Human influenza is a contagious respiratory disease resulting in substantial morbidity and mortality worldwide. With the recent cases of avian influenza infections in humans and the heightened concern for an influenza pandemic arising from these infections, it is essential to understand host responses that would confer protective immunity to influenza. The cell-mediated immune responses to influenza virus play an important role during influenza infection. To analyze the specificity and diversity of memory T-cell responses, we performed a genome-wide screening of T cell epitopes to influenza A virus in healthy adult donors. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-(IFN-γ) enzyme-linked immunosorbent spot assays (ELISPOT) using peripheral blood mononuclear cells (PBMCs) from four healthy adult donors. We found that among 11 influenza viral proteins, hemagglutinin (HA) and matrix protein 1 (M1) had more T-cell epitopes than other viral proteins. The donors were not previously exposed to H5N1 subtype, but we detected H5 HA T cell responses in two of the four donors. To confirm that HA is a major target of T cell responses we also analyzed H1 and H3 HA-specific T-cell responses using PBMC of additional 30 adult donors. Fifteen out of thirty donors gave a positive response to H3 HA peptides, whereas five of thirty donors gave a positive response to H1 HA peptides. Because we detected T cell responses to the H5 HA peptides in donors without prior exposure to H5N1 subtype, we asked if cross-reactive T cells to H5 HA peptides can be attributed to a prior exposure to H2N2 subtype, the closest HA to the H5 based on their phylogeny. We compared younger donors who have no prior exposure to H2N2 subtype and older donors who were likely to be exposed to H2N2 subtype, and both groups responded H2N2 peptides at similar level, suggesting that memory T cells cross-reactive to H5 HA peptides can be generated by prior exposure to the H1N1 and H3N2 subtypes, and the exposure to H2N2 subtype is not necessary. We subsequently identified a CD4+ T cell epitope that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of HA of influenza A and the HA of the influenza B virus. A CD4+ T cell line specific to this epitope recognizes target cells infected with various influenza A viruses including seasonal H1N1 and H3N2, a reassortant H2N1, the 2009 pandemic H1N1, H5N1 and influenza B virus in cytotoxicity assays and intracellular cytokine staining assays. Individuals who have the HLA-DRB1*09 allele have ex vivo IFN-γ responses to this epitope peptide in ELISPOT. Although natural infection or standard vaccination may not induce strong T and B cell responses to this very conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4+ T cells which are cross-reactive to both influenza A and B viruses.

The generation of memory T cells following primary exposure to a pathogen is a critical feature of the vertebrate immune system which has evolved as a protective mechanism in order to defend the host against repeated assaults by the patnogen. Memory T cells are long-lived, undergo rapid proliferation upon re-activation, mediate a robust secondary response and clear the pathogen much more efficiently. These aspects have made the generation of memory T cells an attractive goal for the production of both prophylactic and therapeutic vaccines. However, the degeneracy of the T cell receptor, whereby a given T cell recognizes more than one epitope, allows the T cell to be modulated by epitope variants which could be self-ligands, ligands related to the original epitope but altered in sequence, or completely unrelated epitopes. Experiments in both mice and humans show that such cross-reactive stimulation of memory T cells results in complete, partial, or no activation of T cells, and in some cases, even alters the functional identity of the T cell (for example, T helper 1 cells start secreting IL-4, IL-5 and become part of a T helper 2 response). In the context of secondary infection of immune organisms with pathogens containing mutated or related T cell epitopes, such alterations at the cellular level translate into drastic changes in the overall clinical outcome of the infection. Thus, the presence of cross-reactive T cells in the memory population implies that the protective or pathologic nature of the secondary immune response is a consequence of the host's infection history. Although several murine models of heterologous infection resulting in altered pathological outcome have been studied, the exact immune correlates of protection versus immunopathology are still unclear. This thesis addresses this issue in dengue virus infections in humans. Dengue fever (DF) and Dengue Hemorrhagic Fever (DHF) are two disease manifestations caused by infections of humans by the dengue viruses. These are a group of 4 serologically distinct flaviviruses (D1-4) which often co-circulate among endemic populations. While primary infection with any of the four serotypes can result in the more severe clinical disease characterized by DHF, epidemiological data from several outbreaks show that 80% - 90% of DHF cases occur among individuals with secondary infection. This implies that prior immunity to dengue is actually a risk factor for developing severe disease. In these DHF cases, there are increased numbers of CD69+ CD8+ T cells in circulation, with increases observed in the frequency of epitope-specific T cells, and the serum levels of several T cell produced cytokines, chemokines, and immune receptors are highly elevated. Since the four serotypes share 65% - 75% amino acid sequence homology, the possibility that unconserved T cell epitope sequences stimulated cross-reactive responses was borne out in in vitroexaminations. In these studies, peripheral blood mononuclear cells (PBMC) and cloned T cells from both vaccinated and infected donors contained large populations of memory T cells that were cross-reactive for heterologous viral serotypes in proliferation and CTL assays. These data suggest that the severity of disease seen in DHF patients can be attributed to an immunopathologic secondary response during heterologous infection, and highlight a role for serotype cross-reactive T cells in this process. This thesis addresses the hypothesis that the recognition of the natural variants of dengue virus T cell epitopes by serotype cross-reactive CD8+ T cells of a dengue-immune donor results in an altered secondary response profile, with the changes reflected in both the quantitative and qualitative nature of the response. In order to compare the functional profile of the secondary response of dengue-immune PBMC re-activated with heterologous serotypes, we focused on a panel of 4 donors who were vaccinated with live attenuated monovalent vaccines corresponding to D1, D2, or D4 serotypes. We screened a panel of peptides predicted to bind to HLA-A*0201 for cytokine responses and identified 4 novel epitopes that were highly immunogenic in all four donors. Direct ex vivo stimulation of donor PBMC with the heterologous sequences of these epitopes also showed sizeable serotype cross-reactive T cell populations. CFSE- and intracellular staining for cytokines and chemokines showed that these cross-reactive T cells not only expanded but also produced IFNγ, TNFα, and MIP-1β. Multi-parameter staining revealed functionally diverse populations comprised of single cytokine (IFNγ+, TNFα+, MIP-1β+, double cytokine (IFNγ+TNFα+, IFNγ+MIP-1β+, TNFα+MIP-1β+, and triple cytokine (IFNγ+TNFα+MIP-1β+ secreting sub-sets. Stimulation with the epitope