The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

Coevolution between Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, and the human host has been documented for thousands of years. Interestingly, while T cell immunity is crucial for host protection and survival, T cell antigens are the most conserved region of the Mtb genome. Hypothetically, Mtb adapts under immune pressure to exploit T cell responses for its benefit from inflammation and tissue destruction for ultimately transmission. EsxH, a gene encoding immunodominant TB10.4 protein, however, contains polymorphic regions corresponding to T cell epitopes. Here, I present two complementary analyses to examine how Mtb modulates TB10.4 for immune evasion. First, I use a naturally occurring esxH polymorphic clinical Mtb isolate, 667, to investigate how A10T amino acid exchange in TB10.4 affect T cell immunity. To verify and identify the cause of the immunological differences, I construct isogenic strains expressing EsxHA10T or EsxHWT. In combination with our recent finding that TB10.44-11-specific CD8 T cells do not recognize Mtb-infected macrophages, we hypothesize that TB10.4 is a decoy antigen as it distracts host immunity from inducing other potentially protective responses. I examine whether an elimination of TB10.44-11-specific CD8 T cell response leads to a better host protective immunity. The studies of in vivo infection and in vitro recognition in this dissertation aim to provide a better understanding of the counteraction between immune evasion and protective immunity.

During virus infections, activation of CD8 T cells takes place in secondary lymphoid organs including spleen and lymph nodes. The kinetics of the T cell response in lymphoid tissues has been clearly studied. However, a large number of virus-specific T cells disseminate into various nonlymphoid tissues. As reservoirs for effector and memory cells, nonlymphoid organs play an important role for defending against infections. T cell responses in nonlymphoid organs may differ from lymphoid organs. T cell repertoire usage in lymphoid and nonlymphoid tissues was studied in an acute lymphocytic choriomeningitis virus (LCMV)-infected murine model. The hierarchy of CD8 T cell specificities was examined with cytotoxic T lymphocyte (CTL) sodium 51 chromate (51Cr) release assays and intracellular interferon (IFN)γ assays. T cell receptor (TCR) repertoire usage was determined by complementarity determining region (CDR)3 length spectratyping analysis. Both T cell specificity and TCR repertoire usage revealed some similarities and differences between several organs. Within an epitope-specific CD8 T cell population, the TCR repertoire usage was similar in different organs of the same mouse, but highly heterogeneous between individual mice with genetically identical backgrounds. A very restricted CD4 TCR repertoire was observed in BALB/c mice after secondary respiratory syncytial virus (RSV) infection. Most of the CD4 T cells of BALB/c mice pre-immunized with RSV glycoprotein (GP) predominantly express Vβ14 TCR with discrete oligoclonal CDR3 regions. Depletion of Vβ14 CD4 T cells dramatically reduced immunopathology. The apoptotic phenotype of LCMV-specific CD8 T cells was studied in various lymphoid and nonlymphoid tissues during acute and memory stages of infections. Peripheral tissues (peritoneal cavity (PEC), fat pad, and lung) reacted with a much lower frequency with the early apoptotic marker Annexin V than those in spleen and lymph nodes. This was not due to a TCR-based selection because similar TCR spectratypes were seen in different organs. Activated lymphoid and nonlymphoid T cells from LCMV GP33 transgenic mice, which have identical TCR α and β chains on all T cells, had differential Annexin V binding. When incubated shortly in vitro, most Annexin V+ T cells rapidly fragmented their DNA and became terminal transferase-mediated dUTP nick end-labeling positive (TUNEL+), while much fewer Annexin V- cells became TUNEL+. Therefore, those Annexin-V+ cells were truly in a pre-apoptotic stage. The differential spontaneous apoptosis in different tissues is independent of several death/survival-related molecules, including Fas/Fas ligand (FasL), turner necrosis factor (TNF)α, interleukin (IL-15), perforin, B cell lymphoma (Bcl)-2 and independent of virus tropism. I further investigated the significance of the high Annexin V reactivity of lymphoid T cells. Pre-apoptotic cells were prevented from fragmenting their DNA by anti-CD3 or IL-2 stimulation in vitro. However, this pre-apoptotic phenotype