Au pic de la réponse effectrice des LT CD8, on retrouve deux sous-populations, soit les effecteurs à demi-vie courte (SLEC) ou les effecteurs précurseurs de cellules mémoires (MPEC). La phase de contraction de la réponse effectrice implique l’apoptose des SLEC et la survie des MPEC, qui se différencient en cellules mémoires pour protéger contre une réinfection. La voie Notch est impliquée dans les choix de différenciation binaire et l’interaction ligand-récepteur mène au clivage du domaine intracellulaire de Notch (NICD), qui migrera au noyau afin d'induire l’expression de gènes cibles. Dans un modèle où l’expression des récepteurs Notch1 et Notch2 est absente uniquement dans les LT CD8 (N1N2∆/∆), le laboratoire a démontré que l’absence du signal Notch favorisait la différenciation en MPEC et affectait l’expression de 217 gènes. Cette étude visait à 1) identifier les gènes cibles de Notch contrôlant la différenciation SLEC-MPEC, et 2) évaluer si l’absence du signal Notch permet un meilleur contrôle tumoral par les LT CD8. Nous avons priorisé les 217 gènes en fonction de différents critères et identifié Il2ra comme une cible importante en aval de la voie Notch. Toutefois, nous avons établis que lors d’une infection aiguë, la surexpression rétrovirale de Il2ra dans les LT CD8 N1N2∆/∆ n’influençait pas la différenciation SLEC-MPEC. Nous avons également déterminé qu’une thérapie adoptive de LT CD8 N1N2∆/∆ limitait le contrôle de la croissance tumorale et impliquait une diminution des fonctions effectrices des LT CD8 N1N2∆/∆, qui étaient moins terminalement différenciés. Une meilleure compréhension du rôle de Notch dans la réponse des LT CD8 permettra de développer de nouvelles stratégies de vaccination et de traitement du cancer. / In response to acute infections, effector CD8 T cells differentiate into short-lived effector cells (SLECs) and memory precursor effector cells (MPECs) capable of generating long-lived memory CD8 T cells. The Notch signaling pathway is a key regulator of cell fate decision. Following ligand- receptor interaction, the Notch intracellular domain (NICD) is cleaved and migrates to the nucleus in order to induce the expression of target genes. In a model in which Notch1 and Notch2 expression is inhibited only in mature CD8 T cells, our team has established that Notch deficiency favors MPEC differentiation in CD8 T cells and influences the expression of 217 gens. This study aims to: 1) identify target genes of the Notch pathway regulating SLEC-MPEC differentiation, 2) evaluate if Notch deficiency can augment tumor control by CD8 T cells. We have prioritized the list of genes differentially expressed in the absence of Notch signaling according to various criteria. We hence identified Il2ra as a target gene, but during an acute infection, overexpression of Il2ra in Notch deficient CD8 T cell was insufficient to modulate SLEC-MPEC differentiation. In addition, we have established that adoptive therapy with Notch deficient CD8 T cell impaired tumor control and implicated a diminution of effector function in Notch deficient CD8 T cell, which were less terminally differentiated. A better understanding of Notch signaling pathway’s role in the CD8 T cell response will allow for improvement of vaccinal strategies and cancer treatment.

