Tolerance to self proteins is largely dependent upon the deletion of immature, self-specific T and B cells in the thymus and bone marrow. Although highly efficient, the elimination of these self-reactive lymphocytes is dependent on the expression of their target antigen in these primary lymphoid organs. Many proteins, however, such as hormones, are developmentally regulated and expressed at different stages of life, while other proteins are expressed outside the thymus and marrow. To ensure self-tolerance, other mechanisms must exist to inactivate or prevent the activation of mature, potentially self-reactive lymphocytes and maintain peripheral tolerance. T cell activation requires direct recognition of a specific protein fragment, presented on the surface of an antigen presenting cell (APC), as well as the interaction between various T cell and APC surface molecules. In the absence of the costimulatory signals provided by these ligand-pair interactions and lymphokines, antigen recognition leads to T cell inactivation and tolerance to the protein. Since many autoimmune disorders appear to be based upon the aberrant activation of mature T lymphocytes, it is important to identify and understand the mechanisms of peripheral tolerance. The obvious importance of the APC in initiating the T cell immune response has led our lab to examine one of the many antigen-processing cells, the B lymphocyte. Our studies have shown that B cells are highly efficient APC and can present antigen at very low doses to cultured T cell lines. In addition, we have found that we can induce tolerance, as measured by a reduced antibody response to an immunogenic form of the protein, in naive, normal mice by targeting a foreign protein to their B cells for antigen processing and presentation. Tolerance in the treated mice can be traced to a lesion in the T cell compartment of the animals, thus suggesting that B cells can act as tolerizing APC for peripherally expressed antigens. To further explore this idea and find more direct evidence for the role of B cells in establishing peripheral tolerance, we developed a model system that would more closely resemble in vivo conditions. This thesis tests and provides additional evidence for the hypothesis that B cells are tolerizing antigen presenting cells for peripherally expressed protein antigens. Tolerance to the foreign protein human μ chain, is induced in normal recipient mice by the transfusion of splenocytes from transgenic mice that express the membrane-bound form of μ on their B cells. Tolerance is antigen-specific since the transfused recipients' antibody production to the irrelevant protein chicken IgG is not compromised. Only viable transgenic spleen cells are tolerogenic and even when human μ chain is accessible to other APCs for presentation, tolerance can be induced by the transfusion of live μ transgenic splenoctyes. These data suggested that the transfused μ chain-expressing B cells are the tolerizing APCs which was confirmed by experiments that compared the tolerizing abilities of purified B and T cells from the transgenic mice. Adoptive transfer experiments showed that the recipients' T cell response to human μ was impaired but an analysis of the isotypes produced by tolerized mice did not indicate that either helper T cell subset was specifically compromised. Splenocytes from human μ chain-secreting transgenic B cells also induce tolerance to human μ in nontransgenic mice. Although human μ chain-expressing B cells were not detected in transfused mice, the presence of measurable levels of human IgM in the sera of mice transfused with μ chain-secreting spleen cells suggests that the transfused transgenic B cells persist in their new host. In addition, the tolerizing ability of both resting and activated membrane-bound μ chain B cells was compared. Lipopolysaccharide (LPS)-activated transgenic spleen cells do not tolerize, nor do they prime for antibody to human μ, thus suggesting that the induction of costimulatory molecules on the transgenic B cells inhibits tolerance induction. To more specifically address this, human μ chain-expressing mice were bred to transgenic mice that express the costimulatory molecule, B7-1 (CD80), on their B cells. Double transgenic splenocytes, in which the B cells bear both human μ and B7-1, did not induce tolerance to human μ chain, a result that supports the idea that activated B cells are not tolerogenic. Together the data in this thesis show that resting B cells can process and present a foreign endogenous antigen in a tolerogenic manner to the immune system and suggest a role for the B cell in the maintenance of peripheral tolerance.

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.

