Le diabète auto-immun résulte de la destruction des cellules bêta pancréatiques sécrétrices d’insuline par les lymphocytes T du système immunitaire. Il s’ensuit une déficience hormonale qui peut être comblée par des injections quotidiennes d’insuline d’origine exogène, toutefois il demeure à ce jour impossible de guérir les patients atteints de la maladie. De façon générale, un système immunitaire sain reconnaît une multitude d’antigènes différents et assure ainsi notre défense à l’égard de différents pathogènes ou encore de cellules tumorales. Il arrive cependant que, pour des raisons génétiques et/ou environnementales, les lymphocytes T puissent s’activer de façon aberrante suite à la reconnaissance d’antigènes provenant du soi. C’est ce bris de tolérance qui mène au développement de pathologies auto-immunes telles que le diabète auto-immun. Afin de limiter l’auto-immunité, des mécanismes de sélection stricts permettent d’éliminer la majorité des lymphocytes T présentant une forte affinité envers des antigènes du soi lors de leur développement dans le thymus. Certains de ces lymphocytes réussissent toutefois à échapper à l’apoptose et migrent en périphérie afin d’y circuler en quête d’un antigène spécifiquement reconnu. Il est alors primordial que des mécanismes périphériques assurent le maintien de la tolérance immunitaire en faisant obstacle à l’activation et à la prolifération des lymphocytes T auto-réactifs. L’une des avenues afin d’inhiber le développement de réponses immunitaires aberrantes est la génération de lymphocytes T régulateurs. Ces cellules, d’origine thymique ou périphérique, peuvent arborer différents phénotypes et agissent via de multiples mécanismes afin d’inactiver et/ou éliminer les cellules impliquées dans l’apparition de pathologies auto-immunes. L’utilisation de modèles murins transgéniques a permis la mise en évidence d’une population peu caractérisée de lymphocytes T au potentiel régulateur. En effet, la proportion de ces cellules T n’exprimant pas les corécepteurs CD4 et CD8 (double négatives, DN) a été inversement corrélée à la prédisposition à l’auto-immunité chez ces ii souris. L’objectif principal de cette thèse est de démontrer la fonction immuno-régulatrice des lymphocytes T DN, tout en investiguant les facteurs génétiques responsables du maintien de cette population cellulaire. Nous avons observé que les lymphocytes T DN exercent une activité cytotoxique à l’égard des lymphocytes B de façon spécifique à l’antigène, via la libération de granules cytolytiques contenant du granzyme B et de la perforine. Par ailleurs, nous avons établi qu’un unique transfert adoptif de ces cellules est suffisant afin d’inhiber le développement du diabète auto-immun chez des hôtes transgéniques prédisposés à la maladie. Le recours à des souris déficientes pour l’expression du gène CD47 a permis de constater que la voie de signalisation CD47-Sirp est essentielle dans le maintien de la proportion des lymphocytes T DN. De plus, le locus murin de prédisposition au diabète auto-immun Idd13, qui contient le gène Sirp, a été identifié pour son rôle dans la régulation de la proportion de ces cellules. Finalement, une analyse génétique a révélé que d’autres intervalles génétiques sont impliqués dans le contrôle de la population des lymphocytes T DN. Parmi ceux-ci, un locus situé en région proximale du chromosome 12 a été validé grâce à la création de souris congéniques. Grâce aux résultats présentés dans cette thèse, notre compréhension de la biologie ainsi que de la régulation des lymphocytes T DN est approfondie. Ces connaissances constituent un pas important vers la création de thérapies cellulaires novatrices permettant de prévenir et de guérir diverses pathologies auto-immunes. / Autoimmune diabetes results from the destruction of the insulin-secreting pancreatic beta cells by the T lymphocytes of the immune system. This leads to a hormonal deficiency that can be regulated with daily injections of exogenous insulin. However, to date, there is no cure for autoimmune diabetes. A healthy immune system generally recognizes a multitude of antigens in order to ensure our defence against different pathogens and tumor cells. Yet, depending on genetic and/or environmental factors, individuals may develop T cells that are aberrantly activated following the recognition of self-antigens. This break in tolerance leads to the development of autoimmune pathologies, such as autoimmune diabetes. In order to limit autoimmunity, rigorous selection mechanisms eliminate the vast majority of the T lymphocytes that present a high affinity for self-antigens during their thymic development. However, some of these auto-reactive lymphocytes escape from the elimination processes and migrate to the periphery where they might encounter a self-antigen. It is then essential that peripheral mechanisms maintain the immune tolerance by abrogating the activation and the proliferation of these self-specific T lymphocytes. One of the means to inhibit aberrant immune responses is the generation of regulatory T lymphocytes. These cells, which can be of thymus or peripheral origin, display various phenotypes and can mediate their action through several mechanisms in order to inactivate and/or eliminate the cells that are implicated in the development of autoimmune diseases. The use of transgenic mouse models made it possible to identify a poorly characterized population of T lymphocytes that exhibit a regulatory potential, namely CD4-CD8- (double negative, DN T cells). Indeed, the proportion of DN T cells in lymphoid organs is inversely correlated to autoimmune predisposition. The main objective of this thesis is to determine the immunoregulatory function of the DN T cells, as well as to reveal the genetic factors underlying the regulation of the proportion of DN T cells. iv We observed that through the release of cytolytic granules containing granzyme B and perforin, DN T lymphocytes exert a cytotoxic activity towards B cells in an antigen-specific manner. In addition, we have established that a single injection of those DN T cells is sufficient to inhibit the development of autoimmune diabetes in highly susceptible transgenic mice. The use of CD47 deficient mice also demonstrated that the CD47-Sirp pathway is essential to maintain DN T cell proportion. Also, we identified that the autoimmune diabetes susceptibility locus Idd13, which contains Sirp participates in defining the proportion of DN T cells. Finally, a genetic analysis revealed that other loci are implicated in the control of the DN T cell population. Among those, the role of a locus situated in the proximal region of chromosome 12 has been validated through to the generation of congenic mice. The results presented in this thesis have allowed us to enhance our understanding of the biology and genetic regulation of DN T lymphocytes. This knowledge constitutes an important step towards the creation of innovative cellular therapies that may prevent and cure a diversity of autoimmune pathologies.

