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Mammary tumour-induced dysregulation in hematopoiesis and DC development and the role of Lyn tyrosine… Chehal, Manreet Kaur 2014

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Mammary tumour-induced dysregulation in hematopoiesis and DC development and the role of Lyn tyrosine kinase in DC activation  by Manreet Kaur Chehal  B.Sc., The University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2014  © Manreet Kaur Chehal, 2014   ii Abstract Previous studies have revealed that perturbations in myelopoiesis can lead to the emergence of immature cells, which can facilitate tumorigenesis and metastasis.  Our studies showed that mammary tumours led to a myeloproliferative-like disease, characterized by anemia, leukocytosis, expansion of immature myeloid cells, and defects in the hematopoietic stem and progenitor cell (HSPC) compartment in tumour-bearing mice.  Furthermore, mammary tumours impaired DC development resulting in the accumulation of DC progenitors and the emergence of immunosuppressive DCs with impaired ability to activate NK and T cells.  Mammary tumour-bearing mice exhibited a shift in hematopoiesis from the bone marrow to the spleen, with large numbers of primitive and committed progenitors accumulating in the spleen.  Mammary tumour development was also associated with epigenetic modifications that facilitated the expression of key hematopoiesis and leukemia regulatory genes, such as Hoxa9, a gene that critically controls HSPC differentiation. Using in vitro assays, we identified mammary tumour derived G-CSF as the factor mediating the dysregulation of the HSPC compartment and suppressing DC development.  DCs are critical for the priming/activation of NK cells and cytotoxic T lymphocytes, which together are critical components of the anti-tumour immune response.  Therefore, proper regulation of signaling thresholds is critical for DC activation and function.  Lyn tyrosine kinase is important in regulating signaling thresholds in immune cells, including DCs, and Lyn gain-of-function (Lynup/up) mice show increased sensitivity to PAMPs (i.e. LPS) characterized by high levels of proinflammatory cytokines and increased susceptibility to endotoxin-mediated death. Our studies demonstrated that DCs were necessary for the enhanced LPS-induced inflammation in Lynup/up mice, by priming excessive IFN-γ production by NK cells.  As such, we hypothesized   iii that enhanced Lyn activity, and the associated changes in DC development and activation will evoke improved anti-tumour immune responses and preliminary data from our lab demonstrates that Lynup/up mice develop smaller mammary tumours than wild type mice.  Taken together, our results suggest that targeting mammary tumour derived G-CSF may reverse mammary tumour-induced anemia, leukocytosis, and DC defects, conversely enhancing DC function by increasing Lyn activity may result in in a more robust anti-tumour immune response, leading to increased survival of breast cancer patients.     iv Preface Contributions: A version of chapter 3 has been published in Cancer Research. 2013; 73:5892-904. In Section 3, I was responsible for establishing the tumour project in our lab and carrying out all of the in vivo experiments. I studied the effects of mammary tumour growth on HSPCs, erythropoiesis and myelopoiesis in the spleen, the reconstitution capability of HSPCs, expression of hematopoiesis regulatory genes and histone methylation by flow cytometry. I determined G-CSF concentrations by bead array, effects of G-CSF in BM cultures and the outcome of inhibiting Menin/MLL interactions in MT-CM treated BM cultures. A Sio was responsible for the analysis of the BM compartment of tumour-bearing mice, FLT3L BM cultures treated with MT-CM, and identifying the factor in MT-CM. A Sio aided in experimental design/data analysis. K Tsai conceptualized the idea of epigenetic dysregulation mediated by mammary tumours and performed qPCR for hematopoietic regulatory genes. XL Fan performed ChIP experiments for the G-CSF loss-of-function experiments and did qPCR for hematopoietic regulatory genes. Dr. D Krebs performed qPCR experiments for hematopoietic regulatory genes and ChIP on BM cultures treated with MT-CM. M Roberts measured viability/proliferation of BM cultures treated with MT-CM. Dr. K Harder conceptualized the project, designed experiments, and provided feedback.  A version of chapter 4 is in preparation for submission. In section 4, in mammary tumour-bearing mice, I analyzed the expansion of splenic DC progenitors, NK cell activation, expression of immunosuppressive enzymes in DCs and in vitro I investigated the effects of MT-CM on GM-CSF DC activation, reversibility of MT-CM on DC phenotype/function and the role of G-CSF on DC development/function. A Sio analyzed the effects of MT-CM on FLT3L DC development/activation/function, of G-CSF on DC function/development and analyzed DC   v development in vivo in the BM.  A Sio designed experiments and analyzed data. XL Fan investigated the effects of MT-CM on DCs ability to prime T cell proliferation/function.  Dr. D Krebs measured protein levels and histone methylation status of immunosuppressive enzymes in MT-CM treated DC cultures. Dr. K Harder conceptualized majority of the experiments, designed the experiments and provided feedback.  A version of chapter 5 has been published in J Immunol. 2012; 188:5094-105. In section 5, I performed experiments analyzing DC development/activation post-LPS stimulation in Lyn+/+, Lyn-/- and Lynup/up mice. I confirmed NK cells were responsible for increased IFN-γ production in Lynup/up mice and showed that both DCs and NK cells were necessary for LPS hypersensitivity of Lynup/up mice, and characterized the splenic NK cell compartment. In in vitro experiments, I confirmed the increased ability of Lynup/up DCs to activate NK cell IFN-γ production and the necessity of SHIP-1 for this interaction. A Sio was responsible for shRNA experiments targeting SHIP-1 and SHP-1. Dr. D Krebs analyzed signaling pathways activated in DCs, determined the expression of proinflammatory cytokines in GM-CSF DCs, and aided in experimental design and data analysis. N Huntington analyzed the NK cell compartment in the spleen and liver. Dr. K Harder did the experiments demonstrating LPS hypersensitivity of Lynup/up mice, necessity of DCs in activating NK cells, the enhanced activation of Lynup/up GM-CSF post-LPS, demonstrated the importance of SHIP-1 and SHP-1 in LPS mediated responses and aided in experimental design/data analysis. Section 6 was based on research and discussion written by Dr. K Harder, Dr. D. Krebs, A Sio and I. Lastly, I contributed to the experimental design, data analysis, making figures and writing/editing of the manuscripts based on Sections 3 - 5. Ethics Approval: Animal experiments were performed in accordance with Canadian Council for Animal Care and the University of British Columbia Animal Care Committee guidelines. Protocol A06-1557 & A09-0814.   vi Table of Contents Abstract .......................................................................................................................................... ii	  Preface ........................................................................................................................................... iv	  Table of Contents ......................................................................................................................... vi	  List of Tables ............................................................................................................................... xii	  List of Figures ............................................................................................................................. xiii	  List of Abbreviations ............................................................................................................... xviii	  Acknowledgements .................................................................................................................. xxiii	  Dedication ................................................................................................................................. xxiv	  Chapter 1: Introduction ................................................................................................................1	  1.1	   Cancer and hematopoiesis .................................................................................................. 1	  1.1.1	   Hematologic abnormalities in neoplastic disease ....................................................... 1	  1.1.1.1	   Anemia in neoplasia ............................................................................................. 2	  1.1.1.2	   Leukocytosis in neoplasia .................................................................................... 3	  1.1.2	   Disruption of hematopoiesis in tumour-bearing hosts ................................................ 4	  1.1.2.1	   Hox gene regulation of hematopoiesis ................................................................. 5	  1.1.2.2	   The role of histone methylation in the regulation of Hox gene expression ......... 6	  1.1.3	   G-CSF and its role in hematopoiesis .......................................................................... 9	  1.1.3.1	   The role of G-CSF in cancer .............................................................................. 10	  1.2	   Cancer and the DC lineage .............................................................................................. 12	  1.2.1	   The DC lineage ......................................................................................................... 12	  1.2.1.1	   DC subsets ......................................................................................................... 12	  1.2.1.2	   DC function ........................................................................................................ 12	    vii 1.2.1.3	   DC development ................................................................................................ 14	  1.2.2	   The role of DCs in neoplasia .................................................................................... 17	  1.2.2.1	   DC localization, numbers and phenotype in neoplasia ...................................... 17	  1.2.2.2	   DC developmental defects in neoplasia ............................................................. 19	  1.3	   Lyn tyrosine kinase and DC/NK cell cross-talk .............................................................. 20	  1.3.1	   Src family kinases ..................................................................................................... 20	  1.3.1.1	   Lyn tyrosine kinase ............................................................................................ 20	  1.3.1.2	   Lyn in DCs ......................................................................................................... 22	  1.3.2	   Innate Immunity ........................................................................................................ 23	  1.3.2.1	   NK cells ............................................................................................................. 24	  1.3.2.2	   DC/NK cell cross talk ........................................................................................ 25	  1.3.2.3	   IFN-γ  in sepsis ................................................................................................... 26	  1.4	   Research Objective .......................................................................................................... 28	  Chapter 2: Materials and methods .............................................................................................30	  2.1	   General reagents ............................................................................................................... 30	  2.2	   Mice ................................................................................................................................. 30	  2.3	   Flow cytometry antibodies ............................................................................................... 31	  2.4	   Tumour cell lines ............................................................................................................. 32	  2.5	   In vitro BM analysis and BM-derived DC culture and splenic NK cell culture .............. 32	  2.6	   Competitive reconstitution and CFU-S12 assays .............................................................. 32	  2.7	   qPCR ................................................................................................................................ 33	  2.8	   Chromatin immunoprecipitation (ChIP) .......................................................................... 33	  2.9	   Methylcellulose assays ..................................................................................................... 34	    viii 2.10	   Western blot ................................................................................................................... 35	  2.11	   Tumour injection and tissue processing ......................................................................... 35	  2.12	   Flow cytometry and FACS ............................................................................................ 35	  2.13	   Menin inhibitor treatment of BM cultures ..................................................................... 36	  2.14	   T cell proliferation, cytokine, phagocytosis and suppression assay .............................. 36	  2.15	   LPS-induced cytokine production and morbidity .......................................................... 36	  2.16	   Microscopy .................................................................................................................... 37	  2.17	   Cell stimulation and lysis ............................................................................................... 37	  2.18	   NK cell co-culture assays ............................................................................................... 38	  2.19	   Design and subcloning of shRNAs targeting SHP-1 and SHIP-1 ................................. 38	  2.20	   Infection of DCs with retrovirus encoding shRNAs ...................................................... 38	  2.21	   Statistical analysis .......................................................................................................... 39	  Chapter 3: Mammary tumour-induced dysregulation in hematopoiesis ...............................40	  3.1	   Mammary cancer induces epigenetic changes and impairs hematopoiesis leading to perturbations in myelopoiesis and erythropoiesis ..................................................................... 41	  3.1.1	   Peripheral blood parameters of mammary tumour-bearing mice ............................. 41	  3.1.2	   Leukemoid reaction in mammary tumour-bearing mice .......................................... 43	  3.1.3	   Dysregulated erythropoiesis in mammary tumour-bearing mice .............................. 45	  3.1.4	   Defective hematopoietic stem/progenitor cell numbers and localization in mammary tumour-bearing mice ............................................................................................................. 50	  3.1.5	   Defects in bone marrow hematopoietic stem/progenitor cell numbers and localization are not due to metastasis of MT-1 cells ............................................................. 55	    ix 3.1.6	   Functional defects in the hematopoietic stem/progenitor cell compartment in mammary tumour-bearing mice ............................................................................................ 56	  3.1.7	   In vitro differentiation of hematopoietic stem/progenitor cells is impaired by mammary tumour secreted factors ........................................................................................ 58	  3.1.8	   Regulation of Hox gene expression and histone modifications at the Hoxa9 locus by mammary tumour secreted factors ........................................................................................ 66	  3.1.9	   Contribution of G-CSF in mammary tumour secreted factor suppression of hematopoietic stem/progenitor cell differentiation ............................................................... 73	  3.1.10	   Role of global H3K4me3 in mammary tumour secreted factor-mediated suppression of hematopoietic stem/progenitor cell defects ...................................................................... 75	  3.1.11	   Discussion ............................................................................................................... 76	  Chapter 4: Mammary tumour-induced dysregulation in DC development ...........................83	  4.1	   Mammary cancer induces changes in DC development, function and phenotype .......... 84	  4.1.1	   Mammary tumour mediated suppression of DC differentiation ............................... 84	  4.1.2	   Role of mammary tumour secreted factors in suppressing in vitro FLT3L derived DCs 90	  4.1.3	   Role of mammary tumour secreted factors in suppressing GM-CSF derived DC development in vitro ............................................................................................................. 96	  4.1.4	   Mammary tumour derived G-CSF is responsible for DC developmental impairment 100	  4.1.5	   MT-1 conditioned media and G-CSF mediate impairment of DC functional activity 105	    x 4.1.6	   Upregulation of immunosuppressive enzymes and markers by MT-1 conditioned media treatment of FLT3L and GM-CSF DCs is accompanied by changes in histone modifications ....................................................................................................................... 111	  4.1.7	   Mammary tumour-conditioned media expands functional DC progenitors that can differentiate into pDCs and cDCs ....................................................................................... 115	  4.1.8	   Discussion ............................................................................................................... 119	  Chapter 5: Role of Lyn tyrosine kinase in DC development and function ...........................124	  5.1	   Lyn-Dependent Signaling Regulates the Innate Immune Response .............................. 125	  5.1.1	   Lynup/up mice exhibit enhanced endotoxin-induced inflammation and morbidity. . 125	  5.1.2	   Lyn promotes production of pro-inflammatory cytokines in response to endotoxin. 127	  5.1.3	   NK cells are the primary source of IFN-γ in LPS-challenged Lynup/up mice. ......... 130	  5.1.4	   BM derived GM-CSF and FLT3L DCs, but not BM derived Mφs, activate NK cells in response to LPS. ............................................................................................................. 132	  5.1.5	   The DC lineage is required for enhanced NK cell IFN-γ production in Lynup/up mice. 134	  5.1.6	   DCs and NK cells are required for LPS-induced morbidity in Lynup/up mice. ........ 135	  5.1.7	   Lyn activity within DCs enhances endotoxin-induced IFN-γ production in NK cells. 137	  5.1.8	   Lyn activity within DCs modulates LPS-induced cytokine production. ................ 141	  5.1.9	   Lyn activity stimulates DC maturation. .................................................................. 144	  5.1.10	   Lyn negatively regulates LPS-induced signal transduction in DCs. ..................... 146	  5.1.11	   Lyn interacts functionally with SHIP-1 and SHP-1 in DCs. ................................ 148	    xi 5.1.12	   Lyn plays a protective role in mammary tumour development. ........................... 154	  5.1.13	   Discussion ............................................................................................................. 157	  Chapter 6: Concluding comments ............................................................................................164	  6.1	   Conclusion and future directions ................................................................................... 164	  6.2	   Summary of implications and applications .................................................................... 165	  References ...................................................................................................................................167	  Appendix .....................................................................................................................................187	  Appendix A ............................................................................................................................. 187	  A.1	   Sub-Appendix – mRNA qPCR primers .................................................................... 187	  A.2	   Sub-Appendix – ChIP qPCR primers ....................................................................... 187	     xii List of Tables Table 3.1    Hematologic analyis of MT-1 bearing mice .............................................................. 42	  Table 3.2    Hematologic analysis of 4T1 tumour-bearing mice .................................................. 49	  Table 5.1        Expression of NK cell markers on NK1.1+ CD49b+ cells from Lyn+/+ and Lynup/up spleen and liver. .......................................................................................................................... 139	  Table 5.2        IFN-γ production from in vitro IL-15-expanded NK cells. ................................. 140	  Table 5.3        Percent of MHC IIhigh, MHC IIint and MHC IIlow DCs following knock down of SHIP-1 or SHP-1 in Lyn+/+ and Lyn-/- GM-CSF DCs. ............................................................... 153	     xiii List of Figures  Figure 1.1    DC differentiation. ................................................................................................... 16	  Figure 3.1    Mammary tumours induce changes in granulocyte and monocyte numbers in mice........................................................................................................................................................ 44	  Figure 3.2    Mammary tumour development is associated with impaired BM erythropoiesis and heightened splenic erythropoiesis. ................................................................................................ 47	  Figure 3.3    Mammary tumour development is associated with reduced numbers of BM erythrocytes and increased numbers of splenic immature erythroid cells. ................................... 48	  Figure 3.4    Melanoma tumour development is not associated with impaired BM erythropoiesis........................................................................................................................................................ 49	  Figure 3.5    4T1 tumor development is associated with impaired BM myelopoiesis and erythropoiesis. ............................................................................................................................... 50	  Figure 3.6    Perturbations in hematopoietic stem/progenitor cell frequency in mammary tumour-bearing mice. ................................................................................................................................. 52	  Figure 3.7    Perturbations in hematopoietic stem/progenitor cell numbers and location in mammary tumour-bearing mice. ................................................................................................... 53	  Figure 3.8    4T1 tumor development disrupts BM hematopoietic stem/progenitor cells. ........... 54	  Figure 3.9    Melanoma tumour development does not disrupt the BM hematopoietic stem/progenitor compartment. ...................................................................................................... 55	  Figure 3.10    Mammary tumour growth is associated with significant functional changes in the stem/progenitor compartments in BM and spleen. ....................................................................... 57	  Figure 3.11    Mammary tumour-conditioned media expands hematopoietic progenitors in in vitro BM cultures supplemented with FLT3L or GM-CSF. ......................................................... 61	    xiv Figure 3.12    Mammary tumour-conditioned media expands hematopoietic progenitors in in vitro BM cultures supplemented with FLT3L, while G-CSF neutralization suppresses mammary tumour-conditioned media-dependent expansion of hematopoietic progenitors. ......................... 62	  Figure 3.13    Mammary tumour-conditioned media expands hematopoietic progenitors in BM cultures supplemented with GM-CSF. .......................................................................................... 63	  Figure 3.14    Identification of cytokines produced by MT-1 cells. ............................................. 64	  Figure 3.15    Mammary tumor-conditioned media increases hematopoietic progenitor cell proliferation and decreases LSK death in in vitro BM cultures supplemented with FLT3L or GM-CSF. .............................................................................................................................................. 65	  Figure 3.16    Mammary tumour growth is associated with enhanced Hoxa gene expression and changes in histone methylation. .................................................................................................... 69	  Figure 3.17    Mammary tumor-conditioned media is associated with changes in histone modifying proteins and histone methylation. ................................................................................ 70	  Figure 3.18    Mammary tumour-conditioned media treatment of BM cultures is associated with enabling changes in histone methylation at the Hoxa9 locus. ...................................................... 71	  Figure 3.19    Mammary tumor growth is associated with changes in histone modifying proteins and G-CSF induced changes in histone methylation at the Hoxa9 locus. .................................... 72	  Figure 3.20    G-CSF enhances Hoxa9 expression and CFC numbers, while Menin inhibitors reverse mammary tumour-conditioned media induced expansion of LSKs, LKs and CFCs in in vitro BM cultures. ......................................................................................................................... 74	  Figure 4.1    DC progenitor gating scheme. .................................................................................. 85	  Figure 4.2    Perturbations in DC progenitor numbers and location in mammary tumour-bearing mice. .............................................................................................................................................. 85	    xv Figure 4.3    Perturbations in DC progenitor numbers and location in 4T1 tumour-bearing mice........................................................................................................................................................ 86	  Figure 4.4    Perturbations in DC progenitor numbers and location in B16 tumour-bearing mice........................................................................................................................................................ 86	  Figure 4.5    Perturbations in DC frequency in mammary tumour-bearing mice. ........................ 88	  Figure 4.6    Perturbations in DC numbers in mammary tumour-bearing mice. .......................... 89	  Figure 4.7    Mammary tumour-conditioned media impairs DC differentiation in in vitro FLT3L DC cultures. .................................................................................................................................. 93	  Figure 4.8    Mammary tumour-conditioned media impairs DC expression of MHC II and co-stimulatory receptors in in vitro FLT3L DC cultures. .................................................................. 94	  Figure 4.9    Mammary tumour-conditioned media enhances DC progenitor generation and impairs essential DC transcription factor mRNA expression in in vitro FLT3L DC cultures. .... 95	  Figure 4.10    Mammary tumour-conditioned media impairs DC activation in in vitro GM-CSF DC cultures. .................................................................................................................................. 97	  Figure 4.11    Mammary tumour-conditioned media impairs DC differentiation in in vitro GM-CSF DC cultures at different concentrations and treated for different periods of time. ............... 98	  Figure 4.12    Mammary tumour-conditioned media impairs cytokine secretion in in vitro GM-CSF DC cultures. .......................................................................................................................... 99	  Figure 4.13    G-CSF impairs DC development and activation in in vitro FLT3L cultures. ...... 102	  Figure 4.14    G-CSF impairs DC activation in in vitro GM-CSF cultures. ............................... 103	  Figure 4.15    Mammary tumour-conditioned media derived G-CSF enhances DC progenitor generation. ................................................................................................................................... 104	  Figure 4.16    Mammary tumours impair DC activation of NK and T cells. .............................. 109	    xvi Figure 4.17    Mammary tumours impair antigen specific DC activation of T cells and induces DC mediated suppression of T cell proliferation. ....................................................................... 110	  Figure 4.18    Mammary tumour-conditioned media induces immunosuppressive protein expression in in vitro and in vivo DCs. ....................................................................................... 113	  Figure 4.19    Mammary tumour conditioned media derived G-CSF contributes to Arg1 expression,. ................................................................................................................................. 114	  Figure 4.20    Mammary tumour-conditioned media treatment of FLT3L DC cultures produce functional DC progenitors that differentiate into pDCs and cDCs. ............................................ 117	  Figure 4.21    Removal of mammary tumour-conditioned media restores ability of FLT3L DCs to activate OTI and OTII cell proliferation. .................................................................................... 118	  Figure 4.22    Removal of mammary tumour-conditioned media reduces expression of immunosuppressive proteins/enzymes and increases MHC II expression. ................................ 118	  Figure 5.1        Lyn activity regulates endotoxin sensitivity in mice. ......................................... 126	  Figure 5.2        Lynup/up mice exhibit enhanced inflammatory cytokine production in response to LPS. ............................................................................................................................................. 128	  Figure 5.3        Elevated levels of chemokines and proinflammatory cytokines in the serum of Lynup/up mice following LPS injection. ....................................................................................... 129	  Figure 5.4        NK cells are the predominant source of IFN-γ in LPS-stimulated mice. ........... 131	  Figure 5.5        DCs and not Mφs are necessary for IFN-γ production by NK cells. .................. 133	  Figure 5.6        IFN-γ production and hypersensitivity to LPS in Lynup/up mice depends on DCs and NK cells. ............................................................................................................................... 136	  Figure 5.7        Lyn polarizes DCs with respect to NK cells to secrete IFN-γ. ........................... 138	  Figure 5.8        Lyn polarizes DCs with respect to LPS-induced cytokine production. .............. 142	    xvii Figure 5.9        Lyn polarizes DCs with respect to LPS- and NK cell-induced cytokine production. .................................................................................................................................. 143	  Figure 5.10        Lyn controls spontaneous and TLR-dependent DC maturation. ...................... 145	  Figure 5.11        Lyn regulates LPS-induced signaling pathways in GM-CSF DCs. ................. 147	  Figure 5.12        Lyn regulates SHIP-1 and SHP-1 phosporylations in GM-CSF DCs. ............. 150	  Figure 5.13        SHIP-1 and SHP-1 regulate GM-CSF DC maturation and function. ............... 151	  Figure 5.14        SHIP-1 and SHP-1 regulate GM-CSF DCs activation in response to LPS. ..... 152	  Figure 5.15         Role of DCs and Lyn in mammary tumour development. .............................. 