Le système immunitaire comporte différents mécanismes de tolérance périphérique permettant de contrôler la réponse des cellules T CD8+. Dans certaines conditions encore peu connues, des cellules T potentiellement auto-réactives peuvent contourner les mécanismes de tolérance et se différencier en cellules effectrices, capables d’attaquer différentes organes de l’organisme, dans un processus d’auto-réactivité. En utilisant une souris transgénique exprimant un antigène modèle dans les cellules bêta du pancréas, j’ai étudié deux processus fondamentaux impliqués dans la différenciation des cellules T CD8+ en réponse aux antigènes du soi.1) Rôle de la translocation des lipopolysaccharides (LPS) dans la rupture de la tolérance. Nous avons préalablement démontré dans le laboratoire que des protocoles de lympho-déplétions, tels l’irradiation, étaient capables d’induire une rupture de la tolérance périphérique dans les cellules T CD8+. L’irradiation provoque la translocation des LPS des bactéries commensales vers la circulation sanguine, ce qui induit une activation du système immunitaire inné. Mes données ont montré que la translocation des LPS était corrélée avec l’activation systémique des cellules dendritiques (DC) CD11c+, en particulier les DC CD8+, responsables de la cross-présentation des auto-antigènes pancréatiques dans les tissus lymphoïdes. Alors que le traitement par des antibiotiques avant l’irradiation permet de prévenir la translocation des LPS, l’activation des DC n’est que partiellement affectée, et le développement de l’auto-immunité résultant d’une rupture de la tolérance périphérique des cellules T CD8+ ne peut pas être empêchée par le traitement.2) Visualisation de la coopération entre cellules T CD4+ et CD8+ effectrices dans la destruction des cellules bêta pancréatiques in vivo. En utilisant la microscopie intra-vitale à 2-photons, j’ai pu analyser, pour la première fois, la dynamiques des cellules T CD4+ et CD8+ auto-réactives exprimant un marqueur fluorescent, lors de l’infiltration du pancréas et du développement du diabète auto-immun. J’ai mis en évidence que l’infiltration des cellules T était accompagnée d’un remodelage de la matrice extracellulaire du pancréas, permettant la migration dirigée des lymphocytes. De plus, j’ai montré que l’arrêt MHC classe II-dépendant des cellules T CD4+, dû à des interactions avec des cellules présentatrices d’antigène recrutées au site d’inflammation et impliquant dans certains cas également les cellules T CD8+, contribuait au maintien des fonctions effectrices des cellules T CD8+. / The immune system has evolved multiple mechanisms of peripheral tolerance to control CD8+ T cell responses. Under particular conditions that are not yet well understood, potentially autoreactive T cells may override tolerance and differentiate into effector cells capable of targeting the own components of the organism resulting in self-reactivity. Utilizing transgenic mice expressing a model antigen in the beta cells of the pancreas, I have studied two important processes involved in CD8+ T cells differentiation in response to self-antigens. 1) Role of lipopolysaccharides (LPS) translocation in the breakdown of CD8+ T cell tolerance. It has been previously shown in our laboratory that lymphodepleting protocols, such as total body irradiation, promote breakdown of peripheral CD8+ T cell tolerance. Irradiation induces translocation of commensal bacteria LPS, a potent innate immune system activator, into the bloodstream. My data demonstrated that LPS translocation correlated with systemic activation of CD11c+ dendritic cells (DC), in particular CD8+ DC, responsible for pancreatic self-antigen cross-presentation, in lymphoid tissue. While antibiotic treatment of mice before irradiation prevented LPS translocation, DC activation was only partially affected, and onset of autoimmunity and breakdown of CD8+ T cell tolerance could not be prevented.2) Intra-vital visualization of effector CD8+ and CD4+ T cell cooperation in beta cell destruction in the pancreas. Using two-photon microscopy, I have been able, for the first time, to simultaneously analyze dynamics of fluorescently tagged autoreactive CD8+ and CD4+ T cells as they infiltrated the pancreas and induced autoimmune diabetes. I found that T cell infiltration promoted extracellular matrix remodeling in the pancreas, which in turn served as a scaffold for T cell migration. In addition, I showed that MHC class II dependent arrest of effector CD4+ T cells, due to interactions with antigen presenting cells, occasionally also implicating CD8+ T cells, provided help to effector CD8+ T cells in maintaining their effector functions.

