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Role of RasGRP1 in T cell development and function Chen, Xiaoxi 2009

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ROLE OF RASGRP1 TN T CELL DEVELOPMENT AND FUNCTION  by  XIAOXI CHEN B. Sc., Wuhan University, Wuhan, P.R.China, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2009 © Xiaoxi Chen, 2009  ABSTRACT  The unique feature of the thymus is to establish the discrimination between self and non-self and providing immunity to foreign peptides, particularly those derived from pathogens, that are presented by self-major histocompatibility complex (MHC) molecules. The development and activation of T lymphocytes require many critical decisions that profoundly affect their differentiation and ability to mount an effective immune response. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP 1 and Sos. RasGRP 1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. In this thesis, by using RasGRP1 null mutant mice, we characterized the role of RasGRP1 in conventional and regulatory T cell (Treg) development, as well as the consequential effects that result from its deficiency in peripheral tolerance, T cell activation, proliferation and immune response against infections. Investigations on RasGRP1 mice expressing a transgenic (Tg) MHC class Il-restricted TCR revealed that RasGRP 1 transmits differentiation signaling critically required for conventional CD4 T cell development. As a result of the impaired developmental program, RasGRPF’ mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion. Interestingly, studies using MHC class I-restricted TCR Tg mice lacking RasGRP 1 suggest that RasGRP1 is essential for lowering activation threshold and achieving optimal cytokine receptor expression on conventional CD8 T cells upon activation. We also investigated the consequence of RasGRP1 deficiency on the development and function of regulatory T cells. We found that RasGRP 1 differentially affected the development and function of CD4 and CD8 regulatory T cells. Overall, our studies provide novel insights to the significance of RasGRP1 signaling pathway in T cell development and function, along with how its deficiency affects the development and function of regulatory T cells.  11  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF FIGURES LIST OF ABBREVIATIONS  viii xi  ACKNOWLEDGEMENTS  xiii  CO-AUTHORSHIP STATEMENT  xiv  CHAPTER 1  INTRODUCTION  1  1.1  Preface  1  1.2  T cell development  2  1.2.1  Generation of CD4CD8 immature thymocytes  2  1.2.2  Fate decision of DP thymocytes  4  1.2.3  Models of CD4/CD8-lineage choice  5  1.2.4  Mechanisms of CD4/CD8-lineage decision  8  1.2.5  Central tolerance in the thymus  1.3  Role of RasGRP family in lymphocyte development and effector function  12 14  1.3.1  RasGRP1 signaling pathway in T cell selection and activation  14  1.3.2  RasGRP1 and RasGRP3 link BCR signaling to Ras in B lymphocyte  17  1.4  Naïve T cell activation and homeostasis-induced proliferation  18  1.4.1  Activation of naïve T cells  18  1.4.2  Homeostatic proliferation of peripheral T cells  19  1.5  T regulatory cell development and function  20  1.5.1  Naturally arising Foxp3-expressing Treg cells  21  1.5.2  Inducible regulatory CD4 T cells  24  1.5.3  Unconventional CD8 T cells with regulatory function  25  1.6  Thesis objectives  26  1.7  Figures  28  1.8  References  37  111  CHAPTER 2 RASGRP1 TRANSMITS PRODIFFERENTIATION TCR SIGNALING 54 THAT IS CRUCIAL FOR CD4 T CELL DEVELOPMENT 2.1  Introduction  54  2.2  Materials and methods  57  Mice  57  Flow cytometry  57  Proliferation assays  58  CTL assays  58  DP thymocyte stimulations  58  Cell signaling studies and immunoblotting  59  Intrathymic injection (IT) experiments  59  Coreceptor re-expression assay  60  2.3  Results  60  CD4 SP thymocytes are critically dependent on RasGRP1 for their development  60  Decreased ERK activity in RasGRP1TcrAND DP thymocytes is correlated with reductions in TCR and Bcl-2 expression  61  RasGRP1 DP thymocytes demonstrate defective P-ERK, CD69, and Bcl-2 upregulation following TCR engagement  62  Tg Bcl-2 expression fails to restore CD4 T cell development in RasGRP 1-deficient mice 63 RasGRP 1-deficient TcrAND CD8 SP thymocytes resemble CD8 SP thymocytes that 65 develop in RasGRP 1 mice expressing the MHC class I-restricted H-Y TCR RasGRPFTcrAND DP thymocytes give rise to a few CD8 but not CD4 SP thymocytes 65 after intrathymic transfer Mutant CD8 SP thymocytes express markers of positive selection, respond to a MHC 67 class Il-specific Ag, and differentiate into cytotoxic effectors RasGRP1TcrAND animals possess significant numbers of peripheral AND TCR CD8 T cells but very small numbers of AND TCR CD4 T cells  69  2.4  Discussion  69  2.5  Acknowledgements  74  2.6  Figures  75  2.7  References  87  CHAPTER 3 CHRONIC IMMUNODEFICIENCY IN MICE LACKING RASGRP1 RESULTS IN CD4 T CELL IMMUNE ACTIVATION AND EXHAUSTION  94  iv  3.1  Introduction. 94  3.2  Materials and methods  97  Mice  97  Flow cytometry  97  Adoptive transfer experiments  98  Bacterial and viral infections  98  Detection of IFN-y production by intracellular flow cytometry  98  Direct ex vivo CTL assays  99  3.3  Results  99  RasGRP 1’ CD4 T cells exhibit markers of acute activation, exhaustion, proliferation, and spontaneous apoptosis 99 RasGRP 1 CD4 SP thymocytes display a naive cell surface phenotype  101  RasGRP1 host animals support rapid donor T cell proliferation  102  RasGRPF’ CD4 T cells proliferate vigorously in chronically immunodeficient RAG-l mice 104 RasGRP 1  mice generate poor pathogen-specific T cell responses  104  Failure of RasGRP 1 mice to generate pathogen-specific T cells is the result of RasGRP1 loss in thymocytes and/or T cells  106  3.4  Discussion  108  3.5  Acknowledgements  113  3.6  Figures  114  3.7  References  125  CHAPTER 4 RASGRP1 REGULATES THE RECRUITMENT OF NArVE CD8 T CELLS TO INITIATE ANTIGEN-INDUCED DEVELOPMENTAL PROGRAMMING 129 4.1  Introduction  129  4.2  Materials and methods  131  Mice  131  Flow cytometry  131  Purification and activation of 2C CD8 T cells  132  2C CD8 T cell stimulation using limiting doses of cognate Ag  132  Cell signaling studies and immunoblotting  133  Generation of a recombinant strain of Listeria monocytogenes expressing the 2C agonist peptide SlY 133  v  Adoptive T cell transfers and bacterial infection experiments 4.3  Results  RasGRP1 2C CD8 T cells exhibit defects in early activation events and IL-2 production  134 134 134  Addition of exogenous IL-2 can support the differentiation of naïve RasGRP 1-deficient 136 CD8 T cells into effector CTLs RasGRP1 2C CD8 T cells display transient ERK activation upon TCR ligation but 138 normal ERK phosphorylation following IL-2 stimulation Recombinant Listeria monocytogenes expressing the SlY epitope induces wild type 2C 139 T cells to proliferate in vivo RasGRP 1-deficient T cells exhibit a reduced rate of proliferation and diminished cytokine production upon cognate antigen stimulation in vivo  140  4.4  Discussion  142  4.5  Acknowledgments  147  4.6  Figures  148  4.7  References  156  CHAPTER 5 PREFERENTIAL DEVELOPMENT OF CD4 ANT) CD8 I REGULATORY 162 CELLS IN RASGRP1-DEFICIENT MICE 5.1  Introduction  162  5.2  Materials and methods  164  Mice  164  Flow cytometry  165  CD4 T cell proliferation and suppression assays  165  Adoptive transfer experiment  166  CFSE labeling  166  CD8 T cell purification and direct ex vivo assays  166  CD8 T cell cytokine and suppression assays  167  5.3  Results  Defective thymic differentiation of CD4Foxp3 T cells in RasGRP 1 mice  167 167  Favored expansion and enhanced suppressive function of peripheral CD4Foxp3 T 168 cells in RasGRP1 mice Preferential development of memory phenotype CD8 T cells in the thymus and periphery of RasGRP 1 mice  170  Elevated expression of NK receptors by CD8 T cells in RasGRP1 mice  171 vi  Activated RasGRP 1’ CD 1 22CD8 T cells are less suppressive than their wild-type counterpart 172 5.4  Discussion  173  5.5  Acknowledgements  178  5.6  Figures  179  5.7  References  191  CHAPTER 6  GENERAL DISCUSSION AND PERSPECTIVE  196  6.1  Summarizing the role of RasGRP1 in T cell development and function  196  6.2  RasGRP1 signaling, positive selection and lineage commitment  198  6.3  Partial T-cell immunodeficiency and immune dysregulation  200  6.3  Concluding remarks  202  6.4  References  203  APPENDIX: BIOSAFETY AND ANIMAL CARE CERTIFICATES  206  vii  LIST OF FIGURES  Figure 1.1 The thymus architecture  31  Figure 1.2 Stages in early T-cell development  32  Figure 1.3 The classical models of CD4/CD8 lineage choice  33  Figure 1.4 The kinetic signaling model of CD4/CD8-lineage choice  34  Figure 1.5 Central tolerance mechanisms  35  Figure 1.6 Switching on Ras in different cellular locations  36  Figure 2.1 RasGRP 1 mice expressing the MHC class 11-restricted AND TCR lack CD4 SP 79 thymocytes Figure 2.2 RasGRP1-deficient TcrAND DP thymocytes display reductions in ERK activity and Bcl-2 expression 80 Figure 2.3 RasGRPF’ DP thymocytes exhibit a decreased capacity to up-regulate P-ERK, CD69, and Bcl-2 levels following TCR engagement 81 Figure 2.4 Tg Bcl-2 expression fails to restore CD4 T cell development in RasGRPF’ TcrAND mice  82  Figure 2.5 RasGRPf’ animals share a similar thymic developmental profile regardless of whether they express either the MHC class Il-restricted or MHC class I-restricted TCR 83 Figure 2.6 RasGRP1TcrAND DP thymocytes exhibit limited developmental potential  84  Figure 2.7 CD8 SP thymocytes that develop in RasGRPF’TcrAND mice respond to MHC class TI-specific peptide and differentiate in cytolytic effectors 85 Figure 2.8 RasGRPF’TcrAND animals possess significant numbers of peripheral AND TCR CD8 T cells but very small numbers of AND TCR CD4 T cells Figure 3.1 Peripheral CD4 T cells from RasGRP 1 mice display signs of activation, exhaustion, proliferation, and spontaneous apoptosis  86  117  viii  Figure 3.2 Aberrant-positive selection in RasGRP 1-deficient mice results in a small population of SP thymocytes that bear a naive cell surface phenotype  118  Figure 3.3 The cellular environment within RasGRP1-deficient animals promotes T cell 119 expansion Figure 3.4 RasGRPF’ T cells expand vigorously after transfer into chronically immunodeficient RAG-F’ hosts  120  Figure 3.5 RasGRP1 mice mount a poor MHC class IT-restricted LLO response upon infection with L. monocytogenes  121  Figure 3.6 RasGRPF’ animals generate a weak T cell response toward LCMV and exhibit delayed viral clearance 122 Figure 3.7 Peripheral RasGRP 1’ T cells possess an altered TCR repertoire  123  Figure 3.8 Failure of RasGRP mice to generate pathogen-specific T cells is the result of 124 RasGRP1 loss in thymocytes and/or T cells Figure 4.1 RasGRPF’ 2C CD8 T cells exhibit defects in TCR-induced proliferation, early 151 activation events and IL-2 production Figure 4.2 RasGRP1-deficient 2C CD8 T cells acquire an impaired effector phenotype after antigenic stimulation 152 Figure 4.3 RasGRP1 regulates the antigen dose necessary to recruit naïve T cells to initiate proliferation and effector cell formation 153 Figure 4.4 RasGRPF’ 2C CD8 T cells display transient ERK activation upon TCR ligation but normal ERK phosphorylation after TL-2 stimulation 154 Figure 4.5 RasGRP 1-deficient T cells exhibit a reduced rate of proliferation and diminished cytokine production upon antigen stimulation in vivo 155 Figure 5.1 Inefficient intrathymic development of CD4Foxp3 Treg cells in RasGRPP’ mice 183 Figure 5.2 RasGRP 1 CD4Foxp3 cells  mutant mice possess a high frequency of activated peripheral  Figure 5.3 Preferential peripheral expansion of CD4 Treg cells in RasGRP 1  184 mice  185  ix  Figure 5.4 Enhanced suppressive activity of peripheral RasGRPF’ CD4CD25 Treg cells in 186 vitro f Figure 5.5 Efficient development 0 periphery ofRasGRPl mice  cells in both thymus and 187  Figure 5.6 Increased expression of NK receptors by peripheral CD8 T cells from RasGRPF’ 188 mice cells proliferate in response to IL-2 or IL-iS Figure 5.7 RasGRP1 189 and produce IFN-y in response to PMA plus ionomycin stimulation Figure 5.8 Activated RasGRP 1 CD8CD 1 22 cells are less suppressive than activated wild 190 type CD8CD122 cells  x  LIST OF ABBREVIATIONS  7-AAD  7-aminoactinomycin D  Ag  antigen  AIRE  autoimmune regulator gene  APC  antigen-presenting cell  BCR  B cell receptor  CCR  CC-chemokine receptor  CFSE  carboxyfluorescein diacetate succinimidyl ester  CTL  cytotoxic T lymphocyte  CTLA-4  cytotoxic T lymphocyte-associated antigen 4  CXCR  CXC-chemokine receptor  DAG  diacylglycerol  DN  double-negative (CD4CDW) thymocyte  DP  double-positive (CD4CD8) thymocyte  ERK  extracellular-regulated kinase  GTP  guanosine-5 ‘-triphosphate  HSA  heat stable antigen  IFN-y  interferon gamma  IL-2  interleukin 2  INK  jun-N-terminal kinase  LAT  linker of activated T cells  LCMV  lymphocytic choriomeningitis virus  LLO  listeriolysin-O  LM  Listeria monocytogenes  mAbs  monoclonal antibodies  MAPK  mitogen activated protein kinase  MHC  major histocompatibility complex  NK  natural killer  OVA  ovalbumin  PD-i  programmed cell death xi  PD-Li  PD-i ligand 1  PLCy1  phospholipase Cyi  PM  plasma membrane  PMA  phorbol 1 2-myristate 13-acetate  RAG  recombinase activating gene  Ras GAPs  Ras GTPase-activating proteins  Ras GEFs  Ras-guanyl nucleotide exchange factors  SLE  systemic lupus erythromatosus  Sos  son-of-sevenless  SP  single positive (CD4 CD8 or CD4 CD8  TCR  T cell receptor  TECs  thymic epithelial cells  Tg  transgenic  TGF-j3  tumor growth factor-(3  TNF  tumor necrosis factor  Treg  T regulatory cell  ZAP-70  zeta-associated protein of 70 kDa  +  -  -  +  ) thymocyte  xii  ACKNOWLEDGEMENTS  I would like to express my enduring gratitude to Prof. Hung-Sia Teh for offering me the opportunity to come to Canada to study immunology and providing me with a strong foundation for my future research.  I want to thank the Department of Microbiology and Immunology for giving me the permission to commence my graduate studies at UBC in the first instance. I thank Soo-Jeet Teh for providing guidance in the principles in immunological research and technical assistance. I owe particular thanks to John Priatel, who has inspired me to continue my work in science and whose penetrating questions taught me to question more deeply. In addition, I thank my thesis committee members, Drs. Pauline Johnson, Ninan Abraham, Megan Levings and Marc Horwitz for their stimulating support. I also thank the CIHR for providing me with funding for my research.  My current and former colleagues in the Teh lab, including Salim Dhanji, Edward Kim, Darryl Oble and Mike Chow, supported me in my research work. I want to thank them for all their help, support, interest and valuable hints. I also want to thank all the people used to and currently work in our department including (but not limited to) Paul Jaschke, Martin Richer, Caylib Durand, Kevin Lin, Michael Hermawan, Justin LeBlanc, Shelley Small, Jacqueline Lai and Nelson Wong.  Special thanks are owed to my parents, who have encouraged and supported me throughout my entire life, both morally and financially. Without your love and guidance, I could not have accomplished anything. To Uncle Douglas and family, thank you for helping me settled down here and taking care of me all the time. To my friends, KC, Neil & Wendy, Teresa, Samuel, George, Winnie, LW, Lynn, Qing, Leo, Junrong, Duo, XK, Afei, Luobo, I always feel blessed to have known you and obliged to your support.  Especially, I would like to give special thanks to my fiancée Eva. It is your love, patience and encouragement that help me overcome difficulties and guide me through the hard times.  xlii  CO-AUTHORSHIP STATEMENT  For the co-authored chapter 2, I contributed 70% of the experimental design, 80% of the research and data analysis, 30% of the manuscript preparation. For the co-authored chapter 3, I contributed to 90% of the experimental design, 70% of the research and data analysis, 50% of the manuscript preparation. For the co-authored chapter 4, I contributed to 60% of the experimental design, 40% of the research and data analysis, 30% of the manuscript preparation. For the co-authored chapter 5, I contributed all of the experimental design, 95% of the research, all the data analysis, and wrote the manuscript.  xiv  CHAPTER 1  1.1  INTRODUCTION  Preface  This thesis represents a culmination of work and learning that has taken place over a period of almost five years (2004-2009) at Department of Microbiology and Immunology, University of British Columbia. The initiative of this study came from our preliminary data published in Immunity (2002) by the senior scientist Dr. John Priatel in our lab. The foundation for this thesis is the strength of signal hypothesis, which argues that weak signals lead to the selective activation of ERK]MAPK and positive selection whereas strong signals lead to the activation of full range of MAPK and negative selection. The Immunity paper suggests that the Ras-guanyl nucleotide exchange factor, RasGRP 1, serving as a critical TCR signaling module, plays a relatively minor role in CD8 T cell development. Based on the strength of TCR signals model, we followed the logical trend of investigating the role of RasGRP1 in CD4 T cell development (chapter 2). Due to the importance of T cell development in establishment of central tolerance and maintenance of functional T cell population in the periphery, studies were performed to understand how the impaired T cell development resulted from RasGRP1 deficiency would affect peripheral T cell homeostasis and function (chapter 3). Furthermore, the relatively normal positive selection of thymocytes expressing a strongly selecting TCR (2C) lacking RasGRP1 offered us the unique opportunity to investigate the role ofRasGRPl in peripheral naïve CD8 T cell activation (chapter 4). Finally, since accumulating evidence suggest that T regulatory cells are an essential force in suppressing autoreactive T lymphocyte and acting as a safeguard for peripheral tolerance, we characterize the role of RasGRP 1 in the development, homeostasis and function of different T regulatory subsets (chapter 5). In this first chapter I provide a general introduction to T cell biology, as well as a survey of the latest scientific discovery.  1  1.2  T cell development  1.2.1  Generation of CD4 CD8 immature thymocytes  +  +.  The thymus is an organ that supports the differentiation and selection of T cells. Mammalian T cells of diverse functional types share a complex developmental history. They originate from pluripotent precursors in the bone marrow or fetal liver and migrate to the thymus to begin a protracted differentiation process in which they gradually acquire T cell characteristics (1). Relatively few T cell progenitors migrate into the thymus per day, but they respond to this new environment by proliferating extensively, while initiating the T cell differentiation transcriptional program (2-4) and gradually turning off genes that allow differentiation to non-T-cell lineages. They then undergo TCR gene rearrangements and assemble TCR complexes, upon which their future survival and functions will depend (1). These cells can then mature into different T cell lineages, including y T cells and c43 T cells. The c3 T cells further diverge into different sub-lineages, such as CD4 T cells, CD8 T cells, natural killer T (NKT) cells and regulatory T cells (Treg cells), each of which have greatly differing functions once they emigrate from the thymus to the periphery.  The thymic development of T cells consists of several processes that require that dynamic relocation of developing lymphocytes into, within and out of the multiple environments of the thymus (Fig. 1.1) (5). It all begins when the lymphoid progenitor cells make their way into the thymus through blood vessels at the cortico-medullary junction (2). In the thymus, these cells lose the potential to become B cells and natural killer (NK) cells (6). The result is a CD4CDW double-negative (DN), committed-T cell precursor. DN thymocytes then go through a sequential differentiation process characterized by the acquisition and loss of their surface expression of CD44 and CD25: DN1, CD44CD25; DN2, CD44CD25; DN3, CD44CD25; and DN4, CD44CD25 (7). This stepwise progress is accompanied with the crosstalk between developing thymocytes and thymic stromal cells, as well as the thymocyte trafficking including the outward migration of DN thymocytes to the capsule regulated by chemokine signals through CXC-chemokine receptor 4 (CXCR4) and CC-chemokine  2  receptor 7 (CCR7) followed by the further migration to subcapsular region mediated by CCR9 signals (5).  Double-negative T cells can give rise to either yó or a13 TCR-expressing cells (8). For cells that proceed along the a TCR pathway, DN3 stage is thought to be a developmental checkpoint (7, 9-11). Development through the first “pro-T-cell” stages early T-cell-lineage progenitor (ETP) stage to DN3 stage  —  —  that is, from the  is independent of the TCR (12).  ETPs and DN2 cells proliferate extensively while acquiring their first T cell characteristics. As the cells reach the DN3 stage, they first express pre-TCR-ct (pTa), which is encoded by a non-rearranging locus and used as a surrogate a chain (7, 9, 10). pTa then pairs with the TCR 3-chain, which is the product of a set of somatic DNA rearrangements that require expression of recombination activating gene 1 (RAG 1) (13) and RAG2 (14) protein. Defects in Rag gene expression prevents -se1ection and hence, pre-TCR assembly, leads to developmental arrest at the DN3 stage (6). At the cell surface, the pre-TCR is then associated with a collection of proteins (the CD3 complex) that is involved in signal transduction (15). Alternatively, DN3 T cells that successfully rearrange TCR y- and ô-chains instead of  -  chains develop into yó T cells (Fig. 1.2).  T cells that emerge from p-selection (late DN3 and DN4) undergo 6-8 cell divisions, after which gene rearrangement at the TCR-a locus produces the second component chain of the mature a3 antigen receptor. The expression of pTa is lost during this stage, which results in the cell-surface display of a low level of a13 TCR assembled with CD3/ proteins. The thymocytes also begin to turn on the expression of both CD4 and CD8 coreceptor proteins most often CD8 first, followed by CD4 (8)  —  and eventually, form a large population of  CD4CD8 double-positive (DP) c43-TCR-expressing immature cells that constitute 90% of the lymphoid compartment in the thymus of young individuals (6, 7). At this time, the DP thymocytes are prepared for the next stage of developmental program: TCR-mediated selection.  3  1.2.2  Fate decision of DP thymocytes  DP thymocytes are unique among T-lineage cells in that they express both CD4 and CD8 coreceptors. CD4 and CD8 coreceptors are transmembrane proteins with extracellular domains that promote TCR engagement of MHC ligands and intracellular domains that enhance TCR signal transduction. As a result, CD4 and CD8 are molecules that promote signaling by MHC-restricted TCRs (16). DP thymocytes that are the product of 3-selection undergo a series of TCR-mediated selection to test if their newly rearranged receptor has some reactivity to self-peptide in the context of self-MHC molecule, so that the subset best suited to function in the host environment is permitted to mature and migrate to peripheral lymphoid tissues. Four distinguishable processes characterize this selection  —  death by  neglect, negative selection, positive selection and lineage-specific development (also referred as lineage commitment) (8, 17). Most DP thymocytes express TCRs that interact so poorly with the available self-peptide-MHC ligands, so that the intracellular signals that are required to sustain viability are not generated, die as a result of death by neglect (18, 19). By contrast, a small proportion of thymocytes that bear TCRs that bind very well to seif-ligands could cause autoimmune pathology if they were permitted to develop and leave the thymus. Thus, signaling on engagement of these TCRs with self-peptide-MHC ligands promotes rapid apoptotic death, a process referred to as negative selection. Only a small fraction of DP thymocytes, which express TCRs of low to intermediate avidity for self-peptide-MHC complexes, initiate a multi-step process known as positive selection that ultimately results in lineage-specific differentiation in either CD4 or CD8 single-positive (SP) mature T cells (Fig. 1.2) (20, 21).  For developing thymocytes to experience the selecting forces at the right places and in the right order, the developing thymocytes and thymic stromal cells have to communicate with each other both in close proximity and remotely (2). In thymus, the cortex contains DP CD4CD8 thymocytes that are subject to T cell selection events, whereas in the thymic medulla, accumulating SP CD4 and CD8 T cells are screened further for self reactivity before their exit from the thymus (5). Stromal cells consisting of various cell types are present in the thymus, including thymic epithelial cells (TECs) and bone marrow-derived 4  cells such as dendritic cells (DC5) and macrophages. The TECs are present in both cortical and medullary thymic areas, both of which can express MHC class I and MHC class II molecules. The cortical TECs (cTEC5) are efficient mediators of positive selection (22). By contrast, abnormal medullary TECs (mTEC5) development and organization are often associated with autoimmunity, which might be due to the impaired development of Treg cells (23, 24) or failed expression of autoimmune regulator gene (AIRE) (2, 25). Cells of bone marrow origin are involved in shaping the developing T cell repertoire through efficient clonal deletion (negative selection) of self-reactive thymocytes by dendritic cells and elimination of apoptotic thymocytes by macrophages (5).  1.2.3  Models of CD4/CD84ineage choice  DP thymocytes that have been positively selected ultimately develop into either CD4 or CD8 SP T cells, with their precise lineage fate being determined by the MHC-restriction specificity of their TCR. In the peripheral lymphoid tissues, mature CD4 T cells function as T helper cells when activated, assisting effector components of the immune system (such as B cells) through cytokine secretion and up-regulation of expression of specific membrane ligands, whereas mature CD8 T cells are can acquire cytotoxic properties after activation, allowing them to directly kill target cells expressing their cognate antigen. As CD4 and CD8 T cells are derived from a common precursor pool of DP thymocytes, the question arises as to how the concordance between MHC specificity and CD4/CD8-lineage choice is established.  The CD4/CD8-lineage choice was classically thought to result in the transcriptional termination of one or the other coreceptor gene as a consequence of the same TCR signaling event that mediates positive selection (6, 16). All classical models of CD4/CD8-lineage choice incorporate this perspective and fall into two main categories, ‘stochastic’ or ‘instructive’, which differ depending on whether termination of coreceptor transcription is random or instructed (16). The stochastic selection model proposes that lineage choice is independent from MHC/TCR signals. According to this model, the termination of coreceptor  5  gene expression during positive selection of DP thymocytes occurs randomly and that a second TCR-dependent rescue step occurs after positive selection, so that only SP thymocytes with matching TCRs and coreceptor survive and differentiate into mature T cells (26-28) (Fig. 1 .3A). Alternatively, the instructive models postulate that during positive selection, TCR signals direct DP thymocytes to specifically terminate the expression of mismatching coreceptor molecule (29, 30). Consequently, instructive models require that MCH class I- and class IT-restricted TCR signals be distinct from one another, that is, MHC +  class TI-induced signals promote positive selection and differentiation into CD4 T cells, whereas MHC class-I-induced signals promote positive selection and differentiation into CD8 T cells. Attempts to validate the selective or instructive models have generated a large amount of evidence that cannot be explained by either model as originally formulated (6, 16, 31, 32).  In the original instructional model, CD4 and CD8 coreceptors were thought to transduce qualitatively different instructional signals (33). However, this idea was subsequently replaced by the proposal that DP thymocytes are instructed by quantitative differences in the intensity of signaling transduced by TCR and coreceptor co-engagement during positive selection (34) (Fig. 1 .3B). The strength of signal model proposes that lineage choice is dictated by the overall strength of signals that are transduced by co-engaged TCR and coreceptor molecules, with strong signals promoting CD4-lineage choice and weak signals promoting CD8-lineage choice (34). This concept is supported by evidence that the cytosolic tail of CD4 binds significantly more intracellular Lck than the cytosolic tail of CD8 (35, 36). TCR and CD4 co-engagement generates strong signals, whereas TCR and CD8 co engagement generates weak signals. It is the relative strength of these signals that induces thymocytes to specifically terminate either cd8 or cd4 gene expression (16). Supporting this view, transgenic expression of CD8 molecules with CD4 cytoplasmic tails allows MHC class I-restricted thymocytes to differentiate into CD4 lineage (33, 34), whereas CD4 disruption causes MHC class IT-restricted thymocytes to differentiate into CD8 T cells (37, 38).  The strength of signal model provided a straightforward explanation for the experiments that manipulate the activity of intracellular kinases, such as Lck, Csk (C-terminal SRC kinase),  6  TEC-family kinases and ERKs in DP thymocytes (16). However, the crucial experiments assessing its core concept by directly altering the signaling intensity of TCR or coreceptor molecules themselves suggested the signal intensity did not determine CD4/CD 8-lineage choice (6). The results indicate that, rather than determining lineage choice, the coreceptor tails control the number of thymocytes being selected, thereby ensuring the numerical predominance of MHC class IT-restricted T cells (signaled through CD4) over MHC Irestricted T cells (signaled through CD8) (16).  An updated version of the original strength of signal model is the duration of signal instructional model, with the important twist that TCR signal duration, perhaps in addition to signal strength, determines the CD4/CD8-lineage choice (39, 40) (Fig. 1 .3C). According to this model, TCR signals of long duration instruct DP thymocytes to terminate cd8 gene expression and to differentiate into CD4 T cells, whereas TCR signals of short duration instruct DP thymocytes to terminate cd4 gene expression and to differentiate into CD8 T cells (40). Experiments that restrict the expression of ZAP7O, a tyrosine kinase required for TCR-signal transduction to DP thymocytes, thereby genetically limiting the duration of TCR signaling during positive selection, favor this hypothesis (41). When TCR signals are confined to DP thymocytes, only CD8-lineage cells are permitted to develop. Furthermore, thymocytes expressing MHC class IT-restricted TCRs were redirected to the CD8 lineage, suggesting the duration of signal directly affects lineage choice.  Although it was originally unclear why MHC class I- and MHC class Il-restricted TCR signals would be of different duration (40), an explanation adopted from the kinetic signaling model (39, 42) provided a solution: all TCR-signaled DP thymocytes selectively decrease the surface expression of CD8, which disrupts MHC class I-restricted TCR signaling but does not affect MHC class Il-restricted TCR signaling. The kinetic signaling model relies on three postulates. First, that TCR signaling in DP thymocytes, regardless of its MHC specificity, down-regulates cd8 gene expression (39, 43). Second, that CD4-lineage commitment does not occur at DP stage, but rather after TCR signaling has transcriptionally down-regulated CD8, as in CD4+CD8b0 thymocytes. Finally, that in CD4+CD&0 cells, the persistence of TCR signaling blocks interleukin-7 (IL-7)-mediated signaling and induces differentiation  7  into mature CD4 T cells. By contrast, cessation or disruption of TCR signaling allows IL-7mediated signaling, which induces CD4+CD8b0. intermediate thymocytes to undergo coreceptor reversal and differentiate into CD8 T cells (16) (Fig. 1.4). Conclusively, the kinetic signaling model proposes that CD4/CD8-lineage choice is determined by TCR-signal duration and that cytokines, such as IL-7, serves as sensors to detect the duration of the TCR signal (39, 42, 44). This model is based on a different set of fundamental principles than those that underlie classical models, as it was promoted by experimental observations that could not be reconciled with the concepts on which classical models are based (16).  1.2.4  Mechanisms of CD4/CD8-lineage decision  Instead of analyzing the function of TCR signaling (the “top-down” approach), an alternative approach for determining lineage-choice signals stems from the fact that lineage differentiation signals eventually lead to the transcriptional regulation of Cd4 and Cd8. By defining first the cis-acting elements required for lineage-specific control of Cd4 and Cd8 and then the functionally critical trans-acting factors that bind to these elements, this approach aims to define the control of lineage commitment from the “bottom-up” (45). Important insights have emerged from this approach, including fundamental distinctions between the lineage-specific regulation of Cd4 and Cd8 and the identification of a crucial factor controlling lineage-specific Cd4 expression (46).  Cd4 gene transcription is regulated differently from Cd8 gene transcription (16). Cellspecific expression of Cd4 results from the activity of a silencer element that abrogates Cd4 gene transcription in CD4 cells (47, 48). The absolute requirement for this element in initiation of Cd4 silencing was demonstrated by the fact that germline deletion results in Cd4 derepression in all T cells (49). By contrast, cell-specific expression of Cd8 is the result of stage-specific enhancer elements that actively induce its expression in CD8 T cells (50, 51). Five enhancer elements that regulate the expression of Cd8a gene have been identified (known as E8 -E8v) (50-53), and two of these enhancer elements might be particularly 1 relevant to our understanding of CD4/CD8-lineage choice, as the E8 111 enhancer is active only  8  in DP thymocytes and the E8 1 enhancer is active in CD8 SP thymocytes and CD8 T cells (16). No trans-acting factors have yet been identified that control lineage-specific expression of Cd8.  RIJNX (runt-related transcription factor) proteins are members of the runt-domain family of transcription factors, which have similar structural organization and conserved DNA-binding sites (54). In the thymus, RUNX1 is mainly expressed by DN thymocytes and RUNX3 is expressed by post-selection CD8 thymocytes. RIJNX1 and RUNX3 both bind to the Cd4 silencer and silence Cd4 gene transcription, indicating a role for RUNX proteins in CD4/CD8-lineage choice (54). Importantly, RUNX3 expression is up-regulated during the differentiation of CD4+CD8b0. thymocytes into CD8 I cells, when it provides two crucial functions: first, RUNX3 binds to the Cd4 silencer element and silences Cd4 gene transcription (54); and second, RUNX3 binds to the E8 1 Cd8 enhancer element and reinitiates Cd8 gene transcription (55). So, RIJNX3 may be the transcriptional mediator of coreceptor reversal by silencing Cd4 and reinitiating Cd8 gene expression during the differentiation of CD4+CD8b0 intermediate thymocytes into CD8 mature T cells (16).  GATA3 is an enhancer-binding zinc-finger protein that functions as a lineage-specific transcription factor in T cells at various stages of development (56, 57). GATA3 is expressed in the earliest progenitor T cells and is required for thymocytes to differentiate beyond the DN stage (56). GATA3 may also have an important role in CD4-lineage choice, as suggested by observations that GATA3 is preferentially expressed by CD4 T cells (57), that GATA3 expression is up-regulated by TCR signaling in DP thymocytes and that sustained expression of GATA3 blocks the generation of CD8 T cells (58). In addition, conditional deletion of Gata3 in DP thymocytes markedly decreases CD4 T cell number without affecting CD8 T cell generation, indicating a crucial role for GATA3 in the survival and/or differentiation of positively selected thymocytes into CD4-lineage T cells (16). However, enforced expression of GATA3 fails to redirect MHC class I-restricted thymocytes to differentiate into CD4 T cells (57), suggesting that GATA3 might not be a CD4-lineage regulator. Since it is expressed in the early stage of thymocyte development, GATA3 might function upstream of other lineage-specific factors and play a crucial role in CD4-lineage choice.  9  A recently identified transcription factor has provided novel insight on the mechanisms of CD4/CD8-lineage choice. Th-POK (T helper-inducing POZ-Kruppel factor, also called cKrox) is a zinc-finger protein that is encoded by the Zbtb 7b (zinc-finger-and-BTB-domain containing 7B) gene, which is responsible for the phenotype of the “helper-deficient” (HD) mice (59, 60). In an exciting series of experiments, two laboratories discovered that Th-POK was important for CD4-lineage choice and CD4 T cell differentiation (59, 60). The HD mice were originally identified by virtue of their specific lack of mature SP thymocytes and peripheral T cells (61). Notably, in those mice, MHC class IT-signaled thymocytes failed to differentiate into CD4 cells and instead differentiated into mature CD8 T cells. In one of the studies, the HD mice were found to have a point mutation in the second zinc-finger domain of Th-POK that presumably disrupts DNA binding (59). Therefore, the phenotype of RD mice suggested that a functional Th-POK molecule is necessary for CD4 T cell differentiation. The reciprocal experiments done by the other group (60) revealed that the expression of transgene-encoded Th-POK proteins throughout thymocyte development forced virtually all positively selected thymoyctes to differentiate into CD4 T cells, regardless of their MHC specificity. Hence, Th-POK seemed to be the “master switch” of CD4 T cell development, as it seemed to be both necessary and sufficient to impose the CD4 fate on TCR-signaled DP thymocytes (46, 62). It seems reasonable to conclude that Th-POK serves both as a lineage-specification factor, turning on CD4 lineage-specific genes, as well as a commitment factor, preventing the expression of CD8 lineage-specific genes. Consistent with this perspective, retroviral transduction of mature CD8 T cells with Th-POK led to decreased cytotoxic T cell lineage function and induced some CD4 T-helper cell characteristics, which indicates that even mature CD8 T cells are susceptible to the CD4-lineage-promoting effects of Th-POK (63).  