Le contrôle immunitaire des infections virales est effectué, en grande partie, par les lymphocytes T CD8+ cytotoxiques. Pour y parvenir, les lymphocytes T CD8+ doivent être en mesure de reconnaître les cellules infectées et de les éliminer. Cette reconnaissance des cellules infectées s’effectue par l’interaction du récepteur T (TCR) des lymphocytes T CD8+ et des peptides viraux associés au complexe majeur d’histocompatibilité (CMH) de classe I à la surface des cellules hôtes. Cette interaction constitue l’élément déclencheur permettant l’élimination de la cellule infectée. On comprend donc toute l’importance des mécanismes cellulaires menant à la génération des peptides antigéniques à partir des protéines virales produites au cours d’une infection. La vision traditionnelle de cet apprêtement protéique menant à la présentation d’antigènes par les molécules du CMH propose deux voies cataboliques distinctes. En effet, il est largement admis que les antigènes endogènes sont apprêtés par la voie dite ‘‘classique’’ de présentation antigénique par les CMH de classe I. Cette voie implique la dégradation des antigènes intracellulaires par le protéasome dans le cytoplasme, le transport des peptides résultant de cette dégradation à l’intérieur du réticulum endoplasmique, leur chargement sur les molécules du CMH de classe I et finalement le transport des complexes peptide-CMH à la surface de la cellule où ils pourront activer les lymphocytes T CD8+. Dans la seconde voie impliquant des antigènes exogènes, le dogme veut que ceux-ci soient apprêtés par les protéases du compartiment endovacuolaire. Les peptides ainsi générés sont directement chargés sur les molécules de CMH de classe II à l’intérieur de ce compartiment. Par la suite, des mécanismes de recyclage vésiculaire assurent le transport des complexes peptide-CMH de classe II à la surface de la cellule afin de stimuler les lymphocytes T CD4+. Cependant, cette stricte ségrégation des voies d’apprêtement antigénique a été durement éprouvée par la capacité des cellules présentatrices d’antigènes à effectuer l’apprêtement d’antigènes exogènes et permettre leur présentation sur des molécules de CMH de classe I. De plus, l’identification récente de peptides d’origine intracellulaire associés à des molécules de CMH de classe II a clairement indiqué la présence d’interactions entre les deux voies d’apprêtement antigénique permettant de transgresser le dogme préalablement établi. L’objectif du travail présenté ici était de caractériser les voies d’apprêtement antigénique menant à la présentation d’antigènes viraux par les molécules du CMH de classe I lors d’une infection par le virus de l’Herpès simplex de type I (HSV-1). Dans les résultats rapportés ici, nous décrivons une nouvelle voie d’apprêtement antigénique résultant de la formation d’autophagosomes dans les cellules infectées. Cette nouvelle voie permet le transfert d’antigènes viraux vers un compartiment vacuolaire dégradatif dans la phase tardive de l’infection par le virus HSV-1. Cette mise en branle d’une seconde voie d’apprêtement antigénique permet d’augmenter le niveau de présentation de la glycoprotéine B (gB) virale utilisée comme modèle dans cette étude. De plus, nos résultats décrivent la formation d’une nouvelle forme d’autophagosomes dérivés de l’enveloppe nucléaire en réponse à l’infection par le virus HSV-1. Ces nouveaux autophagosomes permettent le transfert d’antigènes viraux vers un compartiment vacuolaire lytique, action également assurée par les autophagosomes dits classiques. Dans la deuxième partie du travail présenté ici, nous utilisons l’infection par le virus HSV-1 et la production de la gB qui en résulte pour étudier le trafic membranaire permettant le transfert de la gB vers un compartiment vacuolaire dégradatif. Nos résultats mettent en valeur l’importance du réticulum endoplasmique, et des compartiments autophagiques qui en dérivent, dans ces mécanismes de transfert antigénique permettant d’amplifier la présentation antigénique de la protéine virale gB sur des CMH de classe I via une voie vacuolaire. L’ensemble de nos résultats démontrent également une étroite collaboration entre la voie classique de présentation antigénique par les CMH de classe I et la voie vacuolaire soulignant, encore une fois, la présence d’interaction entre les deux voies. / Immune control of viral infections is mainly carried out by cytotoxic CD8+ T lymphocytes. To achieve this, CD8+ T lymphocytes must be able to recognize infected cells and eliminate them. This recognition of infected cells occurs by the interaction of the T cell receptor (TCR) of CD8+ T lymphocytes and viral peptides associated with major histocompatibility complex (MHC) class I on the surface of host cells. This interaction is the key element triggering the elimination of infected cells. This emphasizes the major role of cellular mechanisms leading to the generation of antigenic peptides from viral proteins. The traditional view of antigen presentation by MHC molecules proposes two segregated pathways. Indeed, it is widely accepted that endogenous antigens are processed by the ''classical'' MHC class I presentation pathway. This pathway involves the degradation of intracellular antigens by the proteasome complex in the cytoplasm of the cell, the resulting peptides are then translocated in the endoplasmic reticulum where they are loaded on MHC class I molecules, and finally peptide-MHC complex are exported at the cell surface to activate CD8+ T lymphocytes. In contrast, exogenous antigens internalized by endocytosis or phagocytosis are processed by hydrolases in the lytic endovacuolar compartment and the resulting peptides are loaded on MHC class II molecules. Thereafter, vesicle recycling mechanisms transport the peptide-MHC class II complex on the cell surface where they can stimulate CD4+ T lymphocytes. However, the strict segregation of these two pathways has been revisited to account for the ability of antigen presenting cells to present exogenous antigens on MHC class I molecules by a process called cross-presentation. Moreover, the recent finding that intracellular peptides might also be presented by MHC class II molecules clearly emphasized the presence of interactions between these two antigen processing pathways that transgress the previously established dogma. The objective of the work presented here was to characterize the antigen processing pathways leading to antigen MHC class I presentation during herpes simplex type I (HSV-1) infection. In the results reported here, we describe a new antigen processing pathway resulting from the formation of autophagosomes in HSV-1 infected cells. This new pathway allows the transfer of viral antigens in a lytic vacuolar compartment during the late phase of infection. The development and activation of this second pathway of antigen processing leads to an increased MHC class I presentation of the viral glycoprotein B (gB) used as a model in this study. Moreover, our results describe the establishment of a new form of autophagosomes derived from the nuclear envelope in response to HSV-1 infection. This new form of autophagosomes also contributes to viral antigen transfer to lytic vacuolar compartment in parallel to the action of classical autophagy. Our results also show a close collaboration between the classical MHC class I presentation pathway and vacuolar pathway induced by the formation of autophagosomes, still reinforcing the idea that these two pathways interact together to ensure optimal antigens processing during viral infection. In the second part of the work presented here, we use HSV-1 infection and the resulting viral glycoprotein B to study membrane trafficking allowing the transfer of gB to degradative vacuolar compartments. Our results highlight the role of the endoplasmic reticulum in antigen transfer mechanisms that induce an amplified MHC class I presentation of the viral glycoprotein B.

