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The role of SAP in antigen priming and differentiation of T lymphocytes Huang, Yu-Hsuan 2015

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THE ROLE OF SAP IN ANTIGEN PRIMING AND DIFFERENTIATION OF T LYMPHOCYTES by  Yu-Hsuan Huang  M.S., National Taiwan University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (PATHOLOGY AND LABORATORY MEDICINE)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2015  © Yu-Hsuan Huang, 2015  ii ABSTRACT  Mutations in the SH2D1A gene that encodes signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) cause X-linked lymphoproliferative disease (XLP), a congenital immunodeficiency defined by exquisite sensitivity to Epstein-Barr virus (EBV). Upon EBV infection, XLP patients develop fulminant infectious mononucleosis, characterized by massive expansions of EBV-infected B cells and susceptibility to malignant B cell lymphomas. The precise mechanism of how SAP mediates immunity against EBV remains unclear. Here, we utilized SAP-deficient (Sh2d1a-/-) mice to investigate the role of SAP in regulating T cell differentiation and function. We found that SAP-deficient CD4 and CD8 T cells had a diminished capacity to differentiate into IL-17-producing T helper (Th17) and T cytotoxic (Tc17) cells. The use of co-stimulating SLAM antibodies was found to augment the differentiation of IL-17-producing effectors in wild type but not Sh2d1a-/- splenic T cells under IL-17-polarizing conditions. Furthermore, Sh2d1a-/- mice were protected from experimental autoimmune encephalomyelitis (EAE) and exhibited decreased numbers of CNS-infiltrating Th17 and Tc17 effectors. Together, these results demonstrate that SAP signaling drives the differentiation and function of Th17 and Tc17 cells in vitro and in vivo. In addition, we hypothesized that SAP and SLAM family receptors may be critical for B cell-priming of antigen-specific CD8 T cells. To test this hypothesis, purified wild type and Sh2d1a-/- CD8 T cells were stimulated with various types of antigen-presenting cells (APCs) including B cells, B cell-depleted splenocytes and B lymphoma cells. We found that Sh2d1a-/- CD8 T cells exhibited diminished proliferation and effector functions when stimulated with antigen-presenting B cells or B lymphoma cells but not B cell-depleted splenocytes. In addition, wild type and Sh2d1a-/-  iii CD8 T cells proliferated equivalently when cultured with non-SLAM family receptors-expression APCs: antigen-expressing melanoma or carcinoma cells. Together, these results identify a critical role for SAP and SLAM family receptors in the priming of CD8 T cells towards antigen-presenting B cells or B lymphoma cells. Collectively, our findings suggest that the susceptibility of XLP patients to EBV may be a consequence of virus’s B cell tropism and an inability of SAP-deficient naïve CD8 T cells to proliferate and differentiate upon encountering EBV-infected B cells.      iv PREFACE  Contributions: A version of CHAPTER 3 has been published in the Journal of Immunology; 193(12): 5841-53, 2014. Yu-Hsuan Huang, Kevin Tsai, Caixia Ma, Bruce A. Vallance, John J. Priatel and Rusung Tan. SLAM-SAP signaling promotes differentiation of IL-17-producing T cells and progression of experimental autoimmune encephalomyelitis. Professors Tan and Priatel provided supervision for this project and contributed to manuscript editing. Professor Vallance and Ms. Caixia Ma offered technical support and advice for infection experiments using C. rodentium. I conducted all the experiments, completed data analysis and wrote the manuscript.   A version of CHAPTER 4 is currently being prepared for submission. Professor Priatel provided supervision for this project and contributed to manuscript editing. I performed all the experiments, completed data analysis, and wrote the manuscript.  Ethics Approval:  All animal experimentation followed protocols approved by the Animal Care Committee at University of British Columbia in conjunction with the Canadian Council on Animal Care. Protocols: A14-0303, A14-0008, and A14-0007  Figures and table Approval:  Publishers have granted permission for the usage of previously published figures and tables within my thesis.   v TABLE OF CONTENTS  ABSTRACT .................................................................................................................................... ii	  PREFACE ...................................................................................................................................... iv	  TABLE OF CONTENTS .................................................................................................................v	  LIST OF TABLES ......................................................................................................................... xi	  LIST OF FIGURES ...................................................................................................................... xii	  LIST OF SYMBOLS ................................................................................................................... xvi	  LIST OF ABBREVIATIONS ..................................................................................................... xvii	  ACKNOWLEDGEMENTS ....................................................................................................... xxiii	  DEDICATION .............................................................................................................................xxv	   CHAPTER 1: GENERAL INTRODUCTION ................................................................................1	  1.1	   Immunity: Host defenses ................................................................................................... 1	  1.1.1	   Hematopoiesis ............................................................................................................. 3	  1.1.2	   Innate immunity .......................................................................................................... 6	  1.1.2.1	   Innate immune components ................................................................................. 8	  1.1.2.1.1	   Phagocytes .................................................................................................... 8	  1.1.2.1.2	   Natural killer cells and innate lymphoid cells ............................................... 9	  1.1.2.1.3	   Dendritic cells and professional antigen-presenting cells ........................... 11	  1.1.2.2	   Major histocompatibility complex molecules and antigen presentation ............ 12	  1.1.3	   Adaptive immunity ................................................................................................... 16	  1.1.3.1	   T cells ................................................................................................................. 17	   vi 1.1.3.2	   T cell development ............................................................................................. 18	  1.1.3.2.1	   TCR rearrangement and diversity ............................................................... 20	  1.1.3.2.2	   Positive selection and negative selection .................................................... 23	  1.1.3.3	   Natural killer T cells .......................................................................................... 27	  1.1.3.4	   T cell-mediated responses .................................................................................. 28	  1.1.3.5	   T cell tolerance ................................................................................................... 31	  1.1.3.6	   T cell subsets ...................................................................................................... 33	  1.1.3.6.1	   T helper cell (Th) ........................................................................................ 33	  1.1.3.6.2	   T cytotoxic cell (Tc) .................................................................................... 38	  1.1.3.7	   T cell activation pathways ................................................................................. 39	  1.1.3.8	   B cells ................................................................................................................. 41	  1.1.3.8.1	   B cells and antigen-presenting cells ............................................................ 44	  1.2	   Autoimmunity .................................................................................................................. 45	  1.2.1	   Multiple sclerosis and experimental autoimmune encephalomyelitis ...................... 48	  1.2.2	   IL-17-producing T cells in health and disease .......................................................... 50	  1.3	   Epstein-Barr virus ............................................................................................................ 50	  1.3.1	   Immune responses against EBV infection ................................................................ 58	  1.4	   X-linked lymphoproliferative disease .............................................................................. 59	  1.4.1	   Structure of SLAM-associated protein ..................................................................... 64	  1.4.2	   The signaling lymphocyte activation molecule receptor family ............................... 66	  1.4.3	   SLAM family receptor-SAP signaling regulates immune responses ....................... 71	  1.5	   Rationale and Hypothesis ................................................................................................ 76	    vii CHAPTER 2: MATERIALS AND METHODS ...........................................................................78	  2.1	   Mice ................................................................................................................................. 78	  2.2	   In vitro Th1, Th2, and iTreg cell differentiation assays .................................................. 78	  2.3	   In vitro Th17/Tc17 cell differentiation assays ................................................................. 79	  2.4	   Naïve CD4 T cell adoptive transfers, Citrobacter rodentium infections, and donor CD4 T cell analyses .............................................................................................................................. 80	  2.5	   Experimental autoimmune encephalomyelitis induction and the isolation of CNS-infiltrating T cells ...................................................................................................................... 80	  2.6	   Preparation of highly purified OT-1 CD8 T cells ............................................................ 81	  2.7	   Preparation of antigen-presenting B cells and B cell-depleted splenocytes .................... 82	  2.8	   Thymidine incorporation assays ...................................................................................... 82	  2.9	   CFSE labeling .................................................................................................................. 82	  2.10	   Derivation and in vitro culture of B cell lymphomas, melanoma and breast carcinoma cell lines .................................................................................................................................... 83	  2.11	   In vitro stimulations and CFSE-based OT-1 CD8 T cell proliferation assays ................. 83	  2.12	   In vitro cytotoxicity assays .............................................................................................. 84	  2.13	   In vitro blocking of SLAM receptors CD48 and 2B4 ...................................................... 84	  2.14	   Cell signaling studies and phospho-specific flow analyses ............................................. 85	  2.15	   Adoptive CD8 T cell transfers, lymphoma challenges and Listeria monocytogenes infections ................................................................................................................................... 86	  2.16	   Flow cytometry ................................................................................................................ 87	  2.17	   Statistical analyses ........................................................................................................... 87	   viii CHAPTER 3: SLAM-SAP SIGNALING PROMOTES DIFFERENTIATION OF IL-17 PRODUCING T CELLS AND PROGRESSION OF EXPERIMENTAL AUTOIMMUE ENCEPHALOMYELITIS .............................................................................................................89	  3.1	   Introduction ...................................................................................................................... 89	  3.2	   SAP is strongly expressed by Th17 cells. ........................................................................ 92	  3.3	   SAP positively regulates the differentiation of IL-17-producing CD4 and CD8 T cells. 97	  3.4	   SLAM-SAP signaling positively modulates the differentiation of Th17 and               Tc17 cells. ............................................................................................................................... 101	  3.5	   SAP-SLAM controls the differentiation of naïve CD4 and CD8 T cells into Th17 and Tc17 effectors. ........................................................................................................................ 106	  3.6	   SAP-SLAM signaling promotes Th17 cell differentiation through an IFN-γ and IL-4 independent mechanism. ......................................................................................................... 110	  3.7	   CD4 T cell-intrinsic SAP function is required for normal Th17 cell differentiation         in vivo. ..................................................................................................................................... 116	  3.8	   SAP exacerbates the development of experimental autoimmune encephalomyelitis. ... 119	  3.9	   SAP-deficient mice show defective CD4 T cell priming and decreased numbers of CNS-infiltrating Th17 and Th1/Th17 effectors upon MOG immunization. .................................... 121	  3.10	   Discussion ...................................................................................................................... 125	   CHAPTER 4: SAP ENHANCES B CELL-PRIMING OF CD8 T CELL IMMUNE RESPONSES130	  4.1	   Introduction .................................................................................................................... 130	  4.2	   SAP-deficient CD8 T cells respond poorly to antigen-presenting B cells. .................... 132	   ix 4.3	   The role of SAP in CD8 T cells is required to regulate IL-2 production and CD25 expression upon stimulation by antigen-presenting B cells. ................................................... 138	  4.4	   SAP promotes anti-B cell lymphomas CD8 T cell responses in vitro. .......................... 143	  4.5	   SAP-deficient CD8 T cells exhibit weak cytotoxicity towards B cell lymphoma      targets. ..................................................................................................................................... 148	  4.6	   SLAM-family receptor, CD48, enhances B cell-priming of CD8 T cells ..................... 152	  4.7	   CD48 expression by OVA-expressing B cell lymphomas modulates CD8 T cell responses. ................................................................................................................................ 160	  4.8	   CD48-2B4 signaling modulates CD8 T cell responses when B cells act as primary   APCs. ...................................................................................................................................... 162	  4.9	   2B4/SAP signaling regulates CD8 T cell responses towards antigen-presenting B cell lymphomas. ............................................................................................................................. 166	  4.10	   SAP signaling affects TCR signal transduction when CD8 T cells respond to antigen-bearing B cell lymphomas. ..................................................................................................... 168	  4.11	   Intrinsic role of SAP in CD8 T cells activates ERK and AKT signaling pathways when primed by antigen-presenting B cells. .................................................................................... 170	  4.12	   SAP-deficient CD8 T cells exhibit diminished responses upon stimulation with antigen-expressing B cell lymphomas in vivo. ..................................................................................... 172	  4.13	   Discussion ...................................................................................................................... 177	   CHAPTER 5: CONCLUSION ....................................................................................................180	  5.1	   Conclusions and implications ........................................................................................ 180	  5.1.1	   SAP regulation of Th17 differentiation. ................................................................. 181	   x 5.1.2	   SAP modulates CD8 T cell priming by antigen-presenting B cells. ....................... 184	  5.1.3	   SAP is required for cytotoxic CD8 T effectors to recognize antigen-expressing B cell targets. .......................................................................................................................... 187	  5.2	   Future research directions .............................................................................................. 189	   BIBLIOGRAPHY ........................................................................................................................194	  APPENDICES .............................................................................................................................240	  Appendix A ............................................................................................................................. 240	  A.1	   Sorting gates for naïve CD4 and CD8 T cells. .......................................................... 240	  A.2	   Preparation of naïve CD4 T cells for C. rodentium infection experiment. ............... 241	  A.3	   Confirmation of CD48 and Ly108 expression in CD48-/- and Ly108-/- splenocytes. 242	  A.4	   Wild type and Sh2d1a-/- CD8 T cell responses towards antigen-presenting           Ly108-/- B cells. ................................................................................................................... 243	             xi LIST OF TABLES  Table 1-1 SLAM family receptor signaling and phenotype of SLAM family receptor-deficient mice. .............................................................................................................................................. 69	    xii LIST OF FIGURES  Figure 1-1 A schematic view of the hierarchy of hematopoiesis. ................................................... 4	  Figure 1-2 Dendritic cells antigen presentation pathways. ........................................................... 14	  Figure 1-3 Schematic view of αβ T cell development. ................................................................. 19	  Figure 1-4 TCR α and β chain gene rearrangement. ..................................................................... 22	  Figure 1-5 The affinity model of thymocyte selection. ................................................................ 26	  Figure 1-6 Kinetics of T cell responses. ....................................................................................... 30	  Figure 1-7 CD4 T cells differentiation and plasticity. .................................................................. 35	  Figure 1-8 Signaling transduction from the T cell receptor. ......................................................... 40	  Figure 1-9 Etiology of autoimmune disease. ................................................................................ 46	  Figure 1-10 Blood smear from a patient with infectious mononucleosis. .................................... 52	  Figure 1-11 A schematic view of Epstein-Barr virus infection of an immunocompetent host. ... 55	  Figure 1-12 Relationship between T cell immune responses and viral replication during EBV infection. ....................................................................................................................................... 56	  Figure 1-13 X-linked lymphoproliferative disease: Duncan’s disease ......................................... 61	  Figure 1-14 SAP regulation of T cell receptor signal transduction. ............................................. 65	  Figure 1-15 SLAM family receptors in mice and humans. .......................................................... 68	  Figure 1-16 Immune cell deficits in patients with X-linked lymphoproliferative disease. .......... 72	  Figure 1-17 SLAM receptor-SAP signaling controls NKT cell development. ............................. 75	  Figure 3-1 SAP expression in lymphocyte subsets. ...................................................................... 94	  Figure 3-2 SAP is strongly expressed by Th17 cells. ................................................................... 96	  Figure 3-3 SAP positively regulates the differentiation of IL-17-producing CD4 T cells. .......... 98	   xiii Figure 3-4 SAP positively regulates the differentiation of IL-17-producing CD8 T cells. ........ 100	  Figure 3-5 SLAM-SAP signaling positively modulates the differentiation of Th17 cells. ........ 103	  Figure 3-6 SLAM-SAP signaling positively modulates the differentiation of Tc17 cells. ........ 105	  Figure 3-7 SAP-SLAM controls the differentiation of naïve CD4 into Th17 effectors. ............ 107	  Figure 3-8 SAP-SLAM controls the differentiation of naïve CD8 T cells into Tc17 effectors. . 109	  Figure 3-9 SAP-SLAM signaling promotes Th17 cell differentiation through an IFN-γ and IL-4 independent mechanism. ............................................................................................................. 111	  Figure 3-10 Th17 cell differentiation is independent of SAP under stronger TCR signaling conditions. ................................................................................................................................... 114	  Figure 3-11 SAP plays a CD4 T cell-intrinsic role in regulating Th17 cell differentiation in vivo...................................................................................................................................................... 118	  Figure 3-12 SAP exacerbates the development of experimental autoimmune encephalomyelitis...................................................................................................................................................... 120	  Figure 3-13 Sh2d1a-/- mice show defective CD4 T cell priming upon MOG immunization. .... 122	  Figure 3-14 Sh2d1a-/- mice show decreased numbers of CNS-infiltrating Th17 and Th1/Th17 effectors upon MOG immunization. ........................................................................................... 124	  Figure 4-1 SAP-deficient CD8 T cells proliferate poorly towards antigen-presenting B cells. . 134	  Figure 4-2 SAP-deficient CD8 T cells exhibit defective proliferation and cytokine production upon stimulation with antigen-presenting B cells. ...................................................................... 137	  Figure 4-3 SAP-deficient CD8 T cells exhibit decreased IL-2 production and CD25 expression upon stimulation with antigen-presenting B cells. ...................................................................... 140	  Figure 4-4 IL-2 rescues SAP-deficient CD8 T cell proliferation towards antigen-presenting B cells. ............................................................................................................................................ 142	   xiv Figure 4-5 SAP-deficient CD8 T cells mount defective responses towards Ag-presenting B cell lymphomas. ................................................................................................................................. 145	  Figure 4-6 SAP is not essential for CD8 T cell responses towards tumor cells that lack SLAM family receptor expression. ......................................................................................................... 147	  Figure 4-7 SAP is essential for the efficient generation of CD8 T cell effector functions when B cells act as the primary APCs. .................................................................................................... 149	  Figure 4-8 SAP is important for CD8 T cells effector functions against B cell lymphoma targets...................................................................................................................................................... 151	  Figure 4-9 SLAM family receptors are differentially expressed by APCs and T cells. ............. 153	  Figure 4-10 CD48 regulates B cell-priming of CD8 T cell immune responses. ......................... 156	  Figure 4-11 CD48 is not required for CD8 T cell responses when B cell-depleted splenocytes act as antigen-presenting cells. ......................................................................................................... 158	  Figure 4-12 CD48 expression on B cell lymphomas modulates CD8 T cell immune responses...................................................................................................................................................... 161	  Figure 4-13 Wild type and Sh2d1a-/- CD8 T cells rapidly upregulate 2B4 expression upon stimulation by antigen-presenting B cells or B cell lymphomas. ............................................... 163	  Figure 4-14 2B4 regulates CD8 T cell immune responses towards antigen-presenting B cells. 165	  Figure 4-15 2B4 regulates CD8 T cell responses against antigen-expressing B cell lymphoma cells. ............................................................................................................................................ 167	  Figure 4-16 SAP regulates Nur77 expression upon stimulation with antigen-presenting B cell lymphoma cells. .......................................................................................................................... 169	  Figure 4-17 SAP-deficient CD8 T cells exhibit defects in the activation of ERK & AKT signaling pathways when stimulated by antigen-presenting B cells. .......................................... 171	   xv Figure 4-18 SAP expression in CD8 T cells is critical for anti-B cell lymphoma immunity. .... 174	  Figure 4-19 Wild type and SAP-deficient CD8 T cells expand equivalently to Listeria monocytogenes infection. ............................................................................................................ 176	  Figure 5-1 SAP regulation of Th17 cells and EBV infection. .................................................... 183	  Figure 5-2 SAP is essential for the differentiation of EBV-specific CD8 T cells. ..................... 186	  Figure 5-3 SAP expression within CD8 T cell effectors is required for the recognition and killing of B cell targets. .......................................................................................................................... 188	    xvi LIST OF SYMBOLS α alpha β beta γ gamma δ delta ε epsilon ζ zeta θ theta κ kappa λ lambda µ micron/mu M molar concentration  % percent Gene-/- genetic deletion of “gene”    xvii LIST OF ABBREVIATIONS Ab antibody Ag antigen AIDS acquired immune deficiency syndrome AIRE autoimmune regulator αGalCer alpha-galactosylceramide APC  antigen presenting cell B6 C57BL/6J mouse BBB blood-brain barrier BCR B cell receptor CDR complementarity determining region CFA complete Freund’s adjuvant  CFSE      carboxyfluorescein succinimidyl ester CFU colony forming unit CLP common lymphoid progenitor CMP common myeloid progenitor CMV cytomegalovirus CNS central nervous system CRACC CD2-like receptor-activating cytotoxic cell C. rodentium Citrobacter rodentium cTEC cortical thymic epithelial cell CTL cytotoxic T lymphocyte CTLA-4 cytotoxic T-lymphocyte-associated protein 4  xviii d days DAG diacylglycerol DAMP damage-associated molecular pattern DC  dendritic cell DMEM Dulbecco’s Modified Eagle’s Medium DN double negative DP double positive EAE  experimental autoimmune encephalitis EAT-2 Ewing’s sarcoma-associated transcript 2 EBV  Epstein-Barr virus ER endoplasmic reticulum ERT EAT-2-related transducer FBS fetal bovine serum FIM fulminant infectious mononucleosis FOXP3 forkhead box P3 GC germinal center GFP green fluorescent protein GMP granulocyte and macrophage progenitor h hours HCT hematopoietic cell transplantation HCV hepatitis C virus HIV human immunodeficiency virus HLA human leukocyte antigen  xix HLH hemophagocytic lymphohistiocytosis HSCT hematopoietic stem cell transplantation IBD inflammatory bowel disease IEL intraepithelial lymphocyte IFN interferon iGb3 isoglobotriosylceramide ILC innate lymphoid cell  IM infectious mononucleosis IMDM Iscove’s Modified Dulbecco’s Media i.p. intraperitoneal IPEX immunodysregulation polyendocrinopathy enteropathy X-linked syndrome ITAM immunoreceptor tyrosine-based activation motif iTreg induced regulatory T cell ITSM  immunoreceptor tyrosine-based switch motifs i.v. intravenous KIR killer-cell immunoglobulin-like receptor LAT linker for activation of T cells LCK lymphocyte-specific protein tyrosine kinase LCL lymphoblastoid cell line LGP2 laboratory of genetics and physiology 2 L. monocytogenes Listeria monocytogenes LPS lipopolysaccharide MAPK mitogen-activated protein kinase  xx MBP myelin basic protein MCP mast cell progenitor MDA5 melanoma differentiation associated gene 5 MDP macrophage and DC progenitor MDP muramyl dipeptide MEP megakaryocyte and erythroid progenitor MFI mean fluorescence intensity MHC major histocompatibility complex  min minute MLN mesenteric lymph node MOG myelin oligodendrocyte glycoprotein MS  multiple sclerosis mTEC medullary thymic epithelial cell MyD88 Myeloid differentiation primary response gene 88 NK cell  natural killer cell NKT cell  natural killer T cell NLR nucleotide-binding oligomerization domain (NOD)-like receptor NTBA  NK-T-B-antigen nTreg natural regulatory T cell OVA      ovalbumin PAMP pathogen-associated molecular pattern PBS phosphate buffered saline PKC protein kinase C  xxi PMA phorbol 12-myristate 13-acetate PRR pattern-recognition receptor RA rheumatoid arthritis RAG recombination-activating gene RBC red blood cell RLR retinoid acid-inducible gene I (RIG-1)-like receptor RORγt  retinoic acid-related orphan receptor-γt RSS recombination signal sequence SAP      SLAM associated protein s.c.      subcutaneous Ser serine SH2     Src homology 2 SH2D1A  SH2-domain containing 1A SHIP-1 Src homology 2 domain containing inositol phosphatase-1 SHP Src homology 2 domain-containing phosphatase SIV simian immunodeficiency virus SLAM  signaling lymphocyte activation molecule SLE  systemic lupus erythematosus SNP single-nucleotide polymorphism SP single positive STAT3  signal transducer and activator of transcription 3 T1D type I diabetes TAP transporter associated with antigen processing  xxii Tc      T cytotoxic cell TCR  T cell receptor TdT terminal deoxynucleotidyl transferase Tet tetramer Tfh T follicular helper cell TGF transforming growth factor Th    T helper cell Thr    threonine TLR Toll-like receptor TNF tumor necrosis factor Treg regulatory T cell TSA tissue specific antigen tTreg thymus-derived regulatory T cell Tyr tyrosine wk week XIAP X-linked inhibitor of apoptosis protein XLP X-linked lymphoproliferative disease ZAP70  zeta-chain-associated protein kinase 70  xxiii ACKNOWLEDGEMENTS  I would like to thank my supervisors Drs. John J. Priatel and Rusung Tan for giving me the opportunity of working in their labs and supporting me throughout my PhD studies.   I owe my thanks to my PhD committee, Dr. Kenneth W. Harder, Dr. Peter van den Elzen, Dr. Jacqueline Quandt and Dr. Paul Rennie for all of your suggestions and support.   I thank Drs. Cox Terhorst (Harvard University, Boston, MA), José Villadangos (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia), André Veillette (Institut de Recherches Cliniques de Montréal, Montréal, QC) and Brad H. Nelson (British Columbia Cancer Agency, Victoria, BC) for generously providing reagents.   I offer my gratitude to the following agencies for financial support during my studies: Canadian Institutes of Health Research Operating Grant (to Drs. Rusung Tan and John J. Priatel), Child & Family Research Institute Studentship Award, and Multiple Sclerosis Society of Canada Studentship Award.  I offer my thanks to my colleagues, Ashish Marwaha, Brian K Chung, Huilian Qin, I-Fang Lee, Jason Hung, Kevin Tsai, Lixin Xu, Rosemary Delnavine, Sohyeong Kang and Xiaoxia Wang. My PhD journey was more fun and memorable with your company.   xxiv I owe special thanks to my mentor Dr. John J. Priatel for all of your advice, support and patience throughout my research. With your guidance, I not only learned experimental skills, but also developed my critical thinking and working independence. I wish and encourage myself to be a great scientist like you!  Last but not least, I thank my family for all your caring and support during my studies. It’s a long and difficult journey, however, I can smile and proudly say “I have had the best supporters!”   xxv DEDICATION           To my family and friends who supported me throughout this journey!  1 CHAPTER 1: GENERAL INTRODUCTION  1.1 Immunity: Host defenses Humans are bathed in a vast array and massive quantities of microorganisms (about 100 trillion, outnumbering human cells by 10 to 1; at least 1,000 bacterial species) that colonize regions of the body, such as skin, conjunctiva (mucosal surfaces of the eye), the intestine (mouth to anus) and the respiratory tract (1). The relationship of humans with this complex microbiota is varied, ranging from symbiotic (commensal) to disease causing (pathogenic). Commensal (from Latin for “sharing a table”, derived from com meaning “together” and mensa meaning “table” or “meal”) microbes play many beneficial roles for the host. For example, microbes play critical roles in the processing of nutrients, digestion (enzymatic degradation of polysaccharides) and filling environmental “niches” that may otherwise be occupied by pathogens. Thus, the immune system must be able to distinguish between huge varieties of different types of antigens (defined as molecules that elicit immune responses), determining which are dangerous (pathogens such as viruses, bacteria, fungi and parasitic worms) from those that are harmless (healthy tissue, environmental, dietary and commensal microbiota). Consequently, the failure of the immune system to discriminate, either between self-antigens from foreign antigens or harmless antigens from pathogenic antigens, may lead to life-threatening complications from autoimmune-mediated self-destruction, infections associated with immunodeficiency or autoinflammation resulting from immune dysregulation.  The immune system, composed of many structures and processes and encompassing virtually all cells of the body, is essential for protecting against life-threatening infections and disease. The first defensive lines of the immune system are the epithelial surfaces of the body,  2 forming a barrier to prevent microbial entry into the body. In addition, epithelial cells also secrete mucus and antimicrobial peptides to keep microbes at bay. Host immune defense invokes the activities of various types of white blood cells and is often classified as humoral immunity or cell-mediated immunity, distinguishing whether the activity was cell-free or cell-associated respectively. Humoral immunity is mediated by macromolecules secreted by cells, including antibodies, complement proteins and antimicrobial peptides, and is so denoted because it refers to substances that are present within the humors, also known as extracellular body fluids. By contrast, cell-mediated immunity is dependent on the activation of phagocytes, natural killer (NK) cells and antigen-specific T cells and is accompanied by the release of cytokines, serving to regulate other cells, upon pathogen recognition.  The host immune system is also subdivided into the innate and adaptive immune systems and each are comprised of humoral and cell-mediated components. The innate immune system is an evolutionarily ancient defense strategy, and found within all forms of plant and animal life. Cells of the innate immune system recognize and respond rapidly to pathogens in general (antigen-independent) fashion. However, the innate immune system does not confer long-term memory responses upon re-encounter with the same pathogen. The functions of innate immune cells include complement activation, removal of dying and dead cells, clearance of antibody-complexes and antibody-bound cells, recruitment of immune cells to sites of infection and antigen processing and antigen presentation for the priming of T cells. By contrast, adaptive immunity, also known as “acquired immunity”, is newer in evolutionary terms, being only present within vertebrates, and mediated by B and T cells. Adaptive immune responses are slower to generate relative to innate responses but antigen (and pathogen)-specific, in nature, providing long-lasting protection to the host. Moreover, adaptive immunity results in  3 immunological memory upon initial exposure to pathogen, leading to accelerated responses upon reencounter with the same specific pathogen, and is the basis for vaccination.   1.1.1 Hematopoiesis All blood cells are derived from the hematopoietic stem cells (HSCs) that reside in the bone marrow (Figure 1-1). HSCs are both multi-potent, meaning they are capable of self-renewal, giving rise to new HSCs necessary for maintaining their numbers, and can differentiate into a wide variety of specialized blood cells (2). The developmental process of forming blood cells, termed hematopoiesis, requires a complex array of signals, such as cytokines and cell-cell interactions necessary to drive proliferation, differentiation and lineage commitment (3). As hematopoietic cells differentiate, they gradually lose the capacity for self-renewal and their developmental potential becomes progressively restricted.        