precluded generation of memory. Annexin V reactive cells did not give rise to long-lived memory after being transferred into naïve hosts. The pre-apoptotic phenotype is also an intrinsic property of the epitope. Different proportions of apoptotic cells were found in LCMV effector and, memory T cells specific to two different epitopes, nucleoprotein (NP)396 and GP33. Higher Annexin V reactivity of NP396-specific CD8 T cells was independent of virus tropism and duration of encounter with antigen. Higher expression of IL-7R was found in peripheral, Annexin V- and GP33-specific CD8 T cells, indicating that IL-7-dependent signals may inhibit apoptosis. Nonlymphoid T cells were more resistant than lymphoid T cells to activation-induced cell death (AICD). When stimulated with anti-CD3 in vitro for 40 hours (hr), a significantly reduced number of splenic transgenic T cells were recovered with much higher frequency of Annexin V reactivity and TUNEL staining than transgenic T cells from PEC. Consistent with the finding that Fas and FasL regulates AICD, a much lower expression of Fas and FasL was observed in PEC and lung transgenic T cells than spleen and lymph nodes after short time stimulation. FasL blockage largely increased cell-number recovery and reduced Annexin V and TUNEL staining of spleen transgenic T cells. Interestingly, the leukocyte environment played an important role of deciding the fate of transgenic T cells. When placing activated spleen transgenic T cells with excess infected PEC cells, spleen transgenic cells rapidly reduced their Annexin V staining and TUNEL staining and were recovered with greater number after stimulation. Vice versa, PEC transgenic T cells became Annexin V and TUNEL positive with lower numbers of cells recovered when placed with excess splenocytes. Less detection of Annexin V+ cells in peripheral tissues was not due to rapid phagocytosis by macrophages, because Cytochalasin D, which can inhibit phagocytosis, did not induce equal amount of pre-apoptotic cells in spleen and PEC. This reduced death in the periphery may contribute to the long-term maintenance of nondividing nonlymphoid memory T cells, enabling them to efficiently function without being driven into apoptosis. Overall, this study characterizes in detail the different T cell repertoire usage and apoptosis of virus-specific T cells based on their organ localization and specificities and helps to better understand T cell immunity after infections and vaccine design.

T cell development in the thymus produces multiple lineages of cells, including conventional naïve CD4+ and CD8+ T cells, regulatory T cells, and innate T cells. Innate T cells encompass γδ T cells, invariant natural killer (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and H2-M3-restricted cells (Berg, 2007). Although they are a minor subset of all thymocytes, innate T cells develop in the thymus and share characteristics of the innate and adaptive immune systems (Berg, 2007). These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation (Berg, 2007). However, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8+ T cells develop as innate cells that share characteristics with memory T cells (Atherly et al., 2006b; Broussard et al., 2006; Fukuyama et al., 2009; Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). One of these signaling proteins, inducible T cell kinase (Itk) is a nonreceptor protein tyrosine kinase that signals downstream of the T cell receptor (TCR) (Berg et al., 2005). Upon TCR activation, Itk is activated and recruited to the TCR signaling complex, where Itk interacts with Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), linker for activation of T cells (LAT), and phospholipase C γ1 (PLCγ1) (Berg et al., 2005). Thus, in Itk-deficient mice, TCR signaling is disrupted, which results in mature CD4- CD8+ (CD8SP) thymocytes that are CD44high, CD62Lhigh, CD122+, and CXCR3+ and that express high levels of the transcription factor, Eomesodermin (Eomes) (Atherly et al., 2006b; Broussard et al., 2006; Weinreich et al., 2010). Recently, it was determined that the development of these innate CD8SP thymocytes in itk-/- mice is dependent on IL-4 produced in the thymic environment by a poorly characterized subset of CD3+ thymocytes expressing the transcriptional regulator, promyelocytic leukemia zinc finger (PLZF) (Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). Here we show that a sizeable proportion of mature CD4+ CD8- (CD4SP) thymocytes in itk-/- mice also develop as Eomesodermin+ innate T cells. These Eomes+ innate CD4+ T