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The development and function of self-specific CD8 T cells Dhanji, Salim 2006

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T H E D E V E L O P M E N T A N D FUNCTION O F S E L F - S P E C I F I C C D 8 T C E L L S  by Salim Dhanji B . S c , University of British Columbia, 2001  A T H E S I S SUBMITTED IN PARTIAL F U L F I L L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies Microbiology  T H E UNIVERSITY O F BRITISH C O L U M B I A July 2006  © Salim Dhanji, 2006  II  Abstract  A distinguishing feature of conventional T cells is that they are tolerant to selfpeptides that are presented by self-major histocompatibility complex (MHC) molecules even though these cells are positively selected by low affinity interactions between their T C R s and self-peptide/MHC ligands in the thymus. Over the last few years several subsets of unconventional T cells have begun to emerge which often rely on strong interactions between their T C R s and selfantigens for both their development and function. Furthermore, these unconventional T cells often express receptors that are not commonly associated with T cells but rather are associated with cells of the innate immune system. The work in this thesis is focused on the discovery and characterization of a novel subset of C D 8 T cells which demonstrate a high affinity for self-antigens and are thus self-specific. These self-specific C D 8 T cells can be found in normal mice as well as in self-antigen expressing T C R transgenic mice. Self-specific C D 8 T cells are selected by high-affinity interactions either in the thymus or extrathymically and possess a memory-phenotype. A s a consequence of this memoryphenotype, which is associated with high expression of CD122 (IL-2RP), these cells can proliferate in an antigen-independent manner in response to stimulation with interleukin-2 or -15. Upon activation, the cells express several natural killer cell receptors including N K G 2 D , CD94, 2B4, and CD16. These NK receptors can act independently or in concert with the self-specific T C R leading to target cell killing and cytokine production. Self-specific C D 8 T cells require self-antigen interactions not only for their development but also for the maintenance of their memory-phenotype and cytokine responsiveness. These cells become activated in response to infection or inflammation and this activation results in the innate production of cytokines such as IFNy which are protective during infection. Thus, self-specific C D 8 T cells represent a novel C D 8 T cell subset that utilize their T C R s , cytokine receptors as well as N K receptors to provide early protection against infection and the elimination of either stressed or transformed cells.  Table of Contents  Abstract  ii  Table of Contents  iii  List of Tables  viii  List of Figures  ix  List of Abbreviations  xi  Acknowledgements  xiii  Co-Authorship Statement  xiv  Chapter 1 Introduction 1.1  1  Preface  1  1.2 Receptors of the Innate and Adaptive Immune system 1.2.1 Biology of NK cells 1.2.2 Activating and inhibitory receptors of NK cells 1.2.3 Activating and inhibitory receptors of T cells 1.2.4 NK receptor expression by memory-phenotype C D 8 T cells 1.2.5 The role of interleukin-2 and 15 in NK and T cell homeostasis  1 1 4 8 12 15  1.3 T cell development 1. 3.1 Positive and negative selection of the naive T cell repertoire 1.3.2 Extrathymic T cell development  17 17 20  1.4 Positive selection of unconventional T cells 1.4.1 NKT cell development and function 1.4.2 T C R a p C D 8 a a Intestinal intra-epithelial lymphocytes (ilELs) 1.4.3 Agonist selection of unconventional C D 8 T cells in male H-Y T C R transgenic mice  23 23 25  1.5  Thesis objectives  28  1.6  Tables  30  1.7  Figures  32  1.8  References  36  +  27  Chapter 2 IL-2 Activated C D 8 C D 4 4 Cells Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells 66 +  2.1  Abstract  h i g h  66  iv 2.2  Introduction  67  2.3 Materials and Methods Mice Abs and flow cytometry Cell lines Ex vivo staining C D 8 T Cell purification and sorting Natural Killer (NK) cell purification and activation C F S E labeling Proliferation assays C T L Assays RT-PCR  69 69 69 70 71 71 72 72 72 73 73  +  2.4 Results 74 Phenotypic characterization of C D 8 C D 4 4 cells from normal mice 74 C D 8 C D 4 4 T cells proliferate in response to IL-2 stimulation 75 IL-2 activated C D 8 C D 4 4 cells expressed NK receptors 75 CD48 and C D 2 are crucial for IL-2-induced proliferation of C D 8 C D 4 4 cells. 76 Activated C D 8 C D 4 4 cells preferentially kill syngeneic tumors 78 IL-2-activated C D 8 C D 4 4 cells express D A P 12 80 Expression of Rae-18 enhances the lysis of syngeneic tumors by I n activated C D 8 C D 4 4 cells 81 +  +  hl  hi  +  h  +  +  hl  +  +  hl  hi  hi  2.5  Discussion  83  2.6  Acknowledgments  89  2.7  Figures  90  2.8  References  101  Chapter 3 Self-reactive memory-phenotype C D 8 T cells exhibit both M H C restricted and non-MHC restricted cytotoxicity: A role for the T cell receptor and natural killer cell receptors 109 3.1  Abstract  109  3.2  Introduction  110  3.3 Materials and Methods Mice Abs and flow cytometry Cell lines C D 8 T cell purification and sorting Natural Killer (NK) cell purification and activation C F S E Labeling Adoptive transfer Proliferation assays CTL Assays RT-PCR +  :  111 112 112 113 113 114 114 114 115 115 116  V  Immunoblotting Statistical Analysis  116 116  3.4 Results 117 Cell surface phenotype of C D 8 T cells from self-antigen expressing H-Y T C R transgenic mice 117 C D 8 T cells from male H-Y mice possess an increased activation threshold .. 118 IL-2 or IL-15 promotes the proliferation of CD8'° and C D 8 cells 119 Role of antigen and bacterial infection on the expansion of CD8'° cells in vivo 120 Bystander proliferation of CD8'° cells in response to bacterial infection.,... 121 CD8'° cells express N K receptors after activation 122 N K G 2 D is an activating receptor for C D 8 cells 123 Listeria infection induces expression of N K G 2 D on proliferating CD8'° cells and a heightened ability to produce IFNy 125 int  |0  3.5  Discussion  126  3.6  Acknowledgements  130  3.7  Figures  131  3.8  References  142  Chapter 4 The Low Affinity Fc Receptor for IgG Functions as an Effective Cytolytic Receptor for Self-Specific C D 8 T Cells  148  4.1  Abstract  148  4.2  Introduction  149  4.3 Materials and Methods Mice Abs and flow cytometry Cell lines Cell purification and activation RT-PCR C T L Assays Immunopreceipitation and immunoblot analysis  152 152 152 153 153 153 154 154  4.4 Results 155 Expression of FcyRllla/FcRy on activated self-specific CD8+ cells 155 IL-2-activated self-specific CD8+ cells can mediate A D C C 157 The Fc receptor on self-specific CD8+ cells from H-Y male mice functions independently of the T C R 159 Engagement of C D 16 on self-specific CD8+ cells induces cytokine production 160 4.5  Discussion  162  4.6  Acknowledgements  164  vi 4.7  Figures  165  4.8  References  173  Chapter 5 Self-Antigen Maintains the Innate Anti-Bacterial Function of SelfSpecific C D 8 T Cells in Vivo 177 5.1 5.2  Abstract  177  Introduction  178  5.3 Materials and Methods Mice Abs and flow cytometry CD8+ T cell purification N K c e l l purification Adoptive transfers, infections, and bacterial load measurement  182 182 182 183 183 184  5.4 Results 184 Self-specific C D 8 T cells in H-Y T C R transgenic male mice can develop in the absence of a functional thymus 184 Activated self-specific C D 8 T cells produce pro-inflammatory cytokines... 187 IFNy production by self-specific C D 8 T cell provides protection against bacterial infection 188 Memory-phenotype C D 8 C D 4 4 cells and self-specific H-Y male C D 8 T cells provide similar innate protection during infection 189 IL-15-activated cells provide protection against LM infection 190 Self-Ag interactions are important for innate protection by self-specific C D 8 T cells 192 Self-Ag interactions are crucial for maintaining the memory phenotype and cytokine-responsiveness of self-specific C D 8 T cells 193 Self-specific C D 8 T cells provide greater IFNy-dependent protection against LM infection than NK cells 194 +  +  hi  5.5  Discussion  196  5.6  Acknowledgements  202  5.7  Figures  203  5.8  References  215  Chapter 6 General discussion and perspectives  223  Chapter 6 General discussion and perspectives  223  6.1 Self-specific C D 8 T cells in non-TCR transgenic mice 223 6.1.1 The relationship between foreign-antigen specific memory C D 8 T cells and self-specific C D 8 T cells 225 6.1.2 The potential involvement of M H C class lb molecules in the selection of self-specific C D 8 T cells 228 6.2  The in vivo significance of self-specific C D 8 T cells  231  VII  6.2.1 The autoimmune potential of self-specific C D 8 T cells 231 6.2.3 The potential role for self-specific C D 8 T cells in anti-tumor immunity.. 234 6.2.3 Other potential roles for self-specific C D 8 T cells 236 6.3  Concluding remarks  237  6.4  References  238  viii List of Tables  Table 1.1 Inhibitory NK receptors for M H C  30  Table 1.2 Activating NK cell receptor complexes and their ligands  31  ix  List of Figures  Figure 1.1 Overall scheme of T-cell development in the thymus  34  Figure 1.2 Central-tolerance mechanisms  35  Figure 2.1 Cell surface phenotype of C D 8 C D 4 4 and CD8 CD44'° T cells +  hi  +  ex vivo  94  Figure 2.2 Only C D 8 C D 4 4 cells proliferate in response to IL-2 +  hi  95  Figure 2.3 Activated C D 8 C D 4 4 cells express several NK receptors 96 Figure 2.4 CD48/CD2 interactions are required for IL-2-induced proliferation of C D 8 C D 4 4 cells 97 +  +  hl  hi  Figure 2.5 Activated C D 8 C D 4 4 cells +  hl  cells preferential killed syngeneic tumor target 98  Figure 2.6 Activated C D 8 C D 4 4 cells expressDAP12 +  hi  99  Figure 2.7 NKG2D/Rae1 interaction enhances killing of syngeneic tumor targets by IL-2 activated C D 8 C D 4 4 cells 100 +  hi  Figure 3.1 C D 8 T cells from antigen-expressing H-Y T C R transgenic mice possess an activated/memory phenotype +  135  Figure 3.2 C D 8 T cells from male H-Y mice possess a high activation threshold due to a defect in T C R signal transduction 136 +  Figure 3.3 The extent of the memory-phenotype of H-Y C D 8 T cells determines their ability to respond to cytokines in vitro as well as their ability to undergo homeostatic expansion in vivo 137 Figure 3.4 Role of antigen and bacterial infection in the expansion of H-Y male CD8'° cells in vivo 138 Figure 3.5 Activated H-Y male CD8'° cells expressed NK receptors and D A P 12 after activation 139 Figure 3.6 N K G 2 D enhances the killing of target cells by male H-Y CD8'° cells in both an M H C restricted and non-MHC restricted fashion 140 Figure 3.7 Bacterial infection primes CD8'° cells in vivo  141  X  Fig. 4.1 Activation of C D 8 C D 4 4 +  hl  cells results in the expression of CD16  168  Fig. 4.2 Activated self-specific C D 8 T cells express a low affinity Fc receptor similar to N K cells 169 Fig. 4.3 C D 8 C D 4 4 cells efficiently kill antibody-coated targets via an Fcdependent mechanism +  hl  170  Fig. 4.4 CD16-mediated killing of target cells by self-specific C D 8 T cells is T C R independent 171 Fig. 4.5 Cytokine production by self-specific C D 8 T cells in response to CD16 engagement 172 Figure 5.1 Limited development of self-specific C D 8 T cells in athymic (nude) HY T C R transgenic male mice 207 Figure 5.2 Self-specific C D 8 T cells rapidly produce inflammatory cytokines upon activation 208 Figure 5.3 Self-specific C D 8 T cells provide an innate source of IFNy during infection 209 Figure 5.4 Self-specific H-Y male C D 8 T cells and memory-phenotype C D 8 T cells from normal B6 mice provide similar protection against LM infection 210 Figure 5.5 Self-specific C D 8 T cells expanded in IL-15 are protective in vivo whereas IL-2 expanded cells are not  211  Figure 5.6 Self-Ag interactions enhance the protection mediated by self-specific C D 8 T cells during infection 212 Figure 5.7 Self-Ag interactions maintain the memory phenotype and cytokine responsiveness of self-specific C D 8 T cells in vivo 213 Figure 5.8 Self-specific C D 8 T cells provide more IFN-y-dependent protection in vivo against LM infection than NK cells 214  List of Abbreviations a-GalCer  alpha-galactosyl ceramide  ADCC  antibody-dependent cell-mediated cytotoxicity  AICD  activation-induced cell death  Ag  antigen  APC  antigen-presenting cell  CD8  hi  female H-2 H-Y T C R transgenic C D 8 T cell  CD8  int  male H - 2  CD8  10  male H-2 H-Y T C R transgenic C D 8 T cell  b  b/d  H-Y T C R transgenic C D 8 T cell  b  CFSE  carboxyfluorescein diacetate succinimidyl ester  CTL  cytotoxic T lymphocyte  DC  dendritic cell  DN  C D 4 C D 8 " d o u b l e negative  DP  C D 4 C D 8 double positive  DLN  draining lymph node  GM-CSF  granulocyte macrophage colony stimulating factor  HSC  hematopoetic stem cell  \ICOS  inducible co-stimulator  +  +  IEL  intra-epithelial lymphocyte  ig  immunoglobulin  ITAM  immunoreceptor tyrosine-based activation motif  ITIM  immunoreceptor tyrosine-based inhibitory motif  JNK/SAPK  jun-N-terminal kinase/stress-activated protein kinase  KIR  killer Ig-like receptor  LAK  lymphokine-activated killer  LAT  linker of activated T cells  LCMV  lymphocytic choriomeningitis virus  LLO  listeriolysin-0  LM  Listeria monocytogenes  LPS  lipopolysaccharide  MAPK  mitogen activated protein kinase  MHC  major histocompatibility complex  NFAT  nuclear factor of activated T cells  NF-KB  nuclear factor K B  NK  natural killer  NKT  natural killer T cell  NOD  nonobese diabetic  OM  oncostatin M  PI3K  phosphatidylinositol 3-kinase  PMA  phorbol 12-myristate 13-acetate  RAG  recombinase activating gene  T-bet  T-box expressed in T cells  TCR  T cell receptor  TLR  Toll-like receptor  TRAF  T N F R receptor-associated factor  TNF  tumor necrosis factor  TNFR  TNF receptor  T N F R 2 , p75  tumor necrosis factor receptor-2  WT  wild-type  ZAP-70  zeta-associated protein of 70 kDa  XIII  Acknowledgements  I would like to thank Hung-Sia Teh for giving me the opportunity to study immunology and providing me with a strong foundation for my future research. I thank Soo Jeet Teh for teaching me the basic principals in immunological research. I would also like to thank members of my thesis supervisory committee including Michael Gold, Ninan Abraham, and Fumio Takei. In addition I thank Mark Horwitz and Shelley Small for their support. Thank you to N S E R C and M S F H R for providing me with funding for my research. I have been blessed to be surrounded by great people and great scientists. I have learned a lot about science from other members of the Teh lab including John Priatel, Edward Kim, Darryl Oble, Mike Chow, and Xiaoxi Chen and I thank them all immensely for sharing their knowledge. In addition I thank all of the people in our department including (but not limited to) SLT, BR, HK, MR, and LO. To my parents and brother thank you for providing me with everything that I needed to be the person I am today. I could not have accomplished this without your guidance and support. To my beautiful children, Khayali and Kaysan, thank you for making the long days worthwhile. The biggest thank you goes out to my beautiful wife Neilin whose love and support has guided me through all of the hard times. If it was not for you I would never have survived the disappointments of science nor had anyone to share in its triumphs. I dedicate this thesis to you.  xiv  Co-Authorship Statement  For the co-authored chapter 3, I contributed all of the experimental design, 80% of the research, all of the data analysis, and wrote the manuscript. For the coauthored chapter 4, I contributed all of the experimental design, 90% of the research, all of the data analysis, and wrote the manuscript. For the co-authored chapter 5, I contributed all of the experimental design, 90% of the research, all of the data analysis, and wrote the manuscript.  1  Chapter 1 1.1  Introduction  Preface  The foundation for this thesis is the observation that unusual T cell subsets exist which display characteristics of adaptive as well as innate immune cells. Even though the majority of T cells undergo a stringent selection process in order to eliminate any self-reactive cells the immune system still retains some selfreactive cells to perform functions distinct from those performed by the majority of conventional T cells. From this foundation it is reasonable to believe that the C D 8 T cell population that is normally thought to be specific for foreign antigens may in fact contain some cells that display self-specificity. This thesis consists of work which clearly demonstrates the existence of C D 8 T cells expressing selfreactive T cell receptors whose selection and maintenance depend on selfantigen interactions. Much of the following work describes the development and function of these self-specific C D 8 T cells including their expression of natural killer cell receptors and their responses to infection.  1.2  Receptors of the Innate and Adaptive Immune system  1.2.1  Biology of NK cells  2 Natural killer (NK) cells are an integral part of the innate immune system. Activation of NK cells leads to the rapid production of cytokines as well as the destruction of infected or transformed cells. NK cells also have the ability to alter the adaptive immune response by either promoting the maturation of dendritic cells (DCs) that can prime T cells or by destroying immature D C s before they can present antigen to T cells (1, 2). The effector pathways used by N K cells are very similar to those used by T cells. NK cells, like cytotoxic C D 8 T cells, use perforin and granzymes to lyse cells (3). Both cell types are also capable of producing IFNy that has multiple effects on the immune response. Unlike T cells, NK cells can immediately kill target cells and produce cytokines without the need for prior restimulation, thus leading to their classification as cells of the innate immune system. NK cell precursors are derived from hematopoetic stem cells (HSCs). The development of NK cells in adults is thought to occur mainly in the bone marrow where stromal cells provide cell-cell contact mediated signals as well as cytokine mediated signals important for NK cell development (reviewed in (3)). Most of what is known about NK cell development has been learned through the in vitro generation of NK cells from H S C s or NK precursors. Figure 1.1 provides a good overview about what is known about NK cell development from these mainly in vitro studies. The stromal cells that drive NK cell development can actually be replaced in these in vitro cultures by cytokines such as stem-cell factor (SCF), fetal liver kinase 2 ligand (FLK2), and IL-7 suggesting that these cytokines are required for NK cell development and are normally provided by stromal cells (4,  3 5). These cytokines drive the development of NK precursor cells whose further maturation can be driven by IL-2 or IL-15 (6, 7). NK precursor cells become immature NK cells and finally mature NK cells, a process characterized by the sequential induction of NK receptors. The acquisition of NK receptors is not random and is thought to be required to prevent the erroneous activation of maturing NK cells in response to normal host cells (reviewed in (8)). NK cells use several germ-line encoded receptors to recognize infected, stressed or transformed cells. N K receptors can transduce inhibitory, activating, or costimulatory signals and NK cell activation is regulated through the integration of the signals from several NK receptors (9). The finding that NK cells were tightly regulated by inhibitory receptors for self-MHC class I molecules was a breakthrough in the field and resulted in the "missing-self hypothesis" which stated that NK cells could recognize and kill target cells that had lost expression of M H C class I, an event that is known to occur both during infection and transformation (10). Recently, the missing-self hypothesis has been modified to account for the fact that NK cells cannot kill cells that have lost M H C class I without the engagement of activating receptors (9). Thus NK cells do not recognize erythrocytes which lack M H C class I molecules because they do not express any ligands for activating NK receptors. In order for a target cell to be killed by an NK cell the signals from activating receptors have to outweigh signals from the inhibitory receptors. The two ways for NK activation to occur are through either decreasing inhibitory receptor engagement or increasing activating  4 receptor engagement either of which can happen during infection or transformation.  1.2.2 Activating and inhibitory receptors of NK cells  Inhibitory NK receptors have been extensively characterized in both mice and humans. Table 1.1 shows a few common human and mouse inhibitory receptors and their M H C class I ligands. These receptors can be organized into two structural categories: killer Ig-like type I integral membrane receptors (KIRs), encoded in the leukocyte receptor complex, and lectin-like type II integral membrane receptors, encoded in the NK gene complex (11-14). The later category includes Ly49 homodimers in mice and C D 9 4 - N K G 2 A heterodimers in both mice and humans (9, 15). Despite their structural differences, the inhibitory NK receptors share many functional characteristics. Similar to T cell receptors (TCRs), these inhibitory receptors bind to major histocompatibility (MHC) class I molecules (9). The KIR and Ly49 family members interact with particular allelic variants of classical M H C class I molecules. In contrast, the ligand for the CD94N K G 2 A receptor is the non-classical M H C class I molecule HLA-E, or its murine ortholog Q a - 1 , complexed to a nonapeptide, Qdm, derived from the leader b  sequence of particular M H C class I heavy chains (16). Interaction of inhibitory receptors with M H C class I. molecules prevents activation of NK effector function by eliciting a strong inhibitory signal mediated by an immunoreceptor tyrosinebased inhibitory (ITIM) motif in the cytoplasmic domains of these receptors.  5 When these inhibitory receptors are engaged, the ITIM is tyrosine phosphoryiated and SHP-1 phosphatase is recruited and activated, which presumably dephosphorylates signaling molecules involved in the activation cascade (17-20). Until recently, the molecular structures involved in NK triggering have long been a matter of debate. Thus, the identification of a panel of recognition structures expressed on NK cells directly involved in NK triggering, is a major breakthrough in the field. Table 1.2 provides a summary of common human and mouse activating NK receptors along with their ligands and the signaling pathways which they use. Individual NK cells express multiple different activating NK receptors of both Ig and lectin-like structural families that lack cytoplasmic ITIMs (21, 22). These activating receptors include the natural cytotoxicity receptors, the activating NK receptors, which include stimulatory KIRs in humans and certain Ly49 receptors, i.e. Ly49D (23) in the mouse, C D 9 4 - N K G 2 C heterodimers in both species (24), and as described later, N K G 2 D and 2B4 receptors. Most of these receptors contain charged amino acids in their transmembrane domains, permitting interaction with distinct signaling chains containing immunoreceptor tyrosine-based activation motifs (ITAMs). NK cells express multiple ITAM signaling chains, including DAP12, CD3C,, and FcsRIy (25). Ly-49D and Ly-49H associate with D A P 12 (22), NKR-P1 associates with FcsRIy (26), and CD16 associates with CD3C, (27, 28) and FcsRIy (29). When activating receptors are cross-linked, the ITAMs in the associated signaling chain become tyrosine phosphoryiated. These ITAMs then recruit downstream  6 signaling kinases such as Syk and ZAP-70, triggering activation of cytotoxic, proliferative, and/or secretory responses (30, 31). One recently characterized activation molecule expressed on both human and mouse NK cells is the lectin-like N K G 2 D molecule (32). N K G 2 D has recently been shown to exist in two alternatively spliced isoforms, N K G 2 D - S and N K G 2 D L (33). Both isoforms can pair with the signaling molecule, DAP10 (33), which contains a YxxM motif thought to recruit phosphatidylinositol 3-kinase (PI3-K) (34). Only N K G 2 D - S pairs with D A P 12 (33). This differential recruitment of DAP10 versus DAP12 provides N K G 2 D with both directly stimulatory and costimulatory functions, respectively (33, 35). Human N K G 2 D recognizes the stress-inducible MIC family (MICA and MICB) (32). In addition, it interacts with the U L B P family of molecules, which also bind the cytomegalovirus-encoded UL16 molecule (36). The U L B P s stimulate cytokine and chemokine production from NK cells, and expression of U L B P s in NK cell-resistant target cells confers susceptibility to N K cell cytotoxicity (37). Mouse N K G 2 D binds the minor histocompatibility molecule H60, as well as members of the retinoic acidinducible (Rae-1) family of molecules (38, 39). These N K G 2 D ligands are not expressed by most normal cells but are up-regulated on numerous tumor cells upon infection or in response to DNA damage. Expression of an N K G 2 D ligand by target cells triggers NK cell cytotoxicity, IFNy secretion, as well as nitric oxide release and T N F - a and (3 transcription by macrophages (37, 40). Studies have demonstrated that N K G 2 D serves as a co-stimulatory molecule for T C R activated C D 8 T cells due to the lack of DAP12 expression; however, it functions +  7 as a primary activation receptor on NK cells which do express DAP12 (33-35, 41). One of the first activating receptors described on N K cells was FcyRllla or CD16. C D 1 6 is a low affinity Fc receptor (FcR) that binds to IgG and is involved in antibody dependent cell-mediated cytotoxicity (ADCC) in which an antibody coated target cell is destroyed by NK cells (42). Stimulation of C D 1 6 on NK cells also results in the production of cytokines such as IFNy, T N F a and G M - C S F (43). FcyRllla associates mainly with the ITAM-containing homo or heterodimers of CD3C and FcsRIy (FcRy) in humans (28) or solely with FcRy homodimers in mice (44). The binding of IgG to CD16 results in the phosphorylation of the ITAMs in the signaling chains leading to the recruitment of kinases such as ZAP-70 and Syk (45). These kinases initiate a signaling cascade resulting in the lysis of antibody-coated target cells and cytokine production. 2B4 (CD244) is an N K receptor that is expressed by all NK cells in both humans and mice (9). Both 2B4 and its ligand CD48 are members of the C D 2 family of Ig-related proteins (46). 2B4 contains a binding-site within its intracellular domain that upon phosphorylation can bind to the protein-tyrosine phosphatases SHP-1 and S H P - 2 or to the adaptor molecule S A P (SH2D1A) (47). The function of 2B4 is quite controversial with reports suggesting that the receptor is either activating, co-stimulatory or inhibitory. The strongest evidence for an inhibitory role for 2B4 comes from studies with 2B4-deficient mice (48, 49). The NK cells from these animals display enhanced responses against CD48positive targets. The evidence for 2B4 being involved in activation comes from  studies showing that cross-linking of 2B4 on IL-2-activated NK cells leads to stimulation of lytic activity (50-52), IFNy secretion (52), and granule exocytosis (53) . 2B4 is constitutively associated with the linker for activation of T cells (LAT) (54) and antibody-mediated engagement of 2B4 resulted in tyrosine phosphorylation not only of 2B4 but also of the associated LAT molecules. Furthermore, tyrosine phosphorylation of LAT leads to the recruitment of signaling molecules that include PLCy and Grb2 (54). A recent report showed that NK cells express another adaptor protein related to S A P called EAT-2 that can associate with 2B4. Unlike S A P , EAT-2 transduces an inhibitory signal (55). Thus the seemingly opposite functions of 2B4 are probably regulated by the relative amounts of the adaptor proteins expressed by the cell. The regulation of NK cell activation is complex and multifaceted. For N K cells, several receptors are likely involved in triggering and it is the sum of the signals which dictates whether or not an NK cell will respond. N K cells, unlike T cells are not restricted to one particular ligand and can potentially respond to several different target cells expressing different combinations of ligands for activating and inhibitory NK receptors.  1.2.3 Activating and inhibitory receptors of T cells  T cells are an integral part of the adaptive immune system. In contrast to NK cells, which provide early protective immune responses, T cells are more important in providing protection against microbial infections during the later  9 stages of the immune response. T cell activation is a complex process that involves activation through the T C R and co-stimulatory receptors (56-58). The a p T C R on T cells consists of the peptide/MHC-binding T C R a and p chains along with the C D 3 signaling complex consisting of one CD3y, one CD38, two CD3e, and two C D 3 ^ chains (reviewed in (59)). The C D 3 chains all contain one ITAM motif in their cytoplasmic tail with the CD3C, chains containing 3 ITAMs each. When the T C R binds to peptide/MHC, the ITAMs in the C D 3 chains become phosphoryiated by src-family kinases. Lck, a member of the src-family kinases, is thought to be the major contributor to ITAM phosphorylation in T cells (60). The phospho-tyrosine residues within the ITAMs on  then recruit the  kinases ZAP-70 and Syk (61). These kinases then act on several downstream targets including LAT eventually leading to the activation of the M A P kinase pathway and E R K activation (reviewed in (62)). Ultimately the signals emanating from the T C R lead to the activation of several transcription factors including cjun, c-fos, and NF-AT, which result in the transcription of genes involved in cell division and differentiation (reviewed in (63)). Full activation of naive T cells requires simultaneous engagement of the T C R and co-stimulatory molecules, resulting in triggering of T cell effector function, survival and proliferation (64). The best-characterized T cell costimulatory molecule is CD28, which recognizes B7.1 and B7.2 (65, 66). The expression of B7.1 and B7.2 is restricted mainly to the professional antigenpresenting cells (APCs) that are required for naive T cell activation. CD28 recruits and activates various tyrosine-phosphorylated proteins, including the  10 kinases, Lck and Itk, and the phosphoinositol lipase P L C y l (57). CD28 signals through a YxxM motif present in its cytoplasmic domain, which is a consensus binding site for the p85 subunit of PI3-K (67-69). CD28-engagement leads to the induction of survival molecules as well as to the efficient production of interleukin2 in antigen-activated T cells (70). Another member of the CD28-family of co-stimulatory molecules expressed by T cells is ICOS (71). The ligand for ICOS, ICOS-L, is also expressed primarily by professional A P C s (72, 73). ICOS also contains the PI3-K binding motif but unlike CD28, ICOS is induced on activated T cells and plays a role in T cell survival but has little influence on IL-2 production (71, 74). ICOS does however play a role in the production of IL-10 by C D 4 T cells (71) and ICOS-L expression by tumor cells augments tumor-specific C D 8 T cell responses (75). T cells can also express co-stimulatory molecules that are not a part of the CD28-family of molecules. These other co-stimulatory molecules include members of the tumor necrosis factor receptor (TNFR) family. Members of this family include OX40, 4-1BB, CD27, and T N F R 2 . The expression of T N F R family members on T cells can be either constitutive, as is the case for CD27 and T N F R 2 , or inducible, like for 4-1 BB and OX40 (76, 77). These receptors all share the ability of activating T N F receptor associated factors (TRAFs) and all of the receptors can activate  NFKB  (76, 78). The ligand for CD27, CD70, is expressed  by activated A P C s (79). The ligands for OX40 and 4-1 BB, O X 4 0 L and 4-1BBL respectively, are also induced on activated A P C s (80, 81). T N F R 2 binds to T N F a  11 which is produced by activated T cells themselves and acts in an autocrine manner (77). CD27 and T N F R 2 are involved early during T cell activation and OX40 and 4-1BB seem to be involved more in the survival of activated T cells (82-85). Nevertheless all of the T N F R family members play important roles in full T cell activation. T cells also express inhibitory receptors such as CTLA-4 and PD-1 (reviewed in (86)). Both of these receptors are induced upon T cell activation and both are capable of dampening the T cell response. CTLA-4 binds to B7.1 and B7.2, the same ligands that bind CD28, but it does so with a much higher affinity than CD28 (87, 88). Thus one mechanism by which CTLA-4 may inhibit T cell activation is through competition with CD28 for binding to the B7 molecules (89). CTLA-4 has also been shown to recruit the phosphatase S H P - 2 which leads to the dephosphorylation of proteins in the T cell activation pathway (90). The importance of CTLA-4 is clear when one looks at mice lacking this receptor. CTLA-4"'" mice develop severe immunopathology where all of their T cells display an activated phenotype and the mice die within a few weeks of birth (91, 92). P D 1-deficiency also leads to autoimmunity although it seems to be less severe than that seen with CTLA-4 (93). PD-1 contains an ITIM motif that can potentially signal the recruitment of phosphatases that can dampen T C R signaling (94). The ligands for PD-1 are PD-L1 (B7-H1) and PD-L2 (B7-DC) (95-98). These ligands are expressed in both lymphoid and non-lymphoid tissue suggesting that PD-1 is involved in regulating T cell responses in secondary lymphoid organs and in the periphery (95, 96). P D -  12 1 is expressed only on activated T cells and is predominant on chronically stimulated T cells where it plays a role in their inability to respond to further stimulation (94, 99). In fact blocking of PD-1/PD-L1 interactions can rescue these exhausted T cells and allow them to respond normally to T C R stimulation (99). PD-1 engagement can cause cell-cycle arrest in T cells which can be overcome by the addition of exogenous IL-2 (100). Interestingly, PD-L1 and PD-L2 can also costimulate T cell responses suggesting that perhaps there are other receptors capable of binding to these ligands (97, 98). Regardless, it is clear that PD-1 plays an important role in dampening T cell activation and more importantly its function is not redundant with CTLA-4. Although T cell activation is generally dictated by the recognition of peptide/MHC by the T C R it is clear that full T cell activation is regulated by other receptors which can either modulate T C R signaling or act independently of the T C R through the induction of survival molecules. Unlike for NK cells where no single activating receptor dictates a particular NK cells' specificity, the T C R is the sole determinant of a T cells' specificity.  1.2.4 NK receptor expression by memory-phenotype CD8 T cells  Even though N K cells and T cells perform quite distinct functions they appear to be related. Both cell types are derived from a common progenitor cell (101). NK cells are more like C D 8 T cells in the sense that they both use perforin and granzymes to kill target cells and both cell types are efficient producers of  13 IFNy (9). NK cells can immediately exert their function in the absence of prior activation differentiating them from C D 8 T cells which need prior antigenactivation before they can produce cytokines or kill target cells. Another commonality between NK cells and T cells is the observation that both cell types use similar signaling molecules and signaling pathways for their activation. A s discussed in the previous sections, both NK cells and T cells have activating, costimulatory, and inhibitory receptors and both cell types use the same proteins to transmit the signals from these receptors. It is possible then, that if T cells were to express NK receptors, that these receptors would in fact be functional. In fact, there is potential for NK receptors to either amplify or abrogate T C R signals depending on whether the NK receptor involved is inhibitory or activating. This possibility would require the expression of either inhibitory and/or activating NK receptors by T cells. Interestingly, various T cell subsets, B cells and myeloid cells do in fact express inhibitory as well as activating NK receptors (102). N K G 2 D is expressed by all human C D 8 T cells and y5T cells (32). In mice, N K G 2 D is expressed by +  activated, but not resting C D 8 T cells, macrophages, and subsets of T C R a | 3 +  +  and N K 1 . 1 T cells (39). In contrast to NK cells, cross-linking of N K G 2 D on activated C D 8 T cells failed to mediate redirected lysis of FcR-expressing target +  cells suggesting that N K G 2 D does not directly stimulate C D 8 T cells (103). +  Furthermore, the activated C D 8 T cells did not respond to N K G 2 D cross-linking +  by producing IFNy or by fluxing C a  + 2  (103). However, N K G 2 D cross-linking  augmented the proliferative response of C D 8 T cells to limiting doses of anti+  14 T C R mAb (41, 103). Thus, N K G 2 D can only provide a costimulatory signal for the activation of N K G 2 D C D 8 cells due to their lack of expression of DAP12. In +  +  this regard, the transgenic expression of DAP12 into C D 8 T cells is sufficient to convert N K G 2 D into a directly activating receptor confirming that the function of N K G 2 D on C D 8 T cells is governed by the adaptor molecules which the T cell expresses (104). In addition to NK cells, 2B4 is expressed on monocytes, basophils, and subsets of y5T cells and C D 8 T cells (46, 52, 105). The biological function of +  2B4 on C D 8 T cells remains largely unclear. 2B4 is preferentially expressed on +  C D 8 T cells with an activated/memory phenotype (106) and by T cells involved +  in non-MHC restricted cytotoxicity (106, 107). A recent study suggests that 2B4 on activated/memory C D 8 T cells serves as a ligand for CD48, and by its ability +  to interact with CD48 provides costimulatory function for neighboring T cells (106). 2B4 engagement on C D 8 T cells can also enhance the killing of CD48positive target cells (108). Thus it seems that when 2B4 is expressed on memory-phenotype C D 8 T cells it does in fact play a role in both their activation and their effector functions. In both mice and humans, the expression and function of inhibitory NK receptors on memory-phenotype C D 8 T cells has been described extensively (reviewed in (109, 110)). Expression of a Ly49A transgene has been shown to effect both activation and effector functions of C D 8 T cells (111-113). Strikingly, the expression of a Ly49A transgene in CTLA-4-deficient mice can prevent the T cell lymphoproliferative disease in these animals demonstrating that the signals  15 mediated by these distinct receptors are similar (114). The expression of NK receptors has also been shown to be important for the survival and development of memory-phenotype C D 8 T cells (115). It has been suggested that C D 8 T cells can acquire the expression of inhibitory NK receptors only upon recognition of cognate antigen as C D 8 T cells that are chronically activated such as those in healthy carriers of Epstein Barr virus or cytomegalovirus often exhibit a memory phenotype and express NK receptors (116). The fact that NK receptor expression is not detected on C D 8 T cells in the thymus but is induced in the periphery is consistent with this idea (117). Although memory-phenotype C D 8 T cells have been shown to express NK receptors their physiological roles during normal immune responses remain largely unclear. Thus it remains possible that memory-phenotype C D 8 T cells may express NK receptors in order to dampen signaling by their T C R s which may be specific for self-antigens. Consistent with this observation is the fact that T cells specific for non-mutated self-antigens expressed by melanomas often express inhibitory NK receptors, even in healthy donors (118).  1.2.5 The role of interleukin-2 and 15 in NK and T cell homeostasis  CD122 is a cytokine receptor shared by both interleukin (IL)-2 and IL-15. A high level of C D 122 expression is characteristic of both memory-phenotype C D 8 T cells and NK cells and thus it is not surprising that IL-2 and IL-15 play pivotal roles in the activation and homeostasis of both NK cells and T cells. The  16 receptors for these cytokines share two subunits, CD132 or the common gamma chain (yc) and CD122 (IL-2R0) (119, 120). CD122 expressed with CD132 makes up the low affinity receptor for both IL-2 and IL-15. The high affinity receptor for IL-2 consists of an added chain CD25 (IL-2Rot) that is capable of binding to IL-2 with a much higher affinity than CD132 and CD122 alone (121). The same is true for IL-15 where a third receptor component IL-15Ra increases the affinity of the IL-15R for IL-15 (122). CD122 is the signaling chain for both the IL-2 receptor as well as the IL-15 receptor and its expression along with CD132 is sufficient to confer both NK cells and T cells with the ability to respond to IL-2 or IL-15 (reviewed in (123)). IL-2 is produced by activated T cells where it binds to the high affinity IL-2 receptor in an autocrine manner (reviewed in (121)). Signaling through IL-2 is involved in the expansion phase of T cell activation but it is also crucial for programming T cells to undergo contraction upon further stimulation, a process termed activation-induced cell death (AICD) (124). Provision of high doses of IL-2 to NK or T cells is capable of inducing the activation and proliferation of the cells and these lymphokine activated killer (LAK) cells exhibit a high degree of killer activity towards several self- and non-self target cells that are normally resistant to NK cells (125, 126). IL-15 is produced by multiple cell types (122) and plays a major role in the development and/or homeostasis of N K cells, C D Id-restricted NKT cells, which will be discussed later, and memoryphenotype C D 8 T cells all of which express high levels of CD122 (127, 128). IL15 and IL-15Ra-deficient mice have a drastic reduction in the number of NK, NKT, and memory-phenotype C D 8 T cells due partly to the inability of the cells to  17 divide in vivo. One oddity of IL-15 signaling is the fact that IL-15Ra seems to be involved mainly in presenting IL-15 to CD122 and CD132 (129, 130). In fact this presentation of IL-15 by IL-15Ra can occur in trans where the IL-15Rocexpressing cell provides IL-15 to a neighboring cell that expresses only CD122 and CD132 (131). Thus in IL-15Ra-deficient C D 8 T cells undergo normal expansion in an IL-15Ra-sufficient host. IL-15, like IL-2, induces the activation and proliferation of C D 8 T cells but unlike IL-2, induces the survival rather than contraction of the responding cells (132). Thus although IL-2 and IL15 share similar signaling components the outcome of IL-2 and IL-15 stimulation varies significantly. However, it is important to remember that both IL-2 and IL-15 are capable of activating both NK cells and memory-phenotype C D 8 T cells without the need for T C R or NK receptor stimulation and this cytokine-dependent activation likely plays an important role during the majority of immune responses.  