L’immunothérapie tumorale à médiation cellulaire est un traitement qui utilise le système immunitaire des patients afin d’induire une réponse des lymphocytes T CD8+ (T CD8+) contre la tumeur. Cette réponse est produite suite à la reconnaissance des antigènes par les T CD8+. Ces cibles sont appelées antigènes tumoraux (TAA) et définies comme des protéines exprimées par les cellules cancéreuses mais absentes des tissus normaux. Par une approche bio-informatique, notre laboratoire a identifié Dickkopf-1 (DKK1), une protéine inhibitrice de la voie de Wnt, comme un TAA potentiel. Une immunothérapie à médiation cellulaire efficace requiert l’identification de TAA candidats pertinents. Le traitement de patients par immunothérapie pourrait également être améliorées par l’augmentation de la puissance d’action anti-tumorale ainsi que la persistante des T CD8+ spécifiques aux TAA. Ce projet de doctorat se divise en deux parties : 1- La caractérisation de l’expression de DKK1 dans les cancers communs et la détermination de son immunogénicité afin de valider sa candidature comme TAA. 2- La reprogrammation des T CD8+, de patients atteints d’un cancer commun, vers un phénotype moins différentié afin d’augmenter leur potentiel anti-tumoral et leur persistance. Dans le premier objectif, nous avons caractérisé l’expression de DKK1 dans le cancer du sein et dans d’autres cancers communs. Le profil d’expression de DKK1 a été étudié par RT-PCR et par ELISA dans plusieurs lignées cellulaires de cancer et dans les tissus normaux. L’expression de DKK1 a aussi été étudiée dans des échantillons cliniques provenant de cancers du sein, du poumon et du rein. Trente pourcents (30%) des tumeurs provenant d’un cancer du sein exprimaient DKK1. La moitié des tumeurs DKK1(+) était triple négative, donc pas de récepteurs d’œstrogène et de progestérone et était Her-2/neu(-) (ces patientes ont des possibilités de traitements très restreintes). De plus, 50% des échantillons cliniques de tumeurs du poumon et 30% des tumeurs de rein exprimaient DKK1. Les observations effectuées dans le cancer du poumon ont été, par la suite, corroborées par d'autres groupes qui ont montré une corrélation entre l'expression de DKK1 et un mauvais pronostic. Après avoir confirmée l’expression de DKK1 dans les cancers communs, justifiant ainsi sa candidature comme TAA, nous avons évalué l’immunogénicité de DKK1. Pour ce faire, nous avons effectué des stimulations in vitro de cellules mononucléées du sang périphérique (PBMC) de patient(e)s atteint(e)s d’un cancer du sein ou du poumon avec des peptides dérivés de DKK1 pouvant être présentés par les complexes majeurs d’histocompatibilité (CMH) HLA-A*0201. Des clones de T CD8+ reconnaissant un peptide de DKK1 ont été identifiés et isolés. Par essai multiplex et cytométrie de flux intracellulaire, la polyfonctionnalité d’un ces clones T CD8+ spécifiques à DKK1 a été étudiée et a révélée un profil effecteur, renforçant ainsi la candidature de DKK1 comme TAA. Dans l’ensemble, les résultats obtenus dans cette première partie de thèse suggèrent une possible utilisation de DKK1 en immunothérapie contre les cancers communs, attribuable à son expression dans ces cancers et la possibilité de faire proliférer des T CD8+ effecteurs spécifiques à DKK1 à partir de sang de patients. Dans la seconde partie de cette thèse, je décrirai la manipulation in vitro des T CD8+ de patients atteints d’un cancer commun, afin d’augmenter la force et la durée de leurs fonctions anti-tumorales. Il a été démontré que des lymphocytes moins différentiés sont capables d’une réponse immunologique plus efficace et durable. Nous avons basé ce projet sur l’utilisation d’un inhibiteur pharmacologique de la GSK-3, pour activer de la voie de Wnt chez les T CD8+ et ainsi leur conférer un phénotype moins différentié, partageant des caractéristiques de la cellule naïve et de la cellule mémoire. Des cultures de T CD8+, spécifiques à des antigènes viraux, en présence de l’inhibiteur ont permis d’augmenter la sécrétion d’interféron (IFN)- et leur activité cytotoxique. Ces résultats indiquent un effet de l’activation de la voie de Wnt sur la fonction des T CD8+. Ces observations sont rapportées pour la première fois chez les T CD8+ humains et suggèrent une nouvelle stratégie, applicables à l’immunothérapie du cancer, afin de prolonger la persistance des cellules ainsi que leur activité anti-tumorale. En conclusion, ces travaux de recherche ont mené à la réalisation d’une étape très importante dans la validation de la candidature de DKK1 comme TAA pour les cancers communs, soit la démonstration de son expression dans ces cancers et son absence dans les tissus normaux dérivés d’organes importants. Ces travaux ont également mené à la démonstration de l’immunogénicité de DKK1, par l’identification d’un peptide de DKK1 reconnu par les T CD8+. De plus, l’étude de la polyfonctionnalité des T CD8+ spécifiques à DKK1 a révélée un profil effecteur favorable pour l’obtention d’une réponse anti-tumorale efficace. Ces découvertes pourraient servir à l’élaboration d’une stratégie d’immunothérapie à médiation cellulaire pour les cancers communs. Pour sa part, l’étude phénotypique et fonctionnelle de la modulation de la voie de Wnt dans les T CD8+ a donné lieu à l’observation d’un phénotype encore jamais rapporté chez l’humain, conférant aux T CD8+ un aspect moins différentié avec des caractéristiques propre à un phénotype mémoire. Ces résultats sont pertinents dans l’amélioration de l’immunothérapie du cancer, passant par l’augmentation de la persistance des lymphocytes. En résumé, les résultats présentés dans