cells are CD44high, CD62Lhigh, CD122+, and CXCR3+ (Atherly et al., 2006b; Broussard et al., 2006; Dubois et al., 2006; Weinreich et al., 2010). Surprisingly, neither CD4SP nor CD8SP innate thymocytes in itk-/- mice are dependent on γδ T cells for their development as was previously hypothesized (Alonzo and Sant'Angelo, 2011). Instead, both subsets of innate itk-/- T cells require the presence of a novel PLZF-expressing, SAP-dependent thymocyte population that is essential for the conversion of conventional CD4+ and CD8+ T cells into Eomesodermin-expressing innate T cells with a memory phenotype. This novel subset of PLZF-expressing SAP-dependent innate T cells preferentially home to the spleen and mesenteric lymph nodes and have a restricted TCR repertoire. Thus, we have christened this subset as CD4+ PLZF + MAIT-like cells. We have characterized multiple subsets of innate T cells that expand in the absence of Itk. Therefore, we were interested in how innate T cells respond to infection. Although Itk KO mice have defects in cytolytic function and cytokine production during an acute infection, these mice are able to clear viral infections (Atherly et al., 2006a; Bachmann et al., 1997). Hence, we hypothesized that Itk-deficient memory CD8+ T cells would be able to provide protection upon a challenge infection. Conversely, we found this not to be true although Itk-deficient memory CD8+ T cells were present in similar frequencies and cell numbers as WT memory CD8+ T cells at 42 days post-infection. Furthermore, Itk-deficient memory CD8+ T cells were able to produce IFNγ and exert cytolytic function upon stimulation. Although the function of Itk-deficient memory CD8+ T cells appeared to be intact, we found that these cells were unable to expand in response to a challenge infection. Remarkably, conventional memory CD8+ T cells lacking Itk were able to expand and form protective memory responses upon challenge. Thus, the inability of Eomes+ innate CD8+ T cells to form protective memory responses does not appear to be intrinsic to cells deficient in Itk. This thesis is divided into six major chapters. The first chapter will provide an introduction to T cell development and the role of Itk in T cell development. Additionally, it will introduce a variety of innate T cell subsets that will be discussed throughout this thesis and will provide an overview of CD4+ and CD8 + T cell differentiation during infection. This section will explain the role of Itk in CD4+ helper T cell differentiation and describe how Itk-deficient CD8+ T cells respond to acute infection. The introduction will also discuss the generation of conventional memory CD8+ T cells. The second chapter will provide the details of the experimental procedures used in this thesis. The third chapter will describe the characterization and development of Eomes+ innate CD4+ T cells that develop in the absence of Itk. Additionally, this chapter will address the subset of PLZF+ innate T cells that induce the expression of Eomes in innate T cells. The fourth chapter will further characterize and explore the development of itk-/- CD4+ PLZF+ MAIT-like T cells. The fifth chapter will examine the role of Eomes + innate CD8+ T cells in protective memory responses. Chapters three through five will display work that is in preparation to be submitted to a peer-reviewed journal. The sixth chapter will discuss the results of this thesis and their implications.

Activation and proliferation of antigen-specific T cells is the hallmark of an anti-viral immune response. Effector T cells generated during an immune response are heterogeneous in regards to their ability to populate the memory pool once the immune response has resolved. Initial T cell activation takes place in the lymphoid organs, after which T cells migrate into the non-lymphoid tissues. The presence of memory T cells at non-lymphoid tissue sites has been shown to be critical for protection against secondary virus challenge. Our lab has previously demonstrated that during and after the resolution of the immune response to Lymphocytic choriomeningitis virus (LCMV) CD8+T cells in the nonlymphoid tissues are more resistant to apoptosis than those in the lymphoid organs. This stability of T cells in the non-lymphoid tissues may be critical in ensuring protection against a secondary virus challenge. Mechanisms regulating tissue-dependent differences in CD8+T cell apoptosis were studied in an acute LCMV infection model. Virus-specific CD8+T cells from lymphoid (spleen, mesenteric lymph nodes (MLN), inguinal lymph nodes (ILN)) and