variants altered the magnitude of the overall response as well as the relative sizes of these sub-sets. The patterns of responses revealed the effects of epitope immunogenicity, infection history and donor-specific variability. All 4 donors showed the highest cytokine response to a -single epitope (NS4b 2353). The same two peptide variants (D2 NS4a 2148 and D3 NS4b 2343) induced the highest response in all 4 donors regardless of the serotype of primary dengue infection. Interestingly, the epitope variants which showed the highest immunogenecity in our donors corresponded to the D2 and D3 serotypes which have been documented as being more virulent as well as a viral risk factor for DHF. In one donor, the response to all peptide variants was dominated by the same cytokine sub-sets. These data suggested that the dengue-immune memory T cell repertoire was functionally diverse and underwent alterations in size after secondary stimulation. Therefore, we also investigated the effect of epitope variants on dengue-specific CD8+T cell clones isolated from vaccinated and infected donors in order to determine if epitope variants induced altered functional outcomes at the clonal level. The epitope variants functioned either as strong agonists (particularly the D2 and D3 sequences), partial agonists, or null ligands. Some variants were able to induce cytolysis but not other effector functions at low concentrations. The variant ligands also influenced the hierarchy of cytokine responses within each clone. The third part of this thesis focused on the characterization of the frequency and phenotypic profile of epitope-specific CD8+ T cells in patients with DHF and DF at different times in the disease course in order to better understand the kinetics of the response and delineate any differences between the immune profile of severe vs. moderate disease. Tetramer staining for a previously identified HLA-B*07 restricted epitope was combined with staining for activation markers (CD69, CD38, HLA-DR), homing receptors (CCR7, CD62L), and programmed death receptor 1 (PD-1). The DHF subjects had early T cell activation with higher frequencies of tetramer+CD69+ cells as compared to DF subjects, in whom T cell frequencies peaked around the time of defervescence. While each subject had a unique phenotypic profile of tetramer+ cells, there was a difference between DF and DHF subjects in terms of CCR 7 expression; all subjects expressed low levels of CCR7 during acute illness but only the DHF subjects did not show upregulation of CCR7 on tetramer+ cells during convalescence. These data suggest that there is a sustained alteration in memory phenotype in those who recovered from severe dengue disease. A majority of the tetramer+cells also expressed PD-1 during acute illness but not during convalescence. Double-staining with variant tetramers allowed us to directly visualize serotype cross-reactivity of the epitope-specific population, and showed that secondary stimulation did induce the expansion of cells with low avidity for that secondary serotype and higher avidity to the variant. Furthermore, the ratios of these sub-sets changed during the course of the response. Taken together, these studies suggest that the immune response to heterologous secondary dengue infection is mediated by a heterogeneous population of serotype-cross reactive T cells that have different functional avidities to epitope variants and is influenced by the serotype of the secondary infection as well as the prior infection history of the individual. The preferential expansion of clones which secrete IFNγ but not inflammatory MIP-1β or TNFα or a repertoire characterized by a higher ratio of cytolytic to cytokine producing clones could limit immune mediated damage while efficiently clearing the virus. This information will be useful in the design of vaccine strategies aimed at inducing protective cross-reactive responses against all 4 dengue serotypes while preventing immunopathological outcomes following secondary infection.

T cells play a central role in cellular-mediated immunity and must become activated to participate as effector cells in the immune response. The activation process is highly intricate and involves stimulation of a number of downstream signaling pathways enabling T cells to proliferate and produce cytokines that are vital for proper effector function. This increase in protein production and protein folding activity adds to the normal physiological strain on cellular machinery. One cellular compartment that has generated a mechanism to mitigate the stress induced by increased protein production is the endoplasmic reticulum (ER). In general, an increase in cellular production of proteins that overwhelms a cell’s protein folding capability can alter ER homeostasis and lead to ER stress. To counteract this stress, an adaptive cellular mechanism known as the ER stress response (ERSR) is initiated. The ERSR allows a cell to cope with normal physiological stress within the ER caused by increased protein translation. In this dissertation, we show that in vitro and in vivoT cell activation involving T cell receptor (TCR) ligation in the presence of costimulation initiates the physiological ERSR. Interestingly, the ERSR was also activated in T cells exposed only to TCR ligation, a treatment known to induce the ‘non-responsive’ states of anergy and tolerance. We further identified a key component of the downstream TCR signaling pathway, protein kinase C (PKC), as an initiator of physiological ERSR signaling, thus revealing a previously unknown role for this serine/threonine protein kinase in T cells. Therefore, induction of the physiological ERSR through PKC signaling may be an important ‘preparatory’ mechanism initiated during the early activation phase of T cells. If ER stress is persistent and ER homeostasis is not reestablished, physiological ER stress becomes pathological and initiates cellular death pathways through ER stress-induced apoptotic signaling. We further present data demonstrating that absence of functional Gimap5, a putative GTPase implicated to play a role in TCR signaling and maintenance of overall T cell homeostasis, leads to pathological ER stress and apoptosis. Using the BioBreeding diabetes-prone (BBDP) rat, a model for type 1 diabetes (T1D), we link pathological ER stress and ER stress-induced apoptotic signaling to the observed T cell lymphopenic phenotype of the animal. By depleting the ER stress apoptotic factor CHOP with siRNA, we were able to protect Gimap5-/-BBDP rat T cells from ER stress-induced death. These findings indicate a direct relationship between Gimap5 and maintenance of ER homeostasis for T cell survival. Overall, our findings suggest that the ERSR is activated by physiological and pathological conditions that disrupt T cell homeostasis. TCR signaling that leads to PKC activation initiates a physiological ERSR, perhaps in preparation for a T cell response to antigen. In addition, we also describe an example of pathological ERSR induction in T cells. Namely, we report that the absence of functional Gimap5 protein in T cells causes CHOP-dependent ER stress-induced apoptosis, perhaps initiated by deregulation of TCR signaling. This indicates a dual role for TCR signaling and regulation in the initiation of both the physiological and pathological ERSR. Future research that provides insights into the molecular mechanisms that govern ERSR induction in TCR signaling and regulation may lead to development of therapeutic modalities for treatment of immune-mediated diseases such as T1D.