Les cellules dendritiques plasmacytoïdes (pDC) sont spécialisées dans la lutte antivirale, notamment grâce à leur capacité à sécréter des IFN de type I. Néanmoins, elles sont aussi impliquées dans Pactivation des réponses immunitaires adaptatives, et des lymphocytes T (LT) en particulier. C'est pourquoi, lors d'épisodes inflammatoires chroniques ou incontrôlés, les pDC sont à l'origine de l'initiation ou du maintien de syndromes inflammatoires et du développement de pathologies auto-immunes. Il doit donc exister des mécanismes permettant de contrôler l'activité de ces cellules. À l'aide d'un modèle in vivo d'inflammation non virale induite par l'injection d'un anticorps anti-CD3 (Ac aCD3), nous avons observé une apoptose des pDC dans différents organes lymphoïdes, et ce de façon dépendante de l'activation des lymphocytes T. De plus, nous avons pu observer que la diminution de la survie des pDC dans ce contexte inflammatoire n'était pas associée à l'orage cytokinique induit par l'efièt mitogénique de l'Ac aCD3. En revanche nos résultats montrent que les LT CD8* et la voie cytotoxique de la perforine dans ce contexte inflammatoire aigu sont responsables de la déplétion des pDC. Nous avons également étendu ces résultats à d'autres situations inflammatoires stériles comme lors de la maladie du greffon contre l'hôte. Ces données suggèrent que cette voie de régulation pourrait être utilisée à des fins thérapeutiques, afin de contrôler la survie des pDC impliquées dans la physiopathologie de syndromes auto-immuns comme le lupus érythémateux disséminé, le psoriasis, la sclérose en plaques ou encore le diabète de type I. / Plasmacytoid dendritic cells (pDC) are specialized in type I interferons (IFN-I) secretion to control viral infections. However, these cells can also activate adaptive immune responses, and polarize T cells. Indeed, during chronic or uncontrolled inflammatory episodes, pDC can induce or maintain inflammatory syndromes and autoimmune diseases. So some mechanisms should exist to control the fonction of these cells. In an in vivo modcl of non viral inflammation induced by the injection a CD3-specific antibody (aCD3 Ab), we could observed pDC's apoptosis dependent of T cell activation in different lymphoid organs. Moreover, we could observe that this depletion of pDC was not associated with the cytokinic storm induced by the mitogenic effect after aCD3 Ab treatment. On the other hand our data shovved that CD8+ T cells and the perforin pathway in this acute inflammatory context are responsible for pDC depletion We also obtained the same results in other non viral inflammation settings such as graft versus host disease. Overall, these data suggesi that this regulation pathway could be used for therapeutic purposes, to control pDC survival and avoid their involvement in the physiopathology of autoimmune disorders like systemic lupus erythematosus, psoriasis, multiple sclerosis or type I diabetes.

Abstract : CD8 + T cells can be programmed in their naïve state with pro-inflammatory cytokines such as IL-15 and IL-21. IL-15 induces the proliferation of CD8 + T cells and allows the generation of memory cells. IL-21 programs CD8 + T cells to become more cytotoxic while retaining a memory type phenotype. In the laboratory, we studied the effect of these two cytokines in different contexts by using the mouse model MHC-I-restricted Pmel-1 transgenic TCR specific to the melanoma-derived gp10025-33 antigen, which is also expressed by normal melanocytes. First, we elucidated the effect of IL-15 on CD8 + T cells in the Pmel-1 transgenic model lacking the protein Suppressor of cytokine signaling 1 (SOCS1). SOCS1 is a critical regulator of T cell homeostasis. We have found that these mice have CD8 + T-cells expressing surface proteins characteristic of memory T cells (CD44, Ly6C, CD122 and CD62L). However, these cells decrease the expression of the TCR and increase that of CD5, indicative of TCR activation in vivo. When stimulated in vitro, these cells displayed a highly cytotoxic phenotype but very low proliferation. Adoptive cell transfer studies in Rag1 - / - mice showed that these cells can undergo homeostatic proliferation under lymphopenic conditions. This proliferation was characterized by severe inflammatory lesions in the skin, extremities and eyes. This study demonstrates the importance of IL-15 and SOCS1 in the regulation of self-reactive cells that can be activated under lymphopenic conditions and can cause autoimmune diseases. Second, we studied the synergistic effect of IL-15 and IL-21 in native CD8+ T cells for cancer immunotherapy. We used the mouse melanoma model B16-F10 (B16) which expresses very weakly MHC-I molecules. In parallel, we studied the effect of NOD-like receptor CARD domain containing 5 (NLRC5) overexpression, the trans-activator of MHC-I genes, in B16 cells in order to increase their immunogenicity and restore anti-tumor immunity. We generated stable lines of B16 cells expressing NLRC5 (B16-5); the co-stimulatory molecule of T cells, CD80 (B16-CD80), or both (B16-5 / 80). The over-expressing NLRC5 cells positively regulated the MHC-I and LMP2, LMP7 and TAP1 genes. B16-5 cells efficiently presented gp10025-33 to CD8+ Pmel-1 T cells and induced their proliferation. This proliferation was very robust when Pmel-1 naive cells were pre-stimulated with IL-15 and IL-21 prior to activation. In the presence of CD80, B16-5 cells stimulate Pmel-1 cells even without the addition of gp100, indicating that NLRC5 facilitates the treatment and presentation of endogenous tumor antigens. During subcutaneous implantation, B16-5 cells showed a significant reduction in tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that tumor cells expressing NLRC5 generated an anti-tumor immunity. This response is dependent on CD8 + T cells since in mice depleted of these cells, B16-5 cells formed large subcutaneous and pulmonary tumors. Finally, immunization with irradiated B16-5 cells allowed anti-tumor protection during challenge of parental B16 cells. Collectively, our results indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity. / Les cellules T CD8+ peuvent être programmées à leur état naïf avec des cytokines pro-inflammatoires telles que l’IL-15 et l’IL-21. IL-15 induit la prolifération