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

Les lymphocytes T (LT) CD8 sont un sous-type de cellules immunitaires qui participent à l’élimination des certains agents infectieux et de cellules tumorales. La capacité de réponse des LT CD8 est dépendante de leur état de différenciation. Lors d’une réponse immunitaire, les LT CD8 spécifiques à l’agent infectieux sont activés, se différencient et prolifèrent en LT effecteurs (LTE) qui participent à l’élimination de l’agent pathogène. Les LTE peuvent être distingués en effecteurs de courte durée de vie (SLEC), terminalement différenciés et éliminés par apoptose après l’infection, et en précurseurs des cellules mémoires (MPEC) qui survivent et génèrent les LT mémoires (LTM). Par contre, lors d’une infection chronique ou d’une réponse antitumorale, la persistance antigénique et inflammatoire induisent l’épuisement des LT CD8, soit un état de différenciation caractérisé par des fonctions effectrices et prolifératives diminuées ainsi qu’une forte expression de récepteurs inhibiteurs (RI). Afin d’améliorer les thérapies vaccinales, les traitements antitumoraux et les thérapies lors des infections chroniques, il est important de mieux comprendre la différenciation des LT CD8. Nous avons étudié le rôle de NR4A3 dans la différenciation des LT CD8 chez la souris. NR4A3 est un récepteur nucléaire et un facteur de transcription (FT) dont l’expression est rapidement induite par une stimulation antigénique, mais dont le rôle dans les LT CD8 n’a pas été encore déterminé. Nous avons émis l’hypothèse que l’expression de NR4A3 dans ces cellules suite à une stimulation antigénique contrôle leur différenciation. Premièrement nous avons étudié le rôle du NR4A3 dans la différenciation des LT CD8 lors d’une infection aiguë et avons déterminé que la délétion de NR4A3 dans les LT CD8 augmente la différenciation MPEC, la génération des LTM et la production de cytokines. La régulation de la différenciation des LT CD8 par NR4A3 est transcriptionnelle et, lors des premiers jours postinfection, sa délétion induit un programme transcriptionnel associé avec la différenciation des LTM. De plus, dès les premières heures postactivation, la délétion de NR4A3 favorise l’induction d’un état de chromatine plus ouvert avec une prédiction d’activité augmentée des FT bZIP. Deuxièmement, nous avons étudié le rôle de NR4A3 lors d’une réponse immunitaire antitumorale au cours d’une thérapie cellulaire adoptive (ACT) sous traitement d’anti-PD-L1 (ligand d’un RI) où la meilleure fonctionnalité et persistance des LT CD8 NR4A3 déficients ont été mises à l’épreuve. Ainsi, l’ACT avec des LT CD8 NR4A3 déficients augmente la survie de souris porteuses de tumeurs et les lymphocytes T infiltrants les tumeurs (TIL) NR4A3 déficients sont moins terminalement épuisés et présentent des plus fortes proportions et nombres cellulaires intra-tumoraux. Le profil transcriptomique au niveau de cellules uniques a révélé que les TIL NR4A3 déficients favorisent la génération de progéniteurs distincts et des populations fonctionnelles associées avec le traitement anti-PD-L1. En conclusion, NR4A3 est un régulateur de la fonction et la différenciation des LT CD8 dont l’activité pourrait être modulée afin d’améliorer les stratégies de vaccination ou les thérapies cellulaires. / CD8 T cells are an immune cell population involved in the clearance of different types of infections and the elimination of tumor cells. The response capacity of CD8 T cells depends on their differentiation state. During an immune response, antigen-specific CD8 T cells are activated, differentiate and proliferate into effectors that participate in elimination of the pathogen. Among the pool effectors, there are short-lived effector cells (SLEC) that are terminally differentiated and die by apoptosis after the infection clearance, and the memory precursors effector cells (MPEC) that survive to give rise to memory CD8 T cells. However, during chronic infection or an anti-tumor immune response, antigen persistence and inflammation induce CD8 T cell exhaustion, which is a differentiation state characterized by decreased effector functions and proliferative capacity and an increased expression of inhibitory receptors (IR). Thus, to be able to increase the efficiency of CD8 T cells following vaccination, in the context of antitumoral or during treatment against chronic viral infections, it is important to better understand CD8 T cell differentiation. We studied the role of NR4A3 in CD8 T cell differentiation in mice. NR4A3 in a nuclear receptor and transcription factor (TF), which expression is rapidly induced following antigenic stimulation, but the role of its induction in CD8 T cells was not yet identified. We propose that NR4A3 expression in CD8 T cells following antigenic stimulation controls their differentiation. First, we studied the role of NR4A3 in CD8 T cell differentiation during acute infection and determined that its deletion increases MPEC differentiation, memory generation, and cytokines production. NR4A3 regulates CD8 T cell differentiation at the transcriptional level, and its deletion induces a memory-related transcriptional program early during the immune response (day three post-infection). Moreover, a few hours following the CD8 T cell activation, NR4A3 deletion increased chromatin accessibility, particularly to bZIP TF. Secondly, we studied the role of NR4A3 during the antitumoral response in the context of adoptive cell therapy (ACT) and cotreatment with anti-PD-L1 (ligand of an IR), during which we tested the increased functionality and persistence of NR4A3 deficient cells. ACT with NR4A3-deficient cells increases the survival of tumor-bearing mice. In addition, NR4A3 deficiency increased the frequency of tumor-infiltrating lymphocytes (TILs) and decreased T cell exhaustion. Single-cell transcriptional profile of TILs revealed that NR4A3 deficiency induces the generation of a transcriptionally distinct progenitor population and an increase in functional populations associated with the anti-PD-L1 treatment. To conclude, NR4A3 is a new regulator of CD8 T cell differentiation and function whose activity regulation could increase the efficiency of vaccinations and cell therapy treatments.