cette thèse de doctorat fournissent des évidences indéniables quant à la validation de DKK1 comme TAA pour une immunothérapie à médiation cellulaire des cancers communs. Ces résultats fournissent également des preuves quant à la pertinence de la reprogrammation des T CD8+ par l’activation de la voie de la voie de Wnt, afin de générer des lymphocytes médiateurs plus efficaces pour ce type de thérapie. / Cell-mediated cancer immunotherapy is based on the priming of the patient’s CD8+ T lymphocytes (CD8+ T cells) to mediate an immune response directed against the tumour. This anti-tumour response is antigen-specific and directed against tumour associated antigens (TAA), which are defined as proteins expressed principally by cancer cells and absent from non-malignant tissues. By utilizing a bio-informatic approach, we identified the gene DKK1, a Wnt pathway inhibitor, as a potential TAA. This was an important novel finding as the identification of a new TAA is one of the key elements to enhance cell-mediated cancer immunotherapy. Furthermore, patient treatment options may also be improved through the amplification of the force and duration of the anti-tumour immune response mediated by TAA specific T cells. This thesis is divided in two parts: 1- The characterization of DKK1 expression and immunogenicity in common cancers as validation of TAA candidate. 2- The reprogrammation of CD8+ T cells from patient with common cancers to restore a less-differentiated phenotype in an attempt to improve their anti-tumour response. We first characterized DKK1 expression in breast cancer and other common cancers. In order to prove its specificity to malignant tissues, the DKK1 expression profile was initially established by RT-PCR and ELISA assay using cancer cell lines and in RNA panels from normal tissues. DKK1 expression was also described using clinical samples from breast, lung and kidney cancers. We found that 30% of breast cancer clinical samples were positive for DKK1 expression. Interestingly, half of the triple negative breast cancer tumours (negative for the expression of progesterone and estrogen receptors and Her-2/neu) were DKK1 (+). Moreover, 50% of the lung cancer and 30% of the kidney cancer clinical samples were also DKK1 (+). These results have been corroborated by other groups who recently reported similar observations in lung cancer with a correlation with poor prognosis. After confirming that the DKK1 gene expression profile in common cancer qualifies DKK1 as a relevant TAA, we then explored its immunogenicity. To do so, we performed in vitro stimulations of peripheral blood mononuclear cells (PBMC) from lung and breast cancer patients with DKK1-derived synthetic peptides, which were selected for their capacity to be presented by the major histocompatibility complex (MHC) HLA-A*0201. With this method, we identified and isolated CD8+ T cell clones with a specificity unique for one DKK1 peptide. Cytokine secretion profile of anti-DKK1 T cells was established by cytokine mutiplex assay and flow cytometry. This study revealed that DKK1-specitfic CD8+ T cells had an effector profile with polyfunctionality proprieties, thereby reinforcing DKK1 as a TAA candidate. Altogether, the results obtained in this first part of this thesis suggest the possible use of DKK1 in common cancer immunotherapy as it is principally expressed by malignant tissues and can generate the activation of effector CD8+ T cells. In the second part of this thesis, I will describe in vitro manipulations of patients’ CD8+ T cells in the goal of augmenting their longevity and the strength of the anti-tumour response. Previous research revealed that a less-differentiated phenotype correlated with an augmented capacity of persistence and the intensity of the T cell response. For this project, we generated less-differentiated CD8+ T cells by activating the Wnt pathway with a pharmalogical inhibitor of GSK-3. These less-differentiated T cells shared a phenotype of both naive and memory T cells. As for their immune functions, viral antigen specific CD8+ T cells cultured with the inhibtitor showed an elevation in interferon (IFN)-γ production and cytotoxic activity. This represent the first report of such observations in humans CD8+ T cells and suggest a new stategy to prolonge the persistence of T cells in a cancer immunotherapy setting. In conclusion, this work has strongly contributed to the validation of DKK1 as a TAA for common cancers, as it is expressed in malignant tissues and relatively absent in form normal tissues. It demonstrated the immunogenicity of DKK1 with the identification of a DKK1 peptide recognized by CD8+ T cells. Moreover, these DKK1-specific CD8+ T cells appear to be polyfunctional with an effector profil, which is favorable to mount a potent anti-tumour response. These findings could serve in novel strategies to be exploited in cell-mediated immunotherapy against common cancers. Furthermore, the phenotypic and functional study of the Wnt pathway activation resulting in a less-differentiated CD8+ T cells, generated observations that had never been reported in humans. These findings are relevant for cancer immunotherapy because they could help generate less-differentiated cells with augmented persistance and anti-tumorale capacities. Altogether, the results presented in this doctoral thesis provide significant evidence that DKK1 may serve as a TAA in cell-mediated immunotherapy for patients affected by common cancers and that reprogramming of CD8+ T cells through activation of the Wnt pathway could generate more effective mediator for this type of treatment.