de cellules T CD8+ et permet la génération de cellules T CD8+ mémoire. IL-21 programme les cellules T CD8+ à devenir plus cytotoxiques tout en conservant un phénotype de type mémoire. Dans le laboratoire, nous avons étudié l’effet de ces deux cytokines dans différent contextes en utilisant le modèle de souris transgénique Pmel-1 qui possède des récepteurs de cellules T (TCR) spécifiques envers le peptide gp10025-33, exprimé par des cellules de mélanome et aussi par des mélanocytes. Premièrement, nous avons élucidé l’effet de l’IL-15 sur les cellules T CD8+ dans le modèle transgénique Pmel-1 déficient dans la protéine Suppressor of cytokine signaling 1 (SOCS1). SOCS1 est un régulateur critique de l’homéostasie de lymphocytes T. Nous avons trouvé que ces souris ont de cellules T CD8+ exprimant des protéines de surface caractéristique de cellules T mémoire (CD44, Ly6C, CD122 et CD62L). Cependant, ces cellules diminuent l’expression du TCR et augmentent celle de CD5, ce qui témoigne d’une activation du TCR in vivo. Lorsque stimulées in vitro, ces cellules montrent un phénotype hautement cytotoxique mais une prolifération très faible. Lorsque nous avons fait des études de transfert cellulaire adoptive dans de souris Rag1-/-, ces cellules ont proliféré de façon importante causant des lésions inflammatoires sévères dans la peau, les extrémités et les yeux. Cette étude démontre l’importance de l’IL-15 et de SOCS1 dans la régulation de cellules auto-réactives qui peuvent être activées sous des conditions lymphopéniques et qui peuvent causer de maladies auto-immunitaires. Deuxièmement, nous avons étudié l’effet synergique d’IL-15 et d’IL-21 dans les cellules T CD8+ naïves pour l’immunothérapie du cancer. Nous avons utilisé le modèle B16-F10 (B16) de mélanome de souris qui exprime très faiblement des molécules de CMH-I. En parallèle, nous avons étudié l’effet de la surexpression de NOD-like receptor CARD domain containing 5 (NLRC5), le trans-activateur de gènes de CMH-I dans les cellules B16, dans le but d’augmenter leur immunogénicité et de restaurer l’immunité anti-tumorale. Nous avons généré des lignées stables de cellules B16 exprimant NLRC5 (B16-5); la molécule co-stimulatrice de cellules T, CD80 (B16-CD80), ou les deux (B16-5 / 80). Les cellules sur-exprimant NLRC5 ont régulé positivement de manière constitutive les gènes MHC-I et LMP2, LMP7 et TAP1. Les cellules B16-5 ont efficacement présenté gp10025-33 aux cellules T CD8+ Pmel-1 et ont induit leur prolifération. Cette prolifération a été très robuste lorsque les cellules naïves Pmel-1 étaient pré-stimulées avec IL-15 et IL-21 avant leur activation. En présence de CD80, les cellules B16-5 stimulent les cellules Pmel-1 même sans l'addition de gp100, ce qui indique que NLRC5 facilite l’apprêtement et la présentation des antigènes tumoraux endogènes. Lors de l'implantation sous-cutanée, les cellules B16-5 ont montré une réduction significative de la croissance tumorale chez des hôtes C57BL/6, mais pas chez des hôtes immuno-déficients, ce qui indique que les cellules tumorales exprimant NLRC5 ont provoqué une immunité anti-tumorale. Cette réponse est dépendante de cellules T CD8+ puisque chez les souris déplétées de ces dernières, les cellules B16-5 ont formé de grandes tumeurs sous-cutanées et pulmonaires. Enfin, l'immunisation avec des cellules B16-5 irradiées a permis une protection anti-tumorale lors de la réimplantation des cellules B16 parentales. Collectivement, nos résultats indiquent que NLRC5 pourrait être exploité pour restaurer l'immunogénicité tumorale et pour stimuler l’immunité anti-tumorale protectrice.

The ability of our immune system to mount a response against non-self-antigens legitimates the semi-allogenic fetus as a target for maternal immune attack. Yet, in a normal pregnancy the fetus stays well protected due to the concerted action of several diverse mechanisms which either suppress the fetal allogenicity or spatio-temporally inhibit maternal immune cells’ growth and functions. One such factor which aids in the establishment, progression and maintenance of pregnancy is the 28 kDa dimeric sialylated glycoprotein Glycodelin-A (GdA). Synthesized by the endometrium and decidua, this protein has myriad functions, the most important being that of immunosuppression. GdA is inhibitory to all hematopoietic cells and also induces programmed cell death in activated T cells and monocytes via the intrinsic mitochondrial pathway. In the Introductory chapter of this thesis, details about GdA and the other isoforms of the glycodelin family of proteins have been presented which highlight the involvement of glycodelins in primate pregnancy, with emphasis on GdA and its pleiotropic functions associated with reproduction in females. Activated T-lymphocytes against paternal antigens are found in the uterine compartment and in the maternal circulation throughout pregnancy. Activated CD8+ T-lymphocytes have been reported to pre-dominate the uterine T-lymphocyte population during pregnancy and unlike the CD4+ T cells, are retained until term. Studies show that activated CD8+ T-lymphocytes are necessary for the establishment and progression of early pregnancy. However, how these lymphocytes harbouring cytotoxic activity are regulated at the later stages of pregnancy is poorly defined. We attempted to uncover a possible mechanism of regulation of CTL (cytotoxic T lymphocyte) activity (if any) during primate pregnancy by GdA. In the absence of established human CD8+ T cell lines, we first standardized the generation of CTLs in-vitro from hPBMCs (human peripheral blood mononuclear cells) by alloactivating them with an ovarian carcinoma cell line OVCAR-3 utilized as a mimic of an allograft. The details of the rationale behind using this method for generating CTLs and the alloactivation methodology have been put together in the Chapter 1 of this thesis. The activation of hPBMCs was confirmed by the surface expression of an early activation marker CD69 and tritiated thymidine incorporation. Differentiation of CD8+ T cells into effector cells was confirmed by the upregulation of perforin and granzyme transcripts by real time RT-PCR analysis. Target-cell specific cytolytic activity of the CTLs was assessed by using a cytotoxicity measurement