However, surprisingly, latest data from the groups of Taniuchi (64), Bosselut (65) and Littman (66) now collectively demonstrate that Th-POK is not the sole “master regulator” of CD4 lineage and does not reside at the apex of the molecular circuitry controlling CD4versus-CD8 lineage choice (62, 64-66). Instead, they proposed that GATA3 acts upstream of Th-POK to initiate CD4 specification, whereas Th-POK acts later as a commitment factor and serves to “lock down” the CD4 fate (62). Wang et a!. made the unexpected finding that  10  Th-POK is not expressed when GATA3 is absent (65). They further showed that GATA3 binds in the proximal regulatory element of Th-POK encoding gene zbtb 7b, suggesting that GATA3 is necessary, although probably not sufficient, for Th-POK expression in DP thymocytes (62). However, the converse is not true, as thymocytes lacking Th-POK still can express GATA3. Furthermore, transgenic expression of Th-POK could not rescue CD4 T cell development in the absence of GATA3, indicating that Th-POK is not sufficient to impose the CD4 T cell lineage when GATA3 is absent. Together, their findings suggest Th-POK is required at the late phase of CD4 T cell differentiation, downstream of GATA3 (62). Studies by Egawa and Littman (66), using Runx3d reporter mice, successfully showed the derepression of Runx3 expression in MHC class Il-selected Th-POK deficient mice contributes to the redirection of DP thymocytes to CD8 T cell lineage. Strikingly, in the absence of both Th-POK and Runx, this redirection was prevented and cells potentially belonging to the CD4 lineage, presumably specified independently of Th-POK, were generated. Their results suggest that MHC class IT-selected thymocytes are directed toward the CD4 lineage independently of Th-POK but require Th-POK to prevent Runx-dependent differentiation toward the CD8 lineage (66). Muroi et al., however, provide insight into the mechanisms of action of Th-POK late in CD4 T cell differentiation. Their data indicate that Th-POK controls a positive feedback loop, locking down its own expression and consequently the CD4 T cell fate by binding to and antagonizing the activity of both the zbtb7b and Cd4 silencers (64). With these new data, it is becoming apparent how persistent TCR signals communicate with the cell fate determination circuitry (62). It seems likely that Th-POK, acting as a critical CD4-lineage regulator, needs the orchestration from upstream expression of GATA3 and antagonism of Runx expression.  Other molecule such as HMG (high-mobility group) factor TOX has been known to promote the development to the CD8 lineage (67). HMG proteins are DNA-binding proteins that regulate gene expression by modulating local chromatin structure and recruiting other nuclear factor (68). TOX is an 11MG box protein that was first discovered because it was found to be up-regulated in the TCR-signaled DP thymocytes and so was thought to have a role in positive selection and/or lineage choice (16). Experiments in TOX deficient mice revealed that positively selected thymocytes do not become CD4+CD8I0 cells, but instead  11  become  cells that failed to differentiate into CD4 T cells (69). However,  reversal of TOX deficiency through the introduction of a TOX transgene restores this phenotype and rescues the CD4-lineage (69). Thus, TOX seems to be important for maintaining or up-regulating CD4 expression in positively selected DP thymocytes (16).  It has been noted that chromatin organization and chromatin-modifying factors also play an important role in the control Qf coreceptor expression. A proposed model for Cd4 silencing is that BAF, the mammalian chromatin remodeling complex, opens the Cd4 silencer to binding by Runx3, which in turns mediates recruitment of histone deacetylases and corepressors. Sequential deacetylation and CpG methylation accompanied by repositioning to heterochromatin, possibly mediated by transcription factor Ikaros, then leads to a permanently silenced state (46).  1.2.5  Central tolerance in the thymus  Central tolerance refers to those events in the early life of a lymphocyte that focus the adaptive immune system on pathogens and steer it away from healthy tissue. It is induced at the primary sites of lymphocyte development the thymus for a developing T cell  —  —  the bone marrow for a developing B cell and  and it encompasses all of the mechanism by which  antigen-receptor recognition of self-antigen at these sites results in self-tolerance (70). However, central-tolerance mechanisms are not perfect, and self-reactive lymphocytes can escape from this scrutiny and migrate into peripheral tissues. Therefore, peripheral tolerance mechanisms must exist to regulate the activity of self-reactive T cells that have escape central tolerance (70).  The hallmark of T cell central tolerance is clonal deletion: that is, suicide of T-cell progenitors that have high affinity for self-antigen (71). Other processes have been described, including anergy and receptor editing, but these are thought to have a lesser role (70). These three processes impair or eliminate high-affinity self-reactive cells and are considered to be negative selection mechanisms. However, not all central tolerance mechanisms cripple self  12  reactive T cells. Recently, there is growing evidence for a fourth alternative fate, rather than being positively selected for useful T cells, being negatively selected for harmful T cells or death by neglect, during the thymic development (72). It has been proposed that, instead of actively induced cell death, some developing thymocytes expressing high affinity TCR against self-peptide/MHC can be positively selected to become regulatory T cells; this process is also referred to as agonist selection (Fig. 1.5). CD4CD25 regulatory T cells, CD8cLa intestinal epithelial lymphocytes and natural killer T cells have been suggested as T regulatory cells (70). These additional mechanisms are required for reducing the threat of autoinimunity inherent in the adaptive immune responses.  Inherited autoimmune syndromes due to impaired transcriptional factors have emphasize the crucial role for these two distinct central-tolerance mechanisms (73). In one case, mutations in autoimmune regulator (AIRE) protein lead to defective clonal deletion of T cells. AIRE deficiency results a disease known as autoimmune polyendocrinopathy-candidiasis ectodermal-dystrophy syndrome (APECED), a syndrome that is characterized by the presence of autoantibodies that are specific for multiple self-antigens (70, 71); this leads to lymphocytic infiltration of endocrine glands and autoimmune disorders localized at these organs (73). Mice with mutation in the Aire gene have pathological autoimmune features that are similar to those of patients with APECED. Remarkably, mTECs from AIRE-deficient mice have a significant decrease in the expression of multiple transcripts encoding peripheral tissue-specific antigens (TSAs) (74), including the insulin 2 (Ins2) and salivary protein 1 (Sptl) genes, which seem to be under the direct control of AIRE (73). Experiments using TCR transgenic mice that are specific for neo-self antigens have provided compelling evidence that AWE is essential for efficient deletion of self-reactive T cells (75, 76). However, in the same AIRE-deficient mice, aggressive diabetes at birth was observed due to the escape of autoreactive cells expressing transgenic TCR agonist to the antigen expressed by the pancreatic 13-cells (75, 76). This suggests that T cells that escape negative selection are capable of inducing autoimmunity. Thus, the consequence of AIRE deficiency is thought to be due to deficient negative selection of T cells that are specific for self-antigens, which could be caused by defective expression of self-antigens by thymic epithelial cells (73).  13  Another inherited disease results from mutations in the forkhead box P3 (Foxp3) gene which leads to impaired development of Treg cells and cause the syndrome known as immuno dysregulation, polyendocrinopathy and enteropathy, X-linked syndrome (IPEX) (77). The occurrence of IPEX reflects impaired dominant central-tolerance mechanisms which we will cover the details in following chapters. In summary, although there are many peripheral mechanisms that control lymphocyte reactivity, central mechanisms, which operate during development, seem to be essential for the maintenance of self-tolerance (70).  1.3  Role of RasGRP family in lymphocyte development and effector function  1.3.1  RasGRP1 signaling pathway in T cell selection and activation  T cells communicate with their environment through a variety of cell-surface receptors that recognize and bind molecules present in the extracellular environment. After engagement of the T cell receptor (TCR) by antigen-presenting cells, multiple T cell signaling systems come into play, which ultimately alter the cell’s behavior. The simultaneous deployment of several signaling cascades is thought to result in the efficient transcription of genes encoding cytokines and cytokine receptors, which promote further activation and proliferation of mature T lymphocytes (78, 79).  One of these signaling pathways involves the small GTPase Ras, which was originally identified as an oncogene able to induce cell transformation (80, 81). Ras exists as a key binary switch between “off” and “on” by the receptor-transduced incoming stimuli, and propagate signals further downstream. This equilibrium is regulated by the rate of guanyl nucleotide exchange and the rate of GTP hydrolysis. Ras guanyl nucleotide exchange factors (Ras GEFs) catalyze the release of GDP and replacement of GTP. Conversely, Ras proteins can be inactivated by either their own intrinsic GTPase activity or by pairing with GTPase activating proteins (Ras GAPs). In unstimulated cells, including T cells, Ras exists predominantly in its inactive GDP-bound form. However, in response to TCR stimulation,  14  Ras-GTP accumulates, leading to the activation of effectors such as mitogen-activated protein kinase (MAPK) pathway and, ultimately, to changes in gene expression (82).  In T cells, Ras activation is mediated by at least two Ras GEFs, the well-characterized Sos (83) and the recently identified RasGRP1 (82). Recognition of major histocompatibility complex (MHC)-peptide complexes by TCR results in activation of the protein tyrosine kinases Lck (84) and ZAP-70 (85). Phosphorylation of the adaptor LAT (linker for activated T cells) by ZAP-70 leads to the recruitment of numerous proteins. On one hand, LAT serves as a docking site for Grb2 that brings Sos to the membrane leading to an increase in GTP bound Ras. On the other hand, phospho-LAT also recruits phospholipase Cyl (PLOy 1) (86). Activated PLCy1 converts phosphotidylinositol 4,5 bisphosphate (PIP2) into inositol 3,4,5 triphosphate (1P3) and diacyiglycerol (DAG), resulting in membrane localization for RasGRP1 through its DAG binding Cl domain (87, 88). DAG analogs, such as phorbol 12myristate 13-acetate (PMA), can bypass the TCR mediated pathway to directly recruit RasGRP 1 to the membrane which leads to the activation of Ras, as well as the downstream signaling (82). It has recently been demonstrated that PLCy1 selectively activates RasGRP1 on the Golgi membrane and is therefore, distinct from the Grb2/Sos pathway that activates Ras at the Plasma membrane (PM) (89-92) (Fig. 1.6). This surprising finding adds further complexity to Ras signaling, potential cellular responses and the induction of developmental programs. In addition to the differential localization, Ras activation also displays delayed or sustained kinetics at the Golgi relative to the PM (91).  The RasGRP1 protein (Ras guanyl nucleotide-releasing protein, also termed Ca1DAG-GEFII) is the first characterized protein within the RasGRP family (88) (other RasGRP 1 related molecules (93) include RasGRP2, RasGRP3 and Ra5GRP4). All RasGRP family members share similar domain structures such as the catalytic region consisting of the Ras exchange motif (REM), the CDC25 box, the regulatory domains that include a DAG-phorbol ester binding domain and a pair of calcium-binding EF hands (88). Since PMA-induced Ras activation is absent in RasGRPF’ mice (82), this Ras GEF may be the principal molecule of its type acting in thymocytes.  15  Ras signaling can result in the activation of three families of MAPKs, extracellular signalregulated kinases (ERK), c-Jun N-terminal kinases (iNK), and p38. These MAPKs have been implicated in providing different contributions to the thymic selection (94). The Ras-ERK pathway that activates Raf-1, MAPK kinase (MEK1) and ERK has been determined to play an important role in the positive selection of T cells whereas INK and p38 activation have been correlated with negative selection (95-98). More recent studies using Grb2’ mice (83) demonstrated that the activation threshold for ERK was lower than that for JNK and p38. These data suggest that relatively weak TCR signals only activate ERK activation and lead to positive selection whereas strong TCR signals result in activation of the full range of MAPKs and negative selection (93).  RasGRP 1-deficient mice display a block at the DP stage of thymocyte development suggesting a defect in the positive selection of T cells (99). This observation led to the hypothesis that the principal mechanism for distinguishing between positive and negative selection signals is coupled to the utilization of the RasGRP1 and Grb2/Sos pathways and hence, the MAPKs that get activated (100). According to this model, thymocytes that express low affinity TCR for their selecting ligands will activate Ras and ERK via RasGRP1 leading to positive selection; while thymocytes expressing high affinity TCR for the selecting ligands will activate Ras, ERK, INK and p38 via the Grb2/Sos pathway leading to negative selection. To test this hypothesis, we generated two lines of RasGRP 1  MHC class I-restricted TCR Tg  mice, expressing either a weakly or strongly selecting TCR (93). Our findings indicate that .a weakly selecting TCR is critically dependent on RasGRP 1 for its positive selection. However, ERK activation and positive selection of thymocytes expressing a strongly selecting TCR are much less dependent on RasGRP1. By contrast, RasGRP1 deficiency has no effect on INK and p38 MAPK activation or negative selection (93). Therefore, the positive selection of thymocytes expressing a strongly selecting TCR lacking RasGRP1 also offered us the unique opportunity to investigate the role of RasGRP 1 in peripheral T cell homeostasis and function. Our previous studies suggested that RasGRP1 is important for transducing low-grade TCR signals which are necessary for survival and differentiation of naïve peripheral T cells, while strong TCR signals, responsible for the induction of early phase of Ag-driven growth, are RasGRP1 independent (93).  16  As a complement of the loss of function studies, experiments using mice with transgenic overexpression of RasGRP1 have suggested its role in compensating deficient pre-TCR signaling in vivo (101). In this study, it has been found that RasGRP1 expression is rapidly up-regulated in DN thymocytes following pre-TCR ligation. DN thymocytes from RasGRP1 transgenic mice overcome the deficiency of RAG2 expression and are able to mature to the DP stage (101). In addition, RasGRP1 transgenic mice favors thymocyte development towards the CD8 lineage and lowers the threshold of TCR signaling needed to initiate the proliferation of SP thymocytes. Together with studies using RasGRP1-deficient mice, it emphasizes the critical role ofRasGRPl in orchestrating optimal TCR signaling during T cell selection and activation.  1.3.2  RasGRP1 and RasGRP3 link BCR signaling to Ras in B lymphocyte  Although RasGRP1 has been widely studied for its crucial role in T lymphocyte development and function, recent studies using DNA microarray highlight the expression of RasGRP 1 in other cell types both in mice and humans, such as NK cells, dendritic cells and B lymphocytes (102). However, except for B cells, the function of RasGRP 1 in those cell types is unknown. In addition, other members of the RasGRP family have been detected in various cell types other than T and B lymphocyte. RasGRP2 acts on the Ras-related protein Rap and functions in platelet adhesion, whereas RasGRP4 is expressed in mast cells and certain myeloid leukemia cells (103).  Recently, RasGRP1 and RasGRP3 have been found to be coexpressed in primary murine B cells. In B cells, both RasGRP1 and RasGRP3 play a similar role downstream of the B cell receptor (BCR) linking BCR to Ras-ERK signaling (104). By using RasGRP1 and RasGRP3 single and double null mutant mice, recent studies have suggested that RasGRP1 and RasGRP3 both contribute to the BCR-induced Ras activation, although RasGRP3 alone is responsible for maintaining basal Ras-GTP levels in unstimulated cells (104). Surprisingly, in a sharp contrast to T cells, RasGRP-mediated Ras activation is not essential for B cell development since this process occurs normally in double-mutant mice (104). However,  17  RasGRP deficiencies affect humoral responses to specific immunogens and their presence is required for BCR-induced proliferation in vitro (104). Collectively, these data suggest that depending on the strength and duration of signaling event, as well as the developmental stage and contingent signaling through other pathways, various immune cell processes are facilitated by the controlled activation of Ras effector systems (103).  1.4  Naïve T cell activation and homeostasis-induced proliferation  1.4.1  Activation of naïve T cells  After entering the periphery, T cells become the adaptive immune systems’ decisive recipients of messages from the innate immune system. Thus, their effective and efficient mobilization is crucial to the host’s ability to respond to antigenic challenge (105). As naïve T cells, they rest in peripheral lymphoid organs until they encounter activating signals, such as foreign antigen presented by antigen-presenting cells (APC5), and gain the ability to enter sites of inflammation. While a CD4 T cell response emanates from the interaction of a TCR with its cognate peptide bound to an MHC class II molecule on the surface of an antigenpresenting cell, a CD8 T cell response originates from the engagement between a TCR and peptide/MHC class I complex. Stimulation through the TCR alone is insufficient to induce full activation of T cells. To prevent inappropriate activation that might be deleterious to the host, T cells require a second costimulatory signal to link proximal and distal signaling cascades effectively, which results in the transcription of a wide variety of downstream genes (105). However, in the absence of the costimulatory signals derived from the interaction of CD28 on T cells and B7 (CD8O/CD86) on APCs, aberrant activation of TCR can produce a long-lived state of functional unresponsiveness, known as anergy. Specifically, anergized T cell clones produce negligible amounts of IL-2, which is crucial for clonal expansion following the T cell activation (106). Activated T cells then up-regulate the expression of high affinity IL-2 receptor ci. chain (CD25), and respond promptly to IL-2 in autocrine or paracrine fashion. They undergo vigorous clonal expansion, and finally acquire effector functions. With respect to CD8 T cells, the effector T cells become cytolytic and armed with 18  killer function, whereas CD4 T cells produce cytokines that can mediate direct effects on target cells or assist in the activating other arms of the immune system.  After massive proliferation and lineage-specific differentiation into effector cells, most activated T cells undergo activation-induced cell death (AICD), a process in which activated TCR restimulated T cells die after the engagement of cell-death receptors, such as CD95 or tumor necrosis factor receptor (TNFR). Only a small fraction (—5-l0%) of the clonally expanded T cells survive the contraction phase and become long-lived memory cells. They experience a return to homeostasis mediated by balancing a low level of T cell proliferation with survival and death. Upon secondary challenge, those memory T cells can respond in a faster rate and more strongly to eliminate the antigens. Cytokines, especially the common y chain-cytokine family, including IL-2, IL-7 and IL-15 are thought to play critical roles during the four major events of an immune response, that is initiation, clonal expansion, contraction and memory generation (107). After ligation by peptide/MHC, substantial T cell clonal expansion occurs and might be driven, in part by IL-2. IL- 15 might also enhance the proliferation of antigen-specific T cells. During the late phase of clonal expansion, IL-2 is thought to induce T cell death. The massive cell death that occurs during the contraction phase results in the loss of most antigen-specific T cells. Both IL-iS and IL-7 might rescue T cells from cell death and allow memory T cell generation by promoting survival signaling, such as the induction of Bcl-2 expression (107).  1.4.2  Homeostatic proliferation of peripheral T cells  Via the process of selection events during the thymic education, the thymus generates a peripheral repertoire that is largely depleted of overtly autoreactive T cells but retains low but significant reactivity for self-MHC molecules (108). Retaining weak affinity for self MHC/peptide ligands is not only required for T cells to optimally recognize foreign antigens in the context of self-MHC molecules, but also helps to maintain the survival and homeostatic proliferation of naïve T cells (109).  19  Although it has long been realized that mature T cells are regulated at a population level by homeostatic mechanisms that maintain the total size of the T cell pooi at a near-constant level (110-112), the knowledge of homeo static proliferation originally came from experiments in which a small number of naïve T cells were adoptively transferred into immunodeficient hosts (108, 113). However, using host mice deficient in MHC class I or II molecules, this +  slow proliferation was hampered, suggesting the homeostatic proliferation of CD4 and CD8 cells requires contact with self-MHC class II and I molecules, respectively (108). Recently, there is compelling evidence that homeo static proliferation of naïve T cells is driven by the low-affinity interaction with self-MHC molecules loaded with self-peptides (1 14-116). In addition, IL-7 signaling is thought to be important by promoting cell survival during the homeostatic proliferation of naïve T cells (117). In contrast to responses to highaffinity foreign peptides, homeo static proliferation of naïve T cells to self-peptide/MHC complex is relatively slow and is not associated with the up-regulation of acute activation markers such as CD25 and CD69 (114-116). Instead, the dividing cells acquire cell surface markers typically expressed on memory T cells, such as CD44 and Ly6C, although cells become CD44hi only after multiple rounds of cell division (108). However, whether these memory phenotype cells equated with “real” memory cells arising after an immune response to foreign antigens is still uncertain. In homeostasis-induced proliferation, the dividing cells can acquire effector functions, such as cytotoxicity and the capacity to rapidly secrete inflammatory cytokines (108, 117). This may pose risk to lymphopenic hosts, as it may skew the TCR repertoire of resident pooi of T cells toward autoreactivity and furthermore, restrict its diversity.  1.5  T regulatory cell development and function  The random generation of antigen receptors in developing thymocytes results in a considerable risk of autoimmunity. The immune system has evolved several mechanisms to establish and sustain unresponsiveness to self-antigens (immunological self-tolerance), including physical elimination or functional inactivation of self-reactive lymphocytes (clonal deletion and anergy, respectively) (77). There is also substantial evidence that T cell  20  mediated active suppression of self-reactive T cells is another essential mechanism of selftolerance (118, 119). Although the idea ofT cells that negatively control immune responses is not new for immunologists, there has been great controversy as to whether they actually constitute a functionally distinct cellular entity in the immune system and, if they exist, whether they are important in controlling autoimmune diseases (77). Recent years, however, have witnessed resurgent interest in suppressor or regulatory T (Treg) cells in many fields of basic and clinical immunology (118). Foxp3-expressing CD4CD25 Treg cells (77, 120), CD8aa intestinal epithelial lymphocytes (121) and natural killer T (NKT) cells (122) have been suggested to possess the regulatory function. All are thought to be induced by high affmity interactions between self-peptide/MHC with TCRs on developing thymocytes (70). Surprisingly, recent studies indicate a subset of CD8 T cell population expressing CD 122 (IL-2 receptor  1) and high levels of CD44 also possesses regulatory functions since its  deficiency is associated with severe autoimmune diseases (123-126). These results suggest CD8CD 1 22 T cells contain novel population that can function as Treg cells. However, these results are controversial since they cannot be reproduced by other labs.  1.5.1  Naturally arising Foxp3-expressing Treg cells  A crucial experiment for addressing the function of Treg cells in natural self-tolerance is to determine whether their removal from the normal immune system can break self-tolerance, resulting in autoimmune disease (77). Efforts were made in mid-1980s to identify the Treg population by expression of particular cell surface molecules, such as CD5, CD45RB and CD25 (119). It has been shown that the IL-2 receptor-a chain (CD25), which is constitutively expressed by 5-10% of CD4 T cells and 5% of CD4 SP mature thymocytes in normal naïve mice, is able to be used as a marker for autoimmune-preventive Treg cells (118). These “naturally arising” CD4CD25 Treg cells became the best candidates for the T cell population mediating dominant tolerance to self. To emphasize their origin and importance, Treg cell production has been called the third function of the thymus (127).  21  The observation that autoimmunity can be prevented by transfer of peripheral CD4 T cells in neonatally thymectomized mice provided evidence for the thymic origin of Treg cells (119). Subsequent studies suggested that the CD25 CD4 SP thymocytes are capable of suppression in adoptive transfer models and in vitro suppression assay (128). In addition to its suppressive capacity and CD25 expression, this thymocyte subset displays markers characteristics of peripheral Treg cells, including increased expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4), glucocorticoid-inducible tumor necrosis factor receptor (GITR) and 0X40 (129). The essential function for TCR signals in the development of Treg cells was suggested by the finding that TCR Tg mice on a RAG-1/2 deficient background do not develop Treg cells, whereas most TCR Tg mice expressing functional RAG-112 contains varying numbers of Treg cells (130). In addition, it has been shown that the costimulatory signals mediated by CD28 engagement of CD8O or CD86 molecules are important in shaping the repertoire and size of the Treg cell compartment (131). Furthermore, signals mediated by IL-2-IL-2R have been linked to the development, maintenance, survival and expansion of Treg population (132). Deficiency in IL-2 (133, 134), IL-2Ra (135) or IL-2R3 (126) produce similar fatal lymphoproliferative inflammatory disease with an autoimmune component, generally called IL-2 deficiency syndrome (77). Notably, the number of CD4 Tregs is selectively reduced in the thymus and periphery of IL-2-deficient mice (133). IL-2 neutralization also inhibits the physiological proliferation of CD4 Tregs that are presumably responding to normal self-antigens (136). Although CD28 and IL-2 have a considerable effect on the size of developing Treg cell subset, the nature of the putative non-redundant signal involved in Treg cell lineage commitment in the thymus together with the TCR remains to be identified.  A dramatic feature shared by CD4 Treg cells and T cells with autoimmune potential is the ability to recognize self-antigens (137). Recognition of self-antigens by CD4 Tregs was initially suggested by observations indicating that the presence of a particular organ was important for the maintenance of CD4 Tregs-mediated tolerance to that organ (138). Further support comes from studies of mice transgenic for the c43 TCR, in which the development of CD4 Treg cells expressing the transgenic TCRs, is enhanced by thymic expression of the cognate antigen to the Tg TCR (139, 140). Finally, non-regulatory T cells transduced with  22  Treg cell-derived TCRs rapidly expand their populations in vivo and induce autoimmune disease in lymphopenic hosts (137). These data collectively demonstrate that self-reactivity is a prominent feature of naturally arising Treg cells’ TCR repertoire.  Although CD25 expression has been useful in defining the Treg cell population in mice and humans, the most distinguishing feature of CD4 Tregs is the expression of a member of the forkhead family of transcription factor, Foxp3 (141). The discovery of Foxp3 as a critical gene in immune regulation came from the studies of the X-linked immunodeficiency syndrome IPEX and analogous X-linked recessive inflammatory disease in scurfr mutant mouse (131). Foxp3-deficient mice fail to develop CD4CD25 Treg cells and succumb to scurfr-like inflammatory diseases, which can be prevented by transfer of normal CD4 CD25 Treg cells (131). It is suggested that in the absence of CD4 Treg cells, some CD4 T cells expressed self-specific TCRs that are usually expressed by CD4 Tregs and these cells contribute to the pathology of Foxp3-deficient mice (142). Analysis of Foxp3 expression in T cells at both the mRNA and protein level has shown high expression in CD4CD25 Treg cells and low expression of Foxp3 in naïve and activated CD4 T cells (131). Furthermore, analysis of the origin of CD4CD25 Treg cells in chimeric mice containing a 1:1 mixture of allelically marked bone marrow stem cells derived from Foxp3-deficient and wild type mice showed that Foxp3-deficient bone marrow cannot give rise to CD4CD25 Treg cells, thus demonstrating that CD4CD25 Treg cell development is critically dependent on Foxp3 expression (120). These and other studies together suggest that Foxp3 functions as the Treg cell lineage specific factor, which is both necessary and sufficient for their development and function (131, 143).  The precise molecular mechanism by which CD4 Tregs suppress the activation and proliferation of other T cells is controversial and under active investigation (118). Some findings support essential roles of cytokines, such as IL-b, TGF-f3 and IL-35 in the regulation; others suggest the crosslinking of B7 (CD8O and CD86) molecules on the surface of APC or expression of CTLA-4 on Treg cells is required for suppression (131). It is likely the predominant effector mechanism of Treg cell-mediated suppression may vary depending  23  on the specific tissue and inflammation type being studied. Treg cell-specific gene targeting may help to elucidate the suppressive mechanisms operating in vivo.  1.5.2  Inducible regulatory CD4 T cells  Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically (144). Although neonatal thymectomy on day 3 substantially reduces peripheral CD4CD25 T cells number, emphasizing the important function of thymus in the production of Treg cells, intrathymic generation does not seem to represent the only mode by which CD4CD25Foxp3 T cells can be generated. It has been found that Treg cells can also be produced de novo from naïve CD4CD25 T cell by continuous supply of subimmunogenic doses of agonist peptides (145). In those experiments, generation of suppressor T cells was not due to the proliferation and/or local accumulation of preexisting Foxp3 cells, as they used TCR Tg T cells on RAG-2 background, which cannot produce Foxp3 -expressing T cells. A recent report has shown that the conversion of truly naïve CD4 T cells into suppressor cells expressing Foxp3 can be achieved by targeting of peptide-agonist ligands to dendritic cells (146). In addition, the fact that TGF- could help the conversion of in vitro- stimulated peripheral CD4CD25 T cells into suppressor cells suggests that TGF-j3 signaling could be involved in the antigen-driven conversion of suppressor cells in vivo (147, 148). Consistent with that idea, mice deficient in components of the TGF-13-TGF-13 receptor system have reduced numbers of peripheral CD4CD25 T cells, but it is not apparent whether the deficiency in suppressor cells in these mice is exclusively due to reduced survival and/or proliferation or whether a deficit in de novo induction in peripheral lymphoid tissues (149). Overall, these studies suggest that there is mechanism for converting peripheral CD4CD25 T cells possessing high-affinity TCRs into Foxp3-expressing suppressor T cells in the presence of TGF-j3, although the details of this conversion still remain unknown.  24  1.5.3  Unconventional CD8 T cells with regulatory function  TCR Tg mice in which CD8 T cells of defined specificity are generated provides a powerful tool for studying T cell development and function (150). In collaboration with Harald von Boebmer, we used mice that express the Tg H-Y TCR to study the mechanisms of T cells development. The H-Y TCR is specific for the male (H-Y) peptide presented by H2Db (20, 21). In male H-2 b H-Y TCR Tg mice, immature CD4 + CD8 + thymocytes that express the H-Y .  .  TCR are deleted (151). However, surprisingly, large numbers of peripheral T cells that express low levels of CD8 (CD8 °) and exclusively the H-Y TCRs, are present in the spleen 1 and lymph nodes of male H-2” H-Y TCR Tg mice (152). These self-specific CD8’° cells possess a memory phenotype characterized by high expression of CD44, CD 122 and Ly6C (153) and a lower level of TCR expression than naïve CD8 T cells (154). Interaction with cognate self-antigen was required for the optimal expansion of these cells in peripheral lymphoici organs (153). Furthennore, the CD8 ° cells are hyporesponsive to antigen 1 stimulation relative to naïve CD8 T cells, while they can proliferate vigorously in response to either IL-2 or IL- 15 (153). Upon activation by these cytokines, CD8’° cells up-regulate NKG2D and DAP12, thus, are capable to perform killing in a NKG2D dependent fashion.  Although those studies were done using Tg mice, it has been shown that in normal B6 mice, there is a certain population of peripheral CD8 T cells possessing memory phenotype that share many functional similarities with the CD8 ° cells in H-Y mice (154, 155). It is generally 1 assumed that these memory phenotype CD8 T cells in B6 mice are the progenies of conventional CD8 T cells following exposure to foreign antigens. However, similar frequencies of these memory CD8 T cells are also found in germ-free and antigen-free colonies, arguing they are unlikely memory cells. Recent data have identified the Tec kinases, Itk and Rik as crucial regulators of CD8 T cell development into the conventional lymphocyte lineage (156). Studies using Itk, Rik and IL-iS knockout mice support the hypothesis that the CD 1 22 unconventional CD8 cells represent a cell lineage that is distinct from conventional CD8 T cells (157-159). These studies show that whereas IL-15’ mice lack CD44’CD122 CD8 T cells, Itk’Rlk’ lack CD44’°CD12TCD8 T cells. Furthermore, the same defects were observed during thymus development. They also confirmed that these 25  unconventional CD8 T cells express NK receptors and possess innate immune function whereas CD44I0CD12ZCD8+ T cells do not express NK receptors. These findings lead to the hypothesis that altered TCR signaling is the basis for the development of unconventional T cell lineage (156).  While there is a vast amount of literature on the developmental biology, function and TCR repertoires of CD4 Treg cells, little is known about the developmental biology, function and TCR repertoires of regulatory CD8 T cells, Studies in CD 122-deficient mice have implicated the existence of CD8 Tregs that preferentially regulate the immune functions of CD8 T cells. CD 122-deficient mice exhibit severe hyperimmunity (126), which is associated with the expansion of abnormally activated T cells (160). Suzuki el al (161) proposed that the lack of certain CD8 regulatory T cells might be responsible for the abnormally activated T cells, This notion was supported by the observation that the transfer of highly purified CD8CD122 T cells from normal mice into CD 122-deficient neonates prevented the development of the abnormally activated T cells leading to a normal phenotype in the treated mice (124), although this result cannot be reproduced by other labs. This result suggests that CD8CD122 T cells contain novel Tregs that effectively regulate the activity of CD122deficient CD8 T cells. Furthermore, RAG-2 mice that received wild type CD8CD12Z cells die within 10 wk after transfer, suggesting that these normal CD8 T cells subpopulation can become dangerously activated in the absence of CD8CD122 Tregs (124). Follow-up studies indicate that the suppressor activity of CD8CD 1 22 Tregs is mediated by IL- 10 that is produced by these cells (123). Therefore, evidence supporting the existence of CD8 Tregs is beginning to emerge. However, much more work is needed to further characterize the molecular mechanisms how these CD8 Treg cells perform the suppressive function.  1.6  Thesis objectives  Our hypothesis of thesis is that RasGRP1 plays crucial roles in T cell development and peripheral function. Its deficiency will cause partial T-cell immunodeficiency as well as impaired central and peripheral tolerance.  26  The objectives of this thesis are: 1) Demonstrate the role of RasGRP1 in CD4 T cell development; 2) Characterize CD4 T cells with an activated phenotype in the periphery of B6 RasGRP 1’ mice; 3) Investigate potential link between the impaired CD4 T cell development and the activated CD4 phenotype in B6 RasGRPF’ mice; 4) Determine the role of RasGRP1 in naïve CD8 T cell activation; 5) Determine the role of RasGRP1 in the development and function of CD4 and CD8 Treg cells.  Chapter 2 starts by examining the role of RasGRP 1 in CD4 T cell development by using MHC class Il-restricted AND TCR Tg mice. Chapter 3 characterizes the acutely activated and proliferating CD4 T cell population in the periphery of RasGRP 1 mice, elucidates the consequences, such as homeo static proliferation, generation of TCR repertoire and T cell response against infectious pathogen, due to the impaired T cell development in B6 RasGRP1 mice. Chapter 4 describes how RasGRP1 lowers the CD8 T cell activation threshold via controlling the IL-2 signaling, using MHC class I-restricted 2C TCR Tg mice. Chapter 5 describes the analysis of both CD4 and CD8 Treg cell development and function in B6 RasGRP1 mice.  27  1.7  Figures  Permission grantedfrom Nature Publishing Group for reusing the adaptedfigures in this thesis.  Figure 1.1 The thymus architecture. A. The human thymus section from a newborn child was stained with haematoxylin and eosin. B. The thymus section from an adult C57BL/6 mouse was stained for CD4 (green), CD8 (blue), and medullary thymic epithelial cells defined using Ulex europaeus 1 (red). Cells in cyan indicate the coexpression of CD4 and CD8, indicating CD4CD8 DP cells.  Figure 1.2 Stages in early T-cell development. Cross-section through an adult thymic lobule showing the migration path of T-cell precursors during development. Immigrant precursors initially enter the thymus through blood vessels near the cortico—medullary junction, the early T-cell precursors (ETP) subsequently migrate, and differentiate from double negative (DN) to double positive (DP) to single positive (SP) stages, through the distinct microenvironments of the thymus.. ETPs, DN2, DN3 and DN4 T-cell subsets: note that at any given stage of the cell cycle, the ETPs appear to have the options either to continue their expansion with minimal differentiation in the cortico—medullary junction region or to differentiate into DN2 cells (presumably DN2a cells as shown) that can begin their migration from the site of entry deep within the cortex to the outer rim of the cortex.  13— selection occurs  during the accumulation of the DN3 T cells in the extreme outer portion of the thymus (that is, the subcapsular zone). A directional reversal of migration back across the cortex towards the medulla occurs at the later stages of thymocyte development. Broken arrows depict alternative developmental pathways that are still possible for ETPs and for different subsets of DN2 cells that likely correspond to DN2a and DN2b cells before they complete their commitment to the T-cell-lineage. CCR9, CC-chemokine receptor 9; ISP, immature single positive; LMPP, lymphoid primed multipotent progenitor; NK, natural killer; TCR, T-cell receptor.  