L’hépatite autoimmune (HAI) est une maladie grave affectant le foie et présentant un haut taux de mortalité lorsque non traitée. Les traitements disponibles sont efficaces, mais de graves effets secondaires leur sont associés. Ces effets secondaires sont généralement le résultat d'une forte immunosuppression et d’autres sont spécifiques à chaque médicament. Aucune immunothérapie spécifique n’est présentement disponible pour le traitement de l’HAI. Récemment, un modèle murin d’HAI a été développé dans notre laboratoire par xénoimmunisation des souris C57BL/6 avec les antigènes humains de l'HAI de type 2. Ce modèle présente la plupart des caractéristiques biochimiques et cliniques retrouvées chez les patients atteints d'HAI de type 2. Dans cette étude, nous avons évaluée l’efficacité de deux types de traitement pour l’HAI de type 2 à l’aide de notre modèle murin. Dans un premier temps, l’anticorps anti-CD3ε a été étudié en prophylaxie et en traitement. Nous avons montré qu’une posologie de 5µg d’anti-CD3 i.v. par jour pendant 5 jours consécutifs induit une rémission chez les souris avec HAI de type 2 établie (traitement). Cette rémission est caractérisée par une normalisation des niveaux d’alanine aminotransférase et une diminution significative de l’inflammation hépatique. Cette rémission semble être associée à une déplétion partielle et transitoire des lymphocytes T CD3+ dans la périphérie et une augmentation des lymphocytes T régulateurs CD4+, CD25+ et Foxp3+ dans le foie. La même posologie lorsqu’elle est appliquée en prophylaxie n’a pas réussi à prévenir l’apparition de l’HAI de type 2. La deuxième voie de traitement consiste en l’administration par voie intranasale d’un forte dose de formiminotransférase cyclodésaminase murin (mFTCD), un autoantigène reconnu dans l’HAI de type 2. Une administration en prophylaxie par voie intranasale de 100µg de mFTCD par jour durant 3 jours consécutifs arrive à prévenir l’HAI de type 2 en diminuant l’inflammation hépatique au bout de deux semaines post-traitement. / Autoimmune hepatitis (AIH) is a severe liver disease with high mortality rates if left untreated. Current treatments, while effective, are associated with deleterious side-effects. These side effects are specific to each drug and the result of broad immunosuppression. Recently, a murine model of type 2 AIH has been created in our laboratory in wild-type naïve mice. In this model, DNA immunization with type 2 AIH human autoantigens breaks immune tolerance and induces an autoimmune response against the liver. Lately, new therapeutic strategies based on depletion of specific immune cell populations have been proposed for the treatment of several diseases, including autoimmune diseases. Currently, no immunotherapies using biological agents are available for the treatment of autoimmune liver diseases. Therefore, the goal of this project is to study the efficacy of new immunotherapeutic agents for the treatment of type 2 AIH in an experimental model. We evaluated the effectiveness of two approaches for treating type 2 AIH. First, we tested the anti-CD3ε antibody in prophylaxis and in treatment of type 2 AIH. We showed that a dosage of 5µg i.v. of anti-CD3ε antibody per day for 5 consecutive days induced remission in mice with established type 2 AIH. This remission was defined as a normalization of serum alanine aminostransferase levels and a significant decrease of liver inflammation in treated mice. This remission seems to be associated with a transitory depletion of CD3+ T lymphocytes in peripheral blood mononuclear cells and increased CD4+CD25+Foxp3+ regulatory T lymphocytes in the liver. But when this dosage was applied in prophylaxis, it could not prevent the induction of type 2 AIH. The second approach was to induce tolerance by nasal administration of murin formiminotransferase cyclodeaminase (mFTCD), an autoantigen of type 2 AIH. We showed that nasal administration of 100µg of mFTCD for 3 consecutive days prevented development of type 2 AIH in prophylaxis by reducing liver inflammation.

T-lymphocytes are an essential cell type of the adaptive immune system. Due to their importance in immune responses and disorders, the molecular mechanisms leading to T-lymphocyte activation have been the subject of extensive research which has translated into important therapeutic developments. Early signalling events involving tyrosine phosphorylation are well characterised. However, later events involving other post-translational modifications are less well understood. Several studies have provided evidence suggesting a role for protein arginine methylation in T-lymphocyte activation. Arginine methylation is an essential post-translational modification in mammals and yet has not been extensively studied. No large scale analysis of arginine methylation sites has been performed. To gain insight into the role of protein arginine methylation in T-lymphocyte activation, the aims of this work were to: 1. Establish whether levels of arginine methylation are altered during Tlymphocyte activation 2. Use mass spectrometry based proteomics to identify arginine methylated proteins in the T-lymphocyte proteome 3. Further characterise an arginine methylated protein important to Tlymphocyte activation Arginine methylation was found to be induced after long term (>20 hours) stimulation of primary T-lymphocytes. Large increases in the main protein arginine methyltransferase, PRMT1, were also observed. Enrichment and labelling methods were developed to detect arginine methylated peptides from T-lymphocytes by mass spectrometry. This resulted in the identification of 265 unique arginine methylation sites in 141 proteins. 204 of the methylation sites were novel and 103 of the proteins had not previously been described as arginine methylated. Individual arginine methylation sites were characterised before and after activation of T-lymphocytes, with some sites showing significant changes in abundance. Among the novel arginine methylated proteins discovered were Dynamin II, WASp and WIPF1. These proteins are involved in re-organisation of the actin cytoskeleton at the immunological synapse formed between a Tlymphocyte and an antigen presenting cell. The functional consequences of the arginine methylation sites inWASp were characterised. WASp is essential for T-lymphocyte activation and some initial evidence showed that one of the arginine methylation sites is important for WASp activation.

CD73 est un ecto-enzyme qui a été associé à la suppression de l'immunité anti-tumorale. Ses valeurs pronostiques et thérapeutiques ont été mises de l'avant dans plusieurs types de cancer. La première hypothèse du projet est que l'expression de CD73 dans la tumeur prédit le pronostic des patients atteints du cancer de la prostate. L'expression de CD73 a été étudiée par immunofluorescence dans des échantillons de tumeur. Puis, des analyses univariées et multivariées ont été conduites pour déterminer si l'expression de CD73 permet de prédire la récidive biochimique des patients. Nous avons déterminé que CD73 prédit indépendamment le pronostic des patients atteints du cancer de la prostate. De plus, nous avons déterminé que son expression dans le tissu normal adjacent ou dans la tumeur prédit différemment la survenue de la récidive biochimique. La deuxième hypothèse est que l'inhibition de CD73 permet d'améliorer l'efficacité d'un vaccin thérapeutique contre le cancer de la prostate. L'effet d'un vaccin de type GVAX a été étudié dans des souris CD73KO ou en combinaison avec un anticorps ciblant CD73. Nous avons observé que l'efficacité du vaccin était augmentée dans les souris où CD73 était absent. Cependant, la combinaison avec l'anti-CD73 n'a pas permis d'améliorer l'efficacité. / The ectoenzyme CD73 has been associated with suppression of anti-tumor immunity. Its prognostic and therapeutic values have been highlighted in many types of cancer. The first hypothesis of this project is that tumor CD73 expression predicts the prognosis of prostate cancer patients. CD73 expression was determined by immunofluorescence in tumor tissues. Univariates and multivariates analysis were conducted to determine if CD73 expression could predict patients' biochemical recurrence. We determined that CD73 predicts independently the prognosis of prostate cancer patients. Moreover, we determined that CD73 expression in normal adjacent tissue or in the tumor predicts differently the occurrence of biochemical recurrence. The second hypothesis is that CD73 blockade enhances the efficacy of a therapeutic vaccine against prostate cancer. The GVAX-like vaccine was studied in CD73KO mice or in combination with an antibody against CD73. We observed that the vaccine was more efficient in mice where CD73 was absent. However, the combination with the anti-CD73 did not enhances the efficacy of the vaccine.