155	  Figure 5.16         Lyn expression does not reverse the effects of mammary tumour-conditioned media in in vitro GM-CSF DC cultures. ..................................................................................... 156	     xviii List of Abbreviations Ab antibody ADCC antibody dependent cellular cytotoxicity AML acute myeloid leukemia AngII Angiotensin II APC antigen presenting cell Arg1 Arginase I BAL-F bronchoalveolar lavage fluid BCR B cell receptor BM bone marrow cDC conventional dendritic cell CDP common dendritic cell progenitor CFSE carboxyfluorescein succinimidyl ester CFU colony forming unit CFU-G colony forming unit - granulocyte CFU-GM colony forming unit – granulocyte macrophage CFU-M colony forming unit - macrophage ChIP chromatin immunoprecipitation CLP common lymphoid progenitor CMP common myeloid progenitor DAPI 4',6-diamidino-2-phenylindole DC dendritic cell DT diphtheria toxin   xix DTR diphtheria toxin receptor Eed embryonic ectoderm development EZH2 enhancer of zeste homolog 2 Fizz1 found in inflammatory zone 1 FLT3L FMS-like tyrosine kinase 3 ligand  G-CSF granulocyte colony-stimulating factor  G-CSFR granulocyte colony stimulating factor receptor GM-CSF granulocyte-macrophage colony-stimulating factor GMP granulocyte macrophage progenitor GOF gain-of-function GRA granulocyte GrB granzyme B H&E hematoxylin & eosin H3K27 histone 3 lysine 27 H3K4 histone 3 lysine 4 HCT hematocrit HGB hemoglobin HOX Homeobox HSC hematopoietic stem cell HSPC hematopoietic stem/ progenitor cell IFN interferon iMC immature myeloid cell iNOS inducible nitric oxide synthase   xx i.p. intraperitoneal ITAM immunoreceptor tyrosine-based activation motifs ITIM immunoreceptor tyrosine-based inhibitor motifs JMJD3 JmjC domain-containing protein 3 LOF loss-of-function LPS lipopolysaccharide LT-HSC long-term hematopoietic stem cell LYM lymphocyte M-CSF Macrophage colony-stimulating factor MDP monocyte dendritic cell progenitor MDS myelodysplastic syndrome MDSC myeloid derived suppressor cell MEP megakaryocyte erythrocyte progenitor MFP mammary fat pad MHC major histocompatibility complex MI Menin inhibitor MLL mixed lineage leukemia  MON monocyte Mφ macrophage MPP multi-potent progenitors MPS mononuclear phagocyte system MT mammary tumour MT-1-CM mammary tumour 1 conditioned media   xxi MT-CM mammary tumour conditioned media NK cell natural killer cell NOS nitric oxide synthase OVA ovalbumin PAMP pathogen associated molecular pattern PAS protein A sepharose PcG Polycomb Group pDC plasmacytoid dendritic cell PI propidium iodide PLT platelet PRC2 Polycomb Repressive Complex 2 PreDC dendritic cell precursor PRR pattern recognition receptor Rae1 retinoic acid early inducible 1 RBC red blood cell ROS reactive oxygen species SCF stem cell factor SFK src family kinase SH2 Src homology 2 SHIP-1 Src homology 2 domain containing inositol polyphosphate phosphatase-1 SHP Src homology 2 domain-containing phosphatase SQ subcutaneous ST-HSC short-term hematopoietic stem cell   xxii SUZ12 suppressor of zeste 12 TAM tumour associated macrophage TAN tumour associated neutrophil TAP transporter associated with antigen presenting T-CM tumour conditioned media TCR T cell receptor Th T helper TLR Toll like receptor TSLP thymic stromal lymphopoietin Treg regulatory T cell TrxG Trithorax Group VEGFa vascular endothelial growth factor a WBC white blood cell WT wild type     xxiii Acknowledgements I would like to thank… Dr. K Harder, for all of your support, advice, and guidance during my graduate studies. Dr. P Johnson, Dr. C Zaph, and Dr. M Levings for all of your guidance and suggestions. A Sio, M.Sc., for making ‘tumour FACS days’ fun and for being my awesome partner for the wild type tumour project. XL Fan, Ph.D. candidate, for all of your help in getting experiments done and figures made for the wild type tumour project. M Roberts, Ph.D. candidate, for all of your help in the lab and in proofreading my thesis, I cannot imagine how much coffee you had to drink to get through it!!  Dr. D Krebs, for all of your advice and help with experiments, presentations, committee meeting reports, and scholarship applications. D Birkenhead, for all of your advice, guidance, and reassurance.  Graduate students/Post-docs of I3, for making graduate school so much fun!! My husband for all of your support during my graduate studies and listening to me complain when experiments did not work.  My parents for all of their support and making sure I never starved.  Lastly, my son for always putting a smile on my face while writing my thesis.   xxiv Dedication To my: Dad for always prioritizing education in our family, being my #1 supporter and for always reminding me when things got tough, if it was easy then everyone would be going to school and that all I could do was try my best.  Mom for always making sure that I never went hungry, for waking up at 5 am to make me breakfast and drop me off at the skytrain station and helping out with anything (Aikam, food, chores) so I would have more time for my studies.  Sister for always putting things in perspective, highlighting all of the opportunities and helping me take one day at a time.  Brother for giving me an excuse to take a break from my studies.  Husband for always being so supportive, taking over making sure I was fed and always reminding me that I could do it.  Son for making me realize how easy I had it J       1 Chapter 1: Introduction 1.1 Cancer and hematopoiesis 1.1.1 Hematologic abnormalities in neoplastic disease Hematopoietic system abnormalities including anemia and the expansion of immature myeloid cells (iMCs), are commonly observed in cancer patients and are associated with poorer prognosis (Diaz-Montero et al., 2009; Geeganage et al., 2010; Qiu et al., 2010a; Wilcox, 2010).  Qiu et al. (2010a) demonstrated that in patients, including breast cancer patients, anemia was associated with a lower quality of life and was one of several factors that negatively affected their overall survival (Qiu et al., 2010a).  However, it is still not well understood whether anemia precedes cancer and if it is necessary for the development of cancer or whether anemia is the result of cancer.  Furthermore, cancer induced changes in hematopoiesis can result in the expansion of tumour promoting cells associated with poor prognosis, including tumour associated macrophages (TAMs), regulatory T cells (Tregs), a subset of cells broadly termed myeloid derived suppressor cells (MDSCs), as well as functionally impaired tumour-associated dendritic cells (tDCs) (Gabrilovich et al., 2012). The implication and significance of the expansion of MDSCs, also referred to as the leukemoid reaction, in cancer patients has recently become better appreciated.  It is now established that this expansion of MDSCs in cancer patients is linked to higher mortality and poorer prognosis due to the ability of MDSCs to inhibit anti-tumour immune responses and aid in tumour progression (Almand et al., 2001; Chen et al., 2009; Schmidt et al., 2005; Schmielau and Finn, 2001; Shoenfeld et al., 1986; Wilcox, 2010).  Furthermore, breast cancer patients who have also been diagnosed with leukocytosis, thrombocytosis and anemia have diminished survival rates compared to breast cancer patients without any hematological abnormalities (Qiu et al., 2010a).  Since red blood cells (RBCs) and   2 iMCs both originate from the common myeloid progenitor (CMP), it is possible that the cancer-associated dysregulation of erythropoiesis and myelopoiesis is due to alterations in a common pathway culminating in these hematopoietic abnormalities.  Therefore, it is important to understand how tumour cells alter hematopoiesis in order to correct these defects in cell differentiation. 1.1.1.1 Anemia in neoplasia The development of anemia in cancer patients is common and is associated with lower quality of life (Leonard et al., 2005).  Importantly, anemia is also associated with decreased tumour control and therefore survival of cancer patients (Caro et al., 2001).  However, whether the development of anemia precedes cancer or is a result of cancer development is currently not well understood.  Additionally, if the development of anemia is the result of tumour development, it is not known whether the tumour is directly interfering with the production of RBCs or indirectly via the secretion of cytokines (Qiu et al., 2010b).  However, it has been shown that certain tumours can produce/induce the production of various cytokines including interleukins, IFN-γ, and TNF-α, which can inhibit erythropoiesis and the ability of erythroid progenitors to respond to erythropoietin and cause hemolysis (Dowlati et al., 1997; Spivak, 1994).  The development of pre-treatment anemia in breast, and other cancer patients, negatively impacts survival (Gislason and Nou, 1985; Metindir and Bilir Dilek, 2009; Qiu et al., 2010a; Qiu et al., 2010b).  A study by Qiu et al. (2010a) demonstrated that the prevalence of anemia in breast cancer patients was higher compared to the control group with benign diseases (Qiu et al., 2010a).  Furthermore, it has been established that anemia is an independent prognostic factor for survival of breast cancer patients (Qiu et al., 2010a).  Therefore, the negative association between cancer and anemia   3 highlights the importance of understanding the mechanism of anemia development in cancer patients in order to reverse/inhibit it.  1.1.1.2 Leukocytosis in neoplasia Expansion of iMCs, broadly termed MDSCs, have been linked to poor prognosis in cancer patients (Diaz-Montero et al., 2009; Gabitass et al., 2011; Qiu et al., 2010a).  In a healthy individual, iMCs make up less than 1% of the peripheral blood mononuclear cells, however, under disease settings such as cancer, infectious diseases, sepsis, trauma, bone marrow (BM) transplantation and autoimmune diseases, MDSCs expand and accumulate in the BM, peripheral blood, secondary lymphoid organs and in tumours (Gabrilovich and Nagaraj, 2009; Geeganage et al., 2010; Wilcox, 2010).  MDSCs are the result of impaired terminal differentiation of macrophages (Mφs), dendritic cells (DCs) and/or granulocytes from myeloid progenitors (Gabrilovich, 2004; Kusmartsev and Gabrilovich, 2003; Li et al., 2004; Narita et al., 2009). During pathological conditions, MDSC activation results in the upregulation of immunosuppressive factors such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), nitric oxide species (NOS) and reactive oxygen species (ROS), which mediate their ability to suppress various immune cells such as T and natural killer (NK) cells (Gabrilovich, 2004; Gabrilovich et al., 2012).  For example, MDSCs suppress T cell activation through mechanisms such as depletion of L-arginine via Arg1, which is necessary for T cell activation; production of ROS and NOS, which can result in post-translational modifications of the T cell receptor (TCR), impairing its ability to bind major histocompatibility complex (MHC)/peptide complexes; and secretion of factors such as IL-10, that suppress the function of antigen-presenting cells (APCs) (Gabrilovich, 2004; Gabrilovich et al., 2012).  Furthermore, MDSCs can also decrease the   4 number of NK cells and impair NK cell function by presenting TGF-β on the surface of MDSCs (Gabrilovich, 2004; Gabrilovich et al., 2012).  In cancer, MDSC expansion has been linked to tumour burden/clinical grade. Cytokines secreted by tumours such as IL-6, granulocyte colony-stimulating factor (G-CSF), Mφ colony-stimulating factor (M-CSF), stem cell factor (SCF), vascular endothelial growth factor (VEGF), prostaglandins and granulocyte-Mφ colony-stimulating factor (GM-CSF) have all been linked to MDSC expansion (Gabrilovich and Nagaraj, 2009). Interestingly, chemotherapeutic agents such as gemcitabine, 5-fluoruracil and doxorubicin have been shown to deplete or prevent the expansion of MDSCs, which may contribute an additional, tumour extrinsic, mechanism of controlling tumour growth (Alizadeh et al., 2014; Geeganage et al., 2010; Suzuki et al., 2005; Vincent et al., 2010; Wilcox, 2010). MDSCs have also been shown to mediate angiogenesis, which may aide in tumour growth and metastasis (Geeganage et al., 2010; Shojaei et al., 2008; Wilcox, 2010). Together, these studies suggest that MDSC expansion contributes to tumour growth, and links control of tumour growth by chemotherapy with immunosurveillance (Alizadeh et al., 2014; Geeganage et al., 2010; Suzuki et al., 2005; Vincent et al., 2010; Wilcox, 2010). The recognition of the significance of the expansion of immunosuppressive cells during cancer progression provides an important avenue to explore the development of anti-cancer therapeutic strategies. Furthermore, it suggests that for cancer immunotherapy to be successful, the elimination of immunosuppressive factors, such as MDSCs will need to be incorporated into cancer immunotherapy.   1.1.2 Disruption of hematopoiesis in tumour-bearing hosts Hematopoiesis involves the sequential differentiation of BM hematopoietic stem and progenitor cells (HSPCs) into more committed blood cell types.  This is a tightly controlled process that   5 involves DNA-binding transcription factors and epigenetic changes that regulate cell differentiation into mature red and white blood cells as well as the maintenance of HSPCs and their self renewal capacity (Cedar and Bergman, 2011). The ability of tumours to impair leukocyte function has been extensively studied, however their impact on leukocyte development remains unclear.  The extent of tumour induced changes in hematopoiesis, especially leukocytosis and anemia, and the ability of these changes to aid tumour growth need to be better understood. An understanding of these processes will allow for the development of therapies to inhibit these tumour-mediated alterations in HSPCs, with the end goal of development of immune cells better able to eliminate the tumour.   1.1.2.1 Hox gene regulation of hematopoiesis Homeobox (Hox) genes, particularly Hoxa9, have been shown to be important in hematopoiesis by mediating the expansion, differentiation, and maintenance of hematopoietic stem cells (HSCs) (Argiropoulos and Humphries, 2007).  Hoxa9-deficient mice display defects in HSC repopulation, maintenance, and differentiation into more committed hematopoietic lineages. By contrast, gain-of-function mutations in Hoxa9 result in increased HSC regeneration with an inability to differentiate into more restricted cell types and the eventual development of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) (Argiropoulos and Humphries, 2007; Thorsteinsdottir et al., 2002).  In patients with MDS or AML, normal BM cells are replaced with rapidly dividing, undifferentiated cells of the myeloid lineage, and these changes can occur concurrently with anemia and impaired erythropoiesis (Argiropoulos and Humphries, 2007; Tefferi and Vardiman, 2009).  Interestingly, the expansion of leukocytes and development of anemia in patients with MDS and AML have also been documented in patients with solid tumours.  The similarities in the expansion of leukocytes, development of anemia and   6 dysregulation of HSPCs between patients with MDS, AML and solid tumours, suggests the possibility that these diseases share a common mechanism for inducing hematopoietic abnormalities.  Therefore, dysregulation of Hoxa, particularly Hoxa9, genes may be responsible for the changes occurring in patients with solid tumours, similar to MDS/AML patients.  This suggests that therapies used to treat MDS/AML patients may also have efficacy in patients with solid tumours.  1.1.2.2 The role of histone methylation in the regulation of Hox gene expression Polycomb group proteins (PcG) and Trithorax group (TrxG) proteins modify chromatin structure and are important in the epigenetic regulation of cellular development, differentiation and maintenance (Zardo et al., 2008). PcG and TrxG proteins govern HSPC differentiation through the repression or activation of gene expression by the methylation of histone components, histone 3 lysine 27 (H3K27) and histone 3 lysine 4 (H3K4), respectively (Zardo et al., 2008). The epigenetic silencer, Polycomb Repressive Complex 2 (PRC2), part of the PcG complexes, consists of suppressor of zeste 12 (Suz12), embryonic ectoderm development (Eed), and enhancer of zeste homolog 2 (EZH2) and is important in the tri-methylation of H3K27 resulting in silencing of gene transcription (Zardo et al., 2008).  Opposing the activity of the methyltransferase PRC2 are histone demethylases, such as JmjC domain-containing protein 3 (JMJD3), which can reduce methylation at H3K27, resulting in stem cell differentiation (Agger et al., 2007).  The TrxG complexes, also antagonize the activity of PRC2, and include mixed lineage leukemia (MLL) 1-4 and Set1A and Set1B proteins (Zardo et al., 2008). These complexes are involved in the trimethylation of H3K4, resulting in open chromatin structure, which is permissive for gene transcription (Zardo et al., 2008). Furthermore, Menin, encoded by Men1, plays an important role in hematopoiesis through its interaction with MLL, and loss of   7 Menin results in impaired ability of HSCs to repopulate under conditions of hematopoietic stress (Maillard et al., 2009).  The actions of the TrxG complexes, like those of PRC2, are important for the regulation of genes such as Hoxa9 and are key regulators of HSPC physiology (Zardo et al., 2008). Expression of Hoxa9 is epigenetically regulated by PcG and TrxG proteins, thereby connecting the epigenetic regulation of Hoxa9 expression with HSPC homeostasis and leukemic transformation (Cao et al., 2002; Cao and Zhang, 2004; Yan et al., 2006).  PRC2 maintains HSC homeostasis through negative regulation, since the loss of any component of the PRC2 complex, SUZ12, Eed or EZH2, results in increased HSC activity (Majewski et al., 2008; Majewski et al., 2010).  A study by Majewski et al. (2010) demonstrate that the loss of SUZ12 results in an increase in HSPC activity, which correlated with an increase in the expression of Hoxa9 in HSPCs, thereby linking expression of Hoxa9 with the activity of PRC2 in mediating HSPC activity (Majewski et al., 2010).  In another study, loss of Eed resulted in an accumulation of primitive myeloid progenitors whereas overexpression of Ezh2 in HSCs by retroviral transfection maintained their long-term repopulating capacity (Kamminga et al., 2006; Lessard et al., 1999).  These data suggest that PRC2 restricts HSC activity by transcriptional repression of genes that are important regulators of hematopoiesis, such as Hoxa9.     MLL proteins also play an important role in HSC homeostasis. They are important for normal HSC development as well as HSC differentiation into multipotent-progenitors through the regulation of transcription of Hox genes (Ernst et al., 2004a; Ernst et al., 2004b).   Translocation of Mll with various fusion partners in HSCs leads to increased expression of Hoxa9 (Chen et al., 2006; Faber et al., 2009; Hess, 2004; Horton and Huntly, 2012).  In response to this aberrant Hoxa9 expression, HSCs exhibit impaired differentiation but sustained proliferative abilities,   8 resulting in the development of leukemia (Chen et al., 2006; Faber et al., 2009; Hess, 2004; Horton and Huntly, 2012).  Thus, while PRC2 restricts gene transcription by trimethylation of H3K27, MLL opposes this activity by trimethylating H3K4. This results in genes, such as Hox genes, to be “poised” and permissive to transcription.  The interaction of MLL proteins with Menin is important during leukemic transformation and in the maintenance of HSCs, through the modulation of Hoxa9 expression (Chen et al., 2006; Yokoyama et al., 2005).  The deletion of Men1 in conditional Men1-knockout mice results in decreased Hoxa9 expression in BM cells, hematopoietic progenitor colony formation and peripheral blood leukocytes (Chen et al., 2006).  Moreover, blocking the interactions between Menin and MLL in MLL fusion protein-transformed BM cells reduces Hoxa9 expression and proliferation but promotes cellular differentiation, which together contribute to the reversal of leukemic activity (Grembecka et al., 2012).  Overall, the activities of PRC2 and Menin/MLL oppose each other and any dysregulation in their functions results in aberrant Hoxa9 expression, which leads to hematologic abnormalities.         Together, the importance of Hox genes in hematopoiesis and the ability of solid tumours to dysregulate hematopoiesis, suggests that tumours may induce hematologic defects in cancer patients by altering the expression of Hox genes. Furthermore, the established role of histone methylation in regulating expression of Hox genes such as Hoxa9, suggests that tumours may exploit this mechanism to induce the hematologic changes, including leukocytosis and anemia, observed in cancer patients.  Understanding these mechanisms and their importance in driving cancer is critical in preventing hematopoietic dysregulation, thereby possibly leading to better prognosis in cancer patients.   9 1.1.3 G-CSF and its role in hematopoiesis G-CSF, a hematopoietic cytokine, is important in the mobilization of HSPCs and in the maintenance of neutrophils under steady-state and stress conditions (Lieschke et al., 1994; Liu et al., 1996; Panopoulos and Watowich, 2008).  G-CSF is also important for neutrophil function, including phagocytosis, mobilization and survival (Eyles et al., 2006).  Both G-CSF and G-CSF receptor (G-CSFR) knockout mice (G-CSF-/- and G-CSFR-/-) are severely neutropenic.  This neutropenia results in an impaired ability to eliminate Listeria monocytogenes (Lieschke et al., 1994; Zhan et al., 1998), Pseudomonas aeruginosa (Gregory et al., 2007), and Candida albicans (Basu et al., 2000) infections.  This mimics the increased susceptibility to bacterial infections observed in neutropenic patients (Zeidler et al 2003).  Aside from its role in neutrophil homeostasis, G-CSF plays an important role in regulating HSPCs. G-CSF injection alone or in combination with another hematopoietic regulatory cytokine, FMS-like tyrosine kinase 3 ligand (FLT3L) can induce HSPC expansion and mobilization into the peripheral blood of healthy volunteers and primates (Brasel et al., 1997; Papayannopoulou et al., 1997).  The ability of G-CSF to mobilize HSPCs is currently being utilized to increase the yield of HSPCs in the peripheral blood of donors for stem cell transplantation.  Though studies examining the effects of G-CSF on HSC homeostasis are limited, G-CSF is hypothesized to be play a role due to the expression of G-CSFR on HSCs (Alexander, 2000).   In support of this hypothesis, HSPCs isolated from G-CSFR-/- mice are less efficient than wild-type (wt) HSCs in their ability to repopulate irradiated recipients (Liu et al., 2000).  Therefore, G-CSF is important in the regulation of fully differentiated cells such as neutrophils, but also plays a key role in HSPC mobilization from the BM to the periphery.    10 1.1.3.1 The role of G-CSF in cancer Tumours are known to manipulate the immune system in order to aid tumour growth and metastasis (Gabrilovich and Nagaraj, 2009; Gabrilovich et al., 2012). Of the various potential immune mediators that might play a role in tumour growth, G-CSF has been studied as a potentially important cytokine. This is due to its importance in hematopoiesis as well as its potential use as an adjuvant therapy, because of its ability to increase cancer cell susceptibility to radiation and chemotherapy (Altundag et al., 2004).  Studies have identified increased levels of G-CSF in breast tumours compared to the healthy tissues surrounding the tumour and tissues from healthy volunteers and in the serum of breast cancer patients (Dehqanzada et al., 2007; Lawicki et al., 2007; Lawicki et al., 2009; Park et al., 2011).  Importantly, higher G-CSF levels in the serum of breast cancer patients correlated with poorer prognosis, increased tumour grade and metastasis to the lymph nodes (Dehqanzada et al., 2007; Lawicki et al., 2007; Lawicki et al., 2009).  Furthermore, the administration of G-CSF to mice impairs erythropoiesis in the BM, therefore G-CSF production by the tumour may induce the development of anemia (de Haan et al., 1994; de Haan et al., 1992; Nijhof et al., 1994). Studies have also demonstrated the ability of G-CSF to induce the expansion of MDSCs, which have been shown to enhance tumour growth and survival by impairing the anti-tumour immune response and immunosurveillance.  Mice bearing 4T1 mammary tumours, and tumours that had metastasized to the liver and lungs, were infiltrated with immunosuppressive MDSCs.  The increase in MDSCs correlated with the constitutive expression of g-csf, gm-csf and m-csf by the tumours as well as increased levels of G-CSF in the serum of tumour-bearing mice (DuPre et al., 2007).  In another study, neutralization of G-CSF in tumour-bearing mice with an anti-G-CSF antibody resulted in a decrease in MDSCs and tumour size (Shojaei et al., 2009).  Together these   11 studies highlight another possible negative effect of G-CSF in cancer involving expansion of MDSCs.   It has recently been suggested that G-CSF may play an important role in leukemic transformation (Beekman and Touw, 2010).  Therefore, the possibility of healthy volunteers developing leukemia due to G-CSF treatment to mobilize HSCs into the periphery is a major concern.  However, in two separate studies involving over 5000 patients, there was no statistically significant increase in incidence of leukemia in G-CSF treated patients compared to untreated controls (Beekman and Touw, 2010).  However, in these studies, the follow-up period lasted a maximum of 4-5 years, but it has been shown that in patients treated with G-CSF for neutropenia, the risk of developing AML/MDS due to G-CSF increases 6 years after treatment (Beekman and Touw, 2010). Therefore an extended follow-up period is needed to determine whether a similar trend also occurs in patients treated with G-CSF to mobilize HSCs.  In addition, breast cancer patients with severe neutropenia treated with G-CSF to increase white blood cell numbers also had an increased risk of developing MDS/AML (Citron et al., 2003; Cole and Strair, 2010; Hershman et al., 2007).  There was an increased rate of AML/MDS in breast cancer patients undergoing adjuvant cancer therapy consisting of cyclophosphamide who also required G-CSF support (Citron et al., 2003).  Furthermore, another study demonstrated that in breast cancer patients 65 years old or older, the risk of developing AML/MDS doubled when treated with adjuvant chemotherapy concomitantly with G-CSF (Hershman et al., 2007). Overall, G-CSF negatively affects hematopoiesis, myelopoiesis, and erythropoiesis and can result in leukemic transformation, culminating in poor patient survival.  Therefore, the benefits of treating patients with G-CSF need to considered against the risks before this treatment is given to breast cancer patients.       12 1.2 Cancer and the DC lineage 1.2.1 The DC lineage DCs were discovered in 1973 in peripheral lymphoid organs of mice due to their unique morphology of dendrite like appendages (Steinman and Cohn, 1973). DCs have been shown to be the most potent inducers of the mixed leukocyte reaction and of naïve T cell activation (Steinman and Cohn, 1973).  Since their initial discovery, it is now known that multiple subsets of DCs exist with unique functions, which are important in the type of innate and adaptive immune response initiated.  1.2.1.1 DC subsets Lymphoid and nonlymphoid tissues harbor multiple subsets of DCs with specialized functions. Although functionally equivalent DCs exist within different peripheral tissues, they can be differentiated based on the expression of cell surface markers.  In the spleen, there exist three distinct subsets of DCs that can be segregated based on function as well as cell surface marker expression. These include, plasmacytoid DCs (pDCs), CD8+ conventional DCs (cDCs), and CD4+ cDCs, which include CD4+CD8- and CD4-CD8- populations.  Lastly, inflammatory DCs, which arise only under conditions of inflammation such as during infection with L. monocytogenes and B. melitensis, aid in controlling bacterial infections (Coquerelle and Moser, 2010).  Overall, the different subsets of DCs with their specialized functions are critical in mediating a tailored immune response against a wide range of infections and tumours. 1.2.1.2 DC function DCs undergo a maturation process after recognition and binding of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs), by pathogen recognition receptors (PRRs). This maturation process allows for the initiation of an immune   13 response against tumours and pathogens.  The process of DC maturation in response to PRR signaling results in the migration of DCs from peripheral tissues to secondary lymphoid organs, with a concomitant decrease in phagocytosis and antigen uptake as well as increased antigen processing (Banchereau et al., 2000; Coquerelle and Moser, 2010). DC maturation also involves upregulation of surface expression of MHC/peptide complexes; co-stimulatory molecules, such as CD86, CD80 and CD40; and chemokine receptors, such as CCR7, important in mediating DC migration to secondary lymphoid organs (Banchereau et al., 2000; Coquerelle and Moser, 2010).  These changes in DC phenotype and function make them better equipped for activating T cells.  Activated DCs also secrete cytokines such as IL-12, TNF-α, IL-6 and IL-10, which are important in influencing the type of T cell response initiated (Banchereau et al., 2000; Steinman and Idoyaga, 2010).  More recently it has been shown that activated DCs are also necessary in priming NK cells.  Activated DCs secrete IL-12, IL-18 and IL-15, which have all been shown to be important in NK cell activation (Fernandez et al., 1999; Lucas et al., 2007; Steinman and Cohn, 1973)). The different subsets of DCs have unique roles in dictating the type of immune response initiated based on their functions and the types of PRRs they express.  CD8+ cDCs are superior in their ability to cross-present antigens derived from pathogens and tumours to CD8+ T cells and readily produce IL-12, which is important for driving Th1 responses (Kronin et al., 2001; Pooley et al., 2001; Pulendran, 2005; Schnorrer et al., 2006; Shortman and Heath, 2010).  Conversely, CD4+ cDCs are better able to process and present antigens on MHC II molecules and are adept at initiating Th2 responses (Pooley et al., 2001; Pulendran, 2005).  Plasmacytoid DCs are critical in antiviral and anti-tumour immunity through the secretion of type I interferon (Jegalian et al., 2009).  During inflammatory conditions, inflammatory DCs, referred to as TNF-α and iNOS   14 producing (Tip) DCs differentiate from monocyte precursors and are the main source of iNOS and TNF-α and are critical in eliminating bacteria, recruiting neutrophils, and mediating inflammation (Serbina et al., 2003).  Overall, the unique capabilities of the various DC subsets result in a tailored immune response dependent on the type of immunological stimuli.   DCs also play a critical role in inducing immunological tolerance mediated by the expression of immunosuppressive enzymes, such as Arg1 and the ability to activate immunosuppressive immune cells, such as Tregs.  DCs can inhibit T cell activation and proliferation by depleting L-arginine via Arg1 in the extracellular environment (Munder, 2009).  DCs can also activate Tregs by presenting antigens on MHC molecules and tolerogenic receptors such as PD-L1, without the expression of necessary co-stimulatory molecules (Maldonado and von Andrian, 2010).  In turn, Tregs can then suppress and modulate the activation and functions of T helper (Th) cells and DCs by secreting immunosuppressive cytokines such as IL-10 and TGF-β, cytolytic enzymes and disrupting metabolism (Vignali et al., 2008). 1.2.1.3 DC development DC development begins in the BM and occurs through the stepwise differentiation of cell types arising from resident progenitor cells (Fig. 1.1).  At the top of the hierarchy are the long-term hematopoietic stem cells (LT-HSC).  These cells can differentiate towards the myeloid lineage by differentiating into the CMP, which gives rise to all myeloid cells (Liu et al., 2009). The multi-lineage CMP can then give rise to a more restricted progenitor, the macrophage dendritic cell progenitor (MDP), which is the precursor of monocytes and the common dendritic cell progenitor (CDP) (Naik et al., 2007; Onai et al., 2007).  CDPs give rise to pDCs and preDCs, which migrate from the BM into the blood and seed peripheral tissues (Naik et al., 2007; Onai et   15 al., 2007).  PreDCs can then give rise to cDCs that include both CD8+ and CD4+/- subsets (Fogg et al., 2006; Liu et al., 2009; Naik et al., 2007; Onai et al., 2007).   