Indiana University-Purdue University Indianapolis (IUPUI) / Germinal Centers (GCs) are transient lymphoid structures that arise in lymphoid organs in response to T cell-dependent antigen. Within the GC, follicular T helper (TFH) cells promote GC B cell differentiation and in turn the proper antibody production to protect us from invading pathogens. We wished to study the regulation of this process by transcription factors STAT3 and Bcl6. STAT3 is important for both TFH cell differentiation and IL-4 production by Th2 cells. IL-4 is a major functional cytokine produced by TFH cells. To dissect the role of STAT3 in IL-4 production by TFH cells, we generated T cell-specific conditional STAT3 knockout mice (STAT3KO). Compared to WT mice, TFH cell differentiation in STAT3KO mice was partially impaired, both in spleen following sheep red blood cells (SRBC) immunization and in Peyer's patches (PPs). In STAT3KO mice, the numbers of splenic GC B cells were markedly decreased, whereas PP GC B cells developed at normal numbers and IgG1 class switching was greatly increased. Unexpectedly, we found that STAT3 intrinsically suppressed the expression of IL-4 and Bcl6 in TFH cells. Mechanistically, in vitro repression of IL-4 expression in CD4 T cells by Bcl6 required STAT3 function. Apart from TFH cells, the GC reaction is also controlled by regulatory follicular T helper (TFR) cells, a subset of Treg cells. To study the mechanism of how TFR cells regulate the GC reaction, we generated mice specifically lacking TFR cells by specifically deleting Bcl6 in Treg cells. Following immunization, these "Bcl6FC" mice developed normal TFH and GC B cell populations. However, Bcl6FC mice produced altered antigen-specific antibody responses, with reduced titers of IgG and increased IgA. Bcl6FC mice also developed IgG antibodies with significantly decreased avidity to antigen in an HIV-1 gp120 "prime-boost" vaccine model. Additionally, TFH cells from Bcl6FC mice produced higher levels of Interferon-γ, IL-10 and IL-21. Loss of TFR cells therefore leads to highly abnormal TFH and GC B cell responses. Overall, our studies have uncovered unexpected regulatory roles of STAT3 in TFH cell function as well as the novel regulatory roles of TFR cells on cytokine production by TFH cells and on antibody production.