assay- JAM test, details of which also form a part of chapter 1. Having generated effective CTLs in vitro, we tested the effect of GdA on CTL activity. Our findings, on the effect of GdA on CTLs have also been discussed in the Chapter 1. We observed that the cytolytic activity of CTLs was significantly reduced by GdA treatment albeit at a dose three to four times higher than that required for inhibiting CD8+ T cell proliferation, implying that a mechanism of temporal regulation of CTL activity operated at the feto-maternal interface, thereby contributing to the establishment and progression of pregnancy. Interestingly, in our quest to uncover the mechanism of inhibition of CTL activity by GdA, we found that the inhibition of proliferation was comparable in both CD4+ and CD8+ T-lymphocytes at all dosages of GdA, but unlike CD4 + T cells CD8 + T cells were resistant to GdA-induced apoptosis even at high dosage of GdA. Hence we could rule out that the loss of CTL activity upon GdA treatment was due to CD8+ T cell death. Further, we assessed the functional competence of alloactivated CTLs by quantitating the mRNA transcripts of key cytolytic molecules; perforin and granzyme B, in GdA treated alloactivated hPBMCs and found that there was a significant reduction in the mRNA of these cytolytic molecules. Additionally, we also found that GdA treated CD8+ T cells exhibited impaired release of the cytolytic molecules by the process of degranulation, measured by the surface exposure of LAMPs (Lysosome associated membrane proteins) on the surface of cells by flow cytometry and as seen by the retention of perforin protein in them assessed by intracellular staining and flow cytometry. Intrigued by the observations, we probed for the regulators of perforin and granzymes in CTLs. EOMES (Eomesodermin) and T- Bet are well known transcription factors which control the differentiation of CD8+ T cells into effector and memory cell CD8+ T cell type. Interestingly we found that the expression of EOMES was significantly reduced in activated GdA treated hPBMCs, both at the transcriptional and translational level, however T-Bet did not show any variation in expression upon GdA treatment. All the above findings have been compiled in Chapter 2 along with our studies on the possibility of GdA to induce a tolerogenic phenotype in T cells. We found there was no difference in the mRNA level and surface expression of CD103 and CD28 in alloactivated PBMCs, while FOXP3 mRNA did not show any variation upon GdA treatment, indicating that GdA does not induce a tolerogenic phenotype in T-lymphocytes, further confirming our data that the decreased cytolytic activity of CTLs upon GdA treatment was not due to tolerance but due to impaired function Interestingly, IL-2/IL-2R signaling is known to directly regulate perforin and granzyme expression as well as it plays a role in the expression of T-Bet and EOMES. Therefore, as a read out of IL-2 signaling we checked for the surface expression of the high affinity IL-2R subunit, CD25. As expected, CD25 expression was more pronounced in CD4+ T cells and consistent with published reports in literature that GdA suppresses IL-2 synthesis, we also observed a significant reduction in the CD25bright population in both the T cell subsets (CD4+ and CD8+) upon GdA treatment (addressed in Chapter 3). This finding supports a mechanism of action of GdA, wherein the cytolytic activity of CTLs is compromised by the downregulation of EOMES, triggered by the low IL-2 levels. This translates to aberrant synthesis of key cytolytic molecules perforin and granzyme B, leading to low efficiency CTLs, which are further disabled by defective degranulation machinery induced by GdA. We did not look into the mechanistic aspects of how GdA suppresses degranulation, which can be addressed later as a part of another study. Building up on our observations, and taking cues from existing literature, that IL-2 regulates the expression of pro and anti-apoptotic protein levels within activated cells, we looked at the expression profile of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in activated PBMCs upon GdA treatment. There was a significant reduction in the total mRNA and protein level of Bcl-2, while a very significant increase in Bax mRNA and protein was observed. Chapter 3 of the thesis also presents this data and explains a plausible mechanism of the inhibitory effect of GdA on T-lymphocytes. In Chapter 2, we have also addressed the probable reasons for the differences in the responses of CD4+ and CD8+ T-lymphocytes to GdA. Interestingly, surface glycan profile of CD4+ and CD8+ T-lymphocytes upon activation and the surface expression of the most probable receptor for GdA i.e. CD7 was comparable in both the T cell subsets, indicating that possibly the downstream signaling events leading to GdA-induced apoptosis and not the surface binding of GdA may vary in CD4+ and CD8+ T-lymphocytes, due to which we observed a difference in the extent of apoptosis induced in these cell types by GdA although the inhibition of proliferation in both the subsets was comparable. In summary, this study is the first to provide evidence for a possible mechanism of temporal regulation of CTL activity at the feto-maternal interface, where activated CD8+ T cells are abundantly present. We can say with much confidence that binding of GdA to T-lymphocytes causes sub-optimal IL-2 signaling which translates into reduced expression of EOMES and hence downregulation of perforin and granzyme B, leading to impaired CTL activity in CD8+ T-lymphocytes, which is further weakened by the impaired release of the cytolytic molecules from them. Insufficient IL-2 signaling in the presence of GdA can also be a cause of inhibition of proliferation in T-lymphocytes, while the resulting decrease in anti-apoptotic protein Bcl-2 and increase in pro-apoptotic protein Bax seem to contribute to the induction of apoptosis in CD4+ T cell. It will be interesting to explore the mediators involved in the IL-2 signaling pathway that are differentially regulated in CD4+ and CD8+ T cells which confer resistance in CD8+ T cells to GdA-induced apoptosis and also the mechanism by which GdA regulates the degranulation of cytolytic vesicles in CTLs needs to be worked out.