Les cellules dendritiques (CD) et l'interleukine-7 (IL-7) sont deux facteurs essentiels au développement et à l'homéostasie des lymphocytes T. Une anomalie de la production ou de la signalisation de l’IL-7 est associée à un défaut de la thymopoïèse en plus d’une profonde lymphopénie. Parallèlement, la déficience des CD, entraine une rupture de la tolérance au soi menant au développement de maladies auto-immunes et une altération de l’homéostasie des Lymphocyte T. Dans le cadre de cette thèse, nous nous sommes intéressés globalement à la contribution de l’IL-7 et des CD dans la reconstitution et l'homéostasie des lymphocytes dans deux contextes thérapeutiques différents. Premièrement, il a été démontré que le traitement avec l'imatinib (médicament utilisé pour traiter la leucémie myéloïde chronique (LMC)) induisait une diminution du nombre de lymphocytes T chez les patients atteints de LMC. À partir de notre étude in vitro sur des cellules mononuclées du sang périphérique (peripheral blood mononuclear cells) (PBMC) humaines cultivées en présence d’imatinib avant d’être stimuler par l’IL-7 à différentes concentrations, nous avons mis en évidence une perturbation de la signalisation de l’IL-7 dans les lymphocytes T suite à une interférence avec l’imatinib. À l’aide de notre modèle murin traité à l’imatinib, nous avons également montré in vivo, une altération de l’homéostasie des lymphocyte T. La modulation négative de l’homéostasie des LT par l’imatinib est également accompagnée d’une diminution dramatique de la proportion des CD, ce qui pourrait également faire décliner le pool des lymphocyte T. Deuxièmement, dans un contexte de greffe de moelle osseuse (MO) syngénique, nous avons analysé la contribution de l’IL-7 produite par les CD, dans la thymopoïèse et l’homéostasie des lymphocyte T. Pour évaluer la contribution de l’IL-7 produite par les cellules hématopoïétiques dans la reconstitution et l’homéostasie lymphocytaire T, nous avons généré des chimères de MO avec une production d’IL-7 exclusivement limitée aux cellules stromales, par greffe de MO IL-7-/- dans des souris Rag -/-. Les souris Rag -/- transplantées avec des cellules de MO IL-7 - /- développent une maladie auto-immune systémique létale à 4-5 semaines post greffe. Nos résultats suggèrent fortement une contribution cruciale de l’IL-7 produite par les CSH (Cellules Souches Hématopoïétiques) dans la reconstitution immunitaire après greffe de MO. L’ensemble de nos travaux contribue à élargir la compréhension de la biologie de l'axe IL-7/ IL-7Rα et des CD dans le développement et l’homéostasie des lymphocytes T. / Dendritic cells (DCs) and interleukin-7 (IL-7) are two factors essential for the development and homeostasis of T lymphocytes. An abnormality in the production or signaling of IL-7 is associated with a defective thymopoiesis and severe lymphopenia. DCs deficiency leads to a breakdown in self-tolerance leading to the development of autoimmune diseases and impaired T cells homeostasis. In this thesis, we have focused on the contribution of IL-7 and DCs in the reconstitution and homeostasis of lymphocytes in two different therapeutic contexts. Firstly, imatinib (drug used to treat chronic myeloid leukemia (CML)) has been shown to induce a decrease of T lymphocytes number in patients with CML. From our in vitro study on human peripheral blood mononuclear cells (PBMCs) cultured in the presence of imatinib before stimulation by IL-7 at different concentrations, we demonstrated a disruption of IL-7 signaling in T cells following interference with imatinib. Using our imatinib-treated mouse model, we also showed in vivo impaired T cells homeostasis. The negative modulation of T lymphocytes homeostasis by imatinib is also accompanied by a dramatic decrease in the proportion of DCs, which could also decline T cells pool. Secondly, we used murine syngeneic bone marrow transplantation (BMT) models to study the contribution of IL-7 produced by DCs in thymopoiesis and T cells homeostasis. To assess the role of IL-7 produced by DCs in T cell reconstitution and homeostasis, we generated BM chimeras with IL-7 production exclusively limited to stromal cells, by BMT of IL-7 - / - into Rag - / - mice. Rag - / - mice transplanted with IL-7 -/- BM cells develop a lethal systemic autoimmune disease post-transplant at 4-5 weeks post-BMT. Our results strongly suggest a major contribution of Hematopoietic stem cells (HSC)-produced IL-7 in immune reconstitution after BM transplantation. Our work contributes to expanding our understanding of the biology of the IL-7 / IL-7Rα axis and DCs in T cell development and homeostasis.