28  Figure 1.3 The classical models of CD4/CD8 lineage choice. A. The stochastic selection model postulates that positive-selecting T-cell receptor (TCR) signals randomly terminate the expression of one or the other co-receptor molecule, resulting in the generation of short-lived intermediate cells, which undergo programmed cell death unless they are rescued by a second TCR signal. Because the TCR-mediated rescue signal requires TCRs and co-receptors that are matched, 50% of positively selected thymocytes will fail to survive and mature. B. The strength-of-signal instructional model postulates that weak TCR signals terminate Cd4 transcription, whereas strong TCR signals terminate Cd8 transcription. Signaling by CD8 and MHC class I-restricted TCRs is weak and results in mature CD8 T cells, whereas signaling by CD4 and MHC class TI-restricted TCRs is strong, which results in mature CD4 T cells. C. The duration-of-signal instructional model postulates that short and/or weak TCR signals terminate Cd4 transcription, whereas long and/or strong TCR signals terminate Cd8 transcription. MHC class I-restricted and MHC class TI-restricted TCR signals are proposed to differ in both duration and intensity. DP, double positive.  Figure 1.4 The kinetic signaling model of CD4/CD8-lineage choice. Regardless of the specificity of their T-cell receptor (TCR), positively selecting TCR signals induce double positive (DP) thymocytes that are transcriptionally Cd4Cd8 to terminate Cd8 gene expression and to convert into Cd4Cd8 intermediate thymocytes. Because of the absence of Cd8 gene transcription, Cd4Cd8 intermediate thymocytes appear phenotypically as CD4+CD8b0 cells, and these are the cells in which lineage choice is made. Persistence of TCR signalling in Cd4 Cd8 intermediate thymocytes blocks interleukin-7 (IL-7)-mediated  signalling and induces differentiation into mature CD4 T cells. Cessation or disruption of TCR signalling in Cd4Cd8 allows IL-7-mediated signalling, which induces Cd4Cd& intermediate thymocytes to undergo coreceptor reversal, gain a Cd4Cd8 phenotype and differentiate into CD8 T cells.  Figure 1.5 Central tolerance mechanisms. The affinity of the T-cell receptor (TCR) for self peptide—MHC ligands is the crucial parameter that drives developmental outcome in the thymus. Progenitors that have no affinity or very low affinity die by neglect. This is thought  29  to be the fate of most thymocytes. If the TCR has a low affinity for self-peptide—MHC, then the progenitor survives and differentiates, a process that is known as positive selection. If the progenitor has a high affinity for self-peptide—MHC, then several outcomes are possible. First, the progenitor can be selected against, a process that is known as negative selection. The main mechanism of negative selection is clonal deletion, but receptor editing and anergy have also been described. Second, there seem to be mechanisms that select for high-affinity self-reactive cells and result in differentiation into a ‘regulatory’-cell phenotype. It is not known what determines whether a T cell is tolerized by negative selection or is selected to become a regulatory T cell. TEL, intestinal epithelial lymphocyte; NKT cell, natural killer T cell; Treg cell, CD4CD25 regulatory T cell.  Figure 1.6 Switching on Ras in different cellular locations, a, When receptors on the cell surface are activated, they recruit the enzyme Src. Simultaneously, another enzyme, phospholipase C’1 (PLC-yl), is recruited and phosphorylated by Src (represented by a circled ‘P’), activating it. b, PLC-yl leads to the generation of diacyiglycerol (DAG) and an increase in the level of Ca 2 ions. c, DAG and Ca 2 cause a cytoplasmic protein, RasGRP1, to move to the Golgi. This protein activates Golgi-associated Ras, by catalysing the exchange of GTP for GDP. This new pathway coexists in the cell with the ‘textbook pathway that links the cell surface receptor to Grb2 and Sos; the latter protein activates Ras at the plasma membrane (d) ma Ca -independent way. e, The increase in Ca 2 2 levels also leads to the activation of CAPRI, and possibly to its movement to the plasma membrane. f, CAPRI inhibits Ras by stimulating its intrinsic GTPase activity, which hydrolyses GTP into GDP.  30  Subcapsular zone  Cortex  Cortico—medullary junction  Medulla  Figure 1.1 The thymus architecture. Figure adapted from Takahama, Y. 2006. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol 6: 127-135 (5).  31  Subcapsular zone  Monocyte  •  NKceIL Atternatve 4 pathway  Figure 1.2 Stages in early T-cell development. Figure adapted from Rothenberg, E.V., J.E. Moore and M.A. Yui. 2008. Lauching the T-cell-lineage developmental programme. Nat Rev Immunol 8: 9-21 (1).  32  a Stochastic selection model  b Strength-of-signal instructional modeL  Pre-selection DPyt  Short-lived mtermediate cells  Pre selection OP thymocytes  Mature T cells  MHC class I  MHC class I restricted TCR  -  CD4 TC,’  —MHCclassl restricted 7CR  TCR  signal  a  a  MHC class I selection C04 C08  No rescue Cell death  MHC class II selection  MHC class II  TCR signal  d&J  v— MHC class II-  It__ Q o Rescuing TCR signal  a  d, CD4CD8  TCR—co-receptor mismatch  MHC class II selection  Weak TCR signai  ci  MHC class I selection  Mature T cells  restricted 7CR  1< CD4CD8  1  Cd4 Cdt CD4 CDS  Strong TCR signal  tx— MHC class II restricted TCR  (c CD4CDS  a  ‘  CD4 CD8  C04CD8  O C  7CR—co-receptor mismatch No rescue Cell death  Duration-of-signal instructional model Pre-selectiori DR thymocytes  Mature T cei MHC class Irestricted TCR Short and/or weak TCR signal  MHC class I selection  -  Cd4 Cdt  C04 CD8  CD4CD8  MHC class II restricted TCR  2 MHC class II selection  ‘S  c ft  Long and/or strong TCR signal  ‘S  CD4C08  CD4CD8  Figure 1.3 The classical models of CD4/CD8 lineage choice. Figure adapted from Singer, A., S. Adoro and J.-H. Park. 2008. Lineage fate and intense debate: myth, models and mechanisms of CD4- versus CD8-lineage choice. Nat Rev Immunol 8: 788-801(16). 33  CD4 TCR signal  CD4CDSb0w  CD4CD8  CD4CD8  TCRsignalceases U  IL-7 signal]  CD4CD8  CD8  Figure 1.4 The kinetic signaling model of CD4/CD8-lineage choice. Figure adapted from Singer, A., S. Adoro and J.-H. Park. 2008. Lineage fate and intense debate: myth, models and mechanisms of CD4- versus CD8-lineage choice. Nat Rev Immunol 8: 788-801 (16). 34  Thymus  y1 neglect C,)  3)  Positive selection  C) 0  2>‘  Negative selection: clonal deletion receptor editing anergy  G)  -o 2 D z  0.o 000 o %0°oO0oao 6 000 mO 00 OObIO0000 Affinity Jr  rAgonist selection: cells CD8aa IELs NKT cells Lymph node  ‘  OOo 00000 Provide immune 0 0 0 0 responses to 0 00 Oo forgnantigens 00000  ‘00 I 000 Regulate immune responses  Figure 1.5 Central tolerance mechanisms. Figure adapted from Hogquist, K.A., T.A. 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JExp Med 190:1561-1572.  53  CHAPTER 2  RASGRP1 TRANSMITS PRODIFFERENTIATION TCR  SIGNALING THAT IS CRUCIAL FOR CD4 T CELL DEVELOPMENT’  2.1  Introduction  Developing double-positive (DP) thymocytes are subjected to extreme selection pressures since the thymus culls through millions of these cells each day; killing the masses and selecting a precious few for survival (1). The positive outcome of this process, a diverse TCR repertoire that is both self-restricted and self-tolerant, is vital to fight infection and prevent autoimmunity. Current evidence supports the “strength of signaling” hypothesis, proposing that quantitative attributes of TCR signaling instruct cell fate during thymocyte selection (2, 3). The intensity of interaction between the TCR and its ligands on thymic cortical epithelial cells and bone marrow-derived cells determines the magnitude of signaling. According to this model, weak TCR signaling instructs survival and differentiation, also known as positive selection. In contrast, a lack of TCR signaling and strong TCR signaling results in cell death programming, death by neglect, and negative selection, respectively.  Expression of CD4 or CD8 defines two distinct T cell lineages that differ both by their MHC specificity and function. Most CD4 T cells are MHC class IT-restricted and function as Th cells, whereas CD8 T cells are MHC class I-restricted and differentiate into cytolytic effectors after TCR engagement. Because CD4 and CD8 T cells arise from a common precursor pooi of DP thymocytes, the question arises as to how the concordance between MHC specificity and lineage is established. Experiments manipulating TCR signaling, such as mutations affecting the coreceptor (4-8) and the tyrosine kinase Lck (9, 10) function in developing thymocytes, have provided strong support for the notion that CD4/CD8-lineage choice is also dictated by quantitative attributes of TCR signaling. The strength of signaling  ‘A version of this chapter has been published. Priatel, J.J., X. Chen, S. Dhanji, N. Abraham, and H. S. Teh. (2006) RasGRP1 Transmits Prodifferentiation TCR Signaling That Is Crucial for CD4 T Cell Development. I Immunol. 177: 1470-1480. Copyright 2006. TheAmerican Associateion of Immunologists, Inc.  54  model as applied to lineage selection states that stronger TCR signaling promotes CD4 T cell fate, whereas weaker TCR signaling directs CD8 T cell fate (2, 3).  The expression of either constitutively active or dominantly interfering forms of Ras (11), Raf- 1 (12), and MEK1 (13) in transgenic (tg) mice have demonstrated the critical role of the Ras/MAPK pathway in positive but not negative selection. However, recent in vitro studies suggest that the strength and/or duration of Ras/ERK signaling may be critical in determining whether a thymocyte adopts a positive or negative selection program (14-16). Positively selecting ligands were found to induce a sustained, low-level ERK activity, whereas negatively selecting ones brought about stronger but transient signals. Furthermore, these findings have now been corroborated to also occur in fetal thymic organ cultures (17). One function for sustained ERK signaling in positive selection may be to provide survival signaling. Studies using MAPK inhibitors have demonstrated that ERK activity is critical for the up-regulation of the antiapoptotic protein Bcl-2 (18, 19). In addition, ERK has been shown to phosphorylate the BH3-only Bcl-2 family member Bim, preventing its association with Bax and disabling it from inducing cell death (20). Therefore, sustained ERK activation may also facilitate T cell maturation by phosphorylating Bim and subsequently blocking its proapoptotic activities.  The role of ERK signaling in lineage commitment has been the center of some controversy. Although studies using Tg mice expressing either gain-of- or loss-of-function forms of Ras, Raf, and MEK1 were not found to sway CD4 vs CD8 differentiation (11-13), mice bearing a hypersensitive mutant form of ERK2 gene favor the development of CD4 single-positive (SP) thymocytes (21). Further support for ERK signaling directing thymocyte fate has come from investigations using MEK1 inhibitors in combination with fetal thymic organ cultures (2123). Therefore, the amplitude and/or kinetics of ERK activation may also be key in determining lineage decision (2 1-23). Moreover, ERK activity has recently been found to positively regulate TCR signaling by modifying the Lck tyrosine kinase and subsequently preventing its inactivation by the Src homology protein-i phosphatase (24). Thus, it is possible that ERK may affect TCR signaling strength by lengthening the duration of Lck activity through a feedback-loop mechanism.  55  In thymocytes, at least two Ras-guanyl nucleotide exchange factors, the well-studied Sos (25) and the more recently identified RasGRP1 (26), mediate Ras activation. The action of these two Ras-guanyl nucleotide exchange factors is dependent on their relocation to membranes by two distinct mechanisms (27). TCR stimulation and subsequent LAT (linker for activated T cells) phosphorylation result in the Src homology 2-domain-mediated targeting of Grb2/Sos complex to the plasma membrane (PM). In contrast, the movement of RasGRP1 is dependent on phospho-LAT’s recruitment and activation of phospholipase Cyl (PLCy1). The action of PLCy1, converting phosphotidylinositol 4,5 bisphosphate into inositol 3,4,5 triphosphate and diacyiglycerol, results in membrane localization for RasGRP1 through its diacylglycerol-binding Cl domain (28, 29). The surprising finding that RasGRP1 is recruited to the Golgi membrane (30-32), activating Golgi-associated Ras, rather than at the PM adds further complexity to Ras signaling, potential cellular responses, and the induction of developmental programs. In addition to the differential localization, Ras activation also displays delayed or sustained kinetics at the Golgi relative to the PM.  RasGRP1 mice bear a defect in late thymic selection, where mutant DP thymocytes undergo rearrangement of their TCRa genes and express normal levels of TCRc /CD3 but are poorly promoted to the mature SP lineages (26, 33). Previously, we have determined that RasGRP 1-dependent and -independent mechanisms exist for ERK activation and positive selection of MHC class I-restricted TCRs (33). To further characterize the role of RasGRP 1 in thymocyte maturation, examining its relative importance in CD4 T cell development and lineage commitment, we generated RasGRP i mice that express a Tg MHC class Il-specific TCR. Here, we show that in mutant mice, the development of CD4 T cells expressing a Tg MHC class Il-restricted TCR is completely abrogated. Therefore, we find that RasGRP1 deficiency affects CD4 T cell development more profoundly than the development of CD8 T cells. From in vitro TCR-stimulation experiments, RasGRP1 DP thymocytes display a diminished capacity to up-regulate Bcl-2. Based on these findings, we hypothesized that an important role for RasGRP1 may be to maintain cell viability and thus enable the differentiation of DP into SP thymocytes. However, the failure of forced Bcl-2 expression to restore CD4 T cell development in RasGRP 1-deficient mice suggests that the RasGRP 1-  56  RasIMAPK pathway may be particularly important for imparting differentiation signals that are essential for CD4 T cell development.  2.2  Materials and methods  Mice RasGRP i-null breeder mice were provided by J. C. Stone (University of Alberta, Edmonton, Alberta, Canada) and bred onto C57BL/6J background at least seven generations before mating with TCR Tg mice. The TcrAND (34) and TcrH-Y (35) Tg mice were bred onto the C57BL/6 (H-2”) background. Breeders bearing the TcrAND transgene were purchased from Jackson ImmunoResearch Laboratories. The Eji-Bcl-2-36 Tg mouse line expresses strongly in thymocytes and has been bred at least six generations onto the C57BL/6J background as described previously (36). The Ej.t-Bcl-2-36 Tg mouse line was provided by C. J. Ong (University of British Columbia, Vancouver, British Columbia, Canada). B6.PL-Thy 1 a/Cy (Thy 1. 1) mice were acquired from The Jackson Laboratory. All studies followed guidelines set by the Animal Care Committee at the University of British Columbia in conjunction with the Canadian Council on Animal Care.  Flow cytometry Abs against CD4 (GK1.5), CD8 (53-6.7), H-Y TCR (T3.70), TCR (H57-597), CD3r (2C1 1), heat-stable Ag (HSA; M1/69), CD127 (A7R34), CD69 (H1.2F3), CD44 (1M7), Thyl.l (H1551), and Thyl.2 (53-2.1) were purchased from eBioscience. Abs against CD5 (53-7.3), TCR Vail (RR8-1), TCR V3 (KJ25), and Bcl-2 (3F1 1) were purchased from BD Biosciences. For phospho-ERK (P-ERK)-specific staining, cells were fixed in methanol-free 2% formaldehyde (Polysciences), washed, and made permeable by incubation in 90% methanol for 30 mm on ice. Intracellular staining was conducted in FACS buffer (2% FCS in PBS) at room temperature for 30 mm. Rabbit anti-P-ERK1/2 Ab (Cell Signaling Technology; catalog no. 9101) was detected with donkey anti-rabbit Ig F(ab’)2-PE (Jackson ImmunoResearch Laboratories; catalog no. 711-116-152). After blocking unbound binding  57  sites of the secondary Ab with rabbit IgG, cells were subsequently incubated with anti-CD4FITC and CD8-PerCP (BD Biosciences). Data were acquired using either a FACScan or FACSCalibur and CellQuest software (BD Biosciences). Data were analyzed with CeliQuest or FCSPress software (<www.fcspress.com>).  Proliferation assays SP thymocytes were identified by staining with anti-CD8-PE and anti-CD4-PE-Cy5 Abs and purified by cell sorting using a BD FACSVantage (BD Biosciences). For Ab stimulation, 96well flat-bottom plates were coated with 10 pg/ml of either ant-CD3E (2C11) or anti- Vail (RR8-i) in PBS for 2 h at 37°C. After washing wells three times with PBS, 40,000 sorted cells were seeded per well in complete IMEM (Invitrogen Life Technologies) containing 20 U/ml IL-2. For peptide stimulation, 20,000 sorted cells were incubated with 5 x C3H (H2k) splenocytes in round-bottom 96-well plates with the indicated pigeon  irradiated  cytochrome c (PCC) concentration and IL-2 (20 U/mi). After 72 h, cultures were pulsed with 1 jiCi of [ H] thymidine for 6 h to assess proliferation. 3  CTL assays SP thymocytes were identified by staining with anti-CD8-PE and anti-CD4-PE-Cy5 Abs and purified by cell sorting using a BD FACSVantage. To generate effector T cells, purified AND CD4CD8 and CD4CD8 SP thymocytes were stimulated for 3—4 days on anti-AND TCR-Ab-coated dishes in the presence of exogenous IL-2 (20 U/mi). The H2kexpressing CH12 lymphoma cells were 51 Cr labeled and subsequently incubated with various numbers of effectors in the presence and absence of 10 .tM PCC peptide. After 5 h, supernatants were assayed for released 51 Cr and specific killing determined as described previously (37).  DP thymocyte stimulations Tissue culture wells (24-well plate; Falcon) were coated with 0.5 ml of either PBS alone, 10 .tg/ml anti-CD3E (2C1 1), or 10 ig/ml anti-CD3E plus 10 jig/ml anti-CD4 (GK1.5) Abs for 2 h at 37°C. After washing the wells three times with PBS, total thymocytes (2 x 106) were  58  placed in culture in complete IMEM for 24 h, harvested, and stained with anti-CD4 (RM4-4), anti-CD8 (53-6.7), and 5 jig/mI 7-aminoactinomycin D (7-AAD; Calbiochem) for dead cell exclusion and various cell surface markers. For Bcl-2 expression, cells were fixed in 2% paraformaldehyde/ PBS solution for 15 mm, made permeable by treatment with 0.2% Tween 20/PBS for 15 mm and stained with either anti-Bci-2 or isotype-control Abs conjugated to PE (BD Biosciences) for 30 mm on ice. Following two washes with PBS, sample data acquisition was performed on a FACSCalibur.  Cell signaling studies and immunoblotting Cell sorting, using a FACSVantage, was used to purify DP thymocytes. For activation, sorted DP thymocytes (10 /mI) were coated with 10 jig/mi hamster anti-CD3E Ab for 20 mm on ice, 7 washed, and suspended in prewarmed (37°C) medium containing 80 jig/ml goat anti-hamster Ab (Jackson ImmunoResearch Laboratories) for the indicated periods. Cells were lysed in 10 mlvi Tris (pH 7.5), 150 mM NaC1, 1% Nonidet P-40, 0.1% SDS, protease, and phosphatase inhibitors. Abs used for immunoblotting that recognize activated and total ERK, LAT, and Zap7O were purchased from Cell Signaling Technology.  Intrathymic injection (IT) experiments DP thymocytes were electronically sorted exactly as described for coreceptor re-expression assay. Approximately 1 million sorted DP thymocytes (10 jil volume) were injected per thymic lobe of (nonirradiated) B6.PL-Thy 1 aJCy (Thy 1. 1) mice. Two days postinjection, injected thymi were recovered and mashed into single-cell suspensions, at a concentration of 1 0 cells/mi. To deplete host cells, thymocytes were labeled with 0.5 jig/mi anti-Thy 1.1 Ab, washed, and incubated with (25 jiu/ml) sheep anti-mouse-Ig-coupled Dynabeads (catalog no. 110.31; Dynal Biotech). Following magnetic separation, the remaining cells were split into two portions: one aliquot for ex vivo coreceptor expression analyses of donor cells, whereas the second was pronase-treated for a coreceptor re-expression assay. Because binding of anti CD4 mAb (GK1.5) may block the ability of pronase to subsequently strip surface CD4 molecules (38), we selected to assess CD4 and CD8 coreceptor re-expression by staining with Abs conjugated to fluorochromes distinct from those used for cell sorting and thus avoid  59  potential residual fluorescence. All samples were stained with anti-CD4-allophycocyanin, anti-CD8-PE, and Thyl.2-FITC-conjugated Abs, and the data were acquired on a BD LSR II benchtop (BD Biosciences) cytometer using FACSDiVa software (BD Biosciences) for both acquisition and analysis.  Coreceptor re-expression assay Thymic single-cell suspensions were stained with PE-Cy7-conjugated anti-CD4 (GK1 .5) and APC-Cy7-conjugated anti-CD8 (53-6.7) Abs and electronically sorted using either a FACSVantage or FACSAria flow cytometer (BD Biosciences). To cleave off surface coreceptor expression, purified DP thymocytes were pronase-treated as described previously (38, 39). Briefly, thymocytes were extensively washed with PBS and incubated in 0.0 1% pronase (Calbiochem) and 100 tg/ml DNase I (Sigma-Aldrich) for 15 mm at 37°C. The enzymatic reaction was quenched by washing with medium containing 10% FCS and placed in overnight cultures at 37°C in complete medium. After culture, thymocytes were stained with anti-CD4-APC, anti-CD8-PE, and anti-Thyl.2-FITC Abs, and sample data were acquired with a BD LSR II cytometer (BD Biosciences) and FACSDiVa software (BD Biosciences). FCS used in these experiments had been depleted of endogenous steroids by pretreatment with 0.5% Norit A Charcoal (Sigma-Aldrich) and 0.05% dextran (SigmaAldrich).  2.3  Results  CD4 SF thymocytes are critically dependent on RasGRP] for their development To address the role of RasGRP 1 in the development of thymocytes expressing a MHC class IT-restricted TCR, we crossed the AND TCR transgene (TcrAND) onto RasGRP 1-null animals. In C57BL/6 (H-2”) mice, the AND TCR (Vail, Vj33) selects DP thymocytes on the MHC class II molecule I-A” and directs the development of large numbers of CD4 SP thymocytes (34). Strikingly, analyses of thymic subpopulations, as visualized by labeling with anti-CD4 and anti-CD8 Abs, revealed that RasGRPF’TcrAND mice are virtually  60  devoid of CD4 SP thymocytes (Fig. 2. 1A). Associated with impaired thymic selection, RasGRP 1 TcrAND DP thymocytes possess reduced TCR expression, as judged by staining with anti-Tg TCRa, anti-TCR3 or anti-CD3E Abs, and appear slightly immature by HSA and CD5 levels (Fig. 2. 1B). By contrast with the MHC class IT-restricted AND TCR, our previous results with RasGRP 1-deficient mice expressing MHC class I-restricted TCRs exhibited a more modest effect on CD8 T cell development (33). Moreover, mutant mice expressing the weakly selecting H-Y TCR resulted in about a 4-fold reduction in CD8 SP thymocyte number, whereas the recovery of CD8 SP thymocytes bearing the strongly selecting 2C TCR resembled wild-type animals. These results indicate that RasGRP 1-independent mechanisms cannot compensate for the positive selection of CD4 SP thymocytes by the AND TCR despite being able to mediate the development of CD8 T cells expressing either the H-Y or 2C TCRs (33).  Decreased ERK activity in RasGRPT’TcrAND DP thymocytes is correlated with reductions in TCR and Bcl-2 expression We have previously observed that RasGRP 1-deficient DP thymocytes bearing MHC class Irestricted TCRs exhibit reduced levels of active ERK as compared with wild type (33). To examine the contribution of RasGRP 1 on ERK activation under the defined TCR signaling conditions of a MHC class II TCR, we used a flow cytometric assay based on a phospho specific ERK1/2 Ab as described previously (33). Thymocyte single-cell suspensions were rapidly generated in serum-free medium, fixed, and assayed for ERK activity (Fig. 2.2A). Analysis of RasGRPF’TcrAND DP thymocytes revealed a considerable reduction in the frequency  cells (6.8 vs 20%, respectively; Fig. 2.2A). Next, we examined  whether the level of active ERK correlated strongly with TCR up-regulation, an index of positive selection (Fig. 2.2B). Indeed, ERK activity corresponded well with TCR expression regardless of genotype. Wild-type TcrAND DP thymocytes exhibit a large fraction (36%) of PERKl/2h AND high cells. Despite diminished positive selection in RasGRP l TcrAND thymi, mutant DP thymocytes possess a smaller but significant fraction of P ERKl/2’” AND  high  cells (7%). This observation is consistent with previous studies  demonstrating a requirement for sustained ERK activity in T cell development (14-17). However, because our flow cytometric-based assay measures ERK activity during a narrow 61  window in time, it is not possible to determine whether the reductions in ERK signaling are the result of decreased intensity, duration, or both.  Previous studies using MEK1 inhibitors have suggested that ERK signaling is important for inducing Bcl-2 expression during thymic selection (18, 19). To examine whether RasGRP1 facilitates T cell differentiation by influencing Bcl-2 levels, we assayed Bcl-2 expression in TcrAND DP thymocytes via intracellular flow cytometry (Fig. 2C). As noted earlier (40), wild-type TcrAND DP thymocytes have elevated Bcl-2 levels as compared with DP thymocytes from a normal (non-TCR Tg) mouse, a likely consequence of the fact that every thymocyte in a TCR Tg mouse expresses a selectable TCR. By contrast, RasGRP 1 ‘TcrAND DP thymocytes have considerably lower Bcl-2 expression as compared with wild-type TcrAND DP thymocytes (33.4 vs 74.1 mean fluorescence intensity (MFI); Fig. 2.2C). These findings suggest that RasGRP1 may transduce survival signaling via a Bcl-2-dependent mechanism.  RasGRPT DP thymocytes demonstrate defective P-ERK, CD69, and Bcl-2 up-regulation following TCR engagement ERK activation is a critical event in T cell development, and its relative activity correlates with the level of positive selection (41). Our analyses of TCR Tg mice demonstrate that RasGRP 1-deficient DP thymocytes possess reduced levels of active ERK in isolated cells ex vivo (Ref. 33; Fig. 2). However, these reductions may be an indirect effect, a consequence of impaired positive selection in mutant animals. Therefore, we sought to investigate the direct effect of RasGRP 1 deficiency on TCR-mediated ERK activation by stimulating DP thymocytes in vitro. A previous study (26) has shown that RasGRP1 thymocytes do not appreciably activate ERK upon either PMA or TCR stimulation. However, since this result was obtained from total thymocytes, it raises a concern that the differential composition of wild-type and mutant thymocyte populations may be responsible for these observations. Therefore, for TCR signaling studies, we sorted DP thymocytes from normal (non-TCR Tg) wild-type and mutant thymi and found that they expressed similar amounts of TCR!CD3E (Fig. 2.3, A and B). Punfied RasGRP1 +1+ and RasGRP1 -I- DP thymocytes were subjected to anti-TCR cross-linking for the indicated times (Fig. 2.3C). Wild-type cells induced strong 62  ERK1/2 phosphorylation after ICR stimulation, whereas mutant DP thymocytes exhibited only a weak signal indicating that optimal TCR-induced ERK1/2 phosphorylation in these cells is dependent on RasGRP1. However, Zap7O and LAT, molecules upstream of RasGRP1, exhibit near-normal phosphorylation following TCR stimulation (Fig. 2.3 C). This finding indicates that TCR-induced ERK activation is diminished in RasGRP1 DP thymocytes, whereas more proximal TCR signaling appears unaffected.  To examine whether RasGRP 1 has direct impact on the expression of various markers of positive selection, we took DP thymocytes from normal (non-TCR Tg) wild-type and mutant animals and cultured them in vitro for 1 day in the presence or absence of TCR stimulation (Fig. 2.3D). Previous studies have shown that TCR engagement of DP thymocytes can induce many aspects of positive selection in vitro (42, 43). For our studies, we used two types of stimulation, either anti-TCR alone or anti-TCR plus anti-CD4 Abs, to mimic conditions of weak or strong TCR signaling. Analysis of CD5 induction revealed that RasGRP1 DP thymocytes behave similarly to wild-type cells under both conditions tested (Fig. 2.3D). In contrast, mutant thymocytes do not efficiently up-regulate CD69 upon stimulation with either TCR agonist. To address the RasGRP1 function in supporting survival signaling, we assessed the ability of mutant thymocytes to elevate Bcl-2 expression following TCR engagement. Using anti-TCR Ab alone, RasGRPF’ DP thymocytes failed to up-regulate Bcl-2 significantly. However, when anti-ICR and anti-CD4 Abs are used in tandem, Bcl-2 can be induced in mutant thymocytes, although these levels are still lower than wild type (16.3 vs 35.8 MFI). These findings suggest that RasGRPF DP thymocytes must receive strong TCR signaling for their positive selection and support the conclusion that RasGRP1 augments Bcl-2 expression.  Tg Bcl-2 expression fails to restore CD4 T cell development in RasGRPI-deficient mice Positive selection entails the rescue of DP thymocytes from programmed cell death and is dependent on the appropriate level of TCR/self-MHC signaling to provide antiapoptotic cues, such as the induction of Bcl-2 (40, 44-46). Because our in vitro findings suggest that RasGRP1 promotes Bcl-2 levels (Fig. 2.3D), we sought to determine whether enforced Bcl-2 expression could rescue CD4 I cell development in RasGRP1-deficient mice. To test our 63  hypothesis, we introduced the Ep.-Bcl-2 transgene into both wild-type and RasGRPF’ TcrAND mice (Fig. 2.4).  In RasGRP 1-null animals, transgene-driven Bcl-2 expression results in a sharp increase in CD4 SP thymocyte number (6.1 lower than wild type (46.0  ±  ±  2.4 x 106 vs 0.31  ±  0.05 x 106), although still considerably  7.6 x 106). Next, we examined whether Ep.-Bcl-2 RasGRP1  CD4 SP thymocytes display signs of differentiation. To address this question, we compared the level of TCR expression in Ei-Bc1-2 RasGRPF’TcrAND DP and CD4 SP thymocytes relative to wild-type TcrAND CD4 SP thymocytes (Fig. 2.4C). These analyses revealed that Ei.t-Bcl-2 RasGRPF’TcrAND CD4 SP thymocytes showed signs of positive selection because they had slightly elevated TCR expression vs DP thymocytes. This increase in TCR expression was observed regardless of whether cytometry was performed with anti-Tg TCRa-, anti-Tg TCR3-, or anti-TCRf3 constant region-specific Abs (Fig. 2.4C and data not shown). However, TCR expression by Eji-Bcl-2 RasGRP1TcrAND CD4 SP thymocytes is considerably lower than the level that is exhibited by wild-type TcrAND CD4 SP thymocytes. Corroborating these studies, the analysis of non-TCR Tg animals lacking RasGRP1 also revealed that Bcl-2 overexpression fails to restore CD4 SP thymocyte proportion or numbers (data not shown). Therefore, we conclude that the developmental arrest of RasGRP i TcrAND DP thymocytes cannot be rescued by provision of Tg Bcl-2 expression.  In agreement with a previous study using 1ck-Bcl-2 TcrAND mice (40), the Eji-Bcl-2 transgene has a dramatic effect on wild-type TcrAND thymi. These changes include an increase in total thymocyte number, the formation of massive numbers of AND TCR CD8 SP thymocytes, and a modest augmentation of the CD4 SP thymocyte number (Fig. 2.4, A and B). Although Linette et al. (40) have suggested that these CD8 SP thymocytes are developmentally arrested, we have found that this population possesses characteristics of mature T cells—expressing high levels of AND TCR, bearing intermediate to low levels of HSA and proliferating when contacted by cognate Ag (data not shown). These findings suggest that limited survival signaling restricts the formation of CD8 SP thymocytes in wild type TerAND animals. However, the fact that enforced Bcl-2 expression results in only a modest increase in RasGRPFTcrAND CD8 SP thymocyte number (8.0 ± 3.8 x 106 vs 3.9  ±  64  0.8 x 106) argues that their inefficient positive selection is not primarily due to deficient Be! 2 expression. Therefore, we conclude that RasGRP1 is necessary for development of massive numbers of CD8 SP thymocytes seen in wild-type Eii-Bcl-2 TcrAND mice and provides developmental cues that are independent of Bcl-2 expression.  RasGRP1-deficient TcrAND CD8 SP thymocytes resemble CD8 SP thymocytes that develop in RasGRPTL mice expressing the MHC class I-restricted H-Y TCR In addition to the lack of CD4 SP thymocytes, another feature of RasGRP 1 TcrAND thymus is a small population of CD8 SP thymocytes that expresses the CD8a3 coreceptor (Figs. 2.1A and 2.5A and data not shown). Uncannily, the thymic CD4/CD8 developmental profile of RasGRP 1 ‘TcrAND mice seems reminiscent of mutant (female) animals expressing the MHC class I-restricted H-Y TCR (Fig. 2.5A). However, in contradistinction to the comparison of wild-type and RasGRP1TcrH-Y mice, there is no change in CD8 SP thymocyte frequency or cell number recovered from RasGRP1TcrAND mice relative to wild type (Fig. 2.5B). To examine whether RasGRPF’TcrAND CD8 SP thymocytes might be a product of positive selection, we compared surface TCR expression and markers of differentiation between RasGRP1-deficient CD8 SP thymocytes expressing either the AND or H-Y TCR (Fig. 2.5C). Regardless of the TCR expressed, mutant CD8 SP thymocytes share an immature cell surface phenotype (HSAh) and possess low levels of TCR, CD5, and CD69. These observations suggest that RasGRP1TcrAND CD8 SP thymocytes may be the product of weak and/or inefficient positive selection.  RasGRPT”TcrAND DP thymocytes give rise to afew CD8 but not CD4 SP thymocytes after intrathymic transfer Some unusual features of RasGRP1TcrAND CD8 SP thymocytes may raise doubt regarding their developmental origin. Their rarity and relative immaturity (HSAI1, CD69b0, CD8b0s) suggests the possibility that these cells may be immature SP thymocytes (ISPs), a  transitional, developmental intermediate that exists between the double-negative (DN) and DP cell stages (47), rather than being progenies of DP thymocytes. To gain insight into the derivation of RasGRP1TcrAND CD8 SP thymocytes, we used electronic cell sorting of DP  65  thymocytes in combination with a previously described coreceptor re-expression assay (38). The ability of coreceptor re-expression assay to assess lineage commitment is based on the fact that developing DP thymocytes committing to either the CD4 or CD8 lineage terminate synthesis of the inappropriate coreceptor molecule. The coreceptor re-expression assay enables one to discern what coreceptor molecules are actively being synthesized by individual cells by first stripping thymocytes of pre-existing coreceptor molecules by treatment with a protease called ‘pronase,” followed by culturing overnight at 37°C to enable cells to re-express coreceptor proteins and finally assessing their newly synthesized CD4/CD8 surface phenotype by flow cytometry.  To assess the developmental potential ofRasGRP1TcrAND DP thymocytes in vivo, we purified TcrAND DP thymocytes (Thy 1 .2) by cell sorting (Cell purity: >98% for wild type, >99% RasGRPF’) before adoptively transferring them into B6 (Thy1.1) host thymi (Fig. 2.6). Two days post-IT, recovered thymi were assessed for CD4ICD8 surface phenotype of donor cells either before (untreated) or after pronase treatment and overnight culture at 37°C (pronase/37°C). Under these in vivo conditions, RasGRP1TcrAND DP thymocytes gave rise to CD8 SP thymocytes (4.4%) but no CD4 SP thymocytes (Fig. 2.6). Importantly, the frequency of RasGRP1TcrAND CD8 SP thymocytes (3.75 thymocytes (0.02  ±  ±  1.73%; n = 7) and CD4 SP  02%; n = 7) observed in seven independent DP thymocyte IT  experiments are similar to what is observed in normal RasGRP1TcrAND thymi (Fig. 2.1). By contrast, wild-type TcrAND DP thymocytes gave rise to larger frequencies of both CD4 (9.9  ±  1.7%; n = 5) and CD8 SP thymocytes (12.3  ±  3.2%; n = 5). The observed increase in  CD8 lineage cells is in line with a recent report, which showed that weakly selecting (or nonselecting) neighboring thymocytes can shift some AND TCR-expressing thymocytes toward the CD8 lineage (48). True to form, the coreceptor re-expression assay revealed an increased proportion of lineage-committed cells and conversely a decreased proportion of DP thymocytes in both wild-type and mutant samples. More importantly, this assay indicated that RasGRPF’TcrAND DP thymocytes could give rise to only CD8 but not CD4 thymocytes. Oddly, a sizeable fraction of RasGRPFTcrAND DP thymocytes failed to express either surface CD4 or CD8 molecules, particularly after pronase treatment, and it is unclear whether the lack of RasGRP1 signaling has either aborted their development or taken  66  them toward a cryptic differentiation program with a CD4CDW destiny. Previously, the derivation of CD4CDW progeny has been associated with DP thymocytes cultured in medium alone (no signal) before their intrathymic transfer as compared with those treated with either PMA!ionomycin or anti-TCR plus anti-CD2 Abs (39). In conclusion, our IT experiments indicate that RasGRPFTcrAND DP thymocytes yield a similar percentage of CD8 SP thymocytes as observed in the thymi of unmanipulated RasGRPF’TcrAND mice. Although these findings do not exclude an alternate origin, our observations are consistent with the notion that CD8 SP thymocytes present in RasGRP1TcrAND animals are derived from DP thymocytes.  Mutant CD8 SF thymocytes express markers ofpositive selection, respond to a MHC class II-specfIc Ag, and dfferentiate into cytotoxic effectors We have previously observed that the formation of CD8 SP thymocytes is associated with an elevated level of active ERK relative to DP thymocytes (33). Therefore, we sought to determine whether TcrAND CD8 SP thymocytes possess a similarly heightened ERK activity directly ex vivo (Fig. 2.7A). Indeed, both wild-type and mutant TcrAND CD8 SP thymocytes exhibited an elevated level of ERK activity, typical of positive selection. However, the nonconformity of CD8 SP thymocytes expressing a MHC class Il-restricted TCR combined with their immature surface phenotype raises the question of whether these cells have truly undergone the positive selection process. To address this issue, we next examined whether RasGRPFTcrAND CD8 SP thymocytes exhibit two other events associated with the formation of SP thymocytes, IL-7R expression and elevated Bcl-2 levels. Somewhat surprisingly, RasGRP 1-sufficient TerAND CD8 SP thymocytes do not express detectable levels of IL-7R and have atypically low quantities of Bcl-2 (Fig. 2.7B). Apparently, the selective formation of CD4 SP thymocytes impairs CD8 SP thymocyte development in wild-type TcrAND mice (34, 48) and results in very few peripheral CD8 T cells (34). In contrast, RasGRPFTcrAND CD8 SP thymocytes express IL-7R and display Bcl-2 amounts characteristic of SP thymocytes, suggesting that these cells have undergone positive selection. Again, RasGRPF’ CD8 SP thymocytes expressing the AND TCR share quite similar attributes to those bearing H-Y TCRs. Therefore, in contradistinction to CD8 SP thymocytes  67  from wild-type TcrAND mice, RasGRP1TcrAND CD8 SP thymocytes bear hallmarks of mature SP thymocytes.  A more definitive measure of positive selection is acquisition of mature T cell function. Therefore, we sought to establish whether the CD8 SP thymocytes that develop in RasGRPF ‘TcrAND mice could proliferate upon TCR engagement. Upon placement into tissue culture wells coated with anti-TCR Ab (either anti-CD3E or anti-TCR Vail Ab), mutant TcrAND CD8 SP thymocytes proliferated at least as strongly as wild-type TcrAND CD4 and CD8 SP thymocytes (Fig. 2.7C). Next, we tested the capacity of these subpopulations to react with the cognate Ag for the AND TCR, the PCC peptide (88—1 04) presented by MHC class II H2Ek molecule (34). Equivalent numbers of responder T cells were incubated with various doses of peptide plus irradiated, H2Ekexpressing stimulator splenocytes. In contrast to the proliferation assays based on plate-bound anti-TCR Abs, RasGRPf’TcrAND CD8 SP thymocytes proliferated less vigorously than wild-type TcrAND CD4 SP thymocytes, particularly at low peptide concentrations (Fig. 2.7D). A plausible reason for the poorer response to peptide stimulation is that TcrAND CD8 SP thymocytes lack the appropriate coreceptor for corecognition of the I-E’ MHC class II molecule.  