4   Figure 1-1 A schematic view of the hierarchy of hematopoiesis. Diagram illustrates the developmental relationship of various types of mature blood cells derived from hematopoietic stem cells (HSCs). HSCs maintain their numbers through balancing replication via self-renewal and differentiation into the common myeloid progenitors (CMP) and common lymphoid progenitors (CLP). The CMP can give rise to megakaryocytes, erythrocytes, mast cells, granulocytes (neutrophils, basophils and eosinophils), monocytes, conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs). The CLP are restricted to differentiate into natural killer cells (NK cells), T cells, B cells and some DCs. Figure adapted from Gabrilovich et al. Nat Rev Immunol. 2012 with permission from Nature Publishing Group (4).  5 HSCs initially give rise to two different lineages: the common myeloid progenitor (CMP) and common lymphoid progenitor (CLP) as shown in figure 1-1 (5, 6). Subsequently, common myeloid progenitors can give rise to megakaryocyte and erythroid progenitors (MEP), mast cell progenitors (MCP), granulocyte and macrophage progenitors (GMP) and macrophage and DC progenitors (MDP). MEPs differentiate into megakaryocytes and erythrocytes (also known as red blood cells or RBCs). Ultimately, megakaryocytes form platelets (also known as thrombocytes) that are essential for blood clotting and the RBCs that serve to deliver oxygen to tissues. MCPs produce mast cells, containing many granules rich in histamine and heparin and playing important roles in allergy and wound healing. GMPs are restricted to differentiating into one of three types of granulocytes which are characterized by the presence of large granules within their cytoplasm: neutrophils, basophils or eosinophils. MDPs give rise to monocytes that circulate within the bloodstream and eventually, infiltrate within tissues where they differentiate into macrophages or dendritic cells (DCs). Macrophages function by engulfing and digesting dying cells, infected cells or harmful particles. In addition, MDPs can differentiate into common DC progenitors (CDP) that give rise to conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Both granulocytes and macrophages function as innate immune cells that patrol and immediate general (non-specific antigen) defense. The function of DCs is critical for adaptive immune responses by processing antigens and presenting them on the cell surface for recognition by T cells. In addition, DCs can also produce inflammatory cytokines to further activate T cells as well as other immune cells such as natural killer (NK) cells and innate lymphoid cells (ILCs).    The CLPs are largely committed to differentiating into lymphocytes although some may give rise to the DC lineage. Resting lymphocytes typically have a single large nucleus surrounded by a thin-layer of non-granular cytoplasm and are chief cellular components found in  6 the lymph. Classically, lymphocytes have been subdivided into three cell types: NK cells, T cells and B cells. NK cells are innate cytotoxic cells that respond rapidly to infection through germline-encoded receptors, destroying target cells and releasing vast quantities of pro-inflammatory cytokines. By contrast, T cells and B cells are the principal cellular components of the adaptive immune system, reacting through germline-rearranged receptors to generate antigen-specific responses and form immunological memory.   1.1.2 Innate immunity  The innate immune system is a host defense strategy that is found in nearly all forms of life. The sensing of infectious foreign invaders by the innate immune system relies on a limited number of germline-encoded receptors called pattern-recognition receptors or PRRs (7). Toll-like receptors (TLRs) are a well-characterized subtype of PRRs that are mainly expressed on the cell surface of sentinel cells such as macrophages, neutrophils and DCs (the role of this subset in shaping adaptive immunity will be discussed in Section 1.1.2.1.3 and 1.1.2.2). There are ten types of TLRs found in humans (TLR1-TLR10) whereas mice have all of the aforementioned TLRs plus an additional three members: TLR11, TLR12 and TLR13 (8, 9). Each TLR recognizes specific conserved molecular patterns that are broadly shared by microbial pathogens, called pathogen-associated molecular patterns (PAMPs) (10, 11). Some examples of PAMPs include lipopolysaccharide (LPS), peptidoglycan, flagellin, virus-associated double-stranded RNA and unmethylated CpG oligodeoxynucleotide (CpG DNA). TLR1, TLR2 and TLR6 recognize bacterial peptidoglycans and lipoproteins. TLR3 and TLR4 bind double-stranded RNA and LPS, respectively. TLR5 and TLR9 recognize bacterial flagella and CpG DNA, respectively. TLR7 and TLR8 bind single-stranded RNA and their functions are thought to be critical for viral  7 immunity through detection of intracellular viral genomes. In addition, TLRs can also sense stress signals released from infected or damaged host cells, termed damage-associated molecular patterns (DAMPs) (12). Examples of various types of DAMPs include proteins, DNA, uric acid, ATP and glycoconjugates.  The recognition of PAMPs and DAMPs by TLRs triggers the activation of intracellular signaling pathways through Myeloid differentiation primary response gene 88 (MyD88)-dependent and –independent pathways, resulting in the activation of innate immune cells (13). Activated macrophages and neutrophils secrete anti-microbial molecules to destroy pathogens and produce pro-inflammatory cytokines and chemokines to attract additional immune cells to the infected site (13, 14). In addition, activated macrophages and DCs engulf pathogens into enzyme-filled vesicles and digest them into peptides in preparation for antigen-presentation. The processing of antigens by macrophages and DCs and their subsequent presentation to T cells acts as a bridge connecting innate and adaptive immunity. The presentation of pathogenic-associated antigens on the cell surface along with the upregulated expression of co-stimulatory molecules enables activated macrophages and DCs to prime robust pathogen-specific T cell immune responses (15, 16).   Retinoid acid-inducible gene I (RIG-1)-like receptors (RLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are PRRs expressed in the cytoplasm that detect intracellular PAMPs (17, 18). RLRs include three members, RIG-I, melanoma differentiation associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) and these receptors recognize single-stranded RNA, double-stranded viral RNA and viral DNA specifically. The RLR family is essential for sensing viral replication and promoting anti-viral responses. NLRs are expressed in macrophages, DCs, lymphocytes and epithelial cells (19).  8 These receptors exhibit specificity for bacterial-derived diaminopimelic acid and muramyl dipeptide (MDP). Following microbial detection, NLR signaling induces the production of pro-inflammatory cytokines and anti-microbial molecules. Collectively, these cytosolic PRRs play a pivotal role in sensing intracellular viruses and bacteria and stimulating immune responses towards these pathogens.  1.1.2.1 Innate immune components The main components of innate immunity are: phagocytic white blood cells (phagocytes: neutrophils, monocytes and macrophages), NK cells, ILCs, DCs and circulating plasma complement proteins (9, 20). These cells circulate the body and are positioned near body surfaces to rapidly sense and attack pathogens and limit the spread of infection.  1.1.2.1.1 Phagocytes The ancient Greek word “phagein” means to eat. Phagocytes such as neutrophils, monocytes and macrophages engulf foreign particles into vesicles filled with enzymes and chemical substances that aid in the digestion of these pathogenic agents (21, 22). In addition, these cells can secrete cytokines to increase vessel dilation and cause inflammation by recruiting more immune cells to migrate to the infection site and destroy pathogenic targets (23). The typical clinical signs of inflammation are redness, heat, swelling, and pain, all of which serve as warnings that our body is under attack.     9 1.1.2.1.2 Natural killer cells and innate lymphoid cells NK cells, defined as large granular cytotoxic lymphocytes, were named “natural killer cells” because of the early notion that they did not require prior activation to recognize and are capable of destroying their targets in the absence of antibodies. NK cells are innate effector lymphocytes that attack infected or malignant cells rather than combating directly with invading microbes themselves (24). NK cells are generated from common lymphoid progenitors, sharing the same progenitor cells as B cells and T cells. However, rather than employing antigen-specific receptors based on somatic gene rearrangement, NK cells use germline-encoded receptors to mount rapid responses towards “stressed” host cells. Upon target cell recognition, NK cells release granules, such as perforin and granzymes, causing target cell apoptosis, and can produce type I cytokines, such as IFN-γ and TNF-α, to help limit microbial infections and tumor growth (24).  NK cell activation is regulated through checks and balances of activating and inhibitory cell surface receptor stimulation (25). Inhibitory receptors suppress the cytotoxic activity of NK cells whereas activating receptors promote NK cell cytotoxic activity. NK cell inhibitory receptors include Ly49 family of C-type lection receptors in mice and killer-cell immunoglobulin-like receptors (KIRs) in humans and CD94/NKG2A heterodimer found in both rodents and primates (26). Upon recognition of self-molecules, major histocompatibility complex (MHCs) molecules on target cell surface, NK cell responses are inhibited although they remain functionally competent. Consequently, NK cell inhibitory receptors sense normal levels of self-MHC molecules on healthy cells and are protected from NK cell attack. Tumors and virally-infected cells may evade antigen-presentation and T cell-mediated immunity (a subject described  10 in more detail in Section 1.1.2.2) by down-regulation of the expression of MHC molecules, a condition termed “missing self” (27). The lack of appropriate MHC class I molecules on the surface of target cells results in the activation of NK cells, and NK cells and T cells collaborate to eliminate the virally-infected or cancerous target cells that down-regulate MHC class I molecules.  NKG2D, CD94/NKG2C and CD16 function as dedicated NK cell activating receptors, playing key roles in the regulation of NK cell function (26). Interestingly, Ly49 (mice) and some KIR (humans) family receptors can also function as NK cell activating receptors. The recognition by NK cell activating receptors of stimulatory ligands on target cell results in NK cell activation, NK cell cytokine production and NK cell killing of the target cell (28, 29). For example, NKG2D and CD94/NKG2C mediate the recognition MHC class I or MHC class I-like molecules to attack “stressed” or malignant targets whereas CD16, a low affinity receptor for the Fc constant region of antibodies, mediates antibody-dependent cell-mediated cytotoxicity or ADCC (30).  ILCs resemble NK cells in that they belong to the lymphoid lineage and lack germline-rearranged (antigen-specific) receptors (31, 32). ILCs are prominent at the mucosal surfaces, such as intestine and lung, and some ILC subsets may function in a way analogous to helper T cells (33-35). Moreover, several ILC subsets express transcription factors and cytokines often associated with helper CD4 T cells (CD4 T cell lineages will be described in Section 1.1.3.6.1). By producing various cytokines, ILCs relay communication with neighbouring surrounding cells, such as epithelial cells and innate/adaptive immune cells, contribute to tissue homeostasis and regulate immune responses to infections (31, 32). On the basis of signature transcription factors  11 and cytokine profiles, ILCs are defined into three subgroups: group 1, group 2 and group 3 ILCs (31). Group 1 ILCs are comprised of NK cells and ILC1s, which produce the type 1 cytokines IFN-γ and TNF-α and provide protections against viruses. By contrast to NK cells, ILC1s are weakly cytotoxic. Group 2 ILCs produce cytokine IL-5 and IL-13 and play roles in controlling parasite infection and wound healing. Group 3 ILCs secrete IL-17 and IL-22, promoting responses against extracellular bacteria. Collectively, ILCs play key roles in protective immunity and regulation of homeostasis and inflammation.  1.1.2.1.3 Dendritic cells and professional antigen-presenting cells A bridging function of innate immune cells is to process pathogen-associated molecules and present these foreign antigens on the cell surface to direct the activation of the adaptive immune system (15, 36). DCs and macrophages are considered professional antigen-presenting cells (APCs), initiating adaptive T cell responses (37, 38). DCs, characterized as star-like shaped cells that grow branched-like projections called dendrites, and considered as the most efficient and effective type of APCs. DCs are often present at sites that contact the external environment sampling antigens and upon activation, migrate to lymph nodes to present antigens with T cells and B cells. Macrophages, meaning “big eater” in Greek, digest anything that does not appear to be healthy tissue including microbes, cellular debris, dying cells and cancerous cells. DCs and macrophages are able to process these antigens, coupling peptides to MHC class I and class II molecules, display them on their cell surface for recognition by T cells.  Professional APCs play key roles in providing three signals necessary for conventional naïve (antigen-inexperienced) T cells to become fully activated and differentiate into various types of effectors (39). Besides antigen processing and antigen presentation (“Signal 1”),  12 professional APCs upregulate co-stimulatory molecules on their surface that are important for amplifying antigen receptor signaling ("Signal 2"). In addition, professional APCs and neighboring cells supply pro-inflammatory and suppressive cytokines that are key in dictating the magnitude and directing the type of immune response generated (“Signal 3”) (40). Consequently, professional APCs perform essential functions in regulating an adaptive immune response, instructing the appropriate response for the type of pathogen challenge and preventing self-destructive autoimmune attack.   1.1.2.2 Major histocompatibility complex molecules and antigen presentation MHC is a set of cell surface molecules encoded in a large cluster of genes: histocompatibility-2 (H2) complex in mice and human leukocyte antigens (HLAs) in humans. MHCs are highly polymorphic, and a mismatch between donor and recipient MHCs is a primary cause of graft rejection after transplantation (41, 42). The function of MHC is to present self or foreign antigens to T cells, which is necessary for T cell activation (43, 44). This process involves antigen processing, where antigens are broken down into peptides, and antigen presentation, where MHC molecules present these peptides on their cell surface for T cell recognition (42, 43).  Importantly, T cells are restricted to recognizing peptides bound by self-MHC, a phenomenon called MHC restriction (45). There are two classical MHC molecules that are responsible for delivering peptides from different sources to the cell surface: MHC class I and class II molecules (Figure 1-2) (46, 47). MHC class I molecules are found on all nucleated cells, and present peptides of 8-15 amino acids derived from cytosolic proteins (48). Intracellular pathogens, such as viruses, enter the cytosol during infection, where their proteins are degraded by proteasomes and transported to the  13 endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP) (49, 50). Peptide fragments are loaded onto MHC class I molecules in the ER, and then displayed on the cell surface for recognition by CD8 T cells. CD8 T cells, which are also known as cytotoxic T cells, will be discussed in depth in Section 1.1.3.6.2.         14   Figure 1-2 Dendritic cells antigen presentation pathways. Dendritic cells (DCs) have both functional major histocompatibility complex (MHC) class I and MHC class II presentation pathways. MHC class I molecules present endogenous antigen that are derived from proteins in the cytosol. The precursor proteins are degraded into peptide fragments through proteasome cleavage, transported to endoplasmic reticulum by transporter-associated with antigen processing (TAP) protein and bound by MHC class I molecules. MHC class II molecules present peptide that is generated within intracellular vesicles. The precursor proteins include exogenous material that is endocytosed from the extracellular environment, and also endogenous components that access the endosome by autophagy. In addition, DCs, especially the CD8+ subset, have a unique cross-presentation pathway that delivers exogenous antigens to MHC class I molecules for the stimulation of naïve CD8 T cells. Cross-presentation is especially important for immunity against most tumours and against viruses that do not infect antigen-presenting cells. Figure reprinted from Villadangos and Schnorrer. Nat Rev Immunol. 2007 with permission from Nature Publishing Group (47).         15 MHC class II molecules are predominantly expressed by professional APCs, and present antigens derived from extracellular sources, such as bacteria and parasites (46). Extracellular pathogens are phagocytosed and enzymatically digested into small peptides of ~11-30 amino acids (51). Subsequently, these peptide fragments are loaded onto MHC class II molecules within the endosomal compartments, and exported to cell surface, where they can be recognized by CD4 T cells. CD4 T cells, which are also known as helper T cells, will be described in detail in Section 1.1.3.6.1.  Antigen processing pathways for MHC class I and class II molecules are less restricted than originally thought. Extracellular antigens can also be presented on MHC class I molecules via a process called “cross-presentation”.  This process has been mainly described in DCs, but has also been suggested to occur in other APCs. During cross-presentation, phagocytosed extracellular proteins are digested and redirected into the MHC class I pathway for presentation to CD8 T cells (52, 53). Although mechanistically unclear, this process plays an essential role in immune responses against viruses that do not target APCs, and tumors that evade the classical MHC class I restricted antigen presentation (54, 55). CD1 family of transmembrane glycoproteins are classified as MHC-class-I-like glycoproteins that share sequence homology and overall structure with MHC class I molecules: CD1 molecules are heterodimers comprised of a heavy chain, possessing three extracellular domains that are non-covalently linked with beta-2 microglobulin (56). However, rather than present peptides, CD1 molecules survey the endocytic pathway and are involved in the presentation of foreign and self lipid and glycolipid antigens to T cells. CD1 family has five human isoforms (CD1a, CD1b, CD1c, CD1d and CD1e) whereas rodents have only CD1d. Based on sequence information, CD1 isoforms are classified into three groups: group 1 contains  16 CD1a, CD1b and CD1c, group 2 contains CD1d, and group 3 contains CD1e. Group 1 CD1 molecules present to T cells with a diverse T cell receptor repertoire, whereas group 2 CD1 molecules present to a population of T cells with a semi-invariant TCR known as natural killer T or NKT cells (further details will be described in Section 1.1.3.3). Consequently, group 1 CD1 molecules are thought to mediate adaptive T cell responses towards a vast array of microbial lipid antigens. By contrast, group 2 CD1 molecules function in innate responses by activating NKT cells. Group 3 CD1 molecules are thought to not be expressed on the cell surface of DCs, and may function in lipid antigen processing within late endosomes and lysosomes (56).    1.1.3 Adaptive immunity Adaptive immune cells rely on receptor-mediated recognition of specific antigens, including proteins, peptides and carbohydrates. Adaptive immune cells remain silent (naïve) under normal conditions, but they initiate antigen specific responses against infectious agents upon activation (57). Although slower than innate immune responses, the adaptive immune response is potent and can control pathogens more efficiently. Another unique feature of adaptive immunity is the development of memory, which enables cells to remember and recall their previous encounters with specific pathogens, and thus provide faster and stronger responses upon repeat infections.  The two main lineages of adaptive immunity are T cells and B cells, discovered 50 years ago by Dr. Max Dale Cooper (58). By removing the thymus and bursa of chickens, he found that thymus-derived cells (T cells) are critical in cell-mediated immunity, such as skin-graft rejection (59, 60). In addition, he found that bursa-derived cells (B cells) are responsible for humoral responses, and antibodies are critical for combating extracellular pathogens (59, 61).  17 1.1.3.1 T cells T cells are generated in the thymus, and each cell expresses a unique T cell receptor (TCR) that recognizes specific antigens bound to an MHC molecule (62). The TCR is composed of two different protein chains. Around 95% of T cells are αβ T cells, which have TCRs consisting of an α chain and a β chain, and 5% of T cells are γδ T cells, which have TCRs consisting of a γ chain and a δ chain (63, 64). In order to generate diverse TCR specificities, T cells undergo genetic rearrangement, called somatic recombination, resulting in each T cell bearing a unique TCR (65-70). In addition, T cells also express either CD4 or CD8 co-receptors, which recognize MHC class II or MHC class I, respectively (71). These co-receptors increase the binding affinity of TCR to MHC molecules by recognizing a region of MHC molecules from the TCR, stabilizing the reaction, and thus prolonging the engagement between T cells and APCs and enhancing T cell activation (72).    A key function of T cells is to promote appropriate immune responses for a given type of infectious challenge. Immune specificity is mediated in part through the actions of the innate immune system, which recognizes the presence of invading pathogens and provides signals to promote the differentiation of naïve T cells into various types of effector cells, each with unique functional properties tailored to eliminate the invading pathogen (73). To prevent unnecessary or excessive immune responses that can be deleterious to the host, T cells have to remain unresponsive to self-antigens. Thus, T cells are educated to discriminate between self- and non-self-antigens (74). Furthermore, certain subsets of T cells are equipped with the ability to suppress inflammatory responses mounted by other immune cells. This regulatory capacity is essential for immune tolerance, which is important for the well being of the host.  18 1.1.3.2 T cell development T cell development occurs in the thymus, where T cell progenitors develop into mature naïve T cells, and become educated to discriminate between self and foreign antigens. To support the importance of the thymus for T cell development, patients with DiGeorge Syndrome, which are born without thymi, are T cell deficient (75). Similarly, nude mice or athymic mice, which have defects in thymic epithelium, lack T cells (76). The thymus is composed of cortical and medullary areas surrounded by a capsule layer. T cell progenitors, which develop from hematopoietic stem cells in the bone marrow, migrate into the subcapsular cortical area (Figure 1-3). Each early T cell progenitor receives signals from the thymic epithelial cells (TECs), and go through a series of developmental stages to generate a unique TCR and one of the two co-receptors, CD4 or CD8 (77).         19   Figure 1-3 Schematic view of αβ T cell development. Developing thymocytes undergo a series of distinct steps of differentiation within the thymus to select T cells that express a functional, self-restricted and self-tolerant T cell receptor (TCR) along with the expression of the appropriate co-receptor, CD4 or CD8. The early T cell precursors lack CD4 and CD8 expression and are called double-negative (DN, CD4-CD8-) thymocytes. DN thymocytes are subdivided into four stages of differentiation on the bases of CD25 and CD44 expression (DN1, CD44+CD25−; DN2, CD44+CD25+; DN3, CD44−CD25+; and DN4, CD44−CD25−). Upon successful TCRβ rearrangement and pairing with the pre-TCRα chain, DN thymocytes initiate massive proliferation and transition to double-positive (DP, CD4+CD8+) thymocytes and then pre-TCRα chain is replaced with newly rearranged TCRα chain to form a complete αβ TCR. Subsequently, DP thymocytes undergo positive and negative selection through interactions with thymic epithelial cells and hematopoietic APCs that express MHC class I and MHC class II molecules associated with self-peptides. DP thymocytes expressing TCRs with high affinity for self-peptides bound to MHC class I or class II complex are deleted (negative selection) whereas DP thymocytes expressing TCRs with appropriate levels of affinity for these ligands are positively selected into mature CD8- or CD4-expressing single positive (SP) thymocytes respectively and exit the thymus.  Figure reprinted from Germain RN. Nat Rev Immunol. 2002 with permission from Nature Publishing Group (78).    20 These progenitors lack expression of TCR, CD4 and CD8, and are called double-negative thymocytes (DN, CD4-CD8-). Most DN thymocytes pass through four phases of differentiation to become double positive thymocytes (DP, CD4+CD8+), bearing a rearranged αβ TCR and expressing of both co-receptors CD4 and CD8. A minor population of the DN thymocytes goes through rearrangement to express γδ TCR, and remains CD4 and CD8 negative. The four differentiation stages are characterized as DN1 to DN4 based on the expression of CD25 and CD44 (DN1: CD25-CD44+; DN2: CD25+CD44+; DN3: CD25+CD44-; DN4: CD25-CD44-) (79). The DN thymocyte bears the pre-TCR that consists of a rearranged TCRβ chain and a surrogate pre-TCRα chain (66). The assembled pre-TCR leads to successful differentiation from DN to DP, which involves the replacement of the pre-TCRα chain with a newly rearranged TCRα chain and the expression of both CD4 and CD8 co-receptors. These αβ TCR DP thymocytes then interact with thymic epithelial cells and hematopoietic APCs expressing MHC class I and class II molecules loaded with self-peptides, and undergo positive and negative selection, which will be described in detail in section 1.1.3.2.2. Cells that survive the selection process mature to become CD8 or CD4 single positive (SP) cells depending on whether they interact with MHC class I or class II molecules, respectively, and then exit the thymus.     1.1.3.2.1 TCR rearrangement and diversity Most TCRs are composed of an α chain and a β chain. Both chains contain a variable region that forms a unique antigen-binding site for the recognition of peptide loaded on the MHC molecules. The variable regions are encoded by recombined and rearranged gene segments (Figure 1-4) (80). TCRα chain genes contain Vα (variable) and Jα (joining) segments, and TCRβ chain genes contain Vβ, Jβ and Dβ (diversity) segments. During TCRβ chain gene  21 rearrangement, Dβ segments are joined to Jβ segments to form a DJβ region. Vβ segments are then joined to the DJβ block by DNA ligase. The rearranged VDJβ segment is transcribed and spliced with the constant region (Cβ). Similarly, Vα segments are joined to Jα segments during TCRα chain gene rearrangement, and subsequently, the VJα block and Cα transcripts are assembled together.     22   Figure 1-4 TCR α and β chain gene rearrangement. The V-regions (variable region) of TCRα- and β-chain genes are composed of discrete segments that are joined by somatic recombination during development of the T cell. TCRα chain genes contain Vα (variable) and Jα (joining) segments and TCRβ chain genes contain Vβ, Jβ and Dβ (diversity) segments. Rearrangement of these gene segments generates a functional Vα- and Vβ-region exon that is transcribed and spliced to join to Cα and Cβ (constant region) respectively. The resulting mRNAs are translated to yield the T-cell receptor α and β chains and are paired to generate the αβ TCR heterodimer. Figure reprinted with permission from Garland Science (Charles Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Publications).       23 The recombination process is mediated by several enzymes, including recombination activation genes 1 and 2 (RAG1 and RAG2) (81, 82), terminal deoxynucleotidyl transferase (TdT) (83) and DNA ligase. The TCR gene segments are flanked by recombination signal sequences (RSS) to mark the points where the recombination occurs. RAG1: RAG2 complexes recognize the RSS regions and cleave DNA fragments at a random adjacent site. Subsequently, the DNA repair enzyme, TdT, modifies the cleavage sites by inserting or deleting nucleotides, and DNA ligase join gene segments together. After transcription and splicing with the constant regions, the completed VJCα and VDJCβ mRNA are translated to TCR α and β chains, respectively, and both chains are paired to form the αβ TCR. The random joining of gene segments along with random insertion or deletion of nucleotides at V, D and J junctions generates tremendous diversity for the assembled αβ TCR. It is estimated that about 1014 different TCR specificities can be generated through this process.  The variable domains of both the TCR α and β chains each have three hypervariable regions, termed complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 (84). These regions consist of hypervariable amino acid sequences in discrete loops, forming a binding site for a specific antigen. Between the three variable regions, CDR3 has the greatest variability, and is the primary region for binding the unique peptide presented on MHC molecules. Thus, the combination of six CDRs (three from α-chain and three from β-chain) contributes to the diversity of the TCR.    1.1.3.2.2 Positive selection and negative selection After successful rearrangement of their TCRs, DP thymocytes undergo thymic selection, including positive and negative selection. Positive selection ensures that T cells have the ability  24 to recognize self-peptides in the context of self-MHC molecules (self-antigens). On the other hand, negative selection is critical for promoting tolerance to self-antigens by eliminating self-destructive T cells (85, 86). Both positive and negative selection processes are dependent upon interactions between thymocytes and various types of thymic epithelial cells and APCs (78, 87). In addition, lineage commitment of the developing T cells is determined by whether their TCR recognizes MHC class I or MHC class II molecules. Moreover, DP thymocytes that recognize self-peptides loaded on MHC class I molecules commit to the CD8 SP T cell lineage, whereas DP thymocytes that recognize self-peptides loaded on MHC class II molecules differentiate into CD4 SP T cell lineage (78). Cortical thymic epithelial cells (cTECs) play essential roles as APCs during the positive selection. After successful rearrangement of their TCRs, DP thymocytes migrate to the cortical-medullary junction, where they interact with cTECs expressing self-peptides loaded on MHC class I or MHC class II molecules. Thymocytes receive survival signal if they have the capability to recognize self-MHC molecules expressed on cTECs (85, 88). By contrast, thymocytes undergo apoptosis if they fail to recognize any MHC molecules.  Negative selection, also known as clonal deletion, is a process where DP or SP thymocytes that express TCRs with high affinities for self-antigens are eliminated (85, 88). This process is critical for central tolerance to prevent self-reactive T cells from going to the periphery, resulting in detrimental autoimmune responses. Cortical thymic epithelial cells, medullary thymic epithelial cells (mTECs) and bone marrow-derived APCs, especially DCs and macrophages, all contribute to the presentation of self-antigens to developing thymocytes, and thus, function to mediate negative selection.  25 mTECs play an essential role in presenting non-thymic tissue-specific antigen (TSAs), such as insulin from the pancreas (89, 90). The expression of TSAs in mTECs is partially driven by their expression of autoimmune regulator (AIRE) (91), a transcriptional activator that causes the transcription of a wide array of genes that are usually expressed in peripheral tissues (92, 93). In addition, terminally differentiated mTECs undergo apoptosis, and the apoptotic fragments can then be taken up by surrounding DCs for cross-presentation (94). This process results in the indirect-presentation of TSAs by thymic APCs. Thus, both direct and indirect presentation of TSAs likely contributes to central tolerance (94). Self-reactive thymocytes that strongly recognize TSAs receive apoptotic signals and are eliminated (95).  It has also been proposed that some self-reactive thymocytes become anergic and unable to generate immune responses (96), and some receive signals to undergo receptor editing to change their TCR specificity (97).  The affinity of the interaction between TCR and peptide-MHC complex is the key determinant of thymic selection (Figure 1-5) (98). Most thymocytes have no affinity for self-peptide-MHC complexes, and die by neglect. On the other hand, thymocytes with low affinity for self-peptide complexes receive survive signals, and are induced to differentiate into CD4 or CD8 lineages. High affinity binding of the TCR to self-peptide-MHC complexes results in negative selection and cell death by apoptosis. Interestingly, some high affinity, self-reactive T cells survive negative selection and develop into regulatory T cells (Treg) (99, 100), which are able to suppress immune responses and provide another mechanism of immunological tolerance towards self-antigens (101). With the successful completion of both positive and negative selection, functional naïve T cells leave the thymus and migrate into the periphery.   26   Figure 1-5 The affinity model of thymocyte selection. The strength (affinity) of interaction between the T cell receptor (TCR) and self-peptides-MHC complexes (self-antigens) is critical for thymocyte fate. Many thymocytes express TCRs having no affinity for self-antigens and die by neglect. Thymocytes expressing TCRs that interact weakly with self-antigens are positively selected, receiving survival signaling and differentiate into mature, functional T cells. Thymocytes expressing TCRs that interact strongly with self-peptides/self-MHC molecules undergo clonal deletion (negative selection), receptor editing or anergy. Some thymocytes expressing high-affinity (self-reactive) TCRs differentiate into “regulatory-phenotype” T cells such as regulatory FOXP3-positive CD4 T cells (TReg cells), CD8αα+ intestinal epithelial lymphocytes (IELs) and natural killer T cells (NKT cells). It is not known how signals that select thymocytes to form regulatory T cells differ from signals that mediate negative selection. Figure reprinted from Hogquist et al. Nat Rev Immunol. 2005 with permission from Nature Publishing Group (98).    27 1.1.3.3 Natural killer T cells NKT cells are a subset that differs from conventional T cells in that they express markers characteristic of NK cells and function like innate-like effector cells (102). The majority of NKT cells carry a semi-invariant αβ TCR that is consists of an invariant α chain paired with a limit set of β chains (103). In addition, NKT cells possess a natural memory phenotype and are capable of liberating vast amounts of cytokine rapidly upon TCR stimulation. Rather than the recognition of peptides bound to MHC class I or II molecules, the TCRs of NKT cells recognize glycolipid antigens presented on the non-polymorphic MHC-like molecule, CD1d (104). To date, the most powerful activator of NKT cells is a marine sponge-derived lipid called α-galactosylceramide (α-GalCer) and is thought to mimic the action of natural endogenous or exogenous ligands (105). Importantly, the use of α-GalCer has been invaluable for stimulating, phenotyping, enumerating and assessing their cellular functions. More recently, NKT cells have been found to be reactive against related, but likely more physiological antigens, such as the self-lipid isoglobotrihexosylceramide (iGb3) (106) and microbial cell wall glycolipids from Sphingomonas (107, 108).  NKT cells develop within the thymus like conventional T cells however their positive selection is mediated by glycolipids presented by CD1d molecules on developing thymocytes rather than peptides in the context of MHC molecules on thymic cortical epithelial cells (109). Recent studies have found that NKT cells can adopt polarized effector programs, resembling helper T cells of the Th1, Th2 or Th17 lineage, during their development within the thymus and subsequently, likely migrate to different tissues depending on their chosen fate: NKT1 to spleen and liver, NKT2 to the lung and NKT17 to dermis and peripheral lymph nodes (110). However, it is unclear how developing NKT cells choose a given polarized fate although early data suggest  28 that the strength of TCR signaling and recognition of specific glycolipids may be a determining factor (110, 111). The function of NKT cells is to generate rapid and robust responses upon receiving signals from infected cells or antigen-presenting DCs that have sensed infection, helping prime the innate and adaptive immune systems (112). NKT cells respond en bloc by secreting vast amounts of cytokines (IL-2, IL-4, IFN-γ, IL-17) and consequently, exhibit potent immunoregulatory properties by polarizing or augmenting immune responses. Moreover, NKT cells have been shown to be critical for promoting the optimal activation of NK cells to induce cytolysis and IFN-γ production (113, 114), stimulating antigen-processing and antigen-presentation and enhancing the priming of antigen-specific B and T cells (115, 116). Altogether, the physiological functioning of NKT cells has been linked to autoimmunity (117, 118), immunity against bacterial and viral infections (119, 120), and immune surveillance of cancers (121, 122).     