L’épuisement des lymphocytes T CD8+ (LT CD8) est une voie de différentiation unique survenant lors de contextes pathologiques particuliers ayant en commun la persistance d’antigènes dans l’hôte, tel que les infections virales chroniques (expl : VIH, hépatites B et C) et différents types de cancers. Il apparait aujourd’hui très clairement que ce mécanisme est à l’origine de l’échec de l’immunité adaptative face à ces pathologies particulièrement néfastes pour l’homme. L’étude de ce processus a mené à la découverte de cible thérapeutiques d’un grand intérêt (« immune checkpoints ») pouvant être ciblées pour corriger et/ou reverser l’épuisement. Les essais thérapeutiques ayant découlés de ces découvertes ont donné des résultats extrêmement prometteurs dans le traitement de plusieurs cancers. Cependant, bien que ces thérapies ciblées permettent un regain temporaire de la fonction des LT CD8+, elles ne permettent pas d’inverser le processus d’épuisement. Il est donc crucial aujourd’hui de se tourner vers les agents causateurs de cet état d’épuisement qui restent très méconnues à ce jour. La famille de cytokines partageant la chaine commune gamma (cytokines gamma c) comprenant l’IL-2 -4 -7 -9 -15 et -21 sont des acteurs solubles clés de l’immunité adaptative. Ces cytokines sont intimement liées aux processus de développement, d’homéostasie, de différenciation et de maintenance des lymphocytes T. Parmi elles, l’IL-2 et l’IL-15 ont un rôle majeur dans le processus de différenciation des LT CD8+ au cours d’une infection virale aigue. Malgré cela, l’implication de ces cytokines dans l’épuisement des LT CD8+ dans un contexte d’infection virale chronique n’a jamais été investiguée. En se basant sur les connaissances actuelles des rôles de l’IL-2 et de l’IL-15 sur la différenciation des LT CD8+ au cours d’une infection virale aigue, nous avons émis l’hypothèse que ces cytokines pourraient promouvoir l’épuisement dans un contexte d’infection virale chronique. Dans un premier temps, nous avons démontré chez l’homme (patients atteints d’hépatite C chronique) et la souris (modèle LCMV Clone 13) que la chaîne beta du récepteur à l’IL-2 (IL2R beta[CD122]) qui se lie à l’IL-2 et l’IL-15 reste sélectivement exprimée à la surface des LT CD8+ épuisés au cours d’une infection virale chronique. De plus, une expression élevée de cette chaîne de récepteur corrèle avec un épuisement plus sévère des LT CD8+ chez l’homme et la souris. En développant un modèle murin dans lequel les LT CD8+ sont déficients pour cette chaîne, nous avons démontré que l’IL-2 et IL-15 contrôlent plusieurs aspects clés du processus d’épuisement. Ces cytokines augmentent l’expression de plusieurs récepteurs inhibiteurs (caractéristiques de l’épuisement) et contrôlent même directement l’expression de certains d’entre eux (notamment 2B4 et TIM-3). L’IL-2 et l’IL-15 dirigent également la différenciation terminale des LT CD8+ vers un état d’épuisement extrême et abrogent de manière irréversible leur potentiel de différenciation en cellules mémoires. Nous montrons donc pour la première fois un rôle clé de l’IL-2 et l’IL-15 dans l’épuisement des LT CD8+ au cours d’une infection virale chronique. Dans un deuxième temps nous avons investigué les fonctions individuelles et redondantes de l’IL-2 et l’IL-15 dans l’épuisement des LT CD8+. Nous avons également déterminé les fenêtres d’actions déterminantes de ces cytokines et les mécanismes intracellulaires clés par lesquels elles contrôlent le processus d’épuisement. L’IL-2 et l’IL-15 coopèrent pour promouvoir l’expression de 2B4 et TIM-3 à la surface des LT CD8+ et ces cytokines semblent collaborer pour diriger leur différenciation terminale. En revanche, les signaux médiés par l’IL-2 pendant la phase de « priming » abrogent sélectivement leur potentiel de différenciation en cellules T centrales mémoires (Tcm) alors que l’IL-15 semble plutôt supprimer celle des T effecteurs mémoires (Tem) pendant la phase chronique. Pour finir, nous avons identifié la voie JAK3/STAT5 comme étant la principale voie intracellulaire par laquelle l’IL-2 et l’IL-15 dirigent l’épuisement des LT CD8+. Au cours de cette thèse, nous avons donc mis en évidence un nouveau rôle de l’IL-2 et l’IL-15 dans l’épuisement des LT