Proper DC development and differentiation depends on the expression of specific set of transcription factors, such as IRF-4, IRF-8, Zbtb46, E2-2, Batf3, and Id2, important for DC lineage commitment.  IRF-4 is an essential transcription factor for the development of CD4+ cDCs (Tamura et al., 2005). A loss-of-function (LOF) mutation in IRF-4 significantly impacted CD4+ cDC numbers however, other DC subsets were not affected (Tamura et al., 2005).  Conversely, the development of CD8+ cDCs depends on the expression of IRF-8, since the loss of IRF-8 expression results in impaired CD8+ cDC development in the lymphoid organs of mice (Aliberti et al., 2003; Schiavoni et al., 2002).  The development of pDCs requires the expression of IRF-8 and E2-2 and to a lesser extent IRF-4, since the knockout of IRF-8 and E2-2 in mice results in a significant loss of pDCs, however, there is only a modest decrease in pDCs in IRF-4 knockout mice (Cisse et al., 2008; Geissmann et al., 2010; Schiavoni et al., 2002; Steinman and Idoyaga, 2010; Tsujimura et al., 2003).  Recently, the development of a transgenic mouse model that allows for the inducible ablation of Zbtb46+ cells identified that Zbtb46, a zinc finger transcription factor, is specifically expressed in preDCs and cDCs (Meredith et al., 2012; Satpathy et al., 2012).  In this model, the human diphtheria toxin receptor (DTR) is expressed as a fusion protein with mCherry, under the control of the Zbtb46 promoter, therefore injection of mice with diphtheria toxin (DT) results in the depletion of Zbtb46+ cells (Meredith 2012).  Treatment of the Zbtb46 DTR mice resulted in the depletion of preDCs in the BM and cDCs in lymphoid and nonlymphoid tissues (Meredith, 2012).  Therefore, the transcription factors, IRF-4, IRF-8, Zbtb46 and E2-2 are all critical in the development of the various DCs subsets present during steady-state conditions, making them important targets for studying DC development.    16  Figure 1.1    DC differentiation. LT-HSC, long term hematopoietic stem cell; MDP, monocyte dendritic cell progenitor; CDP, common dendritic cell progenitor; pDC, plasmacytoid dendritic cell; preDC, dendritic cell precursor.    17 1.2.2 The role of DCs in neoplasia DCs play a critical role in the activation of innate and adaptive immunity and therefore are important in the immunosurveillance involved in preventing the transition from neoplasia to the development of cancerous tumours (Dunn et al., 2002; Dunn et al., 2004; Schreiber et al., 2011).  However, tumours can impair DC function, inhibit DC maturation and/or prevent DC differentiation resulting in an increase in immature/inhibitory DCs, thereby escaping immune recognition (Gabrilovich, 2004).  Understanding and reversing the effects of tumours on DC differentiation and function will be important for better treatment of cancer patients via the activation of anti-tumour innate and adaptive immune responses mediated by DCs.     1.2.2.1 DC localization, numbers and phenotype in neoplasia DCs can reside both within tumours and in the surrounding tissues. In a study by Bell et al. (1999), over 90% of tumours isolated from breast cancer patients were infiltrated with immature DCs, and in 60% of the patients, mature DCs were found in the peritumoral areas (Bell et al., 1999).  This segregation of immature and mature DCs to different regions of the tumour may inhibit the priming of an anti-tumour T cell response.  Another study demonstrated an overall decrease in the number of mature DCs with a concomitant accumulation of immature DCs in the peripheral blood of patients with breast cancer, malignant glioma, and hormone-refractory prostate cancer, thus suggesting a tumour mediated inhibition of DC differentiation (Pinzon-Charry et al., 2005).  These tumour induced immature DCs upregulated MHC II and co-stimulatory molecules in response to stimulation, but to a lesser degree than control DCs, and were poor activators of T cell proliferation and cytokine production (Pinzon-Charry et al., 2005). Moreover, other studies have demonstrated the accumulation of immature DCs in the peripheral blood of breast, head and neck, and lung cancer patients at the expense of mature DCs (Almand   18 et al., 2000; Della Bella et al., 2003). In these studies, the immature DCs were not only impaired in their ability to induce T cell proliferation but could actively suppress T cell proliferation (Almand et al., 2000; Della Bella et al., 2003).  Based on these studies, it suggests that the ability of tumours to manipulate the location and maturation of DCs may be important immune evasion mechanisms. DCs found in cancer patients and animal models of cancer are usually either immunosuppressive or are skewed towards stimulation of Th2-type immune responses, which promote tumour growth and metastasis over tumour elimination.  A study that utilized a rat colon carcinoma model demonstrated that though tumour-infiltrating DCs expressed MHC I/II, they did not express the co-stimulatory molecules CD80 or CD86, and were therefore poor inducers of T cell activation (Chaux et al., 1997).  Additionally, in a B16 tumour model, DCs were converted into tolerogenic DCs, which produced TGF-β that mediated an expansion of Tregs (Ghiringhelli et al., 2005).  DCs harvested from breast cancer patients were impaired in their ability to induce both T cell proliferation and mixed leukocyte reactions as compared to DCs harvested from healthy volunteers (Satthaporn et al., 2004).  Additional studies have also shown that tumours can manipulate DC function to induce Th2 responses, thereby promoting cancer progression (Ochi et al., 2012; Olkhanud et al., 2011; Pedroza-Gonzalez et al., 2011).  In a study using human breast cancer cells, DCs upregulated OX40L expression in response to tumour derived thymic stromal lymphopoietin (TSLP).  These OX40L expressing DCs activated inflammatory Th2 cells that aided in tumour growth and metastasis by secreting IL-13 and IL-4 (Pedroza-Gonzalez et al., 2011).  Neutralization of either TSLP or OX40L inhibited the activity of the inflammatory Th2 cells and reduced tumour growth in vivo (Pedroza-Gonzalez et al., 2011).    19 Overall tumours can impair DC differentiation and function as a strategy to evade anti-tumour immune responses. This results in reduced DC numbers and accumulation of immature cells that are defective in their ability to activate anti-tumour adaptive immune responses, or that promote immunosuppressive immune responses.  Understanding the mechanism of breast cancer mediated inhibition of DC function and development is important in order to reverse this block on the initiation of an anti-tumour immune response.     1.2.2.2 DC developmental defects in neoplasia The ability of tumours to impair DC function and initiate an anti-tumour immune response is well documented in both animal models and human cancer patients, however how tumours impact DC progenitors is less well understood.  Studies suggest that tumours may halt DC development resulting in the accumulation of immature cells with an immunosuppressive phenotype, although how tumour mediated changes in DC development impact their function still needs to be further investigated (Gabrilovich, 2004).  One study has shown that B16 melanoma tumours recruit preDCs from the BM to the tumour, where they subsequently differentiate into cDCs.  Compared to splenic cDCs, tumour cDCs expressed lower levels of MHC II, however, functionally, tumour cDCs were similar to splenic cDCs in their ability to induce OTII cell proliferation, upregulate co-stimulatory molecule expression and induce a mixed leukocyte reaction (Diao et al., 2010).  Importantly, the function of tumour cDCs and preDC differentiation was compared to splenic cDCs in the same tumour-bearing mouse, therefore if the tumour had systemic effects, then defects in DC phenotype, function and differentiation would not have been detected in this study.  It is also important to note that preDC differentiation and cDC function were measured in mice bearing small tumours of less than 0.5 cm, therefore the impact of a larger tumour burden on DC development was not investigated   20 (Diao et al., 2010).  Although limited studies have looked at the effects of tumours on DC development, the impact of tumours on DC development at the progenitor level, and the effects of tumour growth on the composition and function of DC subsets present in the periphery still needs to be determined.  1.3 Lyn tyrosine kinase and DC/NK cell cross-talk 1.3.1 Src family kinases There are nine Src family kinases (SFKs) that are expressed in various combinations in hematopoietic and non-hematopoietic cells and have been shown to modulate signaling from immunoreceptors such as the TCR, B cell receptor (BCR), Fc receptors, growth factor receptors and integrins (Hibbs and Harder, 2006; Lowell, 2004; Scapini et al., 2009).  Due to their role in signaling downstream of such a diverse repertoire of receptors, SFKs have been implicated in many cell functions including migration, adhesion, phagocytosis, cell survival, and proliferation.  SFKs can also play redundant roles, therefore, making it difficult to study the function/importance of individual SFKs in the various signaling pathways.   1.3.1.1 Lyn tyrosine kinase Lyn, a member of the SFKs, is expressed in all hematopoietic cells except for T cells, and is critical for the generation of an effective immune response against pathogens and perhaps tumours (Lowell, 2004).  Lyn plays an important role in signaling pathways activated by cytokine receptors, growth factor receptors, immunoreceptors and receptors important in mediating adhesion, migration, proliferation and hematopoiesis (Hibbs and Harder, 2006).  In addition, Lyn is important in positively and negatively regulating signal transduction, a role that is dependent on the type of stimulus, the cell’s developmental stage and extracellular environment.  Lyn mediates inhibitory signal transduction by phosphorylating tyrosine residues   21 within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in immunoreceptors such as CD22, FcγRIIB1, PIR-B and Sirpα, thereby creating docking sites for inhibitory molecules such as the phosphatases Src homology 2 domain-containing phosphatase (SHP)-1/2 and Src homology 2 domain containing inositol polyphosphate phosphatase-1 (SHIP-1), which bind the phosphorylated tyrosine residues through their Src homology 2 (SH2) domains.  Conversely, Lyn positively regulates signal transduction by phosphorylating immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of receptors such as Igα/β and CD19, which recruit signaling molecules such as, Syk and Zap70, that propagate down-stream signals (Hibbs and Harder, 2006; Lowell, 2004; Scapini et al., 2009; Xu et al., 2005). Lyn knockout (Lyn-/-) and gain-of-function (Lynup/up) mouse models have been utilized to study the role of Lyn in vivo, where Lyn has been shown to be critical in regulating the function of B cells, proliferation and function of mast cells, signaling through integrins in neutrophils and M2 polarization in Mφs (Ingley, 2012).  Lyn-/- mice develop autoimmune disease and hematopoietic system defects, characterized by an age-dependent increase in extramedullary myelopoiesis, disseminated myeloid neoplasia, and splenomegaly that is characterized by an increase in myeloid and erythroid progenitors (Harder et al., 2004; Hibbs et al., 1995; Nishizumi et al., 1995; Scapini et al., 2009; Xu et al., 2005).  Lyn-/- mice are also characterized by the development of a lupus-like disease with an accumulation of self-reactive antibodies and nephritis, a phenotype that requires MyD88 signaling, IL-6 and BAFF (Hibbs et al., 1995; Nishizumi et al., 1995; Scapini et al., 2010; Silver et al., 2007; Tsantikos et al., 2010; Xu et al., 2005).  Recently, Lamagna et al. (2014) demonstrated that mice with Lyn deficient B cells, similar to Lyn-/- mice, developed spontaneous B cell activation, autoreactive antibodies and glomerulonephritis (Lamagna et al., 2014).  As expected, the deletion of MyD88 in Lyn deficient   22 B cells, as demonstrated in global Lyn-/- mice, reversed the autoimmune phenotype in these mice (Lamagna et al., 2014).  Alternatively, Lynup/up mice express a mutant form of Lyn with a point mutation in the inhibitory tyrosine 508 (Y508F) leading to a constitutive increase in Lyn activity (Harder et al., 2001).  Lynup/up mice are also characterized by perturbations in the immune system and similar to the Lyn-/- mice, Lynup/up mice can develop autoantibodies and glomerulonephritis, however the B cells do not demonstrate increased activation in response to BCR stimulation (Harder et al., 2001; Hibbs et al., 2002; Hibbs et al., 1995; Xu et al., 2005). 1.3.1.2 Lyn in DCs Lyn tyrosine kinase has been shown to be important in modulating activating and inhibitory signaling in mast cells, basophils, eosinophils, neutrophils, Mφs and DCs (Scapini et al., 2009).  With the use of Lyn-/- mice, it has been demonstrated that Lyn is important in the positive and negative regulation of DC differentiation and maturation, and LPS has also been shown to activate Lyn in DCs, monocytes and Mφs (Beavitt et al., 2005; Chu and Lowell, 2005; Herrera-Velit and Reiner, 1996; Keck et al., 2010; Napolitani et al., 2003; Stefanova et al., 1993).  SFKs, specifically Lyn and Src, have been shown to be necessary for the secretion of proinflammatory cytokines such as IL-12, TNF-α and IL-6 in human myeloid DCs in response to stimulation with LPS and CpG (Napolitani et al., 2003).  Interestingly, Lyn and Src were not necessary for the expression of either MHC II or co-stimulatory molecules (Napolitani et al., 2003). Lyn deficiency results in enhanced cell division in in vitro BM GM-CSF DC cultures, and aged Lyn-/- mice possess increased numbers of DCs in vivo.  However, both in vitro and in vivo, Lyn-/- DCs exhibit an immature phenotype, even after stimulation with TLR ligands.  Lyn-/- BM-derived DCs also produce less IL-12 compared to wt DCs, and are therefore impaired in their ability to prime Th1 cell responses. This suggests a positive role for Lyn in mediating DC activation in   23 response to LPS stimulation (Beavitt et al., 2005; Chu and Lowell, 2005).  This diminished ability to produce IL-12 has been linked to lower SHIP-1 activation in Lyn-/- DCs.  SHIP-1 is important in downregulating the PI-3K pathway, which when hyperactivated impairs IL-12 production by DCs (Fukao et al., 2002).  Correlating with the reduced IL-12 production, Lyn-/- DCs are skewed towards inducing Th2 immune responses.  Following airway challenge in an allergy model, Lyn-/- mice displayed increased lung eosinophilia, mast cell hyper-degranulation, and increased IgE production, culminating in prolonged bronchospasms compared to wt mice.  Furthermore, transfer of Lyn-/- DCs into wt mice was sufficient to recapitulate the allergic response measured in Lyn-/- mice.  This demonstrated the sufficiency of Lyn-/- DCs, in driving increased susceptibility to a model of asthma/allergic inflammation (Beavitt et al., 2005).  Recently, Lamagna et al. (2013) generated chimeric mice with Lyn deficient DCs by crossing mice with LoxP sites on either sides of exons 3 and 4 of the lyn gene to Cd11c-cre transgenic mice (Lamagna et al., 2013).  Similar to Lyn-/- mice, mice with Lyn deficient DCs developed T and B cell activation, production of autoreactive antibodies, and development of nephritis, but unlike Lyn-/- mice these mice also developed severe inflammation (Lamagna et al., 2013).  These studies demonstrating that mice with either Lyn deficient B cells or DCs can recapitulate the phenotypes seen in global Lyn-/- mice suggest that multiple cells types can be dysregulated in Lyn-/- mice (Lamagna et al., 2014; Lamagna et al., 2013).   Overall Lyn plays a positive role in DC activation in response to LPS stimulation, with Lyn-/- DCs showing impaired expression of activation markers and secretion of Th1 cytokines post TLR stimulation.   1.3.2 Innate Immunity The innate immune system is the first line of defense against invading pathogens and tumours.  DCs, monocytes, Mφs, and NK cells are key cells of the innate immune system.  These cells are   24 activated by signaling through PRRs such as Toll like receptors (TLRs) that recognize conserved PAMPs such as LPS (Iwasaki and Medzhitov, 2004; Kaisho and Akira, 2003; Reis e Sousa, 2004).  LPS binds to TLR4, activating down-stream signaling cascades culminating in cell activation and secretion of proinflammatory cytokines, including IL-12, TNF-α, IL-6 and type I interferons (IFNs), as well as chemokines and NO.  These inflammatory responses are essential for innate and subsequent adaptive immune responses against pathogens and tumours (Reis e Sousa, 2004).  Deviation in the signaling, development or interactions of innate immune cells can lead to the inability to clear infections or an excessive immune response and can lead to the development of chronic inflammatory diseases, autoimmunity or sepsis. 1.3.2.1 NK cells NK cells are BM derived lymphocytes important in the early innate immune response against virally infected and malignant cells.  However, unlike T and B cells, NK cells do not undergo somatic recombination, instead it is the balance of signals received through activating and inhibitory receptors on NK cells that dictates their effector functions (Cerwenka and Lanier, 2001). NK cell effector responses consist of the secretion of cytotoxic granules containing granzymes and perforin, that induce programmed cell death in the target cell, and secretion of IFN-γ, which is important in activating innate and adaptive immune cells (Schoenborn and Wilson, 2007).  NK cell inhibitory receptors recognize and bind MHC I molecules whereas activating receptors recognize stress-induced ligands such as retinoic acid early inducible 1 (Rae1) and H60 (Concha et al., 1991; Karre et al., 1986; Ljunggren and Karre, 1985).  Malignant and virally infected cells may downregulate the expression of MHC I, in order to avoid detection by T cells and/or upregulate the expression of activating/stress ligands, changes that make these cells more susceptible to NK cells. Furthermore, NK cells also express the activating receptor,   25 FcγRIII, which binds the constant region of IgG, resulting in antibody dependent cellular cytotoxicity (ADCC). This receptor has been exploited in immunotherapies, such as with Herceptin, which binds the HER2/neu oncogene expressed on approximately 20-30% of human breast cancers, coating the tumour and allowing NK cells to target tumour cells (Barok et al., 2007; Clynes et al., 2000; Hudis, 2007).     1.3.2.2 DC/NK cell cross talk Immune cells not only directly protect the host from harmful insults but can also enhance the function of other immune cells.  Recently it has been shown that crosstalk between DCs and NK cells impacts the function of these cells during an immune response (Ferlazzo and Munz, 2009; Moretta et al., 2008).  In order for NK cells to kill target cells, DCs must prime their effector functions, including IFN-γ production and cytotoxicity, both in vitro and in vivo (Andoniou et al., 2005; Borg et al., 2004; Fernandez et al., 1999; Koka et al., 2004; Krug et al., 2004).  Fernandez et al. (1999) demonstrated that DCs primed NK cells, however, it was only demonstrated that DCs were sufficient but not necessary (Fernandez et al., 1999).  Therefore, Lucas et al. (2007) utilized a transgenic mouse model that allowed for the inducible ablation of all conventional CD11chi DCs to study the interaction between DCs and NK cells (Lucas et al., 2007).  In this transgenic mouse model, similar to the Zbtb46 DTR mouse, the simian DTR is expressed as fusion protein with GFP under the control of the CD11c promoter, therefore treatment of mice with DT results in the depletion of CD11chi cells (Jung et al., 2002).  Ablation of DCs in vivo resulted in reduced NK cell IFN-γ production and cytotoxicity after TLR stimulation.  Importantly, though marginal zone and metallophilic Mφs were also depleted in this model, these subsets of Mφs were not important in priming NK cells.  Overall, Lucas et al.   26 (2007) demonstrated that after stimulation with activating agonists, NK cells migrate to secondary lymphoid organs where in response to type I IFNs, DCs transpresent IL-15 on the IL-15 receptor α (IL-15Rα) chain to prime the effector functions of NK cells (Lucas et al., 2007).  However, other studies have demonstrated that cytokines such as IL-12 and IL-18 can also prime IFN-γ production and cytotoxicity by NK cells (Borg et al., 2004; Koka et al., 2004; Krug et al., 2004).  Conversely, NK cells have also been shown to modulate the functions of DCs.  During inflammation, NK cells become capable of eliminating immature DCs with low MHC I expression, as a mechanism to resolve inflammation (Moretta 2008).   Understanding the mechanism of how DCs and NK cells influence each other’s functions is an important component of innate immunity and also provides us with a possible target for immunotherapy approaches against tumour cells.   1.3.2.3 IFN-γ  in sepsis IFN-γ is an important cytokine in mediating immunity against intracellular pathogens and tumours (Schoenborn and Wilson, 2007). NK and NKT cells are the major innate producers of IFN-γ with Th1 and CD8+ T cells also contributing to IFN-γ production once adaptive immune responses are initiated (Schoenborn and Wilson, 2007).  Mice deficient in either IFN-γ production or expression of its receptor have an increased susceptibility to intracellular pathogens, viruses as well as naturally occurring and inducible tumours (Dalton et al., 1993; Harty and Bevan, 1995; Huang et al., 1993; John et al., 2002; Jouanguy et al., 1999; Kaplan et al., 1998).  IFN-γ boosts the immune response by enhancing antigen processing and presentation by increasing the expression of MHC I/II molecules, transporter associated with antigen presenting (TAP) 1/2, invariant chain, and the expression and activity of the proteasome   27 (Schoenborn and Wilson, 2007).  Interestingly, IFN-γ stimulates the synthesis of alternate subunits of the proteasome that form the immunoproteasome, which when compared to the proteasome, provides greater diversity of peptides that are better able to bind MHC I/II molecules (Schroder et al., 2004).  IFN-γ also enhances Mφ phagocytosis and their ability to kill pathogens (Beutler et al., 1986; Boehm et al., 1997; Burchett et al., 1988; Jurkovich et al., 1991).  Overall IFN-γ helps recruit lymphocytes to inflammatory sites and maintains these cells in an activated state (Hill and Sarvetnick, 2002; Savinov et al., 2001).  Therefore, the production of IFN-γ must be carefully regulated because excessive amounts of IFN-γ have been linked to the development of autoimmunity such as experimental autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, insulin-dependent diabetes mellitus and autoimmune nephritis (Awata et al., 1994; Baechler et al., 2003; Billiau et al., 1988; Espejo et al., 2001; Heremans et al., 1978; Jacob and McDevitt, 1988; Lee et al., 2001; Panitch et al., 1987; Sarvetnick et al., 1988; Wang et al., 1997). IFN-γ has been shown to be important in endotoxin-induced lethality, especially in priming mice to the effects of LPS (Heinzel, 1990; Jurkovich et al., 1991; Ozmen et al., 1994).  Therefore, IFN-γ receptor (IFNGR1) knockout mice and mice treated with IFN-γ neutralizing antibodies were resistant to LPS mediated toxicity (Heinzel, 1990; Heremans et al., 1990; Ozmen et al., 1994).  Ozmen et al. demonstrated that a priming dose of LPS results in the secretion of IFN-γ by NK cells, which is mediated by IL-12.  IFN-γ then acts on Mφs to produce IL-1 and TNF-α, cytokines that are important in mediating endotoxin-induced lethality (Ozmen et al., 1994).  Interestingly, the authors suggested that IL-12, which was a newly characterized cytokine at the time of this study, was produced by Mφs in response to LPS, however, given what is now known   28 about IL-12, it is highly probable that DCs contributed significantly to this IL-12 production.  IFN-γ may also aid in enhancing the LPS response by upregulating the expression of TLR4 on the surface of APCs and the expression of proteins important in the LPS signaling cascade resulting in NF-κB activation (Schroder et al., 2004).  Overall, IFN-γ is a critical cytokine in protective immune responses against pathogens and tumours, however, similar to other cytokines, IFN-γ can also be detrimental when produced uncontrollably or inappropriately. 1.4 Research Objective Hematopoietic system abnormalities including anemia and the expansion of iMCs are commonly observed in cancer patients and are associated with poor prognosis (Diaz-Montero et al., 2009; Gabitass et al., 2011; Qiu et al., 2010a).  However, it is not well understood how tumour cells influence hematopoietic cell differentiation.  Furthermore, similarities in the expansion of leukocytes, development of anemia and dysregulation of HSPCs between patients diagnosed with MDS/AML and patients with solid tumours, suggests that these diseases share a common mechanism for inducing hematopoietic abnormalities.  Importantly, HSCs with dysregulated increases in Hoxa9 expression, which is epigenetically regulated by TrxG and PcG proteins, results in increased regeneration leading to the development of MDS/AML (Argiropoulos and Humphries, 2007; Thorsteinsdottir et al., 2002).  Therefore, I hypothesized that mammary tumours induce epigenetic dysregulation of the hematopoietic system leading to abnormalities in HSPC numbers/localization, anemia, and aberrant myelopoiesis. DCs are necessary for the activation of the innate and adaptive immune responses and therefore play an important role in immunosurveillance (Dunn et al., 2002; Dunn et al., 2004; Schreiber et al., 2011).  However, it is well established that tumours can impair DC function, inhibit DC maturation and/or prevent DC differentiation. This can result in an increase in the numbers of   29 immature/inhibitory DCs which helps tumours escape immune recognition (Gabrilovich, 2004).  It is less well understood how tumours impact DC progenitors and their differentiation into DCs, though studies suggest that tumours may halt DC development resulting in the accumulation of immature cells with an immunosuppressive phenotype (Gabrilovich, 2004).  Therefore, the second aim for this project was to examine how mammary tumour-derived factors modify DC development to preferentially give rise to DCs that potentiate immunosuppression/suppress immune activation. Lyn, a member of the SFKs, is expressed in all hematopoietic cells except for T cells, and is critical for the generation of an effective immune response against pathogens and perhaps tumours (Lowell, 2004).  Lynup/up mice were generated by point mutation in the inhibitory tyrosine 508 (Y508F) resulting in a constitutive increase in Lyn activity (Harder et al., 2001).  Lynup/up mice develop perturbations in the immune system and glomerulonephritis, and produce autoantibodies (Harder et al., 2001; Hibbs et al., 2002; Hibbs et al., 1995; Xu et al., 2005).  LPS activates Lyn in the mononuclear phagocyte system (MPS), which includes DCs, monocytes and Mφs, and studies using Lyn-/- GM-CSF derived DCs have shown impaired DC activation and IL-12 secretion post-LPS stimulation (Beavitt et al., 2005; Chu and Lowell, 2005; Herrera-Velit and Reiner, 1996; Keck et al., 2010; Napolitani et al., 2003; Stefanova et al., 1993).  Therefore, I hypothesized that a gain-of-function mutation in Lyn will alter the response of the MPS to TLR agonists and their subsequent interactions with other immune cells.        30 Chapter 2: Materials and methods 2.1  General reagents LPS (from E. coli 0111.B4), diphtheria toxin and polybrene were from Sigma-Aldrich.  CpG-ODN and peptidoglycan (from Staphylococcus aureus) were from InvivoGen.  ELISA kits for TNFα, IL1α, IL-6, IFNγ, IL-12, IL-10 and bead array were from eBioscience, the ELISA kit for IL-1β was from R&D Systems, and lastly the IFNα ELISA kit was from PBL Interferon Source.  Antibody (Ab)-coupled magnetic beads were from Miltenyi Biotec. IL-15 was from Invitrogen.  Recombinant cytokines used in in vitro assays were purchased from Peprotech, eBioscience and R&D Systems, including IL-3, IL-6, IL-10, VEGF, G-CSF, M-CSF, GM-CSF, TPO, TSLP, SCF and IL-15.  GM-CSF, FLT3L and L-cell conditioned medium (a source of M-CSF) were produced and standardized in our laboratory.  CSFE was from Sigma-Aldrich.  Menin inhibitors (MI-2/MI-3) and control inhibitor (MI-nc) have been previously described (Grembecka et al., 2012).  Methocult GF 3534 was purchased from StemCell Technologies. Neutralizing antibodies Abs against G-CSF (9B4CSF) and IL-6 (MP5-20F3) and isotype control Abs were purchased from eBioscience. The Proteome Profiler Mouse Cytokine Array Kit, Panel A, used to determine MT-1 cytokine levels was purchased from R&D Systems.  Anti-NK1.1 antibody Ab (PK136) was from the Biomedical Research Centre (Vancouver, BC).  Lympholyte M was from Cedarlane. 2.2 Mice RAG1-/-, OT-I, and OT-II mice were kindly provided by Dr. Hung-Sia Teh, and B6.FVB-Tg(MMTV-neu/OT-I/OT-II)CBnel Tg(Trp53R172H)8512Jmr/J (MMTV-neu) and B6.SJL-Ptprca Pepcb/BoyJ (BoyJ) mice were from Jackson Labs. Lyn-/- (Hibbs et al., 1995), Lynup/up (Harder et al., 2001), CD11c-DTR/GFP(Jung et al., 2002), Ship-1-/- (Helgason et al., 1998), and   31 MeV/MeV (Tsui et al., 1993) mice have been previously described. Experiments were performed in accordance with guidelines from the Canadian Council for Animal Care and the University of British Columbia Animal Care Committee.  All mice were C57BL/6 genetic background (>10 generations).  Mice were age-, sex- and weight-matched, and were analyzed between 6-14 weeks of age.  For BM-chimeric mice, RAG1 recipient mice were subjected to lethal irradiation (650 rads, 2 doses, 4 hr apart) and injected IV with 5x106 CD11c-DTR/GFP RAG1-/- BM cells. For MT-1 tumour injections in immunosufficient mice, MMTV-neu mice were used due to their tolerance to the NeuOT-1/OT-II protein (Wall et al., 2007). 2.3 Flow cytometry antibodies Abs were from eBioscience, BD biosciences, Beckman Coulter or produced in-house (Biomedical Research Centre): CD11c (N418), CD11b (M1/70), Gr-1 (RB6-8C5), CD115 (AFS98), MHC class II (I-A/I-E), CD19 (eBio1D3), DX5 (DX5), Ter119 (TER119), CD71 (R17217), c-kit (2B8), Flt3 (A2F10), CD45.1 (A20), CD45.2 (104), Sca-1 (D7), FcγRII/III (2.4G2), CD34 (RAM34), CD24 (M1/69; 30-F1), SIRPα (P84), CD8α (53-6.7), CD4 (GK1.5; RM4-5), CD40 (HM40-3), CD80 (16-10A1), CD86 (GL-1), PD-L1 (M1H5), Ly6c (AL-21), B220 (RA3-6B2), CD44 (IM7), Thy1.2 (53-2.1), NK1.1 (PK136), IL-7Rα (A7R34), F4/80 (BM8), CD62L (MEL-14), MHC class I (28-8-6), CD69 (H1.2F3), CD44 (IM7), CD45RA (14.8), IFNγ XMG1.2), FcεRI (MAR-1), H3K27me3 (Millipore, #07-449), and H3K4me3 (Cell Signaling, #9571).  The lineage cocktail for HSC analysis contained Abs recognizing Gr-1, CD3, CD11b, CD19, Ter119, DX5, MHC II, CD11c, ± FcεR1. The lineage cocktail for DC progenitor analysis contained Abs recognizing Gr-1, CD3, CD11b, CD19, Ter119, DX5, MHC II, and IL-7Rα.   32 2.4 Tumour cell lines NOP cell lines were derived from spontaneous tumours in mice expressing neu linked to the OVAOTI/OTII peptide sequences and a dominant-negative p53 transgene (Wall et al., 2007; Yang et al., 2009).  NOP cells were maintained in NOP medium (RPMI 1640 (Invitrogen) supplemented with 10% heat inactivated (HI)-FBS, 100 U/mL penicillin-G, 100 µg/mL streptomycin, 2 mM glutaMAX, 55 µM 2-mercaptoethanol and insulin/transferrin/selenium (Lonza). NOP12, 18, 21, 23 cell lines are referred to as mammary tumour (MT) -1, 2, 3, 4, respectively. CMT93, 4T1, and B16 cell lines were maintained in DMEM with 10% HI-FBS. Tumour-conditioned media (T-CM) was produced by culturing cells at 5×106 cells/25 mL for 4 days. Cellular debris was removed by centrifugation and T-CM was stored at -80°C. 2.5 In vitro BM analysis and BM-derived DC culture and splenic NK cell culture BM was cultured in NOP medium without insulin/transferrin/selenium in the presence of FLT3L at 1-2×106 cells/mL for 3-13 days. For GM-CSF experiments, BM was cultured at 2×105 cells/mL unless otherwise indicated for 3-8 days. For T-CM treatments, BM cultures were supplemented with 25% T-CM unless otherwise indicated.  BM-derived macrophages (BMMφs) were derived in medium supplemented with L-cell conditioned medium (source of M-CSF). NKs were purified from spleens by using biotinylated anti-NK1.1 mAb together with anti-biotin magnetic beads, and cultured in medium supplemented with 50 ng/mL IL-15 for 7 days. 2.6 Competitive reconstitution and CFU-S12 assays Recipient congenic BoyJ (CD45.1+) mice were subjected to lethal irradiation (650 rads, 2 doses, 4hr apart) and injected IV with 5×106 BM cells or splenocytes from control or tumour-bearing mice (CD45.2+) the following day. For reconstitution with in vitro cultured cells, BM cells from CD45.2+ UBC-GFP mice were cultured in vitro as described for 8-10 days in the presence of   33 FLT3L and MT-CM.  