1.1.3.4 T cell-mediated responses A T cell-mediated response involves three phases: priming of naïve T cells to proliferate and differentiate into effector cells (123, 124), contraction of the T cell population (125, 126), and memory cell formation (127, 128) (Figure 1-6). Primary T cell activation happens through a complex integration of signals received from antigenic peptides (Signal 1), co-stimulatory molecules (Signal 2), and the cytokine milieu (Signal 3) (40, 125). Each T cell expresses a clonal TCR on their surface that recognizes a specific antigen (specific peptide/MHC complex) on the APC surface. There are various types of APCs that are able to process antigens efficiently and deliver signals to activate naïve T cells, including DCs, B cells, macrophages and some activated  29 epithelial cells (62). In addition, APCs also provide co-stimulatory signals, such as CD28 co-stimulation, for effective T cell activation. The binding of TCRs to peptide-MHC complexes, together with co-stimulation, triggers a series of intracellular signaling cascades, resulting in the activation of naïve T cells. These activated, antigen-specific T cells then rapidly proliferate (clonal expansion phase), differentiate into different T cell subsets, and acquire effector functions based on the cytokine milieu. The different T cell subsets and their functions are discussed in section 1.1.3.6. Effector cells perform cell-mediated cytotoxicity and produce various cytokines to control infection and clear pathogens. This rapid clonal expansion is a crucial element in host defense. T cells can divide approximately every 4 to 6 hours after the initial priming (129), and the effector T cell pool can increase 5×105-fold to provide robust antigen-specific protection against pathogens (130, 131).     30   Figure 1-6 Kinetics of T cell responses. (a) During an acute viral infection, viral-specific naïve CD8 T cells undergo massive expansion, culminating in their differentiation into effectors and viral clearance. Subsequently, the large majority (~90 %) of T cell effectors die during the contraction phase while a minority (5 to 10 %) give rise to long-lived memory T cells. (b) A naïve T cell differentiates into diverse subsets of effectors (based on gene expression), varying in effector functions and long-term cell fate. Effector cells (shown in blue) are terminally differentiated, possessing effector function but lacking proliferative potential short-lived whereas effectors with self-renewal potential (shown in red) are maintained and differentiate into long-lived memory T cells. Figure reprinted from Kaech and Cui. Nat Rev Immunol. 2012 with permission from Nature Publishing Group (132).          31 After the pathogenic agent is eliminated, most effector T cells disappear by going through an apoptotic process (contraction phase), and only a few antigen-specific T cells (5-10%) remain to form memory cells (124, 126). One of the key features of memory T cells is their self-renewal ability, whereby cells survive and persist in circulation within the body for several years. Moreover, these antigen-experienced memory T cells can perform immediate effector functions or undergo clonal expansion more rapidly to provide efficient secondary responses when encountering the same pathogen again (133-136).   1.1.3.5 T cell tolerance In addition to playing a pivotal role in generating specific responses against a broad range of pathogens, T cells also mediate self-tolerance to avoid excessive immune reactions that would be harmful to the host. There are two types of mechanisms involved in T cell tolerance: central tolerance and peripheral tolerance (137). Central tolerance is an early checkpoint in the regulation of T cell tolerance that occurs in the thymus through negative selection, where self-reactive T cells are eliminated before they develop into fully immunocompetent cells (as discussed in section 1.1.3.2.2).  Although most self-reactive T cells are effectively eliminated in the thymus, some may escape from negative selection and go into the periphery (138), where mechanisms of peripheral tolerance is needed to regulate them. T cells with low avidity TCRs for self-peptide-MHC complexes, which are unaffected by negative selection, have also been shown to contribute to autoimmune diseases (diseases caused by immune responses mounted against self-antigens) (139). More importantly, peripheral tolerance is needed to prevent excessive immune responses against some non-harmful foreign antigens, such as those derived from food, inhaled particles  32 and commensal gut bacteria. As a result, peripheral tolerance provides the second checkpoint to restrain T cell responses against self-antigens and non-harmful foreign antigens (140, 141). There are four mechanisms involved in peripheral tolerance: ignorance, anergy, deletion and suppression (142).  One way to mediate tolerance is by ignorance, where a physical barrier prevents the interaction between self-reactive T cells and the tissues that express their self-antigens, thereby creating immune privileged sites. Unless the barrier is broken, these self-reactive T cells thus remain circulating in the periphery, and unaware that their antigens are present in the host (143).  Peripheral anergy is another way to restrain T cell responses. This process is regulated in part by the absence of co-stimulatory molecules on APCs upon peptide-MHC: TCR binding. For example, tolerogenic APCs have low levels of co-stimulatory molecules expression, and T cells failing to receive co-stimulation concurrent to TCR engagement by these APCs become anergic. Another mode of peripheral anergy depends on the expression of co-inhibitory receptors, such as Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), by T cells. Upon association with co-stimulatory molecules CD80 or CD86 on APCs, CTLA-4 transmits inhibitory signals and induces T cell anergy (144, 145). Anergic T cells fail to proliferate and mount an immune response against their antigen. The third mechanism of peripheral tolerance is peripheral deletion, which is mediated via programmed cell death. Chronic engagement of self-reactive TCRs with their self-peptide-MHC complexes triggers the activation of Fas/FasL signaling pathways and Bim-dependent mitochondrial death pathway, resulting in cell death (42). In support, the deficiency of pro-apoptotic molecules, such as Fas and Bim, have been shown to cause autoimmune diseases caused by uncontrolled proliferation of T cells (146, 147).   33 Regulatory T cells (Treg) play an essential role in immunological tolerance through the suppression of autoreactive T cells (74). Treg cells are CD4 T cells that express Foxp3, a key transcription factor for the function of Treg cells (148). This distinct T cell subset can be generated in the thymus (thymus-derived Treg; tTreg or natural Treg; nTreg) or induced from naïve T cells in periphery (induced Treg; iTreg). Treg cells maintain tolerance by actively suppressing the differentiation and function of other effector T cells (149), and a deficit of Treg cells results in T cell-mediated autoimmune diseases and excessive immune responses to non-harmful antigens (150, 151).  1.1.3.6 T cell subsets The two major αβ T cell subpopulations are categorized by their expression of the co-receptor CD4 and CD8. CD4 T cells (T helper cell, Th) and CD8 T cells (T cytotoxic cell, Tc) recognize antigens presented by APCs on MHC class II and I molecules, respectively (72). CD4 T cells “help” the activity of other immune cells in a contact-dependent or cytokine-dependent manner, whereas CD8 T cells mediate cytotoxicity to eliminate infected cells. T cells can be further divided into different subsets, each with its own unique purpose.  1.1.3.6.1 T helper cell (Th) CD4 T cell subsets play crucial roles in mediating adaptive immune responses against invading pathogens and tumor cells, and maintaining tolerance to self-antigens (73). Upon recognition of antigens presented by APCs, naïve CD4 T cells can differentiate into various lineages depending on the cytokine milieu of the microenvironment. These different lineages release different combinations of effector cytokines to help other immune cells mount more  34 efficient immune responses tailored towards the present immune challenge. For example, Th cells can provide help to B cells for antibody generation, enhance CD8 T cell function, promote eosinophil activation, recruit macrophages and neutrophils to control bacterial and fungal infection, and also suppress cell proliferation to maintain self-tolerance (73). The effector Th lineages were initially defined as Th1 or Th2 on the basis of cytokines produced, IFN-γ or IL-4 respectively, and the types of responses elicited, whether promoting clearance of intracellular pathogens or augmenting antibody-mediated humoral responses (152). However, it is now clear that a much greater diversity of effector T cell lineages exists, including T follicular helper cell (Tfh), iTreg and Th17 subsets, and that their differentiation fate is determined through a complex integration of signals received from the TCR, the context of a particular cytokine milieu, and the expression of master transcription factors as indicated in figure 1-7 (153).              35   Figure 1-7 CD4 T cells differentiation and plasticity. Upon interaction with cognate antigen on the surface of antigen-presenting cells, naïve CD4 T cells can differentiate into Th1, Th2, T follicular helper cell (Tfh), induced regulatory T cell (iTreg), and Th17 cell lineages depending on the cytokine milieu of the microenvironment and induction of master transcription factors (T-bet, GATA3, RORγt, FOXP3 and Bcl-6) controlling cell fate. Recent work suggests that Th lineage commitment is not fixed as previously thought and Th programming can convert from one lineage to another depending on the cellular environment. Figure adapted from Zhou et al. Immunity. 2009 (154).          Th1T-betIFN-γTh2GATA3IL-4IFN-γIL-12IL-4IL-21TfhiTregNaiveBcl-6FOXP3Th17RORγtIL-6TGF-βIL-23TGF-βIL-2IL-21IL-10IL-17IL-21IL22 36 Th1 cells mainly produce the cytokine IFN-γ and promote cellular immunity against intracellular pathogens such as viruses, and against tumor cells (155, 156). Th1 cell differentiation is regulated by the cytokines IL-12 and IFN-γ, and the master transcription factor T-bet (157, 158). Activation of T-bet drives T cells into the Th1 lineage and upregulates the transcription of the Ifng gene (158). By contrast, T-bet deficient cells exhibit defects in Th1 differentiation in vitro, and T-bet deficient mice exhibit poor protection against the intracellular pathogen Leishmania major (158).  The Th2 subset is characterized by the production of IL-4, and is responsible for promoting antibody generation and eosinophil activation against extracellular parasites, bacteria, and allergens (155). Th2 differentiation is driven by exposure of T cells to IL-4, which promotes the expression of transcription factor GATA3, and subsequently directs cells to become IL-4-producing Th2 cells (159, 160). Evidence for the importance of GATA3 in Th2 differentiation is found in the observation that GATA3 deficient CD4 T cells fail to generate Th2 responses both in vitro and in vivo (161-163).  Tfh is a newly defined Th subset that expresses the chemokine receptor CXCR5, and reside within B cell follicles of secondary lymphoid organs (164, 165). The function of the Tfh subset is to provide cognate help to B cells for the generation of T cell-dependent B cell responses, including germinal center (GC) formation, and the development of long-lived memory B cells and plasma cells (166, 167). Tfh differentiation is highly dependent on the exposure of T cells to IL-21, which can elevate the expression of CXCR5 and increased antibody production by co-cultured B cells (168). An absence of Tfh cells has been observed in Bcl-6 deficient mice, suggesting that Bcl-6 is the main transcription factor for Tfh differentiation (169, 170).  37 Treg cells are often characterized as CD4 T cells that co-express CD25 and Foxp3. This subset plays an essential role in the maintenance of immune homeostasis by suppressing the responses of other effector immune cells. Mutations in gene Foxp3 result in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), causing overwhelming systemic autoimmune diseases (150, 171, 172). The cytokines TGF-β and IL-2, along with activation of the transcription factor Foxp3, are essential for the differentiation of iTregs in the periphery, and the maintenance of Treg function in the periphery (173-175). Since TGF-β induce Foxp3 expression and maintain the suppressive potential of Treg cells, TGF-β deficient mice developed excessive inflammatory responses (176, 177).  Th17 effector cells are a relatively newly described lineage that possesses unique developmental requirements and immunological functions (178). The formation of Th17 cells is dependent on a milieu of cytokines (TGF-β and IL-6 in mice and TGFβ, IL-1β and IL-6, IL-21 or IL-23 in humans) that cooperates to activate STAT3 and induce the master transcription factor retinoic acid-related orphan receptor-γt (RORγt) (179-182). By contrast, cytokines IFN-γ and IL-4 secreted by Th1 and Th2 cells respectively play important roles in suppressing the development of Th17 cells (183). Besides their hallmark cytokine, Th17 cells also secrete IL-21, IL-22, and TNF-α, and mediate critical functions in controlling microbial defense and leukocyte recruitment (184-188). Although Th1 and Th2 are considered terminally differentiated lineages, recent reports suggest that the Th lineages are “plastic”, especially in Treg and Th17 subsets, allowing for their conversion to other lineages. For example, upon stimulation with IL-6 and IL-21, Treg cells can differentiate into Th17 cells (189, 190). Interestingly, upon the interaction of B cells through CD40, Treg cells in the gut can convert into Tfh and regulate GC formation and IgA synthesis  38 (191). Furthermore, upon exposure to IL-12 or IL-4 in vitro, Th17 cells can become Th1 and Th2 cells, respectively (192, 193). Notably, Th17 cells that has been converted into Th1-like subsets sustain the ability to induce both colitis and diabetes in adoptive transfer models (193, 194). These findings highlight the flexibility between Th lineages, help us improve our mechanistic understanding of T cell differentiation, and can provide useful information for designing immune intervention.  1.1.3.6.2 T cytotoxic cell (Tc) CD8 T cells are pivotal in controlling intracellular pathogens and malignant cells through cytokine production and cell-mediated cytotoxicity for direct killing of target cells (195). Upon receiving cognate antigen stimulation by APCs, naïve CD8 T cells undergo massive clonal expansion and differentiation into effector cells. IL-2 has been shown to positively regulate this process (196, 197). Activated CD8 T cells display upregulated expression of IL-2 and CD25, a high affinity IL-2R subunit, in vitro and in vivo (196, 198, 199), and exogenous IL-2 can sustain the proliferation and function of cytotoxic CD8 T cells both in vitro and in vivo (197, 200). Remarkably, impaired IL-2 signaling (IL-2-deficient or CD25-deficient) results in defective CD8 T cell expansion upon viral infection or tumor induction (196).    Similar to CD4 T cells, naïve CD8 T cells can differentiate into various subsets upon activation, including Tc1, Tc2, and the recently defined Tc17 cells, which secrete cytokines IFN-γ, IL-4, and IL-17, respectively (201). These CD8 T cell subsets have distinct requirements for their differentiation. The differentiation of Tc1, for example, requires exposure to IL-12 and IL-2, along with expression of the transcription factor T-bet. Tc1 plays a critical role in providing protection against intracellular pathogens and tumors (132, 202). The Tc2 lineage can be induced  39 by IL-4 and the expression of the transcription factor GATA3, and has been shown to mediate allergy and autoimmune diseases, such as type I diabetes (T1D), multiple sclerosis (MS) (203-205). Tc17 differentiation is induced by IL-6, TGF-β, and expression of the transcription factor RORγt, while IL-23 stabilizes their phenotype. Similar to the Th17 lineage, the Tc17 lineage is involved in regulating autoimmune disease, viral infection and anti-tumor responses (206-208). Cytotoxic T cells play an essential role in host defense against viral infection. The anti-viral function of CD8 T cells is highlighted in Epstein-Barr virus (EBV)-induced infectious mononucleosis, where massive clonal expansions of CD8 T cells can be observed upon EBV infection (209). Furthermore, these cells have been shown to specifically recognize EBV peptides and respond to EBV-infected B cells (210). In addition, the magnitude of the anti-viral CD8 T cell response correlates with lower human immunodeficiency virus (HIV) load (211), and increased simian immunodeficiency virus (SIV) load has been observed in macaques after CD8 T cell-depletion (212).   1.1.3.7 T cell activation pathways The TCR complex is an octamer composed of the TCRα and β chains, CD3 co-receptor chains (a CD3γ chain, a CD3δ chain and two CD3ε chains) and two ζ chains and the association of these components is necessary for its stable cell surface expression (213). Engagement of the TCR complex by a peptide-MHC complex leads to the activation of downstream signaling cascades that regulates cell proliferation, differentiation, survival and cytokine production (Figure 1-8) (214). In this section, the molecular events of T cell activation are briefly described.    40   Figure 1-8 Signaling transduction from the T cell receptor. Engagement of the TCR to its cognate peptide-MHC complex leads to the activation of LCK (lymphocyte-specific protein tyrosine kinase) and ZAP70 (zeta-chain-associated protein kinase 70), phosphorylation of the adaptor docking protein LAT (linker for activation of T cells) and assembly of a signaling complex including GADS, SLP76, NCK, ITK, PAK, VAV and PLCγ1. PLCγ1 activation leads to hydrolysis of phosphatidylinositol-4, 5-bisphosphate (PIP2) to the second messengers inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). The generation of IP3 causes rises in intracellular calcium levels leading to NFAT (nuclear factor of activated T cells) activation whereas DAG stimulates the Ras exchange factor RasGRP1 to activate Ras and its downstream MAPK/ERK kinase pathway. Figure reprinted from Tybulewicz et al. Nat Rev Immunol. 2009 with permission from Nature Publishing Group (215).     41 The recognition of cognate antigen by the TCR results in the recruitment of lymphocyte-specific protein tyrosine kinase (LCK) (216). LCK then phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) found on the tails of TCR complex, allowing Zeta-chain-associated protein kinase 70 (ZAP70) to bind to the ζ chains (217). Subsequently, LCK phosphorylates and activates ZAP70, which phosphorylates the T lymphocyte adaptor protein (LAT) that serves to recruit other signaling molecules such as PLCγ1 (218) and initiates various signaling cascades, including the Ras pathway (219), the AKT pathway (220), the protein kinase Cθ (PKCθ) pathway (221), and calcium mobilization (222). The activation of these signaling pathways results in enhanced cell proliferation, increased IL-2 production and cell survival.  1.1.3.8 B cells B cells are named after the organ, bursa, where the development of these cells was first observed in chicken (58). In mammals, B cells are generated from pluripotent hematopoietic stem cells in the bone marrow. Similar to TCRs expressed on T cells, each B cell has a unique B cell receptor (BCR) expressed on its surface that allows it to specifically bind to a particular antigen (223, 224). However, instead of detecting antigens that have been processed and presented on the MHC molecules by APCs, BCRs recognize native proteins that can be found on the surface of or are secreted by pathogens.  The BCR consists of two heavy chains and two light chains, with each chain consisting of variable (V) and constant (C) regions (225). There are two types of light chains, lambda (λ) and kappa (κ), and five types of heavy chains, alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (µ). Each of which could be chosen as a part of the BCR. The V-regions of heavy-chain and light-chain genes are composed of discrete segments that are joined by somatic recombination during  42 B cell development. Along with the BCR gene rearrangement, B cells receive signals from stromal cells in the bone marrow to undergo differentiation, which directs the expression of other surface proteins displayed on B cells (226).  After successful gene rearrangement, B cells further undergo selection to eliminate self-reactive BCRs in the bone marrow (227). If the developing B cells recognize self-molecules during selection, they can receive signals to undergo cell death by apoptosis, receptor editing, permanent anergy or ignorance. The selection outcome depends on the nature of the ligands and the binding affinity of the BCRs. B cells that recognize multivalent self-molecules would undergo either clonal deletion or receptor editing to change the receptor specificity. On the other hand, binding with soluble self-molecules that weakly cross-link the BCR usually leads to unresponsiveness and short life span. B cells that recognize non-cross-linking self-molecules with low affinities remain temporarily ignorant. B cells that survive the selection process will subsequently go into the periphery. Upon antigen recognition and receiving stimulatory signals from T helper cells, naïve B cells proliferate and differentiate into antibody-secreting plasma cells (228, 229). Antibodies are the effector products of adaptive humoral immunity, which share the same structure and antigen specificity with BCRs. There are several different antibody isotypes, including IgM, IgD, IgG, IgA, and IgE (230). Based on the cytokines secreted by T helper cells, B cells are instructed to undergo isotype switching and produce different types of antibodies. In addition, activated B cells undergo somatic hypermutation to generate higher affinity/avidity antibodies (231). This process introduces variations in the variable region of rearranged immunoglobulin, and leads to a selection process where B cells with deleterious mutations that cause them to lose their ability to bind their antigens fail to thrive. By contrast, B cells with mutations that improve their antigen- 43 binding affinity will survive through positive selection. Meanwhile, a small proportion of B cells are differentiated into long-lived memory B cells, which have the ability to survive for years. Memory B cells remain quiescent in normal conditions, but are able to generate much faster and more effective humoral responses if re-exposed to the same antigen (232-234).  Antibodies bind to the extracellular targets to neutralize and eliminate the antigens (235). Antibodies provide protection through three mechanisms: neutralization, opsonization and complement activation. Neutralization is the process in which antibodies bind to the infective pathogen, and prevent them from entering host cells. Antibody binding also promotes opsonization, where the coating of foreign particles with antibodies promotes their phagocytosis and subsequent destruction by macrophages and neutrophils. In addition, the binding of pathogens with antibodies activates the complement system, which triggers the degradation of the pathogens.  Although the primary function of antibodies is to eliminate pathogens, they have been widely used for medical diagnosis (236) and therapies in clinic (237). Detection of antibody titers or level of immunoglobulin classes against particular antigens is a very common method of medical diagnostics for bacterial infections, viral infections, blood typing and autoimmune disease. In addition, since transfer of antigen-specific antibodies into patients can provide a short-term immunity against specific diseases, antibody-based therapies have been employed as therapy against autoimmune diseases, infectious diseases and cancers.      44 1.1.3.8.1 B cells and antigen-presenting cells In addition to playing an essential role in humoral immune responses, B cells are also recognized as professional APCs that play a role in the initiation of T cell activation (238, 239). B cells constitutively express MHC class II molecules, and are able to upregulate the expression of co-stimulatory molecules upon activation. Extracellular proteins from bacteria or antigens bound by BCRs are internalized into intracellular vesicles and degraded into peptide fragments, where they are then loaded onto MHC class II molecules and transported to the B cell surface for the recognition by CD4 T cells. Previous reports have indicated that B cells are able to efficiently present antigens and promote antigen-specific T cell differentiation and function in vitro (240) and in vivo (239, 241).  The antigen-specific interactions between B cells and T cells are critical for CD4 T cell-priming. Importantly, the antigen-specific CD4 T cells also provide signals that help B cells to produce antibodies (242, 243). Upon recognizing the presented antigens and receiving co-stimulatory signals from B cells, naïve CD4 T cells become activated T helper effector cells. These activated CD4 T cells produce various cytokines and deliver contact-dependent signals to elicit B cell responses, inducing them to become antibody-producing plasma cells. Overall, the cognate interaction between B cells and T cells promote the responses of both cell types.  B cells also exhibit relatively higher expression of MHC class I molecules compared to other nucleated cells. They are able to process endogenous antigens and display them on MHC class I molecules for CD8 T cell-priming. In addition, B cells have the ability of “cross-presentation”, which allows them to process exogenous proteins and load peptides onto MHC class I molecules for the recognition by CD8 T cells. Previous reports have suggested that B cells can act as APCs to prime CD8 T cells in vitro, resulting in increased IL-2 production and  45 enhanced killing activity (244, 245). The role of B cells in CD8 T cell-priming in vivo remains controversial, where antigen-presenting B cells have been shown to induce immunogenic (246) or tolerogenic CD8 T cell responses (247, 248).  This discrepancy may result from differences in antigen concentrations and expression levels of co-stimulatory molecules.    1.2 Autoimmunity Autoimmune diseases occur when our immune system is unable to discriminate self from foreign antigens, causing an immune reaction that attacks and destroys self-tissues. These diseases are characterized by autoantibody-mediated (humoral) and T cell-mediated responses towards self-antigens. There are more than eighty types of autoimmune disease, including T1D, MS, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). The precise etiology of autoimmune diseases remains unknown; however, genetic, environmental and endogenous factors play important roles in disease development (Figure 1-9). These contributing factors are briefly discussed in this section using T1D as a main example of an autoimmune disease.        46   Figure 1-9 Etiology of autoimmune disease. Autoimmune disease is characterized as an abnormal immune response against self-antigens and self-tissue destruction. The development of autoimmune disease is thought to involve convergence of one or more influences that may vary between affected individuals and various diseases. Genome-wide association studies have identified key immunoregulatory genes being linked with several autoimmune conditions, establishing that immune dysregulation is a critical component of disease causation. In addition, environmental and endogenous factors are also key contributors to autoimmune pathogenesis. Venn diagram was adapted from a figure presented by Ermann and Fathman. Nat Immunol. 2001 (249).      EnvironmentAutoimmunediseaseGeneEndogenous FactorsGenderHormonesPregnancyAgeGut floraInfectionsDietVitamin DHLA-DRCTLA-4IL-7RIL-2R 47 Based on studies in T1D patients, which is characterized by autoimmune destruction of insulin-producing β-cells, associations between certain HLA genes and disease progress have been reported (250, 251). Interestingly, there is only a 35% risk of developing T1D in identical twins, even though they share the same genetic background. These findings imply that other factors besides genetics contribute to the onset of disease in susceptible individuals.  Environmental factors such as viral infections and altered gut flora, have been reported in triggering autoimmune diseases, such as T1D (252, 253). Upon viral infection, the immune system attacks virus-infected cells, causing damage to surrounding tissues and subsequently the release of autoantigens that can be recognized by autoreactive cells (254). It has also been proposed that molecular mimicry, in which viral antigens sharing sequence similarities with self-antigens trigger the cross-activation of autoreactive immune cells, can prompt the onset of an autoimmune disease (255). Interestingly, the presence of commensal gut flora is critical for the regulation of immune homeostasis (253). For example, increased T1D incidence has been observed in mice housed in the germ-free conditions (256), and aberrant composition of microbiome significantly influences the onset of T1D (257-259). A previous study has indicated that the microbiome of children with T1D genetic susceptibility is less diverse and stable compared to healthy controls (259). Therefore, gut microbiota-induced immunoregulation may also contribute to the control of T1D.  Gender differences have a great impact in the incidence of autoimmune disease, for example, many autoimmune diseases are more prevalent in women than in men (260). Although the mechanism behind these gender differences are largely unknown, endogenous hormones have been suggested to play a role in regulating the susceptibility of autoimmune diseases. For example, a previous report has indicated that the sexual hormone testosterone can influence the  48 composition of gut microbiome and in turn confers protection from autoimmune T1D (261). In summary, many factors have been suggested to contribute to the development of autoimmune diseases. Below, we will discuss another example of an autoimmune disease, multiple sclerosis, and particularly how Th17 plays a role in the progression of this disease and other autoimmune diseases.  1.2.1 Multiple sclerosis and experimental autoimmune encephalomyelitis MS is an autoimmune neurodegenerative disorder that affects the central nervous system (CNS), resulting in myelin destruction and axonal damage. Patients with MS may develop a wide range of symptoms, and experience physical and mental problems depending on the affected parts of the nervous system (262, 263). To date, there is no known cure for MS, and current medical treatments for MS are aimed at delaying disease progression. The underlying cause of MS remains unclear, but the combination of endogenous, genetic and environmental factors have been proposed to contribute to the development of the disease. The incidence of MS is approximately three times more common in women than men (264). In addition, genetic variations in several HLA regions have been linked to the susceptibility of MS (263, 265). Based on genome-wide association studies, single nucleotide polymorphisms (SNPs) in IL-2 and IL-7 receptors also influence the risks of MS (266, 267). Interestingly, the exposure of sunlight and the intake of vitamin D are strongly correlated to lower risk of MS (268, 269). Infectious agents have also been suggested to trigger the development of MS. People with no history of EBV infection have reduced risks of MS, and by contrast, individuals who have had EBV infections as young adults have increased risks of MS (270, 271).   49  Experimental autoimmune encephalomyelitis (EAE) is the most common mouse model for studying MS (272-275). Immunization with myelin peptide (myelin oligodendrocyte glycoprotein peptide, MOG) or myelin protein (myelin basic protein, MBP) along with adjuvants, including Complete Freund’s Adjuvant (CFA) and pertussis toxin, induces the migration of activated autoreactive T cells into CNS. Subsequently, inflammation, demyelination and neuronal death usually occur at 1 to 2 weeks after the induction of disease. During the progression of EAE, abnormal muscle tensions are observed starting from the tail towards the body, and a clinical scale is usually given based on the severity of this symptom. EAE shares some commonalities with MS in disease pathophysiology, including the characteristic of the inflammatory environment, and the distribution and mechanisms of myelin damage (276, 277). Some dissimilarity has also been pointed out between MS and EAE. For example, the incidence of MS is higher in women than in men, whereas gender does not influence the susceptibility to EAE (264, 278). Furthermore, MS is a complicated autoimmune disease that is driven by a combination of T cells, B cells and various immune cells (279, 280), whereas CD4 T cells primarily drive disease in EAE (281).       Several reports have shown that there are positive correlations between the cytokine IL-17, the number of Th17 cells, and disease progression (282-284). Th17 cells have been shown to infiltrate the CNS through the blood brain barrier (BBB), and subsequently promote lymphocyte infiltration and inflammatory cytokine production (285). The use of neutralizing IL-17 antibody (286) or IL-17a-/- mice (287) has strongly implied that IL-17 plays a causative role in the T cell-mediated model of MS called EAE. Although dispensable for the generation of IL-17-secreting T cell effectors, IL-23 has been shown to be essential for maintaining the stability and pathogenicity of Th17 effectors in both EAE and autoimmune diabetes, (186, 288).  50 Understanding the pathogenic role of Th17 cells in autoimmune disease may provide valuable information for improving therapeutic options.   1.2.2 IL-17-producing T cells in health and disease Th17 cells are pro-inflammatory cells providing protection against bacterial and fungal infections (289). However, Th17 effector cells have also been suggested to be pathogenic given their associations with sites of tissue inflammation and autoimmunity in psoriasis, RA, IBD, SLE, MS, and T1D (186, 187, 206, 208, 283, 290-296). Although much of the literature has focused on the role of IL-17-secreting effectors in the clearance of bacterial and fungal infections (185), recent evidence suggests that IL-17-secreting T cell effectors may mediate control of viral infections (188, 206, 297). Experimental infections of mice with influenza have shown that IL-17-producing T cells are generated upon repeat challenge and are protective against a lethal viral dose (206). In addition, IL-17 effector T cells have been found to be expanded by vaccinia virus and to mediate viral clearance through acquisition of a cytotoxic phenotype (188). Furthermore, patients with chronic hepatitis C virus (HCV) were found to have HCV-specific IL-17-producing T cells and those individuals with the highest cell frequencies of these T cells exhibited low inflammatory activity, suggesting a protective role for IL-17 in chronic HCV infection (297). Together, these findings indicate that IL-17-secreting T cells are expanded by viral infections and may play key functions in protective anti-viral immunity.  1.3 Epstein-Barr virus EBV is a prominent family member of Herpesviridae, derived from the Greek herpein "to creep" symbolizing this group’s pattern to cause latent and recurrent infections, and its  51 double stranded DNA genome is 172 kilobases in length, encoding about 85 genes (298). EBV is one of the most common viruses within humans, with greater than 90% of the adult population having been previously infected. EBV is a γ-herpesvirus that replicates and persists primarily in lymphocytes. Virus acquisition is common in childhood and subsequently, establishes lifelong residence.  EBV infection is often referred to colloquially as the “kissing disease” because it is most frequently transmitted by salivary contact although transmission may sometimes occur via genital secretions (299-301). Primary infection in children may be asymptomatic or produce subclinical flu-like symptoms. However, people infected with EBV in adolescence or early adulthood often develop infectious mononucleosis (IM) (302-304). IM, first described by Sprunt and Evans in 1920 as an infectious disease, consists of fever, sore throat, fatigue, rash, pharyngitis and cervical lymphadenopathy. In addition, IM is associated with a massive expansion of atypically blasting peripheral blood lymphocytes from where it gets its name (i.e. mononucleosis). Normally, mononuclear cells are vastly outnumbered by polymorphonuclear granulocytes) (Figure 1-10) (305). In 1967, EBV was determined to be the cause of IM (306) and later, its associated atypical lymphocytes were characterized as EBV-specific CD8 T cells (209, 210). The duration of IM is often 2 to 6 weeks and most people recover spontaneously (307). However, some people develop complications, such as airway obstruction and splenic rupture, during the primary infection, whereas others experience illness, fatigue and malaise, for many months (308, 309).      52   Figure 1-10 Blood smear from a patient with infectious mononucleosis. Blood smear showing atypical activated lymphocytes from patient with infectious mononucleosis. The morphology of these atypical mononuclear cells resembles abnormal lymphocytes or monocytes. “This image was originally published in ASH Image Bank. John Lazarchick. Infectious mononucleosis-2. ASH Image Bank. 2010; #00001049. © the American Society of Hematology.” Figure reprinted with permission from American Society of Hematology.       