Les lymphocytes T sont au coeur du système immunitaire adaptatif et leur dérégulation est à la base de pathologies. Les cellules T se développent dans le thymus et passent par de nombreuses étapes de maturations identifiables par l'expression des corécepteurs CD4+/CD8+ à la surface des cellules. À leur sortie du thymus, les cellules T sont divisées en deux sous-types principaux: les cellules T auxiliaires CD4+ spécifique aux antigènes présentés sur complexe majeur d'histocompatibilité (CMH) de classe II et les cellules T cytotoxiques CD8+ reconnaissant un antigène présenté sur un CMH-I. Toutes les cellules T proviennent d’un précurseur commun. Leur différenciation en cellule T CD4+ et T CD8+ est influencée par l'intensité et la durée de la signalisation du récepteur des cellules T (RCT) et des cytokines. Cette signalisation résulte en l’expression des facteurs de transcription ThPOK pour la différenciation de cellule T CD4+ et Runx3 pour les cellules T CD8+. Il a été démontré que ThPOK est à la fois nécessaire et suffisant pour le développement des lymphocytes T CD4+, puisque le gain et la perte de la fonction de ThPOK favorise le développement de cellules lymphocytes T CD4+ et CD8+, respectivement. Ma thèse vise à approfondir notre compréhension du choix de la lignée CD4+/CD8+ en explorant les mécanismes moléculaires de la voix de signalisation de ThPOK et du RCT. Dans cette étude, nous avons étudié l'impact d'un gain-de-fonction de ThPOK sur la différenciation des thymocytes, en utilisant trois lignées transgéniques exprimant des niveaux variables de ThPOK. Une analyse approfondie de ces transgènes chez des souris dont le RCT est restreint soit au CMH de classe I ou de classe II, a démontré que, comparés aux thymocytes restreints au CMH-II, les thymocytes restreints au CMH-I requéraient des niveaux plus importants de ThPOK pour se différencier en CD4+. L’introduction d’un transgène exprimant un niveau moins élevé de ThPOK comparé aux deux autres transgènes, mais un niveau plus élevé de ThPOK par rapport au niveau endogène dans les cellules CD4+ WT, n'induit qu'une réorientation partielle des cellules T CD8+ en CD4+, ce qui a mené à la génération, à la fois de lymphocytes T CD4+, DN (doubles négatifs) et CD8+ matures. L'analyse génotypique, plus précisément celle des cellules DN chez les souris porteuses du transgène ThPOK et dont le RCT est restreint au CMH-I, a révélé que l’inhibition des gènes spécifiques à la lignée CD8+ 2 nécessitait des niveaux d'expression différents de ThPOK comparés à ceux requis pour l’induction des gènes spécifiques à la lignée CD4+. En effet, cette étude nous a permis de démontrer que l’intensité du signal dérivé du RCT ainsi que sa spécificité pour un CMH donné jouent un rôle essentiel dans le choix de différentiation CD4+/CD8+ induit par ThPOK. Ainsi, la réorientation CD8+/CD4+ chez les souris exprimant le transgène ThPOK-H est significativement augmentée par l'amplification de l’intensité du signal dérivé du RCT dans les cellules spécifiques aux CMH-I. De plus, la fréquence des cellules CD4+ était plus élevée lorsqu’une quantité identique de ThPOK était exprimée dans des lymphocytes T spécifiques au CMH-II, suggérant qu’il existe un aspect qualitatif quant à la régulation de la différenciation des lymphocytes T CD4+ par la signalisation induite par le RCT. Nous avons également tenté d’étudier la voie de différenciation CD4+ en l’absence de ThPOK, à la suite de la perturbation physiologique de la voie de signalisation induite par le RCT, par rapport à la perte de fonction de ThPOK. Bien que nous ayons observé une réorientation des thymocytes spécifiques au CMH-II vers la lignée CD8+, aussi bien à la suite d'une délétion de Thpok, qu’à la perturbation de la signalisation RCT les deux modes de redirections semblent toutefois être différents. En effet, notre investigation a démontré qu’en l’absence de ThPOK, la signalisation induite par le RCT dans les cellules restreintes au CHM-II induit l’activation de certains gènes, suggérant ainsi leur implication dans la voie de différenciation CD4+. Ces résultats suggèrent également que la contribution de la signalisation du RTC dans la différenciation des thymocytes restreints au CMH-II ne se limitait pas à l'induction de ThPOK. Étonnamment, seul un effet synergique limité a été observé sur la différenciation des thymocytes restreints au CMH-I, lorsque Gata3, un autre facteur de transcription également induit dans les thymocytes restreints au CMH-II, et ThPOK étaient surexprimés en même temps dans ces cellules, suggérant peu de chevauchement fonctionnel entre ces deux facteurs de transcription. L’ensemble de ces résultats indique que ThPOK et la signalisation induite par le RCT fonctionnent en synergie durant le développement des lymphocytes T CD4+. / T lymphocytes are at the core of the adaptive immune system and their dysfunction is associated with several disorders and pathologies, which are at times fatal. The two main types of T-cells in mice and man are: the major histocompatibility complex (MHC) class-II-restricted CD4+ helper T-cells, and the MHC-I-restricted CD8+ cytotoxic T-cells. Developmental stages of the two types of T-cells occurs in the thymus in multiple sequential maturation stages that are identified by cell-surface CD4+/CD8+ co-receptor expression. Differentiation of the two types of T-cells in the thymus from a common precursor is influenced by the intensity and duration of signals derived from the T-cell receptor (TCR) and cytokines secreted by the thymic stromal cells. These signals lead to the activation of ThPOK or Runx/CBF transcription factors, which control the transcriptional network regulating CD4+ and CD8+ lineage fate, respectively. Studies have demonstrated that ThPOK is both necessary and sufficient for CD4+ T-cell development as gain- and loss-of-ThPOK function redirects positively selected MHC-I- and MHC-II-restricted thymocytes into CD4+ and CD8+ T-cell lineage fate, respectively. However, the role of TCR signaling and the extent