Taquizoítos de T. gondii esterilizados por radiação ionizante é uma vacina interessante para induzir uma imunidade semelhante à infecção, mas sem a formação de cistos. Neste estudo avaliamos as populações celulares do sangue e do baço induzidas pela imunização, a resposta imune humoral, celular e a proteção após desafio com parasitas viáveis. Camundongos foram imunizados com taquizoítos de T. gondii irradiados por v.o. ou i.p.. Os animais foram desafiados com 10 cistos da cepa ME-49 ou VEG por via oral e apresentaram altos níveis de proteção com baixa carga parasitária. Camundongos imunizados por i.p. e v.o. apresentaram anticorpos específicos no soro e o aumento das populações de células B, plasmócitos, células TCD4+ e TCD8+ tanto no sangue como no baço. As células esplênicas de camundongos imunizados por i.p. mostraram a produção de IL-10, IFN-γ e IL-4. Células TCD4+ e células B do baço de camundongos imunizados por i.p. proliferaram após a estimulação com antígeno. A imunização com esse modelo vacinal induziu uma resposta imune mediada com células B, TCD4+ e TCD8+, com aumento da resposta imune humoral e celular que são necessárias para proteção do hospedeiro após uma infecção. Essa resposta imune induzida é uma resposta semelhante a uma infecção natural, sendo assim o desenvolvimento de vacinas utilizando a radiação ionizante como uma ferramenta, pode ser um modelo atrativo e eficiente para testar novos imunógenos no futuro. / Tachyzoites of T. gondii sterilized by ionizing radiation is an interesting vaccine for inducing immunity to infection similarly but without the formation of cysts. In this study we evaluated the cell populations from blood and spleen induced by immunization, the humoral immune response, cellular and protection after challenge with viable parasites. Mice were immunized with irradiated tachyzoites of T. gondii by v.o. or i.p.. The animals were challenged with 10 cysts of the ME-49 or VEG strain orally and showed high levels of protection with low worm burden. Immunized mice by i.p. and v.o. present specific antibodies in the serum and increased populations of B cells, plasma cells, CD4+ and CD8+ T cells in blood and spleen. The spleen cells of immunized mice by i.p. showed the production of IL-10, IFN-γ and IL-4. CD4+ T cells and B cells in the spleen of immunized mice i.p. proliferated upon stimulation with antigen. The immunization with this vaccine model induced an immune response mediated by B cells, CD4+ and CD8+ with increased humoral and cellular immune response are necessary for host protection after infection. This induced immune response is a response similar to natural infection, therefore the development of vaccines using ionizing radiation as a tool, can be an attractive and efficient model for testing new immunogens in the future.

Les rythmes circadiens contrôlent de nombreux aspects de la physiologie chez les mammifères. Parmi ces processus physiologiques, les horloges circadiennes contrôlent entre autres la réponse immunitaire innée et adaptative. Depuis des décennies, de nombreuses études ont commencé à couvrir ce sujet. Cependant, le contrôle circadien de la réponse adaptative reste peu étudié. Dans le cadre de ce projet de recherche de doctorat, nous avons exploré le rôle des rythmes circadiens dans la réponse des lymphocytes T CD8 à la présentation antigénique par des cellules dendritiques. Des travaux du laboratoire publiés par Erin E. Fortier et al. ont mis en évidence une différence jour/nuit dans l’expansion des lymphocytes T CD8 dont le récepteur T (TCR) est spécifique au complexe KbOVA exprimé par les cellules dendritiques ainsi que dans le nombre de lymphocytes T CD8 CD44hi IFN+ spécifiques pour l’antigène de l’ovalbumine (OVA)1. En effet, la réponse des lymphocytes T CD8 est plus importante après une vaccination faite en milieu de jour (zeitgeber time (ZT) 6) par rapport à une vaccination faite en milieu de nuit (ZT18). Cependant, ces travaux de recherche n’ont pas démontré le rôle des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Mes travaux de recherche de doctorat ont dans un premier temps confirmé l’implication des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Nous avons ensuite démontré la contribution des horloges circadiennes des cellules dendritiques ainsi que le rôle essentiel des horloges circadiennes des lymphocytes T CD8 dans ce rythme de réponse. De plus, nous avons montré que ce rythme avait un impact sur la capacité des lymphocytes T CD8 à contrôler une infection bactérienne (Listeria monocytogenes). En effet, les variations jour/nuit de la charge bactérienne dans la rate et le foie des souris de type sauvage étaient abolies dans les souris déficientes pour le gène des horloges circadiennes Bmal1 dans les lymphocytes T CD8. Dans un second temps, nous avons mis en évidence suite à l’analyse du transcriptome des lymphocytes T CD8 de souris naïves collectés toutes les 4 heures sur 48 heures que ces cellules sont plus enclines à être activées le jour et à l’opposé plus enclines à être inhibées la nuit. Ces résultats corrèlent avec le rythme de réponse des lymphocytes T CD8 à la vaccination. Dans un dernier temps, nous avons confirmé que le rythme de réponse des lymphocytes T CD8 à la présentation antigénique agissait de manière précoce dans l’activation de ces cellules. Pour cela nous avons irradié des souris de type sauvage et nous les avons ensuite reconstituées avec une moelle osseuse contenant 1% de précurseurs de cellules OT-I (lymphocytes T CD8 spécifiques au complexe KbOVA, exprimant la chaîne du TCR V5. Après vaccination de ces souris en milieu de jour subjectif (circadian time (CT) 6) ou en milieu de nuit subjective (CT18), nous avons observé un rythme de la réponse des lymphocytes CD8 pour différents marqueurs impliqués dans la réponse précoce des lymphocytes T CD8, telles que CD69, CD5, IRF4, et la phosphorylation de S6 (marqueur de l’activité de mTOR) et de AKT. L’ensemble des recherches de mon doctorat ont permis de mettre en évidence un tout nouveau mécanisme impliquant les horloges circadiennes dans la réponse des lymphocytes T CD8 en réponse à une vaccination. Une meilleure compréhension du fonctionnement des horloges circadiennes dans la réponse immunitaire permettra de mettre en place des nouveaux traitements personnalisés en fonction du type d’infection et du type de maladie, délivré à un certain moment de la journée dans le but d’améliorer l’efficacité