Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) with characteristic multifocal lesions or ‘ plaques’ of demyelination mainly in the white matter of the brain ( involving cerebral cortex, cerebellar, brain stem and spinal cord). These MS plaques vary in size and shape, and are composed of infiltrates of lymphocytes and macrophages - which contain myelin debris. CD8 T cells are more prevalent in CNS lesions and display oligoclonal expansion. However, their role in disease remains unclear with studies showing both protective and pathogenic roles for myelin-specific CD8 T cells in the experimental autoimmune encephalomyelitis (EAE) model. Our studies have demonstrated a disease-suppressive function for CNS-specific CD8 T cells in a model where the antigen is exogenously administered in vivo and used for in vitro CD8 activation. My studies focus on probing the nature of the CD8 response elicited by endogenously presented myelin antigens in vivo utilizing a novel approach, infection with Listeria monocytogenes (LM) encoding for myelin proteolipid protein peptide PLP178-191 (LM-PLP). I show that LM-PLP infection preferentially induces PLP-specific CD8, but not CD4, T cell responses. Despite this, infection does not result in autoimmunity. In fact, routinely induced EAE is significantly ameliorated in LM-PLP-infected mice, compared to controls. Disease suppression is dependent on the presence of CD8 T cells, and the effector molecules IFN-g and perforin. CNS T cell infiltration and inflammatory responses are reduced in LM-PLP-protected mice, and CD4 T cells from LM-PLP-protected mice are less inflammatory than those from controls. Importantly, infection with LM-PLP ameliorates already established disease. My studies indicate that myelin-specific CD8 T cells induced by endogenous presentation of antigen attenuate CNS autoimmunity in multiple mouse models of EAE, implicating the potential of this approach as a novel immunotherapeutic strategy.

Memory CD8 T cells offer increased protection to immune hosts by rapidly eliminating pathogen-infected cells during re-infection. Generating and sustaining a protective memory CD8 T cell response is considered a hallmark of adaptive immunity. Extensive research has been devoted to understanding the parameters affecting memory CD8 T cell generation after infection or immunization in order to design the most effective vaccines. An accepted notion in the field is that increased protection from re-infection is afforded by the generation of a large number of memory CD8 T cells. Consecutive prime-boost immunization strategies that elicit secondary responses are often used to increase the absolute numbers of memory CD8 T cells. While parameters affecting the generation of primary memory CD8 T cells are well known, the factors influencing the development of re-stimulated secondary CD8 T cell responses remain understudied. Here, I addressed the mechanisms involved in the generation and development of secondary memory CD8 T cells. I found that the time at which primary memory CD8 T cells enter into an immune response during re-infection impacts their fate and differentiation into secondary memory CD8 T cells. Late-entry of primary memory CD8 T cells into an immune response (relative to the initiation of infection) not only facilitates expression of transcription factors associated with memory formation in secondary effector CD8 T cells, but also influences the ability of secondary memory CD8 T cells to localize within the lymph nodes, produce interleukin-2 cytokine (IL-2), and undergo robust antigen-driven proliferation. The timing of stimulation of primary memory CD8 T cells also impacts the duration of expression of the high-affinity IL-2 receptor (CD25) on secondary effector CD8 T cells and their sensitivity to IL-2 signaling. Importantly, by blocking or enhancing IL-2 signaling in developing secondary CD8 T cells, I verify the role of IL-2 in controlling the differentiation of secondary CD8 T cell responses. The data I present herein suggest that the process of primary memory-to-secondary memory CD8 T cell differentiation is not fixed and can be manipulated, a notion with implications in the design of future prime-boost vaccination approaches. Although vaccines are designed and intended to benefit a range of individuals, at times the efficacy of a vaccination regime depends on the overall health status of a host. Thus, in another portion of my thesis work I explored the extent to which obesity compromises the differentiation and maintenance of protective memory CD8 T cell responses. I found that diet-induced obesity did not impact the maintenance of pre-existing memory CD8 T cells, including their acquisition of a long-term memory phenotype (i.e., CD27hi, CD62Lhi, KLRG1low) and function (i.e., cytokine production, antigen-driven secondary expansion, and memory CD8 T cell-mediated protection). Additionally, diet-induced obesity did not influence the differentiation and maintenance of newly evoked memory CD8 T cell responses, in inbred and outbred hosts, that were generated in response to different types of systemic (LCMV, L. monocytogenes) and/or localized (influenza virus) infections. Interestingly, I found that the rate of naïve-to-memory CD8 T cell differentiation after a peptide-coated dendritic cell immunization was similar in lean and obese hosts. This suggests that obesity-associated inflammation is unlike pathogen- or adjuvant-induced inflammation, and does not influence the development of an endogenous memory CD8 T cell response. My studies reveal that the obese environment does not influence the development or maintenance of memory CD8 T cell responses that are either primed before or after obesity is established. This is a surprising notion with implications for future studies aiming to elucidate the role of obesity in susceptibility to infection and vaccine efficacy. Collectively, the data presented here further the understanding of memory CD8 T cell responses in contexts of health and disease.

Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants. In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values. Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation. Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.