A profilaxia pré-exposição (PrEP) ao HIV se consolidou como uma importante estratégia de combate ao avanço da epidemia. Ainda assim, a incidência de casos da infecção vem aumentando na população jovem, assim como a de outras infecções sexualmente transmissíveis (ISTs), que atuam como importante fator de risco para transmissão do HIV-1. Entre as infecções mais frequentemente diagnosticadas estão Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG). A presença de lesões na mucosa genital e anal são fatores de risco estabelecidos para a transmissão do HIV-1, porém o impacto das infecções assintomáticas ainda é pouco conhecido. Dados recentes mostram que a ativação sistêmica de linfócitos T é um fator de risco para a aquisição da infecção pelo HIV-1. Nesse estudo, estudamos a ativação de linfócitos T na presença de infecção anorretal assintomática por CT e/ou NG. Células mononucleares do sangue periférico de voluntários do PrEP Brasil, um estudo clínico demonstrativo de PrEP ao HIV em homens que fazem sexo com homens, foram descongeladas para análise da ativação de linfócitos T. Trinta e quatro participantes com swab anorretal positivo para CT e/ou NG foram selecionados, enquanto assintomáticos e negativos para outras ISTs. Trinta e cinco controles foram selecionados randomicamente. Encontramos uma maior frequência de linfócitos T CD8+ HLA-DR+CD38+ (1,5 vs. 0,9% p < 0,005) no grupo com infecção assintomática. Os linfócitos T CD8+ de memória também apresentaram uma maior expressão dos marcadores de ativação. Os marcadores de exaustão e senescência foram significantemente mais expressos no grupo com a infecção. Não foi observado aumento ou diferença nos níveis de CD14 solúvel no plasma. Nossos achados demonstram que as infecções anorretais assintomáticas por CT e NG induzem a ativação sistêmica de linfócitos T CD8+. Considerando a alta prevalência dessas infecções e o risco associado de aquisição da infecção pelo HIV-1, o rastreamento periódico e o tratamento sistemático devem sem explorados em conjunto com as estratégias de prevenção ao HIV / Oral antiretroviral pre-exposure prophylaxis (PrEP) has been established as a pivotal strategy in the prevention against HIV epidemic. However, the incidence of HIV-1 infections has been rising among the youth, as well as other sexually transmitted infections (STIs), acting as an important risk factor for HIV-1 acquisition. Infection by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most diagnosed. Although the presence of mucosal lesions is a known risk factor for HIV-1 acquisition, the potential increase in risk associated with asymptomatic STIs is not completely understood. Recent data defined higher T cell activation as a single risk factor for sexually acquired HIV-1 infection. We examined the effect of asymptomatic CT and/or NG anorectal infection on immune activation. Peripheral blood mononuclear cells from participants of PrEP Brasil, a study of daily oral PrEP among healthy men who have sex with men, were analyzed for T cell activation by flow cytometry. Thirty-four participants with positive anorectal swab for CT and/or NG were selected, while negative for other STIs and without any reported symptoms. Thirty-five controls were randomly selected. We found a higher frequency of CD8+ HLA-DR+CD38+ T cells (1.5 vs. 0.9% p < 0.005) in the group with CT and/or NG infection and a greater median proportions of activation markers expression in CD8+ T cells with memory phenotype. Exhaustion and senescence markers were also significant higher in the infected group. No difference was observed in the soluble CD14 levels. Our findings suggest that asymptomatic CT and NG anorectal infection lead to a systemic activation of the T cell compartment. Considering the high prevalence of asymptomatic infection and the risk of HIV-1 acquisition associated, regular screening and treatment should be explored as an adjuvant tool for HIV prevention

Neuere Untersuchungen legen eine kardioprotektive Rolle für den Angiotensin AT2-Rezeptor nahe, welcher die Postinfarkt-Entzündungsreaktion vermindert, wobei der zelluläre Mechanismus noch wenig verstanden ist. Das Ziel dieser Arbeit war es deshalb, die potentielle Rolle des AT2-Rezeptors in der zellulären Immunantwort auf ischemische Herzverletzungen zu ergründen. Sieben Tage nach myokardialem Infarkt in Ratten wurde der AT2-Rezeptor mittels Immunfluoreszenzfärbung von Gewebeschnitten in einer CD8 T-Zellfraktion detektiert, die das Peri-Infarkt-Myokard infiltiert hatte. Wir haben eine Methode entwickelt, die es mittels kombinierter MACS und FACS Technilogie ermöglicht, CD8+AT2R+ T-Zellen aus dem Myokard zu isolieren und zu analysieren. Im Gegensatz zu den CD8+AT2R- T-Zellen, die in Kultur sowohl auf adulte als auch auf fötale Kardiomyozyten stark zytotoxisch wirkten, zeigten die CD8+AT2R+ T-Zellen keinerlei Zytotoxizität. Die CD8+AT2R+ T-Zellen zeigten eine erhöhte Expression von IL-10 und eine geringere mRNA Expression von IL-2 und IFN-gamma im Vergleich zu CD8+AT2R-T-Zellen. Weiterhin konnten wir zeigen, dass in vitro Stimulation des AT2-Rezeptors zur Hochregulation der IL-10-Expression von CD8+ T-Zellen führt. Entsprechend führt die in vivo Aktivierung des AT2-Rezeptors zur Vergrößerung der CD8+AT2R+ T-Zellpopulation und erhöhter IL-10-Produktion im ischemischen Myokard. Diese CD8+AT2R+ T-Zellen konnten auch in humanem periphärem Blut detektiert werden. Wir haben eine CD8+AT2+T-Zellpopulation definiert, welche sich während ischemischer Herzverletzung vergrößert und das Kardiomyocytenüberleben mittels kardioprotektivem IL-10 aufrechterhält. Somit konnten wir einen neuartigen AT2-Rezeptorvermittelten zellulären Mechanismus aufdecken, welcher die adaptive Immunantwort im Herzen moduliert. / One important aspect of cardiac remodeling after myocardial infarction is the activation of an immune response, which removes death cardiomyocytes and initiates scar formation. On the other hand, activation and infiltration of immunocompetent cells are responsible for augmenting damage in non-infarcted areas. Emerging evidence suggests a cardioprotective role of the angiotensin AT2R by attenuating this post-infarct inflammatory reaction, albeit