non-lymphoid tissues (peritoneal exudate cells (PEC), fat-pads) were compared for expression of surface antigenic markers known to correlate with a memory phenotype. Non-lymphoid tissues were enriched in IL-7Rhi, KLRG-1lo, CD27hi and CXCR3hi virus-specific CD8+ T cells, and the presence of these antigenic markers correlated with increased memory potential and survival. Transcription factors in addition to cell surface antigens were assessed as correlates of resistance to apoptosis. Virus-specific CD8+T cells in the nonlymphoid tissues were enriched in cells expressing T cell factor-1 (TCF-1), which correlated with increased memory potential and survival. CD8+T cells in the peritoneum of TCF-1-deficient mice had decreased survival during resolution of the immune response to LCMV, suggesting a role for TCF-1 in promoting survival in the non-lymphoid tissues. As an additional mechanism, I investigated whether apoptosis-resistant CD8+T cells migrate to non-lymphoid tissues and contribute to tissue-dependent apoptotic differences. CXCR3+ CD8+T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into the peripheral tissues. The PECs expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may have recruited the non-apoptotic cells from the lymph nodes. By adoptively transferring splenic T cells into the spleen or PEC environment I showed that the peritoneal environment through a yet undefined factor promoted survival of CD8+T cells. In this study I have elucidated the mechanisms by which CD8+T cells preferentially survive in the non-lymphoid tissues. I found that non-lymphoid tissues were enriched in memory-phenotype CD8+T cells which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosisresistant CD8+T cells may preferentially migrate into the non-lymphoid tissues where the availability of tissue-specific factors may enhance memory cell survival. Few transcription factors have been identified that regulate CD8+T cell effector-memory differentiation during an immune response. In this thesis, I have also studied the mechanism by which the transcription factor Blimp-1 regulates the generation of effector and memory CD8+T cells. Blimp-1 is known to repress a large number of target genes, and ChIP (chromatin immunoprecipitation) sequencing analysis done by Dr. HyunMu Shin in the lab of Dr. Leslie J. Berg identified CD25 (IL-2Rα) and CD27 as potential targets of Blimp-1. I found that Blimp-1-deficient CD8+T cells had sustained expression of CD25 (IL-2Rα) and CD27 during peak and resolution of the immune response to LCMV. By performing adoptive transfers of CD25hi and CD27hi CD8+T cells I showed that CD25 and CD27 expression on CD8+T cells during resolution of the immune response correlates with enhanced survival. Silencing Il2rα and Cd27 expression reduced the Blimp-1-deficient CD8+T cell response, suggesting that sustained expression of CD25 and CD27 was in part responsible for the enhanced CD8+T cell response seen in the Blimp-1-deficient mice. Furthermore, our collaborator Dr. HyunMu Shin showed that CD25 and CD27 are direct targets of Blimp-1, and that Blimp-1 recruits histone modifying enzymes to Il2rα and Cd27 loci to suppress their expression during the peak of the anti-viral immune response. This study identifies one of the mechanisms by which Blimp-1 regulates the balance between generation of effector and memory CD8+T cells. In this thesis work I also studied the function of the transcription factor ROG (Repressor of GATA-3) in regulating in vivo T cell responses during both acute and chronic LCMV infection. ROG-deficient mice had increased CD8+T cell responses during an acute LCMV infection. ROG deficiency also led to the generation of memory T cells with an enhanced recall response compared to WT controls. By using LCMV-specific P14+ TCR transgenic ROG-deficient CD8+T cells these defects were shown to be T cell intrinsic. ROG-deficient mice had enhanced CD8+T cell responses and viral clearance during a persistent high dose LCMV Clone 13 infection. During chronic LCMV infection ROG-deficient mice also had increased lung pathology and mortality. The results indicate that ROG negatively regulates T cell responses and memory generation during both acute and chronic LCMV infection. The studies highlighted in this thesis elucidate the mechanisms promoting CD8+T cell survival in non-lymphoid tissues as well as transcription factormediated regulation of memory CD8+T cell differentiation. Knowledge of this will help us better understand T cell immunity after infections and may eventually help develop better vaccines.