Cytomegalovirus (CMV) is a β-herpesvirus that, after primary infection, establishes a life-long persistence in the human host. Up to 90% of humans are infected with CMV, that is kept under control by CMV-specific CD8+ and CD4+ T cells. In patients with an impaired cellular immunity, however, CMV infections can be life-threatening. Thus, it is vital to identify risk factors and target high-risk patients. In this thesis we have evaluated low-dose valacyclovir prophylaxis in renal transplant patients and studied CMV-specific T cell immunity in healthy and renal transplant patients. In renal transplant patients, the CMV serostatus of both the recipient (R) and the donor (D) has a major impact on the risk of developing CMV disease. In the high-risk D+/R- population, >50% are likely to develop CMV disease in the absence of prophylaxis and/or pre-emptive therapy. We have used low-dose valacyclovir prophylaxis for high-risk renal transplant patients, and graft and patient survival up to 5 years after transplantation was comparable to data reported for other prophylactic protocols. The incidence of CMV disease and graft rejection during the first year after transplantation was also comparable to that achieved with other protocols, and without the adverse effects reported for other therapies. In the D+/R+ population, with a 15-35% risk of developing CMV disease, it is important to identify those individuals that are subject to a higher risk because of risk factors other than CMV serostatus. We therefore measured several immunologic parameters in renal transplant patients and in immunocompetent individuals with latent and primary CMV infection. In patients with a primary symptomatic CMV infection, CMV-specific CD8+ T cells peaked within a month after onset of symptoms but declined rapidly. In renal transplant patients, we found that the reduction in IFNγ-producing CMV-specific CD4+ T cells at 2 months post-transplantation may predict high-grade CMV DNAemia.

Introduction: Virus-specific CD8+ T cells are crucial to suppress the viral replication in HIV infection. Their functional status is important as well. Also, the chronic nonspecific immune activation of T and B lymphocytes plays an important role in the immunopathogenesis of HIV infection. Aim of the study: To analyze the frequency and functional status of HIV-specific CD8+ T cells and the expression of nonspecific activation markers on B and T cells in HIV+ patients and to assess the effect of combined antiretroviral therapy (cART) on these parameters. Patients and methods: Our cohort included 80 HIV+ patients: 36 HIV+ patients on cART, 18 patients without therapy, in whom cART was introduced during our study, 9 patients without therapy, 10 patients with primary HIV infection, 3 long-term non-progressors and 4 patients initially on cART, in whom the therapy was discontinued. Control group consisted of 34 HIV- healthy individuals. We examined CD4+ a CD8+ T cell counts, viral load, expression of nonspecific activation markers on T cells and the frequency of HIV-specific CD8+ T cells by ELISpot method and flow cytometry using MHC tetramers and intracellular cytokine detection. Results: No significant differences in HIV-specific CD8+ T cells were found between treated and untreated HIV+ patients. The frequency...

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Introduction: Virus-specific CD8+ T cells are crucial to suppress the viral replication in HIV infection. Their functional status is important as well. Also, the chronic nonspecific immune activation of T and B lymphocytes plays an important role in the immunopathogenesis of HIV infection. Aim of the study: To analyze the frequency and functional status of HIV-specific CD8+ T cells and the expression of nonspecific activation markers on B and T cells in HIV+ patients and to assess the effect of combined antiretroviral therapy (cART) on these parameters. Patients and methods: Our cohort included 80 HIV+ patients: 36 HIV+ patients on cART, 18 patients without therapy, in whom cART was introduced during our study, 9 patients without therapy, 10 patients with primary HIV infection, 3 long-term non-progressors and 4 patients initially on cART, in whom the therapy was discontinued. Control group consisted of 34 HIV- healthy individuals. We examined CD4+ a CD8+ T cell counts, viral load, expression of nonspecific activation markers on T cells and the frequency of HIV-specific CD8+ T cells by ELISpot method and flow cytometry using MHC tetramers and intracellular cytokine detection. Results: No significant differences in HIV-specific CD8+ T cells were found between treated and untreated HIV+ patients. The frequency...

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.

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.