The acquisition of effector function is another stringent evaluation of positive selection. To address this question, TcrAND SP thymocytes were activated with anti-TCR Vail Ab and examined for hallmarks of CD8 T cell effector function, namely the expression of the cytotoxic-associated molecule granzyme B and cytotoxicity. In contrast to TcrAND CD4 SP thymocytes, TcrAND CD8 SP thymocytes, regardless of genotype, rapidly expressed high levels of granzyme B upon activation (Fig. 2.7K). Subsequently, we investigated whether activated TcrAND SP thymocytes could mediate peptide-specific cytotoxicity (Fig. 2.7K). Indeed, RasGRP1TcrAND CD8 SP thymocytes could kill 51 Cr-labeled targets in a MHC class TI-restricted (PCC)-dependent fashion. Wild-type TcrAND CD4 SP thymocytes were at least 10-fold less potent killers (mutant cells have a greater kill at 1:1 E:T ratio compared with 10:1 for wild type). Thus, despite expressing a relatively immature cell surface phenotype, RasGRPFTcrAND CD8 SP thymocytes can efficiently kill Ag-coated target cells and thus are functionally mature.  68  RasGRPT’TcrAND animals possess significant numbers ofperipheralAND TcR cD8 T cells but very small numbers ofAND TCR CD4 T cells To investigate the consequences of altered thymic selection on the peripheral lymphoid compartments, we examined the splenic T cell composition in these animals (Fig. 2.8A). In RasGRP1 -I- TcrAND mice, there is a 35-fold reduction in AND TCR+ CD4 T cell numbers, .  .  .  .  whereas the recovery of AND TCR CD8 T cells is increased 1.8-fold relative to wild-type mice (splenic cell recovery: RasGRP1’TcrAND, 98.7 ± 11.2 x 106, n = 3; RasGRP1 TcrAND, 78.1 ± 7.1 x 106, n = 3). Curiously, mutant CD8 T cells express slightly lower levels of CD8a- and CD8f3-chain (Fig. 2.8A and data not shown). It is also noteworthy that only 23% of peripheral CD4 T cells in RasGRP1 mice express high levels of the AND TCR, suggesting that the majority of these cells exploit endogenous TCRa-chains for positive selection (Fig. 2.8B). By contrast, peripheral CD8 T cells from RasGRP 1 ‘TcrAND mice exclusively express the AND TCR (Fig. 2.8B), consistent with the notion that these cells use the AND TCR for their development. Moreover, RasGRPPTcrAND CD8 T cells share a comparable cell surface phenotype to fUnctionally mature female RasGRP1’TcrH-Y CD8 T cells (Fig. 2.8C). These observations support the conclusion that the few CD8 T cells that develop in RasGRP 1 TcrAND mice are the product of the positive selection process mediated by the AND TCR.  2.4  Discussion  Experiments with TCR Tg mice have demonstrated that thymocyte cell fate is dependent on whether the TCR of a developing DP thymocyte recognizes self-MHC class I or -MHC class II molecules. The strength of signaling model of lineage commitment proposes that intensity and/or duration of a TCR signaling determines whether a developing DP thymocyte chooses a helper CD4 or cytotoxic CD8 T cell fate; stronger and/or sustained TCR signaling results in CD4 SP thymocytes, whereas weaker and/or transient signaling produces CD8 SP thymocytes. Defining the molecular mechanisms controlling this cell fate decision has been a central focus of many investigations. Roles of the Ras/MAPK signaling pathway in positive selection have been implied by studies using either pharmacologic inhibitors of MEK1/2 in  69  vitro or Tg mice expressing copious amounts of either constitutively active or DN forms of Ras and downstream MAPK family members (49, 50). In addition, some studies have suggested that ERK signaling strength may also be deterministic in lineage commitment (2123). Moreover, studies based on either transgene overexpression or pharmacologic inhibitors may be especially prone to pleiotropic effects. Thus, many questions remain on how surface TCR signal transduction connects to Ras activation, links up to MAPK signaling pathways, and contributes to signaling cues necessary for the survival and differentiation of DP thymocytes. Using RasGRP 1-null mice, we have previously shown that RasGRP1-dependent and -independent mechanisms exist for ERK activation and positive selection of CD8 SP thymocytes (33). Because the formation of CD4 SP thymocytes is thought to be dependent on prolonged TCR signaling (51) and RasGRP 1 induces Ras activation with sustained and/or delayed kinetics (30-32), we sought to investigate the relative importance of RasGRP1 in the development of thymocytes expressing a MHC class TI-restricted TCR. Herein, we report that RasGRP1 is a crucial TCR signaling component for CD4 T cell development.  The surprising finding of CD8 SP thymocytes, instead of CD4 SP thymocytes, in RasGRPF’ TcrAND raised questions regarding their nature and origin. The CD8 dull-phenotype of these cells suggests the possibility that they may belong to the y T cell lineage, because premature expression of the cL3 TCR in mice bearing the TcrH-Y and TcrAND transgenes causes aberrant y T cell development (52, 53). Moreover, male TcrH-Y (H-2 ’) mice notably 1 contain large numbers of mature H-Y TCR CD8b0 and H-Y TCR DN T cells with y T cell-like properties (52, 53), whereas TcrAND transgene is normally associated with an abundance of mature AND TCR DN T cells possessing such traits (53). However, TcrH-Y ’) mice only develop CD8b0 H-Y TCR T cells when nominal Ag (male, H2b) is 1 (H-2 present because they are absent in the positively selecting (female, H-2”) and nonselecting (male or female, H-2’) backgrounds (54). Because TcrAND mice on a H-2” background lack nominal Ag, RasGRPF’TcrAND CD8 SP thymocytes are unlikely to be yô-lineage “wannabe’ cells analogous to those present in H-Y H-2 ’ male mice. Another possibility 1 regarding the origin of RasGRPF’TcrAND CD8 SP thymocytes is raised by their immature phenotype (HSAh, CD69b0) that is often characteristic of ISPs rather than “truer’ (mature) CD8 SP thymocytes. In contrast to ISPs, RasGRP1TcrAND CD8 SP thymocytes exhibit  70  mature functional attributes and can be derived from RasGRPF’TcrAND DP thymocytes. Therefore, together with our intrathymic transfer experiments (Fig. 2.6), indicating that RasGRPF’TcrAND DP thymocytes can give rise to some CD8 but not CD4 SP thymocytes, it is reasonable to presume that CD8 SP thymocytes observed in RasGRPF’i’crAND mice are the product of inefficient positive selection and of the c43 lineage.  The fact that RasGRP 1’ DP thymocytes expressing a MHC class 11-restricted TCR fail to develop into CD4 SP thymocytes but can form functional CD8 SP thymocytes suggests that CD4 T cell development may be much more dependent on RasGRP1 than the development of CD8 T cells. This conclusion is corroborated with our MHC class I-restricted TCR Tg studies (33), defining significant RasGRP 1-independent mechanisms for the positive selection of CD8 SP thymocytes, and reduced ratio of CD4 vs CD8 SP thymocytes in both normal and TCR Tg RasGRP1 mice. The findings are also consistent with previous studies using thymocyte cultures in combination with MEK1 inhibitors to analyze the significance of the Ras/ERK pathway (21-23). These studies revealed that the formation of CD4 SP thymocytes is more dependent on ERK activation than CD8 SP thymocytes. Reinforcing these observations, a recent report on ERK-deficient mice has found that the elimination of ERK1 and ERK2 protein preferentially affects the CD4 lineage (55). Therefore, we propose that RasGRP 1-deficiency reduces the propensity of TCR signaled-DP thymocytes to take the CD4 SP developmental path rather than directly influencing lineage commitment per Se.  To examine whether RasGRP1 promotes CD4 T cell development by inducing Bcl-2 expression, we introduced the E.t-Bcl-2 transgene into RasGRP1TcrAND animals. In Eji Bcl-2 RasGRP 1 ‘TcrAND mice, we observed a substantive (20-fold) recovery of CD4 SP thymocytes vs RasGRPFTcrAND animals lacking the Bcl-2 transgene. Because enforced Bcl-2 expression cannot transform DP thymocytes into SP thymocytes in the absence of MHC molecules (40), the prosurvival effect of Bcl-2 without TCR signaling is insufficient for the formation of SP thymocytes. Therefore, the increased presence of CD4 SP thymocytes in E.t-Bcl-2 RasGRPF’TcrAND mice argue that RasGRP1 assists CD4 T cell development by providing survival signaling to DP thymocytes. However, the facts that the CD4 SP thymocyte number is still much less than wild type (7.5-fold decrease) and that the  71  cells that develop have lower TCR expression indicates that RasGRP1 transmits differentiation signals independent of those necessary for Bcl-2 induction. Collectively, our data suggest that positive selection in RasGRPF’TcrAND is inefficient, with only a small proportion of DP thymocytes receiving TCR signaling strength necessary for differentiation.  The impaired T cell development in RasGRP 1-deficient animals results in a large number of discernable alterations including decreased ERK activation, altered surface marker expression, and reduced Bcl-2 levels. Suëh changes may result either directly from RasGRP1 -deficiency or indirectly, a consequence of delayed positive selection. To determine whether RasGRP1 function was unequivocally connected to some of these changes, we cultured DP thymocytes from non-TCR Tg mice in the presence or absence of anti-TCR Ab stimulation to mimic the positive selection process in vitro as described previously (42, 43). Although CD5 induction is not affected, RasGRP i DP thymocytes fail to up-regulate the positive selection marker CD69. This observation is consistent with a previous study demonstrating that this TCR-mediated event is dependent on Ras activation (56). Our experiment also revealed that RasGRP l DP thymocytes require conditions of stronger TCR signaling to elevate Bcl-2 levels. As a consequence, these data suggest that RasGRPF’ DP thymocytes must be strongly self-reactive to overcome their signaling deficits and differentiate into SP thymocytes. Notably, a recent report has linked T cells that develop in the absence of RasGRP1 to autoimmune consequences in a novel mouse strain, bearing a spontaneous mutation in RasGRP1 (57).  The relative rarity of TcrAND CD8 SP thymocytes in the RasGRP1 mice may be attributable to a couple of factors. First, the lack of concordance between TCR!MHC specificity and coreceptor expression has been postulated to result in reduced avidity for MHC class II molecules, inefficient TCR signaling, and subsequent failure of such mismatched cells to develop. The facts that MHC class 11-restricted CD8 T cells can be positively selected and function in CD4-deficient mice argues against this conclusion (6-8). However, although these experiments demonstrate that MHC class Il-specific CD8 T cells can develop, their formation and maintenance may still be compromised. Second, RasGRP1 is necessary for the efficient development of CD8 SP thymocytes (33). It is possible that the  72  down-regulation of CD4 and lack of RasGRP 1 expression may collaborate to limit the formation of RasGRPF’TcrAND CD8 SP thymocytes. The inability of Tg Bcl-2 expression to greatly improve CD8 SP thymocyte numbers in RasGRPF’TcrAND mice suggests that their limited quantity is not the result of inadequate levels of Bcl-2 but rather inefficient TCR signaling required for CD8 T cell differentiation. Since T cells require optimal TCR and CD4/CD8 coreceptor stimulation for their differentiation and survival, it serves not only to facilitate self-restriction but also maintains concordance between MHC specificity, effector function, and the CD4/CD8 phenotype of T cells in normal mice.  It is noted that small numbers of MHC class TI-restricted CD8 T cells have been observed in wild-type MHC class Il-restricted TCR Tg animals (6, 58). One potential explanation for the origin of these cells is that they are a product of limiting niches for selection of CD4 SP thymocytes in wild-type TcrAND mice. Using the strength of signal model of lineage commitment, some DP thymocytes may receive weakened TCR signaling due to the intense competition for positively selecting ligands (self-peptides/self-MHC class II) and thus be diverted to the CD8 lineage. Previous studies have shown that the development of MHC class TI-restricted CD8 T cells in these animals requires MHC class TI molecules but is independent of endogenous TCRa-chains (6, 58). Although we cannot formally rule out the participation of endogenous TCRct-chains in the derivation of RasGRPF’TcrAND CD8 SP thymocytes, the fact that virtually all RasGRPFTcrAND peripheral CD8 T cells express the AND TCR (Fig. 2.8B) implies that endogenous TCRa-chains are not critical for their development.  The emerging role of subcellular compartmentalization in cell signaling has impacted the Ras/MAPK pathway (59). Recent work has demonstrated that PLCy1 selectively activates RasGRP1 on the Golgi and is therefore distinct from the Grb2/Sos pathway that activates Ras at the PM (3 0-32). Differential localization may serve to alter the duration of signaling and/or pair-activated Ras with a different subset of effectors. Pointedly, Golgi-activated Ras has been found to have a delayed or sustained kinetics relative to PM-activated Ras (30-32). Such prolonged signaling by RasGRP1 may be critical to efficiently induce the expression of Th-POK (also known as cKrox), a Kruppel transcription factor family member that is both  73  necessary and sufficient to direct thymocytes undergoing positive selection into CD4 lineage (60, 61). Collectively, our results position RasGRP 1 and its product, Golgi-activated Ras, to play a crucial role in initiating the CD4 T cell differentiation program.  2.5  Acknowledgements  We thank Ken W. Harder and Mike Chow for valuable discussions; Soo-Jeet Teh for technical assistance; James C. Stone for RasGRP 1-null breeder mice; Wilfred A. Jefferies for C3H  112 ( k)  mice; Christopher J. Ong for Eji-bcl-2-36 Tg mice; Michael R. Gold for CH12 B  cell line; Andy Johnson and Jeff Duenas for cell-sorting at the University of British Columbia MultiUser Flow Cytometry Facility; and Bill Masin for animal husbandry.  74  2.6  Figures  Figure 2.1 RasGRPF’ mice expressing the MHC class IT-restricted AND TCR lack CD4 SP thymocytes. A, Thymocytes from wild-type and mutant mice expressing MHC class IIrestricted AND TCR were stained with anti-CD4 and anti-CD8 Abs. Percentage of cells residing within each quadrant is shown on the dot plots. Thymocyte recovery from wild-type and RasGRP1-deficient TCR Tg animals were as follows: RasGRPf ’TcrAND, 131.2 1 x 106 (n = 6); and RasGRPF’TcrAND, 95.4 ± 17.3 x 106 (n 7). B, Flow cytometric  ±  20.1  analyses of TCR and developmental marker expression displayed by wild-type (shaded, thin line) and mutant (bold-line) DP thymocytes.  Figure 2.2 RasGRP 1-deficient TcrAND DP thymocytes display reductions in ERK activity and Bcl-2 expression. A, P-ERK reactivity in wild-type (+7+) and RasGRPF’ (-I-) TcrAND DP thymocytes was assayed by flow cytometry directly ex vivo. Numbers within the high 2 / 1 histogram represent the proportion of p..J( cells. Shaded histogram represents  staining observed with secondary Ab alone. B, Up-regulation of the AND TCR on DP thymocytes correlates with elevations in ERK activity. DP thymocytes from wild-type (+1+) and RasGRP1 (-I-) TcrAND mice were stained with anti-P-ERK1/2 and anti-AND TCR Abs as described in Materials and Methods. Percentage of cells residing within each quadrant is shown on the dot plots. C, Bcl-2 histograms representing RasGRP1TcrAND, RasGRP1TcrAND, and isotype-Ab control DP thymocyte staining (shaded) are shown. MFIs are indicated within the histogram.  Figure 2.3 RasGRP1 DP thymocytes exhibit a decreased capacity to up-regulate P-ERK, CD69, and Bcl-2 levels following TCR engagement. A, Normal (non-TCR Tg) RasGRP1’, and RasGRPF’ thymocytes were stained with anti-CD4 and anti-CD8 Abs and subjected to cell sorting. Percentage of cells residing within each quadrant is shown on the density plots. Boxed area indicates the gate location used to sort DP thymocytes for signaling studies. B, Sorted RasGRP1 (thin line) and RasGRP1 DP thymocytes (bold line) express similar  75  levels of CD3 8. C, Sorted DP thymocytes were stimulated for indicated times (mm) with anti-CD3E. Data from one representative experiment of four are shown. D, Total thymocytes from wild-type and mutant mice were incubated for 24 h in the presence (bold line) or absence (thin, dashed line) of plate-bound anti-TCR (aTCR) or anti-TCR plus anti-CD4 Abs (aTCR + cCD4). Data presented were electronically gated on viable DP thymocytes (CD4CD8 7-AAIX). Numbers within Bcl-2 histograms indicate the MFI with (right) and without (left) TCR stimulation. Shaded histograms indicate intracellular staining observed with an isotype-control Ab.  Figure 2.4 Tg Bcl-2 expression fails to restore CD4 T cell development in RasGRP1 TcrAND mice. A, Thymic developmental profiles for wild-type TcrAND and RasGRP1deficient TcrAND thymocytes bearing the Ei-Bcl-2 transgene were ascertained by staining with anti-CD4 an4 anti-CD8 Abs. Percentage of cells residing within each quadrant is shown on the density plot. B, Cell numbers present within thymic subpopulations among various mice shown in A. C, RasGRPlTcrAND CD4 SP thymocytes (thin line) express intermediate TCR levels as compared with wild-type TcrAND CD4 SP thymocytes (shaded, thin line) and RasGRPFTcrAND DP SP thymocytes (bold line). Dotted, gray line represents background autofluorescence staining.  Figure 2.5 RasGRPF’ animals share a similar thymic developmental profile regardless of whether they express either the MHC class TI-restricted or MHC class I-restricted TCR. A, Thymocyes from RasGRPF’TcrH-Y or TcrAND were stained with anti-CD4 and anti-CD8 Abs. Percentage of cells residing within each quadrant is shown on the density plot. B, Cell numbers present within thymic subpopulations from both wild-type and RasGRP 1-deficient TcrAND and TcrH-Y were determined. To facilitate comparison between various thymi, cell numbers present within thymic subpopulations were plotted in bar graph format as the percentage of cells relative to wild type. C, RasGRPlTcrAND CD8 SP thymocytes express comparable levels of various maturation markers as RasGRPF’TcrH-Y CD8 SP thymocytes.  76  Figure 2.6 RasGRP1TcrAND DP thymocytes exhibit limited developmental potential. Wild-type and RasGRPFTcrAND thymocytes were stained with anti-CD4 and CD8 Abs and subjected to electronic cell-sorting to purify DP thymocytes. The sorting gate is shown as a box superimposed on original thymocyte populations (left). To assess the potential of TcrAND DP thymocytes to differentiate in vivo, purified DP thymocytes were injected directly into the thymus of congenic (Thy1.1) B6 mice. Two days after injection, host thymi were harvested, and the coreceptor expression of donor cells (Thy 1 .2k) was determined either directly ex vivo (Untreated) or after coreceptor re-expression assay (Pronase/37°C). In this assay, cells are treated with pronase extracellularly to strip pre-existing CD4 and CD8 coreceptor molecules and cultured overnight. After culture at 37°C, surface coreceptor expression is a consequence of coreceptor molecules being actively synthesized by the cell. Frequency of cells present within each quadrant is indicated.  Figure 2.7 CD8 SP thymocytes that develop in RasGRPF’TcrAND mice respond to MHC class IT-specific peptide and differentiate in cytolytic effectors. A, TcrAND CD8 SP thymocytes possess elevated ERK activity. Numbers within histograms represent background subtracted MFTs. Shaded histograms represent staining observed with secondary alone. B, CD 127 (IL-7Ra) and Bcl-2 histograms representing RasGRP1 +1+ , RasGRP1 -l , and isotype control staining of CD8 SP thymocytes. C, Sorted RasGRP1TcrAND CD4 SP, RasGRP1’TcrAND CD8 SP, and RasGRPF’TcrAND CD8 SP thymocytes were cultured on plates coated with either anti-CD3E or anti-Va 11 (AND TCR; clone RR8-1) Ab plus exogenous IL-2 as described in Materials and Methods. D, Sorted SP thymocytes, either RasGRP1TcrAND CD4 SP, RasGRP1’TcrAND CD8 SP, or RasGRPF’TcrAND CD8 SP, were stimulated with various doses of PCC and exogenous of IL-2 as described in Materials and Methods. E, Using intracellular flow cytometry, RR8- 1-activated RasGRP1TcrAND CD4, RasGRP1TcrAND CD8, and RasGRP1TcrAND CD8 SP thymocytes were stained with anti-granzyme B Ab. F, RR8-1-activated RasGRP1’TcrAND CD4 SP and RasGRPFTcrAND CD8 SP thymocytes were tested for their ability to kill the CH12 (IEk expressing) lymphoma either in the presence or absence of PCC. Assays were  77  done in triplicate, and the error bars represent the SD. Data from one representative experiment of three are shown.  Figure 2.8 RasGRPlTcrAND animals possess significant numbers of peripheral AND TCR CD8 T cells but very small numbers of AND TCR CD4 T cells. A, Lymph node cells were stained with anti-CD4 and anti-CD8 Abs. Percentage of cells residing within each quadrant is shown on the density plots. B, Clonotypic TCR (anti-Vail Ab; clone RR8-i) expression by peripheral CD4 and CD8 T cells in with wild-type and RasGRPF’TcrAND animals. Numbers within histograms indicate the proportion of T cells expressing high levels of the AND TCR. C, Comparison of differentiation markers and TCR expression by CD8 T cells from the lymph nodes of female RasGRP1 +1+ TcrH-Y (shaded), female RasGRP1 -I- TcrH Y (thin-line), and RasGRPlTcrAND (bold-line) mice. Data from one representative experiment of three is shown.  78  A  +1+  AND  -I-  B  CD69  Figure 2.1 RasGRPIt mice expressing the MHC class IT-restricted AND ICR lack CD4 SP thymocytes. 79  A  +1+  DP  -I..  20.0  ;.  A6.8 P-ERK1/2  8 j 36  -I-  DP  Bt___  7  .  41  :  15  81  1  AND TCR  C  +1+  DP  -I  74.1  Be1-2  Figure 2.2 RasGRP 1-deficient TcrAND DP thymocytes display reductions in ERK activity and Bcl-2 expression. 80  4—  +1÷  A  87.4 0.7  93.8  D  C)  B  -/-DP  CD&  C  +1+  -IP-ERK1/2 ERK2 P-ZAP7O  _a.  — .i — — — — ZAP7O P-LAT  Figure 2.3 RasGRPF’ DP thymocytes exhibit a decreased capacity to up-regulate P-ERK, CD69, and Bcl-2 levels following TCR engagement.  81  A  Normal ‘  AND 65 10  Ea-BcI-2 15  B  AND • -I-AND  -I-I-f- AND Bcl-2 Tg -I- AND Bcl-2 Tg  100  +1+  (I,  0  14 0 0.2  851  2.7  89  j  F  —I.  1D8’> C  •  -I- Bcl-2 CD4  +1+ CD4  • ....E...LL T9TCRI3  COB SP  TgTCRa  Figure 2.4 Tg Bcl-2 expression fails to restore CD4 T cell development in RasGRPF’ TcrAND mice. 82  A  B 140 120 100 80 a 60 40 20 0  DN  DP  CD8SP  CD4SP  CD&  >  CD69  )  Figure 2.5 RasGRPF’ animals share a similar thymic developmental profile regardless of whether they express either the MHC class Il-restricted or MHC class I-restricted TCR. 83  Sorting Gate 4389—  Cell Purity  552 ‘1.3  IT inject sorted DP  Pronase/ 370 C  Untreated  984  0.3  71.9  23.7  15.9  3.9  31.3  0 cc’:  o II  S  Sf!4 i04m,  I  837 ‘01  4i  99.2  I  1.9  Analyze donor cells 2 days post-transfer  9  I  72.8  311{ II  M’  0  -  29  0  It 4t0,  22.8  4.4  8.4  22.  CDS-APC-Cy7  Figure 2.6 RasGRPFLTc7AND DP thymocytes exhibit limited developmental potential.  84  A  +1+ -  CD8SP 17.6  •-ICD8  -.1-.  D  20.7  ALL  P-ERK1I2  B  0  E  F -i-1÷ CD4  +1+  0.1  1  +1+ 0D4 + PCC 50 —-J CD8+PCCA °‘  E’  -/-cD8,,/  *1  Granzyme B  E/T0.3  1  3.3  10  Figure 2.7 CD8 SP thymocytes that develop in RasGRPF’TcrAND mice respond to MHC class Il-specific peptide and differentiate in cytolytic effectors. 85  A  +1+  CD8  B  +1+  CD4 TceII .1*y  CD8 Teell  LI  .  CD8 TceII  ..  .  23j  j96  26  lA -AND TCR  C  -I-  AND  I  -+/+  —  H-Y  AND ....  HSA CD8 TceII  CD44  CD69  I  TCR(3  Figure 2.8 RasGRP1TcrAND animals possess significant numbers of peripheral AND  TCR CD8 T cells but very small numbers of AND TCR CD4 T cells. 86  2.7  References  1.  von Boehmer, H.. 1994. Positive selection of lymphocytes. Cell 76: 219-228.  2.  Hogquist, K. A.. 2001. Signal strength in thymic selection and lineage commitment. Curr. Opin. Immunol. 13: 225-231.  3.  Germain, R. 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The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433: 826-833.  61.  Sun, G., X. Liu, P. Mercado, S. R. Jenkinson, M. Kypriotou, L. Feigenbaum, P. Galera, R. Bosselut. 2005. The zinc fmger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat. Immunol. 6: 373-381.  93  CHAPTER 3  CHRONIC IMMUNODEFICIENCY IN MICE LACKING RASGRP1  1 RESULTS IN CD4 T CELL IMMUIE ACTIVATION AND EXHAUSTION  3.1  Introduction  The two dominant forces maintaining homeo static control on the size of naive T cell compartment are the availability of self-peptides/self-MHC ligands and the cytokine IL-7 (1, 2). Under normal T cell-sufficient conditions, low-avidity TCR interactions with self peptides/self-MHC molecules along with IL-7R signaling promote T cell survival. During T cell lymphopenia, the increased abundance of these same elements is thought to induce slow T cell (homeostatic) proliferation in an attempt to increase peripheral T cell numbers. Therefore, homeostatic expansion is beneficial for preserving the size of the T cell population but it may pose risks because T cell proliferation is accompanied by acquisition of effector function, such as cytotoxicity and the capacity to rapidly secrete inflammatory cytokines (1, 2). Because this slow cellular division only applies to a subset of T cells, it is thought that the T cells recruited into cell cycle are the ones expressing TCRs with greater avidity for self Ags (1, 2), Therefore, lymphopenia-induced T cell expansion could be dangerous as it may skew the TCR repertoire of the resident pooi of T cells toward autoreactivity and, further, restrict its diversity.  The term “homeostatic proliferation” was originally coined to describe the slow T cell expansion observed when naive TCR-transgenic T cells were adoptively transferred into lymphopenic recipients, such as congenitally T cell-deficient RAG’ mice or normal mice rendered lymphopenic by irradiation (2-4). Because donor TCR-transgenic T cells were RAG deficient, it ensured that these T cells were monoclonal, expressing a single c43 TCR, and verified that this slow proliferation occurred in the absence of agonist peptides. Moreover,  ‘A version of this chapter has been published. Priatel, J.J., X. Chen, L.A. Zenewicz, H. Shen, K.W. Harder, M.S. Horwitz, and H. S. Teh. (2007) Chronic Immunodeficiency in Mice Lacking RasGRP1 Results in CD4 T cell Immune Activation and Exhaustion. I Immunol. 179: 2143-2152. Copyright 2007. The American Associateion ofImmunologists, Inc.  94  the product of this cell division possesses a memory T cell phenotype resembling the functional attributes and gene expression profiles of conventional memory T cells (2, 5). Despite these observations, recent work has suggested that foreign Ags may be responsible for some donor T cell proliferation observed following transplantation into congenitally T cell-deficient RAG’, TCRcC’, or SCID hosts (6, 7). Moreover, because mice lacking any c4 T cells are severely immunocompromised, these hosts are highly susceptible to developing chronic infections and, as a result, may present a broad array of microbial Ags to donor T cells. In support of this hypothesis, the adoptive transfer of normal (polyclonal) T cells into a congenitally T cell-deficient host (chronically immunodeficient) results in a subset of T cells undergoing massive T cell expansion whereas only slow division is apparent when such T cells are transferred into wild-type hosts made lymphopenic by irradiation (acutely immunodeficient). The observation that donor TCR-transgenic (monoclonal) T cells undergo similar rates of division in these two disparate recipients demonstrates that rapid T cell proliferation only applies to a subset of T cells and suggests that it may be dependent on TCR specificity (6, 7). Because rapid T cell proliferation is lost when T cells are transferred into congenitally T cell-deficient, gnotobiotic (germfree) hosts (7), it argues that foreign Ags, likely derived from commensal microbes within the gut, are driving fast T cell proliferation in congenitally T cell-deficient animals.  The loss of T cell immunity is a common occurrence during chronic viral infections in both mice and humans (8). Recent evidence suggests that continual exposure to cognate Ag results in the overstimulation of viral-specific T cells and the development of an “exhausted” memory T cell phenotype. In contrast to acute infection, memory T cells derived from chronic infection exhibit Ag dependency, limited self-renewal capacity, diminished cytokine production, and reduced cytotoxicity (9). Therefore, these functional impairments displayed by chronically activated T cells may contribute to the failure to clear virus. Programmed death-i (PD-i), a negative regulator of activated T cells (10), is strongly up-regulated on exhausted viral-specific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice and HIV infection in humans (ii, 12). Because blockade of PD-i interaction with its ligand PD-Li can restore function in exhausted CD8 T cells (11), it  95  makes the case that PD-i is not simply indicative of an exhausted state but also plays a key role in its maintenance.  In thymocytes, Sos (13) and RasGRP1 (14), two Ras-guanyl-nucleotide exchange factors, link Ras and MAPK activation to ICR signal transduction with their respective functions dependent on relocating to membranes by two distinct mechanisms (15). RasGRP1 mobilizes to membranes by binding the phospholipase Cyl product diacyiglycerol through its Cl domain whereas Sos is recruited to the phosphorylated adaptor molecule linker for activated T cells by way of its association with the Src homology 2-domain-containing protein Grb2. RasGRPF’ thymocytes show signs of reduced TCR signaling (14, 16, 17) and a selective impairment of positive but not negative selection (16). Therefore, mice lacking RasGRP1 serve as a model lacking a positive regulator of TCR signaling. RasGRPF’ mice exhibit a marked T cell lymphopenia (14, 16), likely a consequence of decreased single-positive (SP) thymocyte maturation and T cell hyporesponsiveness (14, 16, 17). Paradoxically, a recently described novel mouse strain called  (lymphoproliferation-autoimmunity  glomerulonephritis), bearing a spontaneous mutation in RasGRP1, develops an autoimmune syndrome resembling systemic lupus erythematosus (SLE), exhibiting massive lymphoproliferation, high levels of serum autoantibodies and, eventually, advanced disease that required euthanasia (18). Therefore, RasGRP1 signaling may be critical for both thymocyte maturation and T cell tolerance.  In this study, we report that although RasGRPF’ mice remain T cell lymphopenic and free of overt disease until at least 1 year of age, they possess a population of proliferating CD4 T cells that display an exhausted phenotype, characteristic of chronic infection (8). Adoptive transfer experiments suggest that chronic immunodeficiency and foreign Ags might be responsible for inducing RasGRPF’ CD4 T cell proliferation rather than autoreactivity to self-Ags. Supporting the notion of a dysfunctional immune system, bacterial and viral challenge experiments revealed that RasGRP i mice exhibited impaired T cell responses and a delay in pathogen clearance. Lastly, we find that chronic T cell immunodeficiency in RasGRPF’ mice is likely a consequence ofRasGRPi protein loss in thymocytes and/or T cells rather than due to defects in innate immunity. In conclusion, these investigations  96  highlight the roles of RasGRP1 in determining a normal immune status and as an essential regulator of adaptive T cell immunity toward experimental infection.  3.2  Materials and methods  Mice C57BL/6J (B6), B6.PL-ThylaJCy (Thy l.1), B6.SJL-Ptprca Pep3b/BoyJ (Ly 5.l), B6.RAG-l, and B6.TCRcC’ mice were acquired from The Jackson Laboratory. RasGRP1 breeder mice were provided by J. C. Stone (University of Alberta, Alberta, Canada) and bred onto a B6 background at least seven generations. To generate Thyl .1 2C TCR-transgenic 112 ( animals, the 2C TCR transgene was bred onto the B6.PL-ThylaJCy background b,  Thy 1. 1k). All studies followed guidelines set by the Animal Care Committee at the University of British Columbia in conjunction with the Canadian Council on Animal Care.  Flow cytometry Abs against CD4 (GK1.5), CD8 (53-6.7), CD5 (53-7.3), TCR (H57-597), CD62L (MEL 14), CD25 (PC61.5), CD69 (Hl.2F3), CD45.i (A20), Thyl.1 (HIS51), CD44 (1M7), CD62L (MEL-14), CD127 (AKR34), PD-i (J43), PD-Li (MIH5), PD-L2 (TY25), TNF-a (MP6XT22), and IFN-y (XMG1 .2) were purchased from eBioscience. Annexin VPE, anti-Fas (DX2), anti-FasL (MFL3), anti-Ki-67 Abs (B56), and anti-TCR Vf3 screening panel (no. 0143KK) reagent sets were purchased from BD Biosciences. For anti-Ki-67 staining, cells were fixed with 2% formaldehyde (Polysciences) for 10 mm, permeabilized with 90% methanol, washed with 2% FCS/PBS and subsequently, incubated with anti-Ki-67 Ab for 30 mm at room temperature. Isotype-control Ab (clone MOPC-2i; BD Biosciences) staining was negligible (> 0.2%). Annexin V-PE staining was conducted as described previously (19). Data were acquired using either a FACScan or FACSCalibur and CeliQuest software (BD Biosciences). Data were analyzed either with CellQuest, FCSPRESS, or FlowJo (Tree Star) software.  97  Adoptive transfer experiments Wild-type splenic and lymph node T cells were purified from Thy 1.1 + animals and labeled with 1 jiM CFSE (Molecular Probes) as previously described (20). Approximately 2 x 106 purified wild-type (polyclonal) Thy 1.1 + T cells or 1 x 10 6 Thy 1.1 + 2C TCR CD8 T cells were i.v. injected into Thyl .2 recipients, either irradiated (600 rad) wild-type B6 or nonirradiated wild-type B6, B6.RasGRPF’, B6.RAG-F’, and B6.TCRcC’ mice. Conversely, 2 x 106 CFSE-labeled RasGRP 1 T cells (Thy 1 .2k) were transferred into either B6.Thy 1.1k, irradiated B6.Thyl .1, or B6.RAG-1 hosts. Spleens were recovered 1 wk posttransfer and proliferation of donor cells assessed by flow cytometry using a FACSCalibur (BD Biosciences).  Bacterial and viral infections Mice were infected i.v. with —10,000 CFU of a recombinant strain of Listeria monocytogenes engineered to express the 2C TCR agonist peptide SIYRYYGL (J. Priatel, L. Zenewicz, H. Shen, and H. Teh, manuscript in preparation). For viral infection, mice were injected i.p. with --400,000 PFU of LCMV-Armstrong. Splenic viral titers were determined as described previously (21). For wild-type I cell infusion into RasGRPF’ (Ly5.2) animals, purified (10 million) Ly5.1 T cells (—55:45% ratio of CD4 vs CD8 T cells) were i.v. injected into RasGRP 1-deficient animals 1 day before infection with either rLM-SIY or LCMV.  Detection ofIFN-yproduction by intracellularflow cytometry Spleens were harvested from mice at either day 7 (rLM-SIY) or day 8 (LCMV) postinfection, pressed through metal mesh to generate single-cell suspensions, and subjected to RBC lysis by ammonium chloride treatment. Splenocytes were cultured for 5 h in 96-well, flat-bottom plates, at a concentration of 2-4 x 106 cells/well, in 0.2 ml of complete medium supplemented with 1 jil/mi Golgi Plug (contains brefeldin A; BD Biosciences) to block cytokine secretion. Cells were stimulated with a concentration of 1 jiM for the MHC class I peptides (SlY, SIYRYYGL; GP , KAVYNFATC; GP 3341 , FQPQNGQFI; 3904 , AVYNFATCGI; NP 33 , YTVKYPNL; 0VA 205212 , SIINFEKL) and 10 jiM 257264 GP SGVENPGGYCL; NP , 27286 for the IAb MHC class II peptides , 201 NEKYAQAYPNVS; , _ 190 (LL0 80 _ 61 GP  98  GLKGPDIYKGVYQFKSVEFD). For anti-TCR stimulations, 2 x 106 splenocytes were incubated for 5 h in a 24-well plate that had been precoated with 10 .tg/ml anti-CD3E (1452C1 1) Ab. After culture, cells were fixed for 15 mm in 2% paraformaldehyde/PBS solution, permeabilized for 15 mill with 0.2% Tween 20/PBS and stained with anti-CD4allophycocyanin, anti-CD8-PE-Cy5, and anti-IFN-y-FITC Abs (eBioscience). Data were acquired on a FACSCalibur using CellQuest software (BD Biosciences) and analyzed with FCSPress (www.fcspress.com). All peptides were synthesized at the University of British Columbia’s Nucleic Acid Protein Service Unit.  Direct ex vivo CTL assays After 7 days postinfection with rLM-SIY, splenic CD8 T cell effectors were isolated by staining total splenocytes with rat anti-mouse CD4 (GK1 .5) Abs and subsequently depleted of CD4 and surface Ig cells with anti-mouse (and rat-reactive) Ig-liriked Dynabeads (catalog no. 110.02; Dynal Biotech). The target EL-4 cell line was labeled with 51 Cr, pulsed with STY peptide, washed, and incubated with various numbers of effectors as previously described (20).  3.3  Results  RasGRPT CD4 T cells exhibit markers ofacute activation, exhaustion, proflferation, and spontaneous apoptosis The impaired T cell development results in a T cell lymphopenia, exhibiting a 10-fold decreased abundance of peripheral T cells, in 1-mo-old RasGRPF’ mice (14). To account for the limited positive selection in RasGRP1 animals, it had been proposed that thymocytes capable of being selected without RasGRP 1 must express strongly self-reactive TCRs to overcome their signaling deficits (16-18). Notably, autoreactive CD4 T cells were suspected to be the root cause of massive lymphoproliferation and an underlying autoimmune disorder in RasGRP1’ mice (18). However, although we observed some incidents of massive lymphoproliferation, splenomegaly, generalized lymphadenopathy, and a 20- to 30-fold  99  increase in total cellularity, in mice homozygous for the targeted mutation on a mixed C57BL/6J:129 background (our unpublished observations), this phenotype seems to have vanished after sequential backcrossing of the RasGRP1 gene-knockout allele to the C57BL/6J (B6) genetic background. In this report, we focus on RasGRPf’ mice that have been bred at least seven generations onto the B6 mouse background.  B6-backcrossed RasGRPl mice appear healthy until at least 1 year of age and do not develop massive lymphoproliferation. Sampling of 2- to 4-mo-old RasGRP 1 mice revealed that they remain T cell lymphopenic, the recovery of both CD4 and CD8 T cells from spleens and pooled lymph nodes (LNs) were reduced vs age-matched wild-type mice (Fig. 3.1A). Comparison of secondary lymphoid organs revealed that RasGRPl mice have similar-sized spleens with respect to wild type (Fig. 3.1 B). Curiously, RasGRP 1 .1. mice possess small peripheral LN such as axillary, brachial and inguinal LNs whereas mesenteric ENs (MEN) from the mutant mice were enlarged as compared with age-matched wild-type animals (Fig. 3.1B). Next, flow cytometric analyses revealed that RasGRPF’ CD4 and CD8 T cells express very high levels of CD44 as compared with wild type regardless of whether they were isolated from the spleen, peripheral LN, or MLN (Fig. 3.1C and our unpublished observations). In addition, a large proportion ofRasGRPF CD4 T cells also display signs of 69 CD127b0, CD62Lb0, ( acute activation hih  h1h FasEhish) 5  The activated and  memory phenotype for RasGRP1 T cells is particularly conspicuous because our TCR transgenic studies demonstrated that central tolerance was not affected by RasGRP 1 deficiency and that RasGRPl T cells displayed diminished capacities to undergo homeostatic expansion and respond to cognate Ag (16).  