FACS sorted lineage-Sca-1+c-kit+ (LSK) cells, or unfractionated cells from in vitro BM cultures, were injected into sublethally irradiated RAG1-/- (500 rads, 1 dose) or lethally irradiated BoyJ (650 rads, 2 doses, 4hr apart) mice.  For peripheral blood analysis to assess hematopoietic reconstitution, RBCs were lysed and blood was analyzed by flow cytometry at the indicated times.  Donor cells were distinguished by expression of CD45.2 and/or GFP expression. For the CFU-S12 assay, 5×105 BM cells or splenocytes from control or tumour-bearing mice were injected IV into lethally irradiated (900 rads, 1 dose) recipient C57BL/6 mice. Recipients were euthanized 12 days post-transfer and spleens were collected and fixed in Carnoy’s solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for colony enumeration. 2.7 qPCR Total RNA was isolated using the RNeasy kit (Qiagen) or TRI Reagent (Invitrogen). Reverse transcription was performed using the iScript cDNA Synthesis Kit (BioRad).  Blood collected from control and tumour-bearing mice via cardiac puncture was lysed in TRI Reagent (Invitrogen) after red cell lysis. qPCR was performed using the SsoFast EvaGreen real-time PCR mix (BioRad) on a Bio-Rad CFX96 instrument, and analyzed using CFX Manager software. Target genes were normalized to Gapdh or Rps29. For primers, see appendix A1. 2.8 Chromatin immunoprecipitation (ChIP) 1×107 cells were suspended in 5mL medium and cross-linked by adding 1% formaldehyde for 10min at room temperature. 125 mM glycine was added for 5min to stop the cross-linking reaction.  Cells were rinsed and lysed at 4×107 cells/mL in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1, protease inhibitor cocktail (Roche)).  Lysate was diluted with 3 volumes of ChIP dilution buffer (0.01% SDS, 1.1% Triton-X 100, 1.2 mM EDTA, 16.7 mM   34 Tris-HCl, 167 mM NaCl, protease inhibitor cocktail (Roche)), incubated on ice 15 min, transferred to 15 mL polystyrene tubes (BD Biosciences) and sonicated 10-15 min using a Bioruptor (Diagenode), high setting, 30 sec on 30 sec off pulse.  To prepare “Input” DNA, 50 µL sonicated lysate was mixed with 350 µL elution buffer (1% SDS, 100 mM NaHCO3) containing 25 µg proteinase K (Invitrogen) and incubated at 65ºC for 4-5 hrs to reverse cross-links. DNA was purified using the phenol chloroform method and DNA concentration was determined by nanodrop.  For ChIP, lysates were further diluted 2-fold with ChIP dilution buffer.  Lysates containing 12.5 µg DNA were precleared on 40 µL protein A Sepharose (PAS) beads.  Lysates were transferred to new tubes and incubated with precipitating Abs overnight at 4ºC with rocking.  Precipitates were collected for 1.5 hrs with 40 µL PAS beads coated with sheared salmon sperm DNA (75 ng/µL beads) and BSA (0.1 mg/µL beads).  Precipitates were washed 3X with low salt wash buffer (20 mM Tris HCl pH 8, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, protease inhibitor cocktail) and 1X with 1mL high salt buffer (20 mM Tris HCl pH 8, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 500 mM NaCl, protease  inhibitor cocktail).  DNA was eluted in 400 µL elution buffer by incubating at 30ºC for 15 min.  Eluate was transferred to new tubes and incubated with 25 µg proteinase K at 65ºC for 4-5 hrs. DNA was purified using the phenol chloroform method and analyzed by qPCR. Abs used: H3K4me3 and H3K27me3. For primers, see appendix. 2.9 Methylcellulose assays The methylcellulose assay was carried out according to manufacturer’s instructions (Stemcell Technologies). Briefly, cell samples were resuspended in 2% HI-HBS, IMDM, and a 1:10 v/v ratio of cells to Methocult were mixed and plated in 3 cm plates. CFCs were typed and enumerated 12 days later.   35 2.10 Western blot Abs used: H3K4me3 (Cell Signaling #9751S), H3K27me3 (Millipore, #07-449), histone H3 (Cell Signalling #9715), EZH1 (abcam #13665), EZH2 (Cell Signalling #4905S), Arg1 (BD Biosciences, #610709), p-p38 (Cell Signaling  #9215), p-Akt (Cell Signaling Thr 308, #4056), p-Akt (Cell Signaling Ser 473, #4058), p-Erk1/2, (Cell Signaling #4377), p-Jnk (Cell Signaling #9251), p-Ikkα/b (Cell Signaling #2681), p-p65RelA (Cell Signaling #3033), p-IkB (Cell Signaling #9246), p-Gsk3α/b (Cell Signaling #9331), p-Bad (Cell Signaling #9291), p-Tbk1 (Cell Signaling  #5483), P-S6 (Cell Signaling #4856), T-IkBα (Cell Signaling #9242), T-p65 (Cell Signaling #3034), T-Akt (Cell Signaling #9272), T-Erk1/2 (Cell Signaling #4695).  Anti-SHP-1 was from Dr. Cheng, U. of Melbourne, Australia, anti-SHP-2 was from Santa Cruz Biotechnologies. Anti P-Tyr (clone 4G10) was from Millipore, anti-iNOS was from BD Biosciences. 2.11 Tumour injection and tissue processing Mice were injected with 1-5×106 MT cells subcutaneously (SQ) in the left flank or mammary fat-pad (MFP), or 7.5×105 B16 cells (SQ) or 1x105 4T1 cells (SQ).  Spleens, femurs, tumours and blood were analyzed once tumours reached 1cm3. Blood (cardiac punctures) was analyzed on the scil Vet abc hematology analyzer (scil animal care company) or treated with ammonium chloride before flow cytometry. 2.12 Flow cytometry and FACS Cells were incubated with 2.4G2 mAb (Fc block) and stained with fluorochrome-labeled antibodies (Abs). Data was acquired on an LSRII (BD Biosciences) using FACS Diva software and analyzed with FlowJo software (TreeStar).  Dead cells were excluded based on propidium   36 iodide (PI) or 4',6-diamidino-2-phenylindole (DAPI) uptake, and red cells were excluded by lysis and/or size. 2.13 Menin inhibitor treatment of BM cultures BM cells were treated with Menin inhibitors, MI-2 or MI-3, on day 0 of culture and analyzed on day 3 for CFCs.  Alternatively, inhibitors were added on day 0 and cells were analyzed on day 8 by flow cytometry. For Hoxa9 qPCR, cells were treated for 24 hrs with the MI-2/MI-3 prior to analysis. 2.14 T cell proliferation, cytokine, phagocytosis and suppression assay FLT3L or GM-CSF BM derived DCs (BMDCs) were cultured for 8-10 days. For phagocytosis assays, GM-CSF DCs were co-cultured CFSE (carboxyfluorescein succinimidyl ester) labeled tumour cells (B16-OVA or MT-1), treated with 0.5-5 µg/ml of doxorubicin for 24 hr, for 16 hr. Cells were analyzed by flow cytometry. For DC:T cell co-culture experiments, GM-CSF or FLT3L DCs (control or 25% MT-1-CM treated) were incubated with SIINFEKL peptide, ovalbumin (OVA) protein, or doxorubicin-treated B16-OVA or MT-1 cells for 16 hr, then co-cultured with OTI, OTII or B3Z cells for 2-3 days. Supernatant was collected and analyzed for cytokine content by ELISA. To measure T cell proliferation in DC:T cell co-culture experiments, OTI or OTII cells were labeled with CFSE prior to co-culture with DCs, and proliferation was measured by CFSE dilution. For T cell suppression assays, GM-CSF or FLT3L DCs (control, 25% MT-1-CM treated, or 20 ng/mL G-CSF treated) were co-cultured with OTI or OTII splenocytes in the presence of OVA protein for 2-3 days. T cell proliferation was measured by flow cytometry analysis of CFSE dilution.  2.15 LPS-induced cytokine production and morbidity Mice were treated with LPS by intraperitoneal (i.p.) injection, as indicated in figure legend. To   37 deplete DCs, CD11c-DTR/GFP-background mice were injected i.p. with 5 ng/g DT, 24 hrs before LPS injection.  To deplete NKs, mice were injected i.p. with 200 µg anti-NK1.1 antibody, 48 and 24 hrs before LPS injection.  Serum cytokine concentrations were determined by ELISA analysis from blood obtained by cardiac puncture or from the retro-orbital venous plexus.  To determine IFN-γ and granzyme B production by NK or T cells, splenocytes were harvested and surface stained for NK1.1 and DX5 (NKs), or Thy1 and CD4/CD8 (T cells).  Splenocytes were fixed and permeabilized and stained intracellularly with anti-IFN-γ, -granzyme B or isotype control antibodies. To assess morbidity, mice were monitored hourly and sacrificed when moribund. Alternatively, core body temperature was measured using an ELAMS subcutaneous transponder (BioMedic Data Systems) and mice euthanized 10 hrs after LPS injection. 2.16 Microscopy All bright field microscopy images were captured using the Olympus BX61 microscope (Olympus, Center Valley, PA), using 4X and 10X apochromatic objective lenses, and an Olympus colour CCD camera with Olympus CellSens Dimension software. 2.17 Cell stimulation and lysis BM derived DCs were stimulated with 100ng/ml LPS for the indicated times.  For ELISA assays, culture supernatant was collected and analyzed according to the manufacturer’s instructions.  For Western blot experiments, cells were starved for 3 hrs in DC medium containing 0.5% serum, prior to stimulation with 100ng/ml LPS.  Cells were then washed with ice-cold RPMI (no additives), and lysed on ice in buffer containing 1% Triton X-100, 0.1% SDS, 1% glycerol, 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, supplemented with a protease inhibitor cocktail (Roche).  Insoluble   38 material was removed by centrifugation, and protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). 2.18 NK cell co-culture assays The indicated number of in vitro propagated NK cells were co-cultured with 5.0x105 GM-BMDCs, FLT3L-BMDCs (sorted or un-sorted populations) or BMMφs.  Cells were stimulated + 100 ng/ml LPS for 16 hrs. IFN-γ, IL-10 or IL-12 levels in culture medium were determined by ELISA.  2.19 Design and subcloning of shRNAs targeting SHP-1 and SHIP-1 ShRNAs targeting SHP-1, SHIP-1 or control shRNAs were designed using an in-house algorithm based on characteristics of effective siRNAs (Khvorova et al., 2003). Three shRNAs (minimum) were designed for each target, and specificity was assessed by searching the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags (EST) database. Annealed oligonucleotides were directionally subcloned into the Pme1/Mfe1-digested pQCXIP vector (BD/Clonetech) containing the mU6 promoter, a puromycin resistance gene and an eGFP reporter gene. To generate retrovirus, 3.5x106 phoenix cells were transfected using the CaPO4 method with 21 µg pCL-Eco and 21 µg pQCXIPmU6shRNA-eGFP plasmids. 2.20 Infection of DCs with retrovirus encoding shRNAs For infections, 2x105 BM cells were cultured in 1ml DC medium for 2 days. 1mL of fresh retrovirus-containing medium containing 8µg/mL polybrene was added, and cells were centrifuged at 800xg for 2 hrs at room temperature. A second infection was immediately performed, as above.  Cells were then re-plated in DC medium. For biochemical analyses, infected DCs (day 6) were treated with puromycin (1 µg/ml) for 48 hrs, and dead cells were   39 removed.  For flow cytometry, infected DCs were gated based on eGFP fluorescence and examined for LPS-induced maturation marker expression.  2.21 Statistical analysis Statistical comparisons were performed using unpaired t-test. Error bars and ± symbols represent SEM or SD as stated. For chapters 4 and 5: *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 and chapter 6: *p < 0.05, **p < 0.01, ***p < 0.001.    40 Chapter 3: Mammary tumour-induced dysregulation in hematopoiesis Hypothesis: Mammary tumours induce epigenetic dysregulation of the hematopoietic system leading to abnormalities in HSPC numbers and localization, anemia, and aberrant myelopoiesis Objectives: Aim A: To understand the effects of mammary tumours on the hematopoietic system, erythropoiesis and myelopoiesis Aim B: To investigate the nature and extent of mammary tumour-induced impairment of hematopoiesis by thorough analysis of hematopoietic stem/progenitor function, localization and subset composition  Aim C: To develop an in vitro system to investigate tumour-induced changes in hematopoietic differentiation  Aim D: To determine the factor(s) in mammary tumour conditioned media mediating dysregulation of hematopoiesis  Aim E: To understand the mammary tumour-mediated changes in the epigenetic regulation of hematopoietic transcription factors in hematopoietic stem/progenitor cells Aim F: To test whether inhibiting mammary tumour-conditioned media-induced elevation of activating epigenetic marks restores hematopoietic cell differentiation       41 3.1 Mammary cancer induces epigenetic changes and impairs hematopoiesis leading to perturbations in myelopoiesis and erythropoiesis 3.1.1 Peripheral blood parameters of mammary tumour-bearing mice Due to the presence of hematologic abnormalities in cancer patients, our studies focused on the blood compartment of tumour-bearing mice. Analysis of hematologic parameters in the blood of mammary tumour (MT)-bearing immunodeficient (RAG1-/-) and immunosufficient syngeneic (MMTV-neu) mice showed that MTs induced an ~10-fold expansion of granulocytes (leukocytosis) and a significant reduction in RBCs (anemia) and platelets (PLT) (thrombocytopenia) (Table 3.1).  The development of leukocytosis, anemia and thrombocytopenia in MT-1 bearing mice correlated with changes that occur in cancer patients (Caro et al., 2001; Diaz-Montero et al., 2009; Gabitass et al., 2011; Leonard et al., 2005; Qiu et al., 2010a).  Interestingly, MTs did not impact lymphocyte numbers in immunosufficient mice (Table 3.1).             42 Table 3.1    Hematologic analysis of MT-1 bearing mice  RAG1-/- MMTVneu  control MT-1 control MT-1 WBC, 103/µL 3.7 ± 0.5 19.8 ± 2.6**** 7.9 ± 0.9 30.8 ± 6.8* RBC, 106/µL 9.9 ± 0.1 8.3 ± 0.4*** 10.3 ± 0.1 9.2 ± 0.4* HGB, g/dL 14.9 ± 0.1 12.6 ± 0.5*** 15.0 ± 0.2 13.5 ± 0.5* HCT, % 46.4 ± 0.6 39.1 ± 1.6*** 46.6 ± 1.1 41.7 ± 1.7 PLT, 103/µL) 1103 ± 59 884 ± 57* 1119 ± 30 866 ± 46** LYM, 103/µL 0.8 ± 0.2 0.7 ± 0.1 4.7 ± 0.6 5.5 ± 0.8 MON, 103/µL 0.3 ± 0.0 0.7 ± 0.1*** 0.8 ± 0.1 1.5 ± 0.2* GRA, 103/µL 2.5 ± 0.3 18.3 ± 2.5**** 2.4 ± 0.2 23.9 ± 6.1*  RAG1-/-: Control n=13, MT-1 n=12; MMTVneu: Control n=4, MT-1 n=4. Blood was obtained from cardiac puncture and analyzed on a scil Vet abc hematology analyzer, scil animal care company, Gurnee, IL. *, P < .05; **, P < .01; **, P < .001; ****, P < .0001. WBC indicates white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; PLT, platelet; LYM, lymphocyte; MON, monocyte; GRA, granulocyte.               43 3.1.2 Leukemoid reaction in mammary tumour-bearing mice To further characterize the changes occurring in the myeloid compartment in MT-1 bearing mice, frequency and absolute numbers of monocytes (CD11b+ CD115+) and granulocytes (CD11b+Gr1+) were measured by flow cytometry (Fig. 3.1).  In contrast to control and B16 (melanoma) tumour-bearing mice, MT-1 bearing mice developed splenomegaly and cellularity was reduced in the BM (Fig 3.1B).  There was an expansion of the CD11b+Gr1+ population in the spleen, blood and BM of MT-1 bearing mice compared to both control and B16 tumour-bearing mice (Fig 3.1).  Monocytes were decreased in the BM and increased in the spleens of MT-1 bearing mice compared to controls and B16 tumour-bearing mice (Fig 3.1).  Overall, MT-1 tumour development resulted in BM hypocellularity, splenomegaly and an expansion in the CD11b+Gr1+ granulocyte population, correlating with the development of leukocytosis.   44  Figure 3.1    Mammary tumours induce changes in granulocyte and monocyte numbers in mice. (A) Frequencies of CD11b+Gr-1hi and CD11b+Gr-1int cells in BM, blood and spleen of control, MT-1 and B16 tumour-bearing mice. Mouse genetic backgrounds include MMTVneu/C57BL/6 for immunosufficient models, and RAG1-/- for immunodeficient models. (B) Absolute numbers of unfractionated cells, monocytes (gated as CD115hiCD11b+Gr-1low/neg) and granulocytes (gated as CD115low/negCD11b+Gr-1hi) in spleen and BM of control and MT-1 bearing mice. Data are representative of a minimum of three independent experiments, n=3. *p < 0.05, **p < 0.01, ***p < 0.001.        45 3.1.3 Dysregulated erythropoiesis in mammary tumour-bearing mice Next, we wanted to further understand the development of anemia in MT-1 bearing mice and determine if impaired erythropoiesis was responsible for the reduction of RBCs.  Macroscopic and histological analysis (H&E) of femurs from MT-1 bearing mice showed pale bones and a reduction of RBC precursors, respectively (Fig. 3.2A).   Compared to the femurs harvested from control mice, BM cells in MT-1 bearing mice were replaced with cells that resembled granulocytes and cells with a blast-like morphology (Fig. 3.2A).  There was also a decrease in the frequencies and absolute numbers of erythroid cells, (CD71+Ter119+ and CD71-Ter119+), but no change in the absolute number of CD71+Ter119- cells (Fig. 3.2B, 3.3A,B).  These changes in immature erythroid cells suggest that MT development impairs erythrocyte production.  In contrast, all of the immature erythroid cells were increased in the spleens of MT-1 bearing mice compared to controls (Fig. 3.2C, 3.3C).  This suggests that MT-1 growth impairs erythropoiesis in the BM, however, increases erythropoiesis in the spleen.  Importantly, erythropoiesis in the spleen cannot compensate for the impaired erythropoiesis in the BM, as demonstrated by the development of anemia in MT-1 bearing mice (Table 3.1).  In comparison, B16 tumours did not induce changes in erythroid progenitors in the BM, although there was a variable increase in erythroid progenitors in the spleen of C57BL/6 B16 tumour-bearing mice (Figs. 3.3C, 3.4).  In order to determine if perturbed myelopoiesis and erythropoiesis was a general characteristic of mammary tumour growth, we utilized the well-characterized 4T1 mammary tumour cell line.  Similar to MT-1 development, 4T1 tumour growth was associated with peripheral blood WBC expansion and reduction in RBC, HGB and PLT numbers (Table 3.2, Fig. 3.5).  Also, similar to MT-1 tumour-bearing mice, 4T1 bearing mice developed splenomegaly, which was associated   46 with an increase in granulocytes and monocytes, whereas BM was hypocellular with a corresponding reduction in monocytes, but a slight increase in granulocytes (Fig. 3.5).                         47  Figure 3.2    Mammary tumour development is associated with impaired BM erythropoiesis and heightened splenic erythropoiesis.     (A) Femurs from control and MT-1 bearing mice (left), H&E stained BM sections imaged at ×10 (middle) and ×40 (right) original magnification. (B) Frequencies of CD71+Ter119-, CD71+Ter119+ and CD71-Ter119+ erythroid populations in the BM of control and MT-1 bearing mice. SQ, subcutaneous; MFP, mammary fat-pad. (C) Frequencies of splenic CD71+Ter119-, CD71+Ter119+ and CD71-Ter119+ erythroid populations in control and MT-1 bearing RAG1-/- and MMTV-neu mice. Data are representative of three independent experiments, n=3-4.     48  Figure 3.3    Mammary tumour development is associated with reduced numbers of BM erythrocytes and increased numbers of splenic immature erythroid cells.     (A) Absolute numbers of erythroid populations in the BM of RAG1-/- mice, or (B) MMTV-neu mice.  (C) Absolute numbers of CD71+Ter119-, CD71+Ter119+ and CD71-Ter119+ erythroid populations in the spleens of control, MT-1 and B16 tumour-bearing mice. SQ, subcutaneous; MFP, mammary fat-pad. Data are representative of three independent experiments, n=3-4. *p < 0.05, **p < 0.01, ***p < 0.001.     49  Figure 3.4    Melanoma tumour development is not associated with impaired BM erythropoiesis.     Absolute numbers of CD71+Ter119-, CD71+Ter119+ and CD71-Ter119+ erythroid populations in the BM of C57BL/6 or RAG1-/- mice with B16 tumours.  Data are representative of three independent experiments, n=3.   Table 3.2    Hematologic analysis of 4T1 tumour-bearing mice  RAG1-/-: Control n=9, 4T1 n=3. Blood was obtained from cardiac puncture and analyzed on a scil Vet abc hematology analyzer, scil animal care company, Gurnee, IL. *, P < .05; **, P < .01; **, P < .001; ****, P < .0001. WBC indicates white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; PLT, platelet; LYM, lymphocyte; MON, monocyte; GRA, granulocyte.   50  Figure 3.5    4T1 tumour development is associated with impaired BM myelopoiesis and erythropoiesis.   Absolute cell numbers of monocytes, granulocytes and CD71+Ter119-, CD71+Ter119+ and CD71-Ter119+ erythroid populations in the BM and spleen of RAG1-/- mice with 4T1 tumors.  Data are representative of two independent experiments, n=2-3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.  3.1.4 Defective hematopoietic stem/progenitor cell numbers and localization in mammary tumour-bearing mice The changes in multiple mature hematopoietic lineages suggested that MT-1 tumours were impairing the functions of HSPCs that give rise to RBCs, PLTs and leukocytes.   We examined the HSPC compartment in the BM and spleen by flow cytometry using an antibody cocktail to identify and exclude differentiated cells (lineage: CD3+CD19+CD11b+CD11c+DX5+Gr-1+Ter119+MHCII+FcεRI+).  In the BM of MT-1 bearing mice, LSKs (lineage-Sca-1+c-kit+) were increased, whereas LKs (lineage- Sca-1-c-kit+) were reduced (Fig. 3.6, 3.7A).  These results were consistent in both immunodeficient and immunosufficient backgrounds (Fig. 3.7A).  Further analysis of subpopulations of HSPCs within the LSK compartment revealed that short-term hematopoietic stem cells (ST-HSCs; LSK, CD34+Flt3-) and multi-potent progenitors (MPP;   51 LSK, CD34+Flt3+) were increased in the BM of MT-1 bearing mice, whereas LT-HSC (LSK, CD34-Flt3-) were unchanged or reduced, depending on the strain of mice analyzed (Fig. 3.7A).  The reduction in BM LKs was mainly due to a reduction in the numbers of megakaryocyte erythrocyte progenitors (MEPs; LK, FcγRII/III-CD34lo) (Fig. 3.7A).  The numbers of granulocyte macrophage progenitors (GMP; LK, FcγRII/IIIhiCD34+) were either unchanged or increased, depending on the strain of mice analyzed and CMP (LK, FcγRII/IIIintCD34+) were unchanged (Fig. 3.7A).  All of the HSPC subsets that we investigated by flow cytometry were increased in the spleen of MT-1 bearing mice (Fig. 3.7B).  These results suggest that hematopoiesis in MT-1 bearing mice has shifted from the BM to the spleen, with the spleen attempting to compensate for the impaired hematopoiesis in the BM.  4T1, similar to MT-1, tumour-bearing mice possessed increased numbers of HSPCs in the spleen and defects in the BM, with the exception of CMPs and GMPs, which were also significantly reduced in the BM (Fig. 3.8). B16 tumour growth did not alter BM HSCPs, suggesting that abnormalities in the hematopoietic system in MT-bearing mice may be specific to mammary tumours (Fig. 3.9).     52  Figure 3.6    Perturbations in hematopoietic stem/progenitor cell frequency in mammary tumour-bearing mice.     Gating scheme for classification of HSPCs. BM from control and MT-1 or B16 tumour-bearing RAG1-/- mice was stained with antibodies recognizing lineage-specific antigens (lineage-), as well as Sca-1, c-kit, CD34, FcγRII/III, and Flt3, and analyzed by flow cytometry. Lineage-DAPI- cells were analyzed.           53  Figure 3.7    Perturbations in hematopoietic stem/progenitor cell numbers and location in mammary tumour-bearing mice.     (A) BM total cell numbers and numbers of the HSPC subsets; LSK (lineage-Sca-1+c-kit+), LK (lineage-Sca-1-c-kit+), LT-HSC (LSK, Flt3-CD34-), ST-HSC (LSK, Flt3-CD34+), MPP (LSK, Flt3+CD34+), CMP (LK, FcγRII/IIIintCD34+), GMP (LK, FcγRII/IIIhiCD34+), and MEP (LK, FcγRII/IIIloCD34-). (B) Splenic total cell numbers and stem/progenitors were identified and enumerated as in A.  Data are representative of a minimum of three independent experiments, n=3. *p < 0.05, **p < 0.01.      54  Figure 3.8    4T1 tumour development disrupts BM hematopoietic stem/progenitor cells.                                                                   Total BM and splenic cellularity and absolute numbers of the following stem/progenitor cell subsets in 4T1 tumor-bearing mice: LSK (Lineage-Sca-1+c-kit+), LK (Lineage-Sca-1-c-kit+), LT-HSC (LSK, Flt3-CD34-), ST-HSC (LSK, Flt3-CD34+), MPP (LSK, Flt3+CD34+), CMP (LK, FcγRII/IIIintCD34+), GMP (LK, FcγRII/IIIhiCD34+), and MEP (LK, FcγRII/IIIloCD34-). Data are representative of two independent experiments, n=2-3. *p < 0.05, **p < 0.01, ***p < 0.001.    55  Figure 3.9    Melanoma tumour development does not disrupt the BM hematopoietic stem/progenitor compartment.  Total BM cellularity and absolute numbers of the following stem/progenitor cell subsets in B16 tumour-bearing mice: LSK (lineage-Sca-1+c-kit+), LK (lineage-Sca-1-c-kit+), LT-HSC (LSK, Flt3-CD34-), ST-HSC (LSK, Flt3-CD34+), MPP (LSK, Flt3+CD34+), CMP (LK, FcγRII/IIIintCD34+), GMP (LK, FcγRII/IIIhiCD34+), and MEP (LK, FcγRII/IIIloCD34-). Data are representative of two independent experiments, n=3. *p < 0.05.  3.1.5 Defects in bone marrow hematopoietic stem/progenitor cell numbers and localization are not due to metastasis of MT-1 cells The changes occurring in BM, which is distant to the actual tumour, in MT-1 bearing mice suggested the possibility of metastasis of MT-1 cells to the BM where they could directly interact and modify BM cells.  However, tumour cells were not detected by histology in the BM sections from MT-1 bearing mice (Fig. 3.2A).  Furthermore, no expression of the neu transgene (uniquely expressed in the MT-1 cell line) could be detected in the BM harvested from MT-1 bearing mice (data not shown).  This data suggests that the effects mediated by MT-1 cells in the BM were not due to metastasis of tumour cells but instead due to tumour-secreted factors that were acting at distant sites from the tumour.     56 3.1.6 Functional defects in the hematopoietic stem/progenitor cell compartment in mammary tumour-bearing mice  In order to determine if MT development influenced HSPC function in addition to numbers, we utilized assays to measure the colony forming potential and reconstitution capabilities of HSPCs from MT-1 bearing mice compared to control mice.  To determine if splenocytes and BM from MT-1 bearing mice possessed myeloid progenitor colony forming activity (CFU-G, granulocytic colony; CFU-M, macrophage colony; CFU-GM, granulocyte-macrophage colony), cells were analyzed by methylcellulose colony forming assay. Myeloid progenitor colony numbers were largely unchanged in the BM of MT-1 bearing mice, with the exception of an increase in CFU-M (Fig. 3.10A). Conversely, splenic CFUs were significantly increased in MT-1 bearing mice (Fig. 3.10A).  The CFU-S12 assay identifies primitive progenitors able to form colonies on the spleens of irradiated recipients.  This assay showed that MT-1 bearing mice had a reduction in primitive progenitors in the BM, which correlated with the reduction in LKs, and an increase in these primitive progenitors in the spleen corresponding with the increase in LKs (Fig. 3.7A, 3.10B).   Lastly, we employed a competitive reconstitution assay using 1:1 ratios of CD45.2+ BM or splenocytes from control or MT-1 bearing mice to CD45.1+ control BM or splenocytes, respectively.  The relative reconstitution capability of BM harvested from MT-1 bearing mice was similar compared to BM from control mice at 3, 8, and 20 weeks post-reconstitution (Fig. 3.10C).  Although, splenocytes from control mice only provided short-term reconstitution and were unable to reconstitute the hematopoietic system long term, splenocytes from MT-1 bearing mice provided short- and long-term reconstitution measured at 3, 8 and 20 weeks, correlating with the increased number of LT-HSCs (Fig. 3.7B, 3.10C).    57  Figure 3.10    Mammary tumour growth is associated with significant functional changes in the stem/progenitor compartments in BM and spleen.     (A) Total numbers of colony-forming cells (CFU-G/M or CFU-GM) in spleen (left) and BM (right) of control and MT-1 bearing MMTV-neu mice were determined by methylcellulose assay. (B) Numbers of primitive multi-potent colony forming cells in the BM and spleen of control and MT-1 bearing RAG1-/- mice were determined by CFU-S12 assay. (C) CD45.2+ splenocytes or BM cells from MMTV-neu control or MT-1 bearing mice were transplanted into lethally-irradiated CD45.1+ recipients at a 1:1 ratio together with CD45.1+ carrier cells. Engraftment efficiency was determined by assessing the relative frequency of CD45.2+ versus CD45.1+ cells in the blood at 3, 8, and 20 weeks post-reconstitution by flow cytometry. Percentages of CD45.2+ nucleated PI- cells are shown. †, euthanized due to lack of reconstitution. Data represent a minimum of two independent experiments, n=3-5. *p < 0.05, **p < 0.01, ***p < 0.001.          58 3.1.7 In vitro differentiation of hematopoietic stem/progenitor cells is impaired by mammary tumour secreted factors  In order to investigate the mechanism behind MT-induced hematopoietic abnormalities, an in vitro BM culture system using hematopoietic cytokines was used to determine the effects of MT-1 conditioned media (MT-1-CM) on progenitor content.  The combination of FLT3L or GM-CSF, but not other cytokines, with MT-1-CM resulted in increased cellularity of BM cultures at day 9 (Fig. 3.11A, 3.12A).  In the FLT3L cultures treated with MT-1-CM, LSKs, LKs, CMPs and GMPs were increased by day 3, whereas increase in HSPCs could only be detected on day 3 in the GM-CSF cultures (Fig. 3.11A, 3.12A, 3.13).  Furthermore, MT-1-CM treatment of FLT3L BM cultures resulted in an increase in myeloid cells, CD11b+CD115hi cells and a transient increase in CD11b+Gr1hi cells which were detected on day 3 (Fig. 3.12A).  Surprisingly, CD11b+Gr1hi cells did not change in MT-1-CM treated GM-CSF BM cultures compared to control GM-CSF BM cultures, however there was an increase in CD11b+CD115hi cells in GM-CSF BM cultures supplemented with MT-1-CM (Fig. 3.13).  Interestingly, only T-CM from MT lines increased total cellularity and LSKs in the in vitro BM cultures treated with FLT3L, whereas B16 melanoma and CMT93 colon cancer T-CM had no effect (Fig. 3.11B).   To determine the cytokine/cytokines in MT-1-CM that were responsible for the increased progenitor content in in vitro BM cultures treated with FLT3L, FLT3L BM cultures were treated with the following cytokines with known roles in tumour physiology, IL-3, IL-6, TGF-β, VEGF, IL-10, M-CSF, G-CSF, GM-CSF, TPO and TSLP.  Out of the cytokines that were tested, G-CSF and IL-6 both synergized with FLT3L resulting in increased cellularity and LSKs in the in vitro BM cultures similar to MT-1-CM (Fig. 3.11C).  However, in a cytokine array performed on MT-1-CM, G-CSF, but not IL-6, was detected, indicating that G-CSF (and possibly other   59 chemokines/cytokines such as CXCL1, M-CSF, MIP-2, MIG, TNF-α and CXCL10) may be responsible for the increase in cellularity and progenitor content in the in vitro BM cultures treated with FLT3L (Fig. 3.14A).  Furthermore, only mammary tumour cell lines MT1-4 and 4T1 secreted G-CSF (Fig. 3.14B).  Also, G-CSF levels were elevated in the serum of MT-1 bearing mice (Fig. 3.14B).     In order to determine if G-CSF in MT-1-CM was mediating the effects of MT-1-CM in FLT3L BM cultures, G-CSF was neutralized by the addition of antibody to the in vitro FLT3L BM cultures treated with MT-1-CM.  Neutralizing G-CSF, but not IL-6, GM-CSF or M-CSF, reduced culture cellularity, LKs, LSKs and CFUs in the in vitro FLT3L BM cultures (Fig. 3.12B-D).  These results show that MT-1-CM derived G-CSF is necessary to mediate the increase in total cellularity and progenitor content in in vitro FLT3L BM cultures. Next, we wanted to determine if treating in vitro BM cultures supplemented with FLT3L or GM-CSF with MT-1-CM again influenced HSPC function in addition to numbers.  Using the methylcellulose assay, we observed increased numbers of CFU-G/M/GM colonies in the MT-1-CM treated day 9 FLT3L BM cultures compared to control FLT3L BM cultures and freshly harvested BM cells (Fig. 3.11D).  Methylcellulose assay with day 3 GM-CSF cultures treated with MT-1-CM showed a similar, though a less drastic, increase in CFU’s compared to control cultures (Fig. 3.11D).   Interestingly, transfer of whole or LSKs from day 9 FLT3L BM cultures treated with MT-1-CM into lethally irradiated recipient mice did not result in reconstitution of recipient mice (data not shown).  We also studied if MT-1-CM was impacting progenitor survival, apoptosis and/or proliferation in day 3 FLT3L and GM-CSF BM cultures by measuring Ki67, Annexin V and DAPI staining (Fig. 3.15).  In FLT3L cultures, addition of MT-1-CM resulted in increased Ki67 expressing LSKs and LKs, however did not alter levels of apoptosis   60 (Fig. 3.15A,B).  There was also a reduction in DAPI+ LSKs in MT-1-CM treated FLT3L and GM-CSF cultures (Fig. 3.15C).        61  Figure 3.11    Mammary tumour-conditioned media expands hematopoietic progenitors in in vitro BM cultures supplemented with FLT3L or GM-CSF.     (A) Total cells (left) and LSKs (right) at day 9 in BM cultured in media containing the indicated cytokines and supplemented ± MT-1-CM (designated as MT-1). (B) BM was cultured in the presence of FLT3L supplemented with conditioned media from the indicated tumour cell lines, and total cellularity and LSK numbers were determined. (C) As in A, except that BM was cultured with FLT3L in combination with the indicated cytokines or MT-1-CM. (D) Total CFCs in FLT3L or GM-CSF BM cultures treated ± MT-1-CM. BM cells were plated in methylcellulose after 8 days (FLT3L) or 3 days (GM-CSF) of culture, and colonies were scored 12 days later. Freshly isolated BM was also plated to compare relative CFCs in the cultures and equivalent numbers of plated uncultured BM. Data are representative of a minimum of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.   62  Figure 3.12    Mammary tumour-conditioned media expands hematopoietic progenitors in in vitro BM cultures supplemented with FLT3L, while G-CSF neutralization suppresses mammary tumour-conditioned media-dependent expansion of hematopoietic progenitors.     (A) Total cells and absolute numbers of HSPCs (LSK, LK, CMP, and GMP) in FLT3L BM cultures treated with MT-1-CM over a period of 9 days. Numbers of CD11b+CD115hi and CD11b+Gr-1hi cells in the cultures are also shown. (B) Total cells and numbers of LSKs and LKs in FLT3L BM cultures supplemented with a reduced dose (1.6%) of MT-1-CM, treated with G-CSF or IL-6 neutralizing Abs (or isotype control Abs, iso). (C) Numbers of CFCs were enumerated in control and G-CSF or IL-6 neutralizing Ab treated FLT3L BM cultures treated ± 1.6% MT-1-CM. BM cells were plated in methylcellulose after 8 days of culture, and colonies were scored 12 days later. (D) Total cells and absolute numbers of LSKs and LKs in FLT3L BM cultures supplemented with a reduced dose (1.6%) of MT-1-CM, treated with G-CSF, GM-CSF or M-CSF neutralizing Abs (or isotype control Abs, iso).  Data are representative of a minimum of at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.   63  Figure 3.13    Mammary tumour-conditioned media expands hematopoietic progenitors in BM cultures supplemented with GM-CSF.     Total cells and absolute numbers of HPSCs and myeloid cells in GM-CSF BM cultures treated with MT-1-CM over a period of 9 days. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.        64   Figure 3.14    Identification of cytokines produced by MT-1 cells.     (A) A mouse cytokine array containing a panel of 40 different cytokines/chemokines was used to detect cytokine/chemokine expression by MT-1 cells. Top panel, control medium; bottom panel, MT-1-CM.  For this experiment, all medium contained 0.5% serum.  Duplicate spots at the upper and lower left, and upper right, are positive controls. Factors showing differential expression are boxed. The position of IL-6 on the array is also shown although no expression was detected. (B) Levels of G-CSF protein in various tumor-conditioned media (top) and serum of control and MT-1 bearing mice (bottom). **p < 0.01.           65  Figure 3.15    Mammary tumor-conditioned media increases hematopoietic progenitor cell proliferation and decreases LSK death in in vitro BM cultures supplemented with FLT3L or GM-CSF.                                                                                                                                                                (A-C) BM cells were cultured in media for 3 days containing the indicated cytokines supplemented with or without MT-1-CM. (A) LK and LSK proliferation was assessed by Ki67 expression. Cells were stained with a viability stain prior to fixation (only live cells are shown). Numbers on histograms represent frequencies of Ki67 positive cells. (B-C) Cell death of LK and LSK populations was analyzed by flow cytometry as indicated by positive staining for (B) Annexin V and (C) DAPI, graphs represent mean frequency of DAPI or Annexin V+ LKs or LSKs pooled from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.         66 3.1.8 Regulation of Hox gene expression and histone modifications at the Hoxa9 locus by mammary tumour secreted factors The HSPC abnormalities detected in MT-1 bearing mice and the increase in progenitor content in FLT3L and GM-CSF BM cultures treated with MT-1-CM, along with the associated leukocytosis and anemia in MT-1 bearing mice reflects similar changes to those observed in mice with myeloproliferative diseases induced by the over-expression of certain Hox genes or MLL fusion proteins (Argiropoulos and Humphries, 2007; Chen et al., 2006; Faber et al., 2009; Hess, 2004; Horton and Huntly, 2012; Thorsteinsdottir et al., 2002).  This warranted further analysis into the genetic regulation of hematopoiesis and Hoxa gene expression.  Interestingly, the mRNA levels of Hoxa7, Hoxa9 and Hoxa10 were all increased in both FLT3L and GM-CSF BM cultures supplemented with MT-1-CM, especially the levels of Hoxa9 (Fig. 3.16A). Importantly, there were similar perturbations in Hoxa9 expression in MT-1 bearing mice with increased Hoxa9 expression in sorted lineage- (lin-) BM progenitors in MT-1 bearing mice compared to control mice (Fig. 3.16B).    Since Hoxa9 is under the control of both activating and inhibitory epigenetic regulation, we further investigated the effects of MT-1-CM and MT-1 on the expression of proteins involved in the epigenetic regulation of Hoxa9 expression.  Expression of Ezh2, the methyltransferase component of the PRC2 epigenetic silencing complex, was decreased in MT-1-CM treated FLT3L and GM-CSF BM cultures compared to control cultures (Fig. 3.16A).  As expected, expression levels of other components of the PRC2 complex, including Ezh1, Suz12 and Eed, were all decreased in MT-1-CM treated FLT3L BM cultures (Fig. 3.17A).  Expression levels of Jmjd3, which counteracts PRC2 by demethylating H3K27me3, and Mll1-5, H3K4 methyltransferases, and Rbbp5, a Mll complex component, were also decreased in MT-1-CM   67 treated FLT3L BM cultures (Fig. 3.16A, 3.17A).  Unexpectedly, all of the epigenetic regulators, with the exception of Ezh2, Jmjd3, Mll1 and Mll3, were unchanged in MT-1-CM treated GM-CSF BM cultures (Fig. 3.16A, 3.17A).  Ezh2 mRNA levels were also decreased in sorted lin- BM cells from MT-1 bearing mice compared to control mice, which correlated with the lower levels of the EZH2 product, H3K27me3 (inhibitory) in BM LKs, as measured by intracellular flow cytometry (Fig. 3.16B).  However, H3K4me3 (activating) in BM LKs was unchanged between control and MT-1 bearing mice (Fig. 3.16B).   To determine if MT-1-CM derived G-CSF mediated Hoxa9 expression in the in vitro BM FLT3L cultures, anti-G-CSF neutralizing antibodies were added to in vitro BM cultures supplemented with a lower dose of 1.6% of MT-1-CM to ensure complete G-CSF neutralization. Neutralization of G-CSF, but not GM-CSF or M-CSF, partially restored Ezh2 and decreased Hoxa9 expression to levels comparable to control cultures (Fig. 3.16C,D).  This data demonstrates the importance of MT-1-CM derived G-CSF in altering the expression of Hoxa9 and Ezh2.   MT-1-CM mediated dysregulation of Ezh2 mRNA suggested to us that MT-1-CM may alter the histone methylation status of the in vitro BM cultures in a genome-wide manner.  Correlating with the changes in H3K27me3 occurring in BM LKs in MT-1 bearing mice, global levels of H3K27me3 in GM-CSF BM cultures were reduced in a dose dependent manner (Fig. 3.16B,E).  Moreover, H3K27me3 levels were reduced in the total population and in the LKs in FLT3L BM cultures treated with MT-1-CM, which correlated with reduced EZH2/1 protein (Fig. 3.17B,C).  In addition, H3K4me3 levels were also decreased in the total population and in the LKs in MT-1-CM treated FLT3L BM cultures (Fig. 3.17B).   To further understand the epigenetic regulation of the Hoxa9 locus, ChIP was used to assess the levels of H3K4me3 and H3K27me3 associated with the Hoxa9 locus (Fig. 3.18, 3.19B).  MT-1-  68 CM treatment of FLT3L or GM-CSF BM cultures showed reduced H3K27me3 modifications of the Hoxa9 locus at amplicon A and was variably reduced at other amplicons, consistent with reduced Ezh2 expression, and elevated H3K4me3 modifications of the Hoxa9 locus (amplicon A-E) (Fig. 3.18B, 3.19B).  This data correlated with the increased expression of Hoxa9 in the MT-1-CM treated FLT3L and GM-CSF BM cultures, suggesting the possibility that MT-1-CM may induce Hoxa9 expression through histone modifications in the Hoxa9 locus (Fig. 3.18B, 3.19B).  The neutralization of G-CSF in in vitro FLT3L BM cultures treated with 1.6% MT-1-CM reduced H3K4me3 at amplicons A and C, however, this low dose of MT-1-CM did not attenuate H3K27me3 at amplicon A, nor was neutralization of G-CSF able to restore H3K27me3 levels at amplicon C (Fig. 3.19A).       69  Figure 3.16    Mammary tumour growth is associated with enhanced Hoxa gene expression and changes in histone methylation. (A) Expression of Ezh2, Jmjd3, Hoxa7, Hoxa9 and Hoxa10 mRNA in FLT3L and GM-CSF BM cultures treated ± MT-1-CM. (B) Hoxa9 and Ezh2 expression in sorted Lin- BM cells (left), and H3K4me3 and H3K27me3 levels in BM LKs from control and MT-1 bearing mice analyzed by intracellular flow cytometry (right). (C) Expression of Hoxa9 and Ezh2 mRNA in FLT3L BM cultures treated with a reduced dose of MT-1-CM (1.6%) together with isotype control Abs (iso) or neutralizing Abs against G-CSF. (D) Expression of Hoxa9 mRNA in FLT3L BM cultures supplemented with a reduced dose (1.6%) of MT-1-CM, treated with G-CSF, GM-CSF or M-CSF neutralizing Abs (or isotype control Abs, iso).  (E) Lysates from GM-CSF BM cells treated with an increasing dose of MT-1-CM were analyzed by Western blot for H3K27me3, histone H3 and β-actin (loading control). Data represents a minimum of at least two independent experiments.    70  Figure 3.17    Mammary tumor-conditioned media is associated with changes in histone modifying proteins and histone methylation.                                                                                                                 (A) Expression of Eed, Suz12, Rbbp5, Ezh1, and Mll1-5 mRNA in FLT3L and GM-CSF BM cultures treated with or without MT-1-CM. (B) Levels of global H3K4me3 and H3K27me3 in total cells and LKs in FLT3L BM cultures supplemented with or without MT-1-CM measured by intracellular flow cytometry. (C) Western blot analysis of lysates from FLT3L BM cultures treated with increasing amounts of MT-1-CM.  Blots were probed with antibodies recognizing EZH1, EZH2 and β-actin (loading control).  Band intensities (relative to β-actin) are indicated above each band.  Densitometry was performed using Image J software Data are representative of a minimum of two independent experiments.  MT-1-CM is designated as “MT” and MT-1-CM treated cells are represented by dashed lines.    71  Figure 3.18    Mammary tumour-conditioned media treatment of BM cultures is associated with enabling changes in histone methylation at the Hoxa9 locus. (A) Depiction of amplicons (A–E) in the Hoxa9 locus for ChIP analysis. (B) Repressive (H3K27me3-amplicon A) and activating (H3K4me3-amplicons B–E) histone modifications in FLT3L or GM-CSF BM cultures treated with + MT-1-CM were investigated by ChIP. Amplicons A–E were analyzed by qPCR. MT-1-CM is designated as MT-1. Data represents 3 experiments.    72  Figure 3.19    Mammary tumor growth is associated with changes in histone modifying proteins and G-CSF induced changes in histone methylation at the Hoxa9 locus.                                                                                  (A) Repressive (H3K27me3) and activating (H3K4me3) histone modifications in the Hoxa9 locus were investigated by ChIP of chromatin isolated from FLT3L or GM-CSF BM cultures treated with MT-1-CM together with isotype control (iso) or neutralizing Abs against G-CSF. Chromatin was immunoprecipitated using anti-H3K27me3, anti-H3K4me3 or IgG control antibodies and amplicons A and C were analyzed by qPCR. (B) As in (A), except amplicons A-E were analyzed by qPCR.             73 3.1.9 Contribution of G-CSF in mammary tumour secreted factor suppression of hematopoietic stem/progenitor cell differentiation The neutralization of G-CSF in MT-1-CM restored progenitor content and Hoxa9 expression to control levels, in order to better understand the role of G-CSF in mediating the effects of MT-1-CM in in vitro BM cultures, FLT3L and GM-CSF BM cultures were treated with G-CSF.  The addition of G-CSF decreased H3K27me3 levels, increased Hoxa9 expression and expanded myeloid progenitors in a dose dependent manner, similar to MT-1-CM (Fig. 3.20A, B, data not shown).  This data demonstrates the significance of G-CSF in MT-1-CM in mediating the dysregulation of MT-1-CM in in vitro BM cultures.             74  Figure 3.20    G-CSF enhances Hoxa9 expression and CFC numbers, while Menin inhibitors reverse mammary tumour-conditioned media induced expansion of LSKs, LKs and CFCs in in vitro BM cultures.  (A-B) Expression of Hoxa9 mRNA in FLT3L and GM-CSF BM cultures treated with MT-1-CM or G-CSF (1, 5 or 20 ng/mL), and total numbers of CFCs from FLT3L and GM-CSF BM cultures treated with MT-1-CM or 20 ng/mL G-CSF. (C-E) Total cells and absolute numbers of LSKs and LKs in BM cultures supplemented with FLT3L and MT-1-CM, and treated with Menin inhibitors MI-2 or MI-3 or control drug (MI-nc) (3.1 µM, 6.3 µM, or 12.5 µM). (F) Total CFCs in BM cultures supplemented with GM-CSF and MT-1-CM and treated with 3.1 µM or 6.3 µM of control drug (MI-nc) or Menin inhibitors (MI-2 or MI-3). (G) Hoxa9 mRNA expression in FLT3L bone marrow cultures supplemented with MT-1- CM, treated with MI-2/MI-3 or MI-nc (6 and 12.5 µM). MT-1-CM is designated as MT-1. Data represents a minimum of two experiments. *p < 0.05, **p < 0.01, ***p < 0.001.    75 3.1.10 Role of global H3K4me3 in mammary tumour secreted factor-mediated suppression of hematopoietic stem/progenitor cell defects Menin and MLL proteins are part of the TrxG complex that catalyzes the tri-methylation of H3K4, which is an activating epigenetic mark for permissive gene expression.  In order to determine the significance of tri-methylation of H3K4 in the effects mediated by MT-1-CM, we inhibited the interaction between Menin and MLL using the recently created Menin inhibitors, MI-2 and MI-3, which have been shown to inhibit Hoxa9 expression and prevent leukemic transformation (Grembecka et al., 2012).  First, we confirmed that these Menin inhibitors inhibited trimethylation of H3K4 by Western blotting (Fig. 3.20C inset).  Treatment of GM-CSF BM cultures with MI-3 attenuated H3K4me3 in a dose dependent manner (Fig. 3.20C inset). The addition of MI-2 and MI-3 to MT-1-CM treated FLT3L BM cultures reduced total cellularity, LSK/LKs and Hoxa9 expression compared to in vitro cultures treated with the control drug, MI-nc, in a dose dependent manner (Fig. 3.20 C-E, G).  Utilizing the methycellulose assay to enumerate myeloid progenitors, treatment of day 3 GM-CSF BM cultures supplemented with MT-1-CM with MI-2 and MI-3 reduced CFCs compared to control cultures (Fig. 3.20 F).  These results link the expansion of progenitor content by MT-1-CM with the increase in H3K4me3 activating marks in the Hoxa9 locus.  Importantly, Menin inhibitor mediated reduction in LSKs, LKs, CFCs in MT-1-CM treated FLT3L and GM-CSF BM cultures resembled the results where G-CSF was neutralized in MT-1-CM treated in vitro BM cultures.  These results link G-CSF in MT-1-CM, with the enrichment of H3K4me3 activating marks in BM progenitor cells, resulting in increased Hoxa9 expression and progenitor content.   76 3.1.11 Discussion Perturbations in myelopoiesis are commonly detected in neoplastic diseases and may be important in disease aetiology.  However, tumours alter the components of both the innate and adaptive immune systems in order to aid in their growth and metastasis.  Tumours have been shown to alter the development of immune cells resulting in the differentiation of TAMs, MDSCs and/or immature DCs (Gabrilovich, 2004; Gabrilovich et al., 2012).  These subsets of immunosuppressive cells can mediate important facets of tumour growth, therefore, understanding how tumours mediate these changes in the development of immune cells is critical in order to reverse or inhibit them (Bayne et al., 2012; Pylayeva-Gupta et al., 2012).  In breast cancer, the immune status of the patient may predict treatment outcome, therefore a better understanding of the impact of cancer on the immune system may provide a better indicator of disease severity and progression. Understanding this phenomenon may also aid in the selection of more appropriate treatments as well as the development of novel therapies (DeNardo et al., 2011; West et al., 2011). Our data demonstrates that mammary tumours affect the hematopoietic system resulting in a myeloproliferative-like disease, characterized by an expansion of iMCs, anemia and defects in HSPCs.  Importantly, we found similar perturbations in the HSPC compartment using five different mammary tumour cell lines, however, the B16 melanoma tumours did not share this phenotype.  Mammary tumour induced perturbations in the HSPC compartment occurred in immunosufficient and immunodeficient background mice, which suggests that the adaptive immune system does not play a critical role in mediating the effects of mammary tumours in vivo.  Furthermore, we did not detect any mammary tumour mediated perturbations in the lymphocyte compartment of the peripheral blood of immunosufficient background mice.    77 However, in human breast, ovarian, lung and other cancers, a decrease in lymphocyte levels in the peripheral blood has been detected as compared to healthy individuals (Milne et al., 2012). This discrepancy between our results may be due to the long tumour latency in humans.  Unlike the short time period of weeks required for cancer development in animal models, it can take years for cancer to develop in humans.  This difference in the time required for disease progression may be responsible for the conflicting results pertaining to lymphocyte development.  The effects of mammary tumours were measurable at distant sites, such as the BM and we confirmed that these changes were not due to tumour metastases but were instead due to tumour-secreted factors.  To investigate this further, we established an in vitro system to study the impact of mammary tumour secreted factors on HSPCs.  We found that both GM-CSF and FLT3L synergized with MT-CM, resulting in increased BM cell proliferation and progenitor cell expansion.  Importantly, the T-CM from all of the mammary tumour cell lines that we tested synergized with FLT3L and GM-CSF resulting in the expansion of BM cells and progenitors, however T-CM from a melanoma and a colon cancer cell line did not have this effect.  Overall, our in vivo and in vitro data suggests that different types of tumours interact with and alter the hematopoietic system in unique ways, with mammary tumours resulting in the disruption of the hematopoietic system.         To further explore the tumour-mediated expansion of HSPCs in vitro and in vivo, we performed functional assays to test the reconstitution capability of the HSPCs.  The HSPC competitive reconstitution capability of BM LT-HSCs from mammary tumour-bearing mice was intact, correlating with the phenotypic analysis.  However, there was a decrease in the number of restricted primitive BM progenitors in mammary tumour-bearing mice measured through the CFU-S12 assay.  Conversely, the spleens harvested from mammary tumour-bearing mice,   78 contained increased numbers of restricted primitive progenitors and the LSK/LKs could reconstitute the myeloid and lymphoid compartments of lethally-irradiated recipients.  Additionally, more restricted myeloid progenitors were intact in the BM of mammary tumour-bearing mice and were increased in the spleen.  This data supports that mammary tumour development results in extramedullary hematopoiesis, with fully functional splenic HSPCs.  Though we found increased numbers of LSK/LKs in our in vitro BM cultures treated with MT-CM and FLT3L, which also possessed abundant myeloid progenitors, these progenitors were unable to reconstitute either lethally or sub-lethally irradiated recipients.  This suggested that these progenitor cells were either functionally impaired or were not bona fide progenitors. However, these cells may in fact be true progenitor cells but may mimic actively cycling HSCs or HSCs located in inflammatory conditions in vivo, that have been shown to be defective in their ability to reconstitute irradiated hosts (Passegue et al., 2005).                   The identification of functional HSPCs in the spleens of mammary tumour-bearing mice suggests that mammary tumours induce the mobilization and potentially the proliferation of the HSPCs from the BM to the spleen.  A study by Cortez-Retamozo et al. (2013) demonstrated that murine lung adenocarcinoma induced the mobilization of HSPCs from the BM to the spleen, via tumour-produced angiotensin II (AngII). Once in the spleen these HSPCs proliferated and preferentially gave rise to TAMs and tumour-associated neutrophils (TANs), which went onto aid in tumour progression.  In splenectomized mice, there was a decrease in TAM and TANs located in the lungs, which correlated with decreased tumour burden, demonstrating the significance of extramedullary hematopoiesis in tumour progression (Cortez-Retamozo et al., 2013).  In our study, there was an accumulation of HSPCs in the spleens of mammary tumour-bearing mice, however these changes are likely due to G-CSF.  Though the two models used   79 different mechanisms to induce extramedullary hematopoiesis, in both models the alternate location of HSPCs resulted in the development of leukocytosis.  This highlights the importance of directing therapies at eliminating leukocytes versus targeting signaling pathways responsible for leukocytosis. Tumours can produce factors and cytokines important for tumour growth, survival and metastases. The mouse 4T1 mammary tumour cell line expresses GM-CSF, G-CSF and M-CSF, all of which were important in inducing the leukemoid reaction in tumour-bearing mice (DuPre and Hunter, 2007).  Accordingly, neutralization of 4T1 tumour-derived G-CSF resulted in a decrease in splenomegaly and tumour growth, whereas systemic administration of G-CSF lead to an increase in MDSCs and the development of splenomegaly (Waight et al., 2011).  G-CSF levels have also been shown to be higher in breast cancer tissue and in patient serum compared to surrounding healthy tissue and tissues and serum from control volunteers (Chavey et al., 2007). Although G-CSF may play a negative role in breast cancer it is an important cytokine in granulocyte differentiation, mobilization of HSPCs and treatment of neutropenia.  The combination of G-CSF and FLT3L can mobilize HSPCs into peripheral blood and G-CSF is currently being used in the clinic for this purpose in BM donors (Brasel et al., 1997).  However, the use of G-CSF has also been linked to promoting tumour angiogenesis and patients with tumours that secrete G-CSF have poorer prognosis (Matsumoto et al., 2010; Park et al., 2011).  There is also a positive correlation between the levels of G-CSF and the clinical grade and lymph node metastasis in breast cancer patients, demonstrating the positive correlation between G-CSF levels and tumour progression and disease severity (Dehqanzada et al., 2007; Lawicki et al., 2007; Lawicki et al., 2009).  We identified increased levels of G-CSF in the serum of mammary tumour-bearing mice and confirmed that the mammary tumour cells were capable of producing   80 G-CSF.  In our in vitro cultures, G-CSF synergized with FLT3L or GM-CSF, and using loss- and gain-of-function experiments we demonstrated that MT-CM derived G-CSF was responsible for the increase in cellularity and progenitor content. Furthermore, studies have shown that treatment of mice with G-CSF impairs erythropoiesis in the BM, however, it does not result in the development of anemia as measured by RBC levels in the peripheral blood, due to compensatory splenic erythropoiesis (de Haan et al., 1994; de Haan et al., 1992; Nijhof et al., 1994).  In our mammary tumour-bearing mice, erythropoiesis in the spleen could not compensate for the impaired erythropoiesis in the BM, resulting in the development of anemia.  This suggests that factors in addition to G-CSF secreted by mammary tumours may be responsible for the development of anemia.  Pre-existing anemia in cancer patients before therapy has been associated with reduced survival post treatment for breast, lung, prostate and other cancers, therefore anemia is considered an independent prognostic indicator of disease progression (Caro et al., 2001; Qiu et al., 2010a).  Therefore, treating tumour-mediated anemia may be an alternative method to increase survival in cancer patients by neutralizing anemia-inducing factors such as G-CSF.  Overall, the effects of G-CSF on hematopoiesis, erythropoiesis, metastasis and anti-tumour immune responses needs to be better understood before G-CSF is used in the clinic for cancer therapy and mobilization of stem cells. The similarities between mice with myeloproliferative diseases and HSPC abnormalities and the development of leukocytosis and anemia in mammary tumour-bearing mice suggest that mammary tumours may also dysregulate key hematopoietic regulatory genes. Hox genes are important regulators of HSPC renewal, expansion and differentiation. This is evidenced by studies that have demonstrated that loss of Hoxa9 expression results in hematopoietic cell differentiation whereas its overexpression leads to the development of AML (Thorsteinsdottir et   81 al., 2002).  Furthermore, loss-of-function mutations in Ezh2, a component of PRC2, which negatively regulates Hoxa9 expression, were also associated with myeloid disorders due to overexpression of Hoxa9 (Ernst et al., 2010; Nikoloski et al., 2010).  Mammary tumour development and the addition of MT-CM to in vitro BM cultures resulted in increased expression of Hoxa9 and a corresponding decrease in Ezh2.  This enhanced expression of Hoxa9 in HSPCs correlated with a histone methylation pattern on the Hoxa9 locus known to promote gene expression.  These results suggest that mammary tumours may mediate their effects on HSPCs by dysregulating epigenetic marks at the Hoxa9 locus, resulting in its over-expression and culminating in leukocytosis and anemia in mammary tumour-bearing mice.  To determine if the acquisition of the activating mark, H3K4me3, mediated by MLL and Menin was needed for the effects of MT-CM in in vitro BM cultures, we used Menin inhibitors that attenuate H3K4 trimethylation and suppress Hoxa9 expression in transformed leukemic cells (Grembecka et al., 2012).  These inhibitors reduced total cellularity, progenitor content, and Hoxa9 expression in MT-CM treated BM cultures, demonstrating the importance of trimethylation of H3K4 at the Hoxa9 locus for MT-CM-mediated changes in BM cultures.  Therefore, the development of Menin inhibitors could be important in suppressing tumour progression by targeting the epigenetic changes mediated by the mammary tumours (Grembecka et al., 2012).    Overall, we have found that mammary tumours dysregulate HSPCs leading to myeloproliferative-like disease consisting of the accumulation of iMCs and the development of anemia.  The effects of mammary tumours were mediated through changes in global epigenetic regulation resulting in increased Hoxa9 expression.  We found that many of these changes in the HSPC compartment and gene regulation were mediated by tumour-derived G-CSF.  Future   82 studies will focus on the necessity of G-CSF by knocking down G-CSF expression in mammary tumour cell lines and injecting mammary tumour cells into G-CSF receptor deficient mice.      83 Chapter 4: Mammary tumour-induced dysregulation in DC development Hypothesis: Mammary tumour-derived factors modify DC development to preferentially give rise to DCs that potentiate immunosuppression/suppress immune activation Objectives: Aim A: To characterize mammary tumour-induced changes in DC development and maturation Aim B: To study the effects of mammary tumour-conditioned media on DC development in vitro  Aim C: To determine the factor(s) responsible in mammary tumour-conditioned media responsible for inhibiting DC development Aim D: To understand the functional impact of mammary tumours on DCs  Aim E: To determine the relationship between mammary tumour induction of immunosuppressive enzymes in DCs and corresponding histone modifications Aim F: To understand the reversibility of the effects of mammary tumour-conditioned media on DC development, activation and function          84 4.1 Mammary cancer induces changes in DC development, function and phenotype 4.1.1 Mammary tumour mediated suppression of DC differentiation It has been well established that tumours can skew the numbers and subsets of DCs in humans and mice (Gabrilovich, 2004).  Therefore, we set out to determine whether these defects were caused by aberrant DC development. By excluding differentiated cell types expressing lineage markers (lineage: CD3+CD19+CD11b+DX5+Gr-1+Ter119+MHCII+FcεRI+), we analyzed BM and splenic compartment of MT-1 bearing mice for MDPs (lineage-CD11c-c-kit+M-CSFR+Flt3hi), CDPs (lineage-CD11c-c-kitintM-CSFR+Flt3+) and preDCs (lineage-CD11c+c-kitloM-CSFR+Flt3+) (Fig. 4.1, 4.2).  We found a decrease in BM preDCs, and an increase in MDP, CDP and preDC progenitors in the spleens of immunodeficient and immunosufficient MT-1 bearing mice (Fig. 4.2).  Interestingly, MDPs and CDPs were also significantly reduced in the BM of MT-1 bearing RAG1-/- mice only, suggesting that impaired lymphocyte development may have a detrimental effect on DC development in MT-1 bearing mice (Fig. 4.2).  In order to determine if mammary tumours in general perturbed DC development, we analyzed DC progenitors in the BM and spleen of 4T1 tumour-bearing mice.  Similar to changes in MT-1 bearing mice, 4T1 development was associated with reduced DC progenitors in the BM and an increase in the spleen (Fig. 4.3).  Furthermore, B16 melanoma tumour-bearing mice did not develop similar perturbations in DC progenitors (Fig. 4.4).  Our data shows that DC development is impaired in the BM of MT-1 bearing mice, with a concomitant increase in DC progenitors in the spleen.   85  Figure 4.1    DC progenitor gating scheme. Gating scheme for classification of DC progenitors. BM from control mice were stained with antibodies recognizing lineage-specific antigens, MHC II, CD11c, c-kit, M-CSFR, and Flt3, and analyzed by flow cytometry. Lineage-PI- cells were analyzed. MDP, monocyte dendritic cell progenitor; CDP, common dendritic cell progenitor; preDC, DC precursor.   Figure 4.2    Perturbations in DC progenitor numbers and location in mammary tumour-bearing mice. BM and splenic numbers of DC progenitor subsets in control or MT-1 bearing mice; MDP (lineage-CD11c-c-kithiM-CSFR +Flt3+), CDP (lineage-CD11c-c-kitintM-CSFR+Flt3+) and preDC (lineage-CD11c+c-kitloM-CSFR+Flt3+). Data are representative of a minimum of two independent experiments, n=3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.    86  Figure 4.3    Perturbations in DC progenitor numbers and location in 4T1 tumour-bearing mice. BM and splenic numbers of DC progenitor subsets in control or 4T1 tumour-bearing mice; MDP (lineage-CD11c-c-kithiM-CSFR+Flt3+), CDP (lineage-CD11c-c-kitintM-CSFR+Flt3+) and preDC (lineage-CD11c+c-kitloM-CSFR+Flt3+). Data are representative of a minimum of two independent experiments, n=3. *p < 0.05, **p < 0.01, ***p < 0.001.      Figure 4.4    Perturbations in DC progenitor numbers and location in B16 tumour-bearing mice. BM and splenic numbers of DC progenitor subsets in control or B16 tumour-bearing mice; MDP (lineage-CD11c-c-kithiM-CSFR+Flt3+), CDP (lineage-CD11c-c-kitintM-CSFR+Flt3+) and preDC (lineage-CD11c+c-kitloM-CSFR+Flt3+). Data are representative of a minimum of two independent experiments, n=3.       87 pDCs fully differentiate in the BM prior to extravasation and migration into the blood, therefore we investigated the impact of reduced DC progenitors in the BM on the number of pDCs by flow cytometry (Diao et al., 2004; Fogg et al., 2006; Naik et al., 2007).  Consistent with the reduction in CDPs in the BM, the frequency and absolute numbers of pDCs (CD45RA+CD11c+) were reduced in MT-1 bearing mice, regardless of the site of tumor development (SQ or MFP) (Fig. 4.2, 4.5A, 4.6).  Conversely, the increase in DC progenitors in the spleen of MT-1 bearing mice suggested an increase in peripheral DC development.  