII\u00C2\u00A0 cell\u00C2\u00A0 lines,\u00C2\u00A0 enhance\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s.\u00C2\u00A0 \u00C2\u00A0 5.2.5.2 TAMs\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 isolated\u00C2\u00A0 TAMs\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 compared\u00C2\u00A0 their\u00C2\u00A0 abilities\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0in\u00C2\u00A0vitro.\u00C2\u00A0At\u00C2\u00A0each\u00C2\u00A0ratio\u00C2\u00A0tested,\u00C2\u00A067NR\u00C2\u00A0TAMs\u00C2\u00A0were\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0 immune\u00C2\u00A0 suppressors,\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 per\u00C2\u00A0 cell\u00C2\u00A0 basis\u00C2\u00A0 (Fig.\u00C2\u00A0 5.10).\u00C2\u00A0 This\u00C2\u00A0 was\u00C2\u00A0 somewhat\u00C2\u00A0 surprising,\u00C2\u00A0 since\u00C2\u00A0 4T1\u00C2\u00A0 TAMs,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 67NR\u00C2\u00A0 TAMs,\u00C2\u00A0 expressed\u00C2\u00A0 Arg1\u00C2\u00A0 (Fig.\u00C2\u00A0 5.4B),\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 often\u00C2\u00A0 associated\u00C2\u00A0 with\u00C2\u00A0 immune\u00C2\u00A0 suppression.\u00C2\u00A0 However,\u00C2\u00A0 this\u00C2\u00A0 finding\u00C2\u00A0 could\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 our\u00C2\u00A0 understanding\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 roles\u00C2\u00A0 of\u00C2\u00A0TAMs\u00C2\u00A0 in\u00C2\u00A0normoxic\u00C2\u00A0 (i.e.\u00C2\u00A0 67NR)\u00C2\u00A0 versus\u00C2\u00A0hypoxic\u00C2\u00A0 (i.e.\u00C2\u00A0 4T1)\u00C2\u00A0 primary\u00C2\u00A0tumors.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 5.2.5.3 SpM\u00CF\u0086s\u00C2\u00A0 \u00C2\u00A0 Studies\u00C2\u00A0 quantifying\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0 of\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 they\u00C2\u00A0were\u00C2\u00A0only\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0at\u00C2\u00A0high\u00C2\u00A0concentrations\u00C2\u00A0(Fig.\u00C2\u00A05.11).\u00C2\u00A0 As\u00C2\u00A0 the\u00C2\u00A0 ratio\u00C2\u00A0 of\u00C2\u00A0 SpM\u00CF\u0086s:\u00C2\u00A0 splenocytes\u00C2\u00A0 was\u00C2\u00A0 decreased,\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 no\u00C2\u00A0 longer\u00C2\u00A0 immunosuppressive\u00C2\u00A0(Fig.\u00C2\u00A05.11A).\u00C2\u00A0Interestingly,\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0exhibited\u00C2\u00A0 very\u00C2\u00A0similar\u00C2\u00A0suppressive\u00C2\u00A0properties\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A05.11A).\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0neither\u00C2\u00A04T1\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 tumors\u00C2\u00A0 altered\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 consistent\u00C2\u00A0 manner\u00C2\u00A0(Fig.\u00C2\u00A05.11B).\u00C2\u00A0 104 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.9\u00C2\u00A0 Tumors\u00C2\u00A0augment\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control,\u00C2\u00A0 4T1,\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 PM\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0 splenocyte)\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0B,\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 (o),\u00C2\u00A0 4T1\u00C2\u00A0 (\u00E2\u0096\u00A0),\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 (\u00E2\u0088\u0086)\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0ratios\u00C2\u00A0(PM\u00CF\u0086s:\u00C2\u00A0splenocytes;\u00C2\u00A01:2,\u00C2\u00A01:4,\u00C2\u00A01:8,\u00C2\u00A01:16,\u00C2\u00A01:32).\u00C2\u00A0Proliferation\u00C2\u00A0was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0C,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0 cultured\u00C2\u00A0alone\u00C2\u00A0(white\u00C2\u00A0bar)\u00C2\u00A0or\u00C2\u00A0with\u00C2\u00A0control\u00C2\u00A0(grey\u00C2\u00A0bar)\u00C2\u00A0or\u00C2\u00A0SCCVII\u00C2\u00A0(black\u00C2\u00A0bar)\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0 ratios\u00C2\u00A0 (PM\u00CF\u0086s:\u00C2\u00A0 splenocytes;\u00C2\u00A0 1:4,\u00C2\u00A0 1:8,\u00C2\u00A0 1:16,\u00C2\u00A0 1:32)\u00C2\u00A0 and\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 *,p<\u00C2\u00A0 0.05;\u00C2\u00A0 **,p<0.01;\u00C2\u00A0 NS,\u00C2\u00A0 not\u00C2\u00A0 significant\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0\u00C2\u00A0 105 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.10\u00C2\u00A0 67NR\u00C2\u00A0TAMs\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0immunosuppressive\u00C2\u00A0than\u00C2\u00A04T1\u00C2\u00A0TAMs.\u00C2\u00A0\u00C2\u00A0 TAMs\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 (open\u00C2\u00A0 square)\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 (closed\u00C2\u00A0 square)\u00C2\u00A0mice\u00C2\u00A0 using\u00C2\u00A0 F4/80+\u00C2\u00A0 selection\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0concentrations\u00C2\u00A0 (TAMs:\u00C2\u00A0splenocytes;\u00C2\u00A01:1,\u00C2\u00A01:2,\u00C2\u00A01:4,\u00C2\u00A01:8).\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 duplicate.\u00C2\u00A0\u00C2\u00A0 106 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.11\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0only\u00C2\u00A0immunosuppressive\u00C2\u00A0at\u00C2\u00A0high\u00C2\u00A0concentrations.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0control\u00C2\u00A0 (\u00CE\u00BF)\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0(\u00E2\u0096\u00A0)\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 concentrations\u00C2\u00A0 (SpM\u00CF\u0086s:\u00C2\u00A0 splenocytes;\u00C2\u00A0 2:1,\u00C2\u00A0 1:1,\u00C2\u00A0 1:2,\u00C2\u00A0 1:4,\u00C2\u00A0 1:8,\u00C2\u00A0 1:16,\u00C2\u00A0 1:32,\u00C2\u00A0 1:64).\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0B,\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control\u00C2\u00A0(white\u00C2\u00A0bars),\u00C2\u00A04T1\u00C2\u00A0(black\u00C2\u00A0bars),\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0(grey\u00C2\u00A0bars)\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090 cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 concentrations\u00C2\u00A0 (SPM\u00CF\u0086s:\u00C2\u00A0 splenocytes;\u00C2\u00A0 1:1,\u00C2\u00A0 1:2).\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0\u00C2\u00A0 107 5.2.5.4 Splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0we\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells.\u00C2\u00A0 Although\u00C2\u00A0 our\u00C2\u00A0 phenotypic\u00C2\u00A0 data\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 most\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 cells,\u00C2\u00A0 particularly\u00C2\u00A0 those\u00C2\u00A0 isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice,\u00C2\u00A0express\u00C2\u00A0both\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0Gr1\u00C2\u00A0(Fig.\u00C2\u00A05.7),\u00C2\u00A0before\u00C2\u00A0we\u00C2\u00A0could\u00C2\u00A0define\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0 as\u00C2\u00A0 MDSCs\u00C2\u00A0 we\u00C2\u00A0 first\u00C2\u00A0 had\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities.\u00C2\u00A0\u00C2\u00A0 Interestingly,\u00C2\u00A0we\u00C2\u00A0observed\u00C2\u00A0an\u00C2\u00A0immense\u00C2\u00A0difference\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0whether\u00C2\u00A0they\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control,\u00C2\u00A04T1,\u00C2\u00A0 or\u00C2\u00A067NR\u00C2\u00A0mice.\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Fig.\u00C2\u00A05.12A,\u00C2\u00A0while\u00C2\u00A04T1\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0potently\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 proliferation,\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0much\u00C2\u00A0less\u00C2\u00A0suppressive\u00C2\u00A0at\u00C2\u00A0all\u00C2\u00A0cell\u00C2\u00A0ratios\u00C2\u00A0tested.\u00C2\u00A0 We\u00C2\u00A0also\u00C2\u00A0assayed\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0cytokine\u00C2\u00A0production.\u00C2\u00A0Consistent\u00C2\u00A0 with\u00C2\u00A0the\u00C2\u00A0proliferation\u00C2\u00A0data,\u00C2\u00A04T1\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0inhibited\u00C2\u00A0 T\u00C2\u00A0cell\u00E2\u0080\u0090produced\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0 increased\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 IL\u00E2\u0080\u00902\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0culture\u00C2\u00A0medium\u00C2\u00A0(Fig.\u00C2\u00A05.12B),\u00C2\u00A0 which\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0model\u00C2\u00A0in\u00C2\u00A0which\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0produce\u00C2\u00A0IL\u00E2\u0080\u00902\u00C2\u00A0but\u00C2\u00A0are\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0utilize\u00C2\u00A0 it\u00C2\u00A0 for\u00C2\u00A0 cell\u00C2\u00A0division\u00C2\u00A0 (Strickland\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 1996).\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0all\u00C2\u00A0 three\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0Gr1+\u00C2\u00A0 cells\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 IL\u00E2\u0080\u009010\u00C2\u00A0production\u00C2\u00A0 to\u00C2\u00A0an\u00C2\u00A0equal,\u00C2\u00A0albeit\u00C2\u00A0minor,\u00C2\u00A0extent\u00C2\u00A0(Fig.\u00C2\u00A05.12B).\u00C2\u00A0 Finally,\u00C2\u00A0we\u00C2\u00A0assessed\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0NO\u00C2\u00A0and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0none\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0 subsets\u00C2\u00A0 produced\u00C2\u00A0 considerable\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 NO\u00C2\u00A0 upon\u00C2\u00A0 co\u00E2\u0080\u0090culture\u00C2\u00A0 with\u00C2\u00A0 activated\u00C2\u00A0 splenocytes\u00C2\u00A0 (Fig.\u00C2\u00A0 5.12B).\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 4T1\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 characterized\u00C2\u00A0 as\u00C2\u00A0 MDSCs,\u00C2\u00A0 since\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 potently\u00C2\u00A0 suppressing\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses,\u00C2\u00A0 whereas\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 67NR\u00C2\u00A0 or\u00C2\u00A0 control\u00C2\u00A0 mice\u00C2\u00A0 lack\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 therefore\u00C2\u00A0 not\u00C2\u00A0MDSCs.\u00C2\u00A0 These\u00C2\u00A0 findings\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 idea\u00C2\u00A0 that\u00C2\u00A0many\u00C2\u00A0tumors\u00C2\u00A0 induce\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0and\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0 MDSCs,\u00C2\u00A0 yet\u00C2\u00A0 also\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 not\u00C2\u00A0 all\u00C2\u00A0 tumors\u00C2\u00A0 induce\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 acquire\u00C2\u00A0 immunosuppressive\u00C2\u00A0abilities.\u00C2\u00A0 108 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.12\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 enhance\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 splenic\u00C2\u00A0Gr1+\u00C2\u00A0 cells.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 Splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 (o),\u00C2\u00A0 4T1\u00C2\u00A0 (\u00E2\u0096\u00A0),\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 (\u00E2\u0088\u0086)\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090 cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0ratios\u00C2\u00A0(Gr1+\u00C2\u00A0cells:\u00C2\u00A0splenocytes;\u00C2\u00A0 2:1,1:1,\u00C2\u00A0 1:2,\u00C2\u00A0 1:4,\u00C2\u00A0 1:8,\u00C2\u00A0 1:16,\u00C2\u00A0 1:32).\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 experiments,\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 B,\u00C2\u00A0 Splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control,\u00C2\u00A0 4T1,\u00C2\u00A0 or\u00C2\u00A0 67NR\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090 cultured\u00C2\u00A0with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0Gr1+\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes).\u00C2\u00A0 Supernatants\u00C2\u00A0 were\u00C2\u00A0collected\u00C2\u00A0and\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0IL\u00E2\u0080\u00902,\u00C2\u00A0or\u00C2\u00A0NO\u00C2\u00A0were\u00C2\u00A0determined.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0 mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0independent\u00C2\u00A0experiments,\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0 109 5.2.5.5 Gr1+\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0different\u00C2\u00A0tissues\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0accumulate\u00C2\u00A0in\u00C2\u00A0multiple\u00C2\u00A0organs\u00C2\u00A0and\u00C2\u00A0tissues\u00C2\u00A0(Ilkovitch\u00C2\u00A0 and\u00C2\u00A0Lopez,\u00C2\u00A02009;\u00C2\u00A0Youn\u00C2\u00A0and\u00C2\u00A0Gabrilovich,\u00C2\u00A02010),\u00C2\u00A0we\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0of\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0various\u00C2\u00A0sites.\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0spleens\u00C2\u00A0or\u00C2\u00A0 lungs\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 significantly\u00C2\u00A0 more\u00C2\u00A0 than\u00C2\u00A0 their\u00C2\u00A0 counterparts\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0mice,\u00C2\u00A0 and\u00C2\u00A0did\u00C2\u00A0 so\u00C2\u00A0 to\u00C2\u00A0 an\u00C2\u00A0 equal\u00C2\u00A0 extent\u00C2\u00A0 (Fig.\u00C2\u00A0 5.13A).\u00C2\u00A0 Similarly,\u00C2\u00A0 splenic\u00C2\u00A0 and\u00C2\u00A0 pulmonary\u00C2\u00A0 4T1\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 both\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 and\u00C2\u00A0 IL\u00E2\u0080\u009010\u00C2\u00A0 production\u00C2\u00A0 and\u00C2\u00A0 did\u00C2\u00A0 so\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 similar\u00C2\u00A0 degree\u00C2\u00A0 (Fig.\u00C2\u00A0 5.13B).\u00C2\u00A0 Finally,\u00C2\u00A0 we\u00C2\u00A0 compared\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 harvested\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 spleens,\u00C2\u00A0 lungs,\u00C2\u00A0 tumors,\u00C2\u00A0 kidneys,\u00C2\u00A0 and\u00C2\u00A0 livers\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 they\u00C2\u00A0 all\u00C2\u00A0 potently\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0degree\u00C2\u00A0(Fig.\u00C2\u00A05.13C).\u00C2\u00A0Consequently,\u00C2\u00A0from\u00C2\u00A0this\u00C2\u00A0point\u00C2\u00A0forward\u00C2\u00A0we\u00C2\u00A0 will\u00C2\u00A0refer\u00C2\u00A0to\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0as\u00C2\u00A0MDSCs.\u00C2\u00A0 110 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.13\u00C2\u00A0 4T1\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 different\u00C2\u00A0 tissues\u00C2\u00A0 are\u00C2\u00A0 equally\u00C2\u00A0 immunosuppressive.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bar)\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 spleens\u00C2\u00A0or\u00C2\u00A0 lungs\u00C2\u00A0of\u00C2\u00A0 control\u00C2\u00A0 (grey\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0 (black\u00C2\u00A0bars)\u00C2\u00A0 (1\u00C2\u00A0Gr1+\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes)\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 B,\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 spleens\u00C2\u00A0or\u00C2\u00A0lungs\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0Gr1+\u00C2\u00A0 cell:\u00C2\u00A02\u00C2\u00A0splenocytes).\u00C2\u00A0Supernatants\u00C2\u00A0were\u00C2\u00A0collected,\u00C2\u00A0and\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0IL\u00E2\u0080\u009010\u00C2\u00A0were\u00C2\u00A0measured.\u00C2\u00A0Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0performed\u00C2\u00A0 in\u00C2\u00A0duplicate.\u00C2\u00A0C,\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0spleens,\u00C2\u00A0lungs,\u00C2\u00A0tumors,\u00C2\u00A0kidneys,\u00C2\u00A0or\u00C2\u00A0livers\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 Gr1+\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes)\u00C2\u00A0 and\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 experiments,\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 **,p<0.01;\u00C2\u00A0 ***,p<\u00C2\u00A00.001\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0Gr1+\u00C2\u00A0cells.\u00C2\u00A0 111 5.2.6 Summary\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 myeloid\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00ADbearing\u00C2\u00A0mice\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0 previously\u00C2\u00A0 mentioned,\u00C2\u00A0 few\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 directly\u00C2\u00A0 compared\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0potencies\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0types.\u00C2\u00A0To\u00C2\u00A0address\u00C2\u00A0this\u00C2\u00A0we\u00C2\u00A0plated\u00C2\u00A0 equal\u00C2\u00A0numbers\u00C2\u00A0of\u00C2\u00A0splenic\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0their\u00C2\u00A0 abilities\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 polyclonal\u00C2\u00A0 and\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00C2\u00A0 stimulated\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation.\u00C2\u00A0 Our\u00C2\u00A0 results\u00C2\u00A0 clearly\u00C2\u00A0 demonstrated\u00C2\u00A0 that\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 much\u00C2\u00A0 more\u00C2\u00A0 immunosuppressive\u00C2\u00A0 than\u00C2\u00A0 splenic\u00C2\u00A0 MDSCs,\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 per\u00C2\u00A0 cell\u00C2\u00A0 basis\u00C2\u00A0 (Fig.\u00C2\u00A0 5.14A).\u00C2\u00A0 Specifically,\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 the\u00C2\u00A0 following:\u00C2\u00A0 1)\u00C2\u00A0 control\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 control\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 possess\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties,\u00C2\u00A02)\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0immunosuppressive\u00C2\u00A0than\u00C2\u00A04T1\u00C2\u00A0MDSCs,\u00C2\u00A0and\u00C2\u00A03)\u00C2\u00A0the\u00C2\u00A0 presence\u00C2\u00A0of\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 increases\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0Gr1+\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 5.14A).\u00C2\u00A0 As\u00C2\u00A0 Figure\u00C2\u00A0 5.14B\u00C2\u00A0 illustrates,\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 more\u00C2\u00A0 than\u00C2\u00A0 32\u00E2\u0080\u0090fold\u00C2\u00A0 (polyclonal)\u00C2\u00A0 or\u00C2\u00A0 10\u00E2\u0080\u0090fold\u00C2\u00A0 (Ag\u00E2\u0080\u0090specific)\u00C2\u00A0 more\u00C2\u00A0 immunosuppressive\u00C2\u00A0 than\u00C2\u00A0 MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A04T1\u00C2\u00A0mouse.\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0performed\u00C2\u00A0a\u00C2\u00A0direct\u00C2\u00A0comparison\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0 the\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 we\u00C2\u00A0 isolated\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 the\u00C2\u00A0 following\u00C2\u00A0 immunosuppressive\u00C2\u00A0 hierarchy\u00C2\u00A0 (listed\u00C2\u00A0 from\u00C2\u00A0most\u00C2\u00A0suppressive\u00C2\u00A0to\u00C2\u00A0least\u00C2\u00A0suppressive):\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0>\u00C2\u00A04T1\u00C2\u00A0TAMs\u00C2\u00A0\u00E2\u0089\u0088\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0>\u00C2\u00A0 4T1\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0>\u00C2\u00A0control\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0>\u00C2\u00A04T1\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0>\u00C2\u00A0control\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0these\u00C2\u00A0trends\u00C2\u00A0were\u00C2\u00A0 comparable\u00C2\u00A0whether\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0activated\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0polyclonal\u00C2\u00A0or\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00C2\u00A0manner\u00C2\u00A0 (Fig.\u00C2\u00A05.14C).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 112 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.14\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0suppressors\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0than\u00C2\u00A04T1\u00C2\u00A0 MDSCs\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0Splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090\u00C2\u00A0 (left)\u00C2\u00A0 or\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0 (right)\u00C2\u00A0 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 myeloid\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes).\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0B,\u00C2\u00A0 Splenic\u00C2\u00A0Gr1+\u00C2\u00A0 cells\u00C2\u00A0 (black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 (white\u00C2\u00A0bars)\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090 cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090\u00C2\u00A0 or\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 myeloid\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes).\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0\u00C2\u00A0 C,\u00C2\u00A0Splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0TAMs,\u00C2\u00A0or\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090 cultured\u00C2\u00A0with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090\u00C2\u00A0 (left)\u00C2\u00A0 or\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090\u00C2\u00A0 (right)\u00C2\u00A0 splenocytes.\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<\u00C2\u00A00.001\u00C2\u00A0 113 These\u00C2\u00A0 results\u00C2\u00A0 are\u00C2\u00A0 also\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0data\u00C2\u00A0 comparing\u00C2\u00A0 the\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 cytokine\u00C2\u00A0 production.\u00C2\u00A0 As\u00C2\u00A0 Figure\u00C2\u00A0 5.15A\u00C2\u00A0 shows,\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0TAMs\u00C2\u00A0 inhibited\u00C2\u00A0T\u00C2\u00A0 cell\u00E2\u0080\u0090produced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 and\u00C2\u00A0 IL\u00E2\u0080\u009010\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 extent\u00C2\u00A0 than\u00C2\u00A0MDSCs\u00C2\u00A0or\u00C2\u00A0 SpM\u00CF\u0086s.\u00C2\u00A0 Interestingly,\u00C2\u00A0we\u00C2\u00A0also\u00C2\u00A0measured\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0NO\u00C2\u00A0 in\u00C2\u00A0 culture\u00C2\u00A0 supernatants\u00C2\u00A0and\u00C2\u00A0 found\u00C2\u00A0that\u00C2\u00A0only\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0which\u00C2\u00A0we\u00C2\u00A0had\u00C2\u00A0previously\u00C2\u00A0determined\u00C2\u00A0 suppress\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0NO\u00E2\u0080\u0090mediated\u00C2\u00A0mechanism\u00C2\u00A0(Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010),\u00C2\u00A0produced\u00C2\u00A0NO\u00C2\u00A0upon\u00C2\u00A0co\u00E2\u0080\u0090 culture\u00C2\u00A0with\u00C2\u00A0activated\u00C2\u00A0splenocytes\u00C2\u00A0(Fig.\u00C2\u00A05.15B).\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0none\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A04T1\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 exerted\u00C2\u00A0 their\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 via\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0 previous\u00C2\u00A0 report\u00C2\u00A0 that\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 mammary\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 produce\u00C2\u00A0less\u00C2\u00A0NO\u00C2\u00A0upon\u00C2\u00A0LPS\u00C2\u00A0stimulation\u00C2\u00A0than\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(Di\u00C2\u00A0Napoli\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.15\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0 suppressors\u00C2\u00A0 of\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 cytokine\u00C2\u00A0production\u00C2\u00A0 than\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0TAMs,\u00C2\u00A0and\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0myeloid\u00C2\u00A0cell:\u00C2\u00A02\u00C2\u00A0splenocytes)\u00C2\u00A0and\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0and\u00C2\u00A0IL\u00E2\u0080\u009010\u00C2\u00A0 were\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 culture\u00C2\u00A0 supernatants.\u00C2\u00A0B,\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0control\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0TAMs,\u00C2\u00A0and\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice.\u00C2\u00A0Myeloid\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 myeloid\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes).\u00C2\u00A0 Supernatants\u00C2\u00A0 were\u00C2\u00A0 collected\u00C2\u00A0 and\u00C2\u00A0 NO\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments,\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0 114 Finally,\u00C2\u00A0 we\u00C2\u00A0 corroborated\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 flow\u00C2\u00A0 cytometric\u00C2\u00A0 approach.\u00C2\u00A0 We\u00C2\u00A0 labelled\u00C2\u00A0 responder\u00C2\u00A0 splenocytes\u00C2\u00A0 with\u00C2\u00A0 CFSE,\u00C2\u00A0 a\u00C2\u00A0 fluorescent\u00C2\u00A0 dye\u00C2\u00A0 that\u00C2\u00A0 becomes\u00C2\u00A0 stably\u00C2\u00A0 trapped\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 cytoplasm\u00C2\u00A0 of\u00C2\u00A0 individual\u00C2\u00A0 cells.\u00C2\u00A0 Using\u00C2\u00A0 this\u00C2\u00A0 method,\u00C2\u00A0 we\u00C2\u00A0 were\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 visualize\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation,\u00C2\u00A0since\u00C2\u00A0each\u00C2\u00A0time\u00C2\u00A0a\u00C2\u00A0cell\u00C2\u00A0divides\u00C2\u00A0its\u00C2\u00A0fluorescence\u00C2\u00A0intensity\u00C2\u00A0is\u00C2\u00A0 halved,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0peaks\u00C2\u00A0that\u00C2\u00A0each\u00C2\u00A0represent\u00C2\u00A0one\u00C2\u00A0cell\u00C2\u00A0division\u00C2\u00A0(Lyons,\u00C2\u00A02000).\u00C2\u00A0 We\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0with\u00C2\u00A0 stimulated\u00C2\u00A0CFSE\u00E2\u0080\u0090labeled\u00C2\u00A0 splenocytes\u00C2\u00A0 and\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0analyzed\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0CD4+\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0undergone\u00C2\u00A0cell\u00C2\u00A0 division,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0divisions\u00C2\u00A0(Fig.\u00C2\u00A05.16).\u00C2\u00A0However,\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0 viable\u00C2\u00A0cells\u00C2\u00A0we\u00C2\u00A0were\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0obtain\u00C2\u00A0data\u00C2\u00A0 for\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A04T1\u00C2\u00A0Gr1+\u00C2\u00A0 cells,\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0is\u00C2\u00A0discussed\u00C2\u00A0further\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A05.3.\u00C2\u00A0In\u00C2\u00A0general,\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0were\u00C2\u00A0consistent\u00C2\u00A0 with\u00C2\u00A0our\u00C2\u00A03H\u00E2\u0080\u0090thy\u00C2\u00A0incorporation\u00C2\u00A0data;\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A04T1\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A04T1\u00C2\u00A0TAMs\u00C2\u00A0exhibited\u00C2\u00A0 strong\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects,\u00C2\u00A0 reducing\u00C2\u00A0 the\u00C2\u00A0 maximal\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 divisions\u00C2\u00A0 (Fig.\u00C2\u00A05.16)\u00C2\u00A0and\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0divided\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0(Fig.\u00C2\u00A05.17),\u00C2\u00A0while\u00C2\u00A0control\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0control\u00C2\u00A0 SpM\u00CF\u0086s,\u00C2\u00A0and\u00C2\u00A04T1\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0did\u00C2\u00A0not.\u00C2\u00A0 In\u00C2\u00A0both\u00C2\u00A0proliferation\u00C2\u00A0assays,\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0 immunosuppressive,\u00C2\u00A0completely\u00C2\u00A0preventing\u00C2\u00A0all\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0division\u00C2\u00A0(Fig.\u00C2\u00A05.14\u00C2\u00A0and\u00C2\u00A0Fig\u00C2\u00A05.17).\u00C2\u00A0 Finally,\u00C2\u00A0we\u00C2\u00A0compared\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0CD4+\u00C2\u00A0versus\u00C2\u00A0 CD8+\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0they\u00C2\u00A0did\u00C2\u00A0so\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0similar\u00C2\u00A0extent\u00C2\u00A0(Fig.\u00C2\u00A05.18).\u00C2\u00A0 115 \u00C2\u00A0 Figure\u00C2\u00A05.16\u00C2\u00A0 Visualization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 populations.\u00C2\u00A0\u00C2\u00A0 Responder\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 labelled\u00C2\u00A0 with\u00C2\u00A0 fluorescent\u00C2\u00A0 CFSE\u00C2\u00A0 dye\u00C2\u00A0 and\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells,\u00C2\u00A0 TAMs,\u00C2\u00A0 and\u00C2\u00A0 SpM\u00CF\u0086s\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 or\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 polyclonal\u00C2\u00A0stimulation.\u00C2\u00A0After\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0for\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0CD4,\u00C2\u00A0CD8,\u00C2\u00A0and\u00C2\u00A0CFSE.\u00C2\u00A0The\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0CD4+\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0shown,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0undergone\u00C2\u00A0cell\u00C2\u00A0division.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0 experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0N/A;\u00C2\u00A0assay\u00C2\u00A0could\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0performed\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0insufficient\u00C2\u00A0 live\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 116 \u00C2\u00A0 \u00C2\u00A0 117 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.17\u00C2\u00A0 Summary\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0to\u00C2\u00A0inhibit\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0division\u00C2\u00A0as\u00C2\u00A0determined\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometric\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0CFSE\u00C2\u00A0intensity.\u00C2\u00A0\u00C2\u00A0 Proportion\u00C2\u00A0of\u00C2\u00A0CFSE\u00E2\u0080\u0090labelled\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0CD4+\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0 T\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0divided\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0once\u00C2\u00A0during\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 populations.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0n/a;\u00C2\u00A0assay\u00C2\u00A0 could\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0performed\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0insufficient\u00C2\u00A0live\u00C2\u00A0cells.\u00C2\u00A0 118 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.18\u00C2\u00A0 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(lower)\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<\u00C2\u00A00.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0 relative\u00C2\u00A0to\u00C2\u00A0RC\u00C2\u00A0\u00C2\u00A0 119 5.2.7 4T1\u00C2\u00ADinduced\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0TAMs,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0decrease\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 subsequently\u00C2\u00A0 performed\u00C2\u00A0 a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 experiments\u00C2\u00A0 to\u00C2\u00A0 elucidate\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 immunosuppression\u00C2\u00A0 utilized\u00C2\u00A0 by\u00C2\u00A0 different\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types.\u00C2\u00A0We\u00C2\u00A0 first\u00C2\u00A0 investigated\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0viability.\u00C2\u00A0We\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0or\u00C2\u00A0TAMs\u00C2\u00A0significantly\u00C2\u00A0decreased\u00C2\u00A0 the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0splenocytes,\u00C2\u00A0but\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0or\u00C2\u00A0with\u00C2\u00A0 control\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0(Fig.\u00C2\u00A05.19A).\u00C2\u00A0However,\u00C2\u00A0when\u00C2\u00A0we\u00C2\u00A0specifically\u00C2\u00A0assayed\u00C2\u00A0 the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0viability,\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0were\u00C2\u00A0less\u00C2\u00A0clear.\u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0 that\u00C2\u00A0control\u00C2\u00A0or\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0greatly\u00C2\u00A0 increased\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0CD4+\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0 cells,\u00C2\u00A0while\u00C2\u00A0 control\u00C2\u00A0 or\u00C2\u00A0 4T1\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 both\u00C2\u00A0 decreased\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 viability,\u00C2\u00A0 and\u00C2\u00A0 4T1\u00C2\u00A0 TAMs\u00C2\u00A0 decreased\u00C2\u00A0 CD4+\u00C2\u00A0 and\u00C2\u00A0 increased\u00C2\u00A0 CD8+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 (Fig.\u00C2\u00A0 5.19B).\u00C2\u00A0 These\u00C2\u00A0 results\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 have\u00C2\u00A0 contrasting\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 and\u00C2\u00A0 suggest\u00C2\u00A0that\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0MDSCs,\u00C2\u00A0and\u00C2\u00A0TAMs\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0mechanisms.\u00C2\u00A0\u00C2\u00A0 120 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.19\u00C2\u00A0 Effect\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0on\u00C2\u00A0cell\u00C2\u00A0viability.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 for\u00C2\u00A0 72\u00C2\u00A0 h\u00C2\u00A0 with\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 different\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 populations\u00C2\u00A0 and\u00C2\u00A0A,\u00C2\u00A0 Bulk\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 (total\u00C2\u00A0 splenocytes)\u00C2\u00A0 and\u00C2\u00A0B,\u00C2\u00A0 CD4+\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0 and\u00C2\u00A0 CD8+\u00C2\u00A0 (white\u00C2\u00A0 bars)\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 was\u00C2\u00A0 determined\u00C2\u00A0 by\u00C2\u00A0 exclusion\u00C2\u00A0 of\u00C2\u00A0 PI\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0 For\u00C2\u00A0 B,\u00C2\u00A0 data\u00C2\u00A0 are\u00C2\u00A0 reported\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 fold\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 alone\u00C2\u00A0 (responder\u00C2\u00A0 control).\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0duplicate.\u00C2\u00A0***,p<\u00C2\u00A00.001\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0RC\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 121 5.2.8 4T1\u00C2\u00ADinduced\u00C2\u00A0 MDSCs\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 contact\u00C2\u00ADindependent\u00C2\u00A0 mechanism\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 further\u00C2\u00A0 explore\u00C2\u00A0 the\u00C2\u00A0 different\u00C2\u00A0 suppressive\u00C2\u00A0 mechanisms\u00C2\u00A0 used\u00C2\u00A0 by\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0we\u00C2\u00A0used\u00C2\u00A0a\u00C2\u00A0Transwell\u00C2\u00A0assay\u00C2\u00A0system\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0direct\u00C2\u00A0cell\u00E2\u0080\u0090cell\u00C2\u00A0contact\u00C2\u00A0 was\u00C2\u00A0 required.\u00C2\u00A0We\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 either\u00C2\u00A0 the\u00C2\u00A0 spleen\u00C2\u00A0 or\u00C2\u00A0 lungs\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 could\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0whether\u00C2\u00A0 or\u00C2\u00A0 not\u00C2\u00A0 they\u00C2\u00A0were\u00C2\u00A0 separated\u00C2\u00A0 from\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0semi\u00E2\u0080\u0090permeable\u00C2\u00A0membrane\u00C2\u00A0(Fig.\u00C2\u00A05.20A),\u00C2\u00A0suggesting\u00C2\u00A0contact\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0 MDSC\u00C2\u00A0immunosuppression.\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0only\u00C2\u00A0immunosuppressive\u00C2\u00A0when\u00C2\u00A0 in\u00C2\u00A0 direct\u00C2\u00A0 contact\u00C2\u00A0 with\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 promoted\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 when\u00C2\u00A0 contact\u00C2\u00A0 was\u00C2\u00A0 removed\u00C2\u00A0(Fig.\u00C2\u00A05.20A).\u00C2\u00A0However,\u00C2\u00A0when\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0proliferation\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0 ratios,\u00C2\u00A0we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0at\u00C2\u00A0high\u00C2\u00A0 concentrations\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 could\u00C2\u00A0 inhibit\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0even\u00C2\u00A0when\u00C2\u00A0contact\u00C2\u00A0with\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0prevented,\u00C2\u00A0but\u00C2\u00A0this\u00C2\u00A0was\u00C2\u00A0 not\u00C2\u00A0true\u00C2\u00A0at\u00C2\u00A0lower\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A05.20B).\u00C2\u00A0One\u00C2\u00A0hypothesis\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0 with\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0produce\u00C2\u00A0one\u00C2\u00A0or\u00C2\u00A0more\u00C2\u00A0secreted\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 function\u00C2\u00A0 along\u00C2\u00A0a\u00C2\u00A0concentration\u00C2\u00A0gradient,\u00C2\u00A0and\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0increases,\u00C2\u00A0so\u00C2\u00A0does\u00C2\u00A0the\u00C2\u00A0level\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 factor(s),\u00C2\u00A0 which\u00C2\u00A0 enables\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 distance.\u00C2\u00A0 However,\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0the\u00C2\u00A0secreted\u00C2\u00A0factor,\u00C2\u00A0are\u00C2\u00A0low,\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 must\u00C2\u00A0 be\u00C2\u00A0 in\u00C2\u00A0 close\u00C2\u00A0 contact\u00C2\u00A0 with\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0 their\u00C2\u00A0 activation.\u00C2\u00A0 Finally,\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 whether\u00C2\u00A0there\u00C2\u00A0were\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0contact\u00C2\u00A0requirement\u00C2\u00A0for\u00C2\u00A0control,\u00C2\u00A04T1,\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0PM\u00CF\u0086\u00C2\u00A0 immunosuppression\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0even\u00C2\u00A0at\u00C2\u00A0high\u00C2\u00A0concentrations,\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0required\u00C2\u00A0 contact\u00C2\u00A0 to\u00C2\u00A0 effectively\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation,\u00C2\u00A0 while\u00C2\u00A0 4T1\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 could\u00C2\u00A0 suppress\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0control\u00C2\u00A0and\u00C2\u00A0Transwell\u00C2\u00A0systems\u00C2\u00A0(Fig.\u00C2\u00A05.20C).\u00C2\u00A0However,\u00C2\u00A0all\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 types\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 degree\u00C2\u00A0 when\u00C2\u00A0 contact\u00C2\u00A0 was\u00C2\u00A0 allowed\u00C2\u00A0between\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0test\u00C2\u00A0cells\u00C2\u00A0(Fig.\u00C2\u00A05.20).\u00C2\u00A0\u00C2\u00A0 122 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.20\u00C2\u00A0 At\u00C2\u00A0 high\u00C2\u00A0 concentrations,\u00C2\u00A0 both\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 via\u00C2\u00A0 contact\u00C2\u00ADindependent\u00C2\u00A0mechanisms.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0MDSCs\u00C2\u00A0(splenic\u00C2\u00A0and\u00C2\u00A0pulmonary)\u00C2\u00A0and\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A02\u00C2\u00A0splenocytes\u00C2\u00A0or\u00C2\u00A01\u00C2\u00A0PM\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0in\u00C2\u00A0 control\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0Transwell\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0wells.\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 B,\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090 stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 concentrations\u00C2\u00A0 (PM\u00CF\u0086:\u00C2\u00A0 splenocytes;\u00C2\u00A0 1:2,\u00C2\u00A0 1:8)\u00C2\u00A0 in\u00C2\u00A0 control\u00C2\u00A0 (black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0Transwell\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0wells.