The activated state ofRasGRPF’ CD4 T cells is reminiscent of the exhausted phenotype described in mice and humans suffering from chronic viral infections (8). Therefore, we sought to determine whether RasGRP 1  T cells expressed PD-i, a molecule that is strongly  expressed by exhausted but not normal T cells (10). Strikingly, a large fraction ofRasGRPF’ CD4 T cells possess high levels of PD-i on their surface (Fig. 3.1D). In addition, PD-Li, a PD-i ligand that is constitutively expressed by most splenocytes (10), is markedly up regulated by RasGRPF CD4 T cells whereas it is more modestly elevated by mutant CD8 T  100  cells (Fig. 3.1D). By contrast, expression of PD-L2 does not appear to be altered in RasGRP1 mice (our unpublished observations). Next, we examined whether T cell activation in RasGRP1 mice was linked with cellular proliferation (Fig. 3.1E). Indeed, RasGRP 1 CD4 T cells exhibit an elevated frequency of cells bearing the proliferationassociated nuclear Ag Ki-67 as compared with wild type (13.3 vs 5.0%). Because elevations in CD44 levels are intimately linked with peripheral T cell expansion (2), Ki-67 staining associates with increased CD44 expression in both wild-type and mutant mice (Fig. 3.1E). The difference in Ki-67 expression between wild-type and mutant CD4 T cells is significantly less pronounced when comparisons are based on the =  13.4%; mutant  hih 44  subsets (wild type  15.7%). Notably, the CD44 expression profiles shown for wild-type and  mutant T cells (Fig. 3.1E) is altered as compared with Fig. 3.1C because it was performed after formaldehyde fixation and permeabilization with methanol to facilitate the detection of the intracellular Ag Ki-67 (see Materials and Methods). Studies have shown that formaldehyde/methanol treatment of cells results in a reduction in the intensity of CD44 staining, as detected by the clone 1M7 mAb, and an increase in background cell staining levels (22). Subsequently, we sought to determine whether RasGRPF CD4 T cells show signs of accelerated cell death because elevated Fas/FasL levels and exhausted T cell phenotypes are associated with increased rates of T cell apoptosis (Fig. 3. iF). Staining with the apoptotic marker annexin V revealed that RasGRP1 CD4 T cells are strongly reactive toward this reagent (RasGRP1  =  76.0% vs RasGRP1’ = 24.1%). Collectively, these  findings suggest that RasGRPF’ CD4 T cells are highly activated, cycling, and apoptotic as compared with wild type.  RasGRPT’ CD4 SF thymocytes display a naive cell surface phenotype Mice deficient in RasGRP1 exhibit severely diminished numbers of mature SP thymocytes demonstrating that this molecule plays a critical role in thymopoiesis (14, 16, 18) (Fig. 3.2A). To examine whether RasGRP1 CD4 and CD8 T cells spontaneously acquire a memory phenotype from their development in the thymus, we stained thymocytes with Abs specific for CD4, CD8, and TCR to identify mature (TCR13) SP (CD4CDW or CD4CD8) thymocyte subpopulations. In stark contrast to peripheral RasGRPF’ T cells, RasGRP1 SP thymocytes express abnormally low amounts of CD44 and CD69 as compared with their 101  wild-type counterparts (Fig. 3.2B). However, in concordance with SP thymocyte maturation, RasGRPF’ CD4 SP thymocytes bear equivalent expression of CD5, a marker of TCR signaling during positive selection (23), as well as similar levels of CD62L and Bcl-2 as compared with wild-type CD4 SP thymocytes (Fig. 3.2B and our unpublished observations). Strikingly, the CD44 ” phenotype of most RasGRPF’ CD4 SP thymocytes contrasts with 1 the elevated CD44 expression levels previously reported for  CD4 SP thymocytes  (18). To explain the contradiction between these findings, we hypothesize that the massive lymphoproliferation and lymphocytic tissue infiltration observed in RasGRP1’ animals (18) results in activated peripheral CD4 T cells also infiltrating the thymus. Because B6backcrossed RasGRPF’ mice remain lymphopenic, fewer RasGRPF’ CD4 T cells likely traffic to the thymus and contaminate the CD4 SP electronic gate. Therefore, we argue that RasGRP 1 deficiency supports the development of naive CD4 SP thymocytes and that their hih conversion to a 44 phenotype in the periphery may result from homeostatic pressures.  RasGRPT’ host animals support rapid donor T cell proliferation A hypothesis for the origin of memory phenotype (CD44) RasGRP 1-deficient T cells is that they are the product of slow homeostatic proliferation that result in the conversion of naive T cells into memory T cells. Because a substantially reduced number of SP thymocytes develop and are exported to the peripheral lymphoid organs in RasGRPF’ mice (16), the few mature SP thymocytes that immigrate to the periphery are subjected to a T cell lymphopenic environment. Therefore, the availability of IL-7 and self-MHC interactions in RasGRP 1-/ mice may be well-suited for inducing the peripheral T cell expansion. To test this hypothesis, we adoptively transferred equivalent numbers of wild-type T cells (Thy1.1), labeled with the mitotic tracker CFSE, into RasGRPF’, wild-type B6, irradiated B6 (600 rad) and TCRcC host animals for a 1-wk period (Fig. 3.3A). As expected, the majority of CD4 and CD8 T cells recovered from normal (lymphoreplete) mice had failed to divide whereas those from irradiated recipients had undergone slow expansion that is characteristic of homeo static proliferation, still retaining some fluorescence imparted by CFSE. In contrast, the outcome is substantially different when T cells are transferred into in congenitally T cell-deficient TCRcC” or RAG-1 mice (6, 7). In these chronically immunodeficient animals, which are  102  +  completely devoid of any aj3 TCR cells, some donor T cells underwent I? typical •  homeostatic proliferation, cycling one to four times per week, while other donor T cells divide rapidly, greater than eight times within a week, and completely lost their CFSE fluorescence. The massive growth of these rapidly dividing cells results in their increased numbers and representation when looking at the distribution of donor cell CFSE fluorescence l-wk posttransplantation (TCRcc null; Fig. 3.3A). Interestingly, the donor cell division history in RasGRPF recipients revealed a unique CFSE profile bearing similarities to both TCRcC and irradiated B6 hosts: a rapidly dividing population (52% of CD4 and 37% of CD8 T cells) and a slowly dividing population (21% of CD4 and 50% of CD8 T cells), respectively. These findings demonstrate that the lymphopenia present within RasGRP1 mice promotes spontaneous peripheral T cell expansion.  Foreign Ags, derived from commensal microbes, are thought to be responsible for the rapid naive T cell expansion observed after transfer of naive T cells into chronically immunodeficient mice RAG-F’ and TCRcC’ mice (7). To support the idea that these rapidly dividing T cells in RasGRP1 recipients are undergoing an Ag-driven differentiation program, we charted CD62L (L-selectin), a marker that is down-modulated upon acute activation, vs CFSE fluorescence after 1-wk residence in vivo (Fig. 3.3B). Strikingly, a large proportion of the CFSEb0 CD4 (69%) and CD8 (47%) T cells recovered from RasGRPF’ mice have low CD62L expression. By comparison, T cells residing in irradiated B6 recipients maintain high levels of CD62L while the majority of T cells recovered from RAG 1 hosts have down-regulated this marker (Fig. 3.3B). Next, we examined whether RasGRP 1 mice could also promote expansion of TCR-transgenic T cells by infusing wildtype 2C TCR-transgenic CD8 T cells (Thyl.1), labeled with CFSE, into normal B6, irradiated B6, RAG-F’, and RasGRP1 host animals and measuring cellular proliferation 1 wk later (Fig. 3.3 C). Notably, RasGRPF’ recipients induce slow 2C T cell proliferation but this amount is considerably less than both RAG-1 and irradiated B6 mice (97 and 87%, respectively). Moreover, the observation that 2C T cells do not undergo rapid proliferation in either RasGRPF or RAG-F’ hosts suggests that this type of T cell expansion may apply to only a subset of T cells with unique TCR specificity. Together, these results suggest that  103  RasGRPF’ mice are chronically immunodeficient and that forces driving wild-type T cell expansion in RasGRPF’ hosts could be a combination of self- and foreign-Ags.  RasGRPT” CD4 T cells prolferate vigorously in chronically immunodeficient RAG-] mice The previous experiments examined how wild-type T cells respond following transplant into RasGRPF’ hosts and therefore may not be reflective of how RasGRP1 T cells react to environmental cues. To define how mutant T cells respond in these different settings, CFSE labeled RasGRP 1 T cells (Thy 1 .2k) were i.v. injected into normal B6 (Thy 1 .1), irradiated B6 (Thy 1 .l), and RAG-l animals (Fig. 3.4A). Strikingly, the majority of RasGRPF’ CD4 T cells recovered from either normal or irradiated B6 animals were not recruited into cell cycle (63 and 65%, respectively) while the large fraction of RasGRP i CD8 T cells isolated from irradiated recipients were proliferating slowly (87%). By contrast, RasGRPF’ CD4 and CD8 T cells transplanted into RAG-1 hosts proliferated vigorously. Furthermore, RasGRP l T cells, particularly the CD4 T cells, recovered from RAG- 1 recipients strongly down-regulated CD62L expression whereas those placed in irradiated B6 hosts resembled the surface phenotype before adoptive transfer (Figs. 3.4B and 3.1C). Next, we addressed whether the residence of RasGRP i T cells in different hosts influenced effector function (Fig. 3.4C). Upon TCR stimulation, RasGRPF’ T cells recovered from RAG-I’ hosts possessed an increased frequency of cells capable of producing the proinflammatory cytokines TNF-a and IFN-y vs those residing in irradiated wild-type recipients. In summation, these studies demonstrate that chronically immunodeficient hosts are capable of inducing RasGRPl T cells to proliferate rapidly and boost effector function.  RasGRP] mice generate poor pathogen-specfIc T cell responses To test the hypothesis that RasGRPI’ mice are immunodeficient, wild-type and mutant mice were infected with a novel recombinant strain of L. monocytogenes (rLM-SIY) expressing a MHC class I K”-restricted peptide SIYRYYGL (STY), an agonist for the 2C TCR (24). One week postinfection, splenocytes were stimulated with either the endogenous MHC class IT restricted peptide LLO 19201 (listeriolysin 0 (LLO); Fig. 3.5A) or SlY (Fig. 3.5B) and Ag specific T cell responses were monitored by IFN-y production using intracellular flow  104  cytometry. As an additional control, splenocytes were also cultured on anti-TCR Ab-coated plates to test for the capacity to produce IFN-y. Strikingly, RasGRPf’ mice mounted a barely detectable immune response toward the LLO peptide (Fig. 3.5C). The fact that RasGRPF’ CD4 T cells can respond to anti-TCR Abs suggests that the weak response by RasGRP i mice is the result of a failure to generate LLO-reactive T cells rather than to secrete IFN-y. By contrast, RasGRP 1  CD8 T cells generate a strong anti-SlY response that  is modestly reduced in Ag-specific T cell numbers as compared with wild type (Fig. 3.5C). In addition, we tested the function of RasGRP i CD8 T cell effectors in a standard assay and found that these cells displayed cytotoxicity similar to their wild-type counterparts (Fig. 3 .5D). These rLM-SIY infection studies suggest that RasGRP 1 is particularly critical for generating MHC class Il-restricted immune responses.  To address the possibility that the importance of RasGRP1 on mounting MHC class IIrestricted immune responses may be restricted to the ELO peptide or bacterial infection, we used LCMV to infect wild-type and RasGRPF’ mice. Eight days postinfection with a sublethal dose of LCMV, Ag-specific T cells were enumerated to viral immunodominant ,,, 33 MHC class TI-restricted GP 80 (Fig. 3.6A) and MHC class I-restricted peptides (GP _ 61 ,, 3 GP 286 NP _ 276 GP 212 and NP _ 205 ; Fig. 3 .6B). Similar to the bacterial infection, 4 ,o 396 RasGRP 1  ‘  mice generated a weak MHC class TI-restricted response although this time a  reduced fraction of mutant CD4 T cells produced IFN-’’ upon TCR stimulation (3.6 vs 13.0%). However, in contrast to rLM-STY results, RasGRPf’ mice mounted weak immune responses toward all five MHC class I-restricted viral peptides analyzed despite the fact that a similar proportion of wild-type vs mutant CD8 T cells can secrete IFN-’’ upon TCR stimulation (48%). The fact that total T cell numbers were also sharply reduced in RasGRPF ‘  animals makes the drop in Ag-specific T cell numbers even more dramatic (Fig. 3.6C). To  evaluate whether the diminished T cell responses were physiologically relevant, splenic tissue, harvested 8 days postinfection, was screened for virus using plaque-forming assays (Fig. 3 .6D). Indeed, 3 of 4 RasGRP 1” spleens in this experiment (9 of 13 total) contained readily detectable virus at this late period postinfection, at a time point when virus is undetectable among wild-type mice. In conclusion, the weakened immune responses by RasGRP 1 1 mice is associated with delayed viral clearance.  105  The ability of RasGRPF’ mice to respond strongly to the STY peptide but only weakly to immunodominant LCIvIV peptides led us to speculate whether the differential responses were intrinsic to the peptides or the pathogen. To resolve this issue, wild-type and RasGRPF’ mice were infected with two other rLMs, either rLM-OVA or rLM-GP33, and the frequency of MHC class I-restricted 264 and anti-GP _ 257 anti-0VA i-specific T cells assessed 7 days 33 postinfection. In stark contrast to the SlY peptide, RasGRPl mice mounted weak, barely 264 and GP _ 257 331 MHC class I-restricted peptides detectable responses toward both 0VA (our unpublished observations). These findings that RasGRPF’ mice can respond to a limited set of immunodominant peptides suggest that its resident T cells may possess an altered TCR repertoire. To look for differences in the TCR repertoire between wild-type and RasGRPl mice, V3 TCR chain usages were determined by staining splenocytes with various anti-Vf3 TCR chain-specific Abs in conjunction with Abs specific for CD4, CD8, and the constant region of the TCR chain (Fig. 3.7). Although we observed differences among the V13 TCRs used by both RasGRP1 CD4 and CD8 T cells, the CD4 T cell population exhibited fewer statistically significant changes, due in part to a large deviation between RasGRP l mice. It is not clear whether the TCR repertoire changes in RasGRP i mice results from aberrant T cell development, homeostatic strain, or a mixture of both processes. Moreover, the possibility exists that CD4 T cell immune activation, resulting in persistent rounds of cellular proliferation and death, could contribute to great variability exhibited by RasGRPF” CD4 T cell TCR repertoire. Collectively, these studies raise the prospect that changes in the TCR repertoire in RasGRP1 mice could be responsible for the impaired generation of pathogenspecific T cells.  Failure ofRasGRPT’ mice to generate pathogen-specific T cells is the result ofRasGRPJ loss in thymocytes and/or T cells Because the engineered mutation in RasGRP1 results in a systemic loss of RasGRP1 function (14), it is possible that RasGRP1 deficiency in another cell type, besides T cells, may contribute to defective pathogen-specific responses. To investigate whether the innate immune system within RasGRPF’ mice is capable of nurturing T cell responses, mutant mice (Ly5 .2k) were infused with purified wild-type T cells (Ly5.1) and infected the next day  106  with either rLM-SIY or LCMV (Fig. 3.8A). Seven days post-rLM-SIY infection, a sizable proportion (7.1%) of the wild-type CD4 T cells were LLO reactive whereas few RasGRP1’ CD4 T cells produced IFN-y upon peptide stimulation (Fig. 3.8B). Interestingly, RasGRPf’ CD8 T cells (Ly5T) mounted a weaker anti-STY response in the presence of wild-type T cells (Figs. 3.8B and 3.5B), implying that they may not compete well for Ag. These findings suggest that the innate immune system in RasGRP 1  mice is not to blame for the impaired  generation of rLM-specific T cells.  To determine whether the wild-type T cells could also respond in mutant mice after viral infection, we performed an analogous wild-type T cell infusion experiment except this time we infected with LCMV. Consistent with our rLM-SIY observations, wild-type CD4 and CD8 T cells generate strong anti-LCMV responses toward immunodominant viral peptides (Fig. 3.8C). Interestingly, RasGRPF’ mice seem to mount a better IFN-y response to the NP396 peptide in the presence of wild-type donor T cells (12 vs 0.8%; Figs. 3.8C and 3.6B). A plausible explanation for this observation is that wild-type CD4 T cells help the mutant CD8 T cell response by producing cytokines or providing costimulation. Notably, we have previously shown that the addition of the cytokine IL-2 can restore Ag-driven proliferation by RasGRPF’ 2C CD8 T cells in vitro (16). These experiments argue that poor T cell responses observed in RasGRP1 mice are the consequence ofRasGRPl deficiency in thymocytes and/or T cells.  The finding that the “empty” lymphoid compartment in RasGRPF’ mice can induce spontaneous T cell expansion suggests the possibility that it could initiate the differentiation of donor T cells into pathogen-specific T cell effectors without infection. To explore the likelihood of this possibility, we compared cohorts ofRasGRP1 mice receiving wild-type T cell infusions that were left untreated with those infected with LCMV. Nine days postdonor T cell infusion, mice that were left untreated did not exhibit significant numbers of viral specific T cells (our unpublished observations) and the donor cell recovery was substantially lower than those infected with LCMV (3.4-  ±  0.7-fold-decrease for CD4 T cells; 7.3-  ±  1.2-  fold-decrease for CD8 T cells). These experiments demonstrate that the adoptive transfer of  107  wild-type T cells into RasGRP 1’ mice does not induce their spontaneous differentiation into anti-LCMV T cell effectors.  3.4  Discussion  The prime directives of thymocyte development are to generate a TCR repertoire that is selfrestricted, self-tolerant, and diverse, enabling responses toward a vast array of foreign peptides associated self-MHC molecules. Because the generation of the TCR repertoire is dependent on TCR signaling, mutations affecting signaling molecules downstream of the TCR may have deleterious effects on both T cell function and TCR repertoire. In this study, we report the consequences of RasGRP1 deficiency and reduced TCR-induced Ras signaling on peripheral T cell homeostasis and T cell immunity.  A recently described mouse strain called  suffers from massive  lymphoproliferation and an autoimmune syndrome sharing similarities with SLE (18). Although young mice appeared normal, older  mice developed massive  lymphoproliferation, displaying splenomegaly and lymphadenopathy, with an excess of 10fold larger lymph node size and cell numbers as compared with age-matched controls (18). By 5—8 mo of age, RasGRP 1 lag mice were found to be so anorexic and lethargic that it necessitated euthanasia (18). Although we observed RasGRP1’ mice that developed substantial splenomegaly and lymphadenopathy (our unpublished observations), the penetrance of this phenotype disappeared after successive backcrossing of the targeted RasGRP1 mutation to the B6 background. Our B6 backcrossed RasGRPF mice remain T cell lymphopenic and appear healthy until at least 1 year of age. However, despite the absence of massive lymphoproliferation, these RasGRP i mice do possess elevated levels of serum autoantibodies (our unpublished observations). Because autoimmune disease often requires a complex mixture of genetics and environmental factors (25, 26), it is perhaps not surprising that a change in genetic background may be responsible for the contradictions between our findings and those previously reported (18). Moreover, a recent study has found that SLE can simply develop from a hybrid 129:B6 background rather than targeted gene  108  disruption (27). Therefore, we suspect that genetic modifiers from the 1 29/SvJ mouse strain may synergize with RasGRP1 deficiency to cause massive lymphoproliferation and exacerbate autoimmune disease.  It has been proposed that RasGRP 1-deficient thymocytes capable of maturing into SP thymocytes need to express more strongly self-reactive TCRs to overcome their signaling deficits (16-18). Because central tolerance does not appear to be affected by RasGRP 1 deficiency (16), it has led us to hypothesize that self-reactivity of TCRs mediating positive selection of RasGRPF’ double-positive thymocytes (DP) must bridge the boundary between positive and negative selection. The question of why immune activation selectively affects the CD4 T cell lineage in RasGRPF’ mice is unknown. Because CD4 T cell development has been proposed to be more highly dependent on RasGRP1/ERK signaling (17, 28), RasGRP 1 deficiency may affect thymic ontogeny by selecting more strongly self-reactive CD4 than CD8 SP thymocytes. Alternatively, RasGRP 1-dependent mechanisms preserving peripheral tolerance or a relentless homeostatic strain may preferentially induce the activation of RasGRP i CD4 T cells. However, because RasGRP i T cells are severely hyporesponsive as compared with wild-type cells (16), their autoimmune potential may be counterbalanced by their inefficient TCR signaling and reduced proliferation upon Ag encounter.  Homeostatic mechanisms that function to regulate peripheral T cell numbers may be basis for the association between autoimmunity and T cell lymphopenia observed in both animals and humans (4, 25). Our studies of the homeostatic mechanisms operating in RasGRPF’ mice suggest that both self- and foreign-Ags could be driving T cell proliferation (Fig. 3.3). However, because the RasGRP1 mutation was made in 129/SvJ embryonic stem cells (14), it is plausible that 129/SvJ-derived alloantigens may be responsible for some donor wild-type B6 T cell proliferation observed after their transfer into RasGRP  mice (Fig. 3.3). Although  the RasGRPF’ mice used in this study have been bred at least seven generations onto the B6 background, this mouse line may still contain a significant amount of 129/SvJ DNA that is likely closely linked to the targeted locus. To identify the forces driving the cell cycling of RasGRP 1 CD4 T cells in vivo (Fig. 3.1 K), we initiated a series of adoptive transfer  109  experiments using RasGRPF’ donor T cells. Notably, it had been hypothesized that RasGRP1 deficiency allows autoreactive T cells to escape the thymus, proliferate upon encounter with peripheral self-Ags, and initiate autoimmunity (18). However, the failure of the majority of RasGRPF’ CD4 T cells to proliferate after placement in wild-type hosts, either unmanipulated recipients or ones made lymphopenic through irradiation, suggests that self-Ags may not be responsible for their expansion (Fig. 3.4). By contrast, the observation that a subset ofRasGRPF’ CD4 T cells can undergo massive expansion in RAG-deficient hosts suggests that foreign Ags could be stimulating CD4 T cell proliferation in RasGRPF’ animals (Fig. 3.4). Moreover, foreign Ags may be ideally suited to provoke weakly responsive RasGRP 1-deficient CD4 T cells to proliferate vigorously because they can simultaneously act as a direct TCR stimulus, an activator of APCs and an inducer of inflammatory cytokine production. Therefore, these studies demonstrate that the environment within irradiated wild-type mice, possessing increased availability to both self-Ags and cytokines, is insufficient to recruit most RasGRP 1  CD4 T cells into cell cycle.  The observation that RasGRPF’ mice exhibit diminished T cell responses and delayed pathogen clearance suggests that they could be prone to developing chronic infections. Notably, RasGRP1 CD4 T cells share some attributes with functionally exhausted memory T cells found in both mice and humans during chronic infections (8, 9). First, RasGRP1 CD4 T cells possess markers of acute activation and T cell memory like exhausted T cells (Fig. 3.1 C). Second, RasGRP 1 .1- CD4 T cells seem to have limited self-renewal capacity because they expand poorly in irradiated wild-type recipients, a cellular environment where the availability of the common ‘-chain-linked cytokines IL-7 and IL-15 is increased (Fig. 3.4). RasGRP1 4 CD4 T cells also have reduced IL-7R expression (Fig. 3.1C). Third, RasGRP 1 CD4 T cells strongly express PD-i, an inhibitory receptor that is coupled with exhausted viral-specific CD8 T cells in both mice and humans (11, 12), as well as its ligand PD-Li (Fig. 3.1D). In contrast to the CD4 T cell phenotype, RasGRPF’ CD8 T cells do not possess an exhausted phenotype because they do not exhibit signs of acute activation (CD69b0w, CD62Lhigh CDi27; Fig. 3.iC), fail to express elevated levels of PD-i (Fig.  3.iE) and can mount a significant anti-SlY T cell response (Fig. 3.5, B—D). A complication of housing chronically activated CD4 T cells is that it could promote autoimmunity in  110  RasGRPF mice, perhaps through elevated FasL expression, inducing nonspecific cell death, or proinflammatory TNF-a production (Figs. 3.1C and 3.4C). Therefore, T cell immunodeficiency could predispose RasGRPF’ mice to both chronic infections and autoimmunity.  The failure ofRasGRPF’ mice to generate normal numbers of Ag-specific CD4 and CD8 T cells after bacterial and viral infection likely results from a mixture of direct (T cell hyporesponsiveness) and indirect (altered thymic development and T cell homeostasis) influences of RasGRP1 deficiency. Decreased T cell responsiveness would be predicted to reduce the probability that a given T cell undergoes Ag-induced developmental programming whereas changes to T cell development and peripheral T cell homeostasis could alter the TCR repertoire and T cell differentiation. Notably, studies on RasGPRF’ 2C CD8 T cells have found that RasGRP 1 regulates homeostatic proliferation (16), TCR-signaling thresholds, and augments cytokine production (J. Priatel, X. Chen, L. Zenewicz, H. Shen, J. Coughlin, J. Stone, and H. Teh. manuscript in preparation). Because the precursor frequency of Ag-specific T cells is a critical parameter for the generation of effector and memory T cells (29, 30), we assessed Vj3 TCR usage among splenic T cells from naive (uninfected) mice to look for alterations in the TCR repertoire between normal and mutant mice. Although our results demonstrate that the TCR repertoire of RasGRPl mice is significantly different, they do not provide a measure of TCR diversity. Moreover, the  hih 44  surface  phenotype of RasGRP l T cells (Fig. 3.1 C) suggests the possibility that these cells may be derived from a considerable amount of peripheral expansion, a phenomena thought to restrict the TCR repertoire because it could result from the selective outgrowth of cells expressing a given TCR. In addition, the exhausted phenotype ofRasGRPF” CD4 T cells suggests that their state of T cell differentiation could also contribute to their inaction. Therefore, reduced generation of Ag-specific T cells by RasGRP 1 mice could result from changes in T cell differentiation, T cell function, and/or T cell TCR repertoire.  The fact that numerous T cell lymphopenic animals and humans exhibit T cell activation argues that an “empty” T cell compartment and changes to T cell homeostasis play a major role in the phenotype observed in RasGRPF’ mice rather than being solely attributable to  lii  RasGRP 1 loss in peripheral T cells. Interestingly, T cell lymphopenia is also often associated with T cell hyporesponsiveness and autoimmunity (25) and as a consequence, it raises the question of what are common denominators between these phenomena. The knowledge that TCR signaling is critically important for both T cell development and T cell survival has provided insight into the pairing of these occurrences (1, 31). Moreover, mutations affecting TCR signaling may cause T cell activation by impacting central and/or peripheral tolerance (32). In addition, immunodeficiency may lead to disruptions in T cell homeostasis, development of chronic infections, persistent T cell activation, and T cell exhaustion (8, 10). Additionally, studies on two models of spontaneous autoimmune diabetes, the NOD mouse and BioBreeding diabetic rat, suggest that T cell lymphopenia may initiate disease by altering T cell homeostasis (33, 34).  In conclusion, our study highlights the dangers associated with RasGRP1 loss, including changes to T cell development, peripheral T cell homeostasis and T cell immunity. Because our findings suggest that chronic immunodeficiency promotes persistent CD4 T cell activation and constitutive proinflammatory cytokine production, they provide insight into why the conditions of T cell lymphopenia and T cell hyporesponsiveness are linked to autoimmunity in humans (25). An added complication of persistent T cell activation is that it may result in functional impairments by inducing a state of T cell exhaustion, a phenomenon commonly observed during chronic infection in mice and humans (8). Recently, PD-i, an inhibitory receptor that functions to put a brake on TCR signaling (10), has been shown to impair function of exhausted viral-specific CD8 T cells during chronic LCMV infection (ii). Because CD4 T cells in HIV-infected individuals (12) and RasGRPi mice express high levels of PD-i (Fig. 3.1 D), it leads us to speculate whether these exhausted states arise from similar forces despite the fact that CD4 T cell lymphopenia in these scenarios has disparate origins. In HIV-infected individuals, it has been hypothesized that CD4 T cell immune activation results from a combination of homeostatic strain and immunodeficiency at mucosal sites, facilitating infections by opportunistic pathogens (35, 36). Thus, it is possible that chronic immunodeficiency in RasGRPF’ mice allows for translocation of commensal microflora across intestinal epithelium, causing local or systemic infections and CD4 T cell immune activation. Further study of RasGRP 1” mice will provide additional mechanistic  112  insights into consequences of T cell immunodeficiency and contribute to understanding how such alterations predispose to autoimmune disease.  3.5  Acknowledgements  We thank Salim Dhanji and Michael Chow for helpful discussions, Soo-Jeet Teh for technical assistance, Iryna Shanina and Diane Fang for LCMV plaque titer determinations, James C. Stone for RasGRPF’ breeder mice, Ninan Abraham for providing B6.SJL-Ptprca Pep3b/BoyJ (Ly5.1) mice, Hermann J. Ziltener for providing additional B6.RAG-F mice, the Wesbrook Animal Unit for animal husbandry, and the Life Sciences Flow Cytometry Facility for cell sorting.  113  3.6  Figures  Figure 3.1 Peripheral CD4 T cells from RasGRPF’ mice display signs of activation, exhaustion, proliferation, and spontaneous apoptosis. A, RasGRPF’ animals possess a reduced frequency and numbers of peripheral CD4 and CD8 T cells. Decreased splenic CD4 and CD8 c4 TCR T cell numbers in mice lacking RasGRP1. The single (*) and double asterisk (* *) represent p values, using an unpaired, two-tailed Student’s t test, calculated at p <0.02 and p  <  0.005, respectively. B, Comparison of spleens, PLNs (brachial LN is shown),  and MLN between wild-type and age-matched RasGRPf’ mice. C, RasGRPF’ CD4 and CD8 a3 TCR T cells (bold line) possess markers of memory and acute activation. Thin line, shaded histograms indicate staining pattern of wild-type T cell counterparts. D, RasGRPF’ CD4 T cells (bold line) express elevated levels of PD-i and its ligand PD-Li as compared with wild type (thin line). Shaded histograms represent background autofluorescence of unstained cells. E, An increased frequency of RasGRP 1’ CD4 T cells exhibit expression of the proliferation-associated nuclear Ag Ki-67. F, RasGRPL CD4 T cells bind high levels of apoptotic marker armexin V.  Figure 3.2 Aberrant-positive selection in RasGRP 1-deficient mice results in a small population of SP thymocytes that bear a naive cell surface phenotype. A, Regions used for gating are indicated in the developmental profiles from wild-type and mutant mice. B, Expression of the CD5, CD44, CD62L, and CD69 differentiation markers are shown for wild-type (thin line) and RasGRPF’ (bold line) DP, CD4 SP, and CD8 SP thymocyte subpopulations. Shaded histograms represent autofluorescence of unstained thymocyte subpopulations.  Figure 3.3 The cellular environment within RasGRP 1-deficient animals promotes T cell  expansion. A, Purified wild-type T cells (Thy 1.i) were labeled with CFSE and adoptively transferred into normal C57BL/6J (B6), irradiated B6, normal B6. RasGRPF’, and B6.TCRC’ mice. Splenocytes were harvested 1-wk posttransfer and the proliferation of  114  donor CD4 and CD8 T cells measured by flow cytometry. B, Same type of experiment as in A except that contour plots are presented, displaying CD62L expression as a function of CFSE fluorescence. C, Same experiment as in A except that 2C CD8 T cells (Thy 1.1) were adoptively transferred into the indicated host animals and donor T cell proliferation tracked +  +  +  by gatmg on Thyl.1 CD8 2C TCR cells.  Figure 3.4 RasGRP1 T cells expand vigorously after transfer into chronically immunodeficient RAG-F’ hosts. A, CFSE-labeled RasGRPF’ T cells (Thy l.2) were adoptively transferred into normal B6 (Thy l.1), irradiated B6 (Thy 1.1), or B6.RAG-F’ host animals. After residing within recipient animals for 1 wk, donor T cell proliferation was measured by staining splenocytes with specific Abs to Thyl .2, CD4, CD8, and TCRI3 and electronic gating. B, Same type of experiment as in A except that the relationship between cellular proliferation and CD62L expression is presented as contour plots. C, RasGRPF’ I cells were recovered from either B6.RAG- l or irradiated wild-type B6 recipients after 1 -wk residence, stimulated on anti-TCR Ab-coated plates, and assessed for cytokine production.  Figure 3.5 RasGRPI’ mice mount a poor MHC class IT-restricted LLO response upon infection with L. monocytogenes. Wild-type and RasGRPF’ animals were infected i.v. with rLM-STY. Spleens of infected mice were harvested 7 days postinfection and assayed for immune responses toward the immunodominant MHC class Il-restricted peptide LL0 201 _ 190 (A) or the MHC class I-restricted peptide SlY (B) by measuring IFN-y production using intracellular flow cytometry. Numbers within the plot reflects the frequency of CD8 or CD4 T cells responding to a particular condition. C, RasGRPF’ mice possess reduced numbers of anti-LLO-reactive CD4 T cells. Error bars, SD. D, RasGRP1 CD8 T cell effectors display potent cytotoxic activity. Various numbers of splenic CD8 T cells from wild-type and mutant mice were incubated with a 51 Cr-labeled EL-4 target.  Figure 3.6 RasGRPF’ animals generate a weak T cell response toward LCMV and exhibit delayed viral clearance. Wild-type and mutant mice were infected i.p. with the LCMV. At  115  day 8 postinfection, splenocytes were cultured in either medium alone (no peptide), stimulated with the indicated immunodominant viral peptide or placed in a well coated with anti-TCR Ab (aTCR). Anti-LCMV T cells were enumerated by IFN-y production by gating on either CD4 (A) or CD8 (B) T cells. Numbers within the plot reflects the frequency of CD8 or CD4 I cells responding to a particular condition. C, RasGRPF’ animals possess greatly reduced numbers of Ag-specific T cells. Error bars represent the SD. D, At day 8 postinfection, LCMV can still be detected in the spleens of some RasGRP 1 animals. Dashed line indicates the approximate detection level of the assay.  Figure 3.7 Peripheral RasGRP1 T cells possess an altered TCR repertoire. The TCR V(3 usage of wild-type (n = 5) and mutant T cells (n  7) was analyzed by using a panel of anti  TCR V Abs, flow cytometry and electronic gating on either CD4 TCR13 (A) or CD8 TCR(3 cells (B). To determine the significant differences observed between the two sets of animals, a two-tailed Student’s t test (two-sample, unequal variance; heteroscedastic) was performed. The single asterisk (*) and double asterisk (**) represent values of p < 0.02 and p <0.005, respectively.  Figure 3.8 Failure of RasGRPI’ mice to generate pathogen-specific T cells is the result of RasGRP1 loss in thymocytes and/or T cells. A, RasGRPF’ mice (Ly 5.2k) were infused with l 0 million wild-type (Ly 5. 1) T cells and infected 1 day later with either rLM-SIY or LCMV. Splenocytes were recovered from animals after infection with either rLM-SIY (B) or LCMV (C), stimulated with the indicated peptides in vitro, and IFN-y production measured by flow cytometry. Data were electronically gated on either CD4 or CD8 and the frequency of responding wild-type (Ly5 .1k; right) or RasGRP 1  T cells (Ly5.1 ; left) is indicated  within the density plot.  116  A  +1+  -I-  A  16  ** —  SPI.61  o 1r 3i LN  ML. ::  8  HEI7 26 ;I,.  4’  **  12  CD4  0  k.  CD8  ‘I)  0 C  Cb8’  PL .PLL CD4  CD8  E CD4  10.8  PD-i  —PD-L1  +1+  -I-  j••2.  1.0  2,3  1.3  0g  >  123  21 -4  CD8  C CD4  CD J 8 H —CD44-—* —CD69  —  4  ..  JA\J  F  —CDi27—--  CD4 CD8  /\JZ:7 .  -..J —CD62L  —  —  FasL—*  ArrnexinV  Figure 3.1 Peripheral CD4 T cells from RasGRPl mice display signs of activation, exhaustion, proliferation, and spontaneous apoptosis. 117  +1+  A  -1-  CD8  B  DP  CD4 SP  CD44  CD8SP  —  / S —CD69  CD5  Figure 3.2 Aberrant-positive selection in RasGRP 1-deficient mice results in a small  population of SP thymocytes that bear a naive cell surface phenotype.  118  A  CD4 Host  5  11  -  C  CD8  Wild Type  4  Thyl.1 2CCD8 Host  7  Wild Type  Wild Type  2 j j  rTrrv.N  12  16  Wild Type 600 R  )  —TA 11T  Ras GRP1 null  21  T  1  37  7  50  F2  5  TCRcZ null  I  0  Wild Type 600 R  L  ...  52  Ii  13  Ras GRP1 i255 null  16 RAG-i null  I  11  LThfT11.I1.T  r.  cr  _J  r.  CFSE  —.  Host  B  RasGRP1 null  Wild Type 600 R -  44  2\/1  •0  CD4  C  RAG-i null  (ç14  2  84  0  /y)) $  4  -J  3  1••UV•__3  C’:,  C)  CD8  j  c)  .__-z  -  16  -  J67  /  C)  ‘Ji 4  L.  14  3  3\ hI  0  Figure 3.3 The cellular environment within RasGRP 1-deficient animals promotes T cell  expansion.  119  A  Host  CD4 4  33  Mutant 6  C  CD8 6  CD4  88  Wild Type  2.5  Mutant 0.3 21  CD8 0,8  Wild  Type 600 R 21  14 Wild  1  .1- ‘-  87  ,:. .  L  null  U  12  Type 600 R  RAG-i  ...  I  —..I  I—  RAG-I null  A.  .1  13.4.  9  0  ‘r’—’---  3.4  F  Mutant  6138  1.3  -n’*r—  5.2  0.3 IFN-y CD4  B  z F  ..  4.2  2.4  CD8 1 92  Wild Type 600 R -J C” Co  a  :;  16 10  C) RAG-i  null  ‘N”-2oAIr. /  F.  L,—))  ji)  ‘J  r-’  0  I  /  ,->c  463  CFSE  Figure 3.4 RasGRPF’ T cells expand vigorously after transfer into chronically immunodeficient RAG-l hosts.  120  A  _•P eptide A •0 06  LLO  cLTCR  ¶  (N)  +1+  112.0 r\  z  hI  -I-  o.iSJ  .  .  .  .  CD4 NoPepttde  +1 z  —1-  aTCFI  SlY  47ç,  012Q  I  I  41.1  5.55  -CD8-  io 0 C  F  0) 0  F C)  D50 40  — -I- plus SlY -o- +1+ plus SlY /  —.-  F°  0) C.  04  20  (I) °  0  LLO Sly  10 0 3.7  11 33 Ei Ratio  100  Figure 3.5 RasGRPF’ mice mount a poor MHC class IT-restricted LLO response upon infection with L. monocytogenes.  121  A A  No Peptide  C  •‘TCR  GP61  -.J0  .-  Lj --  .O 3  03  I  $  +1+ s-I  L 1  0.5  o 108  0.4  10  I  0.3 0.2  36  •  Th  •i  0.1 0  1  10  (0 0  +1+ -I  I  B +1+  No Peptide • S,O•1  0P33  GP34 19.’  ..  N•  GP276 11’  \.  N P205 2.7  N,  N P396 36  L. 1)  —‘  aTCR  48 fl  .  E  F—fl  Figure 3.6 RasGRPF’ animals generate a weak T cell response toward LCMV and exhibit delayed viral clearance.  122  A20 ÷15 cr 0 I-  —  : I Irili 2  3  4 5.1 6 78.18.39 lObli 12 13 14 17a 5.2 8.2  1 Repertoire TCR V  B  20  °15  &  **  C) io  *_ —  i :1 11 iii i 1 2  3  **  ,1IIilIIIJz1 1417a  451 6 7 8183 9 lObil 1213 5,2 8.2 TCR V Repertoire  Figure 3.7 Peripheral RasGRP1’ T cells possess an altered TCR repertoire. 123  A  0  Wild-Type Ly5.1 TceIIsAz  Mutant  (S”  / Next Day  oPe ptide 1.0 TCR 1.0 10.6 7.1 9.9 33  CD4±  H  No Poptide 0.3 2.4  ..  SlY .  CDz  L  •.  No Peotide GP33 A 0.3 1.0 Li  38  NP205 27 A  -..-..-..,.  G P34 11  9.9  CD8  uTCR 8.8.57  *4 Ly5.l  No Poptide GP61 cLTCR 1.2!0.1 6.61.5  rLM-StY or LCMV  LyS.2  B  C  NP396 57 12  :  1536  -  65  CD8Z1JAZ —.—  ——..——--  Ly5.1—-——  Figure 3.8 Failure of RasGRPF’ mice to generate pathogen-specific T cells is the result of  RasGRP1 loss in thymocytes and/or T cells. 124  3.7  References  1.  Surh, C. D., J. 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Med. 12: 13651371.  