La reconnaissance du peptide lié au complexe majeur d’histocompatibilité (pCMH) par les cellules T CD8+ et leur récepteur spécifique (TCR) est une étape cruciale dans l’établissement d’une réponse immunitaire protectrice adéquate et de longue durée. L’engagement du TCR engendre une cascade d’évènements de signalisation qui contrôle la prolifération, la différenciation et les fonctions effectrices des lymphocytes T CD8+ (LT8). Différentes études suggèrent un rôle crucial du zinc dans la biologie des LT8, particulièrement l’observation d’un influx de zinc suivant l’engagement du TCR. De plus, la multitude de transporteurs contrôlant homéostasie du zinc au sein des LT8, couplé aux multiples rôles de cet ion dans les processus cellulaires nous a amené à concentrer notre recherche sur le rôle du zinc dans les cellules T CD8+, qui à ce jour n’est que peu décrit. ZIP13, un des 14 transporteurs contrôlant l’homéostasie du zinc chez les LT8 murins, régule l’activation, la prolifération et la différenciation de ces cellules. En effet, une diminution de l’expression de ZIP13 (ZIP13-KD) en utilisant un petit ARN en épingle à cheveux (shRNA) entraîne une activation précoce exacerbée des LT8 se traduisant par une plus grande prolifération. Ceci a été démontré in vitro dans des lignées d’hybridomes CD8+, mais également chez les LT8 primaires murins (OT-I). De plus, cette hyperactivation est suivie par une perte substantielle des cellules, démontrant un rôle pour ZIP13 de régulateur positif de la survie des cellules T CD8+. L’étude d’une modulation de l’expression de ZIP13 chez les cellules primaires OT-I in vivo dans un modèle d’infection aiguë à Listeria monocytogenes souligne des rôles additionnels de ce gène dans la différenciation des populations effectrices des LT8. Les LT8 ZIP13-KD montrent un biais de différenciation vers la population de cellules effectrices précoces (EEC) au détriment des autres populations effectrices, ainsi qu’une diminution de Ly6C dans les organes lymphoïdes secondaires (SLO). Au site de l’infection, les cellules ZIP13-KD sont enrichies en précurseur de cellules T CD8+ résidentes mémoires (pré-Trm). ZIP13 joue également un rôle dans la formation de la mémoire immunologique puisque les cellules ZIP13-KD ne forment pas de cellules T CD8+ centrales mémoires (Tcm) dans les ganglions lymphatiques. Ces observations sont, corrélées avec une diminution drastique du marqueur de surface CD62L. Ceci se traduit par des cellules OT-I ZIP13-KD formant une mémoire immunologique uniquement composée LT8 effecteurs mémoires (Tem) et de LT8 résidents mémoires (Trm). Finalement, des analyses transcriptomiques des OT-I ZIP13-KD au stade effecteur de la réponse à une infection à Lm-OVA identifient des signatures de gènes associées aux LT8 épuisés et aux cellules Tc17. Nos résultats soulignent l’importance d’une régulation stricte et précise de l’homéostasie du zinc dans les cellules T CD8+ par des transporteurs spécifiques pour une réponse immunitaire adéquate. ZIP13 est le premier transporteur étudié dans le contexte d’une infection aiguë et identifié comme crucial pour la différenciation des populations effectrices ainsi que la formation de la mémoire immunitaire, ajoutant de l’importance à la régulation du zinc dans la biologie des LT8. Une connaissance précise de l’homéostasie spatiotemporelle du zinc dans les LT8 identifierait de nouveaux mécanismes de régulation de ces cellules qui pourrait mener potentiellement au développement de nouveaux outils pour des immunothérapies à base de cellules T génétiquement modifiées. / CD8+ T cell activation via the recognition of peptide-loaded major histocompatibility complex (pMHC) by the T cell receptor (TCR) is crucial for the establishment of a long lasting and protective immune response. TCR triggering induces a cascade of signaling events controlling T cell effector function and differentiation. The multitude of genes controlling zinc homeostasis in T cells, and the observation of an influx of zinc following TCR activation point towards a link between the regulation of zinc homeostasis and CD8+ T cell biology. This growing body of evidence led us to focus our research on zinc homeostasis in T cells, which remains poorly understood. With this study, we have shown that ZIP13, one of the many transporters controlling zinc homeostasis, significantly regulates T cell activation and differentiation following activation. Knockdown (KD) of ZIP13 by shRNA led to a substantial increase in early activation, both in murine CD8+ T cell lines and primary mouse OT-I cells in vitro. Subsequently, this increase in T cell activation is followed by a massive loss of these cells days after activation, highlighting an addition role for ZIP13 in T cell survival. Study of ZIP13 in primary CD8+ T cells in an in vivo acute infection model of Listeria monocytogenes highlights new roles of ZIP13 in the control of effector T cell differentiation. ZIP13-KD in CD8+ T cells alter the normal effector cell differentiation pattern; favoring the early effector cells (EEC) subset and induces a decrease in the surface marker Ly6C in secondary lymphoid organs. At the site of infection, ZIP13-KD CD8+ T cells are enriched in resident memory T cells precursor (pre-Trm). ZIP13 has also an impact on memory T cell formation, since ZIP13-KD cells are lacking the central memory population (Tcm), through the drastic decrease of the surface marker CD62L. This results in ZIP13-KD cells at the memory stage that are mostly composed of effector memory (Tem) and resident memory (Trm) cells. Finally, transcriptomic analyses of WT CD8+ T cells and ZIP13-KD CD8+ T cells at the effector stage demonstrates ZIP13’s role in driving an exhausted T cell and Tc17 cell gene expression signature. Taken together, our results point to the importance of a strict and fine-tuned regulation of zinc homeostasis in T cells through specific zinc transporters for an effective immune response. ZIP13 is the first zinc transporter reported as essential for T cell differentiation and memory formation, adding weight to the importance of zinc regulation in CD8+ immune responses. Having a clear picture of zinc homeostasis in CD8+ T cells would reveal new mechanisms regulating the activity of these cells that could potentially lead to the development of new tools for T cell-based immunotherapy.