1.3  T cell development  1. 3.1 Positive and negative selection of the naive T cell repertoire  T cell development is a complicated process involving the seeding of the thymus with bone marrow derived progenitor cells followed by selection of T lineage committed thymocytes that have successfully rearranged their T cell receptor (TCR). It all begins when progenitor cells from the bone marrow make their way  18 into the thymus. These CD4"CD8" double negative (DN) progenitors then go through a stepwise process characterized by the acquisition and loss of the cell surface molecules CD44 and CD25 (133). The cells progress from the DN1 (CD44 CD25-) stage to the DN2 ( C D 4 4 C D 2 5 ) stage and then the DN3 (CD44" +  +  +  CD25 ) stage which are all accompanied by the stepwise movement of the cells +  from the cortico-medullary junction of the thymus towards the cortex and subcapsular regions. At the DN3 stage is where the thymocytes initiate the rearrangement of their T C R p genes which upon successful pairing with the surrogate light chain, pTa, allows the cells to progress to the DN4 stage (CD44" CD25"). At this point the cells undergo extensive proliferation accompanied by the expression of both the C D 4 and C D 8 coreceptors. These C D 4 C D 8 double +  +  positive (DP) thymocytes then undergo rearrangement of their T C R a loci in order to prepare them for the next stages of development (134). Thymocytes expressing rearranged T C R s undergo another round of selection that ensures that their newly rearranged receptor has some reactivity to self-peptide in the context of self-MHC. The selection of weakly self-reactive thymocytes, or positive selection, leads to their survival and maturation into C D 4 or C D 8 single positive (SP) T cells. If a T C R cannot bind to self-peptide/MHC the thymocyte expressing that T C R will not receive any survival signals and thus will die by neglect. When developing thymocytes react too strongly with self-peptide/MHC they are eliminated by negative selection in order to ensure that they cannot harm the host. Thus there are several checkpoints to ensure that mature T cells can recognize foreign peptides in the context of self-MHC without being overtly  19 autoreactive. Figure 1.1 provides an overview of T cell development from the seeding of progenitor cells in the thymus through to their positive or negative selection. Positive selection of T cells is crucial for the function of mature T cells. During the positive selection process C D 4 C D 8 + double positive (DP) +  thymocytes expressing a rearranged T C R are screened for reactivity with selfpeptide/MHC complexes expressed by thymic cortical epithelial cells (135). If the T C R has weak to moderate affinity for a self-peptide/MHC complex, the thymocyte will receive a signal through the T C R that induces survival and differentiation. Weak stimulation of the T C R on D P thymocytes results in the activation of NFAT and E R K which are thought to be critical for efficient positive selection (136, 137). The self-peptide/MHC complexes that mediate positive selection react weakly with the T C R and these weak signals are insufficient to activate mature T cells (134). In addition to mediating survival, positive selection also commits D P thymocytes to either the helper (CD4) lineage or killer (CD8) lineage depending on whether the T C R binds to either M H C class II or M H C class I molecules respectively (138). Thus positive selection is not only important for the survival of T cells but it also ensures that the T cell adopts the appropriate functional phenotype. Positive selection ensures that T cells can recognize self-peptide/MHC with a low affinity. Negative selection, on the other hand, eliminates T cells in the thymus that react too strongly with self-peptide/MHC. Negative selection is thought to occur in the thymic medulla where thymic medullary epithelial cells  20 scans positively selected thymocytes for strong reactivity with self-peptide/MHC complexes (139). Strong T C R signaling in these thymocytes results in the phosphorylation and activation of E R K , P38, and J N K which leads to cell death. It is important that thymic medullary epithelial cells express a wide array of selfpeptides derived from organ specific proteins in order to prevent autoimmunity as a consequence of inefficient negative selection. Perhaps the most important mediator of this self-peptide presentation is a recently described gene, aire. Mice and humans with a disruption in aire develop autoimmune disease characterized by multiorgan cell infiltrates (140). The cause of disease in a/re-deficient mice is not only the inability of medullary epithelial cells to express organ-specific peptides but also in their inability to efficiently present these peptides to developing T cells (141). Thus it is clear that negative selection against a variety of self-peptides, including those restricted to specific organs, is crucial for maintaining tolerance to self. Emphasizing the importance of purging autoreactive T cells is the fact that even if negative selection fails in the thymus, peripheral (extrathymic) mechanisms exist that can induce T cell tolerance to self -antigens (142).  1.3.2 Extrathymic T cell development  The thymus has evolved as an organ dedicated to T cell development. It contains all of the supporting cells and has the appropriate architecture and microenvironment for the development and selection of T cells. The thymus is not  21 the only location of T cell development, however, as certain secondary lymphoid organs have been shown to support T cell development. The mesenteric lymph nodes are a particularly effective site of extrathymic T cell development although their contribution to T cell lymphopoesis is minimal in the presence of an intact thymus (143). The problem with extrathymic T cell development seems to be due to the inability of the LNs to attract T cell progenitors. Overexpression of the cytokine oncostatin M (OM) can overcome this problem and converts the LNs into potent sites of T cell development (144, 145). Extrathymic T cell development involves the positive and negative selection of D P T cells but the major difference between conventional thymic and unconventional extrathymic positive selection is the nature of the selecting cell (146). In the thymus, cortical epithelial cells are the main cell type mediating positive selection whereas in the LNs, positive selection is mediated by hematopoetic cells (147). This difference in the positively selecting cell type imparts functional differences in the selected T cells, including those that express the same T C R . Extrathymically selected T cells are functionally distinct from intrathymically selected T cells. Extrathymic T cells express lower levels of T C R and have a natural memory phenotype characterized by expression of high levels of CD44 and CD122 (147, 148). These cells also undergo excessive homeostatic proliferation in response to lymphodepletion which results in the availability of excess cytokines such as IL-15. Another unusual characteristic of extrathymic T cells that may stem from their memory phenotype is their ability to respond rapidly to infection. In fact extrathymic T cell numbers peak at around day 4 post-  22 infection whereas conventional T cells peak at day 8 (149). Even though these cells proliferate rapidly they also undergo a greater degree of apoptosis when compared to thymus-derived T cells. Extrathymic T cells are able to rapidly produce cytokines such as IFNy upon stimulation without the need for the cells to proliferate (149). In addition, extrathymic T cells are not as efficient as conventional T cells when it comes to eliminating pathogens or generating memory (149). Many, but not all, C D 8 T cells restricted to the nonclassical M H C class lb molecules can be selected by hematopoetic cells (150). Extrathymic T cells provide early protection against pathogens before classical C D 8 T cells are activated to eliminate the infection. Consistent with this hypothesis is the fact that the M H C class lb-restricted C D 8 T cell response peaks 2-3 days before the classical M H C class la-restricted C D 8 T cell response during infection with Listeria monocytogenes (151). At least some extrathymic T cells appear to be dependent on selection by self-antigens for their development (152).OM transgenic mice normally develop autoimmunity but fail to do so if the mice are either deficient for both C D 4 and C D 8 T cells or if the T cells express a transgenic T C R specific for a foreign antigen suggesting that TCR-specificity plays a role in disease (153). The observation that the in vivo delivery of IL-2 to athymic Balb/c nu/nu mice led to the development of an autoimmune disease suggests that these cells are self-reactive and can contribute to autoimmunity (154). It is unknown what proportion of memory-phenotype C D 8 T cells in normal mice may actually develop extrathymically. This is an important question, however, since extrathymic C D 8 T cells are clearly different from thymic C D 8 T  23 cells in terms of their high affinity for self-antigens. Moreover it is important to determine if extrathymically derived T cells play a significant, non-redundant, role during immune responses.  1.4  Positive selection of unconventional T cells  A s one can imagine there is a range of affinities that a particular T C R can display for self-peptide/MHC complexes that can lead to positive selection without causing negative selection and thus T cells inherently vary in the extent of their "self-reactivity". The majority of conventional naive T cells have a low to intermediate affinity for self-peptide/MHC but several unconventional T cells, such as natural killer T (NKT) cells and C D 8 a a lELs, display a high affinity for self-peptide/MHC making them self-reactive. Importantly, these agonist selected T cells behave differently than conventional T cells and usually perform regulatory functions. Figure 1.2 provides a schematic representation of T C R affinity and the selection of conventional as well as unconventional T cells.  1.4.1  NKT cell development and function  NKT cells in mice express an a p T C R that recognizes lipid antigens in the context of the nonclassical M H C class lb molecule CD1d. In mice these T cells have an invariant Va14-Ja18 rearrangement which restricts them to CD1d (155). Due to  their CD1d restriction, NKT cells can be identified by staining with CD1d tetramers bound to the NKT cell ligand a-galactosyl ceramide (aGalCer) (156). These cells also express several NK receptors including NK1.1 (NKRP1) and are either C D 4 C D 8 " or C D 4 (156). NKT cells are unlike conventional n a i v e T c e l l s in +  that they exhibit an activated or memory phenotype even in germfree animals and respond very rapidly to infection without the need to divide and differentiate (157). a G a l C e r is a very potent agonist for NKT cells but it is not naturally found in any mouse or human pathogen. Under more physiological conditions, NKT cells can recognize other lipids derived from bacteria in the context of CD1d (158, 159). These cells can also be activated in response to self-lipids/CD1d in conjunction with inflammatory cytokines like IL-12 differentiating them from conventional T cells that solely recognize foreign antigens (160). Upon activation NKT cells are thought to play a major role in regulating the immune response through the early production of IFNy or IL-4, which can promote either TH1 or TH2 responses, respectively (161). NKT cells are not only capable of cytokine production but they can also kill target cells that express the appropriate ligands (162). NKT cells can induce the maturation of D C s (163) and cause NK cells to proliferate and produce cytokines (164). These cells have been implicated in everything from protection against bacterial infections, to promoting anti-tumor immunity and preventing diabetes (reviewed in (165)). Thus NKT cells seem to have a special multifaceted role during immune responses. The special role of NKT cells seems to stem from their unconventional selection in the thymus. Like conventional T cells, NKT cells are thought to be  25 derived from D P thymocytes that have undergone random gene-rearrangements (166, 167). They are also thought to undergo positive selection by selflipids/CD1d. What makes the selection of NKT cells different from conventional T cells is the cell type that mediates their positive selection. Unlike conventional T cells that are selected by thymic cortical epithelial cells, NKT cells require D P thymocytes for their selection (156). In addition, the ligands that mediate the positive selection of NKT cells are actually agonist ligands and provide a positive selection signal that is at the threshold of negative selection (168). NKT cells also differ from conventional T cells in their requirements for specific genes that have relatively little effect on conventional T cells. For example, NKT cell development requires  NF-KB1  expression (169) and also relies on the expression of the T-box  family transcription factor T-bet (T-box expressed in T cells) (170). Thus it is clear that NKT cells are a distinct lineage from conventional T cells and that their functions are somehow tied to their agonist selection in the thymus.  1.4.2  TCRaB* CD8aa Intestinal intra-epithelial lymphocytes (ilELs)  T C R a p CD8aa ilELs are another unconventional T cell type that plays a huge +  role in regulating the homeostasis of the immune system. These cells make up a very large part of both the human and mouse immune system. They reside in the lining of the gut lumen where they play a large role in maintaining gut integrity as they are the first line of defense against ingested particles (reviewed in (171)). Similar to NKT cells, TCRap CD8aa ilELs have a natural memory phenotype +  26 (172) and express functional NK receptors (173). TCRocp CD8aa ilELs, +  however, are more heterogeneous in terms of their M H C reactivity when compared to NKT cells. These cells can be restricted to classical M H C class la, non-classical M H C class lb, or to M H C class II molecules although it seems that the majority of the cells are in fact restricted to non-classical M H C class lb (174). Even though these cells are selected by a wide range of M H C they are all thought to be selected by high affinity interactions between self-peptide/MHC. TCRap CD8aa ilELs often express overtly autoreactive T C R s that are deleted +  from the conventional T cell repertoire (175). In fact, using T C R transgenic models of TCRap CD8aa ilELs development, it is clear that these cells are in +  fact selected by high affinity agonist self-peptides (176, 177). The function of TCRap CD8aa lELs is regulatory in nature as the cells +  have been shown to be crucial for preventing uncontrolled immune responses in the gut. In double-transgenic mice that express a M H C class la-restricted T C R specific for an L C M V peptide (GP33-41) as well as the cognate peptide under control of the M H C class I promoter there are large numbers of TCRap  +  CD8aa lELs that can be activated in response to L C M V infection (177). Instead of these self-reactive lELs causing disease, the virus-activated cells produced TGFp, an important immunoregulatory cytokine that negatively regulates immune responses. In another T C R transgenic system it was clearly demonstrated that TCRap CD8aa lELs expressing a self-reactive T C R could actively prevent the +  development of colitis by suppressing pathogenic CD4 T cells (178). This suppression was dependent on IL-10 production by the lELs and perhaps most  27 strikingly was completely dependent on the expression of cognate self-antigen in the protected mice. Thus it is clear that agonist selection of T C R a P C D 8 a a lELs +  is crucial for their special regulatory function and that the presence of self-antigen plays a role in not only the selection of these cells but is also important for their function.  1.4.3 Agonist selection of unconventional C D 8 T cells in male H-Y T C R transgenic mice  H-Y T C R transgenic mice have been widely used as an animal model system for the determination of mechanisms of positive and negative selection of T cells. In these mice, C D 8 T cells that express the H-Y T C R are positively selected in the thymus of B6 (H-2 ) female mice (179). In B6 male mice, the H-Y T C R is b  negatively selected, leading to the massive deletion of double positive (DP) thymocytes (180). Interestingly, there is a population of H-Y T C R C D 8 T cells, +  +  which express a low level of C D 8 (referred to as CD8'°) (181), that are resistant to deletion in male mice (181). The self-specific CD8'° cells can develop via an extrathymic pathway (152) and express high levels of CD44 and CD122 that are characteristic of memory T cells. However, they differ from conventional memory T cells in that they are more refractory to activation by antigen as compared to naive T cells (182, 183). Another unusual feature of these self-specific CD8'° T cells is that they can develop extrathymically and can proliferate in response to cytokines such as IL-2 and IL-15 in an antigen-independent manner (184).  28 However, the C D 8  10  cells are similar to conventional memory T cells with regard  to the ability to rapidly produce IFNy upon T C R stimulation (185) and with respect to the killing of susceptible target cells without the need for additional reactivation with antigen (186, 187). The work done in the H-Y T C R transgenic mice is complemented by studies in non-TCR transgenic mice, which suggest that a similar population of C D 8 T cells exists in normal mice (152, 183, 184). These unconventional C D 8 T cells exhibit a memory-phenotype and appear to be selected by cognate selfantigen (152). Interestingly, they respond to cytokines produced during infection (184). In addition, these cells maintain the ability to rapidly produce cytokines and kill target cells without the need for prior stimulation (187). These characteristics are very similar to those of the other unconventional T cells described in the previous sections. These results suggest that perhaps the normal immune system contains a population of self-antigen specific C D 8 T cells that display characteristics of both innate and adaptive cells.  1.5  Thesis objectives  The objectives of this thesis are: 1) Demonstrate self-reactivity in the memory-phenotype C D 8 T cell population in normal mice. 2) Look for the expression of NK receptors on self-reactive C D 8 T cells and determine if these NK receptors can alter T C R signaling.  29 3) Determine the relationship between self-specific C D 8 T cells in H-Y male T C R transgenic mice and memory-phenotype C D 8 T cells in normal mice. 4) Use the self-specific H-Y male C D 8 T cells to learn more about the development, maintenance, and activation of self-specific C D 8 T cells. 5) A s s e s s the role of self-specific C D 8 T cells in vivo during infection.  Chapter 2 starts by examining the characteristics of self-reactive memoryphenotype C D 8 T cells from normal mice. Chapter 3 examines the development and function of self-specific C D 8 T cells in male H-Y T C R transgenic mice and demonstrates that these cells are nearly identical to the self-reactive memoryphenotype C D 8 T cells described in chapter 2. Chapter 4 describes the expression and function of an activating NK receptor, CD16, on self-specific C D 8 T cells from non-transgenic B6 as well as from male H-Y T C R transgenic mice. This chapter also shows that NK receptors on self-specific C D 8 T cells can work either independently or together with the T C R to induce killing and cytokine production. Chapter 5 is a series of in vivo experiments that demonstrates the ability of self-specific C D 8 T cells to provide innate protection from infections. This chapter also demonstrates that the self-reactive T C R on self-specific C D 8 T cells plays a role in the maintenance and function of this cell type in vivo.  30  1.6  Tables  Table 1 | Inhibitory NK receptors for MHC Species  Receptor  Ligands  Mouse  Ly49  H-2K,  Mouse'  CD94/NKG2A Qa-1  Human  KIR2DL  HLA-C  Human  KIR3DL  HLA-Bw4, HLA-A  Human  CD94/NKG2A (CD159a)  HLA-E  ^Human-; • " ^ G m S j , GD85d  H-2D b  HLA class I  ;  ;;,  ;  Inhibitory receptors contain immunoreceptor tyrosine-based Inhibitory motifs OH Ms) in their cytoplasmic domains that recruit the intracellular phosphatases SHP-1 and/or SHP-2. The human KIR and mouse Ly49 receptors are encoded by several genes that demonstrate extensive allelic polymorphism. They recognize polymorphic determinants on their major histocompatibility complex (MHC) class I ligands. CD94/NKG2A (CD 159a) receptors bind to ligands with limited polymorphism and CD85J reacts with a region In thea3domaln of human human leukocyte antigen (HLA) class I that Is highly conserved in class I molecules. KIR, killer cell Immunologlobulln-like receptors; NK, natural killer.  Table 1.1 Inhibitory NK receptors for M H C . Table from Cerwenka, A., and L.L. Lanier. 2001. Natural killer cells, viruses, and cancer. Nat Rev Immunol 1:41-49. (188).  31  Table 2 | Activating NK cell receptor complexes and their ligands Receptor  Species  Signalling adaptor  Signalling Pathway  CD16  Mouse, human  FcsRIy or CD3£  ZAFYO/Syk  Ligand* igG  NKp30  H u m a n ...  FceRlyOrCD3?"  ZAP70/Syk  ?••  Influenza haem agglutinin, others?  •  NKp46  Mouse, human  FccRly Ol C D 3 ^  2AP70/Syk  NKR-P1C  Mouse  FceRly or C D 3 I ;  ZAP70/Syk  KIR2DS  Human  D A P 12  ZAP70/Syk  H L A - C , others?  CD94/NKG2C  Mouse, human  D A P 12  ZAP70/Syk  H L A - E (Qa-1)  Ly49D  Mouse  ZAP70/Syk  H-2D  Ly49H  Mouse  ZAP70/Syk  MCMV-induced?  NKp44  Human  NKG2D  Mouse, human  DAP12 D A P 12 D A P 12 DAP10  CD2-M  Mouse, human  SAP  3  ZAP70/Syk  Influenza haem agglutinin, others?  P13K  MIC, U L B P ( R A E - 1 . H 6 0 )  ?  CDA8  •Molecules In parentheses are the ligands for the mouse natural kflier(NK) receptors. HLA human leukocyte antigen; KIR, Wlercea rnmunologlotxillrvllke receptors; MCMV, mouse cylomsgalovtrus; MIC. MHC-dass-l-chal^refetedmotecutes; PBK, pnospnatwylinositoi 3-wnase; RAE-1, retinoic acu early Inducible 1; ULBP. ULi6-dind*ig protein.  Table 1.2 Activating NK cell receptor complexes and their ligands. Table from Cerwenka, A., and L.L. Lanier. 2001. Natural killer cells, viruses, and cancer. Nat Rev Immunol 1:41-49. (188).  32  1.7  Figures  Figure 1.1 Overall scheme of T-cell development in the thymus. Committed lymphoid progenitors arise in the bone marrow and migrate to the thymus. Early committed T cells lack expression of T-cell receptor (TCR), C D 4 and C D 8 , and are termed double-negative (DN; no C D 4 or CD8) thymocytes. DN thymocytes can be further subdivided into four stages of differentiation (DN1, C D 4 4 + C D 2 5 - ; DN2,CD44+CD25+; DN3, C D 4 4 - C D 2 5 + ; and DN4, C D 4 4 - C D 2 5 - ) (189). A s cells progress through the DN2 to DN4 stages, they express the pre-TCR, which is composed of the non-rearranging pre-Ta chain and a rearranged T C R B-chain (190). Successful pre-TCR expression leads to substantial cell proliferation during the DN4 to double positive (DP) transition and replacement of the preT C R a-chain with a newly rearranged T C R a-chain, which yields a complete ap T C R . The aB-TCR+CD4+CD8+ (DP) thymocytes then interact with cortical epithelial cells that express a high density of M H C class I and class II molecules associated with self-peptides. The fate of the D P thymocytes depends on signaling that is mediated by interaction of the T C R with these self-peptide-MHC ligands (191, 192). Too little signaling results in delayed apoptosis (death by neglect). Too much signaling can promote acute apoptosis (negative selection); this is most common in the medulla on encounter with strongly activating selfligands on hematopoietic cells, particularly dendritic cells (193). The appropriate, intermediate level of T C R signaling initiates effective maturation (positive selection). Thymocytes that express T C R s that bind self peptide-MHC-class-l  33 complexes become CD8+ T cells, whereas those that express T C R s that bind self-peptide-MHC-class-ll ligands become CD4+ T cells; these cells are then ready for export from the medulla to peripheral lymphoid sites. S P , single positive. Figure from Germain, R.N. 2002. T-cell development and the C D 4 - C D 8 lineage decision. Nat Rev Immunol 2:309-322. (194).  Figure 1.2 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 to be the fate of most thymocytes. If the T C R 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 Yegulatory'-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 (195). IEL, intestinal epithelial lymphocyte; NKT cell, natural killer T cell; T  cell, C D 4 C D 2 5 regulatory T cell. Figure from Hogquist, K.A., +  R e g  +  T.A. Baldwin, S . C . Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat Rev Immunol 5:772-782. (196).  34  Nature Reviews | immunology  Figure 1.1 Overall scheme of T-cell development in the thymus. Figure from Germain, R.N. 2002. T-cell development and the C D 4 - C D 8 lineage decision. Nat Rev Immunol 2:309-322. (194).  Thymus  Death b y I negtoct negl  If®• e t\  o oo  Positive  selection K  % , i  o°o°go  Negativfi salection: cioraJ deletion receptor editing anergy  © OO  t ooo  G  *©V©©©°< Affinity  Lymph node  OOoO  w  Prowrte immune  responses to  :  © © ©  O  I© ©© © 0  fof&gn antigens j QQQQ  Q  Regulate  immune responses  Copyright © 2005 Nature Publishing Group Nature Reviews j Immunology  Figure 1.2 Central-tolerance mechanisms. Figure from Hogquist, K.A., T.A. Baldwin, S.C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat Rev Immunol 5:772-782. (196).  36  1.8  References  1.  Gerosa, F., B. Baldani-Guerra, C. Nisii, V. Marchesini, G . Carra, and G . Trinchieri. 2002. Reciprocal Activating Interaction between Natural Killer Cells and Dendritic Cells. J. Exp. Med. 195:327-333.  2.  Piccioli, D., S. Sbrana, E. Melandri, and N. M. Valiante. 2002. Contactdependent Stimulation and Inhibition of Dendritic Cells by Natural Killer Cells. J. Exp. Med. 195:335-341.  3.  Colucci, F., M. A . Caligiuri, and J . P. Di Santo. 2003. What does it take to make a natural killer? Nat Rev Immunol 3:413-425.  4.  Mrozek, E., P. Anderson, and M. A . Caligiuri. 1996. Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. Blood 87:2632-2640.  5.  Williams, N. S., T. A. Moore, J . D. Schatzle, I. J . Puzanov, P. V. Sivakumar, A . Zlotnik, M. Bennett, and V. Kumar. 1997. Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates. J Exp Med 186:1609-1614.  6.  Jaleco, A. C , B. Blom, P. Res, K. Weijer, L. L. Lanier, J . H. Phillips, and H. Spits. 1997. Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells. J Immunol 159:694-702.  7.  Miller, J . S., K. A . Alley, and P. McGlave. 1994. Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-  based long-term culture system: identification of a CD34+7+ NK progenitor. Blood 83:2594-2601. 8.  Veinotte, L. L , B. T. Wilhelm, D. L. Mager, and F. Takei. 2003. Acquisition of MHC-specific receptors on murine natural killer cells. Crit Rev Immunol 23:251-266.  9.  Lanier, L. L. 2005. NK cell recognition. Annu Rev Immunol 23:225-274.  10.  Ljunggren, H. G., and K. Karre. 1990. In search of the 'missing self: M H C molecules and NK cell recognition. Immunol Today 11:237-244.  11.  Ciccone, E., D. Pende, O. Viale, A. Than, C. Di Donato, A. M. Orengo, R. Biassoni, S. Verdiani, A. Amoroso, A. Moretta, and et al. 1992. Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2). J Exp Med 176:963-971.  12.  Colonna, M., and J . Samaridis. 1995. Cloning of immunoglobulinsuperfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268:405-408.  13.  Karlhofer, F. M., R. K. Ribaudo, and W. M. Yokoyama. 1992. The interaction of Ly-49 with H-2Dd globally inactivates natural killer cell cytolytic activity. Trans Assoc Am Physicians 105:72-85.  14.  Moretta, A., M. Vitale, C. Bottino, A. M. Orengo, L. Morelli, R. Augugliaro, M. Barbaresi, E. Ciccone, and L. Moretta. 1993. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I  38 molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of M H C class l-protected cells in NK clones displaying different specificities. J Exp Med 178:597-604. 15.  Moretta, A., C. Bottino, M. Vitale, D. Pende, R. Biassoni, M. C. Mingari, and L. Moretta. 1996. Receptors for HLA class-l molecules in human natural killer cells. Annu Rev Immunol 14:619-648.  16.  Vance, R. E., J . R. Kraft, J . D. Altman, P. E. Jensen, and D. H. Raulet. 1998. Mouse C D 9 4 / N K G 2 A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Q a 1(b). J Exp Med 188:1841-1848.  17.  Burshtyn, D. N., A. M. Scharenberg, N. Wagtmann, S. Rajagopalan, K. Berrada, T. Y i , J . P. Kinet, and E. O. Long. 1996. Recruitment of tyrosine phosphatase H C P by the killer cell inhibitor receptor. Immunity 4:77-85.  18.  Campbell, K. S., M. Dessing, M. Lopez-Botet, M. Cella, and M. Colonna. 1996. Tyrosine phosphorylation of a human killer inhibitory receptor recruits protein tyrosine phosphatase 1C. J Exp Med 184:93-100.  19.  Nakamura, M. C , E. C. Niemi, M. J . Fisher, L. D. Shultz, W. E. Seaman, and J . C. Ryan. 1997. Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J Exp Med 185:673-684.  20.  Vely, F., S. Olivero, L. Olcese, A. Moretta, J . E. Damen, L. Liu, G . Krystal, J . C. Cambier, M. Daeron, and E. Vivier. 1997. Differential association of  phosphatases with hematopoietic co- receptors bearing immunoreceptor tyrosine-based inhibition motifs. EurJ Immunol 27:1994-2000. 21.  Biassoni, R., C. Cantoni, M. Falco, S. Verdiani, C. Bottino, M. Vitale, R. Conte, A. Poggi, A. Moretta, and L. Moretta. 1996. The human leukocyte antigen (HLA)-C-specific "activatory" or "inhibitory" natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions. J Exp Med 183:645650.  22.  Smith, K. M., J . Wu, A . B. Bakker, J . H. Phillips, and L L. Lanier. 1998. Ly49D and Ly-49H associate with mouse DAP12 and form activating receptors. J Immunol 161:7-10.  23.  Nakamura, M. C , P. A. Linnemeyer, E. C. Niemi, L. H. Mason, J . R. Ortaldo, J . C. Ryan, and W. E. Seaman. 1999. Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity. J Exp Med 189:493500.  24.  Carretero, M., M. Llano, F. Navarro, T. Bellon, and M. Lopez-Botet. 2000. Mitogen-activated protein kinase activity is involved in effector functions triggered by the C D 9 4 / N K G 2 - C N K receptor specific for HLA-E.  EurJ  Immunol 30:2842-2848. 25.  Lanier, L. L. 2000. Turning on natural killer cells. J Exp Med 191:12591262.  26.  Arase, N., H. Arase, S. Y . Park, H. Ohno, C. R a , and T. Saito. 1997. Association with FcRgamma is essential for activation signal through  NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells. J Exp Med 186:1957-1963. 27.  Anderson, P., M. Caligiuri, C. O'Brien, T. Manley, J . Ritz, and S. F. Schlossman. 1990. Fc gamma receptor type III (CD16) is included in the zeta NK receptor complex expressed by human natural killer cells. Proc Natl Acad Sci USA  28.  87:2274-2278.  Lanier, L. L , G . Y u , and J . H. Phillips. 1989. Co-association of C D 3 zeta with a receptor (CD 16) for IgG Fc on human natural killer cells. Nature 342:803-805.  29.  Wirthmueller, U., T. Kurosaki, M. S. Murakami, and J . V. Ravetch. 1992. Signal transduction by Fc gamma RIM (CD16) is mediated through the gamma chain. J Exp Med 175:1381-1390.  30.  Lanier, L. L., B. C. Corliss, J . Wu, C. Leong, and J . H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:703-707.  31.  McVicar, D. W., L. S. Taylor, P. Gosselin, J . Willette-Brown, A. I. Mikhael, R. L. Geahlen, M. C. Nakamura, P. Linnemeyer, W. E. Seaman, S. K. Anderson, J . R. Ortaldo, and L. H. Mason. 1998. DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk proteintyrosine kinase. J Biol Chem 273:32934-32942.  32.  Bauer, S., V. Groh, J . Wu, A. Steinle, J . H. Phillips, L L. Lanier, and T. Spies. 1999. Activation of N K cells and T cells by N K G 2 D , a receptor for stress- inducible MICA. Science 285:727-729.  33.  Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J . K. Hsia, E. Vivier, and D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of N K G 2 D . Nat Immunol 3:1142-1149.  34.  Wu, J . , Y . Song, A. B. Bakker, S. Bauer, T. Spies, L. L. Lanier, and J . H. Phillips. 1999. An activating immunoreceptor complex formed by N K G 2 D and DAP10. Science 285:730-732.  35.  Gilfillan, S., E. L. Ho, M. Cella, W. M. Yokoyama, and M. Colonna. 2002. N K G 2 D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat Immunol 3:1150-1155.  36.  Cosman, D., J . Mullberg, C . L. Sutherland, W. Chin, R. Armitage, W. Fanslow, M. Kubin, and N. J . Chalupny. 2001. U L B P s , novel M H C class Irelated molecules, bind to C M V glycoprotein UL16 and stimulate NK cytotoxicity through the N K G 2 D receptor. Immunity 14:123-133.  37.  Sutherland, C. L , N. J . Chalupny, and D, Cosman. 2001. The UL16binding proteins, a novel family of M H C class l-related ligands for N K G 2 D , activate natural killer cell functions. Immunol Rev 181:185-192.  38.  Cerwenka, A., A. B. Bakker, T. McClanahan, J . Wagner, J . Wu, J . H. Phillips, and L. L. Lanier. 2000. Retinoic acid early inducible genes define a ligand family for the activating N K G 2 D receptor in mice. Immunity 12:721-727.  39.  Diefenbach, A., A. M. Jamieson, S. D. Liu, N. Shastri, and D. H. Raulet. 2000. Ligands for the murine N K G 2 D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat Immunol 1:119-126.  40.  Diefenbach, A., E. R. Jensen, A. M. Jamieson, and D. H. Raulet. 2001. Rae1 and H60 ligands of the N K G 2 D receptor stimulate tumour immunity. Nature 413:165-171.  41.  Ho, E. L., L. N. Carayannopoulos, J . Poursine-Laurent, J . Kinder, B. Plougastel, H. R. Smith, and W. M. Yokoyama. 2002. Costimulation of multiple NK cell activation receptors by NKG2D. J Immunol 169:36673675.  42.  Perussia, B., and J . V. Ravetch. 1991. Fc gamma RIM (CD16) on human macrophages is a functional product of the Fc gamma RIII-2 gene. EurJ Immunol 21:425-429.  43.  Cassatella, M. A., I. Anegon, M. C. Cuturi, P. Griskey, G . Trinchieri, and B. Perussia. 1989. Fc gamma R(CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2+ in Fc gamma R(CD16)-induced transcription and expression of lymphokine genes. J Exp Med 169:549-567.  44.  Kurosaki, T., and J . V. Ravetch. 1989. A single amino acid in the glycosyl phosphatidylinositol attachment domain determines the membrane topology of Fc gamma Rill. Nature 342:805-807.  45.  Vivier, E., A. J . da Silva, M. Ackerly, H. Levine, C. E. Rudd, and P. Anderson. 1993. Association of a 70-kDa tyrosine phosphoprotein with the  43 CD16: zeta: gamma complex expressed in human natural killer cells. Eur J Immunol 23:1872-1876. 46.  Boles, K. S., S. E. Stepp, M. Bennett, V. Kumar, and P. A. Mathew. 2001. 2B4 (CD244) and C S 1 : novel members of the C D 2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes. Immunol Rev 181:234-249.  47.  Tangye, S. G., S. Lazetic, E. Woollatt, G . R. Sutherland, L. L. Lanier, and J . H. Phillips. 1999. Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase S H P - 2 and the adaptor signaling protein S A P . J Immunol 162:6981-6985.  48.  Lee, K. M., M. E. McNerney, S. E. Stepp, P. A. Mathew, J . D. Schatzle, M. Bennett, and V. Kumar. 2004. 2B4 acts as a non-major histocompatibility complex binding inhibitory receptor on mouse natural killer cells. J Exp Med 199:1245-1254.  49.  McNerney, M. E., D. Guzior, and V. Kumar. 2005. 2B4 (CD244)-CD48 interactions provide a novel M H C class l-independent system for NK-cell self-tolerance in mice. Blood 106:1337-1340.  50.  Boles, K. S., H. Nakajima, M. Colonna, S. S. Chuang, S. E. Stepp, M. Bennett, V. Kumar, and P. A. Mathew. 1999. Molecular characterization of a novel human natural killer cell receptor homologous to mouse 2B4. Tissue Antigens 54:27-34.  51.  Nakajima, H., and M. Colonna. 2000. 2B4: an NK cell activating receptor with unique specificity and signal transduction mechanism. Hum Immunol 61:39-43.  52.  Valiante, N. M., and G . Trinchieri. 1993. Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes. J Exp M e d 178:1397-1406.  53.  Garni-Wagner, B. A., A. Purohit, P. A. Mathew, M. Bennett, and V. Kumar. 1993. A novel function-associated molecule related to non-MHC-restricted cytotoxicity mediated by activated natural killer cells and T cells. J Immunol 151:60-70.  54.  Bottino, C , R. Augugliaro, R. Castriconi, M. Nanni, R. Biassoni, L. Moretta, and A. Moretta. 2000. Analysis of the molecular mechanism involved in 2B4-mediated NK cell activation: evidence that human 2B4 is physically and functionally associated with the linker for activation of T cells. Eur J Immunol 30:3718-3722.  55.  Roncagalli, R., J . E. Taylor, S. Zhang, X . Shi, R. Chen, M. E. Cruz-Munoz, L. Yin, S. Latour, and A. Veillette. 2005. Negative regulation of natural killer cell function by EAT-2, a SAP-related adaptor. Nat Immunol 6:10021010.  56.  Bretscher, P. 1992. The two-signal model of lymphocyte activation twentyone years later. Immunol Today 13:74-76.  57.  Chambers, C. A., and J . P. Allison. 1997. Co-stimulation in T cell responses. Curr Opin Immunol 9:396-404.  58.  Lenschow, D. J . , T. L. Walunas, and J . A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu Rev Immunol 14:233-258.  59.  Love, P. E., and E. W. Shores. 2000. ITAM multiplicity and thymocyte selection: how low can you go? Immunity 12:591-597.  60.  Zamoyska, R., A. Basson, A. Filby, G. Legname, M. Lovatt, and B. Seddon. 2003. The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation. Immunological  Reviews  191:107-118. 61.  Bu, J . Y., A. S. Shaw, and A. C. Chan. 1995. Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance. Proc Natl Acad Sci USA  62.  92:5106-5110.  Jordan, M. S., A. L. Singer, and G . A. Koretzky. 2003. Adaptors as central mediators of signal transduction in immune cells. Nat Immunol 4:110-116.  63.  Lucas, J . A., A. T. Miller, L. O. Atherly, and L. J . Berg. 2003. The role of Tec family kinases in T cell development and function. Immunological Reviews  64.  191:119-138.  Allison, J . P. 1994. CD28-B7 interactions in T-cell activation. Curr Opin Immunol 6:414-419.  65.  Aruffo, A., and B. Seed. 1987. Molecular cloning of a CD28 c D N A by a high-efficiency C O S cell expression system. Proc Natl Acad Sci  USA  84:8573-8577. 66.  June, C. H., J . A. Bluestone, L. M. Nadler, and C. B. Thompson. 1994. The B7 and CD28 receptor families. Immunol Today 15:321-331.  67.  August, A., and B. Dupont. 1994. Activation of src family kinase Ick following CD28 crosslinking in the Jurkat leukemic cell line. Biochem Biophys Res Commun 199:1466-1473.  68.  Prasad, K. V., Y. C. Cai, M. Raab, B. Duckworth, L. Cantley, S. E. Shoelson, and C. E. Rudd. 1994. T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-MetXaa-Met motif. Proc Natl Acad Sci  69.  USA91:2834-2838.  Truitt, K. E., C. M. Hicks, and J . B. Imboden. 1994. Stimulation of CD28 triggers an association between CD28 and phosphatidylinositol 3-kinase in Jurkat T cells. J Exp Med 179:1071-1076.  70.  Boise, L. H., A. J . Minn, P. J . Noel, C. H. June, M. A. Accavitti, T. Lindsten, and C. B. Thompson. 1995. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3:87-98.  71.  Hutloff, A., A. M. Dittrich, K. C . Beier, B. Eljaschewitsch, R. Kraft, I. Anagnostopoulos, and R. A. Kroczek. 1999. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 397:263-266.  72.  Yoshinaga, S. K., J . S. Whoriskey, S. D. Khare, U. Sarmiento, J . Guo, T. Horan, G . Shih, M. Zhang, M. A. Coccia, T. Kohno, A. Tafuri-Bladt, D. Brankow, P. Campbell, D. Chang, L. Chiu, T. Dai, G . Duncan, G . S. Elliott, A . Hui, S . M. M c C a b e , S. Scully, A . Shahinian, C. L. Shaklee, G . V a n , T. W. Mak, and G . Senaldi. 1999. T-cell co-stimulation through B7RP-1 and ICOS. Nature 402:827-832.  73.  Mages, H. W., A. Hutloff, C. Heuck, K. Buchner, H. Himmelbauer, F. Oliveri, and R. A. Kroczek. 2000. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. EurJ Immunol 30:1040-1047.  74.  Coyle, A. J . , S. Lehar, C. Lloyd, J . Tian, T. Delaney, S. Manning, T. Nguyen, T. Burwell, H. Schneider, J . A. Gonzalo, M. Gosselin, L. R. Owen, C. E. Rudd, and J . C. Gutierrez-Ramos. 2000. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 13:95-105.  75.  Wallin, J . J . , L. Liang, A. Bakardjiev, and W. C. Sha. 2001. Enhancement of CD8+ T cell responses by ICOS/B7h costimulation. J Immunol 167:132139.  76.  Croft, M. 2003. Co-stimulatory members of the T N F R family: keys to effective T-cell immunity? Nat Rev Immunol 3:609-620.  77.  Ware, C. F., P. D. Crowe, T. L. Vanarsdale, J . L. Andrews, M. H. Grayson, R. Jerzy, C. A. Smith, and R. G . Goodwin. 1991. Tumor necrosis factor (TNF) receptor expression in T lymphocytes. Differential regulation of the type I T N F receptor during activation of resting and effector T cells. J Immunol 147:4229-4238.  78.  Kim, E. Y., and H. S. Teh. 2004. Critical role of T N F receptor type-2 (p75) as a costimulator for IL-2 induction and T cell survival: a functional link to CD28. J Immunol 173:4500-4509.  79.  Hintzen, R. Q., S. M. Lens, M. P. Beckmann, R. G . Goodwin, D. Lynch, and R. A. van Lier. 1994. Characterization of the human CD27 ligand, a novel member of the T N F gene family. J Immunol 152:1762-1773.  80.  Ohshima, Y., Y. Tanaka, H. Tozawa, Y. Takahashi, C. Maliszewski, and G. Delespesse. 1997. Expression and function of OX40 ligand on human dendritic cells. J Immunol 159:3838-3848.  81.  Pollok, K. E., Y. J . Kim, J . Hurtado, Z. Zhou, K. K. Kim, and B. S. Kwon. 1994. 4-1 BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. EurJ Immunol 24:367-374.  82.  Bertram, E. M., P. Lau, and T. H. Watts. 2002. Temporal segregation of 41BB versus CD28-mediated costimulation: 4-1 BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection. J Immunol 168:3777-3785.  83.  Gramaglia, I., A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft. 2000. The OX40 costimulatory receptor determines the development of C D 4 memory by regulating primary clonal expansion. J Immunol 165:3043-3050.  84.  Hendriks, J . , L. A. Gravestein, K. Tesselaar, R. A. van Lier, T. N. Schumacher, and J . Borst. 2000. CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol 1:433-440.  85.  