128  CHAPTER 4  RASGRP1 REGULATES THE RECRUITMENT OF NAIVE CD8 T  CELLS TO INITIATE ANTIGEN-INDUCED DEVELOPMENTAL PROGRAMMING’  4.1  Introduction  The TCR plays a fundamental role in both the development of mature T cells in the thymus and their function in the periphery (1, 2). During their ontogeny, the recognition of self peptides/self-MHC molecules (self-Ags) by the TCR of a developing thymocyte is translated into a directive for either apoptosis or survival and differentiation. After maturation and export to the periphery, the interaction of a naïve CD8 T cell’s TCR with seif-Ags promotes their survival and peripheral maintenance whereas encounter with a specific foreign Ag (foreign peptide associated with self-MHC molecules) instructs a developmental program consisting of massive clonal expansion, effector cell formation, contraction through apoptosis and differentiation into long-lived memory cells. The basis by which the TCR instructs different outcomes onto a cell is mediated through graded signaling strengths, the affinity/avidity for its ligands determining the magnitude of signaling. Tight regulation of T cell homeostasis is essential for productive immune responses necessary for the clearance of pathogen and to prevent autoimmune-mediated self-destruction (3). The molecular mechanisms that regulate the recruitment of naïve T cells into cognate Ag-induced developmental program and navigate their transition from naïve to effector to memory cells are poorly understood.  TCR stimulation induces the rapid activation of small GTPase Ras whose signals are critical for orchestrating the positive selection of developing thymocytes (4, 5). Ras functions to link membrane receptor signaling to the internal MAPK cascades, cycling between an “off’ (GDP-bound) and an “on” (GTP-bound) form. Ras guanine nucleotide exchange factors (Ras  1  A version of this chapter has been submitted for publication. Priatel, J.J., X. Chen, L.A. Zenewicz, J.J. Coughlin, H. Shen, J.C. Stone, R. Tan, and H. S. Teh. (2008) RasGRP1 regulates the recruitment of naïve CD8 T cells to initiate antigen-induced developmental programming. 129  GEFs) greatly accelerate the release of GDP, facilitating Ras to bind GTP and the assumption of the active state. Conversely, Ras activity can be shut off by either its slow intrinsic GTPase activity or by pairing with GTPase-activating proteins (Ras GAPs). In thymocytes, the regulation of Ras activity by TCR signal transduction invokes at least two Ras GEFs, Sos and RasGRP1 (6-8). The respective functions of both Ras GEFs are dependent on relocating to membranes by two distinct mechanisms. RasGRP1 is recruited to membranes by binding the phospholipase Cyl-product diacylglycerol (DAG) through its Cl domain whereas Sos is recruited to the phosphorylated adaptor molecule linker for activated T cells by way of its constitutive association with the SH2-domain-containing protein Grb2. RasGRP1 is likely the primary Ras GEF responsible for well-documented Ras activation observed following treatment of thymocytes with DAG analogs such as PMA (6, 9, 10).  The role of Ras activation in mature T cell function is less clear since transgenic mouse approaches of knocking down either Ras or its downstream MAPK cascade results in blocked thymocyte development accompanied with very few peripheral T cells and technical limitations of introducing genes into primary T cells (4). At least two distinct mechanisms have been postulated to regulate Ras activation upon TCR ligation in both human and mouse peripheral T cells (11, 12). Similar to the case of regulation of Ras in thymocytes, mature T cells are also thought to utilize the RasGRP1 and Sos for TCR-induced Ras/ERK activation (13, 14). However, the relative contribution of these two Ras GEFs to TCR-induced Ras signaling and their resulting impact on the activation and differentiation of mature CD8 T cells is unclear. Notably, defects in both Ras and ERK activation following TCR stimulation have been associated with anergized CD4 T cells and the inability to produce the T cell growth factor IL-2 (15, 16). A causal relationship between defective Ras signaling and T cell anergy has recently been demonstrated using novel techniques to transfect a constitutively active Ras into anergic T cells (17). Moreover, gain-of-function studies using the human Jurkat T cell line suggest that RasGRP1 promotes IL-2 transcription and thus, may stave off an anergic state (13).  We have previously generated two lines of TCR transgenic mice to study the role of RasGRP1 in CD8 T cell development under conditions of defined TCR signaling (9). Our  130  results found that RasGRP 1 is critical for thymocytes expressing the weakly selecting H-Y TCR whereas those that express the strongly selecting 2C TCR are much less dependent on RasGRP1 for ERK activation and positive selection (9). The relatively efficient thymic development of 2C TCR CD8 T cells in mice lacking RasGRP1 along with their naïve peripheral phenotype and near-normal cell numbers afforded us with the unique opportunity to study the role ofRasGRPl in Ag-specific T cell responses. Here, we report that RasGRP1 plays a key role in the early activation events, such as CD25 up-regulation and IL-2 secretion, following exposure to cognate Ag. RasGRP1 2C CD8 T cells exhibit greatly reduced sensitivity to TCR stimulation, failing to proliferate to either immobilized anti-TCR Abs or limiting doses of cognate Ag, and reduced burst size upon encounter with cognate Ag both in vitro or in vivo. These investigations highlight RasGRP l’s function in regulating the growth of mature T cells and suggest that RasGRP1 may prove valuable as a potential drug target, suppressing T cell responses in transplantation and autoimmune disease settings.  4.2  Materials and methods  Mice RasGRP 1-deficient mice have been created and described previously by Dower et al. (6). C57BL/6J (B6) and B6.PL-Thy]”/Cy (Thy 1.1k) were acquired from The Jackson Laboratory (Bar Harbor, ME). RasGRP1-deficient 2C TCR transgenic mice were created by crossing with gene-targeted RasGRPF’ mice that had been bred onto B6 background at least 10 generations. To generate Thyl .1/Thy1 .2 2C TCR transgenic animals, B6 mice bearing the 2C TCR transgene was mated with B6.PLThy1a/Cy mice (H2b, Thyl .1k). All studies followed guidelines set by the Animal Care Committee at the University of British Columbia in conjunction with the Canadian Council on Animal Care.  Flow cytometry Antibodies against CD3 (145-2C1 1), CD4 (GK1.5), CD8a (53-6.7), CD813 (53-5.8), TCRI3 (H57-597), CD25 (PC61.5), CD44 (1M7), CD62L (MEL-14), CD69 (H1.2F3), CD122 (TM  131  hi), CD127 (AKR34), Thyl.l (HIS51), Thyi.2 (53-2.1), HSA (Mi/69), PD-i (J43), PD-Li (MIH5), IL-2 (JES6-5H4), IL-4 (1 iBi i), IL-b (JES5-16E3), TNF-a (MP6-XT22) and IFN y (XMG1 .2) were purchased from eBioscience. The clonotypic anti-2C TCR Ab was purified from the iB2 hybridoma (from Dr. Herman Eisen, M.I.T.) and conjugated to either FITC or biotin using standard methods. For detection of intracellular cytokine, cells were stimulated in the presence of GolgiPlug (BD Biosciences; contains Brefeldin A) to block cytokine secretion as previously described (18). Anti-Ki-67 (B56) and isotype control (MOPC-21) Abs were purchased from BD Biosciences and the staining protocol employed was as published (18). Data was acquired with either a BD LSRII benchtop cytometer using FACSDiVa software (BD Biosciences) or FACSCalibur and CELLQUEST software (BD Biosciences). Data was analyzed with either CELLQUEST or FlowJo (Tree Star, Inc) software.  Purification and activation of2C CD8 T cells CD8 T cells were purified from spleens and lymph nodes using indirect magnetic bead-based separation according to manufacturer’s instructions (MACS, Miltenyi Biotec). Briefly, CD8 T cells were labeled with anti-CD83 (53-5.8) Ab coupled to biotin, followed by incubation with Streptavidin-microbeads (Miltenyi Biotec) and column purification. For plate-bound anti-TCR Ab stimulations, tissue culture wells were coated with either 10 ig/ml of anti CD3E (145-2Ci 1) Ab alone or 10 tg/ml of anti-CD3s plus 5 tg/ml of anti-CD28 (37.51) mAbs. For cognate Ag stimulations, 2C CD8 T cells were incubated with irradiated B6 splenocytes that had been pulsed with 0.1 pg/ml of SlY peptide.  2C CD8 T cell stimulation using limiting doses ofcognate Ag CFSE-labeled wild type and RasGRPF’ 2C CD8 T cells (50,000; Thyl .2k) were cultured in vitro for 4 days with one million congenic CD8-depleted splenocytes (feeder; Thy 1 .1k) and limiting numbers of SlY-coated APCs in flat-bottom 96-well plates. For preparation of SlY coated APCs, CD8-depleted splenocytes from congenic (H2b; Thyi.i) mice were incubated with 0.1 ig/ml SlY peptide for 1 h at RT and subsequently, washed 3 times with PBS. For assessment of IFN-y production, one million STY-loaded splenocytes (Thy 1.1k) and the 132  golgi-transport inhibitor Golgi-Plug (BD Biosciences) were added to cultures 4 days poststimulation for a 5 h period. Samples were stained and electronically gated on 2C CD8 (Thy 1 .2 CD8) cells to assess proliferation and markers of differentiation. All wild type and RasGRP1 -I- Thyl .2 + CD8 + T cells expressed high levels of the 2C TCR as measured by anti clonotypic 2C TCR mAb (1B2; data not shown).  Cell signaling studies and immunoblotting 2C CD8 T cells were purified by labeling pooled splenic/lymph node single cell suspensions with biotinylated anti-CD8f3 (53-5.8) Ab, incubated with streptavidin microbeads (Miltenyi Biotec) and subjected to magnetic separation. For TCR stimulations, CD8 T cells (10 /ml) 7 were prewarmed at 37 °C for at least 15 mm in serum-free media and subsequently, treated with 10 ig/ml of anti-CD3E Ab (145-2C11). Antibodies used to detect phosphorylated forms of ERK, LAT and ZAP-70 and total amounts of ERK2 have been described previously (19). For IL-2 signaling, T cell blasts were generated by activating purified 2C CD8 T cells for 2 d with 0.1 tg/ml SlY peptide plus irradiated splenocytes for APCs and subsequently, expanding them for 3-4 d with 20 U/ml IL-2. T cell blasts were serum-starved for 4 h prior to stimulation with either IL-2 (100 U/ml) or PMA (100 tM). At the indicated time, cells were rapidly precipitated by centrifugation, washed with ice-cold PBS and lysed in 10 mlvi Tris (pH 7.5), 150 mM NaC1, 1% NP4O, 0.1% SDS, protease inhibitors and phosphatase inhibitors. Ras pulldown assays using Raf-GST fusion protein bound to glutathione-coupled beads were performed as described elsewhere (6, 13). A mixture of a pan-Ras (#R02120, Transduction Labs) and anti-K-ras (#3 0, Santa Cruz Biotec) Abs were used to measure Ras in both GST-Raf precipitates and total lysates. Blots were developed using ECL system (Amersham).  Generation ofa recombinant strain ofListeria monocytogenes expressing the 2C agonist peptide SlY A recombinant Listeria monocytogenes (rLM-SIY) was engineered to express the SlY peptide within a secreted DHFR fusion protein. The Ag cassette containing the SIY-peptide with flanking OVA and a-KG amino acid residues has been previously shown to induce  133  strong anti-SlY responses in B6 mice (20). After PCR amplification and cloning of the Ag cassette into the suicide vector pJJD 180, the construct was introduced into the bacterial genome by homologous recombination as described previously (21, 22, 23).  Adoptive T cell transfers and bacterial infection experiments To assess the capacity of rLM-SIY to induce 2C TCR CD8 T cell proliferation, one million purified wild type (Thy 1 .2/1 .2) 2C TCR CD8 T cells were labeled with CFSE and adoptively transferred into congenic (Thy 1.1/1.1) B6 mice via intravenous injection. Twenty-four h later, mice were either left untreated, infected with 10,000 CFU wild type EM (strain 10403S) or infected with 10,000 CFU rLM-SIY. One-week post-infection, donor 2C CD8 T cells were identified by staining with anti-Thy 1.2 and anti-CD8 Abs and their proliferation measured by flow cytometry using a BD FACSCalibur or BD LSRJI benchtop cytometer using CeliQuest and FACSDiVa software respectively. For co-adoptive transfers experiments, a 50:50 mixture (10,000 of each genotype) of wild type (Thyl .1/l.2) and RasGRPF’ (Thy1.2/1.2) 2C CD8 T cells were infused into syngenic (Thy1.1/1.1) B6 mice and infected the next day by intravenous injection using a dose of either 1,000 or 10,000 CFU of rEM-STY in PBS. Bacterial doses were verified by plating the injectant on brain-heart infusion agar (BD Biosciences).  4.3  Results  RasGRP] 2C CD8 T cells exhibit defects in early activation events and IL-2 production To investigate the role of RasGRP1 in peripheral CD8 T cell function, we analyzed CD8 T cells from RasGRP 1-deficient mice expressing the MHC class I-restricted 2C TCR transgene (24). For the study of cognate Ag-driven CD8 T cell responses, the 2C TCR recognizes the agonist peptide SIYRYYGL (SlY) bound to syngeneic H-2K” MHC class I molecules with high affinity (25). Previously, we have observed that CD8 SP thymocytes were present at similar numbers in wild type and RasGRPF’ 2C TCR thymi demonstrating that RasGRP1 is not critical for the development of mature cells by this TCR (9). Although peripheral  134  RasGRP1 2C CD8 T cells are modestly reduced (about 50 %) relative to wild type, they possess a mature and naïve cell surface phenotype as determined by expression levels of 2C TCR, PD-i, PD-Li, CD3, CD24 (HSA), CD8a, CD83, CD25, CD44, CD62L, CD69, CD122 and CD 127 [(9), and data not shown]. To investigate for impairments in TCR signaling, we tested the ability of wild type and RasGRP1 2C CD8 T cells to proliferate when cultured in wells coated with anti-TCR Ab. Proliferation was tracked 3 d post-stimulation by either analyzing dilution of CFSE-endowed fluorescence by flow cytometry (Fig. 4. 1A) or H]-thymidine during a 6 h pulse period 3-days post-stimulation 3 measuring incorporated [ (Fig. 4.1B). By contrast to the SIY/APC response, RasGRPF’ 2C CD8 T cells failed to even enter cell cycle when cultured immobilized anti-TCR Ab alone. Furthermore, complementing TCR signals with anti-CD28 Ab-mediated costimulation also failed to induce significant proliferation by RasGRP1-deficient 2C CD8 T cells (Fig. 4.iA, 4.1B). However, the proliferative responses by RasGRPF’ 2C CD8 T cells resembled wild type T cells when either plate-bound anti-TCR Ab or anti-TCR plus anti-CD28 Abs stimulations were supplemented with exogenous IL-2. These data demonstrate RasGRP1-deficient 2C CD8 T cells display an anergic phenotype that is reversible upon the addition of exogenous IL-2.  The findings made with RasGRPF’ 2C CD8 T cells using immobilized anti-TCR Ab as an agonist argue that RasGRP1 is important for early T cell activation and recruitment into cell cycle (Fig. 4. 1A, 4. 1B). However, our previous experiments, using soluble SlY peptide presented by syngeneic splenocytes to stimulate RasGRPi 2C CD8 T cells, had suggested early activation and proliferation were RasGRP 1-independent events (9). It is possible that the apparent discrepancy between these observations may be a consequence of the fact that APCs deliver additional signals, such as coreceptor engagement, costimulatory molecule interactions and cytokines, that anti-TCR plus anti-CD28 Abs alone do not provide. To investigate the respective role of RasGRP 1 in mediating early T cell activation events and whether this role differs depending on the type of stimulation, wild type and RasGRPF’ 2C CD8 T cells were stimulated with either SlY-pulsed splenocytes or anti-TCR Ab for 16 hours and subsequently, assessed for the induction of activation markers (Fig. 4.1 C). A comparison of histograms and mean fluorescence intensities (MFIs) revealed that RasGRPF’ 2C CD8 T cells exhibited impaired up-regulation of the activation markers as compared to wild type  135  (CD25: 201 vs. 1280, 6.4x lower MFI; CD69: 295 vs. 776, 2.6x lower MFI; and PD-i: 295 vs. 630, 2.2 x lower MFI). By contrast, RasGRPF’ 2C CD8 T cells exhibited a more normal activation marker expression profile when cells were treated with peptide/APC (CD25: 1182 vs. 1748, 1.5x lower MFI; CD69: 642 vs. 893, 1.4x lower MFI; PD-i: 370 vs. 464, i.3x lower MFI). These findings argue that Ag receptor-induced expression of activation markers is heavily dependent on RasGRP1 when costimulatory or supplementary signals are limiting, such as the case with plate-bound anti-TCR Ab stimulation alone. Next, we sought to determine whether RasGRP 1 2C T cells show defects in the induction of IL-2 since its production by T cells is an early event upon encounter with cognate Ag and exogenously added cytokine rescues mutant T cell proliferation (Fig. 4.1D). Wild type and RasGRPF’ 2C T cells were stimulated with STY-pulsed splenocytes and monitored hourly for IL-2 expression by intracellular flow cytometry. At each time point post-stimulation, RasGRP1 2C T cells displayed a significant reduction in the proportion of IL-2-positive cells. This finding implies that RasGRP 1 affects T cell growth through the regulation of IL-2 transcription.  Addition ofexogenous IL-2 can support the differentiation ofnaïve RasGRPJ-deJicient CD8 T cells into effector CTLs Previously, we had observed that RasGRP 1-deficient 2C CD8 T cells undergo early proliferation following exposure to cognate Ag, soluble STY peptide and irradiated syngeneic splenocytes, but failed to sustain their growth (9). To investigate whether the impaired Agdriven proliferation and developmental programming of RasGRPF’ 2C CD8 T cells may be related to their decreased capacity to produce IL-2, we compared the differentiation status of wild type and mutant naïve 2C CD8 T cells stimulated with cognate antigen in vitro either in the absence or presence of exogenous IL-2 (Fig. 4.2A, 4.2B). After four days of culture with SlY-pulsed irradiated syngeneic splenocytes alone, wild type 2C CD8 T cells underwent considerable remodeling of their cells surface, elevating CD25, CD44 and the cytotoxic effector molecule Granzyme B while simultaneously down-regulating CD62L (Fig. 4.2A). Although RasGRP i-deficient 2C CD8 T cells lost CD62L expression, they exhibited markedly reduced expression of activation and differentiation markers relative to wild type (CD25: 91 vs. 301, 3.3x lower MFT; CD44: 450 vs. 1646, 3.6x lower MFI; Granzyme B: 138 136  vs. 294, 2.lx lower MFI). Next, we tested whether culturing in the presence of both SlYpulsed irradiated syngeneic splenocytes and exogenous IL-2 could rescue the differentiation of RasGRP1 CD8 T cells (Fig. 4.2B). Wild type and RasGRPF’ CD8 T cell effector profiles exhibited much less pronounced differences when exogenous IL-2 is added to cultures (CD25: 354 vs. 777, 2.2x lower MFI; CD44: 1431 vs. 1715, l.2x lower MFI; Granzyme B: 384 vs. 392, similar MFI). Therefore, the differentiation of RasGRPF’ CD8 T cells, as measured by surface markers and Granzyme B expression, can largely be restored by supplementation of medium with IL-2. Collectively, these experiments demonstrate that RasGRP 1 plays a critical role in the efficient generation of CTL effectors and that its function can be compensated for by the addition of exogenous IL-2.  The stimulation of naïve T cells with cognate Ag results in their commitment to a developmental program linked with the production of effector cytokines such as IFN-y (26). To determine whether signaling by RasGRP1 contributes to cytokine production elicited upon TCR engagement, CD8 T cell blasts, generated by culture with cognate Ag and exogenous IL-2 (Fig. 4.2B), were stimulated with STY-pulsed splenocytes in vitro for 5 h. Regardless of genotype, the majority (‘—‘99 %) of CD8 T cell effectors produce IFN-y after contact with cognate Ag (Fig. 4.2C). CD8 T cell effectors can be subdivided into IFN-y producing (Tcl) and IL-4-producing (Tc2) populations (27). As shown for naive RasGRPF’ 2C T cells, RasGRP 1  “  T cell blasts secrete less IL-2 than their wild-type counterparts. To  investigate if RasGRP1 -deficiency affects cytokine polarization of CD8 T cells, we also assessed the production of the Tc2 cytokines IL-4 and IL-lO (Fig. 4.2C). These results demonstrate that RasGRP1 CD8 T cell effectors expanded by cognate Ag and exogenous IL-2 produce IFN-y and suggest that RasGRP1 function does not appear to influence Tcl versus Tc2 polarization.  RasGRP 1 regulates the antigen dose necessary to recruit naïve T cells to initiate proliferation and effector cell formation. The poor responsiveness to immobilized anti-TCR antibodies and decreased capacity to synthesize IL-2 suggest that RasGRP1 2C T cells may be less sensitive to cognate Ag stimulation. To test this hypothesis, a constant number of CF SE labeled wild type and RasGRP1 2C T cells were cultured with limiting numbers of STY  137  pulsed APCs (Fig. 4.3A). After four days of culture, 150,000 total events were collected and CFSE fluorescence of 2C T cells plotted on histograms with cell numbers’ scale indicated on the y-axis. As has been observed previously in an analogous experiment with P14 TCR CD8 T cells (28), increasing the number of cognate Ag-loaded APCs resulted in an increased frequency of TCR transgenic CD8 T cells recruited into cell cycle and correlated with an increased burst size (larger scale on y-axis). RasGRPF’ 2C T cells were almost 10-fold less sensitive to cognate Ag relative to wild type since 2,000 SlY-loaded APCs were required to recruit greater than 40 % of cells into cycle while only 200 SIY-APCs were necessary for wild type T cells to reach this level of recruitment. In addition, recruited RasGRP 1  2C T  cells retained more CFSE-endowed fluorescence suggesting these cells underwent fewer divisions than wild type (Fig. 4.3A). These findings imply that RasGRP1 strongly influences activation threshold of naïve CD8 T cells.  We next examined the relationship between the T cell differentiation marker CD44 and cell division in wild type and mutant 2C T cells stimulated with the indicated doses of SlYcoated APCs (Fig. 4.3B). Regardless of cell genotype, Ag-driven proliferation was accompanied with elevated levels of CD44. However, RasGRP1 2C T cells had fewer CD44hi cells particularly at lower doses of cognate antigen. To detennine whether CD44 upregulation correlated with effector function in mutant T cells, cultures were prepared as above (Fig. 4.3A, 4.3B), stimulated with one million SlY-coated splenocytes for additional 5 h and assessed for IFN-’’ production (Fig. 4.3C). Again, a reduced fraction of RasGRP1 2C T cells were capable of secreting IFN-y since a meager number of cells were mobilized to proliferate and differentiate when cognate antigen was limiting. Collectively, these findings argue that RasGRP1 regulates the recruitment of naïve CD8 T cells to initiate Ag-induced developmental programming.  RasGRP1-- 2C CD8 T cells display transient ERK activation upon TCR ligation but normal ERKphosphorylation following IL-2 stimulation Prior studies have shown that thymocytes lacking a functional RasGRP1 protein exhibit a greatly diminished capacity to activate ERK upon treatment with either DAG analogs (6, 9,  138  10) or anti-TCR antibodies (6, 10, 19). To examine the consequences of RasGRP 1-deficiency on the ability of mature peripheral T cells to activate ERK, 2C CD8 T cells were purified using magnetic separation from wild type and mutant mice and stimulated with anti-TCR antibody for the indicated times (Fig. 4.4A). By contrast to the hypo-responsiveness of RasGRPF’ immature thymocytes (6, 19), RasGRP1-deficient 2C CD8 T cells strongly phosphorylate ERK at an early time (3 mm) post-stimulation at levels resembling wild type (Fig. 4.4A). However, ERJ( activation in mutant 2C CD8 T cells is shorter in duration, exhibiting significant deficits at 30 and 60 mill post TCR stimulation. Analysis of the upstream signaling molecules argues that LAT and ZAP-70 phosphorylation are not substantially affected by RasGRP 1-deficiency. These findings suggest that RasGRP1 may be critical for sustaining the kinetics of ERK activation following cognate antigen stimulation and that such prolonged signaling may be necessary for Ag-stimulated T cells to enter cell cycle.  Activation of the Ras/MAPK cascade in T cells not only accompanies Ag receptor ligation but is also invoked by IL-2 receptor signaling (29, 30). To examine whether RasGRP1 plays a role in IL-2-mediated Ras signaling, activated wild type and RasGRPl 2C CD8 T cell blasts were serum-starved for 4 h prior to incubation with IL-2 for 10 mm (Fig. 4.4B). Assessment of the activation status of Ras and ERK post-IL-2 stimulation revealed that the Ras/ERK cascade seemed largely intact in RasGRP 1  ‘  cells despite the observation that they  exhibited a 2-fold reduction in levels of the IL-2 receptor a chain CD25 (Fig. 4.4B; Fig. 4.2B). By contrast, RasGRPP’ 2C T cell blasts displayed very weak Ras activation upon PMA treatment relative to wild type (Fig. 4.4B). These findings suggest that activation of Ras initiated by IL-2 receptor signaling does not directly depend on RasGRP1.  Recombinant Listeria monocytogenes expressing the SlY epitope induces wild type 2C T cells to prolferate in vivo To study the consequences of RasGRP 1-deficiency on Ag-driven proliferation, differentiation and memory formation of 2C CD8 T cells in vivo, we engineered Listeria monocytogenes, a well-characterized pathogen model for eliciting T cell responses (31, 32), to express the 2C agonist STY peptide (Fig. 4.5A). The recombinant strain (called rLM-SIY), 139  derived by integration of an Ag cassette into the bacterial chromosome between the lecithinase and lactate dehydrogenase operons using techniques described previously (21-23), expresses the SlY peptide as a secreted DHFR fusion under the control of a virulence promoter (Phly). To test the capacity of rLM-SIY to induce 2C T cell proliferation, one million CFSE-labeled wild type 2C T cells were infused to B6 mice (non-irradiated; Th1.1) and left untreated (naïve) or infected with 10,000 CFU of either rLM-SIY or wild type LM 10403S (Fig. 4.5B). One-week post-infection, the vast majority of splenic 2C CD8 T cells residing in either naïve mice or those infected with wild type LM failed to proliferate (95 % and 90 % respectively). By contrast, analysis of donor 2C CD8 T cells in rLM-SIY-infected mice revealed that these cells had strongly proliferated, virtually completely losing their CFSE-endowed fluorescence. In addition, donor 2C T cell proliferation induced by infection with rLM-SIY was accompanied by a greater than 100-fold increased recovery of donor 2C T cells relative to either naïve B6 hosts or those infected with wild type LM (data not shown). These findings demonstrate that rLM-SIY can effectively prime and induce the expansion of naïve wild type 2C T cells in vivo.  RasGRP]-deficient T cells exhibit a reduced rate ofproliferation and diminished cytokine production upon cognate antigen stimulation in vivo Naïve T cells encountering cognate Ag and the appropriate costimulatory signals initiate a development program, undergoing massive clonal expansion (26, 28). To test the proliferative capacity and function of RasGRPF’ 2C CD8 T cells in vivo, equivalent numbers of CFSE-labeled wild type (Thy1.1/1.2) and mutant (Thy1.2/1.2) 2C CD8 T cells were infused into the same mouse, ensuring cells of each genotype were exposed to a similar environment and an equivalent amount of cognate Ag, and recipients infected the next day with 1,000 CFU of rLM-SIY (Fig. 4.5C). Since recent experiments have shown that naïve T cell precursor frequency profoundly affects the differentiation program taken by CD4 and CD8 T cells (33, 34), we injected greatly reduced numbers of 2C T cells (10,000/genotype) per mouse in comparison to the experiments presented in Figure 5B to more accurately resemble physiological levels of naïve T cells specific for a given Ag (35, 36). Using anti-Thy 1.2 Ab to identify donor cells and anti-Thy 1.1 Ab to discriminate genotype, RasGRPF’ 2C CD8 T cells were found to be represented at a 4-fold decreased 140  proportion and total numbers as compared to wild type counterparts one-week post-infection (Fig. 4.5D, 4.5E). However, the majority of 2C T cells had undergone at least 7-8 rounds of proliferation regardless of genotype and therefore, lacked fluorescence bestowed by CFSE (Fig. 4.5F). Since tracking of cell division using CFSE fails to resolve additional information once the fluorescence of CF SE-labeled cells approaches the autofluorescence of unstained cells (37), expression of the proliferation-associated nuclear antigen Ki-67 was used to investigate whether decreased levels of cellular proliferation was associated with the reduced burst size by mutant CD8 T cells (Fig. 4.5G). A lower proportion of RasGRPl 2C CD8 T cells expressed the Ki-67 marker as compared to wild type (10.7  ±  2.4 % vs. 26.9 ± 5.9 %),  implying that their decreased representation is a consequence of slower cell cycling rates by this population of cells. These in vivo findings suggest that RasGRP1 is important in regulating the burst size of CD8 T cells responding towards cognate Ag. The differentiation of naïve T cells resulting from Ag-driven clonal expansion is tightly linked with phenotypic and functional changes. This transformation includes increased expression of effector/memory markers, like CD44, and the acquisition of effector functions such as the capacity to produce the pro-inflammatory cytokines, like IFN-y and TNF-a, and cytotoxicity (28, 26, 38). To investigate the cell surface phenotype of donor 2C T cells after cognate Ag exposure in vivo, splenocytes of host mice 7 days post-infection with rLM-SIY were stained with antibodies against various markers in addition to Thy 1.1, Thy 1.2 and CD8 (Fig. 4.5G). Analysis of donor 2C T cells (filled, black histograms) revealed that cells of either genotype had also strongly up-regulated CD44 expression as compared to naïve 2C cells (grey histograms), albeit the mutant cells expressed modestly reduced CD44 levels relative to wild type (2720 vs. 3790 MFI). To test the function of donor 2C T cell effectors, splenocytes isolated from host mice 7 days post-infection were placed in culture, stimulated for 5 h with the SlY peptide and subsequently, assessed for the production of the proinflammatory cytokines IFN-y and TNF-a (Fig. 4.51). By contrast to wild type, a significant fraction of RasGRPF’ 2C CD8 T cell effectors failed to produce cytokine (21 %) and a diminished percentage co-expressed IFN-y and TNF-ct relative to wild type (38 % vs. 83 %). However, RasGRPF’ 2C CD8 T cell effectors derived by expressed wild type levels of Granzyme B and were efficient killers of EL-4 targets pulsed with the SlY peptide (data  141  not shown). Next, we wanted to test whether the expression of RasGRP 1 influenced the capacity of 2C T cell effectors to differentiate into memory. However, to our dismay, donor 2C T cells, either wild type or mutant, completely disappeared within 2 weeks post-rLM-SIY infection (data not shown). In addition, we were unable to visualize long-lived memory 2C T cells regardless of number of 2C T cells (1 x 1 —2 x 106) adoptively transferred, the infectious dose of rLM-SIY (2 x 1  —  1 x1  CFU) and the number of events acquired (?:2  x 10). Since analogous experiments with OT-I TCR CD8 T cells and rLM-OVA infections yielded abundant numbers of long-lived memory OT-I CD8 T cells (data not shown), we believe that the inability to detect memory 2C CD8 T cells is not a technical issue. Moreover, the fact that rLM-SIY infection gives rise to long-lived anti-STY CD8 memory T cells in normal (non-TCR transgenic) B6 mice [data not shown and (39)] leads us to suspect that the inability to find long-lived memory 2C CD8 T cells following rLM-SIY infection is somehow linked to the 2C TCR.  4.4  Discussion  Understanding the molecular controls of the immune system will yield insight into the basis of immunological diseases, improve the efficacy of vaccination and lead to the identification of molecular targets suitable for therapeutic intervention. Ras signaling pathways are known to play key roles in both the development of thymocytes and the regulation of mature T cell function. At least two important Ras GEFs, RasGRP1 and Sos, are known to play critical roles in TCR signal transduction in developing thymocytes and mature T cells (5). The findings that thymocyte positive selection is relatively inefficient in the absence of RasGRP1 demonstrate that these Ras GEFs are not functionally redundant (6, 9, 10). Moreover, aberrant positive selection in RasGRP 1-deficient mice is associated with an abundance of activated- and memory-phenotype peripheral CD4 and CD8 T cells (10, 18, 40), a likely result of chronic immunodeficiency (18) and/or a breakdown in T cell tolerance (10). Therefore, studies of mature T cells in non-TCR transgenic RasGRP1 mice are tenuous since their differentiation status, a factor that influences TCR signal transduction (41), does not match wild type. Here, we report the use of RasGRP1 2C TCR mice to investigate how  142  this Ras GEF participates in peripheral T cell function and differentiation since these animals produce sufficient quantities of 2C CD8 T cells, exhibiting a mature, naïve cell surface phenotype resembling those recovered from wild type 2C TCR animals (9).  Sufficient exposure of naïve T cells to cognate Ag commits them to an Ag-independent phase of proliferation that is intimately coupled to differentiation, composing of chromatin remodeling, gene expression changes and the acquisition of effector functions (26). We have found that RasGRP 1-deficient 2C CD8 T cells require a much greater dose of cognate Ag to divide and when recruited into cell cycle undergo fewer rounds of proliferation (Fig. 4.3A). A net consequence of decreased sensitivity to TCR-stimulation is that a very low frequency of RasGRPF’ 2C CD8 T cells differentiates into effectors at limiting doses of cognate Ag. A possible mechanism by which RasGRP1 may lower activation threshold and promote the growth of naïve T cells is through its regulation of IL-2 receptor a chain (CD25) expression and IL-2 production (Fig. 4.1C, 4.1D) since it has been shown that blocking of IL-2 signaling in Ag-stimulated TCR transgenic CD8 T cells can reduce the proportion of naïve T cells recruited to proliferate and the extent to which they divide (28). In addition, we found that the addition of exogenous IL-2 can promote both the proliferation and effector cell formation of TCR-stimulated RasGRPF’ 2C CD8 T cells (Fig. 4.1, Fig. 4.2). These findings support a role of RasGRP 1 in which it impacts T cell activation and growth, at least in part, through regulation of IL-2 production.  Anergy is a peripheral tolerance mechanism in which a T cell is rendered functionally inactive following encounter with cognate Ag. The hyporesponsive state of anergic T cells is acquired and maintained through intrinsic wholesale changes in TCR signaling. Moreover, a number of characteristics displayed by RasGRP 1 2C CD8 T cells are reminiscent of T cell anergy including hypo-responsiveness to cognate Ag stimulation, decreased IL-2 production and the apparent reversibility of this phenotype by the addition of exogenous IL-2. In addition, analyses of TCR signaling pathways in anergic T cells have often revealed defects in Ras/ERK activation and AP-1-induced gene transcription (42). Gene expression profiling experiments pinpointed the overexpression of diacylglycerol kinases (DGK5), negative regulators of Ras signaling that convert DAG to phosphatidic acid, as a possible cause of  143  aberrant TCR signaling in anergic T cells (17). Supporting this notion, enforced DGK in Jurkat cells has been found to inhibit TCR-induced activation of Ras, ERK and AP-1 (43). A cause-and-effect relationship between the overexpression of DGKs and anergy was demonstrated by findings that the introduction of DGKa into mouse T cells inhibited MAPK signaling, IL-2 production and the recruitment of RasGRP1 to membranes (17). Our data is consistent with the hypothesis that DGKs suppress T cell responsiveness by blocking RasGRP1 ‘s ability to activate Ras.  Cellular proliferation may be a critical factor in the differentiation of T cells since it may provide the opportunity for chromatin remodeling and thus, facilitate changes in gene expression (44, 45). To investigate whether the decreased proliferative potential of naïve RasGRP 1 2C CD8 T cells in vitro affected their ability to differentiate into effector and memory CD8 T cells in vivo, we engineered the model pathogen Listeria monocytogenes to express the 2C TCR agonist STY peptide (Fig. 4.5A). Although RasGRPF’ 2C CD8 T cells were found to exhibit a 4-fold reduction in the generation of Day 7 effectors relative to wild type (Fig. 4.5D), it is possible that the experimental conditions seen signaling defects in these T cells may have been partially masked under the experimental conditions since the presence of wild type host CD4 T cells and their accompanying helper-associated function of IL-2 secretion may compensate for RasGRP1 loss. Moreover, decreased numbers ofRasGRPF’ 2C T cell effectors was associated with reduced rates of proliferation (Fig. 4.5G) and inflammatory cytokine effector function (Fig. 4.5]), suggesting the possibility that signaling by RasGRP 1 may be important for the formation of T cell memory. However, we were unable to test this hypothesis since we could not detect long-lived memory 2C T cells after rLM-STY infection despite the observations that these cells had been induced to undergo numerous rounds of proliferation (Fig. 4.5F), elevate CD44 expression (Fig. 4.51]) and acquire the capacity to produce proinflammatory cytokines (Fig. 4.5]). Critical parameters mediating the differentiation of naïve CD8 T cells into effector and eventually, memory T cells are thought to be the Ag dose, Ag duration and the affinity of the TCR for a given cognate Ag (26). The findings that rLM-SIY infection in normal B6 mice results in the formation of long-lived memory anti-STY CD8 T cells suggest that the 2C TCR may be somehow problematic for the development of CD8 T cell memory (39). However, a recent  144  study has demonstrated that 2C T cells can differentiate into long-lived memory T cells, persisting at least 225 days post-infection, using a recombinant influenza virus expressing the SlY peptide, named WSN-SIY (46). Perhaps it is the dose or duration of Ag that is a crucial factor for the formation of memory 2C CD8 T cells. After WSN-SIY infection, Ag is detectable for at least 8 days post-infection (Jianzhu Chen, personal communication). By contrast, rLM-SIY likely results in a contracted Ag exposure since it exhibits greatly reduced virulence (ability to divide in vivo), a trait observed for multiple strains of recombinant LM (47), as compared to the wild type strain 10403S. For example, intravenous tail injection of 10,000 CFUs of wild type LM 10403 S into B6 mice will yield millions of CFUs in both the spleen and liver 3 days post-infection whereas the bacterial load following the same dose of rLM-SIY will border the limit of detection (data not shown). However, rLM-SIY still induces strong anti-SlY CD8 T cell responses in naïve B6 mice despite its reduced pathogenicity (18, 39). In addition, an alternate explanation for the disappearance of 2C CD8 T cells following rLM-SIY infection could be the rejection of donor T cells by the host. Although we have bred our 2C TCR transgenic mice 10 generations onto the B6 background, it is plausible that some flanking DNA closely linked to the 2C TCR transgene could be facilitating graft rejection.  One intriguing question is why TCR signaling engages two Ras GEFs, each using intricate, independent mechanisms for their membrane recruitment and mobilization (5). Analyses on wild type and mutant 2C CD8 T cells revealed that RasORP1 is likely crucial for sustaining TCR-induced ERK activation (Fig. 4.4A) and that such a prolonged signal may be required for recruitment of naïve T cells into cell cycle (Fig. 4.1A, 4.1B). Since Ras can also be activated through the IL-2 receptor (29, 30), we also sought to investigate whether IL-2 signaling induced Ras activation via RasGRP 1. Consistent with the finding that cytokine receptors activate the Ras/MAPK pathway after their phosphorylation through binding of Shc adaptor protein and subsequently, the recruitment of Grb2/Sos (48), we found that IL-2 stimulations of RasGRPF’ 2C T cell blasts induced Ras activation at levels resembling wild type (Fig. 4.4B) and restored proliferation to immobilized anti-TCR Ab (Fig. 4.1A, 4.1B). Therefore, we suspect that RasGRP1 does not directly mediate Ras activation through the IL 2 receptor. However, our findings suggest that RasGRP1 may indirectly affect IL-2 receptor  145  signaling by influencing CD25 expression and IL-2 secretion (Fig. 4.1C, 4.1D). Recent work in the human Jurkat T cell line suggests that an unusual type of cooperation exists between RasGRP1 and Sos whereby RasGRP 1-derived Ras-GTP boosts Sos’s activity by binding an allosteric pocket on the molecule, creating a positive feedback loop (49). Therefore, it has been hypothesized that RasGRP1 and Sos collaborate to induce optimal Ras activation following low-level physiological stimulation (49). Fitting this hypothesis, our results demonstrate that RasGRP 1 is essential for the exquisite sensitivity of T cells in responding to a very limited number of peptide/MHC complexes on the surface of an APC.  