\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0 of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0C,\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control,\u00C2\u00A04T1,\u00C2\u00A0or\u00C2\u00A067NR\u00C2\u00A0mice\u00C2\u00A0and\u00C2\u00A0co\u00E2\u0080\u0090 cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 PM\u00CF\u0086:\u00C2\u00A0 2\u00C2\u00A0 splenocytes)\u00C2\u00A0 in\u00C2\u00A0 control\u00C2\u00A0 (black\u00C2\u00A0 bars)\u00C2\u00A0or\u00C2\u00A0Transwell\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0wells.\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0 123 5.2.9 PM\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 and\u00C2\u00A067NR\u00C2\u00A0 tumor\u00C2\u00ADbearing\u00C2\u00A0mice\u00C2\u00A0 suppress\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0NO\u00C2\u00AD dependent\u00C2\u00A0mechanism\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0our\u00C2\u00A0previous\u00C2\u00A0work,\u00C2\u00A0presented\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03,\u00C2\u00A0had\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0mice\u00C2\u00A0 suppressed\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0 via\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0 (Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010),\u00C2\u00A0we\u00C2\u00A0 tested\u00C2\u00A0 whether\u00C2\u00A0 any\u00C2\u00A0 other\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 suppressed\u00C2\u00A0 by\u00C2\u00A0 this\u00C2\u00A0mechanism.\u00C2\u00A0We\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 control\u00C2\u00A0and\u00C2\u00A067NR,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0produced\u00C2\u00A0NO\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 (Fig.\u00C2\u00A05.21A).\u00C2\u00A0Consistent\u00C2\u00A0with\u00C2\u00A0 this,\u00C2\u00A0 inhibiting\u00C2\u00A0NO\u00C2\u00A0production\u00C2\u00A0via\u00C2\u00A0 iNOS\u00C2\u00A0 inhibitors\u00C2\u00A0 (i.e.\u00C2\u00A0L\u00E2\u0080\u0090 NMMA,\u00C2\u00A0 L\u00E2\u0080\u0090NIL)\u00C2\u00A0 or\u00C2\u00A0 iNOS\u00C2\u00A0 induction\u00C2\u00A0 by\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 (i.e.\u00C2\u00A0 anti\u00E2\u0080\u0090IFN\u00E2\u0080\u0090\u00CE\u00B3)\u00C2\u00A0 reversed\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 control\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 5.21B).\u00C2\u00A0 Conversely,\u00C2\u00A0 although\u00C2\u00A0 67NR\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 produced\u00C2\u00A0moderate\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0NO\u00C2\u00A0upon\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0these\u00C2\u00A0inhibitors\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 eradicate\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0suppression\u00C2\u00A0by\u00C2\u00A067NR\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A05.21B),\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A067NR\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0may\u00C2\u00A0suppress\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0or\u00C2\u00A0additional\u00C2\u00A0mechanisms.\u00C2\u00A0 124 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.21\u00C2\u00A0 Control\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0but\u00C2\u00A0no\u00C2\u00A0other\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0tested,\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0 via\u00C2\u00A0NO\u00C2\u00A0production.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0control\u00C2\u00A0mice;\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0splenic\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0TAMs,\u00C2\u00A0and\u00C2\u00A0SpM\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0 isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice;\u00C2\u00A0and\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0TAMs\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A067NR\u00C2\u00A0mice.\u00C2\u00A0Each\u00C2\u00A0cell\u00C2\u00A0type\u00C2\u00A0 was\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 myeloid\u00C2\u00A0 cell:\u00C2\u00A0 2\u00C2\u00A0 splenocytes)\u00C2\u00A0 and\u00C2\u00A0 supernatant\u00C2\u00A0 NO\u00C2\u00A0 production\u00C2\u00A0 measured.\u00C2\u00A0B,\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 PM\u00CF\u0086:\u00C2\u00A0 8\u00C2\u00A0 splenocytes)\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 L\u00E2\u0080\u0090NMMA,\u00C2\u00A0 L\u00E2\u0080\u0090NIL,\u00C2\u00A0 or\u00C2\u00A0 \u00CE\u00B1IFN\u00E2\u0080\u0090\u00CE\u00B3.\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 125 5.2.10 The\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0abrogated\u00C2\u00A0by\u00C2\u00A0 TLR\u00C2\u00A0stimulation\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 we\u00C2\u00A0 had\u00C2\u00A0 previously\u00C2\u00A0 elucidated\u00C2\u00A0 the\u00C2\u00A0 mechanism\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 control\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses,\u00C2\u00A0 we\u00C2\u00A0 were\u00C2\u00A0 particularly\u00C2\u00A0 interested\u00C2\u00A0 in\u00C2\u00A0 investigating\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0mechanism(s)\u00C2\u00A0used\u00C2\u00A0by\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0to\u00C2\u00A0gain\u00C2\u00A0insight\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0tumors\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 phenotypes\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 \u00C2\u00A0 Furthermore,\u00C2\u00A0 our\u00C2\u00A0 results\u00C2\u00A0 had\u00C2\u00A0 revealed\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0potent\u00C2\u00A0 immunosuppressive\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0those\u00C2\u00A0we\u00C2\u00A0tested,\u00C2\u00A0which\u00C2\u00A0made\u00C2\u00A0 them\u00C2\u00A0 particularly\u00C2\u00A0 intriguing.\u00C2\u00A0 Since\u00C2\u00A0 we\u00C2\u00A0 had\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 control\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 reversed\u00C2\u00A0 by\u00C2\u00A0 pre\u00E2\u0080\u0090treatment\u00C2\u00A0 with\u00C2\u00A0 LPS\u00C2\u00A0 or\u00C2\u00A0 dsRNA\u00C2\u00A0 we\u00C2\u00A0 first\u00C2\u00A0 determined\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0TLR\u00C2\u00A0agonists\u00C2\u00A0on\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086\u00C2\u00A0immunosuppression.\u00C2\u00A0Unlike\u00C2\u00A0control\u00C2\u00A0 PM\u00CF\u0086s,\u00C2\u00A0pre\u00E2\u0080\u0090treating\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0inhibit\u00C2\u00A0their\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities;\u00C2\u00A0in\u00C2\u00A0fact,\u00C2\u00A0 pre\u00E2\u0080\u0090treatment\u00C2\u00A0with\u00C2\u00A0LPS\u00C2\u00A0 and\u00C2\u00A0dsRNA\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086\u00E2\u0080\u0090induced\u00C2\u00A0 suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0(Fig.\u00C2\u00A05.22).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.22\u00C2\u00A0 The\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0not\u00C2\u00A0 reversed\u00C2\u00A0 by\u00C2\u00A0 pre\u00C2\u00ADtreatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0ligands.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(1\u00C2\u00A0PM\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0been\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0LPS,\u00C2\u00A0CpG,\u00C2\u00A0dsRNA,\u00C2\u00A0or\u00C2\u00A0PGN\u00C2\u00A0for\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 washed,\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0*,p<\u00C2\u00A00.05;\u00C2\u00A0***,p<0.001;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0 relative\u00C2\u00A0to\u00C2\u00A0untreated\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0\u00C2\u00A0 126 5.2.11 4T1\u00C2\u00A0PM\u00CF\u0086\u00C2\u00A0suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0reversed\u00C2\u00A0by\u00C2\u00A0N\u00C2\u00ADacetyl\u00C2\u00ADcysteine\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0we\u00C2\u00A0utilized\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0panel\u00C2\u00A0of\u00C2\u00A0inhibitors\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0to\u00C2\u00A0elucidate\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086\u00E2\u0080\u0090induced\u00C2\u00A0immune\u00C2\u00A0suppression.\u00C2\u00A0Consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0previous\u00C2\u00A0 results\u00C2\u00A0 (Fig.\u00C2\u00A0 5.21B),\u00C2\u00A0 inhibitors\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 NO\u00C2\u00A0 pathway\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 reverse\u00C2\u00A0 suppression\u00C2\u00A0 (Fig.\u00C2\u00A0 5.23A).\u00C2\u00A0Similarly,\u00C2\u00A0blocking\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0immunosuppressive\u00C2\u00A0cytokines\u00C2\u00A0also\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0abrogate\u00C2\u00A0 suppression\u00C2\u00A0(Fig.\u00C2\u00A05.23B),\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 by\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0IL\u00E2\u0080\u00904,\u00C2\u00A0IL\u00E2\u0080\u009010,\u00C2\u00A0IL\u00E2\u0080\u009013,\u00C2\u00A0or\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2.\u00C2\u00A0Next\u00C2\u00A0we\u00C2\u00A0tested\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0IL\u00E2\u0080\u00902\u00C2\u00A0or\u00C2\u00A0L\u00E2\u0080\u0090Arg\u00C2\u00A0 supplementation,\u00C2\u00A0 inhibiting\u00C2\u00A0 Arg1,\u00C2\u00A0 or\u00C2\u00A0 blocking\u00C2\u00A0 the\u00C2\u00A0 membrane\u00E2\u0080\u0090bound\u00C2\u00A0 inhibitory\u00C2\u00A0 molecules\u00C2\u00A0CTLA4\u00C2\u00A0or\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0but\u00C2\u00A0none\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0 treatments\u00C2\u00A0restored\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 (Fig.\u00C2\u00A0 5.23C).\u00C2\u00A0 Finally,\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 the\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 ROS\u00C2\u00A0 inhibitors\u00C2\u00A0 on\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086\u00C2\u00A0 immunosuppression.\u00C2\u00A0Catalase\u00C2\u00A0catalyzes\u00C2\u00A0the\u00C2\u00A0conversion\u00C2\u00A0of\u00C2\u00A0H2O2\u00C2\u00A0to\u00C2\u00A0H2O\u00C2\u00A0and\u00C2\u00A0O2,\u00C2\u00A0and\u00C2\u00A0SOD\u00C2\u00A0 catalyzes\u00C2\u00A0 the\u00C2\u00A0dismutation\u00C2\u00A0of\u00C2\u00A0O2\u00E2\u0080\u0090\u00C2\u00A0 into\u00C2\u00A0O2\u00C2\u00A0and\u00C2\u00A0H2O2,\u00C2\u00A0while\u00C2\u00A0NAC,\u00C2\u00A0a\u00C2\u00A0derivative\u00C2\u00A0of\u00C2\u00A0cysteine,\u00C2\u00A0 has\u00C2\u00A0 both\u00C2\u00A0 indirect\u00C2\u00A0 and\u00C2\u00A0 direct\u00C2\u00A0 antioxidant\u00C2\u00A0 functions;\u00C2\u00A0 NAC\u00C2\u00A0 acts\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 scavenger\u00C2\u00A0 of\u00C2\u00A0 free\u00C2\u00A0 radicals\u00C2\u00A0and\u00C2\u00A0also\u00C2\u00A0serves\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0precursor\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0antioxidant\u00C2\u00A0glutathione\u00C2\u00A0 (GSH)\u00C2\u00A0 (Zhang\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 While\u00C2\u00A0 catalase\u00C2\u00A0 and\u00C2\u00A0 SOD\u00C2\u00A0 had\u00C2\u00A0 no\u00C2\u00A0 effect\u00C2\u00A0 in\u00C2\u00A0 our\u00C2\u00A0 assay,\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 NAC\u00C2\u00A0 completely\u00C2\u00A0 eliminated\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086\u00C2\u00A0 immunosuppression\u00C2\u00A0 (Fig.\u00C2\u00A0 5.23).\u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 stimulated\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0NAC.\u00C2\u00A0 Since\u00C2\u00A0NAC\u00C2\u00A0 can\u00C2\u00A0 also\u00C2\u00A0act\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0source\u00C2\u00A0of\u00C2\u00A0cysteine\u00C2\u00A0we\u00C2\u00A0supplemented\u00C2\u00A0cultures\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0concentrations\u00C2\u00A0 of\u00C2\u00A0L\u00E2\u0080\u0090cysteine\u00C2\u00A0 to\u00C2\u00A0 see\u00C2\u00A0 if\u00C2\u00A0 this\u00C2\u00A0mimicked\u00C2\u00A0 the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0NAC,\u00C2\u00A0but\u00C2\u00A0L\u00E2\u0080\u0090cysteine\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 reverse\u00C2\u00A0 4T1\u00C2\u00A0 PM\u00CF\u0086\u00E2\u0080\u0090induced\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown).\u00C2\u00A0 Taken\u00C2\u00A0 together\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0responses\u00C2\u00A0by\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0ROS\u00C2\u00A0other\u00C2\u00A0than\u00C2\u00A0H2O2\u00C2\u00A0 or\u00C2\u00A0O2\u00E2\u0080\u0090.\u00C2\u00A0 127 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.23\u00C2\u00A0 NAC\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0 4T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(1\u00C2\u00A0PM\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0A,\u00C2\u00A00.5\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090NMMA,\u00C2\u00A01\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090NIL,\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IFN\u00E2\u0080\u0090\u00CE\u00B3;\u00C2\u00A0 B,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0rat\u00C2\u00A0IgG,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u00904,\u00C2\u00A02\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u009010,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u009013,\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0 anti\u00E2\u0080\u0090TGF\u00E2\u0080\u0090\u00CE\u00B2;\u00C2\u00A0C,\u00C2\u00A0100\u00C2\u00A0U/well\u00C2\u00A0mIL\u00E2\u0080\u00902,\u00C2\u00A02\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090Arg,\u00C2\u00A0200\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0BEC,\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090CTLA4,\u00C2\u00A0or\u00C2\u00A0250\u00C2\u00A0 ng/ml\u00C2\u00A0LAP;\u00C2\u00A0or\u00C2\u00A0D,\u00C2\u00A01\u00C2\u00A0mg/ml\u00C2\u00A0catalase,\u00C2\u00A0200\u00C2\u00A0U/ml\u00C2\u00A0SOD,\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mM\u00C2\u00A0NAC\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0 measured.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0five\u00C2\u00A0independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0 128 5.2.12 The\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 pulmonary\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 inhibited\u00C2\u00A0by\u00C2\u00A0catalase\u00C2\u00A0 \u00C2\u00A0 Next,\u00C2\u00A0we\u00C2\u00A0performed\u00C2\u00A0a\u00C2\u00A0similar\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0experiments\u00C2\u00A0to\u00C2\u00A0investigate\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0 by\u00C2\u00A0which\u00C2\u00A0MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses.\u00C2\u00A0We\u00C2\u00A0 tested\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0pre\u00E2\u0080\u0090treating\u00C2\u00A0MDSCs\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0TLR\u00C2\u00A0ligands\u00C2\u00A0but\u00C2\u00A0none\u00C2\u00A0decreased\u00C2\u00A0 MDSC\u00E2\u0080\u0090induced\u00C2\u00A0immunosuppression\u00C2\u00A0(Fig.\u00C2\u00A05.24).\u00C2\u00A0We\u00C2\u00A0then\u00C2\u00A0used\u00C2\u00A0our\u00C2\u00A0panel\u00C2\u00A0of\u00C2\u00A0inhibitors\u00C2\u00A0to\u00C2\u00A0 try\u00C2\u00A0to\u00C2\u00A0elucidate\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0suppression.\u00C2\u00A0The\u00C2\u00A0only\u00C2\u00A0inhibitor\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0 4T1\u00C2\u00A0 MDSC\u00E2\u0080\u0090induced\u00C2\u00A0 suppression\u00C2\u00A0 was\u00C2\u00A0 catalase\u00C2\u00A0 (Fig.\u00C2\u00A0 5.25).\u00C2\u00A0 Although\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 catalase\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 completely\u00C2\u00A0 eliminate\u00C2\u00A0 4T1\u00C2\u00A0MDSC\u00C2\u00A0 suppressive\u00C2\u00A0 function,\u00C2\u00A0 it\u00C2\u00A0 significantly\u00C2\u00A0 reduced\u00C2\u00A0suppression\u00C2\u00A0and\u00C2\u00A0increased\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0(Fig.\u00C2\u00A05.25C),\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0 pulmonary\u00C2\u00A0 MDSCs\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects,\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0 part,\u00C2\u00A0 by\u00C2\u00A0 H2O2\u00C2\u00A0 production.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.24\u00C2\u00A0 The\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0reversed\u00C2\u00A0by\u00C2\u00A0 pre\u00C2\u00ADtreatment\u00C2\u00A0with\u00C2\u00A0TLR\u00C2\u00A0ligands.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0 4T1\u00C2\u00A0splenic\u00C2\u00A0MDSCs\u00C2\u00A0(i.e.\u00C2\u00A0Gr1+\u00C2\u00A0cells)\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A02\u00C2\u00A0splenocytes)\u00C2\u00A0that\u00C2\u00A0had\u00C2\u00A0been\u00C2\u00A0pre\u00E2\u0080\u0090treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0LPS,\u00C2\u00A0 CpG,\u00C2\u00A0 dsRNA,\u00C2\u00A0 or\u00C2\u00A0 PGN\u00C2\u00A0 for\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 washed,\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0 129 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.25\u00C2\u00A0 Catalase\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 Polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0with\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0 4T1\u00C2\u00A0pulmonary\u00C2\u00A0MDSCs\u00C2\u00A0(1\u00C2\u00A0PM\u00CF\u0086:\u00C2\u00A02\u00C2\u00A0splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0A,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0rat\u00C2\u00A0IgG,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090IL\u00E2\u0080\u00904,\u00C2\u00A0100\u00C2\u00A0 U/well\u00C2\u00A0mIL\u00E2\u0080\u00902,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090CTLA4,\u00C2\u00A0or\u00C2\u00A0250\u00C2\u00A0ng/ml\u00C2\u00A0LAP\u00C2\u00A0+\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/ml\u00C2\u00A0anti\u00E2\u0080\u0090TGF\u00E2\u0080\u0090\u00CE\u00B2;\u00C2\u00A0B,\u00C2\u00A00.5\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090 NMMA,\u00C2\u00A0 1\u00C2\u00A0 mM\u00C2\u00A0 L\u00E2\u0080\u0090NIL,\u00C2\u00A0 10\u00C2\u00A0 \u00CE\u00BCg/ml\u00C2\u00A0 anti\u00E2\u0080\u0090IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0 2\u00C2\u00A0 mM\u00C2\u00A0 L\u00E2\u0080\u0090Arg,\u00C2\u00A0 or\u00C2\u00A0 200\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0 BEC;\u00C2\u00A0 or\u00C2\u00A0 C,\u00C2\u00A0 1\u00C2\u00A0 mg/ml\u00C2\u00A0 catalase,\u00C2\u00A0 ,\u00C2\u00A0 10\u00C2\u00A0 mM\u00C2\u00A0 NAC,\u00C2\u00A0 or\u00C2\u00A0 200\u00C2\u00A0 U/ml\u00C2\u00A0 SOD\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 measured.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 ***,p<\u00C2\u00A0 0.001\u00C2\u00A0 relative\u00C2\u00A0to\u00C2\u00A0untreated\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0\u00C2\u00A0 130 5.2.13 M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0 suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 responses\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0ROS\u00C2\u00ADmediated\u00C2\u00A0 mechanisms\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 follow\u00C2\u00A0 up\u00C2\u00A0 on\u00C2\u00A0 these\u00C2\u00A0 experiments,\u00C2\u00A0 we\u00C2\u00A0 determined\u00C2\u00A0 whether\u00C2\u00A0 our\u00C2\u00A0 results\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 different\u00C2\u00A0 suppressive\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 valid\u00C2\u00A0for\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0from\u00C2\u00A0different\u00C2\u00A0tissues.\u00C2\u00A0\u00C2\u00A0Given\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0lung\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0metastatic\u00C2\u00A0 target\u00C2\u00A0 organ\u00C2\u00A0 for\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 we\u00C2\u00A0 were\u00C2\u00A0 particular\u00C2\u00A0 interested\u00C2\u00A0 in\u00C2\u00A0 pulmonary\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 Importantly,\u00C2\u00A0the\u00C2\u00A0lungs\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0contain\u00C2\u00A0both\u00C2\u00A0CD11b\u00E2\u0080\u0090CD11c+F4/80+\u00C2\u00A0resident\u00C2\u00A0alveolar\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0CD11b+CD11c\u00E2\u0080\u0090F4/80+\u00C2\u00A0infiltrating\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0Although\u00C2\u00A0both\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0populations\u00C2\u00A0are\u00C2\u00A0 isolated\u00C2\u00A0 by\u00C2\u00A0 F4/80\u00C2\u00A0 positive\u00C2\u00A0 selection,\u00C2\u00A0 the\u00C2\u00A0majority\u00C2\u00A0 of\u00C2\u00A0 lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0 are\u00C2\u00A0 likely\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0 tumor\u00C2\u00A0 cells,\u00C2\u00A0 given\u00C2\u00A0 the\u00C2\u00A0 high\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 splenomegaly\u00C2\u00A0 (Fig.\u00C2\u00A0 5.1B)\u00C2\u00A0 and\u00C2\u00A0 myelopoiesis\u00C2\u00A0 (Fig.\u00C2\u00A05.3)\u00C2\u00A0we\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0 these\u00C2\u00A0mice.\u00C2\u00A0We\u00C2\u00A0 first\u00C2\u00A0 tested\u00C2\u00A0whether\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0suppressed\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0mechanism.\u00C2\u00A0We\u00C2\u00A0 found\u00C2\u00A0that,\u00C2\u00A0 like\u00C2\u00A0PM\u00CF\u0086s,\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0decreased\u00C2\u00A0by\u00C2\u00A0NAC,\u00C2\u00A0 but\u00C2\u00A0not\u00C2\u00A0catalase\u00C2\u00A0(Fig.\u00C2\u00A05.26A).\u00C2\u00A0However,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0interesting\u00C2\u00A0that\u00C2\u00A0NAC\u00C2\u00A0completely\u00C2\u00A0eliminated\u00C2\u00A0 PM\u00CF\u0086\u00C2\u00A0 suppression,\u00C2\u00A0 but\u00C2\u00A0 only\u00C2\u00A0 partially\u00C2\u00A0 reduced\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 5.26A).\u00C2\u00A0 Importantly,\u00C2\u00A0 neither\u00C2\u00A0 NAC,\u00C2\u00A0 nor\u00C2\u00A0 any\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 inhibitors\u00C2\u00A0 we\u00C2\u00A0 tested\u00C2\u00A0 significantly\u00C2\u00A0 lessened\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 TAMs\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown),\u00C2\u00A0 which\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0TAMs\u00C2\u00A0 inhibit\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0mechanism\u00C2\u00A0than\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0 lung\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 These\u00C2\u00A0data\u00C2\u00A0may\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0site\u00C2\u00A0function\u00C2\u00A0differently\u00C2\u00A0than\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 periphery.\u00C2\u00A0 We\u00C2\u00A0 also\u00C2\u00A0 tested\u00C2\u00A0 whether\u00C2\u00A0 MDSCs\u00C2\u00A0 from\u00C2\u00A0 different\u00C2\u00A0 tissues\u00C2\u00A0 utilized\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 suppressive\u00C2\u00A0mechanism\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0catalase,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0NAC,\u00C2\u00A0significantly\u00C2\u00A0lessened\u00C2\u00A0the\u00C2\u00A0 suppressive\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0splenic\u00C2\u00A0and\u00C2\u00A0pulmonary\u00C2\u00A04T1\u00C2\u00A0MDSCs,\u00C2\u00A0and\u00C2\u00A0did\u00C2\u00A0so\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0equal\u00C2\u00A0 extent\u00C2\u00A0(Fig.\u00C2\u00A05.26B).\u00C2\u00A0All\u00C2\u00A0 together,\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0model\u00C2\u00A0 in\u00C2\u00A0which\u00C2\u00A04T1\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0exert\u00C2\u00A0extremely\u00C2\u00A0potent\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0via\u00C2\u00A0contact\u00E2\u0080\u0090dependent\u00C2\u00A0ROS\u00C2\u00A0 production,\u00C2\u00A0 control\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 strongly\u00C2\u00A0 inhibit\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 via\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090induced\u00C2\u00A0 NO\u00C2\u00A0 production,\u00C2\u00A0and\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0contact\u00E2\u0080\u0090independent\u00C2\u00A0ROS\u00C2\u00A0production,\u00C2\u00A0 albeit\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0lesser\u00C2\u00A0extent\u00C2\u00A0than\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0(Fig.\u00C2\u00A05.27).\u00C2\u00A0 131 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.26\u00C2\u00A0 4T1\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 via\u00C2\u00A0 different\u00C2\u00A0 ROS\u00C2\u00AD mediated\u00C2\u00A0mechanisms.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 Lung\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 or\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090 stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0M\u00CF\u0086:\u00C2\u00A08\u00C2\u00A0splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010\u00C2\u00A0mM\u00C2\u00A0NAC\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A01\u00C2\u00A0mg/ml\u00C2\u00A0catalase\u00C2\u00A0 (grey\u00C2\u00A0bars).\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0B,\u00C2\u00A0 Splenic\u00C2\u00A0 or\u00C2\u00A0 pulmonary\u00C2\u00A0 Gr1+\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0MDSC:\u00C2\u00A02\u00C2\u00A0splenocytes)\u00C2\u00A0\u00C2\u00B1\u00C2\u00A010\u00C2\u00A0mM\u00C2\u00A0NAC\u00C2\u00A0(black\u00C2\u00A0bars),\u00C2\u00A0or\u00C2\u00A01\u00C2\u00A0 mg/ml\u00C2\u00A0 (light\u00C2\u00A0 grey\u00C2\u00A0 bars)\u00C2\u00A0 or\u00C2\u00A0 2\u00C2\u00A0 mg/ml\u00C2\u00A0 (dark\u00C2\u00A0 grey\u00C2\u00A0 bars)\u00C2\u00A0 catalase.\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0experiments\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0triplicate.\u00C2\u00A0*,p<0.05;\u00C2\u00A0**,p<0.01;\u00C2\u00A0***,p<\u00C2\u00A00.001\u00C2\u00A0relative\u00C2\u00A0 to\u00C2\u00A0untreated\u00C2\u00A0cells\u00C2\u00A0\u00C2\u00A0 132 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.27\u00C2\u00A0 Myeloid\u00C2\u00A0cells\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0mechanisms\u00C2\u00A0and\u00C2\u00A0 to\u00C2\u00A0different\u00C2\u00A0degrees.\u00C2\u00A0\u00C2\u00A0 4T1\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 via\u00C2\u00A0 contact\u00E2\u0080\u0090dependent\u00C2\u00A0 ROS\u00C2\u00A0 production\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 inhibited\u00C2\u00A0 by\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 NAC.\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 mice\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0 effects\u00C2\u00A0 via\u00C2\u00A0 contact\u00E2\u0080\u0090dependent\u00C2\u00A0 NO\u00C2\u00A0 production,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 prevented\u00C2\u00A0 by\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 L\u00E2\u0080\u0090 NMMA,\u00C2\u00A0 L\u00E2\u0080\u0090NIL,\u00C2\u00A0 and/or\u00C2\u00A0 \u00CE\u00B1\u00E2\u0080\u0090IFN\u00CE\u00B3.\u00C2\u00A0 Unlike\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 4T1\u00C2\u00A0 MDSCs\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 contact\u00E2\u0080\u0090independent\u00C2\u00A0mechanism\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 inhibited\u00C2\u00A0 by\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 catalase,\u00C2\u00A0 indicating\u00C2\u00A0 a\u00C2\u00A0 role\u00C2\u00A0for\u00C2\u00A0H2O2.\u00C2\u00A0These\u00C2\u00A0cells\u00C2\u00A0also\u00C2\u00A0differ\u00C2\u00A0in\u00C2\u00A0their\u00C2\u00A0relative\u00C2\u00A0suppressive\u00C2\u00A0strengths;\u00C2\u00A04T1\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0exhibit\u00C2\u00A0 the\u00C2\u00A0most\u00C2\u00A0potent\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities,\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0control\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A04T1\u00C2\u00A0MDSCs.\u00C2\u00A0 133 5.2.14 ATRA\u00C2\u00A0increases\u00C2\u00A0lung\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A04T1\u00C2\u00A0tumor\u00C2\u00ADbearing\u00C2\u00A0mice\u00C2\u00A0 \u00C2\u00A0 After\u00C2\u00A0 thoroughly\u00C2\u00A0 characterizing\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 immunosuppressive\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0next\u00C2\u00A0examined\u00C2\u00A0the\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0 Specifically,\u00C2\u00A0we\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0distinct\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 and\u00C2\u00A0metastasis.\u00C2\u00A0We\u00C2\u00A0 conducted\u00C2\u00A0 a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 experiments\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 drug\u00C2\u00A0 ATRA,\u00C2\u00A0which\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0into\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0DCs\u00C2\u00A0(Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2003;\u00C2\u00A0Nefedova\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007).\u00C2\u00A0We\u00C2\u00A0 treated\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0with\u00C2\u00A0 or\u00C2\u00A0without\u00C2\u00A0 placebo\u00C2\u00A0 or\u00C2\u00A0 slow\u00E2\u0080\u0090 release\u00C2\u00A0ATRA\u00C2\u00A0pellets\u00C2\u00A0over\u00C2\u00A0a\u00C2\u00A0three\u00C2\u00A0week\u00C2\u00A0timeframe.\u00C2\u00A0We\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0ATRA\u00C2\u00A0treatment\u00C2\u00A0did\u00C2\u00A0 not\u00C2\u00A0have\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0growth\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0primary\u00C2\u00A0tumors,\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0very\u00C2\u00A0similar\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0 groups\u00C2\u00A0(Fig.\u00C2\u00A05.28A).\u00C2\u00A0Since\u00C2\u00A0the\u00C2\u00A0lungs\u00C2\u00A0are\u00C2\u00A0a\u00C2\u00A0principal\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A04T1\u00C2\u00A0model,\u00C2\u00A0 we\u00C2\u00A0 assayed\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 ATRA\u00C2\u00A0 treatment,\u00C2\u00A0 especially\u00C2\u00A0 the\u00C2\u00A0 higher\u00C2\u00A0 10\u00C2\u00A0mg\u00C2\u00A0 dose,\u00C2\u00A0 greatly\u00C2\u00A0 increased\u00C2\u00A0 lung\u00C2\u00A0metastases\u00C2\u00A0 (Fig.\u00C2\u00A0 5.28B).\u00C2\u00A0Furthermore,\u00C2\u00A0we\u00C2\u00A0performed\u00C2\u00A0 these\u00C2\u00A0same\u00C2\u00A0studies\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A04TO7\u00C2\u00A0tumor\u00C2\u00A0model,\u00C2\u00A0 which\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0much\u00C2\u00A0less\u00C2\u00A0aggressive\u00C2\u00A0metastatic\u00C2\u00A0cell\u00C2\u00A0line\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A04T1\u00C2\u00A0cells\u00C2\u00A0(i.e.\u00C2\u00A0although\u00C2\u00A0 4TO7\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 recovered\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 blood\u00C2\u00A0 and\u00C2\u00A0 lungs,\u00C2\u00A0 visible\u00C2\u00A0 metastases\u00C2\u00A0 never\u00C2\u00A0 develop)\u00C2\u00A0 (Aslakson\u00C2\u00A0 and\u00C2\u00A0 Miller,\u00C2\u00A0 1992;\u00C2\u00A0 Heppner\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2000),\u00C2\u00A0 and\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 ATRA\u00C2\u00A0 treatment\u00C2\u00A0increased\u00C2\u00A0metastatic\u00C2\u00A0lung\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0even\u00C2\u00A0greater\u00C2\u00A0extent\u00C2\u00A0(Fig.\u00C2\u00A05.28C).\u00C2\u00A0These\u00C2\u00A0 data\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0although\u00C2\u00A0ATRA\u00C2\u00A0treatment\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0 impact\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0growth,\u00C2\u00A0 it\u00C2\u00A0 enhances\u00C2\u00A0metastasis\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A04TO7\u00C2\u00A0models.\u00C2\u00A0 134 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.28\u00C2\u00A0 ATRA\u00C2\u00A0 treatment\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 alter\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 but\u00C2\u00A0 increases\u00C2\u00A0 lung\u00C2\u00A0metastasis.\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0ATRA\u00C2\u00A0 (5\u00C2\u00A0mg\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg)\u00C2\u00A0or\u00C2\u00A0placebo\u00C2\u00A0pellets\u00C2\u00A0 and\u00C2\u00A0A,\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0and\u00C2\u00A0B,\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 tumor\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0over\u00C2\u00A0 time.\u00C2\u00A0C,\u00C2\u00A04T07\u00C2\u00A0mice\u00C2\u00A0 were\u00C2\u00A0treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0ATRA\u00C2\u00A0(5\u00C2\u00A0mg\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg)\u00C2\u00A0or\u00C2\u00A0placebo\u00C2\u00A0pellets\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 lungs\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0over\u00C2\u00A0time.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SEM\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0independent\u00C2\u00A0experiments,\u00C2\u00A0 n>5\u00C2\u00A0mice\u00C2\u00A0per\u00C2\u00A0group.\u00C2\u00A0\u00C2\u00A0 135 5.2.15 ATRA\u00C2\u00A0 increases\u00C2\u00A0 the\u00C2\u00A0 proportion\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 much\u00C2\u00A0 more\u00C2\u00A0 immunosuppressive\u00C2\u00A0 than\u00C2\u00A0 MDSCs\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00C2\u00ADbearing\u00C2\u00A0 mice\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 next\u00C2\u00A0 performed\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 tests\u00C2\u00A0 to\u00C2\u00A0 elucidate\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 ATRA\u00C2\u00A0treatment\u00C2\u00A0 increased\u00C2\u00A0 lung\u00C2\u00A0metastasis.\u00C2\u00A0First,\u00C2\u00A0we\u00C2\u00A0considered\u00C2\u00A0whether\u00C2\u00A0ATRA\u00C2\u00A0might\u00C2\u00A0 influence\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells.\u00C2\u00A0We\u00C2\u00A0assayed\u00C2\u00A0the\u00C2\u00A0abilities\u00C2\u00A0 of\u00C2\u00A0pulmonary\u00C2\u00A0and\u00C2\u00A0splenic\u00C2\u00A0MDSCs\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0ATRA\u00C2\u00A0 to\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0ATRA\u00C2\u00A0treatment\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0drastically\u00C2\u00A0alter\u00C2\u00A0 the\u00C2\u00A0immunosuppressive\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0pulmonary\u00C2\u00A0or\u00C2\u00A0splenic\u00C2\u00A0MDSCs\u00C2\u00A0(Fig.\u00C2\u00A05.29A).\u00C2\u00A0Similarly,\u00C2\u00A0 ATRA\u00C2\u00A0treatment\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0have\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities\u00C2\u00A0of\u00C2\u00A0lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 5.29B).\u00C2\u00A0However,\u00C2\u00A0 in\u00C2\u00A0 performing\u00C2\u00A0 these\u00C2\u00A0 experiments\u00C2\u00A0we\u00C2\u00A0observed\u00C2\u00A0 that,\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0our\u00C2\u00A0previous\u00C2\u00A0results,\u00C2\u00A0lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0mice\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0treatment\u00C2\u00A0groups\u00C2\u00A0were\u00C2\u00A0much\u00C2\u00A0more\u00C2\u00A0 potent\u00C2\u00A0 suppressors\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 responses\u00C2\u00A0 than\u00C2\u00A0 lung\u00C2\u00A0MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0mice\u00C2\u00A0 (Fig.\u00C2\u00A05.29C).\u00C2\u00A0In\u00C2\u00A0fact,\u00C2\u00A0when\u00C2\u00A0assayed\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0ratio,\u00C2\u00A0pulmonary\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A050\u00E2\u0080\u0090fold\u00C2\u00A0 more\u00C2\u00A0suppressive\u00C2\u00A0than\u00C2\u00A0pulmonary\u00C2\u00A0MDSCs\u00C2\u00A0(Fig.\u00C2\u00A05.29C).\u00C2\u00A0 136 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.29\u00C2\u00A0 ATRA\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0change\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0or\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 Pulmonary\u00C2\u00A0 or\u00C2\u00A0 splenic\u00C2\u00A0 MDSCs\u00C2\u00A0 were\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 treated\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 ATRA\u00C2\u00A0 (5\u00C2\u00A0 mg)\u00C2\u00A0 or\u00C2\u00A0 placebo\u00C2\u00A0 pellets\u00C2\u00A0 and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0 with\u00C2\u00A0 polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0 (1\u00C2\u00A0 MDSC:\u00C2\u00A0 1\u00C2\u00A0 splenocyte).\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0 and\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Significance\u00C2\u00A0is\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0untreated\u00C2\u00A0cells.\u00C2\u00A0B,\u00C2\u00A0Lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 ATRA\u00C2\u00A0(5\u00C2\u00A0mg)\u00C2\u00A0or\u00C2\u00A0placebo\u00C2\u00A0pellets\u00C2\u00A0and\u00C2\u00A0 co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0 splenocytes\u00C2\u00A0at\u00C2\u00A0 different\u00C2\u00A0ratios\u00C2\u00A0(M\u00CF\u0086:\u00C2\u00A0splenocytes;\u00C2\u00A01:1\u00C2\u00A0or\u00C2\u00A01:2).\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0 of\u00C2\u00A0 RC\u00C2\u00A0 proliferation\u00C2\u00A0 calculated.\u00C2\u00A0 Significance\u00C2\u00A0 is\u00C2\u00A0 relative\u00C2\u00A0 to\u00C2\u00A0 untreated\u00C2\u00A0 cells.\u00C2\u00A0C,\u00C2\u00A0 MDSCs\u00C2\u00A0 or\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0lungs\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0ATRA\u00C2\u00A0(5\u00C2\u00A0mg\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg)\u00C2\u00A0or\u00C2\u00A0placebo\u00C2\u00A0pellets\u00C2\u00A0 and\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A0polyclonal\u00E2\u0080\u0090stimulated\u00C2\u00A0splenocytes\u00C2\u00A0(1\u00C2\u00A0myeloid\u00C2\u00A0cell:\u00C2\u00A01\u00C2\u00A0splenocyte).\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 proliferation\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0RC\u00C2\u00A0proliferation\u00C2\u00A0calculated.\u00C2\u00A0Data\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 *,p<0.05;\u00C2\u00A0 ***,p<0.001;\u00C2\u00A0 NS,\u00C2\u00A0 not\u00C2\u00A0significant\u00C2\u00A0\u00C2\u00A0 137 Because\u00C2\u00A0ATRA\u00C2\u00A0induces\u00C2\u00A0MDSC\u00C2\u00A0differentiation,\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0alters\u00C2\u00A0the\u00C2\u00A0proportions\u00C2\u00A0of\u00C2\u00A0 different\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0examined\u00C2\u00A0the\u00C2\u00A0impact\u00C2\u00A0of\u00C2\u00A0ATRA\u00C2\u00A0treatment\u00C2\u00A0on\u00C2\u00A0myelopoiesis.\u00C2\u00A0As\u00C2\u00A0 expected,\u00C2\u00A0control\u00C2\u00A0and\u00C2\u00A0placebo\u00E2\u0080\u0090treated\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0exhibited\u00C2\u00A0extreme\u00C2\u00A0splenomegaly\u00C2\u00A0(Fig.\u00C2\u00A0 5.30A).\u00C2\u00A0However,\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0ATRA\u00C2\u00A0exhibited\u00C2\u00A0much\u00C2\u00A0smaller\u00C2\u00A0spleens\u00C2\u00A0than\u00C2\u00A0control\u00C2\u00A0 mice\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0size\u00C2\u00A0primary\u00C2\u00A0 tumors\u00C2\u00A0 (Fig.\u00C2\u00A05.30A),\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0ATRA\u00C2\u00A0treatment\u00C2\u00A0 decreased\u00C2\u00A0tumor\u00E2\u0080\u0090induced\u00C2\u00A0myelopoiesis.\u00C2\u00A0Furthermore,\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0 to\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs\u00C2\u00A0 was\u00C2\u00A0 significantly\u00C2\u00A0 reduced\u00C2\u00A0 by\u00C2\u00A0 ATRA\u00C2\u00A0 treatment\u00C2\u00A0 (Fig.\u00C2\u00A0 5.30B),\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 reports\u00C2\u00A0 that\u00C2\u00A0 ATRA\u00C2\u00A0 treatment\u00C2\u00A0 induces\u00C2\u00A0 MDSC\u00C2\u00A0 differentiation,\u00C2\u00A0 and\u00C2\u00A0 consequently\u00C2\u00A0 reduces\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 increases\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 (Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003;\u00C2\u00A0Nefedova\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02007).\u00C2\u00A0Taken\u00C2\u00A0together,\u00C2\u00A0our\u00C2\u00A0data\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0 with\u00C2\u00A0a\u00C2\u00A0model\u00C2\u00A0 in\u00C2\u00A0which\u00C2\u00A0ATRA\u00C2\u00A0enhances\u00C2\u00A0metastasis\u00C2\u00A0by\u00C2\u00A0promoting\u00C2\u00A0 the\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0which\u00C2\u00A0 are\u00C2\u00A0much\u00C2\u00A0more\u00C2\u00A0 potent\u00C2\u00A0 suppressors\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 (Fig.\u00C2\u00A0 5.31).