Kim, E. Y., J . J . Priatel, S. J . Teh, and H. S. Teh. 2006. T N F receptor type 2 (p75) functions as a costimulator for antigen-driven T cell responses in vivo. J Immunol 176:1026-1035.  49 86.  Saito, T., and S. Yamasaki. 2003. Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors. Immunol Rev 192:143-160.  87.  Chambers, C. A., M. S. Kuhns, J . G . Egen, and J . P. Allison. 2001. C T L A 4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol 19:565-594.  88.  Salomon, B., and J . A. Bluestone. 2001. Complexities of CD28/B7: C T L A 4 costimulatory pathways in autoimmunity and transplantation. Annu Rev Immunol 19:225-252.  89.  Ostrov, D. A., W. Shi, J . C. Schwartz, S. C. Almo, and S. G . Nathenson. 2000. Structure of murine CTLA-4 and its role in modulating T cell responsiveness. Science 290:816-819.  90.  Marengere, L. E., P. Waterhouse, G . S. Duncan, H. W. Mittrucker, G . S. Feng, and T. W. Mak. 1996. Regulation of T cell receptor signaling by tyrosine phosphatase S Y P association with CTLA-4. Science 272:11701173.  91.  Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J . A. Bluestone, and A. H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541-547.  92.  Waterhouse, P., J . M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, and T. W. Mak. 1995.  Lymphoproliferative disorders with early lethality in mice deficient in Ctla4. Science 270:985-988. 93.  Nishimura, H., T. Okazaki, Y . Tanaka, K. Nakatani, M. Hara, A. Matsumori, S. Sasayama, A. Mizoguchi, H. Hiai, N. Minato, and T. Honjo. 2001. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291:319-322.  94.  Ishida, Y., Y. Agata, K. Shibahara, and T. Honjo. 1992. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. Embo J 11:3887-3895.  95.  Dong, H., G. Zhu, K. Tamada, and L. Chen. 1999. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5:1365-1369.  96.  Freeman, G. J., A. J . Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura, L. J . Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, H. F. Horton, L. Fouser, L. Carter, V. Ling, M. R. Bowman, B. M. Carreno, M. Collins, C. R. Wood, and T. Honjo. 2000. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192:1027-1034.  97.  Latchman, Y., C. R. Wood, T. Chernova, D. Chaudhary, M. Borde, I. Chernova, Y. Iwai, A. J . Long, J . A. Brown, R. Nunes, E. A. Greenfield, K. Bourque, V. A. Boussiotis, L. L. Carter, B. M. Carreno, N. Malenkovich, H. Nishimura, T. Okazaki, T. Honjo, A. H. Sharpe, and G. J . Freeman. 2001.  PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2:261-268. 98.  Tseng, S. Y., M. Otsuji, K. Gorski, X . Huang, J . E. Slansky, S. I. Pai, A. Shalabi, T. Shin, D. M. Pardoll, and H. Tsuchiya. 2001. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 193:839-846.  99.  Barber, D. L , E. J . Wherry, D. Masopust, B. Zhu, J . P. Allison, A. H. Sharpe, G. J . Freeman, and R. Ahmed. 2006. Restoring function in exhausted C D 8 T cells during chronic viral infection. Nature 439:682-687.  100.  Carter, L , L. A. Fouser, J . Jussif, L. Fitz, B. Deng, C. R. Wood, M. Collins, T. Honjo, G . J . Freeman, and B. M. Carreno. 2002. PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur J Immunol 32:634-643.  101.  Spits, H., B. Blom, A. C. Jaleco, K. Weijer, M. C. Verschuren, J . J . van Dongen, M. H. Heemskerk, and P. C. Res. 1998. Early stages in the development of human T, natural killer and thymic dendritic cells. Immunol R e v 165:75-86.  102.  McQueen, K. L , and P. Parham. 2002. Variable receptors controlling activation and inhibition of NK cells. CurrOpin  103.  Immunol 14:615-621.  Jamieson, A. M., A. Diefenbach, C. W. McMahon, N. Xiong, J . R. Carlyle, and D. H. Raulet. 2002. The role of the N K G 2 D immunoreceptor in immune cell activation and natural killing. Immunity 17:19-29.  104.  Teng, M. W. L , M. H. Kershaw, Y . Hayakawa, L. Cerutti, S. M. Jane, P. K. Darcy, and M. J . Smyth. 2005. T Cells Gene-engineered with D A P 12 Mediate Effector Function in an NKG2D-dependent and Major Histocompatibility Complex-independent Manner. J. Biol. Chem. 280:38235-38241.  105.  Schuhmachers, G., K. Ariizumi, P. A. Mathew, M. Bennett, V. Kumar, and A. Takashima. 1995. 2B4, a new member of the immunoglobulin gene superfamily, is expressed on murine dendritic epidermal T cells and plays a functional role in their killing of skin tumors. J Invest Dermatol 105:592596.  106.  Kambayashi, T., E. Assarsson, B. J . Chambers, and H. G . Ljunggren. 2001. Cutting edge: Regulation of CD8(+) T cell proliferation by 2B4/CD48 interactions. J Immunol 167:6706-6710.  107.  Costello, R. T., S. Sivori, F. Mallet, D. Sainty, C. Arnoulet, D. Reviron, J . A. Gastaut, A. Moretta, and D. Olive. 2002. A novel mechanism of antitumor response involving the expansion of CD3+/CD56+ large granular lymphocytes triggered by a tumor-expressed activating ligand. Leukemia 16:855-860.  108.  Lee, K. M., S. Bhawan, T. Majima, H. Wei, M. I. Nishimura, H. Yagita, and V. Kumar. 2003. Cutting Edge: The NK Cell Receptor 2B4 Augments Antigen-Specific T Cell Cytotoxicity Through CD48 Ligation on Neighboring T Cells. J Immunol 170:4881-4885.  109.  McMahon, C. W., and D. H. Raulet. 2001. Expression and function of NK cell receptors in CD8+ T cells. CurrOpin  110.  Immunol 13:465-470.  Ugolini, S., and E. Vivier. 2000. Regulation of T cell function by NK cell receptors for classical M H C class I molecules. CurrOpin  Immunol 12:295-  300. 111.  Coles, M. C , C. W. McMahon, H. Takizawa, and D. H. Raulet. 2000. Memory C D 8 T lymphocytes express inhibitory MHC-specific Ly49 receptors. Eur J Immunol 30:236-244.  112.  Ortaldo, J . R., R. Winkler-Pickett, A. T. Mason, and L. H. Mason. 1998. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J Immunol 160:1158-1165.  113.  Zajac, A. J . , R. E. Vance, W. Held, D. J . Sourdive, J . D. Altman, D. H. Raulet, and R. Ahmed. 1999. Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor. J Immunol 163:5526-5534.  114.  Chambers, C. A., J . Kang, Y . Wu, W. Held, D. H. Raulet, and J . P. Allison. 2002. The lymphoproliferative defect in CTLA-4-deficient mice is ameliorated by an inhibitory NK cell receptor. Blood 99:4509-4516.  115.  Ugolini, S., C. Arpin, N. Anfossi, T. Walzer, A. Cambiaggi, R. Forster, M. Lipp, R. E. Toes, C. J . Melief, J . Marvel, and E. Vivier. 2001. Involvement of inhibitory N K R s in the survival of a subset of memory- phenotype CD8+ T cells. Nat Immunol 2:430-435.  116.  Mingari, M. C , F. Schiavetti, M. Ponte, C. Vitale, E. Maggi, S. Romagnani, J . Demarest, G. Pantaleo, A. S. Fauci, and L. Moretta. 1996. Human  CD8+ T lymphocyte subsets that express HLA class l-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations. Proc Natl Acad Sci USA 117.  93:12433-12438.  Mingari, M. C , M. Ponte, C. Cantoni, C. Vitale, F. Schiavetti, S. Bertone, R. Bellomo, A. T. Cappai, and R. Biassoni. 1997. HLA-class l-specific inhibitory receptors in human cytolytic T lymphocytes: molecular characterization, distribution in lymphoid tissues and co-expression by individual T cells. Int Immunol 9:485-491.  118.  Huard, B., and L. Karlsson. 2000. A subpopulation of CD8+ T cells specific for melanocyte differentiation antigens expresses killer inhibitory receptors (KIR) in healthy donors: evidence for a role of KIR in the control of peripheral tolerance. Eur J Immunol 30:1665-1675.  119.  Bamford, R. N., A. J . Grant, J . D. Burton, C. Peters, G . Kurys, C. K. Goldman, J . Brennan, E. Roessler, and T. A. Waldmann. 1994. The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Proc Natl Acad Sci  USA  91:4940-4944. 120.  Grabstein, K. H., J . Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J . Richardson, M. A. Schoenborn, M. Ahdieh, and et al. 1994. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 264:965-968.  121.  Waldmann, T. A. 1991. The interleukin-2 receptor. J. Biol. Chem. 266:2681-2684.  122.  Giri, J . G., S. Kumaki, M. Ahdieh, D. J . Friend, A. Loomis, K. Shanebeck, R. DuBose, D. Cosman, L. S. Park, and D. M. Anderson. 1995. Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. Embo J 14:3654-3663.  123.  Gaffen, S. L. 2001. SIGNALING DOMAINS O F T H E INTERLEUKIN 2 R E C E P T O R . Cytokine 14:63.  124.  Van Parijs, L , Y . Refaeli, J . D. Lord, B. H. Nelson, A. K. Abbas, and D. Baltimore. 1999. Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death. Immunity 11:281-288.  125.  Grimm, E. A., A. Mazumder, H. Z. Zhang, and S. A. Rosenberg. 1982. Lymphokine-activated killer cell phenomenon. Lysis of natural killerresistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 155:1823-1841.  126.  Kalland, T., H. Belfrage, P. Bhiladvala, and G. Hedlund. 1987. Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells. J Immunol 138:36403645.  127.  Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J . L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, K. Brasel, P. J .  Morrissey, K. Stocking, J . C. Schuh, S. Joyce, and J . J . Peschon. 2000. Reversible defects in natural killer and memory C D 8 T cell lineages in interleukin 15-deficient mice. J Exp M e d 191:771-780. 128.  Lodolce, J . P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, and A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669-676.  129.  Burkett, P. R., R. Koka, M. Chien, S. Chai, F. Chan, A. Ma, and D. L. Boone. 2003. IL-15R alpha expression on CD8+ T cells is dispensable for T cell memory. Proc Natl Acad Sci USA  130.  100:4724-4729.  Koka, R., P. R. Burkett, M. Chien, S. Chai, F. Chan, J . P. Lodolce, D. L. Boone, and A. Ma. 2003. Interleukin (IL)-15R[alpha]-deficient natural killer cells survive in normal but not IL-15R[alpha]-deficient mice. J Exp Med 197:977-984.  131.  Dubois, S., J . Mariner, T. A. Waldmann, and Y . Tagaya. 2002. IL15Ralpha recycles and presents IL-15 In trans to neighboring cells. Immunity 17:537-547.  132.  Ku, C. C , M. Murakami, A. Sakamoto, J . Kappler, and P. Marrack. 2000. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 288:675-678.  133.  Lind, E. F., S. E. Prockop, H. E. Porritt, and H. T. Petrie. 2001. Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development. J Exp Med 194:127-134.  134.  Starr, T. K., S. C. Jameson, and K. A. Hogquist. 2003. Positive and negative selection of T cells. Annu Rev Immunol  135.  I'lASQ-MQ.  Anderson, G., and E. J . Jenkinson. 2001. Lymphostromal interactions in thymic development and function. Nat Rev Immunol 1:31-40.  136.  Alberola-lla, J . , and G. Hernandez-Hoyos. 2003. The R a s / M A P K cascade and the control of positive selection. Immunological Reviews 191:79-96.  137.  Oukka, M., I. C. Ho, F. C. de la Brousse, T. Hoey, M. J . Grusby, and L. H. Glimcher. 1998. The transcription factor NFAT4 is involved in the generation and survival of T cells. Immunity 9:295-304.  138.  Aliahmad, P., and J . Kaye. 2006. Commitment issues: linking positive selection signals and lineage diversification in the thymus. Immunol Rev 209:253-273.  139.  Pircher, H., K. Burki, R. Lang, H. Hengartner, and R. M. Zinkernagel. 1989. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 342:559-561.  140.  Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. P. Berzins, S. J . Turley, H. von Boehmer, R. Bronson, A. Dierich, C. Benoist, and D. Mathis. 2002. Projection of an immunological self shadow within the thymus by the aire protein. Science 298:1395-1401.  141.  Anderson, M. S., E. S. Venanzi, Z. Chen, S. P. Berzins, C. Benoist, and D. Mathis. 2005. The cellular mechanism of Aire control of T cell tolerance. Immunity 23:227-239.  142.  Hammerling, G . J . , G . Schonrich, F. Momburg, N. Auphan, M. Malissen, B. Malissen, A. M. Schmitt-Verhulst, and B. Arnold. 1991. Non-deletional mechanisms of peripheral and central tolerance: studies with transgenic mice with tissue-specific expression of a foreign M H C class I antigen. Immunol Rev 122:47-67.  143.  Guy-Grand, D., O. Azogui, S. Celli, S. Darche, M. C. Nussenzweig, P. Kourilsky, and P. Vassalli. 2003. Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice. J Exp M e d 197:333-341.  144.  Shen, M. M., R. C. Skoda, R. D. Cardiff, J . Campos-Torres, P. Leder, and D. M. Omitz. 1994. Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morphologies. Embo J 13:1375-1385.  145.  Clegg, C. H., J . T. Rulffes, P. M. Wallace, and H. S. Haugen. 1996. Regulation of an extrathymic T-cell development pathway by oncostatin M. Nature 384:261-263.  146.  Blais, M. E., I. Louis, and C. Perreault. 2006. T-cell development: an extrathymic perspective. Immunol Rev 209:103-114.  147.  Terra, R., N. Labrecque, and C. Perreault. 2002. Thymic and extrathymic T cell development pathways follow different rules. J Immunol 169:684692.  148.  Boileau, C , M. Houde, G . Dulude, C. H. Clegg, and C. Perreault. 2000. Regulation of extrathymic T cell development and turnover by oncostatin M. J Immunol 164:5713-5720.  149.  Blais, M. E., G . Gerard, M. M. Martinic, G . Roy-Proulx, R. M. Zinkernagel, and C. Perreault. 2004. Do thymically and strictly extrathymically developing T cells generate similar immune responses? Blood 103:31023110.  150.  Urdahl, K. B., J . C. Sun, and M. J . Bevan. 2002. Positive selection of M H C class lb-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol 3:772-779.  151.  Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, and E. G . Pamer. 1999. H2-M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. J Exp M e d 190:195-204.  152.  Yamada, H., T. Ninomiya, A. Hashimoto, K. Tamada, H. Takimoto, and K. Nomoto. 1998. Positive selection of extrathymically developed T cells by self- antigens. J Exp Med 188:779-784.  153.  Clegg, C. H., H. S. Haugen, J . T. Rulffes, S. L. Friend, and A. G . Farr. 1999. Oncostatin M transforms lymphoid tissue function in transgenic mice by stimulating lymph node T-cell development and thymus autoantibody production. Exp Hematol 27:712-725.  154.  Gutierrez-Ramos, J . C , I. Moreno de Alboran, and C. Martinez. 1992. In vivo administration of interleukin-2 turns on anergic self-reactive T cells and leads to autoimmune disease. Eur J Immunol 22:2867-2872.  60 155.  Lantz, O., and A. Bendelac. 1994. A n invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class Ispecific CD4+ and CD4-8- T cells in mice and humans. J Exp Med 180:1097-1106.  156.  Bendelac, A. 1995. Positive selection of mouse NK1+ T cells by C D 1 expressing cortical thymocytes. J Exp Med 182:2091-2096.  157.  Park, S. H., K. Benlagha, D. Lee, E. Balish, and A. Bendelac. 2000. Unaltered phenotype, tissue distribution and function of Valpha14(+) NKT cells in germ-free mice. EurJ Immunol 30:620-625.  158.  Fischer, K., E. Scotet, M. Niemeyer, H. Koebernick, J . Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. Kaufmann, and U. E. Schaible. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for C D Id-restricted T cells. Proc Natl Acad Sci USA  159.  101:10685-10690.  Wu, D. Y., N. H. Segal, S. Sidobre, M. Kronenberg, and P. B. Chapman. 2003. Cross-presentation of disialoganglioside G D 3 to natural killer T cells. J Exp Med 198:173-181.  160.  Brigl, M., L. Bry, S. C. Kent, J . E. Gumperz, and M. B. Brenner. 2003. Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat Immunol 4:1230-1237.  161.  Stetson, D. B., M. Mohrs, R. L. Reinhardt, J . L. Baron, Z. E. Wang, L. Gapin, M. Kronenberg, and R. M. Locksley. 2003. Constitutive cytokine m R N A s mark natural killer (NK) and NK T cells poised for rapid effector function. J Exp M e d 198:1069-1076.  162.  Kawano, T., J . Cui, Y . Koezuka, I. Toura, Y . Kaneko, H. Sato, E. Kondo, M. Harada, H. Koseki, T. Nakayama, Y . Tanaka, and M. Taniguchi. 1998. Natural killer-like nonspecific tumor cell lysis mediated by specific ligandactivated Valpha 14 NKT cells. PNAS 95:5690-5693.  163.  Vincent, M. S., D. S. Leslie, J . E. Gumperz, X . Xiong, E. P. Grant, and M. B. Brenner. 2002. CD1-dependent dendritic cell instruction. Nat Immunol 3:1163-1168.  164.  Carnaud, C , D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, and A. Bendelac. 1999. Cutting edge: Cross-talk between cells of the innate immune system: N K T cells rapidly activate NK cells. J Immunol 163:46474650.  165.  Kronenberg, M. 2005. Toward an understanding of NKT cell biology: progress and paradoxes. Annu Rev Immunol 23:877-900.  166.  Gadue, P., N. Morton, and P. L. Stein. 1999. The Src family tyrosine kinase Fyn regulates natural killer T cell development. J Exp Med 190:1189-1196.  167.  Shimamura, M., T. Ohteki, U. Beutner, and H. R. MacDonald. 1997. Lack of directed V alpha 14-J alpha 281 rearrangements in NK1+ T cells. EurJ Immunol 27:1576-1579.  168.  Bendelac, A., M. N. Rivera, S. H. Park, and J . H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 15:535-562.  169.  Stanic, A. K., J . S. Bezbradica, J . J . Park, N. Matsuki, A. L. Mora, L. Van Kaer, M. R. Boothby, and S. Joyce. 2004. NF-kappa B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes. J Immunol 172:2265-2273.  170.  Townsend, M. J . , A. S. Weinmann, J . L. Matsuda, R. Salomon, P. J . Farnham, C. A. Biron, L. Gapin, and L. H. Glimcher. 2004. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 20:477-494.  171.  Cheroutre, H. 2005. lELs: enforcing law and order in the court of the intestinal epithelium. Immunol Rev 206:114-131.  172.  Cheroutre, H., and L. Madakamutil. 2004. Acquired and natural memory T cells join forces at the mucosal front line. Nat Rev Immunol 4:290-300.  173.  Guy-Grand, D., B. Cuenod-Jabri, M. Malassis-Seris, F. Selz, and P. Vassalli. 1996. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur J Immunol 26:2248-2256.  174.  Das, G . , and C. A. Janeway, Jr. 2003. M H C specificity of ilELs. Trends Immunol 24:88-93.  175.  Leishman, A. J . , L. Gapin, M. Capone, E. Palmer, H. R. MacDonald, M. Kronenberg, and H. Cheroutre. 2002. Precursors of functional M H C class I- or class ll-restricted CD8alphaalpha(+) T ceils are positively selected in the thymus by agonist self-peptides. Immunity 16:355-364.  176.  Cruz, D., B. C. Sydora, K. Hetzel, G. Yakoub, M. Kronenberg, and H. Cheroutre. 1998. An opposite pattern of selection of a single T cell antigen receptor in the thymus and among intraepithelial lymphocytes. J Exp Med 188:255-265.  177.  Saurer, L , I. Seibold, S. Rihs, C. Vallan, T. Dumrese, and C. Mueller. 2004. Virus-induced activation of self-specific T C R alpha beta C D 8 alpha alpha intraepithelial lymphocytes does not abolish their self-tolerance in the intestine. J Immunol 172:4176-4183.  178.  Poussier, P., T. Ning, D. Banerjee, and M. Julius. 2002. A unique subset of self-specific intraintestinal T cells maintains gut integrity. J Exp Med 195:1491-1497.  179.  Kisielow, P., H. S. Teh, H. Bluthmann, and H. von Boehmer. 1988. Positive selection of antigen-specific T cells in thymus by restricting M H C molecules. Nature 335:730-733.  180.  Kisielow, P., H. Bluthmann, U. D. Staerz, M. Steinmetz, and H. von Boehmer. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333:742-746.  181.  Teh, H. S., H. Kishi, B. Scott, and H. Von Boehmer. 1989. Deletion of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal T C R levels and low levels of C D 8 molecules. J Exp Med 169:795-806.  182.  Pihlgren, M., C. Arpin, T. Walzer, M. Tomkowiak, A. Thomas, J . Marvel, and P. M. Dubois. 1999. Memory CD44(int) C D 8 T cells show increased  64 proliferative responses and IFN-gamma production following antigenic challenge in vitro. Int Immunol 11:699-706. 183.  Yamada, H., G . Matsuzaki, Q. Chen, Y. Iwamoto, and K. Nomoto. 2001. Reevaluation of the origin of CD44(high) "memory phenotype" C D 8 T cells: comparison between memory C D 8 T cells and thymus-independent C D 8 T cells. Eur J Immunol 31:1917-1926.  184.  Yamada, H., T. Nakamura, G . Matsuzaki, Y. Iwamoto, and K. Nomoto. 2000. TCR-independent activation of extrathymically developed, self antigen- specific T cells by IL-2/IL-15. J Immunol 164:1746-1752.  185.  Harada, M., H. Yamada, K. Tatsugami, and K. Nomoto. 2001. Evidence of the extrathymic development of tyrosinase-related protein-2- recognizing CD8+ T cells with low avidity. Immunology 104:67-74.  186.  Opferman, J . T., B. T. Ober, and P. G . Ashton-Rickardt. 1999. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283:1745-1748.  187.  Yamada, H., G . Matsuzaki, Y . Iwamoto, and K. Nomoto. 2000. Unusual cytotoxic activities of thymus-independent, self-antigen- specific CD8(+) T cells. Int Immunol 12:1677-1683.  188.  Cerwenka, A., and L. L. Lanier. 2001. Natural killer cells, viruses and cancer. Nat Rev Immunol 1:41-49.  189.  Godfrey, D. I., J . Kennedy, T. Suda, and A. Zlotnik. 1993. A developmental pathway involving four phenotypically and functionally  65 distinct subsets of C D 3 - C D 4 - C D 8 - triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol 150:4244-4252. 190.  von Boehmer, H., and H. J . Fehling. 1997. Structure and function of the pre-T cell receptor. Annu Rev Immunol 15:433-452.  191.  Robey, E., and B. J . Fowlkes. 1994. Selective events in T cell development. Annu Rev Immunol 12:675-705.  192.  von Boehmer, H., H. S. Teh, and P. Kisielow. 1989. The thymus selects the useful, neglects the useless and destroys the harmful. Immunol Today 10:57-61.  193.  Matzinger, P., and S. Guerder. 1989. Does T-cell tolerance require a dedicated antigen-presenting cell? Nature 338:74-76.  194.  Germain, R. N. 2002. T-cell development and the C D 4 - C D 8 lineage decision. Nat Rev Immunol 2:309-322.  195.  Baldwin, T. A., K. A. Hogquist, and S. C. Jameson. 2004. The fourth way? Harnessing aggressive tendencies in the thymus. J Immunol 173:65156520.  196.  Hogquist, K. A., T. A. Baldwin, and S. C. Jameson. 2005. C E N T R A L T O L E R A N C E : L E A R N I N G S E L F - C O N T R O L IN T H E T H Y M U S . Nature Reviews Immunology 5:772.  66  Chapter 2  IL-2 Activated C D 8 C D 4 4 +  h i g h  Cells Express Both Adaptive and  Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells  2.1  1  Abstract  C D 8 T cells depend on the ap T C R for antigen recognition and function. +  However, antigen-activated C D 8 T cells can also express receptors of the innate +  immune system. In this study, we examined the expression of NK receptors on a population of C D 8 T cells expressing high levels of CD44 ( C D 8 C D 4 4 cells) +  +  hi  from normal mice. These cells are distinct from conventional memory C D 8 T +  cells and they proliferate and become activated in response to Interleukin 2 (IL-2) via a CD48/CD2-dependent mechanism. Prior to activation they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and N K G 2 D . Interesting, the IL-2 activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the N K G 2 D ligand, Rae-1, on the target cell. In contrast to conventional C D 8 T cells, IL-2-activated C D 8 C D 4 4 +  +  hi  cells express DAP12, an  adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated C D 8 C D 4 4 cells express receptors of both +  hl  the adaptive and innate immune system and may play a unique role in the surveillance of host cells, which have been altered by infection or transformation.  1  A version of this chapter has been published as:  Dhanji, S., and H.-S. Teh. 2003. IL-2-Activated CD8+CD44high Cells Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells. J Immunol 171:344  67  2.2  Introduction  The cells of the adaptive immune system utilize functionally rearranged receptors to recognize foreign antigens. By contrast, cells of the innate immune system primarily use germ line encoded receptors to defend against infected or transformed cells. Interestingly, cells of the adaptive immune system can express some of these germ line encoded receptors (1). Furthermore, recent studies have indicated that in addition to providing immediate immune effector mechanisms to contain the spread of infections, the innate immune system also serves to prime the adaptive immune system to defend against infections that cannot be contained by the innate immune system (1). Natural killer (NK) cells are an integral part of the innate immune system, producing cytokines to activate other cells of the immune system as well as directly recognizing and killing infected or transformed cells (2). NK cells use a combination of activating and inhibitory receptors in order to perform these functions (3-5). These activating and inhibitory receptors set a threshold for the activation of NK cells, with inhibitory signals predominating in the absence of infection (6). When a host cell is transformed or infected, the balance shifts towards activation, allowing NK cells to eliminate these hazardous host cells (6). ap T cell receptor ( T C R ) C D 8 T cells play a crucial role in the adaptive +  +  immune system. The primary function of C D 8 T cells is the lysis of virally +  infected target cells, via recognition of viral peptides that are presented by M H C class I molecules. Naive C D 8 T cells require two distinct signals for activation: +  68 signal one is provided by engagement of the T C R with its cognate ligand and signal two is provided by interaction of costimulatory receptors with their respective ligands on the antigen presenting cells (7, 8). Recent studies showed that normal mice possess a subset of C D 8  +  a p T C R T cells that express very high levels of CD44 (herein referred to as +  CD8 CD44 +  hi  cells). These C D 8 C D 4 4 cells possess other markers of +  hi  activated/memory cells such as the high expression of CD122 (IL-2 receptor p) and Ly6C and they can proliferate in response to IL-2 and IL-15 independently of T C R stimulation (9-11). The development of these C D 8 T cells appears to be +  driven by the interaction of the a p T C R with self-antigen and they can develop in the absence of a functional thymus (9). C D 8 C D 4 4 cells have also been shown to express receptors +  hl  characteristic of NK cells (12). Both activating and inhibitory NK receptors can be found on these cells. Receptors such as 2B4, which can be either activating or inhibitory (13), and whose expression on T cells can be induced by activation with various cytokines (14), have been found on C D 8 C D 4 4 cells (14). Inhibitory +  hl  receptors such as the killer cell immunoglobulin-like receptor (KIR) in humans and the lectin-like Ly49 family in mice, have also been implicated in both the development and function of memory phenotype C D 8 T cells (reviewed in (15)). The observation that NK receptor expression by C D 8 T cells is restricted to cells with an activated/memory phenotype and the fact that T cells do not express N K receptors in the thymus (16), strongly suggests that only cells that have encountered cognate antigen are capable of their expression.  69 In this report, we characterized the phenotype and function of C D 8 C D 4 4 +  hl  cells from normal mice. We showed that these cells only require IL-2 for proliferation and the acquisition of cytolytic activity. Interestingly, these IL-2activated cells express both D A P 10 and D A P 12 adaptor molecules. D A P 12 is normally expressed by activated NK, but not conventional C D 8 T cells (17). The +  activated cells demonstrate preferential killing of syngeneic tumor cells. Expression of the N K G 2 D ligand, Rae-15, on the tumor cells led to greatly enhanced killing of the target cells.  2.3  Materials and Methods  Mice Breeders for C57BL/6 (B6), Balb/c, and DBA/2 were obtained from the Jackson Laboratories (Bar Harbor, ME). These mice were bred at the Animal Unit in our department. Mice 8 to 12 weeks of age were used for the experiments described.  Abs and flow cytometry The following mAbs were used: anti-CD4 (GK1.5), anti-CD8a (53-6.7), antiCD8p (53.58), anti-CD3e (2C11), anti-CD44 (PGP1), anti-TCRp (H57.597), antiy S T C R , anti-CD43 (1B11), anti-CD94 (18D3), anti-NK-1.1 (PK136), anti-CD122 (TM-p1), anti-Ly6C (AL-21), anti-CD244.2 (2B4), and anti-NKG2D (18)]. Biotinylated mAbs were detected using streptavidin-PE. All Abs were obtained  70 from BD PharMingen (San Diego, California) except anti-NKG2D (18), which was a kind gift from Dr. Wayne M. Yokoyama (Howard Hughes Medical Institute, Washington University, St. Louis). Staining for N K G 2 D ligands was performed using the mNKG2D-lg fusion protein (19), which was a kind gift from Dr. Lewis L. Lanier (University of California, San Francisco) followed by staining with a FITClabeled anti-human Ig mAb. Cell staining and flow cytometry were performed according to standard procedures. The CellQuest software program (Becton Dickinson, Mountain View, CA) was used for data acquisition and analysis. For three-color analysis, a total of 20,000 live events were collected and analyzed.  Cell lines Cell lines used were the R M A lymphoma (H-2 , Rae-15), RMA-Rae-15 b+  transfectant (H-2 , Rae-18 ), TAP-deficient R M A S (H-2 ", Rae-15-), R M A S - R a e b+  +  b  15 transfectant (H-2 ", Rae-15 ), A20 lymphoma (H-2 ) and P815 mastocytoma b  +  d  (H-2 ). The cell lines were cultured in IMEM (Life Technologies, Burlington, d  Canada), supplemented with 10% (v/v) F B S (Life Technologies), 5x10 u.M 2-ME 5  and antibiotics (l-medium). The RMA-Rae15 and R M A S - R a e 1 5 transfectants (20) were kind gifts from Dr. Lewis L. Lanier (University of California, San Francisco); these cell lines were passaged in l-medium and G418 (800u,g/ml) to maintain high levels of Rae15 expression.  71 Ex vivo staining Single cell suspensions from the lymph nodes (LN) of mice were treated with anti-CD4 mAb and then depleted of C D 4 l g cells using Dynabeads M-450 +  +  Sheep anti-mouse IgG (Dynal Biotech, Lake Success, New York), according to manufacturers instructions. The cells were >95% C D 8 and were then stained +  with the appropriate mAbs and analyzed by F A C S .  CD8 T Cell purification and sorting +  Single cell suspensions from the lymph nodes (LN) of mice were treated with biotinylated anti-CD8p mAb followed by positive selection using the MiniMACS system (Miltenyi Biotech, Auburn, California), according to the manufacturers specifications. The resulting cells were >95% pure C D 8 a p T C R p T cells. C D 8 +  +  +  T cells purified by this method contained - 1 0 % C D 4 4 cells. For purification of hl  CD8 CD44 +  hi  and CD8 CD44'° T cells, MiniMACS purified C D 8 T cells were +  +  stained with anti-CD8a-FITC and anti-CD44-PE and sorted on a Becton Dickinson F A C S Vantage S E Turbo sort cell sorter. Cell sorting was performed by Andrew Johnson (University of British Columbia) and the sorted C D 8 C D 4 4 +  hl  or CD8 CD44'° cells were >98% pure. For some assays the following purification +  method was used to provide a source of CD8 CD44'° cells: lymph node cells +  were incubated with mAbs specific for CD4, mouse Ig and C D 4 4 followed by depletion with anti-mouse Ig-coated D Y N A beads (Dynal). This method yielded a population of >98% C D 8 C D 4 4 " cells. +  72 Natural Killer (NK) cell purification and activation Natural killer cells were isolated by treating spleen cells with anti-CD4, anti-CD8, and anti-CD3 mAbs followed by treatment with sheep anti-mouse Ig in order to deplete C D 3 C D 4 C D 8 l g cells. The cells were about 60% CD3" +  +  +  +  D X 5 N K 1 . 1 C D 1 2 2 and were then cultured in l-medium supplemented with IL-2 +  +  +  (200U/ml) for 5 days. On day 5 the cells were >99% C D 3 D X 5 N K 1 . 1 2 B 4 , +  +  +  CD122 . +  CFSE  labeling  Purified C D 8 T cells (1x10 /ml) were labeled with 1 uM C F S E (Molecular +  7  Probes, Eugene, OR) in P B S for 8 min at room temperature. After stopping the reaction with the addition of an equal volume of F C S , cells were washed four times with complete media prior to use.  Proliferation  assays  For IL-2 proliferation, 1x10 purified C D 8 T cells of the indicated CD44 5  +  phenotype were cultured in l-medium + IL-2 (200U/ml) in 96-well U-bottom plates for 5 days. Blocking mAbs were added to the indicated cultures at 10 u,g/ml. The cells were pulsed with 1u.ci of [ H]thymidine for the final 6 hours to assess 3  proliferation. Foranti-CD3s (2C11)-induced proliferation, 1x10 purified C D 8 T 4  +  cells of the indicated CD44 phenotype were cultured in 96-well flat bottom plates coated with 2C11 (10u.g/ml) and IL-2 (20U/ml) with or without the indicated  73 blocking mAbs (10uig/ml) for 4 days and pulsed with 1uCi of [ H]thymidine for the 3  final 6 hours to assess proliferation. For C F S E proliferation assays, the same conditions as above were used except the cells were CFSE-labeled and analyzed on day 4 by F A C S .  CTL Assays Target cells (RMA, R M A S , RMA-Rae18, RMAS-Rae18, A20, or P815) were labeled with  5 1  Cr (100u.Ci) for 1 hour at 37°C and then washed. 1x10 labeled 4  targets were added to 96-well U-bottom plates containing activated C D 8 or NK cells at the indicated ratios in a final volume of 200ul After a 5-hour incubation, the supernatants were collected and counted. Spontaneous release varied from 8-15% of the maximum. All assays were performed in triplicate. Percent specific lysis was calculated as 100% x [cpm (experimental well) - cpm (spontaneous release)]/[(cpm (maximum release) - cpm (spontaneous release)].  RT-PCR NK cells and C D 8 C D 4 4 T cells were activated with IL-2 (200U/ml) and +  hi  CD8 CD44'° cells were activated with anti-CD3 and IL-2 (20U/ml) for 5 days. +  Cells were then harvested, and total R N A was prepared according to the manufacturer's recommendations using the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNAs were generated from total R N A using the Protoscript c D N A synthesis kit according to manufacturers recommendations (NEB, Beverly, MA).  74 Left and right primer sequences, respectively, were as follows: DAP10: 5'C A G G C T A C C T C C T G T T C C T G - 3 ' and 5 ' - G C C A G G C A T G T T G A T G T A G A - 3 ' ; DAP12: 5 ' - C T G G T G T A C T G G C T G G G A T T - 3 ' and 5'C T G G T C T C T G A C C C T G A A G C - 3 ' ; Glyceraldehyde-3-phosphate dehydrogenase (GPADH): 5 ' - T G C ( A / C ) T C C T G C A C C A C C A A C T - 3 ' and 5'( C / T ) G C C T G C T T C A C C A C C T T - 3 ' . The P C R products were subjected to electrophoresis on a 2% Agarose gel and visualized by ethidium bromide.  2.4  Results  Phenotypic characterization  of CD8 CD44 +  hl  cells from normal mice.  C D 8 T cells from normal mice express varying levels of CD44. We first +  determined the cell surface phenotype of C D 8 T cells from B6 mice, which +  expressed either high (CD44 ) or low (CD44'°) levels of CD44. The C D 8 C D 4 4 hi  +  hi  T cells comprised about 10 to 20% of total C D 8 T cells in B6 mice with older +  mice possessing an increased proportion of these cells (n=40; the mice vary between 6 and 12 weeks of age). Figure 1 shows the relative expression of various cell surface markers by the C D 8 C D 4 4 and CD8 CD44'° cells from the +  hi  +  same B6 mouse. These data indicate that the C D 4 4 cells expressed elevated hl  levels of CD44, Ly6C, CD122 and 1B11, a CD43 isoform characteristic of activated C D 8 T cells (21). This activated/memory cell surface phenotype +  suggests that there is prior recognition of cognate antigen by the C D 4 4 cells. hl  Interestingly, C D 4 4 cells expressed lower levels of the a p T C R than the CD44'° hl  75 cells. The lower expression of the ap T C R is characteristic of NKT cells (22) as well as the self-specific C D 8 T cells that can develop via an extrathymic +  pathway (11). However, unlike NKT cells, immediately ex vivo C D 8 C D 4 4 cells +  hl  do not express NK1.1 or significant levels of other NK receptors such as CD94, 2B4 or N K G 2 D (Fig. 2.1).  CD8 CD44 +  hl  T cells proliferate in response to IL-2 stimulation.  The expression of CD122 (IL-2Rp) on C D 8 C D 4 4 T cells suggests that they +  hi  might be capable of proliferating in response to cytokines such as IL-2 or IL-15. To test for this possibility we purified C D 8 C D 4 4 and CD8 CD44'° T cells from +  hi  +  B6 mice by cell sorting and labeled these cells with the fluorescent dye, C F S E and then cultured them with IL-2. The C F S E data in Fig. 2.2A indicate that only the C D 8 C D 4 4 +  hi  proliferated in response to IL-2. Proliferation is relatively rapid,  as some of the cells have undergone more than 6 rounds of cell division by 72 hr. By contrast, the vast majority of IL-2-activated CD8 CD44'° cells did not divide +  during the 48 to 96 hr observation period. Measurement of cell proliferation by following the incorporation of H-thymidine during the last 6hr of a 96 hr culture 3  period also indicate that only the C D 8 C D 4 4 cells are capable of IL-2-induced +  hl  proliferation (Fig. 2.2B). These results are consistent with a previous report showing that only C D 8 T cells expressing high levels of CD44, but not antigen+  specific memory C D 8 T cells, can proliferate in response to IL-2 or IL-15 (23). +  IL-2 activated CD8 CD44 +  h  cells expressed NK receptors  76 Several studies have shown that activated C D 8 T cells can express various +  types of N K receptors (24). However, these studies do not distinguish between the expression of NK receptors by either activated C D 8 C D 4 4 cells or +  CD8 CD44 +  10  cells. To determine whether C D 8 C D 4 4 +  hi  hl  cells express NK receptors  after IL-2 activation, we examined the expression of NK receptors on IL-2activated C D 8 cells. Since only C D 8 C D 4 4 +  +  hi  but not CD8 CD44'° cells can be +  activated by IL-2 (Fig. 2.2) these results were equated with IL-2-activated C D 8 C D 4 4 T cells. Fig. 2.3 shows that IL-2 activated C D 8 C D 4 4 cells +  hi  +  hi  expressed high levels of 2B4 and CD94 but relatively low levels of DX5 and NK1.1. The activated cells also uniformly expressed a low level of NKG2D. This pattern of NK receptor expression is distinct from IL-2-activated N K cells, which express high levels of these NK receptors (data not shown). This pattern is also distinct from antigen-activated conventional CD8 CD44'° T cells, which with the +  exception of N K G 2 D , do not express the other NK receptors (25). Previous studies have shown that both the S and L isoforms of 2B4 are induced upon IL-2 activation of C D 8 T cells (14). Here we showed that IL-2-activated C D 8 C D 4 4  hi  cells expressed high levels of 2B4. By contrast, anti-TCR activated C D 8 C D 4 4  l0  +  +  +  cells did not express 2B4 (data not shown).  CD48 and CD2 are crucial for IL-2-induced proliferation of CD8 CD44 +  hi  cells  2B4 is a member of the C D 2 subset of Ig superfamily molecules and is the high affinity ligand for CD48 (26, 27). CD48 is also a member of the C D 2 subset and is expressed by lymphocytes, monocytes and endothelial cells (28). Members of  77 the C D 2 subset have been observed to interact with themselves or with other family members (28). CD48 has also been shown to function as a costimulatory molecule for T cells (27, 29). Engagement of 2B4 on NK cell surfaces with CD48 can trigger cell-mediated cytotoxicity, interferon-y secretion, phosphoinositol turnover and NK-cell invasiveness (28). During the course of our experiments we noticed that IL-2-induced proliferation of C D 8 C D 4 4 +  hl  cells was dependent on  cell-cell contact (data not shown). However, since highly purified C D 8 C D 4 4 +  hl  cells proliferated in response to IL-2 it is unlikely that contact with other lymph node cell types is required for IL-2-induced proliferation. A s C D 8 C D 4 4 cells +  hl  express high levels of CD48 and C D 2 (Fig. 2.4A) and these cells expressed high levels of 2B4 upon activation (Fig. 2.3), we determined whether CD48, 2B4 or CD2 are important in mediating IL-2-induced proliferation. To test the role of these molecules in IL-2-induced proliferation we cultured purified C D 8 T cells in +  IL-2 in the presence or absence of anti-CD48, anti-CD2, or anti-2B4 mAbs. Proliferation was determined either by measuring C F S E fluorescence (Fig. 2.4B) or by the incorporation of radiolabeled thymidine (Fig. 2.4C). The data indicate that anti-CD48 and anti-CD2 inhibited IL-2-induced proliferation to the same extent whereas the anti-2B4 mAb did not have any effect on the proliferative response. This observation indicates that CD48 and C D 2 , but not 2B4, are important in mediating IL-2-induced proliferation. W e also determined the antiproliferative effect of the anti-CD48 mAb on anti-CD3 + IL-2-induced proliferation, as assessed by C F S E dilution (Fig. 2.4B) or the incorporation of radiolabeled thymidine (Fig. 2.4C). Under these conditions, both the C D 8 C D 4 4 +  hi  as well as  78 the C D 8 C D 4 4 +  populations will be activated. However, since the CD8 CD44'°  10  +  cells comprise ~90% of the starting population these cells constitute the vast majority of the responding cells. By contrast to IL-2-induced proliferation, the antiC D 3 induced proliferation was not inhibited by the anti-CD48 mAb demonstrating that anti-CD3-induced activation of CD8 CD44'° cells is resistant to inhibition by +  anti-CD48 mAb. These data support the hypothesis that IL-2-induced activation of C D 8 C D 4 4 +  hi  cells and anti-CD3-induced activation of C D 8 C D 4 4 +  |0  cells occur  via CD48-dependent and CD48-independent mechanisms, respectively.  Activated CD8 CD44 +  cells preferentially kill syngeneic tumors.  hl  Previous studies suggested that the development of C D 8 C D 4 4 +  hl  cells is  dependent on interaction with self-antigens in peripheral lymphoid organs (9, 30, 31). Therefore, it was of interest to determine whether IL-2-activated C D 8 C D 4 4 +  hl  cells demonstrate self-specificity as would be suggested by the killing of syngeneic tumor cells. R M A tumor cells are syngeneic to B6 mice and are lethal when injected into these mice (32). W e first compared the ability of activated CD8 CD44 +  hi  and CD8 CD44'° cells from a B6 mouse to kill R M A target cells. +  Since only C D 8 C D 4 4 +  CD8 CD44 +  hi  hl  cells can be activated by IL-2 whereas both the  as well as C D 8 C D 4 4 +  |0  cells can be activated by anti-CD3 + IL-2, we  used anti-CD3 + IL-2 as a common means of activating F A C S purified CD8 CD44 +  hi  and C D 8 C D 4 4 +  |0  cells. The purpose of this experiment was to  determine whether activated C D 8 C D 4 4 +  hi  and C D 8 C D 4 4 +  l0  cells differ in their  ability to kill syngeneic tumor targets. We found that only anti-CD3-activated  79 CD8 CD44 +  hi  cells demonstrate significant killing of R M A target cells (Fig. 2.5A)  even though the C D 8 C D 4 4 cells were activated to the same extent as +  |0  assessed by the expression of CD25 and CD69 (data not shown). These results indicate that these two activated C D 8 subsets do indeed differ in their ability to +  kill syngeneic tumor target cells. We then determined whether this preferential killing of syngeneic tumors applied to IL-2-activated C D 8 C D 4 4 cells from other mouse strains. Balb/c and +  hi  DBA/2 mice are of the H-2 haplotype but differ in many non-MHC class I genetic d  loci. We determined the ability of IL-2-activated C D 8 cells from Balb/c and +  DBA/2 mice to kill A20 and P815 tumor targets, which are derived from Balb/c and DBA/2 mice, respectively. Since IL-2 only activates C D 8 C D 4 4 +  hl  cells we  concluded that results derived from activation of purified C D 8 T cells to be +  indicative of IL-2-activated C D 8 C D 4 4 +  CD8 CD44 +  hi  hi  cells. We found that IL-2-activated  cells from Balb/c mice preferentially kill A20 targets (Fig. 2.5B, left  panel) whereas P815 target cells were killed to a greater extent by IL-2-activated CD8 CD44 +  hi  cells from DBA/2 mice (Fig. 2.5C, left panel). These data  demonstrate that there is a preference for the killing of syngeneic tumors by IL-2activated C D 8 C D 4 4 +  hi  cells from Balb/c and DBA/2 mice.  