The discovery of subcellular compartmentalization of Ras activation has garnered great attention and revealed another dimension of complexity to cellular signaling and possible cellular responses (5, 50). Although conventional wisdom had placed the site of Ras activity at the plasma membrane (PM), recent studies suggest that Ras activation also occurs at the Golgi in a PLC’-dependent fashion by way of RasGRP1 (5 1-53). The rationale for alternate cellular sites of Ras activity may be to subject it to differential regulation and/or serve to pair it with a unique subset of effectors. Comparison of Golgi versus PM has found that Ras activation at the former locale appears to have delayed or sustained kinetics relative to the latter (5 1-53). A possible mechanism for prolonged Ras signaling at the Golgi may be through calcium signals, serving to recruit the RasGAP CAPRI to the PM to inactivate Ras at this site while at the same time mediating the translocation of RasGRP1 to the Golgi (51, 54). Recently, subcellular compartmentalization of the Ras/RasGRP1/ERK pathway has been suggested to be pivotal in developing thymocytes undergoing selection (55). Using OT-I TCR transgenic pre-selection CD4CD8 (DP) thymocytes and specific peptides spanning the boundary of positive and negative selection, it was found that negatively selecting peptides recruited Ras, Raf- 1 and RasGRP 1 to the PM whereas these molecules co-localized to the Golgi when thymocytes were stimulated with a peptide mediating positive selection (55). Collectively, our findings hint at the possibility that subcellular compartmentalization of Ras/RasGRP1/ERK pathway may also be critical for the commitment of naïve CD8 T cells to Ag-induced developmental programming.  146  4.5  Acknowledgments  We thank Soo-Jeet Teh for technical assistance and the Wesbrook Animal Facility for animal husbandry. We are also grateful to Dr. D. H. Loh (Nippon Roche Research Center) for providing breeders of the 2C TCR transgenic mouse and Dr. Herman Eisen (M.I.T.) for the gift of the 1B2 hybridoma. The authors have no competing financial interests.  147  4.6  Figures  Figure 4.1 RasGRP 1 2C CD8 T cells exhibit defects in TCR-induced proliferation, early activation events and IL-2 production. A, Purified wild type and RasGRPF’ 2C CD8 T cells were cultured on either anti-TCR or anti-TCR and anti-CD28 antibody coated wells in the presence or absence of exogenous IL-2. After 3 days of in vitro culture, plates were pulsed with [ Hj-thymidine for a 6 h period and subsequently, incorporated radioactivity measured. 3 B, CFSE-labeled RasGRPF’ 2C CD8 T cells were cultured in the same manner as in A. Proliferation was measured 3 days post-stimulation by flow cytometry. RasGRP1-deficient 2C CD8 T cells exhibit diminished IL-2 production and decreased activation marker expression. C, Naïve wild type and RasGRPF’ 2C CD8 T cells (Thy1.2) were cultured with either anti-TCR Ab or SlY-pulsed splenocytes (Thy1.1). After 16 h culture, induction of activation markers was assessed on stimulated T cells (CD8 Thyl .2; filled histograms). Dotted, thin-line histograms represent marker expression on unstimulated (naïve) cells. D, Naïve wild type and RasGRPF’ 2C CD8 T cells were cultured with STY-pulsed splenocytes (Thy 1 .1) for the indicated times and the frequency of IL-2-positive 2C T cells (CD8 Thyl .2k) determined by flow cytometry.  Figure 4.2 RasGRP 1-deficient 2C CD8 T cells acquire an impaired effector phenotype after antigenic stimulation. Wild type and RasGRP1 4 peripheral 2C CD8 T cells (Thy1.2) were purified using and stimulated with the 2C TCR agonist peptide SlY and irradiated splenocytes (B6.PL, Thyl .1) either in absence (A) or presence of exogenous IL-2 (B). After 4 days of in vitro culture, 2C T cell effectors (filled histograms) were analyzed for surface markers (CD25, CD44 and CD62L) and intracellular Granzyme B expression by electronically gating on Thy 1 .2 CD8 events. For comparison sake, naïve 2C T cell profiles are also shown (thin, gray line). C, 2C T cell effectors generated by exposure to STY peptide and exogenous TL-2 (as in B) were stimulated with STY-coated splenocytes for 5 h and cytokine production assessed by intracellular flow cytometry.  148  Figure 4.3 RasGRP 1 regulates the antigen dose necessary to recruit naïve T cells to initiate proliferation and effector cell formation CFSE-labeled wild type and RasGRPF’ 2C CD8 T cells (50,000; Thy1.2) were cultured in vitro for 4 days with one million (feeder; Thyl.lj CD8-depleted splenocytes and limiting numbers of SlY-coated APCs (0, 200, 1000, 2000, 5000 or 10000; shown on left). A, After acquisition of 150,000 total events, CFSE fluorescence of 2C T cells was determined by electronically gating on Thyl .2 CD8 cells. The maximum scale for each histogram is shown vertically at the left. At the top right of each histogram, the frequency of 2C CD8 T cells that have not divided is indicated. B, The expression of CD44 as a function of cell division is shown for wild type and RasGRPF’ 2C CD8 T cells. C, To assess the frequency of 2C T cells capable of producing IFN-y, one million SlY-coated splenocytes (Thy 1.1) were added to each well and cultures were incubated for an additional 4 h. Within each density plot, the frequency of cells residing within each quadrant is indicated.  Figure 4.4 RasGRP1 2C CD8 T cells display transient ERK activation upon TCR ligation but normal ERK phosphorylation after IL-2 stimulation. A, Wild type and RasGRP1 2C CD8 T cells were stimulated with anti-TCR antibodies for the indicated times (mi. Whole lysates were inimunoblotted with anti-phospho-ERK, anti-ERK2, anti-phospho-LAT and anti-phospho-ZAP-70 antibodies. B, After 4 h serum starvation, wild type and RasGRP1 2C CD8 T cell effectors were either left untreated (NS; non-stimulated) or incubated with IL-2 or PMA for 10 mi  Ras activation was assessed by Ras-GTP puildown assay using Raf-GST  and Western blotting precipitates with an anti-Ras specific antibody. Whole lysates were probed for with anti-pan-Ras, anti-phospho-ERK and anti-ERK antibodies. Both long and short exposures of immunoblots are shown.  Figure 4.5 RasGRP 1-deficient T cells exhibit a reduced rate of proliferation and diminished cytokine production upon antigen stimulation in vivo. A, A recombinant strain of Listeria monocytogenes, named rLM-SIY, was engineered to express the 2C TCR agonist peptide SIYRYYGL. To ensure proper cleavage, SIYRYYGL was flanked by 5’ and 3’ sequences known to be cut in murine cells, liberating naturally occurring peptides (SIINFEKL from  149  ovalbumin [OVA] and LSPFPFDL from a-ketoglutaraldehyde dehydrogenase [a—KG]). B, CFSE-labeled wild type Thy1.2 2C CD8 T cells (1 x 106) were adoptively transferred into naïve B6 (Thy 1.1k) mice, which were then left untreated or infected with either rLM-SIY or wild type Listeria monocytogenes. One week post-infection, proliferation history of donor 2C T cells was assessed. C-I, An equal 50:50 mixture (10,000 cells of each genotype) of wild+  type (Thy 1.1/1.2  +  ) and mutant 2C T cells (Thy 1.2  +  /1.2  +  ) was generated, labeled with CFSE  and co-adoptively transferred into naïve B6 (Thy1.1/1.1) mice one day prior to infection with rLM-SIY. Spleens were recovered from infected mice one-week post-infection, donor T cells (Thy 1 .2k) detected with anti-Thy 1.2 Ab and their genotype identified by reactivity to anti-Thy 1.1 Ab. D, The frequency of wild type versus mutant 2C T cells are presented. E, Numbers of 2C T cells recovered from spleens. F, The majority of wild type and RasGRPl 2C T cells lose their CFSE-endowed fluorescence seven days post-infection with rLM-SIY (filled histogram). Open histogram represents CFSE fluorescence of non-dividing 2C T cells residing in uninfected B6 mice. G, A reduced frequency of RasGRP1-deficient 2C T cells possesses the proliferation-associated nuclear antigen Ki-67. H, Wild type and RasGRP1deficient 2C T cells (filled histogram) up-regulate CD44 during Ag-driven T cell expansion. Open histogram represents CD44 staining on naïve 2C T cells. I, Splenocytes were incubated for 5 h with SlY peptide and donor T cell cytokine production assessed by FACS. All error bars represent the SD.  150  A  B  -I-  +1+ c’.J  cJ -J  C)  i::i +1+ .-I  200  +  1•  AL  F  150 0  ++  ,100  +  -+  50  LJL +++  JLA  TCR CD28  CFSE  C  i280  ø776  630  :  +1+  I L-2  D  +  +  +  +  -  +  -  +  -  +  +  -  50  •  aTCR ‘  I V  295  201  v,  40  -.  295  .  30  /  /  CJ  fli +1+ S IY/APC  I  ‘893  17481  20  ‘  ;  1182’;  /  464  *_/  p642  Q  1.370  10  N.  -I0’  -  — CD25 —)‘ — CD69 —b- — PD-i  .  0  1  2  Time (h)  5  6  Figure 4.1 RasGRPF’ 2C CD8 T cells exhibit defects in TCR-induced proliferation, early activation events and IL-2 production. 151  A  36  +1÷ sly  91  ‘  I  j69  450  138  .A  I  —I—  JGranzyme B—>  L  I.  CD25  >  CD44  >  CD62L  >  —  B  2:::  +1+ sly IL-2  h  —  +  354  —I -  CD25  C  ‘ >  99  i CD44  :.  >  CD62L  29  >  —  L Granzyme B—*  23  3.8  +1+  z U-  0  99  9.0  -J  2.9  23  —I—  CD8  Figure 4.2 RasGRP1 -deficient 2C CD8 T cells acquire an impaired effector phenotype after antigenic stimulation.  152  A  B  Mutant  Wild Type  Number SIY-APC  95  Mutant 315  Wild Type  94  C  Wild Te 3  Mutant  l  0 59  :  9  •10 2 87 2  8O 29  C  F•i  8  200 -  23  84 C  1,000 5 2,000  AL  51  ‘!: •;4;  :  :59.  ,  4 84 7,,  F  .4 .•  79  40  15t 4 2  -  t$4:,’  5,000 -  ‘a  1  ._  10,000  § i’:-CFSE  CFSE  CFSE  Figure 4.3 RasGRP 1 regulates the antigen dose necessary to recruit naïve T cells to initiate proliferation and effector cell formation.  153  A  Wild Type  B  Mutant  Wild Type  Mutant II  o  3  5  30  60  0  3  5  30  NS  60 Mm ERK  IL-2  PMA  NS  lL-2  PMA  i  Ishort I Ras JGTP ‘-Jul long  —‘--  —  ERK2 L.. —  -  —  P-ZAP70  pan  1 Ras  F  Figure 4.4 RasGRP1 2C CD8 T cells display transient ERK activation upon TCR ligation but normal ERK phosphorylation after IL-2 stimulation.  154  A  ,SIYRYYGL OVA V a-KG cLLKEAY IKVSG LEQ LE  D  80  o  2O 0  B  naive  wt LM  rLM-SIY 90  >1  — r1  C)  Cl)  2O  a)  oC chromosorm  -?3O  =  4O ‘;:  G  °  i60  —EHssI’  E  4  +1+  +  r’-.  CD  10 0  0,  --  I  0 ÷1+ -I-  H  F ÷1+  CFSE  C  —I— -I-  =  Li +1÷  50%  V\  =  50%  CFSE  Thyl.1  ÷/+Thyl.1/1.2 Thyl -  Jst Thyil  r\,_—\  Day  rLM-SIY  a  >  CD44  +1+  -I831  1  > 38  :  6 ——“‘-r-  :“  .  n..’r..r.’  :  41  .  I  I  Figure 4.5 RasGRP 1-deficient T cells exhibit a reduced rate of proliferation and diminished cytokine production upon antigen stimulation in vivo. 155  4.7  References  1.  Germain, R. N. 2002. T-cell development and the CD4-CD8 lineage decision. Nat Rev Immunol 2: 309-322.  2.  Hogquist, K. A. 2001. Signal strength in thymic selection and lineage commitment. Curr Opin Immunol 13: 225-3 1.  3.  Surh, C. D., and J. Sprent. 2005. Regulation of mature T cell homeostasis. Semin Immunol 17: 183-191.  4.  Alberola-Ila, J., and G. Hernandez-Hoyos. 2003. The Ras/MAPK cascade and the control of positive selection. Immunol Rev 191: 79-96.  5.  Stone, J. C. 2006. Regulation of Ras in lymphocytes: get a GRP. Biochem Soc Trans 34: 858-861.  6.  Dower, N. A., S. 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Teixeiro, J. Gill, B. Hausmann, D. Roubaty, K. Holmberg, G. Werlen, G. A. Holländer, N. R. Gascoigne, and E. Palmer. 2006. Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling. Nature 444: 724-729.  161  CHAPTER 5  PREFERENTIAL DEVELOPMENT OF CD4 AND CD8 T  REGULATORY CELLS IN RASGRP1-DEFICIENT MICE’  5.1  Introduction  During the process of thymic education, only thymocytes expressing TCRs capable of distinguishing between self and nonseif are selected for survival and differentiate into mature T cells. According to the strength of signal hypothesis, thymocytes expressing TCRs with weak or modest affinity for self Ags undergo positive selection and become mature T cells. By contrast, developing T cells expressing high affinity receptors for self Ags are clonally deleted, a process referred to as negative selection. However, negative selection is imperfect because autoreactive lymphocytes escape clonal deletion and can be detected within the peripheral lymphocyte compartment (1, 2). To keep these autoreactive T cell clones in check, a dedicated lineage of T regulatory (Treg) cells functions as an important fail-safe mechanism to prevent catastrophic effect of unchecked immune responses (3, 4). Three main cell types have been considered as potential Treg cell subsets, as follows: CD4CD25 Treg cells (5, 6), CD8aa intestinal epithelial lymphocytes (7), and NKT cells (3). All are thought to be induced by high affinity interactions between self peptide:MHC with the TCR on developing T cells in the thymus (1, 8). In addition to these three subsets, recent studies f T cells that function indicate normal mice also possess a population 0 as regulatory cells and can perform roles distinct from CD4 Treg cells in suppressing T cell activation (9-12).  Of all the lineages of Treg cells, the CD4CD25 subset has been the most extensively characterized. CD4CD25 T cells develop naturally in normal individuals and are readily detectable in the thymus and secondary lymphoid organs in mice, rats, and humans, where they make up 2—10% of the total CD4 cells (13). In addition to CD25, CD4 Treg cells also  1  A version of this chapter has been published. Chen, X., J.J. Priatel, M.T. Chow, and H. S. Teh. (2008) Preferential Development of CD4 and CD8 T Regulatory Cells in RasGRP1-Deficient Mice. I Immunol. 180: 5973-5982. Copyright 2008. The American Associateion ofImmunologists, Inc. 162  express high levels of CTLA-4 and glucocorticoid-induced tumor necrosis factor receptorrelated protein (14). However, the most distinguishing feature of CD4 Treg cells is their expression of Foxp3, a member of the forkhead family of transcription factor (8). The expression of Foxp3 is both necessary and sufficient for the development and function of CD4 Treg cells (5, 15). Additionally, studies have found the TCR repertoire of CD25 CD4 T cells is highly self-reactive, a conclusion supported by the observation that when T cells transduced with TCR genes derived from CD25, but CD25 CD4 T cells, they rapidly expand in lymphopenic hosts and induce autoimmune disease (16). Furthermore, studies have shown that TCR transgenic T cells can undergo conversion into Treg cells following exposure to either cognate Ag or peptide-agonist ligands on dendritic cells, respectively (1719).  Although a vast amount of literature exists on the developmental biology, function, and TCR repertoires of CD4 Treg cells, details about CD8 Treg cells are only beginning to emerge. Studies on CD 122-deficient mice have implicated the existence of CD8 Treg cells that function to preferentially regulate the immune functions of CD8 T cells. CD 122-deficient mice exhibit severe hyperimmunity (9), which is associated with the expansion of abnormally activated T cells (10). However, the transfer of highly purified CD8CD122 T cells, from wild-type mice to CD 122-deficient neonates, prevented the aberrant T cell phenotype from developing in the treated mice (11). Moreover, RAG-2 mice that received wild-type CD8CD122 cells die within 10 wk after cell transfer, suggesting CD8CD122 T cells become dangerously activated in the absence of CD8CD122 T cells (11). Follow-up studies indicate the suppressor activity of CD8CD 1 22 T cells was mediated by IL- 10 (12). Collectively, these results suggest CD8CD 1 22 T cells contain novel populations that can function as Treg cells.  RasGRP 1 is one of two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation (20, 21). Upon TCR stimulation, RasGRP1 mobilizes to the Golgi membrane by binding the phospholipase C-yl product diacylglycerol (DAG) through its Cl domain (22-24). Thymocytes from RasGRPF’ mice are defective in TCR- and DAG-induced activation of Ras-ERK signaling (20). Furthermore, mutant mice  163  exhibit a defect in positive selection, as evidenced by reduced numbers of single-positive (SP) thymocytes and T cell lymphopenia (25). By contrast, strong TCR signals responsible for negative selection and the induction of Ag-driven growth appear to be RasGRP 1 independent (21). Because most Treg cells characterized to date express high affinity TCRs for self Ags, our objectives with regard to the analysis of the role ofRasGRPl in the development and function of Treg cells are 2-fold, as follows: 1) determine the role of RasGRP1 in the intrathymic development of CD4 and CD8 Treg cells, and 2) determine the role of RasGRP 1 in peripheral homeostasis and function of CD4 and CD8 Treg cells. Our results indicate that although intrathymic development of CD4Foxp3 cells is severely impaired in the absence of RasGRP1, there exist an elevated frequency and large numbers of Foxp3-expressing CD4 Treg cells in the peripheral lymphoid tissues of mutant mice. This may be attributable to both the massive expansion of RasGRPI’ Foxp3CD4 T cells and increased death rate of mutant Foxp3CD4 T cells. Additionally, RasGRP1 CD4 Treg cells were found to be functional because they could suppress the proliferation of wild-type CD25 CD4 T cells in vitro. In contrast to the CD4 Treg cells, the development of CD8 Treg cells is not affected by RasGRP 1 loss. However, the suppressor function of CD8 Treg cells is dependent on RasGRP1. The implications of these findings on peripheral T cell homeostasis and the development of autoimmune diseases in RasGRPF’ mice are discussed.  5.2  Materials and methods  Mice C57BL/6J (B6) mice were obtained from The Jackson Laboratory. RasGRP1 breeder mice (20) were provided by J. Stone (University of Alberta, Edmonton, Alberta, Canada) and bred onto B6 background for more than seven generations. Mice 6—12 wk of age were used for the experiments described. All studies followed guidelines set by the Animal Care Committee at the University of British Columbia in conjunction with the Canadian Council on Animal Care.  164  Flow cytometry Abs against CD4 (GK1.5), CD8a (53-6.7), CD813 (53.58), TCRI3 (H57-597), CD3s (2C11), CD25 (PC61.5), CD44 (1M7), CD62L (MEL-14), CD69 (H1.2F3), CD94 (18d3), CD122 (5H4), CD127 (A7R34), Foxp3 (FJK-16s), NK1.1 (PK136), NKG2AB6 (16a1 1), NKG2D (CX5), Thyl.1 (HIS51), Thyl.2 (53-2.1), and IL-b (JES5-16E3) were purchased from eBioscience. Annexin V-PE and Abs against 2B4, CD5 (53-7.3), Ly6C (AL-21), and Ki-67 (B56) were purchased from BD Biosciences. Foxp3 staining was performed following the protocol recommended on eBioscience web site (www.ebioscience.com!ebioscience/specs/ antibody_i 2/12-5773 .htm). For anti-IL- 10 staining, cells were stained for surface markers, washed, and fixed with 2% paraformaldehyde and 0.2% Tween 20 in PBS for 20 mm on ice, followed by washing with PBS. Fixed cells were then stained with anti-IL-lO Ab in 0.2% Tween 20/PBS for 30 mm on ice. For anti-Ki-67 staining (26), cells were fixed and permeabilized using the same protocol as for Foxp3 staining, and incubated with anti-Ki-67 Ab for 30 mm at 4°C. Data were acquired using either FACScanJCellQuest software or LSRII/FACSDiva software (BD Biosciences). Data were analyzed with FlowJo (Tree Star) software.  CD4 T cell proflferation and suppression assays Cell sorting with the FACSAria flow cytometer (BD Biosciences) was used to purify CD4CD25 or CD4CD25 cells. The purities of sorted CD4CD25 wild-type or mutant suppressor cells used for experimental studies are 94.2 and 95.4%, respectively. Proliferation and suppression assays were performed, as described (27). Briefly, for proliferation assays, T cells (2 x 1 0 cells/well) were stimulated for 72 h with titrated amounts of Con A in the presence of T cell-depleted, irradiated APCs (8 x 1  per well) in 96-well round-bottom  plates, and pulsed with 1 iCi per well of [ H]thymidine for the final 8 h. Suppression assays 3 were performed under the same conditions using 2 x 1 0 CD4CD25 T cells as responders, 8 x i0 4 irradiated APCs, and a 1:2 titration of the indicated suppressor T cell population (CD4CD25) at a starting concentration of 4 x 1  cells/well in the presence of Con A at 2  ig/ml final concentration. All data are shown as mean [ H] thymidine incorporation in 3 triplicate cultures.  165  Adoptive transfer experiment Cell suspension was prepared from Thy1.1 B6 or Thy1.2 RasGRPF’ animals. Cells were then stained with PE-conjugated anti-CD4 (GK1 .5) and allophycocyanin-conjugated anti CD25 (PC61.5) Abs and sorted for the CD4CD25 or CD4CD25 population (purity> 95%), respectively, using FACSAria. Sorted CD4CD25 (2 x 10) or CD4CD25 (1 x 106) Thy 1.1 + wild-type cells were adoptively transferred into naive B6 Thy 1 .2 wild-type or RasGRPF’ hosts. Similarly, sorted CD4CD25 (2 x 10) or CD4CD25 (1 x 106) Thy1.2 RasGRP 1 cells were adoptively transferred into naive Thy 1.1 + wild-type or RasGRP 1-/ hosts. Spleens of recipients were recovered 3 wk posttransfer, and frequencies of donor cells of the indicated cell surface phenotype were quantified by flow cytometry.  CFSE labeling Purified CD8 T cells (1 x 10 /ml) were labeled with 1 iM CFSE (Molecular Probes) in PBS 7 for 10 mm at room temperature. After stopping the reaction with the addition of an equal volume of FCS, cells were washed four times with complete medium.  CD8 T cell purification and direct ex vivo assays Single-cell suspensions from lymph nodes and spleens of mice were prepared and then treated with biotinylated anti-CD8 (53-6.7) mAb, followed by positive selection using MiniMACS system (Miltenyi Biotec), according to the manufacturer’s specifications. The resulting cells were> 95% pure CD8 TCR T cells. For cytokine proliferation assay, purified wild-type or RasGRP1 CD8 cells were CFSE labeled and cultured in IL-2 (200 U/mi) or IL-iS (100 ng/ml). Proliferation of gated CD8 cells was then analyzed by FACS at 72 h. For IFN-y production assay, 2 x 106 purified wild-type or RasGRPF’ CD8 cells were stimulated with PMA (10 ng/ml) and ionomycin (100 ng/ml) in medium containing Golgi plug (BD Pharmingen) for 5 h at 37°C. Following stimulation, cells were stained for surface Ags and then stained intracellularly for IFN-y.  166  CD8 T cell cytokine and suppression assays CD8 T cells from B6 wild-type and RasGRPF’ mice (Thy1.2) were purified, as described above. Cells were then stained with PE-conjugated anti-CD 122 (5H4) and PE-Cy5conjugated anti-CD8 (53-6.7) Abs and electronically sorted using FACSAria flow cytometer. 4 per Sorted wild-type CD8CD122, CD8CDl22, or RasGRPF’ CD8CD122 cells (5 x i0 well) were stimulated by plate-bound anti-CD3 (10 jig/mi, 1 h incubation at 37°C, washed twice) and 10 jig/ml soluble anti-CD28 plus 100 U/ml IL-2 (final concentration) for 72 h in 24-well flat-bottom plates. For assessment of IL- 10 production by the cultured CD8 cells, Golgi-plug was added to the cell cultures during the last 24 h of culture and IL- 10 production was evaluated by intracellular staining. For suppression assays, sorted wild-type CD8CD122, CD8CD122, or RasGRPF’ CD8CD122 cells (2 x 106) were stimulated using the same condition above. Three days later, activated suppressor cells from the indicated population were added at various suppressor to responder ratios to 6.5 x  CFSE  labeled sorted B6 Thy 1.1 CD8CD 1 22 cells, and cultured with anti-CD3 and anti-CD28 plus IL-2 (same stimulation condition as above) in 96-well flat-bottom plate. Two days later, the proliferation of Thy1.1 CD8CD122 wild-type cells was assessed by CFSE dilution analysis.  5.3  Results  Defective thymic dfferentiation of CD4Foxp3 Tcells in RasGRPT” mice Because Foxp3 is a unique marker for naturally arising CD4CD25 Treg cells, we first examined the development of Foxp3 CD4 SP thymocytes in the thymus ofRasGRPF’ mice. We found that although the proportion of CD4 SP thymocytes in mutant mice is 14-fold lower than in wild-type mice (0.44 vs 6.29%), the proportion of CD4 SP thymocytes that are also CD25Foxp3 in mutant mice is fairly similar to wild-type (2.02 vs 3.08%) (Fig. 5.1A). However, cell number comparison of Foxp3 or Foxp3 lineages in CD4 SP thymocytes between wild-type and RasGRP1 mice revealed that both CD4 Treg (Foxp3) and non-Treg (Foxp3) populations are severely affected by RasGRP1 deficiency, with both populations accounting for 4.5 and 4.2% of wild-type numbers, respectively (Fig. 5.1B). Next, we  167  examined the differentiation of Foxp3-expressing precursors in RasGRPF’ thymus by evaluating the distribution of Foxp3 -expressing thymocytes among the thymocyte subpopulations as defined by CD4 and CD8 expression. A recent report suggests that although Foxp3 induction can occur at the double-positive stage, it is preferentially induced at the CD4 SP stage during the development (28). We found that there is a significant increase in the proportion of CD4CD8Foxp3 thymocytes in RasGRPF’ mice relative to wild type (51 vs 13%) (Fig. 5.1C). This result is consistent with the notion that there is a block in transition from Foxp3 double-positive to Foxp3 CD4 SP thymocytes in RasGRPF “  mice. Alternatively, this observation could be due to a defect in Foxp3 up-regulation by  RasGRPF’ CD4 SP thymocytes. Interestingly, RasGRPF’ Foxp3 -expressing cells within the CD4 SP population display altered expression of TCRI3, CD3E, CD5, and CD69 (Fig. 5.1D). The distribution of TCR and CD5 in the Foxp3 CD4 SP population is bimodal, with a minor population that expresses fairly normal levels of TCR and CD5 and a major population that expresses very low levels of these molecules (Fig. 5.1 D). If only the TCR population is representative of CD4 Treg cells, this would imply that the development of CD4 Treg cells is more greatly affected by the RasGRP1 mutation than implied by the analysis of Foxp3CD4 SP thymocytes. In summary, these observations indicate that naturally arising CD4Foxp3 Treg cell development in the thymus is severely impaired in RasGRPF’ mice.  Favored expansion and enhanced suppressive function ofperipheral CD4Foxp3 T cells in RasGRPT’ mice In contrast to the impaired development of CD4CD25Foxp3 Treg cells in the thymus,we found that the spleen of RasGRPF mice possessed a markedly increased proportion of CD25Foxp3 cells within the CD4 population as compared with wild type (24 vs 8.6%) (Fig. 5 .2A). Furthermore, there is a preferential increase in splenic CD4Foxp3 cell number relative to the CD4Foxp3 population in RasGRPF’ mice (32 vs 14% of wild-type numbers; Fig. 5.2B). We noted in Fig. 5.1D that the majority of CD4Foxp3 thymocytes express very low levels of TCR and CD5. In striking contrast to the thymus, RasGRPF’ peripheral CD4 Treg cells express near wild-type levels of TCRI3, CD3 , and CD5 (Fig. 5 .2C). Interestingly, both Foxp3 and Foxp3 CD4 T cells in RasGRPF’ mice display signs of acute activation  168  (CD44, CD62Lb0,  hih) 69  with a higher proportion of Foxp3 CD4 cells from  RasGRP i mice expressing increased levels of acute activation markers (Fig. 5 .2C).  The huge increase in peripheral Foxp3 CD4 T cell numbers in RasGRPF’ mice could be due to the preferential expansion of these cells and/or the preferential death of Foxp3 CD4 T cells in mutant mice. To test this hypothesis, we performed direct ex vivo staining of Foxp3 and Foxp3 CD4 T cells to determine the proportion of proliferating and dying cells in the wild-type and mutant animals (Fig. 5.3A). We found that both CD4 Foxp3 and Foxp3 cells in RasGRP 1 mice exhibit an elevated frequency of cells bearing the proliferation-associated nuclear Ag Ki-67 (26) as compared with wild-type animals (Foxp3, 24.5 vs 7.2%; Foxp3, 31.1 vs 15.6%) (Fig. 5.3A, middle panel). Costaining of the apoptotic marker annexin V and Foxp3 revealed that Foxp3 and Foxp3 CD4 T cells in RasGRP1 mice undergo different rates of cell death. It is noted that mutant Foxp3 CD4 T cells undergo a higher degree of cell death relative to their wild-type counterpart (13% annexin V vs 4%; Fig. 5.3A). By contrast, +  +  there is a dramatic decrease in the proportion of mutant CD4 Foxp3 cells that are annexrn V, as compared with wild-type mice (7.4% annexin V vs 22%; Fig. 5.3A). To provide an explanation for the increased numbers/frequency of Foxp3 T cells in the mutant periphery relative to the thymus, we performed a series of adoptive transfer experiments to distinguish between the cell intrinsic and extrinsic effects of the RasGRP 1 mutation on the peripheral +  .  .  +  .  +  expansion of Foxp3 CD4 T cells (Fig. 5.3B). First, sorted wild-type CD4 CD25 or CD4CD25 T cells (Thy1.1) were transferred into either wild-type or mutant Thyl.2 hosts, respectively (Fig. 5.3B, top panel). Three weeks postadoptive transfer, significant frequencies of donor CD4 T cells were detected only in RasGRP1, but not wild-type recipients. This result suggests that it is the peripheral environment in RasGRP 1’ mice that favors the •  +  +  +  .  expansion of both wild-type CD4 CD25 and CD4 CD25 cells. This is most likely a consequence of lymphopenia associated with RasGRP 1 .1- mice. We also determined whether there is increase conversion of donor CD4CD25 into CD4CD25 T cells in RasGRPF’ mice. We found that there is no conversion of either wild-type or mutant CD4CD25 into CD4CD25 T cells in wild-type hosts (Fig. 5.3C). Interestingly, 6.8% of donor wild-type CD4CD25 cells developed into CD4CD25 cells in RasGRPF’ host, and these CD4 +  + + CD25 cells were also Foxp3 (Fig. 5.3C). Similarly, 4.9 0/o of donor mutant CD4 CD25 T .  .  .  -  169  cells developed into CD4CD25Foxp3 cells in mutant hosts (Fig. 5.3 C). Collectively, these findings suggest that the elevated frequency of CD4CD25Foxp3 T cells in RasGRPF’ mice is most likely due to a combination of decreased cell death and a cellular environment that favors their peripheral expansion. To evaluate the function of CD4Foxp3 cells in the periphery of RasGRP i mice, we purified both wild-type and mutant peripheral CD4CD25 T cells using FACS and compared their ability to suppress the proliferation of Con A-activated wild-type CD4CD25 cells in vitro (Fig. 5.4). On a per cell basis, CD4CD25 T cells from RasGRPF’ mice were more suppressive compared with wild-type CD4CD25 cells. This increased suppressor activity of CD4CD25 T cells from RasGRP 1’ mice correlates with the increased expression of CD44 and CD69 on these cells compared with their wild-type counterpart (Fig. 5.2C). In summary, these studies demonstrate that there is preferential accumulation of functionally active CD4Foxp3 Treg cells in the periphery of RasGRP1 mice. They also indicate that the suppressor function of CD4Foxp3 Treg cells is independent of RasGRP1.  Preferential development ofmemoiy phenotype CD8 T cells in the thymus and periphery of RasGRPT’ mice The Tec family tyrosine kinases Itk and Rlk are required for full TCR-induced activation of phospholipase C-yl, Ca2 mobilization, and ERK activation (29, 30). Itk and Rlk perform important functions during T cell development, and, in particular, have been implicated in setting the thresholds of positive and negative selection (31-33). Interestingly, although Itk and Rlk are critical for the development of conventional CD8 T cells, Itk’ and Rlk’itk mice possess a large population of memory phenotype CD8 T cells that bear striking similarity to lineages of innate-like lymphocytes (34-3 6). Because RasGRP 1 mice are defective in TCR- and DAG-induced activation of Ras-ERK signaling (20) and play a vital role in the positive selection of conventional T cells (20, 21), we question whether RasGRP 1 is also dispensable for the development of these innate-like CD8 T cells that bear a memory phenotype. We found that there is a large increase in the proportion of CD44’CD122 CD8 SP thymocytes in RasGRP1 mice relative to wild type (Fig. 5.5A, top panel). In addition, 0w, suggesting their mature status 1 these CDl22 cells in the RasGRP1’ thymus are CD24  170  (Fig. 5.5A, middle panel). There is also a slight increase in the total number of CD44 CD122 cells in the thymus of RasGRPF’ mice (115% of wild type) (Fig. 5.5D). All of these observations contrast with the greatly reduced numbers and immature phenotype of CD4Foxp3 cells found in the thymus of RasGRPF’ mice (Fig. 5.1B). Consistent with the thymic data for this CD8 subset, there is 5-fold increase in the proportion of CD122 cells in the spleen of RasGRP1 mice relative to wild type (Fig. 5.5A, bottom panel). We also found that the proportions of CD8 SP thymocytes and splenocytes that are NK1.1 are greatly increased in the thymus and spleen of mutant mice relative to the wild-type counterpart (Fig. 5.5B). In absolute numbers, there is a 1.7- and 9-fold increase in the number of CD8+CD44h1CD122+ and CD8NK1.1 cells, respectively, in the spleen of RasGRPF’ mice as compared with wild type (Fig. 5.5D). This observation contrasts with the ”’CD8 cells that are recovered from both the thymus 1 significantly lower numbers of CD44  and spleen of RasGRPF’ mice relative to wild-type mice (Fig. 5.5D). However, unlike CD4 + high + Treg cells, there is no evidence that the elevation in the numbers of CD8 CD44 CD122 T  cells in RasGRP 1  mice is due to either enhanced proliferation and/or decreased cell death  of these cells relative to wild-type mice (Fig. 5 .5C). These observations suggest that CD8 CD44CD1 22 cells are more resistant to the effects of RasGRP 1 deficiency than CD8 CD44IOWCD 1 22 T cells. Collectively, they indicate that there is a preferential development of  memory phenotype CD8 + CD44 high CD 122 + T cells in the thymus and spleen of RasGRP1 .  mice.  Elevated expression ofNK receptors by CD8 T cells in RasGRPT mice Functional differences between lymphocyte populations are often accompanied by changes in receptor expression patterns. It has been reported that CD8+CD44h cells from normal mice expressed significant levels of NK receptor upon IL-2 activation (37). In addition, CD44 CD 1 22 and CD44b0CD 1 22 CD8 T cells from Itk and IL-i 5 mice, respectively, have high + + distinct patterns of NK receptor expression (34). To distinguish the CD44 CD 122 CD8 T  cells found in RasGRP1 mice from conventional memory CD8 T cells, we compared the expression of NK receptors and memory markers on CD8+ cells from wild-type and RasGRP 1 mice. We found that the proportion of CD8CD 1 22 cells expressing high levels of Ly6C, CD94, NKG2A/C/E, NKG2D, 2B4, and NK1 .1 is dramatically increased in 171  RasGRP1 mice compared with their wild-type counterparts (Fig. 5.6). This finding is 22CD8 T cells in Itk mice (34).  similar to that previously reported for  Notably, despite this dramatic difference in the expression of NK receptors, there are only minor differences in the expression of memory makers such as CD62L and CD 127 between wild-type and RasGRP 1’ CD8CD 1 22 spleen cells (Fig. 5.6). Therefore, it is likely that the CD44 high CD 122 + CD8 + T cells present in RasGRP1 .  mice are similar to those described for .  Itk mice and represent a cell lineage(s) that is distinct from conventional memory CD8 T cells.  Activated RasGRPT’ CD122CD8 T cells are less suppressive than their wild-type counterpart Recent studies (35, 36) have shown that CD44hCDl 22CD8 cells in Itk mice can proliferate in IL-2 or IL-15 without TCR stimulation and produce IFN-y directly ex vivo. To further characterize functional similarities of CD44hhtCD122+CD8+ T cells from RasGRPl and Itk mice, we determined whether these cells from RasGRP i mice can proliferate in response to IL-iS or IL-2. As expected, naive conventional CD8 T cells expressing low levels of CD122 did not proliferate when cultured with either IL-iS or IL-2. By contrast, CD8CD122 T cells from either wild-type or RasGRP1 mice proliferated vigorously when cultured with either IL-15 or IL-2 (Fig. 5.7A). However, mutant CD8CD122 T cells proliferate less well than wild type in response to these cytokines, suggesting a role for RasGRP1 in the transmission of cytokine-dependent growth signals. Furthermore, only CD8 CD44’, but not CD8+CD44b0 T cells from both thymus and spleen of wild-type or RasGRP1’ mice can produce IFN-y after PMA plus ionomycin stimulation ex vivo (Fig. 5.7B). These data indicate that the development of  T cells that possess  innate immune functions is independent of RasGRP 1.  Recent studies show that CD8CDl22 cells from normal mice can also function as Treg cells via an IL-lO-dependent mechanism (11, 12). We sought to investigate whether CD44h1 CD 1 22CD8 T cells present in RasGRP i mice possess similar suppressor function. To test this hypothesis, we compared the ability of CD8CD122 cells from RasGRP1 mice to  172  produce IL-lO using wild-type CD8CD122 cells as a negative control. Sorted CD8CD122 cells from wild-type or RasGRPF’ mice were stimulated with anti-CD3 plus anti-CD28 and exogenous IL-2 for 3 days. IL-b production was then evaluated by intracellular staining with anti-IL-lO mAb. As expected, wild-type CD8CD122 cells failed to produce IL-b after in vitro activation. By contrast, CD8CD 1 22 cells from either wild-type or RasGRP 1 mice produced significant amounts of IL-lO compared with wild-type CD8CD122 cells (Fig. 5.8A). We then compared the ability of activated CD8CD122 cells from wild-type or RasGRP1 mice to suppress the proliferation of wild-type CD8CD12Z cells (Fig. 5.8B). Sorted CD8CDl22 cells from wild-type or RasGRPL’ mice were stimulated with anti-CD3 plus anti-CD28 and exogenous IL-2 for 3 days and used as a source of suppressor cells. Supematants from these cultures were also collected and assessed for their suppressor activity at 1/2 dilution. CD8CD122 cells and culture supernatants from wild-type mice activated in a similar manner were used as negative controls. CFSE-labeled wild-type CD8 CD 1 2T (Thy 1 .1) cells were used as responder cells. The Thy 1 .2 activated suppressor (CD8CD122) cells and negative control (CD8CDl2T) cells were added at various suppressor to responder ratios and stimulated with plate-bound anti-CD3, anti-CD28 (10 ig/ml), plus 100 U/ml IL-2 for another 2 days. We found that activated CD8CD122 cells and culture supernatants from wild-type mice completely suppressed the proliferation of wild-type CD8CD122 cells. By contrast, activated CD8CD122 cells from RasGRP1 mice were only suppressive when used at a high (1:5), but not lower ratios (1:15 and 1:45). Furthermore, the culture supematants of activated mutant CD8CD 1 22 cells were not suppressive. These data suggest that although the development of memory phenotype CD8 CD44CDl 22 Treg cells is independent of RasGRP 1, their suppressor function is much more dependent on RasGRP 1.  5.4  Discussion  We have previously concluded that RasGRP1 is especially crucial for transducing low-grade TCR signals because its absence preferentially affected the positive selection of the weakly selecting H-Y TCR (21). The development of CD4 T cells is also critically dependent on  173  RasGRP1 (20, 25). By contrast, the development of thymocytes expressing the more strongly selecting 2C TCR and negative selection was much less sensitive to RasGRP 1 loss (21). Because recent studies have suggested that a common denominator for the development of multiple Treg lineages is their dependence on high affinity TCR!self ligand interactions for their development (1, 8), we sought to determine how the development of Treg lineages was affected in RasGRP1’ mice. In this study, we found that RasGRP1 mice can support the development of both CD4 and CD8 Treg cells, albeit with differences in efficacy in the generation of CD4 and CD8 Treg cells in the thymus of RasGRP1 mice.  