Surprisingly, we found a reduction in the splenic frequencies of CD11c+MHC II+ cDCs, CD4+ and CD8+ cDCs, and pDCs in MT-1 bearing mice (Fig. 4.5B).  There was a corresponding reduction in the absolute numbers of CD4+ cDCs and pDCs, however, CD8+ cDCs and MHC II+ cDCs were not consistently significantly reduced (Fig. 4.6).  This discrepancy between the frequency and absolute numbers of DC subsets in the spleens of MT-1 tumour-bearing mice is due to the 3- and 5-fold increase in total splenic cellularity in MMTV-neu and RAG1-/- MT-1 bearing mice, respectively, offsetting the reduction in frequencies (Fig. 3.7B, 4.5B, 4.6).             88  Figure 4.5    Perturbations in DC frequency in mammary tumour-bearing mice. (A) Frequencies of CD11c+CD45RA+ cells in the BM of control and MT-1 tumour-bearing mice. (B) Frequencies of CD11c+MHC II+, CD11c+CD4+, CD11c+CD8+, CD11c+CD45RA+ cells in the spleen and tumour of control and MT-1 bearing mice. SQ, subcutaneous; MFP, mammary fat pad. Data was acquired on the RAG1-/- background. Data are representative of a minimum of three independent experiments, n=3.     89   Figure 4.6    Perturbations in DC numbers in mammary tumour-bearing mice. Absolute numbers of pDCs in the BM, and CD11c+MHC II+, CD11c+ CD8+, CD11c+ CD4+ and pDCs in the spleen of control and MT-1 bearing mice on RAG1-/- background. Data are representative of a minimum of three independent experiments, n=3.  Our results demonstrate that mammary tumours impair DC development in the BM, resulting in a shift in DC progenitors from the BM to the spleen.  However, the increase in splenic DC progenitors in MT-1 bearing mice did not result in an increase in mature DC subsets, instead, there was a reduction in mature DC frequencies (pDCs, CD8+ DCs, CD4+ DCs) and numbers (CD4+ DCs, pDCs).  This data suggests that mammary tumours impair the differentiation of DCs from DC progenitors, resulting in overall decrease in DCs.            90 4.1.2 Role of mammary tumour secreted factors in suppressing in vitro FLT3L derived DCs  To elucidate the molecular mechanism behind mammary tumour induced impairment of DC development, we used an in vitro method for culturing DCs using FLT3L, a cytokine that induces the differentiation of pDCs (CD11cloCD45RA+), CD8+-like cDCs (CD11c+CD45RA-CD24+SIRPα-) and CD8--like cDCs (CD11c+CD45RA-CD24-SIRPα+) from BM cells that resemble in vivo splenic DCs subsets (Naik et al., 2005; Naik et al., 2007; Onai et al., 2007).  Based on our previous results demonstrating that soluble, tumor-derived factors impaired in vitro HSC differentiation, we examined the effects of T-CM on DC development in vitro.  We supplemented FLT3L DC cultures with T-CM from multiple mammary tumour cell lines including MT-1-4 and 4T1, as well as T-CM from B16 melanoma cells and CMT93 colon carcinoma cells (Fig. 4.7A and data not shown). Interestingly, only T-CM derived from mammary tumours impaired the differentiation of pDCs and CD8+- and CD8--like cDCs into an indistinct, CD24intSIRPαint subset of cells, whereas B16 and CMT93 T-CM had no effect (Fig. 4.7A and data not shown). Due to the unique ability of MT-CM to suppress the differentiation of DCs, we further studied the effects of MT-1-CM on FLT3L DCs at different stages of DC development. Addition of MT-1-CM from day 0-9 or day 3-9 to FLT3L DC cultures led to impaired DC differentiation, whereas addition from day 6-9 and 8-9 had no effect (Fig. 4.7B). This suggests that MT-1-CM impacts the differentiation of early DC progenitors into mature DCs, corroborating the in vivo effects of MT-1 on DC progenitors in tumour-bearing mice (Fig. 4.2, 4.7B).   Additionally, DC activation was impaired by early treatment with MT-1-CM, with baseline and LPS-induced expression of maturation markers, including MHC II, CD80, CD86 and CD40, impaired in the   91 FLT3L cDCs (Fig. 4.8).  Importantly, MT-1-CM treatment of FLT3L DC cultures did not suppress responsiveness to TLR stimulation, as evidenced by the upregulation of PD-L1, the immunomodulatory receptor, which plays an important role in innate and adaptive immunity, and is important for the induction of regulatory T cells (Tregs) and maintenance of peripheral tolerance and can be exploited in a tumour setting (Francisco et al., 2009; Yao and Chen, 2006; Zou and Chen, 2008), upon LPS stimulation (Fig. 4.8B). Due to the effects of MT-1-CM on DC subsets we next analyzed the impact of MT-1-CM on DC progenitors. Total cellularity was increased in FLT3L DC cultures treated with MT-1-CM, mimicking the splenomegaly observed in MT-1 bearing mice (Fig. 4.9A). Flow cytometry analysis of DC progenitors showed an increase in MDPs and CDPs in FLT3L DC cultures treated with MT-1-CM over 9 days (Fig. 4.9A). Surprisingly, total preDCs either remained unchanged or decreased in the presence of MT-1-CM compared to control FLT3L DC cultures and there was a decrease in CD11c+ cells in MT-1-CM treated cultures (Fig. 4.9A). These results show a similar block in preDC development between in vitro MT-1-CM treated FLT3L DC cultures and the BM of MT-1 bearing mice.  This data suggests that MT-1-CM blocks the differentiation of CDPs into pDCs and preDCs.    To further investigate the effects of MT-1-CM on DC development, expression of transcription factors important in DC differentiation were measured in MT-1-CM treated FLT3L DC cultures.  IRF-4 and E2-2 are essential transcription factors for CD8- cDC and pDC development, respectively, whereas IRF-8 is involved in CD8+ cDC and pDC differentiation (Aliberti et al., 2003; Cisse et al., 2008; Schiavoni et al., 2002; Tamura et al., 2005; Tsujimura et al., 2003).  Reduced expression of these transcription factors in MT-1-CM treated FLT3L DC cultures correlated with the impaired DC differentiation measured by flow cytometry.  Reduced   92 expression of Irf-4 and E2-2 was measured in FLT3L DC cultures treated with MT-1-CM from days 0-9, 3-9 and 6-9, whereas Irf-8 expression was reduced at all time points treated with MT-1-CM (Fig. 4.9B).  Recently it has been shown that a new transcription factor, Zbtb46, is specifically expressed in preDCs and cDCs (Meredith et al., 2012; Satpathy et al., 2012). Similar to the impaired expression of the other DC transcription factors in MT-1-CM treated FLT3L DC cultures, expression of Zbtb46 was reduced when treated with MT-1-CM from day 0-9 and 3-9 (Fig. 4.9B). The effects of MT-1-CM on the expression of transcription factors important for DC differentiation further confirms that DC development is compromised by mammary tumor secreted factors (Figure 4.9B). Our results show that MT-1-CM treatment of FLT3L DC cultures led to a defect in DC development closely resembling the effects observed in MT-1 bearing mice, with a concurrent reduction in the expression of essential transcription factors required for DC subset differentiation.        93  Figure 4.7    Mammary tumour-conditioned media impairs DC differentiation in in vitro FLT3L DC cultures. (A) Frequencies of pDCs (CD11c+CD45RA+), cDCs (CD11c+CD45RA-), CD8+-like (CD11c+CD45RA-CD24+SIRPα-), and CD8--like (CD11c+CD45RA-CD24-SIRPα+) DCs from FLT3L DC cultures treated with T-CM from MT-1, 4T1, B16, and CMT93 cell lines. (B) Frequencies of pDCs, cDCs, CD8+-like, CD4+-like DCs from FLT3L DC cultures treated with MT-1-CM for different periods of time (day 0-9, 3-9, 6-9, 8-9). Data are representative of three independent experiments.    94   Figure 4.8    Mammary tumour-conditioned media impairs DC expression of MHC II and co-stimulatory receptors in in vitro FLT3L DC cultures. (A) Proportion of CD11c+CD45RA- cells expressing low, intermediate and high levels of MHC II in FLT3L DC cultures treated with MT-1-CM for different periods of time (day 0-9, 3-9, 6-9, 8-9). Cells were stimulated with (+) or without (-) LPS. Numbers below graphs represent frequencies of cells in each sector. (B) Frequency of cells expressing CD80, CD86, CD40, PD-L1, and CD8 in FLT3L DC cultures treated with MT-1-CM for different periods of time (day 0-9, 3-9, 6-9, 8-9).  Cells were stimulated with (black bars) or without (white bars) LPS. Data are representative of two experiments.    95  Figure 4.9    Mammary tumour-conditioned media enhances DC progenitor generation and impairs essential DC transcription factor mRNA expression in in vitro FLT3L DC cultures. (A) Absolute numbers of total cells, MDPs, CDPs, preDCs, and CD11c+ cells in FLT3L DC cultures treated with MT-1-CM over a period of 9 days. (B) Expression of transcription factors Irf4, Irf8, E2-2, and Zbtb46 in FLT3L DC cultures treated with MT-1-CM over different periods of time (day 0-9, 3-9, 6-9, 8-9). Expression was analyzed by qPCR. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.            96 4.1.3 Role of mammary tumour secreted factors in suppressing GM-CSF derived DC development in vitro GM-CSF is a classical cytokine used to culture in vitro DCs (Inaba et al., 1992). Therefore, we tested the effects of T-CM on GM-CSF DC cultures. Similar to FLT3L DCs, T-CM from mammary tumour cell lines (MT-1-4, 4T1) impaired expression of MHC II in GM-CSF DC cultures at baseline and post-LPS stimulation, whereas T-CM from B16 and CMT93 cell lines had no effect (Fig. 4.10). Furthermore, MT-1-CM mediated inhibition of MHC II, CD86, CD80 and CD40 expression titrated with the dose of MT-1-CM from 6.25% to 25% (Fig. 4.11A and data not shown). Additionally, MT-1-CM treatment of GM-CSF DCs early in development suppressed MHC II expression more at baseline and post-LPS stimulation then when added at later time points (Fig. 4.11B). PAMP stimulation of PRRs on DCs such as TLRs can induce the production of important cytokines for lymphocyte activation, including IL-12, IL-10, IL-6 and TNF-α (Steinman and Idoyaga, 2010). T-CM from mammary tumour cell lines MT-1 to 4 impaired LPS-induced IL-12 production in GM-CSF DC cultures compared to control cultures, while IL-10 production was induced by T-CM from MT-1 and 2 only after LPS stimulation (Fig. 4.12). No changes in IL-6 and TNF-α production were detected in MT-CM treated GM-CSF DCs (data not shown).  These changes in cytokine production mediated by MT-CM suggest that DCs are skewed towards an immunosuppressive phenotype. Our data demonstrates that T-CM from mammary tumours inhibits DC maturation/activation at baseline and after LPS stimulation.  This suppression of DC activation by T-CM from mammary tumours suggests that mammary tumours may have an enhanced ability to impair host immunity against tumours compared to other tumour types.    97  Figure 4.10    Mammary tumour-conditioned media impairs DC activation in in vitro GM-CSF DC cultures. (A) Frequencies of CD11c+MHC II+ DCs in GM-CSF DC cultures treated with T-CM from MT-1, CMT-93, 4T1, and B16 cell lines. Cells were stimulated ± LPS. (B) Frequencies of CD11c+MHC II+ DCs in GM-CSF DC cultures treated with T-CM from MT-1 through 4. Cells were stimulated ± LPS. Data are representative of a minimum of two independent experiments.      98  Figure 4.11    Mammary tumour-conditioned media impairs DC differentiation in in vitro GM-CSF DC cultures at different concentrations and treated for different periods of time. (A) Frequencies of CD11c+MHC II+ DCs in GM-CSF DC cultures treated with MT-1-CM at concentrations of 6.25%, 12.5% and 25%. Cells were stimulated ± LPS. (B) Frequencies of CD11c+MHC II+ DCs in GM-CSF DC cultures treated with MT-1-CM for different periods of time (day 0-9, 3-9, 6-9, 8-9). Cells were stimulated ± LPS. Data are representative of a minimum of two independent experiments.      99  Figure 4.12    Mammary tumour-conditioned media impairs cytokine secretion in in vitro GM-CSF DC cultures. Expression of IL-12 and IL-10 in GM-CSF DC cultures treated with T-CM from MT-1 through 4. Cytokine levels were measured by ELISA. Cells were stimulated ± LPS. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.                 100 4.1.4 Mammary tumour derived G-CSF is responsible for DC developmental impairment Our studies on the hematopoietic changes induced by mammary tumours showed that these changes were predominantly mediated by tumour-secreted G-CSF. Mammary tumour-derived G-CSF impaired HSPC differentiation and maintenance. Other studies have shown that G-CSF treatment of healthy individuals skews DC differentiation towards a DC2 phenotype, with subsequent induction of Th2 immune responses (Arpinati et al., 2000; Klangsinsirikul and Russell, 2002). However, the question of whether G-CSF affects DCs at a matured stage or at the progenitor level has been largely unexplored. To determine the effects of mammary tumour derived G-CSF on DC differentiation, FLT3L and GM-CSF in vitro DC cultures were supplemented with G-CSF and analyzed for the differentiation of pDC and cDC or CD11c+MHC II+ populations, respectively, by flow cytometry.  G-CSF impaired cDC and pDC differentiation in FLT3L DC cultures, but interestingly whereas MT-1-CM skewed cDC differentiation towards CD8--like cDCs, G-CSF instead induced the differentiation of CD8+-like cDCs (Fig. 4.13A).  This suggests that other factors in MT-1-CM dominate over the effects of G-CSF on cDC differentiation.  Both at baseline and after LPS stimulation, similar to MT-1-CM, the addition of G-CSF to FLT3L cultures inhibited the expression of MHC II, CD86, and CD40 in the cDC population as shown by reduced MFI compared to control cultures (Fig. 4.13B).    The addition of G-CSF to GM-CSF DC cultures also impaired MHC II, CD86, CD40, and CD80 expression on CD11c+ DCs in a dose dependent manner (Fig. 4.14).  Conversely, neutralization of G-CSF in FLT3L DC cultures treated with a reduced dose (1.6%) of MT-1-CM to facilitate complete G-CSF neutralization restored cDC and pDC development and partially restored MHC II   101 expression on cDCs (Fig. 4.13C).  These results suggest that MT-1-CM derived G-CSF impairs DC differentiation in FLT3L DC cultures and inhibits FLT3L and GM-CSF DC activation.  Our work demonstrating the effects of MT-1-CM derived G-CSF on HSPCs suggested the possibility that G-CSF may also be acting on DC progenitors.  G-CSF treatment of FLT3L DC cultures, similar to MT-1-CM, resulted in an expansion of DC progenitors MDPs and CDPs (Fig. 4.15A).  However, unlike MT-1-CM, G-CSF also resulted in the accumulation of preDCs, suggesting that other components of MT-1-CM are acting on DC progenitors and impairing the differentiation of CDPs into preDCs (Fig. 4.15A).  Conversely, analysis of DC progenitors in 1.6% MT-1-CM supplemented FLT3L DC cultures treated with anti-G-CSF antibodies revealed that G-CSF neutralization partially restored MDPs and CDPs to control levels, but not preDCs (Fig. 4.15B and data not shown). These results indicate that MT-1-CM derived G-CSF partially suppresses DC development and is important in the accumulation of MDPs and CDPs.         102  Figure 4.13    G-CSF impairs DC development and activation in in vitro FLT3L cultures. (A) Frequencies of pDCs and cDCs (left) and absolute numbers of pDCs (right) in FLT3L DC cultures treated with MT-1-CM or G-CSF for 9 days. (B) MFI of CD86, MHC II and CD40 in cDCs from FLT3L DC cultures treated with MT-1-CM or G-CSF for 9 days. (C) Frequencies of pDCs and cDCs (left) and expression levels of MHC II in cDCs (right) from FLT3L DC cultures treated with 1.6% MT-1-CM for 9 days. Cultures were treated with neutralizing G-CSF Abs (αG-CSF) or isotype control Abs (iso). *p < 0.05, **p < 0.01, ***p < 0.001.   103  Figure 4.14    G-CSF impairs DC activation in in vitro GM-CSF cultures. (A) Frequencies of MHC II+ CD11c+ cells at baseline and post LPS stimulation in GM-CSF DC cultures treated with MT-1-CM or G-CSF for 9 days. (B) MFI of CD86, MHC II, CD40 and CD40 in CD11c+ cells from GM-CSF DC cultures treated with MT-1-CM or G-CSF for 9 days. *p < 0.05, **p < 0.01.   104  Figure 4.15    Mammary tumour-conditioned media derived G-CSF enhances DC progenitor generation. Numbers of MDPs, CDPs, and preDCs from FLT3L DC cultures treated with (A) MT-1-CM or G-CSF (1, 5, 20 ng/mL) and (B) FLT3L DC cultures treated with 1.6% MT-1-CM and neutralizing G-CSF or isotype control Abs (iso) for 9 days.  Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.             105 4.1.5 MT-1 conditioned media and G-CSF mediate impairment of DC functional activity DCs are critical in activating both the adaptive and innate immune systems to respond to neoplasia (Dunn et al., 2002; Dunn et al., 2004; DuPage et al., 2012; Quezada et al., 2011; Schreiber et al., 2011). Since MT-1-CM impaired DC development and differentiation, we next investigated the effects of MT-1-CM on DC function.   NK cells require priming by activated DCs for their effector functions (Fernandez et al., 1999; Kobayashi et al., 2005; Lucas et al., 2007).  To investigate whether mammary tumours impacted the ability of DCs to prime NK cells, splenic NK cells from LPS-stimulated control and MT-1 bearing mice were assessed for IFN-γ and granzyme B production by intracellular flow cytometry.  MT-1 development impaired NK cell IFN-γ and granzyme B expression after LPS stimulation (Fig. 4.16A,B).  Impaired NK cell activation is likely due to defective DC development and activation in MT-1 bearing mice. The adaptive immune system is critical for the rejection of neoplasia. In order for DCs to activate T cells, immature DCs must take up tumour antigens and process and present them, with necessary co-stimulatory molecules, to T cells as mature DCs.  In order to determine the impact of MT-1-CM on the ability of DCs to phagocytose antigens, GM-CSF derived DCs treated with or without MT-1-CM were co-cultured with doxorubicin-treated, CFSE-labeled MT-1 or B16 tumour cells. Frequencies of CD11c+CFSE+ cells, representing DCs that have phagocytosed CFSE+ dying tumour cells, were greater in DCs treated with MT-1-CM compared to control DCs (Fig. 4.16C).  These results show that MT-1-CM treatment of GM-CSF DCs increases DC phagocytosis, keeping DCs in an immature state, since phagocytosis decreases as DCs mature. Next we investigated whether MT-1-CM treatment impaired GM-CSF DCs ability to prime T cell proliferation.  GM-CSF DCs treated with or without MT-1-CM were co-cultured with   106 doxorubicin-treated, OVA protein expressing B16 or MT-1 cells.  CD11c+ cells were purified and then co-cultured with CFSE-labeled OTI cells in order to measure the ability of these DCs to induce antigen-specific T cell proliferation.  Compared to control DCs, GM-CSF DCs treated with MT-1-CM were impaired in their ability to induce OTI cell proliferation, possibly due to impaired cross-presentation ability, co-stimulatory molecule expression or cytokine production (Fig. 4.16D).  Therefore, in order to measure if the addition of MT-1-CM impaired the ability of GM-CSF DCs to cross-present, OTI or B3Z cells, a MHC class I (H-2b)-restricted OVA-specific hybridoma, were co-cultured with GM-CSF DCs treated with or without MT-1-CM and pulsed with doxorubicin-treated B16-OVA or MT-1 cells.  Cross-presentation was indirectly measured by the production of IFN-γ and IL-2 by OTI and B3Z cells, respectively.  GM-CSF DCs treated with MT-1-CM induced less IFN-γ and IL-2 production from OTI and B3Z cells, respectively compared to control DCs, suggesting that the addition of MT-1-CM to GM-CSF DCs inhibited cross-presentation of tumour antigens  (Fig. 4.16E). For an optimal immune response against tumours, both CD4+ and CD8+ T cell activation is required.  Therefore we next measured the ability of MT-1-CM treated GM-CSF DCs to activate OTI (CD8+) and OTII (CD4+) cell proliferation by CFSE dilution.  Stimulated with either SIINFEKL peptide or OVA protein, GM-CSF DCs treated with MT-1-CM induced lower OTI cell proliferation (Fig. 4.17A).  These results show that even with the use of SIINFEKL peptide, which bypasses antigen processing, GM-CSF DCs treated with MT-1-CM were impaired in their ability to activate T cell proliferation.  GM-CSF DCs treated with MT-1-CM were similarly impaired in their ability to induce OTII cell proliferation (Fig. 4.17A).   Finally, we investigated the effects of MT-1-CM on the ability of DCs to activate T cell cytokine production by measuring IFN-γ and IL-2 production by OTI and B3Z cells, respectively, after   107 co-culture with MT-1-CM treated GM-CSF DCs pulsed with OVA protein or SIINFEKL peptide (Fig. 4.17B).  MT-1-CM treatment of GM-CSF DCs impaired their ability to activate cytokine production by both OTI and B3Z cells after stimulation by OVA and SIINFEKL compared to control GM-CSF DCs (Fig. 4.17B).  This data demonstrates that DC activation of T cells was impaired by MT-1-CM treatment. MT-1-CM treatment of GM-CSF DCs results in reduced MHC II and co-stimulatory molecule expression, increased PDL1 expression, and skewing of cytokine production towards a Th2 response away from a Th1 response.  Overall, MT-1-CM treatment of DCs induces an immunosuppressive phenotype.  These observations lead us to hypothesize that MT-1-CM treated DCs may actively suppress endogenous antigen-specific DC activation of T cell proliferation.  To test this, we co-cultured CFSE labeled OTI or OTII splenocytes with FLT3L or GM-CSF DCs supplemented with or without MT-1-CM and OVA protein.  FLT3L DCs treated with MT-1-CM suppressed OTI and OTII cell proliferation at DC:splenocyte ratios of 1:2 and 1:1, respectively, compared to control FLT3L DCs (Fig. 4.17C and data not shown).   Surprisingly, GM-CSF DCs treated with MT-1-CM suppressed OTII proliferation at a ratio of 1:8, but did not inhibit OTI cell proliferation (Fig. 4.17C and data not shown).  Therefore, MT-1-CM treatment of FLT3L and GM-CSF DCs results in a tolerogenic phenotype, resulting in suppression of OTII proliferation and OTI proliferation by FLT3L DCs treated with MT-1-CM.  Next we wanted to determine if MT-1-CM derived G-CSF, in addition to mediating the accumulation of DC progenitors and skewing of DC differentiation in MT-1-CM treated FLT3L cultures, induced DC mediated suppression of T cell proliferation.  Therefore, FLT3L and GM-CSF DCs treated with G-CSF were co-cultured with control or OVA-treated CFSE-labeled OTI and OTII splenocytes.  FLT3L DCs treated with G-CSF, similar to MT-1-CM, suppressed OTI   108 and OTII proliferation at DC:splenocyte ratios of 1:2 and 1:8 (Fig. 4.17D).  GM-CSF DCs treated with G-CSF also inhibited OTII cell proliferation at a ratio of 1:4, however, similar to MT-1-CM treated GM-CSF DCs, G-CSF treated GM-CSF DCs did not inhibit OTI cell proliferation (Fig. 4.17D).  These results show that tumour secreted G-CSF may contribute to the ability of MT-1-CM to suppress DC activation of T cells.  Overall, our data shows impaired DC development and maturation mediated by MT-1-CM results in impaired lymphocyte activation.             109  Figure 4.16    Mammary tumours impair DC activation of NK and T cells. (A) Frequencies of splenic NK1.1+IFN-γ+ NK cells in control and MT-1 bearing mice stimulated ± LPS. Cells were gated on NK1.1+ DX5+ populations. (B) Frequencies of granzyme B+ (GrB+) NK cells in control and MT-1 bearing mice stimulated ± LPS. Cells were gated on NK1.1+ DX5+ populations. (C). Frequencies of CFSE+CD11c+ cells in GM-CSF DC cultures treated ± MT-1-CM co-cultured with CFSE labeled, doxorubicin treated B16-OVA or MT-1 cells. (D) Frequencies of CFSE- cells in GM-CSF DC cultures treated ± MT-1-CM incubated with doxorubicin treated B16-OVA or MT-1 cells, then co-cultured with CFSE labeled OT-I cells. (E) Expression of IFN-γ in OTI cells (left) or IL-2 in B3Z cells (right) co-cultured with GM-CSF DCs treated ± MT-1-CM, after incubation with doxorubicin treated B16-OVA or MT-1 cells. IFN-γ and IL-2 levels were measured by ELISA. Data are representative of two. *p < 0.05, **p < 0.01, ***p < 0.001.    110  Figure 4.17    Mammary tumours impair antigen specific DC activation of T cells and induce DC mediated suppression of T cell proliferation. (A) Frequencies of CFSE- cells in GM-CSF DC cultures treated ± MT-1-CM incubated with OVA protein or SIINFEKL peptide, then co-cultured with CFSE labeled OTI or OTII cells. (B) Expression of IFN-γ in OT-I cells (left) or IL-2 in B3Z cells (right) co-cultured with GM-CSF DCs treated ± MT-1-CM, incubated with OVA protein or SIINFEKL peptide. IFN-γ and IL-2 levels were measured by ELISA. (C) Frequencies of CFSE- cells in GM-CSF DC cultures treated ± MT-1-CM co-cultured with CFSE labeled OTII splenocytes in the presence of OVA protein. Cells were gated on Thy1.1+CD4+ populations. (D) Frequencies of CFSE- cells in GM-CSF and FLT3L DC cultures treated ± MT-1-CM or G-CSF (20 ng/mL) co-cultured with CFSE labeled OTI or OTII splenocytes in the presence of OVA protein. Cells were gated on Thy1.1+CD8+ populations for OTI splenocytes, and Thy1.1+CD4+ populations for OTII splenocytes. *p < 0.05, ***p < 0.001.   111 4.1.6 Upregulation of immunosuppressive enzymes and markers by MT-1 conditioned media treatment of FLT3L and GM-CSF DCs is accompanied by changes in histone modifications Tumours have been shown to induce immunosuppressive DCs, which instead of targeting tumour cells, aid in tumour progression by inhibiting the immune system.  These immunosuppressive DCs are characterized by the expression of inhibitory markers and enzymes including Arg1, found in inflammatory zone 1 (Fizz1), vascular endothelial growth factor a (VEGFa) and Ym1, collectively referred to as type 2 markers.  Therefore, we measured the expression of Arg1, Ym1, Fizz1 and VEGFa by qPCR and found that all of these type 2 immunosuppressive markers were upregulated in MT-1-CM treated GM-CSF DCs as compared to control DCs (Fig. 4.18A). Recently it has been discovered that M2 marker expression is regulated by JMJD3, a catalytic enzyme that demethylates H3K27, resulting in permissive gene expression (Ishii et al., 2009; Satoh et al., 2010).  ChIP analysis showed a reduction in the repressive H3K27me3 mark associated with the Arg1, Ym1 and Fizz1 locus in MT-1-CM treated GM-CSF DCs, correlating with the increase in mRNA expression of these genes (Fig. 4.18B).    Surprisingly, compared to control FLT3L DCs, only Arg1 expression was upregulated in MT-1-CM treated FLT3L DCs, whereas expression of Fizz1, Vegfa and Ym1 was decreased (Fig. 4.18C and data not shown). BM cells cultured in GM-CSF and FLT3L give rise to DC precursors that are CD11c- prior to differentiating into CD11c+ DCs. Due to the presence of CD11c- cells in FLT3L and GM-CSF cultures treated with MT-1-CM, we wanted to investigate whether the CD11c- DC precursors contributed to the expression of Arg1.  Therefore, we sorted the CD11c- and the CD11c+ fractions from GM-CSF and FLT3L DCs supplemented with or without MT-1-CM.  Interestingly, Arg1 expression was upregulated in the CD11c- and CD11c+ fractions of GM-CSF and FLT3L DCs treated with MT-1-CM (Fig. 4.18D).  Western blot analysis of Arg1   112 protein confirmed the increase in mRNA expression in MT-1-CM treated DCs (Fig. 4.18F).  An increasing dose of MT-1-CM correlated with increased expression of Arg1 protein in both GM-CSF and FLT3L DCs (Fig. 4.18F).  Importantly, increased expression of Arg1 was also measured in CD11c+ cells purified from the spleens of MT-1 bearing mice compared to control mice by qPCR (Fig. 4.18E) To determine if MT-1-CM derived G-CSF polarized DCs, we compared Arg1 expression in DCs cultured with G-CSF to MT-1-CM.  We found that the expression of Arg1 in GM-CSF and FLT3L DCs correlated with the increasing dose of G-CSF, however, G-CSF induced Arg1 expression to a lesser extent then MT-1-CM (Fig. 4.19A). Neutralization of G-CSF in FLT3L DC cultures treated with MT-1-CM resulted in reduced Arg1 expression compared to FLT3L DCs treated with isotype control antibody (Fig. 4.19B).  This data shows that MT-1-CM derived G-CSF does contribute to Arg1 expression in DCs.              113  Figure 4.18    Mammary tumour-conditioned media induces immunosuppressive protein expression in in vitro and in vivo DCs. (A) Expression of Fizz1, Arg1, Vegfa and Ym1 mRNA in GM-CSF DC cultures treated ± MT-1-CM. (B) Repressive H3K27me3 histone modifications were investigated by ChIP of chromatin isolated from GM-CSF DC cultures treated ± MT-1-CM. Chromatin was immunoprecipitated using anti-H3K27me3 or IgG control Abs and amplicons were analyzed by qPCR. (C) As in A, except with FLT3L DC cultures. (D) Expression of Arg1 in CD11c- and CD11c+ fractions from sorted FLT3L and GM-CSF DC cultures treated ± MT-1-CM. (E) Arg1 expression in CD11c+ sorted DCs from the spleens of control and MT-1 bearing mice. (F) Lysates from GM-CSF and FLT3L DC cultures treated with an increasing dose of MT-1-CM were analyzed by Western blot for Arg1 and total Stat3 (T-Stat3, loading control). Data represents a minimum of two independent experiments.   114  Figure 4.19    Mammary tumour conditioned media derived G-CSF contributes to Arg1 expression. (A) Expression of Arg1 in FLT3L and GM-CSF DC cultures treated with MT-1-CM or G-CSF (1, 20 ng/mL). (B) Expression of Arg1 in FLT3L DC cultures treated with 1.6% MT-1-CM and G-CSF neutralizing or isotype control Abs (iso). Data are representative of a minimum of three independent experiments.                  115 4.1.7 Mammary tumour-conditioned media expands functional DC progenitors that can differentiate into pDCs and cDCs In the in vitro FLT3L DC cultures, MDPs, CDPs and preDCs differentiate into pDCs, CD8+-like and CD8--like cDCs (Naik et al., 2007; Onai et al., 2007). Therefore, we hypothesized that the removal of MT-1-CM from FLT3L cultures would result in the differentiation of the expanded DC progenitors into pDCs and cDCs.  To test this hypothesis, on day 6 of FLT3L DC culture, MT-1-CM was either removed or maintained and then cultures were analyzed by flow cytometry, qPCR and functional assays on day 13.  Compared to FLT3L cultures maintained in MT-1-CM, removal of MT-1-CM resulted in the differentiation of pDCs and cDCs (CD8+-like and CD8--like DCs) (Fig. 4.20A).  Moreover, the absolute numbers of pDCs and CD8+-like cDCs were significantly increased in FLT3L cultures with the removal of MT-1-CM on day 6 (Fig. 4.20B).  Correlating with the differentiation of the DC progenitors, removal of MT-1-CM lead to increased expression of transcription factors important in DC differentiation, E2.2, Irf4, Irf8 and Zbtb46 (Fig. 4.20C).  Correspondingly, the absolute number of DC progenitors, MDPs, CDPs and preDCs, decreased with the removal of MT-1-CM compared to FLT3L cultures maintained in MT-1-CM (Fig. 4.20D).  These results demonstrate that MT-1-CM expands DC progenitors in culture that retain the ability to differentiate into pDCs and cDCs when MT-1-CM is removed.  Therefore, MT-1-CM inhibits the differentiation of DC progenitors into mature DC subsets and does not just delay the differentiation of DC progenitors. Next we investigated the functional significance of removing MT-1-CM in FLT3L DC cultures by measuring their ability to activate OTI and OTII cell proliferation by CFSE dilution.  Compared to DC FLT3L cultures maintained in MT-1-CM, MT-1-CM removal increased FLT3L DC mediated proliferation of OTI and OTII cells after OVA stimulation (Fig. 4.21).  Removal of   116 MT-1-CM in FLT3L DC cultures also resulted in decreased Arg1 expression, which correlated with an increase in the repressive H3K27me3 mark in the Arg1 promoter (Fig. 4.22A,B).  Furthermore, removal of MT-1-CM resulted in increased MHC II MFI post-LPS stimulation (10 and 100 ng/ml) compared to FLT3L DCs maintained in MT-1-CM (Fig. 4.22C).  On the other hand, PDL1 MFI was significantly higher both at baseline and post-LPS stimulation in FLT3L DCs maintained MT-1-CM (Fig. 4.22C).  Surprisingly, we did not detect any changes in the MFI of CD86 or CD40 after the removal of MT-1-CM from the FLT3L DC cultures (Fig. 4.22C).  Our data demonstrates that MT-1-CM leads to an expansion of DC progenitors and impairs DC differentiation.  Furthermore, MT-1-CM induced immunosuppressive genes and markers in DCs, and impaired their ability to activate lymphocytes.  MT-1-CM derived G-CSF partially mediated the effects of MT-1-CM in the in vitro DC cultures, suggesting that other components of MT-1-CM may also play an important role in the dysregulation of DC differentiation and function.  Our studies of histone methylation in DCs treated with MT-1-CM revealed an association between epigenetic regulation and DC immunosuppressive gene expression mediated by mammary tumour secreted factors.  Importantly, the effects of MT-1-CM on DCs were reversible, with the removal of MT-1-CM leading to the differentiation of cDCs and pDCs capable of activating OTI and OTII cells.     117  Figure 4.20    Mammary tumour-conditioned media treatment of FLT3L DC cultures produce functional DC progenitors that differentiate into pDCs and cDCs.                                                          (A-D) FLT3L DC cultures were treated with MT-1-CM for 6 days and then transferred to either control media (MT-CM to 0%) or maintained in MT-1-CM (MT-CM media) for 6 days (day 12). Cells were analyzed by flow cytometry and qPCR. (A) Frequencies of pDCs and cDCs.  cDCs were further differentiated into CD8+-like, and CD8--like cDCs. (B) Absolute numbers of pDCs and CD8+-like cDCs from part (A).  (C) Expression of transcription factors Irf4, Irf8, E2-2, and Zbtb46. (D) Absolute numbers of MDPs, CDPs, and preDCs.  Data represent three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.      118  Figure 4.21    Removal of mammary tumour-conditioned media restores ability of FLT3L DCs to activate OTI and OTII cell proliferation. CFSE dilution in co-cultures of OTI/OTII cells with FLT3L DC cultures in which MT-1-CM was either removed or maintained.  FLT3L DCs were incubated with OVA protein then co-cultured with CFSE labeled OTI or OTII cells. Cells were gated on Thy1.1+CD8+ (OTI) or Thy1.1+CD4+ (OTII) populations.  Data represents two independent experiments.     Figure 4.22    Removal of mammary tumour-conditioned media reduces expression of immunosuppressive proteins/enzymes and increases MHC II expression.  (A-C) FLT3L DC cultures were treated with MT-1-CM for 6 days then transferred to either control media (MT-CM to 0%) or maintained in MT-1-CM (MT-CM media) for 6 days (day 12). Cells were analyzed for (A) Arg1 expression by qPCR and (B) repressive H3K27me3 histone modifications were investigated by ChIP of chromatin. Chromatin was immunoprecipitated using anti-H3K27me3 or IgG control Abs and amplicons were analyzed by qPCR (C) Lastly, MFI of MHC II, PDL1, CD40 and CD86 was measured in FLT3L DC cultures where MT-1-CM was removed or maintained.  Data are representative of a minimum of two independent experiments. *p < 0.05, **p < 0.01.     119 4.1.8 Discussion Tumours can impair DC function leading to the emergence of DCs that promote tolerogenic and/or Th2 immune responses, which induce immunosuppression and aid in tumour growth instead of tumour rejection.  However, the effects of tumour growth on DC development are less well understood. We found that in the presence of mammary tumours, there was an expansion of cells with an immature phenotype, including lower expression of co-stimulatory and MHC II molecules, and decreased secretion of Th1 cytokines.  Instead, these DCs exhibited a higher expression of immunosuppressive enzymes and cytokines, were poor activators of NK and T cells, and inhibited endogenous DC activation of T cell proliferation.  It is critical to understand the mechanism of how tumours dysregulate DC development and differentiation, giving rise instead to immunosuppressive DCs and MDSCs, in order to inhibit or reverse these changes (Gabriolovich 2012).  We demonstrated that mammary tumour growth was associated with impaired DC development.  In in vitro FLT3L DC cultures, the addition of MT-CM resulted in decreased expression of transcription factors, Irf4, Irf8, E2-2 and Zbtb46, important in DC development.  Furthermore, the addition of MT-CM to FLT3L DC cultures resulted in the accumulation of DC progenitors including MDPs and CDPs, but not preDCs, suggesting a possible block in the transition from CDPs to preDCs. This was further supported by the lack of pDCs in the MT-CM treated DC cultures which also arise from CDPs.  In vivo, DC development was also impaired in the BM of mammary tumour-bearing mice, with a decrease in DC progenitors in the BM and a corresponding increase in the spleen.  However, this expansion of DC progenitors in the spleens of mammary tumour-bearing mice did not correlate with significant increases in mature DC subsets in the spleens. Instead there were reductions in splenic cDCs and pDCs, where CD4+   120 cDCs and pDCs were significantly reduced.  A study by Diao et al. (2010) showed that B16 melanoma, Lewis lung carcinoma and CT26 colon carcinoma growth had no impact on preDCs or their differentiation into cDCs, nor did the tumours impair the ability of cDCs in the tumour environment to activate T cells, when compared to cDCs in the spleens of tumour-bearing mice (Diao et al., 2010).  However, this lack of detectable effects on DC development and function could have been due to small tumour size and to the comparison of splenic and tumour preDCs and cDCs in tumour bearing mice instead of comparison between splenic DCs in non-tumour-bearing mice.  Furthermore, previously published studies have already established that tumours impair the ability of DCs to further activate innate and adaptive immunity (Gabrilovich, 2004; Preynat-Seauve et al., 2006; Vicari et al., 2002).  Importantly, in our studies we showed that mammary tumours, but not B16 melanoma growth, resulted in dysregulation of DC differentiation, demonstrating that not all tumours impair DC differentiation.     Next we sought to determine the MT-derived factors responsible for the impairment in DC differentiation and development.  Similar to its ability to impair hematopoiesis, G-CSF partially inhibited DC development and expression of MHC II and co-stimulatory molecules post-LPS stimulation.  Addition of exogenous G-CSF to FLT3L DC cultures impaired pDC development similar to the addition of MT-CM, however, whereas MT-CM impaired the development of CD8+-like cDCs, G-CSF suppressed CD8--like cDCs.  This suggests that additional factors in MT-CM are responsible for suppressing DC development in in vitro BM cultures.  G-CSF also lead to the accumulation of MDPs and CDPs, in addition to preDCs, which may be suppressed by another factor in MT-CM. Neutralization of G-CSF in MT-CM treated FLT3L DC cultures restored MDPs, CDPs and pDC development, however only partially restored MHC II expression and did not restore preDC levels.  This suggests that another factor(s) in MT-CM   121 suppresses the ability of G-CSF to increase the numbers of preDCs or directly blocks the transition of CDPs to preDCs, therefore neutralizing G-CSF in MT-CM did not have an effect on the preDC population.  Furthermore, addition of G-CSF to in vitro DC cultures partially impaired T cell activation.  However, it has already been established that G-CSF treatment of humans and mice results in DC activation of Th2 immune responses, which preferentially secrete IL-4 and IL-10 over IFN-γ (Arpinati et al., 2000).  The mobilization of HSCs from the BM to the peripheral blood of donors treated with G-CSF preferentially invokes a Th2 immune response compared to BM aspirates of untreated patients (Klangsinsirikul and Russell, 2002).  Therefore, future studies will focus on if the neutralization of G-CSF in MT-CM DC cultures will restore Th1 responses.  Overall, MT-CM derived G-CSF is responsible for the accumulation of MDPs and CDPs and the impaired differentiation of cDCs and pDCs.  Future studies will focus on identifying the other potential factors in MT-CM that are important in suppressing DC development and differentiation. FLT3L and GM-CSF DCs treated with MT-CM and CD11c+ DCs sorted from the spleens of mammary tumour-bearing mice all over-expressed Arg1.  Arg1 is an enzyme that is known to inhibit T cell proliferation and activation and instead induce tolerance (Munder, 2009).  This increase in Arg1 expression in MT-CM treated FLT3L and GM-CSF DC cultures correlated with reduced trimethylation of H3K27 at the Arg1 promoter, enabling gene expression.  Both loss- and gain-of-function experiments demonstrated the importance of MT-CM derived G-CSF in mediating the increased expression of Arg1. Interestingly, expression of other immunosuppressive factors, Retnla (Fizz1), Chi3l3 (Ym1) and Vegfa, were increased in MT-CM treated GM-CSF DCs but not FLT3L DCs. However since both FLT3L and GM-CSF DCs treated with MT-CM were impaired in their ability to prime T cell proliferation and IFN-γ   122 production, these other immunosuppressive factors may be dispensable for the induction of T cell tolerance by MT-CM treated DCs.  Future studies will focus on inhibiting Arg1 expression and function in order to determine its significance in MT-CM treated DC cultures.   We confirmed that MT-CM treatment of FLT3L DC cultures resulted in the accumulation of DC progenitors. This block in differentiation was reversible, since the removal of MT-CM in FLT3L DC cultures resulted in the differentiation of pDCs and cDCs.  Following MT-CM removal, there were significant increases in the numbers of CD8+ cDCs and pDCs, with a corresponding decrease in DC progenitors.  This was associated with increased expression of transcription factors important in DC differentiation, Irf4, Irf8, E2-2 and Zbtb46.  Removal of MT-CM also resulted in the ability of FLT3L DCs to activate T cell proliferation, which may be linked to the decrease in Arg1 expression associated with increased trimethylation of H3K27 in its promoter and increase in cell surface MHC II expression. Overall, the effects of MT-CM in the in vitro DC cultures were not permanent, since the removal of MT-CM resulted in DC differentiation with the concomitant ability to prime T cell proliferation.  The effects of tumours on DC development and subsequently on function are largely unknown.  A recent study in animals demonstrated that the progression of ovarian cancer was dependent on phenotypic and functional changes in tumour infiltrating DCs (Scarlett et al., 2012).  Late stage tumour infiltrating DCs were characterized as having high arginase activity and PDL1 expression and low MHC II and CD40 expression compared to DCs from early stage tumours (Scarlett et al., 2012).  In our studies, we observed some of these changes in DCs in mammary tumour-bearing mice and preliminary data suggests that DCs may play an immunosuppressive role, since DC depletion in mammary tumour-bearing mice lead to slower tumour growth.  Interestingly, it was proposed that as ovarian cancer progressed, tumour-infiltrating DCs changed from   123 immunostimulatory to tumour promoting, since depletion of DCs during early stages of tumour development accelerated tumour growth whereas depletion of DCs during late stages had the opposite effect (Scarlett et al., 2012).  It was proposed that the tumour itself manipulated the function of DCs from immunostimulatory to immunosuppressive in order to aid tumour growth, supporting our hypothesis that tumours can manipulate the function of DCs to promote tumour growth potentially by modifying DC development.  Therefore, a greater understanding of how tumour progression impacts DC function will be important during immunotherapy, such that it may be necessary to deplete immunosuppressive DCs that are promoting tumour growth. Overall, we have found that mammary tumours resulted in the accumulation of DC progenitors, but impaired their differentiation leading to a reduction in the number of mature DCs.  Mammary tumours inhibited DC function, which may be linked to the increased expression of the immunosuppressive enzyme, Arg1.  We found that many of these changes in DC differentiation, phenotype, and function were mediated by tumour-derived G-CSF.  Interestingly, the effect of mammary tumour secreted factors were reversible, with the removal of these secreted factors restoring DC differentiation and function.  Future studies will again focus on the necessity of G-CSF for the effects on the DC compartment in vivo.         124 Chapter 5: Role of Lyn tyrosine kinase in DC development and function Hypothesis: A gain-of-function mutation in Lyn will alter the response of the MPS, which includes DCs, monocytes and Mφ to TLR agonists and their interactions with other cells of the immune system, such as NK cells.  Objectives: Aim A: To compare the response of Lyn+/+, Lyn-/- and Lynup/up mice to LPS challenge Aim B:  To determine the cell(s) responsible for the increased IFN-γ production in Lynup/up mice post LPS challenge Aim C: To determine the myeloid cell responsible for priming NK cell IFN-γ production in vitro and in vivo  Aim D: To characterize splenic and BM derived GM-CSF DCs from Lyn+/+, Lyn-/- and Lynup/up mice  Aim E: To understand the differences in NF-κB signaling pathways post-LPS challenge in GM-CSF DCs from Lyn+/+, Lyn-/- and Lynup/up mice  Aim F: To determine the importance of SHIP-1 and SHP-1 for the hypersensitivity of Lynup/up GM-CSF DCs to LPS and their superior ability to prime NK cell IFN-γ production       125 5.1 Lyn-Dependent Signaling Regulates the Innate Immune Response 5.1.1 Lynup/up mice exhibit enhanced endotoxin-induced inflammation and morbidity. In order to study the role of Lyn in sepsis, mice were injected with LPS to emulate the symptoms associated with sepsis, including tissue necrosis, multi-organ failure and death. Lyn+/+ and Lynup/up mice were injected i.p. with 1 µg/g of LPS.  Surprisingly, while the Lyn+/+ mice appeared largely unaffected at this does of LPS, the Lynup/up mice became moribund between 12-24 hours post-injection (Fig. 5.1A).  Since it is well established that in rodents, sepsis is associated with a drop in body temperature, core body temperature was measured in Lyn+/+ and Lynup/up mice injected i.p. with 50 µg of LPS.  Correlating with the morbidity data, 10 hours post-LPS treatment, temperature dropped 10-12OC in Lynup/up mice whereas it remained constant in Lyn+/+ mice (Fig. 5.1B).  As expected, mice carrying one Lynup allele (Lyn+/up) were also hyper-responsive to LPS compared to Lyn+/+ mice but required a higher dose of LPS for a 10OC temperature drop compared to Lynup/up mice (Fig. 5.1C).  Furthermore, whereas only 50 µg of LPS was required for a 10OC drop in body temperature in Lynup/up mice, 4000 µg of LPS was required for a similar drop in body temperature in Lyn+/+ and Lyn-/- mice (Fig. 5.1D).         126  Figure 5.1        Lyn activity regulates endotoxin sensitivity in mice.  (A) Lyn+/+ and Lynup/up mice were injected i.p. with 1µg/g of LPS and were monitored carefully for 24 h and were sacrificed when moribund. (B) Lyn+/+ and Lynup/up mice were injected i.p. with 50 µg LPS and core body temperature was measured over 10 h. (C) Lyn+/+ and Lyn+/up mice were injected with PBS (0) or 100 µg LPS, and core body temperature was measured after 10 h. (D) Lyn+/+ and Lyn-/- mice were treated as in C. The LPS dose was increased incrementally until a 10oC temperature drop in 10 h was achieved.  This required 4000 µg LPS.  For A-D n > 5 mice/group.  All experiments were performed a minimum of 3 times.  Error bars represent SD  (C, D). *p < 0.05, **p < 0.01, ***p < 0.001.            127 5.1.2 Lyn promotes production of pro-inflammatory cytokines in response to endotoxin.  Since the production of pro-inflammatory cytokines is associated with endotoxin-induced mortality, we next investigated the role of Lyn in mediating endotoxin-induced cytokine production.  Cytokine levels were measured in Lyn+/+ and Lynup/up mice injected i.p. with 50 µg of LPS at 1.5, 3, and 6 hours post-LPS stimulation.  Serum collected from Lynup/up mice contained elevated levels of pro-inflammatory cytokines, IL-12, IFN-γ, IL-1α, IL-1β, TNF-α, IFN-α and IL-6 and lower levels of the anti-inflammatory cytokine IL-10 compared to Lyn+/+ mice (Fig. 5.2A).   A cytokine array performed on the serum harvested from Lyn+/+ and Lynup/up mice confirmed the increased production of pro-inflammatory cytokines and chemokines in Lynup/up mice post-LPS treatment (Fig. 5.3).  Correlating with the increased hypersensitivity of Lynup/up mice to LPS, protein expression of the proinflammatory mediator, iNOS was also greater in the kidney, spleen, lung and liver of Lynup/up mice compared to Lyn+/+ mice 6 hours post-LPS stimulation (Fig. 5.2B).  Furthermore, after LPS stimulation, myeloid cells from the BM of Lynup/up mice had increased expression of MHC I (Fig. 5.2C).            128        Figure 5.2        Lynup/up mice exhibit enhanced inflammatory cytokine production in response to LPS.  (A) Lyn+/+ and Lynup/up mice (n=5 mice/group) were injected i.p. with PBS (0) or 50 µg LPS.  Serum cytokine concentrations were then determined by ELISA after 0, 1.5, 3 and 6 h. Error bars = SD. (B) At 0 and 6 h post-injection (+LPS), kidney, spleen, lung and liver were removed and lysates were subjected to Western blot analysis for iNOS and Erk1/2 (loading control).  (C) At 6 h post-injection BM was analyzed by flow cytometry to assess upregulation of MHC I and Mac1.  The MFI (X and Y coordinates) for the Mac1+ MHC I+ populations are shown. All experiments were performed a minimum of 3 times. *p < 0.05, **p < 0.01.         129  Figure 5.3        Elevated levels of chemokines and proinflammatory cytokines in the serum of Lynup/up mice following LPS injection.                                                                                                                       (A) Lyn+/+ or Lynup/up mice were injected with LPS (2 µg/g mouse) or PBS (-LPS).  After 6 h, serum was obtained and incubated with blocked cytokine array membranes (RayBio).  Membranes were developed according to the manufacturers instructions.  Groups of 4 spots (upper left) and 2 spots (lower right) were used to orient the filter.  Soluble TNF receptor 1 (sTNFR1), keratinocyte derived chemokine (KC), monocyte chemotactic protein (MCP), macrophage inflammatory protein 2 (MIP2).  (B) Key to the cytokine array.      130 5.1.3 NK cells are the primary source of IFN-γ  in LPS-challenged Lynup/up mice. IFN-γ has been shown to play an important role in innate immunity in response to LPS and the neutralization of IFN-γ can prevent LPS-induced septicemia (Heinzel, 1990; Heremans et al., 1990; Jurkovich et al., 1991; Ozmen et al., 1994).  Therefore, we investigated the importance of Lyn in mediating the IFN-γ response by NK, CD4+ T, and CD8+ T cells.  Firstly, we confirmed that the increase in production of IFN-γ in Lynup/up mice was not due to more NK and T cells and found that the absolute numbers of NK, CD4+ T, and CD8+ T cells were similar in Lyn+/+ and Lynup/up mice (data not shown).  This ruled out the possibility that the higher levels of IFN-γ in Lynup/up mice were simply due to increased numbers of NK and T cells.  The increase in frequency of NK cells in the spleens of Lynup/up mice was due to the decrease in B cells (Fig. 5.4A) (Hibbs et al., 2002).  Interestingly, through intracellular flow cytometry, we found that NK cells in the spleens of Lynup/up mice, and not CD4+ and CD8+ T cells, were the major producers of IFN-γ post-LPS stimulation, with approximately 85% of NK cells producing IFN-γ in Lynup/up mice compared to only 20% in Lyn+/+ mice (Fig. 5.4A).  Moreover, the NK cells in Lynup/up mice produced more IFN-γ per cell compared to the NK cells in Lyn+/+ mice, as indicated by the higher MFI (Fig. 5.4A).  To further exclude a role for NKT, CD4+ and CD8+ T cells in the hypersensitivity of Lynup/up mice to LPS and IFN-γ production, Lynup/up mice on the RAG1-/- background treated with LPS also demonstrated increased morbidity and IFN-γ production by NK cells (Fig. 5.4B,C).    131  Figure 5.4        NK cells are the predominant source of IFN-γ  in LPS-stimulated mice.                                  (A) Lyn+/+ and Lynup/up mice were injected i.p. with sterile PBS (-LPS) or 50 µg LPS (+LPS).  6 h later spleens were harvested and stained with mAbs defining NK cells (NK1.1+ DX5+), CD4+ T cells (Thy1+ CD4+) or CD8+ T cells (Thy1+ CD8+).  Splenocytes were then fixed, permeabilized and stained with anti-IFN-γ mAbs.  Upper panel: DX5+ cells from Lyn+/+ and Lynup/up spleens were gated and analyzed for NK1.1 and IFN-γ expression.  Numbers indicate the percentage of IFN-γ-expressing NK cells or the MFI of this population, + SD.  Lower panel: Thy1+ cells from Lynup/up spleens were gated and analyzed for either CD4 or CD8, and IFN-γ. (B) RAG1-/- and RAG1-/- Lynup/up mice were injected i.p. with PBS (-LPS) or 25 µg LPS (+LPS).  After 6 h splenocytes were analyzed by flow cytometry to assess NK cell IFN-γ production as in A.  The percentage + SD and MFI for the NK1.1+ IFN-γ+ population is shown. (C) Mice were injected i.p. with 10 µg LPS, were carefully monitored for 24 h and were sacrificed when moribund.  For all experiments, n > 4 mice/group and experiments were performed a minimum of 3 times.      132 5.1.4 BM derived GM-CSF and FLT3L DCs, but not BM derived Mφs, activate NK cells in response to LPS.  Recently it has been shown that NK cells do not exist in an activated state, and instead must be primed by either DCs or Mφs in order to acquire their effector functions, including IFN-γ production (Fernandez et al., 1999; Lucas et al., 2007).  Therefore, we next sought to determine if various DC subsets or Mφs were responsible for the hyper-activation of IFN-γ production by NK cells in Lynup/up mice post-LPS treatment.  NK cells, purified from spleens of Lyn+/+ mice, were co-cultured with BM-derived GM-CSF DCs, FLT3L DCs or M-CSF Mφs for 16 hours before culture supernatants were harvested and analyzed for IFN-γ by ELISA.  Our results demonstrated that both GM-CSF and FLT3L DCs, but not M-CSF Mφs, could activate IFN-γ production by NK cells after LPS stimulation (Fig. 5.5A).  Importantly, we did not detect IFN-γ in unstimulated DC/NK cell co-cultures, and NK cell or DC only cultures stimulated with LPS (Fig. 5.5).  Interestingly, in FLT3L DC cultures, CD8--like cDCs were the most potent activators of IFN-γ production by NK cells whereas pDCs were ineffective (Fig. 5.5B).              133  Figure 5.5        DCs and not Mφs are necessary for IFN-γ  production by NK cells.                                     (A) GM-CSF and FLT3L DCs or M-CSF Mφs were co-cultured with NK cells (5.0x105 DCs/Mφs with 2.0x105 NK cells).  Cell mixtures were stimulated with 0, 1, 10 or 100 ng/ml LPS for 16 h.  As controls, DCs (-NK) and NK cells were cultured alone and stimulated with 100 ng/ml LPS.  Culture medium was collected and IFN-γ levels were determined by ELISA. P values indicate a comparison with BM Mφs. (B) FLT3L DCs were sorted into three populations (upper right): CD8--like  [SIRPa+CD24low (CD4-CD8-CD11bhigh) (box 1), CD8+-like [SIRPa-CD24high (CD4-CD8+CD11blow) (box 2) and pDCs (CD45RA+) (box 3).  Populations were co-cultured with NK cells and stimulated as in A. *p < 0.05, **p < 0.01, ***p < 0.001.                134 5.1.5 The DC lineage is required for enhanced NK cell IFN-γ  production in Lynup/up mice.  We established in our model that DCs, not Mφs, were important in inducing IFN-γ by NK cells, therefore we were interested in determining if DCs were also necessary for the increased production of IFN-γ by NK cells in Lynup/up mice.  To determine this, we generated Lynup/up mice on the CD11c-DTR/GFP background.  Lyn+/+- and Lynup/up-CD11c-DTR/GFP mice were injected with either PBS or DT and then IFN-γ production was measured by intracellular flow cytometry and in the serum by ELISA 6 hours after LPS treatment (Fig. 5.6A,B).  As expected, there were more IFN-γ+ NK cells in Lynup/up-CD11c-DTR/GFP mice post-LPS as measured by intracellular flow cytometry as compared to NK cells in Lyn+/+-CD11c-DTR/GFP treated with LPS (Fig. 5.6A).  Also, serum IFN-γ levels were greater in LPS treated Lynup/up-CD11c-DTR/GFP mice than in Lyn+/+-CD11c-DTR/GFP mice (Fig. 5.6B).  By contrast, depletion of DCs in both Lyn+/+- and Lynup/up-CD11c-DTR/GFP mice by DT treatment resulted in a decrease in IFN-γ+ NK cells and in serum IFN-γ levels post-LPS treatment (Fig. 5.6A,B).  DC depletion in the spleen was confirmed by flow cytometry (data not shown).  This demonstrates that DCs are necessary for the increased levels of IFN-γ in Lynup/up mice.               135 5.1.6 DCs and NK cells are required for LPS-induced morbidity in Lynup/up mice.  IFN-γ has been shown to mediate LPS-induced septicemia.  Our results have shown that Lynup/up mice are hypersensitive to LPS and produce excessive amounts of IFN-γ in response to LPS, a response that requires the priming of NK cells by DCs.  Therefore, we next investigated if DCs and NK cells mediated LPS-induced morbidity in Lynup/up mice.  Depletion of DCs in Lynup/up-CD11c-DTR/GFP mice with DT treatment reduced LPS-induced morbidity (Fig. 5.6C).  Furthermore, depletion of NK cells in Lynup/up mice using a NK1.1 antibody completely rescued Lynup/up mice from LPS induced morbidity (Fig. 5.6D).  These results strongly demonstrate the significance of the interaction between DCs and NK cells in the hypersensitivity of Lynup/up mice to LPS.      136  Figure 5.6        IFN-γ  production and hypersensitivity to LPS in Lynup/up mice depends on DCs and NK cells.  (A) Lyn+/+ and Lynup/up mice on the CD11c-DTR/GFP genetic background were injected i.p. with PBS (-DT) or diphtheria toxin (+DT, 5 ng/g mouse).  48 h later, mice were injected i.p. with 25 µg LPS. After 6 h, splenocytes were analyzed by flow cytometry to assess NK cell IFN-γ production.  The percentage and MFI of the NK1.1+ IFN-γ+ population + SD is shown. (B) Mice were treated as in A, except that cardiac punctures were performed to collect blood and serum and serum IFN-γ levels were determined by ELISA. (C) Lyn+/+ and Lynup/up mice on the CD11c-DTR/GFP genetic background were injected i.p. with PBS or DT (5ng/g mouse). 24 h later, mice were injected with 10 µg LPS, were monitored carefully for 100 h and were sacrificed when moribund. (D) Lyn+/+ and Lynup/up mice were injected twice over 48 h with an anti-NK1.1 Ab or an isotype control.  24 h after the second injection, mice were injected i.p. with 10 µg LPS and were monitored as in E.  Error bars = SD, and all experiments were performed a minimum of two times.  For C-F, n > 4 mice/group.  *p < 0.05, **p < 0.01, ***p < 0.001.        137 5.1.7 Lyn activity within DCs enhances endotoxin-induced IFN-γ  production in NK cells.   To better understand the role of DCs in priming increased IFN-γ production by NK cells in Lynup/up mice post-LPS stimulation, we co-cultured Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs with WT NK cells and measured NK cell activation by IFN-γ production after LPS stimulation (Fig. 5.7A).  Correlating with the in vivo results, Lynup/up DCs induced the greatest levels of IFN-γ production by NK cells, whereas Lyn-/- DCs induced the least amount of IFN-γ, post-LPS stimulation (Fig. 5.7A).  Similar results were obtained when Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs were stimulated with E. coli or L. monocytogenes (Fig. 5.7B).   In order to rule out the possibility that the differences in IFN-γ in Lyn+/+ and Lynup/up mice were due to differences in NK cells and not DCs, Lyn+/+, Lyn-/- and Lynup/up NK cells were co-cultured with WT GM-CSF DCs.  Interestingly, after LPS stimulation, Lynup/up NK cells produced less IFN-γ than Lyn+/+ NK cells and Lyn-/- NK cells produced the greatest amount of IFN-γ (Fig. 5.7C).  Also, Lyn+/+, Lyn-/- and Lynup/up NK cell surface marker expression profile was similar and when stimulated with IL-15, IL-12, IL-18 and anti-NK1.1 antibody produced similar amounts of IFN-γ (Tables 5.1, 5.2).   These results confirm that DCs and NK cells are responsible for the hypersensitivity of Lynup/up mice to LPS.       138  Figure 5.7        Lyn polarizes DCs with respect to NK cells to secrete IFN-γ .                                                                                                                              (A) 5x105 Lyn+/+, Lyn-/- or Lynup/up GM-CSF DCs were co-cultured with 2x105 WT NK cells.  Co-cultures were stimulated with 0, 1, 10 or 100 ng/ml LPS.  Alternatively, DCs or NK cells were cultured alone and stimulated with 100 ng/ml LPS.  After 16 h culture medium was collected and IFN-γ levels were determined by ELISA. (B) 5x105 Lyn+/+, Lyn-/- or Lynup/up GM-CSF DCs were co-cultured with 2x105 WT NK cells, and stimulated with the indicated number of bacteria.  After 6 h, culture medium was removed and analyzed for IFN-γ levels by ELISA (C) 2x105 Lyn+/+, Lyn-/- or Lynup/up NK cells were co-cultured with 5x105 WT GM-BMDCs.  Co-cultures were stimulated as in A, and IFN-γ levels were determined by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001.               139 Table 5.1        Expression of NK cell markers on NK1.1+ CD49b+ cells from Lyn+/+ and Lynup/up spleen and liver.  Spleen Liver  Lyn+/+ (%) Lynup/up (%) Lyn+/+ (%) Lynup/up (%) Surface antigen       Ly49A 22.6+1.4 23.0+4.0 * *  Ly49C+I 58.0+1 52.0+3.8 53 53  Ly49D 56.1+2.5 41.0+3.1 53 49  Ly49G2 48.7+2.4 51.3+3.7 * *  Ly49F 13.1+0.2 15.7+2.7 * *  FcRγII 82.4+0.1 79.8+3.9 * *  CD94 51.0+1.9 50.3+1.4 * *  NKG2A/C/E 53.9+2.5 49.3+1.9 * *  NKG2D 97.3+1.2 97.3+1.8 90 91  KLRG1 45.0+2.1 51.0+2.4 60 72  CD27 49.3+3.8 46.3+6.2 47 40  2B4 98.3+0.9 98.0+0.8 * *  CD11b 96.1+0.6 91.0+2.0 96 97  CD43 98.3+0.5 98.7+0.9 * *  * not detected       140 Table 5.2        IFN-γ  production from in vitro IL-15-expanded NK cells.  Stimuli Lyn+/+ Lynup/up IL-15 (pg) <100 <100 Anti-NK1.1 (ng) 7.0+2.4 6.2+1.0 IL-12 plus IL-18 (ng) 223+36 206+30                   141 5.1.8 Lyn activity within DCs modulates LPS-induced cytokine production.   DCs modulate NK cell activity though the secretion of cytokines.  DCs enhance NK cell activity through the secretion of pro-inflammatory cytokines such as IL-12, IL-18, type-I IFNs and IL-15, however, DCs can also down-regulate NK cell activity through the secretion of inhibitory cytokines such as IL-10 and TGF-β.  Therefore, we next wanted to determine if Lyn modulated DC cytokine secretion, specifically IL-12 and IL10 post-LPS stimulation.  In response to increasing concentrations of LPS, Lynup/up GM-CSF DCs produced more IL-12 and less IL-10, whereas Lyn-/- GM-CSF DCs produced more IL-10 and less IL-12, compared to Lyn+/+ GM-CSF DCs (Fig. 5.8A). Similar results were obtained when Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs were stimulated with E. coli or L. monocytogenes instead of LPS (Fig. 5.8B).     We investigated the effect of Lyn activity on the expression of additional cytokines at various time-points post-LPS stimulation by qPCR in Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs.  Similar to the secretion of IL-12, expression of IL-12p35, IL-12p40, IL-15 and IFNβ mRNAs was greater in Lynup/up GM-CSF DCs post-LPS, but lower in Lyn-/- GM-CSF DCs, as compared to Lyn+/+ GM-CSF DCs (Fig. 5.8C).   As described above NK cells require priming by DCs for their effector function, however, NK cells can also play a role in DC activation.  Therefore, we investigated the impact of Lyn activity on the cross-talk between NK cells and DCs.  GM-CSF DCs were co-cultured with increasing number of Lyn+/+ NK cells and stimulated with LPS.  Similar to the earlier results, Lynup/up GM-CSF DCs produced more IL-12 and less IL-10 whereas Lyn-/- GM-CSF DCs produced more IL-10 and less IL-12 compared to Lyn+/+ GM-CSF DCs (Fig. 5.9).     142  Figure 5.8        Lyn polarizes DCs with respect to LPS-induced cytokine production.                               (A) 5.0x105 Lyn+/+, Lyn-/- or Lynup/up GM-CSF DCs were stimulated with 0, 1 or 100 ng/ml of LPS.  After 16 h, IL-12 and IL-10 levels in the culture medium were measured by ELISA. (B) 5.0x105 Lyn+/+, Lyn-/- or Lynup/up GM-CSF DCs were co-cultured with 2x105 wt NK cells, and stimulated with the indicated number of bacteria.  After 6 h, culture medium was removed and subjected to ELISA analysis to determine levels of IL-12 and IL-10.  (C) Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs were stimulated with 100 ng/ml LPS for the times shown.  At each time point RNA was harvested and subjected to qPCR to assess synthesis of cytokine mRNAs (normalized to GAPDH mRNA).  P values indicate a comparison with WT DCs.  For all experiments, error bars = SD and experiments were performed a minimum of 2 times. *p < 0.05, **p < 0.01, ***p < 0.001.   143  Figure 5.9        Lyn polarizes DCs with respect to LPS- and NK cell-induced cytokine production.  2.5x105 Lyn+/+, Lyn-/- or Lynup/up GM-CSF DCs were co-cultured with 0, 1x104 or 5x104 wt NK cells and were stimulated + 100 ng/ml LPS for 16 h.  IL-10 and IL-12 concentrations in the culture medium were determined by ELISA.  P values indicate a comparison with Lyn+/+ DCs.  For all experiments, error bars = SD and experiments were performed a minimum of 3 times. *p < 0.05, **p < 0.01, ***p < 0.001.             144 5.1.9 Lyn activity stimulates DC maturation.  Based on our result that Lynup/up DCs produced more pro-inflammatory cytokines and were better at priming NK cells after stimulation than Lyn+/+ DCs, we further investigated the influence of Lyn on the expression of DC activation markers.  Therefore, we stimulated Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs with LPS (a TLR4 ligand), CpG-ODN (a TLR9 ligand) or peptidoglycan (a TLR2/6 ligand) and measured expression of MHC II by flow cytometry for DC activation.  