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.30\u00C2\u00A0 ATRA\u00C2\u00A0increases\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0reduces\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0 4T1\u00C2\u00A0mice.\u00C2\u00A0\u00C2\u00A0 A,\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0ATRA\u00C2\u00A0 (5\u00C2\u00A0mg\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg)\u00C2\u00A0or\u00C2\u00A0placebo\u00C2\u00A0pellets\u00C2\u00A0 and\u00C2\u00A0primary\u00C2\u00A0 tumor\u00C2\u00A0 weight\u00C2\u00A0and\u00C2\u00A0spleen\u00C2\u00A0weight\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0over\u00C2\u00A0time.\u00C2\u00A0B,\u00C2\u00A04T1\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0ATRA\u00C2\u00A0(5\u00C2\u00A0mg\u00C2\u00A0 or\u00C2\u00A010\u00C2\u00A0mg)\u00C2\u00A0or\u00C2\u00A0placebo\u00C2\u00A0pellets,\u00C2\u00A0and\u00C2\u00A0lung\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0harvested\u00C2\u00A0and\u00C2\u00A0quantified.\u00C2\u00A0\u00C2\u00A0Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0 mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 SEM\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 independent\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 duplicate.\u00C2\u00A0 *,p<0.05;\u00C2\u00A0 **,p<0.01;\u00C2\u00A0NS,\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0untreated\u00C2\u00A0mice\u00C2\u00A0\u00C2\u00A0 138 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.31\u00C2\u00A0 ATRA\u00C2\u00A0 induces\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 into\u00C2\u00A0 more\u00C2\u00A0 immunosuppressive\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0promotes\u00C2\u00A0 lung\u00C2\u00A0metastasis\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A04TO7\u00C2\u00A0 tumor\u00C2\u00A0 models.\u00C2\u00A0\u00C2\u00A0 139 5.3 Discussion\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0this\u00C2\u00A0chapter,\u00C2\u00A0we\u00C2\u00A0characterized\u00C2\u00A0the\u00C2\u00A0phenotypes\u00C2\u00A0of\u00C2\u00A0immunosuppressive\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 normal\u00C2\u00A0 and\u00C2\u00A0 neoplastic\u00C2\u00A0 tissues,\u00C2\u00A0 elucidated\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 they\u00C2\u00A0 inhibit\u00C2\u00A0 immune\u00C2\u00A0 responses,\u00C2\u00A0 and\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 roles\u00C2\u00A0 they\u00C2\u00A0play\u00C2\u00A0 in\u00C2\u00A0 tumorigenesis.\u00C2\u00A0Our\u00C2\u00A0 results\u00C2\u00A0reveal\u00C2\u00A0that\u00C2\u00A0both\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0possess\u00C2\u00A0immunosuppressive\u00C2\u00A0abilities.\u00C2\u00A0However,\u00C2\u00A0 we\u00C2\u00A0also\u00C2\u00A0found\u00C2\u00A0these\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0differ\u00C2\u00A0with\u00C2\u00A0regard\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0potency\u00C2\u00A0and\u00C2\u00A0mechanisms\u00C2\u00A0by\u00C2\u00A0 which\u00C2\u00A0they\u00C2\u00A0exert\u00C2\u00A0their\u00C2\u00A0suppressive\u00C2\u00A0effects,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0functions\u00C2\u00A0they\u00C2\u00A0perform\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0 Our\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 characteristics\u00C2\u00A0 are\u00C2\u00A0 governed\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 contextual\u00C2\u00A0 factors\u00C2\u00A0 including\u00C2\u00A0 exposure\u00C2\u00A0 to\u00C2\u00A0 different\u00C2\u00A0 tumor\u00E2\u0080\u0090derived\u00C2\u00A0 factors,\u00C2\u00A0 location\u00C2\u00A0 in\u00C2\u00A0 different\u00C2\u00A0 tissues,\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0state.\u00C2\u00A0 In\u00C2\u00A0general,\u00C2\u00A0 the\u00C2\u00A0studies\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0reveal\u00C2\u00A0a\u00C2\u00A0 number\u00C2\u00A0of\u00C2\u00A0key\u00C2\u00A0findings\u00C2\u00A0regarding\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0roles\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0play\u00C2\u00A0both\u00C2\u00A0ex\u00C2\u00A0vivo\u00C2\u00A0 and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0and\u00C2\u00A0highlight\u00C2\u00A0the\u00C2\u00A0importance\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0and\u00C2\u00A0metastasis.\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0 important\u00C2\u00A0 trend\u00C2\u00A0 that\u00C2\u00A0we\u00C2\u00A0 saw\u00C2\u00A0 throughout\u00C2\u00A0 our\u00C2\u00A0 studies\u00C2\u00A0was\u00C2\u00A0 the\u00C2\u00A0 contrasting\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 tumor\u00C2\u00A0 models,\u00C2\u00A0 specifically\u00C2\u00A0 metastatic\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 versus\u00C2\u00A0 non\u00E2\u0080\u0090 metastatic\u00C2\u00A067NR\u00C2\u00A0tumors,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0phenotype\u00C2\u00A0and\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0We\u00C2\u00A0found\u00C2\u00A0 that\u00C2\u00A04T1\u00C2\u00A0 tumors\u00C2\u00A0 induce\u00C2\u00A0 immense\u00C2\u00A0expansion\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0 cells,\u00C2\u00A0 including\u00C2\u00A0both\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0 (Fig.\u00C2\u00A0 5.3),\u00C2\u00A0 and\u00C2\u00A0 skew\u00C2\u00A0 their\u00C2\u00A0 phenotype\u00C2\u00A0 towards\u00C2\u00A0 M2\u00C2\u00A0 activation\u00C2\u00A0 (Fig.\u00C2\u00A0 5.8).\u00C2\u00A0 Furthermore,\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 enhanced\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 function\u00C2\u00A0 of\u00C2\u00A0 all\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 we\u00C2\u00A0 investigated\u00C2\u00A0(Fig.\u00C2\u00A05.14).\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0induce\u00C2\u00A0myelopoiesis\u00C2\u00A0(Fig.\u00C2\u00A05.3)\u00C2\u00A0 or\u00C2\u00A0 alter\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells.\u00C2\u00A0 The\u00C2\u00A0 only\u00C2\u00A0 exception\u00C2\u00A0 to\u00C2\u00A0 this\u00C2\u00A0 trend\u00C2\u00A0 was\u00C2\u00A0 with\u00C2\u00A0 regard\u00C2\u00A0to\u00C2\u00A0TAMs,\u00C2\u00A0which\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0were\u00C2\u00A0more\u00C2\u00A0immunosuppressive\u00C2\u00A0from\u00C2\u00A067NR\u00C2\u00A0mice\u00C2\u00A0than\u00C2\u00A0 4T1\u00C2\u00A0mice\u00C2\u00A0(Fig.\u00C2\u00A05.10).\u00C2\u00A0Taken\u00C2\u00A0together,\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0support\u00C2\u00A0two\u00C2\u00A0ideas:\u00C2\u00A01)\u00C2\u00A0Different\u00C2\u00A0tumors\u00C2\u00A0 have\u00C2\u00A0 contrasting\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 induction\u00C2\u00A0 and\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 and\u00C2\u00A0 2)\u00C2\u00A0 there\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0important\u00C2\u00A0differences\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0tumors\u00C2\u00A0on\u00C2\u00A0cells\u00C2\u00A0located\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0tumor\u00C2\u00A0microenvironment\u00C2\u00A0versus\u00C2\u00A0the\u00C2\u00A0periphery.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0we\u00C2\u00A0report\u00C2\u00A0significant\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0 on\u00C2\u00A0 immunosuppressive\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0 the\u00C2\u00A0underlying\u00C2\u00A0principles\u00C2\u00A0remain\u00C2\u00A0unclear.\u00C2\u00A0 It\u00C2\u00A0has\u00C2\u00A0 140 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inflammatory\u00C2\u00A0 conditions,\u00C2\u00A0 including\u00C2\u00A0 autoimmune\u00C2\u00A0 disease,\u00C2\u00A0 trauma,\u00C2\u00A0 burns,\u00C2\u00A0 and\u00C2\u00A0 sepsis\u00C2\u00A0 (Cuenca\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Indeed,\u00C2\u00A0pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0factors\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0tumor,\u00C2\u00A0tumor\u00C2\u00A0 stroma,\u00C2\u00A0 and\u00C2\u00A0 infiltrating\u00C2\u00A0 T\u00C2\u00A0 cells,\u00C2\u00A0 including\u00C2\u00A0 cytokines,\u00C2\u00A0 chemokines,\u00C2\u00A0 and\u00C2\u00A0 S100\u00C2\u00A0 proteins,\u00C2\u00A0 induce\u00C2\u00A0accelerated\u00C2\u00A0myelopoiesis\u00C2\u00A0and\u00C2\u00A0drive\u00C2\u00A0the\u00C2\u00A0mobilization\u00C2\u00A0of\u00C2\u00A0mature\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0PMNs,\u00C2\u00A0and\u00C2\u00A0 immature\u00C2\u00A0populations\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0BM\u00C2\u00A0and\u00C2\u00A0blood\u00C2\u00A0to\u00C2\u00A0inflammatory\u00C2\u00A0sites\u00C2\u00A0(Ueda\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0 Interestingly,\u00C2\u00A0it\u00C2\u00A0has\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0these\u00C2\u00A0same\u00C2\u00A0factors\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0drive\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0 expansion\u00C2\u00A0 and\u00C2\u00A0 activation,\u00C2\u00A0 but\u00C2\u00A0 also\u00C2\u00A0 prevent\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 immature\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0thus\u00C2\u00A0promoting\u00C2\u00A0MDSC\u00C2\u00A0accumulation.\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0studies\u00C2\u00A0by\u00C2\u00A0Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 have\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 from\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 injected\u00C2\u00A0 into\u00C2\u00A0 healthy\u00C2\u00A0 animals\u00C2\u00A0 quickly\u00C2\u00A0lose\u00C2\u00A0their\u00C2\u00A0immunosuppressive\u00C2\u00A0phenotype\u00C2\u00A0and\u00C2\u00A0terminally\u00C2\u00A0differentiate\u00C2\u00A0into\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0DCs\u00C2\u00A0(Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003),\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0the\u00C2\u00A0potential\u00C2\u00A0to\u00C2\u00A0mature,\u00C2\u00A0 but\u00C2\u00A0are\u00C2\u00A0trapped\u00C2\u00A0by\u00C2\u00A0their\u00C2\u00A0environment\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0immature\u00C2\u00A0state.\u00C2\u00A0Therefore,\u00C2\u00A0our\u00C2\u00A0finding\u00C2\u00A0that\u00C2\u00A0 67NR\u00C2\u00A0 tumors\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0promote\u00C2\u00A0myelopoiesis\u00C2\u00A0has\u00C2\u00A0 a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 interesting\u00C2\u00A0 implications.\u00C2\u00A0 First,\u00C2\u00A0it\u00C2\u00A0suggests\u00C2\u00A0that,\u00C2\u00A0unlike\u00C2\u00A0the\u00C2\u00A0vast\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0tumors,\u00C2\u00A067NR\u00C2\u00A0tumors\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0induce\u00C2\u00A0an\u00C2\u00A0 inflammation\u00C2\u00A0 response.\u00C2\u00A0 Further,\u00C2\u00A0 it\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 67NR\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 produce\u00C2\u00A0 factors\u00C2\u00A0that\u00C2\u00A0promote\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0expansion\u00C2\u00A0or\u00C2\u00A0activation.\u00C2\u00A0It\u00C2\u00A0seems\u00C2\u00A0unusual\u00C2\u00A0that\u00C2\u00A067NR\u00C2\u00A0 tumors\u00C2\u00A0 can\u00C2\u00A0 grow\u00C2\u00A0 and\u00C2\u00A0 thrive\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 mammary\u00C2\u00A0 fat\u00C2\u00A0 pad,\u00C2\u00A0 yet\u00C2\u00A0 not\u00C2\u00A0 induce\u00C2\u00A0 a\u00C2\u00A0 systemic\u00C2\u00A0 inflammatory\u00C2\u00A0 response.\u00C2\u00A0 One\u00C2\u00A0 possible\u00C2\u00A0 explanation\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 related\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 non\u00E2\u0080\u0090 metastatic\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A067NR\u00C2\u00A0tumors;\u00C2\u00A0perhaps\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0promote\u00C2\u00A0metastasis\u00C2\u00A0 also\u00C2\u00A0 induce\u00C2\u00A0myelopoiesis\u00C2\u00A0or\u00C2\u00A0perhaps\u00C2\u00A0disseminated\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0 initiate\u00C2\u00A0 an\u00C2\u00A0 inflammatory\u00C2\u00A0 response.\u00C2\u00A0 Alternatively,\u00C2\u00A0 the\u00C2\u00A0 inability\u00C2\u00A0 of\u00C2\u00A0 67NR\u00C2\u00A0 tumors\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 or\u00C2\u00A0 modify\u00C2\u00A0 their\u00C2\u00A0 phenotypes\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 related\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 high\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0 vascularization\u00C2\u00A0in\u00C2\u00A067NR\u00C2\u00A0tumors(Fig.\u00C2\u00A05.2).\u00C2\u00A0Perhaps\u00C2\u00A0the\u00C2\u00A0lack\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0induction\u00C2\u00A0and\u00C2\u00A0 infiltration\u00C2\u00A0 in\u00C2\u00A067NR\u00C2\u00A0 tumors\u00C2\u00A0 is\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 lack\u00C2\u00A0of\u00C2\u00A0hypoxic\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A067NR\u00C2\u00A0 tumors,\u00C2\u00A0which\u00C2\u00A0 have\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 the\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 stimulate\u00C2\u00A0angiogenesis\u00C2\u00A0and\u00C2\u00A0 myeloid\u00C2\u00A0cell\u00C2\u00A0infiltration\u00C2\u00A0(Corzo\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Doedens\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Qian\u00C2\u00A0 141 and\u00C2\u00A0 Pollard,\u00C2\u00A0 2010).\u00C2\u00A0 Additional\u00C2\u00A0 studies\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 panel\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 lines,\u00C2\u00A0 including\u00C2\u00A0 metastatic\u00C2\u00A0and\u00C2\u00A0non\u00E2\u0080\u0090metastatic\u00C2\u00A0tumors,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0tumors\u00C2\u00A0that\u00C2\u00A0exhibit\u00C2\u00A0different\u00C2\u00A0degrees\u00C2\u00A0 of\u00C2\u00A0vascularization\u00C2\u00A0and\u00C2\u00A0hypoxia\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0integral\u00C2\u00A0to\u00C2\u00A0gaining\u00C2\u00A0a\u00C2\u00A0better\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0infiltration\u00C2\u00A0and\u00C2\u00A0function.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0mentioned\u00C2\u00A0earlier,\u00C2\u00A0there\u00C2\u00A0appear\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0substantial\u00C2\u00A0tissue\u00E2\u0080\u0090specific\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0MDSCs.\u00C2\u00A0 Most\u00C2\u00A0 notably,\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 TAMs\u00C2\u00A0 and\u00C2\u00A0 tumor\u00C2\u00A0 MDSCs\u00C2\u00A0 differs\u00C2\u00A0 from\u00C2\u00A0 their\u00C2\u00A0 peripheral\u00C2\u00A0 tissue\u00C2\u00A0 counterparts.\u00C2\u00A0 For\u00C2\u00A0 example,\u00C2\u00A0we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 in\u00C2\u00A0 peripheral\u00C2\u00A0 lymphoid\u00C2\u00A0 organs\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 express\u00C2\u00A0 iNOS\u00C2\u00A0 or\u00C2\u00A0 Arg1,\u00C2\u00A0 while\u00C2\u00A0 tumor\u00C2\u00A0 MDSCs\u00C2\u00A0expressed\u00C2\u00A0both\u00C2\u00A0Arg1\u00C2\u00A0and\u00C2\u00A0Ym1\u00C2\u00A0(Fig.\u00C2\u00A05.8B).\u00C2\u00A0We\u00C2\u00A0also\u00C2\u00A0discovered\u00C2\u00A0 that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 periphery\u00C2\u00A0 (i.e.\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 lung\u00C2\u00A0 M\u00CF\u0086s)\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 different\u00C2\u00A0 mechanism\u00C2\u00A0 than\u00C2\u00A0TAMs.\u00C2\u00A0 Intriguingly,\u00C2\u00A0most\u00C2\u00A0 studies\u00C2\u00A0 investigating\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0performed\u00C2\u00A0 using\u00C2\u00A0cells\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0peripheral\u00C2\u00A0lymphoid\u00C2\u00A0organs\u00C2\u00A0(i.e.\u00C2\u00A0spleen),\u00C2\u00A0while\u00C2\u00A0much\u00C2\u00A0of\u00C2\u00A0what\u00C2\u00A0 we\u00C2\u00A0 know\u00C2\u00A0 about\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 studies\u00C2\u00A0 with\u00C2\u00A0 TAMs.\u00C2\u00A0 Consequently,\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0tumor\u00E2\u0080\u0090associated\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0peripheral\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0how\u00C2\u00A0 they\u00C2\u00A0 compare\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0 differently\u00E2\u0080\u0090located\u00C2\u00A0 counterparts,\u00C2\u00A0 remains\u00C2\u00A0 largely\u00C2\u00A0 unknown\u00C2\u00A0 (Corzo\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010;\u00C2\u00A0 Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009).\u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 past\u00C2\u00A0 few\u00C2\u00A0 years,\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 groups\u00C2\u00A0 have\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 phenotypes\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 peripheral\u00C2\u00A0 lymphoid\u00C2\u00A0 organs\u00C2\u00A0 versus\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 site\u00C2\u00A0 and\u00C2\u00A0 published\u00C2\u00A0 results\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0own\u00C2\u00A0data\u00C2\u00A0(Haverkamp\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011),\u00C2\u00A0including\u00C2\u00A0a\u00C2\u00A0recent\u00C2\u00A0report\u00C2\u00A0from\u00C2\u00A0 Corzo\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0that\u00C2\u00A0presented\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0important\u00C2\u00A0findings.\u00C2\u00A0Using\u00C2\u00A0an\u00C2\u00A0EL\u00E2\u0080\u00904\u00C2\u00A0ascites\u00C2\u00A0model,\u00C2\u00A0 they\u00C2\u00A0noted\u00C2\u00A0major\u00C2\u00A0differences\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function\u00C2\u00A0between\u00C2\u00A0 tumor\u00C2\u00A0and\u00C2\u00A0 splenic\u00C2\u00A0MDSCs.\u00C2\u00A0Specifically,\u00C2\u00A0 they\u00C2\u00A0 found\u00C2\u00A0splenic\u00C2\u00A0MDSCs\u00C2\u00A0could\u00C2\u00A0only\u00C2\u00A0suppress\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00C2\u00A0 CD8+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 function,\u00C2\u00A0 while\u00C2\u00A0 tumor\u00C2\u00A0 MDSCs\u00C2\u00A0 could\u00C2\u00A0 suppress\u00C2\u00A0 both\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00C2\u00A0 and\u00C2\u00A0 non\u00E2\u0080\u0090 specific\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses,\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 differences\u00C2\u00A0 were\u00C2\u00A0 principally\u00C2\u00A0 regulated\u00C2\u00A0 by\u00C2\u00A0 hypoxia\u00C2\u00A0and\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0hypoxia\u00E2\u0080\u0090inducible\u00C2\u00A0 factor\u00C2\u00A0(HIF)\u00E2\u0080\u00901\u00CE\u00B1\u00C2\u00A0(Corzo\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0 \u00C2\u00A0They\u00C2\u00A0 also\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0HIF\u00E2\u0080\u00901\u00CE\u00B1\u00C2\u00A0expression\u00C2\u00A0induced\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0iNOS\u00C2\u00A0and\u00C2\u00A0Arg1\u00C2\u00A0in\u00C2\u00A0MDSCs,\u00C2\u00A0 decreased\u00C2\u00A0 ROS\u00C2\u00A0 production,\u00C2\u00A0 and\u00C2\u00A0 promoted\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 into\u00C2\u00A0 TAMs\u00C2\u00A0 (Corzo\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0shed\u00C2\u00A0light\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0issues\u00C2\u00A0relevant\u00C2\u00A0to\u00C2\u00A0our\u00C2\u00A0work\u00C2\u00A0 and\u00C2\u00A0 may\u00C2\u00A0 help\u00C2\u00A0 explain\u00C2\u00A0 the\u00C2\u00A0 relatively\u00C2\u00A0 low\u00C2\u00A0 potency\u00C2\u00A0 of\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 we\u00C2\u00A0 142 observed.\u00C2\u00A0 Since\u00C2\u00A0 the\u00C2\u00A0 necessary\u00C2\u00A0mice\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 available\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 BALB/c\u00C2\u00A0 background,\u00C2\u00A0we\u00C2\u00A0 were\u00C2\u00A0 unable\u00C2\u00A0 to\u00C2\u00A0 assay\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00C2\u00A0 CD8+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses,\u00C2\u00A0 and\u00C2\u00A0 instead\u00C2\u00A0 only\u00C2\u00A0 tested\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00C2\u00A0 CD4+\u00C2\u00A0 and\u00C2\u00A0 polyclonal\u00C2\u00A0(non\u00E2\u0080\u0090specific)\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0functions.\u00C2\u00A0Therefore,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0possible\u00C2\u00A0that\u00C2\u00A0our\u00C2\u00A0finding\u00C2\u00A0that\u00C2\u00A0 splenic\u00C2\u00A0 and\u00C2\u00A0 lung\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 less\u00C2\u00A0 potent\u00C2\u00A0 immune\u00C2\u00A0 suppressors\u00C2\u00A0 than\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 may\u00C2\u00A0 not\u00C2\u00A0 be\u00C2\u00A0 applicable\u00C2\u00A0 to\u00C2\u00A0 tumor\u00C2\u00A0 MDSCs\u00C2\u00A0 and/or\u00C2\u00A0 CD8+\u00C2\u00A0 Ag\u00E2\u0080\u0090specific\u00E2\u0080\u0090stimulated\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses.\u00C2\u00A0 Furthermore,\u00C2\u00A0 the\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0hypoxia\u00C2\u00A0 regulates\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function,\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 MDSC\u00C2\u00A0 differentiation,\u00C2\u00A0 may\u00C2\u00A0 explain\u00C2\u00A0 some\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 differences\u00C2\u00A0 we\u00C2\u00A0 observed\u00C2\u00A0 in\u00C2\u00A0 suppressive\u00C2\u00A0 ability\u00C2\u00A0 between\u00C2\u00A0 4T1\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 67NR\u00E2\u0080\u0090\u00C2\u00A0 induced\u00C2\u00A0myeloid\u00C2\u00A0 cells.\u00C2\u00A0 Importantly,\u00C2\u00A0 this\u00C2\u00A0 phenomenon\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 appear\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 restricted\u00C2\u00A0 to\u00C2\u00A0 MDSCs,\u00C2\u00A0 since\u00C2\u00A0 hypoxia\u00C2\u00A0 and\u00C2\u00A0 HIF\u00E2\u0080\u00901\u00CE\u00B1\u00C2\u00A0 expression\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0implicated\u00C2\u00A0in\u00C2\u00A0regulating\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0as\u00C2\u00A0 demonstrated\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 recent\u00C2\u00A0 study\u00C2\u00A0 from\u00C2\u00A0 Johnson\u00E2\u0080\u0099s\u00C2\u00A0 group\u00C2\u00A0 (Doedens\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 these\u00C2\u00A0 findings\u00C2\u00A0 may\u00C2\u00A0 highlight\u00C2\u00A0 an\u00C2\u00A0 integral\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0 hypoxia\u00C2\u00A0 in\u00C2\u00A0 regulating\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 and\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0 and,\u00C2\u00A0 in\u00C2\u00A0 turn,\u00C2\u00A0 tumor\u00C2\u00A0 progression\u00C2\u00A0 and\u00C2\u00A0 metastasis.\u00C2\u00A0 \u00C2\u00A0 Unlike\u00C2\u00A0 TAMs,\u00C2\u00A0 the\u00C2\u00A0 roles\u00C2\u00A0 of\u00C2\u00A0 peripheral\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 tumorigenesis\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 yet\u00C2\u00A0 clearly\u00C2\u00A0 elucidated\u00C2\u00A0(Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0Our\u00C2\u00A0results\u00C2\u00A0demonstrate\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 4T1\u00C2\u00A0 tumors\u00C2\u00A0 dramatically\u00C2\u00A0 alters\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 and\u00C2\u00A0 enhances\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0peripheral\u00C2\u00A0tissues,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0PC\u00C2\u00A0and\u00C2\u00A0lung\u00C2\u00A0(Fig.\u00C2\u00A05.14).\u00C2\u00A0 \u00C2\u00A0Our\u00C2\u00A0finding\u00C2\u00A0 that\u00C2\u00A0increased\u00C2\u00A0numbers\u00C2\u00A0of\u00C2\u00A0lung\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0correlate\u00C2\u00A0with\u00C2\u00A0increased\u00C2\u00A0metastatic\u00C2\u00A0growth\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 lungs\u00C2\u00A0 raises\u00C2\u00A0 questions\u00C2\u00A0 about\u00C2\u00A0 whether\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 pre\u00E2\u0080\u0090 metastatic\u00C2\u00A0 niche.\u00C2\u00A0 To\u00C2\u00A0 date,\u00C2\u00A0most\u00C2\u00A0 groups\u00C2\u00A0 have\u00C2\u00A0 focused\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 involvement\u00C2\u00A0 of\u00C2\u00A0 VEGF\u00E2\u0080\u0090R1\u00C2\u00A0 expressing\u00C2\u00A0BM\u00E2\u0080\u0090derived\u00C2\u00A0HPCs\u00C2\u00A0 in\u00C2\u00A0niche\u00C2\u00A0development\u00C2\u00A0(Kaplan\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02005),\u00C2\u00A0but\u00C2\u00A0our\u00C2\u00A0data\u00C2\u00A0 suggest\u00C2\u00A0a\u00C2\u00A0potential\u00C2\u00A0role\u00C2\u00A0for\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0studies\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0integral\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0their\u00C2\u00A0 contribution\u00C2\u00A0 to\u00C2\u00A0 this\u00C2\u00A0 process.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 addition,\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 context\u00C2\u00A0 of\u00C2\u00A0 cancer\u00C2\u00A0 remains\u00C2\u00A0unclear.\u00C2\u00A0It\u00C2\u00A0is\u00C2\u00A0possible\u00C2\u00A0that\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0function\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0activated\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0PC,\u00C2\u00A0 and\u00C2\u00A0may\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 ascites\u00C2\u00A0 formation\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0manner.\u00C2\u00A0 Alternatively,\u00C2\u00A0 the\u00C2\u00A0 increased\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 systemic\u00C2\u00A0 immunosuppressive\u00C2\u00A0 state.\u00C2\u00A0 Taken\u00C2\u00A0 together,\u00C2\u00A0 these\u00C2\u00A0 results\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 both\u00C2\u00A0 TAMs\u00C2\u00A0 and\u00C2\u00A0 143 peripheral\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0central\u00C2\u00A0players\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0network\u00C2\u00A0and\u00C2\u00A0their\u00C2\u00A0roles\u00C2\u00A0 should\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0neglected\u00C2\u00A0or\u00C2\u00A0minimized.\u00C2\u00A0Rather,\u00C2\u00A0 further\u00C2\u00A0studies\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0performed\u00C2\u00A0to\u00C2\u00A0 clarify\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0play\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0stages\u00C2\u00A0of\u00C2\u00A0carcinogenesis\u00C2\u00A0and\u00C2\u00A0validate\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0as\u00C2\u00A0 potential\u00C2\u00A0therapeutic\u00C2\u00A0targets.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0 we\u00C2\u00A0 have\u00C2\u00A0 discussed,\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 display\u00C2\u00A0 immense\u00C2\u00A0 heterogeneity.\u00C2\u00A0As\u00C2\u00A0Figure\u00C2\u00A05.7\u00C2\u00A0 shows,\u00C2\u00A0we\u00C2\u00A0analyzed\u00C2\u00A0MDSC\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0CD11b\u00C2\u00A0and\u00C2\u00A0Gr1\u00C2\u00A0 and\u00C2\u00A0found\u00C2\u00A0some\u00C2\u00A0degree\u00C2\u00A0of\u00C2\u00A0variation\u00C2\u00A0in\u00C2\u00A0Gr1\u00C2\u00A0expression.\u00C2\u00A0However,\u00C2\u00A0these\u00C2\u00A0subpopulations\u00C2\u00A0 were\u00C2\u00A0most\u00C2\u00A0evident\u00C2\u00A0in\u00C2\u00A0control\u00C2\u00A0and\u00C2\u00A067NR\u00C2\u00A0Gr1+\u00C2\u00A0cells,\u00C2\u00A0while\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0appeared\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0a\u00C2\u00A0 more\u00C2\u00A0uniform\u00C2\u00A0population\u00C2\u00A0(Fig.\u00C2\u00A05.7).\u00C2\u00A0Moreover,\u00C2\u00A0we\u00C2\u00A0analyzed\u00C2\u00A0splenic\u00C2\u00A0and\u00C2\u00A0pulmonary\u00C2\u00A04T1\u00C2\u00A0 MDSCs\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0only\u00C2\u00A0a\u00C2\u00A0very\u00C2\u00A0small\u00C2\u00A0proportion\u00C2\u00A0(<5%)\u00C2\u00A0expressed\u00C2\u00A0 Ly6C\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0vast\u00C2\u00A0majority\u00C2\u00A0were\u00C2\u00A0Ly6G+\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown),\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0 morphological\u00C2\u00A0 analysis\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 4\u00C2\u00A0 (Fig.\u00C2\u00A0 4.3).\u00C2\u00A0 \u00C2\u00A0 Consequently,\u00C2\u00A0 we\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 analyze\u00C2\u00A0 different\u00C2\u00A0 subpopulations\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0MDSCs,\u00C2\u00A0 but\u00C2\u00A0 rather\u00C2\u00A0 chose\u00C2\u00A0 to\u00C2\u00A0 study\u00C2\u00A0 them\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 single\u00C2\u00A0 \u00E2\u0080\u0098bulk\u00E2\u0080\u0099\u00C2\u00A0 population.\u00C2\u00A0 \u00C2\u00A0 However,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 reports\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 different\u00C2\u00A0 subpopulations\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0vary\u00C2\u00A0with\u00C2\u00A0regard\u00C2\u00A0to\u00C2\u00A0immunosuppressive\u00C2\u00A0function\u00C2\u00A0(Movahedi\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 Related\u00C2\u00A0 to\u00C2\u00A0 this,\u00C2\u00A0 a\u00C2\u00A0 recent\u00C2\u00A0 study\u00C2\u00A0 by\u00C2\u00A0 Bronte\u00E2\u0080\u0099s\u00C2\u00A0 group\u00C2\u00A0 investigating\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 splenic\u00C2\u00A0 MDSC\u00C2\u00A0 subpopulations,\u00C2\u00A0 isolated\u00C2\u00A0 by\u00C2\u00A0 their\u00C2\u00A0 differential\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 Gr1,\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 CD11b+/Gr1int\u00C2\u00A0 subset,\u00C2\u00A0 which\u00C2\u00A0 was\u00C2\u00A0 comprised\u00C2\u00A0 of\u00C2\u00A0 monocytes\u00C2\u00A0 and\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 progenitors,\u00C2\u00A0 was\u00C2\u00A0 highly\u00C2\u00A0 suppressive,\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 CD11b+/Gr1high\u00C2\u00A0subset,\u00C2\u00A0made\u00C2\u00A0up\u00C2\u00A0of\u00C2\u00A0mostly\u00C2\u00A0granulocytes,\u00C2\u00A0was\u00C2\u00A0only\u00C2\u00A0immunosuppressive\u00C2\u00A0 in\u00C2\u00A0some\u00C2\u00A0tumor\u00C2\u00A0models\u00C2\u00A0and\u00C2\u00A0under\u00C2\u00A0certain\u00C2\u00A0conditions\u00C2\u00A0(Dolcetti\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0Interestingly,\u00C2\u00A0 they\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0more\u00C2\u00A0immunosuppressive\u00C2\u00A0subsets\u00C2\u00A0was\u00C2\u00A0 dependent\u00C2\u00A0 on\u00C2\u00A0 tumor\u00E2\u0080\u0090produced\u00C2\u00A0GM\u00E2\u0080\u0090CSF\u00C2\u00A0 (Dolcetti\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 These\u00C2\u00A0 data,\u00C2\u00A0 along\u00C2\u00A0with\u00C2\u00A0 reports\u00C2\u00A0from\u00C2\u00A0several\u00C2\u00A0other\u00C2\u00A0groups\u00C2\u00A0(Corzo\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010;\u00C2\u00A0Cuenca\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0 2011;\u00C2\u00A0 Peranzoni\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010;\u00C2\u00A0 Youn\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2010),\u00C2\u00A0 highlight\u00C2\u00A0 the\u00C2\u00A0 extreme\u00C2\u00A0 heterogeneity\u00C2\u00A0 encompassed\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 MDSC\u00C2\u00A0 classification\u00C2\u00A0 and\u00C2\u00A0 reinforce\u00C2\u00A0 the\u00C2\u00A0 idea\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 cannot\u00C2\u00A0 be\u00C2\u00A0 identified\u00C2\u00A0 by\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 particular\u00C2\u00A0markers,\u00C2\u00A0 but\u00C2\u00A0 rather\u00C2\u00A0 by\u00C2\u00A0 their\u00C2\u00A0 immunosuppressive\u00C2\u00A0nature\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 144 Since\u00C2\u00A0accurate\u00C2\u00A0detection\u00C2\u00A0of\u00C2\u00A0immunosuppressive\u00C2\u00A0function\u00C2\u00A0was\u00C2\u00A0critical\u00C2\u00A0both\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 accurate\u00C2\u00A0 identification\u00C2\u00A0 of\u00C2\u00A0 MDSCs,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 for\u00C2\u00A0 our\u00C2\u00A0 studies\u00C2\u00A0 comparing\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 immunosuppressive\u00C2\u00A0potencies\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0used\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0assays\u00C2\u00A0to\u00C2\u00A0quantify\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 part,\u00C2\u00A0 whether\u00C2\u00A0 we\u00C2\u00A0 assayed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 by\u00C2\u00A0 3H\u00E2\u0080\u0090thy\u00C2\u00A0 incorporation\u00C2\u00A0 or\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 CFSE,\u00C2\u00A0 the\u00C2\u00A0 results\u00C2\u00A0 were\u00C2\u00A0very\u00C2\u00A0consistent.\u00C2\u00A0However,\u00C2\u00A0we\u00C2\u00A0observed\u00C2\u00A0two\u00C2\u00A0important\u00C2\u00A0discrepancies\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0 of\u00C2\u00A0these\u00C2\u00A0assays\u00C2\u00A0that\u00C2\u00A0reveal\u00C2\u00A0important\u00C2\u00A0clues\u00C2\u00A0about\u00C2\u00A0immunosuppressive\u00C2\u00A0mechanism.\u00C2\u00A0The\u00C2\u00A0 first\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0 while\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 control\u00C2\u00A0 mice\u00C2\u00A0 strongly\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 as\u00C2\u00A0measured\u00C2\u00A0 by\u00C2\u00A0 3H\u00E2\u0080\u0090thy\u00C2\u00A0 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only\u00C2\u00A0 measures\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 final\u00C2\u00A0 18\u00C2\u00A0 h.\u00C2\u00A0 Therefore,\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0may\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0immediately\u00C2\u00A0suppressive,\u00C2\u00A0 but\u00C2\u00A0 require\u00C2\u00A0 time\u00C2\u00A0and/or\u00C2\u00A0activation\u00C2\u00A0 to\u00C2\u00A0acquire\u00C2\u00A0 suppressive\u00C2\u00A0 function.\u00C2\u00A0This\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 our\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0 control\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 only\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 produce\u00C2\u00A0 NO\u00C2\u00A0 and\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 following\u00C2\u00A0 exposure\u00C2\u00A0 to\u00C2\u00A0 T\u00C2\u00A0 cell\u00E2\u0080\u0090produced\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3,\u00C2\u00A0 which\u00C2\u00A0 induces\u00C2\u00A0 iNOS\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 (Fig\u00C2\u00A0 3.13).\u00C2\u00A0Moreover,\u00C2\u00A0 Figure\u00C2\u00A0 5.18\u00C2\u00A0 provides\u00C2\u00A0 further\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0possess\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties,\u00C2\u00A0since\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0significantly\u00C2\u00A0 reduced\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0CD4+\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0culture\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0other\u00C2\u00A0noteworthy\u00C2\u00A0difference\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0 two\u00C2\u00A0methods\u00C2\u00A0 is\u00C2\u00A0 that,\u00C2\u00A0 at\u00C2\u00A0 first\u00C2\u00A0 glance,\u00C2\u00A0 4T1\u00C2\u00A0MDSCs\u00C2\u00A0 seem\u00C2\u00A0much\u00C2\u00A0more\u00C2\u00A0 immunosuppressive\u00C2\u00A0 by\u00C2\u00A0 CFSE\u00E2\u0080\u0090flow\u00C2\u00A0 cytometric\u00C2\u00A0analysis\u00C2\u00A0 than\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 3H\u00E2\u0080\u0090thy\u00C2\u00A0assay\u00C2\u00A0(Fig.\u00C2\u00A05.16).\u00C2\u00A0However,\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0the\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0dead\u00C2\u00A0(98.1%)\u00C2\u00A0following\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0with\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0 (Fig.\u00C2\u00A05.16),\u00C2\u00A0which,\u00C2\u00A0 in\u00C2\u00A0 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determined\u00C2\u00A0the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0following\u00C2\u00A04T1\u00C2\u00A0MDSC\u00C2\u00A0co\u00E2\u0080\u0090culture\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0non\u00E2\u0080\u0090 145 CFSE\u00C2\u00A0 flow\u00C2\u00A0cytometric\u00C2\u00A0approach,\u00C2\u00A0we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0 reduced\u00C2\u00A0 the\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0 viable\u00C2\u00A0CD4+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 from\u00C2\u00A037.4%\u00C2\u00A0to\u00C2\u00A020.3%\u00C2\u00A0and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0 from\u00C2\u00A022.3%\u00C2\u00A0to\u00C2\u00A04.35%\u00C2\u00A0(Fig.\u00C2\u00A0 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support\u00C2\u00A0 our\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0are\u00C2\u00A0 less\u00C2\u00A0 immunosuppressive\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis\u00C2\u00A0 than\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0Further\u00C2\u00A0 studies\u00C2\u00A0 specifically\u00C2\u00A0 investigating\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 CFSE\u00C2\u00A0 pre\u00E2\u0080\u0090treatment\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 viability\u00C2\u00A0 of\u00C2\u00A0 splenocytes\u00C2\u00A0co\u00E2\u0080\u0090cultured\u00C2\u00A0with\u00C2\u00A04T1\u00C2\u00A0MDSCs\u00C2\u00A0will\u00C2\u00A0reveal\u00C2\u00A0the\u00C2\u00A0mechanisms\u00C2\u00A0involved.\u00C2\u00A0 \u00C2\u00A0 Related\u00C2\u00A0 to\u00C2\u00A0 this,\u00C2\u00A0 when\u00C2\u00A0 we\u00C2\u00A0 investigated\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 and\u00C2\u00A0 MDSC\u00C2\u00A0 populations\u00C2\u00A0 on\u00C2\u00A0 bulk\u00C2\u00A0 splenocyte\u00C2\u00A0 and\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0we\u00C2\u00A0made\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 interesting\u00C2\u00A0 observations.\u00C2\u00A0First,\u00C2\u00A0we\u00C2\u00A0noted\u00C2\u00A0that\u00C2\u00A0although\u00C2\u00A0control\u00C2\u00A0and\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0potently\u00C2\u00A0suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 proliferation,\u00C2\u00A0 they\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A0 proportion\u00C2\u00A0 of\u00C2\u00A0 viable\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 culture\u00C2\u00A0 (Fig.\u00C2\u00A0 5.19B).\u00C2\u00A0 In\u00C2\u00A0 contrast,\u00C2\u00A0 co\u00E2\u0080\u0090culture\u00C2\u00A0 with\u00C2\u00A0 either\u00C2\u00A0 control\u00C2\u00A0 Gr1+\u00C2\u00A0 cells\u00C2\u00A0 (which\u00C2\u00A0 were\u00C2\u00A0 not\u00C2\u00A0 immunosuppressive)\u00C2\u00A0 or\u00C2\u00A0 4T1\u00C2\u00A0MDSCs\u00C2\u00A0 significantly\u00C2\u00A0 promoted\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 death\u00C2\u00A0 (Fig.\u00C2\u00A0 5.19B).\u00C2\u00A0 These\u00C2\u00A0data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0MDSCs\u00C2\u00A0have\u00C2\u00A0opposing\u00C2\u00A0effects\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0 and\u00C2\u00A0 also\u00C2\u00A0provide\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0suppression\u00C2\u00A0and\u00C2\u00A0death\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0always\u00C2\u00A0directly\u00C2\u00A0related.\u00C2\u00A0 In\u00C2\u00A0 general,\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 achieved\u00C2\u00A0 via\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 anergy,\u00C2\u00A0 apoptosis,\u00C2\u00A0 or\u00C2\u00A0 necrosis.\u00C2\u00A0 \u00C2\u00A0 However,\u00C2\u00A0 further\u00C2\u00A0 studies\u00C2\u00A0 will\u00C2\u00A0 be\u00C2\u00A0 required\u00C2\u00A0 to\u00C2\u00A0 accurately\u00C2\u00A0 distinguish\u00C2\u00A0 which\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 mechanisms\u00C2\u00A0 is\u00C2\u00A0 utilized\u00C2\u00A0 by\u00C2\u00A0 different\u00C2\u00A0 immunosuppressive\u00C2\u00A0 myeloid\u00C2\u00A0cells.\u00C2\u00A0It\u00C2\u00A0seems\u00C2\u00A0probable\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0we\u00C2\u00A0see\u00C2\u00A0is\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0 removal\u00C2\u00A0 of\u00C2\u00A0 dead\u00C2\u00A0 or\u00C2\u00A0 dying\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 culture\u00C2\u00A0 by\u00C2\u00A0 phagocytic\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 but\u00C2\u00A0 studies\u00C2\u00A0 using\u00C2\u00A0 specific\u00C2\u00A0inhibitors\u00C2\u00A0of\u00C2\u00A0phagocytosis\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0substantiate\u00C2\u00A0this\u00C2\u00A0hypothesis.\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0key\u00C2\u00A0findings\u00C2\u00A0from\u00C2\u00A0these\u00C2\u00A0studies\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0elucidation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mechanisms\u00C2\u00A0 by\u00C2\u00A0which\u00C2\u00A04T1\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0exert\u00C2\u00A0their\u00C2\u00A0immunosuppressive\u00C2\u00A0functions.