tout en réduisant les effets secondaires. / Circadian rhythms control various aspects of the physiology in mammals. Among these processes, circadian clocks control the innate and the adaptive immune responses. Since few decades, numerous studies started to uncover the role of the circadian system in the immune response. However, the circadian control of the adaptive immune response remains poorly studied. My PhD work focused on the circadian control of the CD8 T cell response to vaccination by dendritic cells. Erin E. Fortier et al. published in The Journal of Immunology that wild type mice vaccinated with antigen presenting cells loaded with the OVA peptide present a day/night variation of the CD8 T cell response after a vaccination done during the middle of the day (zeitgeber time (ZT) 6) compared to a vaccination done during the middle of the night (ZT18)1. Indeed, the proportion of CD8 KbOVA+ cells and the proportion of CD8 CD44hi IFN+ T cells were higher during the middle of the day than the middle of the night. However, this work showed a diurnal but not a circadian rhythm, that remained to be confirmed. The first part of my PhD research confirmed a role of the circadian system in the rhythm of the CD8 T cell response to vaccination. We showed a contribution of the dendritic cell clock as well as an essential role of the CD8 T cell clock. Moreover, this rhythm impacts the ability to control an infectious challenge as shown by a circadian variation in bacterial load (Listeria monocytogenes) in wild type but not in mice lacking clock in mature CD8 T cells. The second part of my research focused on the analysis of the transcriptome of CD8 T cells from naive mice collected every 4 hours over 48 hours. We showed that CD8 T cells are more prone to be activated during the day and at the opposite are more prone to be inhibited during the night. These results are correlated with the rhythm of the CD8 T cell response to vaccination. Finally, during the third part of my PhD, we confirmed that the rhythm of the CD8 T cell response to antigen presentation was acting at the early stage of the CD8 T cell activation. We used wild type mice reconstituted with bone marrow cells containing 1% of OT-I precursor cells (CD8 T cells restricted for the KbOVA complex, expressing the receptor chain of the TCR for the OVA peptide, V5) and vaccinate these mice during the middle of the subjective day (CT6) or during the middle of the subjective night (CT18). At the early stage of the CD8 T cell response to vaccination, we showed a higher expression of several activation markers after a vaccination done during the middle of the day than during the middle of the night, such as CD5, CD69, IRF4 and the phosphorylation of S6 (marker of the mTOR activity) and AKT. Altogether, my PhD work highlights a new mechanism involving the circadian system in the control of the immune response. A better understanding of how circadian clocks act on the immune response will allow implementing new treatment strategies in order to increase their efficacy as well as to decrease side effects.

Les myosites sont des maladies auto-immunes, caractérisées par des atteintes musculaires et extra musculaires. Le diagnostic des myosites peut être difficile et nécessite de l’expertise, afin d’éviter l’administration de thérapie inapproprié. Les mécanismes impliqués au cours des myosites sont peu connus. Notre but était de décrire le profil immunitaire des patients, afin d’identifier des biomarqueurs. Nous avons utilisé un panel de 36 marqueurs pour caractériser les PBMC issus de patients actifs (MIs, SAS anti-Jo1, myopathies anti-SRP et anti-HMGCR) et de sujets sains par cytométrie de masse combiné au « barcoding ». Tout d’abord, nous avons mis au point une procédure technique pour la détection simultanée de cibles extracellulaires et intracellulaires. En utilisant différents outils bio-informatiques, nous avons isolé une fréquence de lymphocytes CD8+T-bet+ > 51.5% comme étant un biomarqueur spécifique de la MIs en comparaison aux autres myosites, avec une sensibilité de 94,74% et une spécificité de 88,46%. De plus, nous avons identifié un profil immunitaire CD8+T-bet+ CD57- activé, potentiellement capable de prolifération et de maintien de mécanismes auto-immuns chez les patients atteints de MIs. Chez les patients anti-Jo1, nous avons observé une dérégulation de l’homéostasie des lymphocytes B, caractérisée par une diminution des lymphocytes B mémoires circulants. La présence de ces derniers dans le muscle des patients suggère qu’ils se nichent dans le muscle afin d’éviter l’action des immunosuppresseurs. Ces travaux ont permis l’identification de biomarqueurs et de phénotypes cellulaires potentiellement impliqués au cours de la MIs et du SAS anti-Jo1. / Myositis is an autoimmune disease characterized by muscular and extra-muscular disorders. In the early stages of the disease, the diagnosis of myositis can be misleading and requires expertise, in order to avoid the administration of inappropriate treatment. The mechanisms involved in these diseases are poorly understood. Our aim was to describe the immune profile specific to each patient group, in order to identify biomarkers that may be useful for diagnosis and management of patients. We used a panel of 36 markers by mass cytometry to characterize PBMCs derived from active patients (sIBM, anti-Jo1 ASyS, anti-SRP and anti-HMGCR myopathies) and healthy subjects. First, we developed and optimized a technical procedure for the simultaneous detection of extracellular and intracellular targets by mass cytometry. Using different bioinformatics tools, we isolated a frequency of CD8 +T-bet + cells > 51.5% as a specific biomarker for sIBM compared to other myositis, with a sensitivity of 94.74% and a specificity of 88. , 46%. In addition, we identified an activated CD8 + T-bet + CD57- immune profile, potentially capable of proliferation and the maintenance of autoimmune mechanisms in patients with sIBM. In anti-Jo1 patients, we observed a dysregulation of B cell homeostasis, characterized by a decrease in circulating memory B cells. The presence of the latter in the muscle of patients suggests that they nest in the muscle to avoid immunosuppressants. This work allowed the identification of a biomarker that could enhance the diagnosis of MIs compared to other myositis and the identification of cells potentially involved during sIBM and anti-Jo1 ASyS.