Heterologous immunity is a mechanism by which immunological memory within an individual, developed in response to a previous infection, plays a role in the immune response to a subsequent unrelated infection. In murine studies, heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial (protective immunity) or detrimental effects (e.g. enhanced lung and adipose immunopathology and enhanced viral titers) (Selin et al., 1998; Chen et al., 2001; Welsh and Selin, 2002; Nie et al., 2010; Welsh et al., 2010). Protective heterologous immunity results in enhanced clearance of virus during a subsequent infection with an unrelated pathogen. Such is the case when mice are immunized with lymphocytic choriomeningitis virus (LCMV) and subsequently challenged with Pichinde virus (PV) or vaccinia virus (VACV) (Selin et al., 1998). However, heterologous immunity may also mediate enhanced immunopathology as mice immunized with influenza A virus (IAV) and challenged with LCMV show increased viral titers and enhanced lung immunopathology (Chen et al., 2003). The role heterologous immunity plays during infection is not limited to the murine system. In fact, there have now been several reports of enhanced immunopathology due to heterologous immunity during human infections, involving viruses such as IAV, Epstein-Barr Virus (EBV), hepatitis C virus (HCV), and dengue virus (DENV) (Mathew et al., 1998; Wedemeyer et al., 2001; Acierno et al., 2003; Nilges et al., 2003; Clute et al., 2005; Urbani et al., 2005). Interestingly, in all reported cases in humans, heterologous immunity mediated enhanced immunopathology. Upon infection with EBV the clinical presentation can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (AIM) (Crawford et al., 2006b; Luzuriaga and Sullivan, 2010) which is marked by a massive CD8 lymphocytosis. This lympho-proliferative effect in AIM was shown to be partially mediated by reactivation of cross-reactive IAV-M1 58-66 (IAV-GIL) specific CD8 memory T-cells in HLA-A2 patients reacting to the EBV-BMLF1 280 (EBV-GLC) epitope (Clute et al., 2005). Interestingly, EBV infects ~90% of individuals globally by the third decade of life, establishing a life-long infection (Henle et al., 1969). However, it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact that the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection (Hadinoto et al., 2009). Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL cross-reactive memory CD8 T-cells preferentially expand and produce cytokines to EBV antigens at high functional avidity. Additionally, they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue, where infection is thought to initiate, by CD103 expression. This protective capacity of these cross-reactive memory CD8 T-cells may be explained by a unique T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. The composition of the CD8 T-cell repertoire is a dynamic process that begins during the stochastic positive selection of the T-cell pool during development in the thymus. Thus, upon egress to the periphery a naïve T-cell pool, or repertoire, is formed that is variable even between genetically identical individuals. This T-cell repertoire is not static, as each new infection leaves its mark on the repertoire once again by stochastically selecting and expanding best-fit effectors and memory populations to battle each new infection while at the same time deleting older memory CD8 T-cells to make room for the new memory cells (Selin et al., 1999). These events induce an altered repertoire that is unique to each individual at each infection. It is this dynamic and variable organization of the T-cell repertoire that leads to private specificity even between genetically identical individuals upon infection with the same pathogens and thus a different fate (Kim et al., 2005; Cornberg et al., 2006a; Nie et al., 2010). It is this private specificity of the TCR repertoire that helps explain why individuals with the same epitope specific cross-reactive response, but composed of different cross-reactive T-cell clones, can either develop AIM or never become infected with EBV. Our results suggest that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and potentially be the first demonstration of protective T-cell heterologous immunity between unrelated pathogens in humans. Our results also suggest that CD8 T-cell immunity can be sterilizing and that an individual’s TCR repertoire ultimately determines their fate during infection. To conclusively show that heterologous immunity is actively protecting EBV-SN adults from the establishment of a productive EBV infection, one would have to deliberately expose an individual to the virus. Clearly, this is not an acceptable risk, and it could endanger the health of an individual. A humanized mouse model could allow one to address this question. However, before we can even attempt to address the question of heterologous immunity mediating protection from EBV infection in humanized mice, we must first determine whether these mice can be infected with, and build an immune response to the two viruses we are studying, EBV and IAV. We show here that these mice can indeed be infected with and also mount an immune response to EBV. Additionally, these mice can also be infected with IAV. However, at this time the immune responses that are made to these viruses in our established humanized mouse model are not substantial enough to fully mimic a human immune response capable of testing our hypothesis of heterologous immunity mediating protection from EBV infection. Although the immune response in these mice to EBV and IAV infection is not suitable for the testing of our model the data are promising, as the humanized mouse model is constantly improving. Hopefully, with constant improvements being made there will be a model that will duplicate a human immune system in its entirety. This thesis will be divided into 5 major chapters. The first chapter will provide an introduction to both general T-cell biology and also to the role of heterologous immunity in viral infection. The second chapter will provide the details of the experimental procedures that were performed to test our hypothesis. The third chapter will describe the main scientific investigation of the role of heterologous immunity in providing natural resistance to infection in human subjects. This chapter will also consist of the data that will be compiled into a manuscript for publication in a peer-reviewed journal. The fourth chapter will consist of work performed pertaining to the establishment of a humanized mouse model of EBV and IAV infection. The establishment of this model is important for us to be able to show causation for protection from EBV infection mediated by heterologous 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.