Durant l’hématopoïèse, les cellules souches hématopoïétiques peuvent soit s’autorenouveler soit se différencier dans les divers types de cellules matures constituant le système hématopoïétique. Un des modèles prédominants sur le développement du système hématopoïétique postule une différenciation par étape des différentes lignées le constituant. Ce modèle débute avec les cellules souches hématopoïétiques qui donnent naissance à des précurseurs multipotents qui peuvent à leur tour se différencier en précurseurs dédiées à la lignée lymphoïde ou myéloïde. Bien que la dernière décennie ait apporté de nombreuses connaissances sur les principales signalétiques transcriptionnelles impliquées dans le développement hématopoïétique, le détail des mécanismes moléculaires en jeu expliquant comment les cellules souches hématopoïétiques sont initialement amorcées puis complètement engagées vers une voie de différenciation reste toujours à élucider. Le travail de notre laboratoire indique que l’assemblage combinatoire du complexe de remodelage de la chromatine SWI/SNF est un élément clé parmi les mécanismes épigénétiques qui gouvernent le destin cellulaire et notamment la famille de sous-unités Smarcd qui comporte 3 membre alternatifs Smarcd1/2/3. Des analyses du transcriptome par séquençage haut débit ont montré que l’expression de la sous-unité Smarcd1 du complexe est élevée dans le compartiment des cellules souches, les précurseurs multipotents et les progénitures lymphoïdes tandis que la sous-unité Smarcd2 est principalement exprimée dans les précurseurs myéloïdes. En utilisant des modèles de délétion conditionnelle dans des modèles murins, nous avons démontré que Smarcd1 et Smarcd2 jouent des rôles critiques et lignés spécifiques durant l’hématopoïèse. Dans un premier temps, nous avons pu montrer que Smarcd1 collabore avec le facteur de transcription de la famille bHLH E2A pour spécifier le destin lymphoïde des précurseurs multipotents et qu’elle est donc absolument essentielle pour la lymphopoïèse. Notre travail sur les mécanismes moléculaires en jeu a pu montrer que Smarcd1 interagit directement avec E2A et est nécessaire pour l’accessibilité de la chromatine sur un ensemble de régions enrichies avec les modifications d’histones H3K27ac/H3K3me1 qui marquent des régions activatrices (« enhancer ») impliquées dans l’activation d’une signature lymphoïde dans les précurseurs multipotents. Le blocage de l’interaction entre Smarcd1 et E2A inhibe l’amorce de cette signature lymphoïde et bloque l’émergence de précurseurs destinés à la voie lymphocytaire. Concernant la fonction de Smarcd2, nous avons pu montrer que cette sous-unité est absolument nécessaire pour la granulopoïèse. Les souris ayant subi une délétion génétique de Smarcd2 deviennent très rapidement neutropéniques. Ce phénotype découle d'un blocage au stade de différenciation myélocyte/métamyélocyte, tandis que les autres lignées hématopoïétiques restent non affectées par la délétion. Nous avons pu identifier le facteur de transcription C/ebpƐ comme un partenaire essentiel de Smarcd2 qui interagit avec le complexe SWI/SNF sur les promoteurs de gènes de granules secondaires afin d’en activer la transcription. Les analyses du transcriptome que nous avons effectué lorsque l’interaction de Smarcd2 et C/ebpƐ est interrompue dans des précurseurs de granulocytes ont montré une diminution de l’expression des gènes de granules secondaires liée à une maturation incomplète des granulocytes menant au développement d’un syndrome de myélodysplasie au court du temps. / During hematopoiesis, hematopoietic stem cells (HSCs) either selfrenew or differentiate into all mature blood cell types through successive rounds of binary cell fate decisions. The prevailing model of hematopoiesis predicts a step-by-step model of lineage differentiation in which HSCs first give rise to multipotent progenitors that subsequently differentiate into myeloid and lymphoid restricted progenitors. Although key transcriptional pathways controlling hematopoietic development are beginning to be deciphered, detailed molecular mechanisms explaining how HSCs and progenitors are initially primed and then commit to the different hematopoietic cell lineages are lacking. Work from our laboratory indicates that combinatorial assembly of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex is a key epigenetic mechanism that governs cell fate decisions. Transcriptomics analyses revealed that expression of the Smarcd1 subunit is enriched in hematopoietic stem/progenitors and early lymphoid cells, while Smarcd2 is mainly expressed in myeloid progenitors. Using conditional knock-out mouse models, we demonstrated that Smarcd1 and Smarcd2 subunits perform critical and lineage-specific roles during hematopoiesis. First, we found that Smarcd1 collaborates with the bHLH transcription factor E2A to specify lymphoid cell fate during hematopoiesis and, therefore, is absolutely required for lymphopoiesis. Mechanistically, we showed that Smarcd1 physically interacts with E2A and is required for chromatin accessibility of a set of H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hematopoietic stem cells. Impairing the interaction between Smarcd1 and E2A inhibits lymphoid lineage determination and the emergence of lymphoid-primed multipotent progenitors. Conversely, we showed that Smarcd2 is absolutely required for granulopoiesis. Smarcd2-deficient mice quickly become neutropenic due to a XIII block at the myelocyte/metamyelocyte stage of granulocyte maturation while other lineages remain unaffected. We discovered that Smarcd2 interacts with the transcription factor C/ebpε to recruit the mSWI/SNF complex on the promoter of secondary granule genes, thus inducing their transcriptional activation. As shown by transcriptomic analysis, impairing this interaction results in decreased expression of secondary granule genes, improper granulopoietic maturation, and development of a myelodysplastic-like syndrome over time. Altogether, this work identifies the Smarcd1 and Smarcd2 subunits of SWI/SNF complexes as master chromatin remodelers allowing the recruitment of lineage-specific transcription factors at key regulatory loci controlling lymphoid lineage priming and granulocyte development, respectively. More globally, these studies highlight that combinatorial assembly of alternative subunits of mSWI/SNF complexes is a key epigenetic mechanism controlling cell fate decisions during hematopoiesis.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / In order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes.