We also examined the sensitivity of these tumor targets to killing by antiC D 3 + IL-2-activated CD8 CD44'° T cells. To minimize the contribution of +  CD8 CD44 +  hi  cells in these studies we depleted the C D 8 cells of C D 4 4 cells +  +  prior to culture (see Materials and Methods). We found that anti-CD3-activated CD8 CD44'° T cells from either Balb/c (Fig. 2.5B, right panel) or DBA/2 (Fig. +  80 2.5C, right panel) did not kill either target, even though these cells were highly activated. This observation likely reflects differences in the T C R repertoire of the CD8 CD44 +  hi  and CD8 CD44'° cells. Our data also supports the hypothesis that +  the C D 8 C D 4 4 +  hl  population expresses T C R s with a strong bias towards self-  antigens. By contrast, the T C R s of the conventional C D 8 C D 4 4 +  10  population are  expected to be purged of anti-self (H-2 ) reactivity and therefore are unable to kill d  syngeneic (H-2 ) tumor targets. These data also implied that the C D 8 C D 4 4 d  +  hl  and C D 8 C D 4 4 cells have differential requirements for self-antigens for their +  |0  selection and development.  IL-2-activated CD8 CD44 +  hi  cells express DAP12  The expression of N K G 2 D on IL-2 activated NK and C D 8 C D 4 4 +  hi  cells as well as  anti-CD3 + IL-2-activated CD8 CD44'° cells were determined by staining with an +  anti-NKG2D mAb (18). Consistent with previous reports, we found that IL-2activated NK cells expressed high levels of N K G 2 D whereas anti-CD3 + IL-2activated CD44'° cells expressed lower levels (Fig. 2.6). IL-2-activated C D 4 4  hi  cells also expressed lower levels of N K G 2 D (Fig. 2.6). Recent studies showed that activated NK cells express two alternative splice variants of N K G 2 D that associate differentially with D A P 10 and D A P 12 (17, 33). In NK cells, association of N K G 2 D with DAP10 provides a costimulatory signal whereas association of N K G 2 D with D A P 12 confers a direct stimulatory signal (17, 33). Furthermore, activated C D 8 cells from DAP10-deficient mice lack N K G 2 D expression +  suggesting that only DAP10 but not D A P 12 is expressed by conventional C D 8 T +  81 cells (33). W e determined the expression of D A P 10 and D A P 12 in activated NK cells, IL-2-activated C D 8 C D 4 4 , and anti-CD3 + IL-2-activated C D 8 C D 4 4 +  hi  +  l0  cells from B6 mice. Consistent with previous reports, activated NK cells expressed both D A P 10 and D A P 12 whereas anti-CD3-activated C D 8 C D 4 4 +  |0  cells expressed only D A P 10. Interestingly, IL-2-activated C D 8 C D 4 4 cells +  expressed both D A P 10 and DAP12. Thus, C D 8 C D 4 4 +  hi  hi  cells are more like NK  cells with regard to DAP12 expression.  Expression of Rae-15 enhances the lysis of syngeneic tumors by IL-2-activated CD8 CD44 +  hi  cells.  We next determined whether NK receptors participate in this propensity to kill syngeneic tumor cells by IL-2-activated C D 8 C D 4 4 +  hl  cells. Recent studies have  shown that the activating N K G 2 D receptor plays a crucial role in the killing of syngeneic tumor cells (20, 32). The ligands for N K G 2 D , Rae-1 and H60, are expressed on infected or transformed cells (34). However, R M A tumor cells from B6 mice do not express Rae-1 (34). Rae-15 is normally expressed in B6 mice (20) and we used RMA-Rae18 transfectants (20) to determine whether N K G 2 D participates in the killing of syngeneic tumor cells. W e tested the ability of activated C D 8 C D 4 4 +  hi  In-  and anti-CD3 + IL-2-activated CD8 CD44'° cells from B6 +  mice to kill R M A and R M A - R a e 1 6 target cells. W e also used the peptidetransporter (TAP)-deficient cell lines, R M A S and RMAS-Rae-18, as sources of M H C class l-deficient target cells that do or do not express Rae-18. The expression of Rae-18 on transfectant cell lines was confirmed by staining with a  82 murine NKG2D-lg fusion protein (19)(data not shown). A s an additional control for the specificity of killing we determined the cytolytic activity of IL-2-activated NK cells against these same target cells. NK cells were enriched by depleting B6 spleen cells of C D 4 , C D 8 , l g and C D 3 cells by negative selection and the +  +  +  +  negatively selected cells were activated with IL-2. These activated cells were of the C D 4 C D 8 " C D 3 " N K G 2 D D X 5 N K 1 . 1 2 B 4 C D 9 4 cell surface phenotype (data +  +  +  +  +  not shown), consistent with the conclusion that this purification and activation scheme led to a pure population of effector NK cells. The data in Fig. 2.7 indicate that the activated N K cells killed R M A S target to a greater extent than R M A cells. This is consistent with the conclusion that engagement of M H C class I molecules by inhibitory NK receptors likely contribute to the poorer killing of R M A target cells. More interestingly, and consistent with the observations of others (20, 25, 32), expression of Rae-15 on either R M A or R M A S cells greatly increased their susceptibility to NK killing (Fig. 2.7). By contrast, IL-2-activated C D 8 C D 4 4 +  hl  cells were relatively inefficient in  killing either R M A or R M A S target cells (Fig. 2.7). This observation indicates that the T A P mutation has differential effects on target cell susceptibility to killing by NK or IL-2-activated C D 8 C D 4 4 cells. Interestingly, the presence of Rae16 on +  hi  either R M A or R M A S target cells also led to greatly enhanced killing by IL-2activated C D 8 C D 4 4 cells. This observation suggests that the interaction of +  hl  N K G 2 D with its ligand, Rae-18, greatly enhanced killing of syngeneic tumor cells by IL-2-acitvated C D 8 C D 4 4 cells. +  hi  83 By contrast, anti-CD3 + IL-2-activated C D 8 C D 4 4 +  l0  cells are very  inefficient killers of R M A and R M A S targets and Rae-18 transfectants of these cell lines (Fig. 2.7). These data indicate that IL-2-activated C D 8 C D 4 4 +  hi  cells  possess the ability to kill syngeneic tumor targets and this killing is greatly enhanced by the expression of Rae-18.  2.5  Discussion  In this report we described a population of C D 8 C D 4 4 +  hl  cells in normal mice that  possess properties distinct from conventional memory T cells. These cells comprise between 10 and 2 0 % of total C D 8 T cells of normal mice. They proliferated in response to stimulation by exogenous IL-2 and differentiated into potent killer cells that demonstrate specificity for syngeneic tumors. These cells also upregulate several NK receptors including N K G 2 D upon activation with IL-2. Interestingly, IL-2-activated C D 8 C D 4 4 +  hi  cells express DAP12, an adaptor  molecule that is normally found in activated NK cells (35). Furthermore, tumor targets expressing a N K G 2 D ligand are exquisitely sensitive to killing by IL-2activated C D 8 C D 4 4 +  hi  cells.  Other studies also demonstrate that the C D 8 C D 4 4 +  hl  cells that are present  in normal mice are distinct from conventional memory T cells in many aspects. In contrast to M H C class l-restricted C D 8 T cells, the development of C D 8 C D 4 4 +  +  cells can be thymus-independent (9, 11). The frequency of C D 8 C D 4 4 cells +  hl  increases with age even when the mice are maintained under germ-free  hi  84 conditions suggesting that the development of these cells is not foreign antigendriven (36, 37). Memory C D 8 T cells and C D 8 C D 4 4 +  +  hi  cells also differ in their  activation threshold. Whereas memory C D 8 cells possess a lower activation +  threshold compared to naive T cells, C D 8 C D 4 4 +  hl  cells were shown to require a  higher activation threshold relative to naive C D 8 T cells (11). Memory C D 8 T +  +  cell also expressed lower levels of CD44 and CD122 compared to the CD8 CD44 +  hi  (38) cells and the growth of C D 8 C D 4 4 +  hi  cells, but not memory C D 8  T cells, can be supported by IL-2 or IL-15 (11). These differences between CD8 CD44  hi  cells and memory C D 8 T cells strongly suggest that the  CD8 CD44  hl  cells are of a lineage that is distinct from conventional C D 8 T cells.  +  +  +  +  T C R transgenic mice provide a defined system for determining the developmental requirement of C D 8 C D 4 4 +  hl  cells. In T C R transgenic mice these  cells express only the transgenic T C R and can be easily tracked. It was found that the development of C D 8 C D 4 4 +  hl  cells in T C R transgenic mice is independent  of a thymus but is dependent on interaction with self-antigen in extrathymic tissues (9). Our observation that IL-2-activated C D 8 C D 4 4 +  hl  cells demonstrate a  preference in the killing of syngeneic tumor cells is also consistent with the notion that the development of these cells is dependent on selection by self-antigens. It was shown that in vivo delivery of large amounts of IL-2 to athymic nude mice results in an autoimmune disease (39). This observation is consistent with the hypothesis that the autoimmune disease is mediated by IL-2 activation of selfspecific extrathymic C D 8 C D 4 4 +  hl  cells. Large numbers of extrathymic T cells  developed in oncostatin M transgenic mice (31). Interestingly, the presence of  85 cognate antigen is required for these C D 8 T cells to acquire a memory +  phenotype (31). In normal mice, the self-ligands that are required for the development of C D 8 C D 4 4 +  numbers of C D 8 C D 4 4 +  hl  cells remain to be determined. We found that small  cells are also present in p2-microglobulin and T A P -  hl  deficient mice (our unpublished observations) suggesting that these cells may be restricted to non-classical M H C molecules. The report by Urdahl, et al. demonstrating that C D 8 T cells with a memory phenotype can be positively +  selected on M H C class l molecules by hematopoietic cells is consistent with this b  notion (40). Several reports have also suggested that C D 8 T cells that are +  specific for self-antigens, such as melanocyte differentiation antigens, can be isolated from healthy donors (41, 42). Interestingly, it was found that C D 8 T cells +  that are capable of initiating tumor regression could also induce autoimmune reactions. This finding supports the hypothesis that self-specific C D 8 C D 4 4 +  hl  cells may also contribute to autoimmune diseases (43). The preferential killing of syngeneic tumor target cells by IL-2-activated CD8 CD44 +  hi  but not anti-CD3-activated CD8 CD44'° cells may reflect differences +  in the T C R repertoire of these two cell populations. However, analysis of T C R Vp usage using the BD Pharmingen mouse Vp T C R screening panel (a collection of mAbs to 17 VP's) by these two populations before and after activation reveals no significant differences in T C R Vp usage by these cells (data not shown). This result indicates that there is no preferential usage of Vp gene segments by these two populations. More importantly, they suggest that the T C R repertoire of CD8 CD44 +  hl  cells is likely to be very heterogeneous and more sophisticated  analysis are required to determine whether there is a bias towards self-antigens in this heterogeneous T C R repertoire. Immediately ex vivo C D 8 C D 4 4 cells do not express 2B4 (Fig. 2.1), but +  hi  express high levels of 2B4 upon activation with IL-2 (Fig. 2.3). 2B4 has been shown to be expressed primarily on NK cells and a small subset of memory phenotype C D 8 T cells (44-46), some of which can mediate non-MHC-restricted +  cytotoxicity (44, 45). The expression of 2B4 by C D 8 T cells has been shown to +  correlate with the acquisition of effector functions (46) and was involved in proliferation (14). Since 2B4 is a high affinity receptor for CD48 it may serve as a receptor for CD48 and participate in IL-2-induced proliferation. However, we found that IL-2-induced proliferation was only inhibited by the anti-CD48 but not the anti-2B4 mAb (Fig. 2.4). A trivial explanation for the lack of inhibition by the anti-2B4 mAb is that this mAb does not block CD48/2B4 interaction. Arguing against this explanation is the observation that either the anti-CD48 or the same anti-2B4 mAb suppressed antigen-induced proliferation of C D 8 T cells to the +  same extent; anti-CD2 mAb has no inhibitory effect in this system (14). Furthermore, there was no additive effect of the anti-CD48 and anti-2B4 mAbs in suppressing antigen-induced proliferation of C D 8 T cells in this system (14). +  These observations suggest that the anti-2B4 mAb acts by inhibiting CD48/2B4 interaction. Therefore, a more likely explanation of our data for the lack of inhibitory effect of the anti-2B4 mAb is the late induction of 2B4 in IL-2-activated cells. By contrast, C D 2 is expressed at a high level in ex vivo C D 8 C D 4 4 cells +  hl  and anti-CD2 mAb inhibited IL-2-induced proliferation to the same extent as the  87 anti-CD48 mAb. This observation is consistent with the hypothesis that CD48 and/or C D 2 are important in mediating IL-2-induced proliferation. CD48 is a glycosylphosphatidylinositol (GPI)-anchored molecule and it can aggregate lipid rafts when it is engaged (29). Thus, the anti-CD48 mAb could potentially exert its inhibitory effects by preventing the aggregation of lipid rafts. Mouse C D 2 has also been shown to constitutively associate with lipid rafts (47) and so the antiC D 2 mAb may also exert its effect by preventing the aggregation of lipid rafts. Alternatively, the anti-CD48 mAb could serve as a ligand for the C D 2 receptor. C D 2 is implicated as an important co-stimulatory molecule in lymphocyte activation and proliferation (48). Furthermore, proline residues in the C D 2 cytoplasmic domain have been shown to activate kinase activity such as PI3kinase and the Tec-family tyrosine kinase, ITK (49, 50). In this alternative model, the anti-CD48 or the anti-CD2 mAb inhibits IL-2-induced proliferation by interfering with the C D 2 signaling pathway. It is noted that the anti-CD48 mAb has no effect on anti-CD3-driven proliferation of C D 8 T cells (Fig. 2.4). This +  observation provides independent support that C D 8 C D 4 4 cells and +  hl  conventional C D 8 T cells are dependent on distinct signaling pathways for +  growth. Our data clearly shows the activating NK receptor, N K G 2 D , plays an important role in the lysis of syngeneic tumor targets by IL-2 activated CD8 CD44 +  hi  cells (Fig. 2.7). The expression of the ligands for N K G 2 D , Rae-1  and H60 on tumor cells, leads to their rejection in vivo by both NK cells and C D 8 T cells (20, 32). Interestingly, the expression of the ligands for N K G 2 D on tumor  +  88 cells in vivo, results in protection from subsequent challenge from parental (ligand-negative) tumors, suggesting a role for N K G 2 D in the activation of tumorspecific C D 8 T cells (32). N K G 2 D has been shown to be exclusively +  costimulatory in C D 8 T cells and directly stimulating in NK cells (17, 33). These +  differences have been attributed to the differential recruitment of adaptor molecules in NK cells versus C D 8 T cells. Stimulation of N K G 2 D on NK cells +  primarily results in recruitment of DAP12 while stimulation in C D 8 T cells, leads to recruitment of DAP10 (17, 33). Interestingly, we found that in contrast to antiCD3-activated CD8 CD44'° cells, which express only DAP10, IL-2-activated +  C D 8 C D 4 4 cells express both D A P 10 and DAP12. Thus, the IL-2-activated +  CD8 CD44 +  hi  hl  cells are more NK-like in this regard. In this study we have shown  that IL-2-activated C D 8 C D 4 4 cells from B6 mice can kill Rae-1" R M A target +  hi  cells, albeit with low efficiency. This observation suggests that the killing of syngeneic tumor target cells may be mediated in part by the a(3 T C R . The expression of Rae-15 on R M A cells resulted in greatly enhanced lysis by i n activated C D 8 C D 4 4 cells suggesting that the N K G 2 D receptor plays an +  hi  important role in the killing of syngeneic tumor cells by these cells. Interestingly, deficiency in the T A P peptide transporter did not enhance killing of syngeneic tumor target cells by IL-2-activated C D 8 C D 4 4 +  hi  cells. This observation is  consistent with the notion that these activated cells either lack the inhibitory receptors that are expressed by NK cells and/or have a distinct combination of activating/inhibitory receptors from the activated NK cells. Alternatively, the ligands that are recognized by the ap T C R on IL-2-activated C D 8 C D 4 4 cells +  hi  89 may be independent of the T A P peptide transporter. The existence of CD8 CD44 +  hl  cells in TAP-deficient mice (our unpublished observations) is  consistent with this notion. Collectively, our data support the hypothesis that i n activated C D 8 C D 4 4 cells express ap T C R s that are specific for syngeneic +  hl  tumor target cells. In the absence of Rae-1 expression, the tumor targets are killed at relatively low efficiency. When the tumor targets express Rae-1, then there is synergy between the ap T C R and the N K G 2 D in the killing of syngeneic tumor target cells. Such a synergy between the ap T C R and activating N K receptors would render these cells particularly adept in the surveillance of host cells, which have been altered through infection or transformation.  2.6  Acknowledgments  We thank Soo-Jeet Teh for excellent technical assistance. We are grateful to Dr. Lewis Lanier (UCSF) for providing us with cell lines (RMA, RMA-Rae18, R M A S Rae-18) and the NKG2D-lg fusion protein and Dr. Wayne Yokoyama (HHMI, Washington University, St. Louis) for providing us with the anti-NKG2D mAb.  90  2.7  Figures  Figure 2.1 Cell surface phenotype of C D 8 C D 4 4 +  hi  and CD8 CD44'° T cells ex +  vivo. Lymph node cells from B6 mice were depleted of C D 4 and lg cells and +  +  stained with antibodies against various cell surface markers. The filled histograms represent expression of the indicated cell surface molecule by gated CD8 CD44 +  hl  cells. The unfilled histograms represent expression of the indicated  cell surface molecule by gated CD8 CD44'° cells from the same mouse. +  Figure 2.2 Only C D 8 C D 4 4 +  hi  cells proliferate in response to IL-2. (A) F A C S  sorted C D 8 C D 4 4 and C D 4 4 T cells from B6 mice were labeled with C F S E +  hi  10  and cultured with IL-2-supplemented media. Cell division, as assessed by C F S E dilution, was determined at 48, 72, and 96 hours by F A C S analysis. (B) Sorted C D 8 C D 4 4 and CD44 T cells were cultured with IL-2 for 4 days and pulsed +  hi  10  with H-thymidine for the last 6 hours of culture. Error bars represent standard 3  deviations of triplicate cultures.  Figure 2.3 Activated C D 8 C D 4 4 +  hi  cells express several NK receptors. C D 8 T +  cells were purified from B6 lymph nodes as described in Materials and Methods. The purified C D 8 T cells were cultured with IL-2-supplemented media for 5 days +  and then stained with antibodies against various cell surface markers that are characteristic of NK cells. The filled histograms represent expression levels of the  91 indicated NK receptor by IL-2 activated C D 8 C D 4 4 cells and the unfilled +  hi  histograms represent unstained controls.  Figure 2.4 CD48/CD2 interactions are required for IL-2-induced proliferation of C D 8 C D 4 4 cells. A) The expression of CD48 and C D 2 on B6 C D 8 C D 4 4 cells +  hi  +  hi  ex vivo (filled histograms) vs. unstained controls (unfilled histograms). B) Purified C D 8 T cells from B6 mice were labeled with C F S E and cultured in either IL-2+  supplemented media (left panel) or plate-bound anti-CD3s antibody (2C11) plus 20U/ml IL-2 (right panel) in the presence of 10u.g/ml anti-CD48 mAb (solid line), anti-CD2 (dashed line), anti-2B4 (dotted line) or without any antibody (filled histogram) for 4 days and analyzed by F A C S . C) Purified C D 8 T cells from B6 +  mice were cultured in either IL-2-supplemented media (left panel) or plate-bound anti-CD3s antibody (2C11) plus 20U/ml IL-2 (right panel) in the presence or absence of 10u.g/ml anti-CD48 mAb for 4 days and then pulsed with H-thymidine 3  for the final 6 hours of culture. The error bars represent the standard deviation of triplicate cultures.  Figure 2.5 Activated C D 8 C D 4 4 +  hi  cells preferential killed syngeneic tumor target  cells. A) FACS-purified C D 8 C D 4 4 +  hi  and CD8 CD44'° cells from a B6 mouse +  were activated with anti-CD3 (2C11) and IL-2 (20U/ml) for 3 days and then used as effectors in a Cr-release assay against R M A target cells, which are 51  syngeneic to B6 mice. Error bars represent the standard deviation for triplicate cultures. B) (Left panel) Purified C D 8 T cells from Balb/c mice were cultured in +  92 IL-2-supplemented media for 5 days in order to activate C D 8 C D 4 4 cells. The +  hl  cytolytic activities of the activated cells against A20 (syngeneic to Balb/c) or P815 (syngeneic to DBA/2) targets were then determined. (Right panel) Lymph node cells from Balb/c mice were depleted of lg , C D 4 and C D 4 4 cells. Cells purified +  +  +  in this manner were ~98% CD8 CD44'°. They were cultured with plate-bound +  2C11 in IL-2-supplemented media for 5 days and the cytolytic activities of the activated cells against A20 or P815 targets were then determined.) C) Same as (B) except that purified C D 8 or CD8 CD44'° lymph node cells from DBA/2 mice +  +  were used as the responding population. Error bars represent the standard deviation for triplicate cultures.  Figure 2.6 Activated C D 8 C D 4 4 +  hi  cells express DAP12. NK cells were enriched  by depleting B6 spleen cells of C D 4 , C D 8 \ l g and C D 3 cells by negative +  +  +  selection and the negatively selected cells were activated with IL-2supplemented media. Purified C D 8 cells from B6 mice were activated with IL-2+  supplemented media and provided a source of IL-2-activated C D 8 C D 4 4 cells. +  hl  CD8 CD44'° cells were purified as described in Fig. 2.5 and activated with anti+  C D 3 + IL-2. m R N A was extracted from activated cells and R T - P C R for DAP10, D A P 12 and G A P D H were performed. The data indicate that activated NK and CD8 CD44 +  hi  cells express both D A P 10 and D A P 12 whereas activated  CD8 CD44'° cells only express DAP10. +  Figure 2.7 NKG2D/Rae1 interaction enhances killing of syngeneic tumor targets by IL-2 activated C D 8 C D 4 4 +  hi  cells. NK, C D 8 C D 4 4 and C D 8 C D 4 4 +  hi  +  |0  cells were  purified and activated as described in Fig. 2.6. The ability of IL-2 activated NK cells (upper panel), C D 8 C D 4 4 +  hi  cells (middle panel) or 2C11 + IL-2 activated  CD8 CD44'° cells to kill syngeneic target cells expressing a ligand for N K G 2 D +  was determined. The cells were >99% C D 3 " N K 1 . 1 D X 5 2 B 4 , C D 9 4 N K G 2 D +  +  +  +  +  for the purified NK cells and >98% a p T C R C D 8 for both groups of C D 8 T cells. +  +  +  The cells were used as effectors in a Cr-release assay against R M A , R M A S , 51  RMA-Rae18, and RMAS-Rae18 tumor cells. Error bars represent the standard deviation for triplicate cultures.  94 T  Shaded hisotogram = CD8 CD44 cells Unshaded histogram = CD8 CD44'° cells +  hi  +  CD44  Figure 2.1 Cell surface phenotype of CD8 CD44 and CD8 CD44 T cells ex +  vivo.  HI  +  L0  95  340000  E  a <j c o  "S o a. 8  c c  120000  CD8+CD44hi  Figure 2.2 Only C D 8 C D 4 4 +  hi  CD8+CD44IO  cells proliferate in response to IL-2.  96  Fluorescence Intensity  Figure 2.3 Activated C D 8 C D 4 4 +  hl  cells express several NK receptors.  97  C) 2C11  IL-2 60000  200000  30000  100000  o 5  £ si  no All  aim CD48  no Al>  anti-CD48  Figure 2.4 CD48/CD2 interactions are required for IL-2-induced proliferation of CD8 CD44 +  hi  cells.  98  A) 20.0  —•— CD44lo • - A - CD44hi  C/l  >. —I  £  o d> Q.  10.0  .... 0.0  l ^ ^  1  * —  •  —  3  rrrn*-  * 30  10  E/T Ratio  IL-2 Activated DBA/2 CD8 CD44 +  10. E/T Ratio  Figure 2.5 Activated C D 8 C D 4 4 +  cells.  hi  hi  2C11 Activated DBA/2 CD8 CD44 +  3 E/T Ratio  to  10  cells preferential killed syngeneic tumor target  A) CD44"J CP8+  NK  Figure 2.6 Activated C D 8 C D 4 4 +  hi  cells express DAP12.  CD44'°CD8  +  100  1.00 80  NK -•••-RMA -•--RMA-Rae1 -*--.RMAS. -•—RMAS-Rae1  60 o  CD  CL V)  40 20 0 3  100 .2 "w >. _i 80 6 60 "o a> 4 0Q. u> 20 0100 1 80 60 0 Q. 4 20 0  10  E/T Ratio CD8  3  +  CD44  E/T Ratio  CD8  +  30  hi  10  CD44'  30  0  -••--RMA -•--RMA-Rae1 -A---RMAS —RMAS-Rae1  u  3  10  E/T Ratio  30  Figure 2.7 NKG2D/Rae1 interaction enhances killing of syngeneic tumor targets by IL-2 activated C D 8 C D 4 4 +  hi  cells.  101 2.8  References  1.  Barton, G . M., and R. Medzhitov. 2002. Control of adaptive immune responses by Toll-like receptors. CurrOpin  2.  Immunol 14:380-383.  Cerwenka, A., and L. L. Lanier. 2001. Natural killer cells, viruses and cancer. Nat Rev Immunol 1:41-49.  3.  Lanier, L. L. 1998. NK cell receptors. Annu Rev Immunol 16:359-393.  4.  Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari, R. Biassoni, and L. Moretta. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 19:197-223.  5.  Raulet, D. H., R. E. Vance, and C. W. McMahon. 2001. Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 19:291-330.  6.  McQueen, K. L., and P. Parham. 2002. Variable receptors controlling activation and inhibition of NK cells. Curr Opin Immunol 14:615-621.  7.  Bretscher, P. 1992. The two-signal model of lymphocyte activation twentyone years later. Immunol Today 13:74-76.  8.  June, C. H., J . A. Bluestone, L. M. Nadler, and C. B. Thompson. 1994. The B7 and CD28 receptor families. Immunol Today 15:321-331.  9.  Yamada, H., T. Ninomiya, A. Hashimoto, K. Tamada, H. Takimoto, and K. Nomoto. 1998. Positive selection of extrathymically developed T cells by self- antigens. J Exp Med 188:779-784.  10.  Yamada, H., T. Nakamura, G . Matsuzaki, Y . Iwamoto, and K. Nomoto. 2000. TCR-independent activation of extrathymically developed, self antigen- specific T cells by IL-2/IL-15. J Immunol 164:1746-1752.  11.  Yamada, H., G . Matsuzaki, Q. Chen, Y. Iwamoto, and K. Nomoto. 2001. Reevaluation of the origin of CD44(high) "memory phenotype" C D 8 T cells: comparison between memory C D 8 T cells and thymus-independent C D 8 T cells. Eur J Immunol 31:1917-1926.  12.  Anfossi, N., V. Pascal, E. Vivier, and S. Ugolini. 2001. Biology of T memory type 1 cells. Immunol Rev 181:269-278.  13.  Stepp, S. E., J . D. Schatzle, M. Bennett, V. Kumar, and P. A. Mathew. 1999. Gene structure of the murine NK cell receptor 2B4: presence of two alternatively spliced isoforms with distinct cytoplasmic domains. EurJ Immunol 29:2392-2399.  14.  Kambayashi, T., E. Assarsson, B. J . Chambers, and H. G . Ljunggren. 2001. Cutting edge: Regulation of CD8(+) T cell proliferation by 2B4/CD48 interactions. J Immunol 167:6706-6710.  15.  McMahon, C. W., and D. H. Raulet. 2001. Expression and function of NK cell receptors in CD8+ T cells. CurrOpin  16.  Immunol 13:465-470.  Mingari, M. C , M. Ponte, C. Cantoni, C. Vitale, F. Schiavetti, S. Bertone, R. Bellomo, A. T. Cappai, and R. Biassoni. 1997. HLA-class l-specific inhibitory receptors in human cytolytic T lymphocytes: molecular characterization, distribution in lymphoid tissues and co-expression by individual T cells. Int Immunol 9:485-491.  17.  Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J . K. Hsia, E. Vivier, and D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of N K G 2 D . Nat Immunol 3:1142-1149.  18.  Ho, E. L., L. N. Carayannopoulos, J . Poursine-Laurent, J . Kinder, B. Plougastel, H. R. Smith, and W. M. Yokoyama. 2002. Costimulation of multiple NK cell activation receptors by NKG2D. J Immunol 169:36673675.  19.  Cerwenka, A., A. B. Bakker, T. McClanahan, J . Wagner, J . Wu, J . H. Phillips, and L. L. Lanier. 2000. Retinoic acid early inducible genes define a ligand family for the activating N K G 2 D receptor in mice. Immunity 12:721-727.  20.  Cerwenka, A., J . L. Baron, and L. L. Lanier. 2001. Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cellmediated rejection of a M H C class l-bearing tumor in vivo. Proc Natl Acad Sci USA  21.  98:11521-11526.  Harrington, L. E., M. Galvan, L. G . Baum, J . D. Altman, and R. Ahmed. 2000. Differentiating between memory and effector C D 8 T cells by altered expression of cell surface O-glycans. J Exp Med 191:1241-1246.  22.  Skold, M., A. Rytter, F. Ivars, and S. Cardell. 1999. Characterization of subpopulations of T-cell receptor intermediate (TCRint) T cells. Scand J Immunol 49:611-619.  23.  Gasser, S., P. Corthesy, F. Beerman, H. R. MacDonald, and M. Nabholz. 2000. Constitutive expression of a chimeric receptor that delivers IL-2/IL15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells. J Immunol 164:5659-5667.  24.  Assarsson, E., T. Kambayashi, J . K. Sandberg, S. Hong, M. Taniguchi, L. Van Kaer, H. G . Ljunggren, and B. J . Chambers. 2000. CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo. J Immunol 165:3673-3679.  25.  Bauer, S., V. Groh, J . Wu, A. Steinle, J . H. Phillips, L. L. Lanier, and T. Spies. 1999. Activation of NK cells and T cells by N K G 2 D , a receptor for stress- inducible MICA. Science 285:727-729.  26.  Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, and A. N. Barclay. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J Exp Med 188:20832090.  27.  Latchman, Y., P. F. McKay, and H. Reiser. 1998. Identification of the 2B4 molecule as a counter-receptor for CD48. J Immunol 161:5809-5812.  28.  Boles, K. S., S. E. Stepp, M. Bennett, V. Kumar, and P. A. Mathew. 2001. 2B4 (CD244) and C S 1 : novel members of the C D 2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes. Immunol Rev 181:234-249.  29.  Moran, M., and M. C. Miceli. 1998. Engagement of GPI-linked CD48 contributes to T C R signals and cytoskeletal reorganization: a role for lipid rafts in T cell activation. Immunity 9:787-796.  30.  Poussier, P., H. S. Teh, and M. Julius. 1993. Thymus-independent positive and negative selection of T cells expressing a major histocompatibility complex class I restricted transgenic T cell receptor alpha/beta in the intestinal epithelium. J Exp Med 178:1947-1957.  31.  Terra, R., N. Labrecque, and C. Perreault. 2002. Thymic and extrathymic T cell development pathways follow different rules. J Immunol 169:684692.  32.  Diefenbach, A., E. R. Jensen, A. M. Jamieson, and D. H. Raulet. 2001. Rae1 and H60 ligands of the N K G 2 D receptor stimulate tumour immunity. Nature 413:165-171.  33.  Gilfillan, S., E. L. Ho, M. Cella, W. M. Yokoyama, and M. Colonna. 2002. N K G 2 D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat Immunol 3:1150-1155.  34.  Diefenbach, A., A. M. Jamieson, S. D. Liu, N. Shastri, and D. H. Raulet. 2000. Ligands for the murine N K G 2 D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat Immunol 1:119-126.  35.  Lanier, L. L , B. C. Corliss, J . Wu, C. Leong, and J . H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:703-707.  36.  Ernst, D. N., W. O. Weigle, D. J . Noonan, D. N. McQuitty, and M. V. Hobbs. 1993. The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, C D 4 5 R B , 3G11, and MEL-14 expression. J Immunol 151:575-587.  37.  Lerner, A., T. Yamada, and R. A. Miller. 1989. Pgp-1hi T lymphocytes accumulate with age in mice and respond poorly to concanavalin A. EurJ Immunol 19:977-982.  38.  Pihlgren, M., C. Arpin, T. Walzer, M. Tomkowiak, A. Thomas, J . Marvel, and P. M. Dubois. 1999. Memory CD44(int) C D 8 T cells show increased proliferative responses and IFN-gamma production following antigenic challenge in vitro. Int Immunol 11:699-706.  39.  Gutierrez-Ramos, J . C , I. Moreno de Alboran, and C. Martinez. 1992. In vivo administration of interleukin-2 turns on anergic self-reactive T cells and leads to autoimmune disease. Eur J Immunol 22:2867-2872.  40.  Urdahl, K. B., J . C. Sun, and M. J . Bevan. 2002. Positive selection of M H C class lb-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol 3:772-779.  41.  Huard, B., and L. Karlsson. 2000. A subpopulation of CD8+ T cells specific for melanocyte differentiation antigens expresses killer inhibitory receptors (KIR) in healthy donors: evidence for a role of KIR in the control of peripheral tolerance. Eur J Immunol 30:1665-1675.  42.  Speiser, D. E., M. J . Pittet, D. Valmori, R. Dunbar, D. Rimoldi, D. Lienard, H. R. MacDonald, J . C. Cerottini, V. Cerundolo, and P. Romero. 1999. In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J Exp Med 190:775-782.  43.  Dudley, M. E., J . R. Wunderlich, P. F. Robbins, J . C. Yang, P. Hwu, D. J . Schwartzentruber, S. L. Topalian, R. Sherry, N. P. Restifo, A. M. Hubicki, M. R. Robinson, M. Raffeld, P. Duray, C. A. Seipp, L. Rogers-Freezer, K. E. Morton, S. A. Mavroukakis, D. E. White, and S. A. Rosenberg. 2002. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298:850-854.  44.  Mathew, P. A., B. A. Garni-Wagner, K. Land, A. Takashima, E. Stoneman, M. Bennett, and V. Kumar. 1993. Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells. J Immunol 151:53285337.  45.  Garni-Wagner, B. A., A. Purohit, P.. A. Mathew, M. Bennett, and V. Kumar. 1993. A novel function-associated molecule related to non-MHC-restricted cytotoxicity mediated by activated natural killer cells and T cells. J Immunol 151:60-70.  46.  Speiser, D. E., M. Colonna, M. Ayyoub, M. Cella, M. J . Pittet, P. Batard, D. Valmori, P. Guillaume, D. Lienard, J . C. Cerottini, and P. Romero. 2001. The activatory receptor 2B4 is expressed in vivo by human CD8+ effector alpha beta T cells. J Immunol 167:6165-6170.  47.  Yashiro-Ohtani, Y., X. Y . Zhou, K. Toyo-Oka, X. G . Tai, C. S. Park, T. Hamaoka, R. Abe, K. Miyake, and H. Fujiwara. 2000. Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of T C R with rafts. J Immunol 164:12511259.  48.  Bierer, B. E., A. Peterson, J . C. Gorga, S. H. Herrmann, and S. J . Burakoff. 1988. Synergistic T cell activation via the physiological ligands for C D 2 and the T cell receptor. J Exp Med 168:1145-1156.  49.  King, P. D., A. Sadra, J . M. Teng, G . M. Bell, and B. Dupont. 1998. C D 2 mediated activation of the Tec-family tyrosine kinase ITK is controlled by proline-rich stretch-4 of the C D 2 cytoplasmic tail. Int Immunol 10:10091016.  50.  Kivens, W. J . , S. W. Hunt, 3rd, J . L. Mobley, T. Zell, C. L. Dell, B. E. Bierer, and Y . Shimizu. 1998. Identification of a proline-rich sequence in the C D 2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of phosphoinositide 3-kinase. Mol Cell Biol 18:5291-5307.  109  Chapter 3  Self-reactive memory-phenotype CD8 T cells exhibit both  MHC-restricted and non-MHC restricted cytotoxicity: A role for the T cell receptor and natural killer cell receptors  3.1  1  Abstract  We have recently shown that IL-2-activated C D 8 C D 4 4 cells from normal mice +  hi  express both adaptive and innate immune system receptors and specifically kill syngeneic tumor cells, particularly those that express N K G 2 D ligands. Here we show that C D 8 T cells from antigen-expressing H-Y T C R transgenic mice also +  exhibit characteristics of both T cells and NK cells. Interaction with cognate selfantigen was required for the optimal expansion of these cells in peripheral lymphoid tissues. Although these cells possess a higher activation threshold relative to naive T cells, they can be activated by cytokine alone in vitro. They also undergo bystander proliferation in response to a bacterial infection in vivo. Interestingly, upon activation, the cells express the N K G 2 D receptor as well as the DAP12 adaptor protein. We provide evidence that N K G 2 D can act additively with the T C R in the killing of target cells and it can also function as a directly activating receptor in non-MHC restricted killing of target cells. These properties of C D 8 T cells from H-Y T C R transgenic mice are remarkably similar to +  CD8 CD44 +  hi  cells that are found in normal mice. The H-Y T C R transgenic mice  provide a well-defined system for characterizing the developmental biology and function of these cells.  1  A version of this chapter has been published as:  Dhanji, S., S. J . Teh, D. Oble, J . J . Priatel, and H. S. Teh. 2004. Self-reactive memory-phenotype C D 8 T cells exhibit both MHC-restricted and non-MHCrestricted cytotoxicity: a role for the T-cell receptor and natural killer cell receptors. Blood 104:2116  110  3.2  Introduction  In this study we used mice that express a transgenic T C R that is specific for the male (H-Y) antigen presented by H-2D (1) as a model system for studying the b  developmental requirements and function of a population of self-specific C D 8 T +  cells. H-Y T C R transgenic mice have been widely used as an animal model system for the determination of mechanisms of positive and negative selection of T cells. In these mice, C D 8 T cells that express the H-Y T C R are positively selected in the thymus of female H-2 mice (2). In male H-2 mice, the H-Y T C R b  b  is negatively selected, leading to the massive deletion of double positive (DP) thymocytes (3). Interestingly, there is a population of C D 8 T cells, which express +  a low level of C D 8 (referred to as CD8'°), that are resistant to deletion in male mice (4). The CD8'° cells can develop via an extra-thymic pathway (5) and express high levels of CD44 and IL-2R|3 (CD122) that are characteristic of memory T cells. However, they differ from conventional memory T cells in that they are more refractory to activation by antigen as compared to naive T cells (6, 7). Another unusual feature of these CD8'° T cells is that they proliferate in response to cytokines such as IL-2 and IL-15 in an antigen-independent manner (8). However, the CD8'° cells are similar to conventional memory T cells with regard to the ability to rapidly produce IFN-y upon T C R stimulation (9) and in respect to the killing of susceptible target cells without the need for additional reactivation with antigen (10, 11).  111 C D 8 T cells that develop via an extra-thymic pathway and possessing +  similar functional characteristics are also found in normal (non-TCR transgenic) mice (7). More importantly, cells of this phenotype in normal mice have also been shown to be important in responses towards viral and bacterial infections, suggesting that they may have an important role in the immune response (12, 13). We have recently showed that C D 8 C D 4 4 T cells from normal mice +  hl  possess characteristics of both T cells and NK cells (14). These cells are activated in response to IL-2 alone and show self-reactivity in that they preferentially kill syngeneic tumor cells. Furthermore, C D 8 C D 4 4 T cells +  hl  express NK receptors upon activation and engagement of these NK receptors enhances the ability of the cells to lyse syngeneic tumor cells. In this report we show that C D 8 T cells from antigen-expressing H-Y T C R transgenic mice also +  exhibit characteristics of both T cells and NK cells and are remarkably similar to C D 8 C D 4 4 cells from normal mice with regard to their cell surface and +  hl  functional phenotype. Thus, the H-Y T C R transgenic mice provide a well-defined system for characterizing the developmental biology and function of this interesting cell type.  3.3  Materials and Methods  112 Mice Breeders for C57BL/6 (B6), 66-Tap-l" ", and DBA/2 were obtained from the 7  Jackson Laboratories (Bar Harbor, ME). B D F i mice were Fi mice from the mating of C57BL/6 mice with DBA/2 mice. The H-Y T C R transgenic (tg) mice were bred to the B6 background. Mice 8 to 12 weeks of age were used for the experiments described.  Abs and flow cytometry The following mAbs were used: C D 4 (GK1.5), CD8ot (53-6.7), CD8p (53.58), CD3s (2C11), CD44 (PGP1), H-Y T C R p (F23.1), H-Y TCRoc (T3.70), CD94 (18D3), NK-1.1 (PK136), CD122 (TM-B1), Ly6C (AL-21), CD244.2 (2B4), CD16/32, IL-7Ra (A7R34), N K G 2 D (A10 and CX5), and anti-NKG2D (15). Biotinylated mAbs were detected using streptavidin-PE. C D 8 a , H-Y T C R a , N K G 2 D (A10 and CX5), IL-7Ra, and CD94 were obtained from eBioscience (SanDiego, CA) All other abs were obtained from BD PharMingen (San Diego, CA) except anti-NKG2D (15), which was a kind gift from Dr. Wayne M. Yokoyama (Howard Hughes Medical Institute, Washington University, St. Louis). Cell staining and flow cytometry were performed according to standard procedures. The CellQuest software program (Becton Dickinson, Mountain View, CA) was used for data acquisition and analysis.  113 Cell lines Cell lines used were the R M A lymphoma (H-2 , Rae-15 ), RMA-Rae-15 b+  -  transfectant (H-2 , Rae-15 ) and P815 mastocytoma. The cell lines were b+  +  cultured in IMEM (Life Technologies, Burlington, Canada), supplemented with 10% (v/v) F B S (Life Technologies), 5x10 u,M 2-ME and antibiotics (l-medium). 5  The RMA-Rae-15 transfectant (16) was a kind gift from Dr. Lewis L. Lanier (University of California, San Francisco).  C D 8 T cell purification and sorting +  Single cell suspensions from the lymph nodes (LN) of mice were treated with biotinylated anti-CD8p mAb followed by positive selection using the MiniMACS system (Miltenyi Biotech, Auburn, CA), according to the manufacturers specifications. The resulting cells were >95% pure C D 8 a p T C R p T cells. For +  +  purification of H-Y TCRoc (T3.70) C D 8 T cells single cell suspensions from the +  +  lymph nodes (LN) of H-Y male and female mice were treated with anti-CD4 mAb and then depleted of C D 4 l g cells using Dynabeads M-450 Sheep anti-mouse +  +  IgG (Dynal Biotech, Lake Success, NY), according to manufacturers instructions. After depletion the cells were stained with anti-CD8p-FITC and T3.70-PE and then sorted on a Becton Dickinson F A C S Vantage S E Turbo sort cell sorter. Cell sorting was performed by Andrew Johnson (University of British Columbia) and the resulting cells were >95% pure.  114 Natural Killer (NK) cell purification and activation NK cells were purified and activated as previously described (14).  CFSE  Labeling  Purified C D 8 T cells (1x10 /ml) were labeled with 1 u.M C F S E (Molecular +  7  Probes, Eugene, OR) in P B S for 8 min at room temperature. After stopping the reaction with the addition of an equal volume of F C S , cells were washed four times with complete media prior to use.  Adoptive  transfer  Purified CFSE-labeled C D 8 T cells (1-2x10 ) from H - 2 6  2  b/d  b/b  male and female and H-  male H-Y mice were injected into male or female B6 mice that had received a  sub-lethal dose of irradiation (650 cGy) 24 h earlier. On day 7 spleens of the injected mice were depleted of C D 4 l g cells as above and stained with the +  +  indicated mAbs and analyzed by F A C s . For Listeria infections, purified H-Y C D 8 T cells (3x10 ) were labeled with C F S E and transferred into B6 male or female 6  mice. 24h later the mice were challenged with Listeria monocytogenes  (10,000  CFU). 