53 EBV has a tropism for B cells, preferentially infecting mature B cells that eventually transit into the memory B cell pool (310). The EBV life cycle includes both lytic and latent phases and each phase is associated distinct patterns of viral gene expression (302, 311-314). The lytic phase, taking place in both epithelial cells and B cells, produces infectious virions and facilitates viral spread. By contrast, the latent phase does not yield infectious particles and may occur in epithelial cells or B cells, serving to maintain lifelong persistence in the host. During latency, only a portion of EBV’s genes are expressed, perhaps functioning to avoid host immune surveillance. Moreover, EBV can exhibit three different patterns of latent viral gene expression or programs called latent phase I, II or III (Lat I, Lat II or Lat III).  During primary infection, orally transmitted viruses infect both epithelial cells and local B cells within the oropharynx, and enter a lytic cycle (Figure1-11 and 1-12) (315). The resulting rapid replication leads to high levels of virus shedding in the throat (316). EBV can also initiate a latent cycle, Lat III, which leads to the activation and transformation of B cells (317). The EBV-transformed B cells circulate in blood and lymphoid organs, and induce the activation and expansion of innate NK cells and EBV-specific T cells, resulting in the development of IM. The incubation period between initial exposure and onset of symptoms is approximately 6 weeks, in which patients are infectious and usually unaware of the disease (318). During the convalescence of IM, most of the expanded T cells die through apoptosis, and only a small number of EBV-specific T cells survive to become memory T cells (319). To escape immune surveillance, EBV downregulates their gene expression and enter latent phase 0 (Lat 0) after the primary immune response (317). The EBV-infected B cells that have differentiated into memory B cells then remain quiescent, and they persistently circulate in the host as a long-term carrier. Occasionally, the EBV that latently infect memory B cells reactivates and enters the lytic cycle, resulting in a  54 low level of virus shedding into the oral cavity or the initiation of a new cycle of infection (320-322).           55   Figure 1-11 A schematic view of Epstein-Barr virus infection of an immunocompetent host. Oral transmission of EBV establishes a lytic infection of oropharyngeal epithelial cells and the shedding of virus from this compartment possibly infecting local B cells (blue arrows). Subsequently, EBV initiates growth-transforming infections of B cells (Latency III type viral program, abbreviated “Lat III”) from nearby lymphoid tissues and B cell proliferation ensues. The primary immune response activates natural killer (NK) cells and generates large numbers of CD4 and CD8 T cells, targeting lytic and latent viral antigens (wide gray arrows). Although the anti-viral immune response limits the expansion of EBV-positive B cells, some EBV-infected B cells may escape immune recognition by down-regulating viral antigens and eventually, creating a viral reservoir of memory B cells lacking expression of EBV antigens (Latency 0 viral program, abbreviated “Lat 0”) that may or may not be dependent on a germinal center (GC) reaction. Occasionally,  “Lat 0” latently infected B cells may be reactivated into lytic cycle, perhaps through antigen (Ag)-dependent stimulation or other signals, resulting in new rounds of replication and shedding from oropharyngeal epithelium. Alternatively, newly reactivated lytic B cells may infect adjacent B cells triggering the production more virions and the expansion of transformed B cells. CD4 and CD8 memory T cells, specific for lytic and latent antigens, serve to limit EBV outbreaks and viral spread. Figure reprinted from Taylor et al. Annu Rev Immunol. 2015 with permission from Annual Reviews (311).   56   Figure 1-12 Relationship between T cell immune responses and viral replication during EBV infection. Schematic diagram represents the kinetics of the viral replication and antigen-specific T cell responses during incubation period, primary infection (acute and convalescence infectious mononucleosis, IM), long-term carrier state and following immune suppression. Upon EBV infection, viral replication occurs primarily within throat and only upon development of acute IM does virus become readily detectable among blood cells. A massive expansion of CD8 T cells targeting EBV’s lytic antigens develops early during acute IM whereas a blunted CD8 T cell responses against EBV’s latent antigens peaks later. During convalescence from IM, EBV-positive cells in peripheral blood decline along with the EBV-specific T cell numbers. The frequency of EBV-positive cells remains very low within healthy carriers. Observations that EBV-positive cells climb rapidly upon immunosuppression argue that EBV-specific T cells are critical maintaining viral control. Figure reprinted from Hislop et al. Annu Rev Immunol. 2007 with permission from Annual Reviews (302).  57 EBV has a substantial transforming potential in vitro and in vivo. In vitro, this transforming potential can be utilized to generate immortalized B cells known as lymphoblastoid cell lines (LCLs) (323), whereas in vivo, EBV-induced transformation is thought to cause certain types of cancer, including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and immunoblastic lymphomas (210, 302, 304, 324). In fact, EBV was first discovered from cultured Burkitt’s lymphoma cells (324).  Burkitt’s lymphoma is characterized by the presence of malignant B cell tumors in the lymphatic system, particularly in germinal centers. There is a strong positive correlation between the incidence of Burkitt’s lymphoma and the rate of EBV infection in equatorial Africa, where patients with Burkitt’s lymphoma are all EBV positive (325). Thus, the latent EBV-induced B cell transformation is thought to contribute to tumorigenesis. Although EBV is considered as a benign pathogen for most healthy adults (but not in adolescents, as they develop mononucleosis), it is considered a threat for immunosuppressed or immunocompromised patients (Figure 1-12). Previous reports have indicated that T cell-immunodeficient patients are at high risk of developing EBV-associated B cell lymphomas. These high risk populations include patients with acquired immune deficiency syndrome (AIDS) (326), regimens of immunosuppressive drugs (327), and primary or combined variable immunodeficiencies, such as X-linked lymphoproliferative (XLP) disease (328, 329). Mechanistically, since T cells play an essential role in controlling viral infection, impaired T cell function results in uncontrolled proliferation of EBV-transformed B cell.      58 1.3.1 Immune responses against EBV infection NK cells, CD8 T cells and CD4 T cells all have key roles in the immune response against EBV infection. Previous studies have indicated that NK cells mediate an early innate immune response against EBV infection. Patients with IM have significantly increased numbers of NK cells upon primary EBV infection (Figure 1-11) (330), and there is a strong inverse correlation between NK cell numbers and viral loads. Notably, expanded functional NK cells have been observed to target EBV-infected cells prior to the initiation of CD8 T cell responses in a humanized mouse model (331, 332). Together, these observations suggest that NK cells are responsible for the early control of EBV infection. The role of CD8 T cells in controlling primary EBV infection has been well documented (Figure 1-11 and 1-12). Dramatic expansions of EBV-specific CD8 T cells have been found in the periphery of IM patients. Notably, more than 50% of them recognize antigens expressed in the lytic cycle of EBV, while a small frequency of CD8 T cells respond to latent EBV epitopes (302, 333). This phenomenon is thought to be because EBV is in its lytic cycle during primary infection, and suggest that cytotoxic CD8 T cells mediate cell-contacted killing to eliminate lytically infected B cells (334, 335). Until recently, the role of CD4 T cells in primary EBV infection has not been investigated due to the lack of tools for detecting epitopes on MHC class II molecules. Although EBV-specific CD4 T cells are elevated in IM patients, the size of CD4 T cell expansion is smaller than CD8 T cells (Figure 1-11 and 1-12). In addition, the epitopes that CD4 T cells recognize are distinct from those that CD8 T cell respond to, and CD4 T cells recognize a broader range of antigens displayed in both lytic and latent phases compared to CD8 T cells (336-338).   59 After controlling the primary infection, the expanded CD4 and CD8 T cells are culled, and only few EBV-specific T cells survive to become memory T cells circulating in the blood. During the long-term carrier stage, EBV can enter either a lytic cycle to replicate in the oropharyngeal epithelium or enter latency to induce B cell transformation, and the EBV-specific memory CD4 and CD8 T cells migrate to tonsillar tissues and blood to tightly control the persistent EBV infection (Figure 1-11 and 1-12) (339).  1.4 X-linked lymphoproliferative disease Primary Immunodeficiencies are defined a set of disorders in which the immune system is broken or completely absent from birth and are caused by mutations to genes essential for normal immune function. XLP, known first as Duncan’s disease, is a rare primary immunodeficiency (~1 in million males) that was reported by Dr. David Purtilo and colleagues in 1975, based on the Duncan kindred (Figure 1-13) (328, 329). Within the Duncan kindred, several (6/18) maternal male cousins born within one generation died of a lymphoproliferative disease whereas none of their sisters died of this illness. XLP disease is often brought on by EBV infection resulting in fulminant infectious mononucleosis (FIM), haemophagocytic lymphohistiocytosis (HLH), B cell lymphomas or dysgammaglobulinemia. Strikingly, affected males do not display vulnerabilities to other herpesviruses, specifically cytomegalovirus (CMV) and varicella zoster, that can cause serious complications in immunosuppressed individuals. Consequently, XLP males exhibit a highly exquisite susceptibility to EBV but not other pathogens. Although the article by Purtilo et al. is often considered the original account of XLP, two reports by lead authors Dr. Robert Bar and Dr. Arthur Provisor, published in 1974 and 1975  60 respectively, described similar clinical features associated with EBV infection in several male siblings and maternal cousins from two different families (340, 341).                61   Figure 1-13 X-linked lymphoproliferative disease: Duncan’s disease The pedigree of the Duncan kindred is shown. Of the 18 males born in third generation of the Duncan kindred, 6 died of a lymphoproliferative disease whereas no girls were affected. The penetrance of disease within males but not females suggested that the disease is linked to the X chromosome. Figure reprinted from Putilo et al. Lancet. 1975 with permission from Elsevier (328).           936Fig. 1-Pedigree of the Duncan kindred.affected boys lived in Mas chusetts, T xas, and Cali-fornia. The occurrence of affected half-brothers (nos.36 and 37) from separate fathers (nos. 8 and 10) isnotewo thy. Their mother (no. 9) was married fourtimes (nos. 6, 7, 8, and 10). Clinical findings of 5 ofthe affected males and haematological and pathologicalfindings and a brief clinical description of a malecousin (no. 54) who may be affected are described herein detail.FIRST CASEA 23-month-old White boy (pedigree no. 44) was the3rd of 7 sons in this sibship to die of the fulminatinglymphoproliferative disease. He was admitted to theMemorial Hospital of Worcester in 1973 with fever, cough,a disseminated maculopapular rash, and jaundice. Duringthe previous year he had suffered from recurrent upperrespiratory infections and diarrhoea. He was a well-developed small boy with hepatosplenomegaly, jaundice,and generalised lymphadenomegaly.2 weeks before admission to hospital the patient’shaemoglobin was 11.8 g. per 100 ml., haematocrit 34%,white blood-cells 11,300 per c.mm, (bands 17%, neutro-phils 43%, lymphocytes 37%, moncytes 2%, and eosino-phils 1 %). 2 weeks later his haemoglobin and haematocrithad declined to 10.3 g. per 100 ml. and 30%. The white-blood-cell count was 21,300 per c.mm. (atypical lympho-cytes 61%, lymphocytes 17%, monocytes 8%, eosinophilsFig. 2-Case 1: lymph-node biopsy.Two enlarged germinal centres are separated by a thin fibrousseptum extending from the intact capsule (H. & E., reduced by athird from x 200).1 %, bands 3 %, neutrophils 10%), platelets were adequate.Many of the atypical lymphocytes were Downey type mcells.A MONOS POT ’ determination (Ortho Diagnostics) wasnegative but became strongly positive a week later. SerumIgG was 1200 mg., IgA 620 mg., and IgM 1000 mg. per100 ml. Thus polyclonal hypergammaglobulinsemia waspresent. Cultures of sputum and blood revealed nopathogens and a Coombs’ test was negative.A bone-marrow aspirate showed normal hxmatopoieticactivity. Cervical lymph-node biopsy revealed markedlyexpanded germinal centres, numerous mitotic figures, andlymphoid cells with prominent nucleoli; histiocytes werepresent (fig. 2). Touch imprints revealed numerous plasmacells and plasmacytoid lymphocytes. The immunoperoxi-dase marking technique 8 demonstrated numerous plasmacells in the lymph-node that reacted for IgM and IgG.Treatment with prednisone, mercaptopurine, and vin-cristine was begun because the boy was thought to have arare form of reticuloendotheliosis. His condition steadilyFig. 3-Case 1: post-mortem appearance of cerebral cortex.Perivascular plasma-cell and lymphocyte infiltration areevident (H. & E., reduced by a third from x 450).deteriorated; convulsions progressed to coma; and he diedon the 15th hospital day, 30 days after onset of illness.At necropsy, massive hepatosplenomegaly was present:the liver weighed 850 g. (394 g. normal) and spleen 150 g.(33 g. normal). Lymph-nodes were of normal size, butlacked lymphoid nodules and germinal centres. Prominenterythrophagocytosis and nucleophagocytosis were noted.Numerous plasmacytoid lymphocytes, mature plasma cells,and foamy histiocytes, many containing erythrocytes, werenoted in the lymph-nodes, spleen, and bone-marrow. Thethymus was not studied microscopically, but appeared" normal " on gross examination. The lymphoid sheathsof. the spleen were necrotic, and numerous large plasma-cytoid cells had surrounded the central arterioles. A rimof hyaline was deposited at the junction where the whiteand red pulp normally abut.Mature plasma cells which were secreting IgM andIgG, and lymphocytes, were present in perivascular spacesof the cerebral cortex (fig. 3). The meninges, heart, andliver were cedematous and also infiltrated by lymphoid cells.Within hepatic sinuses, large vacuolated macrophages werepresent.SECOND CASEAn 8-year-old boy (pedigree no. 41) was admitted tothe Boston Children’s Hospital Medical Center in 1969 62 In 1998, three independent groups identified loss of function mutations within SH2D1A (SH2-domain containing 1A) gene, located on the X chromosome, being responsible for the XLP phenotype (342-344). SH2D1A encodes the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), an intracellular adaptor protein that serves to link cell surface SLAM receptors with downstream signaling components. SAP is expressed in most lymphocytes including NK cells, NKT cells, CD4 T cells and CD8 T cells whereas its presence within B cells is controversial. An improved understanding of SAP along with further investigation of XLP patients have revealed that their susceptibility to dysgammaglobulinemia and B cell lymphomas is independent of EBV infection (345-347). Moreover, SAP-deficiency is responsible for dysgammaglobulinemia because SAP expression within helper CD4 T cells is critical for B cell differentiation including GC formation, class Ig switching and formation of memory B cells (348-351). By contrast, the vulnerability of XLP patients to B cell lymphomas is less well understood. SAP has been purported to function as a tumor suppressor gene through interactions with p53 promoting B cell apoptosis (352, 353). Alternatively, it is possible that SAP expression within T cells and NK cells is critical for immune surveillance of pre-cancerous B cells. The diagnosis of XLP at an early age prior to EBV exposure is key to avoid complications and sequelae related to infection. XLP resembles other primary immunodeficiencies in that hematopoietic stem cell transplantation (HSCT) has been considered the only option of curing disease (354). However, the previous development of HLH following EBV infection is a major risk factor for XLP patients surviving HSCT therapy. Clinical management of XLP has employed a range of agents such as conventional anti-viral drugs, immunosuppressive agents, immunoglobulin replacement therapies and chemotherapeutics (to combat lymphoma). However, a novel strategy has incorporated the use of Rituximab (anti- 63 CD20 antibody that targets B cells) to eliminate infected B cells upon primary EBV infection (354). Although this report involved a small sample size, the results so far have shown great promise, reducing viral loads and resolving symptoms of FIM. Together, these findings support the notion that defects in the recognition of infected B cells by NK or T cell effectors underlie the susceptibility of XLP patients to EBV. The role of SAP regulating immune cell development and function will be discussed in more detail in Section 1.4.3. The XLP discussed above is also known as XLP type 1 (XLP-1) to differentiate it from another form of XLP, called XLP type 2 (XLP-2), that shares clinical similarities but is caused by mutations in BIRC4 gene encoding X-linked inhibitor of apoptosis (XIAP) (355, 356). Moreover, XIAP deficiency results in FIM and is linked with a high incidence of HLH (357-359). In addition, XLP-2 patients resemble XLP-1 patients in that they have also been found to lack NKT cells (355), lending additional support to the hypothesis that NKT cell lineage is essential for controlling EBV infection. However, the phenotype of XLP-1 and XLP-2 patients possess some significant discrepancies: (i) XLP-1 males are susceptible to the development of B cell lymphomas whereas XLP-2 patients are not (360) and (ii) XLP-2 patients display an increased incidence of IBD while XLP-1 individual do not show this tendency (361, 362). How mutations in SAP and XIAP lead to abnormal EBV immune responses and whether XIAP may signal downstream of SLAM receptors is unclear. Together, the investigation of SAP and XIAP function should lead to better understanding of immunity towards EBV and improved treatment options for EBV-associated disease.    64 1.4.1 Structure of SLAM-associated protein SAP is a small adaptor, consisting of 128 amino acids and containing a single Src homology 2 (SH2) domain involved in binding phosphorylated tyrosine residues (342-344). SAP expression is present within most lymphocytes including NK cells, NKT cells, CD4 T cells and CD8 T cells but appears to be lacking from most B cells (349, 363). SAP binds to the immunoreceptor tyrosine-based switch motifs (ITSM; TxYxxI/V, “x” signifies any amino acid) found in the cytoplasmic regions of SLAM family receptors. Ligation of SLAM family receptors by self family ligands results in tyrosine phosphorylation of their ITSMs, and the subsequent conscription of SAP may modulate signaling by two distinct means: (i) SAP transmitting positive signaling by recruitment of the tyrosine kinase Fyn and PKC-θ (364-367) and (ii) SAP interfering with negative signaling by sterically impeding the docking of SH2-containing phosphatases SHP-1, SHP-2 and SHIP-1 (344, 364, 366, 368-370) (Figure 1-14). Therefore, SAP may function by both promoting positive signaling and blocking inhibitory signaling by SLAM family receptors.   65   Figure 1-14 SAP regulation of T cell receptor signal transduction. The recruitment of SAP may regulate signaling by two distinct mechanisms: (i) SAP transduction of signaling by binding the tyrosine kinase Fyn and (ii) SAP inhibition of signaling by sterically hindering the docking of SH2-containing phosphatases SHP-1, SHP-2 and SHIP-1. Figure adapted from Dong and Veillette. Trends Immunol. 2010 (366).          66 SLAM family receptors have diverse functions and can act independently of SAP. Moreover, there exists two SAP-like adaptors called Ewing’s sarcoma-associated transcript 2 (EAT-2, encoded by Sh2d1b) and EAT2-related transducer (ERT, encoded by Sh2d1c) that share strong homology and are, comprised of a single SH2 domain and a short C terminus (371, 372). EAT-2 has been found in NK cells, DCs, macrophages and platelets. Murine ERT is expressed only in NK cells while ERT in humans is a pseudogene and not transcribed.  EAT-2 and ERT mediate signaling through phosphorylation of key tyrosine residues within the C-terminal domain, coupling SLAM family receptors to Src family kinases. Consequently, EAT-2 and ERT, trigger signals through mechanisms distinct from those employed by SAP (372-374).   1.4.2 The signaling lymphocyte activation molecule receptor family The SLAM receptor family, which is primarily expressed by hematopoietic cells, consists of seven members: SLAM (SLAMF1, CD150); CD48 (SLAMF2); Ly9 (SLAMF3, CD229); 2B4 (SLAMF4, CD244); CD84 (SLAMF5); NTBA (SLAMF6, NK-T-B-antigen in humans; Ly108 in mice); and CD2-like receptor-activating cytotoxic cells (SLAMF7, CRACC). Genomic studies have determined that genes encoding SLAM family receptors are located in the same cluster in Chromosome 1. Given that the SLAM family receptors SLAM, 2B4 and CD48 are highlighted in the research chapters of this thesis, these receptors are described in depth in this section.  The structure of each member of the SLAM family receptors include an immunoglobulin-like extracellular domain and a cytoplasmic tail, as shown in figure 1-15 (375). The extracellular domain of SLAM receptors is composed of an amino-terminal Ig variable (V)-like domain and a membrane-proximal C2 domain, with the exception of Ly9 (SLAMF3), which contains a duplicated variable-C2 domain (Figure 1-15) (376, 377). The cytoplasmic region of SLAM  67 receptors consists of one or more ITSMs with high binding affinity for SAP or SAP-like adaptors (344, 373).  The extracellular domains of SLAM family receptors are self-ligands, involving homotypic (eg. SLAM-SLAM) interactions except for heterotypic interactions between 2B4 and CD48. These associations result in the recruitment of SAP or SAP-related adaptors and the initiation of signaling cascades that drive immune cell activation and differentiation (364, 366, 378). To study the signal transduction pathways activated downstream of SLAM receptors, SLAM receptor-deficient mice have been produced, and the phenotypes of these knockout mice are listed in table 1-1.     68   Figure 1-15 SLAM family receptors in mice and humans. Mouse (a) and human (b) SLAM receptor family members are type I glycoproteins, composed of SLAM, 2B4, CD84, Ly108/NTB-A, Ly9, and CRACC. SLAM receptors contain a N-terminal Ig V-like domain, a membrane-proximal C2 domain and cytoplasmic tails. The cytoplasmic tails include at least one immunoreceptor tyrosine switch motif (ITSM), involved in the binding of SAP and EAT-2, plus additional tyrosine residues. Figure reprinted from Cannons et al. Annu Rev Immunol. 2011 with permission from Annual Reviews (364).  69 Table 1-1 SLAM family receptor signaling and phenotype of SLAM family receptor-deficient mice.    SLAM receptor family is composed of 7 members and each SLAM receptor, except CD48 (glycosyl-phosphatidylinositol-anchored protein), contains one or more immunoreceptor tyrosine-based switch motif (ITSM) domains (second column). The SLAM family receptors control immune cell development, differentiation and function through regulation of hematopoietic cell: cell interactions and recruitment SAP as well as other downstream effectors (third column). The immune defects in the SLAM receptor-deficient mice (right column) implicate unique (non-redundant) roles for SLAM family receptors in various immune cells. Table reprinted from Cannons et al. Annu Rev Immunol. 2011 with permission from Annual Reviews (364).       IY29CH22-SchwartzbergARI7February201122:42Table 2 Signal transduction pathways activated downstream of SLAM receptor family members and phenotype of mice deficient in SLAM family membersaReceptor ITSM Effectors Phenotype of knockout mice ReferencesSLAMF1SLAMCD150Human: 2Mouse: 2Thymocytes: SAP, Fyn, SHIP, Dok1, Dok2,SHC, Ras-GAPT cells: SAP, Fyn, Akt, PKCθ, NF-κB, SHP-1,SHP-2B cells: Fgr/Lyn, SHIP, SHP-2, AktPlatelets: SAPMacrophages: Vps34-Vps15-beclin complexSlam−/−CD4 T cells: ↓ IL-4 and IL-13, ↑ IFN-γ productionGC TFH cells: ↓ IL-4 productionMacrophages: ↓NO, IL-12, TNF-α production,↑IL-6 production, ↓ phagosome maturationPlatelets: ↓ aggregationNKT development: in conjunction with Ly10814, 18, 44, 59, 61, 67, 68,87, 92, 101, 120, 140,148, 150, 152, 154, 155,161, 211, 299SLAMF2CD48None Cd48−/−T cells: ↓ proliferation and IL-2 production300SLAMF3Ly9CD229Human: 2Mouse: 2T cells: SAP, Fyn, Grb2, µ2 AP-2, ERK,SHP-2Ly9−/−T cells: ↓ proliferation, ↓ IL-4 and IL-2 production63, 66, 116, 140, 153, 180SLAMF42B4CD244Human: 4Mouse: 4NK cells: SAP, EAT-2/ERT, Fyn, LAT Vav1,CBL, PI3K, Ca2+ Flux, ERK1/2, 3BP2, CSK,SHP-1, SHP-2, SHIPCD8 T cells: SAP2b4−/−NK cells: ↑ cytotoxicity52, 62, 78, 80, 82, 85, 118,139, 149, 160, 164, 165,167, 169, 170, 172–174,195SLAMF5CD84Human: 2Mouse: 2T cells: SAPB cells: EAT-2, SHP-1, SHP-2Cd84−/−T-B cell adhesion, impaired GC, ↓ TFH cells,↓ IL-21 production57, 83, 140, 142, 182SLAMF6(CD352)Human:NTB-AMouse: Ly108Human: 2Mouse: 2Thymocytes: SAP, Fyn, Vav1, CBLT cells: Ca2+ flux, SAP, SHP-1B cells: Ca2+ fluxNK cells: SAP, SHP-1Ly108(∆2+3)/(∆2+3)T cells: ↓ IL-4 productionNeutrophils: ↓ ROS production, ↑ IL-12, IL-6,TNF-α production, ↓ bacterial killingNKT cells: ↓ cell numberCD4 T: slight ↓ cell number27, 64, 69, 87, 90, 123,124, 127SLAMF7CRACC, CS1,CD319Human: 1Mouse: 1NK cells: EAT-2, PLCγ1, PLCγ2, Ca2+ FluxBeads: SHP-1, SHP-2, Fyn, SHIP, CSKCracc−/−NK cells: ↓ killing targets, ↓ Ca2+ FluxCD4 T cells: ↓ IL-2, ↓ IFN-γ, ↓ proliferation54, 70, 71, 301aAbbreviations: 3BP2: Abl-SH3 binding protein 2; CBL: casitas B-lineage lymphoma; CD4 T: thymocyte-selected CD4 T cells; CSK: COOH-terminal Src kinase; Dok1/2: Docking protein1/2; EAT-2: Ewing’s sarcomas EWS/FLI1 activated transcript-2; ERT: EAT-2-related transducer; Grb-2: growth factor receptor–bound protein 2; LAT: linker for activated T cells; NF-κB:nuclear factor kappa-light-chain-enhancer of activated B cells; PI3K: phosphoinositide 3-kinases; PLCγ: phospholipase Cγ; SAP: SLAM-associated protein; SHC: Src homology 2 containing;SHIP: SH2-containing inositol polyphosphate 5-phosphatase; SHP-1/2: SH2 domain–containing phosphatase 1/2; SLAM: signal lymphocyte activation molecule.www.annualreviews.org•SLAMFamilyReceptorsandSAPAdaptors669Annu. Rev. Immunol. 2011.29:665-705. Downloaded from www.annualreviews.org Access provided by University of British Columbia on 05/31/15. For personal use only. 70  Both human and mouse SLAM (SLAMF1, CD150) contain 2 ITSMs, and are expressed in thymocytes, T cells, B cells, platelets and macrophages. SLAM demonstrates homotypic binding, and the receptor functions as a co-stimulatory molecule to promote T cell differentiation and function (379, 380). SLAM-deficient CD4 T cells stimulated by anti-CD3 antibodies exhibit a significant reduction in IL-4 production (381). Furthermore, reduced allergic responses have been observed in SLAM-deficient mice as compared to wild type mice, including reduced inflammation and decreased allergen-induced Th2 cytokine production (IL-4, IL-10 and IL-13) (382). Interestingly, similar defects have also been reported in SAP-deficient mice (383), suggesting that SLAM signaling mediates Th2 cell differentiation and function through a SAP-dependent pathway.    2B4 (SLAMF4, CD244) is mainly expressed in NK cells and memory CD8 T cells (384, 385). Murine 2B4 consists of 2 isoforms, a long 2B4 isoform (2B4-L) and a short 2B4 isoform (2B4-S), whereas humans only express 2B4-L (386). The only difference between the 2 isoforms is their cytoplasmic tails, with 2B4-L bearing 4 ITSMs and 2B4-S only 1 motif. The difference in ITSMs between the two isoforms may influence the binding of SAP or SAP-related adaptors, resulting in differences in signal transduction. The ligand for 2B4 is CD48, and their heterotypic interaction has been proposed to either activate or inhibit NK cells. CD48-2B4 engagement increases IFN-γ production and cytotoxicity of NK cells (387). By contrast, 2B4-deficient NK cells exhibit enhanced IFN-γ production and cytotoxicity towards CD48-positive targets, suggesting that 2B4 has an inhibitory role (388). The dual function of 2B4 in mediating NK cell cytotoxicity is dependent on the density of CD48 ligand, the expression of 2B4 receptor, and the presence of SAP or SAP-like adaptors (389). Interestingly, 2B4 also plays a critical role in  71 mediating the cytotoxicity of CD8 T cells against CD48-positive targets in a SAP-dependent manner (390).  CD48 (SLAMF2) is a glycosyl-phosphatidylinositol-anchored protein, which shares homology of its extracellular domain with other SLAM family receptors, but does not contain any ITSMs (391). This receptor is expressed in all hematopoietic cells (392), and its natural ligands are CD2 and 2B4 (393). Interestingly, EBV-derived B cell lymphoblasts have been described to express higher levels of CD48 compared to EBV-negative resting B cells (394). CD48 plays a role in CD4 T cell activation and in hematopoietic progenitor cell function (395, 396). For example, CD48-deficient CD4 T cells have decreased cell proliferation compared to wild type CD4 T cells (396). Moreover, CD48-deficient mice develop autoantibodies, glomerulonephritis and lymphomas at early ages (395).   1.4.3 SLAM family receptor-SAP signaling regulates immune responses XLP patients and SAP-deficient (Sh2d1a-/-) mice share a number of immune cell deficits (Figure 1-16). SAP-deficient mice have severely impaired B cell differentiation resulting in diminished immunoglobulin levels, defective isotype switching, and a lack of GCs and memory B cells relative to wild type mice (364). Interestingly, the impaired B cell differentiation and humoral responses in SAP-deficient mice have been attributed to a fault in CD4 T cell differentiation rather than to an intrinsic B cell defect (348, 349). In support of this hypothesis, adoptive transfer of wild type CD4 T cells can rescue SAP-deficient B cell responses, and generate of long-lived memory B cells and plasma cells. Overall, previous studies have suggested that SLAM family receptor-SAP signaling plays a role in the differentiation and function of CD4 T cells, NKT cells, NK cells and CD8 T cells.  72   Figure 1-16 Immune cell deficits in patients with X-linked lymphoproliferative disease. SLAM receptors and SAP signaling influences lymphocyte: lymphocyte homotypic interactions, development and function. Mutations in the Sh2d1a, encodes SAP adaptor protein, result in a primary immunodeficiency called X-linked lymphoproliferative disease (XLP). Patients with XLP exhibit defects in various types of immune processes, including the development of natural killer T (NKT) cells and T follicular helper (Tfh) cells, formation of germinal centers (GCs) and B cell memory, and cytotoxicity of NK cells and CD8 T cells. Figure reprinted from Cannons et al. Annu Rev Immunol. 2011 with permission from Annual Reviews (364).         73 More recent studies indicate that the expression of SAP in CD4 T cells is important for sustained CD4 T cell-B cell contact, which is necessary for the generation of Tfh cells and the formation of GC (350, 351). Impairments of Tfh differentiation, GC formation and T-B cell adhesion have been reported in SLAM (Slamf1)-, CD84 (Slamf5)- and Ly108 (Slamf6)-deficient mice, implying that SLAM family receptor signaling modulates the interaction between CD4 T cells and B cells, and subsequently regulates the differentiation and function of Tfh (350, 397, 398). In addition, SAP has also been shown to play a critical role in regulating CD4 T cell differentiation both in vitro and in vivo (383). SAP-deficient CD4 T cells have significantly decreased IL-4 production compared to wild type CD4 T cells upon in vitro TCR stimulation, and the levels of Th2 cytokines (IL-4, IL-5 and IL-13) are remarkably reduced in SAP-deficient mice compared to wild type mice upon Leishmania major infection.  In addition to its effect on CD4 T cells, SAP is also essential for the development of NKT cells (399-401), an innate-like T cell subset that is capable of producing IFN-γ and IL-4, and plays a role in tumor immunity (102, 121, 402, 403). The absence of NKT cells has been found in the thymus and peripheral organs of SAP-deficient mice, and in the peripheral blood of XLP patients. Studies involving mice deficient in SLAM (Slamf1), Ly108 (Slamf6), and both SLAM and Ly108 suggest that both of these SLAM family receptors play a role in controlling NKT cell development (381, 404, 405). Mechanistically, the interaction of SLAM receptors between thymocytes recruits SAP and subsequently transduces signaling via the tyrosine kinase Fyn, resulting in the recruitment of molecules to the immunological synapse, and activation of downstream mitogen-activated protein kinase (MAPK), NF-κB and calcium-flux pathways (406).  SLAM receptor-SAP signaling thereby provides the co-stimulatory signals necessary for NKT cell development (Figure 1-17) (406). Similarly, the engagement of TCR-MHC complex  74 along with CD4- or CD8-coreceptor activates the tyrosine kinase, LCK. Both Fyn and LCK, share similar downstream signaling molecules but affect different pathways.         75   Figure 1-17 SLAM receptor-SAP signaling controls NKT cell development. SLAM receptor-SAP signaling affects TCR signal transduction, mediating NKT cell development in the thymus. The engagement of SLAM family receptors between thymocytes functions as co-stimulatory molecules to recruit SAP and activate the tyrosine kinase Fyn. Subsequently, the activation recruits downstream molecules to the TCR immunological synapse and promotes TCR signal transduction. The downstream signaling involves three main pathways, mitogen-activated protein kinase (MAPK), NF-κB and calcium-flux pathways that are critical in regulating positive selection and NKT cell maturation. Figure reprinted from Godfrey et al. Nat Immunol. 2010 with permission from Nature Publishing Group (406).     76 The accumulation of EBV-infected B cells and uncontrolled B cell lymphomas in XLP patients raise the question of whether SAP deficiency affects the cytotoxicity of NK and CD8 T cells. A previous study has shown that NK cells from XLP patients exhibit impaired killing of EBV-positive B cell targets (407). In addition, both 2B4 (Slamf4) and NTB-A (Slamf5, Ly108 in mice) function as co-receptors in the induction of NK cell-mediated cytotoxicity, and engagement of 2B4 or NTB-A on activated NK cells results in increased cytotoxicity (408, 409). A defect in cytotoxicity can also be observed in SAP-deficient CD8 T cells. EBV-specific CD8 T cells from XLP patients exhibit reduced IFN-γ production and cytotoxicity against EBV-positive B cell targets (390, 410). By contrast, the absence of SAP in CTLs doesn’t affect the cytolysis of fibroblast targets (363). Previous reports have suggested that 2B4 (SLAMF4) and Ly108 (SLAMF5) regulate CD8 T cell synapses upon its interaction with B cells, and mediate their cytotoxic activity against cellular targets (370, 410). For example, the blockade of 2B4 has been shown to reduce the cytotoxicity of CTLs (390), and SAP-deficient CTLs exhibit normal cytolysis activity and IFN-γ production towards Ly108-deficient B cell targets (370). Collectively, these findings highlight the crucial role of SLAM family receptor-SAP signaling in regulating cell interaction, differentiation, development and function, and their importance as co-stimulatory molecules.   1.5 Rationale and Hypothesis   EBV causes significant morbidity and mortality worldwide, including infectious mononucleosis, post-transplant lymphomas, immunodeficiency-related lymphomas, Hodgkin’s lymphoma (~ 40%) and two of the most common childhood and adult cancers in tropical Africa (Burkitt’s lymphoma) and East Asia (nasopharyngeal carcinoma), respectively. Investigation of  77 SAP and XLP offers an invaluable opportunity to learn about immunity against this ubiquitous virus. To gain insight into immune control of EBV, my research has focused on investigating SAP’s regulation of CD4 and CD8 T cell differentiation and immune responses.  Helper CD4 T cells play key roles in directing the types of immune responses elicited, promoting clearance of intracellular pathogens and augmenting Ab-mediated humoral responses and parasite removal. In CHAPTER 3, we hypothesized that SAP controls helper CD4 T cell differentiation and immune responses. Through analyses of wild type and SAP-deficient mice, we have found that SLAM and SAP positively regulate the differentiation of Th17 cells in a T cell-intrinsic fashion. In addition, SAP was also found to promote the differentiation of Th17 cell immune and autoimmune responses in vivo.  Immunity against viral infections is dependent on naïve viral-specific CD8 T cells undergoing massive proliferation and differentiation into cytotoxic effectors upon exposure to viral antigens. In CHAPTER 4, we hypothesized that SAP expression within CD8 T cells is critical for mounting responses towards antigen-expressing B cells. Our results demonstrate that SAP is essential for the priming of CD8 T cells when stimulated by antigen-presenting B cells and suggest that the susceptibility of XLP patients to EBV may be a consequence of their naïve CD8 T cells failing to differentiate into cytotoxic effectors upon encountering virally-infected B cells.   78 CHAPTER 2: MATERIALS AND METHODS   2.1 Mice SAP-deficient (Sh2d1a-/-) mice have been previously described (383) and backcrossed onto C57BL/6J (B6) background for at least 10 generations. To generate Sh2d1a-/- OT-1/Thy1.1 mice, SAP-deficient (Sh2d1a-/-) mice were mated with OT-1/Thy1.1 mice. In addition, wild type and Sh2d1a-/- OT-1/Thy1.1 mice were mated with Nur77-GFP mice to generate wild type and Sh2d1a-/- OT-1/Thy1.1/Nur77 mice to report TCR signaling via Nur77 expression (411). C57BL/6J, wild type and Sh2d1a-/- OT-1/Thy1.1 mice were bred and housed in a specific pathogen-free and Helicobacter-free facility at the Child & Family Research Institute (CFRI). B6.PL-Thy1a/Cy (Thy1.1+), B6.SJL-PtprcaPep3b/BoyJ (CD45.1+) and B6.129S4-Cd48tm1Rsr/EpaulJ (CD48-/-) mice were purchased from The Jackson Laboratory.  