Cancer is the second most common cause of death in the world, and the number of people with the disease increases each year. The therapy of the disease currently stands on four pillars; surgery, chemotherapy, radiotherapy, and immunotherapy. Through the past few years, immunotherapy has become the fastest developing treatment modality. However, despite its unprecedented efficacy in some patients, the majority of patients still does not respond to the therapy. Therefore, there is a need to investigate the mechanisms that make immunotherapy inefficient. Cell-based cancer immunotherapy is the treatment modality which uses live ex vivo-produced tumor-targeting immune cells to treat cancer. One of the mechanisms that may compromise its therapeutic efficacy is the expression of inhibitory molecules on the surface of the produced immune cells. Tim-3 is the inhibitory molecule which attracts attention in recent years. Tim-3 expression in the tumor cells and the tumor-infiltrating immune cells is often associated with worse prognosis and more aggressive forms of the disease. However, its role in the in vitro or ex vivo-produced immune cells is difficult to predict. In this work, an in vitro study model which is based on in vitro-produced antigen-specific CD8+ T cells with high expression of Tim-3 has been...

March1 joue un rôle essentiel dans la régulation de la réponse immunitaire. Cette ubiquitine ligase régule à la baisse l’expression de certaines protéines intervenant dans les fonctions des cellules présentatrices des antigènes, telles que le CMH de classe II et le CD86. March1 ubiquitine aussi quelques protéines impliquées dans le métabolisme cellulaire, comme le transporteur des acides aminées CD98 et le récepteur de l’insuline. L'ubiquitination du CMH de classe II et du CD98 par March1 affecte les fonctions de présentation des antigènes par les cellules dendritiques et la capacité de prolifération des TCD8+, respectivement. Cependant, le rôle de l’ubiquitination de CMH de classe II dans le développement et la migration des cellules dendritiques, n’est pas connu, et les implications physiologiques liées au rôle de March1 dans d’autres cellules, telles que les lymphocytes T CD8+, ne sont pas encore claires. Nos travaux démontrent que l'ubiquitination de CMH de classe II par March1 est spécifiquement requise pour la migration des cellules dendritiques dérivées de monocytes (moDCs) de la peau. L’effet de March1 sur la migration est intrinsèque à ces cellules et corrèle avec les niveaux d’expression des protéines impliquées dans la migration, notamment l’IRF4 et le CCR7. Dans un modèle de mélanome chez la souris, la déficience en ubiquitination du CMH de classe II est associée à une exacerbation de la croissance des tumeurs et un défaut de migration des moDCs vers les ganglions drainant les tumeurs. L’utilisation de cellules tumorales exprimant le GM-CSF augmente l’expression de l’IRF4 et du CCR7 dans les moDCs et la migration celles-ci vers les ganglions. D’autre part, nous avons démontré que la déficience en March1 exacerbe la résistance à l’insuline induite par l’obésité. Cet effet est associé à un enrichissement en lymphocytes T CD8+ ayant un phénotype effecteur/mémoire dans le tissu adipeux des souris obèses. Les expériences de transfert adoptif de lymphocytes T CD8+ montrent que March1 exacerbe la résistance à l’insuline en affectant intrinsèquement le phénotype de ces cellules. Nos résultats indiquent une augmentation de l’activité métabolique des lymphocytes T CD8+ est en absence de March1, ce qui est en accord avec le rôle de ce dernier dans l’ubiquitination de CD98 et du récepteur de l’insuline. Dans l’ensemble, nos travaux montrent que March1 régule la capacité migratoire des moDCs et le métabolisme des lymphocytes T CD8+. L’implication de cette régulation dans le développement du cancer de mélanome et du diabète de type de II suggère que March1 pourrait être ciblé dans le cadre de stratégies thérapeutiques contre ces pathologies. / March1 plays a critical role in the immune response regulation. This ubiquitin ligase downregulates the expression of antigen presentation and costimulatory proteins, such as MHC class II and CD86, as well as other proteins involved in the cellular metabolism, such as the amino acid transporter CD98 and the insulin receptor. The ubiquitination of these proteins by March1 alters the antigen presentation and proliferative capacities of dendritic cells and CD8+ T cells, respectively. However, the effect of MHC class II ubiquitination by March1 on the development and the migration of dendritic cells are not known, and the physiological implications of March1 in other cells, such as CD8+ T cells, are not completely understood. In this thesis, we show that MHC class II ubiquitination by March1 is specifically required for the migration of monocyte-derived dendritic cells (moDCs) from skin to skin draining lymph nodes (sdLN). This effect is cell intrinsic and correlates with the expression level of proteins involved in immune cell migration, IRF4 and CCR7. In a melanoma mouse model, the deficiency of MHC class II ubiquitination is associated with exacerbated tumor growth and impaired moDCs migration from tumor to tumors-draining LNs. Using GM-CSF producing tumors, we found that this cytokine increases the expression of IRF4 and CCR7 in moDCs and improves their migration. On the other hand, we show that March1 deficiency exacerbates obesity-induced insulin resistance. Adipose tissue from these mice was enriched in CD8+ T cells with an effector/memory phenotype. Adoptive transfer of splenic CD8+ T cells showed that March1 intrinsically affects the phenotype of these cells in obese adipose tissue and exacerbates insulin resistance. Consistent with the role of March1 in the ubiquitination of CD98 and insulin receptor, the metabolic activity of CD8+ T cells was increased in absence of March1. Overall, we showed that March1 regulates the migratory capacity of moDCs and the metabolic activity of CD8+ T cells. The involvement of these effects in the development of melanoma cancer and type II diabetes suggests that March1 can be a target for therapeutic strategies against these pathologies.