to which ThPOK expression influences CD4+ lineage choice remains to be investigated. My thesis aims to elucidate the fundamental basis the CD4+/CD8+ lineage choice by exploring the molecular mechanism of action of ThPOK and TCR signaling in CD4+ lineage fate of MHC-I- and MHC-II-specific thymocytes. In this study, we have characterized gain-of-function of ThPOK in three independent transgenic mouse lines expressing varying amounts of ThPOK. Extensive analysis of the three ThPOK transgenic lines expressing MHC-I- and MHC-II-specific monoclonal TCR indicated that MHC-I-restricted, compared to MHC-II-restricted, thymocytes required significantly more ThPOK for efficient differentiation into the CD4+ lineage. Interestingly, the founder line with the lowest transgene expression, despite expressing significantly higher amounts of ThPOK compared to the endogenous levels in WT CD4+ T cells, induced a partial CD8+ to CD4+ redirection of MHC-I-restricted cells, leading to the generation of mature CD4+, DN and CD8+ T-cells in the same mouse. Lineage specific gene expression analysis, specifically in DN mature T cells from ThPOK transgenic mice expressing MHC-I-specific TCR, showed that, compared to induction of helper program, suppression of cytotoxic program required lower amount of ThPOK. Further investigation showed that TCR signal strength and MHC specificity of 4 developing thymocytes played a critical role in determining ThPOK-induced CD4+ lineage fate. While increase in TCR signal strength augmented the efficiency of ThPOK-induced CD4+ lineage choice of MHC-I-restricted thymocytes in part via endogenous ThPOK induction, it appeared to have ThPOK independent function as well as judged by significantly different CD4+ T-cell frequencies in OTI mice expressing the same amount of ThPOK but transduced quantitatively different TCR signal. Importantly, the efficiency of CD4+ lineage choice of MHC-I-specific thymocytes with augmented TCR signal strength was still significantly lower compared to the efficiency of CD4+ lineage choice of MHC-II-restricted thymocytes expressing only the transgene-encoded ThPOK suggesting a qualitative role for TCR signaling as well in CD4+ lineage choice. We then evaluated CD4+ lineage fate decision in the absence of ThPOK induction in physiologically relevant alteration in TCR signaling versus loss of ThPOK function. While we observed CD4+ to CD8+ lineage redirection of MHC-II-specific thymocytes due to Thpok-deficiency as well as lack of ThPOK induction due to disruption of TCR signaling, the two modes of lineage redirection appeared to be due to different mechanisms. Our investigation demonstrates that TCR signaling in MHC-II-restricted thymocytes induces the expression of select genes in loss-of-function of ThPOK model suggesting potential role for these genes in establishing the CD4+ helper program. These results also suggest that the contribution of MHC-II-specific TCR signaling in driving CD4+ lineage choice is not limited to Thpok induction. Interestingly, only a limited synergistic effect was observed when both Gata3, which is also induced in MHC-II-signaled thymocytes, and ThPOK were overexpressed in MHC-I-restricted thymocytes suggesting a limited functional overlap between the two transcription factors. Collectively, these data indicate that ThPOK and TCR signaling work synergistically to promote the development of CD4+ T-cells with some ThPOK independent function for TCR signaling.

Syfte: Att ta reda på hur relationen ser ut för B- respektive T-lymfocyter i biopsier av rotfyllda tänder med apikal parodontit med bedömd låggradig inflammation. Denna studie kommer även att analysera hur den inflammatoriska bilden ser ut i förhållande till beskrivna symtom i dessa biopsier. Material & metod: En pilotstudie utfördes på 10 biopsier från rotfyllda tänder med apikal parodontit med bedömd låggradig inflammation enligt Danesh et al 2019 klassificeringssystem. Biopsierna hämtades från Malmö universitets biobank. Immunhistokemisk infärgning av antigenerna CD20+ och CD3+ utfördes samt analyserades med hjälp av digitalmikroskop. Jämförelsen av symtomen från remisserna skedde efter att de histologiska resultaten sammanställts. Resultat: Det fanns fler T-lymfocyter än B-lymfocyter i 6 stycken av biopsierna. I de resterande 4 biopsierna var de lika många. I remisserna hade symtombilden inte angetts föralla biopsier. Slutsats: Enligt vår pilotstudie ser vi tendenser till att T-lymfocyter är fler än B-lymfocyter eller att de är lika många. Inga slutsatser kunde dras gällande symtombilden. Ett större material från olika remittenter krävs för definitiva slutsatser. En vidare infärgning av CD4+ samt CD8+ skulle vara intressant. / Aim: To investigate the relation between B- and T- lymphocytes in biopsies of root-filled teeth with apical periodontitis with assessed low-grade inflammation. This study will also analyse what the inflammation looks like in relation to the symptoms described in these biopsies.  Study design: A pilot study was performed on 10 biopsies of root-filled teeth with apical periodontitis with assessed low-grade inflammation according to the classification system of Danesh et al 2019. The biopsies were collected from Malmö University's biobank. An immunohistochemical staining of antigens CD20+ and CD3+ were performed and analysed by a digital microscope. A comparison of the symptoms from the referrals were performed once the histological results were compiled. Results: There were more T-lymphocytes than B-lymphocytes in 6 of the biopsies. In the remaining 4 biopsies there were an equal amount of B- and T-lymphocytes. The symptoms were not stated for all biopsies in the referrals.  Conclusion: According to our pilot study, we see tendencies that T lymphocytes are more than B lymphocytes or that they are equal. No conclusions could be drawn regarding the symptom picture. Larger material from different referrers is required for definitive conclusions. A further staining of CD4 + and CD8 + would be interesting.