CD8+ au cours d’une infection virale chronique. Nos résultats apportent une meilleure compréhension du processus d’épuisement des LT CD8+ et démontrent pour la première fois une implication des cytokines. Nous espérons que ces travaux contribueront à améliorer les stratégies thérapeutiques actuelles contre le cancer et les infections virales chroniques. / CD8+ T cell exhaustion is a unique differentiation pathway which occurs during particular pathological contexts such as chronic viral infections (i.e. HIV, HCV and HBV) and cancers in which antigen (Ag) persists in the host. It appears clear now that this mechanism provokes the failure of adaptive responses against these pathologies and is particularly harmful to humans. The study of this process has led to the discovery of relevant molecules (“immune checkpoints”) that can be targeted to prevent and/or reverse exhaustion. Ensuing clinical trials have provided extremely promising results in the treatment of several cancers. However, although these targeted therapies allow a temporary regain of CD8+ T cell functions they still fail at reversing the exhaustion process. It is thus crucial to investigate the causative factors of such process that remain to be identified. The common gamma-chain (gamma c) family of cytokines which includes IL-2, -4, -7, -9, -15, and -21 are key soluble mediators involved in the development of adaptive immunity. These cytokines are intimately linked to T cell development, homeostasis, differentiation and maintenance. Among them, IL-2 and IL-15 display important functions on CD8+ T cell differentiation during an acute viral infection. However, impact of these cytokines on CD8+ T cell responses during a chronic viral infection remains to be investigated. Based on current knowledge of the functions of IL-2 and IL-15 on CD8+ T cell differentiation during an acute viral infection, we hypothesized that these cytokines promote CD8+ T cell exhaustion during a chronic viral infection. We first demonstrate in a mouse model of chronic viral infection (LCMV clone 13) and patients with chronic HCV that the IL-2-receptor beta chain (IL2R beta [CD122]) a cytokine receptor chain which binds to both IL-2 and IL-15 is selectively expressed on exhausted CD8+ T cells during a chronic viral infection. The intensity of CD122 expression positively correlates with severe exhaustion of CD8+ T cells in mice and humans. Using a mouse model in which CD8+ T cells lack the expression of the IL2R beta-chain, we demonstrate that IL-2 and IL-15 control several aspects of exhaustion. IL-2 and IL-15-dependent signals sustain the expression of several inhibitory receptors (characteristic of exhaustion) on CD8+ T cells and directly control the expression of some of them (e.g. 2B4 and TIM-3). IL-2 and IL-15 also direct the terminal exhaustion of CD8+ T cells and irreversibly abrogate their developmental plasticity toward memory T cell development. Together, we show for the first time key functions of IL-2 and IL-15 in directing CD8+ T cell exhaustion during a chronic viral infection. Next, we investigated the unique and redundant functions of IL-2 and IL-15 on CD8+ T cell exhaustion. We also determined individual time-frames of these cytokines and intracellular pathways by which they control CD8+ T cell exhaustion. IL-2 and IL-15 cooperate to promote 2B4 and TIM-3 expression on CD8+ T cells, and these cytokines likely collaborate to direct terminal exhaustion. In contrast, IL-2-dependent signals during priming preclude subsequent differentiation into central memory cells (Tcm) while prolonged exposure to IL-15 upon viral persistence likely suppresses effector memory cell (Tem) developmental potential. Finally, we demonstrate that the JAK3/STAT5 pathway is the dominant pathway by which IL-2 and IL-15 direct CD8+ T cell exhaustion. This thesis provides evidence of novel functions of IL-2 and IL-15 in directing CD8+ T cell exhaustion during a chronic viral infection. These results increase our understanding of the CD8+ T cell exhaustion process and demonstrate for the first time the involvement of cytokines. We hope that this work will contribute to the improvement of actual therapeutic strategies against chronic viral infections and cancers.