2.2 In vitro Th1, Th2, and iTreg cell differentiation assays RBC-depleted single-cell suspensions from spleens of 6 to 12 week-old wild type and Sh2d1a-/- mice (5 x 105 per well) were cultured in IMDM supplemented with 10% FBS; 1X non-essential amino acids; 100 U/mL of penicillin/streptomycin; and 55µM β-mercaptoethanol (all reagents from Invitrogen). Splenocytes were stimulated for a 96-hour period with 1 µg/mL plate-bound CD3 (145-2C11) antibody and 10 µg/mL of soluble CD28 (37.51) antibodies in the presence or absence of polarizing cytokines and anti-cytokine antibodies. For Th1- and Tc1-polarizing conditions, cells were treated with IL-2 (100 U/mL), IL-12 (10 ng/mL) and anti-IL-4 antibodies (1 µg/mL, 11B11). To facilitate Th2 differentiation, cells were treated with IL-2 (100 U/mL), IL-4 (10 ng/mL) and anti-IFN-γ (1 µg/mL, XMG1.2) antibodies. For induction of Tregs,  79 splenocytes were treated with the cytokines IL-2 (100 U/mL) and TGF-β (10 ng/mL). All antibodies and cytokines were purchased from eBioscience except for TGF-β (BD Biosciences).  2.3 In vitro Th17/Tc17 cell differentiation assays For the generation of Th17 and Tc17 lineages, wild type and Sh2d1a-/- splenocytes (5 x 105 per well in 96-well flat-bottom plates) were stimulated with 1 µg/mL plate-bound CD3 antibodies, unless otherwise specified, in the presence of IL-6 (10 ng/mL), TGF-β (0.5 ng/mL) and IL-23 (20 ng/mL). Co-stimulation was provided to samples by adding 10 µg/mL of soluble CD28 or SLAM family receptor antibodies. SLAM family receptor antibodies were purchased from eBioscience (CD150, 9D1; 2B4, eBio244F4; CD48, HM48-1) or BioLegend (CD84, mCD84.7; Ly9, Ly9ab3; Ly108, 330-AJ). Neutralizing anti-cytokine antibodies were not added to IL-17-polarizing experimental conditions unless otherwise indicated. In experiments shown in figure 3-9 and 3-10, IL-17-polarizing conditions were performed in the presence of 10 µg/mL anti-IFN-γ (XMG1.2), 10 µg/mL anti-IL-4 (11B11) and 1 µg/mL IL-2 (JES6-1A12) antibodies. For IL-17 differentiation of naïve CD4 and CD8 T cells, splenocytes from wild type and Sh2d1a-/- mice were first labeled with antibodies specific to CD44 (IM7); CD25 (PC61); and CD4 (GK1.5).  CD8α (53-6.7) and naïve (CD25-CD44lo) CD4 and CD8 T cells were sorted using a BD FACSAriaTM flow cytometer. Subsequently, sorted naïve CD4 or CD8 T cells (105 per well in 96-well flat-bottom plates) were activated with the indicated amount of plate-bound CD3 antibody in the absence or presence of soluble CD28 or SLAM antibodies under IL-17-polarizing conditions (IL-6, TGF-β and IL-23 as above, but no neutralizing anti-cytokine antibodies) for 4 d prior to a 4 h PMA/ionomycin stimulation for the assessment of cytokine production.  80 2.4 Naïve CD4 T cell adoptive transfers, Citrobacter rodentium infections, and donor CD4 T cell analyses Naïve CD4 T cells were enriched from spleens and lymph nodes (brachial, inguinal and mesenteric) of wild type (Thy1.1+) and Sh2d1a-/- (Thy1.2+) mice. Briefly, single cell suspensions were incubated with antibodies specific for CD8 (53-6.7), CD25 (3C7), CD44 (KM114), CD19 (eBio1D3) and NK1.1 (PK136).  Antibody-bound cells were removed using anti-mouse Ig/anti-rat Ig coupled Dynabeads (Invitrogen). Residual samples were labeled with antibodies specific for CD44 (IM7), CD25 (PC61), CD4 (GK1.5) and TCRβ (H57-597) to assess naïve CD4 T cell purity. Cell mixtures, containing 6 x 106 CD4 T cells of each genotype, were then generated for their infusion into recipient (CD45.1+) animals. On the following day, mice were administered 100 µL of wild-type C. rodentium biotype 4280 strain DBS100 culture (~2.5 x 108 CFU, grown overnight in Luria broth at 37°C) by oral gavage. Infected mice were monitored daily and no overt signs of distress were observed. At day 10 after infection, lymphocytes recovered from spleens, mesenteric lymph nodes and colons as described (412), were re-stimulated overnight with 50 ng/mL PMA plus 1µg/mL ionomycin. They were then surface-stained with antibodies recognizing CD4 (GK1.5); CD45.1 (A20); CD45.2 (104); Thy1.1 (HIS51); Thy1.2 (53-2.1); and CD3 (145-2C11) before staining with IFN-γ (XMG1.2) and IL-17A (eBio17B7) specific antibodies.  2.5 Experimental autoimmune encephalomyelitis induction and the isolation of CNS-infiltrating T cells To induce EAE disease, 6 to 8 week-old age- and sex-matched wild type and Sh2d1a-/- mice, bred within the CFRI animal facility, were injected subcutaneously with 200 µg myelin  81 oligodendrocyte glycoprotein peptide (MOG35-55) emulsified in a 100 mL volume of a 50:50 mixture of Freund’s Complete Adjuvant (CFA) (Sigma-Aldrich) and PBS. Two hundred nanograms of pertussis toxin (Cedarlane Labs) were also administered via intraperitoneal injection at day 0 and day 2 after MOG immunization. For assessment of cytokine secretion by infiltrating T cells, mononuclear cells were isolated from the CNS when wild type mice reached a mean clinical score of 2. In brief, brains and spinal cords were dissected from mice perfused with PBS and mashed through nylon screens. Subsequently, cell mixtures were suspended in 30% Percoll/PBS solution (GE Healthcare), layered on the top of 70% Percoll/PBS and centrifuged at 2000 rpm for 20 min. After collection of mononuclear cell fraction from the percoll gradient, cells were stimulated for 4 h with 50 ng/mL PMA plus 1µg/mL ionomycin in the presence of 3 µg/mL Brefeldin A (eBioscience), labeled with surface marker antibodies, and fixed, permeabilized and stained intracellularly with antibodies specific for IFN-γ (XMG1.2), IL-17A (eBio17B7) and FOXP3 (FJK-16s) as described above.  2.6 Preparation of highly purified OT-1 CD8 T cells OT-1 CD8 T cells were enriched using direct magnetic-based cell separation (Miltenyi Biotec) prior to cell sorting. Briefly, RBC-depleted single-cell suspensions from lymph nodes and spleens were bound to CD8α microbeads and isolated by magnetic cell separation using Miltenyi MS columns. Subsequently, fractionated CD8 T cells were stained with antibodies against CD8α (53-6.7) and CD11c (N418), and sorted using a BD FACSAriaTM flow cytometer to recover highly purified CD8 T cells (> 98% CD8α+CD11c-). All surface specific antibodies were purchased from eBioscience.   82 2.7 Preparation of antigen-presenting B cells and B cell-depleted splenocytes To obtain B cells, splenic single-cell suspensions were labeled with antibodies specific to CD19 (eBio1D3) and CD11c, and highly purified B cells (> 98% CD19+CD11c-) collected by cell sorting using a BD FACSAriaTM flow cytometer. For preparation of B cell-depleted splenocytes, RBC-depleted splenic single-cell suspensions were coated with CD19 antibody and subsequently, CD19 antibody-bound cells and surface Ig-expressing cells removed using a 50:50 mixture of anti-mouse IgG Dynabeads (Cat: 11031, Invitrogen) and anti-rat IgG Dynabeads (Cat: 11035, Invitrogen) according to the manufacturer’s instructions. All surface specific antibodies were purchased from eBioscience.  2.8 Thymidine incorporation assays To measure CD8 T cell proliferation by thymidine incorporation, indicated numbers of CD8 T cells (> 98% CD8α+CD11c-) were incubated with B cells and B-depleted splenocytes in the presence or absence of various concentrations of OVA (SIINFEKL) peptide. After 3 d, cultures were pulsed with 1µCi of [3H] thymidine for 16 h to access proliferation. Subsequently, radioactive [3H]-thymidine incorporated into cellular DNA was measured using a MicroBeta Trilux counter (PerkinElmer).  2.9 CFSE labeling Purified OT-1 CD8 T cells (> 98% CD8α+CD11c-) (107 cells/mL) were labeled with 1µM CFSE (Sigma) in PBS at room temperature for 10 min. CFSE labeling reactions were quenched by the addition of equivalent volume of FBS. Subsequently, cells were washed three times with  83 complete media prior to in vitro stimulation. For adoptive transfer experiments, CFSE-labeled CD8 T cells were given a final wash with PBS before intravenous injection.  2.10 Derivation and in vitro culture of B cell lymphomas, melanoma and breast carcinoma cell lines Primary parental wild type B lymphoma cells were derived from the lymph nodes of Eµ-myc transgenic mice with terminal disease (413, 414). Subsequently, Eµ-myc B lymphoma cells were infected with retroviruses expressing GFP alone or with membrane bound form of ovalbumin and sorted to isolate the transduced subsets (415). Purified transduced cells, resembling the parental Eµ-myc tumor cells, were grown through two rounds of passaging in mice and cryopreserved. Using these mixed lymph node/lymphoma cell suspensions, we expanded B lymphoma cells expressing GFP alone (B-GFP) or with membrane form of ovalbumin (B-OVA) through in vitro culture using DMEM supplemented with 10% FBS, 1X non-essential amino acids, 100 U/mL of penicillin/streptomycin and 55 µM β-mercaptoethanol (all reagents from Invitrogen). B16 melanoma cells (B16), B16-OVA- melanoma cells (B16-OVA) and mammary carcinoma-OVA cells (NOP12) have been described previously (416-420) and were maintained using the same media as above except for the addition of (500 µg/mL) gentamycin (Sigma-Aldrich) for B16-OVA and insulin/transferrin/selenium (Lonza) for NOP12.  2.11 In vitro stimulations and CFSE-based OT-1 CD8 T cell proliferation assays 104 sorted CD8 T cells (> 98% CD8α+CD11c-) were stimulated with 2 x 105 purified B cells (> 98% CD19+CD11c-), B cell-depleted splenocytes and soluble OVA peptide (SIINFEKL), in the absence or presence of IL-2 (100 U/mL), in the round-bottom 96-well plates  84 for 3 to 4 d. In some experiments, 104 CFSE-labeled purified CD8 T cells were stimulated with 105 Eµ-myc B cell lymphomas (B-GFP) (415) and soluble OVA peptide; or 105 endogenous OVA-expressing Eµ-myc B cell lymphomas (B-OVA) (415); or 104 melanoma (B16); or 104 endogenous OVA-expressing melanoma (B16-OVA); or 105 breast carcinoma (NOP12), in the absence or presence of exogenous IL-2 (100 U/mL) for 4 d. The B-GFP, B-OVA, B16, B16-OVA and NOP12 cell lines have been previously described.  2.12 In vitro cytotoxicity assays Purified wild type and Sh2d1a-/- OT-1 (Thy1.1+) CD8 T cells (> 98% CD8α+CD11c-) were stimulated with 10-8 M OVA peptide in the presence of purified B cells (> 98% CD19+CD11c-) or B cell-depleted splenocytes for 4 d to generate CD8 T cell effectors. Subsequently, CD8 T cell effectors were cultured at 37 °C with targets labeled with Dye eFluor®450 OVA-expressing (B-OVA or B16-OVA cells) or Dye eFluor® 670 OVA-negative targets (B-GFP or B16 cells). After 4 h of incubation, the proportion of live OVA-positive and OVA-negative targets was assessed using the viability exclusion dye 7-AAD (Calbiochem). All antibodies and cell-tracking dyes were purchased from eBioscience.  2.13 In vitro blocking of SLAM receptors CD48 and 2B4 To block CD48 on the surface of APCs from interacting with SLAM family receptors on CD8 T cells, sorted B cells (> 98% CD19+CD11c-), B cell-depleted splenocytes or OVA-expressing B cell lymphomas were treated with 10 µg/mL of anti-mouse CD48 antibody (HM48-1, eBioscience) or control IgG antibody for 1 h at 37 °C and subsequently, washed with media three times prior to incubation with wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98%  85 CD8α+CD11c-). For blocking 2B4 on the surface of CD8 T cells from interacting with SLAM family receptors on APCs, sorted wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were cultured with 10 µg/mL of anti-mouse 2B4 antibody (2B4, BD Biosciences) or control IgG2b antibody upon stimulation with OVA-expressing B cell lymphoma cells or with sorted B cells (> 98% CD19+CD11c-) or B cell-depleted splenocytes in the presence of OVA peptide. After 4 d of culture, samples were stained with antibodies against Thy1.1 (HIS51), CD8α (53-6.7) and TCR Vα2 chain (B20.1) and OT-1 CD8 T cell proliferation was tracked via flow cytometry. All antibodies were purchased from eBioscience unless indicated otherwise.  2.14 Cell signaling studies and phospho-specific flow analyses  To assess T cell receptor signaling strength via the induction of Nur77 expression, wild type or Sh2d1a-/- OT-1 were crossed to the Nur77-GFP reporter mouse described by Moran et al (411). 104 sorted wild type or Sh2d1a-/- OT-1-Nur77-GFP CD8 T cells (> 98% CD8α+CD11c-) were incubated with 2 x 105 splenocytes in the presence of soluble OVA peptide or with 105 OVA-expressing B cell lymphoma cells. OT-1 CD8 T cells were discriminated with antibodies recognizing CD8α and TCR Vα2 chain and GFP expression monitored by flow cytometry at the indicated times. For flow cytometric measurements of phosphorylation, 104 purified wild type or Sh2d1a-/- OT-1 Thy1.1+ CD8 T cells (> 98% CD8α+CD11c-) were incubated with OVA peptide and 2 x 105 purified B cells (> 98% CD19+CD11c-) or splenocytes. At the indicated time post-stimulation, samples were fixed with 4% formaldehyde/PBS for 10 min at 37 °C and permeabilized with ice-cold 90% methanol for 20 min. Subsequently, samples were re-suspended in 2 % FBS in PBS and incubated at room temperature for 30 min with the following  86 phospho-specific antibodies (all from Cell Signaling Technology): phosphorylated ERK1/2 (Thr202/Tyr204, D13.14.4E), phosphorylated NFκB (Ser536, 93H1), and phosphorylated AKT  (Ser473, D9E). Wild type or Sh2d1a-/- OT-1 CD8 T cells were discriminated by staining with antibodies against CD8α (53-6.7) and Thy1.1 (HIS51). Samples were acquired by flow cytometry using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer.   2.15 Adoptive CD8 T cell transfers, lymphoma challenges and Listeria monocytogenes infections 5 x 105 purified CFSE-labeled wild type or Sh2d1a-/- OT-1 CD8 T cells (CD45.2+) were infused intravenously into recipient (CD45.1+) animals. On the next day, mice were given 5 x 105 Eµ-myc-OVA B cell lymphoma cells or simply PBS via intravenous injection. At 9 d post-lymphoma challenge, splenic single cell suspensions were stained with antibodies specific to CD8α (53-6.7), Vα2 (B20.1), CD45.1 (A20), CD45.2 (104), Thy1.1 (HIS51) and Thy1.2 (30-H12). For bacterial infection experiments, host mice (CD45.1+) were first co-administered 104 CFSE-labeled wild type (Thy1.1+CD45.2+) and Sh2d1a-/- (Thy1.2+CD45.2+) OT-1 CD8 T cells via intravenous injection prior to being infected with 2 x 103 CFU of recombinant L. monocytogenes expressing ovalbumin (LM-OVA) one day later.  At 7 d post-bacterial infection, splenocytes were stained with specific antibodies as described above to discriminate donor wild type and Sh2d1a-/- OT-1 CD8 T cells from host CD8 T cells and donor OT-1 T cell proliferation measured by flow cytometry. In addition, splenocytes were re-stimulated with 50 ng/mL PMA and 1 µg/mL ionomycin (Sigma) in the presence of GolgiPlug (BD Bioscience) for 4 h prior to the detection of IFN-γ (XMG1.2) and CD107a (eBio1D4B) expression. Bacteria doses were  87 determined by plating the injectant on brain-heart infusion agar plates (BD Bioscience). All antibodies were purchased from eBioscience.  2.16 Flow cytometry Antibodies (with clone name) against CD3e (145-2C11), CD4 (GK1.5), CD8α (53-6.7), Vα2 (B20.1), Thy1.1 (HIS51), Thy1.2 (30-H12), CD25 (PC61.5), CD44 (IM7), B220 (RA3-6B2), CD49b (DX5), 2B4 (eBio244F4), CD107a (eBio1D4B), IL-2 (JES6-1A12), IFN-γ (XMG1.2), IL-4 (11B11), FOXP3 (FJK-16s) and IL-17A (eBio17B7) were purchased from eBioscience. For detection of NKT cells, alpha-galactosylceramide-loaded mouse CD1d tetramers were acquired from the NIH Tetramer Core Facility (Emory University). For intracellular cytokine staining, samples were stimulated with OVA peptide or 50 ng/mL PMA plus 1 µg/mL ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences), or 3 µg/mL of Brefeldin A (eBioscience). Subsequently, cells were stained with antibodies specific for surface markers, fixed with 2% paraformaldehyde/PBS solution for 15 min, treated with eBioscience permeabilization buffer and stained with antibodies specific for the indicated intracellular antigens. SAP (clone 12C4) antibody and SAP detection by intracellular flow cytometry have been described previously (349, 372). Sample acquisition was performed using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer. Sample data were analyzed with Flowjo software version 8.8.6 (Tree Star, Inc).  2.17 Statistical analyses Statistical significance was determined by performing unpaired, two-tailed student t tests in most of the experiments. For multiple comparisons, statistical significance was calculated  88 using ANOVA test, followed by Tukey’s method. For EAE experiments, non-parametric Mann-Whitney U and Fisher’s exact tests were used to calculate statistical significance between mouse cohorts for disease incidence and disease severity. All statistical analyses were performed using Prism4 software (GraphPad).   89 CHAPTER 3: SLAM-SAP SIGNALING PROMOTES DIFFERENTIATION OF IL-17 PRODUCING T CELLS AND PROGRESSION OF EXPERIMENTAL AUTOIMMUE ENCEPHALOMYELITIS  3.1 Introduction Pathogen clearance and immunity require generating the appropriate adaptive immune responses for a given type of infectious challenge. This immune specificity is mediated in part through the actions of the innate immune system, that recognizes the invading pathogen and tailors specific immune responses through instruction of naïve CD4 and CD8 T cells to differentiate into various types of effector T helper (Th) or T cytotoxic (Tc) cell subsets respectively. The effector Th lineages were initially defined as Th1 or Th2 on the bases of cytokines produced, IFN-γ or IL-4 respectively, and the types of responses elicited, promoting clearance of intracellular pathogens or augmenting Ab-mediated humoral responses and parasite removal (152). However, it is now clear that a much greater diversity of effector T cell lineages exists and that their differentiation is regulated through a complex integration of signals received from the TCR, co-stimulatory molecules and cytokine receptors (153).  Th17 and Tc17 effector lineages have recently been described that possess unique developmental requirements and immunological functions (178). Specific cytokines (TGFβ and IL-6 in mice and TGFβ, IL-1β and IL-6, IL-21 or IL-23 in humans) along with STAT3 activation induce differentiation of IL-17-secreting T cells by inducing the master transcription factor retinoic acid-related orphan receptor-γt (RORγt) essential for this lineage (179-182). Besides their signature cytokine, Th17 and Tc17 cells also produce IL-21, IL-22 and TNF-α and have important immune roles in the regulation of microbial defense and leukocyte recruitment (184- 90 188). However, Th17 and Tc17 effector cells have also been suggested to be pathogenic given their associations with sites of tissue inflammation and autoimmunity in psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus (SLE), multiple sclerosis (MS) and type 1 diabetes (186, 187, 206, 208, 283, 290-296). Murine studies, using neutralizing IL-17 Ab (286) or IL-17a-/- mice (287), strongly imply that IL-17 has a causative role in the T cell-dependent MS model experimental autoimmune encephalitis (EAE). The cytokine IL-23 has been shown to be critical for promoting the stabilization and pathogenicity of Th17 and Tc17 effector cells in models of EAE and autoimmune diabetes although dispensable for the generation of IL-17-secreting T cell effectors (186, 288).  Cell surface SLAM (signaling lymphocytic activation molecule) family receptors are drivers of immune cell activation and differentiation, acting in concert with the intracellular adaptor SAP (SLAM-associated protein) to mediate certain critical immune functions (364, 366, 378). Loss of SLAM-SAP signaling pathways due to mutations within the locus encoding SAP (called SH2D1A, SH2-domain containing 1A) result in X-linked lymphoproliferative disease (XLP), a condition noted for its exquisite susceptibility to EBV but not other pathogens (364, 366). SAP contains a single SH2 domain that interacts with the immunoreceptor tyrosine-based switch motifs (ITSM; TxYxxI/V, “x” representing any amino acid) located in the cytoplasmic regions of SLAM family receptors such as SLAM (CD150), CD48, CD84, Ly9 (CD229), NTBA (NK-T-B-antigen in humans; Ly108 in mouse) and 2B4 (CD244). SLAM family receptors, principally expressed by hematopoietic cells, have immunoglobulin-like extracellular domains involved in homotypic- (eg. SLAM-SLAM) or heterotypic-interactions (eg. 2B4-CD48). Engagement of SLAM family receptors by self family ligands results in tyrosine phosphorylation of their ITSMs and the subsequent recruitment of SAP is thought to regulate signaling by two  91 distinct mechanisms: (i) SAP transduction of signaling by binding the tyrosine kinase Fyn and PKC-θ (364-367) and (ii) SAP inhibition of signaling by sterically hindering the docking of SH2-containing phosphatases SHP-1, SHP-2 and SHIP-1 (344, 364, 366, 368-370). Thus, the absence of SAP may negate positive signaling and reinforce inhibitory signaling by SLAM family receptors.   SAP is expressed by most lymphocytes including NK cells, NKT cells, CD4 T cells and CD8 T cells but absent from most of B cells (349, 363), and plays key roles in lymphocyte development, differentiation and function (364). Studies of XLP patients and SAP-deficient (Sh2d1a-/-) mice reveal a matching range of immune cell deficits including severely impaired B cell differentiation resulting in diminished immunoglobulin levels, defective isotype switching and the lack of germinal centers and memory B cells (364). Defective B cell differentiation and humoral responses in SAP-deficient mice are attributed to a fault in CD4 T cell differentiation rather than a B cell-intrinsic defect (348, 349). More recent work indicates that SAP within CD4 T cells is important for sustained CD4 T-B cell contact, necessary for the generation of T follicular helper (Tfh) cells and the formation of germinal centers (350, 351). Besides its effects on CD4 T cells, SAP has also been found to be essential for the development of NKT cells in both mice and humans (399-401) and for the effector functions of NK cells and CD8 T cells (369, 370).   In this report, we have studied the effect of SAP on the differentiation of Th17 and Tc17 differentiation using wild type and Sh2d1a-/- mice. We show that CD4 and CD8 T cells from SAP-deficient mice have an impaired ability to differentiate into Th17 and Tc17 cells relative to other Th and Tc subsets. In addition, co-stimulation of T cells with SLAM-specific Abs enhances IL-17 production from wild type but not SAP-deficient splenic T cells. Additionally, in vitro and  92 in vivo studies utilizing purified wild type and Sh2d1a-/- naive T cells demonstrated a T cell-intrinsic role for SAP’s control of IL-17 T cell differentiation. Finally, Sh2d1a-/- mice were protected from EAE relative to wild type mice and the protected mice had reduced numbers of Th17 and Tc17 effectors in both the spleen and infiltrates of the CNS. Collectively, these findings suggest that the SLAM/ SAP signaling pathway positively regulates the differentiation of Th17 and Tc17 effector T cells in vitro and can promote autoimmune pathogenicity.    3.2 SAP is strongly expressed by Th17 cells. SAP protein is expressed in NK, NKT and T cells of both humans and mice (349, 363). To characterize SAP expression within other lymphocyte lineages, in particular, its distribution within naïve, effector and memory T cell lineages, we used an anti-SAP mouse antibody, previously shown to detect cells transfected with SAP but not the closely related adaptor molecule EAT-2 (349). We performed intracellular flow cytometry employing SAP-deficient (Sh2d1a-/-) mice as a control to establish background, and autofluorescence staining (Figure 3-1). Virtually all naïve and memory CD4 and CD8 T cells, defined as CD44lo and CD44hi respectively, exhibit detectable SAP levels with memory-phenotype T cells showing an appreciable increase relative to naïve T cells (Figure 3-1A & 1B; CD44hi CD4+ = 1.5-fold, MFI: 4,210 ± 127 vs. 2,885 ± 144, p < 0.01; CD44hiCD8+ = 2.2-fold, MFI: 5,812 ± 202 vs. 2,699 ± 167, p < 0.001). By contrast, the splenic regulatory (tTreg; CD4+FOXP3+) T cell subset displayed a greater variation in SAP expression, with almost one quarter of the population having low or undetectable SAP levels. In agreement with a previous study (349), NK (DX5+CD3-) cells and especially NKT (αGalCer/CD1-tetramer+ CD3+) cells were found to strongly express SAP, whereas we were unable to discern any SAP expression within splenic B  93 (B220+CD3-) cells (Figure 3-1A & 1B).                   94   Figure 3-1 SAP expression in lymphocyte subsets. (A) Splenocytes from wild type (C57BL/6J) mice were labeled with antibodies specific for the indicated surface markers directly ex vivo, fixed, made permeable and stained intracellularly with SAP (clone 12C4) antibody. To account for autofluorescence and non-specific SAP antibody labeling, splenocytes from SAP-deficient (Sh2d1a-/-) mice were used as a negative control, being surface stained, fixed, permeabilized and treated with SAP antibody in an identical fashion (gray shaded histograms). (B) SAP expression is presented in bar-graph format as background-subtracted MFI (wild type SAP MFI - Sh2d1a-/- SAP MFI) and regulatory (tTreg; CD4+ FOXP3+), naïve (nCD4 or nCD8; CD44lo) and memory (mCD4 or mCD8; CD44hi) T cells, NK (CD49b+ CD3-), NKT (αGalCer/CD1d Tet+ CD3+) and B cells (B220+ CD3-) were electronically gated as shown. Data reflect results from 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests and double (**) and triple (***) asterisks represent p values of less than 0.01, and 0.001 respectively. Error bars denote the standard error of mean (SEM).           SAPCD44mCD8nCD8CD8AFOXP3-/- +/+tTregnCD4CD4mCD4SAPB cell B220CD3 SAPTetNKTCD49bNKCD4***BnCD4mCD4tTregnCD8mCD8SAP MFI80006000200040000**NK NKTB cells******** 95 Previous results implicating a role for SAP in Th differentiation (364) led us to investigate SAP expression in other effector T cell lineages. Splenic single-cell suspensions were cultured in vitro under polarizing conditions to induce Th1, Th2, Th17 or induced-regulatory T (iTreg) cells and SAP protein levels were quantified in CD4 and CD8 T cells that expressed IFN-γ, IL-4, IL-17 or FOXP3 (Figure 3-2A & 2B). Notably, Th17 (IL-17+FOXP3-) cells were found to contain significantly higher levels of SAP when compared with both Th1 (IFN-γ+ IL-17-) and Th2 (IL-4+IL-17-) cells (Th17/Th1 = 2.0-fold, MFI: 5,030 ± 102 vs. 2,470 ± 222, p < 0.01; Th17/Th2 = 3.0-fold, 5,030 ± 102 vs. 1,672 ± 436, p < 0.05). By contrast, SAP levels were similar between Tc1 (IFN-γ+IL-17-) and Tc17 (IL-17+IL-4-) effector cells. These results demonstrate that SAP is expressed in most T cells at various stages of differentiation and that the Th17 cell lineage, in particular, maintains high-levels of SAP.       96   Figure 3-2 SAP is strongly expressed by Th17 cells. Splenocytes, treated with the indicated polarizing conditions to generated Th and Tc lineages, were stimulated with PMA/ionomycin for 4 hours to facilitate detection of intracellular cytokine. (A) SAP levels in various Th and Tc cells, distinguished through cytokine expression (shown in contour plots), are presented by open histograms. Gray shaded histograms (Sh2d1a-/- Th and Tc cells) represent background fluorescence. (B) Cumulative data of SAP expression in Th and Tc are shown as bar graphs with MFI calculated as in figure 3-1B. Data reflects results from 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) and double (**) asterisks represent p values of less than 0.05 and 0.01 respectively. Error bars denote the SEM.           A BTh2iTregTh1 IFN-γIL-4FOXP3IL-17ATh17SAPIL-4Tc1 IFN-γTc17IL-17A SAP**Th1Th2Th17Tc1Tc17SAP MFI600040002000iTreg0* 97 3.3 SAP positively regulates the differentiation of IL-17-producing CD4 and CD8 T cells. Given the high levels of SAP expression in Th17 cells, we hypothesized that SAP is especially critical for the formation of Th17 cells. To address whether SAP plays a selective role in the differentiation of Th17 cells, wild type and Sh2d1a-/- splenocytes were cultured with anti-CD3 plus anti-CD28 antibodies under Th1-, Th2-, Th17- or iTreg-polarizing conditions for 4 d in vitro. Cytokine production was then assessed by intracellular cytokine staining (Figure 3-3A & 3B). Strikingly, Sh2d1a-/- CD4 T cells exhibited reduced potential to differentiate into Th17 effectors relative to wild type (Th17: 1.7-fold decrease; 12.5 ± 0.6% vs. 21.7 ± 1.2%, p < 0.01). By contrast, lack of SAP did not affect the generation of Th1 (IFN-γ+, IL-17-), Th2 (IL-4+, IL-17-), or iTreg (FOXP3+IL-17-) cells under respective Th1-, Th2- and iTreg-polarizing conditions respectively. These observations are consistent with previous findings documenting that Sh2d1a-/- CD4 T cells have a similar capacity to wild type cells in their ability to differentiate into Th1 and Th2 cells under Th1- and Th2-polarizing conditions in vitro (383, 421). These findings suggest that SAP plays a selective role in positively regulating the differentiation of Th17 cells.      98   Figure 3-3 SAP positively regulates the differentiation of IL-17-producing CD4 T cells. Splenocytes from wild type and Sh2d1a-/- mice were treated with plate-bound anti-CD3 and soluble anti-CD28 antibodies under various polarizing Th conditions for 4 d. Cells were then stimulated for 4 h with PMA plus ionomycin prior to being labeled for surface markers and stained for intracellular antibodies. (A) Contour plots, generated by electronically gating on CD4+ T cells, present relative amounts of cytokine and FOXP3 expression. (B) Proportions of the indicated Th lineage among total CD4+ T cells are shown after 4 d of in vitro polarization. Data represent findings from 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests and double (**) asterisks represent p values of less than 0.01. Error bars denote the SEM.            +/+-/-IL-17AIFN-γTh1 Th2 iTreg Th17IL-4 FOXP3 IFN-γ42.0 9.7A38.5 6.917.219.122.113.10.00.00.00.00.00.0 0.0 0.0 0.00.10.00.212.34.117.62.4B% Th lineages of total CD4 T cellsTh1 Th2 iTreg Th17605040303020100+/+-/-**Th1/Th17Polarizing Conditions 99 In addition, to address whether SAP also plays a selective role in the differentiation of Tc17 cells, cytokine production by CD8 T cells was then assessed under various polarizing conditions (Figure 3-4). Fewer IL-17-positive CD8 T cells, either IFN-γ- IL-17+ (Tc17) or IFN-γ+ IL-17+ (Tc1/Tc17) subsets, were observed among Sh2d1a-/- relative to wild type splenocytes culturing under IL-17-polarizing conditions (Tc17: 3.2-fold decrease; 2.7 ± 1.3% vs. 8.7 ± 1.1%, p < 0.05; Tc1/Tc17: 2-fold decrease; 4.0 ± 0.2% vs. 7.9 ± 1.4%, p < 0.05; Figure 3-4A & 4B). By contrast, the frequencies of Tc1 cells (IFN-γ+IL-17-) were remarkably similar when wild type and Sh2d1a-/- splenocytes were incubated under Th1-polarizing conditions. These findings suggest that SAP plays a selective role in positively regulating the differentiation of IL-17-producing CD8 T cell effectors.           100   Figure 3-4 SAP positively regulates the differentiation of IL-17-producing CD8 T cells. Splenocytes from wild type and Sh2d1a-/- mice were treated with plate-bound anti-CD3 and soluble anti-CD28 antibodies under various polarizing Tc conditions for 4 d. Cells were then stimulated for 4 h with PMA plus ionomycin before being labeled for surface markers and stained for intracellular antibodies. (A) Contour plots indicate relative amounts of cytokine production by CD8+ T cells after 4 d of in vitro polarization. (B) Proportions of the indicated Tc lineage among total CD8+ T cells are shown after 4 d of in vitro polarization. Data represent findings from 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) asterisks represent p values of less than 0.05. Error bars denote the SEM.           ATc17Tc1IL-17A30.028.01.7+/+-/-IFN-γ0.00.00.0 0.0Tc17BTc1* *151050203040% Tc lineages of total CD8 T cells8.3 7.046.24.063.5Tc1/Tc17Polarizing Conditions+/+-/- 101 3.4 SLAM-SAP signaling positively modulates the differentiation of Th17 and Tc17 cells. SAP regulates lymphocyte differentiation and function by transmitting signals from surface SLAM family receptors. Consequently, we hypothesized that the addition of co-stimulating SLAM family receptor antibodies could drive the differentiation of Th17 and Tc17 lineages. Previous reports have indicated that the SLAM family receptors SLAM, Ly108, Ly9, CD48 and CD84 are expressed on the T cell surface (364) and that SLAM ligation can regulate T cell activation (422). To investigate the potential of SLAM family receptors to promote the differentiation of IL-17-producing T effectors, wild type and Sh2d1a-/- splenocytes were incubated under IL-17-polarizing conditions with anti-CD3 antibody alone (TCR), anti-CD3 plus SLAM family receptor antibodies or anti-CD3 plus CD28 (TCR/CD28) antibodies for comparison (Figure 3-5 & 3-6). After 4 d, cultures were re-stimulated with PMA/ionomycin for 4 h before labeling with surface markers, fixation and intracellular cytokine staining. A similar fraction of wild type and Sh2d1a-/- CD4 T cells were found to express IL-17 when polarized with anti-CD3 antibody alone (Figure 3-5A & 5B). By contrast, treatment with SLAM antibody (CD150; clone 9D1), an activating antibody shown to co-stimulate TCR-induced proliferation in a SAP-independent fashion (378), plus anti-CD3 antibody increased the proportion of Th17 cells among wild type but not Sh2d1a-/- CD4 T cells (WT, 1.6-fold; 20.3 ± 1.2% vs. 12.9 ± 0.5%, p < 0.01). However, antibodies directed against either Ly108 (330-AJ), Ly9 (Ly9ab3), CD48 (HM48-1), CD84 (mCD84.7) or 2B4 (eBio244F4) were unable to synergize with anti-CD3 antibody to boost the frequency of IL-17-positive T cells, regardless of T cell phenotype or genotype (data not shown). Interestingly, anti-CD28 antibody co-stimulation also was found to increase the proportion of wild type, but not of Sh2d1a-/- CD4 T cells producing IL-17 (wild type, 1.7-fold; 21.7 ± 1.2% vs. 12.9 ± 0.5%, p < 0.001). Regardless of genotype or the presence  102 or absence of CD28 or SLAM co-stimulation, very few FOXP3-positive CD4 T cells were detected under tested culture conditions (Data not shown). The failure of other SLAM family receptor antibodies to support the generation of IL-17 effector T cells could be a consequence of the tested antibodies lacking sufficient stimulatory activity under test conditions or non-redundant functioning of SLAM family receptors. These findings suggest that SLAM-SAP signaling can augment the differentiation of Th17 cells.            103   Figure 3-5 SLAM-SAP signaling positively modulates the differentiation of Th17 cells. Splenocytes from wild type and Sh2d1a-/- mice were treated with CD3 antibody alone (TCR); CD3 plus SLAM antibodies (TCR/SLAM); or CD3 plus CD28 antibodies (TCR/CD28) under IL-17-polarizing conditions for 4 d. Subsequently, samples were stimulated for 4 h with PMA plus ionomycin, and IL-17 and IFN-γ cytokine profiles determined for CD4 T cells by electronic gating (A). Cumulative data are presented as bar graphs, representing the frequencies of IL-17-producing CD4 T cells (B). Data denote results from 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests and double (**) and triple asterisks (***) indicate p values of less than 0.01 and 0.001 respectively. Error bars denote the SEM.            A+/+-/-IL-17AIFN-γ13.213.0TCR TCR/SLAM TCR/CD28 B1.98.91.28.518.7 3.510.311.0 1.813.822.1 4.112.313.1 2.417.6% cytokine (+) Th cells25 +/+-/-20151050IL-17AIFN-γ+- ++TCR TCR/SLAM TCR/CD28-++- ++ -+ +- ++ -+*** ******* 104 In addition, by selective gating of CD8+ T cells, we observed that Sh2d1a-/- CD8 T cells stimulated under IL-17-polarizing conditions with anti-CD3 antibody alone exhibited a reduced propensity to differentiate into an IL-17-positive effectors, Tc17 or Tc1/Tc17 phenotype, compared with wild type CD8 T cells (Figure 3-6A & 6B; Tc17, 2.6-fold; 1.8 ± 0.2% vs. 4.6 ± 0.2%, p < 0.01; Tc1/Tc17, 1.9-fold; 3.5 ± 0.3% vs. 6.5 ± 0.9%, p < 0.05). In addition, activation with anti-CD3 plus anti-SLAM antibodies was found to increase the proportion of wild type but not Sh2d1a-/- CD8 T cells that express IL-17, both Tc17 and Tc1/Tc17 subsets (wild type; Tc17, 1.7-fold; 7.8 ± 0.8% vs. 4.6 ± 0.2%, p < 0.05; Tc1/Tc17, 2-fold; 12.9 ± 2.4% vs. 6.5 ± 0.9%). Again, stimulation with anti-CD3 plus anti-CD28 antibodies was found to boost IL-17 production by wild type but not Sh2d1a-/- CD8 T cells (wild type; Tc17, 1.9-fold; 8.7 ± 1.1% vs. 4.6 ± 0.2%, p < 0.05; Tc1/Tc17, 1.2-fold; 7.9 ± 1.4% vs. 6.5 ± 0.9%). These results indicate that SAP-SLAM signaling can also support the differentiation of IL-17-producing CD8 T cell effectors.       105   Figure 3-6 SLAM-SAP signaling positively modulates the differentiation of Tc17 cells. Splenocytes from wild type and Sh2d1a-/- mice were treated with CD3 antibody alone (TCR); CD3 plus SLAM antibodies (TCR/SLAM); or CD3 plus CD28 antibodies (TCR/CD28) under IL-17-polarizing conditions for 4 d. Subsequently, samples were stimulated for 4 h with PMA plus ionomycin, and IL-17 and IFN-γ cytokine profiles determined for CD8 T cells by electronic gating (A). Cumulative data are presented as bar graphs, representing the frequencies of IL-17-producing CD8 T cells (B). Data denote results from 3 independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) and double (**) asterisks indicate p values of less than 0.05 and 0.01 respectively. Error bars denote the SEM.          