Preclinical models of lupus indicate that T cell-B cell collaboration drives antinuclear antibody (ANA) production and sustains T cell activation. Autoreactive B lymphocytes are present in the normal repertoire but persist as ignorant or anergic cells. Mechanisms that normally limit T cell activation of autoreactive B cells remain incompletely resolved, but potentially include the absence of autoreactive effector T cell subsets and/or the presence of autoAgspecific regulatory T cells (Tregs). Several studies have addressed this issue by using experimental systems dependent on transgenic autoreactive B cells, but much less is known about the activation of autoreactive B cells present in a polyclonal repertoire. In the second chapter of this thesis, I have explored the role of effector T cells and Tregs using mice that express an inducible pseudoautoAg expressed on B cells and other antigen presenting cells (APCs). In this system, activated Th2 cells, but not naïve T cells, elicit the production of ANAs, but ANA production is severely limited by autoAg-specific Tregs. Bone marrow chimera experiments further demonstrated that this B cell activation is constrained by radioresistant autoantigen-expressing APCs (rAPC) present in the thymus as well as by non-hematopoietic stromal cells located in peripheral lymphoid tissue. Importantly, peripheral rAPC expression of autoAg induced the expansion of a highly effective subset of CD62L+CD69+ Tregs. The third chapter of this thesis focuses on the contribution of CD8+ T cells to fibrosis resulting from sterile lung injury. Type 2 effector production of IL-13 is v a demonstrated requirement in several models of fibrosis, and is routinely ascribed to CD4+ Th2 cells. However, we now demonstrate a major role for pulmonary CD8+ T cells, which mediate an exaggerated wound healing response and fibrosis through robust differentiation into IL-13-producing pro-fibrotic type 2 effectors (Tc2). Remarkably, differentiation of these Tc2 cells in the lung requires IL-21. We further show that the combination of IL-4 and IL-21 skews naïve CD8+ T cells to produce IL-21, which in turn acts in an autocrine manner to support robust IL-13 production. TGF-β negatively regulates production of IL-13 by suppressing CD8+ T cell responsiveness to IL-21. Our data illuminate a novel pathway involved in the onset and regulation of pulmonary fibrosis, and identify Tc2 cells as key mediators of fibrogenesis.

Adaptive immune response plays a key role in maintaining homeostasis of the organism. T cells use an immense repertoire of T-cell receptors (TCRs) to discriminate between self and foreign antigens with very high sensitivity. Although we have many clues outlining how an ideal TCR repertoire is selected, and a good understanding of the TCR signaling machinery, there are still some key aspects of these processes that remain controversial. The objective of this thesis is to extend our knowledge of the very proximal events of TCR signaling, with special focus on interaction of TCR coreceptors with lymphocyte-specific kinase LCK. Coreceptor-LCK interaction has been described to regulate several aspects of T- cell development and response. We observed dynamic change of this interaction in course of T-cell development. Interestingly, CD4 and CD8 coreceptors displayed differential dynamics of interaction with LCK. Our data suggest that such disparity in coreceptor- LCK interaction leads to selection of more self-reactive TCR repertoire in CD8+ T cells. Moreover, when the highly self-reactive CD8+ T cells get to the periphery, the homeostatic signals drive their differentiation towards a more tolerogenic memory-like phenotype. To finally resolve the role of coreceptor-LCK interaction in the T-cell...