T Zellen können in zwei Hauptpopulationen mit unterschiedlichen Aufgaben unterschieden werden. CD4+ T Zellen exprimieren im Zuge ihrer Aktivierung CD40L, welches ein zentraler kostimulatorischer Rezeptor zur Induktion von B-Zell basierter humoraler Immunität, APC Aktivierung und einer effizienten Effektor CD8+ T Zell Entwicklung ist („Helfer-Funktion“). Im Gegensatz dazu sind die zytotoxischen CD8+ T Zellen dazu vorbestimmt, infizierte oder maligne Zellen direkt abzutöten. Jedoch wurde eine Fraktion von CD8+ T Zellen identifiziert, die nach Aktivierung CD40L exprimiert. Bisher ist nicht verstanden, wie in solchen CD8+ T Zellen a) die CD40L Expression reguliert ist, b) wann und wie die Fähigkeit CD40L zu exprimieren implementiert wird und c) was die Folgen für das Immunsystem sind. In dieser Arbeit konnten wir zeigen, dass sowohl in CD4+ als auch in CD8+ T Zellen die CD40L Expression durch DNA-Methylierung regulatorischer Regionen des CD40LG Lokus reguliert wird. Die Demethylierung zentraler Elemente wird im Thymus implementiert, manifestiert sich mit der T-Zell Reifung und geht mit einer zunehmenden Stabilität der CD40L Expression einher. Erhöhte Expression von CD5 und NUR77 in CD40L+ CD8+ SP Thymozyten weisen auf eine positive Selektion mit hoher Affinität gegen Selbst-peptide während der Reifung im Thymus hin, welche das weitere Schicksal der CD40L exprimierenden CD8+ T Zellen beeinflusst. Naive CD40L+ CD8+ T Zellen besitzen ein anderes TCR Repertoire als CD40L- CD8+ T Zellen und reifen im Zuge ihrer Aktivierung bevorzugt zu Gedächtniszellen mit Zytokin- und Chemokinrezeptorprofilen von Tc2, Tc17 und Tc22 Zellen heran. Mit ihrem nicht-zytotoxischen Phänotyp und ihrer Genexpressionsignatur ähneln diese Zellen stark Helfer-CD4+ T Zellen und können von den klassisch zytotoxischen Tc1 und Tc17+1 Zellen durch ihre IL-6R und fehlende SLAMF7 Expression sowie der Expression von Markern die auf eine Fähigkeit in die Haut zu wandern schließen lassen, unterschieden werden. Zusammenfassend zeigen wir hier, dass naive CD8+ T Zellen von den frühsten Entwicklungsstadien im Thymus an nicht homogen sind und die Fähigkeiten über CD40L Expression eine Helferfunktion auszuüben beziehungsweise über die Sekretion zytolytischer Moleküle Zielzellen abzutöten unabhängig vom CD4+ or CD8+ T-Zell Status sind. Zellen mit Zytokin- und Genexpressionsignaturen, die mit denen der CD8+ Helfer-T Zellen übereinstimmen, wurden von uns und anderen in Geweben (Haut, Lunge) identifiziert und tragen zu den verschiedensten autoinflammatorischen Erkrankungen bei. Diese Arbeit insinuiert daher die Notwendigkeit einer grundlegenen Neubewertung der CD8+ T Zell Fähigkeiten und Funktionen in Immunantworten. / The T cell compartment consists of two major subsets with diverse assignments. CD4+ T cells express CD40L upon activation, a central co-stimulatory receptor to induce B cell mediated humoral immunity, activate APCs and prime efficient effector CD8+ T cell development (“helper function”). In contrast, cytotoxic CD8+ T cells are predetermined to kill infected or malignant cells directly. However, a fraction of CD8+ T cells expressing CD40L upon activation was identified. So far, it is not understood in CD8+ T cells a) how CD40L expression is regulated, b) when and how the ability of CD40L expression is implemented and c) what are the implications for the immune system. In this thesis, we found that CD40L expression is regulated by DNA-methylation of regulatory regions of the CD40LG locus in CD4+ as well as CD8+ T cells. The de-methylation of central elements is implemented in the thymus and increases with T cell maturation reflected by enhanced stability of CD40L expression. Elevated CD5 and NUR77 expression of CD40L+ CD8+ SP thymocytes suggests that high affine detection of self-peptides during positive selection in the thymus implements CD40L expression ability and predetermines the fate of the CD40L imprinted CD8+ T cells. CD40L+ naïve CD8+ T cells differ in their TCR repertoire from their CD40L- counterparts and preferentially mature into memory cell subsets with cytokine and chemokine receptor profiles of Tc2, Tc17 and Tc22 cells. With their non-cytotoxic phenotype and gene expression signatures, the CD40L+ memory CD8+ T cell subsets Tc2, Tc17 and Tc22 widely resemble helper CD4+ T cells and can be distinguished from classical cytotoxic Tc1 and Tc17+1 cells by their IL-6R and absent SLAMF7 expression and their skin migratory phenotype. Altogether, we demonstrate that from the earliest developmental stages in thymus onwards naive CD8+ T cells are not homogenous and the abilites to provide “CD40L based help” or “cytotoxicity mediated killing” are independent of the CD4+ or CD8+ T cell status. Cells with helper-type CD8+ T cell cytokine and gene-expression signatures were found at barrier sites (skin, lung) by us and others where they contribute to multiple autoinflammatory diseases. Therefore, this work insinuates the need to revisite CD8+ T cell capablities and function in immune responses.