TCR TCR/SLAM TCR/CD28A BIFN-γ5.0 7.252.11.8 4.073.39.3 14.541.62.1 3.472.78.3 7.046.21.7 4.063.5% cytokine (+) Tc cells806040200+/+-/-IL-17AIL-17AIFN-γ+-++TCR TCR/SLAM TCR/CD28-++-++-++-++-+** * *** * ***** 106 3.5 SAP-SLAM controls the differentiation of naïve CD4 and CD8 T cells into Th17 and Tc17 effectors. Next, we sought to determine whether SLAM-SAP signaling directly influences the differentiation of naïve CD4 and CD8 T cells into Th17 and Tc17 cells. Naïve wild type and Sh2d1a-/- CD4 and CD8 T cells were sorted based on CD25-CD44lo population (Appendix A.1). Subsequently, cells were cultured with various concentrations of anti-CD3 antibody alone (TCR) or CD3 plus co-stimulating SLAM antibodies for 4 d under IL-17-polarizing conditions. SLAM antibody was found to drive the conversion of wild type but not Sh2d1a-/- naïve CD4 T cells into Th17 effectors at a low TCR antibody (1 µg/mL) concentration (Th17: 3.8-fold, 15.4 ± 1.8% vs. 4.1 ± 0.1%, p < 0.01) (Figure 3-7A & 7B). In addition, colligation of SLAM at intermediate (3 µg/mL) TCR antibody concentrations also fostered wild type naïve CD4 T cells to become IL-17-positive effectors (Th17: 2.7-fold, 16.6 ± 1.1 % vs. 6.2 ± 0.2 %, p < 0.001) whereas this effect was not apparent at higher (10 µg/mL) TCR antibody concentrations. These findings suggest that SLAM-SAP interactions are effective at promoting naïve CD4 T cells into Th17 cells under weaker TCR signaling conditions.  107   Figure 3-7 SAP-SLAM controls the differentiation of naïve CD4 into Th17 effectors. Sorted wild type and Sh2d1a-/- naïve (CD25-CD44lo) CD4 T cells were treated with 1, 3 or 10 µg/mL of plate-bound TCR antibody alone or TCR plus SLAM antibodies under IL-17- polarizing conditions. (A) After 4 d, cells were re-stimulated for 4 h with PMA plus ionomycin and IL-17 and IFN-γ cytokine profiles measured for wild type and Sh2d1a-/- CD4 T cells. (B) Cumulative data are presented as bar graphs, representing the frequencies of IL-17-producing CD4 T cells. Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests and double (**) and triple asterisks (***) indicate p values of less than 0.01 and 0.001 respectively.      18.9 0.40.417.6 0.10.122.8 0.10.116.8 0.20.1A 1 +SLAM4.2 0.00.03.2 0.2B +/+-/-51015202530** ***** ***% IL-17+ IFN-γ- CD4 T cellsIL-17AIFN-γ[TCR][TCR] 1SLAM3 10+- +- +- +- +- +-0** ***0.0-6.2 0.10.23.2 0.00.03.2 0.00.03+-10+-22.5 0.00.03.1 0.00.00.00.023.9+/+-/-CD4 108 Similarly, colligation of SLAM antibody was found to direct the differentiation of wild type but not Sh2d1a-/- naïve CD8 T cells into Tc17 effectors at a low TCR antibody (1 µg/mL) concentration (Tc17: 1.8-fold, 13.9 ± 0.2% vs. 7.8 ± 0.3%, p < 0.001) (Figure 3-8A & 8B). At intermediate (3 µg/mL) TCR antibody concentrations, SLAM antibody also fostered wild type naïve CD8 T cells to become IL-17-positive effectors (Tc17: 1.3-fold, 15.8 ± 0.4 % vs. 11.9 ± 0.6 %, p < 0.001). However, this effect was not apparent at higher (10 µg/mL) TCR antibody concentrations. Thus, these findings suggest that SLAM-SAP interactions promote naïve CD8 T cells into Tc17 cells effectively under weaker TCR signaling conditions.   109   Figure 3-8 SAP-SLAM controls the differentiation of naïve CD8 T cells into Tc17 effectors. Sorted wild type and Sh2d1a-/- naïve (CD25-CD44lo) CD8 T cells were treated with 1, 3 or 10 µg/mL of plate-bound TCR antibody alone or TCR plus SLAM antibodies under IL-17- polarizing conditions. (A) After 4 d, cells were re-stimulated for 4 h with PMA plus ionomycin and IL-17 and IFN-γ cytokine profiles measured for wild type and Sh2d1a-/- CD8 T cells. (B) Cumulative data are presented as bar graphs, representing the frequencies of IL-17-producing CD8 T cells. Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) and triple asterisks (***) indicate p values of less than 0.05 and 0.001 respectively. A 1 +SLAMB +/+-/-IL-17AIFN-γ[TCR]-3+-10+-+/+-/-CD8510152025******% IL-17+ IFN-γ- CD8 T cells[TCR] 1SLAM3 10+- +- +- +- +- +-0******* *****0.10.610.015.6 0.52.313.4 0.52.810.5 0.63.14.9 0.31.61.6 0.00.86.7 0.52.64.0 0.10.61.7 0.00.415.0 0.42.29.7 0.00.73.6 0.00.7 110 3.6   SAP-SLAM signaling promotes Th17 cell differentiation through an IFN-γ and IL-4 independent mechanism. Given the critical role that cytokines play in the programming of Th subsets (153), we next investigated whether SAP’s regulation of Th17 differentiation is mediated through expression of the IFN-γ and IL-4. Wild type and Sh2d1a-/- splenocytes were cultured with two different concentrations of plate-bound anti-CD3 antibody (TCR, 0.3 and 1 µg/mL), with and without co-stimulating SLAM and CD28 antibodies and in the presence (Figure 3-9A) or absence of neutralizing anti-IFN-γ and anti-IL-4 antibodies (Figure 3-9B). As would be expected, the inclusion of neutralizing anti-cytokine antibodies along with TCR/CD28 antibodies resulted in a higher frequency of wild type CD4 T cells differentiating into Th17 cells (0.3 µg/mL: 12.0-fold change, 10.8 ± 0.8 % vs. 0.9 ± 0.2 %, p < 0.001; 1.0 µg/mL: 2.0-fold change, 37.1 ± 0.4 % vs. 18.3 ± 0.3 %, p < 0.001). Moreover, TCR/CD28-stimulated wild type CD4 T cells adopted Th17 fate at an increased frequency relative to Sh2d1a-/- CD4 T cells under the same conditions (0.3 µg/mL: 1.6-fold change, 10.8 ± 0.8 % vs. 6.9 ± 0.9 %, p < 0.05; 1.0 µg/mL: 1.3-fold change, 37.1 ± 0.4 % vs. 28.8 ± 1.4 %, p < 0.01). The use of SLAM antibody enhanced the generation of Th17 cells from wild type CD4 T cells at a higher (1 µg/mL) concentration of plate-bound CD3 antibody but did not affect Sh2d1a-/- CD4 T cells (2.1-fold change, 38.5 ± 3.0 % vs. 18.3 ± 1.3 %, p < 0.001). Furthermore, the addition of SLAM antibody in tandem with CD28 antibody was found to promote Th17 differentiation of wild type CD4 T cells at the lower amount (0.3 µg/mL) of CD3 antibody (CD28/SLAM vs. CD28 alone; 1.6-fold change, 17.5 ± 1.6 % vs. 10.8 ± 0.8 %, p < 0.01). These experiments suggest that SLAM-SAP boosts Th17 differentiation under weaker TCR signaling conditions and that this regulation is independent of the cytokines IFN-γ and IL-4.  111   Figure 3-9 SAP-SLAM signaling promotes Th17 cell differentiation through an IFN-γ and IL-4 independent mechanism. +/+-/-IL-17ATCR alone +SLAM +CD28 +SLAM/CD281 001 1012 0020 007 007 001 001 00A+/+-/-FOXP318 0040 0036 0036 0022 0026 0022 0020 000.31% IL-17+ CD4 T cells05CD28SLAM--++++--***--++++--% IL-17+ CD4 T cells010CD28SLAM--++++--***--++++--1015202520304050[TCR]IL-17A******+/+-/-IL-17ATCR alone +SLAM +CD28 +SLAM/CD28% IL-17+ CD4 T cells0246CD28SLAM--++++--+/+-/-1 004 013 003 011 001 001 001 00***B+/+-/-FOXP311 0019 0122 0019 0010 008 009 0012 000.31--++++--% IL-17+ CD4 T cells05CD28SLAM--++++--***--++++--10152025[TCR]IL-17A ********* 112 Wild type and Sh2d1a-/- splenocytes were treated with 0.3 or 1 µg/mL of plate-bound CD3 antibody alone (TCR); CD3 plus SLAM antibodies (+ SLAM); CD3 plus CD28 antibodies (+ CD28); or CD3, SLAM and CD28 antibodies (+ SLAM/CD28) under IL-17-polarizing conditions (TGF-β, IL-6 and IL-23) in the presence (A) or absence (B) of neutralizing anti-cytokine antibodies (anti-IL-4, anti-IFN-γ and anti-IL-2 antibodies; see MATERIAL AND METHODS).  After 4 d, cells were re-stimulated with PMA/ionomycin and IL-17 and FOXP3 expression measured in CD4 T (CD4+TCRβ+) cells by intracellular flow cytometry. Cumulative data are presented as bar graphs and error bars represent the SD. Statistical significance was calculated using ANOVA test, followed by Tukey’s multiple comparison test and single (*), double (**) and triple asterisks (***) indicate p values of less than 0.05, 0.01 and 0.001 respectively.                      113 In addition, we sought to investigate whether the expression of the IFN-γ and IL-4 alters SAP’s regulation of Th17 differentiation under higher TCR signaling conditions. Wild type and Sh2d1a-/- splenocytes were cultured with two higher concentrations of plate-bound anti-CD3 antibody (TCR, 3 and 10 µg/mL), with and without co-stimulating SLAM and CD28 antibodies and in the presence (Figure 3-10A) or absence of neutralizing anti-IFN-γ and anti-IL-4 antibodies (Figure 3-10B). The impact of costimulatory CD28 and SLAM antibodies was modest or even absent when higher concentrations of plate-bound CD3 antibody was applied in both presence or absence of neutralizing anti-IFN-γ and anti-IL-4 antibodies conditions. These results suggest that Th17 cell differentiation is independent of SAP under stronger TCR signaling conditions.     114   Figure 3-10 Th17 cell differentiation is independent of SAP under stronger TCR signaling conditions. +/+-/-IL-17ATCR alone +SLAM +CD28 +SLAM/CD2853 0061 0056 0053 0050 0051 0055 0052 00A+/+-/-FOXP361 0068 0058 1063 0051 0055 0061 0063 00310% IL-17+ CD4 T cells0CD28SLAM--++++--**--++++--% IL-17+ CD4 T cells0CD28SLAM--++++-- --++++--2040608020406080[TCR]IL-17A+/+-/-IL-17ATCR alone +SLAM +CD28 +SLAM/CD28% IL-17+ CD4 T cells0510CD28SLAM--++++--+/+-/-17 0019 0117 1015 0114 0016 0016 0017 00B+/+-/-FOXP317 0020 0017 0118 0117 0017 0017 0016 00310--++++--% IL-17+ CD4 T cells05CD28SLAM--++++-- --++++--10152025[TCR]IL-17A1520 25 115 Wild type and Sh2d1a-/- splenocytes were treated with 3 or 10 µg/mL of plate-bound CD3 antibody alone (TCR); CD3 plus SLAM antibodies (+ SLAM); CD3 plus CD28 antibodies (+ CD28); or CD3, SLAM and CD28 antibodies (+ SLAM/CD28) under IL-17-polarizing conditions (TGF-β, IL-6 and IL-23) in the presence (A) or absence (B) of neutralizing anti-cytokine antibodies (anti-IL-4, anti-IFN-γ and anti-IL-2 antibodies; see MATERIAL AND METHODS).  After 4 d, cells were re-stimulated with PMA/ionomycin and IL-17 and FOXP3 expression measured in CD4 T (CD4+TCRβ+) cells by intracellular flow cytometry. Cumulative data are presented as bar graphs and error bars represent the SD. Statistical significance was calculated using ANOVA test, followed by Tukey’s multiple comparison test and double (**) asterisks indicate p values of less than 0.01.                    116 3.7 CD4 T cell-intrinsic SAP function is required for normal Th17 cell differentiation in vivo. Previous studies have established that orogastric infection of mice with the natural rodent pathogen Citrobacter rodentium directs strong Th17 responses that are dependent on both IL-23 and innate immune recognition of apoptotic host cells (289, 423, 424). To establish whether SAP plays a T cell-intrinsic role in the differentiation of Th17 cells in vivo, naïve (CD25-CD44lo) CD4 T cells were purified from wild type (Thy1.1+CD45.2+) and Sh2d1a-/- (Thy1.2+CD45.2+) mice, mixed at a 1:1 ratio prior to intravenous infusing into lymphoreplete (non-irradiated; CD45.1+) recipient hosts and either left untreated or infected with C. rodentium by oral gavage (Appendix A.2). At day 10 after infection, the numbers of donor wild type and Sh2d1a-/- CD4 T cells were not found to be significantly different from one another in the spleen or mesenteric lymph nodes (Figure 3-11B). However, donor colonic Sh2d1a-/- CD4 T cell numbers were reduced relative to donor wild type (2.6-fold, 27.4 ± 8.1 % vs. 72.6 ± 8.1 %, p < 0.01; Figure 3-11B). The proportions of donor Sh2d1a-/- Th17 cells were markedly decreased in the spleens, mesenteric lymph nodes (MLN) and colons relative to donor wild type Th17 cells (spleen: 1.8-fold, 35.3 ± 7.1 % vs. 64.7 ± 7.1 %, p < 0.05; MLN: 2.9-fold, 25.6 ± 5.9 % vs. 74.4 ± 5.9 %, p < 0.001; colon: 3.6-fold, 21.6 ± 7.8 % vs. 78.4 ± 7.8 %, p < 0.001; Figure 3-11A & 11C). In addition, donor Sh2d1a-/- Th1 cells were significantly decreased in the colon but not the spleen or mesenteric lymph nodes relative to wild type (Figure 3-11A & 11E). Reduced colonic Sh2d1a-/- Th1 cells could be a consequence of decreased antigen priming or homing of effectors to the gut. Given that Th17 cells may trans-differentiate into Th1 cells (425), fewer Sh2d1a-/- Th17 cells or a diminished conversion rate could also be contributors responsible for a smaller number of Th1 effectors. By contrast, the frequencies of donor wild type and Sh2d1a-/- CD4 T cells in uninfected  117 recipient mice at 15 d post-transfer were found to be similar, suggesting that SAP is not critical for the maintenance of naïve CD4 T cells (wild type/ Sh2d1a-/- CD4 T cell ratio in spleens: 1.1, MLN: 1.0, colons: 1.1). Together, these findings indicate that SAP within naïve CD4 T cells regulates Th17 cell differentiation in vivo.                118   Figure 3-11 SAP plays a CD4 T cell-intrinsic role in regulating Th17 cell differentiation in vivo. Naïve (CD25-CD44lo) CD4 T cells from wild type (Thy1.1+CD45.2+) and Sh2d1a-/- (Thy1.1+CD45.2+) mice were mixed at a 1:1 ratio and injected intravenously into host (CD45.1+) mice one day prior to C. rodentium infection. (A) At day 10 post-infection, mononuclear single cell suspensions from spleens, mesenteric lymph nodes and colons were re-stimulated before detection of intracellular IFN-γ and IL-17 expression by donor CD4 T cells. (B) The representation of wild type and Sh2d1a-/- donor CD4 T cells relative to total donor CD4 T cells is shown. The proportion of wild type and Sh2d1a-/- donor CD4 T cells expressing IL-17+IFN-γ- (C), IL-17+IFN-γ+ (D) and IL-17-IFN-γ+ (E) relative to the total donor CD4 T cells producing the indicated cytokine. Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*), double (**) and triple (***) asterisk indicate p values less than 0.05, 0.01 and 0.001 respectively.      A Spleen+/+ -/-MLNIFN-γColonIL-17A1.4 0.01.50.5 0.00.59.0 1.43.53.5 1.42.030.9 24.115.521.3 11.09.6B 1008040200Spleen MLN Colon% donor CD4 T cells+/+-/-**Spleen MLN Colon1008060200% donor IL-17+ IFN-γ+ cells *CDE601008040200Spleen MLN Colon* ******% donor IL-17+ IFN-γ- cells60% donor IL-17- IFN-γ+ cellsSpleen MLN Colon*10080604004020 119 3.8 SAP exacerbates the development of experimental autoimmune encephalomyelitis. Given the well-described pathogenic role of Th17/Tc17 effectors in experimental autoimmune encephalomyelitis (EAE) (185, 208, 287) and our findings that SLAM-SAP signaling acts to promote generation of IL-17-positive CD4 and CD8 T cells in vitro, we investigated how the absence of SAP expression would affect disease progression (Figure 3-12). Wild type and Sh2d1a-/- mice were injected with MOG35-55 in CFA and disease course was monitored in a blinded fashion for 26 d after immunization. Cohorts of Sh2d1a-/- mice showed delayed disease and decreased disease severity relative to cohorts of wild type mice (Figure 3-12A; mean clinical scores at day 15 after immunization: Sh2d1a-/- mice = 0.9 ± 0.3, wild type mice = 2.3 ± 0.1, p < 0.001). Moreover, groups of Sh2d1a-/- mice had a reduced incidence of disease compared with wild type (Figure 3-12B; 0 % vs. 60 % between days 6-8, p < 0.05; 40 % vs. 100 % at day 10, p < 0.05). Collectively, these experiments demonstrate that SAP contributes to pathogenic immune responses that occur in the EAE model of autoimmunity.       120   Figure 3-12 SAP exacerbates the development of experimental autoimmune encephalomyelitis. Mice (n=8 per group) were immunized with MOG35-55 in CFA and pertussis toxin to induce EAE. Mice were monitored for disease development and severity using the following scale for clinical scoring: 0, no overt disease; 1, tail or hind limb weakness; 2, tail and hind limb weakness; 3, partial (one) hind limb paralysis; 4, complete paralysis. Regardless of genotype, mice were not found in a moribund state (clinical score of 5) during the course of these experiments. (A) Data are presented as the mean clinical scores ± SEM for each group. Mann-Whitney U tests were used to calculate statistical significance with single (*), double (**) and triple (***) asterisks denoting p values of less than 0.05, 0.01 and 0.001 respectively. (B) Data are presented indicating the disease incidence for each group. Statistical significance was determined using Fisher’s exact tests and single (*) and triple (***) asterisks represent p values of 0.05 and 0.001 respectively.          A012 35 10 15 200Days post-immunization+/+-/-Mean EAE score25** ***** **************B0Disease incidence (%)20406080100Days post-immunization5 10 15 20 25 300**** * 121 3.9 SAP-deficient mice show defective CD4 T cell priming and decreased numbers of CNS-infiltrating Th17 and Th1/Th17 effectors upon MOG immunization. Next, we investigated whether SAP expression affected the development of Th1 and Th17 effectors, two T cell lineages implicated in the autoimmune pathogenesis of EAE central nervous system pathology. To assess whether SAP affected antigen priming of naïve CD4 and CD8 T cells, wild type and Sh2d1a-/- mice were treated with MOG35-55 as described above. Splenic T cell numbers and their profiles of cytokine-secreting capacities were determined on day 14 after immunization, at which time wild type mice had reached a mean clinical score of 2. Previous work has shown that naïve Sh2d1a-/- mice have similar splenic CD4 and CD8 T cell numbers as wild type mice (383) and our findings support those observations (data not shown). By contrast, Sh2d1a-/- mice immunized with MOG35-55 had reduced numbers of both splenic CD4 and CD8 T cells relative to wild type (Figure 3-13A; CD4 T cells, 1.4-fold: 7.5 ± 0.3 x 106 vs. 10.7 ± 0.9 x 106, p < 0.05; CD8 T cells, 1.6-fold: 4.7 ± 0.2 x 106 vs. 7.7 ± 0.5 x 106, p < 0.01). To assess their cytokine-secreting potential, splenocytes from MOG35-55-immunized mice were stimulated ex vivo with PMA/ionomycin for 4 h and stained for intracellular cytokine expression (Figure 3-13B). Sh2d1a-/- mice displayed a substantial decrease in Th17 (IL-17+IFN-γ-)-, Th1/Th17 (IL-17+IFN-γ+)- and Th1 (IL-17-IFN-γ+)-effectors compared with wild type mice (Figure 3-13C; Th17, 4.3-fold: 4.4 ± 1.8 x 104 vs. 19.0 ± 4.6 x 104, p < 0.05; Th1/Th17, 14.7-fold: 0.1 ± 0.1 x 104 vs. 2.1 ± 0.5 x104, p < 0.05; Th1, 7.9-fold: 14.2 ± 4.8 x 104 vs. 112.4 ± 13.9 x 104, p < 0.01). By contrast, wild type and Sh2d1a-/- mice possessed similar numbers of Tc1 cells and only scant numbers of IL-17-positive CD8 T cells. These findings show that SAP promotes the priming of antigen-specific CD4 T cells to differentiate into Th17, Th1/Th17 and Th1 effectors.  122   Figure 3-13 Sh2d1a-/- mice show defective CD4 T cell priming upon MOG immunization. On day 14 after EAE induction (wild type mice reached a clinical score of 2), splenic T cells were counted and analyzed for cytokine production. (A) Absolute numbers of splenic T cells from wild type and Sh2d1a-/- mice are shown at day 14 after EAE induction. (B) Representative cytokine production by wild type and Sh2d1a-/- splenic T cells are shown. (C) Cumulative data on numbers of cytokine-secreting wild type and Sh2d1a-/- splenic CD4 and CD8 T cells. Data were represented from 3 independent experiments. Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) and double (**) asterisks indicate p values less than 0.05 and 0.01 respectively.              CD4 CD80.1 0.015.40.0 0.09.7IFN-γIL-17A+/+1.8 0.314.30.8 0.01.4B CASplenic T cells (x 106)-/-CD8CD4151050***+/+-/-Splenic cytokine-(+) T cells (x 104)50251510CD4IL-17AIFN-γ+- ++50100150-+20CD8+- ++ -+0.20.00.60.40.850100150**** 123 To determine whether differences in disease severity between wild type and Sh2d1a-/- mice were associated with changes in the degree of lymphocytic infiltration within the CNS, mononuclear cells isolated from brains and spinal cords of MOG35-55-immunized mice were enumerated and surface stained, and their cytokine-secreting profiles were determined as described above. Sh2d1a-/- mice exhibited markedly decreased numbers of infiltrating CD4 and CD8 T cells relative to wild type mice (Figure 3-14A; CD4 T cells, 5.3-fold: 1.6 ± 0.4 x 104 vs. 7.9 ± 0.5 x 104, p < 0.001; CD8 T cells, 4.2-fold: 1.9 ± 0.5 x 104 vs. 8.0 ± 0.5 x 104, p < 0.001). Moreover, CNS-infiltrates of Sh2d1a-/- mice consisted of greatly diminished frequencies (Figure 3-14B) and absolute numbers of Th17, Th1/Th17 and Th1 effectors as compared to wild type mice (Figure 3-14C; Th17, 21.5-fold: 0.3 ± 0.1 x 103 vs. 6.0 ± 0.4 x103, p < 0.001; Th1/Th17, 63-fold: 0.03 ± 0.01 x 103 vs. 1.9 ± 0.1 x 103, p < 0.001; Th1, 11.8-fold: 1.9 ± 0.5 x 103 vs. 23.4 ± 1.2 x 103, p < 0.001). Additionally, Sh2d1a-/- mice had fewer CNS-infiltrating T cell effectors of the Tc17-, Tc1/Tc17- and Tc1-phenotype relative to wild type animals (Tc17, 33.3-fold: 0.04 ± 0.01 x103 vs. 1.0 ± 0.1 x 103, p < 0.001; Tc1/Tc17, 62.4-fold: 0.02 ± 0.01 x 103 vs. 1.0 ± 0.1 x 103, p < 0.001; Tc1, 7.4-fold: 3.2 ± 0.8 x103 vs. 23.6 ± 1.4 x103, p < 0.001). Together, these findings show that SAP contributes to the generation of IL-17-positive CD4 and CD8 T cell effectors and autoimmune pathology of EAE.   124   Figure 3-14 Sh2d1a-/- mice show decreased numbers of CNS-infiltrating Th17 and Th1/Th17 effectors upon MOG immunization. On day 14 post-EAE induction (wild type mice reached a clinical score of 2), CNS-infiltrating T cells were counted and analyzed for cytokine production. (A) Absolute numbers of CNS-infiltrating CD4 and CD8 T cells from wild type and Sh2d1a-/- mice. (B) Representative cytokine profiles are shown for CNS-CD4 and CD8 T cells. (C) Cumulative data on numbers of cytokine-producing T cells within the CNS. Data were represented from 3 independent experiments. Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests and triple (***) asterisks indicate p values less than 0.001.          *** ***+/+-/-ACD4 CD8CNS T cells (x 104)0.02.55.07.510.0 B+/+1.8 0.213.2CD4 CD8IFN-γIL-17A7.6 2.429.91.3 1.329.50.2 0.117.4C-/-0.0CNS cytokine-(+) T cells (x 103)0.51.01.50.05.07.52.5CD4IL-17AIFN-γ+- ++ -+CD8+- ++ -+102030*********102030********* 125 3.10 Discussion SLAM/SAP pathways are known to regulate the immune system through control of lymphocyte-lymphocyte interactions necessary for the differentiation of Tfh cells, B cells and NKT cells (364) and for optimal effector functions of NK and CD8 T cells (369, 370). Here, we show that another consequence of SAP-deficiency is impaired differentiation of Th17 and Tc17 effectors and reduced susceptibility to T cell-mediated autoimmunity, raising the possibility that IL-17 deficits might also contribute to the phenotype of XLP and in particular to the anti-viral immunity to EBV. IL-17-secreting effectors have been strongly implicated in the clearance of bacterial and fungal infections (185, 290), and interestingly, some evidence suggests also that IL-17-secreting T cell effectors may have a role in the control of viral infections (188, 206, 294). These observations along with our results suggest the possibility that diminished Th17/Tc17-mediated immunity within XLP patients may contribute to the inability to control EBV and virus-induced immune pathology.  Sh2d1a-/- CD4 and CD8 T cells displayed decreased differentiation into Th17 and Tc17 effectors relative to wild type when cultured with TCR and stimulating SLAM antibodies (Figure 3-5 & 3-6) However, Sh2d1a-/- CD4 and CD8 T cells also exhibited marked deficits when stimulations were performed in the absence of SLAM antibody, with TCR plus CD28 antibodies or TCR antibody alone, demonstrating that SAP influences TCR signaling even when SLAM receptors are not apparently engaged (Figure 3-3, 3-4, 3-5 & 3-6). This phenomenon might reflect the recent finding that SAP associates with ITAM sequences of CD3ζ (426, 427) and TCR activation results in the SLAM family receptors and SAP being recruited to the immunological synapse (378, 428). SAP has also been found to sustain TCR signaling through inhibition of diacylglycerol (DAG) kinase α (DGKα), a negative regulator of the DAG- 126 responsive RasGRP1-Ras-ERK pathway via its conversion of DAG to phosphatidic acid (429). These studies argue that SAP regulates cognate antigen-specific T cell responses directly by binding the TCR or by bringing together SLAM family receptors and SAP with the TCR signalosome. The influence of SLAM-SAP signaling pathway on the generation of Th17 and Tc17 cells is unclear. Engagement of SLAM family receptors through their ligand interactions causes SAP to recruit the Src family tyrosine kinase FYN and possibly other effectors to mediate downstream signaling (364). One plausible connection comes from an investigation suggesting that a SAP/FYN/PKCθ-signaling network modulates NF-κB signaling (430). NF-κB activation induces the basic leucine zipper transcription factor ATF-like BATF (431), an inhibitor of AP-1 activity that is itself essential for RORγt expression and Th17 differentiation (432). In addition, SLAM co-stimulation has been proposed to drive IL-17 transcription in human T cells through the activation of NFAT (433). Consequently, SAP may regulate the formation of Th17 and Tc17 lineages through its control of the NF-κB or NFAT pathways. Recently, a study has found that the SAP downstream effector FYN promotes the differentiation of Th17 cells through influences on RORγt expression (434). However, it remains unclear whether FYN’s effects on Th17 differentiation are dependent on SAP or its functions are mediated through a SAP-independent route downstream of the TCR (434).  Previous work has demonstrated that SAP can play a pivotal role in the development of T cell-dependent humoral autoimmunity (435, 436). The loss of SAP expression was found to result in decreased antibody levels, anti-nuclear antibodies and renal pathology, and amelioration of disease in experimental and spontaneous mouse models of SLE (435, 436). This reflects SAP’s mediating of the critical functions in humoral immunity (364). Moreover, Hron and  127 colleagues have reported that a deficiency of SAP exacerbated EAE (436), contrasting their observations with lupus and contradicting our findings documented here (Figure 3-12). It remains unclear whether the discrepant results are a consequence of variation in genetic background heterogeneity, SAP mutagenesis, method used to induce EAE or environmental factors. With respect to genetic background, Sh2d1a-targeted mouse strains used in our study (383) and the one employed by Hron et al. (421) were both derived using 129 embryonic stem (ES) cells and subsequently bred with the C57BL/6 strain. Moreover, 129:C57BL/6 hybrid mice have been shown to be especially autoimmune prone, and may confound experiments assessing autoimmune susceptibility (437). Importantly, our Sh2d1a-/- mouse line has been backcrossed to C57BL/6J mouse line (>10 generations), minimizing the number of potentially meddling genes descended from the 129 strain, whereas Sh2d1a-/- mice used by Hron et al. appear to be of mixed 129:C57BL/6J background (436). Regardless of backcrossing, both gene-targeted mouse lines retain some adjacent 129 genes tightly linked to the mutated Sh2d1a locus on the X-chromosome. With respect to methodological differences, we used a different Sh2d1a-targeted mouse strain [(383) vs. (421)], MOG peptide (MOG35-55 vs. MOG38-55) and amount of peptide (200 µg vs. 300 µg) and we did not administer supplemental heat-killed M. tuberculosis H37RA (0 vs. 1.2 mg) besides what is present within CFA. Finally, cohorts of wild type and Sh2d1a-/- mice used in our study were derived from intercrosses within our animal facility. This is likely to have limited variances in gut microbiota, another factor that influences autoimmunity (438). Natural killer T (NKT) cells have been shown to act (depending on their environment) as important immunoregulatory cells necessary to suppress autoimmune T cell responses (439). Given that a number of studies have demonstrated that NKT cell activation via αGalCer administration protects mice against EAE (440-442), the lack of NKT cells in Sh2d1a-/- mice  128 might be expected to result in more severe EAE than wild type mice. However, NKT cell-deficient mice (CD1d-/- or Jα18-/-) on a C57BL/6J background have been shown to exhibit an EAE course of disease closely resembling wild type mice (440-442), suggesting that NKT cell deficiency in Sh2d1a-/- mice is likely to have little impact on EAE disease course. Collectively, our findings allude to the possibility that diminished Th17 differentiation is responsible for the reduced severity of EAE in Sh2d1a-/- mice. However, Sh2d1a-/- mice also exhibit greatly decreased numbers of IFN-γ-producing CD4 and CD8 T cells relative to wild type mice (Figure 3-13 & 3-14). Moreover, decreased EAE disease in Sh2d1a-/- mice may be a consequence of defective antigen priming or homing, leading to reduced numbers of pathogenic Th1 or Th17 cells at the target site. T-B cell cooperation likely promotes pathogenesis of MS as the depletion of B cells from MS patients has been found to reduce inflammation and myelin loss (443, 444). The importance of SAP functioning in lymphocyte-lymphocyte communication has been established (364). Our findings, documenting roles for SAP in potentiating Th17/Tc17 differentiation and the development of T cell-mediated autoimmunity, lend additional support to the hypothesis that SAP contributes to the pathogenesis of MS. Moreover, elevated levels of Th17 cells and IL-17 have been found in cerebrospinal fluid of MS patients (445) and disease susceptibility is linked to IL-23R and STAT3 genes, known regulators of IL-17 expression (446). Increased SLAM (CD150) and SLAMF3 (CD229 or Ly9)/SLAMF6 (CD352 or NTB-A) expression has been described for T cells of MS- (447) and SLE-patients (448), respectively, suggesting the possibility that the heightened SLAM family receptor-SAP signaling promotes inflammation by amplifying IL-17 expression. The observations reported here suggest that attenuation of SAP signaling through application of SLAM family receptor antibodies or SLAM family receptor/Ig  129 fusion proteins may prove valuable as novel therapeutics for MS and other forms of autoimmunity or inflammatory disease.  130 CHAPTER 4: SAP ENHANCES B CELL-PRIMING OF CD8 T CELL IMMUNE RESPONSES  4.1 Introduction X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency caused by inactivating mutations within the SH2D1A (SH2-domain containing 1A) gene that encodes SLAM-associated protein (SAP) (328, 329). The defining hallmark of XLP is the exquisite sensitivity to the ubiquitous Epstein-Barr virus (EBV), leading to massive lymphoproliferation, fulminant infectious mononucleosis (FIM) and often, haemophagocytic lymphohistiocytosis (HLH). By contrast, XLP males mount protective immune responses to other pathogens including several herpesviruses: cytomegalovirus (CMV), herpes simplex virus and varicella zoster (310). Besides EBV, XLP patients also develop dysgammaglobulinemia and B cell lymphomas although these two phenotypes are independent of EBV infection (364). The extreme susceptibility of XLP patients to a B-lymphotropic virus (EBV) and B cell lymphomas suggest that SAP is essential for the immune surveillance of transformed and malignant B cells.    SAP functions as an adaptor by associating with the cytoplasmic tails of the signaling lymphocyte activation molecule (SLAM) family receptors via its SH2 domain (342-344, 349, 363). SLAM family receptors include SLAM (SLAMF1, CD150), CD48 (SLAMF2), Ly9 (SLAMF3, CD229), 2B4 (SLAMF4, CD244), CD84 (SLAMF5), NTB-A (SLAMF6, NK-T-B-antigen in humans; Ly108 in mice) and CD2-like receptor-activating cytotoxic cells (SLAMF7, CRACC). SAP expression is restricted to NK cells, NKT cells, T cells and some B cells while SLAM family receptors are expressed by most hematopoietic cells. SLAM family receptors engage in homotypic interactions except for 2B4 whose ligand is CD48, regulate cell: cell  131 contact and intercommunication (364, 366, 378). Through the study of XLP patients and SAP-deficient (Sh2d1a-/-) mice, SAP has been implicated in mediating lymphocyte: lymphocyte interactions including signal transduction essential for NKT cell development (399-401), regulation of CD4 T cell: B cell intercommunications required for humoral immunity (350, 351, 397, 398) and modulation of NK cell (407-409) and CD8 T cell effector functions (363, 370, 390, 410). Thus, loss of SAP function in one or a combination of lymphocyte lineages likely underlies the vulnerability of XLP patients to EBV and B cell-associated malignancies. CD8 T cell immunity is thought to be crucial to control EBV infection in healthy individuals (302). During primary EBV infection, virus entry results in lytic viral replication within oral epithelial cells (302, 311-314) and subsequently, infectious EBV virions cause virus-induced transformation and expansion of neighboring oropharyngeal B cells, establishing a pool of latently infected B cell reservoir (310, 315-317). Consequently, EBV-infected B cells are likely pivotal antigen-presenting cells for priming latent antigen-specific CD8 T cells given the population size and unique viral protein expression profile. Importantly, latent antigen-specific CD8 T cells are critical for mediating host control of EBV by limiting the numbers of latent B cells (302). The justifications above along with the observation that EBV causes the super-induction of CD48 expression on virally-infected B cells (449) have led us to speculate that 2B4 and SAP expression by CD8 T cells is critical for priming by EBV-infected B cells. Viral infection experiments of Sh2d1a-/- mice using the arenavirus LCMV Armstrong or murine γ-herpesvirus-68 (MHV-68) have not uncovered overt immunodeficiency (383, 421, 450, 451). Moreover, Sh2d1a-/- mice were found to mount slightly exaggerated CD8 T cell responses but cleared virus similarly to wild type mice. However, the failure to recapitulate the XLP phenotype in the mouse model may be related to the broader viral tropisms of both LCMV  132 Armstrong and MHV-68 relative to EBV. Consequently we have investigated the importance of SAP and CD48 in regulating the priming of CD8 T cells by cognate antigen-expressing B cells by utilizing wild type and SAP-deficient (Sh2d1a-/-) TCR transgenic CD8 T cells, expressing the OT-1 TCR specific to ovalbumin (452), and wild type and CD48-deficient B cells. Here, we show that Sh2d1a-/- CD8 T cells exhibit a selective impairment in proliferation upon activation with antigen-presenting B cells and further, fail to differentiate into functional cytotoxic T cell effector lymphocytes. Furthermore, wild type CD8 T cell responses were found to be dependent on CD48 expression on antigen-presenting B cells. In addition, Sh2d1a-/- CD8 T cells also displayed greatly diminished proliferation against antigen-expressing B lymphoma cells both in vitro and in vivo. By contrast, Sh2d1a-/- CD8 T cell responses towards antigen-presenting-melanoma or -breast carcinoma cells resembled those of wild type CD8 T cells. Collectively, these findings indicate that CD48 on the B cell surface delivers critical positive co-stimulatory signals through 2B4 and SAP to promote CD8 T cell proliferation and differentiation. Additionally, our results indicate that SAP function within CD8 T cells is critical for immune surveillance of B cells, providing insight into the susceptibility of XLP patients to EBV and B cell lymphomas.   4.2 SAP-deficient CD8 T cells respond poorly to antigen-presenting B cells. To determine whether SAP deficiency affects the proliferation of CD8 T cells, wild type and Sh2d1a-/- OT-1/Thy1.1 CD8 T cells were cultured using B cells or B cell-depleted splenocytes as APCs, in the presence of various concentrations of OVA peptide for 3 d, and cell proliferation was measured by [3H]-thymidine incorporation (Figure 4-1). Wild type and Sh2d1a-/- CD8 T cells had equivalent [3H]-thymidine incorporation in the absence of OVA (WT vs.  133 Sh2d1a-/-: 357.7 ± 22.02 vs. 372.5 ± 68.5); however, Sh2d1a-/- CD8 T cells had reduced thymidine incorporation upon B cell stimulation compared to wild type cells (at Ag 10-9 M: 3.0-fold, Sh2d1a-/- vs. WT: 3,583 ± 78.5 vs. 10,576 ± 542.0, p < 0.01; at Ag 10-8 M: 4.6-fold, Sh2d1a-/- vs. WT: 3,552 ± 46.1 vs. 16,451 ± 2,419, p < 0.01). Although increased [3H]-thymidine uptake was observed in Sh2d1a-/- CD8 T cells at the highest Ag (10-7 M) concentration, Sh2d1a-/- CD8 T cells had a three-fold reduction in [3H]-thymidine incorporation compared to wild type cells (Sh2d1a-/- vs. WT: 13,068 ± 1,896 vs. 39,234 ± 4,488, p < 0.05). By contrast, Sh2d1a-/- CD8 T cells had similar or higher [3H]-thymidine uptake upon stimulation with B cell-depleted splenocytes compared to wild type cells. These results suggest that Sh2d1a-/- CD8 T cells exhibit poor cell expansion upon stimulation by antigen-presenting B cells.         134   Figure 4-1 SAP-deficient CD8 T cells proliferate poorly towards antigen-presenting B cells. Sorted wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with OVA peptide and purified antigen-presenting B cells (> 98% CD19+CD11c-) or B cell-depleted splenocytes for 3 d. Subsequently, proliferation was measured by pulsing with [3H]-thymidine for a 16 h period. Line graphs indicate the average of triplicate cultures ± SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) and double (**) indicate p values of less than 0.05 and 0.01 respectively. The data are representative of at least 5 independent experiments.                    +/+-/-B cells[3H] incorporation (104 cpm)OVA (M)510-904321010-8 10-7B-depleted splenocytes[3H] incorporation (104 cpm)OVA (M)2010-90151050 10-8 10-7***** * 135 To investigate the proliferative capacity of antigen-specific SAP-deficient CD8 T cells more closely, wild type and Sh2d1a-/- OT-1/Thy1.1 CD8 T cells were labeled with CFSE, and stimulated with B cells or B cell-depleted splenocytes in the presence of various concentrations of OVA peptide. At 4 d post-stimulation, the proliferation of wild type and Sh2d1a-/- CD8 T cells were compared based on the dilution of CFSE and the relative cell counts of CD8 T cells, as analyzed by flow cytometry. Upon B cell priming in the absence of OVA, the vast majority of wild type and Sh2d1a-/- CD8 T cells remained undivided (WT vs. Sh2d1a-/-: 99% vs. 98%), and about the similar numbers of CD8 T cells were recovered (Figure 4-2A). Strikingly, the proliferation of Sh2d1a-/- CD8 T cells was impaired relative to wild type cells at low Ag (10-9 M) concentration (Sh2d1a-/- vs. WT, undivided: 17% vs. 1%; slow-division: 73% vs. 22%; fast-division: 10% vs. 77%), and less Sh2d1a-/- CD8 T cells were recovered compared to wild type control (cell count, Sh2d1a-/- vs. WT: 361 vs. 912). Similarly, reduced cell proliferation and cell numbers were observed in Sh2d1a-/- CD8 T cells compared to wild type at intermediate Ag (10-8 M) concentration  (Sh2d1a-/- vs. WT, undivided: 1% vs. 0%; slow-division: 19% vs. 8%; fast-division: 80% vs. 92%; cell count: 1,000 vs. 3,081). Although Sh2d1a-/- CD8 T cells had equivalent cell proliferation compared to wild type at high Ag (10-7 M) concentration, they had reduced cell yield (cell count, Sh2d1a-/- vs. WT: 5,415 vs. 13,096). By contrast, Sh2d1a-/- CD8 T cells exhibit equivalent proliferation as wild type when stimulated by B cell-depleted splenocytes at all Ag concentrations (Figure 4-2A). These findings imply that the expression of SAP is critical for the proliferation of CD8 T cells upon stimulation by antigen-presenting B cells, especially at low Ag concentration. Naïve CD8 T cells undergo massive clonal expansion and differentiation into cytokine-producing effector cells in the presence of antigenic stimulation by APCs, appropriate co- 136 stimulatory signals and inflammatory cytokines. To test whether SAP plays a role in promoting cytokine production in CD8 T cells, CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells were cultured with B cells or B cell-depleted splenocytes as APCs, in the presence of various concentrations of OVA peptide. After 4 d, the co-cultures were re-stimulated with PMA/ionomycin for 4 h prior to labeling with surface markers, fixation, and intracellular cytokine staining. Upon stimulation by antigen-presenting B cells, IFN-γ production was significantly reduced in Sh2d1a-/- CD8 T relative to wild type, especially at low Ag concentrations (Figure 4-2B). In the absence of Ag, both wild type and Sh2d1a-/- CD8 T cells remained undivided and incapable of IFN-γ production. At low antigen (10-9 M OVA peptide) concentration, Sh2d1a-/- CD8 T cells underwent fewer cell divisions compared with wild type. In addition, 37% of the proliferated wild type cells produced IFN-γ, whereas none of the proliferated Sh2d1a-/- cells were able to produce cytokine. At higher Ag (10-7 M) concentration, both wild type and Sh2d1a-/- CD8 T cells proliferated and a similar proportion of cells expressed IFN-γ (Sh2d1a-/- vs. WT: 54% vs. 61%). By contrast, the lack of SAP in CD8 T cells did not influence cytokine production upon priming by B cell-depleted splenocytes. These findings suggest that SAP positively regulates cytokine production of B cell-primed CD8 T cells at low Ag concentration.       