Les polynucléaires neutrophiles représentent 50-70% des leucocytes sanguins et possèdent un rôle majeur dans la défense de l’organisme contre les pathogènes. Le Ca2+ est un second messager qui joue un rôle primordial dans le chimiotactisme, la phagocytose, la dégranulation et la production de formes réactives de l’oxygène (FRO) afin de neutraliser l’agent pathogène. Dans ces cellules, l’influx calcique de type SOCE est essentiel pour l'homéostasie calcique. Il est peu étudié en raison du manque d’outils pharmacologiques spécifiques d’où l’importance dans un premier temps de chercher de nouvelles molécules. Les cellules T Jurkat dont le SOCE est largement caractérisé servent de modèle pour la caractérisation initiale de ces molécules. Le 2-APB est parmi les molécules les plus largement utilisées dans la caractérisation du SOCE en raison de sa double activité sur le SOCE avec une potentialisation à [1-10 μM] et une inhibition à [> 20 μM]. En revanche, ce produit manque de spécificité et agit sur d’autres cibles cellulaires comme les récepteurs à l’inositol (1,4,5)-trisphosphate (InsP3Rs). La 1ère étape est de sélectionner à partir d’analogues commerciaux du 2-APB (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB et MDEB), des composés plus spécifiques et également plus efficaces que la molécule mère. Deux molécules se sont distinguées : le MDEB comme uniquement potentialisant du SOCE et le Benzothienyl-APB comme un puissant inhibiteur. En revanche, tous les analogues du 2-APB inhibent les InsP3Rs à l’exception du MDEB qui semble plus spécifique du SOCE. L’effet du MDEB sur le courant calcique, ICRAC, a été étudié grâce à la technique du patch-clamp. Il augmente d’environ 4 fois l’amplitude de ICRAC par rapport à celle enregistrée dans les cellules contrôle. Par ailleurs, le MDEB ralentie l’inactivation rapide de ICRAC due au Ca2+. Sur le plan physiologique, le MDEB à des concentrations croissantes inhibe la synthèse de l’IL-2 dans les cellules Jurkat stimulées et ceci malgré son effet potentialisant du SOCE. Cette activité est liée à son effet pro-apoptotique dans les cellules Jurkat stimulées. Le MDEB et le Benzothienyl-APB caractérisés dans la 1ère partie nous ont servi d’outils potentiels afin d’étudier le SOCE des cellules PLB-985 différenciées en cellules proches de neutrophiles. Le SOCE a été induit soit par un traitement des cellules avec la thapsigargine (Tg) soit de manière physiologique avec les peptides fMLF et le WKYMVm deux chimioattractants, ligands des récepteurs aux peptides formylés FPR et FPRL1 respectivement. En plus, le SOCE induit par la Tg est modulable par le 2-APB, potentialisé par le MDEB et inhibé par le Benzothienyl-APB. La phagocytose des levures par les cellules PLB-985 différenciées ainsi que la production de FRO intraphagosomales ont été inhibées par le MDEB et le Benzothienyl-APB. Les FRO extracellulaires ont été également inhibées par Benzothienyl-APB en revanche à cause de la forte interférence du MDEB avec la technique de mesure nous n’avons pas pu étudier ses activités. En conclusion, le MDEB et le Benzothienyl-APB sont de nouveaux outils pharmacologiques potentialisant ou inhibant le SOCE des leucocytes, qui nous permettront dans l’avenir une meilleure compréhension de l'entrée calcique et ses rôles dans ces cellules. / Neutrophils represent 50-70% of human blood leukocytes; their role is to protect the body against pathogens. Calcium is a second messenger which plays an important role in chemotaxis, phagocytosis, degranulation and the production of reactive oxygen species (ROS) in order to eliminate microbes. In neutrophils, the mechanism of store-operated calcium entry (SOCE) is essential for calcium homeostasis. However, neutrophil SOCE is not well understood because of the lack of specific pharmacological tools. It is necessary to first identify and characterize new molecules using a model of Jurkat T cells in which SOCE was the best characterized. 2-APB is the most widely used molecule in SOCE characterization due to its dual activity with a potentiation at lower concentrations [1-10 μM] and an inhibition at higher concentrations [> 20 μM]. However, this molecule lacks specificity because it acts on other cellular targets such as inositol (1,4,5)-trisphosphate receptors (InsP3Rs). The first step is to select from a library of 8 commercial 2-APB analogues (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB and MDEB) those that are more specific and also more efficient molecules than 2-APB. Two interesting molecules were identified, MDEB as the only SOCE potentiating product currently known and the Benzothienyl-APB, which is a strong inhibitor. Like 2-APB, all these analogues inhibit InsP3Rs except