\u00C2\u00A0We\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0 both\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0by\u00C2\u00A0ROS\u00C2\u00A0production,\u00C2\u00A0but\u00C2\u00A0use\u00C2\u00A0different\u00C2\u00A0 mechanisms.\u00C2\u00A0 Specifically,\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0MDSCs\u00C2\u00A0were\u00C2\u00A0 contact\u00E2\u0080\u0090independent\u00C2\u00A0 (Fig.\u00C2\u00A05.20A)\u00C2\u00A0and\u00C2\u00A0inhibited\u00C2\u00A0by\u00C2\u00A0catalase\u00C2\u00A0(Fig.\u00C2\u00A05.25C),\u00C2\u00A0suggesting\u00C2\u00A0MDSCs\u00C2\u00A0inhibit\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0 146 production\u00C2\u00A0 of\u00C2\u00A0 extracellular\u00C2\u00A0H2O2.\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0MDSCs\u00C2\u00A0 in\u00C2\u00A0 other\u00C2\u00A0 tumor\u00C2\u00A0models\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reported\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0ROS,\u00C2\u00A0this\u00C2\u00A0immunosuppressive\u00C2\u00A0mechanism\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0specifically\u00C2\u00A0 reported\u00C2\u00A0for\u00C2\u00A04T1\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086\u00E2\u0080\u0090induced\u00C2\u00A0suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0 was\u00C2\u00A0 inhibited\u00C2\u00A0 by\u00C2\u00A0 NAC\u00C2\u00A0 (Fig.\u00C2\u00A0 5.23D)\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 were\u00C2\u00A0 much\u00C2\u00A0 more\u00C2\u00A0 potent\u00C2\u00A0 suppressors\u00C2\u00A0when\u00C2\u00A0 in\u00C2\u00A0 contact\u00C2\u00A0with\u00C2\u00A0 target\u00C2\u00A0 cells\u00C2\u00A0 (Fig.\u00C2\u00A0 5.20B).\u00C2\u00A0NAC\u00C2\u00A0 contains\u00C2\u00A0 both\u00C2\u00A0 a\u00C2\u00A0 thiol\u00C2\u00A0 (sulfhydryl;\u00C2\u00A0 SH)\u00C2\u00A0 and\u00C2\u00A0 cysteine\u00C2\u00A0 group,\u00C2\u00A0 each\u00C2\u00A0 of\u00C2\u00A0 which\u00C2\u00A0 contributes\u00C2\u00A0 to\u00C2\u00A0 its\u00C2\u00A0 anti\u00E2\u0080\u0090oxidant\u00C2\u00A0 properties\u00C2\u00A0 (Zhang\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0On\u00C2\u00A0 the\u00C2\u00A0one\u00C2\u00A0hand,\u00C2\u00A0 via\u00C2\u00A0 its\u00C2\u00A0 thiol\u00C2\u00A0 group,\u00C2\u00A0NAC\u00C2\u00A0 serves\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 proton\u00C2\u00A0 donor,\u00C2\u00A0 reducing\u00C2\u00A0 unstable\u00C2\u00A0molecules\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 ROS\u00C2\u00A0 (Zhang\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 On\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 via\u00C2\u00A0 its\u00C2\u00A0 cysteine\u00C2\u00A0 group,\u00C2\u00A0 NAC\u00C2\u00A0 functions\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 rate\u00E2\u0080\u0090limiting\u00C2\u00A0 factor\u00C2\u00A0 in\u00C2\u00A0 GSH\u00C2\u00A0 synthesis,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 potent\u00C2\u00A0 intracellular\u00C2\u00A0 antioxidant\u00C2\u00A0 in\u00C2\u00A0 cells\u00C2\u00A0 (Pompella\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2003).\u00C2\u00A0 Therefore,\u00C2\u00A0 although\u00C2\u00A0NAC\u00C2\u00A0 is\u00C2\u00A0 an\u00C2\u00A0 important\u00C2\u00A0 player\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 neutralization\u00C2\u00A0 of\u00C2\u00A0 free\u00C2\u00A0 radicals\u00C2\u00A0 and\u00C2\u00A0ROS\u00C2\u00A0both\u00C2\u00A0directly\u00C2\u00A0and\u00C2\u00A0indirectly\u00C2\u00A0(Zhang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011),\u00C2\u00A0we\u00C2\u00A0are\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0surmise\u00C2\u00A0from\u00C2\u00A0 our\u00C2\u00A0data\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0ROS\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s.\u00C2\u00A0However,\u00C2\u00A0since\u00C2\u00A0neither\u00C2\u00A0catalase\u00C2\u00A0nor\u00C2\u00A0 SOD\u00C2\u00A0reduced\u00C2\u00A0 their\u00C2\u00A0suppressive\u00C2\u00A0effects,\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A04T1\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0via\u00C2\u00A0intracellular\u00C2\u00A0ROS\u00C2\u00A0other\u00C2\u00A0than\u00C2\u00A0H2O2\u00C2\u00A0or\u00C2\u00A0O2\u00E2\u0080\u0090.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Not\u00C2\u00A0 only\u00C2\u00A0 do\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 via\u00C2\u00A0 different\u00C2\u00A0 mechanisms,\u00C2\u00A0 they\u00C2\u00A0 also\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 distinct\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 tumorigenesis.\u00C2\u00A0To\u00C2\u00A0discriminate\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0different\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0we\u00C2\u00A0used\u00C2\u00A0 ATRA,\u00C2\u00A0which\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0terminal\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0into\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0DCs\u00C2\u00A0(Almand\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02001;\u00C2\u00A0Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003;\u00C2\u00A0Mirza\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006).\u00C2\u00A0A\u00C2\u00A0recent\u00C2\u00A0report\u00C2\u00A0by\u00C2\u00A0Nefedova\u00C2\u00A0et\u00C2\u00A0 al.\u00C2\u00A0investigated\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0ATRA\u00C2\u00A0induced\u00C2\u00A0MDSC\u00C2\u00A0differentiation\u00C2\u00A0and\u00C2\u00A0found\u00C2\u00A0 that\u00C2\u00A0 ATRA\u00C2\u00A0 increased\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 glutathione\u00C2\u00A0 synthase\u00C2\u00A0 (GSS)\u00C2\u00A0 in\u00C2\u00A0 MDSCs,\u00C2\u00A0 which\u00C2\u00A0 in\u00C2\u00A0 turn\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 increased\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 GSH\u00C2\u00A0 and\u00C2\u00A0 decreased\u00C2\u00A0 ROS\u00C2\u00A0 production\u00C2\u00A0 (Nefedova\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007).\u00C2\u00A0These\u00C2\u00A0findings\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0previous\u00C2\u00A0reports\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0addition\u00C2\u00A0 to\u00C2\u00A0mediating\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0MDSCs,\u00C2\u00A0ROS\u00C2\u00A0also\u00C2\u00A0contributed\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0inability\u00C2\u00A0of\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 differentiate\u00C2\u00A0 into\u00C2\u00A0 mature\u00C2\u00A0 cells\u00C2\u00A0 (Kusmartsev\u00C2\u00A0 and\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 2003;\u00C2\u00A0 Kusmartsev\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2004).\u00C2\u00A0 Interestingly,\u00C2\u00A0 ATRA\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 stimulate\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0induced\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0model\u00C2\u00A0of\u00C2\u00A0sepsis\u00C2\u00A0(Cuenca\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011),\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0compatible\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0 idea\u00C2\u00A0that\u00C2\u00A0different\u00C2\u00A0stimuli\u00C2\u00A0induce\u00C2\u00A0MDSCs\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0phenotypes\u00C2\u00A0(i.e.\u00C2\u00A0sepsis\u00E2\u0080\u0090induced\u00C2\u00A0 147 MDSCs\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0produce\u00C2\u00A0ROS\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0affected\u00C2\u00A0by\u00C2\u00A0ATRA\u00C2\u00A0treatment).\u00C2\u00A0Nevertheless,\u00C2\u00A0 a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0studies\u00C2\u00A0have\u00C2\u00A0demonstrated\u00C2\u00A0that\u00C2\u00A0ATRA\u00C2\u00A0potently\u00C2\u00A0eliminates\u00C2\u00A0MDSCs\u00C2\u00A0both\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 (Almand\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02001;\u00C2\u00A0Gabrilovich\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02001)\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0vivo,\u00C2\u00A0 in\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 cancer\u00C2\u00A0 patients\u00C2\u00A0 (Kusmartsev\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2003;\u00C2\u00A0 Kusmartsev\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008;\u00C2\u00A0 Mirza\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006),\u00C2\u00A0 by\u00C2\u00A0 differentiating\u00C2\u00A0 MDSCs\u00C2\u00A0 into\u00C2\u00A0 mature\u00C2\u00A0 myeloid\u00C2\u00A0 cells.\u00C2\u00A0 Furthermore,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0ATRA,\u00C2\u00A0 in\u00C2\u00A0 combination\u00C2\u00A0with\u00C2\u00A0cancer\u00C2\u00A0vaccines\u00C2\u00A0or\u00C2\u00A0 chemotherapy,\u00C2\u00A0 increases\u00C2\u00A0 cancer\u00C2\u00A0treatment\u00C2\u00A0efficacy\u00C2\u00A0(Kusmartsev\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02003;\u00C2\u00A0Mirza\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02006;\u00C2\u00A0Sanz\u00C2\u00A0and\u00C2\u00A0Lo\u00E2\u0080\u0090Coco,\u00C2\u00A0 2011).\u00C2\u00A0 However,\u00C2\u00A0 little\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 about\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 ATRA\u00C2\u00A0 on\u00C2\u00A0 primary\u00C2\u00A0 or\u00C2\u00A0 metastatic\u00C2\u00A0 growth\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 other\u00C2\u00A0 treatments.\u00C2\u00A0 Our\u00C2\u00A0 studies\u00C2\u00A0 reveal\u00C2\u00A0 that\u00C2\u00A0 treating\u00C2\u00A0 tumor\u00E2\u0080\u0090 bearing\u00C2\u00A0mice\u00C2\u00A0with\u00C2\u00A0ATRA\u00C2\u00A0reduces\u00C2\u00A0 the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0MDSCs,\u00C2\u00A0 increases\u00C2\u00A0 the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (Fig.\u00C2\u00A0 5.30),\u00C2\u00A0 and\u00C2\u00A0 increases\u00C2\u00A0 lung\u00C2\u00A0metastasis,\u00C2\u00A0 but\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 affect\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 (Fig.\u00C2\u00A0 5.28).\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 data\u00C2\u00A0 argue\u00C2\u00A0 against\u00C2\u00A0 the\u00C2\u00A0 notion\u00C2\u00A0 that\u00C2\u00A0 inducing\u00C2\u00A0 MDSC\u00C2\u00A0 differentiation\u00C2\u00A0 leads\u00C2\u00A0to\u00C2\u00A0increased\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0immune\u00C2\u00A0responses\u00C2\u00A0and\u00C2\u00A0decreased\u00C2\u00A0tumorigenesis.\u00C2\u00A0Rather,\u00C2\u00A0 they\u00C2\u00A0 suggest\u00C2\u00A0 that,\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0 some\u00C2\u00A0 tumor\u00C2\u00A0models,\u00C2\u00A0 inducing\u00C2\u00A0MDSC\u00C2\u00A0 differentiation\u00C2\u00A0 can\u00C2\u00A0 promote\u00C2\u00A0metastasis.\u00C2\u00A0Interestingly,\u00C2\u00A0we\u00C2\u00A0noted\u00C2\u00A0that\u00C2\u00A0while\u00C2\u00A0ATRA\u00C2\u00A0enhanced\u00C2\u00A0lung\u00C2\u00A0metastasis\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A04T1\u00C2\u00A0and\u00C2\u00A04TO7\u00C2\u00A0models,\u00C2\u00A0 it\u00C2\u00A0 increased\u00C2\u00A04TO7\u00C2\u00A0metastasis\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 extent.\u00C2\u00A0This\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 because\u00C2\u00A0 1)\u00C2\u00A0 the\u00C2\u00A0 baseline\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 4TO7\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 metastasis\u00C2\u00A0 is\u00C2\u00A0 much\u00C2\u00A0 lower,\u00C2\u00A0 allowing\u00C2\u00A0the\u00C2\u00A0metastasis\u00E2\u0080\u0090enhancing\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0ATRA\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0evident,\u00C2\u00A0and/or\u00C2\u00A02)\u00C2\u00A04TO7\u00C2\u00A0 tumors\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0sensitive\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0both\u00C2\u00A0pro\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system,\u00C2\u00A0 i.e.,\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 ATRA\u00C2\u00A0 the\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 is\u00C2\u00A0 sufficient\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0 4TO7\u00C2\u00A0 metastasis,\u00C2\u00A0 but\u00C2\u00A0 the\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0 pro\u00E2\u0080\u0090tumor\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 mediated\u00C2\u00A0 by\u00C2\u00A0 ATRA\u00C2\u00A0 profoundly\u00C2\u00A0increases\u00C2\u00A04TO7\u00C2\u00A0metastatic\u00C2\u00A0growth.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Given\u00C2\u00A0our\u00C2\u00A0finding\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0immune\u00C2\u00A0suppressors\u00C2\u00A0than\u00C2\u00A0MDSCs\u00C2\u00A0on\u00C2\u00A0 a\u00C2\u00A0per\u00C2\u00A0cell\u00C2\u00A0basis\u00C2\u00A0(Fig.\u00C2\u00A05.14),\u00C2\u00A0we\u00C2\u00A0propose\u00C2\u00A0that\u00C2\u00A0inducing\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 increases\u00C2\u00A0overall\u00C2\u00A0 immune\u00C2\u00A0suppression,\u00C2\u00A0which\u00C2\u00A0 in\u00C2\u00A0turn\u00C2\u00A0 fosters\u00C2\u00A0 the\u00C2\u00A0growth\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 lungs.\u00C2\u00A0 In\u00C2\u00A0 contrast,\u00C2\u00A0 altering\u00C2\u00A0 the\u00C2\u00A0 ratio\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 with\u00C2\u00A0 ATRA\u00C2\u00A0 treatment\u00C2\u00A0did\u00C2\u00A0 not\u00C2\u00A0 affect\u00C2\u00A0 primary\u00C2\u00A0 tumor\u00C2\u00A0 growth\u00C2\u00A0 (Fig.\u00C2\u00A0 5.28A),\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0similar\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0primary\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0than\u00C2\u00A0metastatic\u00C2\u00A0 growth.\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0preliminary\u00C2\u00A0data\u00C2\u00A0demonstrating\u00C2\u00A0that\u00C2\u00A0tumor\u00C2\u00A0MDSCs\u00C2\u00A0 148 and\u00C2\u00A0 TAMs\u00C2\u00A0 exhibit\u00C2\u00A0 comparable\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown),\u00C2\u00A0 unlike\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 metastatic\u00C2\u00A0 sites.\u00C2\u00A0 The\u00C2\u00A0 idea\u00C2\u00A0 that\u00C2\u00A0 MDSCs\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 have\u00C2\u00A0 different\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0metastasis\u00C2\u00A0 is\u00C2\u00A0 further\u00C2\u00A0 supported\u00C2\u00A0by\u00C2\u00A0our\u00C2\u00A0 finding\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0located\u00C2\u00A0in\u00C2\u00A0very\u00C2\u00A0different\u00C2\u00A0regions\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0tumor\u00C2\u00A0nodules,\u00C2\u00A0with\u00C2\u00A0MDSCs\u00C2\u00A0lining\u00C2\u00A0the\u00C2\u00A0 periphery\u00C2\u00A0of\u00C2\u00A0 \u00C2\u00A0nodules\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 invading\u00C2\u00A0deep\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 interior\u00C2\u00A0 (Fig.\u00C2\u00A05.2B).\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 also\u00C2\u00A0possible\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0promote\u00C2\u00A0metastatic\u00C2\u00A0growth\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0greater\u00C2\u00A0extent\u00C2\u00A0than\u00C2\u00A0MDSCs\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 other\u00C2\u00A0 mechanisms\u00C2\u00A0 (i.e.\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 support\u00C2\u00A0 cell\u00C2\u00A0 growth\u00C2\u00A0 or\u00C2\u00A0 angiogenesis).\u00C2\u00A0 Although\u00C2\u00A0 our\u00C2\u00A0 hypothesis\u00C2\u00A0 is\u00C2\u00A0 supported\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 critical\u00C2\u00A0 role\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 to\u00C2\u00A0play\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0 stages\u00C2\u00A0of\u00C2\u00A0 tumorigenesis,\u00C2\u00A0 including\u00C2\u00A0metastasis,\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0contribution\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0to\u00C2\u00A0metastatic\u00C2\u00A0growth\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0accurately\u00C2\u00A0determined\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0T\u00C2\u00A0cell\u00E2\u0080\u0090deficient\u00C2\u00A0mouse\u00C2\u00A0model.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0 a\u00C2\u00A0 whole,\u00C2\u00A0 our\u00C2\u00A0 studies\u00C2\u00A0 underscore\u00C2\u00A0 the\u00C2\u00A0 vast\u00C2\u00A0 heterogeneity,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 phenotypic\u00C2\u00A0 similarities,\u00C2\u00A0 of\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00C2\u00A0 cells.\u00C2\u00A0 By\u00C2\u00A0 characterizing\u00C2\u00A0 the\u00C2\u00A0 differences\u00C2\u00A0 between\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 normal\u00C2\u00A0 tissues\u00C2\u00A0 and\u00C2\u00A0 their\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 counterparts\u00C2\u00A0 we\u00C2\u00A0 have\u00C2\u00A0 increased\u00C2\u00A0 our\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 tumors\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0and\u00C2\u00A0 immunosuppressive\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells.\u00C2\u00A0 Importantly,\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0 highlight\u00C2\u00A0 that\u00C2\u00A0 not\u00C2\u00A0 all\u00C2\u00A0 tumor\u00C2\u00A0 models\u00C2\u00A0 have\u00C2\u00A0 equivalent\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 and\u00C2\u00A0 suppressive\u00C2\u00A0 function\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 while\u00C2\u00A0 most\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 possess\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 the\u00C2\u00A0 potency\u00C2\u00A0 varies\u00C2\u00A0 dramatically\u00C2\u00A0 between\u00C2\u00A0 different\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0as\u00C2\u00A0do\u00C2\u00A0the\u00C2\u00A0mechanisms\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0cells\u00C2\u00A0exert\u00C2\u00A0their\u00C2\u00A0suppressive\u00C2\u00A0effects,\u00C2\u00A0 and\u00C2\u00A0finally,\u00C2\u00A0reveal\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0critical\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0promoting\u00C2\u00A0metastasis,\u00C2\u00A0and\u00C2\u00A0may\u00C2\u00A0do\u00C2\u00A0 so\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 extent\u00C2\u00A0 than\u00C2\u00A0MDSCs.\u00C2\u00A0 Together,\u00C2\u00A0 these\u00C2\u00A0 findings\u00C2\u00A0 help\u00C2\u00A0 clarify\u00C2\u00A0 the\u00C2\u00A0 roles\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0cancer,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0targeted\u00C2\u00A0 therapeutics.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 149 CHAPTER\u00C2\u00A06\u00C2\u00A0: SUMMARY\u00C2\u00A0AND\u00C2\u00A0FUTURE\u00C2\u00A0DIRECTIONS\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0overall\u00C2\u00A0objective\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0thesis\u00C2\u00A0was\u00C2\u00A0to\u00C2\u00A0investigate\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0elucidate\u00C2\u00A0the\u00C2\u00A0roles\u00C2\u00A0these\u00C2\u00A0 cells\u00C2\u00A0 play\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 normal\u00C2\u00A0 and\u00C2\u00A0 neoplastic\u00C2\u00A0 tissues.\u00C2\u00A0 Although\u00C2\u00A0 the\u00C2\u00A0 immunomodulatory\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 have\u00C2\u00A0 long\u00C2\u00A0 been\u00C2\u00A0 recognized\u00C2\u00A0 (Holda\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1985;\u00C2\u00A0 Strober,\u00C2\u00A0 1984),\u00C2\u00A0 when\u00C2\u00A0 we\u00C2\u00A0 embarked\u00C2\u00A0 on\u00C2\u00A0 this\u00C2\u00A0 work\u00C2\u00A0 many\u00C2\u00A0 questions\u00C2\u00A0 remained\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0involved,\u00C2\u00A0the\u00C2\u00A0mechanisms\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0they\u00C2\u00A0functioned,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0contexts\u00C2\u00A0 in\u00C2\u00A0 which\u00C2\u00A0 they\u00C2\u00A0 exerted\u00C2\u00A0 their\u00C2\u00A0 effects.\u00C2\u00A0 Our\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 focused\u00C2\u00A0 on\u00C2\u00A0 clarifying\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0 two\u00C2\u00A0mononuclear\u00C2\u00A0cell\u00C2\u00A0populations,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 Overall,\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0of\u00C2\u00A0our\u00C2\u00A0work\u00C2\u00A0reveal\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0important\u00C2\u00A0insights\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0functions\u00C2\u00A0 and\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 physiological\u00C2\u00A0 and\u00C2\u00A0 pathological\u00C2\u00A0 environments,\u00C2\u00A0 highlight\u00C2\u00A0 the\u00C2\u00A0 considerable\u00C2\u00A0 phenotypic\u00C2\u00A0 and\u00C2\u00A0 functional\u00C2\u00A0 heterogeneity\u00C2\u00A0 exhibited\u00C2\u00A0 by\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0MDSCs,\u00C2\u00A0and\u00C2\u00A0underscore\u00C2\u00A0the\u00C2\u00A0considerable\u00C2\u00A0therapeutic\u00C2\u00A0potential\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 Chapter\u00C2\u00A0 3,\u00C2\u00A0 we\u00C2\u00A0 examined\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 under\u00C2\u00A0physiological\u00C2\u00A0conditions\u00C2\u00A0and\u00C2\u00A0obtained\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0interesting\u00C2\u00A0results\u00C2\u00A0(Hamilton\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0Although\u00C2\u00A0CD11b+Gr1+\u00C2\u00A0IMCs\u00C2\u00A0exist\u00C2\u00A0 in\u00C2\u00A0various\u00C2\u00A0murine\u00C2\u00A0tissues,\u00C2\u00A0 including\u00C2\u00A0the\u00C2\u00A0 BM\u00C2\u00A0 and\u00C2\u00A0 spleen,\u00C2\u00A0 they\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 possess\u00C2\u00A0 immunosuppressive\u00C2\u00A0 abilities\u00C2\u00A0 under\u00C2\u00A0 steady\u00E2\u0080\u0090state\u00C2\u00A0 conditions\u00C2\u00A0 and\u00C2\u00A0 therefore\u00C2\u00A0 are\u00C2\u00A0not\u00C2\u00A0 considered\u00C2\u00A0MDSCs\u00C2\u00A0 (Delano\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007;\u00C2\u00A0 Laoui\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 We\u00C2\u00A0 thus\u00C2\u00A0 focused\u00C2\u00A0 on\u00C2\u00A0 exploring\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 We\u00C2\u00A0 determined\u00C2\u00A0 that\u00C2\u00A0 resident\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 PC\u00C2\u00A0 of\u00C2\u00A0 non\u00E2\u0080\u0090tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice,\u00C2\u00A0 potently\u00C2\u00A0 suppressed\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 an\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 and\u00C2\u00A0 NO\u00E2\u0080\u0090dependent\u00C2\u00A0 mechanism\u00C2\u00A0 (Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 These\u00C2\u00A0 results\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 previous\u00C2\u00A0 studies\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 early\u00C2\u00A01990s\u00C2\u00A0 that\u00C2\u00A0 identified\u00C2\u00A0 this\u00C2\u00A0 pathway\u00C2\u00A0 in\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 (Albina\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1991;\u00C2\u00A0Cox\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1992;\u00C2\u00A0 Deng\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 1993).\u00C2\u00A0 However,\u00C2\u00A0 these\u00C2\u00A0 original\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 largely\u00C2\u00A0 overlooked\u00C2\u00A0 in\u00C2\u00A0 recent\u00C2\u00A0years\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0this\u00C2\u00A0pathway,\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0or\u00C2\u00A0 not\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0exert\u00C2\u00A0their\u00C2\u00A0suppressive\u00C2\u00A0effects,\u00C2\u00A0remained\u00C2\u00A0poorly\u00C2\u00A0understood.\u00C2\u00A0We\u00C2\u00A0performed\u00C2\u00A0a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 experiments\u00C2\u00A0 to\u00C2\u00A0 address\u00C2\u00A0 this\u00C2\u00A0 question\u00C2\u00A0 and\u00C2\u00A0 discovered\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 abilities\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0were\u00C2\u00A0eliminated\u00C2\u00A0by\u00C2\u00A0TLR\u00C2\u00A0agonists\u00C2\u00A0that\u00C2\u00A0signal\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0TRIF\u00C2\u00A0cascade\u00C2\u00A0 150 (i.e.\u00C2\u00A0LPS\u00C2\u00A0or\u00C2\u00A0dsRNA),\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0by\u00C2\u00A0those\u00C2\u00A0that\u00C2\u00A0signal\u00C2\u00A0exclusively\u00C2\u00A0through\u00C2\u00A0MyD88\u00C2\u00A0(i.e.\u00C2\u00A0CpG\u00C2\u00A0or\u00C2\u00A0 PGN)\u00C2\u00A0(Hamilton\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0Moreover,\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0these\u00C2\u00A0effects\u00C2\u00A0were\u00C2\u00A0mediated\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 that\u00C2\u00A0 occurs\u00C2\u00A0 following\u00C2\u00A0 TRIF\u00C2\u00A0 recruitment\u00C2\u00A0 and\u00C2\u00A0 IRF\u00E2\u0080\u00903\u00C2\u00A0 activation,\u00C2\u00A0 which\u00C2\u00A0 decreases\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 to\u00C2\u00A0 respond\u00C2\u00A0 to\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 stimulation\u00C2\u00A0 (Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010).\u00C2\u00A0 These\u00C2\u00A0 novel\u00C2\u00A0 findings\u00C2\u00A0 reveal\u00C2\u00A0 a\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0 resident\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 maintenance\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 homeostasis,\u00C2\u00A0 increase\u00C2\u00A0 our\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 tolerance,\u00C2\u00A0 and\u00C2\u00A0 suggest\u00C2\u00A0 a\u00C2\u00A0 key\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0 TLR\u00C2\u00A0 signaling\u00C2\u00A0 and\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 in\u00C2\u00A0 regulating\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 findings\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 offer\u00C2\u00A0 novel\u00C2\u00A0 insights\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0mechanisms\u00C2\u00A0 regulating\u00C2\u00A0M\u00CF\u0086\u00E2\u0080\u0090induced\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 tolerance\u00C2\u00A0 on\u00C2\u00A0multiple\u00C2\u00A0 levels.\u00C2\u00A0 At\u00C2\u00A0 the\u00C2\u00A0molecular\u00C2\u00A0 level,\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0provide\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0can\u00C2\u00A0antagonize\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0signaling;\u00C2\u00A0however,\u00C2\u00A0the\u00C2\u00A0precise\u00C2\u00A0 mechanism(s)\u00C2\u00A0 by\u00C2\u00A0which\u00C2\u00A0 this\u00C2\u00A0 occurs\u00C2\u00A0 remain(s)\u00C2\u00A0 unclear\u00C2\u00A0 and\u00C2\u00A0will\u00C2\u00A0 require\u00C2\u00A0 further\u00C2\u00A0work.\u00C2\u00A0 Specifically,\u00C2\u00A0 activation\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 signaling\u00C2\u00A0 components\u00C2\u00A0 (e.g.\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00C2\u00A0 receptor,\u00C2\u00A0 JAK\u00E2\u0080\u00901,\u00C2\u00A0 JAK\u00E2\u0080\u00902,\u00C2\u00A0 STAT1)\u00C2\u00A0 in\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B3\u00E2\u0080\u0090stimulated\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 pre\u00E2\u0080\u0090treated\u00C2\u00A0 with\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0 would\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 our\u00C2\u00A0 understanding\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 process.\u00C2\u00A0 Our\u00C2\u00A0 data\u00C2\u00A0 support\u00C2\u00A0 the\u00C2\u00A0 concept\u00C2\u00A0 that\u00C2\u00A0 NO\u00C2\u00A0 can\u00C2\u00A0 play\u00C2\u00A0 divergent\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 (Prabhu\u00C2\u00A0 and\u00C2\u00A0 Guruvayoorappan,\u00C2\u00A0 2010)\u00C2\u00A0 and\u00C2\u00A0 demonstrate\u00C2\u00A0 a\u00C2\u00A0 previously\u00C2\u00A0 unappreciated\u00C2\u00A0 link\u00C2\u00A0 between\u00C2\u00A0 TLR\u00C2\u00A0signaling\u00C2\u00A0and\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0immune\u00C2\u00A0suppression.\u00C2\u00A0These\u00C2\u00A0findings\u00C2\u00A0also\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0 our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 suppression\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 tolerance\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0cellular\u00C2\u00A0and\u00C2\u00A0organismal\u00C2\u00A0levels.\u00C2\u00A0Intriguingly,\u00C2\u00A0we\u00C2\u00A0show\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0time\u00C2\u00A0that\u00C2\u00A0viral\u00E2\u0080\u0090\u00C2\u00A0but\u00C2\u00A0 not\u00C2\u00A0 bacterial\u00E2\u0080\u0090derived\u00C2\u00A0 PAMPs\u00C2\u00A0 reduce\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 However,\u00C2\u00A0an\u00C2\u00A0important\u00C2\u00A0caveat\u00C2\u00A0to\u00C2\u00A0these\u00C2\u00A0findings\u00C2\u00A0is\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0stimulation\u00C2\u00A0 of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0with\u00C2\u00A0individual\u00C2\u00A0PAMP\u00C2\u00A0ligands\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0accurately\u00C2\u00A0reflect\u00C2\u00A0the\u00C2\u00A0context\u00C2\u00A0in\u00C2\u00A0 which\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0would\u00C2\u00A0encounter\u00C2\u00A0intact\u00C2\u00A0pathogens\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0Future\u00C2\u00A0experiments\u00C2\u00A0investigating\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 pathogens\u00C2\u00A0 on\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 immunosuppression\u00C2\u00A0 using\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 animal\u00C2\u00A0 infection\u00C2\u00A0 models,\u00C2\u00A0 e.g.,\u00C2\u00A0 Legionella\u00C2\u00A0 pneumophila\u00C2\u00A0 (intracellular\u00C2\u00A0 bacteria),\u00C2\u00A0Mycobacterium\u00C2\u00A0 tuberculosis\u00C2\u00A0 (extracellular\u00C2\u00A0 bacteria),\u00C2\u00A0 influenza\u00C2\u00A0 (virus),\u00C2\u00A0 Candida\u00C2\u00A0 albicans\u00C2\u00A0 (fungus),\u00C2\u00A0 Trichuris\u00C2\u00A0muris\u00C2\u00A0(helminth)\u00C2\u00A0would\u00C2\u00A0validate\u00C2\u00A0our\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0results\u00C2\u00A0and\u00C2\u00A0provide\u00C2\u00A0insights\u00C2\u00A0into\u00C2\u00A0 the\u00C2\u00A0physiological\u00C2\u00A0rationale\u00C2\u00A0for\u00C2\u00A0these\u00C2\u00A0findings.\u00C2\u00A0\u00C2\u00A0 151 \u00C2\u00A0 Importantly,\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 reveal\u00C2\u00A0 specific\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 attractive\u00C2\u00A0 therapeutic\u00C2\u00A0 targets.\u00C2\u00A0 Treatment\u00C2\u00A0with\u00C2\u00A0 IFN\u00E2\u0080\u0090\u00CE\u00B2,\u00C2\u00A0LPS,\u00C2\u00A0dsRNA,\u00C2\u00A0or\u00C2\u00A0small\u00C2\u00A0molecule\u00C2\u00A0 iNOS\u00C2\u00A0 inhibitors\u00C2\u00A0may\u00C2\u00A0reduce\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0 suppression,\u00C2\u00A0 which\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 useful\u00C2\u00A0 following\u00C2\u00A0 infection\u00C2\u00A0 or\u00C2\u00A0 as\u00C2\u00A0 part\u00C2\u00A0 of\u00C2\u00A0 future\u00C2\u00A0 cancer\u00C2\u00A0 therapies.\u00C2\u00A0Alternatively,\u00C2\u00A0small\u00C2\u00A0molecule\u00C2\u00A0Arg1\u00C2\u00A0or\u00C2\u00A0IFN\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0inhibitors,\u00C2\u00A0via\u00C2\u00A0their\u00C2\u00A0enhancement\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 desirable\u00C2\u00A0 in\u00C2\u00A0 patients\u00C2\u00A0 with\u00C2\u00A0 autoimmune\u00C2\u00A0diseases\u00C2\u00A0or\u00C2\u00A0GvHD.\u00C2\u00A0Future\u00C2\u00A0studies\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0test\u00C2\u00A0the\u00C2\u00A0effectiveness\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0potential\u00C2\u00A0 therapies\u00C2\u00A0 in\u00C2\u00A0vivo,\u00C2\u00A0 in\u00C2\u00A0 animal\u00C2\u00A0models\u00C2\u00A0 of\u00C2\u00A0 clinical\u00C2\u00A0 diseases.\u00C2\u00A0Overall,\u00C2\u00A0 the\u00C2\u00A0 findings\u00C2\u00A0presented\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0emphasize\u00C2\u00A0the\u00C2\u00A0dual\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0promotion\u00C2\u00A0and\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 in\u00C2\u00A0 normal\u00C2\u00A0 tissues\u00C2\u00A0 and\u00C2\u00A0 highlight\u00C2\u00A0 their\u00C2\u00A0 considerable\u00C2\u00A0 therapeutic\u00C2\u00A0potential,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0importance\u00C2\u00A0of\u00C2\u00A0increasing\u00C2\u00A0our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0function.\u00C2\u00A0 \u00C2\u00A0 After\u00C2\u00A0investigating\u00C2\u00A0the\u00C2\u00A0regulatory\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0under\u00C2\u00A0physiological\u00C2\u00A0conditions,\u00C2\u00A0we\u00C2\u00A0 sought\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 their\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 tumor\u00C2\u00A0 environment,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 compare\u00C2\u00A0 the\u00C2\u00A0 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properties\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0experiments\u00C2\u00A0are\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A04.\u00C2\u00A0 We\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0MDSCs\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice,\u00C2\u00A0 unlike\u00C2\u00A0 PM\u00CF\u0086s\u00C2\u00A0 or\u00C2\u00A0 TAMs,\u00C2\u00A0 could\u00C2\u00A0not\u00C2\u00A0suppress\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0FCS\u00C2\u00A0and,\u00C2\u00A0in\u00C2\u00A0fact,\u00C2\u00A0promoted\u00C2\u00A0T\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 some\u00C2\u00A0 experiments\u00C2\u00A0 (Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 Investigation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 underlying\u00C2\u00A0mechanisms\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0 serum\u00C2\u00A0 albumin\u00C2\u00A0was\u00C2\u00A0 a\u00C2\u00A0major\u00C2\u00A0 contributor\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 antagonistic\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 FCS\u00C2\u00A0 on\u00C2\u00A0 MDSC\u00E2\u0080\u0090induced\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 and\u00C2\u00A0 did\u00C2\u00A0 so\u00C2\u00A0 by\u00C2\u00A0 restricting\u00C2\u00A0ROS\u00C2\u00A0 production\u00C2\u00A0 from\u00C2\u00A0MDSCs\u00C2\u00A0 (Hamilton\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 These\u00C2\u00A0novel\u00C2\u00A0 findings\u00C2\u00A0 have\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 important\u00C2\u00A0 implications\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 accurate\u00C2\u00A0 detection\u00C2\u00A0 and\u00C2\u00A0 identification\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0\u00C2\u00A0 152 \u00C2\u00A0 MDSCs\u00C2\u00A0represent\u00C2\u00A0an\u00C2\u00A0extremely\u00C2\u00A0heterogeneous\u00C2\u00A0population\u00C2\u00A0of\u00C2\u00A0cells.\u00C2\u00A0 In\u00C2\u00A0fact,\u00C2\u00A0some\u00C2\u00A0 have\u00C2\u00A0 argued\u00C2\u00A0 that\u00C2\u00A0 identifying\u00C2\u00A0 MDSCs\u00C2\u00A0 by\u00C2\u00A0 co\u00E2\u0080\u0090expression\u00C2\u00A0 of\u00C2\u00A0 CD11b\u00C2\u00A0 and\u00C2\u00A0 Gr1\u00C2\u00A0 has\u00C2\u00A0 led\u00C2\u00A0 to\u00C2\u00A0 ambiguity\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0literature\u00C2\u00A0since\u00C2\u00A0this\u00C2\u00A0definition\u00C2\u00A0is\u00C2\u00A0neither\u00C2\u00A0specific\u00C2\u00A0nor\u00C2\u00A0inclusive\u00C2\u00A0(Cuenca\u00C2\u00A0 et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0In\u00C2\u00A0reality,\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mononuclear\u00C2\u00A0phagocyte\u00C2\u00A0lineage\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0particularly\u00C2\u00A0 difficult\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0by\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0markers\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0phenotype\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0depends\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 factors\u00C2\u00A0 including\u00C2\u00A0 inflammatory\u00C2\u00A0 status\u00C2\u00A0 (i.e.\u00C2\u00A0 steady\u00E2\u0080\u0090state\u00C2\u00A0 versus\u00C2\u00A0 inflammatory\u00C2\u00A0 conditions),\u00C2\u00A0 type\u00C2\u00A0 and/or\u00C2\u00A0 duration\u00C2\u00A0 of\u00C2\u00A0 inflammation,\u00C2\u00A0 and\u00C2\u00A0 tissue\u00C2\u00A0 location\u00C2\u00A0 (Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0Therefore,\u00C2\u00A0MDSCs\u00C2\u00A0must\u00C2\u00A0be\u00C2\u00A0 identified\u00C2\u00A0 functionally,\u00C2\u00A0which\u00C2\u00A0requires\u00C2\u00A0 assays\u00C2\u00A0 to\u00C2\u00A0 accurately\u00C2\u00A0 assess\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 ex\u00C2\u00A0 vivo.\u00C2\u00A0 Our\u00C2\u00A0 data,\u00C2\u00A0 which\u00C2\u00A0 demonstrate\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0first\u00C2\u00A0 time\u00C2\u00A0that\u00C2\u00A0different\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0profound\u00C2\u00A0effect\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 MDSCs,\u00C2\u00A0 are\u00C2\u00A0 of\u00C2\u00A0 critical\u00C2\u00A0 importance\u00C2\u00A0 for\u00C2\u00A0 researchers\u00C2\u00A0 regarding\u00C2\u00A0the\u00C2\u00A0appropriate\u00C2\u00A0design\u00C2\u00A0and\u00C2\u00A0interpretation\u00C2\u00A0of\u00C2\u00A0immunosuppression\u00C2\u00A0assays.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0caveat\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0our\u00C2\u00A0studies\u00C2\u00A0were\u00C2\u00A0 limited\u00C2\u00A0 to\u00C2\u00A04T1\u00E2\u0080\u0090induced\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0we\u00C2\u00A0did\u00C2\u00A0 not\u00C2\u00A0test\u00C2\u00A0the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0on\u00C2\u00A0MDSCs\u00C2\u00A0induced\u00C2\u00A0in\u00C2\u00A0any\u00C2\u00A0other\u00C2\u00A0models.\u00C2\u00A0Validation\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 different\u00C2\u00A0 models\u00C2\u00A0 of\u00C2\u00A0 MDSC\u00C2\u00A0 induction\u00C2\u00A0 (e.g.\u00C2\u00A0 different\u00C2\u00A0 tumor\u00C2\u00A0 models,\u00C2\u00A0 trauma,\u00C2\u00A0 parasitic\u00C2\u00A0 infections,\u00C2\u00A0 sepsis)\u00C2\u00A0 will\u00C2\u00A0 reveal\u00C2\u00A0 whether\u00C2\u00A0 this\u00C2\u00A0 phenomenon\u00C2\u00A0 is\u00C2\u00A0 unique\u00C2\u00A0 to\u00C2\u00A0 4T1\u00E2\u0080\u0090 MDSCs\u00C2\u00A0or\u00C2\u00A0more\u00C2\u00A0generally\u00C2\u00A0applicable\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0tumor\u00C2\u00A0or\u00C2\u00A0inflammatory\u00C2\u00A0models.\u00C2\u00A0Similarly,\u00C2\u00A0 we\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 separate\u00C2\u00A0 different\u00C2\u00A0 populations\u00C2\u00A0 of\u00C2\u00A0 MDSCs,\u00C2\u00A0 but\u00C2\u00A0 studied\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 features\u00C2\u00A0 of\u00C2\u00A0 bulk\u00C2\u00A0 pulmonary\u00C2\u00A0 or\u00C2\u00A0 splenic\u00C2\u00A0 Gr1+\u00C2\u00A0 cells.\u00C2\u00A0 Although\u00C2\u00A0 we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 4T1\u00E2\u0080\u0090MDSCs\u00C2\u00A0 were\u00C2\u00A0 a\u00C2\u00A0 relatively\u00C2\u00A0 uniform\u00C2\u00A0 population\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometric\u00C2\u00A0 and\u00C2\u00A0 morphological\u00C2\u00A0analyses\u00C2\u00A0(>95%\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0G\u00E2\u0080\u0090MDSCs),\u00C2\u00A0 further\u00C2\u00A0studies\u00C2\u00A0comparing\u00C2\u00A0 the\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 serum\u00C2\u00A0 and\u00C2\u00A0 albumin\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 functions\u00C2\u00A0 and\u00C2\u00A0 ROS\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 subpopulations\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 will\u00C2\u00A0 reveal\u00C2\u00A0 the\u00C2\u00A0 potential\u00C2\u00A0 applicability\u00C2\u00A0 and/or\u00C2\u00A0 limitations\u00C2\u00A0of\u00C2\u00A0our\u00C2\u00A0findings.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Despite\u00C2\u00A0the\u00C2\u00A0technical\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0work\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A04,\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0may\u00C2\u00A0 shed\u00C2\u00A0light\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0physiological\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0biology.\u00C2\u00A0Our\u00C2\u00A0finding\u00C2\u00A0that\u00C2\u00A0MDSC\u00E2\u0080\u0090 induced\u00C2\u00A0immunosuppression\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0a\u00C2\u00A0fixed\u00C2\u00A0property,\u00C2\u00A0but\u00C2\u00A0varies\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0different\u00C2\u00A0 culture\u00C2\u00A0conditions\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0reports\u00C2\u00A0that,\u00C2\u00A0in\u00C2\u00A0certain\u00C2\u00A0contexts,\u00C2\u00A0MDSCs\u00C2\u00A0can\u00C2\u00A0exert\u00C2\u00A0 pro\u00E2\u0080\u0090inflammatory\u00C2\u00A0 functions\u00C2\u00A0 (Cuenca\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011;\u00C2\u00A0 Nausch\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008;\u00C2\u00A0 Pastula\u00C2\u00A0 and\u00C2\u00A0 153 Marcinkiewicz,\u00C2\u00A02011).