137   Figure 4-2 SAP-deficient CD8 T cells exhibit defective proliferation and cytokine production upon stimulation with antigen-presenting B cells.  CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with indicated concentration of OVA peptide and purified wild type B cells (> 98% CD19+CD11c-) or B cell-depleted splenocytes for 4 d. Subsequently, samples were acquired for 1 min interval and proliferation measured by CFSE dilution analyses (A). The maximum scale (y-axis) for each histogram is shown vertically at the left. To assess IFN-γ production, cells were re-stimulated with 50 ng/mL PMA and 1 µg/mL ionomycin for 4 h prior to fixation and cytokine detection (B). The data are representative from at least 3 independent experiments.      +/+-/-CFSENumbersA+/+-/-OVA 10-7 M10-8 M10-9 M10B cellsB-depletedsplenocytes5001003010300050025020227717731008921198002982593990198021000010000026740287201090013870109001090B+/+-/-OVA 10-7 M 9510-8 M10-9 M0B cells+/+-/-CFSEIFN-γB-depletedsplenocytes3096200633779200584207030039610465498109540052480534704456041590316902674102030 1005003000500250 138 4.3 The role of SAP in CD8 T cells is required to regulate IL-2 production and CD25 expression upon stimulation by antigen-presenting B cells.  IL-2 signals play key roles in CD8 T cell responses upon antigen stimulation (453, 454). Activated naïve CD8 T cells help promote their own proliferation through IL-2 production and upregulated IL-2 receptor expression, providing autocrine stimulation and enhancing proliferation (453, 454). We hypothesized that poor proliferation of Sh2d1a-/- CD8 T cells may be a consequence of decreased IL-2 production or insufficient IL-2α (CD25) receptor, a key component of the high affinity IL-2 receptor. To determine whether SAP regulates IL-2 production and CD25, wild type and Sh2d1a-/- OT-1 CD8 T cells were activated with B cell or B cell-depleted splenocyte APCs plus OVA peptide and their expression of IL-2 and CD25 measured at the indicated times post-stimulation (Figure 4-3). When stimulated with antigen-presenting B cells, the frequency of IL-2-positive cells was reduced among Sh2d1a-/- CD8 T cells relative to wild type CD8 T cells (2.1-fold at 2 h, Sh2d1a-/- vs. WT: 4.9 ± 0.5 % vs. 10.3 ± 0.3 %, p < 0.001; 2.0-fold at 6 h, Sh2d1a-/- vs. WT: 8.9 ± 1.0 % vs. 18.1 ± 0.6 %, p < 0.01) (Figure 4-3A). By contrast, the proportions of IL-2 producing CD8 T cells were similar between wild type and Sh2d1a-/- CD8 T cells when primed by B cell-depleted splenocytes. Next, Sh2d1a-/- CD8 T cells exhibited delayed CD25 induction compared with wild type CD8 T cells when activated by antigen-presenting B cells (Figure 4-3B). Moreover, marked reductions in CD25 levels were also observed by Sh2d1a-/- CD8 T cells relative to wild type CD8 T cells at 4 h and 6 h after stimulation (3.0-fold decrease at 4 h, MFI, Sh2d1a-/- vs. WT: 1,741 ± 88 vs. 5,315 ± 630, p < 0.01; 1.2-fold decrease at 6 h, MFI, Sh2d1a-/- vs. WT: 13,147 ± 252 vs. 15,502 ± 333, p < 0.01). However, SAP-deficiency was not found to affect CD25 upregulation when CD8 T cells were activated by B cell-depleted splenocytes. Together, these findings demonstrate that SAP  139 modulates IL-2 production and CD25 expression by CD8 T cells when antigen-presenting B cells serve as stimulators.               140   Figure 4-3 SAP-deficient CD8 T cells exhibit decreased IL-2 production and CD25 expression upon stimulation with antigen-presenting B cells.  Naïve wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with 10-8 M OVA peptide and purified wild type B cells (> 98% CD19+CD11c-) or B cell-depleted splenocytes. Subsequently, IL-2 production (A) and CD25 expression (B) by wild type and Sh2d1a-/- OT-1 CD8 T cells were assessed at the indicated times by flow cytometry. Line graphs indicate the average of triplicate samples ± SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*), double (**) and triple asterisks (***) indicate p values of less than 0.05, 0.01 and 0.001 respectively. The data are representative from 2 independent experiments.                      A B20% IL-2(+) CD8 T cellsTime (h)0151050 4 6B cells ****20% IL-2(+) CD8 T cellsTime (h)0151050 4 6B-depleted splenocytess +/+-/-20CD25 MFI (103)Time (h)0151050 4 6B cells20CD25 MFI (103)Time (h)0151050 4 6B-depleted splenocytes*******2 2 2 2*0.31 141 Next, we aimed to investigate whether the presence of exogenous IL-2 can rescue the proliferation and cytokine production of Sh2d1a-/- CD8 T cells upon stimulation by antigen-presenting B cells. Interestingly, Sh2d1a-/- CD8 T cells had equivalent or higher [3H]-thymidine uptake compared to wild type upon antigen-presenting B cell stimulation in the presence of exogenous IL-2 (Figure 4-4A). Similarly, the cell division profiles of Sh2d1a-/-CD8 T cells were similar to wild type at all Ag concentrations in the presence of exogenous IL-2, as measured by CFSE dilution (Figure 4-4B). In addition, the cytokine-producing ability of Sh2d1a-/-CD8 T cells upon stimulation by antigen-presenting B cells was restored in the presence of IL-2 (Figure 4-4C). These results suggest that exogenous IL-2 can rescue the ability of Sh2d1a-/-CD8 T cells to proliferate and produce cytokines in response to stimulation by antigen-presenting B cells.      142   Figure 4-4 IL-2 rescues SAP-deficient CD8 T cell proliferation towards antigen-presenting B cells.  Purified wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with the indicated concentration of OVA peptide and purified wild type B cells (> 98% CD19+CD11c-) in the presence of exogenous IL-2. At day 3 post-stimulation, cultures were pulsed for a 16 h period with [3H]-thymidine and proliferation measured by DNA incorporation of radioisotope (A). Line graphs indicate the average of triplicate cultures ± SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) indicate p values of less than 0.05. (B & C) CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with the indicated concentration of OVA peptide and purified wild type B cells (> 98% CD19+CD11c-) in the presence of exogenous IL-2. At day 4 post-stimulation, wild type and Sh2d1a-/- CD8 T cell proliferation and cytokine production was assessed by CFSE dilution analyses (B) and intracellular cytokine staining (C), respectively. Samples were collected for 1 min interval via flow cytometry. The maximum scale (y-axis) for each histogram, shown in (B), is shown vertically at the left. The data are representative from at least 3 independent experiments.     B+/+-/-OVA 10-7 M10-8 M10-9 MNumbers0B cells+IL-2CFSE15001000150020 97129811019904960199049601990397C OVA 10-7 M10-8 M10-9 M0+/+-/-CFSEIFN-γB cells+IL-29820973002674029710217901684012880158520150010001500A B cells + IL-2[3H] incorporation (104 cpm)OVA (M)10-90151050 10-8 10-7+/+-/-* 143 4.4 SAP promotes anti-B cell lymphomas CD8 T cell responses in vitro. The findings that XLP patients can develop B cell lymphomas with or without infection by EBV suggests that SAP may be critical for the immune surveillance of pre-cancerous B cells. Alternatively, SAP may act as a tumor suppressor with its loss of function resulting in upregulated B cell growth (352, 353). To test whether SAP is critical for a B cell-immune surveillance, we investigated whether SAP is required for CD8 T cell responses towards antigen-expressing B cell lymphomas and we utilized the OVA- (Ag positive) and GFP-expressing (Ag negative) Eµ-myc B cell lymphomas provided by Villadangos et al. (415). CFSE-labeled wild type and Sh2d1a-/- CD8 T cells were cultured with Eµ-myc tumors and various concentrations of soluble OVA peptide in the absence or presence of IL-2 for 4 d, and proliferative responses were measured by flow cytometry. In the absence of SAP, CD8 T cells responded poorly to exogenous Ag presented by B cell lymphomas, particularly at low Ag concentrations (Figure 4-5). Reduced proliferation was observed in Sh2d1a-/- CD8 T cells relative to wild type at a low Ag concentration (10-9 M, Sh2d1a-/- vs. WT, undivided: 77% vs. 9%; slow-division: 20% vs. 64%; fast-division: 3% vs. 27%). With increased Ag concentration, Sh2d1a-/- CD8 T cells were able to proliferate, although to a lesser extent compared to wild type cells (10-8 M, Sh2d1a-/- vs. WT, undivided: 49% vs. 2%; slow-division: 42% vs. 32%; fast-division: 9% vs. 66%). In addition, fewer Sh2d1a-/- CD8 T cells were recovered compared to wild type control  (cell count, Sh2d1a-/- vs. WT: 575 vs. 5,879). At a high Ag concentration, an increased proportion of proliferated cells was observed in Sh2d1a-/- CD8 T cells; however, their proliferation remained lower relative to wild type (10-6 M, Sh2d1a-/- vs. WT; undivided: 9% vs. 0%; slow-division: 45% vs. 15%; fast-division: 47% vs. 85%; cell count: 1,724 vs. 10,310). Interestingly, upon the addition of exogenous IL-2, the proliferative responses of Sh2d1a-/- CD8 T cells could be restored, as  144 detected by increased cell divisions and cell numbers. Collectively, these findings suggest that SAP is essential for the immune response of CD8 T cells against antigen-expressing B cell lymphomas, and in addition, SAP signaling can lower the threshold for the activation of CD8 T cells upon their recognition of Ag-presenting B cell lymphomas.            145   Figure 4-5 SAP-deficient CD8 T cells mount defective responses towards Ag-presenting B cell lymphomas.  CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells were activated with the indicated concentration of OVA peptide and B cell lymphoma cells in the absence (top two rows of histograms) or presence of exogenous IL-2 (bottom two rows of histograms). After 4 d of culture, samples were collected for 1 min interval and wild type and Sh2d1a-/- OT-1 CD8 T cell proliferation measured by CFSE dilution analyses. The maximum scale (y-axis) for each histogram is shown vertically at the left. The data are representative from 3 independent experiments.          OVA 10-6 M45018010-7 M+/+-/-16010-8 M2510-9 M400+/+-/-+IL-250035400500700CFSENumbers090077 23 022 64 1489 10 184 15 176 23 191 8 19 42 4966 32 227 64 93 20 7782 17 164 35 10 0 1000 0 1000 0 1000 0 100 1410451586904785403525700160500180400500450 146 SAP transmits signals from surface SLAM family receptors engaged in homotypical- or heterotypical- interactions to regulate lymphocyte development, differentiation and function (364, 455, 456). Thus, we hypothesized that the engagement of SLAM family receptors delivered SAP-mediated to regulate B cell lymphoma-priming of CD8 T cell responses. To address this hypothesis, we acquired endogenous OVA-expressing B cell lymphomas, and OVA-expressing tumors that lack the expression of SLAM family receptors, including melanoma (B16-OVA) and breast carcinoma (NOP12). We then analyzed the proliferative responses and cytokine production of wild type and Sh2d1a-/- CD8 T cells towards those tumors (Figure 4-6). Strikingly, Sh2d1a-/- CD8 T cells remained undivided (100%), whereas most wild type cells were proliferated (slow-division: 28%; fast-division: 71%, Figure 4-6A) upon stimulation by OVA-expressing B cell lymphomas (B-OVA). Upon antigen-negative B cell lymphoma stimulation (B-GFP), both wild type and Sh2d1a-/- CD8 T cells remain undivided, emphasizing CD8 T cells proliferate in response to Ag-specific stimulation. Furthermore, Sh2d1a-/- CD8 T cells had reduced IFN-γ production relative to wild type upon stimulation with OVA-expression B cell lymphomas (Figure 4-6B). 56% of the proliferated wild type cells produced IFN-γ, whereas most of Sh2d1a-/- cells were undivided and non-cytokine producing. In the absence of Ag (B-GFP), both wild type and Sh2d1a-/- CD8 T cells remained undivided and did not produce IFN-γ. These observations indicate that SAP is critical for CD8 T cells to respond to endogenous antigens presented by B cell lymphomas.  Interestingly, Sh2d1a-/- CD8 T cells had a similar capacity to wild type cells in response to melanoma and carcinoma, as indicated by comparable cell divisions, equivalent cell counts and cytokine production (Figure 4-6A & 6B). These findings suggest that SAP is dispensable for CD8 T cell responses towards tumors that do not express SLAM family receptors.  147   Figure 4-6 SAP is not essential for CD8 T cell responses towards tumor cells that lack SLAM family receptor expression.  CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with OVA-expressing B cell lymphomas or tumor cells that lack SLAM family receptor expression: B16-OVA melanoma cells and NOP12-OVA breast carcinoma cells. After 4 d of culture, samples were collected for 1 min interval on a BD LSR II bench-top cytometer using FACSDiVa software (BD Biosciences). Wild type and Sh2d1a-/- CD8 T cell proliferation and cytokine production was assessed by CFSE dilution analyses (A) and intracellular cytokine staining (B), respectively. The maximum scale (y-axis) for each histogram shown in (A) is shown vertically at the left. The data of B cell lymphomas are representative from 5 independent experiments and the data of melanoma cells and breast carcinoma cells are representative from 3 independent experiments.     71 28 1A10040CFSEB-OVA7515 35 43melanomaB16-OVAB16carcinomaNOP12 B-GFP57 044 56 030 68 234 65 10 0 1000 0 1000 0 1000 0 100+/+-/-Numbers 0 0 10040 100 15 75 354938 13BCFSEB-OVA76melanomaB16-OVAB16carcinomaNOP12 B-GFP24 044 56 0962 0963 000 1000 0 10000 10000 100+/+-/-IFN-γ00 1007228 016 93 148 4.5 SAP-deficient CD8 T cells exhibit weak cytotoxicity towards B cell lymphoma targets. Next, we sought to assess the role of SAP in regulating the cytolytic activity of CD8 T cells against antigen-expressing B cell lymphoma or melanoma (B16) targets (See Materials and Methods, Figure 4-7 & 4-8). We did not observe any killing activity towards Ag-negative B cell lymphomas and melanoma (B-GFP and B16), emphasizing the Ag-specificity of CD8 T cell cytotoxicity. Interestingly, B cell-primed Sh2d1a-/- CD8 T cells had impaired cytotoxicity toward Ag-positive B cell tumors and melanoma (B-OVA and B16-OVA) relative to wild type (Figure 4-7). Even at a high E: T ratio (20:1), B cell-primed Sh2d1a-/- CD8 T cells exhibited a 3.9-fold reduction in cytolytic activity against B-OVA, and a 2.3-fold decrease toward B16-OVA compared to wild type (B-OVA, Sh2d1a-/- vs. WT:  7.7± 0.9% vs. 30.0 ± 1.2%, p < 0.001; B16-OVA, Sh2d1a-/- vs. WT:  36.7± 2.4% vs. 83.3 ± 1.8%, p < 0.001). Collectively, these results suggest that SAP is required for the effector function of B cell-primed CD8 T cells.    149   Figure 4-7 SAP is essential for the efficient generation of CD8 T cell effector functions when B cells act as the primary APCs.   Naïve wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8+CD11c-) were stimulated with OVA peptide (10-8 M) and purified B cells (> 98% CD19+CD11c-) for 4 d. Subsequently, activated wild type and Sh2d1a-/- OT-1 CD8 T cells were incubated with OVA-expressing (B-OVA) and OVA-negative B cell lymphoma (B-GFP) cells (A) or OVA-expressing (B16-OVA) and OVA-negative melanoma  (B16) cells (B) at the indicated effector/target ratios (E: T Ratio). After 4 h of incubation, samples were treated with the viability exclusion dye 7-AAD and cytotoxicity determined by flow cytometry. Line graphs indicate the average of triplicate cultures ± SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*), double (**) and triple asterisks (***) indicating p values of less than 0.05, 0.01 and 0.001 respectively. The data are representative from 3 independent experiments.          A% specific killing010203040E:T Ratio0.6 2 6 20*****WT : B-OVAKO : B-OVAWT : B-GFPKO : B-GFPB cell-primed B% specific killing0306090E:T Ratio0.6 2 6 20WT : B16-OVAKO : B16-OVAWT : B16KO : B16B cell-primed ******* 150 In addition, we sought to investigate the role of SAP in regulating the effector function of CD8 T cells primed by B cell-depleted splenocytes. Wild type and Sh2d1a-/- CD8 T cells were stimulated by B cell-depleted splenocytes, plated with different cellular targets, and antigen-specific killing was analyzed using the viability exclusion dye 7-AAD. As expected, B cell-depleted splenocyte-primed wild type and Sh2d1a-/- CD8 T cells displayed similar levels of cytotoxicity toward B16-OVA (Figure 4-8B). Consistent with previously published results (370), B cell-depleted splenocyte-primed Sh2d1a-/- CD8 T cells were impaired in Ag-specific killing against B-OVA relative to wild type (Figure 4-8A; E: T ratio = 20:1, 2.6-fold decrease, Sh2d1a-/- vs. WT:  9.3± 0.7% vs. 24.3 ± 1.5%, p < 0.001). Thus, these results indicate that SAP is important for the effector function of CD8 T cells against B cell lymphoma targets.          151   Figure 4-8 SAP is important for CD8 T cells effector functions against B cell lymphoma targets. Naïve wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8+CD11c-) were activated with OVA (10-8 M) peptide and B cell-depleted splenocytes for 4 d. Subsequently, wild type (open symbols) and Sh2d1a-/- OT-1 CD8 T cell effectors (black symbols) were incubated with OVA-expressing (B-OVA) and OVA-negative B cell lymphoma (B-GFP) cells (A) or OVA-expressing (B16-OVA) and OVA-negative melanoma (B16) cells (B) at the indicated effector/target ratios (E: T Ratio). After 4 h of incubation, samples were treated with the viability exclusion dye 7-AAD and target killing determined by flow cytometry. Line graphs indicate the average of triplicate cultures ± SEM. Statistical significance was calculated using unpaired, two-tailed t tests and single (*) and triple asterisks (***) indicating p values of less than 0.05 and 0.001 respectively. The data are representative from 3 independent experiments.          A B****20620.6E:T Ratio4020100% specific killingnon-B cell-primed309020620.6E:T Ratio60300% specific killingnon-B cell-primed*WT : B-OVAKO : B-OVAWT : B-GFPKO : B-GFPWT : B16-OVAKO : B16-OVAWT : B16KO : B16 152 4.6 SLAM-family receptor, CD48, enhances B cell-priming of CD8 T cells Our results suggest that SAP is critical for CD8 T cell responses towards antigen-presenting B cells and B cell lymphomas, but not SLAM-family receptor negative APCs (i.e. melanoma & carcinoma). We next sought to investigate which SLAM family receptor(s) expressed on B cells and B cell lymphomas conveys the signals to influence CD8 T cell responses. First, we examined the expression of SLAM family receptors on wild type splenic B cells (CD19+CD3-), DCs (MHCII+CD11c+), CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD8+) and B cell lymphomas (CD19+) ex vivo by flow cytometry (Figure 4-9). B cells strongly express three SLAM family receptors: Ly9, Ly108 and CD48 (Figure 4-9A). DCs strongly express Ly108 (Figure 4-9A). CD4 and CD8 T cells express higher expression of Ly9, Ly108 and CD48 compared to other SLAM family receptors (Figure 4-9B). Interestingly, B cell lymphomas and B cells exhibited similar expression patterns of SLAM family receptors, with high expression levels of Ly9, Ly108 and CD48 (Figure 4-9C). These results suggest that B cell and B cell lymphomas show distinct SLAM family receptors expression compared to other APCs, and that these particular SLAM family receptors could be the receptors responsible for mediating the interaction between B cells and CD8 T cells.       153   Figure 4-9 SLAM family receptors are differentially expressed by APCs and T cells. Splenocytes from wild type (C57BL/6J) mice and B cell lymphomas were labeled with antibodies specific for SLAM family receptors, including SLAM, 2B4, Ly9, Ly108, CD48, CD84 and CRACC and antibodies recognized surface markers specific for B cells (CD19+CD3-), DCs (MHCII+CD11c+), CD4 T cells (CD3+CD4+), CD8 T cells (CD3+CD8+) and B cell lymphomas (CD19+) prior to flow cytometry. SLAM receptor expression is presented in bar-graph format as background-subtracted MFI in gated B cells and DCs (A) or CD4 and CD8 T cells (B) or B cell lymphomas (C). The data shown in (A) are representative from 2 independent experiments and the data shown in (B & C) are derived from a single experiment. B cells0Expression (MFI x 103 )246DCsASLAM 2B4 Ly9 Ly108 CD48 CD84 CRACCCD4 T cells0Expression (MFI x 103 )246CD8 T cellsBSLAM 2B4 Ly9 Ly108 CD48 CD84 CRACCB cell lymphomas0Expression (MFI x 103 )4812CSLAM 2B4 Ly9 Ly108 CD48 CD84 CRACC1355311062 154 Next, we sought to determine whether the expressions of Ly9, Ly108 and/or CD48 on B cells contribute to the modulation of CD8 T cell responses. First, we utilized CD48-/- mice, and confirmed that splenocytes from these mice lacked the expression of CD48, as analyzed by flow cytometry (Appendix A.3). Wild type and Sh2d1a-/- CD8 T cells were primed by wild type or CD48-/- B cells in the presence of various concentrations of OVA peptide. Cell proliferation was assessed after 3-4 d of stimulation by thymidine incorporation assay or CFSE dilution (Figure 4-10). In the absence of OVA peptide, similar levels of [3H]-thymidine uptake were found in both wild type and Sh2d1a-/- CD8 T cells upon stimulation with either wild type or CD48-/- B cells (Figure 4-10A & 10B). On the contrary, wild type CD8 T cells had greatly reduced [3H]-thymidine incorporation upon stimulation by antigen-presenting CD48-/- B cells compared with wild type B cells in all antigen concentrations, as shown in figure 4-10A (at Ag 10-9 M: 2.7-fold, CD48-/- vs. WT: 3,974 ± 510.5 vs. 10,576 ± 542.0, p < 0.01; at Ag 10-8 M: 3.8-fold, CD48-/- vs. WT: 4,358 ± 781 vs. 16,451 ± 2,419, p < 0.01; at Ag 10-7 M: 2.4-fold, CD48-/- vs. WT: 16,217 ± 38.5 vs. 39,234 ± 4,488, p < 0.01). By contrast, there was no difference in the [3H]-thymidine incorporation of Sh2d1a-/- CD8 T cells stimulated by wild type or CD48-/- B cells (Figure 4-10B). Consistent with our previous observation, Sh2d1a-/- CD8 T cells exhibited poor cell proliferation relative to wild type as detected by CFSE dilution, when primed by wild type antigen-presenting B cells (Figure 4-10C). Wild type CD8 T cells had reduced cell proliferation and cell count in response to CD48-/- B cells compared to wild type B cells, as shown in figure 4-10C (10-9 M; CD48-/- vs. WT, undivided: 12% vs. 1%; slow-division: 83% vs. 36%; fast-division: 5% vs. 63%; cell count: 277 vs. 5,881). At a high Ag concentration, CD48-/- B cell-primed CD8 T cells had a reduced cell count compared to CD8 T cells primed by wild type B cells (10-7 M;  155 CD48-/- vs. WT, cell count: 17,003 vs. 35,435). Collectively, these results demonstrate that CD48 enhances B cell-priming of CD8 T cell immune responses.          156   Figure 4-10 CD48 regulates B cell-priming of CD8 T cell immune responses.  Sorted wild type (A) and Sh2d1a-/- (B) OT-1 CD8 T cells (> 98% CD8+CD11c-) were stimulated with the indicated concentration of OVA peptide and purified wild type or CD48-/- B cells (> 98% CD19+CD11c-) acting as antigen-presenting cells. At day 3 post-stimulation, cultures were pulsed for a 16 h period with [3H]-thymidine and proliferation measured by DNA incorporation of tritium. Line graphs indicate the average of triplicate cultures ± SEM. Statistical significance was calculated using unpaired, two-tailed t tests and double (**) indicating p values of less than 0.01 respectively. (C) Sorted CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T (> 98% CD8+CD11c-) cells were activated with OVA peptide and purified wild type or CD48-/- B (> 98% CD19+CD11c-) cells acting as antigen-presenting cells. After 4 d culture, samples were acquired for 1 min interval using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer and proliferation measured via CFSE dilution analyses. The maximum scale (y-axis) for each histogram is shown vertically at the left. The data shown in (A&B) are representative from 2 independent experiments and the data shown in (C) are representative from 5 independent experiments. C+/+-/-OVA 10-7 M10-8 M10-9 M0B cells1200350356+/+-/-CFSENumbersCD48-/-B cells6100001000013663128800247606040028720326802674032680396105842128355743010000100006 35 35012001200120035035035356AWT B cellsCD48-/- B cellsWT CD8 T cells[3H] incorporation (104 cpm)OVA (M)510-904321010-8 10-7Sh2d1a-/- CD8 T cells[3H] incorporation (104 cpm)OVA (M)10-90 10-8 10-7****** 543210BWT B cellsCD48-/- B cells 157 To determine whether the expression of CD48 on other APCs is also required for the regulation of CD8 T cell responses, wild type and Sh2d1a-/- CD8 T cells were stimulated by wild type or CD48-/- B cell-depleted splenocytes in the presence of various concentrations of OVA peptides, and cell proliferation was assessed after 4 d by CFSE dilution (Figure 4-11). Both wild type and Sh2d1a-/- CD8 T cells had equivalent responses to priming by wild type or CD48-/- B cell-depleted splenocytes (Figure 4-11), indicating that CD48 signaling is not required for CD8 T cell responses when stimulated by B cell-depleted splenocytes.                158   Figure 4-11 CD48 is not required for CD8 T cell responses when B cell-depleted splenocytes act as antigen-presenting cells. Sorted CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with OVA peptide along with wild type or CD48-/- B-depleted splenocytes acting as antigen-presenting cells. After 4 d of culture, samples were acquired for 1 min interval using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer and proliferation measured via CFSE dilution analyses. The maximum scale (y-axis) for each histogram is shown vertically at the left. The data are representative from 5 independent experiments.           +/+-/-OVA 10-7 M10-8 M10-9 M0B-depletedsplenocytes150010+/+-/-CFSENumbersCD48-/-B-depletedsplenocytes 350060010000100000793099101990298019901990199029801990199059508921000010000101010600600600150015001500350035003500 159 To address whether Ly108 is also involved in mediating CD8 T cell responses towards antigen-presenting B cells, we utilized splenocytes from Ly108-/- mice, which were confirmed to lack the expression of Ly108 by flow cytometry (Appendix A.3). Wild type and Sh2d1a-/- CD8 T cells were stimulated by wild type or Ly108-/- B cells in the presence of various concentrations of OVA peptide. Cell proliferation was assessed after 4 d stimulation by CFSE dilution (Appendix A.4). Neither wild type nor Sh2d1a-/- CD8 T cells showed differences in proliferation when stimulated by Ly108-/- B cells compared with wild type B cells (Appendix A.4). These results suggest that Ly108 is not essential for CD8 T cell responses when B cells act as the primary APCs. Although we observed high expressions of Ly9 in both B cells and B cell lymphomas, we were not able obtain Ly9-/- mice to perform similar in vitro experiments to those as described above. Instead, we utilized anti-Ly9 antibody to block Ly9 expression on B cells. CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells were stimulated with untreated, anti-Ly9 antibody-treated or control antibody-treated B cells in the presence of various concentrations of OVA peptide for 4 d. Subsequently, cell proliferation was measured by CFSE dilution. Wild type and Sh2d1a-/- CD8 T cells exhibited equivalent levels regardless of Ly9 blockade  (Data not shown). The failure to see any difference by using anti-Ly9 antibody could be a consequence of the tested antibody lacking sufficient blocking activity under test condition. Therefore, we cannot rule out the possibility that Ly9 may play a role in regulating CD8 T cell: B cell interaction.     160 4.7 CD48 expression by OVA-expressing B cell lymphomas modulates CD8 T cell responses. Given that CD48 expression is upregulated in EBV-transformed B cells (449), we sought to investigate whether the expression of CD48 on B cell lymphomas mediates CD8 T cell immune responses. To address this question, OVA-expressing B cell lymphomas were left untreated, or were treated with anti-CD48 or control antibodies prior to culture with CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells. At day 4 post-stimulation, cell proliferation was measured based on CFSE dilution by flow cytometry as shown in figure 4-12. Diminished proliferation was observed in wild type CD8 T cells in response to α-CD48 treated OVA-expressing B cell lymphomas compared to untreated or control IgG treated lymphomas (α-CD48 vs. untreated vs. control IgG, undivided: 24% vs. 1% vs. 1%; slow-division: 48% vs. 2% vs. 2%; fast-division: 28% vs. 97% vs. 97%; cell count: 1,477 vs. 21,681 vs. 19,553). These results indicate that the expression of CD48 on B cell lymphomas plays a critical role in controlling CD8 T cell responses.        161   Figure 4-12 CD48 expression on B cell lymphomas modulates CD8 T cell immune responses.  CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with untreated (No Tx), α-CD48 antibody-coated or control (isotype matched) antibody-coated OVA-expressing B cell lymphoma cells. After 4 d of culture, samples were acquired for 1 min interval using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer and proliferation measured via CFSE dilution analyses. The maximum scale (y-axis) for each histogram is shown vertically at the left. The data are representative from at least 3 independent experiments.           +/+-/-+control IgG+α-CD48No Tx800800800CFSENumbers129725472824482899101297194041800800800 162 4.8 CD48-2B4 signaling modulates CD8 T cell responses when B cells act as primary APCs. Two plausible self-ligands for CD48 on the CD8 T cell surface are CD2 and 2B4 and 2B4 is a SLAM family receptor expressed by NK cells, γδ T cells and some activated CD8 T cell subsets (364, 456). Interestingly, 2B4 interactions with CD48 have been found to enhance NK cell proliferation and cytokine production (385, 387). Moreover, we favored the possibility that SAP may be acting through 2B4 to promote CD8 T cell responses to antigen-presenting B cells given that CD2 has not been previously documented to associate with SAP. However, we failed to detect significant 2B4 expression on the surface of either wild type or Sh2d1a-/- naïve CD8 T cells directly ex vivo (data not shown). Next, we examined whether 2B4 expression was upregulated on the CD8 T cell surface upon activation with antigen-presenting-B cells or -B lymphoma cells (Figure 4-13). Strikingly, 2B4 expression was rapidly induced on the cell surface of both wild type and Sh2d1a-/- CD8 T cells as early as 2 h post-stimulation and was maximal at 6 h post-stimulation regardless of the type of APC used. Consequently, these results demonstrate that 2B4 expression on CD8 T cells is strongly upregulated when stimulated by antigen-presenting-B cells or -B lymphoma cells and suggest that the recognition of its ligand CD48 on the B cell surface serves to co-stimulate CD8 T cell responses.     163   Figure 4-13 Wild type and Sh2d1a-/- CD8 T cells rapidly upregulate 2B4 expression upon stimulation by antigen-presenting B cells or B cell lymphomas. Purified wild type and Sh2d1a-/- CD8 T cells (> 98% CD8α+CD11c-) were stimulated with OVA peptide (10-8 M) along with purified B cells (> 98% CD19+CD11c-) or with OVA-expressing B cell lymphoma cells (B-OVA). At the indicated times, 2B4 expression, presented as mean fluorescence intensity (MFI) values, on the surface of CD8 T cells was assessed using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer. The data are representative from 2 independent experiments.            B cell-primed +/+-/-A2B4 MFI10008004000Time (h)0 2 6 12 24 48 72 96B-OVA-primedB2B4 MFI6004501500Time (h)0 2 6 12 24 48 72 96600200300 164 Next, to address whether CD48/2B4 signaling directly influences B cell-primed CD8 T cell responses, CFSE-labeled wild type and Sh2d1a-/- CD8 T cells were stimulated by antigen-presenting B cells in the absence or presence of 2B4 blocking antibody or control antibody for 4 d. Subsequently, the proliferation of CD8 T cells was measured by flow cytometry (Figure 4-14). B cell-primed wild type CD8 T cells exhibited fewer cell divisions and decreased cell counts in the presence of anti-2B4 antibody compared to untreated or control IgG2b antibody-treated groups (Figure 4-14). These reductions were more profound at a low Ag concentration (10-9M; α-2B4 vs. untreated vs. control IgG2b, undivided: 5% vs. 0% vs. 2%; slow-division: 79% vs. 80% vs. 88%; fast-division: 16% vs. 20% vs. 10%; cell count: 666 vs. 1,893 vs. 1,791). These findings suggest that 2B4-SAP signaling by CD8 T cells promotes antigen-driven proliferation upon stimulation with B cells.     165   Figure 4-14 2B4 regulates CD8 T cell immune responses towards antigen-presenting B cells. CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with the indicated concentrations of OVA peptide along with purified B cells (> 98% CD19+CD11c-) in the absence of antibody (No Tx), with anti-2B4 antibody or control (isotype matched) IgG2b antibody. After 4 d of culture, samples were acquired for 1 min interval using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer. Wild type and Sh2d1a-/- OT-1 CD8 T cell proliferation was measured via CFSE dilution analyses and the maximum scale for the y-axis is shown vertically at the left of each histogram. The data are representative from 5 independent experiments.         +/+-/-OVA 10-7 M010-8 M10-9 M0B cellsNo Tx12004506010+/+-/-CFSE+α-2B412004506010+/+-/-Numbers+α-IgG2b10 60 450120025750386209280928024760415905149086142881032680042583871034660085150861408020579163268097309910991099109550982010 60601010 60 450120012004501200450 166 4.9 2B4/SAP signaling regulates CD8 T cell responses towards antigen-presenting B cell lymphomas. To address whether 2B4/SAP signaling modulates CD8 T cell immune responses towards antigen-expressing B cell lymphomas, CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells were cultured with OVA-expressing B cell lymphomas in the absence or presence of anti-2B4 or control antibody. Subsequently, cell proliferation was measured by CFSE dilution at day 4 post-stimulation as shown in figure 4-15. With 2B4 blocking antibody treatment, significantly reduced cell proliferation and cell yield were observed in wild type CD8 T cells relative to no treatment or control antibody treatment (α-2B4 vs. untreated vs. control antibody, undivided: 25% vs. 1% vs. 1%; slow-division: 41% vs. 2% vs. 2%; fast-division: 34% vs. 97% vs. 97%; cell count: 1,449 vs. 22,720 vs. 22,947). These findings indicate that 2B4/SAP signaling enhances CD8 T cell proliferation upon stimulation by B cell lymphomas.      167   Figure 4-15 2B4 regulates CD8 T cell responses against antigen-expressing B cell lymphoma cells. CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells (> 98% CD8α+CD11c-) were stimulated with OVA-expressing B lymphoma cells in the absence of antibody (No Tx), with anti-2B4 antibody or with control (isotype matched) IgG2b antibody. After 4 d of culture, samples were acquired for 1 min interval using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer. Wild type and Sh2d1a-/- OT-1 CD8 T cell proliferation is shown via CFSE dilution analyses and the maximum scale for the y-axis is shown vertically at the left of each histogram. The data are representative from at least 3 independent experiments.          +/+-/-+control IgG2b+α-2B4No Tx800800800CFSENumbers1297254728254134474671297213049800800800 168 4.10 SAP signaling affects TCR signal transduction when CD8 T cells respond to antigen-bearing B cell lymphomas. Previous reports have suggested that SLAM family receptors serve as costimulatory molecules to augment immune cell signaling (457). To determine whether SAP influences TCR signal transduction when CD8 T cells are stimulated by Ag-presenting B lymphoma cells, wild type and Sh2d1a-/- OT-1 mice were crossed onto the previously described Nur77GFP BAC reporter (411). In the Nur77GFP line, green fluorescent protein (GFP) is induced by antigen receptor but not inflammatory stimulation and GFP intensity is reflective of the strength of TCR signaling (411). Purified wild type or Sh2d1a-/- OT-1-Nur77GFP CD8 T cells were stimulated with B lymphoma cells expressing OVA (B-OVA) or with splenocytes plus OVA peptide and GFP fluorescence assessed at the indicated times by flow cytometry (Figure 4-16). Of note, wild type and Sh2d1a-/- OT-1-Nur77GFP CD8 T cells exhibited comparable GFP fluorescence directly ex vivo. However, Sh2d1a-/- OT-1-Nur77GFP CD8 T cells displayed decreased GFP intensities relative to wild type OT-1-Nur77GFP CD8 T cells when stimulated by antigen-expressing B lymphoma cells (at 24 h, MFI, Sh2d1a-/- vs. WT: 2,468 vs. 5,466; at 48 h, MFI, Sh2d1a-/- vs. WT: 1,056 vs. 3,528). By contrast, wild type and Sh2d1a-/- OT-1-Nur77GFP CD8 T cells were found to exhibit similar GFP levels upon stimulation with splenocytes APCs along with OVA peptide. Together, these studies suggest that SAP impacts TCR signal transduction under weaker TCR signaling conditions using lower antigen doses.      