La sclérose en plaques (SP) est une maladie inflammatoire du système nerveux central (SNC) affectant plus de 2,5 millions de personnes dans le monde. Bien que son étiologie soit inconnue, de nombreuses données supportent la contribution des réponses immunitaires à la maladie. La présence de leucocytes au sein des lésions de démyélinisation est un marqueur neuropathologique de la SP. Les traitements actuels diminuent les exacerbations de la SP mais échouent à arrêter sa progression. Ainsi, il est essentiel d’identifier des nouvelles voies contribuant à la pathologie de la SP. NKG2D est un récepteur co-activateur de cellules effectrices qui participent à la surveillance immunitaire. Cependant, cette voie peut contribuer à l’inflammation et aux lésions tissulaires. Le blocage ou l’élimination du NKG2D réduit la gravité de la maladie dans des modèles animaux de SP. Dans la pathologie humaine, des lymphocytes T CD4+ et CD8+ NKG2D+ sont présents dans les lésions et élevés dans le sang périphérique des patients atteints de la forme cyclique de SP durant une poussée. Au moins un ligand du NKG2D (NKG2DL) est exprimé par des oligodendrocytes dans des lésions et les lymphocytes T CD8+ causent la mort in vitro des oligodendrocytes humains de façon NKG2D dépendante. On ignore encore si d’autres cellules neurales expriment des NKG2DL et sont susceptibles à la reconnaissance par le NKG2D. Dans cette thèse, nous avons investigué la contribution de la voie NKG2D à la pathologie de la SP. La première partie présente la caractérisation de l’expression des NKG2DL au sein du SNC. Nous avons trouvé des niveaux élevés de ULBP4, mais aucun autre ligand, dans les lésions et la matière blanche d’apparence normale chez des patients SP. Nous avons identifié des déclencheurs potentiels de l’expression de ULBP4 par les astrocytes. ULBP4 soluble est détecté dans le liquide céphalo-rachidien, et des essais fonctionnels ont démontré sa capacité à renforcer la sécrétion de cytokines inflammatoires par les lymphocytes T CD8+. Dans la seconde partie, nous avons caractérisé les lymphocytes T exprimant NKG2D dans le sang de patients atteints des formes cycliques et progressives de la SP ainsi que chez des sujets témoins. Bien que nous ayons trouvé des proportions similaires, les lymphocytes T NKG2D+CD4+ de patients SP avaient un phénotype mémoire activé associé aux profils Th1 et Th1/Th17. Les lymphocytes T NKG2D+CD8+ étaient diminués chez les patients atteints de la forme cyclique. Cette population affichait une expression prédominante du granzyme B et manifestait des capacités de dégranulation envers les astrocytes exprimant ULBP4. Finalement, les analyses d’imagerie en temps réel ont révélé un rôle pour NKG2D et son ligand ULBP4 dans les interactions durables entre les astrocytes et les lymphocytes T CD8+ humains. Nos travaux identifient un nouveau mécanisme dans le dialogue entre ces types cellulaires. Globalement, ce projet de thèse présente une caractérisation en profondeur de la voie NKG2D dans la SP. De plus, il fournit de nouvelles preuves quant à l’implication de ULBP4 dans la pathophysiologie de la SP. De nouvelles investigations contribueront à élucider la validité de ULBP4 en tant que cible thérapeutique. / Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) that affects more than 2.5 million people worldwide. Despite its unknown etiology, numerous evidences point to aberrant immune responses that contribute to the typical tissue damage. Indeed, the presence of infiltrating immune cells within the characteristic focal demyelinating lesions is a pathological hallmark of MS. Current treatments, which target the immune system, generally control disease exacerbations, but have failed to stop progression. Therefore, it is essential to identify common immune pathways that contribute to MS pathology. NKG2D is a co-activating receptor of immune cells that plays a critical role in immune surveillance. Nevertheless, aberrant NKG2D-mediated responses can contribute to inflammation and tissue damage. Various studies have implicated the NKG2D pathway in MS. NKG2D blocking or depletion reduced disease severity in various EAE models, a commonly used animal model of MS. In the human pathology, NKG2D+CD4+ and CD8+ T lymphocytes have been found in MS lesions and are upregulated in the peripheral blood of RRMS patients under relapse. Moreover, at least one NKG2DL has been observed in oligodendrocytes from MS lesions, which were found near CD8+ T lymphocytes. Furthermore, in vitro studies have demonstrated NKG2D-dependent killing of human oligodendrocytes by CD8+ T lymphocytes. Whether other neural cells express NKG2DL and can thus be susceptible to NKG2D-mediated recognition was still unknown. In this thesis, we investigated further the contribution of the NKG2D pathway to the pathobiology of MS. The first part of this project consisted in the evaluation of NKG2DL expression within the CNS. We found upregulated levels of ULBP4, and no other NKG2DL, in MS lesions and normal appearing white matter from MS patients. Moreover, we identified potential triggers observed in MS lesions that could impact on ULBP4 expression. Soluble ULBP4 was also found in the cerebrospinal fluid, and functional assays demonstrated its capacity to boost inflammatory cytokines secretion by CD8+ T lymphocytes. In the second part, we performed a deep characterization of CD4+ and CD8+ T lymphocytes expressing NKG2D in blood samples from relapsing-remitting and progressive forms of MS as well as age and sex matched healthy controls. Despite finding similar proportions, NKG2D+CD4+ T lymphocytes from MS patients exhibited an activated memory phenotype associated with Th1 and Th1/Th17 responses. In contrast, NKG2D+CD8+ T lymphocytes were reduced in RRMS patients. This subset displayed a predominant granzyme B expression irrespective of the donors’ group, and exhibited degranulating capacities toward ULBP4-expressing astrocytes. Finally, live imaging analysis revealed a role for NKG2D and its ligand ULBP4 in the establishment of long-lasting interaction between astrocytes and CD8+ T lymphocytes. This provides a new mechanism involved in the dialogue between these cell types. Overall, this thesis project provides a deep characterization of the NKG2D pathway in relapsing-remitting and progressive MS patients. Moreover, it provides new evidence for the involvement of ULBP4, a specific NKG2DL, in the pathophysiology of MS. Further investigations will contribute to elucidate the validity of ULBP4 as a therapeutic target in MS.