\u00C2\u00A0Briefly,\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0MDSCs\u00C2\u00A0that\u00C2\u00A0 enable\u00C2\u00A0 them\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 ROS\u00C2\u00A0 and\u00C2\u00A0 inflammatory\u00C2\u00A0 cytokine\u00C2\u00A0 production,\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0anti\u00E2\u0080\u0090microbial\u00C2\u00A0responses\u00C2\u00A0and\u00C2\u00A0increased\u00C2\u00A0immune\u00C2\u00A0surveillance\u00C2\u00A0 (Cuenca\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 Moreover,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 some\u00C2\u00A0 reports\u00C2\u00A0 that\u00C2\u00A0 MDSCs,\u00C2\u00A0 under\u00C2\u00A0 some\u00C2\u00A0 circumstances,\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 immunostimulatory\u00C2\u00A0 (Nausch\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008;\u00C2\u00A0 Pastula\u00C2\u00A0 and\u00C2\u00A0 Marcinkiewicz,\u00C2\u00A0 2011),\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 results\u00C2\u00A0 obtained\u00C2\u00A0 in\u00C2\u00A0 some\u00C2\u00A0 of\u00C2\u00A0 our\u00C2\u00A0 experiments\u00C2\u00A0in\u00C2\u00A0which\u00C2\u00A0serum\u00C2\u00A0was\u00C2\u00A0present.\u00C2\u00A0 In\u00C2\u00A0addition,\u00C2\u00A0our\u00C2\u00A0finding\u00C2\u00A0that\u00C2\u00A0albumin\u00C2\u00A0blunts\u00C2\u00A0 ROS\u00C2\u00A0production\u00C2\u00A0and,\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result,\u00C2\u00A0 immune\u00C2\u00A0suppression,\u00C2\u00A0demonstrates\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 first\u00C2\u00A0 time\u00C2\u00A0 that\u00C2\u00A0 4T1\u00E2\u0080\u0090induced\u00C2\u00A0 MDSCs\u00C2\u00A0 can\u00C2\u00A0 inhibit\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 via\u00C2\u00A0 ROS\u00C2\u00A0 production.\u00C2\u00A0 Although\u00C2\u00A0 these\u00C2\u00A0experiments\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0reveal\u00C2\u00A0the\u00C2\u00A0particular\u00C2\u00A0ROS\u00C2\u00A0involved,\u00C2\u00A0our\u00C2\u00A0findings\u00C2\u00A0from\u00C2\u00A0Chapter\u00C2\u00A0 5\u00C2\u00A0suggest\u00C2\u00A0a\u00C2\u00A0possible\u00C2\u00A0role\u00C2\u00A0for\u00C2\u00A0H2O2,\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0studies\u00C2\u00A0measuring\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0H2O2\u00C2\u00A0and\u00C2\u00A0 other\u00C2\u00A0ROS\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0performed\u00C2\u00A0to\u00C2\u00A0validate\u00C2\u00A0this\u00C2\u00A0finding.\u00C2\u00A0As\u00C2\u00A0previously\u00C2\u00A0mentioned,\u00C2\u00A0these\u00C2\u00A0 data\u00C2\u00A0may\u00C2\u00A0suggest\u00C2\u00A0a\u00C2\u00A0physiological\u00C2\u00A0function\u00C2\u00A0for\u00C2\u00A0albumin\u00C2\u00A0in\u00C2\u00A0restricting\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0and\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0MDSCs.\u00C2\u00A0While\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0not\u00C2\u00A0identified\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0mechanism(s)\u00C2\u00A0at\u00C2\u00A0play,\u00C2\u00A0we\u00C2\u00A0 have\u00C2\u00A0 some\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 albumin\u00E2\u0080\u0090associated\u00C2\u00A0 FAs\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 involved.\u00C2\u00A0 It\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 interesting\u00C2\u00A0to\u00C2\u00A0further\u00C2\u00A0elucidate\u00C2\u00A0the\u00C2\u00A0involvement\u00C2\u00A0of\u00C2\u00A0FAs\u00C2\u00A0in\u00C2\u00A0regulating\u00C2\u00A0MDSC\u00C2\u00A0function\u00C2\u00A0and\u00C2\u00A0 test\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0altering\u00C2\u00A0the\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0FAs\u00C2\u00A0(i.e.\u00C2\u00A0omega\u00E2\u0080\u00903\u00C2\u00A0versus\u00C2\u00A0\u00E2\u0080\u00906)\u00C2\u00A0as\u00C2\u00A0this\u00C2\u00A0might\u00C2\u00A0 be\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0value.\u00C2\u00A0In\u00C2\u00A0summary,\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A04\u00C2\u00A0clearly\u00C2\u00A0highlight\u00C2\u00A0 the\u00C2\u00A0 importance\u00C2\u00A0 of\u00C2\u00A0 testing\u00C2\u00A0 different\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 culture\u00C2\u00A0 conditions\u00C2\u00A0 on\u00C2\u00A0 MDSC\u00C2\u00A0 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Once\u00C2\u00A0we\u00C2\u00A0established\u00C2\u00A0culture\u00C2\u00A0conditions\u00C2\u00A0that\u00C2\u00A0allowed\u00C2\u00A0immune\u00C2\u00A0suppression\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0 accurately\u00C2\u00A0 assayed\u00C2\u00A0 in\u00C2\u00A0 vitro,\u00C2\u00A0 we\u00C2\u00A0 embarked\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 series\u00C2\u00A0 of\u00C2\u00A0 studies\u00C2\u00A0 comparing\u00C2\u00A0 the\u00C2\u00A0 regulatory\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 subtypes\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 control,\u00C2\u00A0 4T1,\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice.\u00C2\u00A0 These\u00C2\u00A0 experiments,\u00C2\u00A0 communicated\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 5,\u00C2\u00A0 revealed\u00C2\u00A0 several\u00C2\u00A0striking\u00C2\u00A0results.\u00C2\u00A0We\u00C2\u00A0demonstrated\u00C2\u00A0that,\u00C2\u00A0contrary\u00C2\u00A0to\u00C2\u00A0currently\u00C2\u00A0accepted\u00C2\u00A0notions,\u00C2\u00A0 not\u00C2\u00A0all\u00C2\u00A0 tumors\u00C2\u00A0 induce\u00C2\u00A0myelopoiesis\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0 immunosuppressive\u00C2\u00A0cells.\u00C2\u00A0 154 We\u00C2\u00A0 noted\u00C2\u00A0 a\u00C2\u00A0 significant\u00C2\u00A0 difference\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 vascularization\u00C2\u00A0 of\u00C2\u00A0 4T1\u00C2\u00A0 and\u00C2\u00A0 67NR\u00C2\u00A0 tumors,\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0reports\u00C2\u00A0suggesting\u00C2\u00A0a\u00C2\u00A0 link\u00C2\u00A0between\u00C2\u00A0hypoxia\u00C2\u00A0and\u00C2\u00A0 induction\u00C2\u00A0 and/or\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0 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responses\u00C2\u00A0via\u00C2\u00A0different\u00C2\u00A0 mechanisms;\u00C2\u00A0 although\u00C2\u00A0 both\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 MDSCs\u00C2\u00A0 produced\u00C2\u00A0 ROS,\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 suppression\u00C2\u00A0 was\u00C2\u00A0 contact\u00E2\u0080\u0090dependent\u00C2\u00A0 and\u00C2\u00A0 inhibited\u00C2\u00A0 by\u00C2\u00A0 NAC\u00C2\u00A0 while\u00C2\u00A0 MDSC\u00C2\u00A0 suppression\u00C2\u00A0 was\u00C2\u00A0 contact\u00E2\u0080\u0090 independent\u00C2\u00A0 and\u00C2\u00A0 inhibited\u00C2\u00A0 by\u00C2\u00A0 catalase.\u00C2\u00A0 There\u00C2\u00A0 a\u00C2\u00A0 few\u00C2\u00A0 potential\u00C2\u00A0 caveats\u00C2\u00A0 that\u00C2\u00A0 must\u00C2\u00A0 be\u00C2\u00A0 considered\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0these\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0results:\u00C2\u00A01)\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0lack\u00C2\u00A0of\u00C2\u00A0appropriate\u00C2\u00A0genetically\u00C2\u00A0 modified\u00C2\u00A0mice\u00C2\u00A0we\u00C2\u00A0were\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0compare\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0MDSCs\u00C2\u00A0to\u00C2\u00A0specifically\u00C2\u00A0 suppress\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0Ag\u00E2\u0080\u0090specific\u00C2\u00A0responses,\u00C2\u00A02)\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0low\u00C2\u00A0cell\u00C2\u00A0yield\u00C2\u00A0we\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0 compare\u00C2\u00A0 the\u00C2\u00A0 suppressive\u00C2\u00A0 abilities\u00C2\u00A0of\u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0 and\u00C2\u00A03)\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0 examining\u00C2\u00A0 precise\u00C2\u00A0 MDSC\u00C2\u00A0 and\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 subsets\u00C2\u00A0 we\u00C2\u00A0 examined\u00C2\u00A0 total\u00C2\u00A0 cell\u00C2\u00A0 populations.\u00C2\u00A0 Identification\u00C2\u00A0of\u00C2\u00A0individual\u00C2\u00A0subpopulations\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0difficult\u00C2\u00A0since\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0 are\u00C2\u00A0highly\u00C2\u00A0related\u00C2\u00A0and\u00C2\u00A0exhibit\u00C2\u00A0variable\u00C2\u00A0phenotypes\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0stimuli\u00C2\u00A0 to\u00C2\u00A0which\u00C2\u00A0 155 they\u00C2\u00A0are\u00C2\u00A0exposed\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011).\u00C2\u00A0\u00C2\u00A0Related\u00C2\u00A0to\u00C2\u00A0this,\u00C2\u00A0we\u00C2\u00A0observed\u00C2\u00A0major\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 strength\u00C2\u00A0 and\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 peripheral\u00C2\u00A0 versus\u00C2\u00A0 tumor\u00C2\u00A0 tissues.\u00C2\u00A0 For\u00C2\u00A0 example,\u00C2\u00A0 we\u00C2\u00A0 found\u00C2\u00A0 that,\u00C2\u00A0 unlike\u00C2\u00A0 peritoneal\u00C2\u00A0 and\u00C2\u00A0 pulmonary\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 TAM\u00E2\u0080\u0090 mediated\u00C2\u00A0 immune\u00C2\u00A0suppression\u00C2\u00A0could\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0reversed\u00C2\u00A0by\u00C2\u00A0NAC,\u00C2\u00A0or\u00C2\u00A0any\u00C2\u00A0other\u00C2\u00A0 inhibitors\u00C2\u00A0 that\u00C2\u00A0we\u00C2\u00A0 tested\u00C2\u00A0 in\u00C2\u00A0 our\u00C2\u00A0 assay.\u00C2\u00A0 These\u00C2\u00A0 findings\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0multiple\u00C2\u00A0 studies\u00C2\u00A0 that\u00C2\u00A0 have\u00C2\u00A0reported\u00C2\u00A0significant\u00C2\u00A0functional\u00C2\u00A0differences\u00C2\u00A0between\u00C2\u00A0cells\u00C2\u00A0located\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0and\u00C2\u00A0 periphery\u00C2\u00A0(Corzo\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02010).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 well\u00C2\u00A0 established\u00C2\u00A0 that\u00C2\u00A0 cancer\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 systemic\u00C2\u00A0 disease,\u00C2\u00A0 inducing\u00C2\u00A0 wide\u00E2\u0080\u0090spread\u00C2\u00A0 immune\u00C2\u00A0 deficiency\u00C2\u00A0 in\u00C2\u00A0 addition\u00C2\u00A0 to\u00C2\u00A0 local\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 (Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009).\u00C2\u00A0 However,\u00C2\u00A0 while\u00C2\u00A0 much\u00C2\u00A0 emphasis\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 placed\u00C2\u00A0 on\u00C2\u00A0 elucidating\u00C2\u00A0 the\u00C2\u00A0 roles\u00C2\u00A0 of\u00C2\u00A0 TAMs\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 progression,\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 peripheral\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 remains\u00C2\u00A0 poorly\u00C2\u00A0 understood\u00C2\u00A0 (Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0Our\u00C2\u00A0data\u00C2\u00A0clearly\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0certain\u00C2\u00A0tumors\u00C2\u00A0 changes\u00C2\u00A0 the\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 peripheral\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 increasing\u00C2\u00A0 their\u00C2\u00A0 suppressive\u00C2\u00A0 capabilities.\u00C2\u00A0 Studies\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0Lopez\u00C2\u00A0laboratory\u00C2\u00A0investigating\u00C2\u00A0the\u00C2\u00A0phenotype\u00C2\u00A0of\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0from\u00C2\u00A0mice\u00C2\u00A0bearing\u00C2\u00A0 D1\u00E2\u0080\u0090DMBA\u00E2\u0080\u00903\u00C2\u00A0mammary\u00C2\u00A0 tumors\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 cells\u00C2\u00A0 exhibit\u00C2\u00A0 decreased\u00C2\u00A0 APC\u00C2\u00A0 ability\u00C2\u00A0 (Watson\u00C2\u00A0 and\u00C2\u00A0Lopez,\u00C2\u00A0 1995)\u00C2\u00A0 and\u00C2\u00A0produce\u00C2\u00A0 lower\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 IL\u00E2\u0080\u00901\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u00906,\u00C2\u00A0 IL\u00E2\u0080\u009012,\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1,\u00C2\u00A0NO,\u00C2\u00A0 CCL2,\u00C2\u00A0and\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0(Dinapoli\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01996;\u00C2\u00A0Handel\u00E2\u0080\u0090Fernandez\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A01997;\u00C2\u00A0Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0Recently,\u00C2\u00A0they\u00C2\u00A0published\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0decreased\u00C2\u00A0pro\u00E2\u0080\u0090 inflammatory\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0PM\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0due,\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0in\u00C2\u00A0part,\u00C2\u00A0to\u00C2\u00A0lower\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0 NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0and\u00C2\u00A0CCAAT/enhancer\u00C2\u00A0binding\u00C2\u00A0protein\u00C2\u00A0 (C/EBP)\u00C2\u00A0TFs\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 cells,\u00C2\u00A0which\u00C2\u00A0 cannot\u00C2\u00A0be\u00C2\u00A0 classified\u00C2\u00A0as\u00C2\u00A0 either\u00C2\u00A0M1\u00C2\u00A0or\u00C2\u00A0M2,\u00C2\u00A0 are\u00C2\u00A0more\u00C2\u00A0 sensitive\u00C2\u00A0 to\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0express\u00C2\u00A0 lower\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0markers\u00C2\u00A0 (i.e.\u00C2\u00A0 F4/80,\u00C2\u00A0CD11b,\u00C2\u00A0CD68,\u00C2\u00A0CD116).\u00C2\u00A0 (Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2005;\u00C2\u00A0Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0These\u00C2\u00A0studies\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0Lopez\u00C2\u00A0laboratory\u00C2\u00A0provide\u00C2\u00A0a\u00C2\u00A0 mechanistic\u00C2\u00A0basis\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0tumors\u00C2\u00A0on\u00C2\u00A0peripheral\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0useful\u00C2\u00A0to\u00C2\u00A0 perform\u00C2\u00A0 these\u00C2\u00A0 experiments\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 to\u00C2\u00A0 reveal\u00C2\u00A0 whether\u00C2\u00A0 these\u00C2\u00A0 mechanisms\u00C2\u00A0 are\u00C2\u00A0 consistent\u00C2\u00A0 between\u00C2\u00A0 different\u00C2\u00A0mammary\u00C2\u00A0 tumor\u00C2\u00A0models,\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 to\u00C2\u00A0 explore\u00C2\u00A0the\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 156 As\u00C2\u00A0 well,\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 5\u00C2\u00A0 we\u00C2\u00A0 show\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 first\u00C2\u00A0 time\u00C2\u00A0 that\u00C2\u00A0 ATRA\u00C2\u00A0 treatment,\u00C2\u00A0 which\u00C2\u00A0 induces\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 MDSCs\u00C2\u00A0 to\u00C2\u00A0 terminally\u00C2\u00A0 differentiated\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 DCs,\u00C2\u00A0 enhances\u00C2\u00A0metastatic\u00C2\u00A0growth.\u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0are\u00C2\u00A0particularly\u00C2\u00A0intriguing\u00C2\u00A0given\u00C2\u00A0that\u00C2\u00A0ATRA\u00C2\u00A0 has\u00C2\u00A0been\u00C2\u00A0proposed\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0possible\u00C2\u00A0cancer\u00C2\u00A0therapy\u00C2\u00A0because\u00C2\u00A0of\u00C2\u00A0its\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0decrease\u00C2\u00A0MDSC\u00C2\u00A0 levels\u00C2\u00A0 (Kusmartsev\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008).\u00C2\u00A0 ATRA\u00C2\u00A0 is\u00C2\u00A0 currently\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 treat\u00C2\u00A0APL\u00C2\u00A0 in\u00C2\u00A0 combination\u00C2\u00A0 with\u00C2\u00A0chemotherapy\u00C2\u00A0(Sanz\u00C2\u00A0and\u00C2\u00A0Lo\u00E2\u0080\u0090Coco,\u00C2\u00A02011);\u00C2\u00A0however,\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0difference\u00C2\u00A0is\u00C2\u00A0that,\u00C2\u00A0in\u00C2\u00A0APL,\u00C2\u00A0 ATRA\u00C2\u00A0targets\u00C2\u00A0the\u00C2\u00A0malignant\u00C2\u00A0cells\u00C2\u00A0directly,\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0acting\u00C2\u00A0indirectly\u00C2\u00A0through\u00C2\u00A0MDSCs.\u00C2\u00A0 Our\u00C2\u00A0results\u00C2\u00A0suggest\u00C2\u00A0that,\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0in\u00C2\u00A0certain\u00C2\u00A0tumor\u00C2\u00A0models,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0promote\u00C2\u00A0metastasis\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0 greater\u00C2\u00A0 degree\u00C2\u00A0 than\u00C2\u00A0 MDSCs.\u00C2\u00A0 Although\u00C2\u00A0 this\u00C2\u00A0 finding\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 our\u00C2\u00A0 data\u00C2\u00A0 demonstrating\u00C2\u00A0 that,\u00C2\u00A0 in\u00C2\u00A0 vitro,\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 suppress\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 degree\u00C2\u00A0 than\u00C2\u00A0 MDSCs,\u00C2\u00A0 these\u00C2\u00A0 experiments\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 sufficient\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 causality.\u00C2\u00A0 \u00C2\u00A0 Related\u00C2\u00A0 to\u00C2\u00A0 this,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 substantial\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 can\u00C2\u00A0 promote\u00C2\u00A0 metastasis\u00C2\u00A0 via\u00C2\u00A0 non\u00E2\u0080\u0090 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0 including\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 IL\u00E2\u0080\u00901,\u00C2\u00A0 TNF,\u00C2\u00A0 IL\u00E2\u0080\u00906,\u00C2\u00A0 VEGF,\u00C2\u00A0 and\u00C2\u00A0 proteases\u00C2\u00A0 that\u00C2\u00A0 breakdown\u00C2\u00A0 the\u00C2\u00A0 ECM\u00C2\u00A0 and\u00C2\u00A0 aid\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 escape\u00C2\u00A0 and\u00C2\u00A0migration\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 cathepsins,\u00C2\u00A0MMPs,\u00C2\u00A0and\u00C2\u00A0serine\u00C2\u00A0proteases\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Mantovani\u00C2\u00A0and\u00C2\u00A0Sica,\u00C2\u00A02010;\u00C2\u00A0 Qian\u00C2\u00A0and\u00C2\u00A0Pollard,\u00C2\u00A02010).\u00C2\u00A0On\u00C2\u00A0 the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0 to\u00C2\u00A0promote\u00C2\u00A0 carcinogenesis\u00C2\u00A0via\u00C2\u00A0multiple\u00C2\u00A0 immune\u00C2\u00A0regulatory\u00C2\u00A0mechanisms,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0IL\u00E2\u0080\u0090 10,\u00C2\u00A0PGE2,\u00C2\u00A0and/or\u00C2\u00A0TGF\u00E2\u0080\u0090\u00CE\u00B2\u00C2\u00A0(Kuang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Torroella\u00E2\u0080\u0090Kouri\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009),\u00C2\u00A0upregulation\u00C2\u00A0 of\u00C2\u00A0PD\u00E2\u0080\u00901L\u00C2\u00A0on\u00C2\u00A0monocytes\u00C2\u00A0(Kuang\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009),\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0Tregs\u00C2\u00A0via\u00C2\u00A0CCL22\u00C2\u00A0production\u00C2\u00A0 (Curiel\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2004),\u00C2\u00A0 and\u00C2\u00A0based\u00C2\u00A0 on\u00C2\u00A0 our\u00C2\u00A0 results,\u00C2\u00A0 ROS\u00C2\u00A0production.\u00C2\u00A0 Given\u00C2\u00A0 the\u00C2\u00A0 key\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 in\u00C2\u00A0 fostering\u00C2\u00A0 tumorigenesis,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 possible\u00C2\u00A0 that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 promote\u00C2\u00A0 4T1\u00C2\u00A0 metastatic\u00C2\u00A0 growth,\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 in\u00C2\u00A0 part,\u00C2\u00A0 via\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 T\u00C2\u00A0 cell\u00E2\u0080\u0090mediated\u00C2\u00A0 immunity.\u00C2\u00A0Further\u00C2\u00A0experiments\u00C2\u00A0using\u00C2\u00A0T\u00C2\u00A0 cell\u00E2\u0080\u0090deficient\u00C2\u00A0mice\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0helpful\u00C2\u00A0 to\u00C2\u00A0 test\u00C2\u00A0 this\u00C2\u00A0 hypothesis;\u00C2\u00A0 however,\u00C2\u00A0 initial\u00C2\u00A0 studies\u00C2\u00A0 comparing\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0wild\u00E2\u0080\u0090type\u00C2\u00A0 and\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 deficient\u00C2\u00A0 mice\u00C2\u00A0 will\u00C2\u00A0 be\u00C2\u00A0 required\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0 the\u00C2\u00A0 validity\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0approach.\u00C2\u00A0Another\u00C2\u00A0question\u00C2\u00A0that\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0addressed\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0DCs\u00C2\u00A0 in\u00C2\u00A0 ATRA\u00E2\u0080\u0090treated\u00C2\u00A0 mice.\u00C2\u00A0 Studies\u00C2\u00A0 analyzing\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 proportions\u00C2\u00A0 and\u00C2\u00A0 immunosuppressive\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0 DCs\u00C2\u00A0 in\u00C2\u00A0 4T1\u00C2\u00A0 mice\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 or\u00C2\u00A0 without\u00C2\u00A0 ATRA,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 studies\u00C2\u00A0 specifically\u00C2\u00A0 targeting\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 or\u00C2\u00A0 DCs,\u00C2\u00A0 should\u00C2\u00A0 be\u00C2\u00A0 performed\u00C2\u00A0 to\u00C2\u00A0 clarify\u00C2\u00A0the\u00C2\u00A0distinct\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0metastasis.\u00C2\u00A0\u00C2\u00A0 157 While\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0demonstrated\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0enhance\u00C2\u00A0metastatic\u00C2\u00A0growth,\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0not\u00C2\u00A0 investigated\u00C2\u00A0 at\u00C2\u00A0which\u00C2\u00A0 stage\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 are\u00C2\u00A0 involved.\u00C2\u00A0Metastasis\u00C2\u00A0 requires\u00C2\u00A0 a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 steps:\u00C2\u00A0 release\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 primary\u00C2\u00A0 tumor,\u00C2\u00A0migration\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 circulatory\u00C2\u00A0 or\u00C2\u00A0 lymphatic\u00C2\u00A0system,\u00C2\u00A0extravasation\u00C2\u00A0in\u00C2\u00A0distant\u00C2\u00A0tissues,\u00C2\u00A0and\u00C2\u00A0subsequent\u00C2\u00A0survival\u00C2\u00A0and\u00C2\u00A0growth\u00C2\u00A0 (Qian\u00C2\u00A0 and\u00C2\u00A0Pollard,\u00C2\u00A0 2010).\u00C2\u00A0Recently,\u00C2\u00A0 important\u00C2\u00A0work\u00C2\u00A0has\u00C2\u00A0 revealed\u00C2\u00A0 a\u00C2\u00A0direct\u00C2\u00A0 and\u00C2\u00A0 critical\u00C2\u00A0 role\u00C2\u00A0 for\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 extravasation,\u00C2\u00A0 survival,\u00C2\u00A0 and\u00C2\u00A0metastatic\u00C2\u00A0 growth\u00C2\u00A0 (Qian\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009).\u00C2\u00A0Moreover,\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0likely\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0establishment\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0pre\u00E2\u0080\u0090metastatic\u00C2\u00A0niche,\u00C2\u00A0 since\u00C2\u00A0 they\u00C2\u00A0are\u00C2\u00A0recruited\u00C2\u00A0 to\u00C2\u00A0 future\u00C2\u00A0sites\u00C2\u00A0of\u00C2\u00A0metastasis\u00C2\u00A0prior\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0arrival\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 greatly\u00C2\u00A0 enhance\u00C2\u00A0 metastatic\u00C2\u00A0 seeding\u00C2\u00A0 and\u00C2\u00A0 growth\u00C2\u00A0 (Qian\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009).\u00C2\u00A0 However,\u00C2\u00A0 the\u00C2\u00A0 precise\u00C2\u00A0 timing,\u00C2\u00A0 cell\u00C2\u00A0 types,\u00C2\u00A0 and\u00C2\u00A0 mechanisms\u00C2\u00A0 involved\u00C2\u00A0 are\u00C2\u00A0 subjects\u00C2\u00A0 of\u00C2\u00A0 current\u00C2\u00A0debate\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0investigation\u00C2\u00A0(Dawson\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009;\u00C2\u00A0Mantovani\u00C2\u00A0and\u00C2\u00A0Sica,\u00C2\u00A02010;\u00C2\u00A0 Psaila\u00C2\u00A0and\u00C2\u00A0Lyden,\u00C2\u00A02009;\u00C2\u00A0Qian\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02009).\u00C2\u00A0 \u00C2\u00A0 While\u00C2\u00A0 we\u00C2\u00A0 successfully\u00C2\u00A0 elucidated\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 pathways\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 immunosuppressive\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells,\u00C2\u00A0 many\u00C2\u00A0 questions\u00C2\u00A0 still\u00C2\u00A0 remain.\u00C2\u00A0 In\u00C2\u00A0 particular,\u00C2\u00A0 we\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 address\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 tumor\u00E2\u0080\u0090derived\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 the\u00C2\u00A0 accumulation\u00C2\u00A0 and\u00C2\u00A0 function\u00C2\u00A0 of\u00C2\u00A0 M\u00CF\u0086s.\u00C2\u00A0 Previous\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 demonstrated\u00C2\u00A0 roles\u00C2\u00A0 for\u00C2\u00A0 a\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 factors\u00C2\u00A0 (i.e.\u00C2\u00A0 IL\u00E2\u0080\u00901\u00CE\u00B2,\u00C2\u00A0 IL\u00E2\u0080\u00906\u00C2\u00A0 (Bunt\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2006;\u00C2\u00A0 Bunt\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007),\u00C2\u00A0 S100A8/A9\u00C2\u00A0 proteins\u00C2\u00A0 (Sinha\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2008),\u00C2\u00A0 PGE2\u00C2\u00A0 (Eruslanov\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010;\u00C2\u00A0 Sinha\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2007b),\u00C2\u00A0VEGF\u00C2\u00A0(Fricke\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02007;\u00C2\u00A0Gabrilovich\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A01998),\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0(Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011),\u00C2\u00A0 GM\u00E2\u0080\u0090CSF\u00C2\u00A0 (Dolcetti\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010)),\u00C2\u00A0 chemokines\u00C2\u00A0 (i.e.\u00C2\u00A0 CCL2\u00C2\u00A0 (Laoui\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011;\u00C2\u00A0 Qian\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011)),\u00C2\u00A0and\u00C2\u00A0signaling\u00C2\u00A0pathway\u00C2\u00A0components\u00C2\u00A0(i.e.\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB,\u00C2\u00A0STAT\u00E2\u0080\u00901,\u00C2\u00A0STAT\u00E2\u0080\u00903,\u00C2\u00A0C\u00E2\u0080\u0090EBP\u00CE\u00B2,\u00C2\u00A0HIF\u00E2\u0080\u00901\u00CE\u00B1\u00C2\u00A0 (Karin\u00C2\u00A0and\u00C2\u00A0Greten,\u00C2\u00A02005;\u00C2\u00A0Laoui\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Mantovani\u00C2\u00A0and\u00C2\u00A0Sica,\u00C2\u00A02010))\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0murine\u00C2\u00A0 and\u00C2\u00A0human\u00C2\u00A0cancers.\u00C2\u00A0In\u00C2\u00A0particular,\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0CCL2\u00E2\u0080\u0090CCR2\u00C2\u00A0axis\u00C2\u00A0(Qian\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A02011;\u00C2\u00A0Si\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010)\u00C2\u00A0 and\u00C2\u00A0M\u00E2\u0080\u0090CSF\u00C2\u00A0 (Abraham\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2010;\u00C2\u00A0 Aharinejad\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009;\u00C2\u00A0 Kubota\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2009;\u00C2\u00A0 Lin\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2001)\u00C2\u00A0 have\u00C2\u00A0 proven\u00C2\u00A0 particularly\u00C2\u00A0 effective\u00C2\u00A0 in\u00C2\u00A0 limiting\u00C2\u00A0 the\u00C2\u00A0 cancer\u00E2\u0080\u0090 promoting\u00C2\u00A0 functions\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cells.\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 likely\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0specific\u00C2\u00A0 factors\u00C2\u00A0 involved\u00C2\u00A0will\u00C2\u00A0 vary\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0tumor\u00C2\u00A0types,\u00C2\u00A0disease\u00C2\u00A0stages,\u00C2\u00A0and\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0person\u00E2\u0080\u0090to\u00E2\u0080\u0090person\u00C2\u00A0level.\u00C2\u00A0Given\u00C2\u00A0 the\u00C2\u00A0 immense\u00C2\u00A0 therapeutic\u00C2\u00A0 potential\u00C2\u00A0 of\u00C2\u00A0 targeting\u00C2\u00A0 these\u00C2\u00A0 pathways\u00C2\u00A0 using\u00C2\u00A0 small\u00C2\u00A0molecule\u00C2\u00A0 inhibitors\u00C2\u00A0 it\u00C2\u00A0would\u00C2\u00A0 be\u00C2\u00A0 extremely\u00C2\u00A0beneficial\u00C2\u00A0 to\u00C2\u00A0 develop\u00C2\u00A0 an\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0 assay\u00C2\u00A0 that\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0 158 used\u00C2\u00A0 to\u00C2\u00A0 screen\u00C2\u00A0 for\u00C2\u00A0 efficacy\u00C2\u00A0 (i.e.\u00C2\u00A0 reduction\u00C2\u00A0 in\u00C2\u00A0 immunosuppressive\u00C2\u00A0 cell\u00C2\u00A0 development\u00C2\u00A0 and/or\u00C2\u00A0function)\u00C2\u00A0on\u00C2\u00A0an\u00C2\u00A0individual\u00C2\u00A0basis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0 the\u00C2\u00A0work\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 each\u00C2\u00A0 chapter\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 thesis\u00C2\u00A0 reveals\u00C2\u00A0 individual\u00C2\u00A0 interesting\u00C2\u00A0and\u00C2\u00A0novel\u00C2\u00A0 findings,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 some\u00C2\u00A0 common\u00C2\u00A0 themes\u00C2\u00A0 that\u00C2\u00A0 exist\u00C2\u00A0 throughout\u00C2\u00A0 the\u00C2\u00A0 entirety\u00C2\u00A0 of\u00C2\u00A0 our\u00C2\u00A0 studies.\u00C2\u00A0 Firstly,\u00C2\u00A0 the\u00C2\u00A0 regulatory\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 fixed,\u00C2\u00A0but\u00C2\u00A0vary\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0exposed.\u00C2\u00A0Examples\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0include\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086\u00C2\u00A0immunosuppression\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0TLR\u00C2\u00A0agonists,\u00C2\u00A0regulation\u00C2\u00A0 of\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0 by\u00C2\u00A0 albumin,\u00C2\u00A0 and\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 M\u00CF\u0086\u00C2\u00A0 and\u00C2\u00A0 MDSC\u00C2\u00A0 immunosuppression\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0 tumor\u00E2\u0080\u0090derived\u00C2\u00A0 factors.\u00C2\u00A0 Importantly,\u00C2\u00A0 the\u00C2\u00A0mechanisms\u00C2\u00A0 that\u00C2\u00A0control\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0immunosuppression\u00C2\u00A0are\u00C2\u00A0complex\u00C2\u00A0and\u00C2\u00A0precise,\u00C2\u00A0i.e.\u00C2\u00A0not\u00C2\u00A0all\u00C2\u00A0TLRs\u00C2\u00A0 or\u00C2\u00A0tumors\u00C2\u00A0have\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0function.\u00C2\u00A0While\u00C2\u00A0the\u00C2\u00A0studies\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0 this\u00C2\u00A0 thesis\u00C2\u00A0 reveal\u00C2\u00A0 some\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0mechanisms\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00E2\u0080\u0090induced\u00C2\u00A0 immune\u00C2\u00A0 suppression,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 great\u00C2\u00A0 deal\u00C2\u00A0 that\u00C2\u00A0 we\u00C2\u00A0 still\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 understand;\u00C2\u00A0 further\u00C2\u00A0 elucidation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00C2\u00A0 immunosuppression\u00C2\u00A0 is\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0important\u00C2\u00A0challenges\u00C2\u00A0in\u00C2\u00A0our\u00C2\u00A0field.\u00C2\u00A0Secondly,\u00C2\u00A0our\u00C2\u00A0findings\u00C2\u00A0emphasize\u00C2\u00A0the\u00C2\u00A0enormous\u00C2\u00A0 heterogeneity\u00C2\u00A0 of\u00C2\u00A0myeloid\u00C2\u00A0 cells.\u00C2\u00A0 The\u00C2\u00A0 phenotypic\u00C2\u00A0 and\u00C2\u00A0 functional\u00C2\u00A0 characteristics\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 and\u00C2\u00A0MDSCs\u00C2\u00A0 are\u00C2\u00A0 incredibly\u00C2\u00A0 diverse\u00C2\u00A0 and\u00C2\u00A0vary\u00C2\u00A0depending\u00C2\u00A0 on\u00C2\u00A0many\u00C2\u00A0 factors\u00C2\u00A0 (e.g.\u00C2\u00A0 cell\u00C2\u00A0 lineage,\u00C2\u00A0maturation\u00C2\u00A0state,\u00C2\u00A0activation\u00C2\u00A0state,\u00C2\u00A0 \u00C2\u00A0duration\u00C2\u00A0and/or\u00C2\u00A0strength\u00C2\u00A0of\u00C2\u00A0stimulation\u00C2\u00A0by\u00C2\u00A0 specific\u00C2\u00A0 factors,\u00C2\u00A0 cell\u00E2\u0080\u0090cell\u00C2\u00A0 interactions,\u00C2\u00A0 tissue\u00C2\u00A0 location,\u00C2\u00A0 inflammatory\u00C2\u00A0 status,\u00C2\u00A0 etc.).\u00C2\u00A0 Additional\u00C2\u00A0 studies\u00C2\u00A0 characterizing\u00C2\u00A0 the\u00C2\u00A0 heterogeneity\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 identifying\u00C2\u00A0 meaningful\u00C2\u00A0ways\u00C2\u00A0to\u00C2\u00A0define\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0essential\u00C2\u00A0 for\u00C2\u00A0advancing\u00C2\u00A0research.\u00C2\u00A0Finally,\u00C2\u00A0 the\u00C2\u00A0findings\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0thesis\u00C2\u00A0demonstrate\u00C2\u00A0the\u00C2\u00A0important\u00C2\u00A0regulatory\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0 M\u00CF\u0086s,\u00C2\u00A0 which\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 largely\u00C2\u00A0 overlooked\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 field\u00C2\u00A0 to\u00C2\u00A0 date.\u00C2\u00A0 Although\u00C2\u00A0 MDSCs\u00C2\u00A0 are\u00C2\u00A0 considered\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0main\u00C2\u00A0 contributors\u00C2\u00A0 to\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 in\u00C2\u00A0 cancer,\u00C2\u00A0 our\u00C2\u00A0work\u00C2\u00A0 reveals\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0potent\u00C2\u00A0immune\u00C2\u00A0suppressors\u00C2\u00A0than\u00C2\u00A0MDSCs\u00C2\u00A0and\u00C2\u00A0suggests\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0 key\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 peripheral\u00C2\u00A0 tolerance\u00C2\u00A0 and\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 immune\u00C2\u00A0 suppression.\u00C2\u00A0 While\u00C2\u00A0we\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0dispute\u00C2\u00A0 the\u00C2\u00A0 influence\u00C2\u00A0of\u00C2\u00A0MDSCs,\u00C2\u00A0we\u00C2\u00A0propose\u00C2\u00A0 that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0are\u00C2\u00A0 important\u00C2\u00A0 regulatory\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0explored\u00C2\u00A0as\u00C2\u00A0potential\u00C2\u00A0therapeutic\u00C2\u00A0targets.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 159 Taken\u00C2\u00A0together,\u00C2\u00A0the\u00C2\u00A0findings\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0thesis\u00C2\u00A0provide\u00C2\u00A0further\u00C2\u00A0evidence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0important\u00C2\u00A0regulatory\u00C2\u00A0roles\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0maintenance\u00C2\u00A0of\u00C2\u00A0homeostasis\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0cancer,\u00C2\u00A0 and\u00C2\u00A0reveal\u00C2\u00A0new\u00C2\u00A0insights\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0control\u00C2\u00A0the\u00C2\u00A0suppressive\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0 cells.\u00C2\u00A0The\u00C2\u00A0relationship\u00C2\u00A0between\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0and\u00C2\u00A0tumors\u00C2\u00A0is\u00C2\u00A0complex.\u00C2\u00A0There\u00C2\u00A0is\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0M\u00CF\u0086s\u00C2\u00A0 play\u00C2\u00A0both\u00C2\u00A0positive\u00C2\u00A0 and\u00C2\u00A0negative\u00C2\u00A0 roles\u00C2\u00A0 and\u00C2\u00A0participate\u00C2\u00A0 in\u00C2\u00A0 all\u00C2\u00A0 three\u00C2\u00A0 stages\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 cancer\u00C2\u00A0 immunoediting\u00C2\u00A0 process\u00C2\u00A0 (i.e.\u00C2\u00A0 elimination,\u00C2\u00A0 equilibrium,\u00C2\u00A0 and\u00C2\u00A0 escape)\u00C2\u00A0 (Qian\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A0 2010;\u00C2\u00A0 Schreiber\u00C2\u00A0 et\u00C2\u00A0 al.,\u00C2\u00A0 2011).\u00C2\u00A0 However,\u00C2\u00A0 data\u00C2\u00A0 from\u00C2\u00A0 both\u00C2\u00A0 animal\u00C2\u00A0models\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 clinic\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 most\u00C2\u00A0 cases\u00C2\u00A0 M\u00CF\u0086s\u00C2\u00A0 promote\u00C2\u00A0 carcinogenesis,\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 fact,\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0 extreme\u00C2\u00A0 diversity\u00C2\u00A0 and\u00C2\u00A0 plasticity,\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 all\u00C2\u00A0 phases\u00C2\u00A0 of\u00C2\u00A0 tumorigenesis\u00C2\u00A0 (Qian\u00C2\u00A0 and\u00C2\u00A0 Pollard,\u00C2\u00A02010).\u00C2\u00A0Further\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0molecular\u00C2\u00A0and\u00C2\u00A0functional\u00C2\u00A0heterogeneity\u00C2\u00A0 of\u00C2\u00A0tumor\u00E2\u0080\u0090induced\u00C2\u00A0myeloid\u00C2\u00A0cells,\u00C2\u00A0using\u00C2\u00A0studies\u00C2\u00A0analogous\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0presented\u00C2\u00A0herein,\u00C2\u00A0will\u00C2\u00A0 be\u00C2\u00A0 critical\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 targeted\u00C2\u00A0 therapies.\u00C2\u00A0 Nevertheless,\u00C2\u00A0 the\u00C2\u00A0 potent\u00C2\u00A0 immunosuppressive\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0M\u00CF\u0086s,\u00C2\u00A0combined\u00C2\u00A0with\u00C2\u00A0our\u00C2\u00A0enhanced\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 factors\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 these\u00C2\u00A0 functions,\u00C2\u00A0make\u00C2\u00A0 them\u00C2\u00A0extremely\u00C2\u00A0 attractive\u00C2\u00A0 therapeutic\u00C2\u00A0 targets.\u00C2\u00A0Although\u00C2\u00A0these\u00C2\u00A0findings\u00C2\u00A0will\u00C2\u00A0need\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0validated\u00C2\u00A0in\u00C2\u00A0patients,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0our\u00C2\u00A0hope\u00C2\u00A0that\u00C2\u00A0 the\u00C2\u00A0 findings\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 thesis\u00C2\u00A0 will\u00C2\u00A0 inform\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 enhanced\u00C2\u00A0 immunotherapies\u00C2\u00A0 to\u00C2\u00A0 treat\u00C2\u00A0human\u00C2\u00A0disease.\u00C2\u00A0 Strategies\u00C2\u00A0 that\u00C2\u00A0 inhibit\u00C2\u00A0 immune\u00C2\u00A0suppression\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0utilized\u00C2\u00A0to\u00C2\u00A0promote\u00C2\u00A0anti\u00E2\u0080\u0090tumor\u00C2\u00A0immunity\u00C2\u00A0and\u00C2\u00A0also\u00C2\u00A0to\u00C2\u00A0treat\u00C2\u00A0infectious\u00C2\u00A0diseases.\u00C2\u00A0 Moreover,\u00C2\u00A0 therapies\u00C2\u00A0 that\u00C2\u00A0 enhance\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0harnessed\u00C2\u00A0 to\u00C2\u00A0 reduce\u00C2\u00A0 autoimmune\u00C2\u00A0disease\u00C2\u00A0and\u00C2\u00A0increase\u00C2\u00A0transplantation\u00C2\u00A0tolerance.\u00C2\u00A0In\u00C2\u00A0summary,\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 mononuclear\u00C2\u00A0 phagocytes\u00C2\u00A0 to\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 immunity\u00C2\u00A0 and\u00C2\u00A0 tolerance,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 promotion\u00C2\u00A0 and\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 responses,\u00C2\u00A0 emphasizes\u00C2\u00A0 their\u00C2\u00A0 considerable\u00C2\u00A0 therapeutic\u00C2\u00A0potential\u00C2\u00A0and\u00C2\u00A0highlights\u00C2\u00A0the\u00C2\u00A0importance\u00C2\u00A0of\u00C2\u00A0increasing\u00C2\u00A0our\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0factors\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0function\u00C2\u00A0under\u00C2\u00A0normal\u00C2\u00A0and\u00C2\u00A0neoplastic\u00C2\u00A0conditions.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 160 REFERENCES\u00C2\u00A0 Abraham,\u00C2\u00A0D.,\u00C2\u00A0K.\u00C2\u00A0Zins,\u00C2\u00A0M.\u00C2\u00A0Sioud,\u00C2\u00A0T.\u00C2\u00A0Lucas,\u00C2\u00A0R.\u00C2\u00A0Schafer,\u00C2\u00A0E.R.\u00C2\u00A0Stanley,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Aharinejad.\u00C2\u00A02010.\u00C2\u00A0 Stromal\u00C2\u00A0 cell\u00E2\u0080\u0090derived\u00C2\u00A0 CSF\u00E2\u0080\u00901\u00C2\u00A0 blockade\u00C2\u00A0 prolongs\u00C2\u00A0 xenograft\u00C2\u00A0 survival\u00C2\u00A0 of\u00C2\u00A0 CSF\u00E2\u0080\u00901\u00E2\u0080\u0090 negative\u00C2\u00A0neuroblastoma.\u00C2\u00A0Int\u00C2\u00A0J\u00C2\u00A0Cancer\u00C2\u00A0126:1339\u00E2\u0080\u00901352.