Correlating with previous results, Lynup/up GM-CSF DCs, both at baseline and after stimulation, had a higher frequency of DCs that were MHC IIhigh and lower frequency of MHC IIlow DCs compared to Lyn+/+ DCs (Fig. 5.10A).  Contrary to the Lynup/up DCs, Lyn-/- GM-CSF DCs had a higher frequency of DCs within the MHC IIlow population and lower frequency of DCs within the MHC IIhigh population (Fig. 5.10A).  Surprisingly, FLT3L DCs did not show a similar trend in MHC II expression between Lyn+/+, Lyn-/- and Lynup/up DCs (Fig. 5.10A).   Importantly, in vivo the absolute number of DCs and the frequencies of CD11c+CCR9+ pDCs and CD11c+CD8+ cDCs were similar between the spleens of Lyn+/+, Lyn-/- and Lynup/up mice (Fig. 5.10B).  Interestingly, though the splenic frequency of CD11c+CD4+ cDCs was similar between Lyn+/+ and Lynup/up mice, CD11c+CD4+ cDC splenic frequency was lower in Lyn-/- mice (Fig. 5.10B).  Expression of CD86 and PDL1 was similar, whereas, as already published, CD80 expression was lower on CD11c+ cells in Lyn-/- spleens compared to Lyn+/+ and Lynup/up spleens (Fig. 5.10C) (Tsantikos et al., 2010).  Similar to the in vitro cultured GM-CSF DCs, both at baseline and after LPS stimulation, splenic Lynup/up DCs expressed more MHC II (indicated by MFI), whereas Lyn-/- DCs expressed less, compared to Lyn+/+ DCs (Fig. 5.10C).  These results indicate that both in vitro and in vivo, Lyn enhances spontaneous and inducible DC maturation.       145  Figure 5.10        Lyn controls spontaneous and TLR-dependent DC maturation.  (A) DCs were derived from BM in either GM-CSF or FLT3L.  Cells were left untreated or treated with LPS, CpG-ODN or peptidoglycan (PGN).  16 hours later DCs were stained for CD11c and MHC II. MHC II expression on CD11c+ DCs fell into three regions: low, intermediate and high.  Percentages of cells in each region are shown.  For FLT3L DCs the MFI for MHC II is shown. (B) Splenocytes from Lyn+/+, Lyn-/- and Lynup/up mice were analyzed by flow cytometry to assess the frequency of CD11c+CCR9+ pDCs and CD11c+CD4+ or CD11c+CD8+ cDCs. Percentages of cells in each region + SD are shown. (C) (Upper panel) The expression of CD86, CD80 and PDL1 on CD11c+ splenic DCs was assessed as in B. (Lower panel) Lyn+/+, Lyn-/- and Lynup/up mice were injected with 25 µg LPS or sterile PBS (Control).  After 1.5, 3 and 6 h, spleens were analyzed by flow cytometry for MHC II expression on CD11c+ cells.   The MFI for MHC II is shown + SD.  For all time points, n>4 mice/group.   ** p < 0.01 and indicates a comparison with Lyn+/+ cells.  All experiments were performed a minimum of 3 times.              146 5.1.10 Lyn negatively regulates LPS-induced signal transduction in DCs. We next investigated whether the hypersensitivity of Lynup/up mice was mediated through changes in TLR4 cell signaling.  Ligation of TL4 activates three different signaling pathways regulated by the MAPKs, PI3K and NF-kB.  Therefore we stimulated Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs with LPS and then the lysates were analyzed by Western blotting.  Surprisingly, in the MAPK pathway, activation of p38, Erk1/2 and Jnk were increased in Lyn-/- DCs and decreased in Lynup/up DCs as compared to Lyn+/+ DCs (Fig. 5.11).  A similar trend was found in the PI3K pathway, where phosphorylation of Akt, Bad, S6 and Gsk3a/b were all elevated again in Lyn-/- DCs and decreased in Lynup/up DCs (Fig. 5.11).  Similarly, activation of the NF-κB signaling pathway was increased in Lyn-/- DCs where phosphorylation of ikBα, p65/RelA and Tbk1 were increased in Lyn-/- DCs and decreased in Lynup/up DCs compared to Lyn+/+ DCs (Fig. 5.11).                  147  Figure 5.11        Lyn regulates LPS-induced signaling pathways in GM-CSF DCs.                                     Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs were stimulated + 100 ng/ml LPS for the times shown (min).  Lysates were prepared and Western blotting was carried out to assess activation of signaling pathways regulated by the MAPKs, PI3K and NF-κB.  Relative band intensities are shown below each band (determined using ImageJ software). All experiments were performed a minimum of 3 times.               148 5.1.11 Lyn interacts functionally with SHIP-1 and SHP-1 in DCs.  To further understand how Lyn regulates signaling pathways, we blotted for anti-phosphotyrosine on lysates from Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs post-LPS stimulation and found that there were more hyper-phosphorylated proteins in Lynup/up DCs of molecular weight of ~145,000 and 70,000, but were hypo-phosphorylated in Lyn-/- DCs (Fig. 5.12A).  Further analysis revealed that these phosphoproteins included SHP-1 (68 kDa) and SHIP-1 (145 kDa) (Fig. 5.12B).  Previously it has been shown that Lyn mediates inhibitory signaling by tyrosine phosphorylating ITIMs, thereby creating docking sites for SHP-1 and SHIP-1.  Furthermore, it has been shown that SHIP-1 is required for TLR-induced DC maturation and IL-12 secretion (Fukao et al., 2002). Therefore, to test whether SHIP-1 and SHP-1 were necessary for the hypersensitivity of Lynup/up DCs to LPS, we used shRNAs to reduce SHP-1 or SHIP-1 expression in Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs and then assessed MHC II expression post-LPS stimulation (Fig. 5.13A,B).  Interestingly, SHP-1 knockdown only impaired both baseline and LPS-induced maturation of Lynup/up GM-CSF DCs, but had no affect on Lyn+/+ and Lyn-/- DCs (Fig. 5.13B and Table 5.3).  However, SHP-1 knock down did not impact LPS-mediated signal transduction in Lynup/up GM-CSF DCs (Fig. 5.13C).  Surprisingly, GM-CSF DCs cultured from Mev/Mev, which lack SHP-1 activity, had impaired spontaneous and LPS-induced MHC II expression (Fig. 5.13C, 5.14).  On the other hand, SHIP-1 knockdown did impair baseline and LPS-induced maturation of Lyn+/+, Lyn-/- and Lynup/up DCs, although Lynup/up DCs were most significantly affected with the enhancement of the PI3K pathway (Fig. 5.13B,C Table 5.3).  Furthermore, SHIP-1 knockdown also impaired the ability of Lynup/up DCs to prime IFN-γ by NK cells (Fig. 5.13D).  Similar to the results obtained with Mev/Mev GM-CSF DCs, DCs derived from SHIP-1-/- BM also had impaired baseline and LPS-induced MHC II expression (Fig.   149 5.14).  This data demonstrates a role for Lyn in regulating SHIP-1, and to a lesser degree SHP-1, and the importance of Lyn in mediating DC activation and priming of NK cell activation.                             150  Figure 5.12        Lyn regulates SHIP-1 and SHP-1 phosphorylation in GM-CSF DCs.  (A) Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs were stimulated with 100 ng/ml LPS for 0 or 15 min and lysates were blotted using anti-phosphotyrosine (P-Tyr) Abs. Arrows indicate phosphoproteins that were hyper-phosphorylated in Lynup/up DCs, and hypo-phosphorylated in Lyn-/- DCs. (B) Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs, stimulated with 100 ng/ml LPS, were immunoprecipitated for SHP-1 and SHIP-1, followed by anti-P-Tyr Western blotting.  Blots were re-probed with anti-SHIP-1 and anti-SHP-1 Abs to confirm equal loading.      151  Figure 5.13        SHIP-1 and SHP-1 regulate GM-CSF DC maturation and function.  (A) Lyn+/+ GM-CSF DCs were infected with retrovirus producing shRNAs targeting SHP-1, SHIP-1 or control shRNA (Ctr).  Two shRNAs per target are shown.  Knockdown was confirmed by Western blotting with anti-SHIP-1 and anti-SHP-1 Abs.  Blots were re-probed with anti-SHP-2 and Erk1/2 Abs to confirm equal loading. (B) Lynup/up GM-CSF DCs were infected with retrovirus as in A.  DCs were stimulated + LPS for 16 h and expression of MHC II on CD11c+ DCs was assessed by flow cytometry. MHC II expression fell into three regions: low (box 3), intermediate (box2) and high (box 1).  Percentages of cells in each region are shown. Lyn+/+ and Lyn-/- DCs were analyzed in the same way (see Table 5.3 for Lyn+/+ and Lyn-/- data). (C) Lynup/up GM-CSF DCs were infected with retrovirus as in A.  DCs were stimulated with 100ng/ml LPS for 0 or 15 min, and lysates were subjected to Western blotting with Abs recognizing p-Ikka/b, p-Akt, p-Erk1/2 and p-S6.  The blot was reprobed with anti-total Akt (T-Akt) Abs (loading control). (D) Lynup/up GM-CSF DCs were infected with control or SHIP-1 shRNA retrovirus.  Infected DCs were then sorted based on eGFP expression, and co-cultured with NK cells (1.0x105 DCs with 4.0x104 NK cells).  Cell mixtures were stimulated with 0, 1, 10 or 100 ng/ml LPS for 16 h. Culture medium was collected and IFN-γ levels were determined by ELISA.  Error bars represent SEM. *p < 0.05, ***p < 0.001.       152   Figure 5.14        SHIP-1 and SHP-1 regulate GM-CSF DCs activation in response to LPS.  GM-CSF DCs derived from Lyn+/+, Lyn-/-, Mev/Mev (lacking SHP-1 activity) or SHIP-1-/- mice were stimulated with + 100 ng/ml LPS for 16 h, and analyzed for the expression of MHC II on CD11c+ DCs by flow cytometry.  The percent of cells in each region is shown.                 153 Table 5.3        Percent of MHC IIhigh, MHC IIint and MHC IIlow DCs following knock down of SHIP-1 or SHP-1 in Lyn+/+ and Lyn-/- GM-CSF DCs.    Control shRNA SHIP-1 shRNA SHP-1 shRNA  -LPS (%) +LPS (%) -LPS (%) +LPS (%) -LPS (%) +LPS (%)   Region*        Lyn+/+ 1 15.3 41.6 15.2 24.4 16.3 39.6  2 40.9 32.2 31.6 35.4 41.7 32.2  3 36.5 17.3 47.9 34.5 34.2 19.9  Lyn-/- 1 10.9 22.5 7.3 12.6 7.0 20.5  2 38.4 38.7 30.0 41.0 35.5 38.4  3 44.5 32.2 56.9 41.1 47.4 31.9  * Region 1 = MHC II high, Region 2 = MHC II intermediate and region 3 = MHC II low                  154 5.1.12 Lyn plays a protective role in mammary tumour development.  Next, we wanted to determine if the enhanced maturation of Lynup/up DCs and ability to activate NK cell IFN-γ production in vivo and in vitro would play a protective role in mammary tumour-bearing mice.  First, we investigated if DCs were immunostimulatory or immunosuppressive in MT-1 bearing mice.  We reconstituted lethally irradiated CD45.1+ RAG1-/- mice with BM from CD45.2+ RAG1-/- CD11c-DTR/GFP mice in order to continuously deplete DCs through DT injections (Zammit et al., 2005).  8 weeks later, mice were injected with MT-1 cells and treated with DT or with PBS (negative control) (Fig. 5.15A-C).  Whether DCs were depleted continuously from day 0, when mice were injected sub-cutaneously with MT-1 cells, or once tumour became palpable, DC depletion was associated with slower tumour growth (Fig. 5.15A,B).  We confirmed DC depletion in the spleens of DT-treated mice versus control mice by flow cytometry for CD11c+MHC II+ and CD11c+GFP+ cells (Fig. 5.15C and data not shown).  In order to study the role of Lyn in tumour growth, MT-1 tumour growth was measured in RAG1-/- and RAG1-/- Lynup/up mice (Fig. 5.15D).  Interestingly, MT-1 tumours were significantly smaller in RAG1-/- Lynup/up mice versus RAG1-/- mice (Fig. 5.15D).  Surprisingly, the addition of MT-1-CM reduced MHC II expression at baseline and post-LPS stimulation in GM-CSF DCs derived from the BM of Lyn+/+/Lyn-/-/Lynup/up mice (Fig. 5.16A).  Correlating with previous data, Lynup/up GM-CSF DCs treated with MT-1-CM still had a greater frequency of CD11c+MHC II+ cells post-LPS stimulation compared to Lyn+/+ and Lyn-/- GM-CSF DCs treated with MT-1-CM (Fig. 5.16A).  However, Lynup/up GM-CSF DCs treated with MT-1-CM had the lowest Arg1 protein expression compared to Lyn+/+ and Lyn-/- GM-CSF derived DCs (Fig. 5.16B).  This data demonstrates that Lyn plays a protective role in tumour development and future studies will focus on determining the significance of DCs in MT-1 bearing RAG1-/- Lynup/up mice and the   155 significance of the reduced expression of Arg1 on the function of Lynup/up GM-CSF DCs treated with MT-1-CM.     Figure 5.15         Role of DCs and Lyn in mammary tumour development.  (A-C) Lethally irradiated RAG1-/- mice reconstituted with RAG1-/- CD11c-DTR/GFP bone marrow were injected with MT-1 tumours sub-cutaneously (day 0).  Mice were injected with 5 ng/g of DT on day 0 or when tumours became palpable or PBS (negative control).  Mice were injected with DT or PBS every three days for the duration of the experiment. (A,B) Tumours were measured every 1-3 day(s). (C) Frequencies of CD11c+GFP+ and CD11c+ MHC II+ cells in the spleen of MT-1 tumour-bearing mice treated with PBS (negative control) or DT.  Experiment was done once, n=4. (D) Mammary tumour measurements in RAG Lyn+/+and RAG Lynup/up mice. Data is representative of a minimum of three independent experiments, n=4.  *p < 0.05, **p < 0.01, ***p < 0.001.    156  Figure 5.16         Lyn expression does not reverse the effects of mammary tumour-conditioned media in in vitro GM-CSF DC cultures.  (A) Frequencies of CD11c+MHC II+ Lyn+/+/Lyn-/-/Lynup/up DCs in GM-CSF DC cultures treated with or without MT-1-CM. Cells were stimulated ± LPS. (B) Lyn+/+, Lyn-/- and Lynup/up GM-CSF DCs were treated with an increasing concentration of MT-CM.  Lysates were prepared and Western blotting was carried out to assess Arg expression. Lanes with – and + IL-4 represent negative and positive controls for Arg, respectively.   Data are representative of a minimum of two independent experiments.     157  5.1.13 Discussion In order to study the role of Lyn in innate immune cells, we utilized Lynup/up mice and found that Lynup/up mice were hypersensitive to LPS challenge, since at a dose of LPS well tolerated by both Lyn+/+ and Lyn-/- mice, Lynup/up mice became hypothermic and moribund.  In response to LPS, there were elevated levels of proinflammatory cytokines, IL-1α, IL-1β, IFN-γ, IL-12, TNFα, IFN-α and iNOS in Lynup/up mice compared to Lyn+/+ mice. The LPS hypersensitivity of Lynup/up mice was dependent on the increased activation of DCs and their superior ability to activate NK cells, since the depletion of either DCs or NK cells protected Lynup/up mice from LPS mediated septicemia.  IFN-γ is an important cytokine in mediating immunity against intracellular pathogens and tumours and has been shown to be important in endotoxin-induced lethality including-priming mice to the effects of LPS (Doherty et al., 1992; Heinzel, 1990; Heremans et al., 1990; Jurkovich et al., 1991; Ozmen et al., 1994; Schoenborn and Wilson, 2007; Schroder et al., 2004).  Therefore, we investigated if Lyn regulated IFN-γ production and if this phenotype was important for the LPS induced septicemia in Lynup/up mice.  NK cells have been shown to be the major producers of IFN-γ in response to LPS in Lyn+/+ mice, and NK cells in Lynup/up mice produced more IFN-γ after LPS treatment than NK cells in Lyn+/+ mice (Andoniou et al., 2005; Borg et al., 2004; Fernandez et al., 1999; Koka et al., 2004; Krug et al., 2004). Correspondingly, depletion of NK cells resulted in a decrease in IFN-γ production and rescued Lynup/up mice from LPS-induced mortality.  This demonstrates the importance of NK cell production of IFN-γ in the hypersensitivity of Lynup/up mice to LPS.      158 NK cell activation requires priming by myeloid cells and, corresponding to previous studies, we found that in response to LPS stimulation, in vitro and in vivo, DCs but not Mφs were the myeloid cells critical for priming IFN-γ production by NK cells (Fernandez et al., 1999; Lucas et al., 2007).  Depletion of CD11c+ cells, the majority of which are DCs, in LPS treated Lynup/up mice blocked IFN-γ production by NK cells and protected Lynup/up mice from LPS-induced mortality.  Therefore our results suggest that in LPS treated Lynup/up mice, DCs prime excessive IFN-γ production by NK cells and that both DCs and NK cells are important in the LPS hypersensitivity of Lynup/up mice.  These results demonstrate that Lyn activity can act as a rheostat for DC maturation, and therefore may be a good target for manipulating DC function.   In vitro Lyn-/- GM-CSF derived DCs stimulated with LPS expressed lower levels of co-stimulatory and MHC II molecules and were poor stimulators of NK cells compared to Lyn+/+ DCs.  Conversely, Lynup/up DCs showed superior activation and NK cell priming capabilities compared to Lyn+/+ DCs.  In response to LPS or bacterial stimulation, Lynup/up DCs produced more pro-inflammatory cytokines such as IL-12, IL-15 and IFN-β, whereas Lyn-/- DCs produced more IL-10.  Impaired ability of Lyn-/- DCs to produce IL-12 and their tendency to induce more of a Th2 immune response has already been published (Chu and Lowell, 2005).   Correlating with the in vitro data, splenic DCs in LPS treated Lynup/up mice expressed more MHC II, whereas Lyn-/- splenic DCs expressed less as compared to Lyn+/+ splenic DCs. Surprisingly, Lynup/up FLT3L DCs did not show enhanced maturation in response to LPS.  In future studies, we will sort the cDC and pDC populations before investigating their response to LPS stimulation, since differences in the cDC population in Lyn-/- mice have already been demonstrated, and this should provide us with a better understanding about the role of Lyn in particular DC subsets (Chu and   159 Lowell, 2005).  Importantly, Lyn did not impact DC development, with the frequencies of CD8+ cDCs and pDCs being similar in the spleens of Lynup/up, Lyn-/- and Lyn+/+ mice, although there was a small decrease in the number of CD4+ cDCs in the spleens of Lyn-/- mice.  However, Chu et al.  (2005) found that there was a reduced number of CD8+ cDCs in the spleens of Lyn-/- mice, with an overall increase in splenic DC numbers with age in Lyn-/- mice (Chu and Lowell, 2005).  However, as Lyn-/- mice age they develop autoimmune disease and hematopoietic system defects, characterized by an age-dependent increase in extramedullary myelopoiesis, myeloid neoplasia, and splenomegaly due to an increase in myeloid and erythroid progenitors (Harder et al., 2004; Hibbs et al., 1995; Nishizumi et al., 1995; Scapini et al., 2009).  Therefore more care needs to be taken into interpreting results using aged Lyn-/- mice and hence in our studies we used mice under 14 weeks of age.   In our studies, we found that Lyn did not impact LPS-induced signaling or cytokine production in BM derived Mφs, except that Lyn-/- Mφs produced more IL-10.  In contrast, Keck et al. (2010) showed that Lyn-/- BM-derived Mφs produced more IL-6 and TNF-α post-LPS stimulation and found increased IL-6, TNF-α and IFN-α/β in the serum of LPS treated Lyn-/- mice compared to Lyn+/+ mice.  Overall they demonstrated that Lyn negatively regulates MyD88- and TRIF-dependent cytokine secretion in BM derived Mφs and in vivo (Keck et al., 2010).  A possibility for the discrepancy between our results could be the method utilized to culture Mφs in vitro.  We used the adherent population of Mφs whereas in the above study Mφs were grown under non-adherent conditions in Teflon coated bags.  Lyn is important in positively and negatively regulating signal transduction, a role that is dependent on the type of stimulus, the cell’s developmental stage and the extracellular   160 environment (Hibbs and Harder, 2006; Lowell, 2004; Scapini et al., 2009).  In our studies, we found that Lyn promoted DC activation and maturation in response to LPS, including cytokine production, expression of MHC II and co-stimulatory molecules and the ability to prime NK cells.  Conversely, Lyn negatively regulated LPS-mediated activation of MAPKs, NK-κB and PI3K.  PI3K is a lipid kinase important in the generation of phosphatidylinositol-3,4,5 triphosphate (PIP3), which then recruits proteins containing pleskstrin homology (PH) domains, such as Akt, which when activated, increase cell survival and proliferation (Koyasu, 2003; Vanhaesebroeck et al., 2001).  Although the Lyn-mediated down-regulation of signaling seems contradictory to the promotion of DC activity by Lyn, previous studies have demonstrated that in certain subsets of cells, such as DCs, monocytes and Mφs, PI3K inhibits MyD88 and TRIF dependent TLR signaling. This results in decreased production of proinflammatory cytokines, including IL-12, TNF-α, IL-6 and IFNα/β (Fukao and Koyasu, 2003; Hazeki et al., 2007; Martin et al., 2005; Xu et al., 2005).  Inhibition of PI3K in DCs or the use of DCs derived from p85α-/-, (a regulatory subunit of PI3K), mice produced more IL-12 post TLR stimulation, thereby, demonstrating that PI3K is a negative regulator of IL-12 production by DCs (Fukao and Koyasu, 2003; Fukao et al., 2002; Hazeki et al., 2007).  The activity of PI3K is negatively regulated in hematopoietic cells by phosphatases including SHIP-1.  Lyn is an important regulator of SHIP-1, a lipid phosphatase, that antagonizes the function of PI3K, by hydrolyzing PIP3 back to PIP2, thereby inhibiting Akt activation (Krystal, 2000; Kuo et al., 2006; Rohrschneider et al., 2000; Sly et al., 2007).  Recently, it has been demonstrated that SHIP-1 negatively regulates the development, proliferation and survival of DCs both in vitro and in vivo, with significantly more CD11c+ DCs in the spleen and DC progenitors in the BM of SHIP-1-/- mice (Antignano et al., 2010; Neill et al., 2007).  In addition,   161 compared to control DCs, SHIP-1-/- splenic and GM-CSF BM derived DCs, exhibited a more immature phenotype and decreased activation after TLR stimulation. These DCs expressed lower levels of MHC II and co-stimulatory molecules; secreted less IL-12; had impaired ability to stimulate antigen specific T cell proliferation; and were prone to inducing Th2 over Th1 T cell responses (Antignano et al., 2010).  Overall, SHIP-1 negatively regulates DC development, proliferation and survival, however, positively regulates their activation and subsequent ability to prime T cells.  The changes in SHIP-1-/- mice were attributed to increased PI3K activity, since inhibition of the PI3K pathway in SHIP-1-/- DCs increased MHC II expression and it was suggested that the activation of Akt in the PI3K pathway was responsible for the enhanced proliferation and survival of the DCs (Antignano et al., 2010).  Since SHIP-1 is a known substrate of Lyn, it is therefore not surprising that phenotypes in SHIP-1-/- mice overlap with Lyn-/- mice including perturbations in myelopoiesis, immature phenotype of DCs and the reduced production of IL-12 by DCs post-TLR stimulation (Antignano et al., 2010; Baran et al., 2003; Chu and Lowell, 2005; Harder et al., 2004; Hernandez-Hansen et al., 2004).  In our studies we found that Lynup/up GM-CSF BM derived DCs were more susceptible to the absence of SHIP-1 than Lyn+/+ or Lyn-/- DCs as evidenced by decreased expression of MHC II and co-stimulatory molecules (data now shown) both at baseline and post-LPS stimulation.  Knockdown of SHIP-1 in Lynup/up GM-CSF BM derived DCs also impaired their ability to prime IFN-γ production by NK cells.  These results demonstrate the importance of SHIP-1 in mediating the effects of Lyn in DCs.   We also studied the effects of the absence of SHP-1 in Lynup/up DCs since, like SHIP-1, SHP-1 was also hyperphosphorylated in Lynup/up DCs and hypophosphorylated in Lyn-/- DCs.  SHP-1 knockdown only impaired maturation of Lynup/up DCs at baseline and after LPS stimulation but did not impact Lyn+/+ or Lyn-/- DCs.  However, GM-CSF BM derived DCs from   162 Mev/Mev mice, which are deficient in SHP-1, showed impaired MHC II expression at baseline and post-LPS stimulation. Overall our data suggests that Lyn can phosphorylate ITIMs within inhibitory proteins, allowing SHIP-1 and SHP-1 to bind and dephosphorylate proteins and/or phospholipids, downregulating signal transduction, thereby influencing DC development, activation and cytokine production. Previous studies have shown a correlation between reduced NF-κB signaling and increased production of proinflammatory cytokines, similar to what we found in LPS stimulated Lynup/up DCs.  Mice with myeloid cells lacking IKKβ, IKKα, p50 or p50/p65 had increased production of proinflammatory cytokines even though there was reduced NF-κB signaling (Gadjeva et al., 2004; Greten et al., 2007; Lawrence et al., 2005). Classical NF-κB signaling involves RelA and c-Rel containing dimers and the ability of RelA to bind promoters is dependent on the stability of RelA, where phosphorylation of serine 536 results in its destabilization and possibly degradation by the proteasome (Bode et al., 2009).  Interestingly, a comparison of DCs stimulated with CpG versus LPS, showed that LPS lead to stronger NF-κB signaling than CpG stimulation, however, DCs stimulated with CpG produced more IL-12. This was due to increased stability of RelA binding to the IL-12p40 promoter associated with decreased phosphorylation of Ser536 in RelA, whereas LPS stimulation resulted in increased phosphorylation of Ser536 (Bode et al., 2009).  Similarly, in our studies we found a reduction in phosphorylation of RelA at Ser536 in Lynup/up DCs, correlating with reduced LPS-induced NK-κB signaling but increased IL-12 production.   DCs play a critical role in mediating innate and adaptive immune responses, therefore it is not surprising that tumours have evolved multiple mechanisms to inhibit and/or dysregulate DC activity.  Preliminary data suggests that DCs play an immunosuppressive role in mammary   163 tumour-bearing mice, as depletion of DCs resulted in slower tumour growth in Lyn+/+ mice.  Based on our data demonstrating that Lynup/up DCs are hyperactive after TLR stimulation led to the hypothesis that Lynup/up DCs will be more resistant to the immunosuppressive effects of the tumour, leading to better activation of NK and T cells, culminating in lower tumour burden.  Interestingly, mammary tumours were significantly smaller in Lynup/up mice compared to Lyn+/+ mice.  Future studies will focus on determining the importance of DCs in the resistance to tumour growth observed in Lynup/up mice.  Furthermore, we will investigate whether the treatment of Lyn+/+ mammary tumour-bearing mice with activated Lynup/up DCs can reinvigorate an anti-tumour immune response due to their production of excessive proinflammatory cytokines and increased priming of IFN-γ production by NK cells.   164 Chapter 6: Concluding comments 6.1 Conclusion and future directions Our data demonstrates that mammary tumours dysregulate HSPCs leading to myeloproliferative-like disease consisting of the accumulation of iMCs and the development of anemia.  Mammary tumour development was also associated with the accumulation of DC progenitors and impaired DC differentiation resulting in DCs with dysregulated functions and a reduction in the numbers of mature DCs.  The effects of mammary tumours on hematopoiesis were associated with changes in global epigenetic regulation in HSPC leading to increased Hoxa9 expression.  I found that many of the changes in the HSPC compartment, including DC differentiation, phenotype and function were mediated by tumour-derived G-CSF.  Future studies will focus on determining the necessity of G-CSF in mediating the effects of mammary tumours on the dysregulation of the HSPC compartment and DC differentiation.  This will be accomplished by injecting mammary tumour cells with G-CSF loss-of-function into WT mice, or by injecting WT mammary tumour cells into G-CSF receptor deficient mice, and monitoring the development of anemia, leukemoid reaction and aberrations in DC development  Due to the important role of DCs in innate and adaptive immune responses, tumours have evolved multiple mechanisms to inhibit and/or dysregulate proper DC function.  Preliminary data from our lab demonstrated that DC depletion in mammary tumour-bearing mice was associated with slower tumour growth, showing that DCs may contribute to immunosuppression in tumour-bearing mice. Interestingly, I found that Lynup/up DCs were hyperactive after TLR stimulation. Therefore, I hypothesized that Lynup/up DCs would be more resistant to the immunosuppressive effects of mammary tumours, which would lead to better NK and T cell activation and suppression of tumour growth. I found that mammary tumours injected into Lynup/up mice grew   165 slower compared to Lyn+/+ mice.  Future studies will focus on determining the role of DCs in tumour progression in mammary tumour-bearing Lynup/up mice.  Furthermore, we will investigate whether the treatment of Lyn+/+ mammary tumour-bearing mice with activated Lynup/up DCs can initiate a strong anti-tumour immune response by secreting proinflammatory cytokines and increasing IFN-γ production in NK cells. 6.2 Summary of implications and applications Our studies have demonstrated that mammary tumours not only impair the function of immune cells, such as DCs, but also dysregulate hematopoiesis.  Our in vitro data suggests that MT-CM derived G-CSF mediated the disruption in hematopoiesis in mammary tumour-bearing mice.  Correlating with this data, there were increased levels of G-CSF in the serum from mammary tumour-bearing mice.  However, previous studies have shown that factors such as AngII can also mediate tumour-induced dysregulation of hematopoiesis resulting in the development of immunosuppressive immune cells, which enhance tumour progression (Cortez-Retamozo et al., 2013).  Therefore understanding how tumours interact with the development of the immune system is important, however, since tumours can utilize different mechanisms to increase the differentiation of immunosuppressive cells, it may be more economical to directly target, and either eliminate or inactivate tumour-induced immunosuppressive cells such as MDSCs, TAMs, Tregs and immature DCs. Our studies also demonstrated that mammary tumours impaired erythropoiesis resulting in the development of anemia.  The positive correlation between anemia and tumour progression culminating in decreased patient survival has been established in cancer patients, therefore treating anemia in cancer patients may increase both quality of life and patient survival (Leonard et al., 2005).  Previous studies in animal models have established a role for G-CSF in reducing   166 RBC levels, therefore normalizing G-CSF levels may also reduce anemia in cancer patients (de Haan et al., 1994; de Haan et al., 1992; Nijhof et al., 1994).      DCs are key players in the immune system and our studies demonstrated that mammary tumours dysregulated DC development, resulting in the generation of DCs with reduced activation and ability to prime NK and T cells.  Our data suggested that tumour-derived G-CSF lead to the accumulation of DC progenitors and blocked their differentiation into mature DCs and impaired their ability to activate T cells.  Furthermore, preliminary data from our lab shows that depletion of DCs is associated with slower tumour growth, providing further support for the immunosuppressive role of DCs in mammary tumour-bearing mice.  Therefore, future therapies directed at eliminating factors that impair DC development and/or function, or even eliminating host derived DCs and replacing them with proinflammatory DCs resistant to the immunosuppressive effects of the tumour may increase patient survival.      Understanding DC biology more thoroughly and how to make DCs resistant to the effects of tumours will be important for any DC-based cancer therapies.  We found that the hypersensitivity of Lynup/up mice to LPS was mediated by DCs and their increased ability to prime IFN-γ production by NK cells.  Splenic DCs and GM-CSF BM-derived DCs from Lynup/up mice exhibited increased maturation and activation post-TLR stimulation compared to Lyn+/+ and Lyn-/- DCs.  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Immunology 8, 467-477.     187 Appendix Appendix A   A.1 Sub-Appendix – mRNA qPCR primers Primer 5’ à  3’ sequence Hoxa9 forward:  AAA CAA TGC CGA GAA TGA GAG GGG Hoxa9 reverse: AAA CTC CTT CTC CAG TTC CAG CGT Ezh2 forward: TCA CGT GTG GAG CTG CTG ACC Ezh2 reverse: AGC CTG CCA CAT CAG ACG GTG IRF-4 forward: GCT GCA TAT CTG CCT GTA TTA CCG IRF-4 reverse: GTG GTA ACG TGT TCA GGT AAC TCG TAG IRF-8 forward: ACG AGG TTA CGC TGT GCT CTG IRF-8 reverse: CAC GCC CAG CTT GCA TTT E2-2 forward: AGA CCA AGC TCC TGA TTC TC E2-2 reverse: AGG CTC TGA GGA CAC CTT CT GAPDH forward: CAT TTG CAG TGG CAA AGT GGA G GAPDH reverse: GTC TCG CTC CTG GAA GAT GGT G Rps29 forward: ACG GTC TGA TCC GCA AAT AC Rps29 reverse: CAT GAT CGG TTC CAC TTG GT A.2 Sub-Appendix – ChIP qPCR primers Primer 3’ à  5’ sequence Hoxa9 amplicon A forward: TCC ACC TTT CTC TCG ACA G Hoxa9 amplicon A reverse: CCT GGC ACG ATT AGG CCT T Hoxa9 amplicon B forward: CAT TAA CAA CAG TGG CTG TGG CCT Hoxa9 amplicon B reverse: AAT TAA CCC GGG AGG AAC ACT GGA Hoxa9 amplicon C forward: AAC GAA TCT GTT GGT CGC TCC TGA Hoxa9 amplicon C reverse: AGC AGC CAA ATC GCA TTC TCA CTC Hoxa9 amplicon D forward: TGC ACA ACT GTT GAT GAC TGG CTG Hoxa9 amplicon D reverse: GCA GAA TGA TCT GCC ACA CGA AGA Hoxa9 amplicon E forward: AGC AGT GGA AAG TCC ACC CTT TCA Hoxa9 amplicon E reverse: ACC ACC CTA CTT CCA AAG GCT TCT   

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