In the absence of RasGRP 1, development of CD4 Treg cells in the thymus is severely impaired, resulting in  <  5% yield relative to wild-type mice (Fig. 5.1B). However, there is  preferential expansion of CD4 Treg cells in the periphery, and the number of splenic CD4 Treg cells is 32% of wild type (Fig. 5.2B). The development of CD4 Treg cells is influenced by at least two factors: availability of IL-2 and self Ag/MHC ligands (1, 38, 39). It remains to be determined whether the poor development of CD4 Treg cells in RasGRP 1 thymus reflects the paucity of one or both of these factors. We have previously shown that T cells expressing relatively high affinity TCRS for self ligands can develop via a RasGRP 1independent mechanism (21). Consistent with this hypothesis is our observation that a large proportion of peripheral CD4 T cells in RasGRP1 mice is actively cycling and undergoing apoptosis, presumably as a result of high affinity TCR/self ligand interactions (40). In this study, we further characterized the CD4 phenotype in mutant mice by discriminating between Treg and non-Treg populations. Our observations suggest that in the periphery of RasGRPF’ mice, although both Treg and non-Treg subsets are undergoing massive proliferation, a smaller proportion of Treg population shows signs of apoptosis as compared with wild type (Fig. 5.3A), which might contribute to the high frequencies of CD4Foxp3 T cells in the mutant spleens. It is also conceivable that the proliferating CD4 non-Treg cells can produce cytokines that include IL-2. Together with the lymphopenia present in RasGRP1 mice, this could provide a favorable environment for peripheral CD4 Treg cell expansion that is mediated by its high affinity IL-2R. This hypothesis is supported by our adoptive transfer experiments demonstrating that there is a preferential expansion of adoptively transferred wild-type CD4CD25 cells in RasGRP 1  mice relative to wild-type  174  recipients (Fig. 5.3B, top panel). Recent studies suggest that in the presence of cognate Ag, peripheral conventional CD4CD25 T cells expressing high affinity TCRs for self ligands can be efficiently converted into Treg cells expressing Foxp3 (19). We therefore evaluated the possibility that mutant mice provide an environment niche that favors the conversion of conventional CD4CD25 T cells to CD4CD25Foxp3 Treg cells by transferring wild-type or mutant CD4CD25 cells into wild-type or mutant recipients, respectively (Fig. 5 .3B). Interestingly, we found that the preferential expansion of transferred CD4CD25 cells (either wild type or mutant) in RasGRPF’ hosts is associated with an increase in the numbers of +  +  +  .  .  CD4 CD25 Foxp3 cells (Fig. 5.3, B and C). This result raises the possibility that the increase in Foxp3 -expressing CD4 T cells in the periphery of RasGRP 1 .1- mice could at least be due in part to the conversion of CD4CD25 cells into CD4CD25Foxp3 cells. However, because a small percentage of donor CD4CD25 cells was Foxp3 before transfer (see Fig. 5.2B), the increase in the number of CD4CD25Foxp3 cells in RasGRPF’ hosts could +  +  +  + + either be due to the conversion of CD4 CD25 Foxp3 cells into CD4 CD25 Foxp3 cells or •  the conversion of CD4CD25Toxp3 cells into CD4CD25Foxp3 cells. However, the fact that neither transferred mutant CD4CD25 or CD4CD25 cells could survive in wild-type hosts (Fig. 5 .3B) indicates it is the mutant environment rather than cell intrinsic effects of the RasGRP1 mutation that plays a dominant role in the preferential increase of peripheral CD4 Treg cells in mutant mice.  In contrast to the development of CD4 Treg cells, intrathymic development of memory phenotype CD8 Treg cells is not affected in RasGRP1 mice. It is likely that the memory phenotype CD8 T cells that developed in RasGRP 1 mice represent multiple lineages of CD8 T cells. Consistent with this hypothesis is the observation that these memory phenotype CD8 T cells are heterogeneous in terms of expression of cell surface markers such as CD44, CD122, NK1.1, Ly6C, CD94, NKG2A!C/E, NKG2D, and 2B4 (Fig. 5.6). These cells are also heterogeneous in IFN-y production, with only 8.4 and 13.3% of total CD8 cells in the thymus and spleen, respectively, producing this cytokine upon PMA and ionomycin stimulation (Fig. 5.7B), whereas 15 and 50.7% of total CD8 cells (thymus and spleen, respectively) possess the memory phenotype (CD44hCD 1 22k) (Fig. 5.5A). The preferential development of CD8 T cells that possess innate-like properties has also been observed in Tec  175  kinase-deficient mice (34-36). The observation that CD8 T cells of similar cell surface phenotype and function develop in both RasGRP1 and Tec-kinase-deficient mice also suggests that RasGRP1 and Tec kinases activate components of the same signaling pathway. It is also likely that a common denominator in the generation of these memory phenotype CD8 T cells is the expression of relatively high affinity TCRs for self ligands, and therefore, these cells can develop via a RasGPR1-independent mechanism. Consistent with a previous report (12), we found that activated wild-type CD8CD122 cells are able to produce IL-b and efficiently suppressed the proliferation of anti-CD3-, anti-CD28-, and IL-2-activated wild-type CD8CD122 cells. Furthermore, culture supematants derived from wild-type +  + CD8 CD 122 cells activated with anti-CD3, anti-CD28, and IL-2 were also highly .  .  .  suppressive. However, similarly activated mutant CD8CD 1 22 cells were much less suppressive at low suppressor. to responder ratio. In addition, supematants of activated mutant CD8CD122 cells failed to show detectable suppressive activity. Because we have previously shown that RasGRP1 plays a fundamental role in the developmental programming of Ag-activated CD8 T cells by providing signals necessary for their survival (21), it is possible that the less efficient suppressive activity of activated mutant CD8CD 1 22 T cells may be due to the poorer survival of these cells. This poorer survival may also affect their ability to sustain the production of suppressor cytokines such as IL- 10. Collectively, these results indicate that although RasGRP1 is not required for the development CD 1 22 cells, the suppressive function of these cells is critically dependent on RasGRP 1.  Consistent with our observation that peripheral lymphoid tissues of RasGRPF’ mice harbor large numbers of both CD4 and CD8 Treg cells, we did not observe any overt autoimmune disorders in these mice, despite our hypothesis that only T cells expressing TCRs with high affinity for self ligands can develop in these mice. This observation contrasts with observations in a mouse strain that harbors a spontaneous mutation in the RasGRP1 (RasGRP 1 lag) gene (41). In RasGRP 1 lag mice, there are massive lymphoproliferation and development of an autoimmune syndrome that share similarities with systemic lupus erythematosus (41). It is possible that the autoimmune phenotype that developed in RasGRP1 mice might be due to contributions of the hybrid 129:B6 background in addition to disruption of RasGRP1 gene function (42). Consistent with this hypothesis, we did  176  observe massive lymphoadenopathy and splenomegaly in RasGRP1 mice in early backcrosses of 129 mutant mice to B6 mice. However, upon more than seven generations of backcross to the B6 background, no overt autoimmune symptoms were observed in RasGRPl mice.  Our studies demonstrate that although the development of CD4 Treg cells in the thymus of RasGRP F’ mice is very inefficient, peripheral mechanisms exist to greatly expand the numbers of CD4 Treg cells. It is likely that these CD4 Treg cells may be involved in preventing the development of overt autoimmune disease in RasGRPF’ mice. By contrast, there is very efficient development of a heterogeneous population of memory phenotype CD8 T cells in the thymus, resulting in higher frequencies of these cells in the periphery of RasGRP1 mice. These memory phenotype CD8 T cells also include cells that can perform innate immune functions. To explain the contrasting functions of these memory phenotype CD8 T cells that exhibit both innate functions and suppressive functions, we propose differential conditions used to activate memory phenotype CD8 T cells from either wild-type or RasGRPF’ mice lead to distinct immunological functions. The development of memory phenotype CD8 T cells in RasGRPF’ mice may be due to high affinity TCR interactions with self Ags, because we have previously demonstrated the high expression levels of the memory markers CD44 and CD 122 found in naive mice are maintained when the self Ag is present (43, 44). These studies complement those conducted in Itk and Itk’Rlk’ mice and support the hypothesis that unconventional CD8 T cells differ from conventional CD8 T cells in the requirement for Itk, Rlk, and RasGRP1 for their development. More importantly, our studies indicate RasGRP1 signaling plays a discriminative role in the intrathymic development of CD4 and CD8 Treg cells, whereas it is not required for peripheral expansion of either lineage.  177  5.5  Acknowledgements  We thank Soo-Jeet Teh for technical assistance, Salim Dhanji for insightful discussions, the Wesbrook Animal Unit for animal husbandry, and the Life Sciences Centre Flow Cytometry Facility for cell sorting.  178  5.6  Figures  Figure 5.1 Inefficient intrathymic development of CD4Foxp3 Treg cells in RasGRPF’ mice. A, CD4/CD8 developmental profiles of wild-type and RasGRP1’ mice. The expression of Foxp3 (detected by intracellular staining) and CD25 by gated CD4 SP thymocytes is indicated. B, Recovery of Foxp3 and Foxp3 CD4 SP thymocytes in wild-type and RasGRPF’ mice. Error bars represent the SD of 3 mice per group. The yield of Foxp3 and Foxp3 cells as a percentage of wild-type numbers is as indicated. C, The CD4/CD8 profiles of Foxp3-expressing cells in thymus. D, Representative flow cytometric analysis of developmental markers on Foxp3 CD4 SP thymocytes. Wild-type and mutant subpopulations are represented in shaded and bold line histograms, respectively. Numbers shown on the plots indicate percentages of gated population.  Figure 5.2 RasGRP 1 mutant mice possess a high frequency of activated peripheral CD4Foxp3 cells. A, Representative flow cytometric analysis of CD25 and Foxp3 expression on wild-type and RasGRPl peripheral CD4 T cells. Plots were gated on total cells and CD4 cells, respectively. B, Recovery of RasGRPF’ CD4Foxp3 or CD4Foxp3 spleen cells as percentages of wild type. Number of animals used is as follows: +1+, n  5; -7-,  n = 6. C, Flow cytometric analyses of developmental and activation markers of gated CD4Foxp3 or CD4Foxp3 spleen cells. Shaded and bold line histograms represent staining of wild-type and mutant cells, respectively. Data shown are representative of three independent analyses.  Figure 5.3 Preferential peripheral expansion of CD4 Treg cells in RasGRPF mice. A, Correlation between annexin V or Ki-67 vs Foxp3 expression of wild-type or RasGRPF’ CD4 T cells by intracellular staining directly ex vivo. Isotype control Ab staining was negligible. Numbers indicate percentages of annexin V- or Ki-67-positive cells within total Foxp3 or Foxp3 subpopulations. Data shown are representative of three independent experiments. B, RasGRPF’ mice provide a favored environment for CD4 Treg cell  179  expansion. Sorted CD4CD25 (2 x 10) or CD4CD25 (1 x 106) Thyl .1 wild-type cells were adoptively transferred into naive B6 Thy1.2 wild-type or RasGRP1 hosts (top panel). Similarly, sorted CD4CD25 (2 x 10) or CD4CD25 (1 x 106) Thyl.2 RasGRP1 cells were adoptively transferred into naive Thy1.1 wild-type or RasGRP1 hosts (bottom panel). Recovery of donor cells was accessed 3 wk postadoptive transfer. Numbers shown are the percentages of donor cells within the recipient’s total spleen. C, Same type of experiment as in B. The data indicate increased Foxp3 expression by transferred wild-type or mutant CD4CD25 donor cells that were transferred into RasGRPF’ hosts. Plots shown are gated on donor CD4 cells.  Figure 5.4 Enhanced suppressive activity of peripheral RasGRPF’ CD4CD25 Treg cells in vitro. Graded doses of sorted B6 wild-type (gray circles) or RasGRP1’ (black squares) mutant CD4CD25 cells were cultured with 2 x 1 0 sorted B6 wild-type CD4CD25 cells for 72 h with 2 ig/ml Con A and 8 x  irradiated B6 APCs. This assay was set up in 96-  H] thymidine (1 iCi per well) was added to the culture for 3 well round-bottom plates, and [ the final 8 h. Numbers on horizontal axis indicate the ratio of CD4CD25 (suppressor) cells H] thymidine incorporation in 3 to CD4CD25 (responder) cells. All data are shown as mean [ triplicate cultures. The asterisks (*) represent p values of <0.05, calculated using unpaired, two-tailed Student’s t test.  Figure 5.5 Efficient development of  22CD8 cells in both thymus and  periphery of RasGRPF’ mice. A, Greatly increased proportion of mature memory phenotype CD8 SP T cells in the thymus of RasGRP1 mice. The thymus of RasGRPF’ mice contains 22+CD24b0 cells. B, Increased a greatly increased percentage of + + +-. . both thymus and spleen of RasGRP1 .1. mice. proportion of CD8 TCRJ3 INK1.1 T cells in  Numbers shown represent the percentages of gated CD8 SP cells. C, Annexin V and Ki-67 vs CD 122 staining of wild-type and RasGRP1 CD8 splenocytes directly ex vivo. Numbers in the plot reflect the frequency of annexin V- or Ki-67-positive CD8 cells within the total CD122 or CD122 CD8 population. D, Increased CD44highCDl22+CD8+ and NK1.1CD8 T cells in contrast to decreased CD44b0CD8+ T cells were observed in both thymus and spleen  180  of RasGRPl mice relative to wild-type mice. Number of animals analyzed is as follows: +1+, n = 6; -I-, n = 7. Error bars, SD. Thy, Gated on CD8 SP thymocytes; Spl, gated on CD8 spleen cells.  Figure 5.6 Increased expression of NK receptors by peripheral CD8 T cells from RasGRPF mice. Representative flow cytometric analysis of splenocytes from wild-type and RasGRP i mice. The histograms denote expression of the indicated cell surface marker by gated CD8 spleen cells. Numbers shown are percentages of gated cells. Number of animals analyzed is as follows: wild type, n = 4; RasGRPF’, n = 5. Phenotypic data from one representative experiment are shown.  Figure 5.7 RasGRPF’  cells proliferate in response to IL-2 or IL-15  and produce IFN-y in response to PMA plus ionomycin stimulation. A, CD8CD122 and CD8CD122 T cells were purified from spleens of wild-type or RasGRPF’ mice, CFSE labeled, and cultured in IL-2 (200 U/mi) or IL-iS (100 ng/ml). After 72 h, CFSE profiles of both cell types were analyzed by FACS. B, IFN-’ production by wild-type or RasGRPF’ CD8 SP thymocytes and CD8 splenocytes was determined after PMA plus ionomycin stimulation for 5 h. Results shown are representative of three experiments.  Figure 5.8 Activated RasGRP 1 CD8CD 1 22 cells are less suppressive than activated wildtype CD8CDl22 cells. A, IL-b production is evaluated by intracellular FACS staining after activating the indicated sorted populations (+/+, wild type; -/-, RasGRPF’ by anti CD3/CD28 plus IL-2 for 3 days. B, Sorted Thyl .2 wild-type (CD8CD122, CD8CD122) or RasGRP1 (CD8CD122j cells were stimulated with anti-CD3/CD28 plus IL-2. Three days later, cultured cells and supematants were harvested. The indicated activated cells (suppressor) were cultured with Thy l.1 CFSE-labeled wild-type CD8CDl22 cells (responder) at the indicated suppressor to responder ratios. The responder cells were activated with anti-CD3/CD28 plus IL-2 for 2 days. At the end of 2 days, proliferation of Thy l.1 CD8CDl22 cells was determined by analyzing the CFSE profiles of the responder  181  cells. Where indicated, culture supernatant from the first culture was added to responder cells at a 1/2 dilution. The CFSE assay was done in triplicate. The CFSE profiles shown are representative of three experiments.  182  A  :6.29  B  RasGRP1  Wild Type 89.3  0.44  10  94.6 Total  2.25  2.08  3.74  I  1.22  CD8 1.68  308  S Wild Type I RasGRPI’  0.1 1.65  2.  2  ..  c.) CI)25  0.01  0.001 CD4Foxp3  CD4Foxp3  C  ildType  D  kasGRP[  .  13  RasGRP1  Wild Type  51  n150  [  f.  I  Foxp3  CD8  t  I  .  TCR  (‘I)3i  ..  (D5  (1)69  Figure 5.1 Inefficient intrathymic development of CD4Foxp3 Treg cells in RasGRPF’ mice.  183  A  Wild Type  4  V  4  i  I  t  B  WT8.6  Wild Type 2 82  .  RasGRP1 lii.  Total  f 2 fj  !  C  RasGRP1  Foxp3  9.41  24  J5 CD25 60  in.: iA AA M c., 4 1  TCRD  40  20  0 CD4i?oxp3  I  Toxp3 1 CD4  CD3e  -  Foxp3  Foxp3  ..  CD5  Foxp3  Foxp3  CD44  CD62L  CD69  Figure 5.2 RasGRP1 mutant mice possess a high frequency of activated peripheral  CD4Foxp3 cells.  184  A 4  Wild Type 4  Host  B  RasGRP1  [  =  I  ;. 4  Donor  RasGRP1  Wild Type  r  22  >1  7.2  15.6:  z  31.1  +1+ CD4CD25  -  I  r  Spleen CD4  L  .  o.iTi  -‘  +1+ CD4CD25  -  r  t 0.:  I  I  4  --  1  0  0.16r\ —I— CD4CD25  “s../’  Foxp3  C  Host Wild lvpe 0.8  I RasGRPI’  3.6  1.2  4.8  6.8  .  0.12iN  .1... CD4CD25  Donor  +1+ CD4CD25  1.9  0.O3uINl  CD4  4.9  —I— CD4CD25  CD2S  Figure 5.3 Preferential peripheral expansion of CD4 Treg cells in RasGRPF mice.  185  50  Wild -W RasGRPi  40 30 20  10  S Only  2  1 0.5 0.25 0.125 0.0625 R Only S (uppressor) : R (esponder)  Figure 5.4 Enhanced suppressive activity of peripheral RasGRP1 CD4CD25 Treg cells in vitro. 186  A  Wild Type  rz  B  RasGRP1  .,.  RasGRP[  —  —  Thymus  CD44  1 0.4  Wild T3pe  cis s  0.98  6.15  i:-i  6.03  Spleen CD8  TCR___ CD24  r  t  Spen  106  ::  (1)44  C 4  Wild Type RasGRP1 5.8 12.8 22.9 18.4  1.  27.3’  37.8  39.4”  34.8  I[ Thy  Spi  CD44CD122  Thy  Spi  NKL1  Thy  SpI  CD44°  CD122  Figure 5.5 Efficient development of  cells in both thymus and  periphery of RasGRP1 mice. 187  RasGRP1  Wild Type CD122  iF  1  / 1  CDl22  CD122  CD122 83.$  20.21  II  H  82.1  17.81  Ly6C  /  H  R  iJ VH --‘-—.  1  d 15  0.51  H 2.06  J 42.3 HI  H  .1  I  NKG2A/C/E  I  i/ ii-’ 5.12  32.1 i  it, 1  4)  H  [.  ,i,  1.33  ‘  12.4 1!  1 I  ?j  i  I  H  ‘J  fI  CD94  Li \  7.28  1 u  .  .  25.6  j’..; 0.76  :‘  .1  NKG2D  7.08 I  I  I  2B4  . .  ....4  7 21 f 2 ’  r  NK1J  pI  1  71.4 j  97.3  H  I  68.2  I  ii  :  I  I ....../  1’  ,i  I  96.5  I  I  Ij  I CD62L  1,  96.4  86.1  1ii  !  CD127  ..  Figure 5.6 Increased expression of NK receptors by peripheral CD8 T cells from RasGRPF ‘  mice. 188  A  I  IL-2  Wild Type  IL-IS  CD122  1°  U.  RasGRPl  0.1  0  13.4  L41.61  2.2  ‘[A  I  I I  0.O3  -  j  PMA+  /39.4 i85.2 0.24  s-.  5.8 0.65  10.02  Control  6 M!L  L1.49.5  2.1  CFSE  5.7 RasGRPl  B  8.4  CDI 22  FT  Figure  0.56  I  I( 58,1  rj  0.55  Controi  583  CD122  1  I  CM  13.31  PMA+ lonomycin  CDI 22 72.  ‘—  2  35.2  48.F  CD44  cells proliferate in response to IL-2 or IL-15  and produce IFN-y in response to PMA plus ionomycin stimulation. 189  A  +1+ CDrCD122  +1+ CD8CD122  CD122 4 CD8  9.7  0.4  17  CD8  B  Suppressor  (S) uppressor: R) esponder  :  I  j.  —  D122  :1  :  A  A1  A  A  Supernatant  1:45  1:15  1:5  CD122  [ -—  • I —,  .1 I  I••.’ ‘S  RasGRP1” CDl22  IA ‘  .1  FSE Responder: CFSE4aheled Wild Type Thyl.l) cDrCDl22 cells  Figure 5.8 Activated RasGRP 1 CD8CD 1 22 cells are less suppressive than activated wild-  type CD8CD122 cells. 190  5.7  References  1.  Hogquist, K., T. Baldwin, and S. Jameson. 2005. Central tolerance: learning selfcontrol in the thymus. Nat Rev Immunol 5:772-782.  2.  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Self-reactive memory-phenotype CD8 T cells that exhibit both MHC-restricted and non-MHC-restricted cytotoxicity: a role for the T cell receptor and natural killer receptors. Blood 104:2116-2123.  44.  Dhanji, S., M. Chow, and H.-S. Teh. 2006. Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo. Jlmmunol 177:138-146.  195  CHAPTER 6  6.1  GENERAL DISCUSSION AND PERSPECTIVE  Summarizing the role of RasGRP1 in T cell development and function  This thesis has focused on how different aspects of immune system are affected by a deficiency in RasGRP 1. Novel roles of RasGRP 1 in T cell development, homeo stasis and peripheral function were identified. The TCR transgenic mouse models provided us with a powerful tool to study the effect of RasGRP 1 null mutation in thymocyte development and T cell function. Chapter 2 was focused on the consequences of RasGRP1 null mutation in CD4 T cell development by using MHC class TI-restricted AND TCR Tg mice (1). We found that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP 1. RasGRP 1 DP thymocytes exhibit marked deficits in TCR-stimulated upregulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2. To evaluate the role of RasGRP 1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1 thymocytes. However, Tg Bcl-2 expression failed to restore CD4 T cell development in RasGRP 1 -deficient mice, indicating that RasGRP 1 transmits differentiation signals independent of those necessary for Bcl-2 induction (1). Following the notion that the RasGRP 1 -Ras/MAPK pathway is particularly important for imparting differentiation signals that are essential for CD4 T cell development, we went on to study how the severely impaired thymic development would affect peripheral T cell homeostasis and T cell activation against pathogens (2) in Chapter 3. The results in that chapter demonstrate that despite apparently normal central tolerance, RasGRPF’ mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion. To elucidate the basis for RasGRP 1 CD4 T cell immune activation, we initiated a series of adoptive transfer experiment using acutely and chronically immunodeficient hosts. Remarkably, the copious amounts of cytokines and self Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP 1 CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and signs of acute activation. These findings imply that the activation and proliferation of RasGRP1 CD4 T  196  cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRPF mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Therefore, our results in Chapter 3 provide a reasonable explanation of the activated phenotype of CD4 T cells in RasGRPF’ mice, which suggest that chronic T cell immunodeficiency in RasGRPF’ mice, resulting from impaired T cell development, may be responsible for CD4 T cell activation, proliferation, and exhaustion.  In Chapter 4, follow-up studies of the Immunity paper (Priatel et al. 2002) were performed to study the impact of RasGRP1 deficiency in naïve CD8 T cell activation by using 2C TCR Tg mouse model lacking RasGRP 1. Our results suggest RasGRP 1 regulates the threshold of T cell activation and Ag-induced expansion, at least partly, through the regulation of IL-2 production. Moreover, RasGRPF’ 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is reversible upon addition of exogenous IL-2. However, the capacity of IL-2/IL-2 receptor interaction to mediate Ras-activation and CD8 T cell expansion and differentiation appears to be largely RasGRP 1-independent. Although our previous studies have uncovered the significant role of RasGRP 1 in the development and function of major T cell subsets, defmed by the expression of coreceptor, we neglected a population that is thought to play an important role in the peripheral tolerance. Numerous studies on Treg cell suggest that this population requires high affinity TCR-MHC/self peptide engagement for development and the production of regulatory cytokines to suppress conventional T cell activation and immune responses (3). As such, the results presented in Chapter 5 (4) provide detailed analysis of CD4 and CD8 Treg cell development, homeostasis and function in the absence of RasGRP 1. Despite impaired CD4 Treg development in the thymus, the periphery of RasGRPF’ mice contained significantly increased frequencies of CD4 Treg cells that possessed a more activated cell surface phenotype. Interestingly, mutant CD4 Treg cells are more suppressive than their wild-type counterpart in inhibiting the proliferation of non-Treg CD4 T cells. In contrast, the intrathymic development of innate-like CD8 Treg cells, defined by CD44hiCD122+, is unaffected in RasGRP1 mice. Moreover, RasGRP i mice contain greater numbers of CD8 Treg cells in the spleen, relative to wild-  197  type mice. Activated CD8 Treg cells from RasGRP 1 mice retained their ability to synthesize IL-lO and suppress the proliferation of wild-type non-Treg CD8 T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells. These results complement our previous studies and point out the crucial role of RasGRP1 in transducing optimal signal in the development and function of different T cell lineages. Overall, this thesis provides comprehensive analysis of the consequence of RasGRP1 deficiency on conventional T cell development, the regulation of T cell homeostasis, the development of Treg cells, and the regulation of immune responses. Our studies suggest that RasGRP1 is a crucial modulator downstream of TCR signaling controlling signal threshold during thymic development and peripheral function, and its presence is essential for the maintenance of peripheral tolerance and T cell homeostasis.  6.2  RasGRP1 signaling, positive selection and lineage commitment  CD8 T cells normally express MHC class I-restricted TCRs. Unexpectedly, our results in Chapter 2 (1) revealed that a small number of functional CD8 SP thymocytes expressing the Tg MHC class IT-restricted TCR exists in RasGRP1 mutant mice. Furthermore, these unusual mutant CD8 SP thymocytes express markers of positive selection and are able to respond to a MHC class IT-specific Ag and differentiate into cytotoxic effectors. These results raised questions regarding their nature and origin, as well as the role of RasGRP1 in lineage commitment during thymocyte development. However, the difficulty of distinguishing consequences of TCR signaling on thymocyte survival and T cell lineage commitment has hindered the assessment of the role of RasGRP 1 in regulating these processes. Recent studies using mice devoid of CD4 helper T cells (HD) and Th-POK Tg mice have suggested that Th P0K is a master regulator of commitment of developing thymocytes to the CD4 T cell lineage during positive selection (5, 6). By collaborating with Bosselut group, we backcrossed Th-POK Tg mice onto the RasGRP1 background to investigate if the overexpression of Th-POK can rescue CD4 T cell development in AND RasGRPf’ mice. One of key discovery in their previous paper (6) is that the up-regulation of zbtb 7b (thpok) expression is tightly correlated with the intrathymic TCR signaling received by the  198  developing thymocytes. Only CD69-expressing DP thymocytes were able to show detectable level of zbtb7b expression in the RT-PCR analyses. Since our data revealed that RasGRPl AND TCR Tg DP thymocytes possess reduced TCR expression, appear slightly immature phenotype and exhibit dramatically decreased CD69 expression (Fig. 2.2B), plus RasGRPl DP thymocytes exhibit marked deficits in CD69 up-regulation against TCR stimulation (Fig. 2.3D), it is interesting to see if the expression of Th-POK is affected by RasGRP 1 deficiency. Our preliminary data (Chen et al., manuscript in preparation) have shown that consistent with reduced CD69 expression level, RasGRPF’ DP from either B6 or AND TCR Tg mice express significantly lower level of zbtb 7b expression, compared to their wild-type counterpart. Furthermore, we have clearly observed that the enforced expression of Th-POK failed to restore CD4 T cell development within the RasGRP1 AND TCR Tg mice, which indicates RasGRP1 may play little or no role in the CD4/CD8-lineage choice while it is extremely crucial for developing thymocytes to translate the positive-selecting TCR signals into pivotal gene expressions, such as zbtb 7b, that are required for T cell lineage commitment.  Although Th-POK was originally thought be both sufficient and necessary for the CD4 lineage during thymic development (5-7), current findings (8-10) challenged this notion and add more complexity to the mechanism of how Th-POK affects the CD4!CD8-lineage choice. The revised model proposes that Th-POK acts downstream of the TCR and GATA-3 to “lock in” the CD4 T cell fate (11). The revised model provides a more superior explanation of the latest data. The new data suggest that upon receiving positive-selecting signals through TCRs, immature DPs up-regulate the expression of GATA-3, which is required for the induction Th-POK expression. Th-POK then commits the developing thymocyte to the CD4 T cell lineage by antagonizing Runx-dependent silencing at both the Cd4 and zbtb7b silencing elements, thereby “locking in” the CD4 T cell fate. As RasGRP1 acts as a signaling intermediate between TCR and crucial nuclear transcription factors such as GATA3 and Th-POK, questions remain as at which stage of this revised model does RasGRP1 fit in. Therefore, future studies will investigate the link between RasGRP1 and those major players in the CD4 lineage commitment, which will provide important novel insight into signaling pathways involved in T cell positive selection and lineage commitment.  199  6.3  Partial T-cdll immunodeficiency and immune dysregulation  Partial T-cell immunodeficiencies constitute a heterogeneous cluster of disorders characterized by an incomplete reduction in T cell number or activity (12). The immune deficiency component of these diseases is less severe than that of the severe T-cell immunodeficiencies and therefore some ability to respond to infectious organisms is retained. Unlike severe T-cell immunodeficiencies, however, partial immunodeficiencies are commonly associated with hyper-immune dysregulation, including autoimmunity and elevated IgE production, both of which are consequences of effector T cell hyperactivity (12). Although the association of effector T cell hyperactivity symptoms with an effector T cell hypoactive disease is counter-intuitive, recent studies in mouse models have shown that hyperactivation of immune system components can be a direct outcome of a partial decrease in T cell number or activity. The ‘population-dependent’ model proposes that tolerance mechanisms evolve in the context of a complete peripheral T cell niche, and these mechanisms start to break down when they are forced to operate in the suboptimal context of a reduction in the functional T cell population size (12). Partial reduction in the number or function of T cells therefore disturbs the balance of tolerance, generating the unfortunate combination of immunodeficiency and immune dysregulation.  Nearly 40 different genes have been identified that cause monogenic severe T-cell immunodeficiencies in human, which can be grouped into five main categories based on the cellular function of the proteins that they encode. The five categories are: antigen presentation, TCR signaling, cytokine signaling, V(D)J recombination and basic cellular processes (12). As in the mouse models, genetic mutations with TCR signaling defects have been well studied. Although ZAP7O deficiency blocks signaling through the TCR and typically results in severe T-cell immunodeficiency (13-15), a comprehensive range of mouse models is available for partial TCR signaling blockades, many of which combine partial immunodeficiency with elevated levels of IgE or autoimmunity (12). Loss of a TCR adaptor (LAT partially impairs TCR signaling, as well as F) protein LAT or a LAT mutant 136  200  accompanied by elevated IgE and IgGi production and multi-organ infiltration of lymphocytes (16, 17). TCR signaling mediator, such as SH2D2A (SRC-homology-2-domain protein 2A; also known as TSAD) or RasGRP1, also belongs to this category.  In Chapter 3, we have provided detailed discussion of a mouse strain call RasGRP1’, which spontaneously developed a lymphoproliferative autoimmune syndrome exhibiting features of systemic lupus erythematosus (SLE) (18). T cells from these mice have been identified to have a spontaneous mutation for Rasgrpl gene, fail to activate Ras and show defects in positive selection. Peripheral  T cells spontaneously adopted a memory  phenotype and were able to transfer disease to lymphopenic recipient mice. CD4 T cells accumulated in the lymphoid tissues of older RasGRP 1 lag mice and were resistant to activation-induced cell death. Consistent with these results, our studies also revealed that RasGRPF’ mice spontaneously acquire an acutely activated and proliferating CD4 population and possess elevated levels of serum autoantibodies (unpublished data) (2). Along with the affected homeostatic proliferation, inflammatory cytokine production, weak T cell responses against pathogenic infection, as well as altered TCR repertoire (2), the RasGRP 1-/ mouse model seems to fit the profile of partial T-cell immunodeficiency and immune dysregulation. Since we have noticed that successive backcrossing of the targeted RasGRP1 mutation to the B6 background alleviated the autoimmune phenotype  mice, it  suggests that genetic factors from strain 129 mice may have contributed to the development of autoimmune disease in RasGRP 1-deficient mice. Our results in Chapter 5 (4) support this explanation.  The ‘population-dependent’ model (12) hypothesizes that under homeostatic conditions, regulatory T cells control autoreactive T cells, through the expression of a complementary TCR repertoire specific for a particular antigen set. In the case of partial thymic immunodeficiency, a reduced number of thymocytes with restricted TCR repertoire develop and enter the periphery. This results in oligoclonal expansion and hence “holes” in the TCR repertoire for both Treg cells and autoreactive T cells, which by chance can create gaps in antigen specific tolerance. However, the lymphopenic compartment in RasGRP1 mice might also provide a beneficial environment for the homeo stasis and/or expansion of Treg  201  cells, as evidenced from our adoptive transfer results (Fig. 5.3) (4). Favored expansion of Treg cells as well as increased conversion from non-Treg cells to Treg cells have both been observed, which suggest that the Treg population might have advantages, compared to conventional T cells, in utilizing cytokines (such as IL-2, IL-7 and IL-15) in an “empty” T cell compartment. Therefore, our data in this chapter argue that it is the Treg cells that help RasGRP 1 mice fight against self-reactive and highly proliferative T cells and remain healthy until at least 1 year of age, providing a reasonable explanation for the question raised in chapter 3.  6.3  Concluding remarks  This thesis characterizes the role of RasGRP 1 in T cell development, homeo stasis and function. With deficiency of the RasGRPI molecule, the development of CD4 T cells is severely impaired, arguing RasGRP1 is critically required for transmitting differentiation signaling for CD4 T cell development. As a result, the chronic T cell immunodeficiency due to the poor T cell development in RasGRP1 null-mutant mice might be responsible for the activation, proliferation and exhaustion of CD4 T cells. We also found that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses. In addition, RasGRP1 signaling plays a discriminative role during the intrathymic development of CD4 and CD8 Treg cells, but is not required for the peripheral expansion of both lineages. Overall, the results presented in this thesis highlight the crucial role for RasGRP1 signaling pathway in various important aspects of the immune system, and provide a potential strategy of targeting RasGRP1 for therapeutics against autoimmune diseases.  202  6.4  References  1.  Priatel, J. J., X. Chen, S. Dhanji, N. Abraham, and H. S. Teh. 2006. RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development. Jlmmunol 177:1470-1480.  2.  Priatel, J. J., X. Chen, L. A. Zenewicz, H. Shen, K. W. Harder, M. S. Horwitz, and H. S. Teh. 2007. Chronic immunodeficiency in mice lacking RasGRP1 results in CD4 T cell immune activation and exhaustion. Jlmmunol 179:2143-2152.  3.  Hogquist, K. A., T. A. Baldwin, and S. C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat Rev Immunol 5:772-782.  4.  Chen, X., J. J. Priatel, M. T. Chow, and H. S. Teh. 2008. Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice. Jlmmunol 180:59735982.  5.  He, X., V. P. Dave, Y. Zhang, X. Hua, E. Nicolas, W. Xu, B. A. Roe, and D. J. Kappes. 2005. The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433:826-833.  6.  Sun, G., X. Liu, P. Mercado, S. R. Jenkinson, M. Kypriotou, L. Feigenbaum, P. Galera, and R. Bosselut. 2005. The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat Immunol 6:373-38 1.  7.  Kappes, D. J., and X. He. 2005. CD4-CD8 lineage commitment: an inside view. Nat Immunol 6:761-766.  8.  Egawa, T., and D. R. Littman. 2008. ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat Immunol 9:1131-1139.  9.  Muroi, S., Y. Naoe, C. Miyamoto, K. Akiyama, T. Ikawa, K. Masuda, H. Kawamoto, and I. Taniuchi. 2008. Cascading suppression of transcriptional silencers by ThPOK seals helper T cell fate. Nat Immunol 9:1113-1121.  203  10.  Wang, L., K. F. Wildt, J. Zhu, X. Zhang, L. Feigenbaum, L. Tessarollo, W. E. Paul, B. J. Fowikes, and R. Bosselut. 2008. Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells. Nat Immunol 9:1122-1130.  11.  Bell, J. J., and A. Bhandoola. 2008. Putting ThPOK in place. Nat Immunol 9:10951097.  12.  Liston, A., A. Enders, and 0. M. Siggs. 2008. Unravelling the association of partial T-cell immunodeficiency and immune dysregulation. Nat Rev Immunol 8:545-558.  13.  Arpaia, E., M. Shahar, H. Dadi, A. Cohen, and C. M. Roifman. 1994. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell 76:947-958.  14.  Chan, A. C., T. A. Kadlecek, M. E. Elder, A. H. Filipovich, W. L. Kuo, M. Iwashima, T. G. Parsiow, and A. Weiss. 1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264:1599-1601.  15.  Elder, M. B., D. Lin, J. Clever, A. C. Chan, T. J. Hope, A. Weiss, and T. G. Parsiow. 1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 264:1596-1599.  16.  Aguado, E., S. Richelme, S. Nunez-Cruz, A. Miazek, A. M. Mura, M. Richelme, X. J. Guo, D. Sainty, H. T. He, B. Malissen, and M. Malissen. 2002. Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 296:2036-2040.  17.  Sommers, C. L., C. S. Park, J. Lee, C. Feng, C. L. Fuller, A. Grinberg, J. A. Hildebrand, E. Lacana, R. K. Menon, E. W. Shores, L. E. Samelson, and P. E. Love. 2002. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 296:2040-2043.  18.  Layer, K., G. Lin, A. Nencioni, W. Hu, A. Schmucker, A. N. Antov, X. Li, S. Takamatsu, T. Chevassut, N. A. Dower, S. L. Stang, D. Beier, J. Buhlmann, R. T.  204  Bronson, K. B. Elkon, J. C. Stone, L. Van Parijs, and B. Lim. 2003. Autoimmunity as the consequence of a spontaneous mutation in Rasgrpl. Immunity 19:243-255. 19.  Dower, N. A., S. L. Stang, D. A. Bottorff, J. 0. Ebinu, P. Dickie, H. L. Ostergaard, and J. C. Stone. 2000. RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol 1:317-321.  205  APPENDIX: BIOSAFETY AND ANIMAL CARE CERTIFICATES  THE UNIVERSITY OF BRITISH COLUMBIA  Department of Health, Safety and Environment  Xiaoj Clteii has successfully completed a course in  Laboratory Biological Safety Chair, Biosafety Committee  Biosafety Officer  Thursday, February 05,2004  Dire&r Health, Safety and Environment  206  Certificate of Training for the Completion of a Course in  Chemical Safety Orientation  UBC  Awarded to  Xiaoxi Chen The course content included WHMIS, chemical handling and storage, safety controls, personal protective equipment, hazardous waste management and emergency procedures. Presented by  Department of Health, Safety and Environment Tuesday, March 16, 2004  David Bell Occupational Hygiene Officer  207  _  THE UNIVERSITY OF BRITISH COLUMBIA  ment Department of Health, Safety and Environ  Xiaoxj Cfien r Safety Commission requirement In has successfully completed the Canadian Nuclea  Radionuclide Safety and Methodology Including receleh.g Class 7 Dangerous Goods  Radiation Hazards s Cbssr, Committee on Radloisotope and  CoorseDate Thursday, March 25, 2004  Radiation Safk Officer  Health, Safety and Environment  208  UBC  THE  UNIVERSITY OF BRITISH COLUMBIA  Xiaoxi Chen has successfully completed the online training requirements of the Canadian Council on Animal Care (CCAC) I National Institutional Animal User Training (NIAUT) Program  Chair, Animal Care Committee  Certificate #: 0955  Veterinarian  Date Issued: September 08, 2005  209  

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