Indiana University-Purdue University Indianapolis (IUPUI) / The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow-derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. β-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic β-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that β-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

Le but de notre étude visait à déterminer si les matrices ECM (extracellular matrix) préparés à partir d'un modèle vertébré (Axolotl) capables de régénérer ses tissus suite à une blessure sont plus efficaces pour stimuler les réponses régénératives chez les animaux non régénérant (par exemple les mammifères). Nous avons testé la capacité de matrice ECM axolotl à améliorer la guérison des plaies cutanées dans des souris et nous les avons comparés à une matrice disponible commercialement (échafaudage Symbios PerioDerm) pour leur efficacité à favoriser la guérison des plaies. Des lésions d'excision ont été créées sur le dos de souris et les animaux ont été regroupés dans différents groupes; a-) ECM de peau axolotl décellularisée (groupe Axolotl), b-) matrice de derme acellulaire Symbios Perioderm (groupe PerioDerm), c-) grillage en titane (groupe témoin); respectivement. Les tissus des plaies ont été récoltés à des moments précis : 7 jours et 30 jours après la blessure pour évaluer la guérison des plaies. La guérison des blessures ayant reçu les différentes matrices a été comparées entre elles en utilisant le test de transillumination et des analyses histologiques. Les résultats indiquent que la ECM de peau d’axolotl décellularisée est bien tolérée par les souris, car aucun rejet n'a été observé. Le groupe qui a reçu l'ECM de la peau axolotl décellularisé a démontré une réépithélialisation, une densité cellulaire, une teneur en collagène (avec une organisation similaire à un tissu intact) et une vascularisation (angiogenèse) élevées par rapport aux groupes PerioDerm et témoins. La présence de follicules pileux était également observé dans le groupe axolotl (qui n'est pas présent dans PerioDerm et groupes de contrôle). Sur la base de nos résultats, l'hypothèse de base semble être correcte en ce qu'une matrice ECM provenant d'un régénérateur puissant semble favoriser la guérison plus efficacement chez les animaux normalement non régénérants. Cependant, des recherches supplémentaires devront être menées pour confirmer ces résultats. / The aim of our study sought to determine whether ECM scaffolds prepared from a vertebrate model (Axolotl) capable of regenerating tissues following injury are more effective at stimulating regenerative responses in non-regenerating animals (e.g., mammals). We tested the ability of axolotl decellularized ECM scaffolds to improve skin wound healing in mammalian models and compare the axolotl skin ECM scaffold to a commercially available one (Symbios PerioDerm scaffold) for efficiency in promoting wound healing. Excisional lesions were created on the back of mice, and animals in different groups were treated by; a-) decellularized axolotl skin ECM (Axolotl group), b-) Symbios Perioderm acellular dermis scaffold (PerioDerm group), d-) Titanized mesh only (Control group); respectively. Wound tissues were harvested at time points: 7- and 30-days post-wounding to assess the scaffolds impact on wound healing. Wound healing was compared between the Axolotl, PerioDerm and Control groups using transillumination test and histological analyses, Results indicate that the decellularized axolotl skin ECM is well tolerated by mammalian models, as no immune rejection was observed. The axolotl group that received the decellularized Axolotl Skin ECM demonstrated high reepithelialization, cellular density, collagen content (in a porous pattern similar to intact skin), vascularization (angiogenesis) compared to PerioDerm and control groups. The presence of hair follicles was also observed in the axolotl group (which is not present in PerioDerm and control groups). Based on our results, the basic hypothesis appears to be correct in that an ECM scaffold from a strong regenerator seems to promote healing more efficiently in non-regenerating animals. However, further research should be conducted to confirm these findings.

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.

No description available.