the underlying cellular mechanisms are not well understood. We aimed here at elucidating a potential role of the cardiac angiotensin AT2R in regulating the cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, immunofluorescence staining of tissue sections showed that AT2R was detected in a fraction of CD8+ T cells infiltrating the peri-infarct myocardium. We developed a method that allowed the isolation and characterization of CD8+AT2R+ T cells infiltrating the myocardium via combined MACS and FACS technology. While the CD8+AT2R- T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes in vitro, the CD8+AT2R+ T cells were non-cytotoxic to these cardiomyocytes. The CD8+AT2R+ T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-gamma gene expression when compared to CD8+AT2R- T cells. We further showed that IL-10 gene expression was enhanced in CD8+ T cells upon in vitro AT2R stimulation. In addition, in vivo AT2R activation leads to an increment of the CD8+AT2R+ T cells and IL-10 production in the ischemic myocardium. Moreover, the CD8+AT2R+ T cell population was also detected in human peripheral blood. We have defined a CD8+ T cell population that expresses AT2R and increases during ischemic heart injury. This population sustains cardiomyocyte viability by providing cardioprotective IL-1 via a novel AT2R-mediated cellular mechanism for modulating adaptive immune response in the heart.

O Diabetes Mellitus tipo 1 (DM1A) é uma doença autoimune caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas, levando à perda progressiva da capacidade de secreção de insulina pelas células beta pancreáticas. A homeostase das células T, ou seja, o desenvolvimento e manutenção apropriados dos números e funções das células T são essenciais para a integridade do sistema imune. Classicamente acreditava-se que as células T CD4+ poderiam se subdividir em duas populações efetoras distintas, T helper 1 e T helper 2. Recentemente, foram descritas duas novas vias de ativação de linfócitos T CD4+: a via Th17, que tem papel fundamental na autoimunidade; a via T regulatória, onde células T CD4+CD25+ high são essenciais na tolerância periférica e proteção contra autoimunidade. As Interleucinas (IL) de cadeia gama comum agem em várias etapas desta diferenciação linfocítica. A IL-21 é o membro mais recente desta família de citocinas, que inclui também: IL-2, IL-4, IL-7 , IL-9 e IL-15. A IL-21 atua através da interação com seu receptor, o IL-21R, apresentando ações pleiotrópicas e, como regra, pró-inflamatórias. Em estudos com modelos animais de diabetes autoimune verificou-se que a IL-21 e seu receptor são essenciais para o desenvolvimento da doença, porém ainda não há estudos sobre a ação desta interleucina no DM1 em humanos. O objetivo de nosso estudo foi avaliar o papel dos receptores das interleucinas de cadeia gama comum na patogênese do DM1A através da determinação da expressão da proteína de superfície e do RNA mensageiro destes receptores em pacientes com DM1A de início recente, em comparação com indivíduos controles normais, e da correlação destes valores com títulos de autoanticorpos pancreáticos. Estudamos a expressão da proteína de superfície do IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) e IL-7R (CD127) em linfócitos T periféricos de 35 pacientes com DM1 e 25 controles sadios utilizando citometria de fluxo. O tempo médio de diagnóstico do DM1 foi de 3 meses, e todos os pacientes estavam em uso de insulina no momento da coleta de sangue. Auto-anticorpos pancreáticos (anti-GAD65 e anti-IA2) foram dosados através de radioimunoensaio. A expressão do RNAm de IL-21R, IL-2R e IL-2R foi quantificada por PCR em tempo real em 23 dos pacientes portadores de DM1A. Detectamos, pela primeira vez, diminuição significativa na expressão proporcional de IL-21R, CD25 e CD122 em linfócitos TCD3+ e TCD4+, além de diminuição na expressão de CD124 em linfócitos T CD4+ e CD127 em linfócitos T CD3+. Verificamos também redução significativa na quantidade de células TCD4+CD25+high (T regulatórias) nos pacientes DM1A. Não houve correlação entre expressão dos receptores de superfície das interleucinas de cadeia gama comum e títulos de autoanticorpos pancreáticos. Realizamos o PCR em tempo real para quantificar a expressão do RNA mensageiro (RNAm) dos receptores de interleucinas de cadeia gama comum, e avaliar se esta correspondia à expressão das proteínas de superfície obtida através de citometria de fluxo. Comparamos a expressão do RNAm de IL-21R, IL-2R e IL-2R nos pacientes DM1A dividindo-os em tercis de acordo com os valores de expressão de proteína de superfície obtidos por citometria de fluxo em linfócitos T CD3+, e verificamos que não houve diferença entre os 3 grupos na expressão relativa dos genes estudados. Portanto, em nossa casuística a redução da expressão da proteína de superfície dos receptores de interleucinas de cadeia gama comum possivelmente decorreu de alterações posteriores à transcrição do RNA mensageiro / Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by pancreatic infiltration of T and B lymphocytes, macrophages and dendritic cells, leading to a progressive destruction of the insulin-producing -cells. Homeostasis of T cells can be defined as the ability of the immune system to maintain normal T-cell counts and to restore T-cell numbers following T-cell depletion or expansion. It was classically believed that the CD4+ T cells could be activated into two distinct effector populations, T helper1 and T helper2. It was recently described two new pathways of CD4+ T lymphocytes activation: the Th17 pathway, that plays a fundamental role in autoimmunity and the regulatory pathway (Treg), where CD4+CD25+high T cells are essential to maintain peripheral tolerance and therefore protect against autoimmunity. The common gamma chain cytokines interfere with several steps of the CD4+ T lymphocytes differentiation. Interleukin-21 (IL-21) is the most recent member of this family, that also includes IL-2, IL-4, IL-7, IL-9 and IL-15, and has pleiotropic effects on the immune system. Interleukin-21 acts through interaction with its receptor, the IL-21R, which is expressed in a great variety of immune cells. Various