La leucémie aigüe myéloïde est un cancer hautement létal notamment parce que le taux de rechutes est élevé, ce qui traduit l’importance du développement de nouvelles thérapies. Ces dernières peuvent tirer avantage du fait que les cellules leucémiques peuvent exprimer des antigènes qui ne sont pas exprimés par les tissus sains, soit les antigènes spécifiques aux tumeurs (TSA). À cet effet, nos collaborateurs ont découvert une source importante « d'aberrantly expressed TSA » (aeTSA) dans les régions non codantes de l’ADN. Ces aeTSA sont présentés par les molécules de CMH I et plusieurs ont provoqué la réactivité des lymphocytes T (LTs) in vitro. En plus d'être spécifiques aux cellules cancéreuses, ces aeTSA sont partagés entre plusieurs patients ce qui fait d'eux des cibles intéressantes dans le cadre d’immunothérapies. Sachant que c’est le récepteur de cellules T (TCR) qui confère la spécificité aux LTs, le but est d'isoler et de caractériser des TCR anti-aeTSA en vue de leur utilisation comme outils thérapeutiques. Pour ce faire, l’expansion de LTs CD8+ naïfs provenant de donneurs sains a été réalisée grâce à une co-culture avec des cellules dendritiques autologues chargées avec l'aeTSA d’intérêt. Les LTs CD8+ spécifiques du aeTSA ont été triés à l’aide d'un marquage dextramères et l’ARN a été isolé afin de réaliser le séquençage du TCR. Ce dernier a révélé que le répertoire de TCR anti-aeTSA est nettement oligoclonal, facilitant l'identification des séquences des chaînes α et β des clonotypes les plus abondants. En revanche, les répertoires de TCR anti-LMP2 426-434 (antigène viral) et anti-WT1 37-45 (antigène associé aux tumeurs) étaient plus diversifiés. De plus, des tests d'avidité fonctionnelle réalisés à l'aide d'ELISpot en concentrations décroissantes de peptides ont révélé que l'avidité fonctionnelle des LTs qui reconnaissent les aeTSA est similaire à celle du peptide LMP2 426-434, ce qui suggère que les aeTSA stimulent des réponses T de hautes avidités. Ensuite, la délétion du TCR endogène a été réalisée à l'aide de la technique CRISPR-Cas9, montrant plus de 90% d'efficacité. À des fins d'optimisation de protocoles, le TCR 1G4 spécifique de NY-ESO-1 a été introduit dans le locus TRAC et, simultanément, le knock-out de la chaîne α du TCR endogène a été réalisé afin de limiter les mésappariements et la compétition entre ces deux TCR. Les prochaines étapes seront d’introduire le gène codant pour le TCR spécifique d'aeTSA dans des LTs et de vérifier que les cellules éditées sont réactives envers ces aeTSA. Finalement, ce projet pourrait ouvrir la voie au ciblage d'aeTSA à l’aide de l’ingénierie du TCR pour rediriger un grand nombre de LTs envers les cellules leucémiques. / Acute myeloid leukemia is a highly lethal cancer for which effective immunotherapies are actively sought. These immunotherapies can take advantage of the fact that leukemia cells can express antigens that are not expressed by healthy tissues, namely tumor-specific antigens (TSA). In this regard, our collaborator's team has discovered an important source of aberrantly expressed TSA (aeTSA) in the non-coding regions of DNA. These aeTSAs are presented by MHC 1 molecules and can elicit T cells reactivity in vitro. In addition to being specific to cancer cells, these aeTSAs are shared between several patients, which makes them interesting targets in the context of immunotherapies. Knowing that the T cell receptor (TCR) is responsible for T cells specificity, the goal is to isolate and characterize anti-aeTSA TCRs for their use as therapeutic tools. To this end, we expanded aeTSA-specific T cells from naive CD8+ T cells obtained from healthy donors through co-culture with autologous dendritic cells loaded with the relevant aeTSA. The aeTSA-specific CD8+ T cells identified by dextramer staining were sorted for RNA extraction TCR sequencing. Amplicon sequencing reveals that the expanded anti-aeTSA TCR repertoire is markedly oligoclonal, facilitating the identification of dominant TCR α and β chains. In contrast, the anti-LMP2 426-434 (viral antigen) and anti-WT1 37-45 (tumor-associated antigen) TCR repertoires were more diverse. In addition, functional avidity tests, performed using ELISpot in decreasing concentrations of peptides, revealed that the functional avidity of T cells recognizing aeTSA is similar to LMP2 426-434 peptide, suggesting that aeTSAs stimulate high-avidity responses. Then, endogenous TCR knock-out was performed using the CRISPR-Cas9 technique, showing more than 90% efficiency. For protocol optimization purposes, the 1G4 TCR specific for NY-ESO-1 was introduced into the TRAC locus and, simultaneously, the knock-out of the α chain of the endogenous TCR was achieved in order to limit mismatches and competition between these two TCRs. The next steps will be to introduce the gene coding for the aeTSA-specific TCR into T cells and to validate that the edited cells are reactive toward these aeTSAs. Ultimately, this project could pave the way for targeting aeTSAs using TCR engineering to redirect large numbers of T cells toward leukemic cells.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