\u00C2\u00A0 Aharinejad,\u00C2\u00A0S.,\u00C2\u00A0M.\u00C2\u00A0Sioud,\u00C2\u00A0T.\u00C2\u00A0Lucas,\u00C2\u00A0and\u00C2\u00A0D.\u00C2\u00A0Abraham.\u00C2\u00A02009.\u00C2\u00A0Targeting\u00C2\u00A0stromal\u00E2\u0080\u0090cancer\u00C2\u00A0cell\u00C2\u00A0 interactions\u00C2\u00A0with\u00C2\u00A0siRNAs.\u00C2\u00A0Methods\u00C2\u00A0Mol\u00C2\u00A0Biol\u00C2\u00A0487:243\u00E2\u0080\u0090266.\u00C2\u00A0 Akira,\u00C2\u00A0S.,\u00C2\u00A0and\u00C2\u00A0K.\u00C2\u00A0Takeda.\u00C2\u00A02004.\u00C2\u00A0Toll\u00E2\u0080\u0090like\u00C2\u00A0receptor\u00C2\u00A0signalling.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Immunol\u00C2\u00A04:499\u00E2\u0080\u0090511.\u00C2\u00A0 Albina,\u00C2\u00A0J.E.,\u00C2\u00A0J.A.\u00C2\u00A0Abate,\u00C2\u00A0and\u00C2\u00A0W.L.\u00C2\u00A0Henry,\u00C2\u00A0Jr.\u00C2\u00A01991.\u00C2\u00A0Nitric\u00C2\u00A0oxide\u00C2\u00A0production\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0 murine\u00C2\u00A0resident\u00C2\u00A0peritoneal\u00C2\u00A0macrophages\u00C2\u00A0to\u00C2\u00A0suppress\u00C2\u00A0mitogen\u00E2\u0080\u0090stimulated\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0 proliferation.\u00C2\u00A0Role\u00C2\u00A0of\u00C2\u00A0IFN\u00E2\u0080\u0090gamma\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0nitric\u00C2\u00A0oxide\u00E2\u0080\u0090synthesizing\u00C2\u00A0 pathway.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0147:144\u00E2\u0080\u0090148.\u00C2\u00A0 Alcon,\u00C2\u00A0V.L.,\u00C2\u00A0C.\u00C2\u00A0Luther,\u00C2\u00A0D.\u00C2\u00A0Balce,\u00C2\u00A0and\u00C2\u00A0F.\u00C2\u00A0Takei.\u00C2\u00A02009.\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0co\u00E2\u0080\u0090receptor\u00C2\u00A0CD72\u00C2\u00A0is\u00C2\u00A0expressed\u00C2\u00A0 on\u00C2\u00A0 NK\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 inhibits\u00C2\u00A0 IFN\u00E2\u0080\u0090gamma\u00C2\u00A0 production\u00C2\u00A0 but\u00C2\u00A0 not\u00C2\u00A0 cytotoxicity.\u00C2\u00A0 Eur\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A039:826\u00E2\u0080\u0090832.\u00C2\u00A0 Allison,\u00C2\u00A0 A.C.\u00C2\u00A0 1978.\u00C2\u00A0 Mechanisms\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 activated\u00C2\u00A0 macrophages\u00C2\u00A0 inhibit\u00C2\u00A0 lymphocyte\u00C2\u00A0 responses.\u00C2\u00A0Immunol\u00C2\u00A0Rev\u00C2\u00A040:3\u00E2\u0080\u009027.\u00C2\u00A0 Almand,\u00C2\u00A0B.,\u00C2\u00A0 J.I.\u00C2\u00A0Clark,\u00C2\u00A0E.\u00C2\u00A0Nikitina,\u00C2\u00A0 J.\u00C2\u00A0van\u00C2\u00A0Beynen,\u00C2\u00A0N.R.\u00C2\u00A0English,\u00C2\u00A0S.C.\u00C2\u00A0Knight,\u00C2\u00A0D.P.\u00C2\u00A0Carbone,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A0 2001.\u00C2\u00A0 Increased\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 immature\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 patients:\u00C2\u00A0 a\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 immunosuppression\u00C2\u00A0 in\u00C2\u00A0 cancer.\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 immunology\u00C2\u00A0166:678\u00E2\u0080\u0090689.\u00C2\u00A0 Antignano,\u00C2\u00A0F.,\u00C2\u00A0M.\u00C2\u00A0Hamilton,\u00C2\u00A0S.\u00C2\u00A0Patterson,\u00C2\u00A0V.\u00C2\u00A0Ho,\u00C2\u00A0C.\u00C2\u00A0Cohen,\u00C2\u00A0M.K.\u00C2\u00A0Levings,\u00C2\u00A0 and\u00C2\u00A0G.\u00C2\u00A0Krystal.\u00C2\u00A0 2011.\u00C2\u00A0SHIP\u00E2\u0080\u0090Deficient\u00C2\u00A0Dendritic\u00C2\u00A0Cells,\u00C2\u00A0Unlike\u00C2\u00A0Wild\u00C2\u00A0Type\u00C2\u00A0Dendritic\u00C2\u00A0Cells,\u00C2\u00A0Suppress\u00C2\u00A0 T\u00C2\u00A0 Cell\u00C2\u00A0 Proliferation\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 Nitric\u00C2\u00A0 Oxide\u00E2\u0080\u0090Independent\u00C2\u00A0 Mechanism.\u00C2\u00A0 PLoS\u00C2\u00A0 One\u00C2\u00A0 6:e21893.\u00C2\u00A0 Aslakson,\u00C2\u00A0C.J.,\u00C2\u00A0and\u00C2\u00A0F.R.\u00C2\u00A0Miller.\u00C2\u00A01992.\u00C2\u00A0Selective\u00C2\u00A0events\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0metastatic\u00C2\u00A0process\u00C2\u00A0defined\u00C2\u00A0 by\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 sequential\u00C2\u00A0 dissemination\u00C2\u00A0 of\u00C2\u00A0 subpopulations\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 mouse\u00C2\u00A0 mammary\u00C2\u00A0tumor.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A052:1399\u00E2\u0080\u00901405.\u00C2\u00A0 Barnes,\u00C2\u00A0D.,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Sato.\u00C2\u00A01980.\u00C2\u00A0Serum\u00E2\u0080\u0090free\u00C2\u00A0cell\u00C2\u00A0culture:\u00C2\u00A0a\u00C2\u00A0unifying\u00C2\u00A0approach.\u00C2\u00A0Cell\u00C2\u00A022:649\u00E2\u0080\u0090 655.\u00C2\u00A0 Bastien,\u00C2\u00A0J.,\u00C2\u00A0and\u00C2\u00A0C.\u00C2\u00A0Rochette\u00E2\u0080\u0090Egly.\u00C2\u00A02004.\u00C2\u00A0Nuclear\u00C2\u00A0retinoid\u00C2\u00A0receptors\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0transcription\u00C2\u00A0 of\u00C2\u00A0retinoid\u00E2\u0080\u0090target\u00C2\u00A0genes.\u00C2\u00A0Gene\u00C2\u00A0328:1\u00E2\u0080\u009016.\u00C2\u00A0 Bettini,\u00C2\u00A0 M.,\u00C2\u00A0 and\u00C2\u00A0 D.A.\u00C2\u00A0 Vignali.\u00C2\u00A0 2009.\u00C2\u00A0 Regulatory\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 inhibitory\u00C2\u00A0 cytokines\u00C2\u00A0 in\u00C2\u00A0 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signaling\u00C2\u00A0pathway.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0160:5729\u00E2\u0080\u00905734.\u00C2\u00A0 Bingle,\u00C2\u00A0L.,\u00C2\u00A0N.J.\u00C2\u00A0Brown,\u00C2\u00A0and\u00C2\u00A0C.E.\u00C2\u00A0Lewis.\u00C2\u00A02002.\u00C2\u00A0The\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0tumour\u00E2\u0080\u0090associated\u00C2\u00A0macrophages\u00C2\u00A0 in\u00C2\u00A0 tumour\u00C2\u00A0 progression:\u00C2\u00A0 implications\u00C2\u00A0 for\u00C2\u00A0 new\u00C2\u00A0 anticancer\u00C2\u00A0 therapies.\u00C2\u00A0 J\u00C2\u00A0 Pathol\u00C2\u00A0 196:254\u00E2\u0080\u0090265.\u00C2\u00A0 Bogdan,\u00C2\u00A0C.\u00C2\u00A02001.\u00C2\u00A0Nitric\u00C2\u00A0oxide\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0response.\u00C2\u00A0Nat\u00C2\u00A0Immunol\u00C2\u00A02:907\u00E2\u0080\u0090916.\u00C2\u00A0 Brem\u00E2\u0080\u0090Exner,\u00C2\u00A0 B.G.,\u00C2\u00A0 C.\u00C2\u00A0 Sattler,\u00C2\u00A0 J.A.\u00C2\u00A0 Hutchinson,\u00C2\u00A0 G.E.\u00C2\u00A0 Koehl,\u00C2\u00A0 K.\u00C2\u00A0 Kronenberg,\u00C2\u00A0 S.\u00C2\u00A0 Farkas,\u00C2\u00A0 S.\u00C2\u00A0 Inoue,\u00C2\u00A0 C.\u00C2\u00A0 Blank,\u00C2\u00A0 S.J.\u00C2\u00A0 Knechtle,\u00C2\u00A0 H.J.\u00C2\u00A0 Schlitt,\u00C2\u00A0 F.\u00C2\u00A0 Fandrich,\u00C2\u00A0 and\u00C2\u00A0 E.K.\u00C2\u00A0 Geissler.\u00C2\u00A0 2008.\u00C2\u00A0 Macrophages\u00C2\u00A0 driven\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 state\u00C2\u00A0 of\u00C2\u00A0 activation\u00C2\u00A0 have\u00C2\u00A0 anti\u00E2\u0080\u0090inflammatory\u00C2\u00A0 properties\u00C2\u00A0in\u00C2\u00A0mice.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0180:335\u00E2\u0080\u0090349.\u00C2\u00A0 Bronte,\u00C2\u00A0 V.,\u00C2\u00A0 P.\u00C2\u00A0 Serafini,\u00C2\u00A0 A.\u00C2\u00A0 Mazzoni,\u00C2\u00A0 D.M.\u00C2\u00A0 Segal,\u00C2\u00A0 and\u00C2\u00A0 P.\u00C2\u00A0 Zanovello.\u00C2\u00A0 2003.\u00C2\u00A0 L\u00E2\u0080\u0090arginine\u00C2\u00A0 metabolism\u00C2\u00A0 in\u00C2\u00A0myeloid\u00C2\u00A0 cells\u00C2\u00A0 controls\u00C2\u00A0 T\u00E2\u0080\u0090lymphocyte\u00C2\u00A0 functions.\u00C2\u00A0Trends\u00C2\u00A0 Immunol\u00C2\u00A0 24:302\u00E2\u0080\u0090306.\u00C2\u00A0 Bunt,\u00C2\u00A0 S.K.,\u00C2\u00A0 P.\u00C2\u00A0 Sinha,\u00C2\u00A0 V.K.\u00C2\u00A0 Clements,\u00C2\u00A0 J.\u00C2\u00A0 Leips,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A0 2006.\u00C2\u00A0 Inflammation\u00C2\u00A0 induces\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 facilitate\u00C2\u00A0 tumor\u00C2\u00A0 progression.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0176:284\u00E2\u0080\u0090290.\u00C2\u00A0 Bunt,\u00C2\u00A0 S.K.,\u00C2\u00A0 L.\u00C2\u00A0 Yang,\u00C2\u00A0 P.\u00C2\u00A0 Sinha,\u00C2\u00A0 V.K.\u00C2\u00A0 Clements,\u00C2\u00A0 J.\u00C2\u00A0 Leips,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A0 2007.\u00C2\u00A0 Reduced\u00C2\u00A0inflammation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0microenvironment\u00C2\u00A0delays\u00C2\u00A0the\u00C2\u00A0accumulation\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 limits\u00C2\u00A0 tumor\u00C2\u00A0 progression.\u00C2\u00A0 Cancer\u00C2\u00A0 research\u00C2\u00A067:10019\u00E2\u0080\u009010026.\u00C2\u00A0 Burnet,\u00C2\u00A0M.\u00C2\u00A0 1957.\u00C2\u00A0 Cancer;\u00C2\u00A0 a\u00C2\u00A0 biological\u00C2\u00A0 approach.\u00C2\u00A0 I.\u00C2\u00A0 The\u00C2\u00A0 processes\u00C2\u00A0 of\u00C2\u00A0 control.\u00C2\u00A0Br\u00C2\u00A0Med\u00C2\u00A0 J\u00C2\u00A0 1:779\u00E2\u0080\u0090786.\u00C2\u00A0 Cesta,\u00C2\u00A0M.F.\u00C2\u00A02006.\u00C2\u00A0Normal\u00C2\u00A0structure,\u00C2\u00A0function,\u00C2\u00A0and\u00C2\u00A0histology\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0spleen.\u00C2\u00A0Toxicol\u00C2\u00A0Pathol\u00C2\u00A0 34:455\u00E2\u0080\u0090465.\u00C2\u00A0 Chitu,\u00C2\u00A0 V.,\u00C2\u00A0 and\u00C2\u00A0 E.R.\u00C2\u00A0 Stanley.\u00C2\u00A0 2006.\u00C2\u00A0 Colony\u00E2\u0080\u0090stimulating\u00C2\u00A0 factor\u00E2\u0080\u00901\u00C2\u00A0 in\u00C2\u00A0 immunity\u00C2\u00A0 and\u00C2\u00A0 inflammation.\u00C2\u00A0Curr\u00C2\u00A0Opin\u00C2\u00A0Immunol\u00C2\u00A018:39\u00E2\u0080\u009048.\u00C2\u00A0 Coghlin,\u00C2\u00A0C.,\u00C2\u00A0and\u00C2\u00A0G.I.\u00C2\u00A0Murray.\u00C2\u00A02010.\u00C2\u00A0Current\u00C2\u00A0and\u00C2\u00A0emerging\u00C2\u00A0concepts\u00C2\u00A0in\u00C2\u00A0tumour\u00C2\u00A0metastasis.\u00C2\u00A0 J\u00C2\u00A0Pathol\u00C2\u00A0222:1\u00E2\u0080\u009015.\u00C2\u00A0 Composto,\u00C2\u00A0 G.,\u00C2\u00A0 D.\u00C2\u00A0 Gonzalez,\u00C2\u00A0 A.\u00C2\u00A0 Bucknum,\u00C2\u00A0 D.\u00C2\u00A0 Silberman,\u00C2\u00A0 J.\u00C2\u00A0 Taylor,\u00C2\u00A0 M.\u00C2\u00A0 Kozlowski,\u00C2\u00A0 T.\u00C2\u00A0 Bloomfield,\u00C2\u00A0T.\u00C2\u00A0Bartlett,\u00C2\u00A0and\u00C2\u00A0J.\u00C2\u00A0Riggs.\u00C2\u00A02011.\u00C2\u00A0Peritoneal\u00C2\u00A0T\u00C2\u00A0lymphocyte\u00C2\u00A0regulation\u00C2\u00A0by\u00C2\u00A0 macrophages.\u00C2\u00A0Immunobiology\u00C2\u00A0216:256\u00E2\u0080\u0090264.\u00C2\u00A0 Condeelis,\u00C2\u00A0 J.,\u00C2\u00A0 and\u00C2\u00A0 J.W.\u00C2\u00A0 Pollard.\u00C2\u00A0 2006.\u00C2\u00A0 Macrophages:\u00C2\u00A0 obligate\u00C2\u00A0 partners\u00C2\u00A0 for\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 migration,\u00C2\u00A0invasion,\u00C2\u00A0and\u00C2\u00A0metastasis.\u00C2\u00A0Cell\u00C2\u00A0124:263\u00E2\u0080\u0090266.\u00C2\u00A0 162 Corzo,\u00C2\u00A0 C.A.,\u00C2\u00A0 T.\u00C2\u00A0 Condamine,\u00C2\u00A0 L.\u00C2\u00A0 Lu,\u00C2\u00A0M.J.\u00C2\u00A0 Cotter,\u00C2\u00A0 J.I.\u00C2\u00A0 Youn,\u00C2\u00A0 P.\u00C2\u00A0 Cheng,\u00C2\u00A0H.I.\u00C2\u00A0 Cho,\u00C2\u00A0 E.\u00C2\u00A0 Celis,\u00C2\u00A0D.G.\u00C2\u00A0 Quiceno,\u00C2\u00A0 T.\u00C2\u00A0 Padhya,\u00C2\u00A0 T.V.\u00C2\u00A0 McCaffrey,\u00C2\u00A0 J.C.\u00C2\u00A0 McCaffrey,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A0 2010.\u00C2\u00A0 HIF\u00E2\u0080\u00901alpha\u00C2\u00A0regulates\u00C2\u00A0function\u00C2\u00A0and\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 microenvironment.\u00C2\u00A0 The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 experimental\u00C2\u00A0 medicine\u00C2\u00A0 207:2439\u00E2\u0080\u00902453.\u00C2\u00A0 Corzo,\u00C2\u00A0C.A.,\u00C2\u00A0M.J.\u00C2\u00A0Cotter,\u00C2\u00A0P.\u00C2\u00A0Cheng,\u00C2\u00A0F.\u00C2\u00A0Cheng,\u00C2\u00A0S.\u00C2\u00A0Kusmartsev,\u00C2\u00A0E.\u00C2\u00A0Sotomayor,\u00C2\u00A0T.\u00C2\u00A0Padhya,\u00C2\u00A0T.V.\u00C2\u00A0 McCaffrey,\u00C2\u00A0 J.C.\u00C2\u00A0 McCaffrey,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A0 2009.\u00C2\u00A0 Mechanism\u00C2\u00A0 regulating\u00C2\u00A0 reactive\u00C2\u00A0 oxygen\u00C2\u00A0 species\u00C2\u00A0 in\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells.\u00C2\u00A0 Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0182:5693\u00E2\u0080\u00905701.\u00C2\u00A0 Cox,\u00C2\u00A0 G.W.,\u00C2\u00A0 G.\u00C2\u00A0 Melillo,\u00C2\u00A0 U.\u00C2\u00A0 Chattopadhyay,\u00C2\u00A0 D.\u00C2\u00A0 Mullet,\u00C2\u00A0 R.H.\u00C2\u00A0 Fertel,\u00C2\u00A0 and\u00C2\u00A0 L.\u00C2\u00A0 Varesio.\u00C2\u00A0 1992.\u00C2\u00A0 Tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00E2\u0080\u0090alpha\u00E2\u0080\u0090dependent\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 reactive\u00C2\u00A0 nitrogen\u00C2\u00A0 intermediates\u00C2\u00A0 mediates\u00C2\u00A0 IFN\u00E2\u0080\u0090gamma\u00C2\u00A0 plus\u00C2\u00A0 IL\u00E2\u0080\u00902\u00E2\u0080\u0090induced\u00C2\u00A0 murine\u00C2\u00A0 macrophage\u00C2\u00A0 tumoricidal\u00C2\u00A0activity.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0149:3290\u00E2\u0080\u00903296.\u00C2\u00A0 Cuenca,\u00C2\u00A0A.G.,\u00C2\u00A0M.J.\u00C2\u00A0Delano,\u00C2\u00A0K.M.\u00C2\u00A0Kelly\u00E2\u0080\u0090Scumpia,\u00C2\u00A0C.\u00C2\u00A0Moreno,\u00C2\u00A0P.O.\u00C2\u00A0Scumpia,\u00C2\u00A0D.M.\u00C2\u00A0Laface,\u00C2\u00A0P.G.\u00C2\u00A0 Heyworth,\u00C2\u00A0P.A.\u00C2\u00A0Efron,\u00C2\u00A0and\u00C2\u00A0L.L.\u00C2\u00A0Moldawer.\u00C2\u00A02011.\u00C2\u00A0A\u00C2\u00A0paradoxical\u00C2\u00A0role\u00C2\u00A0for\u00C2\u00A0myeloid\u00E2\u0080\u0090 derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0sepsis\u00C2\u00A0and\u00C2\u00A0trauma.\u00C2\u00A0Mol\u00C2\u00A0Med\u00C2\u00A017:281\u00E2\u0080\u0090292.\u00C2\u00A0 Curiel,\u00C2\u00A0T.J.,\u00C2\u00A0G.\u00C2\u00A0Coukos,\u00C2\u00A0L.\u00C2\u00A0Zou,\u00C2\u00A0X.\u00C2\u00A0Alvarez,\u00C2\u00A0P.\u00C2\u00A0Cheng,\u00C2\u00A0P.\u00C2\u00A0Mottram,\u00C2\u00A0M.\u00C2\u00A0Evdemon\u00E2\u0080\u0090Hogan,\u00C2\u00A0J.R.\u00C2\u00A0 Conejo\u00E2\u0080\u0090Garcia,\u00C2\u00A0L.\u00C2\u00A0Zhang,\u00C2\u00A0M.\u00C2\u00A0Burow,\u00C2\u00A0Y.\u00C2\u00A0Zhu,\u00C2\u00A0S.\u00C2\u00A0Wei,\u00C2\u00A0I.\u00C2\u00A0Kryczek,\u00C2\u00A0B.\u00C2\u00A0Daniel,\u00C2\u00A0A.\u00C2\u00A0Gordon,\u00C2\u00A0 L.\u00C2\u00A0Myers,\u00C2\u00A0A.\u00C2\u00A0Lackner,\u00C2\u00A0M.L.\u00C2\u00A0Disis,\u00C2\u00A0K.L.\u00C2\u00A0Knutson,\u00C2\u00A0L.\u00C2\u00A0Chen,\u00C2\u00A0and\u00C2\u00A0W.\u00C2\u00A0Zou.\u00C2\u00A02004.\u00C2\u00A0Specific\u00C2\u00A0 recruitment\u00C2\u00A0of\u00C2\u00A0regulatory\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0ovarian\u00C2\u00A0carcinoma\u00C2\u00A0fosters\u00C2\u00A0immune\u00C2\u00A0privilege\u00C2\u00A0 and\u00C2\u00A0predicts\u00C2\u00A0reduced\u00C2\u00A0survival.\u00C2\u00A0Nat\u00C2\u00A0Med\u00C2\u00A010:942\u00E2\u0080\u0090949.\u00C2\u00A0 Dainiak,\u00C2\u00A0N.\u00C2\u00A01985.\u00C2\u00A0Role\u00C2\u00A0of\u00C2\u00A0defined\u00C2\u00A0and\u00C2\u00A0undefined\u00C2\u00A0serum\u00C2\u00A0additives\u00C2\u00A0to\u00C2\u00A0hematopoietic\u00C2\u00A0stem\u00C2\u00A0 cell\u00C2\u00A0culture.\u00C2\u00A0In\u00C2\u00A0Hematopoietic\u00C2\u00A0Stem\u00C2\u00A0Cell\u00C2\u00A0Physiology.\u00C2\u00A0E.P.\u00C2\u00A0Cronkite,\u00C2\u00A0N.\u00C2\u00A0Dainiak,\u00C2\u00A0R.P.\u00C2\u00A0 McCaffrey,\u00C2\u00A0J.\u00C2\u00A0Palek,\u00C2\u00A0and\u00C2\u00A0P.J.\u00C2\u00A0Quesenberry,\u00C2\u00A0editors.\u00C2\u00A0Alan\u00C2\u00A0R.\u00C2\u00A0Liss,\u00C2\u00A0New\u00C2\u00A0York.\u00C2\u00A059\u00E2\u0080\u009076.\u00C2\u00A0 Damen,\u00C2\u00A0J.E.,\u00C2\u00A0H.\u00C2\u00A0Wakao,\u00C2\u00A0A.\u00C2\u00A0Miyajima,\u00C2\u00A0J.\u00C2\u00A0Krosl,\u00C2\u00A0R.K.\u00C2\u00A0Humphries,\u00C2\u00A0R.L.\u00C2\u00A0Cutler,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Krystal.\u00C2\u00A0 1995.\u00C2\u00A0 Tyrosine\u00C2\u00A0 343\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 erythropoietin\u00C2\u00A0 receptor\u00C2\u00A0 positively\u00C2\u00A0 regulates\u00C2\u00A0 erythropoietin\u00E2\u0080\u0090induced\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0and\u00C2\u00A0Stat5\u00C2\u00A0activation.\u00C2\u00A0EMBO\u00C2\u00A0 J\u00C2\u00A014:5557\u00E2\u0080\u0090 5568.\u00C2\u00A0 Dawson,\u00C2\u00A0M.R.,\u00C2\u00A0D.G.\u00C2\u00A0Duda,\u00C2\u00A0S.S.\u00C2\u00A0Chae,\u00C2\u00A0D.\u00C2\u00A0Fukumura,\u00C2\u00A0and\u00C2\u00A0R.K.\u00C2\u00A0 Jain.\u00C2\u00A02009.\u00C2\u00A0VEGFR1\u00C2\u00A0activity\u00C2\u00A0 modulates\u00C2\u00A0 myeloid\u00C2\u00A0 cell\u00C2\u00A0 infiltration\u00C2\u00A0 in\u00C2\u00A0 growing\u00C2\u00A0 lung\u00C2\u00A0 metastases\u00C2\u00A0 but\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 required\u00C2\u00A0for\u00C2\u00A0spontaneous\u00C2\u00A0metastasis\u00C2\u00A0formation.\u00C2\u00A0PLoS\u00C2\u00A0One\u00C2\u00A04:e6525.\u00C2\u00A0 Delano,\u00C2\u00A0M.J.,\u00C2\u00A0 P.O.\u00C2\u00A0 Scumpia,\u00C2\u00A0 J.S.\u00C2\u00A0Weinstein,\u00C2\u00A0D.\u00C2\u00A0Coco,\u00C2\u00A0 S.\u00C2\u00A0Nagaraj,\u00C2\u00A0K.M.\u00C2\u00A0Kelly\u00E2\u0080\u0090Scumpia,\u00C2\u00A0K.A.\u00C2\u00A0 O'Malley,\u00C2\u00A0 J.L.\u00C2\u00A0 Wynn,\u00C2\u00A0 S.\u00C2\u00A0 Antonenko,\u00C2\u00A0 S.Z.\u00C2\u00A0 Al\u00E2\u0080\u0090Quran,\u00C2\u00A0 R.\u00C2\u00A0 Swan,\u00C2\u00A0 C.S.\u00C2\u00A0 Chung,\u00C2\u00A0 M.A.\u00C2\u00A0 Atkinson,\u00C2\u00A0R.\u00C2\u00A0Ramphal,\u00C2\u00A0D.I.\u00C2\u00A0Gabrilovich,\u00C2\u00A0W.H.\u00C2\u00A0Reeves,\u00C2\u00A0A.\u00C2\u00A0Ayala,\u00C2\u00A0J.\u00C2\u00A0Phillips,\u00C2\u00A0D.\u00C2\u00A0Laface,\u00C2\u00A0 P.G.\u00C2\u00A0 Heyworth,\u00C2\u00A0 M.\u00C2\u00A0 Clare\u00E2\u0080\u0090Salzler,\u00C2\u00A0 and\u00C2\u00A0 L.L.\u00C2\u00A0 Moldawer.\u00C2\u00A0 2007.\u00C2\u00A0 MyD88\u00E2\u0080\u0090dependent\u00C2\u00A0 expansion\u00C2\u00A0 of\u00C2\u00A0 an\u00C2\u00A0 immature\u00C2\u00A0 GR\u00E2\u0080\u00901(+)CD11b(+)\u00C2\u00A0 population\u00C2\u00A0 induces\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 suppression\u00C2\u00A0and\u00C2\u00A0Th2\u00C2\u00A0polarization\u00C2\u00A0in\u00C2\u00A0sepsis.\u00C2\u00A0The\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0experimental\u00C2\u00A0medicine\u00C2\u00A0 204:1463\u00E2\u0080\u00901474.\u00C2\u00A0 163 Deng,\u00C2\u00A0W.,\u00C2\u00A0 B.\u00C2\u00A0 Thiel,\u00C2\u00A0 C.S.\u00C2\u00A0 Tannenbaum,\u00C2\u00A0T.A.\u00C2\u00A0Hamilton,\u00C2\u00A0 and\u00C2\u00A0D.J.\u00C2\u00A0 Stuehr.\u00C2\u00A0 1993.\u00C2\u00A0 Synergistic\u00C2\u00A0 cooperation\u00C2\u00A0 between\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 lymphokines\u00C2\u00A0 for\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 nitric\u00C2\u00A0 oxide\u00C2\u00A0 synthase\u00C2\u00A0gene\u00C2\u00A0in\u00C2\u00A0murine\u00C2\u00A0peritoneal\u00C2\u00A0macrophages.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0151:322\u00E2\u0080\u0090329.\u00C2\u00A0 Dexter,\u00C2\u00A0 D.L.,\u00C2\u00A0 H.M.\u00C2\u00A0 Kowalski,\u00C2\u00A0 B.A.\u00C2\u00A0 Blazar,\u00C2\u00A0 Z.\u00C2\u00A0 Fligiel,\u00C2\u00A0 R.\u00C2\u00A0 Vogel,\u00C2\u00A0 and\u00C2\u00A0 G.H.\u00C2\u00A0 Heppner.\u00C2\u00A0 1978.\u00C2\u00A0 Heterogeneity\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 single\u00C2\u00A0 mouse\u00C2\u00A0 mammary\u00C2\u00A0 tumor.\u00C2\u00A0 Cancer\u00C2\u00A0 research\u00C2\u00A038:3174\u00E2\u0080\u00903181.\u00C2\u00A0 Di\u00C2\u00A0 Napoli,\u00C2\u00A0 M.R.,\u00C2\u00A0 M.\u00C2\u00A0 Torroella\u00E2\u0080\u0090Kouri,\u00C2\u00A0 G.\u00C2\u00A0 Perry,\u00C2\u00A0 and\u00C2\u00A0 D.M.\u00C2\u00A0 Lopez.\u00C2\u00A0 2005.\u00C2\u00A0 Diminished\u00C2\u00A0 PKC\u00C2\u00A0 activity\u00C2\u00A0 and\u00C2\u00A0 decreased\u00C2\u00A0 binding\u00C2\u00A0 of\u00C2\u00A0 transcription\u00C2\u00A0 factors\u00C2\u00A0 are\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 impaired\u00C2\u00A0production\u00C2\u00A0of\u00C2\u00A0nitric\u00C2\u00A0oxide\u00C2\u00A0by\u00C2\u00A0macrophages\u00C2\u00A0 from\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice.\u00C2\u00A0 Int\u00C2\u00A0J\u00C2\u00A0Mol\u00C2\u00A0Med\u00C2\u00A015:503\u00E2\u0080\u0090511.\u00C2\u00A0 Diaz\u00E2\u0080\u0090Montero,\u00C2\u00A0 C.M.,\u00C2\u00A0 M.L.\u00C2\u00A0 Salem,\u00C2\u00A0 M.I.\u00C2\u00A0 Nishimura,\u00C2\u00A0 E.\u00C2\u00A0 Garrett\u00E2\u0080\u0090Mayer,\u00C2\u00A0 D.J.\u00C2\u00A0 Cole,\u00C2\u00A0 and\u00C2\u00A0 A.J.\u00C2\u00A0 Montero.\u00C2\u00A02009.\u00C2\u00A0Increased\u00C2\u00A0circulating\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0correlate\u00C2\u00A0 with\u00C2\u00A0 clinical\u00C2\u00A0 cancer\u00C2\u00A0 stage,\u00C2\u00A0 metastatic\u00C2\u00A0 tumor\u00C2\u00A0 burden,\u00C2\u00A0 and\u00C2\u00A0 doxorubicin\u00E2\u0080\u0090 cyclophosphamide\u00C2\u00A0chemotherapy.\u00C2\u00A0Cancer\u00C2\u00A0Immunol\u00C2\u00A0Immunother\u00C2\u00A058:49\u00E2\u0080\u009059.\u00C2\u00A0 Dinapoli,\u00C2\u00A0M.R.,\u00C2\u00A0C.L.\u00C2\u00A0Calderon,\u00C2\u00A0and\u00C2\u00A0D.M.\u00C2\u00A0Lopez.\u00C2\u00A01996.\u00C2\u00A0The\u00C2\u00A0altered\u00C2\u00A0tumoricidal\u00C2\u00A0capacity\u00C2\u00A0of\u00C2\u00A0 macrophages\u00C2\u00A0 isolated\u00C2\u00A0from\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0 is\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0reduce\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0inducible\u00C2\u00A0nitric\u00C2\u00A0oxide\u00C2\u00A0synthase\u00C2\u00A0gene.\u00C2\u00A0The\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0experimental\u00C2\u00A0medicine\u00C2\u00A0 183:1323\u00E2\u0080\u00901329.\u00C2\u00A0 Doedens,\u00C2\u00A0 A.L.,\u00C2\u00A0 C.\u00C2\u00A0 Stockmann,\u00C2\u00A0 M.P.\u00C2\u00A0 Rubinstein,\u00C2\u00A0 D.\u00C2\u00A0 Liao,\u00C2\u00A0 N.\u00C2\u00A0 Zhang,\u00C2\u00A0 D.G.\u00C2\u00A0 DeNardo,\u00C2\u00A0 L.M.\u00C2\u00A0 Coussens,\u00C2\u00A0 M.\u00C2\u00A0 Karin,\u00C2\u00A0 A.W.\u00C2\u00A0 Goldrath,\u00C2\u00A0 and\u00C2\u00A0 R.S.\u00C2\u00A0 Johnson.\u00C2\u00A0 2010.\u00C2\u00A0 Macrophage\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 hypoxia\u00E2\u0080\u0090inducible\u00C2\u00A0 factor\u00E2\u0080\u00901\u00C2\u00A0 alpha\u00C2\u00A0 suppresses\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 function\u00C2\u00A0 and\u00C2\u00A0 promotes\u00C2\u00A0tumor\u00C2\u00A0progression.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A070:7465\u00E2\u0080\u00907475.\u00C2\u00A0 Dolcetti,\u00C2\u00A0L.,\u00C2\u00A0E.\u00C2\u00A0Peranzoni,\u00C2\u00A0S.\u00C2\u00A0Ugel,\u00C2\u00A0I.\u00C2\u00A0Marigo,\u00C2\u00A0A.\u00C2\u00A0Fernandez\u00C2\u00A0Gomez,\u00C2\u00A0C.\u00C2\u00A0Mesa,\u00C2\u00A0M.\u00C2\u00A0Geilich,\u00C2\u00A0G.\u00C2\u00A0 Winkels,\u00C2\u00A0 E.\u00C2\u00A0 Traggiai,\u00C2\u00A0 A.\u00C2\u00A0 Casati,\u00C2\u00A0 F.\u00C2\u00A0 Grassi,\u00C2\u00A0 and\u00C2\u00A0 V.\u00C2\u00A0 Bronte.\u00C2\u00A0 2010.\u00C2\u00A0 Hierarchy\u00C2\u00A0 of\u00C2\u00A0 immunosuppressive\u00C2\u00A0strength\u00C2\u00A0among\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0subsets\u00C2\u00A0is\u00C2\u00A0 determined\u00C2\u00A0by\u00C2\u00A0GM\u00E2\u0080\u0090CSF.\u00C2\u00A0European\u00C2\u00A0journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A040:22\u00E2\u0080\u009035.\u00C2\u00A0 Dunn,\u00C2\u00A0 G.P.,\u00C2\u00A0 A.T.\u00C2\u00A0 Bruce,\u00C2\u00A0 H.\u00C2\u00A0 Ikeda,\u00C2\u00A0 L.J.\u00C2\u00A0 Old,\u00C2\u00A0 and\u00C2\u00A0 R.D.\u00C2\u00A0 Schreiber.\u00C2\u00A0 2002.\u00C2\u00A0 Cancer\u00C2\u00A0 immunoediting:\u00C2\u00A0from\u00C2\u00A0immunosurveillance\u00C2\u00A0to\u00C2\u00A0tumor\u00C2\u00A0escape.\u00C2\u00A0Nat\u00C2\u00A0Immunol\u00C2\u00A03:991\u00E2\u0080\u0090 998.\u00C2\u00A0 Ehrlich,\u00C2\u00A0P.\u00C2\u00A01909.\u00C2\u00A0\u00C3\u009Cber\u00C2\u00A0den\u00C2\u00A0jetzigen\u00C2\u00A0stand\u00C2\u00A0der\u00C2\u00A0karzinomforschung.\u00C2\u00A0Ned\u00C2\u00A0Tijdschr\u00C2\u00A0Geneeskd\u00C2\u00A0 5:273\u00E2\u0080\u0090290.\u00C2\u00A0 Elkabets,\u00C2\u00A0M.,\u00C2\u00A0V.S.\u00C2\u00A0Ribeiro,\u00C2\u00A0C.A.\u00C2\u00A0Dinarello,\u00C2\u00A0S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A0 J.P.\u00C2\u00A0Di\u00C2\u00A0Santo,\u00C2\u00A0R.N.\u00C2\u00A0Apte,\u00C2\u00A0 and\u00C2\u00A0 C.A.\u00C2\u00A0 Vosshenrich.\u00C2\u00A0 2010.\u00C2\u00A0 IL\u00E2\u0080\u00901beta\u00C2\u00A0 regulates\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0cell\u00C2\u00A0subset\u00C2\u00A0that\u00C2\u00A0impairs\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0function.\u00C2\u00A0European\u00C2\u00A0 journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A040:3347\u00E2\u0080\u00903357.\u00C2\u00A0 164 Erler,\u00C2\u00A0 J.T.,\u00C2\u00A0K.L.\u00C2\u00A0Bennewith,\u00C2\u00A0T.R.\u00C2\u00A0Cox,\u00C2\u00A0G.\u00C2\u00A0Lang,\u00C2\u00A0D.\u00C2\u00A0Bird,\u00C2\u00A0A.\u00C2\u00A0Koong,\u00C2\u00A0Q.T.\u00C2\u00A0Le,\u00C2\u00A0and\u00C2\u00A0A.J.\u00C2\u00A0Giaccia.\u00C2\u00A0 2009.\u00C2\u00A0Hypoxia\u00E2\u0080\u0090induced\u00C2\u00A0 lysyl\u00C2\u00A0oxidase\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0critical\u00C2\u00A0mediator\u00C2\u00A0of\u00C2\u00A0bone\u00C2\u00A0marrow\u00C2\u00A0cell\u00C2\u00A0 recruitment\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0the\u00C2\u00A0premetastatic\u00C2\u00A0niche.\u00C2\u00A0Cancer\u00C2\u00A0Cell\u00C2\u00A015:35\u00E2\u0080\u009044.\u00C2\u00A0 Eruslanov,\u00C2\u00A0E.,\u00C2\u00A0 I.\u00C2\u00A0Daurkin,\u00C2\u00A0 J.\u00C2\u00A0Ortiz,\u00C2\u00A0 J.\u00C2\u00A0Vieweg,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Kusmartsev.\u00C2\u00A02010.\u00C2\u00A0Pivotal\u00C2\u00A0Advance:\u00C2\u00A0 Tumor\u00E2\u0080\u0090mediated\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 M2\u00E2\u0080\u0090 polarized\u00C2\u00A0macrophages\u00C2\u00A0by\u00C2\u00A0altering\u00C2\u00A0intracellular\u00C2\u00A0PGE\u00C2\u00A0catabolism\u00C2\u00A0in\u00C2\u00A0myeloid\u00C2\u00A0cells.\u00C2\u00A0 Journal\u00C2\u00A0of\u00C2\u00A0leukocyte\u00C2\u00A0biology\u00C2\u00A088:839\u00E2\u0080\u0090848.\u00C2\u00A0 Eruslanov,\u00C2\u00A0E.,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Kusmartsev.\u00C2\u00A02010.\u00C2\u00A0Identification\u00C2\u00A0of\u00C2\u00A0ROS\u00C2\u00A0using\u00C2\u00A0oxidized\u00C2\u00A0DCFDA\u00C2\u00A0and\u00C2\u00A0 flow\u00E2\u0080\u0090cytometry.\u00C2\u00A0Methods\u00C2\u00A0Mol\u00C2\u00A0Biol\u00C2\u00A0594:57\u00E2\u0080\u009072.\u00C2\u00A0 Fasano,\u00C2\u00A0 M.,\u00C2\u00A0 S.\u00C2\u00A0 Curry,\u00C2\u00A0 E.\u00C2\u00A0 Terreno,\u00C2\u00A0 M.\u00C2\u00A0 Galliano,\u00C2\u00A0 G.\u00C2\u00A0 Fanali,\u00C2\u00A0 P.\u00C2\u00A0 Narciso,\u00C2\u00A0 S.\u00C2\u00A0 Notari,\u00C2\u00A0 and\u00C2\u00A0 P.\u00C2\u00A0 Ascenzi.\u00C2\u00A0 2005.\u00C2\u00A0 The\u00C2\u00A0 extraordinary\u00C2\u00A0 ligand\u00C2\u00A0 binding\u00C2\u00A0 properties\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0 serum\u00C2\u00A0 albumin.\u00C2\u00A0IUBMB\u00C2\u00A0life\u00C2\u00A057:787\u00E2\u0080\u0090796.\u00C2\u00A0 Fidler,\u00C2\u00A0 I.J.\u00C2\u00A02003.\u00C2\u00A0The\u00C2\u00A0pathogenesis\u00C2\u00A0of\u00C2\u00A0 cancer\u00C2\u00A0metastasis:\u00C2\u00A0 the\u00C2\u00A0 'seed\u00C2\u00A0and\u00C2\u00A0soil'\u00C2\u00A0hypothesis\u00C2\u00A0 revisited.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Cancer\u00C2\u00A03:453\u00E2\u0080\u0090458.\u00C2\u00A0 Fitzner,\u00C2\u00A0N.,\u00C2\u00A0 S.\u00C2\u00A0 Clauberg,\u00C2\u00A0 F.\u00C2\u00A0Essmann,\u00C2\u00A0 J.\u00C2\u00A0 Liebmann,\u00C2\u00A0 and\u00C2\u00A0V.\u00C2\u00A0Kolb\u00E2\u0080\u0090Bachofen.\u00C2\u00A02008.\u00C2\u00A0Human\u00C2\u00A0 skin\u00C2\u00A0 endothelial\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 express\u00C2\u00A0 all\u00C2\u00A0 10\u00C2\u00A0 TLR\u00C2\u00A0 genes\u00C2\u00A0 and\u00C2\u00A0 respond\u00C2\u00A0 to\u00C2\u00A0 respective\u00C2\u00A0 ligands.\u00C2\u00A0Clinical\u00C2\u00A0and\u00C2\u00A0vaccine\u00C2\u00A0immunology\u00C2\u00A0:\u00C2\u00A0CVI\u00C2\u00A015:138\u00E2\u0080\u0090146.\u00C2\u00A0 Fricke,\u00C2\u00A0 I.,\u00C2\u00A0 N.\u00C2\u00A0 Mirza,\u00C2\u00A0 J.\u00C2\u00A0 Dupont,\u00C2\u00A0 C.\u00C2\u00A0 Lockhart,\u00C2\u00A0 A.\u00C2\u00A0 Jackson,\u00C2\u00A0 J.H.\u00C2\u00A0 Lee,\u00C2\u00A0 J.A.\u00C2\u00A0 Sosman,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A02007.\u00C2\u00A0Vascular\u00C2\u00A0endothelial\u00C2\u00A0growth\u00C2\u00A0factor\u00E2\u0080\u0090trap\u00C2\u00A0overcomes\u00C2\u00A0defects\u00C2\u00A0in\u00C2\u00A0 dendritic\u00C2\u00A0 cell\u00C2\u00A0 differentiation\u00C2\u00A0 but\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 improve\u00C2\u00A0 antigen\u00E2\u0080\u0090specific\u00C2\u00A0 immune\u00C2\u00A0 responses.\u00C2\u00A0 Clinical\u00C2\u00A0 cancer\u00C2\u00A0 research\u00C2\u00A0 :\u00C2\u00A0 an\u00C2\u00A0 official\u00C2\u00A0 journal\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 American\u00C2\u00A0 Association\u00C2\u00A0for\u00C2\u00A0Cancer\u00C2\u00A0Research\u00C2\u00A013:4840\u00E2\u0080\u00904848.\u00C2\u00A0 Gabrilovich,\u00C2\u00A0D.,\u00C2\u00A0T.\u00C2\u00A0Ishida,\u00C2\u00A0T.\u00C2\u00A0Oyama,\u00C2\u00A0S.\u00C2\u00A0Ran,\u00C2\u00A0V.\u00C2\u00A0Kravtsov,\u00C2\u00A0S.\u00C2\u00A0Nadaf,\u00C2\u00A0and\u00C2\u00A0D.P.\u00C2\u00A0Carbone.\u00C2\u00A01998.\u00C2\u00A0 Vascular\u00C2\u00A0 endothelial\u00C2\u00A0 growth\u00C2\u00A0 factor\u00C2\u00A0 inhibits\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 dendritic\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0dramatically\u00C2\u00A0affects\u00C2\u00A0the\u00C2\u00A0differentiation\u00C2\u00A0of\u00C2\u00A0multiple\u00C2\u00A0hematopoietic\u00C2\u00A0lineages\u00C2\u00A0in\u00C2\u00A0 vivo.\u00C2\u00A0Blood\u00C2\u00A092:4150\u00E2\u0080\u00904166.\u00C2\u00A0 Gabrilovich,\u00C2\u00A0 D.I.,\u00C2\u00A0 M.P.\u00C2\u00A0 Velders,\u00C2\u00A0 E.M.\u00C2\u00A0 Sotomayor,\u00C2\u00A0 and\u00C2\u00A0 W.M.\u00C2\u00A0 Kast.\u00C2\u00A0 2001.\u00C2\u00A0 Mechanism\u00C2\u00A0 of\u00C2\u00A0 immune\u00C2\u00A0 dysfunction\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 mediated\u00C2\u00A0 by\u00C2\u00A0 immature\u00C2\u00A0 Gr\u00E2\u0080\u00901+\u00C2\u00A0 myeloid\u00C2\u00A0 cells.\u00C2\u00A0 Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0166:5398\u00E2\u0080\u00905406.\u00C2\u00A0 Gallina,\u00C2\u00A0 G.,\u00C2\u00A0 L.\u00C2\u00A0 Dolcetti,\u00C2\u00A0 P.\u00C2\u00A0 Serafini,\u00C2\u00A0 C.\u00C2\u00A0 De\u00C2\u00A0 Santo,\u00C2\u00A0 I.\u00C2\u00A0 Marigo,\u00C2\u00A0 M.P.\u00C2\u00A0 Colombo,\u00C2\u00A0 G.\u00C2\u00A0 Basso,\u00C2\u00A0 F.\u00C2\u00A0 Brombacher,\u00C2\u00A0 I.\u00C2\u00A0 Borrello,\u00C2\u00A0 P.\u00C2\u00A0 Zanovello,\u00C2\u00A0 S.\u00C2\u00A0 Bicciato,\u00C2\u00A0 and\u00C2\u00A0V.\u00C2\u00A0 Bronte.\u00C2\u00A0 2006.\u00C2\u00A0 Tumors\u00C2\u00A0 induce\u00C2\u00A0a\u00C2\u00A0subset\u00C2\u00A0of\u00C2\u00A0inflammatory\u00C2\u00A0monocytes\u00C2\u00A0with\u00C2\u00A0immunosuppressive\u00C2\u00A0activity\u00C2\u00A0on\u00C2\u00A0 CD8+\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Invest\u00C2\u00A0116:2777\u00E2\u0080\u00902790.\u00C2\u00A0 Gandhi,\u00C2\u00A0 R.,\u00C2\u00A0 D.E.\u00C2\u00A0 Anderson,\u00C2\u00A0 and\u00C2\u00A0 H.L.\u00C2\u00A0 Weiner.\u00C2\u00A0 2007.\u00C2\u00A0 Cutting\u00C2\u00A0 Edge:\u00C2\u00A0 Immature\u00C2\u00A0 human\u00C2\u00A0 dendritic\u00C2\u00A0cells\u00C2\u00A0express\u00C2\u00A0latency\u00E2\u0080\u0090associated\u00C2\u00A0peptide\u00C2\u00A0and\u00C2\u00A0inhibit\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0in\u00C2\u00A0 a\u00C2\u00A0TGF\u00E2\u0080\u0090beta\u00E2\u0080\u0090dependent\u00C2\u00A0manner.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0178:4017\u00E2\u0080\u00904021.\u00C2\u00A0 165 Geissmann,\u00C2\u00A0 F.,\u00C2\u00A0 C.\u00C2\u00A0 Auffray,\u00C2\u00A0 R.\u00C2\u00A0 Palframan,\u00C2\u00A0 C.\u00C2\u00A0 Wirrig,\u00C2\u00A0 A.\u00C2\u00A0 Ciocca,\u00C2\u00A0 L.\u00C2\u00A0 Campisi,\u00C2\u00A0 E.\u00C2\u00A0 Narni\u00E2\u0080\u0090 Mancinelli,\u00C2\u00A0 and\u00C2\u00A0 G.\u00C2\u00A0 Lauvau.\u00C2\u00A0 2008.\u00C2\u00A0 Blood\u00C2\u00A0monocytes:\u00C2\u00A0 distinct\u00C2\u00A0 subsets,\u00C2\u00A0 how\u00C2\u00A0 they\u00C2\u00A0 relate\u00C2\u00A0 to\u00C2\u00A0 dendritic\u00C2\u00A0 cells,\u00C2\u00A0 and\u00C2\u00A0 their\u00C2\u00A0 possible\u00C2\u00A0 roles\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 responses.\u00C2\u00A0Immunol\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A086:398\u00E2\u0080\u0090408.\u00C2\u00A0 Geissmann,\u00C2\u00A0 F.,\u00C2\u00A0 M.G.\u00C2\u00A0 Manz,\u00C2\u00A0 S.\u00C2\u00A0 Jung,\u00C2\u00A0 M.H.\u00C2\u00A0 Sieweke,\u00C2\u00A0 M.\u00C2\u00A0 Merad,\u00C2\u00A0 and\u00C2\u00A0 K.\u00C2\u00A0 Ley.\u00C2\u00A0 2010.\u00C2\u00A0 Development\u00C2\u00A0of\u00C2\u00A0monocytes,\u00C2\u00A0macrophages,\u00C2\u00A0and\u00C2\u00A0dendritic\u00C2\u00A0cells.\u00C2\u00A0Science\u00C2\u00A0327:656\u00E2\u0080\u0090 661.\u00C2\u00A0 Godfrey,\u00C2\u00A0 D.I.,\u00C2\u00A0 S.\u00C2\u00A0 Stankovic,\u00C2\u00A0 and\u00C2\u00A0 A.G.\u00C2\u00A0 Baxter.\u00C2\u00A0 2010.\u00C2\u00A0 Raising\u00C2\u00A0 the\u00C2\u00A0 NKT\u00C2\u00A0 cell\u00C2\u00A0 family.\u00C2\u00A0 Nat\u00C2\u00A0 Immunol\u00C2\u00A011:197\u00E2\u0080\u0090206.\u00C2\u00A0 Gordon,\u00C2\u00A0S.\u00C2\u00A02003.\u00C2\u00A0Alternative\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0macrophages.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Immunol\u00C2\u00A03:23\u00E2\u0080\u009035.\u00C2\u00A0 Gordon,\u00C2\u00A0S.,\u00C2\u00A0and\u00C2\u00A0F.O.\u00C2\u00A0Martinez.\u00C2\u00A02010.\u00C2\u00A0Alternative\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0macrophages:\u00C2\u00A0mechanism\u00C2\u00A0 and\u00C2\u00A0functions.\u00C2\u00A0Immunity\u00C2\u00A032:593\u00E2\u0080\u0090604.\u00C2\u00A0 Gordon,\u00C2\u00A0 S.,\u00C2\u00A0 and\u00C2\u00A0 P.R.\u00C2\u00A0 Taylor.\u00C2\u00A0 2005.\u00C2\u00A0 Monocyte\u00C2\u00A0 and\u00C2\u00A0 macrophage\u00C2\u00A0 heterogeneity.\u00C2\u00A0 Nature\u00C2\u00A0 reviews.\u00C2\u00A0Immunology\u00C2\u00A05:953\u00E2\u0080\u0090964.\u00C2\u00A0 Gough,\u00C2\u00A0 D.J.,\u00C2\u00A0 N.L.\u00C2\u00A0Messina,\u00C2\u00A0 L.\u00C2\u00A0 Hii,\u00C2\u00A0 J.A.\u00C2\u00A0 Gould,\u00C2\u00A0 K.\u00C2\u00A0 Sabapathy,\u00C2\u00A0 A.P.\u00C2\u00A0 Robertson,\u00C2\u00A0 J.A.\u00C2\u00A0 Trapani,\u00C2\u00A0 D.E.\u00C2\u00A0Levy,\u00C2\u00A0P.J.\u00C2\u00A0Hertzog,\u00C2\u00A0C.J.\u00C2\u00A0Clarke,\u00C2\u00A0and\u00C2\u00A0R.W.\u00C2\u00A0Johnstone.\u00C2\u00A02010.\u00C2\u00A0Functional\u00C2\u00A0crosstalk\u00C2\u00A0 between\u00C2\u00A0 type\u00C2\u00A0 I\u00C2\u00A0 and\u00C2\u00A0 II\u00C2\u00A0 interferon\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 regulated\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 STAT1.\u00C2\u00A0 PLoS\u00C2\u00A0Biol\u00C2\u00A08:e1000361.\u00C2\u00A0 Greenwald,\u00C2\u00A0R.J.,\u00C2\u00A0G.J.\u00C2\u00A0Freeman,\u00C2\u00A0and\u00C2\u00A0A.H.\u00C2\u00A0Sharpe.\u00C2\u00A02005.\u00C2\u00A0The\u00C2\u00A0B7\u00C2\u00A0family\u00C2\u00A0revisited.\u00C2\u00A0Annu\u00C2\u00A0Rev\u00C2\u00A0 Immunol\u00C2\u00A023:515\u00E2\u0080\u0090548.\u00C2\u00A0 Greifenberg,\u00C2\u00A0 V.,\u00C2\u00A0 E.\u00C2\u00A0 Ribechini,\u00C2\u00A0 S.\u00C2\u00A0 Rossner,\u00C2\u00A0 and\u00C2\u00A0 M.B.\u00C2\u00A0 Lutz.\u00C2\u00A0 2009.\u00C2\u00A0 Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0by\u00C2\u00A0combined\u00C2\u00A0LPS\u00C2\u00A0and\u00C2\u00A0 IFN\u00E2\u0080\u0090gamma\u00C2\u00A0treatment\u00C2\u00A0 impairs\u00C2\u00A0 DC\u00C2\u00A0development.\u00C2\u00A0European\u00C2\u00A0journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A039:2865\u00E2\u0080\u00902876.\u00C2\u00A0 Grimshaw,\u00C2\u00A0 M.J.,\u00C2\u00A0 J.L.\u00C2\u00A0 Wilson,\u00C2\u00A0 and\u00C2\u00A0 F.R.\u00C2\u00A0 Balkwill.\u00C2\u00A0 2002.\u00C2\u00A0 Endothelin\u00E2\u0080\u00902\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 macrophage\u00C2\u00A0 chemoattractant:\u00C2\u00A0implications\u00C2\u00A0for\u00C2\u00A0macrophage\u00C2\u00A0distribution\u00C2\u00A0in\u00C2\u00A0tumors.\u00C2\u00A0European\u00C2\u00A0 journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A032:2393\u00E2\u0080\u00902400.\u00C2\u00A0 Guerra,\u00C2\u00A0N.,\u00C2\u00A0Y.X.\u00C2\u00A0Tan,\u00C2\u00A0N.T.\u00C2\u00A0 Joncker,\u00C2\u00A0A.\u00C2\u00A0Choy,\u00C2\u00A0F.\u00C2\u00A0Gallardo,\u00C2\u00A0N.\u00C2\u00A0Xiong,\u00C2\u00A0S.\u00C2\u00A0Knoblaugh,\u00C2\u00A0D.\u00C2\u00A0Cado,\u00C2\u00A0 N.M.\u00C2\u00A0 Greenberg,\u00C2\u00A0 and\u00C2\u00A0 D.H.\u00C2\u00A0 Raulet.\u00C2\u00A0 2008.\u00C2\u00A0 NKG2D\u00E2\u0080\u0090deficient\u00C2\u00A0mice\u00C2\u00A0 are\u00C2\u00A0 defective\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0surveillance\u00C2\u00A0in\u00C2\u00A0models\u00C2\u00A0of\u00C2\u00A0spontaneous\u00C2\u00A0malignancy.\u00C2\u00A0Immunity\u00C2\u00A028:571\u00E2\u0080\u0090580.\u00C2\u00A0 Guth,\u00C2\u00A0A.M.,\u00C2\u00A0W.J.\u00C2\u00A0Janssen,\u00C2\u00A0C.M.\u00C2\u00A0Bosio,\u00C2\u00A0E.C.\u00C2\u00A0Crouch,\u00C2\u00A0P.M.\u00C2\u00A0Henson,\u00C2\u00A0and\u00C2\u00A0S.W.\u00C2\u00A0Dow.\u00C2\u00A02009.\u00C2\u00A0Lung\u00C2\u00A0 environment\u00C2\u00A0 determines\u00C2\u00A0 unique\u00C2\u00A0 phenotype\u00C2\u00A0 of\u00C2\u00A0 alveolar\u00C2\u00A0 macrophages.\u00C2\u00A0 Am\u00C2\u00A0 J\u00C2\u00A0 Physiol\u00C2\u00A0Lung\u00C2\u00A0Cell\u00C2\u00A0Mol\u00C2\u00A0Physiol\u00C2\u00A0296:L936\u00E2\u0080\u0090946.\u00C2\u00A0 \u00C2\u00A0 166 Habibi,\u00C2\u00A0 M.,\u00C2\u00A0 M.\u00C2\u00A0 Kmieciak,\u00C2\u00A0 L.\u00C2\u00A0 Graham,\u00C2\u00A0 J.K.\u00C2\u00A0 Morales,\u00C2\u00A0 H.D.\u00C2\u00A0 Bear,\u00C2\u00A0 and\u00C2\u00A0 M.H.\u00C2\u00A0 Manjili.\u00C2\u00A0 2009.\u00C2\u00A0 Radiofrequency\u00C2\u00A0 thermal\u00C2\u00A0ablation\u00C2\u00A0of\u00C2\u00A0breast\u00C2\u00A0 tumors\u00C2\u00A0combined\u00C2\u00A0with\u00C2\u00A0 intralesional\u00C2\u00A0 administration\u00C2\u00A0 of\u00C2\u00A0 IL\u00E2\u0080\u00907\u00C2\u00A0 and\u00C2\u00A0 IL\u00E2\u0080\u009015\u00C2\u00A0 augments\u00C2\u00A0 anti\u00E2\u0080\u0090tumor\u00C2\u00A0 immune\u00C2\u00A0 responses\u00C2\u00A0 and\u00C2\u00A0 inhibits\u00C2\u00A0 tumor\u00C2\u00A0 development\u00C2\u00A0 and\u00C2\u00A0metastasis.\u00C2\u00A0Breast\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A0Treat\u00C2\u00A0 114:423\u00E2\u0080\u0090 431.\u00C2\u00A0 Hagemann,\u00C2\u00A0 T.,\u00C2\u00A0 T.\u00C2\u00A0 Lawrence,\u00C2\u00A0 I.\u00C2\u00A0 McNeish,\u00C2\u00A0 K.A.\u00C2\u00A0 Charles,\u00C2\u00A0 H.\u00C2\u00A0 Kulbe,\u00C2\u00A0 R.G.\u00C2\u00A0 Thompson,\u00C2\u00A0 S.C.\u00C2\u00A0 Robinson,\u00C2\u00A0 and\u00C2\u00A0 F.R.\u00C2\u00A0 Balkwill.\u00C2\u00A0 2008.\u00C2\u00A0 \"Re\u00E2\u0080\u0090educating\"\u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0 macrophages\u00C2\u00A0by\u00C2\u00A0targeting\u00C2\u00A0NF\u00E2\u0080\u0090kappaB.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0205:1261\u00E2\u0080\u00901268.\u00C2\u00A0 Hamilton,\u00C2\u00A0M.J.,\u00C2\u00A0F.\u00C2\u00A0Antignano,\u00C2\u00A0A.\u00C2\u00A0von\u00C2\u00A0Rossum,\u00C2\u00A0J.L.\u00C2\u00A0Boucher,\u00C2\u00A0K.L.\u00C2\u00A0Bennewith,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Krystal.\u00C2\u00A0 2010.\u00C2\u00A0 TLR\u00C2\u00A0 agonists\u00C2\u00A0 that\u00C2\u00A0 induce\u00C2\u00A0 IFN\u00E2\u0080\u0090beta\u00C2\u00A0 abrogate\u00C2\u00A0 resident\u00C2\u00A0 macrophage\u00C2\u00A0 suppression\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0185:4545\u00E2\u0080\u00904553.\u00C2\u00A0 Hamilton,\u00C2\u00A0M.J.,\u00C2\u00A0 J.P.\u00C2\u00A0 Banath,\u00C2\u00A0 V.\u00C2\u00A0 Lam,\u00C2\u00A0N.E.\u00C2\u00A0 Lepard,\u00C2\u00A0 G.\u00C2\u00A0 Krystal,\u00C2\u00A0 and\u00C2\u00A0K.L.\u00C2\u00A0 Bennewith.\u00C2\u00A0 2011.\u00C2\u00A0 Serum\u00C2\u00A0inhibits\u00C2\u00A0the\u00C2\u00A0immunosuppressive\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0 cells\u00C2\u00A0 isolated\u00C2\u00A0 from\u00C2\u00A0 4T1\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00C2\u00A0 Cancer\u00C2\u00A0 Immunol\u00C2\u00A0 Immunother\u00C2\u00A0 In\u00C2\u00A0 Press.\u00C2\u00A0 Hanahan,\u00C2\u00A0 D.,\u00C2\u00A0 and\u00C2\u00A0R.A.\u00C2\u00A0Weinberg.\u00C2\u00A0 2011.\u00C2\u00A0Hallmarks\u00C2\u00A0 of\u00C2\u00A0 cancer:\u00C2\u00A0 the\u00C2\u00A0 next\u00C2\u00A0 generation.\u00C2\u00A0Cell\u00C2\u00A0 144:646\u00E2\u0080\u0090674.\u00C2\u00A0 Handel\u00E2\u0080\u0090Fernandez,\u00C2\u00A0M.E.,\u00C2\u00A0X.\u00C2\u00A0Cheng,\u00C2\u00A0L.M.\u00C2\u00A0Herbert,\u00C2\u00A0and\u00C2\u00A0D.M.\u00C2\u00A0Lopez.\u00C2\u00A01997.\u00C2\u00A0Down\u00E2\u0080\u0090regulation\u00C2\u00A0 of\u00C2\u00A0IL\u00E2\u0080\u009012,\u00C2\u00A0not\u00C2\u00A0a\u00C2\u00A0shift\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0T\u00C2\u00A0helper\u00E2\u0080\u00901\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0T\u00C2\u00A0helper\u00E2\u0080\u00902\u00C2\u00A0phenotype,\u00C2\u00A0is\u00C2\u00A0responsible\u00C2\u00A0 for\u00C2\u00A0impaired\u00C2\u00A0IFN\u00E2\u0080\u0090gamma\u00C2\u00A0production\u00C2\u00A0in\u00C2\u00A0mammary\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0 immunology\u00C2\u00A0158:280\u00E2\u0080\u0090286.\u00C2\u00A0 Hanson,\u00C2\u00A0 E.M.,\u00C2\u00A0 V.K.\u00C2\u00A0 Clements,\u00C2\u00A0 P.\u00C2\u00A0 Sinha,\u00C2\u00A0 D.\u00C2\u00A0 Ilkovitch,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A0 2009.\u00C2\u00A0 Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0down\u00E2\u0080\u0090regulate\u00C2\u00A0L\u00E2\u0080\u0090selectin\u00C2\u00A0expression\u00C2\u00A0on\u00C2\u00A0CD4+\u00C2\u00A0 and\u00C2\u00A0CD8+\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0183:937\u00E2\u0080\u0090944.