MDEB, which seems to be more specific. The effect of MDEB on the calcium current, ICRAC, was also studied using the patch-clamp technique. MDEB increases ~4 times the ICRAC amplitude in comparison with control. Otherwise, MDEB slows down the fast Ca2 +-dependent inactivation of ICRAC. Functionally, MDEB at increasing concentrations inhibits IL-2 synthesis in stimulated Jurkat T cells despite its potentiating activity on SOCE. The inhibition is due to MDEB induced apoptosis in stimulated Jurkat T cells. MDEB and Benzothienyl-APB were then used as tools to study SOCE in a neutrophil-like cell model, the differentiated PLB-985 cells. SOCE was induced either by treatment of cells with thapsigargin (Tg) or physiologically with the chemotactic peptides fMLF and WKYMVm, ligands of formyl peptide receptors FPR and FPRL1 respectively. In addition, Tg-induced SOCE was modulated by 2-APB, potentiated by MDEB and inhibited by Benzothienyl-APB. The consequences of these analogues on neutrophil functions were also studied. Phagocytosis of yeasts by PLB-985 cells and intraphagosomal ROS production were inhibited by MDEB and Benzothienyl-APB. Furthermore, extracellular ROS were also inhibited by Benzothienyl-APB. However, because of the high interference of MDEB with our techniques, its activities could not be studied. In conclusion, MDEB and Benzothienyl-APB are new analogues of 2-APB potentiating and inhibiting SOCE, which allow us in the future a better understanding of leukocyte SOCE and its cellular roles.

No description available.

Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.

O timo é um órgão linfoide primário, localizado em região mediastinal, cuja importância funcional é a diferenciação e maturação de todas as subpopulações de linfócitos T provenientes da medula óssea assim como a seleção de células autorreativas. Sua hipoplasia ou aplasia resultam em síndromes de imunodeficiência. Embora de vital importância, o estudo clínico de sua função não é rotineiro na prática clínica, o que pode ser atribuído a sua dificuldade de avaliação em razão de sua localização, necessidade de uso de métodos de imagem não inócuos ao paciente (tomografia computadorizada (TC), PET-SCAN) e complexidade das análises em sangue periférico de subpopulações de células T por citometria de fluxo e, mais recentemente, medição de T cell receptor excision circles (TRECs), por PCR. Um possível método da avaliação do timo sem radiação ionizante ou dor ao paciente seria a elastografia de timo por ultrassom e seu uso na prática clínica poderia substituir a TC, como ocorre na avaliação de lesões hepáticas ou mamárias. Objetivo - Este estudo se propõe a 1. Implantar este método na avaliação da função tímica, 2. Estabelecer valores de referência de TRECs na faixa etária estudada, 3. Investigar se há correlação entre os dois parâmetros. Métodos - Foram incluídas sessenta e quatro crianças de 0-5 anos em acompanhamento no ambulatório de cirurgia infantil sem doença sistêmica ou infecção aguda, e que iriam coletar amostra de sangue para exames pré-operatórios. Quarenta e oito destas coletaram amostra de sangue para avaliação de TRECs, vinte e nove realizaram elastografia num mesmo momento, porém apenas 13 destas apresentaram resultado confiável. A média da idade foi de 36 ± 16meses, predomínio do grupo foi masculino (75%), nascidos a termo (72%) e a principal intervenção cirúrgica foi do tipo urológica de pequeno porte. A elastografia mostrou média de 1,21 ± 0,24m/s, sem diferença significativa quando comparada ano a ano. Observamos uma média de TRECs de 195,6 ± 120,5 cópias/µL, mostrando valores significativamente mais altos quando comparados a adolescentes hígidos da base de dados do laboratório. Os valores de TRECs observados mostram uma ampla variabilidade na faixa etária estudada, sem diferença significativa quando separados por idade ano a ano. Não se encontrou correlação significativa entre a dureza do timo analisada à elastografia e valores de TRECs em sangue periférico. Concluímos que a elastografia é um método que possibilita a avaliação das dimensões e função do timo em crianças a partir de 2 anos de idade, entretanto estudos adicionais são necessários para que se possa recomendar a larga implantação deste método com essa finalidade / The thymus is a primary lymphoid gland responsible for the maturation of T cells as well as the immunological central tolerance. It has been a neglected organ by physicians, despite its relevance in early immunity. Thymic function can be indirectly measured by Computerized Tomography imaging and PET SCAN, T cell subpopulation flow cytometry. More recently, in the beginning of this century, a direct measurement represented by TRECs (T cell