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.

DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response. Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge. The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation. One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells. Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.

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.

Les hydrocarbures aromatiques polycycliques (HAPs), tels que le benzo(a)pyrène (B[a]P), sont des contaminants environnementaux ubiquistes générés lors de la combustion de matière organique. Ces composés ont été associés au développement d'effets toxiques sur la santé humaine, notamment des effets cancérigènes et immunotoxiques, principalement liés à l'activation du récepteur aux hydrocarbures aromatiques (RAh). Parmi les cellules du système immunitaire, les lymphocytes T apparaissent comme des cibles majeures des HAPs. Des résultats antérieurs, obtenus au laboratoire, ont montré que l'activation des lymphocytes T humains en culture primaire conduit à l’augmentation de l'expression et de la fonction du RAh, suggérant la capacité accrue de ces cellules à répondre à une exposition aux HAPs. Nos objectifs sont : (1) de déterminer les effets du B[a]P sur les profils d'expression génique dans les lymphocytes humains normaux en utilisant des approches à haut débit telle que l'analyse transcriptomique sur puce à ADN, (2) d’évaluer les effets génotoxiques et immunotoxiques du B[a]P en mesurant respectivement les dommages à l'ADN induits et leurs actions immunosuppressives et (3) d’analyser la modulation de ces effets en présence d'autres HAPs. Notre travail identifie les lymphocytes T humains normaux comme un bon modèle pour étudier les effets génotoxiques et immunotoxiques des HAPs, et pour prédire les problèmes de santé humaine liés à l’exposition à ces contaminants. Il permet également de mieux comprendre la régulation par les HAPs de la réponse immune et propose de nouveaux biomarqueurs potentiels de l'exposition à ces contaminants environnementaux. / Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B[a]P), are ubiquitous environmental contaminants generated during organic matter combustion. These compounds have been associated with the development of toxic effects on human health, including carcinogenic and immunotoxic effects, mainly related to Aryl hydrocarbon Receptor (AhR) activation. Among the immune system cells, T lymphocytes appear as major targets of PAHs. Previous results, obtained in the laboratory, have shown that activation of primary human T lymphocytes leads to a functional AhR expression increase, suggesting their ability to respond to PAH exposure. Our specific aims are: (1) to determine the effects of B[a]P on gene expression profiles in human normal lymphocytes by using large-scale approaches such as microarray-based transcriptome analysis, (2) to monitor the genotoxic and immunotoxic effects of B[a]P by measuring DNA damage and immunosuppressive actions, respectively and, (3) to analyze the modulation of these effects by the presence of other PAHs. Our work propose primary cultures of activated human T lymphocytes as a good model for studying both genotoxic and immunotoxic effects of environmental contaminants such as PAHs and predicting human health issues. It also gains a comprehensive insight into the immune response regulation after PAH exposure and provides potential new biomarkers of exposure to these environmental contaminants.