\u00C2\u00A0 Haverkamp,\u00C2\u00A0 J.M.,\u00C2\u00A0 S.A.\u00C2\u00A0 Crist,\u00C2\u00A0 B.D.\u00C2\u00A0 Elzey,\u00C2\u00A0 C.\u00C2\u00A0 Cimen,\u00C2\u00A0 and\u00C2\u00A0 T.L.\u00C2\u00A0 Ratliff.\u00C2\u00A0 2011.\u00C2\u00A0 In\u00C2\u00A0 vivo\u00C2\u00A0 suppressive\u00C2\u00A0 function\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 is\u00C2\u00A0 limited\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 inflammatory\u00C2\u00A0site.\u00C2\u00A0Eur\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A041:749\u00E2\u0080\u0090759.\u00C2\u00A0 Heppner,\u00C2\u00A0 G.H.,\u00C2\u00A0 F.R.\u00C2\u00A0 Miller,\u00C2\u00A0 and\u00C2\u00A0 P.M.\u00C2\u00A0 Shekhar.\u00C2\u00A0 2000.\u00C2\u00A0 Nontransgenic\u00C2\u00A0 models\u00C2\u00A0 of\u00C2\u00A0 breast\u00C2\u00A0 cancer.\u00C2\u00A0Breast\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02:331\u00E2\u0080\u0090334.\u00C2\u00A0 Ho,\u00C2\u00A0 V.W.,\u00C2\u00A0 and\u00C2\u00A0 L.M.\u00C2\u00A0 Sly.\u00C2\u00A0 2009.\u00C2\u00A0 Derivation\u00C2\u00A0 and\u00C2\u00A0 characterization\u00C2\u00A0 of\u00C2\u00A0murine\u00C2\u00A0 alternatively\u00C2\u00A0 activated\u00C2\u00A0(M2)\u00C2\u00A0macrophages.\u00C2\u00A0Methods\u00C2\u00A0Mol\u00C2\u00A0Biol\u00C2\u00A0531:173\u00E2\u0080\u0090185.\u00C2\u00A0 Holcomb,\u00C2\u00A0I.N.,\u00C2\u00A0R.C.\u00C2\u00A0Kabakoff,\u00C2\u00A0B.\u00C2\u00A0Chan,\u00C2\u00A0T.W.\u00C2\u00A0Baker,\u00C2\u00A0A.\u00C2\u00A0Gurney,\u00C2\u00A0W.\u00C2\u00A0Henzel,\u00C2\u00A0C.\u00C2\u00A0Nelson,\u00C2\u00A0H.B.\u00C2\u00A0 Lowman,\u00C2\u00A0 B.D.\u00C2\u00A0Wright,\u00C2\u00A0 N.J.\u00C2\u00A0 Skelton,\u00C2\u00A0 G.D.\u00C2\u00A0 Frantz,\u00C2\u00A0 D.B.\u00C2\u00A0 Tumas,\u00C2\u00A0 F.V.\u00C2\u00A0 Peale,\u00C2\u00A0 Jr.,\u00C2\u00A0 D.L.\u00C2\u00A0 Shelton,\u00C2\u00A0 and\u00C2\u00A0 C.C.\u00C2\u00A0 Hebert.\u00C2\u00A0 2000.\u00C2\u00A0 FIZZ1,\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 cysteine\u00E2\u0080\u0090rich\u00C2\u00A0 secreted\u00C2\u00A0 protein\u00C2\u00A0 associated\u00C2\u00A0 with\u00C2\u00A0 pulmonary\u00C2\u00A0 inflammation,\u00C2\u00A0 defines\u00C2\u00A0 a\u00C2\u00A0 new\u00C2\u00A0 gene\u00C2\u00A0 family.\u00C2\u00A0 EMBO\u00C2\u00A0 J\u00C2\u00A0 19:4046\u00E2\u0080\u00904055.\u00C2\u00A0 167 Holda,\u00C2\u00A0 J.H.,\u00C2\u00A0 T.\u00C2\u00A0Maier,\u00C2\u00A0 and\u00C2\u00A0H.N.\u00C2\u00A0 Claman.\u00C2\u00A0 1985.\u00C2\u00A0Murine\u00C2\u00A0 graft\u00E2\u0080\u0090versus\u00E2\u0080\u0090host\u00C2\u00A0 disease\u00C2\u00A0 across\u00C2\u00A0 minor\u00C2\u00A0 barriers:\u00C2\u00A0 immunosuppressive\u00C2\u00A0 aspects\u00C2\u00A0 of\u00C2\u00A0 natural\u00C2\u00A0 suppressor\u00C2\u00A0 cells.\u00C2\u00A0 Immunological\u00C2\u00A0reviews\u00C2\u00A088:87\u00E2\u0080\u0090105.\u00C2\u00A0 Hoves,\u00C2\u00A0S.,\u00C2\u00A0S.W.\u00C2\u00A0Krause,\u00C2\u00A0C.\u00C2\u00A0Schutz,\u00C2\u00A0D.\u00C2\u00A0Halbritter,\u00C2\u00A0J.\u00C2\u00A0Scholmerich,\u00C2\u00A0H.\u00C2\u00A0Herfarth,\u00C2\u00A0and\u00C2\u00A0M.\u00C2\u00A0Fleck.\u00C2\u00A0 2006.\u00C2\u00A0 Monocyte\u00E2\u0080\u0090derived\u00C2\u00A0 human\u00C2\u00A0 macrophages\u00C2\u00A0 mediate\u00C2\u00A0 anergy\u00C2\u00A0 in\u00C2\u00A0 allogeneic\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0and\u00C2\u00A0induce\u00C2\u00A0regulatory\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0177:2691\u00E2\u0080\u00902698.\u00C2\u00A0 Huang,\u00C2\u00A0B.,\u00C2\u00A0 P.Y.\u00C2\u00A0Pan,\u00C2\u00A0Q.\u00C2\u00A0 Li,\u00C2\u00A0A.I.\u00C2\u00A0 Sato,\u00C2\u00A0D.E.\u00C2\u00A0 Levy,\u00C2\u00A0 J.\u00C2\u00A0Bromberg,\u00C2\u00A0 C.M.\u00C2\u00A0Divino,\u00C2\u00A0 and\u00C2\u00A0S.H.\u00C2\u00A0Chen.\u00C2\u00A0 2006.\u00C2\u00A0 Gr\u00E2\u0080\u00901+CD115+\u00C2\u00A0 immature\u00C2\u00A0 myeloid\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 mediate\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 T\u00C2\u00A0 regulatory\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 anergy\u00C2\u00A0 in\u00C2\u00A0 tumor\u00E2\u0080\u0090 bearing\u00C2\u00A0host.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A066:1123\u00E2\u0080\u00901131.\u00C2\u00A0 Husemann,\u00C2\u00A0Y.,\u00C2\u00A0J.B.\u00C2\u00A0Geigl,\u00C2\u00A0F.\u00C2\u00A0Schubert,\u00C2\u00A0P.\u00C2\u00A0Musiani,\u00C2\u00A0M.\u00C2\u00A0Meyer,\u00C2\u00A0E.\u00C2\u00A0Burghart,\u00C2\u00A0G.\u00C2\u00A0Forni,\u00C2\u00A0R.\u00C2\u00A0Eils,\u00C2\u00A0 T.\u00C2\u00A0Fehm,\u00C2\u00A0G.\u00C2\u00A0Riethmuller,\u00C2\u00A0and\u00C2\u00A0C.A.\u00C2\u00A0Klein.\u00C2\u00A02008.\u00C2\u00A0Systemic\u00C2\u00A0spread\u00C2\u00A0is\u00C2\u00A0an\u00C2\u00A0early\u00C2\u00A0step\u00C2\u00A0in\u00C2\u00A0 breast\u00C2\u00A0cancer.\u00C2\u00A0Cancer\u00C2\u00A0Cell\u00C2\u00A013:58\u00E2\u0080\u009068.\u00C2\u00A0 Ilkovitch,\u00C2\u00A0 D.,\u00C2\u00A0 and\u00C2\u00A0 D.M.\u00C2\u00A0 Lopez.\u00C2\u00A0 2009.\u00C2\u00A0 The\u00C2\u00A0 liver\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 site\u00C2\u00A0 for\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00E2\u0080\u0090 derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0accumulation\u00C2\u00A0and\u00C2\u00A0immunosuppression.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A0 69:5514\u00E2\u0080\u00905521.\u00C2\u00A0 Inaba,\u00C2\u00A0 K.,\u00C2\u00A0 M.\u00C2\u00A0 Kitaura,\u00C2\u00A0 T.\u00C2\u00A0 Kato,\u00C2\u00A0 Y.\u00C2\u00A0 Watanabe,\u00C2\u00A0 Y.\u00C2\u00A0 Kawade,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Muramatsu.\u00C2\u00A0 1986.\u00C2\u00A0 Contrasting\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 alpha/beta\u00E2\u0080\u0090\u00C2\u00A0 and\u00C2\u00A0 gamma\u00E2\u0080\u0090interferons\u00C2\u00A0 on\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 macrophage\u00C2\u00A0Ia\u00C2\u00A0antigens.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0163:1030\u00E2\u0080\u00901035.\u00C2\u00A0 Joyce,\u00C2\u00A0J.A.,\u00C2\u00A0and\u00C2\u00A0J.W.\u00C2\u00A0Pollard.\u00C2\u00A02009.\u00C2\u00A0Microenvironmental\u00C2\u00A0regulation\u00C2\u00A0of\u00C2\u00A0metastasis.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0 Cancer\u00C2\u00A09:239\u00E2\u0080\u0090252.\u00C2\u00A0 Kaplan,\u00C2\u00A0 R.N.,\u00C2\u00A0 B.\u00C2\u00A0 Psaila,\u00C2\u00A0 and\u00C2\u00A0D.\u00C2\u00A0 Lyden.\u00C2\u00A0 2006.\u00C2\u00A0 Bone\u00C2\u00A0marrow\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 'pre\u00E2\u0080\u0090metastatic\u00C2\u00A0 niche':\u00C2\u00A0within\u00C2\u00A0bone\u00C2\u00A0and\u00C2\u00A0beyond.\u00C2\u00A0Cancer\u00C2\u00A0Metastasis\u00C2\u00A0Rev\u00C2\u00A025:521\u00E2\u0080\u0090529.\u00C2\u00A0 Kaplan,\u00C2\u00A0 R.N.,\u00C2\u00A0 R.D.\u00C2\u00A0 Riba,\u00C2\u00A0 S.\u00C2\u00A0 Zacharoulis,\u00C2\u00A0 A.H.\u00C2\u00A0 Bramley,\u00C2\u00A0 L.\u00C2\u00A0 Vincent,\u00C2\u00A0 C.\u00C2\u00A0 Costa,\u00C2\u00A0 D.D.\u00C2\u00A0 MacDonald,\u00C2\u00A0D.K.\u00C2\u00A0 Jin,\u00C2\u00A0 K.\u00C2\u00A0 Shido,\u00C2\u00A0 S.A.\u00C2\u00A0 Kerns,\u00C2\u00A0 Z.\u00C2\u00A0 Zhu,\u00C2\u00A0D.\u00C2\u00A0Hicklin,\u00C2\u00A0 Y.\u00C2\u00A0Wu,\u00C2\u00A0 J.L.\u00C2\u00A0 Port,\u00C2\u00A0N.\u00C2\u00A0 Altorki,\u00C2\u00A0E.R.\u00C2\u00A0Port,\u00C2\u00A0D.\u00C2\u00A0Ruggero,\u00C2\u00A0S.V.\u00C2\u00A0Shmelkov,\u00C2\u00A0K.K.\u00C2\u00A0 Jensen,\u00C2\u00A0S.\u00C2\u00A0Rafii,\u00C2\u00A0and\u00C2\u00A0D.\u00C2\u00A0Lyden.\u00C2\u00A0 2005.\u00C2\u00A0 VEGFR1\u00E2\u0080\u0090positive\u00C2\u00A0 haematopoietic\u00C2\u00A0 bone\u00C2\u00A0 marrow\u00C2\u00A0 progenitors\u00C2\u00A0 initiate\u00C2\u00A0 the\u00C2\u00A0 pre\u00E2\u0080\u0090metastatic\u00C2\u00A0niche.\u00C2\u00A0Nature\u00C2\u00A0438:820\u00E2\u0080\u0090827.\u00C2\u00A0 Karin,\u00C2\u00A0 M.,\u00C2\u00A0 and\u00C2\u00A0 F.R.\u00C2\u00A0 Greten.\u00C2\u00A0 2005.\u00C2\u00A0 NF\u00E2\u0080\u0090kappaB:\u00C2\u00A0 linking\u00C2\u00A0 inflammation\u00C2\u00A0 and\u00C2\u00A0 immunity\u00C2\u00A0 to\u00C2\u00A0 cancer\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0progression.\u00C2\u00A0Nature\u00C2\u00A0reviews.\u00C2\u00A0Immunology\u00C2\u00A05:749\u00E2\u0080\u0090759.\u00C2\u00A0 Kennedy,\u00C2\u00A0 A.D.,\u00C2\u00A0 and\u00C2\u00A0 F.R.\u00C2\u00A0 DeLeo.\u00C2\u00A0 2009.\u00C2\u00A0 Neutrophil\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 resolution\u00C2\u00A0 of\u00C2\u00A0 infection.\u00C2\u00A0Immunol\u00C2\u00A0Res\u00C2\u00A043:25\u00E2\u0080\u009061.\u00C2\u00A0 Kleinbongard,\u00C2\u00A0P.,\u00C2\u00A0T.\u00C2\u00A0Rassaf,\u00C2\u00A0A.\u00C2\u00A0Dejam,\u00C2\u00A0S.\u00C2\u00A0Kerber,\u00C2\u00A0and\u00C2\u00A0M.\u00C2\u00A0Kelm.\u00C2\u00A02002.\u00C2\u00A0Griess\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0 nitrite\u00C2\u00A0 measurement\u00C2\u00A0 of\u00C2\u00A0 aqueous\u00C2\u00A0 and\u00C2\u00A0 protein\u00E2\u0080\u0090containing\u00C2\u00A0 samples.\u00C2\u00A0 Methods\u00C2\u00A0 Enzymol\u00C2\u00A0359:158\u00E2\u0080\u0090168.\u00C2\u00A0 168 Ko,\u00C2\u00A0J.S.,\u00C2\u00A0P.\u00C2\u00A0Rayman,\u00C2\u00A0J.\u00C2\u00A0Ireland,\u00C2\u00A0S.\u00C2\u00A0Swaidani,\u00C2\u00A0G.\u00C2\u00A0Li,\u00C2\u00A0K.D.\u00C2\u00A0Bunting,\u00C2\u00A0B.\u00C2\u00A0Rini,\u00C2\u00A0J.H.\u00C2\u00A0Finke,\u00C2\u00A0and\u00C2\u00A0P.A.\u00C2\u00A0 Cohen.\u00C2\u00A02010.\u00C2\u00A0Direct\u00C2\u00A0and\u00C2\u00A0differential\u00C2\u00A0suppression\u00C2\u00A0of\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0 cell\u00C2\u00A0 subsets\u00C2\u00A0 by\u00C2\u00A0 sunitinib\u00C2\u00A0 is\u00C2\u00A0 compartmentally\u00C2\u00A0 constrained.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A0 70:3526\u00E2\u0080\u0090 3536.\u00C2\u00A0 Kodumudi,\u00C2\u00A0K.N.,\u00C2\u00A0K.\u00C2\u00A0Woan,\u00C2\u00A0D.L.\u00C2\u00A0Gilvary,\u00C2\u00A0E.\u00C2\u00A0Sahakian,\u00C2\u00A0S.\u00C2\u00A0Wei,\u00C2\u00A0and\u00C2\u00A0J.Y.\u00C2\u00A0Djeu.\u00C2\u00A02010.\u00C2\u00A0A\u00C2\u00A0novel\u00C2\u00A0 chemoimmunomodulating\u00C2\u00A0 property\u00C2\u00A0 of\u00C2\u00A0 docetaxel:\u00C2\u00A0 suppression\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090 derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0bearers.\u00C2\u00A0Clin\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A016:4583\u00E2\u0080\u00904594.\u00C2\u00A0 Kornstein,\u00C2\u00A0 M.J.,\u00C2\u00A0 J.S.\u00C2\u00A0 Brooks,\u00C2\u00A0 and\u00C2\u00A0 D.E.\u00C2\u00A0 Elder.\u00C2\u00A0 1983.\u00C2\u00A0 Immunoperoxidase\u00C2\u00A0 localization\u00C2\u00A0 of\u00C2\u00A0 lymphocyte\u00C2\u00A0subsets\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0host\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0melanoma\u00C2\u00A0and\u00C2\u00A0nevi.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A0 43:2749\u00E2\u0080\u00902753.\u00C2\u00A0 Kryczek,\u00C2\u00A0 I.,\u00C2\u00A0 L.\u00C2\u00A0 Zou,\u00C2\u00A0 P.\u00C2\u00A0 Rodriguez,\u00C2\u00A0 G.\u00C2\u00A0 Zhu,\u00C2\u00A0 S.\u00C2\u00A0Wei,\u00C2\u00A0 P.\u00C2\u00A0Mottram,\u00C2\u00A0M.\u00C2\u00A0 Brumlik,\u00C2\u00A0 P.\u00C2\u00A0 Cheng,\u00C2\u00A0 T.\u00C2\u00A0 Curiel,\u00C2\u00A0L.\u00C2\u00A0Myers,\u00C2\u00A0A.\u00C2\u00A0Lackner,\u00C2\u00A0X.\u00C2\u00A0Alvarez,\u00C2\u00A0A.\u00C2\u00A0Ochoa,\u00C2\u00A0L.\u00C2\u00A0Chen,\u00C2\u00A0and\u00C2\u00A0W.\u00C2\u00A0Zou.\u00C2\u00A02006.\u00C2\u00A0B7\u00E2\u0080\u0090 H4\u00C2\u00A0expression\u00C2\u00A0 identifies\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0suppressive\u00C2\u00A0macrophage\u00C2\u00A0population\u00C2\u00A0 in\u00C2\u00A0human\u00C2\u00A0 ovarian\u00C2\u00A0carcinoma.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0203:871\u00E2\u0080\u0090881.\u00C2\u00A0 Kuang,\u00C2\u00A0 D.M.,\u00C2\u00A0 Q.\u00C2\u00A0 Zhao,\u00C2\u00A0 C.\u00C2\u00A0 Peng,\u00C2\u00A0 J.\u00C2\u00A0 Xu,\u00C2\u00A0 J.P.\u00C2\u00A0 Zhang,\u00C2\u00A0 C.\u00C2\u00A0Wu,\u00C2\u00A0 and\u00C2\u00A0 L.\u00C2\u00A0 Zheng.\u00C2\u00A0 2009.\u00C2\u00A0 Activated\u00C2\u00A0 monocytes\u00C2\u00A0 in\u00C2\u00A0 peritumoral\u00C2\u00A0 stroma\u00C2\u00A0 of\u00C2\u00A0 hepatocellular\u00C2\u00A0 carcinoma\u00C2\u00A0 foster\u00C2\u00A0 immune\u00C2\u00A0 privilege\u00C2\u00A0 and\u00C2\u00A0 disease\u00C2\u00A0 progression\u00C2\u00A0 through\u00C2\u00A0 PD\u00E2\u0080\u0090L1.\u00C2\u00A0The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 experimental\u00C2\u00A0 medicine\u00C2\u00A0206:1327\u00E2\u0080\u00901337.\u00C2\u00A0 Kubota,\u00C2\u00A0Y.,\u00C2\u00A0K.\u00C2\u00A0Takubo,\u00C2\u00A0T.\u00C2\u00A0 Shimizu,\u00C2\u00A0H.\u00C2\u00A0Ohno,\u00C2\u00A0K.\u00C2\u00A0Kishi,\u00C2\u00A0M.\u00C2\u00A0 Shibuya,\u00C2\u00A0H.\u00C2\u00A0 Saya,\u00C2\u00A0 and\u00C2\u00A0T.\u00C2\u00A0 Suda.\u00C2\u00A0 2009.\u00C2\u00A0 M\u00E2\u0080\u0090CSF\u00C2\u00A0 inhibition\u00C2\u00A0 selectively\u00C2\u00A0 targets\u00C2\u00A0 pathological\u00C2\u00A0 angiogenesis\u00C2\u00A0 and\u00C2\u00A0 lymphangiogenesis.\u00C2\u00A0The\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0experimental\u00C2\u00A0medicine\u00C2\u00A0206:1089\u00E2\u0080\u00901102.\u00C2\u00A0 Kung,\u00C2\u00A0J.T.,\u00C2\u00A0S.B.\u00C2\u00A0Brooks,\u00C2\u00A0J.P.\u00C2\u00A0Jakway,\u00C2\u00A0L.L.\u00C2\u00A0Leonard,\u00C2\u00A0and\u00C2\u00A0D.W.\u00C2\u00A0Talmage.\u00C2\u00A01977.\u00C2\u00A0Suppression\u00C2\u00A0of\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0cytotoxic\u00C2\u00A0response\u00C2\u00A0by\u00C2\u00A0macrophages\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0induced\u00C2\u00A0arginase.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0 146:665\u00E2\u0080\u0090672.\u00C2\u00A0 Kuroda,\u00C2\u00A0 E.,\u00C2\u00A0 V.\u00C2\u00A0 Ho,\u00C2\u00A0 J.\u00C2\u00A0 Ruschmann,\u00C2\u00A0 F.\u00C2\u00A0 Antignano,\u00C2\u00A0M.\u00C2\u00A0 Hamilton,\u00C2\u00A0M.J.\u00C2\u00A0 Rauh,\u00C2\u00A0 A.\u00C2\u00A0 Antov,\u00C2\u00A0 R.A.\u00C2\u00A0 Flavell,\u00C2\u00A0 L.M.\u00C2\u00A0 Sly,\u00C2\u00A0 and\u00C2\u00A0 G.\u00C2\u00A0 Krystal.\u00C2\u00A0 2009.\u00C2\u00A0 SHIP\u00C2\u00A0 represses\u00C2\u00A0 the\u00C2\u00A0 generation\u00C2\u00A0 of\u00C2\u00A0 IL\u00E2\u0080\u00903\u00E2\u0080\u0090 induced\u00C2\u00A0 M2\u00C2\u00A0 macrophages\u00C2\u00A0 by\u00C2\u00A0 inhibiting\u00C2\u00A0 IL\u00E2\u0080\u00904\u00C2\u00A0 production\u00C2\u00A0 from\u00C2\u00A0 basophils.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A0183:3652\u00E2\u0080\u00903660.\u00C2\u00A0 Kusmartsev,\u00C2\u00A0S.,\u00C2\u00A0F.\u00C2\u00A0Cheng,\u00C2\u00A0B.\u00C2\u00A0Yu,\u00C2\u00A0Y.\u00C2\u00A0Nefedova,\u00C2\u00A0E.\u00C2\u00A0Sotomayor,\u00C2\u00A0R.\u00C2\u00A0Lush,\u00C2\u00A0and\u00C2\u00A0D.\u00C2\u00A0Gabrilovich.\u00C2\u00A0 2003.\u00C2\u00A0 All\u00E2\u0080\u0090trans\u00E2\u0080\u0090retinoic\u00C2\u00A0 acid\u00C2\u00A0 eliminates\u00C2\u00A0 immature\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 tumor\u00E2\u0080\u0090 bearing\u00C2\u00A0mice\u00C2\u00A0 and\u00C2\u00A0 improves\u00C2\u00A0 the\u00C2\u00A0 effect\u00C2\u00A0 of\u00C2\u00A0 vaccination.\u00C2\u00A0Cancer\u00C2\u00A0 research\u00C2\u00A0 63:4441\u00E2\u0080\u0090 4449.\u00C2\u00A0 Kusmartsev,\u00C2\u00A0S.,\u00C2\u00A0 and\u00C2\u00A0D.I.\u00C2\u00A0Gabrilovich.\u00C2\u00A02003.\u00C2\u00A0 Inhibition\u00C2\u00A0of\u00C2\u00A0myeloid\u00C2\u00A0cell\u00C2\u00A0differentiation\u00C2\u00A0 in\u00C2\u00A0 cancer:\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0reactive\u00C2\u00A0oxygen\u00C2\u00A0species.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0 leukocyte\u00C2\u00A0biology\u00C2\u00A074:186\u00E2\u0080\u0090 196.\u00C2\u00A0 169 Kusmartsev,\u00C2\u00A0 S.,\u00C2\u00A0 Y.\u00C2\u00A0 Nefedova,\u00C2\u00A0 D.\u00C2\u00A0 Yoder,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A0 2004.\u00C2\u00A0 Antigen\u00E2\u0080\u0090specific\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 CD8+\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 response\u00C2\u00A0 by\u00C2\u00A0 immature\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 is\u00C2\u00A0 mediated\u00C2\u00A0by\u00C2\u00A0reactive\u00C2\u00A0oxygen\u00C2\u00A0species.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0172:989\u00E2\u0080\u0090999.\u00C2\u00A0 Kusmartsev,\u00C2\u00A0S.,\u00C2\u00A0Z.\u00C2\u00A0Su,\u00C2\u00A0A.\u00C2\u00A0Heiser,\u00C2\u00A0J.\u00C2\u00A0Dannull,\u00C2\u00A0E.\u00C2\u00A0Eruslanov,\u00C2\u00A0H.\u00C2\u00A0Kubler,\u00C2\u00A0D.\u00C2\u00A0Yancey,\u00C2\u00A0P.\u00C2\u00A0Dahm,\u00C2\u00A0 and\u00C2\u00A0 J.\u00C2\u00A0 Vieweg.\u00C2\u00A0 2008.\u00C2\u00A0Reversal\u00C2\u00A0 of\u00C2\u00A0myeloid\u00C2\u00A0 cell\u00E2\u0080\u0090mediated\u00C2\u00A0 immunosuppression\u00C2\u00A0 in\u00C2\u00A0 patients\u00C2\u00A0with\u00C2\u00A0metastatic\u00C2\u00A0renal\u00C2\u00A0cell\u00C2\u00A0carcinoma.\u00C2\u00A0Clin\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A014:8270\u00E2\u0080\u00908278.\u00C2\u00A0 Laoui,\u00C2\u00A0D.,\u00C2\u00A0E.\u00C2\u00A0Van\u00C2\u00A0Overmeire,\u00C2\u00A0K.\u00C2\u00A0Movahedi,\u00C2\u00A0J.\u00C2\u00A0Van\u00C2\u00A0den\u00C2\u00A0Bossche,\u00C2\u00A0E.\u00C2\u00A0Schouppe,\u00C2\u00A0C.\u00C2\u00A0Mommer,\u00C2\u00A0 A.\u00C2\u00A0 Nikolaou,\u00C2\u00A0 Y.\u00C2\u00A0 Morias,\u00C2\u00A0 P.\u00C2\u00A0 De\u00C2\u00A0 Baetselier,\u00C2\u00A0 and\u00C2\u00A0 J.A.\u00C2\u00A0 Van\u00C2\u00A0 Ginderachter.\u00C2\u00A0 2011.\u00C2\u00A0 Mononuclear\u00C2\u00A0 phagocyte\u00C2\u00A0 heterogeneity\u00C2\u00A0 in\u00C2\u00A0 cancer:\u00C2\u00A0 Different\u00C2\u00A0 subsets\u00C2\u00A0 and\u00C2\u00A0 activation\u00C2\u00A0states\u00C2\u00A0reaching\u00C2\u00A0out\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0site.\u00C2\u00A0Immunobiology\u00C2\u00A0\u00C2\u00A0 Le,\u00C2\u00A0H.K.,\u00C2\u00A0L.\u00C2\u00A0Graham,\u00C2\u00A0E.\u00C2\u00A0Cha,\u00C2\u00A0J.K.\u00C2\u00A0Morales,\u00C2\u00A0M.H.\u00C2\u00A0Manjili,\u00C2\u00A0and\u00C2\u00A0H.D.\u00C2\u00A0Bear.\u00C2\u00A02009.\u00C2\u00A0Gemcitabine\u00C2\u00A0 directly\u00C2\u00A0 inhibits\u00C2\u00A0myeloid\u00C2\u00A0derived\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0BALB/c\u00C2\u00A0mice\u00C2\u00A0bearing\u00C2\u00A04T1\u00C2\u00A0 mammary\u00C2\u00A0 carcinoma\u00C2\u00A0 and\u00C2\u00A0 augments\u00C2\u00A0 expansion\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00C2\u00A0Int\u00C2\u00A0Immunopharmacol\u00C2\u00A09:900\u00E2\u0080\u0090909.\u00C2\u00A0 Leek,\u00C2\u00A0 R.D.,\u00C2\u00A0 C.E.\u00C2\u00A0 Lewis,\u00C2\u00A0 R.\u00C2\u00A0 Whitehouse,\u00C2\u00A0 M.\u00C2\u00A0 Greenall,\u00C2\u00A0 J.\u00C2\u00A0 Clarke,\u00C2\u00A0 and\u00C2\u00A0 A.L.\u00C2\u00A0 Harris.\u00C2\u00A0 1996.\u00C2\u00A0 Association\u00C2\u00A0 of\u00C2\u00A0 macrophage\u00C2\u00A0 infiltration\u00C2\u00A0 with\u00C2\u00A0 angiogenesis\u00C2\u00A0 and\u00C2\u00A0 prognosis\u00C2\u00A0 in\u00C2\u00A0 invasive\u00C2\u00A0breast\u00C2\u00A0carcinoma.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A056:4625\u00E2\u0080\u00904629.\u00C2\u00A0 Levings,\u00C2\u00A0M.K.,\u00C2\u00A0and\u00C2\u00A0M.G.\u00C2\u00A0Roncarolo.\u00C2\u00A02000.\u00C2\u00A0T\u00E2\u0080\u0090regulatory\u00C2\u00A01\u00C2\u00A0cells:\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0subset\u00C2\u00A0of\u00C2\u00A0CD4\u00C2\u00A0T\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A0immunoregulatory\u00C2\u00A0properties.\u00C2\u00A0J\u00C2\u00A0Allergy\u00C2\u00A0Clin\u00C2\u00A0Immunol\u00C2\u00A0106:S109\u00E2\u0080\u0090112.\u00C2\u00A0 Lin,\u00C2\u00A0E.Y.,\u00C2\u00A0A.V.\u00C2\u00A0Nguyen,\u00C2\u00A0R.G.\u00C2\u00A0Russell,\u00C2\u00A0 and\u00C2\u00A0 J.W.\u00C2\u00A0Pollard.\u00C2\u00A02001.\u00C2\u00A0Colony\u00E2\u0080\u0090stimulating\u00C2\u00A0 factor\u00C2\u00A01\u00C2\u00A0 promotes\u00C2\u00A0 progression\u00C2\u00A0 of\u00C2\u00A0 mammary\u00C2\u00A0 tumors\u00C2\u00A0 to\u00C2\u00A0 malignancy.\u00C2\u00A0 The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 experimental\u00C2\u00A0medicine\u00C2\u00A0193:727\u00E2\u0080\u0090740.\u00C2\u00A0 Ling,\u00C2\u00A0P.D.,\u00C2\u00A0M.K.\u00C2\u00A0Warren,\u00C2\u00A0and\u00C2\u00A0S.N.\u00C2\u00A0Vogel.\u00C2\u00A01985.\u00C2\u00A0Antagonistic\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0 interferon\u00E2\u0080\u0090beta\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0interferon\u00E2\u0080\u0090gamma\u00E2\u0080\u0090induced\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0Ia\u00C2\u00A0antigen\u00C2\u00A0in\u00C2\u00A0murine\u00C2\u00A0macrophages.\u00C2\u00A0 J\u00C2\u00A0Immunol\u00C2\u00A0135:1857\u00E2\u0080\u00901863.\u00C2\u00A0 Lorsbach,\u00C2\u00A0R.B.,\u00C2\u00A0and\u00C2\u00A0S.W.\u00C2\u00A0Russell.\u00C2\u00A01992.\u00C2\u00A0A\u00C2\u00A0specific\u00C2\u00A0sequence\u00C2\u00A0of\u00C2\u00A0stimulation\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0 induce\u00C2\u00A0 synthesis\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 antimicrobial\u00C2\u00A0 molecule\u00C2\u00A0 nitric\u00C2\u00A0 oxide\u00C2\u00A0 by\u00C2\u00A0 mouse\u00C2\u00A0 macrophages.\u00C2\u00A0Infect\u00C2\u00A0Immun\u00C2\u00A060:2133\u00E2\u0080\u00902135.\u00C2\u00A0 Luzzi,\u00C2\u00A0K.J.,\u00C2\u00A0I.C.\u00C2\u00A0MacDonald,\u00C2\u00A0E.E.\u00C2\u00A0Schmidt,\u00C2\u00A0N.\u00C2\u00A0Kerkvliet,\u00C2\u00A0V.L.\u00C2\u00A0Morris,\u00C2\u00A0A.F.\u00C2\u00A0Chambers,\u00C2\u00A0and\u00C2\u00A0A.C.\u00C2\u00A0 Groom.\u00C2\u00A01998.\u00C2\u00A0Multistep\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0metastatic\u00C2\u00A0 inefficiency:\u00C2\u00A0 dormancy\u00C2\u00A0of\u00C2\u00A0 solitary\u00C2\u00A0 cells\u00C2\u00A0 after\u00C2\u00A0 successful\u00C2\u00A0 extravasation\u00C2\u00A0 and\u00C2\u00A0 limited\u00C2\u00A0 survival\u00C2\u00A0 of\u00C2\u00A0 early\u00C2\u00A0 micrometastases.\u00C2\u00A0Am\u00C2\u00A0J\u00C2\u00A0Pathol\u00C2\u00A0153:865\u00E2\u0080\u0090873.\u00C2\u00A0 Lyons,\u00C2\u00A0 A.B.\u00C2\u00A0 2000.\u00C2\u00A0 Analysing\u00C2\u00A0 cell\u00C2\u00A0 division\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 using\u00C2\u00A0 flow\u00C2\u00A0 cytometric\u00C2\u00A0 measurement\u00C2\u00A0of\u00C2\u00A0CFSE\u00C2\u00A0dye\u00C2\u00A0dilution.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0Methods\u00C2\u00A0243:147\u00E2\u0080\u0090154.\u00C2\u00A0 170 Mace,\u00C2\u00A0E.M.,\u00C2\u00A0S.J.\u00C2\u00A0Monkley,\u00C2\u00A0D.R.\u00C2\u00A0Critchley,\u00C2\u00A0and\u00C2\u00A0F.\u00C2\u00A0Takei.\u00C2\u00A02009.\u00C2\u00A0A\u00C2\u00A0dual\u00C2\u00A0role\u00C2\u00A0for\u00C2\u00A0talin\u00C2\u00A0in\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0 cytotoxicity:\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0LFA\u00E2\u0080\u00901\u00E2\u0080\u0090mediated\u00C2\u00A0cell\u00C2\u00A0adhesion\u00C2\u00A0and\u00C2\u00A0polarization\u00C2\u00A0of\u00C2\u00A0NK\u00C2\u00A0 cells.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0182:948\u00E2\u0080\u0090956.\u00C2\u00A0 Maltby,\u00C2\u00A0 S.,\u00C2\u00A0 K.\u00C2\u00A0 Khazaie,\u00C2\u00A0 and\u00C2\u00A0 K.M.\u00C2\u00A0 McNagny.\u00C2\u00A0 2009.\u00C2\u00A0 Mast\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 growth:\u00C2\u00A0 angiogenesis,\u00C2\u00A0tissue\u00C2\u00A0remodelling\u00C2\u00A0and\u00C2\u00A0immune\u00E2\u0080\u0090modulation.\u00C2\u00A0Biochim\u00C2\u00A0Biophys\u00C2\u00A0Acta\u00C2\u00A0 1796:19\u00E2\u0080\u009026.\u00C2\u00A0 Mandruzzato,\u00C2\u00A0 S.,\u00C2\u00A0 S.\u00C2\u00A0 Solito,\u00C2\u00A0 E.\u00C2\u00A0 Falisi,\u00C2\u00A0 S.\u00C2\u00A0 Francescato,\u00C2\u00A0 V.\u00C2\u00A0 Chiarion\u00E2\u0080\u0090Sileni,\u00C2\u00A0 S.\u00C2\u00A0 Mocellin,\u00C2\u00A0 A.\u00C2\u00A0 Zanon,\u00C2\u00A0C.R.\u00C2\u00A0Rossi,\u00C2\u00A0D.\u00C2\u00A0Nitti,\u00C2\u00A0V.\u00C2\u00A0Bronte,\u00C2\u00A0and\u00C2\u00A0P.\u00C2\u00A0Zanovello.\u00C2\u00A02009.\u00C2\u00A0IL4Ralpha+\u00C2\u00A0myeloid\u00E2\u0080\u0090 derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0expansion\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0patients.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0182:6562\u00E2\u0080\u00906568.\u00C2\u00A0 Mantovani,\u00C2\u00A0A.\u00C2\u00A02009.\u00C2\u00A0Cancer:\u00C2\u00A0Inflaming\u00C2\u00A0metastasis.\u00C2\u00A0Nature\u00C2\u00A0457:36\u00E2\u0080\u009037.\u00C2\u00A0 Mantovani,\u00C2\u00A0 A.,\u00C2\u00A0 M.A.\u00C2\u00A0 Cassatella,\u00C2\u00A0 C.\u00C2\u00A0 Costantini,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Jaillon.\u00C2\u00A0 2011.\u00C2\u00A0 Neutrophils\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 activation\u00C2\u00A0 and\u00C2\u00A0 regulation\u00C2\u00A0 of\u00C2\u00A0 innate\u00C2\u00A0 and\u00C2\u00A0 adaptive\u00C2\u00A0 immunity.\u00C2\u00A0 Nature\u00C2\u00A0 reviews.\u00C2\u00A0 Immunology\u00C2\u00A011:519\u00E2\u0080\u0090531.\u00C2\u00A0 Mantovani,\u00C2\u00A0 A.,\u00C2\u00A0 and\u00C2\u00A0A.\u00C2\u00A0 Sica.\u00C2\u00A0 2010.\u00C2\u00A0Macrophages,\u00C2\u00A0 innate\u00C2\u00A0 immunity\u00C2\u00A0 and\u00C2\u00A0 cancer:\u00C2\u00A0 balance,\u00C2\u00A0 tolerance,\u00C2\u00A0and\u00C2\u00A0diversity.\u00C2\u00A0Curr\u00C2\u00A0Opin\u00C2\u00A0Immunol\u00C2\u00A022:231\u00E2\u0080\u0090237.\u00C2\u00A0 Mantovani,\u00C2\u00A0 A.,\u00C2\u00A0 A.\u00C2\u00A0 Sica,\u00C2\u00A0 S.\u00C2\u00A0 Sozzani,\u00C2\u00A0 P.\u00C2\u00A0 Allavena,\u00C2\u00A0 A.\u00C2\u00A0 Vecchi,\u00C2\u00A0 and\u00C2\u00A0 M.\u00C2\u00A0 Locati.\u00C2\u00A0 2004.\u00C2\u00A0 The\u00C2\u00A0 chemokine\u00C2\u00A0system\u00C2\u00A0in\u00C2\u00A0diverse\u00C2\u00A0 forms\u00C2\u00A0of\u00C2\u00A0macrophage\u00C2\u00A0activation\u00C2\u00A0and\u00C2\u00A0polarization.\u00C2\u00A0 Trends\u00C2\u00A0Immunol\u00C2\u00A025:677\u00E2\u0080\u0090686.\u00C2\u00A0 Mantovani,\u00C2\u00A0 A.,\u00C2\u00A0 S.\u00C2\u00A0 Sozzani,\u00C2\u00A0 M.\u00C2\u00A0 Locati,\u00C2\u00A0 P.\u00C2\u00A0 Allavena,\u00C2\u00A0 and\u00C2\u00A0 A.\u00C2\u00A0 Sica.\u00C2\u00A0 2002.\u00C2\u00A0 Macrophage\u00C2\u00A0 polarization:\u00C2\u00A0 tumor\u00E2\u0080\u0090associated\u00C2\u00A0 macrophages\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 paradigm\u00C2\u00A0 for\u00C2\u00A0 polarized\u00C2\u00A0 M2\u00C2\u00A0 mononuclear\u00C2\u00A0phagocytes.\u00C2\u00A0Trends\u00C2\u00A0Immunol\u00C2\u00A023:549\u00E2\u0080\u0090555.\u00C2\u00A0 Martin,\u00C2\u00A0C.W.,\u00C2\u00A0and\u00C2\u00A0I.F.\u00C2\u00A0Muir.\u00C2\u00A01990.\u00C2\u00A0The\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0 lymphocytes\u00C2\u00A0in\u00C2\u00A0wound\u00C2\u00A0healing.\u00C2\u00A0Br\u00C2\u00A0 J\u00C2\u00A0Plast\u00C2\u00A0 Surg\u00C2\u00A043:655\u00E2\u0080\u0090662.\u00C2\u00A0 Martinez,\u00C2\u00A0F.O.,\u00C2\u00A0S.\u00C2\u00A0Gordon,\u00C2\u00A0M.\u00C2\u00A0Locati,\u00C2\u00A0and\u00C2\u00A0A.\u00C2\u00A0Mantovani.\u00C2\u00A02006.\u00C2\u00A0Transcriptional\u00C2\u00A0profiling\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 human\u00C2\u00A0 monocyte\u00E2\u0080\u0090to\u00E2\u0080\u0090macrophage\u00C2\u00A0 differentiation\u00C2\u00A0 and\u00C2\u00A0 polarization:\u00C2\u00A0 new\u00C2\u00A0 molecules\u00C2\u00A0 and\u00C2\u00A0 patterns\u00C2\u00A0 of\u00C2\u00A0 gene\u00C2\u00A0 expression.\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 immunology\u00C2\u00A0 177:7303\u00E2\u0080\u0090 7311.\u00C2\u00A0 Martinez,\u00C2\u00A0 F.O.,\u00C2\u00A0 A.\u00C2\u00A0 Sica,\u00C2\u00A0 A.\u00C2\u00A0Mantovani,\u00C2\u00A0 and\u00C2\u00A0M.\u00C2\u00A0 Locati.\u00C2\u00A0 2008.\u00C2\u00A0Macrophage\u00C2\u00A0 activation\u00C2\u00A0 and\u00C2\u00A0 polarization.\u00C2\u00A0Front\u00C2\u00A0Biosci\u00C2\u00A013:453\u00E2\u0080\u0090461.\u00C2\u00A0 Matzinger,\u00C2\u00A0 P.\u00C2\u00A0 1994.\u00C2\u00A0 Tolerance,\u00C2\u00A0 danger,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 extended\u00C2\u00A0 family.\u00C2\u00A0 Annual\u00C2\u00A0 review\u00C2\u00A0 of\u00C2\u00A0 immunology\u00C2\u00A012:991\u00E2\u0080\u00901045.\u00C2\u00A0 McGrath,\u00C2\u00A0 M.S.,\u00C2\u00A0 and\u00C2\u00A0 V.\u00C2\u00A0 Kodelja.\u00C2\u00A0 1999.\u00C2\u00A0 Balanced\u00C2\u00A0 macrophage\u00C2\u00A0 activation\u00C2\u00A0 hypothesis:\u00C2\u00A0 a\u00C2\u00A0 biological\u00C2\u00A0 model\u00C2\u00A0 for\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 drugs\u00C2\u00A0 targeted\u00C2\u00A0 at\u00C2\u00A0macrophage\u00C2\u00A0 functional\u00C2\u00A0 states.\u00C2\u00A0Pathobiology\u00C2\u00A067:277\u00E2\u0080\u0090281.\u00C2\u00A0 171 McKee,\u00C2\u00A0 A.S.,\u00C2\u00A0 M.W.\u00C2\u00A0Munks,\u00C2\u00A0 and\u00C2\u00A0 P.\u00C2\u00A0Marrack.\u00C2\u00A0 2007.\u00C2\u00A0 How\u00C2\u00A0 do\u00C2\u00A0 adjuvants\u00C2\u00A0work?\u00C2\u00A0 Important\u00C2\u00A0 considerations\u00C2\u00A0for\u00C2\u00A0new\u00C2\u00A0generation\u00C2\u00A0adjuvants.\u00C2\u00A0Immunity\u00C2\u00A027:687\u00E2\u0080\u0090690.\u00C2\u00A0 Metzger,\u00C2\u00A0Z.,\u00C2\u00A0J.T.\u00C2\u00A0Hoffeld,\u00C2\u00A0and\u00C2\u00A0J.J.\u00C2\u00A0Oppenheim.\u00C2\u00A01980.\u00C2\u00A0Macrophage\u00E2\u0080\u0090mediated\u00C2\u00A0suppression.\u00C2\u00A0I.\u00C2\u00A0 Evidence\u00C2\u00A0 for\u00C2\u00A0 participation\u00C2\u00A0 of\u00C2\u00A0 both\u00C2\u00A0 hdyrogen\u00C2\u00A0 peroxide\u00C2\u00A0 and\u00C2\u00A0 prostaglandins\u00C2\u00A0 in\u00C2\u00A0 suppression\u00C2\u00A0of\u00C2\u00A0murine\u00C2\u00A0lymphocyte\u00C2\u00A0proliferation.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0124:983\u00E2\u0080\u0090988.\u00C2\u00A0 Miller,\u00C2\u00A0 F.R.,\u00C2\u00A0 B.E.\u00C2\u00A0 Miller,\u00C2\u00A0 and\u00C2\u00A0 G.H.\u00C2\u00A0 Heppner.\u00C2\u00A0 1983.\u00C2\u00A0 Characterization\u00C2\u00A0 of\u00C2\u00A0 metastatic\u00C2\u00A0 heterogeneity\u00C2\u00A0 among\u00C2\u00A0 subpopulations\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 single\u00C2\u00A0 mouse\u00C2\u00A0 mammary\u00C2\u00A0 tumor:\u00C2\u00A0 heterogeneity\u00C2\u00A0in\u00C2\u00A0phenotypic\u00C2\u00A0stability.\u00C2\u00A0Invasion\u00C2\u00A0Metastasis\u00C2\u00A03:22\u00E2\u0080\u009031.\u00C2\u00A0 Mirza,\u00C2\u00A0N.,\u00C2\u00A0M.\u00C2\u00A0Fishman,\u00C2\u00A0I.\u00C2\u00A0Fricke,\u00C2\u00A0M.\u00C2\u00A0Dunn,\u00C2\u00A0A.M.\u00C2\u00A0Neuger,\u00C2\u00A0T.J.\u00C2\u00A0Frost,\u00C2\u00A0R.M.\u00C2\u00A0Lush,\u00C2\u00A0S.\u00C2\u00A0Antonia,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A0 2006.\u00C2\u00A0 All\u00E2\u0080\u0090trans\u00E2\u0080\u0090retinoic\u00C2\u00A0 acid\u00C2\u00A0 improves\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0immune\u00C2\u00A0response\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0patients.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A066:9299\u00E2\u0080\u0090 9307.\u00C2\u00A0 Monette,\u00C2\u00A0F.C.,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Sigounas.\u00C2\u00A01990.\u00C2\u00A0Some\u00C2\u00A0observations\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0growth\u00C2\u00A0requirements\u00C2\u00A0of\u00C2\u00A0 multi\u00E2\u0080\u0090potent\u00C2\u00A0 stem\u00C2\u00A0 cells\u00C2\u00A0 under\u00C2\u00A0 defined\u00C2\u00A0 culture\u00C2\u00A0 conditions.\u00C2\u00A0 In\u00C2\u00A0 The\u00C2\u00A0 Biology\u00C2\u00A0 of\u00C2\u00A0 Hematopoiesis.\u00C2\u00A0 N.\u00C2\u00A0 Dainiak,\u00C2\u00A0 E.P.\u00C2\u00A0 Cronkite,\u00C2\u00A0 R.\u00C2\u00A0 McCaffrey,\u00C2\u00A0 and\u00C2\u00A0 R.K.\u00C2\u00A0 Shadduck,\u00C2\u00A0 editors.\u00C2\u00A0Wiley\u00E2\u0080\u0090Liss,\u00C2\u00A0Inc.,\u00C2\u00A0New\u00C2\u00A0York.\u00C2\u00A037\u00E2\u0080\u009048.\u00C2\u00A0 Moresco,\u00C2\u00A0E.M.,\u00C2\u00A0D.\u00C2\u00A0 Lavine,\u00C2\u00A0 and\u00C2\u00A0B.\u00C2\u00A0Beutler.\u00C2\u00A0 2011.\u00C2\u00A0Toll\u00E2\u0080\u0090like\u00C2\u00A0 receptors.\u00C2\u00A0Curr\u00C2\u00A0Biol\u00C2\u00A0 21:R488\u00E2\u0080\u0090 493.\u00C2\u00A0 Morrison,\u00C2\u00A0D.C.,\u00C2\u00A0and\u00C2\u00A0D.M.\u00C2\u00A0Jacobs.\u00C2\u00A01976.\u00C2\u00A0Binding\u00C2\u00A0of\u00C2\u00A0polymyxin\u00C2\u00A0B\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0lipid\u00C2\u00A0A\u00C2\u00A0portion\u00C2\u00A0of\u00C2\u00A0 bacterial\u00C2\u00A0lipopolysaccharides.\u00C2\u00A0Immunochemistry\u00C2\u00A013:813\u00E2\u0080\u0090818.\u00C2\u00A0 Mosser,\u00C2\u00A0 D.M.,\u00C2\u00A0 and\u00C2\u00A0 J.P.\u00C2\u00A0 Edwards.\u00C2\u00A0 2008.\u00C2\u00A0 Exploring\u00C2\u00A0 the\u00C2\u00A0 full\u00C2\u00A0 spectrum\u00C2\u00A0 of\u00C2\u00A0 macrophage\u00C2\u00A0 activation.\u00C2\u00A0Nature\u00C2\u00A0reviews.\u00C2\u00A0Immunology\u00C2\u00A08:958\u00E2\u0080\u0090969.\u00C2\u00A0 Movahedi,\u00C2\u00A0 K.,\u00C2\u00A0 M.\u00C2\u00A0 Guilliams,\u00C2\u00A0 J.\u00C2\u00A0 Van\u00C2\u00A0 den\u00C2\u00A0 Bossche,\u00C2\u00A0 R.\u00C2\u00A0 Van\u00C2\u00A0 den\u00C2\u00A0 Bergh,\u00C2\u00A0 C.\u00C2\u00A0 Gysemans,\u00C2\u00A0 A.\u00C2\u00A0 Beschin,\u00C2\u00A0 P.\u00C2\u00A0 De\u00C2\u00A0 Baetselier,\u00C2\u00A0 and\u00C2\u00A0 J.A.\u00C2\u00A0 Van\u00C2\u00A0 Ginderachter.\u00C2\u00A0 2008.\u00C2\u00A0 Identification\u00C2\u00A0 of\u00C2\u00A0 discrete\u00C2\u00A0 tumor\u00E2\u0080\u0090induced\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cell\u00C2\u00A0 subpopulations\u00C2\u00A0 with\u00C2\u00A0 distinct\u00C2\u00A0T\u00C2\u00A0cell\u00E2\u0080\u0090suppressive\u00C2\u00A0activity.\u00C2\u00A0Blood\u00C2\u00A0111:4233\u00E2\u0080\u00904244.\u00C2\u00A0 Movahedi,\u00C2\u00A0K.,\u00C2\u00A0D.\u00C2\u00A0Laoui,\u00C2\u00A0C.\u00C2\u00A0Gysemans,\u00C2\u00A0M.\u00C2\u00A0Baeten,\u00C2\u00A0G.\u00C2\u00A0Stange,\u00C2\u00A0J.\u00C2\u00A0Van\u00C2\u00A0den\u00C2\u00A0Bossche,\u00C2\u00A0M.\u00C2\u00A0Mack,\u00C2\u00A0 D.\u00C2\u00A0 Pipeleers,\u00C2\u00A0 P.\u00C2\u00A0 In't\u00C2\u00A0 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X.\u00C2\u00A0 Wang,\u00C2\u00A0 A.A.\u00C2\u00A0 Beg,\u00C2\u00A0 and\u00C2\u00A0 D.I.\u00C2\u00A0 Gabrilovich.\u00C2\u00A0 2007.\u00C2\u00A0 Mechanism\u00C2\u00A0of\u00C2\u00A0all\u00E2\u0080\u0090trans\u00C2\u00A0retinoic\u00C2\u00A0acid\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0tumor\u00E2\u0080\u0090associated\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0cells.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A067:11021\u00E2\u0080\u009011028.\u00C2\u00A0 Nurieva,\u00C2\u00A0 R.I.,\u00C2\u00A0 X.\u00C2\u00A0 Liu,\u00C2\u00A0 and\u00C2\u00A0 C.\u00C2\u00A0 Dong.\u00C2\u00A0 2011.\u00C2\u00A0 Molecular\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 tolerance.\u00C2\u00A0 Immunological\u00C2\u00A0reviews\u00C2\u00A0241:133\u00E2\u0080\u0090144.\u00C2\u00A0 Ochoa,\u00C2\u00A0A.C.,\u00C2\u00A0A.H.\u00C2\u00A0Zea,\u00C2\u00A0C.\u00C2\u00A0Hernandez,\u00C2\u00A0and\u00C2\u00A0P.C.\u00C2\u00A0Rodriguez.\u00C2\u00A02007.\u00C2\u00A0Arginase,\u00C2\u00A0prostaglandins,\u00C2\u00A0 and\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 renal\u00C2\u00A0 cell\u00C2\u00A0 carcinoma.\u00C2\u00A0 Clin\u00C2\u00A0 Cancer\u00C2\u00A0 Res\u00C2\u00A0 13:721s\u00E2\u0080\u0090726s.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A0 S.\u00C2\u00A0 2010.\u00C2\u00A0Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells:\u00C2\u00A0more\u00C2\u00A0mechanisms\u00C2\u00A0 for\u00C2\u00A0 inhibiting\u00C2\u00A0antitumor\u00C2\u00A0immunity.\u00C2\u00A0Cancer\u00C2\u00A0Immunol\u00C2\u00A0Immunother\u00C2\u00A059:1593\u00E2\u0080\u00901600.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A0 S.,\u00C2\u00A0 and\u00C2\u00A0 P.\u00C2\u00A0 Sinha.\u00C2\u00A0 2009.\u00C2\u00A0 Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells:\u00C2\u00A0 linking\u00C2\u00A0 inflammation\u00C2\u00A0and\u00C2\u00A0cancer.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0182:4499\u00E2\u0080\u00904506.\u00C2\u00A0 Paget,\u00C2\u00A0S.\u00C2\u00A01889.\u00C2\u00A0The\u00C2\u00A0distribution\u00C2\u00A0of\u00C2\u00A0secondary\u00C2\u00A0growths\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0breast.\u00C2\u00A0The\u00C2\u00A0Lancet\u00C2\u00A0 133:571\u00E2\u0080\u0090573.\u00C2\u00A0 Parkin,\u00C2\u00A0 J.,\u00C2\u00A0 and\u00C2\u00A0B.\u00C2\u00A0 Cohen.\u00C2\u00A0 2001.\u00C2\u00A0An\u00C2\u00A0 overview\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system.\u00C2\u00A0Lancet\u00C2\u00A0 357:1777\u00E2\u0080\u0090 1789.\u00C2\u00A0 Pastula,\u00C2\u00A0 A.,\u00C2\u00A0 and\u00C2\u00A0 J.\u00C2\u00A0 Marcinkiewicz.\u00C2\u00A0 2011.\u00C2\u00A0 Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells:\u00C2\u00A0 a\u00C2\u00A0 double\u00E2\u0080\u0090 edged\u00C2\u00A0sword?\u00C2\u00A0Int\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Pathol\u00C2\u00A092:73\u00E2\u0080\u009078.\u00C2\u00A0 Peranzoni,\u00C2\u00A0E.,\u00C2\u00A0S.\u00C2\u00A0Zilio,\u00C2\u00A0I.\u00C2\u00A0Marigo,\u00C2\u00A0L.\u00C2\u00A0Dolcetti,\u00C2\u00A0P.\u00C2\u00A0Zanovello,\u00C2\u00A0S.\u00C2\u00A0Mandruzzato,\u00C2\u00A0and\u00C2\u00A0V.\u00C2\u00A0Bronte.\u00C2\u00A0 2010.\u00C2\u00A0Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cell\u00C2\u00A0heterogeneity\u00C2\u00A0and\u00C2\u00A0subset\u00C2\u00A0definition.\u00C2\u00A0Curr\u00C2\u00A0 Opin\u00C2\u00A0Immunol\u00C2\u00A022:238\u00E2\u0080\u0090244.\u00C2\u00A0 173 Pollard,\u00C2\u00A0 J.W.\u00C2\u00A02009.\u00C2\u00A0Trophic\u00C2\u00A0macrophages\u00C2\u00A0 in\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0disease.\u00C2\u00A0Nature\u00C2\u00A0reviews.\u00C2\u00A0 Immunology\u00C2\u00A09:259\u00E2\u0080\u0090270.\u00C2\u00A0 Pompella,\u00C2\u00A0 A.,\u00C2\u00A0 A.\u00C2\u00A0 Visvikis,\u00C2\u00A0 A.\u00C2\u00A0 Paolicchi,\u00C2\u00A0 V.\u00C2\u00A0 De\u00C2\u00A0Tata,\u00C2\u00A0 and\u00C2\u00A0A.F.\u00C2\u00A0 Casini.\u00C2\u00A0 2003.\u00C2\u00A0 The\u00C2\u00A0 changing\u00C2\u00A0 faces\u00C2\u00A0of\u00C2\u00A0glutathione,\u00C2\u00A0a\u00C2\u00A0cellular\u00C2\u00A0protagonist.\u00C2\u00A0Biochem\u00C2\u00A0Pharmacol\u00C2\u00A066:1499\u00E2\u0080\u00901503.\u00C2\u00A0 Porta,\u00C2\u00A0 C.,\u00C2\u00A0M.\u00C2\u00A0 Rimoldi,\u00C2\u00A0 G.\u00C2\u00A0 Raes,\u00C2\u00A0 L.\u00C2\u00A0 Brys,\u00C2\u00A0 P.\u00C2\u00A0 Ghezzi,\u00C2\u00A0 D.\u00C2\u00A0 Di\u00C2\u00A0 Liberto,\u00C2\u00A0 F.\u00C2\u00A0 Dieli,\u00C2\u00A0 S.\u00C2\u00A0 Ghisletti,\u00C2\u00A0 G.\u00C2\u00A0 Natoli,\u00C2\u00A0 P.\u00C2\u00A0 De\u00C2\u00A0 Baetselier,\u00C2\u00A0 A.\u00C2\u00A0 Mantovani,\u00C2\u00A0 and\u00C2\u00A0 A.\u00C2\u00A0 Sica.\u00C2\u00A0 2009.\u00C2\u00A0 Tolerance\u00C2\u00A0 and\u00C2\u00A0 M2\u00C2\u00A0 (alternative)\u00C2\u00A0macrophage\u00C2\u00A0polarization\u00C2\u00A0are\u00C2\u00A0related\u00C2\u00A0processes\u00C2\u00A0orchestrated\u00C2\u00A0by\u00C2\u00A0p50\u00C2\u00A0 nuclear\u00C2\u00A0factor\u00C2\u00A0kappaB.\u00C2\u00A0Proc\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0U\u00C2\u00A0S\u00C2\u00A0A\u00C2\u00A0106:14978\u00E2\u0080\u009014983.\u00C2\u00A0 Prabhu,\u00C2\u00A0 V.,\u00C2\u00A0 and\u00C2\u00A0 C.\u00C2\u00A0 Guruvayoorappan.\u00C2\u00A0 2010.\u00C2\u00A0 Nitric\u00C2\u00A0 oxide:\u00C2\u00A0 pros\u00C2\u00A0 and\u00C2\u00A0 cons\u00C2\u00A0 in\u00C2\u00A0 tumor\u00C2\u00A0 progression.\u00C2\u00A0Immunopharmacol\u00C2\u00A0Immunotoxicol\u00C2\u00A032:387\u00E2\u0080\u0090392.\u00C2\u00A0 Prehn,\u00C2\u00A0R.T.,\u00C2\u00A0and\u00C2\u00A0J.M.\u00C2\u00A0Main.\u00C2\u00A01957.\u00C2\u00A0Immunity\u00C2\u00A0to\u00C2\u00A0methylcholanthrene\u00E2\u0080\u0090induced\u00C2\u00A0sarcomas.\u00C2\u00A0J\u00C2\u00A0 Natl\u00C2\u00A0Cancer\u00C2\u00A0Inst\u00C2\u00A018:769\u00E2\u0080\u0090778.\u00C2\u00A0 Priceman,\u00C2\u00A0S.J.,\u00C2\u00A0J.L.\u00C2\u00A0Sung,\u00C2\u00A0Z.\u00C2\u00A0Shaposhnik,\u00C2\u00A0J.B.\u00C2\u00A0Burton,\u00C2\u00A0A.X.\u00C2\u00A0Torres\u00E2\u0080\u0090Collado,\u00C2\u00A0D.L.\u00C2\u00A0Moughon,\u00C2\u00A0M.\u00C2\u00A0 Johnson,\u00C2\u00A0 A.J.\u00C2\u00A0 Lusis,\u00C2\u00A0 D.A.\u00C2\u00A0 Cohen,\u00C2\u00A0M.L.\u00C2\u00A0 Iruela\u00E2\u0080\u0090Arispe,\u00C2\u00A0 and\u00C2\u00A0 L.\u00C2\u00A0Wu.\u00C2\u00A0 2010.\u00C2\u00A0 Targeting\u00C2\u00A0 distinct\u00C2\u00A0tumor\u00E2\u0080\u0090infiltrating\u00C2\u00A0myeloid\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0inhibiting\u00C2\u00A0CSF\u00E2\u0080\u00901\u00C2\u00A0receptor:\u00C2\u00A0combating\u00C2\u00A0 tumor\u00C2\u00A0evasion\u00C2\u00A0of\u00C2\u00A0antiangiogenic\u00C2\u00A0therapy.\u00C2\u00A0Blood\u00C2\u00A0115:1461\u00E2\u0080\u00901471.\u00C2\u00A0 Psaila,\u00C2\u00A0B.,\u00C2\u00A0R.N.\u00C2\u00A0Kaplan,\u00C2\u00A0E.R.\u00C2\u00A0Port,\u00C2\u00A0and\u00C2\u00A0D.\u00C2\u00A0Lyden.\u00C2\u00A02006.\u00C2\u00A0Priming\u00C2\u00A0the\u00C2\u00A0'soil'\u00C2\u00A0for\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0 metastasis:\u00C2\u00A0the\u00C2\u00A0pre\u00E2\u0080\u0090metastatic\u00C2\u00A0niche.\u00C2\u00A0Breast\u00C2\u00A0Dis\u00C2\u00A026:65\u00E2\u0080\u009074.\u00C2\u00A0 Psaila,\u00C2\u00A0B.,\u00C2\u00A0and\u00C2\u00A0D.\u00C2\u00A0Lyden.\u00C2\u00A02009.\u00C2\u00A0The\u00C2\u00A0metastatic\u00C2\u00A0niche:\u00C2\u00A0adapting\u00C2\u00A0the\u00C2\u00A0foreign\u00C2\u00A0soil.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0 Cancer\u00C2\u00A09:285\u00E2\u0080\u0090293.\u00C2\u00A0 Pulaski,\u00C2\u00A0 B.A.,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A0 2001.\u00C2\u00A0 Mouse\u00C2\u00A0 4T1\u00C2\u00A0 breast\u00C2\u00A0 tumor\u00C2\u00A0 model.\u00C2\u00A0 Curr\u00C2\u00A0 Protoc\u00C2\u00A0Immunol\u00C2\u00A0Chapter\u00C2\u00A020:Unit\u00C2\u00A020\u00C2\u00A022.\u00C2\u00A0 Punturieri,\u00C2\u00A0A.,\u00C2\u00A0R.S.\u00C2\u00A0Alviani,\u00C2\u00A0T.\u00C2\u00A0Polak,\u00C2\u00A0P.\u00C2\u00A0Copper,\u00C2\u00A0J.\u00C2\u00A0Sonstein,\u00C2\u00A0and\u00C2\u00A0J.L.\u00C2\u00A0Curtis.\u00C2\u00A02004.