5d after infection the mice were sacrificed and the spleens of the animals were analyzed. For the analysis of IFNy production, 5x10 splenocytes from 6  infected and uninfected mice (day 5) were cultured in 1ml of I medium containing GolgiStop (BD PharMingen (San Diego, CA)) in the presence or absence of H-Y  115 peptide (1u,M). After 6h incubation the cells were fixed, permeabilized and stained with anti-IFNy, anti-CD8, and anti-H-Y T C R .  Proliferation  assays  Purified H-Y C D 8 T cells (10 ) were cultured with irradiated BS-Tap-V " 4  7  splenocytes (5x10 ) and the indicated concentration of H-Y peptide 5  (KCSRNRQYL)(17) in the presence or absence of IL-2 (20U/ml). The cells were pulsed with 1u.Ci of H-thymidine for the final 6 hours of a 72 h culture. For 3  C F S E - b a s e d proliferation assays, 1X10 CFSE-labeled C D 8 T cells were 5  cultured in 96 well round-bottom plates in the presence of IL-2 (200U/ml), IL-15 (100ng/ml) orTap-1" " splenocytes (10 ), H-Y peptide (1u.M) and IL-2 (20U/ml). 7  6  CFSE-dilution (cell division) was assessed by F A C s at the indicated times.  CTL Assays C T L assays were performed as previously described (14). The C T L activity of activated C D 8 T cells against RMA, RMA-Rae-18 target cells was assessed at a ratio of 10 effector T cells to 1 target cell in a 4 h  51  Cr-release assay. For  redirected lysis experiments, day 4 activated C D 8 T cells were preincubated with the indicated mAb (10u.g/ml) for 15 min. The C T L activity of Ab-coated C D 8 T +  cells against FcR+ P815 target cells was then determined in a 4-5 h Cr-release 51  assay. Spontaneous release varied from 8-15% of the maximum. All assays were performed in triplicate. Percent specific lysis was calculated as 100% x [cpm  116 (experimental) - cpm (spontaneous release)]/[(cpm (maximum release) - cpm (spontaneous release)].  RT-PCR Sorted H-Y female T 3 . 7 0 C D 8 cells and male T 3 . 7 0 C D 8 cells (10 ) were +  +  +  +  6  activated with T a p - r stimulators (10 ), H-Y peptide (1uM) and IL-2 (20U/ml) for /_  7  4 days. NK cells were activated with IL-2 (200U/ml) for 5 days. R N A isolation and P C R primers were described previously (14).  Immune-blotting Purified C D 8 T cells were stimulated for 10mins at 37°C with anti-CD3e (2C11) or with P M A (25ng/ml) plus ionomycin (500ng/ml) and then pelleted and lysed in 10 mM Tris (pH 7.5), 150 mM NaCl, 1% TX-100, 0.1% S D S , and protease and phosphatase inhibitors. The lysates were separated on a 4-15% Tris-HCl polyacrylamide gel and transferred to a P V D F membrane. Blots were developed using E C L system (Amersham). Phospho-ZAP-70, ZAP-70, and phosphoE R K 1 / 2 antibodies used for detection were from Cell Signaling Technologies (Beverly, MD). Anti-ERK mAb was from Santa Cruz biotechnology (Santa Cruz, CA).  Statistical  Analysis  Student's t-test was used to determine p-values in C T L assays.  117  3.4  Results  Cell surface phenotype of CD8 T cells from self-antigen expressing H-Y TCR transgenic mice The H-Y T C R is specific for a male peptide presented by H-2D (1). Fig. 3.1a b  shows the CD4/CD8 thymocyte profiles of H - 2 well as H - 2  b/d  b/b  H-Y male and female mice as  H-Y male mice. It is clear that the presence of cognate antigen in  H-2  b/b  male mice results in the deletion of the vast majority of D P thymocytes. In  H-2  b/d  H-Y male mice, where there is half the number of the deleting H-Y/D  b  complexes, there is incomplete deletion of D P thymocytes. Deletion of D P thymocytes in H - 2  b/b  and H - 2  b/d  male mice results in drastically reduced numbers  of C D 8 single positive (SP) thymocytes relative to H-2 H-Y female mice b  (Fig.3.1a). Although the majority of H-Y T C R C D 8 T cells are deleted in the male H+  2  b / b  and H - 2  b/d  mice, there is a large population of C D 8 H-Y T C R T cells in the +  +  spleen and lymph nodes of these mice. However, C D 8 T cells from male H-Y +  mice differ from C D 8 T cells from female H-Y mice in many aspects (Fig. 3.1b). +  Female H - 2  b/b  C D 8 T cells express the highest level of the C D 8 co-receptor +  (referred to as CD8 ); those from H - 2 hi  b/d  male mice express an intermediate level  (referred to as C D 8 ) and those from H - 2 int  b/b  male cells express the lowest level  (referred to as CD8'°). There is also a correlation between the degree of cognate self-antigen exposure and the level of expression of memory markers in male H-  118 Y mice. In female mice, the lack of cognate self-antigen exposure results in C D 8 T c e l l s with a naive phenotype ( C D 4 4  l0/int  +  , IL-2Rp", IL-7Ra'°, L y 6 C ) . In contrast,  the C D 8 T cells from H-Y male mice exhibit an activated/memory phenotype +  (CD44  int/hi  , IL-2Rp , IL-7Ra , Ly6C ). Notably, the CD8'° cells expressed higher +  hi  +  levels of memory markers compared to C D 8 ' cells. nt  Previous studies show that the C D 8 cells from female H-Y T C R hl  transgenic mice can express both the transgenic as well as an endogenous a chain (18). In stark contrast, the C D 8 T cells from H-Y male mice express exclusively the transgenic T C R a chain (Fig. 3.1b). Using the T C R a expression as a measure of T C R expression, it is clear that the CD8'° and C D 8 ' cells nt  expressed the same intermediate level of the ap T C R compared to C D 8 T cells, hl  which express a high level (Fig. 3.1b). Corroborating results were obtained by analyzing expression of T C R p and CD3s on these cells (data not shown).  C D S T cells from male H-Y mice possess an increased activation threshold In agreement with a previous study (7) we found that the CD8'° cells proliferated poorly to antigen stimulation in the absence of IL-2 (Fig. 3.2a, upper panel). The C D 8 ' cells also showed a similar proliferative defect. This proliferative defect nt  was partially restored by exogenous IL-2 (Fig. 3.2a, lower panel). The increased activation threshold of the C D 8 ' and CD8'° cells may be nt  due to a defect in T C R signal transduction. Signaling through the T C R leads to phosphorylation of CD3<^, leading to the recruitment and subsequent phosphorylation of ZAP-70 (19). ZAP-70 activation eventually leads to the  119 activation of several important signaling pathways including the Ras/mitogenactivated protein kinase (MAPK) pathway. Figure 2b clearly shows that C D 8 '  nt  and CD8'° cells have a defect in early T C R signaling, as they do not efficiently phosphorylate ZAP-70 in response to T C R stimulation as compared to conventional, naive, C D 8 cells. Correlated with diminished early T C R signaling, hl  the cells also have a defect in downstream signaling events, leading to reduced P  42/p44 (ERK1/2) M A P K phosphorylation. The inefficient activation of ZAP-70  correlates with the amount of cognate self-antigen the cells have encountered in vivo; the naive phenotype C D 8 cells exhibit the strongest signaling, followed by hl  the C D 8  int  and then the CD8'° cells. This is in contrast to true memory C D 8 T  cells, which have a lower activation threshold, and more efficiently induce the phosphoryaltion of ZAP-70 and ERK1/2 relative to naive cells (20). Interestingly, E R K 1/2 were efficiently phosphoryiated in response to P M A and ionomycin (Fig. 3.1b), suggesting that there is no intrinsic defect in the ability of these cells to activate the E R K M A P K pathway.  IL-2 or IL-15 promotes the proliferation of CDS' and CDS"' cells 0  Since the CD8'° and C D 8 ' cells are recovered from mice that express different nt  levels of cognate self-antigen they offer an opportunity for determining whether prior antigen history affects their responsiveness to inflammatory cytokines. This was determined by culturing C F S E labeled C D 8 and C D 8 |0  int  cells with an  exogenous source of IL-2 or IL-15. Proliferation at 48, 72, and 96 hours was determined by measuring the C F S E fluorescence level of the cultured C D 8  +  120 cells. C D 8 cells from female H-Y mice do not proliferate in response to IL-2 or hl  IL-15 (8) so we stimulated these cells with antigen + IL-2 to determine their proliferative potential. The results in Fig. 3.3a indicate that stimulation of C D 8 , hl  CD8'° or C D 8  int  cells with a high antigen dose + IL-2 led to similar extent of cell  division at 48, 72 or 96 hr. In contrast, only the CD8'° and C D 8  cells could  int  proliferate in response to cytokine alone (Fig. 3.3a). Interestingly, CD8'° cells proliferated much more rapidly than the C D 8 ' cells in response to IL-2 at the 72 nt  and 96 hr time points (Fig. 3.3a). IL-15 was more efficient than IL-2 in inducing the growth of CD8'° and C D 8  int  cells, particularly at the 48 and 72 hr time points.  These studies indicate that the prior antigen history of C D 8 cells from male H-Y +  mice determine the qualitative and quantitative aspects of their responsiveness to inflammatory cytokines.  Role of antigen and bacterial infection on the expansion of C D S ' cells in wVoThe 0  antigenic requirements for the expansion of C D 8 T cells from male H-Y mice in +  vivo was determined in adoptive transfer experiments where C F S E labeled C D 8 , C D 8 , and CD8'° were injected into irradiated female or male B6 mice. hi  int  Fig. 3.3b indicates that the C D 8  int  and CD8'° cells homed to and expanded  efficiently in the spleen of either cognate-antigen expressing male or non-antigen expressing female mice whereas the C D 8  hl  cells could only expand in the  presence of antigen. A recent report has also shown that CD8'° cells can in fact expand in M H C class l-deficient host (21). In our experiments we showed that CD8'° cells could expand in female recipients and they underwent one extra  121 division in male recipients. C D 8 ' cells, on the other hand, were much more nt  dependent on the male antigen for proliferation since they underwent about 3 fewer divisions in female recipients. In all cases, the presence of cognate antigen affected the clone sizes, suggesting that antigen does in fact play a role in the lymphopenia-induced expansion of these memory-phenotype C D 8 T cells. This expansion in the absence of cognate self-antigen is likely mediated through interaction of IL-2R(3 and or IL-7Ra with IL-15 and IL-7 (22). By contrast, C D 8  hi  cells could not expand in irradiated female mice as interactions between the H-Y T C R and female self-peptides/H-2D alone are insufficient for the expansion of b  CD8  hi  cells in vivo (21, 23). C D 8  hi  cells proliferated most efficiently in irradiated  male mice.  Bystander proliferation of C D S ' cells in response to bacterial infection 0  The above studies suggest that C D 8 ' and CD8'° cells are dependent on nt  interaction with the self (male)-antigen for their development and likely to be of the same lineage. Since C D 8 ' cells consistently showed an intermediate nt  phenotype relative to CD8'° cells, we decided to focus the remainder of our experiments on CD8'° and C D 8  hl  cells to further characterize the difference  between these two cell types. Since CD8'° cells can expand in an antigenindependent manner in irradiated female mice, we determine if these cells could undergo bystander proliferation in response to bacterial infection. To this end we transferred CD8'° cells into non-irradiated B6 female and male mice and then infected the mice with a sublethal dose of Listeria monocytogenes (LM). A s a  control, we also transferred C D 8 cells into non-irradiated B6 female mice and infected them with L M . Figure 3.4 shows that C D 8  10  cells expand in both male  and female recipients during infection, although this expansion was greater in male mice. The C D 8  hl  cells did not expand in either infected or uninfected  recipients and in fact their numbers were depleted in the infected recipients. Consistent with this, a previous study has shown that nonspecific T cells are depleted early during infection with Listeria (24). Our results show that rather than being depleted, C D 8  10  cells expand in infected mice suggesting that  inflammatory cytokines produced in response to infection could promote the growth of these cells in vivo.  CDS'  0  cells express NK receptors after activation  We recently showed that memory phenotype C D 8 C D 4 4 +  hl  cells from normal mice  express NK receptors after activation (14). Since CD8'° cells are similar to CD8 CD44 +  hl  cells in terms of cell surface phenotype and cytokine  responsiveness, we determined whether they also shared the ability to express NK receptors after activation. Immediately ex vivo, C D 8 and CD8'° cells are hl  negative for all of the N K receptors tested, with the exception of a very low level of N K G 2 D and CD94 expression by the C D 8  l0  cells (Fig. 3.5a). Since both C D 8  and CD8'° cells can be activated with antigen + IL-2, this protocol was used to activate both populations. Figure 3.5b shows that activated C D 8  |0  T cells  expressed several NK receptors especially 2B4, DX5, and CD94. By contrast, activated female C D 8  hi  cells do not express DX5 or CD94. A high level of 2B4  hi  123 was expressed by some of the activated female C D 8  hl  cells. N K G 2 D is known to  be expressed by all activated C D 8 T cells (25) and was expressed equivalently by both activated C D 8  hi  and C D 8 cells. Neither cell type expressed the B6 NK-T l0  cell marker NK1.1. Conventional antigen-activated C D 8 T cells express N K G 2 D in association with the D A P 10 adaptor molecule (26, 27). By contrast, N K G 2 D in activated murine N K cells is associated with both the DAP10 and DAP12 adaptor molecules (26, 27). Association of N K G 2 D with DAP10 provides a costimulatory signal whereas association with DAP12 confers a directly sitmulatory signal (26). We recently showed that C D 8 C D 4 4 +  hl  cells from normal mice expressed both  D A P 10 and D A P 12 after IL-2 activation (14). Fig. 3.5c showed that activated CD8'° cells from male mice also expressed both DAP10 and DAP12 whereas antigen-activated C D 8  hl  cells from female mice expressed only DAP10. Similar  results were obtained following activation of CD8'° cells with IL-2 (data not shown).  NKG2D is an activating receptor for CD8 cells 10  We next determined if N K G 2 D could participate in the lysis of target cells expressing self-antigen. Activated C D 8  hl  and CD8'° cells were used as effector  cells in a C T L assay against peptide-pulsed R M A (H-2 ) cells or R M A cells b  transfected with Rae-15 (RMA-Rae-1) (16), a ligand for N K G 2 D (25). It is clear that the N K G 2 D receptor participated in the lysis of R M A target cells by activated CD8'° T cells since RMA-Rae-1 target cells were lysed significantly better than  124 R M A target cells (Fig. 3.6a). This is particularly evident at low H-Y peptide concentrations, where additive effects between the T C R and N K G 2 D in the lysis of RMA-Rae-1 target cells was observed. However, at high concentrations of antigenic peptide, R M A and RMA-Rae-1 target cells were killed to the same extent. This result indicates that N K G 2 D contributes to killing when there is suboptimal stimulation of the T C R by the antigenic ligand. By contrast, there was no difference in the lysis of R M A and RMA-Rae-1 target cells by activated female CD8 cells at all concentrations of added H-Y peptide, consistent with the finding hl  that N K G 2 D is not an activating receptor in conventional C D 8 T cells. W e used a redirected killing assay to provide further evidence that N K G 2 D is an activating receptor in activated CD8'° cells. In this assay, killing of an Fc receptor expressing target (P815; H-2 ) ± an anti-NKG2D mAb was determined. d  We found that only anti-CD3s, but not anti-NKG2D, was able to induce lysis of P815 target cells by activated C D 8 cells (Fig. 3.6b). This is consistent with the hi  conclusion that N K G 2 D does not function as an activating receptor in conventional C D 8 T cells (26, 27). By contrast, anti-NKG2D could enhance the lysis of P815 target cells by CD8'° cells in the redirected killing assay (Fig. 3.6b). It is noted that activated CD8'° cells exhibited significant killing of P815 targets even in the absence of any added antibody. This background killing of P815 targets was not due to N K G 2 D since we could not eliminate this background killing by using a blocking anti-NKG2D mAb (data not shown). It is conceivable that the non-antibody dependent killing of P815 targets by activated CD8'° cells  125 may be due to P815 ligands, which are recognized by other activating NK receptors.  Listeria infection induces expression ofNKG2D  on proliferating CD8  10  cells and a  heightened ability to produce IFNy. Since C D 8  10  cells could expand in response to LM infection (Fig. 3.4), we  determine if LM infection induces expression of N K G 2 D on these cells. In addition, we wanted to determine the effect of bacterial infection on IFNy production by C D 8  10  cells. W e transferred CFSE-labeled C D 8  10  cells into non-  irradiated B6 male mice and then infected them with L M . It is clear that N K G 2 D was expressed by the majority of cells that have divided in response to infection (Fig. 3.7a). Furthermore, cells that have undergone more cell divisions expressed the highest level of N K G 2 D . To test the production of IFNy by the cells, we cultured the splenocytes from infected or uninfected mice for 6 hours in the presence or absence of H-Y peptide. The C D 8  10  cells from both infected and  uninfected mice did not produce any IFNy without any T C R stimulation (Fig. 3.7b). However, upon a 6 hr stimulation with the antigenic peptide, C D 8  10  cells  from uninfected and LM infected mice produced high levels of IFNy. By contrast, CD8  hl  cells from uninfected or infected recipients did not produce any IFNy in  response to similar stimulation (data not shown). C D 8  10  cells from LM-infected  mice produced higher levels of IFNy than those from uninfected mice. Furthermore, C D 8  10  cells that have undergone the most number of cell divisions  in response to LM infection produced the highest level of IFNy (Fig. 3.7b). Our  126 observation that CD8'° cells from uninfected mice can produce IFNy in response to antigen stimulation is consistent with the observation that CD8'° cells can produce IFNy in response to T C R stimulation without the need for prior activation (7).  3.5  Discussion  In this report we have characterized an unusual population of self-antigen specific C D 8 T cells in H-Y T C R transgenic mice. The development of these +  cells is dependent on interaction with cognate self-antigen and the intensity of interaction with cognate-antigen in vivo determines the expression level of C D 8 and memory markers. However, in contrast to conventional memory cells, these cells have an increased threshold for activation relative to naive C D 8 T cells of +  the same antigen specificity. They proliferate in response to cytokines such as IL2 and IL-15. When introduced into lymphopenic mice, these cells can expand in the absence of self-antigen. More interestingly, they proliferate in response to a bystander bacterial infection in normal mice. Immediately ex vivo, these cells are negative or express low levels of NK receptors but upon activation several NK receptors are induced, including NKG2D. Interestingly, activated male H-Y CD8'° cells express DAP12, an adaptor molecule normally expressed by NK cells (26, 27). Furthermore, the N K G 2 D receptor acts additively and also functions independently of the T C R in the killing of N K G 2 D ligand positive cells. W e have recently reported the existence in normal mice of C D 8 C D 4 4 T cells that +  hl  127 possess similar cell-surface phenotype and functional properties as the male H-Y CD8  10  T cells (14). These results support the hypothesis that the H-Y CD8'° cells  are not a transgenic oddity but are a normal component of the murine immune system. The H-Y T C R transgenic mouse provides a well-defined system for studying the developmental biology of this novel cell type. In this study we showed that they could be selected by a conventional antigen, i.e. the male peptide presented by M H C class la molecules. We also showed that the expression level of C D 8 on this cell type is variable and reflects their prior antigen history. The degree of TCR/cognate self-antigen interaction in vivo also affects the response of these cells to IL-2 or IL-15 in vitro (Fig. 3.3). Proliferation in response to IL-2 occurred much more efficiently in CD8'° cells than C D 8  int  cells  presumably as a result of higher IL-2RP expression (Fig. 3.1). C D 4 or CD4"CD8" NKT cells bearing an invariant T C R a chain (Va14 in mice and +  V a 2 4 in humans) that are reactive to CD1d also share many similarities with CD8'° cells. NKT cells are an important component of the innate immune response to infection and can produce cytokines to activate other cells or can directly lyse some targets (reviewed in (28)). Although these NKT cells react strongly to ot-galactosyl-ceramide (a sphingolipid from a marine sponge) presented by C D 1d, it was recently shown that their activation in response to infection is dependent on CD1d/self-antigen and IL-12 (29). Both CD8'° cells and NKT cells exhibit an activated/memory phenotype and are thought to be selfreactive (30). Another striking similarity is that NKT cells are selected by high-  128 affinity interactions with self-antigen on cells other than thymic cortical epithelial cells (31) and this selection can occur in the absence of a thymus (32). M H C class lb-restricted C D 8 T cells are also thought to be selected by relatively high affinity interactions with self-antigens (33) and are also functionally similar to CD8'° and NKT cells in that they mount an early response against bacterial infection (34). It is tempting to speculate that cells such as NKT, M H C class lbrestricted C D 8 and CD8'° cells, which are selected by high affinity interactions +  with self-antigens, may be members of a family of T cells that evolved to provide an early defense mechanism against bacterial infection. Our data showed that NK receptors such as N K G 2 D can function as an activating receptor in male C D 8 cells but not female C D 8 cells. N K G 2 D has l0  hi  recently been shown to be important for the recognition and destruction of tumor cells by NK cells (16, 25, 35). In vivo, the expression of the ligands for N K G 2 D , Rae-1 and H60, results in the rapid clearance of ligand-positive tumors with the generation of protective immunity against subsequent challenge with parental, ligand-negative, tumors (35). N K G 2 D has been demonstrated to associate with two different adaptor molecules, D A P 10 and DAP12 (26, 27). In C D 8 T cells, +  which only express DAP10, N K G 2 D engagement results in a costimulatory signal whereas in activated NK cells, the expression of DAP12 allows N K G 2 D to provide an activating signal (26, 27). In addition it has been shown that the ectopic expression of D A P 12 in C D 8 T cells results in the ability of N K G 2 D to transduce a directly stimulatory signal (27). Interestingly, we have found that  129 activated CD8'° cells express both D A P 10 and DAP12, unlike C D 8  hi  cells, making  CD8'° cells similar to NK cells in this regard. Despite the fact that H-Y CD8'° cells are self-reactive, the presence of large number of these self-reactive cells in male H-2 H-Y mice did not lead to b  autoimmune disease. It is conceivable that the increased activation threshold of these cells is sufficient to prevent autoimmunity. W e have shown that CD8'° and C D 8 ' cells have a defect in T C R signaling which can be partially compensated nt  for by the addition of exogenous IL-2. This defect stems from the inability of C D 8 ' and CD8'° cells to efficiently phosphorylate signaling proteins such as nt  ZAP-70 and E R K , in response to T C R stimulation (Fig 3.2b). A recent study supports the hypothesis that the lowering of T cell activation threshold can lead to autoimmune diseases in male H-Y T C R transgenic mice. Murga et al found that T cells with a null mutation in the E2F2 transcription factor have a lowered activation threshold (36). Interestingly, male H-2 H-Y T C R transgenic mice with b  this null mutation develop an accelerated and much more severe lupus-like autoimmune syndrome than normal mice with this mutation. The unique combination of high activation threshold, ability to respond to inflammatory cytokines such as IL-15 and undergo bystander proliferation, and the expression of activating N K G 2 D receptor in response to activation, would render these selfspecific C D 8 T cells particularly adept at sensing infected and transformed cells. +  The observed cooperation between the T C R and N K G 2 D in the destruction of R a e - 1 target cells would also allow these cells to focus on normal cells that +  express stress ligands in response to infection or transformation. These cells  130 would also differ from NK cells in their function since the expression of M H C class la antigens on target cells does not inhibit their function. For the CD8'° cells, M H C class la could in fact serve as an activating antigen. Thus, infected or transformed cells that are not susceptible to killing by NK cells, by virtue of high M H C class la expression, would be susceptible to lysis by activated self-specific C D 8 T cells. The fact that a bystander infection results in the proliferation of +  CD8'° as well as the upregulation of N K G 2 D suggests that these cells could use this receptor to eliminate infected cells early during an infection. This non-MHC dependent function of CD8'° cells broadens the range of target cells and increases their versatility in surveillance against infected and transformed cells.  3.6  Acknowledgements  We thank Dr. Lewis Lanier (University of California, San Francisco) for providing us with the RMA-Rae-15 cell line. We also thank Dr. Wayne Yokoyama for providing the anti-NKG2D mAb and Dr. Hao Shen (University of Pennsylvania) for the gift of wild type Listeria monocytogenes strain 10403s.  131  3.7  Figures  Figure 3.1 C D 8 T cells from antigen-expressing H-Y T C R transgenic mice +  possess an activated/memory phenotype. a) The dot plots depict the C D 4 and C D 8 profile of thymocytes from H-2 H-Y female, H-2 H-Y male, and H - 2 b  b  b/d  H-Y  male T C R transgenic mice. The bar graph depicts the mean number of double positive (DP), double negative (DN) and C D 4 and C D 8 single positive (SP) cells recovered from these mice with the error bars representing 1 standard deviation, b) The expression of C D 8 and the H-Y T C R a by lymph node cells from the mice in part (a). The histograms depict the expression of the indicated cell surface markers by gated H-Y T C R C D 8 T cells. +  +  Figure 3.2 C D 8 T cells from male H-Y mice possess a high activation threshold +  due to a defect in T C R signal transduction, (a) Purified C D 8 T cells (1x10 ) from +  female H-2 H-Y (CD8 ), male H-2 H-Y (CD8'°), or male H - 2 b  hi  b  4  b/d  H-Y (CD8 ) mice int  were cultured with irradiated B6-Tap-1" " splenocytes (5x10 ) + IL-2 and the 7  5  indicated concentration of H-Y peptide. Proliferation was determined after 3 days and the error bars represent the standard deviation of triplicate cultures, (b) Western blot analysis of C D 8 , C D 8 , and C D 8 hi  int  |0  cells immediately ex vivo or  after stimulation for 10mins with anti-CD3 (10u.g/ml) or P M A (25ng/ml) and ionomycin (500ng/ml). Blots were probed with anti-phospho-ZAP-70 and phospho-ERK and then stripped and re-probed with unphosphorylated Z A P - 7 0 and E R K 2 .  132  Figure 3.3 The extent of the memory-phenotype of H-Y C D 8 T cells determines their ability to respond to cytokines in vitro as well as their ability to undergo homeostatic expansion in vivo, a) Purified C D 8 T cells from female H-2 H-Y +  (CD8 ), male H-2 H-Y (CD8'°), or male H - 2 hi  b  b/d  b  H-Y (CD8 ) mice were labeled int  with C F S E and cultured with IL-2 (200U/ml), IL-15 (100ng/ml), or H-Y peptide (1u.M), B S - T a p l ' s p l e n o c y t e s (1x10 ), and IL-2 (20U/ml). Proliferation of gated 7  H-Y T C R a C D 8 cells was analyzed by F A C S at 48 (filled histogram), 72 (dark +  +  line), and 96 hours (light line) with each C F S E peak representing one cell division, b) Purified C D 8 , C D 8 hi  int  and C D 8  l0  cells were CFSE-labeled and  transferred into sublethally irradiated B6 female (filled histogram) or male (unfilled histogram) recipients. C F S E profiles of gated C D 8 H-Y T C R cells 7 +  +  days post-transfer are shown in the histograms.  Figure 3.4 Role of antigen and bacterial infection in the expansion of H-Y male CD8'° cells in vivo. Purified C D 8  hi  and CD8'° cells were labeled with C F S E and  injected into the indicated non-irradiated Thy1.1 congenic B6 male or female mice. The mice were then infected with Listeria monocytogenes (right column) or left uninfected (left column). On day 5 the expansion of labeled C D 8  hi  and CD8'°  cells in the spleens of infected or uninfected mice were analyzed by F A C s . The numbers in the histograms represent the percentage of undivided cells.  133 Figure 3.5 Activated H-Y male CD8'° cells expressed NK receptors and D A P 12 after activation, a) Lymph node cells from H-Y male and female mice were depleted of C D 4 l g cells and stained with the indicated antibodies. The +  +  histograms represent the expression of the indicated cell surface markers by gated female H-Y T C R a C D 8 , male H-Y T C R a C D 8 or unstained cells, b) and +  +  +  +  c) Sorted C D 8 H-Y T C R a male or female cells (1x10 ) were cultured with +  +  6  irradiated B6-Tap- (1x10 ) splenocytes + H-Y peptide (1u.M) and IL-2 (20U/ml) /_  7  for 4 days, b) Histograms represent the expression of the indicated cell surface markers by gated C D 8 , CD8'° or unstained cells on day 4. c) P C R using primers hi  specific for DAP10, D A P 12 or p-Actin on c D N A from antigen + IL-2-activated CD8  hi  and CD8'° cells and IL-2-activated B6 NK cells.  Figure 3.6 N K G 2 D enhances the killing of target cells by male H-Y CD8'° cells in both an M H C restricted and non-MHC restricted fashion. Purified C D 8  hl  and  CD8'° T cells (1x10 ) were cultured with irradiated B6-Tap " (1x10 ) splenocytes 6  -/  7  + H-Y peptide (1 uJvl) and IL-2 (20U/ml) for 4 days, a) Day 4 activated cells were used as effectors in a C T L assay against R M A (H-2 , Rae-15 ) or RMA-Rae-15 b  -  (H-2 , Rae-16 ) cells at an effector to target ratio of 10 to 1 in the presence of the b  +  indicated concentrations of H-Y peptide, b) Day 4 activated cells were used in a redirected C T L assay against F c R P815 targets in the presence of the indicated +  mAbs. Part (a) was repeated 5 times with similar results. Error bars represent the standard deviation of triplicate cultures. * p-value < .002 ** p-value < .04  134 Figure 3.7 Bacterial infection primes CD8'° cells in vivo. Purified CD8'° cells were labeled with C F S E and injected into non-irradiated B6.Thy1.1 male recipients. 24h post-transfer the mice were infected with Listeria monocytogenes (LM) or left uninfected, a) The plots represent the C F S E and N K G 2 D profiles of gated CD8'° T cells on day 5 in either uninfected (left) or LM infected (right) mice, b) Spleens from day 5 infected (left) or uninfected (right) mice were cultured with a Golgiinhibitor in the presence or absence of H-Y peptide (1u.M). After 6h incubation the cells were fixed and stained with anti-IFNy, anti-H-Y T C R , and anti-CD8 mAbs. The dot plots depict the C F S E and IFNy profiles of gated H-Y T C R C D 8 cells. +  +  135  a) H-2b H-Y Female  au  H-2b H-Y Male  H-2b/d H-Y Male  Hi" • r |t.J  to  10°  1  10  z  10  *  10"  10°  IO  1  10  2  10  3  10"  CD8 700 ~ § «  1  600 5  0  0  -  400  0 300  o  1  200  °  100 0  DN  • H-2b H-Y Female • H-2b H-Y Male 0H-2b/d H-Y Male  y/mm  DP  CD4  CDB  Thymocyte Population  b) H-2b H-Y female (filled histogram)  o  H-2b H-Y male (light line)  H-2b/d H-Y male (dark line)  1  hX  W  10'  tff  !»' if  CD8  Figure 3.1 C D 8 T cells from antigen-expressing H-Y T C R transgenic mice +  possess an activated/memory phenotype.  136  a) 40000  IL-2  30000 -20000 10000 -  0 0.01  40000  0.1  1.0  + IL-2  30000 20000  o  10000  CD8  0  CD8  hi  C D B ID  0.01 0.1 H-Y peptide concentration  int  1.0  b) Unstimulated CD8  HI  CD8  INT  CD8  Anti-CD3 TO  CD8  HI  CD8  INT  P M A + lonomycin CD8'°  CD8 CD8 HI  INT  CD&° P-ZAP-70  ZAP-70  P-ERK ERK  Figure 3.2 C D 8 T cells from male H-Y mice possess a high activation threshold +  due to a defect in T C R signal transduction.  137  a) IL-2  Antigen + IL-2  IL-15  CD8  • ir  ro  1  i<r  j  i vr J  hi  10  CD8  KT  10  1  to*  int  ir  CD8'°  CFSE  b) CD8  CD8  hi  io  u  10  CD8"  int  to  -1  icr  10  • 1......'  -7  io  u  10  1  .r.»«T'"r »••—|  t<r  ic*  icr  CFSE  Figure 3.3 The extent of the memory-phenotype of H-Y C D 8 T cells determines their ability to respond to cytokines in vitro as well as their ability to undergo homeostatic expansion in vivo.  138  Figure 3.4 Role of antigen and bacterial infection in the expansion of H-Y male CD8'° cells in vivo.  139  a)  HY Female  HY Male HY Female T C R a C D 8 - Filled histogram hl  hi  HY Male TCRa CD8'° - Solid line +  Unstained Cells - Dashed line TCRa (T3.70) NKG2D  5 E  F°  to  to  1  2  lO  2B4  10**  3  10  10°  to  1  2  to  CD94 ^  3  ST  1  DX5  NK1.1  CD 16/32  0)  O io*  to  3  ID"  to  IO°  To  1  2  to  3  10*  «<  Fluorescence Intensity b)  2B4  NKG2D .Q  E  II  10"  10"  IO  2  IO  3  10  4  10°  10  10'  1  I0  1 04" l1n 00"  J  NK1.1  CD94  ,„1 10'  DX5  10'  10  10  J  CD 16/32  0)  O  to  0  io  1  io^  To  3  io  4  io°  lo  \o  1  z  io  3  to** to  0  io  1  ro io 2  3  Fluorescence Intensity Peptide/APC Activated CD8 - Filled histogram hi  Peptide/APC Activated CD8'° - Solid line Unstained Peptide/APC Activated Cells - Dashed line  c)  Activated HY CD8 to  Activated HY CD8  Activated B6 NK Cells  hi  DAP 10  DAP 12  $• Actin  Figure 3.5 Activated H-Y male C D 8 after activation.  |0  cells expressed NK receptors and D A P 12  140  a) CD8  CD8'°  hi  b)  E/T Ratio  Figure 3.6 N K G 2 D enhances the killing of target cells by male H-Y C D 8 cells in |0  both an M H C restricted and non-MHC restricted fashion.  141  a) LM Infected  Uninfected o (D 10  10  u  10  10*  10"  10°  10  10  2  10  10"  CFSE  b) LM Infected  Uninfected  10  z  u_  u  10'  10^  10  J  10  Uninfected + H-Y Peptide (6hr)  LM Infected + H-Y Peptide (6hr) **  io° io  1  id  2  id io~ 3  10°  10*  10'  10"  •  10 •+•.  CFSE  Figure 3.7 Bacterial infection primes CD8'° cells in vivo.  142 3.8  References  1.  Teh, H. S., P. Kisielow, B. Scott, H. Kishi, Y. Uematsu, H. Bluthmann, and  H. von Boehmer. 1988. Thymic major histocompatibility complex antigens and the alpha beta T- cell receptor determine the C D 4 / C D 8 phenotype of T cells. Nature 335:229-233. 2.  Kisielow, P., H. S. Teh, H. Bluthmann, and H. von Boehmer. 1988.  Positive selection of antigen-specific T cells in thymus by restricting M H C molecules. Nature 335:730-733. 3.  Kisielow, P., H. Bluthmann, U. D. Staerz, M. Steinmetz, and H. von  Boehmer. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333:742-746. 4.  Teh, H. S., H. Kishi, B. Scott, and H. Von Boehmer. 1989. Deletion of  autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal T C R levels and low levels of C D 8 molecules. J Exp Med 169:795-806. 5.  Yamada, H., T. Ninomiya, A. Hashimoto, K. Tamada, H. Takimoto, and K.  Nomoto. 1998. Positive selection of extrathymically developed T cells by selfantigens. J Exp Med 188:779-784. 6.  Pihlgren, M., C. Arpin, T. Walzer, M. Tomkowiak, A. Thomas, J . Marvel,  and P. M. Dubois. 1999. Memory CD44(int) C D 8 T cells show increased proliferative responses and IFN-gamma production following antigenic challenge in vitro. Int Immunol 11:699-706.  143 7.  Yamada, H., G . Matsuzaki, Q. Chen, Y. Iwamoto, and K. Nomoto. 2001.  Reevaluation of the origin of CD44(high) "memory phenotype" C D 8 T cells: comparison between memory C D 8 T cells and thymus-independent C D 8 T cells. Eur J Immunol 31:1917-1926. 8.  Yamada, H., T. Nakamura, G . Matsuzaki, Y . Iwamoto, and K. Nomoto.  2000. TCR-independent activation of extrathymically developed, self antigenspecific T cells by IL-2/IL-15. J Immunol 164:1746-1752. 9.  Harada, M., H. Yamada, K. Tatsugami, and K. Nomoto. 2001. Evidence of  the extrathymic development of tyrosinase-related protein-2- recognizing CD8+ T cells with low avidity. Immunology 104:67-74. 10.  Opferman, J . T., B. T. Ober, and P. G . Ashton-Rickardt. 1999. Linear  differentiation of cytotoxic effectors into memory T lymphocytes. Science 283:1745-1748. 11.  Yamada, H., G . Matsuzaki, Y. Iwamoto, and K. Nomoto. 2000. Unusual  cytotoxic activities of thymus-independent, self-antigen- specific CD8(+) T cells. Int Immunol 12:1677-1683. 12.  Tsunobuchi, H., H. Nishimura, F. Goshima, T. Daikoku, Y . Nishiyama, and  Y. Yoshikai. 2000. Memory-type CD8+ T cells protect IL-2 receptor alphadeficient mice from systemic infection with herpes simplex virus type 2. J Immunol 165:4552-4560. 13.  Lertmemongkolchai, G., G . Cai, C. A. Hunter, and G . J . Bancroft. 2001.  Bystander activation of CD8+ T cells contributes to the rapid production of IFNgamma in response to bacterial pathogens. J Immunol 166:1097-1105.  14.  Dhanji, S., and H.-S. Teh. 2003. IL-2-Activated CD8+CD44high Cells  Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells. J Immunol 171:3442-3450. 15.  Ho, E. L , L. N. Carayannopoulos, J . Poursine-Laurent, J . Kinder, B.  Plougastel, H. R. Smith, and W. M. Yokoyama. 2002. Costimulation of multiple NK cell activation receptors by NKG2D. J Immunol 169:3667-3675. 16.  Cerwenka, A., J . L. Baron, and L. L. Lanier. 2001. Ectopic expression of  retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a M H C class l-bearing tumor in vivo. Proc Natl Acad Sci  USA  98:11521-11526. 17.  Markiewicz, M. A., C. Girao, J . T. Opferman, J . Sun, Q. Hu, A. A. Agulnik,  C. E. Bishop, C. B. Thompson, and P. G . Ashton-Rickardt. 1998. Long-term T cell memory requires the surface expression of self- peptide/major histocompatibility complex molecules. Proc Natl Acad Sci USA 18.  95:3065-3070.  von Boehmer, H., H. S. Teh, and P. Kisielow. 1989. The thymus selects  the useful, neglects the useless and destroys the harmful. Immunol Today 10:5761. 19.  Zhang, W., J . Sloan-Lancaster, J . Kitchen, R. P. Trible, and L. E.  Samelson. 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83-92. 20.  Kersh, E. N., S. M. Kaech, T. M. Onami, M. Moran, E. J . Wherry, M. C.  Miceli, and R. Ahmed. 2002. T C R Signal Transduction in Antigen-Specific Memory C D 8 T Cells. J Immunol 170:5455-5463.  145 21.  Kieper, W. C , J . T. Burghardt, and C. D. Surh. 2004. A role for T C R  affinity in regulating naive T cell homeostasis. J Immunol 172:40-44. 22.  Tan, J . T., B. Ernst, W. C. Kieper, E. LeRoy, J . Sprent, and C. D. Surh.  2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J Exp Med 195:1523-1532. 23.  Tanchot, C , F. A. Lemonnier, B. Perarnau, A. A. Freitas, and B. Rocha.  1997. Differential requirements for survival and proliferation of C D 8 naive or memory T cells. Science 276:2057-2062. 24.  Jiang, J . , L. L. Lau, and H. Shen. 2003. Selective depletion of nonspecific  T cells during the early stage of immune responses to infection. J Immunol 171:4352-4358. 25.  Diefenbach, A., A. M. Jamieson, S. D. Liu, N. Shastri, and D. H. Raulet.  2000. Ligands for the murine N K G 2 D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat Immunol 1:119-126. 26.  Gilfillan, S., E. L. Ho, M. Cella, W. M. Yokoyama, and M. Colonna. 2002.  N K G 2 D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat Immunol 3:1150-1155. 27.  Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J . K. Hsia, E.  Vivier, and D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat Immunol 3:1142-1149.  146 28.  Kronenberg, M., and L. Gapin. 2002. The unconventional lifestyle of NKT  cells. Nat Rev Immunol 2:557-568. 29.  Brigl, M., L. Bry, S. C. Kent, J . E. Gumperz, and M. B. Brenner. 2003.  Mechanism of C D Id-restricted natural killer T cell activation during microbial infection. Nat 30.  Immunol4:1230-1237.  Park, S. H., K. Benlagha, D. Lee, E. Balish, and A. Bendelac. 2000.  Unaltered phenotype, tissue distribution and function of Valpha14(+) N K T cells in germ-free mice. EurJ Immunol 30:620-625. 31.  Ernst, B., D. S. Lee, J . M. Chang, J . Sprent, and C. D. Surh. 1999. The  peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity 11:173-181. 32.  Taniguchi, M., and T. Nakayama. 2000. Recognition and function of  Valpha14 N K T cells. Semin Immunol 12:543-550. 33.  Urdahl, K. B., J . C. Sun, and M. J . Bevan. 2002. Positive selection of M H C  class lb-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol 3:772-779. 34.  Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, and E. G . Pamer.  1999. H2-M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. J Exp M e d 190:195-204. 35.  Diefenbach, A., E. R. Jensen, A. M. Jamieson, and D. H. Raulet. 2001.  Rae1 and H60 ligands of the N K G 2 D receptor stimulate tumour immunity. Nature 413:165-171. 36.  Murga, M., O. Fernandez-Capetillo, S. J . Field, B. Moreno, L. R. Borlado,  Y. Fujiwara, D. Balomenos, A. Vicario, A. C. Carrera, S. H. Orkin, M. E.  Greenberg, and A. M. Zubiaga. 2001. Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity. Immunity 15:959-970.  