Les allergies aux produits chimiques et aux médicaments constituent un problème majeur de santé publique. L’objectif de ce travail est de mieux comprendre l’interaction entre les cellules dendritiques (DC) et les lymphocytes T (LT) dans les allergies induites par les haptènes métalliques (nickel et cobalt) et les médicaments [benzylpénicilline (BP)]. En présence des signaux de danger, les DC acquièrent un phénotype dit « mature » et présentent l’antigène apprêté aux LT spécifiques de cet antigène. Les LT représentent les cellules effectrices responsables d’une manière directe ou non des symptômes observés lors des réactions allergiques. Dans un premier temps, nous montrons que le nickel est capable d’induire un ratio d’interleukines (IL) IL-23/IL-12p70 élevé dans les DC favorisant ainsi la polarisation Th17 qui est détectée chez la majorité des patients allergiques au nickel. Nous montrons aussi pour la première fois une production de l’IL-27 par les DC activées par le nickel. Nous avons ensuite montré l’implication du TLR4 et de la voie Jak-STAT dans la régulation des cytokines membres de la famille de l’IL-12. L’activation de la voie Jak-STAT est nécessaire pour la réponse Th1 en favorisant la production de l’IL-12p70 et en inhibant la production de l’IL-23 par les DC activées par du nickel. Par ailleurs, nous avons identifié et, pour la première fois, une activation du facteur de transcription NFIL-3, au sein des DC, par le nickel et le cobalt voire d’une manière plus intense avec ce dernier. D’autre part, nous avons mis en évidence l’existence d’un répertoire de LT naïfs CD4+ et CD8+, provenant de la population générale, spécifiques du nickel. L’activation de ces LT requiert les molécules du complexe majeur d’histocompatibilité (CMH) et ils présentent un faible taux de réactivité croisée avec le cobalt. Simultanément, nous avons mis en évidence la possibilité de détecter des LT naïfs CD8+ spécifiques de la BP. L’activation de ces LT dépend des molécules du CMH de classe I et du protéasome. D’une manière générale, notre travail contribue à une meilleure compréhension des mécanismes des réactions allergiques d’une part, en montrant la fine régulation des cytokines membres de la famille de l’IL-12 dans les DC et d’autre part en élucidant les mécanismes de l’immunisation contre les molécules allergisantes. / Drug and chemical allergy is a major public health concern. The aim of this work is to understand the interaction between dendritic cells (DCs) and T-lymphocytes (LT) in allergic manifestations induced by metallic haptens (nickel and cobalt) and benzylpenicillin (BP). DCs capture the antigen, start maturation, migrate to the regional lymph node and activate hapten-specific T-cells. The latter will represent the effector cells responsible directly or not for the symptoms observed during allergic reactions. We showed that nickel induced a high ratio of interleukin (IL) IL-23 compared to IL-12p70 in DCs leading to Th17 polarization as seen in allergic patients. We also showed for the first time the production of IL-27 by nickel-activated DCs. Moreover, we showed the involvement of TLR4 and Jak-STAT pathways in IL-12 cytokine family regulation. The activation of the Jak-STAT pathway seems to maintain the IL-23/IL-12p70 balance by limiting IL-23 production and promoting Th1 polarization. Furthermore, we identified for the first time the activation of NFIL-3 in DC by nickel and cobalt, more intensely with the latter. In addition, nickel-recognizing CD4+ and CD8+ naïve T-cells repertoire was identified from the general population. These positive T-cells were shown to recognize nickel in the context of major histocompatibility complex (MHC) molecules. We also showed that a low frequency of nickel-recognizing CD4+ naïve T-cells cross-reacted with cobalt. Simultaneously, we showed the possibility of detecting a naïve CD8+ T-cells repertoire for BP. The activation of these specific T-cells requires MHC class I molecule and proteasome. In resume, our work contributes to a better understanding of allergic reactions, on one hand, by studying the fine regulation of the IL-12 cytokines family in DCs and on the other hand, by clarifying the mechanisms of immunization against drugs and chemicals.

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