\u00C2\u00A0Specific\u00C2\u00A0 engagement\u00C2\u00A0of\u00C2\u00A0TLR4\u00C2\u00A0or\u00C2\u00A0TLR3\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0 lead\u00C2\u00A0to\u00C2\u00A0IFN\u00E2\u0080\u0090beta\u00E2\u0080\u0090mediated\u00C2\u00A0innate\u00C2\u00A0signal\u00C2\u00A0 amplification\u00C2\u00A0 and\u00C2\u00A0 STAT1\u00C2\u00A0 phosphorylation\u00C2\u00A0 in\u00C2\u00A0 resident\u00C2\u00A0 murine\u00C2\u00A0 alveolar\u00C2\u00A0 macrophages.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0173:1033\u00E2\u0080\u00901042.\u00C2\u00A0 Qian,\u00C2\u00A0B.,\u00C2\u00A0Y.\u00C2\u00A0Deng,\u00C2\u00A0 J.H.\u00C2\u00A0Im,\u00C2\u00A0R.J.\u00C2\u00A0Muschel,\u00C2\u00A0Y.\u00C2\u00A0Zou,\u00C2\u00A0 J.\u00C2\u00A0Li,\u00C2\u00A0R.A.\u00C2\u00A0Lang,\u00C2\u00A0and\u00C2\u00A0J.W.\u00C2\u00A0Pollard.\u00C2\u00A02009.\u00C2\u00A0A\u00C2\u00A0 distinct\u00C2\u00A0 macrophage\u00C2\u00A0 population\u00C2\u00A0 mediates\u00C2\u00A0 metastatic\u00C2\u00A0 breast\u00C2\u00A0 cancer\u00C2\u00A0 cell\u00C2\u00A0 extravasation,\u00C2\u00A0establishment\u00C2\u00A0and\u00C2\u00A0growth.\u00C2\u00A0PLoS\u00C2\u00A0One\u00C2\u00A04:e6562.\u00C2\u00A0 Qian,\u00C2\u00A0B.Z.,\u00C2\u00A0 J.\u00C2\u00A0Li,\u00C2\u00A0H.\u00C2\u00A0Zhang,\u00C2\u00A0T.\u00C2\u00A0Kitamura,\u00C2\u00A0 J.\u00C2\u00A0Zhang,\u00C2\u00A0L.R.\u00C2\u00A0Campion,\u00C2\u00A0E.A.\u00C2\u00A0Kaiser,\u00C2\u00A0L.A.\u00C2\u00A0Snyder,\u00C2\u00A0 and\u00C2\u00A0 J.W.\u00C2\u00A0 Pollard.\u00C2\u00A0 2011.\u00C2\u00A0 CCL2\u00C2\u00A0 recruits\u00C2\u00A0 inflammatory\u00C2\u00A0 monocytes\u00C2\u00A0 to\u00C2\u00A0 facilitate\u00C2\u00A0 breast\u00E2\u0080\u0090tumour\u00C2\u00A0metastasis.\u00C2\u00A0Nature\u00C2\u00A0475:222\u00E2\u0080\u0090225.\u00C2\u00A0 Qian,\u00C2\u00A0 B.Z.,\u00C2\u00A0 and\u00C2\u00A0 J.W.\u00C2\u00A0 Pollard.\u00C2\u00A0 2010.\u00C2\u00A0Macrophage\u00C2\u00A0 diversity\u00C2\u00A0 enhances\u00C2\u00A0 tumor\u00C2\u00A0 progression\u00C2\u00A0 and\u00C2\u00A0metastasis.\u00C2\u00A0Cell\u00C2\u00A0141:39\u00E2\u0080\u009051.\u00C2\u00A0 174 Raes,\u00C2\u00A0G.,\u00C2\u00A0P.\u00C2\u00A0De\u00C2\u00A0Baetselier,\u00C2\u00A0W.\u00C2\u00A0Noel,\u00C2\u00A0A.\u00C2\u00A0Beschin,\u00C2\u00A0F.\u00C2\u00A0Brombacher,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Hassanzadeh\u00C2\u00A0Gh.\u00C2\u00A0 2002.\u00C2\u00A0Differential\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0FIZZ1\u00C2\u00A0and\u00C2\u00A0Ym1\u00C2\u00A0in\u00C2\u00A0alternatively\u00C2\u00A0versus\u00C2\u00A0classically\u00C2\u00A0 activated\u00C2\u00A0macrophages.\u00C2\u00A0J\u00C2\u00A0Leukoc\u00C2\u00A0Biol\u00C2\u00A071:597\u00E2\u0080\u0090602.\u00C2\u00A0 Rauh,\u00C2\u00A0M.J.,\u00C2\u00A0V.\u00C2\u00A0Ho,\u00C2\u00A0C.\u00C2\u00A0Pereira,\u00C2\u00A0A.\u00C2\u00A0Sham,\u00C2\u00A0L.M.\u00C2\u00A0Sly,\u00C2\u00A0V.\u00C2\u00A0Lam,\u00C2\u00A0L.\u00C2\u00A0Huxham,\u00C2\u00A0A.I.\u00C2\u00A0Minchinton,\u00C2\u00A0A.\u00C2\u00A0Mui,\u00C2\u00A0 and\u00C2\u00A0 G.\u00C2\u00A0 Krystal.\u00C2\u00A0 2005.\u00C2\u00A0 SHIP\u00C2\u00A0 represses\u00C2\u00A0 the\u00C2\u00A0 generation\u00C2\u00A0 of\u00C2\u00A0 alternatively\u00C2\u00A0 activated\u00C2\u00A0 macrophages.\u00C2\u00A0Immunity\u00C2\u00A023:361\u00E2\u0080\u0090374.\u00C2\u00A0 Rayamajhi,\u00C2\u00A0M.,\u00C2\u00A0J.\u00C2\u00A0Humann,\u00C2\u00A0K.\u00C2\u00A0Penheiter,\u00C2\u00A0K.\u00C2\u00A0Andreasen,\u00C2\u00A0and\u00C2\u00A0L.L.\u00C2\u00A0Lenz.\u00C2\u00A02010.\u00C2\u00A0Induction\u00C2\u00A0of\u00C2\u00A0 IFN\u00E2\u0080\u0090alphabeta\u00C2\u00A0 enables\u00C2\u00A0 Listeria\u00C2\u00A0 monocytogenes\u00C2\u00A0 to\u00C2\u00A0 suppress\u00C2\u00A0 macrophage\u00C2\u00A0 activation\u00C2\u00A0by\u00C2\u00A0IFN\u00E2\u0080\u0090gamma.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0207:327\u00E2\u0080\u0090337.\u00C2\u00A0 Roche,\u00C2\u00A0 M.,\u00C2\u00A0 P.\u00C2\u00A0 Rondeau,\u00C2\u00A0 N.R.\u00C2\u00A0 Singh,\u00C2\u00A0 E.\u00C2\u00A0 Tarnus,\u00C2\u00A0 and\u00C2\u00A0 E.\u00C2\u00A0 Bourdon.\u00C2\u00A0 2008.\u00C2\u00A0 The\u00C2\u00A0 antioxidant\u00C2\u00A0 properties\u00C2\u00A0of\u00C2\u00A0serum\u00C2\u00A0albumin.\u00C2\u00A0FEBS\u00C2\u00A0letters\u00C2\u00A0582:1783\u00E2\u0080\u00901787.\u00C2\u00A0 Rodriguez,\u00C2\u00A0 P.C.,\u00C2\u00A0 C.P.\u00C2\u00A0 Hernandez,\u00C2\u00A0 D.\u00C2\u00A0 Quiceno,\u00C2\u00A0 S.M.\u00C2\u00A0 Dubinett,\u00C2\u00A0 J.\u00C2\u00A0 Zabaleta,\u00C2\u00A0 J.B.\u00C2\u00A0 Ochoa,\u00C2\u00A0 J.\u00C2\u00A0 Gilbert,\u00C2\u00A0and\u00C2\u00A0A.C.\u00C2\u00A0Ochoa.\u00C2\u00A02005.\u00C2\u00A0Arginase\u00C2\u00A0I\u00C2\u00A0in\u00C2\u00A0myeloid\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0induced\u00C2\u00A0 by\u00C2\u00A0COX\u00E2\u0080\u00902\u00C2\u00A0in\u00C2\u00A0lung\u00C2\u00A0carcinoma.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0202:931\u00E2\u0080\u0090939.\u00C2\u00A0 Rodriguez,\u00C2\u00A0 P.C.,\u00C2\u00A0 A.H.\u00C2\u00A0 Zea,\u00C2\u00A0 K.S.\u00C2\u00A0 Culotta,\u00C2\u00A0 J.\u00C2\u00A0 Zabaleta,\u00C2\u00A0 J.B.\u00C2\u00A0 Ochoa,\u00C2\u00A0 and\u00C2\u00A0 A.C.\u00C2\u00A0 Ochoa.\u00C2\u00A0 2002.\u00C2\u00A0 Regulation\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0receptor\u00C2\u00A0CD3zeta\u00C2\u00A0chain\u00C2\u00A0expression\u00C2\u00A0by\u00C2\u00A0L\u00E2\u0080\u0090arginine.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A0 277:21123\u00E2\u0080\u009021129.\u00C2\u00A0 Rodriguez,\u00C2\u00A0P.C.,\u00C2\u00A0A.H.\u00C2\u00A0Zea,\u00C2\u00A0J.\u00C2\u00A0DeSalvo,\u00C2\u00A0K.S.\u00C2\u00A0Culotta,\u00C2\u00A0J.\u00C2\u00A0Zabaleta,\u00C2\u00A0D.G.\u00C2\u00A0Quiceno,\u00C2\u00A0J.B.\u00C2\u00A0Ochoa,\u00C2\u00A0and\u00C2\u00A0 A.C.\u00C2\u00A0 Ochoa.\u00C2\u00A0 2003.\u00C2\u00A0 L\u00E2\u0080\u0090arginine\u00C2\u00A0 consumption\u00C2\u00A0 by\u00C2\u00A0 macrophages\u00C2\u00A0 modulates\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0CD3\u00C2\u00A0zeta\u00C2\u00A0chain\u00C2\u00A0in\u00C2\u00A0T\u00C2\u00A0lymphocytes.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0171:1232\u00E2\u0080\u00901239.\u00C2\u00A0 Sansone,\u00C2\u00A0P.,\u00C2\u00A0and\u00C2\u00A0J.\u00C2\u00A0Bromberg.\u00C2\u00A02011.\u00C2\u00A0Environment,\u00C2\u00A0 inflammation,\u00C2\u00A0and\u00C2\u00A0cancer.\u00C2\u00A0Curr\u00C2\u00A0Opin\u00C2\u00A0 Genet\u00C2\u00A0Dev\u00C2\u00A021:80\u00E2\u0080\u009085.\u00C2\u00A0 Sanz,\u00C2\u00A0M.A.,\u00C2\u00A0 and\u00C2\u00A0F.\u00C2\u00A0 Lo\u00E2\u0080\u0090Coco.\u00C2\u00A02011.\u00C2\u00A0Modern\u00C2\u00A0approaches\u00C2\u00A0 to\u00C2\u00A0 treating\u00C2\u00A0acute\u00C2\u00A0promyelocytic\u00C2\u00A0 leukemia.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A029:495\u00E2\u0080\u0090503.\u00C2\u00A0 Schmidt\u00E2\u0080\u0090Kittler,\u00C2\u00A0 O.,\u00C2\u00A0 T.\u00C2\u00A0 Ragg,\u00C2\u00A0 A.\u00C2\u00A0 Daskalakis,\u00C2\u00A0 M.\u00C2\u00A0 Granzow,\u00C2\u00A0 A.\u00C2\u00A0 Ahr,\u00C2\u00A0 T.J.\u00C2\u00A0 Blankenstein,\u00C2\u00A0 M.\u00C2\u00A0 Kaufmann,\u00C2\u00A0J.\u00C2\u00A0Diebold,\u00C2\u00A0H.\u00C2\u00A0Arnholdt,\u00C2\u00A0P.\u00C2\u00A0Muller,\u00C2\u00A0J.\u00C2\u00A0Bischoff,\u00C2\u00A0D.\u00C2\u00A0Harich,\u00C2\u00A0G.\u00C2\u00A0Schlimok,\u00C2\u00A0G.\u00C2\u00A0 Riethmuller,\u00C2\u00A0R.\u00C2\u00A0Eils,\u00C2\u00A0and\u00C2\u00A0C.A.\u00C2\u00A0Klein.\u00C2\u00A02003.\u00C2\u00A0From\u00C2\u00A0latent\u00C2\u00A0disseminated\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0overt\u00C2\u00A0 metastasis:\u00C2\u00A0 genetic\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 systemic\u00C2\u00A0 breast\u00C2\u00A0 cancer\u00C2\u00A0 progression.\u00C2\u00A0 Proc\u00C2\u00A0 Natl\u00C2\u00A0 Acad\u00C2\u00A0Sci\u00C2\u00A0U\u00C2\u00A0S\u00C2\u00A0A\u00C2\u00A0100:7737\u00E2\u0080\u00907742.\u00C2\u00A0 Schreiber,\u00C2\u00A0 R.D.,\u00C2\u00A0 L.J.\u00C2\u00A0 Old,\u00C2\u00A0 and\u00C2\u00A0 M.J.\u00C2\u00A0 Smyth.\u00C2\u00A0 2011.\u00C2\u00A0 Cancer\u00C2\u00A0 immunoediting:\u00C2\u00A0 integrating\u00C2\u00A0 immunity's\u00C2\u00A0roles\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0suppression\u00C2\u00A0and\u00C2\u00A0promotion.\u00C2\u00A0Science\u00C2\u00A0331:1565\u00E2\u0080\u00901570.\u00C2\u00A0 Schroder,\u00C2\u00A0 K.,\u00C2\u00A0 P.J.\u00C2\u00A0 Hertzog,\u00C2\u00A0 T.\u00C2\u00A0 Ravasi,\u00C2\u00A0 and\u00C2\u00A0 D.A.\u00C2\u00A0 Hume.\u00C2\u00A0 2004.\u00C2\u00A0 Interferon\u00E2\u0080\u0090gamma:\u00C2\u00A0 an\u00C2\u00A0 overview\u00C2\u00A0of\u00C2\u00A0signals,\u00C2\u00A0mechanisms\u00C2\u00A0and\u00C2\u00A0functions.\u00C2\u00A0J\u00C2\u00A0Leukoc\u00C2\u00A0Biol\u00C2\u00A075:163\u00E2\u0080\u0090189.\u00C2\u00A0 \u00C2\u00A0 175 Seifert,\u00C2\u00A0C.F.,\u00C2\u00A0and\u00C2\u00A0B.H.\u00C2\u00A0Resman\u00E2\u0080\u0090Targoff.\u00C2\u00A02006.\u00C2\u00A0Clinical\u00C2\u00A0laboratory\u00C2\u00A0tests\u00C2\u00A0and\u00C2\u00A0interpretation.\u00C2\u00A0 In\u00C2\u00A0 Textbook\u00C2\u00A0 of\u00C2\u00A0 Therapeutics:\u00C2\u00A0 Drug\u00C2\u00A0 and\u00C2\u00A0 Disease\u00C2\u00A0 Management.\u00C2\u00A0 R.A.\u00C2\u00A0 Helms,\u00C2\u00A0 D.J.\u00C2\u00A0 Quan,\u00C2\u00A0 E.T.\u00C2\u00A0 Herfindal,\u00C2\u00A0 and\u00C2\u00A0 D.R.\u00C2\u00A0 Gourley,\u00C2\u00A0 editors.\u00C2\u00A0 Lippincott\u00C2\u00A0Williams\u00C2\u00A0 &\u00C2\u00A0Wilkins,\u00C2\u00A0 Philadelphia.\u00C2\u00A091\u00E2\u0080\u0090115.\u00C2\u00A0 Serafini,\u00C2\u00A0P.\u00C2\u00A02010.\u00C2\u00A0Editorial:\u00C2\u00A0PGE2\u00E2\u0080\u0090producing\u00C2\u00A0MDSC:\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0progression?\u00C2\u00A0Journal\u00C2\u00A0 of\u00C2\u00A0leukocyte\u00C2\u00A0biology\u00C2\u00A088:827\u00E2\u0080\u0090829.\u00C2\u00A0 Serafini,\u00C2\u00A0P.,\u00C2\u00A0 S.\u00C2\u00A0Mgebroff,\u00C2\u00A0K.\u00C2\u00A0Noonan,\u00C2\u00A0and\u00C2\u00A0 I.\u00C2\u00A0Borrello.\u00C2\u00A02008.\u00C2\u00A0Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0 cells\u00C2\u00A0 promote\u00C2\u00A0 cross\u00E2\u0080\u0090tolerance\u00C2\u00A0 in\u00C2\u00A0 B\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma\u00C2\u00A0 by\u00C2\u00A0 expanding\u00C2\u00A0 regulatory\u00C2\u00A0 T\u00C2\u00A0 cells.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A068:5439\u00E2\u0080\u00905449.\u00C2\u00A0 Shankaran,\u00C2\u00A0V.,\u00C2\u00A0H.\u00C2\u00A0Ikeda,\u00C2\u00A0A.T.\u00C2\u00A0Bruce,\u00C2\u00A0J.M.\u00C2\u00A0White,\u00C2\u00A0P.E.\u00C2\u00A0Swanson,\u00C2\u00A0L.J.\u00C2\u00A0Old,\u00C2\u00A0and\u00C2\u00A0R.D.\u00C2\u00A0Schreiber.\u00C2\u00A0 2001.\u00C2\u00A0 IFNgamma\u00C2\u00A0 and\u00C2\u00A0 lymphocytes\u00C2\u00A0 prevent\u00C2\u00A0 primary\u00C2\u00A0 tumour\u00C2\u00A0 development\u00C2\u00A0 and\u00C2\u00A0 shape\u00C2\u00A0tumour\u00C2\u00A0immunogenicity.\u00C2\u00A0Nature\u00C2\u00A0410:1107\u00E2\u0080\u00901111.\u00C2\u00A0 Shiku,\u00C2\u00A0 H.,\u00C2\u00A0 P.\u00C2\u00A0 Kisielow,\u00C2\u00A0M.A.\u00C2\u00A0 Bean,\u00C2\u00A0 T.\u00C2\u00A0 Takahashi,\u00C2\u00A0 E.A.\u00C2\u00A0 Boyse,\u00C2\u00A0 H.F.\u00C2\u00A0 Oettgen,\u00C2\u00A0 and\u00C2\u00A0 L.J.\u00C2\u00A0 Old.\u00C2\u00A0 1975.\u00C2\u00A0 Expression\u00C2\u00A0 of\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 differentiation\u00C2\u00A0 antigens\u00C2\u00A0 on\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 cell\u00E2\u0080\u0090 mediated\u00C2\u00A0cytotoxicity\u00C2\u00A0 in\u00C2\u00A0vitro.\u00C2\u00A0Evidence\u00C2\u00A0 for\u00C2\u00A0 functional\u00C2\u00A0heterogeneity\u00C2\u00A0 related\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0surface\u00C2\u00A0phenotype\u00C2\u00A0of\u00C2\u00A0T\u00C2\u00A0cells.\u00C2\u00A0The\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0experimental\u00C2\u00A0medicine\u00C2\u00A0141:227\u00E2\u0080\u0090 241.\u00C2\u00A0 Si,\u00C2\u00A0Y.,\u00C2\u00A0C.L.\u00C2\u00A0Tsou,\u00C2\u00A0K.\u00C2\u00A0Croft,\u00C2\u00A0and\u00C2\u00A0I.F.\u00C2\u00A0Charo.\u00C2\u00A02010.\u00C2\u00A0CCR2\u00C2\u00A0mediates\u00C2\u00A0hematopoietic\u00C2\u00A0stem\u00C2\u00A0and\u00C2\u00A0 progenitor\u00C2\u00A0cell\u00C2\u00A0trafficking\u00C2\u00A0to\u00C2\u00A0sites\u00C2\u00A0of\u00C2\u00A0inflammation\u00C2\u00A0in\u00C2\u00A0mice.\u00C2\u00A0The\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0clinical\u00C2\u00A0 investigation\u00C2\u00A0120:1192\u00E2\u0080\u00901203.\u00C2\u00A0 Sims,\u00C2\u00A0G.P.,\u00C2\u00A0D.C.\u00C2\u00A0Rowe,\u00C2\u00A0S.T.\u00C2\u00A0Rietdijk,\u00C2\u00A0R.\u00C2\u00A0Herbst,\u00C2\u00A0and\u00C2\u00A0A.J.\u00C2\u00A0Coyle.\u00C2\u00A02010.\u00C2\u00A0HMGB1\u00C2\u00A0and\u00C2\u00A0RAGE\u00C2\u00A0in\u00C2\u00A0 inflammation\u00C2\u00A0and\u00C2\u00A0cancer.\u00C2\u00A0Annual\u00C2\u00A0review\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A028:367\u00E2\u0080\u0090388.\u00C2\u00A0 Singh,\u00C2\u00A0D.,\u00C2\u00A0P.G.\u00C2\u00A0Febbo,\u00C2\u00A0K.\u00C2\u00A0Ross,\u00C2\u00A0D.G.\u00C2\u00A0Jackson,\u00C2\u00A0J.\u00C2\u00A0Manola,\u00C2\u00A0C.\u00C2\u00A0Ladd,\u00C2\u00A0P.\u00C2\u00A0Tamayo,\u00C2\u00A0A.A.\u00C2\u00A0Renshaw,\u00C2\u00A0 A.V.\u00C2\u00A0D'Amico,\u00C2\u00A0J.P.\u00C2\u00A0Richie,\u00C2\u00A0E.S.\u00C2\u00A0Lander,\u00C2\u00A0M.\u00C2\u00A0Loda,\u00C2\u00A0P.W.\u00C2\u00A0Kantoff,\u00C2\u00A0T.R.\u00C2\u00A0Golub,\u00C2\u00A0and\u00C2\u00A0W.R.\u00C2\u00A0 Sellers.\u00C2\u00A0 2002.\u00C2\u00A0 Gene\u00C2\u00A0 expression\u00C2\u00A0 correlates\u00C2\u00A0 of\u00C2\u00A0 clinical\u00C2\u00A0 prostate\u00C2\u00A0 cancer\u00C2\u00A0 behavior.\u00C2\u00A0 Cancer\u00C2\u00A0Cell\u00C2\u00A01:203\u00E2\u0080\u0090209.\u00C2\u00A0 Sinha,\u00C2\u00A0P.,\u00C2\u00A0V.K.\u00C2\u00A0Clements,\u00C2\u00A0S.K.\u00C2\u00A0Bunt,\u00C2\u00A0S.M.\u00C2\u00A0Albelda,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A02007a.\u00C2\u00A0Cross\u00E2\u0080\u0090 talk\u00C2\u00A0 between\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 macrophages\u00C2\u00A0 subverts\u00C2\u00A0 tumor\u00C2\u00A0immunity\u00C2\u00A0toward\u00C2\u00A0a\u00C2\u00A0type\u00C2\u00A02\u00C2\u00A0response.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0179:977\u00E2\u0080\u0090983.\u00C2\u00A0 Sinha,\u00C2\u00A0 P.,\u00C2\u00A0 V.K.\u00C2\u00A0 Clements,\u00C2\u00A0A.M.\u00C2\u00A0 Fulton,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A02007b.\u00C2\u00A0 Prostaglandin\u00C2\u00A0 E2\u00C2\u00A0promotes\u00C2\u00A0 tumor\u00C2\u00A0progression\u00C2\u00A0by\u00C2\u00A0 inducing\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells.\u00C2\u00A0 Cancer\u00C2\u00A0research\u00C2\u00A067:4507\u00E2\u0080\u00904513.\u00C2\u00A0 Sinha,\u00C2\u00A0P.,\u00C2\u00A0V.K.\u00C2\u00A0Clements,\u00C2\u00A0S.\u00C2\u00A0Miller,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A02005a.\u00C2\u00A0Tumor\u00C2\u00A0immunity:\u00C2\u00A0a\u00C2\u00A0 balancing\u00C2\u00A0 act\u00C2\u00A0 between\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 activation,\u00C2\u00A0 macrophage\u00C2\u00A0 activation\u00C2\u00A0 and\u00C2\u00A0 tumor\u00E2\u0080\u0090 induced\u00C2\u00A0 immune\u00C2\u00A0 suppression.\u00C2\u00A0 Cancer\u00C2\u00A0 immunology,\u00C2\u00A0 immunotherapy\u00C2\u00A0 :\u00C2\u00A0 CII\u00C2\u00A0 54:1137\u00E2\u0080\u00901142.\u00C2\u00A0 176 Sinha,\u00C2\u00A0P.,\u00C2\u00A0V.K.\u00C2\u00A0Clements,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A02005b.\u00C2\u00A0Interleukin\u00E2\u0080\u009013\u00E2\u0080\u0090regulated\u00C2\u00A0M2\u00C2\u00A0 macrophages\u00C2\u00A0 in\u00C2\u00A0 combination\u00C2\u00A0 with\u00C2\u00A0 myeloid\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 block\u00C2\u00A0 immune\u00C2\u00A0 surveillance\u00C2\u00A0against\u00C2\u00A0metastasis.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A065:11743\u00E2\u0080\u009011751.\u00C2\u00A0 Sinha,\u00C2\u00A0 P.,\u00C2\u00A0 V.K.\u00C2\u00A0 Clements,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A0 2005c.\u00C2\u00A0 Reduction\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090 derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 M1\u00C2\u00A0 macrophages\u00C2\u00A0 facilitate\u00C2\u00A0 the\u00C2\u00A0 rejection\u00C2\u00A0of\u00C2\u00A0established\u00C2\u00A0metastatic\u00C2\u00A0disease.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0174:636\u00E2\u0080\u0090645.\u00C2\u00A0 Sinha,\u00C2\u00A0P.,\u00C2\u00A0C.\u00C2\u00A0Okoro,\u00C2\u00A0D.\u00C2\u00A0Foell,\u00C2\u00A0H.H.\u00C2\u00A0Freeze,\u00C2\u00A0S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Srikrishna.\u00C2\u00A02008.\u00C2\u00A0 Proinflammatory\u00C2\u00A0 S100\u00C2\u00A0 proteins\u00C2\u00A0 regulate\u00C2\u00A0 the\u00C2\u00A0 accumulation\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0181:4666\u00E2\u0080\u00904675.\u00C2\u00A0 Siveen,\u00C2\u00A0K.S.,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Kuttan.\u00C2\u00A02009.\u00C2\u00A0Role\u00C2\u00A0of\u00C2\u00A0macrophages\u00C2\u00A0in\u00C2\u00A0tumour\u00C2\u00A0progression.\u00C2\u00A0Immunol\u00C2\u00A0 Lett\u00C2\u00A0123:97\u00E2\u0080\u0090102.\u00C2\u00A0 Sly,\u00C2\u00A0L.M.,\u00C2\u00A0M.J.\u00C2\u00A0Hamilton,\u00C2\u00A0E.\u00C2\u00A0Kuroda,\u00C2\u00A0V.W.\u00C2\u00A0Ho,\u00C2\u00A0F.L.\u00C2\u00A0Antignano,\u00C2\u00A0S.L.\u00C2\u00A0Omeis,\u00C2\u00A0C.J.\u00C2\u00A0van\u00C2\u00A0Netten\u00E2\u0080\u0090 Thomas,\u00C2\u00A0 D.\u00C2\u00A0Wong,\u00C2\u00A0 H.K.\u00C2\u00A0 Brugger,\u00C2\u00A0 O.\u00C2\u00A0Williams,\u00C2\u00A0M.E.\u00C2\u00A0 Feldman,\u00C2\u00A0 B.T.\u00C2\u00A0 Houseman,\u00C2\u00A0 D.\u00C2\u00A0 Fiedler,\u00C2\u00A0 K.M.\u00C2\u00A0 Shokat,\u00C2\u00A0 and\u00C2\u00A0 G.\u00C2\u00A0 Krystal.\u00C2\u00A0 2009.\u00C2\u00A0 SHIP\u00C2\u00A0 prevents\u00C2\u00A0 lipopolysaccharide\u00C2\u00A0 from\u00C2\u00A0triggering\u00C2\u00A0an\u00C2\u00A0antiviral\u00C2\u00A0response\u00C2\u00A0in\u00C2\u00A0mice.\u00C2\u00A0Blood\u00C2\u00A0113:2945\u00E2\u0080\u00902954.\u00C2\u00A0 Sly,\u00C2\u00A0 L.M.,\u00C2\u00A0 J.\u00C2\u00A0 Kalesnikoff,\u00C2\u00A0 V.\u00C2\u00A0 Lam,\u00C2\u00A0 D.\u00C2\u00A0 Wong,\u00C2\u00A0 C.\u00C2\u00A0 Song,\u00C2\u00A0 S.\u00C2\u00A0 Omeis,\u00C2\u00A0 K.\u00C2\u00A0 Chan,\u00C2\u00A0 C.W.\u00C2\u00A0 Lee,\u00C2\u00A0 R.P.\u00C2\u00A0 Siraganian,\u00C2\u00A0 J.\u00C2\u00A0 Rivera,\u00C2\u00A0 and\u00C2\u00A0 G.\u00C2\u00A0 Krystal.\u00C2\u00A0 2008.\u00C2\u00A0 IgE\u00E2\u0080\u0090induced\u00C2\u00A0 mast\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 requires\u00C2\u00A0 the\u00C2\u00A0 prolonged\u00C2\u00A0 generation\u00C2\u00A0 of\u00C2\u00A0 reactive\u00C2\u00A0 oxygen\u00C2\u00A0 species.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A0 181:3850\u00E2\u0080\u00903860.\u00C2\u00A0 Soehnlein,\u00C2\u00A0 O.,\u00C2\u00A0 and\u00C2\u00A0 L.\u00C2\u00A0 Lindbom.\u00C2\u00A0 2010.\u00C2\u00A0 Phagocyte\u00C2\u00A0 partnership\u00C2\u00A0 during\u00C2\u00A0 the\u00C2\u00A0 onset\u00C2\u00A0 and\u00C2\u00A0 resolution\u00C2\u00A0of\u00C2\u00A0inflammation.\u00C2\u00A0Nature\u00C2\u00A0reviews.\u00C2\u00A0Immunology\u00C2\u00A010:427\u00E2\u0080\u0090439.\u00C2\u00A0 Solinas,\u00C2\u00A0G.,\u00C2\u00A0S.\u00C2\u00A0 Schiarea,\u00C2\u00A0M.\u00C2\u00A0Liguori,\u00C2\u00A0M.\u00C2\u00A0Fabbri,\u00C2\u00A0 S.\u00C2\u00A0Pesce,\u00C2\u00A0L.\u00C2\u00A0Zammataro,\u00C2\u00A0F.\u00C2\u00A0Pasqualini,\u00C2\u00A0M.\u00C2\u00A0 Nebuloni,\u00C2\u00A0 C.\u00C2\u00A0 Chiabrando,\u00C2\u00A0 A.\u00C2\u00A0 Mantovani,\u00C2\u00A0 and\u00C2\u00A0 P.\u00C2\u00A0 Allavena.\u00C2\u00A0 2010.\u00C2\u00A0 Tumor\u00E2\u0080\u0090 conditioned\u00C2\u00A0 macrophages\u00C2\u00A0 secrete\u00C2\u00A0 migration\u00E2\u0080\u0090stimulating\u00C2\u00A0 factor:\u00C2\u00A0 a\u00C2\u00A0 new\u00C2\u00A0 marker\u00C2\u00A0 for\u00C2\u00A0 M2\u00E2\u0080\u0090polarization,\u00C2\u00A0 influencing\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 motility.\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 immunology\u00C2\u00A0 185:642\u00E2\u0080\u0090652.\u00C2\u00A0 Srivastava,\u00C2\u00A0M.K.,\u00C2\u00A0P.\u00C2\u00A0Sinha,\u00C2\u00A0V.K.\u00C2\u00A0Clements,\u00C2\u00A0P.\u00C2\u00A0Rodriguez,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Ostrand\u00E2\u0080\u0090Rosenberg.\u00C2\u00A02010.\u00C2\u00A0 Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 inhibit\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 activation\u00C2\u00A0 by\u00C2\u00A0 depleting\u00C2\u00A0 cystine\u00C2\u00A0 and\u00C2\u00A0cysteine.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A070:68\u00E2\u0080\u009077.\u00C2\u00A0 Stein,\u00C2\u00A0 M.,\u00C2\u00A0 S.\u00C2\u00A0 Keshav,\u00C2\u00A0 N.\u00C2\u00A0 Harris,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Gordon.\u00C2\u00A0 1992.\u00C2\u00A0 Interleukin\u00C2\u00A0 4\u00C2\u00A0 potently\u00C2\u00A0 enhances\u00C2\u00A0 murine\u00C2\u00A0 macrophage\u00C2\u00A0 mannose\u00C2\u00A0 receptor\u00C2\u00A0 activity:\u00C2\u00A0 a\u00C2\u00A0 marker\u00C2\u00A0 of\u00C2\u00A0 alternative\u00C2\u00A0 immunologic\u00C2\u00A0 macrophage\u00C2\u00A0 activation.\u00C2\u00A0 The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 experimental\u00C2\u00A0 medicine\u00C2\u00A0 176:287\u00E2\u0080\u0090292.\u00C2\u00A0 Strickland,\u00C2\u00A0D.,\u00C2\u00A0U.R.\u00C2\u00A0Kees,\u00C2\u00A0and\u00C2\u00A0P.G.\u00C2\u00A0Holt.\u00C2\u00A01996.\u00C2\u00A0Regulation\u00C2\u00A0of\u00C2\u00A0T\u00E2\u0080\u0090cell\u00C2\u00A0activation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0lung:\u00C2\u00A0 alveolar\u00C2\u00A0macrophages\u00C2\u00A0 induce\u00C2\u00A0 reversible\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 anergy\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 associated\u00C2\u00A0with\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 interleukin\u00E2\u0080\u00902\u00C2\u00A0 receptor\u00C2\u00A0 signal\u00C2\u00A0 transduction.\u00C2\u00A0 Immunology\u00C2\u00A0 87:250\u00E2\u0080\u0090 258.\u00C2\u00A0 177 Strober,\u00C2\u00A0S.\u00C2\u00A01984.\u00C2\u00A0Natural\u00C2\u00A0suppressor\u00C2\u00A0(NS)\u00C2\u00A0cells,\u00C2\u00A0neonatal\u00C2\u00A0tolerance,\u00C2\u00A0and\u00C2\u00A0total\u00C2\u00A0lymphoid\u00C2\u00A0 irradiation:\u00C2\u00A0 exploring\u00C2\u00A0 obscure\u00C2\u00A0 relationships.\u00C2\u00A0 Annual\u00C2\u00A0 review\u00C2\u00A0 of\u00C2\u00A0 immunology\u00C2\u00A0 2:219\u00E2\u0080\u0090237.\u00C2\u00A0 Stutman,\u00C2\u00A0O.\u00C2\u00A01974.\u00C2\u00A0Tumor\u00C2\u00A0development\u00C2\u00A0after\u00C2\u00A03\u00E2\u0080\u0090methylcholanthrene\u00C2\u00A0in\u00C2\u00A0immunologically\u00C2\u00A0 deficient\u00C2\u00A0athymic\u00E2\u0080\u0090nude\u00C2\u00A0mice.\u00C2\u00A0Science\u00C2\u00A0183:534\u00E2\u0080\u0090536.\u00C2\u00A0 Stutman,\u00C2\u00A0O.\u00C2\u00A01975.\u00C2\u00A0Immunodepression\u00C2\u00A0and\u00C2\u00A0malignancy.\u00C2\u00A0Adv\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A022:261\u00E2\u0080\u0090422.\u00C2\u00A0 Suit,\u00C2\u00A0H.D.,\u00C2\u00A0R.S.\u00C2\u00A0Sedlacek,\u00C2\u00A0G.\u00C2\u00A0Silver,\u00C2\u00A0and\u00C2\u00A0D.\u00C2\u00A0Dosoretz.\u00C2\u00A01985.\u00C2\u00A0Pentobarbital\u00C2\u00A0anesthesia\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 response\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 and\u00C2\u00A0 normal\u00C2\u00A0 tissue\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 C3Hf/sed\u00C2\u00A0mouse\u00C2\u00A0 to\u00C2\u00A0 radiation.\u00C2\u00A0 Radiat\u00C2\u00A0Res\u00C2\u00A0104:47\u00E2\u0080\u009065.\u00C2\u00A0 Taylor\u00E2\u0080\u0090Robinson,\u00C2\u00A0A.W.,\u00C2\u00A0 F.Y.\u00C2\u00A0 Liew,\u00C2\u00A0A.\u00C2\u00A0 Severn,\u00C2\u00A0D.\u00C2\u00A0 Xu,\u00C2\u00A0 S.J.\u00C2\u00A0McSorley,\u00C2\u00A0 P.\u00C2\u00A0Garside,\u00C2\u00A0 J.\u00C2\u00A0 Padron,\u00C2\u00A0 and\u00C2\u00A0 R.S.\u00C2\u00A0 Phillips.\u00C2\u00A0 1994.\u00C2\u00A0 Regulation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 response\u00C2\u00A0 by\u00C2\u00A0 nitric\u00C2\u00A0 oxide\u00C2\u00A0 differentially\u00C2\u00A0 produced\u00C2\u00A0 by\u00C2\u00A0 T\u00C2\u00A0 helper\u00C2\u00A0 type\u00C2\u00A0 1\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 helper\u00C2\u00A0 type\u00C2\u00A0 2\u00C2\u00A0 cells.\u00C2\u00A0 Eur\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A024:980\u00E2\u0080\u0090984.\u00C2\u00A0 Torroella\u00E2\u0080\u0090Kouri,\u00C2\u00A0 M.,\u00C2\u00A0 X.\u00C2\u00A0 Ma,\u00C2\u00A0 G.\u00C2\u00A0 Perry,\u00C2\u00A0 M.\u00C2\u00A0 Ivanova,\u00C2\u00A0 P.J.\u00C2\u00A0 Cejas,\u00C2\u00A0 J.L.\u00C2\u00A0 Owen,\u00C2\u00A0 V.\u00C2\u00A0 Iragavarapu\u00E2\u0080\u0090 Charyulu,\u00C2\u00A0and\u00C2\u00A0D.M.\u00C2\u00A0Lopez.\u00C2\u00A02005.\u00C2\u00A0Diminished\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0transcription\u00C2\u00A0factors\u00C2\u00A0 nuclear\u00C2\u00A0 factor\u00C2\u00A0 kappaB\u00C2\u00A0 and\u00C2\u00A0CCAAT/enhancer\u00C2\u00A0 binding\u00C2\u00A0protein\u00C2\u00A0 underlies\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 tumor\u00C2\u00A0 evasion\u00C2\u00A0mechanism\u00C2\u00A0 affecting\u00C2\u00A0macrophages\u00C2\u00A0 of\u00C2\u00A0 mammary\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00C2\u00A0Cancer\u00C2\u00A0research\u00C2\u00A065:10578\u00E2\u0080\u009010584.\u00C2\u00A0 Torroella\u00E2\u0080\u0090Kouri,\u00C2\u00A0M.,\u00C2\u00A0R.\u00C2\u00A0 Silvera,\u00C2\u00A0D.\u00C2\u00A0Rodriguez,\u00C2\u00A0R.\u00C2\u00A0 Caso,\u00C2\u00A0A.\u00C2\u00A0 Shatry,\u00C2\u00A0 S.\u00C2\u00A0Opiela,\u00C2\u00A0D.\u00C2\u00A0 Ilkovitch,\u00C2\u00A0 R.A.\u00C2\u00A0 Schwendener,\u00C2\u00A0 V.\u00C2\u00A0 Iragavarapu\u00E2\u0080\u0090Charyulu,\u00C2\u00A0 Y.\u00C2\u00A0 Cardentey,\u00C2\u00A0 N.\u00C2\u00A0 Strbo,\u00C2\u00A0 and\u00C2\u00A0 D.M.\u00C2\u00A0 Lopez.\u00C2\u00A0 2009.\u00C2\u00A0 Identification\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 subpopulation\u00C2\u00A0 of\u00C2\u00A0 macrophages\u00C2\u00A0 in\u00C2\u00A0 mammary\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 neither\u00C2\u00A0 M1\u00C2\u00A0 nor\u00C2\u00A0 M2\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 less\u00C2\u00A0 differentiated.\u00C2\u00A0 Cancer\u00C2\u00A0research\u00C2\u00A069:4800\u00E2\u0080\u00904809.\u00C2\u00A0 Ueda,\u00C2\u00A0Y.,\u00C2\u00A0D.W.\u00C2\u00A0Cain,\u00C2\u00A0M.\u00C2\u00A0Kuraoka,\u00C2\u00A0M.\u00C2\u00A0Kondo,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Kelsoe.\u00C2\u00A02009.\u00C2\u00A0IL\u00E2\u0080\u00901R\u00C2\u00A0type\u00C2\u00A0I\u00E2\u0080\u0090dependent\u00C2\u00A0 hemopoietic\u00C2\u00A0stem\u00C2\u00A0cell\u00C2\u00A0proliferation\u00C2\u00A0is\u00C2\u00A0necessary\u00C2\u00A0for\u00C2\u00A0inflammatory\u00C2\u00A0granulopoiesis\u00C2\u00A0 and\u00C2\u00A0reactive\u00C2\u00A0neutrophilia.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0182:6477\u00E2\u0080\u00906484.\u00C2\u00A0 Ueha,\u00C2\u00A0 S.,\u00C2\u00A0 F.H.\u00C2\u00A0 Shand,\u00C2\u00A0 and\u00C2\u00A0 K.\u00C2\u00A0 Matsushima.\u00C2\u00A0 2011.\u00C2\u00A0 Myeloid\u00C2\u00A0 cell\u00C2\u00A0 population\u00C2\u00A0 dynamics\u00C2\u00A0 in\u00C2\u00A0 healthy\u00C2\u00A0 and\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice.\u00C2\u00A0 International\u00C2\u00A0 immunopharmacology\u00C2\u00A0 11:783\u00E2\u0080\u0090 788.\u00C2\u00A0 Valenti,\u00C2\u00A0 R.,\u00C2\u00A0 V.\u00C2\u00A0 Huber,\u00C2\u00A0 P.\u00C2\u00A0 Filipazzi,\u00C2\u00A0 L.\u00C2\u00A0 Pilla,\u00C2\u00A0 G.\u00C2\u00A0 Sovena,\u00C2\u00A0 A.\u00C2\u00A0 Villa,\u00C2\u00A0 A.\u00C2\u00A0 Corbelli,\u00C2\u00A0 S.\u00C2\u00A0 Fais,\u00C2\u00A0 G.\u00C2\u00A0 Parmiani,\u00C2\u00A0and\u00C2\u00A0L.\u00C2\u00A0Rivoltini.\u00C2\u00A02006.\u00C2\u00A0Human\u00C2\u00A0tumor\u00E2\u0080\u0090released\u00C2\u00A0microvesicles\u00C2\u00A0promote\u00C2\u00A0 the\u00C2\u00A0 differentiation\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 with\u00C2\u00A0 transforming\u00C2\u00A0 growth\u00C2\u00A0 factor\u00E2\u0080\u0090beta\u00E2\u0080\u0090 mediated\u00C2\u00A0suppressive\u00C2\u00A0activity\u00C2\u00A0on\u00C2\u00A0T\u00C2\u00A0lymphocytes.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A066:9290\u00E2\u0080\u00909298.\u00C2\u00A0 van\u00C2\u00A0't\u00C2\u00A0Veer,\u00C2\u00A0L.J.,\u00C2\u00A0H.\u00C2\u00A0Dai,\u00C2\u00A0M.J.\u00C2\u00A0van\u00C2\u00A0de\u00C2\u00A0Vijver,\u00C2\u00A0Y.D.\u00C2\u00A0He,\u00C2\u00A0A.A.\u00C2\u00A0Hart,\u00C2\u00A0M.\u00C2\u00A0Mao,\u00C2\u00A0H.L.\u00C2\u00A0Peterse,\u00C2\u00A0K.\u00C2\u00A0van\u00C2\u00A0 der\u00C2\u00A0Kooy,\u00C2\u00A0M.J.\u00C2\u00A0Marton,\u00C2\u00A0A.T.\u00C2\u00A0Witteveen,\u00C2\u00A0G.J.\u00C2\u00A0Schreiber,\u00C2\u00A0R.M.\u00C2\u00A0Kerkhoven,\u00C2\u00A0C.\u00C2\u00A0Roberts,\u00C2\u00A0 P.S.\u00C2\u00A0 Linsley,\u00C2\u00A0 R.\u00C2\u00A0 Bernards,\u00C2\u00A0 and\u00C2\u00A0 S.H.\u00C2\u00A0 Friend.\u00C2\u00A0 2002.\u00C2\u00A0 Gene\u00C2\u00A0 expression\u00C2\u00A0 profiling\u00C2\u00A0 predicts\u00C2\u00A0clinical\u00C2\u00A0outcome\u00C2\u00A0of\u00C2\u00A0breast\u00C2\u00A0cancer.\u00C2\u00A0Nature\u00C2\u00A0415:530\u00E2\u0080\u0090536.\u00C2\u00A0 178 Van\u00C2\u00A0Ginderachter,\u00C2\u00A0J.A.,\u00C2\u00A0A.\u00C2\u00A0Beschin,\u00C2\u00A0P.\u00C2\u00A0De\u00C2\u00A0Baetselier,\u00C2\u00A0and\u00C2\u00A0G.\u00C2\u00A0Raes.\u00C2\u00A02010.\u00C2\u00A0Myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 parasitic\u00C2\u00A0 infections.\u00C2\u00A0 European\u00C2\u00A0 journal\u00C2\u00A0 of\u00C2\u00A0 immunology\u00C2\u00A0 40:2976\u00E2\u0080\u00902985.\u00C2\u00A0 Van\u00C2\u00A0Pel,\u00C2\u00A0A.,\u00C2\u00A0and\u00C2\u00A0T.\u00C2\u00A0Boon.\u00C2\u00A01982.\u00C2\u00A0Protection\u00C2\u00A0against\u00C2\u00A0a\u00C2\u00A0nonimmunogenic\u00C2\u00A0mouse\u00C2\u00A0leukemia\u00C2\u00A0 by\u00C2\u00A0an\u00C2\u00A0 immunogenic\u00C2\u00A0variant\u00C2\u00A0obtained\u00C2\u00A0by\u00C2\u00A0mutagenesis.\u00C2\u00A0Proc\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0U\u00C2\u00A0S\u00C2\u00A0A\u00C2\u00A0 79:4718\u00E2\u0080\u00904722.\u00C2\u00A0 Vesely,\u00C2\u00A0M.D.,\u00C2\u00A0M.H.\u00C2\u00A0 Kershaw,\u00C2\u00A0 R.D.\u00C2\u00A0 Schreiber,\u00C2\u00A0 and\u00C2\u00A0M.J.\u00C2\u00A0 Smyth.\u00C2\u00A0 2011.\u00C2\u00A0 Natural\u00C2\u00A0 innate\u00C2\u00A0 and\u00C2\u00A0 adaptive\u00C2\u00A0immunity\u00C2\u00A0to\u00C2\u00A0cancer.\u00C2\u00A0Annual\u00C2\u00A0review\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A029:235\u00E2\u0080\u0090271.\u00C2\u00A0 Vieweg,\u00C2\u00A0 J.,\u00C2\u00A0 Z.\u00C2\u00A0 Su,\u00C2\u00A0 P.\u00C2\u00A0 Dahm,\u00C2\u00A0 and\u00C2\u00A0 S.\u00C2\u00A0 Kusmartsev.\u00C2\u00A0 2007.\u00C2\u00A0 Reversal\u00C2\u00A0 of\u00C2\u00A0 tumor\u00E2\u0080\u0090mediated\u00C2\u00A0 immunosuppression.\u00C2\u00A0Clin\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A013:727s\u00E2\u0080\u0090732s.\u00C2\u00A0 von\u00C2\u00A0 Bergwelt\u00E2\u0080\u0090Baildon,\u00C2\u00A0 M.S.,\u00C2\u00A0 A.\u00C2\u00A0 Popov,\u00C2\u00A0 T.\u00C2\u00A0 Saric,\u00C2\u00A0 J.\u00C2\u00A0 Chemnitz,\u00C2\u00A0 S.\u00C2\u00A0 Classen,\u00C2\u00A0 M.S.\u00C2\u00A0 Stoffel,\u00C2\u00A0 F.\u00C2\u00A0 Fiore,\u00C2\u00A0 U.\u00C2\u00A0 Roth,\u00C2\u00A0 M.\u00C2\u00A0 Beyer,\u00C2\u00A0 S.\u00C2\u00A0 Debey,\u00C2\u00A0 C.\u00C2\u00A0 Wickenhauser,\u00C2\u00A0 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of\u00C2\u00A0lymphocyte\u00C2\u00A0subpopulations\u00C2\u00A0infiltrating\u00C2\u00A0breast\u00C2\u00A0carcinomas\u00C2\u00A0and\u00C2\u00A0benign\u00C2\u00A0lesions.\u00C2\u00A0 Int\u00C2\u00A0J\u00C2\u00A0Cancer\u00C2\u00A040:18\u00E2\u0080\u009023.\u00C2\u00A0 Waltenbaugh,\u00C2\u00A0C.,\u00C2\u00A0T.\u00C2\u00A0Doan,\u00C2\u00A0R.\u00C2\u00A0Melvold,\u00C2\u00A0and\u00C2\u00A0S.\u00C2\u00A0Viselli.\u00C2\u00A02008.\u00C2\u00A0Immunology.\u00C2\u00A0Wolters\u00C2\u00A0Kluwer\u00C2\u00A0 Health/Lippincott\u00C2\u00A0Williams\u00C2\u00A0&\u00C2\u00A0Wilkins,\u00C2\u00A0Philadelphia.\u00C2\u00A0\u00C2\u00A0 Wang,\u00C2\u00A0 Q.,\u00C2\u00A0 H.\u00C2\u00A0 Ni,\u00C2\u00A0 L.\u00C2\u00A0 Lan,\u00C2\u00A0 X.\u00C2\u00A0 Wei,\u00C2\u00A0 R.\u00C2\u00A0 Xiang,\u00C2\u00A0 and\u00C2\u00A0 Y.\u00C2\u00A0 Wang.\u00C2\u00A0 2010.\u00C2\u00A0 Fra\u00E2\u0080\u00901\u00C2\u00A0 protooncogene\u00C2\u00A0 regulates\u00C2\u00A0IL\u00E2\u0080\u00906\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0macrophages\u00C2\u00A0and\u00C2\u00A0promotes\u00C2\u00A0the\u00C2\u00A0generation\u00C2\u00A0of\u00C2\u00A0M2d\u00C2\u00A0 macrophages.\u00C2\u00A0Cell\u00C2\u00A0Res\u00C2\u00A020:701\u00E2\u0080\u0090712.\u00C2\u00A0 Watson,\u00C2\u00A0 G.A.,\u00C2\u00A0 and\u00C2\u00A0 D.M.\u00C2\u00A0 Lopez.\u00C2\u00A0 1995.\u00C2\u00A0 Aberrant\u00C2\u00A0 antigen\u00C2\u00A0 presentation\u00C2\u00A0 by\u00C2\u00A0 macrophages\u00C2\u00A0 from\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0 mice\u00C2\u00A0 is\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 down\u00E2\u0080\u0090regulation\u00C2\u00A0 of\u00C2\u00A0 their\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 responses.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0immunology\u00C2\u00A0155:3124\u00E2\u0080\u00903134.\u00C2\u00A0 Wei,\u00C2\u00A0X.Q.,\u00C2\u00A0I.G.\u00C2\u00A0Charles,\u00C2\u00A0A.\u00C2\u00A0Smith,\u00C2\u00A0J.\u00C2\u00A0Ure,\u00C2\u00A0G.J.\u00C2\u00A0Feng,\u00C2\u00A0F.P.\u00C2\u00A0Huang,\u00C2\u00A0D.\u00C2\u00A0Xu,\u00C2\u00A0W.\u00C2\u00A0Muller,\u00C2\u00A0S.\u00C2\u00A0Moncada,\u00C2\u00A0 and\u00C2\u00A0F.Y.\u00C2\u00A0Liew.\u00C2\u00A01995.\u00C2\u00A0Altered\u00C2\u00A0immune\u00C2\u00A0responses\u00C2\u00A0in\u00C2\u00A0mice\u00C2\u00A0lacking\u00C2\u00A0inducible\u00C2\u00A0nitric\u00C2\u00A0 oxide\u00C2\u00A0synthase.\u00C2\u00A0Nature\u00C2\u00A0375:408\u00E2\u0080\u0090411.\u00C2\u00A0 Weinstein,\u00C2\u00A0 S.L.,\u00C2\u00A0 A.J.\u00C2\u00A0 Finn,\u00C2\u00A0 S.H.\u00C2\u00A0 Dave,\u00C2\u00A0 F.\u00C2\u00A0 Meng,\u00C2\u00A0 C.A.\u00C2\u00A0 Lowell,\u00C2\u00A0 J.S.\u00C2\u00A0 Sanghera,\u00C2\u00A0 and\u00C2\u00A0 A.L.\u00C2\u00A0 DeFranco.\u00C2\u00A0 2000.\u00C2\u00A0 Phosphatidylinositol\u00C2\u00A0 3\u00E2\u0080\u0090kinase\u00C2\u00A0 and\u00C2\u00A0 mTOR\u00C2\u00A0 mediate\u00C2\u00A0 lipopolysaccharide\u00E2\u0080\u0090stimulated\u00C2\u00A0 nitric\u00C2\u00A0 oxide\u00C2\u00A0 production\u00C2\u00A0 in\u00C2\u00A0 macrophages\u00C2\u00A0 via\u00C2\u00A0 interferon\u00E2\u0080\u0090beta.\u00C2\u00A0Journal\u00C2\u00A0of\u00C2\u00A0leukocyte\u00C2\u00A0biology\u00C2\u00A067:405\u00E2\u0080\u0090414.\u00C2\u00A0 Whiteside,\u00C2\u00A0 T.L.\u00C2\u00A0 2006.\u00C2\u00A0 Immune\u00C2\u00A0 suppression\u00C2\u00A0 in\u00C2\u00A0 cancer:\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 immune\u00C2\u00A0 cells,\u00C2\u00A0 mechanisms\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0therapeutic\u00C2\u00A0intervention.\u00C2\u00A0Semin\u00C2\u00A0Cancer\u00C2\u00A0Biol\u00C2\u00A016:3\u00E2\u0080\u009015.\u00C2\u00A0 179 Whiteside,\u00C2\u00A0 T.L.\u00C2\u00A0 2008.\u00C2\u00A0 The\u00C2\u00A0 tumor\u00C2\u00A0microenvironment\u00C2\u00A0 and\u00C2\u00A0 its\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 promoting\u00C2\u00A0 tumor\u00C2\u00A0 growth.\u00C2\u00A0Oncogene\u00C2\u00A027:5904\u00E2\u0080\u00905912.\u00C2\u00A0 Whiteside,\u00C2\u00A0 T.L.\u00C2\u00A0 2010.\u00C2\u00A0 Immune\u00C2\u00A0 responses\u00C2\u00A0 to\u00C2\u00A0 malignancies.\u00C2\u00A0 The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 allergy\u00C2\u00A0 and\u00C2\u00A0 clinical\u00C2\u00A0immunology\u00C2\u00A0125:S272\u00E2\u0080\u0090283.\u00C2\u00A0 Xia,\u00C2\u00A0Y.,\u00C2\u00A0 L.J.\u00C2\u00A0Roman,\u00C2\u00A0B.S.\u00C2\u00A0Masters,\u00C2\u00A0 and\u00C2\u00A0 J.L.\u00C2\u00A0 Zweier.\u00C2\u00A01998.\u00C2\u00A0 Inducible\u00C2\u00A0nitric\u00E2\u0080\u0090oxide\u00C2\u00A0 synthase\u00C2\u00A0 generates\u00C2\u00A0 superoxide\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 reductase\u00C2\u00A0 domain.\u00C2\u00A0 The\u00C2\u00A0 Journal\u00C2\u00A0 of\u00C2\u00A0 biological\u00C2\u00A0 chemistry\u00C2\u00A0273:22635\u00E2\u0080\u009022639.\u00C2\u00A0 Yang,\u00C2\u00A0L.,\u00C2\u00A0L.M.\u00C2\u00A0DeBusk,\u00C2\u00A0K.\u00C2\u00A0Fukuda,\u00C2\u00A0B.\u00C2\u00A0Fingleton,\u00C2\u00A0B.\u00C2\u00A0Green\u00E2\u0080\u0090Jarvis,\u00C2\u00A0Y.\u00C2\u00A0 Shyr,\u00C2\u00A0L.M.\u00C2\u00A0Matrisian,\u00C2\u00A0 D.P.\u00C2\u00A0 Carbone,\u00C2\u00A0 and\u00C2\u00A0 P.C.\u00C2\u00A0 Lin.\u00C2\u00A0 2004.\u00C2\u00A0 Expansion\u00C2\u00A0 of\u00C2\u00A0 myeloid\u00C2\u00A0 immune\u00C2\u00A0 suppressor\u00C2\u00A0 Gr+CD11b+\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0host\u00C2\u00A0 directly\u00C2\u00A0 promotes\u00C2\u00A0 tumor\u00C2\u00A0 angiogenesis.\u00C2\u00A0 Cancer\u00C2\u00A0Cell\u00C2\u00A06:409\u00E2\u0080\u0090421.\u00C2\u00A0 Yang,\u00C2\u00A0L.,\u00C2\u00A0J.\u00C2\u00A0Huang,\u00C2\u00A0X.\u00C2\u00A0Ren,\u00C2\u00A0A.E.\u00C2\u00A0Gorska,\u00C2\u00A0A.\u00C2\u00A0Chytil,\u00C2\u00A0M.\u00C2\u00A0Aakre,\u00C2\u00A0D.P.\u00C2\u00A0Carbone,\u00C2\u00A0L.M.\u00C2\u00A0Matrisian,\u00C2\u00A0A.\u00C2\u00A0 Richmond,\u00C2\u00A0 P.C.\u00C2\u00A0 Lin,\u00C2\u00A0 and\u00C2\u00A0H.L.\u00C2\u00A0Moses.\u00C2\u00A0 2008.\u00C2\u00A0 Abrogation\u00C2\u00A0 of\u00C2\u00A0 TGF\u00C2\u00A0 beta\u00C2\u00A0 signaling\u00C2\u00A0 in\u00C2\u00A0 mammary\u00C2\u00A0 carcinomas\u00C2\u00A0 recruits\u00C2\u00A0 Gr\u00E2\u0080\u00901+CD11b+\u00C2\u00A0 myeloid\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 promote\u00C2\u00A0 metastasis.\u00C2\u00A0Cancer\u00C2\u00A0Cell\u00C2\u00A013:23\u00E2\u0080\u009035.\u00C2\u00A0 Yoshida,\u00C2\u00A0 R.,\u00C2\u00A0 H.W.\u00C2\u00A0 Murray,\u00C2\u00A0 and\u00C2\u00A0 C.F.\u00C2\u00A0 Nathan.\u00C2\u00A0 1988.\u00C2\u00A0 Agonist\u00C2\u00A0 and\u00C2\u00A0 antagonist\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 interferon\u00C2\u00A0alpha\u00C2\u00A0and\u00C2\u00A0beta\u00C2\u00A0on\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0human\u00C2\u00A0macrophages.\u00C2\u00A0Two\u00C2\u00A0classes\u00C2\u00A0of\u00C2\u00A0 interferon\u00C2\u00A0gamma\u00C2\u00A0receptors\u00C2\u00A0and\u00C2\u00A0blockade\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0high\u00E2\u0080\u0090affinity\u00C2\u00A0sites\u00C2\u00A0by\u00C2\u00A0interferon\u00C2\u00A0 alpha\u00C2\u00A0or\u00C2\u00A0beta.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A0167:1171\u00E2\u0080\u00901185.\u00C2\u00A0 Youn,\u00C2\u00A0 J.I.,\u00C2\u00A0 and\u00C2\u00A0D.I.\u00C2\u00A0Gabrilovich.\u00C2\u00A02010.\u00C2\u00A0The\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0suppressor\u00C2\u00A0cells:\u00C2\u00A0 the\u00C2\u00A0blessing\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0curse\u00C2\u00A0of\u00C2\u00A0morphological\u00C2\u00A0and\u00C2\u00A0functional\u00C2\u00A0heterogeneity.\u00C2\u00A0Eur\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A040:2969\u00E2\u0080\u00902975.\u00C2\u00A0 Youn,\u00C2\u00A0J.I.,\u00C2\u00A0S.\u00C2\u00A0Nagaraj,\u00C2\u00A0M.\u00C2\u00A0Collazo,\u00C2\u00A0and\u00C2\u00A0D.I.\u00C2\u00A0Gabrilovich.\u00C2\u00A02008.\u00C2\u00A0Subsets\u00C2\u00A0of\u00C2\u00A0myeloid\u00E2\u0080\u0090derived\u00C2\u00A0 suppressor\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0181:5791\u00E2\u0080\u00905802.\u00C2\u00A0 Young,\u00C2\u00A0 M.R.,\u00C2\u00A0 and\u00C2\u00A0 D.M.\u00C2\u00A0 Lathers.\u00C2\u00A0 1999.\u00C2\u00A0 Myeloid\u00C2\u00A0 progenitor\u00C2\u00A0 cells\u00C2\u00A0 mediate\u00C2\u00A0 immune\u00C2\u00A0 suppression\u00C2\u00A0 in\u00C2\u00A0 patients\u00C2\u00A0 with\u00C2\u00A0 head\u00C2\u00A0 and\u00C2\u00A0 neck\u00C2\u00A0 cancers.\u00C2\u00A0 Int\u00C2\u00A0 J\u00C2\u00A0 Immunopharmacol\u00C2\u00A0 21:241\u00E2\u0080\u0090252.\u00C2\u00A0 Zea,\u00C2\u00A0 A.H.,\u00C2\u00A0 P.C.\u00C2\u00A0 Rodriguez,\u00C2\u00A0 M.B.\u00C2\u00A0 Atkins,\u00C2\u00A0 C.\u00C2\u00A0 Hernandez,\u00C2\u00A0 S.\u00C2\u00A0 Signoretti,\u00C2\u00A0 J.\u00C2\u00A0 Zabaleta,\u00C2\u00A0 D.\u00C2\u00A0 McDermott,\u00C2\u00A0 D.\u00C2\u00A0 Quiceno,\u00C2\u00A0 A.\u00C2\u00A0 Youmans,\u00C2\u00A0 A.\u00C2\u00A0 O'Neill,\u00C2\u00A0 J.\u00C2\u00A0 Mier,\u00C2\u00A0 and\u00C2\u00A0 A.C.\u00C2\u00A0 Ochoa.\u00C2\u00A0 2005.\u00C2\u00A0 Arginase\u00E2\u0080\u0090producing\u00C2\u00A0myeloid\u00C2\u00A0suppressor\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0renal\u00C2\u00A0cell\u00C2\u00A0carcinoma\u00C2\u00A0patients:\u00C2\u00A0a\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0evasion.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A065:3044\u00E2\u0080\u00903048.\u00C2\u00A0 Zhang,\u00C2\u00A0F.,\u00C2\u00A0S.S.\u00C2\u00A0Lau,\u00C2\u00A0and\u00C2\u00A0T.J.\u00C2\u00A0Monks.\u00C2\u00A02011.\u00C2\u00A0The\u00C2\u00A0cytoprotective\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0N\u00E2\u0080\u0090acetyl\u00E2\u0080\u0090L\u00E2\u0080\u0090cysteine\u00C2\u00A0 against\u00C2\u00A0 ROS\u00E2\u0080\u0090induced\u00C2\u00A0 cytotoxicity\u00C2\u00A0 is\u00C2\u00A0 independent\u00C2\u00A0 of\u00C2\u00A0 its\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 enhance\u00C2\u00A0 glutathione\u00C2\u00A0synthesis.\u00C2\u00A0Toxicol\u00C2\u00A0Sci\u00C2\u00A0120:87\u00E2\u0080\u009097.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0"@en . "Thesis/Dissertation"@en . "2012-05"@en . "10.14288/1.0072452"@en . "eng"@en . "Experimental Medicine"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "Attribution-NonCommercial-NoDerivatives 4.0 International"@en . "http://creativecommons.org/licenses/by-nc-nd/4.0/"@en . "Graduate"@en . "Immunosuppressive myeloid cells under normal and neoplastic conditions"@en . "Text"@en . "http://hdl.handle.net/2429/39661"@en .