receptors excision circles) was developed. Classical thymic imaging has used ionized radiation, which poses a major risk for the pediatric patient and new techniques are needed. Objectives and methods - In this work, we tested the use of elastography ultrasound for the evaluation of the thymus in a group of < 5-year- old healthy children. In parallel, we measured TRECs in peripheral blood and compared the values obtained from both methods. We have reached sixty-four children at the pediatric surgery outpatients ambulatory, scheduled for minor surgeries. A sample of blood was taken during pre operatory and then patients were sent to the imaging service for elastography. Of all, sixty-four had undertaken TRECs and seventeen, elastography. The median age was 36 ±16 months and we had 75% of boys for surgical correction of urologic minor defects. The elastography results showed a median of 1.2 ± 0.24 m/s in all ages, the same stiffness as the liver, as shown in other works. Our median TREC/µL value was 195.6 ± 120.5 copies/µL showing a trend of reduction in older ages, and with statistical significance when compared with healthy teenagers\' values from the lab database. We concluded that elastography may be a good diagnostic tool for thymus evaluation, and additional works are needed for its recommendation in clinical practice. Our TRECs values showed a large variability, as also demonstrated in previous works, and a trend of reduction over age. We could not observe any significant correlation between elastography and TRECs values

Abordagens atuais no desenho de vacinas contra o HIV-1 estão focadas em imunógenos que codificam proteínas inteiras do HIV-1 e visam induzir respostas citotóxicas específicas. É concebível que vacinas bem-sucedidas devem induzir respostas contra múltiplos epitopos do HIV-1, coincidindo com seqüências das cepas circulantes do vírus, conhecido por sua grande variabilidade genética. Sabe-se que células T CD4+ são necessárias para indução de respostas efetivas de linfócitos T CD8+ citotóxicos. Neste trabalho, nós avaliamos a imunogenicidade de uma vacina de DNA codificando 18 epitopos para linfócitos T CD4+, conservados e ligadores de múltiplas moléculas HLA-DR em camundongos BALB/c e em quatro linhagens de camundongos transgênicos para moléculas de HLA classe II. Os camundongos imunizados apresentaram respostas de amplitude e magnitude significativas com proliferação e secreção de citocinas por linfócitos T CD4+ e T CD8+. Onze dos 18 epitopos para linfócitos T CD4+ presentes na vacina foram reconhecidos pelas linhagens de camundongos transgênicos para moléculas de HLA classe II. Em suma, 17 dos 18 epitopos codificados pela vacina foram reconhecidos. As células induzidas pela vacina apresentaram um perfil polifuncional com tipo 1 de citocinas, incluindo produção de IFN- , TNF- e IL-2. A vacina também induziu células T CD4+ de memória central de longa duração, capazes de fornecer auxílio contínuo para células T CD8 +. Pela capacidade da vacina HIVBr18 de induzir respostas contra múltiplos epitopos de linfócitos T CD4+ conservados que podem ser reconhecidos no contexto de múltiplas moléculas de HLA classe II, esse conceito vacinal pode solucionar o problema da variabilidade genética viral assim como aumentar a cobertura populacional. Portanto, essa vacina, pode ser útil se utilizada isoladamente ou como fonte de auxílio cognato para células T CD8+ HIV-específicas induzidas por outros imunógenos gerando resposta em uma grande proporção dos vacinados / Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that elicit cytotoxic CD8+ responses. It is conceivable that successful vaccines have to elicit responses to multiple epitopes, to match circulating strains of HIV, a virus known for its high genetic variability. It is known that CD4+ T cell responses are necessary for effective CD8+ antiviral responses. Here we assessed the immunogenicity of a DNA vaccine encoding 18 conserved, multiple HLA-DR-binding HIV CD4 epitopes in BALB/c and four strains of HLA class II-transgenic mice. Immunized mice displayed CD4+ and CD8+ proliferative and cytokine T cell responses of significant breadth and magnitude. Eleven out of the 18 encoded epitopes were recognized by CD4+ T cells from HLA class IItransgenic strain. Overall, 17 out of the 18 encoded peptides were recognized. The induced T cell response had a polyfunctional type 1 cytokine profile, including IFN- , TNF- and IL-2. The vaccine also induced long-lived central memory CD4+ T cells, which might provide sustained help for CD8+ T cells. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be usefull either as a standalone approach or as a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, eliciting responses in a wide proportion of vaccinees