studies with animal models of autoimmune diabetes demonstrated that IL-21 and its receptor are essential for the development of the disease, but there are no studies evaluating the role of this interleukin and its receptor in T1DM in humans. The aim of our study was to assess the role of common gamma chain-dependent cytokine receptors in the pathogenesis of T1D, by determining the expression of the surface protein and mRNA of these receptors in recent-onset T1D patients and correlating these values with titles of pancreatic autoantibodies. We studied the surface protein expression of IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) and IL-7R (CD127) in peripheral T lymphocytes of 35 patients with T1D and 25 healthy controls using flow cytometry. Mean T1D duration was 3 months and all patients were using insulin at the time of blood withdraw. Pancreatic autoantibodies (anti-GAD65 and anti-IA2) were assessed by radioimmunoassay. The mRNA expression of IL-21R, IL-2R and IL-2R was quantified by real time PCR in 23 of the T1D patients. We detected for the first time a statistically significant decrease in the proportional expression of IL-21R, CD25 and CD122 on CD3+ and CD4+ T lymphocytes, a decrease in the expression of CD124 on CD4+ T cells and CD127 on CD3+ T lymphocytes. We also observed a significant reduction in the amount of CD4+ CD25+high (T regulatory cells) in T1D patients. There was no correlation between the expression of the surface receptors of common gamma chain cytokines and titles of pancreatic autoantibodies. We performed real-time PCR to quantify RNA expression of common gamma-chain interleukin receptors, and evaluate if these values corresponded to those of surface proteins obtained using flow cytometry. We compared the mRNA expression of IL-21R, IL-2R and IL-2R in T1D patients by dividing them into tertiles according to the expression values of surface protein obtained by flow cytometry in CD3+T lymphocytes. We observed that there was no difference in the relative expression of mRNA among the 3 groups of patients. Therefore, in our study, the reduction of surface protein expression of common gamma chain cytokines receptors was possibly due to alterations that occurred after the transcription of mRNA

A profilaxia pré-exposição (PrEP) ao HIV se consolidou como uma importante estratégia de combate ao avanço da epidemia. Ainda assim, a incidência de casos da infecção vem aumentando na população jovem, assim como a de outras infecções sexualmente transmissíveis (ISTs), que atuam como importante fator de risco para transmissão do HIV-1. Entre as infecções mais frequentemente diagnosticadas estão Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG). A presença de lesões na mucosa genital e anal são fatores de risco estabelecidos para a transmissão do HIV-1, porém o impacto das infecções assintomáticas ainda é pouco conhecido. Dados recentes mostram que a ativação sistêmica de linfócitos T é um fator de risco para a aquisição da infecção pelo HIV-1. Nesse estudo, estudamos a ativação de linfócitos T na presença de infecção anorretal assintomática por CT e/ou NG. Células mononucleares do sangue periférico de voluntários do PrEP Brasil, um estudo clínico demonstrativo de PrEP ao HIV em homens que fazem sexo com homens, foram descongeladas para análise da ativação de linfócitos T. Trinta e quatro participantes com swab anorretal positivo para CT e/ou NG foram selecionados, enquanto assintomáticos e negativos para outras ISTs. Trinta e cinco controles foram selecionados randomicamente. Encontramos uma maior frequência de linfócitos T CD8+ HLA-DR+CD38+ (1,5 vs. 0,9% p < 0,005) no grupo com infecção assintomática. Os linfócitos T CD8+ de memória também apresentaram uma maior expressão dos marcadores de ativação. Os marcadores de exaustão e senescência foram significantemente mais expressos no grupo com a infecção. Não foi observado aumento ou diferença nos níveis de CD14 solúvel no plasma. Nossos achados demonstram que as infecções anorretais assintomáticas por CT e NG induzem a ativação sistêmica de linfócitos T CD8+. Considerando a alta prevalência dessas infecções e o risco associado de aquisição da infecção pelo HIV-1, o rastreamento periódico e o tratamento sistemático devem sem explorados em conjunto com as estratégias de prevenção ao HIV / Oral antiretroviral pre-exposure prophylaxis (PrEP) has been established as a pivotal strategy in the prevention against HIV epidemic. However, the incidence of HIV-1 infections has been rising among the youth, as well as other sexually transmitted infections (STIs), acting as an important risk factor for HIV-1 acquisition. Infection by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most diagnosed. Although the presence of mucosal lesions is a known risk factor for HIV-1 acquisition, the potential increase in risk associated with asymptomatic STIs is not completely understood. Recent data defined higher T cell activation as a single risk factor for sexually acquired HIV-1 infection. We examined the effect of asymptomatic CT and/or NG anorectal infection on immune activation. Peripheral blood mononuclear cells from participants of PrEP Brasil, a study of daily oral PrEP among healthy men who have sex with men, were analyzed for T cell activation by flow cytometry. Thirty-four participants with positive anorectal swab for CT and/or NG were selected, while negative for other STIs and without any reported symptoms. Thirty-five controls were randomly selected. We found a higher frequency of CD8+ HLA-DR+CD38+ T cells (1.5 vs. 0.9% p < 0.005) in the group with CT and/or NG infection and a greater median proportions of activation markers expression in CD8+ T cells with memory phenotype. Exhaustion and senescence markers were also significant higher in the infected group. No difference was observed in the soluble CD14 levels. Our findings suggest that asymptomatic CT and NG anorectal infection lead to a systemic activation of the T cell compartment. Considering the high prevalence of asymptomatic infection and the risk of HIV-1 acquisition associated, regular screening and treatment should be explored as an adjuvant tool for HIV prevention