148  Chapter 4  The Low Affinity Fc Receptor for IgG Functions as an Effective  Cytolytic Receptor for Self-Specific CD8 T Cells  4.1  1  Abstract  We have recently described a population of self-antigen specific murine C D 8 T +  cells with a memory phenotype, which utilize receptors of both the adaptive and innate immune systems in the detection of transformed and infected cells. Here we show that upon activation with IL-2 ± A g , between 10 and 20% of the activated self-specific C D 8 T cells express the low affinity Fc receptor for IgG. +  By contrast, all IL-2-activated NK cells express high levels of this Fc receptor. The Fc receptor comprises the FcyRllla and FcRy subunits. However, the FcRy subunit also associates with the C D 3 complex and this association likely contributes to the low expression of the Fc receptor in the activated cells. Although the Fc receptor is expressed at a low level on activated self-specific C D 8 T cells, it functions very efficiently as a cytolytic receptor in A D C C . Fc +  receptor-dependent killing occurred in the absence of T C R stimulation but could be augmented by concurrent stimulation of the T C R . In addition to mediating A D C C , engagement of the FcR on self-specific C D 8 T cells results in the +  production of both IFNy and T N F a . This is the first report of an activating Fc receptor on self-specific murine C D 8 a p T C R T cells and further establishes the +  +  importance of innate immune system receptors in the function of these selfspecific C D 8 T cells. +  1  A version of this chapter has been published as:  Dhanji, S., K. Tse, and H. S. Teh. 2005. The low affinity Fc receptor for IgG functions as an effective cytolytic receptor for self-specific C D 8 T cells. J Immunol 174:1253  149  4.2  Introduction  Natural killer (NK) cells represent a highly specialized lymphoid population characterized by potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors (1). The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of M H C class I (e.g., tumor cells). Inhibitory receptors such as those belonging to the Ly49 family in mice and the killer Ig-like receptors in humans contain immunoreceptor tyrosine based inhibitory (ITIM) motifs in their cytoplasmic domains (2). Engagement of these receptors results in the recruitment of inhibitory phosphatases that prevent NK cell activation. The activating receptors responsible for NK cell triggering include NKp46, NKp30, NKp44 and N K G 2 D in humans and NKp46 and N K G 2 D in mice (3, 4). The activating receptors, some of which belong to the same families, do not contain activation motifs but rather associate with adaptor molecules or signaling partners important for signal transduction (5). One of the first activating receptors described on NK cells is FcyRllla or CD16. C D 1 6 is a low affinity Fc receptor (FcR) that binds to IgG and is involved in antibody dependent cell-mediated cytotoxicity (ADCC) in which an antibody coated target cell is destroyed by NK cells (6). Stimulation of CD16 on NK cells also results in the production of cytokines such as IFNy, T N F a and G M - C S F (7). FcyRllla associates mainly with immunoreceptor tyrosine based activating motif  150 (ITAM)-containing homo or heterodimers of CD3<^ and FcsRIy (FcRy) in humans (8) or solely with FcRy homodimers in mice (9). The binding of IgG to CD16 results in the phosphorylation of the ITAMs in the signaling chains leading to the recruitment of kinases such as ZAP-70 and Syk (10). These kinases initiate a signaling cascade resulting in the lysis of antibody-coated target cells. T cells also use ITAM containing receptors and Syk and ZAP-70 for their signal transduction [reviewed in (11)]. Thus the signals transduced by the engagement of C D 1 6 on NK cells are very similar to those transduced via the engagement of the T C R on T cells. C D 16 expression is not limited to just NK cells as other cell types have been described which also express this receptor. y8 T cells have been shown to express C D 16 as have a population of large granular lymphocytes (mostly a(5TCR CD4"CD8" T cells) in humans (12, 13). In addition some memory+  phenotype a(5TCR CD8 T cells in humans have been shown to express C D 16 +  +  (14). W e have previously described a population of a p T C R C D 8 a p T cells in +  +  normal B6 mice that exhibit a memory phenotype characterized by the expression of high levels of CD44, IL-2Rp, and Ly6C (15). These cells can be activated by cytokines such as IL-2 and IL-15 and upon activation they express several functional NK receptors including 2B4, CD94, and N K G 2 D as well as the NK adaptor protein DAP12. Using an H-Y T C R transgenic model, we have shown that the development of C D 8 C D 4 4 T cells is driven by the high affinity +  hi  interaction of the a p T C R with cognate self-antigen (16). The H-Y T C R is specific for a male antigen (H-Y) presented by H-2D and in H-2 H-Y female mice, the b  b  151 lack of male antigen results in the development of H-Y C D 8 T cells with a naive phenotype (CD44 ). In H-2 H-Y male mice, on the other hand, the presence of 10  b  the cognate (H-Y) antigen results in the development of a population of C D 8 T +  cells that are virtually identical in cell surface and functional phenotypes with the memory phenotype C D 8 C D 4 4 +  hl  cells in normal B6 mice (16). Since memory  phenotype C D 8 T cells in normal and H-Y T C R transgenic mice are specific for +  self-antigens we will refer to these cells as self-specific C D 8 T cells to +  distinguish them from conventional memory C D 8 T cells. Self-specific C D 8 T +  +  cells from both H-Y T C R transgenic (16) and non-transgenic (15) mice preferentially kill syngeneic tumor cells. This killing of syngenic tumors involves the MHC-restricted ap T C R as well as the activating NK receptor, N K G 2 D , which results in non-MHC-restricted lysis of target cells that express the N K G 2 D ligand, Rae-1 (16). In this report we have described the expression and function of CD16 in self-specific C D 8 cells from B6 and male H-Y T C R transgenic mice. We showed +  that this Fc receptor, comprising the FcyRllla/FcRy subunits, is similar in composition to the NK Fc receptor. Although this Fc receptor is expressed at a low level in self-antigen specific C D 8 T cells, it is particularly efficient at initiating +  the destruction of antibody coated target cells and can induce the production of two key inflammatory cytokines, IFNy and T N F a . These observations underscore the importance of innate immune system receptors in the function of these selfspecific C D 8 T cells. +  152 4.3  Materials and Methods  Mice Breeders for C57BL/6 (B6) mice were obtained from the Jackson Laboratories (Bar Harbor, ME). The H-Y T C R transgenic mice (17) were bred to the B6 background. Mice 6 to 12 weeks of age were used for the experiments described.  Abs and flow cytometry The following antibodies were used: anti-CD4 (GK1.5), anti-CD8a (53.67), antiCD8p (53.38), anti-NK1.1 (PK136), anti-CD3s (2C11), anti-CD90 (T24, Rat lgG)(18), anti-CD16/32 (2.4G2, Rat lgG2b), anti-CD44 (PGP1), anti-FcRy, antiCD3<; (19) and anti-H-Y T C R a (T3.70) (17). All antibodies were purchased from BD PharMingen (San Diego, CA) except anti-FcRy (Upstate Biotechnology, Lake Placid, NY), anti-CD16/32 (American Type Tissue Culture Collection, Rockville, MD) and anti-H-Y T C R a (eBioscience, San Diego, CA). The CellQuest software program (Becton Dickinson, Mountain View, CA) was used for data acquisition and analysis.  153  Cell lines Cell lines used were the R M A lymphoma (H-2 , Rae-15") and Tap-deficient b+  R M A S (H-2 ~, Rae-15"). The cell lines were cultured in IMEM (Life Technologies, b  Burlington, Canada), supplemented with 10% (v/v) F B S (Life Technologies), 5x10 |iM 2-ME and antibiotics (l-medium). 5  Cell purification and activation CD8 CD44 +  hi  T cells from B6 mice were purified and cultured in IL-2 (200U7ml) for  5 days as previously described (15). NK cells were enriched by depletion of CD4 CD8 CD3 lg +  +  +  +  cells using Dynabeads and then cultured in IL-2 for 5 days  resulting in a pure population of activated NK cells (15). Purified naive C D 8  +  (CD44 ) cells do not respond to IL-2 alone and were activated for 5 days on l0  plate-bound anti-CD3s (10ug/ml) and IL-2 (20U/ml). Purified H-Y T C R C D 8 T +  +  cells (1x10 ) were activated by culturing with 1x10 B6 splenocytes, 1uM H-Y 6  7  peptide and 20U/ml IL-2 for 5-6 days.  RT-PCR R N A was extracted from activated cells and reverse-transcribed as previously described (15). P C R was preformed using previously described primers and reaction conditions (20).  154 CTL Assays C T L assays against R M A and R M A S target cells were performed as previously described (15). For antibody-dependent cell-mediated cytoxicity (ADCC), the target cells were pre-treated with anti-CD90 mAb (10u.g/ml) for 15min at room temperature prior to use. For the FcR-blocking experiment, anti-FcR mAb (2.4G2 used at 15|ag/ml) was added to the effector cells 15 min prior to the addition of targets and was present throughout the assay. Spontaneous release varied from 8-15% of the maximum. All assays were performed in triplicate. Percent specific lysis was calculated as 100% x [cpm (experimental well) - cpm (spontaneous release)]/[(cpm (maximum release) - cpm (spontaneous release)].  Immunopreceipitation  and immunoblot analysis  Cells were activated as described above and pelleted/lysed in 10 mM Tris (pH 7.5), 150 mM NaCl, 1% TX-100, 0.1% S D S , and protease and phosphatase inhibitors. The lysates were separated on a 4-15% Tris-HCl polyacrylamide gel and transferred to a P V D F membrane. Blots were developed using E C L system (Amersham). C D 3 immunoprecipitation was preformed by treating lysates with pre-conjugated anti-CD3s/Protein G-sepharose beads for 2 hours at 4°C followed by several washes. Complexes were then removed from the beads by resuspension in 2X protein sample buffer followed by boiling for 5 mins. The samples were then run in a 4-15% Tris-HCl gel and immunoblotted as described above.  155 4.4  Results  Expression of FcyRllla/FcRy on activated self-specific CD8+ cells. We have previously shown that self-specific C D 8 T cells express an +  activated/memory phenotype (15, 16). Immediately ex vivo self-specific C D 8  +  cells from B6 and male H-2 H-Y mice do not express significant levels of C D 1 6 b  (Fig. 4.1a and data not shown). However, upon activation with IL-2 alone (B6) or antigen and IL-2 (H-Y male) for 5-6 days, between 10 and 15% of the activated self-specific C D 8 cells from B6 ( C D 8 C D 4 4 ) and male H-2 H-Y mice express +  +  hi  b  CD16 (Fig. 4.1a). In contrast, all IL-2-activated NK cells express high levels of CD16 (Fig. 4.1a). Since naive (CD8 CD44'°) cells from B6 and female H-Y T C R +  transgenic mice do not proliferate in response to stimulation with IL-2 alone these cells were activated with anti-CD3 + IL-2 (B6) or antigen plus IL-2 (H-Y female) to induce activation and proliferation. It is clear that activated CD8 CD44'° cells from +  B6 and female H-Y T C R transgenic mice do not express CD16 (Fig. 4.1a) consistent with the conclusion that conventional C D 8 T cells do not express CD16 upon activation. We have previously described the bystander expansion and activation of self-specific C D 8 T cells in response to infection with the bacterial pathogen Listeria monocytogenes (LM) (15, 16). Therefore we wanted to determine whether the frequency of C D 1 6 C D 8 C D 4 4 +  +  hl  increased upon infection with L M .  In uninfected B6 mice there is a small percentage of C D 8 T cells that express +  CD16 but this number increases by about 3 fold upon infection with Listeria (Fig.  156 4.1b). In addition all of the C D 8 C D 1 6 cells in both infected and uninfected mice +  +  express high levels of CD44. The antibody used to detect CD16 expression also binds to CD32 and it was important to determine which Fc receptor subunits were actually expressed by the self-specific C D 8 cells. R T - P C R with primers specific for various Fc +  receptor subunits was used to determine the composition of the expressed Fc receptor. It is clear from this analysis that activated self-specific C D 8 cells from +  B6 and male H-2 H-Y mice as well as NK cells from B6 mice express the m R N A b  for only FcyRllla (CD16) and FcRy but not FcyRI or FcyRIIB (Fig.4.2a). A macrophage cell line was used as a positive control for the expression of FcyRI and FcyRIIB (Fig. 4.2a). In murine NK cells, CD16 can only pair with FcRy homodimers (9). Furthermore, the forced expression of CD3d; in murine NK cells actually interferes with the surface expression and function of CD16 through the formation of CD3c7FcRy heterodimers, which cannot associate with CD16 (21).This finding suggests that the low cell-surface expression of C D 16 on self-specific C D 8 cells +  may be due to the high expression of CD3t; in these cells. To address this possibility, we compared the total amount of CD3<^ and FcRy in activated NK and self-specific C D 8 cells by western blot. Figure 4.2b shows that activated NK +  cells express an undetectable level of CD3t; and the most FcRy when compared to self-specific C D 8 cells from B6 ( C D 8 C D 4 4 ) and H-Y male mice. By +  +  hi  contrast, self-specific C D 8 cells from B6 and H-Y male mice express both CD3C; +  and FcRy (Fig. 4.2b). A s expected, activated conventional C D 8 (CD8 CD44'°) +  157 cells only express CD3C,. These findings suggest that the low levels of C D 16 surface expression in self-specific C D 8 cells may be due to the expression of +  CD3C in these cells. In conventional T cells the ap T C R pairs with the CD3-family of signaling chains including CD3s and CD3<; (22). Since FcRy is expressed in self-specific C D 8 cells we wanted to determine if this signaling molecule could associate with +  the T C R / C D 3 complex in these cells. To determine whether FcRy associates with the C D 3 complex, we immunoprecipitated the C D 3 complex from IL-2-activated self-specific C D 8 cells from B6 and male H-Y mice using an anti-CD3e antibody +  and immunoblotted with either an anti-CD3C; or anti-FcRy antibody. Figure 4.2c clearly shows that anti-CD3s precipitates both CD3<; and FcRy in self-specific C D 8 cells from B6 and male H-Y mice. A s expected, anti-CD3s precipitates only +  CD3<; in conventional C D 8 C D 4 4 +  |0  cells. This association of FcRy with the C D 3  complex in self-specific C D 8 cells likely interferes with the association of CD16 +  with FcRy, resulting in low expression of C D 16 in these cells.  IL-2-activated self-specific CD8+ cells can mediate ADCC. After observing the expression of CD16 on IL-2-activated self-specific C D 8 cells +  we determined whether this receptor could mediate A D C C . To this end, we activated self-specific C D 8 cells and NK cells from B6 mice with IL-2 and then +  tested their ability to kill antibody-coated R M A S targets cells. Anti-CD3 + IL-2 activated naive C D 8 ( C D 8 C D 4 4 ) cells were included as a negative control. +  |0  TAP-deficient R M A S cells were used as target cells to rule out contribution by  158 M H C class I molecules in the killing reaction. The R M A S cells were left untreated or pretreated with anti-CD90 (clone T24; 10^g/ml) mAb prior to use as target cells. Figure 4.3a clearly shows that IL-2-activated self-specific C D 8 and NK +  cells can efficiently kill antibody-coated R M A S cells whereas anti-CD3 +  In-  activated conventional C D 8 ( C D 8 C D 4 4 ) cells show absolutely no activity. W e +  |0  noted that anti-CD90 was more efficient in promoting the killing of R M A S targets by self-specific C D 8 cells compared to NK cells. This finding is remarkable +  considering that only a small fraction of the self-specific C D 8 cells express +  CD16 and the level of CD16 expressed per cell is significantly lower than for NK cells (Fig 4.1a). Since R M A S cells are killed efficiently by activated NK cells, the lack of killing of untreated R M A S cells by activated self-specific C D 8 cells also +  indicates the lack of contaminating NK cells in the killing assay. After observing that self-specific C D 8 cells from B6 mice could efficiently +  lyse R M A S targets we determined if the presence of M H C class I had any effect on lysis by using TAP-sufficient R M A targets. In addition we wanted to determine whether blocking of the CD16 receptor with an anti-CD16 mAb on the activated self-specific C D 8 cells could block killing of antibody-coated target cells. Figure +  4.3b demonstrates that self-specific C D 8 cells efficiently killed antibody-coated +  R M A cells. Furthermore, the killing of antibody-coated target cells was greatly reduced by blocking the CD16 receptor on self-specific C D 8 cells prior to +  culturing with antibody-coated R M A targets. For NK cells the lysis of antibodycoated R M A cells was only partially inhibited by blocking C D 1 6 on the NK cells. This is probably due to the high expression of CD16 on NK cells, which could not  159 be blocked completely by the anti-CD16 antibody treatment. A n alternative explanation for the inefficient blocking of killing of antibody coated target cells by NK cells is that the anti-CD16 mAb functions as an agonistic mAb. However, this is unlikely since the treatment of NK cells with anti-CD16 mAb did not have any effect on the lysis of untreated R M A target cells (data not shown). These results clearly demonstrate that self-specific C D 8 cells express a functional FcR, which +  can mediate A D C C . Furthermore, expression of M H C class I molecules on the target cells does not affect FcR-mediated killing.  The Fc receptor on self-specific CD8+ cells from H-Y male mice functions independently of the TCR. Since self-specific C D 8 cells from male H-Y mice express only the male+  specific H-Y T C R we used these cells to determine whether the Fc receptor can function independently of the T C R . Self-specific C D 8 cells from male H-Y mice +  and conventional C D 8 cells from female H-Y mice were activated with antigen +  and IL-2 for 6 days. The activated cells were then assessed for cytolytic activity against untreated or anti-CD90-treated R M A (H-2 ) target cells. The killing of b  anti-CD90-coated target cells in the absence of exogenous H-Y peptide was used as a measure for the contribution of the Fc receptor in the killing reaction. Inclusion of the H-Y peptide in the assay allows an estimation of the contribution of the H-Y T C R in the killing reaction. It is clear from the data in Fig. 4.4 that selfspecific C D 8 cells from male H-Y mice killed antibody-coated R M A targets very +  efficiently even in the absence of the H-Y peptide. The killing of anti-CD90-  160 coated target cells was slightly enhanced by the addition of H-Y peptide (Fig. 4.4). The activated self-specific C D 8 cells from male H-Y mice required almost +  10nM exogenous H-Y peptide to attain the same level of killing as seen with antiCD90-coated target cells in the absence of H-Y peptide. This is remarkable since the entire population of H-Y male cells expressed the H-Y T C R whereas at most 20 percent of the cells expressed CD16. By contrast, activated conventional C D 8 T cells from female H-Y mice could only kill peptide-loaded target cells and +  the presence of antibody on the targets had no effect on this killing. These results suggest that C D 1 6 functions independently of the T C R as an effective cytolytic receptor on self-specific C D 8 cells. Furthermore, the Fc receptor and the T C R +  can act in an additive manner in the killing reaction.  Engagement  of CD 16 on self-specific  CD8+ celts induces cytokine  production.  CD16 engagement on NK cells has been shown to induce the expression of several cytokines in addition to being able to induce A D C C (7). In order to test whether CD16 engagement on self-specific C D 8 T cells could also mediate the production of cytokines we cultured antigen-activated H-Y male C D 8 cells with R M A targets which had been pre-treated with anti-CD90 or left untreated. We found that these cells showed a significant increase in IFN-y production in response to anti-CD90-coated R M A cells (7.2% IFN-y ) over untreated R M A cells +  (1.3%). Furthermore, this increase in IFN-y production was reduced to near basal level by the inclusion of soluble anti-CD16 mAb in this assay (Fig. 4.5a). This  result suggests that the Fc portion of the bound anti-CD90 mAb on R M A cells induces the production of IFN-y by H-Y male C D 8 cells. In order to obtain more direct evidence for the Fc receptor-mediated cytokine production by H-Y male C D 8 cells, we used antibody cross-linking to stimulate CD16 directly. We cultured day 6 antigen-activated H-Y male C D 8 T cells in wells coated with either no antibody (upper row), anti-CD16 (middle row), or anti-CD3s (bottom row). After a 5 hr incubation period, we fixed and stained the cells for C D 8 and IFN-y (left column), T N F - a (middle column) or G M - C S F (right column). CD16 engagement resulted in a large increase in both IFN-y and T N F - a production with the percentage of cytokine positive cells being similar to the percentage of C D 1 6 cells in the sample; CD16 engagement did not induce +  the production of G M - C S F (Fig. 4.5b). We also found that soluble anti-CD16 is inefficient in inducing IFN-y production by H-Y male C D 8 cells (data not shown). It is likely that soluble anti-CD 16 mAb is less efficient than plate-bound anti-CD 16 mAb in aggregating Fc receptors and receptor aggregation is required for efficient activation. By contrast to Fc receptor stimulation, stimulation of the C D 3 complex resulted in the majority of the cells expressing IFN-y and T N F - a . In addition, anti-CD3 stimulated cells produced significant amount of G M - C S F (Fig. 4.5b). These results indicate that the Fc and the ap T C R function as directly activating receptors and which transduce qualitatively and quantitatively distinct signals upon activation.  162 4.5  Discussion  In this study, we showed that upon activation with IL-2 or antigen plus IL-2, selfspecific C D 8 cells express an Fc receptor that is similar in composition to the +  low affinity Fc receptor for IgG that is found on NK cells. Even though these cells express only a relatively low level of this receptor when compared to NK cells, it functions very efficiently in the lysis of antibody-coated target cells. Fc receptormediated killing is independent of antigen expression on the target cells. Furthermore, expression of M H C class I molecules on the target cells did not affect the efficiency of Fc receptor-mediated killing. The Fc receptor can also act in an additive manner with the T C R in the lysis of the susceptible target cells. This Fc receptor not only mediates efficient lysis of susceptible targets, but it also induces the production of both IFNy and T N F a . The combination of these properties would enable self-specific C D 8 T cells to detect infected or +  transformed target cells which might not be detected by NK cells. It is interesting to note that even though the H-Y male cells represent a clonal population in which all cells should have been activated equivalently, only a fraction of the activated cells express CD16. The low cell-surface expression of CD16 on self-specific C D 8 cells is likely due to the high expression of C D 3 ^ in +  these cells. In murine NK cells, CD16 can only pair with FcRy homodimers (9). Furthermore, the expression of CD3d; in murine NK cells interferes with the surface expression and function of CD16 through the formation of CD3£/FcRy heterodimers, which cannot associate with CD16 (21). We found that the FcRy  163 chain in self-specific C D 8 T cells is coprecipitated with CD3<^ by the anti-CD3s +  mAb. It is likely that this association of FcRy with the CD3C, chain contributes to the low expression of the Fc receptor on IL-2-activated self-specific C D 8 T cells. +  The association of the FcRy chain with CD3C, is not unique to self-specific C D 8 T +  cells since in large granular lymphocytes and in T cells from tumor-bearing mice, the C D 3 complex has been shown to associate with FcRy (23, 24). The association of the ap T C R with the FcRy chain would enable the ap T C R of these cells to be linked to additional signaling pathways. We have previously shown that self-specific C D 8 cells undergo bystander +  expansion in vivo in response to Listeria infection, likely as a consequence of the high expression of IL-2RP by these cells, which endowed these cells to proliferate in response to IL-2 or IL-15 (16). Furthermore, self-specific C D 8 T +  cells that proliferate in response to bacterial infection exhibit a heightened ability to produce IFN-y (16). These properties of self-specific C D 8 T cells would +  enable them to detect infected cells and provide an early source of IFN-y. The expression of a self-specific T C R and N K G 2 D on these cells would enable them to focus on host cells that expressed ligands induced by infection or transformation. Here we have provided evidence for the expression and functional significance of another activating NK receptor that adds to the arsenal of self-specific C D 8 cells. W e have shown that CD16 is expressed on a +  significant fraction of C D 8 C D 4 4 T cells upon Listeria infection in vivo or upon +  h l  activation with IL-2 or antigen and IL-2 in vitro. Furthermore, engagement of CD16 on self-specific C D 8 T cells results in efficient lysis of antibody-coated +  164 targets as well as in the production of inflammatory cytokines. The possession of activating receptors of the innate as well as the adaptive immune system distinguished these cells from NK cells and suggests that this interesting cell type may be particularly adept in providing an early response to infected and transformed cells. These cells will also provide an early source of cytokines such as IFN-y and T N F a , which would prime the adaptive immune system in the elimination of infected and transformed cells.  4.6  Acknowledgements  We thank Soo-Jeet Teh for excellent technical assistance and Dr. Hao Shen (University of Pennsylvania) for the kind gift of the wildtype Listeria monocytogenes  strain 10403s.  165  4.7  Figures  Fig. 4.1 Activation of C D 8 C D 4 4 cells results in the expression of CD16. a) +  Purified C D 8 C D 4 4 +  hi  hi  cells from B6 and C D 8 cells from male H-2 H-Y T C R b  transgenic mice were used as a source of self-specific C D 8 T cells. Purified CD8 CD44'° cells from B6 and female H-2 H-Y T C R transgenic mice were used +  b  as a source of naive C D 8 T cells. Self-specific B6 C D 8 T cells and NK cells +  were activated by culturing with IL-2 for 5 days. Naive C D 8 T cells from B6 mice were activated by culturing with anti-CD3 + IL-2 for 5 days and C D 8 cells from male and female H-Y mice were activated with Ag + IL-2 for 6 days. The activated cells were stained for the expression of CD16/CD32. The filled histograms represent the CD16/CD32 profile of the indicated cell type and the unfilled histograms represent unstained controls, b) Naive B6 mice were infected with 10,000 C F U of Listeria monocytogenes.  On day 5 the mice were sacrificed  and the spleens removed and stained. The histograms depict the expression of C D 8 and CD16 on gated C D 8 T cells from infected (right) or uninfected (left) mice and the percentages of C D 1 6 C D 8 T cells are indicated. Three-color +  +  analysis revealed that the C D 8 C D 1 6 cells also expressed high levels of CD44 +  +  (data not shown).  Fig. 4.2 Activated self-specific C D 8 T cells express a low affinity Fc receptor similar to NK cells. Cells were activated as described in Fig. 4.1. a) R T - P C R analysis of FcyRllla, FcRy, FcgRI, and FcgRIIB transcripts present in activated  cells. R N A from the J774 macrophage cell line was used as a positive control for FcyRI and FcyRIIB. b) Whole cell lysates of the activated cells were subjected to immunoblot analysis for the detection of FcRy and CD3<; protein. Blots were stripped and re-probed with anti-ERK2 as a loading control, c) Activated cells were lysed and the lysates were immunoprecipitated (IP) with antiCD3s antibody. The immunoprecipitates were then immunoblotted (IB) with antiCD3<; or anti-FcRy mAbs. (N.D. - not determined)  Fig. 4.3 C D 8 C D 4 4 +  hl  cells efficiently kill antibody-coated targets via an Fc-  dependent mechanism, a) NK, self-specific C D 8 ( C D 8 C D 4 4 ) and naive C D 8 +  hi  ( C D 8 C D 4 4 ) cells from B6 mice were activated as described in Fig. 4.1 and +  l0  used as effectors in a standard chromium release assay against TAP-deficient R M A S target cells that had been treated with anti-CD90 mAb (10|ig/ml) or left untreated, b) Activated NK and C D 8 C D 4 4 +  hl  cells were used as effectors in a  chromium release assay against MHC-sufficient R M A target cells that had been treated with anti-CD90 mAb (10ug/ml) or left untreated. The effector cells were also pretreated with anti-CD16 mAb (10|j.g/ml) or left untreated in order to block the Fc receptor. Error bars represent the standard deviation of triplicate cultures.  Fig. 4.4 CD16-mediated killing of target cells by self-specific C D 8 T cells is T C R independent. Self-specific C D 8 T cells from H-2 H-Y male mice and naive C D 8 b  T cells from female H-2 H-Y mice were activated as described in Fig. 4.1 and b  used in a standard chromium release assay against R M A targets either pre-  treated with anti-CD90 mAb (10u:g/ml) or left untreated. The assay was done at a constant 10 to 1 effector to target ratio with the addition of the indicated concentrations of H-Y peptide. Error bars represent the standard deviation of triplicate cultures.  F i g . 4.5 Cytokine production by self-specific C D 8 T cells in response to C D 1 6 engagement, a) Day 6 antigen + IL-2 activated H-Y male C D 8 cells were cultured with R M A cells treated with anti-CD90 (10u:g/ml) or left untreated. Soluble antiCD16 (10p:g/ml) was added to some cultures to block Fc binding. IFN-y production was assessed by intracellular staining after a 5 hr incubation period, b) Day 6 antigen + IL-2 activated H-Y male C D 8 cells were cultured with plate bound anti-CD16 (10u:g/ml), anti-CD3s (10p;g/ml), or without any antibody for 5 hours. The cells were then stained for intracellular IFN-y, TNF-a, and G M - C S F . The numbers in the dot plots represent the percentage of cytokine-positive C D 8 T cells.  +  168  a)  CD16  F i g . 4.1 Activation of C D 8 C D 4 4 cells results in the expression of C D 1 6 +  hl  169  a)  B6 CD8 B6 CD8 H-Y Male CD44' CD44 CD8 B6NK +  MO  +  0  H I  FcyRllla FcRy FcyRI FcyRIIB p-actin  b)  B6 N K  B6CD8< B6 CD8< HY Male CD44*i CD44°' CD8 CD3^ FcRy ERK2  c)  B6CD8< CD44^  B6 CD8 CD44°' +  HY Male  CD8  IP: CD3s IB: CD3i; IB: FcRy  F i g . 4.2 Activated self-specific CD8 T cells express a low affinity Fc receptor similar to NK cells  170  1008060402004  in 'in >i 50-  _j 40o 30'o 2010Q. w 0-  Q O +  oo Q O  50- • • 30-  None Anti-CD90  10-103  10  4 30  Q O + 00  Q O  E/T Ratio 90 70 50 30 10  Z  50' • None 40' • Anti-CD90 30 • Anti-CD90 + Anti- FcR 20 • 10 0'  D  3  O +  10  oo Q O  E/T Ratio  F i g . 4.3 C D 8 C D 4 4 +  hi  cells efficiently kill antibody-coated targets via an Fc-  dependent mechanism  171  80  H-Y Peptide (nM)  F i g . 4.4 CD16-mediated killing of target cells by self-specific C D 8 T cells is T C R independent  172  F i g . 4.5 Cytokine production by self-specific C D 8 T cells in response to CD16 engagement  173 4.8  References  1.  Lanier, L. L. 1998. NK cell receptors. Annu Rev Immunol 16:359-393.  2.  Long, E. 0 . 1998. Regulation of immune responses by inhibitory receptors. Adv Exp Med Biol 452:19-28.  3.  Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari, R. Biassoni, and L. Moretta. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 19:197-223.  4.  Cerwenka, A., and L. L. Lanier. 2001. Natural killer cells, viruses and cancer. Nat Rev Immunol 1:41-49.  5.  Tomasello, E., L. Olcese, F. Vely, C. Geourgeon, M. Blery, A. Moqrich, D. Gautheret, M. Djabali, M. G . Mattei, and E. Vivier. 1998. Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12. J Biol Chem 273:3411534119.  6.  Perussia, B., and J . V. Ravetch. 1991. Fc gamma RIM (CD16) on human macrophages is a functional product of the Fc gamma RIII-2 gene. EurJ Immunol 21:425-429.  7.  Cassatella, M. A., I. Anegon, M. C. Cuturi, P. Griskey, G . Trinchieri, and B. Perussia. 1989. Fc gamma R(CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells.  Role of Ca2+ in Fc gamma R(CD16)-induced transcription and expression of lymphokine genes. J Exp Med 169:549-567. 8.  Lanier, L. L., G . Y u , and J . H. Phillips. 1989. Co-association of C D 3 zeta with a receptor (CD 16) for IgG Fc on human natural killer cells. Nature 342:803-805.  9.  Kurosaki, T., and J . V. Ravetch. 1989. A single amino acid in the glycosyl phosphatidylinositol attachment domain determines the membrane topology of Fc gamma RIM. Nature 342:805-807.  10.  Vivier, E., A. J . da Silva, M. Ackerly, H. Levine, C. E. Rudd, and P. Anderson. 1993. Association of a 70-kDa tyrosine phosphoprotein with the CD16: zeta: gamma complex expressed in human natural killer cells. Eur J Immunol 23:1872-1876.  11.  Cantrell, D. 1996. T cell antigen receptor signal transduction pathways. Annu Rev Immunol 14:259-274.  12.  Lanier, L. L., T. J . Kipps, and J . H. Phillips. 1985. Functional properties of a unique subset of cytotoxic CD3+ T lymphocytes that express Fc receptors for IgG (CD16/Leu-11 antigen). J Exp Med 162:2089-2106.  13.  Oshimi, K., Y. Oshimi, O. Yamada, M. Wada, T. Hara, and H. Mizoguchi. 1990. Cytotoxic T lymphocyte triggering via C D 16 is regulated by C D 3 and C D 8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes. J Immunol 144:3312-3317.  14.  Zupo, S., L. Azzoni, R. Massara, A. D'Amato, B. Perussia, and M. Ferrarini. 1993. Coexpression of Fc gamma receptor IMA and interleukin-2 receptor beta chain by a subset of human CD3+/CD8+/CD11b+ lymphocytes. J Clin Immunol 13:228-236.  15.  Dhanji, S., and H.-S. Teh. 2003. IL-2-Activated CD8+CD44high Cells Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells. J Immunol 171:34423450.  16.  Dhanji, S., S. J . Teh, D. Oble, J . J . Priatel, and H. S. Teh. 2004. SelfReactive Memory-Phenotype C D 8 T Cells Exhibit Both MHC-Restricted and Non-MHC Restricted Cytotoxicity: A Role for the T Cell Receptor and Natural Killer Cell Receptors. Blood.  17.  Teh, H. S., P. Kisielow, B. Scott, H. Kishi, Y. Uematsu, H. Bluthmann, and H. von Boehmer. 1988. Thymic major histocompatibility complex antigens and the alpha beta T- cell receptor determine the C D 4 / C D 8 phenotype of T cells. Nature 335:229-233.  18.  Dennert, G . , R. Hyman, J . Lesley, and I. S. Trowbridge. 1980. Effects of cytotoxic monoclonal antibody specific for T200 glycoprotein on functional lymphoid cell populations. Cell Immunol 53:350-364.  19.  van Oers, N., S. Teh, A. Garvin, K. Forbush, R. Perlmutter, and H. Teh. 1993. C D 8 inhibits signal transduction through the T cell receptor in C D 4 C D 8 - thymocytes from T cell receptor transgenic mice reconstituted with a transgenic C D 8 alpha molecule. J Immunol 151:777-790.  176 20.  Arase, N., H. Arase, S. Hirano, T. Yokosuka, D. Sakurai, and T. Saito. 2003. IgE-Mediated Activation of NK Cells Through Fc{gamma}RIII. J Immunol 170:3054-3058.  21.  Arase, H., T. Suenaga, N. Arase, Y . Kimura, K. Ito, R. Shiina, H. Ohno, and T. Saito. 2001. Negative regulation of expression and function of Fc gamma RIM by C D 3 zeta in murine NK cells. J Immunol 166:21-25.  22.  Weiss, A., and D. R. Liftman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263-274.  23.  Mizoguchi, H., J . J . O'Shea, D. L. Longo, C. M. Loeffler, D. W. McVicar, and A. C. Ochoa. 1992. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science 258:1795-1798.  24.  Koyasu, S., L. D'Adamio, A. R. Arulanandam, S. Abraham, L. K. Clayton, and E. L. Reinherz. 1992. T cell receptor complexes containing Fc epsilon RI gamma homodimers in lieu of C D 3 zeta and C D 3 eta components: a novel isoform expressed on large granular lymphocytes. J Exp Med 175:203-209.  177  Chapter 5  Self-Antigen Maintains the Innate Anti-Bacterial Function of  Self-Specific CD8 T Cells in Vivo  5.1  1  Abstract  Self-specific C D 8 T cells, which are selected by high affinity interactions with selfantigens, develop into a lineage distinct from conventional C D 8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. Here we show that these selfspecific cells have the ability to produce large amounts of IFNy in response to infection with Listeria monocytogenes  in a bystander fash ion. The rapid  production of IFNy is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific C D 8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific C D 8 T cells are also more efficient in the production of IFNy and T N F a , thus providing more cytokine-dependent protection against bacterial infection when compared to NK cells. These findings illustrate that self-reactive C D 8 T cells can provide an important innate function in the early defense against bacterial infection.  1  A version of this chapter has been accepted for publication as:  Dhanji, S., M.T. Chow, and H. S. Teh. 2006. Self-Antigen Maintains the Innate Anti-Bacterial Function of Self-Specific C D 8 T Cells in Vivo. J Immunol.  178  5.2  Introduction  Successful immune protection against pathogens relies on cooperation between the innate and adaptive arms of the immune system. Cells of the innate immune system provide the first line of defense against pathogens and are usually able to clear infections before they spread. Innate cells use germ line encoded receptors, which either recognize pathogens directly or recognize changes on host cells as a result of infection. In cases where the innate immune system is overwhelmed, the adaptive immune system is usually able to provide the additional immune responses that are required for the elimination of the pathogen. However, the response of adaptive immune cells is slow and requires the expansion of T cells and B cells that express rearranged receptors specific for pathogen derived epitopes. Another distinguishing feature of the adaptive immune system is the production of memory T and B lymphocytes, which are very effective in protection against subsequent infections by the same pathogen. The distinction between innate and adaptive immunity is not absolute. Several adaptive lymphocytes have been shown to express antigen (Ag)-specific receptors of limited diversity in conjunction with the expression of receptors associated with innate cells. For instance, B1-B cells express rearranged B cell receptors (BCRs) that display limited diversity and show specificity for self-Ags such as oxidized phospholipids (1). T cells expressing y8 T cell receptors (TCRs) also show limited diversity in their T C R rearrangements and some of these cells express innate system receptors which are specific for stress-induced Ags (2). Intraepithelial lymphocytes or iELs can express T C R s specific for self-Ags and  179 this interaction is crucial for their function (3, 4). Natural killer T cells (NKT) restricted to the non-classical M H C class I molecule, C D 1 d , also express invariant T C R s (Va14/ in mice) (5). This cell type is abundant in the spleen and liver where the cells are thought to become activated in response to inflammatory cytokines with concurrent recognition of lipids in the context of CD1d. They are then able to rapidly secrete large amounts of cytokines such as IFNy or IL-4, which play a role in biasing the immune response towards the Th1 or Th2 lineage, respectively (6). IFNy is crucial for protection against various intracellular pathogens (7). Binding of IFNy to its receptor on macrophages induces bactericidal activity in infected macrophages resulting in the production of reactive oxygen and nitrogen intermediates as well as the efficient fusion of lysosomes with phagosomes containing pathogens (8). IFNy also has an impact on shaping the adaptive immune response and leads to the expression of host genes involved in A g processing and presentation (9). In addition, this cytokine plays an important role in the polarization of C D 4 T cells to the Th1 lineage which can then activate macrophages (10). IFNy signaling early during infection enhances C D 4 and C D 8 T cell responses (11, 12) and also programs contraction of Ag-specific C D 8 T +  cells thus controlling the level of immunological memory (13). Listeria monocytogenes  (LM) is a gram-positive intracellular pathogen which  is the cause of listeriosis in humans (14). Efficient protection against LM infection relies heavily on early IFN-y production by innate cells (15). IFNy production during infection with LM was thought to come mainly from natural killer (NK) cells  180 but recent studies suggest that memory-phenotype C D 8 T cells are also a major +  contributor (16-20). Memory-phenotype C D 8 T cells include those that are +  specific for foreign-Ags and which have been generated during a previous encounter with these Ags. These bona fide foreign-Ag specific memory C D 8 T +  cells have been shown to provide an innate source of IFNy in the absence of cognate Ag through IL-12 and IL-18 signaling (17). Interestingly, memory C D 8 T +  cells have been shown to be more protective than NK cells during LM infection (18). However, not all memory-phenotype C D 8 T cells are specific for foreign+  Ags. C D 8 T cells that are restricted to non-classical M H C class lb molecules +  also have a memory-phenotype (21). Some M H C class lb-restricted C D 8 T cells +  have been shown to be selected by hematopoetic cells and can respond to L M infection in both an Ag-dependent and Ag-independent fashion by producing IFNy (22, 23). Thus, memory-phenotype C D 8 T cells have been shown to be a +  very significant source of early IFN-y production during infections. We have shown that memory-phenotype C D 8 T cells in normal mice contain +  a subset of T cells that demonstrate a very high reactivity for self-peptide/MHC (24). By virtue of high expression of CD122 (IL-2Rp), these cells respond to IL-2 and IL-15 both in vitro and in vivo. Upon activation, they express several NK receptors including CD16, N K G 2 D and the adaptor protein DAP12. C D 1 6 engagement on these cells results in both the production of inflammatory cytokines and the lysis of antibody-coated target cells (25). N K G 2 D engagement also results in the lysis of NKG2D-ligand expressing target cells. These cells  181 comprise ~10% of peripheral C D 8 T cells in normal mice and they demonstrate +  specificity for syngeneic tumor cells. We have also characterized self-specific C D 8 T cells in H-Y T C R transgenic +  mice (26). The H-Y T C R is specific for the male (H-Y) peptide presented by H2 D . In female H-2 mice C D 8 T cells expressing the H-Y T C R are positively b  b  +  selected. In male H-2 H-Y T C R transgenic mice, the deletion of virtually all of the b  double positive thymocytes greatly affected the development of conventional CD8 as well as CD4 T cells (27, 28). However, large numbers of T cells which expressed low levels of C D 8 and exclusively the H-Y T C R are present in the peripheral lymphoid organs of these male mice (29). More interestingly, these cells have a similar memory phenotype and functional properties as the selfspecific C D 8 T cells that are found in normal mice. They also express NK +  receptors that function in cooperation with the T C R . Most importantly, we have shown that these cells become activated in vivo in response to LM infection (26). In this report we determined the role of self-specific C D 8 T cells in protection +  against LM infection and assessed the role of self-Ag interactions in the maintenance and function of this cell type. W e show that these cells are protective immediately ex vivo but more so after IL-15 activation. We also demonstrate that self-Ag interactions are crucial for the early protection of IFNydeficient mice from LM infection and for maintaining the memory-phenotype and cytokine responsiveness of these self-specific cells. Finally, we show that activated self-specific C D 8 T cells produce more IFNy and T N F a than NK cells +  and are more protective than NK cells during infection with L M .  