Le système immunitaire se doit d’être étroitement régulé afin d’éviter que des réponses immunologiques inappropriées ou de trop forte intensité ne surviennent. Ainsi, différents mécanismes permettent de maintenir une tolérance périphérique, mais aussi d’atténuer la réponse lorsque celle-ci n’est plus nécessaire. De tels mécanismes sont cependant aussi exploités par les tumeurs, qui peuvent ainsi échapper à une attaque par le système immunitaire et donc poursuivre leur progression. Ces mécanismes immunosuppresseurs nuisent non seulement à la réponse naturelle contre les cellules tumorales, mais font aussi obstacle aux tentatives de manipulation clinique de l’immunité visant à générer une réponse anti-tumorale par l’immunothérapie. L’un des mécanismes par lesquels les tumeurs s’évadent du système immunitaire est l’expression d’enzymes responsables du métabolisme des acides aminés dont l’une des principales est l’indoleamine 2,3-dioxygénase (IDO). Cette dernière dégrade le tryptophane et diminue ainsi sa disponibilité dans le microenvironnement tumoral, ce qui engendre des effets négatifs sur la prolifération, les fonctions et la survie des lymphocytes T qui y sont présents. Bien que la régulation de l’expression de cette enzyme ait été largement étudiée chez certaines cellules présentatrices d’antigènes, dont les macrophages et les cellules dendritiques, peu est encore connu sur sa régulation dans les cellules tumorales humaines. Nous avons posé l’hypothèse que différents facteurs produits par les cellules immunitaires infiltrant les tumeurs (TIIC) régulent l’expression de l’IDO dans les cellules tumorales. Nous avons effectivement démontré qu’une expression de l’IDO est induite chez les cellules tumorales humaines, suite à une interaction avec des TIIC. Cette induction indépendante du contact cellulaire résulte principalement de l’interféron-gamma (IFN-g) produit par les lymphocytes T activés, mais est régulée à la baisse par l’interleukine (IL)-13. De plus, la fludarabine utilisée comme agent chimiothérapeutique inhibe l’induction de l’IDO chez les cellules tumorales en réponse aux lymphocytes T activés. Cette observation pourrait avoir des conséquences importantes en clinique sachant qu’une forte proportion d’échantillons cliniques provenant de tumeurs humaines exprime l’IDO. Enfin, les lymphocytes B, qui sont retrouvés également dans certaines tumeurs et qui interagissent étroitement avec les lymphocytes T, sont aussi susceptibles à une induction transcriptionnelle et traductionnelle de l’IDO. Cette enzyme est cependant produite sous une forme inactive dans les lymphocytes B, ce qui rend peu probable l’utilisation de l’IDO par les lymphocytes B comme mécanisme pour freiner la réponse immunitaire. Nos travaux apportent des informations importantes quant à la régulation de l’expression de la molécule immunosuppressive IDO dans les cellules cancéreuses. Ils démontrent que l’expression de l’IDO est influencée par la nature des cytokines présentes dans le microenvironnement tumoral. De plus son expression est inhibée par la fludarabine, un agent utilisé pour le traitement de certains cancers. Ces données devraient être prises en considération dans la planification de futurs essais immunothérapeutiques, et pourraient avoir un impact sur les réponses cliniques anti-tumorales. / The immune system is under tight control to avoid inappropriate and excessive immunological responses. Many mechanisms allow the maintenance of peripheral tolerance and mediate attenuation of the immune response after pathogen clearance. Such mechanisms are also exploited by tumors, thereby favoring their escape from assault by the immune system. These immunosuppressive mechanisms hamper host natural immune responses against tumor cells, but also represent an obstacle to the successful clinical manipulation of the immune system in attempts to generate an anti-tumor response through immunotherapy. One immune escape mechanism used by tumors is the production of enzymes responsible for amino acid metabolism, amongst which indoleamine 2,3-dioxygenase (IDO) is of major importance. IDO degrades tryptophan, thus leading to its depletion from intracellular pools and local microenvironments. This culminates in multi-pronged negative effects on T lymphocytes neighboring IDO-expressing cells, notably on proliferation, function and survival. The regulation of IDO expression has been largely studied in antigen-presenting cells such as macrophages and dendritic cells, but its regulation in human tumor cells must still be characterized. We hypothesized that different factors produced by tumor-infiltrating immune cells (TIIC) regulate IDO expression in tumor cells. Accordingly, we have demonstrated that IDO expression is induced in human tumor cells upon interaction with TIIC. This induction is cell contact-independent, and results mainly from interferon-gamma (IFN-g) produced by activated T lymphocytes, while being antagonised by interleukin (IL)-13. Moreover, the chemotherapeutic agent fludarabine inhibits activated T lymphocyte-dependent IDO induction in tumor cells. This observation could have major clinical consequences, considering the large proportion of human cancer clinical samples expressing IDO. Finally, B lymphocytes, which interact closely with T lymphocytes and are found infiltrating human tumors, are also susceptible to transcriptional and translational IDO induction. This enzyme is however produced in an inactive form, suggesting that B lymphocytes do not exploit this mechanism to impede the immune response. In conclusion, our work brings crucial information on the regulation of the immunosuppressive molecule IDO in human tumor cells. We demonstrate that IDO expression is dependent on the nature of cytokines present in the tumor microenvironment. Furthermore, its expression is inhibited by fludarabine, a compound used to treat some types of cancer. These data should be taken into consideration in planning future immunotherapy trials and could impact anti-tumor clinical responses.