169   Figure 4-16 SAP regulates Nur77 expression upon stimulation with antigen-presenting B cell lymphoma cells. Purified wild type and Sh2d1a-/- OT-1 Nur77-GFP reporter CD8 T cells were stimulated with OVA-expressing B cell lymphoma cells (B-OVA) or with OVA peptide (10-8 M) along with antigen-presenting splenocytes. At the indicated times, GFP expression levels were monitored in wild type (open circles) and Sh2d1a-/- (filled circles) OT-1 CD8 T cells using FACSDiVa software (BD Biosciences) and BD LSR II bench-top cytometer. GFP expression is plotted as mean fluorescence intensity (MFI) values relative to time in hours. The data are representative from 2 independent experiments.          +/+-/-B-OVAGFP MFI (103)Time (h)604202 8 12 24 48 72splenocytesGFP MFI (103)Time (h)0502 8 12 24 48 7210152025 170 4.11 Intrinsic role of SAP in CD8 T cells activates ERK and AKT signaling pathways when primed by antigen-presenting B cells. To investigate the signaling mechanisms by which SAP regulates CD8 T cell proliferation towards antigen-presenting B cells, wild type and Sh2d1a-/- CD8 T cells were stimulated with purified splenic B cells or whole splenocytes as APCs plus OVA peptide for various times. Subsequently, the activation of ERK, AKT and NF-κB in CD8 T cells were measured using phospho-flow cytometry (458-460). Sh2d1a-/- OT-1 CD8 T cells displayed decreased ERK activation relative to wild type CD8 T cells upon stimulation by antigen-presenting B cells (Figure 4-17A; at 1 h, MFI, Sh2d1a-/- vs. WT: 264 vs. 499; at 3 h, MFI, Sh2d1a-/- vs. WT: 408 vs. 690; at 6 h, MFI, Sh2d1a-/- vs. WT: 479 vs. 772). In addition, B cell-primed Sh2d1a-/- CD8 T cells also exhibited reduced AKT activation relative to wild type CD8 T cells (Figure 4-17B; at 1 h, MFI, Sh2d1a-/- vs. WT: 433 vs. 829; at 3 h, MFI, Sh2d1a-/- vs. WT: 629 vs. 803). By contrast, wild type and Sh2d1a-/- CD8 T cells induced NF-κB activation equally well regardless whether B cells or splenocytes were used as APCs (Figure 4-17C). Together, our preliminary results indicate that SAP expression in CD8 T cells may be required for optimal activation of the ERK and AKT pathways upon stimulation by antigen-presenting B cells.  171    Figure 4-17 SAP-deficient CD8 T cells exhibit defects in the activation of ERK & AKT signaling pathways when stimulated by antigen-presenting B cells. Naïve wild type and Sh2d1a-/- OT-1 CD8 T cells were stimulated with OVA peptide (10-8 M) and purified B cells or whole splenocytes as antigen-presenting cells for the indicated times. Samples were surface stained and subsequently, phosphorylation status of ERK, AKT and NF-κB monitored using phosphorylation-dependent antibodies. The data are representative from 2 independent experiments.    B cell-primed+/+-/-ApERK MFI10007502500Time (h)0 1 6 16500splenocytes-primedpERK MFI10007502500Time (h)0 1 6 16500B cell-primedBpAKT MFI10007502500Time (h)0 1 6 16500splenocytes-primedpAKT MFI10007502500Time (h)0 1 6 16500B cell-primedCpNFκB MFI6002000Time (h)0 1 6 16400splenocytes-primedpNFκB MFI6002000Time (h)0 1 6 16400333333 172 4.12 SAP-deficient CD8 T cells exhibit diminished responses upon stimulation with antigen-expressing B cell lymphomas in vivo. Previously, OT-1 T cells have been shown to see cognate Ag on the surface of B lymphoma (Eµ-myc-OVA described above) and that OT-1 CTL can provide some protection (415). However, some B lymphoma antigens may also be seen on the surface of DCs through phagocytosis of apoptotic lymphoma cells and cross-presentation. To investigate whether SAP is critical for B cell-primed CD8 T cell responses in vivo, equivalent numbers of CFSE-labeled wild type or Sh2d1a-/- (Thy1.1+CD45.2+) CD8 T cells were adoptively transferred into lymphoreplete (non-irradiated; CD45.1+) host mice by intravenous injection and the next day, infused with B lymphoma cells (B-OVA, CD45.2+) via intravenous administration. At 9 days post-lymphoma challenge, spleens were harvested and the proliferation history and effector function of donor CD8 T cells and B lymphoma load were assessed by flow cytometry. Sh2d1a-/- CD8 T cells exhibited decreased expansion relative to wild type CD8 T cells as shown by CFSE profiles, reduced representation and cell numbers (Figure 4-18A). About 20% of recovered donor Sh2d1a-/- CD8 T cells remained undivided while the majority of donor wild type CD8 T cells had divided more than once (Figure 4-18A). In addition, the absolute numbers of Sh2d1a-/- CD8 T cells were decreased almost 5-fold relative to wild type (Figure 4-18A, Sh2d1a-/- vs. WT: 10,283 ± 1,144 vs. 49,960 ± 7,313, p < 0.001). Together, these findings argue that SAP is critical for priming of CD8 T cells by Ag-expressing B cell lymphoma cells in vivo. To investigate the effector function of donor wild type and Sh2d1a-/- CD8 T cells, samples were stimulated with PMA and ionomycin and the expression of IFN-γ and CD107a, a marker expressed transiently on the cell surface of degranulating cytotoxic T cells (Figure 4-18B). Corresponding with decreased proliferation, Sh2d1a-/- CD8 T cells exhibited a 4.8-fold  173 reduction in the frequency of CD107a+IFN-γ+ cells relative to wild type CD8 T cells (Figure 4-18B, Sh2d1a-/- vs. WT: 5.1 ± 1.0 % vs. 24.8 ± 1.4%, p < 0.001). Collectively, our results show that SAP is critical for CD8 T cells to acquire effector functions upon recognition of Ag-expressing B cell lymphoma cells in vivo. OT-1 CD8 T cell effectors have previously been shown to provide limited B-OVA lymphoma protection (415). Nevertheless, the B-OVA lymphoma has been documented to rapidly inactivate cytotoxic OT-1 CD8 T cells and modify antigen expression to evade the host immune system (461). Given the difference in proliferation and effector function between wild type and Sh2d1a-/- CD8 T cells, we sought to examine whether genotype of adoptively transferred OT-1 CD8 T cells influenced lymphoma numbers (CD19+CD45.2+) within recipient mice (CD45.1+). However, comparisons of lymphoma burdens between the two cohorts of mice were similar (Hosts receiving Sh2d1a-/- OT-1 CD8 T cells = 1.7 x 107 vs. Hosts receiving wild type OT-1 CD8 T cells = 1.6 x 107, p = 0.6457). Consequently, OT-1 CD8 T cells were unable to hinder the growth of B-OVA lymphoma cells under the test conditions employed regardless of genotype.          174   Figure 4-18 SAP expression in CD8 T cells is critical for anti-B cell lymphoma immunity. CFSE-labeled wild type (Thy1.1+ CD45.2+) or Sh2d1a-/- OT-1 (Thy1.1+ CD45.2+) CD8 T cells were adoptive transferred into congenic B6.BoyJ (Thy1.2+ CD45.1+) host mice via i.v. injection (n=6 per group). One day later, recipient host mice were infused with OVA-expressing B cell lymphoma cells (B-OVA). At day 9 post-lymphoma challenge, spleens were harvested and donor wild type and Sh2d1a-/- OT-1 CD8 T cell proliferation histories and effector functions were assessed. (A) Representative CFSE profiles and absolute numbers of donor wild type and Sh2d1a-/- CD8 T cells are shown. (B) Splenocytes were left untreated (unstimulated) or stimulated with PMA and ionomycin for 4 h. Subsequently, samples were stained to identify donor wild type and Sh2d1a-/- CD8 T cells and detect surface CD107a expression, a marker of degranulation, prior to fixation and labeling of intracellular cytokine. Error bars represent SEM and statistical significance was calculated using unpaired, two-tailed t tests and triple asterisks (***) indicate p values of less than 0.001. The data shown in (A) are representative from at least 3 independent experiments and the data shown in (B) are representative results (n=6 mice) from a single experiment.           ACFSE+/+-/-numbers96 480 20+B-OVA1515BCD107a+/+-/-IFN-γ930unstimulated +PMA/Iono16 4523239514 7161994020CD8 T cells (104)468+/+ -/-***0% IFN-γ+CD107a+ CD8 T cells102030 ***+/+ -/- 175 XLP patients generate sufficient immune responses against most pathogens besides EBV. Previous experiments have shown that CD8 T cells from SAP-deficient mice can match or exceed the capacity of CD8 T cells from wild type mice to expand upon antigen challenge in vivo, specifically LCMV viral epitopes upon LCMV infection (383, 421). Consequently, we tested whether Sh2d1a-/- OT-1 CD8 T cells could respond equivalently to wild type OT-1 CD8 T cells when cognate antigen is presented by a broader array of APCs. CFSE-labeled wild type (Thy1.1+CD45.2+) and Sh2d1a-/- (Thy1.2+CD45.2+) OT-1 CD8 T cells were co-adoptively transferred into recipient hosts (CD45.1+) prior to infection with recombinant Listeria monocytogenes-expressing OVA (LM-OVA). Listeria monocytogenes exhibits an intracellular life-cycle often infecting phagocytic cells like DCs, macrophages and kupffer cells, however, this pathogen can also invade non-phagocytic cells (462). 7 days post-infection, the frequency of donor splenic wild type and SAP-deficient CD8 T cells and their CFSE proliferation profiles were analyzed by flow cytometry. In contrast to the lymphoma challenge, Sh2d1a-/- OT-1 CD8 T cells expanded to a similar extent as wild type OT-1 CD8 T cells 7 d post-LM-OVA infection (Figure 4-19). Moreover, the proportion of donor wild type versus Sh2d1a-/- CD8 T cells were about equal and their CFSE proliferation histories resembled each other (Figure 4-19). Together, these results suggest that SAP is dispensable for CD8 T cell responses when stimulated by mixed group of APCs.      176   Figure 4-19 Wild type and SAP-deficient CD8 T cells expand equivalently to Listeria monocytogenes infection. CFSE-labeled wild type (Thy1.1+ CD45.2+) and Sh2d1a-/- OT-1 (Thy1.2+ CD45.2+) CD8 T cells were adoptively co-transferred into congenic B6.BoyJ (Thy1.2+ CD45.1+) host mice (n=3). One day later, recipient host mice were infected with recombinant L. monocytogenes-expressing OVA (LM-OVA). At 1 wk post-infection, proliferation of donor wild type and Sh2d1a-/- CD8 T cells (black-filled histograms) residing in the spleen was analyzed by flow cytometry. Cumulative data on the proportion of donor wild type versus Sh2d1a-/- OT-1 CD8 T cells are shown. Gray-shaded histograms show representative CFSE profiles of donor wild type and Sh2d1a-/- OT-1 CD8 T cells recovered from naïve (uninfected) recipient mice 1 wk after adoptive transfer. Bar graphs indicated the mean ± SEM and statistical significance determined using unpaired, two-tailed student t tests. The data shown are representative results (n=3 mice) from a single experiment.    CFSE-/-% of Max100 0100 0+LM-OVA+/+200% total CD8 T cells4060+/+ -/- 177 4.13 Discussion Mutations in SAP result in XLP, a primary immunodeficiency characterized by extreme vulnerability to EBV but not other pathogens. SAP associates with SLAM family receptors, regulating lymphocyte development, differentiation and function. However, the fundamental understanding of how SAP confers immunity against EBV infection remains unclear. In this study, we provide compelling evidence that SAP signaling is especially important for CD8 T cell responses when they are stimulated by antigen-presenting B cells. Given that EBV exhibits a unique B cell-tropism and that B cells are likely a major APC for presenting EBV antigens, SAP may be crucial for EBV-specific CD8 T cell responses against virally-infected B cells. Furthermore, we have shown that 2B4 on the CD8 T cell surface interacting with CD48 on the B cell are critical mediators of this CD8 T cell response. Previous work has shown that engagement of SLAM family receptors may confer stimulatory or inhibitory signaling between cells (364). Here, we provide a novel function for SLAM receptors and SAP in regulating interactions between naïve CD8 T cells and antigen-presenting B cells. Moreover, manipulation of SLAM receptors and intercommunications between B cells and CD8 T cells may prove invaluable for modulating CD8 T cell immunity. Collectively, these findings provide a provocative explanation for the inability of XLP patients to be particularly susceptible to EBV infection.  Successful priming of naïve T cells is crucially dependent on cumulative signals from TCR, co-stimulatory molecules and cytokines provided by extracellular milieu (40, 125, 463). Various co-stimulatory molecules have been described, such as B7, PD-1, SLAM and inducible T cell co-stimulator ligand (ICOSL) families (464-468) and they convey activating or inhibitory signaling to regulate T cell function and to determine T cell fate. In this study, we report that SAP is critical for B cell-primed CD8 T cell responses. However, the priming of CD8 T cells by  178 other types of APCs appears to be SAP independent. The variable dependence of CD8 T cell responses on SAP may be a consequence of the repertoire of co-stimulatory molecules expressed (both the level of expression and the classes of co-stimulatory molecules) or the types of cytokines secreted by APC.  Throughout our experiments, we have found that SAP is pivotally important for CD8 T cell when they are stimulated with B cells and B lymphoma cells and low concentrations of antigen (i.e. weak TCR signaling conditions) (Figure 4-2 & Figure 4-5). Previous studies have shown that co-stimulatory molecules augment signaling and lower the threshold for T cell activation (469). Thus, we propose that 2B4-CD48 interactions between CD8 T cells and B cells, respectively, serve a positive co-stimulatory role to enhance TCR signaling and promote CD8 T cell activation. 2B4 on NK cells may act as an activating or inhibitory receptor (470), transducing positive or negative signaling, and its action may be dependent on the type of signaling molecule recruited to its cytoplasmic tail (i.e. SAP or an inhibitory phosphatase like SHP-1, SHP-2 or SHIP). Notably, CD48 has been found to be strongly expressed on the surface of EBV-transformed B cells (449). Therefore, 2B4-CD48 interactions may be particularly relevant for immunity against EBV infection. Moreover, EBV undergoes latency within B cells, limiting the portion of EBV genome that is expressed, likely to evade immune detection. Consequently, responses by naïve EBV-specific CD8 T cells may be highly dependent on SLAM-SAP co-stimulation when the number and amounts of viral antigens presented by latent B cells is very low or limiting. Cell extrinsic factors like cytokines and TCR ligands affect T cell homeostasis and expansion through changes to cellular metabolism (471). Quiescent naïve T cells have modest demands for cell maintenance, principally utilizing oxidative phosphorylation to supply their  179 energy demands (472). However, upon activation, naïve T cells transition to glycolytic metabolism necessary to meet the demands for massive cell growth and differentiation through the actions of the PI3K/AKT pathway inducing surface Glut1 expression to increase glucose uptake (472). The observation that SAP-deficient CD8 T cells exhibit delayed/impaired AKT activation upon B cell-priming suggests that SAP may support naïve T cell proliferation through impacting cellular metabolism. Consequently, it would be interesting to perform glycolysis and glucose uptake assays, measuring oxygen consumption rates and extracellular acidification rates as described (473, 474). Future studies will provide important information on how SAP supports naïve CD8 T cell proliferation.   180 CHAPTER 5: CONCLUSION  5.1 Conclusions and implications In CHAPTER 3, we assessed the role of SAP on the differentiation of Th17 and Tc17 using wild type and Sh2d1a-/- mice. We found that SAP-deficient CD4 and CD8 T cells from mice have an impaired ability to differentiate into Th17 and Tc17 cells but not other Th and Tc subsets relative wild type CD4 and CD8 T cells. Furthermore, SLAM (CD150) receptor co-stimulation of wild type T cells enhanced the differentiation of IL-17-producing T cell effectors whereas this treatment had no effects on SAP-deficient T cells. In addition, experiments using purified wild type and Sh2d1a-/- naive T cells demonstrated a T cell-intrinsic role for SAP’s control of IL-17 T cell differentiation both in vitro and in vivo. Collectively, these findings suggest that the SLAM/ SAP signaling pathway could be modulated to promote or suppress IL-17 mediated T cell immunity and autoimmunity.  In CHAPTER 4, we examined the role of SAP on regulating CD8 T cell immunity towards antigen-presenting B cells and B cell lymphomas. We found that wild type but not SAP-deficient CD8 T cells could respond robustly and differentiate into cytotoxic effectors upon stimulation by antigen-expressing B cells or B cell lymphomas in vitro. In addition, wild type CD8 T cell responses were shown to be dependent on the expression of SLAM receptor CD48 on the B cell or B cell lymphoma surface. Given that 2B4 is a ligand for CD48, we determined that B cell-priming of wild type CD8 T cells could be halted by application of a 2B4 blocking antibody. Together, these findings suggest that 2B4-CD48 interactions and SAP signaling are essential for priming of CD8 T cells by antigen-presenting B cells and B lymphoma cells.   181 Defects in one or more immune subsets may be responsible for XLP patients being unable to mount an effective response towards EBV. To summarize our work, SAP may confer immunity against EBV infection through at least three different mechanisms: (i) SAP promotes Th17 differentiation that contributes to anti-viral responses, (ii) SAP is required for CD8 T cells to proliferate and differentiate upon encountering viral antigens presented by B cells, and (iii) SAP is required for viral-specific CD8 T cell effectors to kill antigen-expressing B cell targets. The details regarding possible immune regulation by SAP are described in the next section and shown schematically in figures 5-1, 5-2 and 5-3.   5.1.1 SAP regulation of Th17 differentiation.  IL-17 is a pro-inflammatory cytokine that plays many key roles in host defense and inflammatory diseases. Previous studies have shown that IL-17 is protective against bacteria and fungi infections (289). In addition, IL-17-secreting effector T cells have been implicated in the pathogenesis of various autoimmune diseases (186, 187, 206, 208, 283, 290-296). However, IL-17-secreting effector T cells have recently been suggested to play important roles in viral clearance (188, 206, 297). IL-17 binding with IL-17 receptor (IL-17R) on epithelial cells, endothelial cells, fibroblast cells and hematopoietic cells promotes the generation of cytokines (IL-6, G-CSF, TNF-α) and chemokines (CXCL1, CXCL5) that recruit neutrophils and macrophages to the sites of infection (475). Subsequently, infiltrating neutrophils and macrophages produce inflammatory cytokines and mediate phagocytosis of infected cells that may be critical to control early viral infection. In addition, elevated numbers of Th17 cells are often found at sites of inflammation and through secretion of IL-22, may stimulate tissue repair (476).   182 EBV establishes a lytic infection of oral epithelial cells and local B cells upon oral transmission (Figure 5-1). Lytic EBV antigens may be presented directly by epithelial cells, lytically infected B cells or cross-presenting DCs (that have engulfed infected apoptotic cells). SLAM/SAP signaling may enhance the generation of viral-specific Th17 cells producing IL-17 and IL-22. IL-17 stimulation of epithelial cells induces the production of pro-inflammatory cytokines and chemokines to attract neutrophils, macrophages and other immune cells, promoting viral clearance. In addition, IL-22, produced by Th17 cells, helps promote epithelial repair and may limit viral spread. Without SAP, Th17 cell generation is less efficient resulting in weakened immune responses and leaving damaged and infected epithelial cells and infectious virions.      183    Figure 5-1 SAP regulation of Th17 cells and EBV infection. IL-17 mediates immune responses that may serve to combat viral infection. IL-17 receptor (IL-17R) is widely expressed in epithelial cells and innate immune cells. Engagement of IL-17 by IL-17R induces the expression of cytokines (IL-6, G-CSF) and chemokines that leads to the recruitment of neutrophils and macrophages to sites of inflammation. SLAM/SAP signaling stimulates the differentiation of Th17 cells. In the healthy controls, Th17 cells and associated IL-17 and IL-22 production may function to limit EBV infection and mediate tissue repair.              Healthy ControlSLAMB cellsDCsSLAMSAPIL-17RIL-17IL-6, G-CSFchemokinesneutrophilsmacrophagesIL-22epitheliumXLPSLAMB cellsDCsIL-17RIL-17IL-22epitheliumCD4CD4SAPTh17CD4CD4Th17SLAM 184 5.1.2 SAP modulates CD8 T cell priming by antigen-presenting B cells. CD8 T cells are a key component of protective immunity against most viral infections (124). The presentation of viral antigens by APCs initiates the activation of viral-specific CD8 T cells, triggering massive clonal expansion and differentiation into cytotoxic and inflammatory cytokine producing effector T cells. Subsequently, viral-specific CD8 T cells promote anti-viral immunity by secreting cytokines and mediating cell-contact dependent killing. Thus, the antigen-priming of CD8 T cells is a critical step to stimulate anti-viral immune responses. The antigen-priming of CD8 T cells require APCs to present sufficient levels of cognate antigen along with costimulatory molecules and cytokines to induce a program of cell activation, proliferation and differentiation (40, 125).  EBV-infected B cells are likely the key APC for priming latent-antigen specific CD8 T cells that in turn are necessary for controlling the expansion of latent B cells. By contrast, EBV-infected epithelial cells, and perhaps cross-presenting DCs, may predominantly prime CD8 T cells specific for lytic antigens and thus, in turn target cells undergoing lytic viral replication. Based on our findings that SAP is critical for priming of naive CD8 T cells by antigen-presenting B cells, we hypothesize that latent antigen-specific CD8 T cells within healthy controls are primed efficiently by EBV-infected B cells, generating a large population of latent-antigen specific T cells with potent effector functions. Viral-specific CD8 T cells may serve to control the reactivation of latently infected-B cells (Figure 5-2). By contrast, SAP-deficient CD8 T cells respond poorly to EBV-infected B cells, failing to expand and differentiate into effector CD8 T cells. Coincidently, CD48 was first described as a surface antigen super-induced by B cells upon infection by EBV (449) and we have found that CD48 expression by B cells is essential for their capacity to prime naïve wild type CD8 T cells. Finally, we speculate that XLP patients may be  185 prone to B cell lymphomas because of a role of SAP in CD8 T cell immune surveillance of pre-cancerous or cancerous B cells.             186    Figure 5-2 SAP is essential for the differentiation of EBV-specific CD8 T cells. Cytotoxic CD8 T cells play a pivotal role in controlling viral infection. Upon antigen-priming by APCs, viral-specific CD8 T cells undergo activation followed by massive clonal expansion and differentiation. EBV-infected B cells may be the key APC for priming latent antigen-specific CD8 T cells and that the generation of latent-antigen specific CD8 T cell effectors is important for limiting latently infected B cells. Given that SAP expression within CD8 T cells is required for the optimal priming by antigen-presenting B cells, we hypothesize that latent-antigen specific CD8 T cells within XLP patients would not be primed efficiently by latently-infected B cells, allowing for latent B cells to expand unchecked by CD8 T cell immune surveillance.          2B4CD48EBV infected B cell  CD8Healthy ControlXLPPriming DifferentiationIFN-γ TNF-αSAPEBV infected B cell CD8EffectorCD8EffectorCD8IFN-γ TNF-α2B4CD48 187 5.1.3 SAP is required for cytotoxic CD8 T effectors to recognize antigen-expressing B cell targets.  CD8 T cell effector-induced target destruction requires the establishment of cell contacts between effector and target cell, resulting in stable immunological synapses and the release of cytolytic granules into target cell. Previous work has documented SLAM receptors/SAP signaling are playing important roles in modulating lymphocyte: lymphocyte interactions and immune responses. Moreover, SLAM family receptors regulate interactions between CD4 T cells and B cells that are critical for Tfh cell differentiation, germinal centers and memory B cell formation (364). In addition, SLAM family receptors and SAP are necessary to support optimal effector functions of NK cells towards lymphocyte targets (369, 370). Cytotoxic T lymphocytes from XLP patients have been shown to exhibit impaired killing towards B cell targets (363). In murine studies, Ly108 (SLAMF6)/SAP signaling is indispensible for stabilizing CD8 T cell: B cell interactions and the killing of LPS-activated B cell blasts (370). Based on the results from CHAPTER 4, SAP-deficient CD8 T cell effectors exhibit poor cytotoxicity against B cell lymphoma targets under defined TCR signaling conditions. However, SAP-deficient CD8 T cell effectors do possess potent cytotoxicity as they can kill melanoma targets as efficiently as wild type CD8 T cell effectors. Together, these findings suggest that SLAM family receptors/SAP signaling is critical for the recognition of EBV-infected B cells by effector CD8 T cells (Figure 5-3). In the healthy controls, recognition of infected B cell targets by viral-specific effector CD8 T cells limits EBV infection. By contrast, impaired ability of SAP-deficient CD8 T cell effectors to kill EBV-infected B cells enables EBV to evade immune recognition and for infected B cells to expand.   188   Figure 5-3 SAP expression within CD8 T cell effectors is required for the recognition and killing of B cell targets.  SAP is required for the efficient killing of B cell targets by effector CD8 T cells. Moreover, SLAM-SAP signaling promotes a stable T cell: B cell immunological synapse, lowering the threshold for TCR signaling enhancing lytic function. In the healthy controls, effector CD8 T cells mediate efficient cytotoxicity against EBV-infected B cells. By contrast, SAP-deficiency results in weakened CD8 T cell cytotoxicity against EBV-infected B cells, enabling these targets to escape and undergo further expansion. Healthy ControlEBV infected B cellSAPEffectorCD8SAPEffectorCD8XLPEBV infected B cellEffectorCD8EffectorCD8SLAMfamilyEBV infected B cellEBV infected B cellExpansionSLAMfamilyIFN-γIFN-γUnder control 189 5.2 Future research directions In CHAPTER 3, we observed that SAP signaling positively regulates the differentiation of Th17 and Tc17 cells and furthermore, promotes the progression of EAE. In addition, co-stimulation with SLAM antibodies was found to enhance IL-17 production by wild type but not SAP-deficient CD4 and CD8 T cells. Furthermore, SAP-deficient mice exhibited reduced propensity to develop EAE relative to wild type mice and protection from disease corresponded with greatly reduced numbers of CNS-infiltrating Th17 and Tc17 cells. Consequently, these findings along with pathogenic role IL-17 is thought to play in MS suggest that the attenuation of SAP signaling may prove valuable as novel therapy for this disease and perhaps, other forms of autoimmune or inflammatory disease. Thus, to further explore the roles of SLAM family receptor and SAP signaling in EAE, we propose to investigate the following:  AIM 1. Are there specific SLAM family receptors expressed or up-regulated on splenic and CNS-infiltrating CD4 and CD8 T cells upon priming and development of EAE? In addition, we will also compare the repertoire of SLAM family receptors expressed by IL-17 and IFN-γ producing CD4 and CD8 T cells. Collectively, these experiments may help identify which SLAM family receptors are expressed by pathogenic T cells and may implicate specific SLAM family receptors in the pathogenesis of EAE. AIM 2. Are SLAM family receptor deficient mice protected from EAE? Our studies showing that SAP-deficient mice are protected from EAE suggest that one or more types of SLAM receptors may be actively engaging SAP to promote disease. Consequently, we will investigate whether SLAM family receptor-deficient mice are also protected from the development of EAE and whether the numbers and cytokine expression of CNS-infiltrating CD4 and CD8 T cells affected in these mice relative to wild type mice. Given our in vitro findings  190 (CHAPTER 3) that SLAM receptor (CD150) antibody can promote the differentiation of IL-17-producing CD4 and CD8 T cells in wild type but not SAP-deficient T cells, we would first examine whether SLAM (CD150)-deficiency affects progression of EAE and the generation of Th17 and Tc17 cells in vivo. Furthermore, our studies with CD48-/- antigen-presenting B cells (CHAPTER 4) indicate that CD48 plays an important co-stimulatory role in generation of antigen-specific CD8 T cells. Consequently, we would also be interested in testing whether CD48-deficiency affects T cell differentiation and EAE pathogenesis. Collectively, these studies along with findings of AIM 1 will help identify which SLAM family receptors contribute to the formation of pathogenic T cells and EAE development.  AIM 3. Can wild type mice be protected from EAE through administration of SLAM receptor-Ig fusion proteins or blocking SLAM family receptor antibodies? Our findings from AIMS 1 and 2 will help focus attention on which SLAM family receptors are the most beneficial for blocking experiments (i.e. attenuate SAP signaling). First experiments will being treatment of mice with blocking SLAM receptor antibody (or SLAM receptor-IgG fusion protein) at time of MOG immunization. If initial experiments show promise, the selected SLAM family receptor antibody (or SLAM family receptor-IgG fusion protein) will be tested for therapeutic benefit after onset of EAE. In CHAPTER 4, we investigated the role of SAP in regulating CD8 T cells responses. Previous studies have shown that CD8 T cell responses were modestly elevated in Sh2d1a-/- mice upon LCMV (arenavirus) infection relative to wild type mice, suggesting recapitulation of the XLP phenotype (i.e. lymphoproliferation) but viral clearance was normal (383, 421). Later studies challenging Sh2d1a-/- mice with the EBV-like (γ-herpesvirus) murine pathogen MHV-68 also did not discriminate immunodeficiency (i.e. inability to clear pathogen), revealing better  191 control of viral latency, elevated numbers of anti-viral CD8 T cells and enhanced CTL activity compared to wild type animals (450, 451). Altogether, we wondered whether the reason XLP patients are exceptionally vulnerable to EBV is related to its B cell tropism and put forward the following rationale why this might be the case. First, the B cell tropism of EBV likely makes it a critical APC during infection: it primes latent antigen (Ag)-specific CD8 T cells that are thought to be critical for controlling infected B cells (302). Second, SAP expression in CD4 T cells is essential for optimal interactions with cognate B cells and necessary for help for T-dependent B cell-responses (351). Consequently, we hypothesized that SAP within CD8 T cells is required for efficient priming by Ag-presenting B cells. Our findings from CHAPTER 4 discovered that SAP signaling is required for optimal CD8 T cell responses towards antigen-presenting B cells or B lymphoma cells in vitro and B lymphoma cells in vivo. To determine whether specific SLAM family receptors were connected with this CD8 T cell phenotype, we performed in vitro experiments utilizing either blocking anti-SLAM family receptor antibodies or SLAM receptor-deficient APCs. Strikingly, wild type CD8 T cell responses were found to be highly dependent on the expression of SLAM receptor CD48 on B cell or B cell lymphoma surface. Given that 2B4 is a ligand for CD48, we next tested whether B cell-priming of wild type CD8 T cells could be halted by application of a blocking 2B4 antibody. Although preliminary observations using anti-2B4 antibody blocking suggest that 2B4 is important for CD8 T cell-priming (Figure 4-14), antibodies can deliver confounding results related to variable blocking efficiency, crosslinking initiated signaling or cross-reactivity to other antigens and thus, further studies are needed to establish the role of 2B4 in co-stimulating CD8 T cell responses. In addition, it is unclear how SAP signaling enhances the capacity of CD8 T cells to respond to antigen-presenting-B cells and -B lymphoma cells.  192 Consequently, to better define the roles of SAP and SLAM family receptors in regulating CD8 T cell responses, we propose to investigate the following:  AIM 1. Do 2B4/CD48 interactions regulate naïve CD8 T cell proliferation to Ag-presenting B cells? To establish 2B4 in mediating naïve CD8 T cell responses towards antigen-presenting B cells or B lymphoma cells, 2B4-/- OT-1 mice would need to be generated so that the proliferation and effector functions of wild type, 2B4-/- and Sh2d1a-/- OT-1 CD8 T cells responding to Ag-presenting B cells or Ag-presenting B lymphoma cells could be compared. If 2B4-/- and Sh2d1a-/- OT-1 CD8 T cells exhibited similarly defective proliferation specifically towards B cell antigen-presenting cells regardless of CD48 expression, these observations would strongly implicate 2B4-SAP signaling in regulating CD8 T cell differentiation upon stimulation with antigen-presenting B cells. By contrast, if responses of 2B4-/- OT-1 CD8 T cells more closely resembled wild type CD8 T cells, these findings would suggest a 2B4-independent means by which SAP regulates CD8 T cell proliferation. Together, these experiments should establish the function of 2B4 and SAP in modulating the proliferation, differentiation and fate of naïve CD8 T cells upon B cell antigen presentation. AIM 2. Does CD48-deficiency impact CD8 T cell immune surveillance of B cells? Our SAP findings along with the observation that CD48 is strongly induced in EBV-infected B cells (449) suggests that CD48 on the B cell surface is critical for recognition by CD8 T cells in vivo. Consequently, to address whether CD48 on the B cell surface is critical for immune system recognition, CD48-/- (Ly5.2+)/ WT (Ly5.1+) mixed bone marrow chimera mice, avoiding confounding influences from a general immune system phenotype, could be infected with model pathogens LCMV clone 13 or MHV-68. At various times post-infection, viral loads within sorted wild type and CD48-/- B cells could be assessed and quantitated using a FACS-based assay to  193 detect LCMV-infected B cells (477) and real-time PCR to detect MHV-68 viral genomes (478) respectively. Collectively, these experiments will determine if CD48-deficiency enables virally-infected B cells to escape detection by host immune system.  AIM 3. How does SAP signaling regulate the proliferation of naïve CD8 T cells?  SAP co-stimulation of CD8 T cells is particularly important for driving proliferation under weaker TCR signaling conditions (lower antigen concentrations). Moreover, decreased IL-2 production by SAP-deficient CD8 T cells may contribute to their diminished responses (Figure 4-3).  However, SAP-deficiency may affect CD8 T cells in other ways leading to diminished proliferation and function. Consequently, it would be interesting to determine whether SAP function controls CD8 T cell responses by regulating cell death, examining expression of Fas/Fas ligand (FasL) and apoptosis markers (Annexin V and Caspase-3 activation) on wild type and Sh2d1a-/- CD8 T cells at various times post-stimulation. In addition, our preliminary observations suggest that the activation of ERK and AKT are decreased upon stimulation by antigen-presenting B cells (Figure 4-17). Consequently, SAP may support naïve T cell proliferation through impacting cellular metabolism given that ERK and AKT pathways switch naïve T cells from oxidative phosphorylation to glycolysis necessary to meet the demands for massive cell growth and differentiation (472). To address this question, glycolysis and glucose uptake assays and measurements of OCR and ECAR using CFRI’s Seahorse XF24 Analyzer could be performed on wild type and Sh2d1a-/- CD8 T cells as described previously (473, 474). 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Gammaherpesvirus latency accentuates EAE pathogenesis: relevance to Epstein-Barr virus and multiple sclerosis. PLoS Pathog. 2012;8(5):e1002715.  240 APPENDICES  Appendix A    A.1 Sorting gates for naïve CD4 and CD8 T cells.     Expression of CD25 and CD44 on bulk and sorted naïve (CD25-CD44lo) CD4+ and CD8+ T cells from wild type and Sh2d1a-/- mice are shown.          CD44+/+-/-ex vivo sorted14.8 7.110.5 10.85.0 0.03.3 0.0CD25+/+-/-16.5 1.016.5 0.56.9 0.55.4 0.0CD4 CD8CD25CD44ex vivo sorted 241 A.2 Preparation of naïve CD4 T cells for C. rodentium infection experiment.    Expression of CD25 and CD44 on CD4+TCRβ+ T cells from wild type and Sh2d1a-/- mice is shown before (A) and after CD25/CD44 antibody-mediated depletion (see Materials and Methods) of regulatory and memory/effector T cells (B). (C) Naïve (CD25-CD44lo) CD4 T cells from wild type (Thy1.1+CD45.2+) and Sh2d1a-/- (Thy1.1+CD45.2+) mice were mixed at a 1:1 ratio and injected intravenously into host (CD45.1+) mice prior to C. rodentium infection.                             A +/+ -/-CD25CD44B14.574.5CThy1.2Thy1.1Ex vivo10.113.777.58.20.397.0Ab-Depleted2.20.297.32.1+/+ -/-CD25CD44CD4TCRβCD4+TCRβ+84Live gated4949 242 A.3 Confirmation of CD48 and Ly108 expression in CD48-/- and Ly108-/- splenocytes.     Histograms indicated CD48 or Ly108 expression in CD19+TCR- population from wild type or CD48-/- or Ly108-/- splenocytes or FMO control.           % of MaxCD48FMOCD48-/-+/+% of MaxLy108FMOLy108-/-+/+ 243 A.4 Wild type and Sh2d1a-/- CD8 T cell responses towards antigen-presenting Ly108-/- B cells.    CFSE-labeled wild type and Sh2d1a-/- OT-1 CD8 T cells were cultured with soluble OVA peptides and purified wild type or Ly108-/- B cells as antigen-presenting cells. Cell proliferation was measured after 4 d based on CFSE dilution by flow cytometry. Samples were acquired for 1 min interval.   +/+-/-OVA 10-7 M10-8 M10-9 M0B cells3501204520+/+-/-CFSENumbersLy108-/-B cells20972196221068221872103178054154229625932296269251283736571067232366119811981120 45 120350350350120120454520

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