182  5.3  Materials and Methods  Mice Breeders for C57BL/6 (B6), B6.foxn1  nu  (athymic nude), B6.Thy1.1, and B6.IFNY"'  -  were obtained from the Jackson Laboratories (Bar Harbor, ME). The H-Y T C R transgenic (tg) mice were bred to the B6 or B6.foxn1  nu  background. Mice 8 to 12  weeks of age were used for the experiments described.  Abs and flow cytometry The following mAbs specific for the indicated molecule were used: CD8cc (536.7), CD8p (53.58), CD44 (Pgp-1), H-Y T C R a (T3.70), CD94 (18D3), NK1.1 (PK136), N K G 2 D (CX5), CD122 (TM-B1), CD127 (A7R34), CD132 (4G3), IFNy (XMG1.2), and T N F a (MP6-XT22). All mAbs were purchased from eBioscience (San Diego, CA) except anti-CD132 (BD PharMingen, San Diego, CA). For intracellular flow cytometry cells were stained for surface markers, washed, fixed with 2% paraformaldehyde and 0.2% Tween-20 in P B S for 20mins followed by one wash with P B S . The cells were then stained with mAbs specific for the intracellular cytokine in 0.2% Tween-20 in P B S . The CellQuest software program (Becton Dickinson, Mountain View, CA) was used for data acquisition and analysis.  183  CD8+ T cell purification Single cell suspensions from the lymph nodes (LN) of H-Y male mice were treated with biotinylated anti-CD8p mAb followed by positive selection using the MiniMACS system (Miltenyi Biotech, Auburn, CA), according to the manufacturer's specifications. The resulting cells were >95% pure C D 8 a p H-Y +  T C R a T cells. For purification of B6 C D 8 C D 4 4 +  +  hi  and CD8 CD44'° T cells, +  C D 8 p T cells from B6 lymph nodes were first enriched by MiniMACS. MiniMACS +  purified B6 C D 8 T cells were then stained with anti-CD8cc-FITC and anti-CD44+  P E and sorted on a Becton Dickinson F A C S Vantage S E Turbo sort cell sorter. Cell sorting was performed by Andrew Johnson (University of British Columbia) and the sorted C D 8 C D 4 4 +  hi  or C D 8 C D 4 4 cells were >98% pure (24). +  l0  NK cell purification Single cell suspensions from the spleens of B6 mice were treated with mAbs against CD4, C D 8 , and T C R p , followed by depletion of Ab-coated cells using Dynabeads M-450 Sheep anti-mouse IgG (Dynal Biotech, Lake Success, NY), according to manufacturers instructions. The resulting cells were ~50-60% CD3" N K 1 . 1 prior to culture and >95% C D 3 N K 1 . 1 * after culture in IL-15. +  184 Adoptive transfers, infections, and bacterial load measurement Purified H-Y C D 8 T cells or sorted B6 C D 8 T cells were injected into the lateral tail vein of IFNy" " or B6-Thy1.1 mice. For IL-2 or IL-15 cultured C D 8 T cells and 7  for IL-15 cultured NK cells, the cells were cultured in cytokine (100ng/ml) for 4 days prior to transfer. The number of cells adoptively transferred is stated in the figure legends. For infection experiments, mice were infected with ~10,000 C F U of wild type LM (strain 10403s) via the lateral tail vein one day after receiving adoptively transferred cells. On day 3 post-infection mice were sacrificed and their spleen and liver were homogenized in P B S . The resulting cell suspensions were mixed with an equal volume of 1% Triton X-100 in P B S and plated on brainheart infusion agar plates in serial 10-fold dilutions and incubated at 37°C overnight prior to counting. Fold reduction was used to assess relative protection in some experiments and was calculated as ( C F U control mouse/CFU experimental mouse).  5.4  Results  Self-specific CD8 T cells in H-Y TCR transgenic male mice can develop in the +  absence of a functional thymus There is some controversy regarding whether the thymus is essential for the development of self-specific C D 8 T cells in the H-Y model. In the first description  185 of the H-Y T C R C D 8 cells in H-Y male mice it was suggested that these cells +  +  were conventional C D 8 T cells that had escaped negative selection by +  decreasing the expression of the C D 8 coreceptor and thus lowering their avidity +  for self-peptide/MHC (27, 29). Consistent with the requirement for the thymus for their development was the observation that these cells failed to develop in athymic nude mice (29). However, this conclusion was challenged by a later report, which showed using adult thymectomy followed by bone marrow reconstitution that self-specific H-Y C D 8 T cells can develop in the absence of a +  functional thymus (30). In view of our findings that these self-specific C D 8 T +  cells are functionally distinct from conventional C D 8 T cells, we decided to re+  evaluate whether self-specific C D 8 T cells can in fact develop in athymic nude (foxn1 ) mice. We first compared the C D 4 and C D 8 populations in the lymph nu  nodes of euthymic H-Y male, athymic H-Y male, euthymic H-Y female, and athymic H-Y female mice. The top panel in Fig. 5.1 A shows that euthymic H-Y female mice have the largest population of both C D 4 and C D 8 cells making up 19.0% and 30.7% of the lymph nodes, respectively. Consistent with previous reports (27, 29) lymph nodes from euthymic H-Y male mice contain very few peripheral C D 4 T cells (3.1%) but possess larger numbers of CD8'° cells (16.7%). By contrast, the lymph nodes of athymic H-Y male mice contain fewer numbers of these CD8'° cells (3.7%) and possess virtually no C D 4 T cells (0.6%). Consistent with previous reports (27, 29) virtually all of the CD8'° cells (96.7%) in euthymic H-Y male mice express exclusively the H-Y T C R , as indicated by the same level of staining with either a mAb specific for the T C R (3 chain or the H-Y  186 T C R (Fig. 5.1 A, lower panel). Also consistent with previous reports these CD8'° T cells express exclusively the CD8a(3 heterodimer (29 and data not shown). By contrast, only 34.9% of the C D 8 T cells in euthymic H-Y female mice express +  the H-Y T C R ; this is consistent with previous reports that the majority of peripheral C D 8 T cells from euthymic female H-Y T C R mice utilize endogenous +  T C R a chains for their positive selection (31). The majority (62.6%) of CD8'° cells in athymic male H-Y mice also express the H-Y T C R . It is noted that about onethird of the CD8'° cells from athymic H-Y male mice did not express the T C R (3 chain suggesting that these cells are unlikely to be of the T C R a p lineage. In athymic H-Y female mice there is very poor development of C D 8 cells (1.0%) |0  consistent with our previous observation that the presence of self-Ag is required for their development (26). In terms of absolute cell numbers, there is a 26-fold increase in the frequency of CD8'° T C R p H-Y TCRct cells in athymic nude H-Y +  +  male mice compared to athymic nude H-Y female mice. There is also a 7-fold reduction in the recovery of CD8'° H-Y T C R cells in athymic nude H-Y male mice +  relative to euthymic H-Y male mice. This suggests that additional cells of this lineage may develop in the thymus or that some thymus-dependent cell(s) may provide selection or survival cues for these cells. W e also noted that the H-Y T C R CD8'° T cells that developed extrathymically in athymic H-Y male mice +  expressed high levels of the memory markers CD44 and CD122 as well as the NK receptors N K G 2 D and CD94 (Fig. 5.1B). These observations support the following conclusions: (1) self-specific CD8'° H-Y T C R T cells can develop in the +  absence of the thymus, (2) thymus-independent development of these cells is  187 relatively inefficient as indicated by a seven-fold reduction in absolute numbers relative to euthymic mice, (3) the development of self-specific C D 8 T cells in athymic mice is also dependent on their interaction with self-Ags, and (4) the selfspecific C D 8 T cells that develop in athymic mice express high levels of memory markers (CD44 and CD122) as well as NK receptors (NKG2D and CD94).  Activated self-specific CD8 T cells produce pro-inflammatory  cytokines  Va14/' NKT cells are an innate T cell type which has been shown to produce both Th1 (IFNy) and Th2 (IL-4) cytokines upon activation (6). Since self-specific C D 8 T cells can produce cytokines immediately ex vivo we determine if these cells were similar to NKT cells in their cytokine production profile. We compared the cytokine production of self-specific C D 8 T cells from H-Y male mice to conventional Ag-specific C D 8 T cells from H-Y female mice. CD8'° cells from euthymic H-Y male mice were used as a source of self-specific C D 8 T cells since they provide a more convenient and larger source of these cells. We also included memory-phenotype (containing self-specific C D 8 T cells of unknown specificity) and naive C D 8 T cells from normal B6 mice in these analyses. Splenocytes from H-Y male, H-Y female and B6 mice were stimulated with P M A and ionomycin for 4 hours. We then measured the production of IFNy, T N F a , IL2, and IL-4 by intracellular flow cytometry after gating on the cell type of interest. It is clear that self-specific H-Y male C D 8 T cells were only capable of producing the pro-inflammatory Th1 cytokines IFNy, T N F a , and IL-2 but not IL-4. The naive H-Y female C D 8 T cells were only capable of producing T N F a and a small  188 amount of IL-2. These results were mirrored by those obtained from the nontransgenic C D 8 T cells from normal B6 mice where the memory-phenotype CD8 CD44 +  hi  T cells were capable of producing IFNy, TNFoc, and IL-2 during a  short stimulation and the naive C D 8 C D 4 4 T cells could only produce T N F a and +  |0  IL-2. In addition, IFNy-production by self-specific H-Y and memory-phenotype B6 C D 8 C D 4 4 T cells was maintained even at 24 hours post-stimulation whereas +  hl  the naive H-Y female and naive B6 C D 8 T cells still could not produce IFNy (data not shown). IL-4 production by all the cell types was never detected even at 24 hours post stimulation. These results further emphasize the functional similarity of self-specific C D 8 T cells from H-Y male mice and the C D 8 C D 4 4 |0  +  hi  cells from  B6 mice.  IFNy production by self-specific CD8 T cell provides protection against  bacterial  infection Memory-phenotype C D 8 T cells have been shown to produce large amounts of IFNy early during infection with Listeria and these cells have been shown to be protective during infection (16-20). To test the ability of self-specific C D 8 T cells to produce IFNy in vivo and to address whether this cytokine production has biological significance we measured the ability of this cell type to protect IFNy  /_  mice against LM infection. Two previous studies have used this approach to demonstrate that C D 8 T cells and NK cells capable of producing IFNy during infection can protect IFNy mice during LM infection due to their ability to provide /_  189 a source of IFNy (17, 18). W e transferred purified self-specific C D 8 T cells into male IFNy"'" mice, which were infected with 10 C F U of Listeria 1 day post 4  transfer. At day 3 post-infection we sacrificed the animals and assessed cytokine production by the transferred cells in the absence of any restimulation. Figure 5.3A depicts IFNy production by gated self-specific C D 8 T cells recovered from the spleens of infected or uninfected IFNy"'" mice. It is clear that a significant proportion (~15%) of the transferred cells was actively producing IFNy and that this cytokine production was a consequence of infection. We also determined if IFNy production by self-specific C D 8 T cells was associated with a decrease in +  bacterial burden in the spleens of infected IFNy"'" mice. Figure 5.3B clearly shows that IFNy"'" mice that had received self-specific C D 8 T cells prior to infection had at least a 10-fold reduction in bacterial load in the spleen relative to mice which did not. Therefore the ability of self-specific C D 8 T cells to protect against Listeria infection is directly associated with their ability to produce IFNy.  Memory-phenotype  CD8 CD44 +  hi  cells and self-specific H-Y male CD8 T cells  provide similar innate protection during infection After determining the ability of self-specific C D 8 T cells from H-Y male mice to protect IFNy"'" mice from infection with L M we decided to test the relative efficacy of self-specific C D 8 T cells from H-Y male mice, memory-phenotype C D 8 C D 4 4 +  hl  T cells from B6 mice and naive CD8 CD44'° T cells from B6 mice to confer +  protection against LM infection in IFNy"'" mice. It is noted that memory-phenotype  190 C D 8 T cells from B6 mice, like self-specific C D 8 T cells from H-Y male mice, have the ability to produce IFNy rapidly upon activation whereas naive C D 8 T cells do not (Fig. 5.2). We sorted memory and naive-phenotype C D 8 T cells from B6 mice and we purified self-specific C D 8 T cells from H-Y male mice. We then transferred 9x10 cells into male IFNy" mice and infected the mice the following 5  /_  day with L M . The results in Fig. 5.4 demonstrate that both self-specific H-Y male C D 8 T cells and memory-phenotype B6 C D 8 T cells, but not naive B6 C D 8 T cells, provide a small but significant degree of protection against L M infection in IFNy  _/_  mice. This protection was observed in both the spleen and liver of IFNy'"  that had received relatively few transferred cells (9x10 ), with both the H-Y male 5  and B6 C D 8 C D 4 4 +  hi  cells reducing the bacterial load by more than 3-fold. This  result indicates that self-specific C D 8 T cells from H-Y male mice and CD8 CD44 +  hl  cells from B6 mice are functionally similar in terms of their ability to  confer protection against LM infection in IFNy mice. /_  IL-15-activated cells provide protection against LM infection Previous reports have shown that self-specific C D 8 T cells can proliferate and become activated in vitro in response to IL-2 or IL-15 (32). The expression of IL2Rp (CD122) in conjunction with yc (CD132) is sufficient to confer lymphocytes with the ability to respond to both IL-2 and IL-15 (33, 34). Activation of NK cells or memory-phenotype C D 8 T cells with either of these cytokines is able to enhance both the cytotoxicity as well as cytokine production by these cells (35-37). Since immediately ex vivo self-specific C D 8 T cells provide protection against LM  191 infection in IFNy" " mice we wanted to see if activation of the cells with IL-2 or IL7  15 could enhance this function. We cultured purified and CFSE-labeled selfspecific C D 8 T cells with either 100 ng/ml of IL-2 or IL-15 for 4 days. Both IL-2 and IL-15 induced the same amount of proliferation of these cells (Fig. 5.5A). W e also determined the potential of cells cultured in either IL-2 or IL-15 to produce IFNy, T N F a and the cytolytic marker, granzyme B. We found that P M A and ionomycin activated IL-2 and IL-15 cultured cells produced equivalent amounts of IFNy. However, activated IL-2-cultured cells produced a greater amount of granzyme B than did IL-15-cultured cells. By contrast, IL-15-cultured cells produced more T N F a than IL-2 cultured cells. We next determined the ability of IL-2 or IL-15-cultured cells to protect IFNy" ' mice from LM infection. IL-2 or IL-15 7  cultured cells were transferred into IFNy" " mice and the mice were infected 1 day 7  later with L M . At day 3 post-infection we assessed the total numbers of transferred cells recovered as well as the bacterial load in the spleens of infected mice. Figure 5.5B clearly shows that self-specific C D 8 T cells cultured in IL-2 were much less efficient at homing to and/or surviving in the spleens of infected animals as we recovered only about 10 IL-2 cultured cells 3 days post-infection. 5  In addition the few IL-2 cultured cells that were present were completely ineffective in protecting IFNy" " mice from L M infection, causing no reduction in 7  bacterial load (Fig. 5.5C). By contrast, IL-15 cultured cells did either home to and/or survive better in the spleen as we recovered about 7x10 cells and the 5  cells were extremely protective against infection, reducing the bacterial load by greater than 100-fold compared to control IFNy" " mice. The protection by IL-15 7  192 cultured cells was greater than that achieved with immediately ex vivo cells (Fig. 5.3B) even though protection was observed in both cases. Therefore consistent with results using IL-15 transgenic mice (38) culturing self-specific C D 8 T cells with IL-15 provides a novel method for the expansion of cells with increase effectiveness in providing protection against bacterial infection.  Self-Ag interactions are important for innate protection by self-specific CD8 T cells We next determined whether the effectiveness of self-specific C D 8 T cells in controlling LM infection is dependent on interaction with self-Ag. IL-15 cultured cells were used since they were more protective than immediately ex vivo cells. We transferred 1 x 10 IL-15 cultured self-specific C D 8 T cells into either male 6  (self-Ag ) or female (self-Ag ) IFNy"'" recipients and then infected the mice with +  -  10 C F U of LM the following day. We then compared the relative reduction in 4  bacterial load in mice that had either received or not received cells. This was done to eliminate any differences in bacterial load due to the sex of the recipient mice. Figure 5.6 shows that self-specific C D 8 T cells were protective in both male and female recipients. However, protection offered by the transferred cells in female recipients was modest, resulting in only a 2-fold reduction in bacterial load in the spleen and liver of female IFNy"'" mice receiving self-specific C D 8 T cells relative to female IFN-y"'" mice that did not. By contrast, the transferred cells offered much greater protection in male IFNy"'" recipients, resulting in about a 10fold reduction in bacteria in the spleen and about a 100-fold reduction in bacteria  193 in the liver relative to male IFNy"'" mice that did not receive any cells. Protection conferred by self-specific C D 8 cells from H-Y male mice against LM infection in female IFNy" mice was increased by transferring more cells (data not shown). /_  This result indicates that these cells can confer protection against LM infection in the absence of self-Ag. However, their protective function is greatly increased in the presence of self-Ag.  Self-Ag interactions are crucial for maintaining the memory phenotype and cytokine-responsiveness  of self-specific CD8 T cells  Previous studies had shown that the expansion of self-specific C D 8 T cells in response to either IL-2 or IL-15 in vitro was independent of self-Ag interactions (32). Our studies have suggested that self-Ag interactions played a role in the proliferation of self-specific C D 8 T cells in vivo during LM infection (26). Since we had also shown that self-Ag interactions dictate the extent of the memoryphenotype in these self-specific C D 8 T cells (26) we decided to test the idea that self-Ag interactions control the expansion of these cells in vivo in part by helping to maintain a memory phenotype and high levels of the associated cytokine receptors. To address this possibility we transferred self-specific C D 8 T cells into congenic Thy-1.1 male or female recipient mice and determined their cell surface phenotype in the spleens of recipient mice 7 days post transfer. There was no difference in the frequency of self-specific C D 8 T cells in the spleen or livers of male or female recipients (Fig. 5.7A) suggesting that survival and homing are unaffected by self-Ag 7 days post transfer. However, we noticed striking  194 differences in the expression of the T C R and CD44 as well as the cytokine receptors CD122 and CD127. Self-Ag interactions in male recipients led to lower T C R expression and higher expression of CD44 (Fig. 5.7B). CD122 and CD127 were also maintained at higher levels in male recipients over female recipients suggesting that self-Ag interaction is crucial for the high expression of these receptors in self-specific C D 8 T cells. The lower expression of CD122 on selfspecific C D 8 T cells that are maintained in female recipients likely leads to their decreased ability to respond to IL-15. To test this possibility more directly we purified self-specific C D 8 T cells which had been rested in either male or female recipients for 7 days, CFSE-labeled these cells and tested their ability to proliferate in response to IL-15 in vitro. Figure 5.7C clearly demonstrates that self-specific C D 8 T cells maintained in Ag" female recipients are less responsive to IL-15 as only 2 1 % of the cells have undergone greater than 4 division events compared to about 5 1 % of the cells maintained in male mice. These observations provide an explanation for the decreased proliferation of these cells in vivo in response to LM infection in female recipient mice.  Self-specific CD8 T cells provide greater IFNy-dependent protection against LM infection than NK cells Several recent reports have focused on the ability of memory-phenotype C D 8 T cells to provide innate protection during infection. One report compared the ability of N K cells and foreign-Ag specific memory C D 8 T cells to protect IFNy"'" mice against infection with LM (18). This study found that memory C D 8 T cells are  195 more protective than NK cells, in part due to their ability to localize to areas of the spleen bearing LM-infected macrophages. W e compared the relative efficacy of self-specific C D 8 T cells and NK cells in providing protection against LM infection by first comparing the ability of self-specific C D 8 T cells and NK cells to produce IFNy, T N F a and granzyme B. This was done by culturing both cell types in IL-15 for 4 days and re-stimulating the cells with P M A and ionomycin for 4 hours. W e found that self-specific C D 8 T cells were much more efficient producers of both IFNy and T N F a than NK cells; however, NK cells were much more efficient in producing granzyme B (Fig. 5.8A). These differences seen after IL-15 activation were also true of these cell types immediately ex vivo (data not shown). To compare the ability of self-specific C D 8 T cells and NK cells in conferring protection against LM infection in IFNy"'" mice equal numbers of day 4 IL-15 cultured self-specific C D 8 T cells and NK cells were transferred into male IFNy" " 7  recipients. We then infected the mice with LM the next day and measured the reduction in bacterial load in the spleens and livers of mice receiving self-specific C D 8 cells or NK cells. The results in Fig. 5.8B indicate that NK cells offered a small degree of protection against LM infection, reducing the bacterial load in the spleen and liver by ~two- and three-fold, respectively. By contrast, self-specific C D 8 T cells were much more effective in reducing bacterial load in IFNy"'" mice, reducing the bacterial load in spleen and liver by ~30- and 700-fold, respectively. These results indicate that self-specific C D 8 T cells are more effective than N K cells in providing protection against LM infection in IFNy"'" mice.  196  5.5  Discussion  In this report we have clearly demonstrated an important role for self-specific C D 8 T cells in early protection of mice from LM infection. We have shown that these self-specific cells are potent producers of IFNy and T N F a immediately ex vivo. We also showed that self-specific C D 8 T cells offered significant protection against L M infection in IFNy"'" mice, particularly in Ag-expressing mice. This protection was greatly enhanced by culturing self-specific C D 8 T cells in IL-15 prior to transfer. We also demonstrate an important role for self-Ag interactions in maintaining the memory phenotype of these cells and their responsiveness to IL15. Finally, we found that self-specific C D 8 T cells were much more effective than NK cells in protecting IFNy"'" mice against LM infection. We have also confirmed in this study that self-specific C D 8 T cells from H-Y male mice can develop in the absence of a functional thymus. W e did however notice a significant reduction in the frequency of H-Y C D 8 T cells in athymic male mice compared to euthymic mice (Fig. 5.1 A). This suggests that thymusdependent mechanisms or thymus-dependent T cells can improve the survival, selection, or expansion of this cell type. The fact that these self-specific C D 8 T cells can be selected by cognate self-Ag outside the thymus indicates that these cells constitute a lineage that is distinct from conventional C D 8 T cells (29, 30). The memory markers CD44 and CD122 as well as the NK receptors CD94 and N K G 2 D are significantly higher on self-specific C D 8 T cells from athymic nude  197 mice relative to their euthymic counterparts. We have seen the expression of CD94 and N K G 2 D increase on self-specific C D 8 T cells upon activation by A g or IL-15 and thus the increased expression of these receptors on athymic C D 8 T cells likely reflects their activation history (26). This may be due in part to increased availability of IL-15 due to the smaller numbers (one-seventh) of selfspecific C D 8 T cells in the athymic nude mice. Consistent with this hypothesis IL15 has been shown to play a role in the induction of both N K G 2 D and CD94 in C D 8 T cells (39, 40). The significance of the high level of C D 9 4 expression in athymic C D 8 T cells is unknown but it may play a role in their survival as one study showed that CD94 expression is associated with protection from activation induced cell death and correlates with increased survival in C D 8 T cells (41). Whether athymic C D 8 T cells preferentially express CD94 to enhance their survival remains to be determined. Although this current study focused mainly on the role of a homogenous population of self-specific C D 8 T cells from male H-Y T C R transgenic mice we have previously shown that memory-phenotype C D 8 T cells from normal B6 mice share many similarities to this cell type (24). In this study, we have provided further confirmation that memory-phenotype C D 8 T cells are very similar to selfspecific H-Y C D 8 T cells in their cytokine production profiles as well as in their ability to provide innate protection to IFNy~ mice against LM infection. The main /_  problem with non-TCR transgenic mice for these studies is the heterogeneity of the memory-phenotype population, which makes it impossible to dissect the relative contributions of conventional memory C D 8 T cells from self-specific C D 8  198 T cells. The H-Y model is not unique when it comes to the development of memory-phenotype C D 8 T cells specific for a self-Ag. For instance, in doubly transgenic mice, which express a transgenic T C R specific for the gag protein from Friend murine leukemia virus (FMuLVgag) as well as the transgenic cognate Ag, FMuLVgag, C D 8 T cells expressing the transgenic T C R still develop. Furthermore, these cells exhibit a memory-phenotype similar to the self-specific C D 8 T cells from H-Y male mice; these mice also did not develop detectable autoimmune diseases even though the self-specific C D 8 T cells retain effector function and are specific for the self-Ag (43). A recent report on these cells also demonstrated their ability to proliferate in response to IL-2 or IL-15 alone (42). Another T C R transgenic model that shares similarities to ours is the P14 T C R transgenic in which C D 8 T cells are specific for a peptide derived from the lymphocytic choriomeningitis virus gp (LCMV-gp). When P14 T C R transgenic mice are crossed to mice ubiquitously expressing cognate antigen the developing P14 C D 8 T cells share a nearly identical cell surface phenotype with the selfspecific C D 8 T cells from H-Y male mice (Pamela S. Ohashi, personal communication). Whether the C D 8 T cells expressing these self-specific T C R ' s behave the same way as those from H-Y male mice remains to be determined although based on their phenotype it is reasonable to believe that they will also share similarities in function. Together, these observations emphasize the importance of TCR/cognate self-Ag interactions for the development of selfspecific C D 8 T cells.  199 Self-reactivity is also characteristic of some other cell types of the adaptive immune cells. For instance NK-T cells exhibit a high degree.of self-reactivity and function through the recognition of self-lipid in the context of CD1d (44). These cells also have an unusual requirement for positive selection in the thymus in that they are selected by bone marrow derived double positive thymocytes rather than cortical epithelial cells which select conventional T cells (5). CD8aoc iELs are also selected by agonist self-peptides (3). Studies using H-Y T C R transgenic mice demonstrate strong interaction with self-Ag is required for the development of C D 8 a a iELs whereas weaker selection leads to the development of C D 8 a p T cells (45). Interestingly, the strength of selection also correlates with the expression of genes associated with innate immune cells. Although the selfspecific CD8 T cells are unrelated to CD8aot ilELs, it is conceivable that their unconventional functions may also be a consequence of their positive selection by agonist self-Ags. One phenotypic similarity between self-specific CD8 T cells and other innate T cells is their memory phenotype. This phenotype is associated with expression of high levels of CD122, which confers IL-2 and IL-15 responsiveness. IL-15 responsiveness seems to be crucial for the maintenance of NK, NK-T, and memory phenotype CD8 T cells (which include self-specific CD8 T cells) as all of these cell types are either drastically reduced in number or virtually absent in mice lacking either IL-15 or its receptor (46, 47). A n interesting finding of the present study is that self-specific CD8 T cells require self-Ag for the maintenance of high levels of CD122, which then makes them more responsive to cytokine  200 stimulation. Two T-box family transcription factors T-bet and eomesodermin have recently been shown to be crucial for the maintenance of IL-15-dependent cell types including NK, NK-T and memory phenotype C D 8 T cells (48). Notably, these transcription factors act directly on the CD122 promoter, inducing transcription and expression of CD122. It remains to be determined whether Tbet or eomesodermin expression by self-specific C D 8 T cells is induced or maintained through self-Ag interactions. We have compared the ability of IL-2 or IL-15 cultured self-specific C D 8 T +  cells to protect INFy" mice from LM infection. The ineffectiveness of IL-2 cultured /_  cells in conferring protection could be due to their inability to home to and/or survive in the spleen since IL-2 is limiting in vivo. IL-15 cultured cells, which are smaller and look more like naive cells compared to IL-2 cultured cells, either home more efficiently or survive better in the spleen of recipient mice. Furthermore, these cells offered superior protective function against LM infection, presumably as a consequence of their ability to respond to IL-15 produced in vivo upon infection. The ability of self-specific C D 8 T cells to respond to IL-15 in vivo allows the cells to rapidly become activated during infection or inflammation and results in the production of cytokines such as IFNy and T N F a . W e have demonstrated in this report that these self-specific C D 8 T cells do produce IFNy early during infection and provide protection against LM infection whether or not the cells were pretreated with IL-15. The mechanism for the induction of IFNy by these cells likely involves responsiveness to IL-12 produced by infected macrophages. IL-12 in conjunction with IL-2, IL-15, or IL-18 has been shown to  201 be sufficient to induce large amounts of IFNy production from self-specific C D 8 T cells (32). There is also the possibility that N K G 2 D may play a role in inducing IFNy production by self-specific C D 8 T cells. We have shown previously that these cells do in fact increase expression of N K G 2 D upon infection (26) and reports suggest that infected macrophages express the ligands for N K G 2 D (49). It is quite possible that N K G 2 D stimulation may play an important role in inducing IFNy by these cells. The requirement for interaction of self-specific C D 8 T cells with their cognate Ag in conferring better protection against LM infection indicates that T C R interactions play a role in the function of these cells. We have shown in this study that continuous interaction with self-Ag in vivo is required for maintaining the high responsiveness of these cells to IL-15. Thus, the increased protective function of self-specific C D 8 T cells in cognate A g expressing mice is likely due to the requirement for self-Ag in maintaining high expression of cytokine receptors such as CD122, which allows them to sense bacterial infection more effectively by responding to physiological levels of IL-15. The self-specific C D 8 cells are still protective in the absence of self-Ag (Fig. 5.6) and still retain function after being in an Ag" female recipient for 7 days. It is clear however, that self-specific C D 8 T cells are less efficient at responding to IL-15 in Ag" female recipients but this inefficiency can be partially overcome by increasing the numbers of these cells. NK cells are a source of early IFNy and T N F a that are involved in protection from pathogens as well as tumor cells (50). Our study clearly demonstrates that self-specific C D 8 T cells are much more potent producers of both IFNy and T N F a  202 when compared to NK cells. Consequently self-specific C D 8 T cells provide more protection than NK cells to IFNy"'" mice during infection with L M . Another difference between self-specific C D 8 T cells and NK cells is their localization. NK cells are relatively abundant in the spleen yet are virtually absent in the lymph nodes of mice (51). Self-specific C D 8 T cells on the other hand are present in larger numbers in both the spleen and lymph nodes and are more efficient than NK cells in homing to either location (our unpublished observations). NK cells have been shown to be involved in many steps of T cell priming either by acting on dendritic cells (52) or by producing IFNy that can participate in C D 4 T cell priming (51). Since T cell priming occurs primarily in secondary lymphoid organs where self-specific C D 8 T cells are abundant it remains possible that these cells may also be involved in this aspect of immunity. Notably, memory phenotype C D 8 T cells, which serve as a source of early IFNy, were shown to polarize C D 4 T cells to the Th1 lineage (38, 53). It remains to be determined whether priming of C D 4 T cells towards the Th1 lineage is another function of self-specific C D 8 T cells.  5.6  Acknowledgements  We thank Soo-Jeet Teh for excellent technical assistance and John Priatel for helpful discussion.  203  5.7  Figures  Figure 5.1 Limited development of self-specific C D 8 T cells in athymic (nude) HY T C R transgenic male mice. A) Lymph node cells from age-matched male and female H-Y T C R transgenic mice on either a wildtype B6 or athymic nude B6 background were stained with mAbs against CD4, C D 8 , T C R p and the H-Y T C R a . The T C R p vs. H-Y T C R a plots in the lower panel are for gated C D 8 C D 4 " +  cells from the upper panel. The cell recovery was similar for all of the mice and the data shown is representative of at least 4 mice per group. B) Expression of memory markers and NK receptors on gated self-specific male H-Y T C R  +  CD8  +  cells from euthymic (black histogram) or athymic H-Y male mice (grey line).  Figure 5.2 Self-specific C D 8 T cells rapidly produce inflammatory cytokines upon activation. Splenocytes from euthymic H-Y male, H-Y female, or B6 mice were stimulated with P M A (10 ng/ml) and ionomycin (500 ng/ml) for 4 hours in the presence of a Golgi inhibitor. The histograms depict intracellular cytokine staining on gated C D 8 H-Y T C R a cells from H-Y male and female mice and from +  +  C D 8 C D 4 4 a n d C D 8 C D 4 4 T cells from B6 mice. Cytokine production by +  hi  +  |0  unstimulated cells (black line) was compared to P M A and ionomycin stimulated cells (grey line) with the numbers in the plots representing the percentage of cytokine-positive cells after subtracting the background cytokine production.  204 Figure 5.3 Self-specific C D 8 T cells provide an innate source of IFN-y during infection. 3x10 purified self-specific C D 8 T cells were transferred into B6 male 6  IFNy" " recipients, which were infected the following day with wildtype L M . A) 7  Intracellular IFNy staining in the absence of restimulation on gated donor cells ( C D 8 H-Y TCRa ) isolated 3 days post infection from IFNy" " recipients infected +  7  +  with LM or left uninfected. The cells were cultured for 5 hours in vitro in the presence of a golgi inhibitor prior to staining. B) Bacterial load in the spleens of IFNy" " mice on day 3 post infection that had received 3x10 self-specific C D 8 7  6  cells (black) or that did not receive any cells (white). Error bars represent the standard deviation from 3-4 mice per group.  Figure 5.4 Self-specific H-Y male C D 8 T cells and memory-phenotype C D 8 T cells from normal B6 mice provide similar protection against LM infection. 9x10  5  sorted B6 C D 8 C D 4 4 , B6 CD8 CD44'°, and male H-Y TCRa C D 8 cells were +  hi  +  +  +  transferred into male IFNy" " mice one day prior to infection with wildtype LM (10 7  4  CFU). Bacterial load in the spleen and liver of infected mice that received either no cells (black bar), B6 CD8 CD44'° (white bar), B6 C D 8 C D 4 4 +  +  hi  (dark grey bar)  or male H-Y TCRa C D 8 (light grey) cells is shown. The error bars represent the +  +  standard deviation from 3 mice per group.  Figure 5.5 Self-specific C D 8 T cells expanded in IL-15 are protective in vivo whereas IL-2 expanded cells are not. (A) Purified self-specific C D 8 T cells were CFSE-labeled and cultured in either IL-2 (100 ng/ml) or IL-15 (100 ng/ml) for 4  205 days. The C F S E profiles of the cultured cells on day 4 are as indicated. The CFSE-labeled cells cultured in IL-2 (filled histogram) or IL-15 (black line) were stimulated with P M A (10 ng/ml) and ionomycin (500 ng/ml) for 4 hours in the presence of a Golgi inhibitor. The cells were then stained intracellular^ for IFNy, T N F a , and granzyme. (B) 3x10 day 4 IL-2 or IL-15 cultured cells were 6  transferred into male IFNy" " mice that were infected the next day with L M . 7  Absolute numbers of transferred cells previously expanded in IL-2 (white) or IL15 (black) and recovered from the spleen of recipient mice at day 3 post infection are indicated. C) Bacterial load on day 3 post infection in spleen of IFNy" " mice 7  that had received IL-2 (white) or IL-15 (black) cultured cells 1 day prior to infection compared to IFNy" " mice that did not receive any cells (gray). Error bars 7  represent the standard deviation from 3-4 mice per group.  Figure 5.6 Self-Ag interactions enhance the protection mediated by self-specific C D 8 T cells during infection. Purified self-specific C D 8 T cells were cultured with IL-15 (100 ng/ml) for 4 days and then 10 cells were transferred into either male 6  or female IFNy" " mice which were infected the next day with 10 C F U wild type 7  4  LM. The data represent the relative reduction in bacterial load on day 3 postinfection in the spleens and livers of male (black) and female (white) IFNy" " mice 7  that had received self-specific C D 8 cells relative to male and female IFN-y" " mice 7  not receiving any cells. Error bars represent the standard deviation from 3-4 mice per group.  206 Figure 5.7 Self-Ag interactions maintain the memory phenotype and cytokine responsiveness of self-specific C D 8 T cells in vivo. 3x10 purified self-specific 6  C D 8 T cells (Thy-1.2 ) were transferred into male and female B6-Thy-1.1 +  congenic mice. A) 7 days post transfer the frequency of donor cells (Thy-1.2 ) +  was quantified in the spleen and liver of recipient mice. B) Surface marker expression on gated transferred C D 8 cells (Thy-1.2 ) 7 days post-transfer into +  male (black line) or female (filled histogram) mice. The numbers in the plots represent the MFI of cells from male (M) or female (F) mice. C) 7 days post transfer into male or female mice self-specific C D 8 T cells were enriched for, labeled with C F S E , and cultured in IL-15 (100 ng/ml) in vitro for 3 days. The histograms represent the cell division of donor self-specific C D 8 T cells from male (black line) or female (filled histogram) recipients in response to IL-15.  Figure 5.8 Self-specific C D 8 T cells provide more IFN-y-dependent protection in vivo against LM infection than NK cells. Purified self-specific C D 8 T cells and NK cells were cultured in IL-15 (100 ng/ml) for 4 days. A) Intracellular staining for IFNy, T N F a and granzyme B of self-specific C D 8 (black line) and NK (filled histogram) cells stimulated with P M A (10 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Numbers in the plots are the MFI. B) Bacterial load reduction day 3 post infection in the spleen and liver of IFNy" " mice receiving 1x10 activated self7  6  specific C D 8 (black) or NK (white) cells one day prior to infection relative to control IFNy" " mice not receiving any cells. Error bars represent the standard 7  deviation of data from 3-4 mice per group.  207  Figure 5.1 Limited development of self-specific C D 8 T cells in athymic (nude) HY T C R transgenic male mice  208  activation  209 A)  Infected  Uninfected 10  0.7  10  15.9  10' ••• V  10  z  LL  io  :  10' 10  :  10  1  £  io id iff id 0  to*  10  (  10  0  id  trj  10  3  io*  H-Y TCR  B)  10  8  10  7  •  No C e l l s  •  H-YCD8  Q.  10  6  10  5  10  4  Figure 5.3 Self-specific C D 8 T cells provide an innate source of IFN-y during infection  210  10  7  Figure 5.4 Self-specific H-Y male C D 8 T cells and memory-phenotype C D 8 T cells from normal B6 mice provide similar protection against LM infection  Figure 5.5 Self-specific CD8 T cells expanded in IL-15 are protective in vivo whereas IL-2 expanded cells are not  Figure 5.6 Self-Ag interactions enhance the protection mediated by self-specific C D 8 T cells during infection  213  A)  Female  Male Spleen  CM  Liver  H-Y TCR B)  M:215  F:79  LJJ  E 3  itf  H-Y TCR  O  O  u  M:110  CD44  id iff  id  CD122  F:54  >  s <D  tx  id  id  icf  id  irf  CD127  C)  4S 40  > E  3S 30 25 20 15 10  CD132  Male - 50.6 Female-21.0  I  10*  CFSE Figure 5.7 Self-Ag interactions maintain the memory phenotype and cytokine responsiveness of self-specific CD8 T cells in vivo  214  A)  Spleen  Figure 5.8  Liver  Self-specific C D 8 T cells provide more IFN-y-dependent protection in  vivo against LM infection than NK cells  215  5.8  References  1.  Shaw, P. X., S. Horkko, M. K. Chang, L. K. Curtiss, W. Palinski, G . J . Silverman, and J . L. Witztum. 2000. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest 105:1731-1740.  2.  Groh, V., A. Steinle, S. Bauer, and T. Spies. 1998. Recognition of stressinduced M H C molecules by intestinal epithelial gammadelta T cells. Science 279:1737-1740.  3.  Cruz, D., B. C. Sydora, K. Hetzel, G . Yakoub, M. Kronenberg, and H. Cheroutre. 