Vitiligo is an autoimmune skin disease in which the pigment producing cells of the epidermis, melanocytes, are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. Previous work has identified IFNg as the central cytokine driving disease pathogenesis in both human patients and in our mouse model of vitiligo. IFNg signaling induces production of the chemokines CXCL9 and CXCL10, which trigger autoreactive T cell migration into the epidermis where effector T cells can target and destroy melanocytes. However, both IFNg and type I IFN signaling through activation of STAT1 proteins can induce transcription of the chemokines CXCL9 and CXCL10. Therefore, it seems reasonable that type I IFN signaling may also contribute to disease pathogenesis. The role of type I IFN in vitiligo is still unclear. Genome wide association studies identified multiple genes within the type I IFN pathway including TICAM1 and IFIH1 as susceptibility loci in vitiligo. One additional study reported increased epidermal staining of CD123, a marker expressed by pDCs, and the type I IFN induced gene MX1 in vitiligo patient skin. However, this study did not show any functional data to support the role of type I IFN signaling in vitiligo pathogenesis. Since the role of type I IFN in vitiligo is ill-defined, we used two different mouse models of vitiligo to functionally determine the role of type I IFN in disease by inducing vitiligo in hosts which lack the type I IFN receptor (IFNaR). In the first model, we induced vitiligo by adoptive transfer of melanocyte-specific CD8 T cells, which are activated in vivo by infection with recombinant vaccinia virus (VACV) expressing their cognate antigen. Vitiligo induction in IFNaR-deficient mice led to the development of severe disease compared to wild type mice. Acceleration and severity of disease was characterized by increased early recruitment of melanocyte-specific CD8 T cells to the skin, increased production of effector cytokines TNFa and IFNg, and reduced PD-1 expression. Increased production of IFNg by CD8 T cells in the skin of IFNaR-deficient mice led to increased expression of the chemokines CXCL9 and CXCL10 driving disease progression. IFNaR-deficient mice also displayed significantly increased VACV titters compared to wild type hosts. This data reveals a role of type I IFN in the clearance of recombinant VACV. This data also suggests that persistent VACV infection and prolonged antigen exposure in IFNaR deficient hosts is likely driving enhanced activation of melanocyte specific CD8 T cells and the subsequent development of severe vitiligo. Since melanocytes and melanoma cells express shared antigens that can be recognized by CD8 T cells, and because the development of vitiligo after melanoma immunotherapy is a positive prognostic factor for patients, we asked whether VACV vaccine therapy in IFNaR deficient mice would enhance the anti-tumor response to melanoma. B16-F10 inoculated wild type and IFNaR-deficient mice received adoptive transfer of melanocyte-specific CD8 T cells in combination with vaccinia virus expressing their cognate antigen to activate the cells in vivo. Treatment of adoptive T cell transfer and infection with VACV in IFNaR-deficient mice revealed significantly reduced tumor burden compared to wild type mice. Improved tumor regression in IFNaR-deficient hosts was characterized by increased infiltrating cytotoxic T lymphocytes and reduced PD-1 expression. These results further demonstrate that in the absence of type I IFN, hosts mount a robust cytotoxic CD8 T cell response against melanocyte/melanoma antigens and this is likely a result of persistent VACV that leads to prolonged CD8 T cell priming. As a result, IFNaR deficient hosts kill tumor cells more efficiently. To determine whether type I IFN regulates disease pathogenesis in the absence of virus infection, we generated a model of vitiligo in which bone marrow derived dendritic cells (BMDCs) pulsed with the cognate antigen were used to prime melanocyte-specific T cells in place of the viral vector. Induction of vitiligo in IFNaR-deficient hosts using BMDCs revealed no significant differences in disease score compared to wild type hosts. This data clearly demonstrates that type I IFN, in contrast to IFNg, is not required during the effector stage of vitiligo pathogenesis in mice. However, since we intentionally activate transferred melanocyte-specific CD8 T cells with VACV or BMDCs expressing their cognate antigen, our mouse models may circumvent the role of type I IFNs in initiating activation of autoreactive cells and driving autoimmunity. Type I IFN is critical for providing innate immune signals that drive the priming of autoreactive T cells through maturation of DCs by inducing antigen presentation, co-stimulatory molecule expression, and migration to the lymph nodes to encounter naïve T cells. Our mouse models of vitiligo may not capture this process. We have addressed this question by using a TLR ligand to activate BMDCs before transfer into hosts. In fact, activation of BMDCs before transfer leads to significantly enhanced vitiligo in mice and this is partially a result of type I IFN signaling on host cells. Thus, we provide evidence that type I IFNs can enhance the activation of melanocyte-specific CD8 T cells and drive autoimmunity. Collectively, our results show that type I IFN signaling has disparate effects on autoreactive T cell priming in a context dependent manner. We reveal that although type I IFN is not required for the effector phase of vitiligo in mice, maturation of DCs and subsequent type I IFN production can enhance the priming of autoreactive T cells and enhance vitiligo severity. Our studies also reveal that type I IFN is required to clear recombinant attenuated VACV infection and vaccine administration in IFNaR deficient hosts led to a robust autoreactive and anti-tumor response. These insights describing the role of type I IFN in autoimmunity and tumor immunology could have important implications for T cell dependent tumor immunotherapy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21303号 / 医博第4392号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.