O timo é um órgão linfoide primário, localizado em região mediastinal, cuja importância funcional é a diferenciação e maturação de todas as subpopulações de linfócitos T provenientes da medula óssea assim como a seleção de células autorreativas. Sua hipoplasia ou aplasia resultam em síndromes de imunodeficiência. Embora de vital importância, o estudo clínico de sua função não é rotineiro na prática clínica, o que pode ser atribuído a sua dificuldade de avaliação em razão de sua localização, necessidade de uso de métodos de imagem não inócuos ao paciente (tomografia computadorizada (TC), PET-SCAN) e complexidade das análises em sangue periférico de subpopulações de células T por citometria de fluxo e, mais recentemente, medição de T cell receptor excision circles (TRECs), por PCR. Um possível método da avaliação do timo sem radiação ionizante ou dor ao paciente seria a elastografia de timo por ultrassom e seu uso na prática clínica poderia substituir a TC, como ocorre na avaliação de lesões hepáticas ou mamárias. Objetivo - Este estudo se propõe a 1. Implantar este método na avaliação da função tímica, 2. Estabelecer valores de referência de TRECs na faixa etária estudada, 3. Investigar se há correlação entre os dois parâmetros. Métodos - Foram incluídas sessenta e quatro crianças de 0-5 anos em acompanhamento no ambulatório de cirurgia infantil sem doença sistêmica ou infecção aguda, e que iriam coletar amostra de sangue para exames pré-operatórios. Quarenta e oito destas coletaram amostra de sangue para avaliação de TRECs, vinte e nove realizaram elastografia num mesmo momento, porém apenas 13 destas apresentaram resultado confiável. A média da idade foi de 36 ± 16meses, predomínio do grupo foi masculino (75%), nascidos a termo (72%) e a principal intervenção cirúrgica foi do tipo urológica de pequeno porte. A elastografia mostrou média de 1,21 ± 0,24m/s, sem diferença significativa quando comparada ano a ano. Observamos uma média de TRECs de 195,6 ± 120,5 cópias/µL, mostrando valores significativamente mais altos quando comparados a adolescentes hígidos da base de dados do laboratório. Os valores de TRECs observados mostram uma ampla variabilidade na faixa etária estudada, sem diferença significativa quando separados por idade ano a ano. Não se encontrou correlação significativa entre a dureza do timo analisada à elastografia e valores de TRECs em sangue periférico. Concluímos que a elastografia é um método que possibilita a avaliação das dimensões e função do timo em crianças a partir de 2 anos de idade, entretanto estudos adicionais são necessários para que se possa recomendar a larga implantação deste método com essa finalidade / The thymus is a primary lymphoid gland responsible for the maturation of T cells as well as the immunological central tolerance. It has been a neglected organ by physicians, despite its relevance in early immunity. Thymic function can be indirectly measured by Computerized Tomography imaging and PET SCAN, T cell subpopulation flow cytometry. More recently, in the beginning of this century, a direct measurement represented by TRECs (T cell receptors excision circles) was developed. Classical thymic imaging has used ionized radiation, which poses a major risk for the pediatric patient and new techniques are needed. Objectives and methods - In this work, we tested the use of elastography ultrasound for the evaluation of the thymus in a group of < 5-year- old healthy children. In parallel, we measured TRECs in peripheral blood and compared the values obtained from both methods. We have reached sixty-four children at the pediatric surgery outpatients ambulatory, scheduled for minor surgeries. A sample of blood was taken during pre operatory and then patients were sent to the imaging service for elastography. Of all, sixty-four had undertaken TRECs and seventeen, elastography. The median age was 36 ±16 months and we had 75% of boys for surgical correction of urologic minor defects. The elastography results showed a median of 1.2 ± 0.24 m/s in all ages, the same stiffness as the liver, as shown in other works. Our median TREC/µL value was 195.6 ± 120.5 copies/µL showing a trend of reduction in older ages, and with statistical significance when compared with healthy teenagers\' values from the lab database. We concluded that elastography may be a good diagnostic tool for thymus evaluation, and additional works are needed for its recommendation in clinical practice. Our TRECs values showed a large variability, as also demonstrated in previous works, and a trend of reduction over age. We could not observe any significant correlation between elastography and TRECs values