1998. An opposite pattern of selection of a single T cell antigen receptor in the thymus and among intraepithelial lymphocytes. J Exp Med 188:255-265.  4.  Poussier, P., T. Ning, D. Banerjee, and M. Julius. 2002. A unique subset of self-specific intraintestinal T cells maintains gut integrity. J Exp Med 195:1491-1497.  5.  Bendelac, A. 1995. Positive selection of mouse NK1+ T cells by C D 1 expressing cortical thymocytes. J Exp Med 182:2091-2096.  6.  Bendelac, A., M. Bonneville, and J . F. Kearney. 2001. Autoreactivity by design: innate B and T lymphocytes. Nat Rev Immunol 1:177-186.  7.  Farrar, M. A., and R. D. Schreiber. 1993. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 11:571-611.  8.  Paulnock, D. M. 1992. Macrophage activation by T cells.  CurrOpin  Immunol 4:344-349. 9.  Steimle, V., C. A. Siegrist, A. Mottet, B. Lisowska-Grospierre, and B. Mach. 1994. Regulation of M H C class II expression by interferon-gamma mediated by the transactivator gene CIITA. Science 265:106-109.  10.  Fernandez-Botran, R., V. M. Sanders, T. R. Mosmann, and E. S. Vitetta. 1988. Lymphokine-mediated regulation of the proliferative response of clones of T helper 1 and T helper 2 cells. J Exp Med 168:543-558.  11.  Whitmire, J . K., N. Benning, and J . L. Whitton. 2005. Cutting Edge: Early IFN-{gamma} Signaling Directly Enhances Primary Antiviral CD4+ T Cell Responses. J Immunol 175:5624-5628.  12.  Whitmire, J . K., J . T. Tan, and J . L. Whitton. 2005. lnterferon-{gamma} acts directly on CD8+ T cells to increase their abundance during virus infection. J. Exp. M e d 201:1053-1059.  13.  Badovinac, V. P., A. R. Tvinnereim, and J . T. Harty. 2000. Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferongamma. Science 290:1354-1358.  14.  Gellin, B. G., and C. V. Broome. 1989. Listeriosis. Jama 261:1313-1320.  15.  Tripp, C. S., S. F. Wolf, and E. R. Unanue. 1993. Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc Natl Acad SciUSA  90:3725-3729.  16.  Berg, R. E., C. J . Cordes, and J . Forman. 2002. Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL18. EurJ Immunol 32:2807-2816.  17.  Berg, R. E., E. Crossley, S. Murray, and J . Forman. 2003. Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. J Exp Med 198:1583-1593.  18.  Berg, R. E., E. Crossley, S. Murray, and J . Forman. 2005. Relative contributions of NK and C D 8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes. J Immunol 175:17511757.  19.  Bregenholt, S., P. Berche, F. Brombacher, and J . P. Di Santo. 2001. Conventional alpha beta T cells are sufficient for innate and adaptive immunity against enteric Listeria monocytogenes. J Immunol 166:18711876.  20.  Lertmemongkolchai, G., G . Cai, C. A. Hunter, and G . J . Bancroft. 2001. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. J Immunol 166:10971105.  21.  Kurepa, Z., J . S u , and J . Forman. 2003. Memory phenotype of CD8+ T cells in M H C class la-deficient mice. J Immunol 170:5414-5420.  22.  S u , J . , R. E. Berg, S. Murray, and J . Forman. 2005. Thymus-dependent memory phenotype C D 8 T cells in naive B6.H-2Kb-/-Db-/- animals  218 mediate an antigen-specific response against Listeria monocytogenes. J Immunol 175:6450-6457. 23.  Urdahl, K. B., J . C. Sun, and M. J . Bevan. 2002. Positive selection of M H C class lb-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol 3:772-779.  24.  Dhanji, S., and H.-S. Teh. 2003. IL-2-Activated CD8+CD44high Cells Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells. J Immunol 171:34423450.  25.  Dhanji, S., K. Tse, and H. S. Teh. 2005. The low affinity Fc receptor for IgG functions as an effective cytolytic receptor for self-specific C D 8 T cells. J Immunol 174:1253-1258.  26.  Dhanji, S., S. J . Teh, D. Oble, J . J . Priatel, and H. S. Teh. 2004. Selfreactive memory-phenotype C D 8 T cells exhibit both MHC-restricted and non-MHC-restricted cytotoxicity: a role for the T-cell receptor and natural killer cell receptors. Blood 104:2116-2123.  27.  Teh, H. S., H. Kishi, B. Scott, and H. Von Boehmer. 1989. Deletion of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal T C R levels and low levels of C D 8 molecules. J Exp Med 169:795-806.  28.  von Boehmer, H., H. S. Teh, and P. Kisielow. 1989. The thymus selects the useful, neglects the useless and destroys the harmful. Immunol Today 10:57-61.  29.  von Boehmer, H., J . Kirberg, and B. Rocha. 1991. An unusual lineage of alpha/beta T cells that contains autoreactive cells. J Exp Med 174:10011008.  30.  Yamada, H., T. Ninomiya, A. Hashimoto, K. Tamada, H. Takimoto, and K. Nomoto. 1998. Positive selection of extrathymically developed T cells by self- antigens. J Exp Med 188:779-784.  31.  Scott, B., H. Bluthmann, H. S. Teh, and H. von Boehmer. 1989. The generation of mature T cells requires interaction of the [alpha][beta] T-cell receptor with major histocompatibility antigens. Nature 338:591.  32.  Yamada, H., T. Nakamura, G . Matsuzaki, Y. Iwamoto, and K. Nomoto. 2000. TCR-independent activation of extrathymically developed, self antigen- specific T cells by IL-2/IL-15. J Immunol 164:1746-1752.  33.  Gasser, S., P. Corthesy, F. Beerman, H. R. MacDonald, and M. Nabholz. 2000. Constitutive expression of a chimeric receptor that delivers IL-2/IL15 signals allows antigen-independent proliferation of CD8+CD44high but not other T cells. J Immunol 164:5659-5667.  34.  Zhang, X., S. Sun, I. Hwang, D. F. Tough, and J . Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8:591-599.  35.  Gamero, A. M., D. Ussery, D. S. Reintgen, C. A. Puleo, and J . Y . Djeu. 1995. Interleukin 15 induction of lymphokine-activated killer cell function against autologous tumor cells in melanoma patient lymphocytes by a CD18-dependent, perforin-related mechanism. Cancer Res 55:4988-4994.  36.  Waldmann, T. A., and Y . Tagaya. 1999. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annu Rev Immunol 17:19-49.  37.  Perussia, B. 1991. Lymphokine-activated killer cells, natural killer cells and cytokines. Curr Opin Immunol 3:49-55.  38.  Yajima, T., H. Nishimura, R. Ishimitsu, K. Yamamura, T. Watase, D. H. Busch, E. G . Pamer, H. Kuwano, and Y. Yoshikai. 2001. Memory phenotype CD8(+) T cells in IL-15 transgenic mice are involved in early protection against a primary infection with Listeria monocytogenes.  EurJ  Immunol 31:757-766. 39.  Mingari, M. C , M. Ponte, S. Bertone, F. Schiavetti, C. Vitale, R. Bellomo, A. Moretta, and L. Moretta. 1998. HLA class l-specific inhibitory receptors in human T lymphocytes: Interleukin 15-induced expression of C D 9 4 / N K G 2 A in superantigen- or alloantigen-activated CD8+ T cells. PNAS 95:1172-1177.  40.  Roberts, A. I., L. Lee, E. Schwarz, V. Groh, T. Spies, E. C. Ebert, and B. Jabri. 2001. Cutting Edge: N K G 2 D Receptors Induced by IL-15 Costimulate CD28-Negative Effector C T L in the Tissue Microenvironment. J Immunol 167:5527-5530.  41.  Gunturi, A., R. E. Berg, and J . Forman. 2003. Preferential Survival of C D 8 T and NK Cells Expressing High Levels of CD94. J Immunol 170:17371745.  42.  Teague, R. M., B. D. Sather, J . A. Sacks, M. Z. Huang, M. L. Dossett, J . Morimoto, X . Tan, S. E. Sutton, M. P. Cooke, C. Ohlen, and P. D. Greenberg. 2006. Interleukin-15 rescues tolerant CD8(+) T cells for use in adoptive immunotherapy of established tumors. Nat Med 12:335-341.  43.  Ohlen, C , M. Kalos, L. E. Cheng, A. C. Shur, D. J . Hong, B. D. Carson, N. C. Kokot, C. G . Lerner, B. D. Sather, E. S. Huseby, and P. D. Greenberg. 2002. CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function. J Exp Med 195:1407-1418.  44.  Brigl, M., L. Bry, S. C. Kent, J . E. Gumperz, and M. B. Brenner. 2003. Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat Immunol 4:1230-1237.  45.  Yamagata, T., D. Mathis, and C. Benoist. 2004. Self-reactivity in thymic double-positive cells commits cells to a C D 8 alpha alpha lineage with characteristics of innate immune cells. Nat Immunol 5:597-605.  46.  Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J . L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, K. Brasel, P. J . Morrissey, K. Stocking, J . C. Schuh, S. Joyce, and J . J . Peschon. 2000. Reversible defects in natural killer and memory C D 8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191:771-780.  47.  Lodolce, J . P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, and A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669-676.  48.  Intlekofer, A. M., N. Takemoto, E. J . Wherry, S. A. Longworth, J . T. Northrup, V. R. Palanivel, A. C. Mullen, C. R. Gasink, S. M. Kaech, J . D. Miller, L. Gapin, K. Ryan, A. P. Russ, T. Lindsten, J . S. Orange, A. W. Goldrath, R. Ahmed, and S. L. Reiner. 2005. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol 6:1236-1244.  49.  Hamerman, J . A., K. Ogasawara, and L. L. Lanier. 2004. Cutting edge: Toll-like receptor signaling in macrophages induces ligands for the N K G 2 D receptor. J Immunol 172:2001-2005.  50.  Cerwenka, A., and L. L. Lanier. 2001. Natural killer cells, viruses and cancer. Nat Rev Immunol 1:41-49.  51.  Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A. Lanzavecchia, and F. Sallusto. 2004. Induced recruitment of N K cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nat Immunol 5:1260-1265.  52.  Walzer, T., M. Dalod, S. H. Robbins, L. Zitvogel, and E. Vivier. 2005. Natural-killer cells and dendritic cells: 'Tunion fait la force". Blood 106:2252-2258.  53.  Das, G . , S. Sheridan, and C. A. Janeway, Jr. 2001. The source of early IFN-gamma that plays a role in Th1 priming. J Immunol 167:2004-2010.  223  Chapter 6  6.1  General discussion and perspectives  Self-specific CD8 T cells in non-TCR transgenic mice  This thesis has focused on the unusual properties of self-specific C D 8 T cells from both normal and T C R transgenic mice. Chapter 2 was focused on the description of C D 8 T cells in normal mice that demonstrated a significant degree of self-reactivity (1). These cells all had a memory-phenotype in naive animals characterized by the expression of high levels of CD44 and CD122 and upon activation the cells expressed several NK receptors. The function of one of these NK receptors, N K G 2 D , was assessed in more detail and shown to be involved in the recognition of syngeneic tumor cells which expressed a ligand for this receptor. Chapter 3 went on to show that the self-specific CD8'° T cells found in male H-Y T C R transgenic mice shared a similar cell surface phenotype, NK receptor expression, and functional properties with self-specific C D 8 T cells from non-TCR transgenic mice (2). The results in that chapter made it clear that the CD8'° cells found in male H-Y T C R transgenic mice were in fact a good model system to study the properties of self-specific C D 8 T cells in non-TCR transgenic mice. A s such, the H-Y model system was used to determine the role of selfantigen in the development and homeostasis of self-specific C D 8 T cells. The use of CD8'° T cells from male H-Y T C R transgenic mice made it possible to determine the relative contributions of the self-specific T C R and NK receptors for target recognition by self-specific C D 8 T cells. The experiments from chapter 4  showed that self-specific C D 8 T cells from both H-Y male mice and from normal B6 mice could use the activating Fc receptor, CD16, for both cytokine production as well as the killing of antibody-coated target cells (3). In addition, this chapter uncovered another unusual feature of self-specific C D 8 T cells that differentiate them from conventional C D 8 T cells; the expression of FcRy as part of the T C R complex. Chapter 5 was perhaps the most exciting chapter as it provided in vivo relevance for self-specific C D 8 T cells by showing that they could provide innate protection during infection with L M . It also confirmed that the development of these cells was unconventional in that it could occur outside of the thymus. Perhaps most importantly this chapter showed that the expression of a selfreactive T C R allowed the cells to respond more vigorously to infections through the maintenance of CD122 expression. Many of the findings presented in this thesis have been based on results obtained from using a homogenous population of self-specific CD8'° T cells from male H-Y T C R transgenic mice. This system was used because it provides a great degree of control during experiments since the T C R has a known specificity. The work in chapter two was aimed at describing the self-specificity of memory-phenotype C D 8 T cells in normal non-TCR transgenic mice. This work clearly demonstrated that a significant proportion of C D 8 C D 4 4 T cells in the +  hl  B6, Balb/c, and DBA/2 strains of mice demonstrate self-reactivity. More importantly these cells share an almost identical cell surface phenotype and functional properties with the self-specific CD8'° cells from male H-Y T C R transgenic mice. The biggest problem with the use of non-TCR transgenic mice  225 for these studies is the fact that the C D 8 C D 4 4 +  hl  population in these mice is  heterogeneous and consists of self-specific C D 8 T cells, true foreign-antigen specific memory C D 8 T cells, and M H C class lb-restricted C D 8 T cells. Thus, it was necessary to complement studies in normal mice with studies in T C R transgenic mice to gain novel insight regarding their developmental biology and function.  6.1.1 The relationship between foreign-antigen specific memory CD8 T cells and self-specific CD8 T cells  When naive C D 8 T cells encounter their cognate antigen for the first time they undergo several rounds of division and differentiation in order to generate a large number of effector cells capable of eliminating pathogens for which they are specific. The majority of these effector cells will then undergo a contraction phase leaving a small population of memory C D 8 T cells specific for the original antigen. These memory C D 8 T cells will then persist for several years at a greater precursor frequency and these cells will be capable of expanding more rapidly than the original naive C D 8 T cell (4). The maintenance of these memory T cells is independent of antigen but is heavily dependent on cytokines, especially IL-15 (5). Aside from their cell-surface phenotype there are other similarities between foreign-antigen specific memory C D 8 T cells and selfspecific C D 8 T cells including their requirement for IL-15 and their ability to rapidly provide effector functions. There are also differences between foreign-  226 antigen specific memory C D 8 T cells and self-specific C D 8 T cells the biggest of which is their responsiveness to T C R stimulation. One of the hallmark characteristics of foreign-antigen specific memory C D 8 T cells is their ability to respond more rapidly during a reencounter with antigen. Memory C D 8 T cells have been shown to have a decreased T C R signaling threshold and thus can activate signaling pathways downstream of their T C R much more efficiently than naive T cells (6). This decreased T C R threshold is in stark contrast to the memory-phenotype self-specific C D 8 T cells described in this thesis. In fact self-specific C D 8 T cells actually have an increased signaling threshold relative to naive C D 8 T cells with the same specificity (2). When Yamada et al compared the phenotype of self-specific H-Y male C D 8 T cells to foreign-antigen specific H-Y female memory C D 8 T cells they found that the self-specific male cells were much less responsive to T C R stimulation (7). In addition, the self-specific H-Y male C D 8 T cells were less efficient producers of IL-2 even though they could produce IFNy. This difference in T C R signaling threshold differentiates self-specific C D 8 T cells from foreign-antigen specific memory C D 8 T cells and is probably in place to prevent the autoimmune potential of C D 8 T cells bearing self-specific T C R s . Memory C D 8 T cells and self specific C D 8 T cells both rely heavily on IL15 for their maintenance and homeostasis. In fact mice deficient in either IL-15 or its receptor IL-15Ra have a large reduction in memory-phenotype C D 8 T cells (8, 9). Homeostatic expansion of memory-phenotype C D 8 T cells in response to lymphodepletion is heavily dependent on the availability of IL-15 (10, 11). IL-15 is  227 produced by a wide array of cell types (12) and is rapidly produced during infection resulting in the expansion of memory-phenotype C D 8 T cells which express CD122. Both self-specific C D 8 T cells and true foreign-antigen specific memory C D 8 T cells can respond to IL-15 both in vitro and in vivo. In fact IL-15 can allow the survival of memory C D 8 T cells in the complete absence of any peptide/MHC interactions (10). Self-specific H-Y male C D 8 T cells are very responsive to IL-15 both in vitro and in vivo and this responsiveness is maintained by their interactions with self-antigen. It is interesting to note that foreign-antigen specific memory C D 8 T cells from H-Y female mice, bearing the same T C R as self-specific H-Y male CD8'° T cells, are relatively unresponsive to IL-15 (7). It appears that this difference in IL-15 responsiveness is likely due to lower expression of CD122 by H-Y female memory C D 8 T cells relative to selfspecific male cells. Foreign-antigen specific memory C D 8 T cells have the ability to rapidly mediate effector functions such as target cell killing and cytokine production without the need for prior restimulation (13). Self-specific C D 8 T cells can also kill target cells and produce cytokines without prior restimulation (14). In terms of cytokine production both foreign-antigen specific memory C D 8 T cells and selfspecific C D 8 T cells can rapidly produce IFNy upon T C R stimulation or upon stimulation with cytokines like IL-12, IL-15, and IL-18 and both of these memoryphenotype C D 8 T cell populations can provide innate protection during bacterial infections (15-19). Whether differences exist in the amount of IFNy production or  228 the stimuli that induce IFNy production will need to be addressed more directly in the future. It is clear from the studies presented in this thesis that foreign-antigen specific memory C D 8 T cells and self-specific C D 8 T cells share some similarities and it is also clear that these two cell types are also different. It would be of great interest to test the phenotype and function of these two cell types directly by comparing foreign-antigen specific memory C D 8 T cells with several known specificities to self-specific C D 8 T cells from H-Y male mice. In addition it would be interesting if one could determine the relative proportions of foreign antigen-specific versus self-specific C D 8 T cells in memory phenotype C D 8 T cells in normal mice.  6.1.2 The potential involvement of MHC class lb molecules in the selection of self-specific CD8 T cells  The vast majority of C D 8 T cells are selected by the highly polymorphic M H C class la molecules. However, C D 8 T cells can also be selected by the less polymorphic M H C class lb family of molecules. There are several different M H C class lb molecules in mice (reviewed in (20)). Two of these M H C class lb molecules, H2-M3 and Qa-1, are known to be involved in the activation of C D 8 T cells during infection (21-23). There are several characteristics of M H C class lbrestricted C D 8 T cells that are similar to self-specific C D 8 T cells and it remains possible that at least some self-specific C D 8 T cells in non-TCR transgenic mice  229 are in fact restricted to M H C class lb molecules. Similar to self-specific C D 8 T cells, M H C class lb-restricted C D 8 T cells have a memory-phenotype even in naive mice which allows them to become activated very rapidly during infections (16, 24). The cells can respond to LM infection in either an antigen-dependent or antigen-independent manner through the production of I F N y (16). M H C class lbrestricted C D 8 T cells can be selected by hematopoetic cells and some of these cells can develop in the absence of a functional thymus (16, 24). Thus, there are many similarities between M H C class lb-restricted C D 8 T cells and self-specific C D 8 T cells. H2-M3 is an M H C class lb molecule that binds and presents formylated peptides found in prokaryotic cells. The only formylated peptides found in eukaryotic hosts are those derived from mitochondrial proteins and thus the peptides capable of selecting H2-M3-restricted T cells is limited (20). One of the peptides involved in the positive selection of H2-M3-restricted C D 8 T cells, ND1, is actually a weak agonist for mature H2-M3-restricted T cells (25). H2-M3restricted C D 8 T cells are very unusual in the sense that they have a memoryphenotype even in naive mice and they can be selected by hematopoietic cells in the thymus (24). Qa-1b-is another M H C class lb molecule capable of selecting C D 8 T cells. Qa-1b normally binds to the leader sequence of several M H C class la molecules (26) and acts as a ligand for the N K G 2 A / C / E NK receptors (27). Qa-1b is also capable of presenting other unconventional peptides including those derived from bacterial heat shock proteins like the GroEL peptide from several Gram-positive  230 and Gram-negative bacteria (28), peptides derived from the T C R chains of C D 4 T cells (29), peptides from mouse heat shock protein 60 (HSP60) (30), as well as a peptide derived from insulin (31). Qa-1b-restricted C D 8 T cells that recognize peptides from the T C R chains of C D 4 T cells play an important role in regulating the C D 4 response (32). Qa-1b restricted C D 8 T cells specific for GroEL have been shown to expand during infection with Salmonella. Interestingly, these GroEL-specific C D 8 T cells can also recognize self-derived H S P 6 0 peptides presented by Qa-1b providing a potential link between Qa-1b, infection, and autoimmunity (30). It is very tempting to speculate that there is in fact a relationship between both H2-M3 and Qa-1b-restricted C D 8 T cells and self-specific C D 8 T cells. Both cell types exhibit a memory-phenotype and both subsets respond rapidly during infections. Perhaps the reason that M H C class lb molecules are so highly conserved is that they act similarly to the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) found on several innate cells. In fact the literature is consistent with the hypothesis that M H C class lb-restricted C D 8 T cells can recognize self-peptides that are induced or upregulated on infected or transformed cells. Consistent with this idea is the recognition of self-HSP60 on stressed cells by GroEL-specific C D 8 T cells (30). Also consistent with this idea is the fact that mice lacking H2-M3-molecules are more susceptible to infection with LM (33) yet mice infected with a mutant strain of LM incapable of producing formylated peptides clear infection normally (34). These two potentially conflicting results can be reconciled by the fact that H2-M3 molecules present self-derived  231 formylated peptides to H2-M3-restricted C D 8 T cells during infection with LM and that this process plays a key role in controlling bacterial infection. This hypothesis, if true, would confirm that M H C class lb-restricted C D 8 T cells are in fact self-specific and may behave similarly to the self-specific C D 8 T cells described in this thesis. It will be of great interest to determine whether there is in fact a relationship between these seemingly unrelated cell types and perhaps the first place to look would be at the C D 8 T cells found in M H C class la-deficient mice.  6.2  The in vivo significance of self-specific CD8 T cells  6.2.1 The autoimmune potential of self-specific CD8 T cells  Self-specific C D 8 T cells express self-reactive T C R s and as such they have the potential to damage normal cells if they are not properly controlled. One of the main characteristics of self-specific C D 8 T cells is their inability to proliferate and become activated in response to normal physiological levels of their cognate selfantigens (2). This antigen-unresponsiveness was shown in chapter 3 to be due to T C R signaling defects in the cells leading to an inability to efficiently activate pathways downstream of the T C R . If this high T C R signaling threshold is in effect  232 reversed, then self-specific C D 8 T cells can cause autoimmunity as seen in E2F2-deficient mice (35). The E 2 F family of transcription factors is important for regulating many aspects of T cell function including proliferation, apoptosis, and differentiation. A report by Murga et al (2001) described the autoimmune phenotype of mice lacking E2F2. These mice develop a late-onset autoimmune disease characterized by the expansion of memory-phenotype C D 8 T cells leading to widespread inflammation and immunocomplex deposition. These C D 8 C D 4 4 +  hl  cells displayed overt self-reactivity proliferating in response to syngeneic stimulator cells. This study went on to show that when E2F2-deficient mice transgenically expressed the H-Y T C R , male mice developed autoimmunity faster and with a more severe phenotype compared to non-TCR transgenic mice. The cause of the disease was the large population self-specific H-Y male CD8'° cells described in this thesis. The lack of E2F2 had lowered the T C R threshold of the cells allowing them to proliferate and become activated leading to disease. Thus it is clear that self-specific C D 8 T cells have the capability of causing disease but they are normally held in check by their inability to efficiently become activated by T C R stimulation alone. Self-specific C D 8 T cells can become activated in response to cytokines such as IL-15 and this activation results in the expression of NK receptors (1, 2). IL-15-responsiveness is tightly controlled through the action of members of the suppressor of cytokine signaling (SOCS) family which dampen cytokine signaling (36). It remains possible however, that uncontrolled cytokine signaling may lead  233 to autoimmunity through the activation of self-specific C D 8 T cells. In fact S O C S 1-deficient mice display a partially T cell-dependent autoimmune disease mediated by overproduction of IFNy (37). IL-15-hypersensitivty in these animals plays a key role in the activation of the memory-phenotype C D 8 T cells and these animals have an increase in the proportion of autoreactive C D 8 T cells (38). The development of disease in SOCS-1-deficient mice depends heavily on the ability of the C D 8 T cells to undergo homeostatic expansion suggesting that selfspecific C D 8 T cells, which display a remarkable ability to homeostatically expand, may play a role in disease in SOCS-1-deficient mice. NK receptor expression by self-specific C D 8 T cells confers the cells with the ability to recognize target cells in the absence of T C R stimulation. This generates cells which are no longer MHC-restricted and can thus potentially destroy host cells expressing the appropriate ligands. N K G 2 D was one of the N K receptors, which is central to the findings in this thesis. Self-specific C D 8 T cells express both N K G 2 D and the adaptor DAP12 when activated with either antigen or cytokine alone (1, 2). Thus the N K G 2 D receptor on these cells has the potential to cause damage to host cells if they overexpress any N K G 2 D ligand. There are several reports in the literature which suggest that the uncontrolled expression of N K G 2 D coupled with expression of NKG2D-ligands does play a role in autoimmunity. In fact, the non-obese diabetic (NOD) strain of mice, was shown to over-express the Rae-1-family of NKG2D-ligands in pancreatic islet cells and that N K G 2 D expression on auto-reactive C D 8 T cells was key in the progression towards diabetes (39). In this system the blocking of N K G 2 D was  234 sufficient to prevent the expansion of autoreactive C D 8 T cells and could prevent disease. There may also be other conditions where N K G 2 D signaling is out of control. Bana Jabri's group showed that TCR-independent killing mediated by N K G 2 D may play a role in celiac disease (40). They found that IL-15 produced in celiac patients was sufficient to convert N K G 2 D on C D 8 T cells into a directly activating receptor. This conversion led to MHC-independent killing activity by C D 8 T cells from celiac patients. Thus it is possible that even in their system that self-specific C D 8 T cells may cause disease if NK receptor expression or signaling is altered.  6.2.3 The potential role for self-specific CD8 T cells in anti-tumor immunity  It is clear that self-specific C D 8 T cells have the potential to be harmful if left uncontrolled. On the flip side, self-specific C D 8 T cells may actually be beneficial if they can be used to eliminate tumor cells which often express normal selfantigens. It is possible that self-specific C D 8 T cells may in fact respond to tumor cells if they over-express particular self antigens or if they express ligands for activating NK receptors like NKG2D. For example if self-specific C D 8 T cells exist for peptides from a particular tumor oncogene, then over-expression of this oncogene in transformed cells may be reflected by over-expression of peptides capable of activating self-specific C D 8 T cells. Since self-specific C D 8 T cells have an increased activation threshold they would require over-expression of cognate antigen for them to be activated. Similarly tumor cells may express one  235 or more N K G 2 D ligands allowing self-specific C D 8 T cells to recognize and destroy these "stressed" cells in the absence of strong T C R stimulation. There are several reports in the literature regarding the expression of NK receptors on melanoma-specific C D 8 T cells, even those recovered from healthy donors (41, 42). It is possible that inhibitory receptors may prevent autoimmunity but allow a response if the target lacks the ligand for the receptor. In fact, inhibitory NK receptor expression by human melanoma-specific C D 8 T cells has been shown to play a role in the killing of tumor cells that have lost expression of the M H C class I ligand for the receptor (43). In mice, one group has shown that C D 8 T cells specific for a non-mutated melanoma antigen, tyrosinase-related protein 2 (TRP-2), have a natural memoryphenotype identical to that of the self-specific C D 8 T cells discussed in this thesis (44). In fact, these TRP-2-specific C D 8 T cells can develop extrathymically suggesting that perhaps self-specific C D 8 T cells described in this thesis can be specific for tumors. It would be of great interest to determine if self-specific C D 8 T cells specific for tumor antigens did behave the same way as self-specific H-Y male CD8'° T cells. There is one system that may be of particular interest. In doubly transgenic mice, which express a transgenic T C R specific for the gag protein from Friend murine leukemia virus (FMuLVgag) as well as the transgenic cognate Ag, FMuLVgag, C D 8 T cells expressing the transgenic T C R still develop (45). Furthermore, these cells exhibit a memory-phenotype similar to the self-specific CD8'° T cells from H-Y male mice; these mice also did not develop detectable  autoimmune diseases even though the self-specific C D 8 T cells retain effector function and are specific for the self-Ag (46). A recent report on these cells also demonstrated their ability to proliferate in response to IL-2 or IL-15 alone and more importantly showed that these self-specific C D 8 T cells could eliminate tumor cells that expressed the self-antigen (47). Whether or not these cells express any NK receptors or function similarly to the self-specific C D 8 T cells in this thesis is unclear although it seems likely that the cells will share some similarities.  6.2.3 Other potential roles for self-specific CD8 T cells  It is very tempting to speculate that self-specific C D 8 T cells play many other roles in the immune response other than the innate production of cytokines to impede bacterial growth. NK cells have been shown to be involved in the production of cytokines such as IFNy that can help to polarize the C D 4 response (48). I have shown that self-specific C D 8 T cells can actually produce significantly more IFNy than NK cells. It is therefore conceivable that they can also be involved in this aspect of immunity. The literature is consistent with the idea that memory-phenotype C D 8 T cells can actually provide a source of IFNy for the priming of C D 4 T cells (49). It should be relatively simple to test the ability of self-specific C D 8 T cells to provide this function.  237  6.3  C o n c l u d i n g remarks  This thesis defines the developmental and functional properties of a novel subset of C D 8 T cells specific for self-antigens. These cells express receptors characteristic of both T cells and NK cells and this property allows the cells to rapidly focus on infected, stressed or transformed cells. The results presented demonstrate a potential role for self-specific C D 8 T cells in various important aspects of immunity ranging from innate immune protection, to tumor immunology, and autoimmunity.  238  6.4  References  1.  Dhanji, S., and H.-S. Teh. 2003. IL-2-Activated CD8+CD44high Cells Express Both Adaptive and Innate Immune System Receptors and Demonstrate Specificity for Syngeneic Tumor Cells. J Immunol 171:34423450.  2.  Dhanji, S., S. J . Teh, D. Oble, J . J . Priatel, and H. S. Teh. 2004. Selfreactive memory-phenotype C D 8 T cells exhibit both MHC-restricted and non-MHC-restricted cytotoxicity: a role for the T-cell receptor and natural killer cell receptors. Blood 104:2116-2123.  3.  Dhanji, S., K. Tse, and H. S. Teh. 2005. The low affinity Fc receptor for IgG functions as an effective cytolytic receptor for self-specific C D 8 T cells. J Immunol 174:1253-1258.  4.  Rocha, B., and C. Tanchot. 2004. C D 8 T cell memory. Seminars in Immunology 16:305.  5.  Schluns, K. S., and L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat Rev Immunol 3:269-279.  6.  Kersh, E. N., S. M. Kaech, T. M. Onami, M. Moran, E. J . Wherry, M. C. Miceli, and R. Ahmed. 2002. T C R Signal Transduction in Antigen-Specific Memory CD8 T Cells. J Immunol 170:5455-5463.  7.  Yamada, H., G. Matsuzaki, Q. Chen, Y. Iwamoto, and K. Nomoto. 2001. Reevaluation of the origin of CD44(high) "memory phenotype" C D 8 T  cells: comparison between memory C D 8 T cells and thymus-independent C D 8 T cells. Eur J Immunol 31:1917-1926. 8.  Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J . L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, K. Brasel, P. J . Morrissey, K. Stocking, J . C. Schuh, S. Joyce, and J . J . Peschon. 2000. Reversible defects in natural killer and memory C D 8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191:771-780.  9.  Lodolce, J . P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, and A . Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669-676.  10.  Tan, J . T., B. Ernst, W. C. Kieper, E. LeRoy, J . Sprent, and C. D. Surh. 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J Exp Med 195:1523-1532.  11.  Zhang, X., S. Sun, I. Hwang, D. F. Tough, and J . Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8:591-599.  12.  Giri, J . G . , S. Kumaki, M. Ahdieh, D. J . Friend, A . Loomis, K. Shanebeck, R. DuBose, D. Cosman, L. S. Park, and D. M. Anderson. 1995. Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. Embo J 14:3654-3663.  240 13.  Barber, D. L , E. J . Wherry, and R. Ahmed. 2003. Cutting edge: rapid in vivo killing by memory C D 8 T cells. J Immunol 171:27-31.  14.  Yamada, H., G . Matsuzaki, Y. Iwamoto, and K. Nomoto. 2000. Unusual cytotoxic activities of thymus-independent, self-antigen- specific CD8(+) T cells. Int Immunol 12:1677-1683.  15.  Berg, R. E., E. Crossley, S. Murray, and J . Forman. 2005. Relative contributions of NK and C D 8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes. J Immunol 175:17511757.  16.  S u , J . , R. E. Berg, S. Murray, and J . Forman. 2005. Thymus-dependent memory phenotype C D 8 T cells in naive B6.H-2Kb-/-Db-/- animals mediate an antigen-specific response against Listeria monocytogenes. J Immunol 175:6450-6457.  17.  Berg, R. E., E. Crossley, S. Murray, and J . Forman. 2003. Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. J Exp M e d 198:1583-1593.  18.  Berg, R. E., C. J . Cordes, and J . Forman. 2002. Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL18. EurJ Immunol 32:2807-2816.  19.  Lertmemongkolchai, G., G . Cai, C. A. Hunter, and G . J . Bancroft. 2001. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. J Immunol 166:10971105.  20.  Rodgers, J . R., and R. G . Cook. 2005. M H C class lb molecules bridge innate and acquired immunity. Nat Rev Immunol 5:459-471.  21.  Lenz, L. L , and M. J . Bevan. 1996. H2-M3 restricted presentation of Listeria monocytogenes antigens. Immunol Rev 151:107-121.  22.  Urdahl, K. B., D. Liggitt, and M. J . Bevan. 2003. CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice, but provide minimal protection. J Immunol 170:1987-1994.  23.  Bouwer, H. G., M. S. Seaman, J . Forman, and D. J . Hinrichs. 1997. M H C class lb-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific C T L s . J Immunol 159:2795-2801.  24.  Urdahl, K. B., J . C. Sun, and M. J . Bevan. 2002. Positive selection of M H C class lb-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol 3:772-779.  25.  Berg, R. E., S. Irion, S. Kattman, M. F. Princiotta, and U. D. Staerz. 2000. A Physiological Ligand of Positive Selection Is Recognized as a Weak Agonist. J Immunol 165:4209-4216.  26.  DeCloux, A., A. S. Woods, R. J . Cotter, M. J . Soloski, and J . Forman. 1997. Dominance of a single peptide bound to the class l(B) molecule, Qa-1 b. J Immunol 158:2183-2191.  27.  Vance, R. E., J . R. Kraft, J . D. Altman, P. E. Jensen, and D. H. Raulet. 1998. Mouse C D 9 4 / N K G 2 A is a natural killer cell receptor for the  nonclassical major histocompatibility complex (MHC) class I molecule Q a 1(b). J Exp Med 188:1841-1848. 28.  Lo, W. F., H. Ong, E. S. Metcalf, and M. J . Soloski. 1999. T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of M H C class lb molecules. J Immunol 162:5398-5406.  29.  Jiang, H., R. Ware, A. Stall, L. Flaherty, L. Chess, and B. Pernis. 1995. Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing V beta 8 T C R : a role of the Qa-1 molecule. Immunity 2:185194.  30.  Lo, W. F., A. S. Woods, A. DeCloux, R. J . Cotter, E. S. Metcalf, and M. J . Soloski. 2000. Molecular mimicry mediated by M H C class lb molecules after infection with gram-negative pathogens. Nat Med 6:215-218.  31.  Li, L., B. A. Sullivan, C. J . Aldrich, M. J . Soloski, J . Forman, A. G . Grandea, III, P. E. Jensen, and L. Van Kaer. 2004. Differential Requirement for Tapasin in the Presentation of Leader- and InsulinDerived Peptide Antigens to Qa-1b-Restricted CTLs. J Immunol 173:37073715.  32.  Jiang, H., and L. Chess. 2000. The specific regulation of immune responses by CD8+ T cells restricted by the M H C class lb molecule, Qa-1. Annu Rev Immunol 18:185-216.  33.  X u , H., T. Chun, H. J . Choi, B. Wang, and C. R. Wang. 2006. Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of M H C class lb-specific T cells in host defense. J Exp Med 203:449-459.  34.  D'Orazio, S. E., C. A. Shaw, and M. N. Starnbach. 2006. H2-M3-restricted CD8+ T cells are not required for M H C class lb-restricted immunity against Listeria monocytogenes. J Exp M e d 203:383-391.  35.  Murga, M., O. Fernandez-Capetillo, S. J . Field, B. Moreno, L. R. Borlado, Y. Fujiwara, D. Balomenos, A. Vicario, A. C. Carrera, S. H. Orkin, M. E. Greenberg, and A. M. Zubiaga. 2001. Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity. Immunity 15:959-970.  36.  Ilangumaran, S., S. Ramanathan, and R. Rottapel. 2004. Regulation of the immune system by S O C S family adaptor proteins. Semin Immunol 16:351-365.  37.  Marine, J . C , D. J . Topham, C. McKay, D. Wang, E. Parganas, D. Stravopodis, A. Yoshimura, and J . N. Ihle. 1999. S O C S 1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell 98:609-616.  38.  Davey, G. M., R. Starr, A. L. Cornish, J . T. Burghardt, W. S. Alexander, F. R. Carbone, C. D. Surh, and W. R. Heath. 2005. S O C S - 1 regulates IL-15driven homeostatic proliferation of antigen-naive C D 8 T cells, limiting their autoimmune potential. J Exp Med 202:1099-1108.  39.  Ogasawara, K., J . A. Hamerman, L. R. Ehrlich, H. Bour-Jordan, P. Santamaria, J . A. Bluestone, and L. L. Lanier. 2004. N K G 2 D blockade prevents autoimmune diabetes in NOD mice. Immunity 20:757-767.  40.  Meresse, B., Z. Chen, C. Ciszewski, M. Tretiakova, G . Bhagat, T. N. Krausz, D. H. Raulet, L. L. Lanier, V. Groh, T. Spies, E. C. Ebert, P. H. Green, and B. Jabri. 2004. Coordinated induction by IL15 of a T C R independent N K G 2 D signaling pathway converts C T L into lymphokineactivated killer cells in celiac disease. Immunity 21:357-366.  41.  Huard, B., and L. Karlsson. 2000. A subpopulation of CD8+ T cells specific for melanocyte differentiation antigens expresses killer inhibitory receptors (KIR) in healthy donors: evidence for a role of KIR in the control of peripheral tolerance. Eur J Immunol 30:1665-1675.  42.  Speiser, D. E., M. J . Pittet, D. Valmori, R. Dunbar, D. Rimoldi, D. Lienard, H. R. MacDonald, J . C. Cerottini, V. Cerundolo, and P. Romero. 1999. In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J Exp Med 190:775-782.  43.  Ikeda, H., B. Lethe, F. Lehmann, N. van Baren, J . F. Baurain, C. de Smet, H. Chambost, M. Vitale, A. Moretta, T. Boon, and P. G . Coulie. 1997. Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by C T L expressing an NK inhibitory receptor. Immunity 6:199-208.  44.  Harada, M., H. Yamada, K. Tatsugami, and K. Nomoto. 2001. Evidence of the extrathymic development of tyrosinase-related protein-2- recognizing CD8+ T cells with low avidity. Immunology 104:67-74.  45.  Ohlen, C , M. Kalos, D. J . Hong, A. C. Shur, and P. D. Greenberg. 2001. Expression of a tolerizing tumor antigen in peripheral tissue does not preclude recovery of high-affinity CD8+ T cells or C T L immunotherapy of tumors expressing the antigen. J Immunol 166:2863-2870.  46.  Ohlen, C , M. Kalos, L. E. Cheng, A. C. Shur, D. J . Hong, B. D. Carson, N. C. Kokot, C. G . Lerner, B. D. Sather, E. S. Huseby, and P. D. Greenberg. 2002. CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function. J Exp Med 195:1407-1418.  47.  Teague, R. M., B. D. Sather, J . A. Sacks, M. Z. Huang, M. L. Dossett, J . Morimoto, X. Tan, S. E. Sutton, M. P. Cooke, C. Ohlen, and P. D. Greenberg. 2006. Interleukin-15 rescues tolerant CD8(+) T cells for use in adoptive immunotherapy of established tumors. Nat Med 12:335-341.  48.  Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A. Lanzavecchia, and F. Sallusto. 2004. Induced recruitment of N K cells to lymph nodes provides IFN-gamma forT(H)1 priming. Nat Immunol 5:1260-1265.  49.  Das, G., S. Sheridan, and C. A. Janeway, Jr. 2001. The source of early IFN-gamma that plays a role in Th1 priming. J Immunol 167:2004-2010.  

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