Le lymphocyte B (LB) des patients lupiques est réfractaire à tous les procédés décrits à ce jour pour activer une fonction régulatrice B (Breg). Il constitue de ce fait un modèle intéressant d’étude de la déficience Breg chez l’Homme et soulève de nombreuses interrogations. Est-il possible de restaurer un défaut d’activation de la régulation LB? Si oui est-il possible d'agir à temps et le plus efficacement possible, et comment s'y prendre? Ou au contraire, est-ce un état irréversible de la cellule B? Ce travail de thèse a pour objectif principal de répondre à cette problématique essentielle à notre compréhension du Breg. Grace à un polypeptide de synthèse le Glatiramer acetate (GA), nous montrons que la restauration de la fonction régulatrice d’un Breg chez les patients lupiques est possible. Le compartiment LB mémoire fixe fortement le GA et la pré-sensibilisation par le GA permet d’augmenter le potentiel régulateur des LB mémoires mais n’affecte aucunement celui des LB matures. Le GA exerce deux actions majeures sur le LB mémoire. D’une part, il génère une meilleure capacité d’inhibition de la prolifération T, dont le mécanisme est associé à un contact cellulaire impliquant les molécules HLA-DR. D’autre part, il favorise un contrôle plus efficace de la polarisation Th1 qui est très probablement associé à sa capacité à induire la production d’IL-10 dans ces LB. Enfin, le GA modifie le phénotype des LB mémoires puisque l’expression de CD5, IL-21R, ou encore PD-1 est significativement augmentée, autant de molécules impliquées dans la fonction suppressive et dans la production d’IL-10. En conclusion, nous montrons qu’amplifier une fonction régulatrice et surtout la restaurer lorsqu’elle est défaillante chez les malades, est parfaitement possible in vitro. Face à l’engouement suscité par le développement de procédés favorisant l’expansion des Bregs chez la souris à des fins thérapeutiques, l’enjeu est aujourd’hui d’être en mesure d’extrapoler de telles démarches chez l’Homme. Ce travail, avec toute la modestie requise, contribuera à faire naître un nouvel élan vers de telles perspectives. / B cell in systemic lupus erythematous (SLE) is unresponsive to all methods described to date, to activate B cells regulatory (Breg) function. Therfore, it is an interesting model to study the Breg deficiency in Human, and highlights many questions: is there a way to restore a defect of the Breg activation ? If yes, how can we act more efficiently ? Or in contrast, is it an irreversible state of the B cell? Glatiramer Acetate (GA) is a synthetic polypeptide used in the treatment of multiple sclerosis. We show that Breg activity of SLE B cells can be restored after stimulation with GA. Interestingly, memory B cells bound high level of FITC-conjuated GA in contrast to mature B cells. We desmonstrate that GA can increased specifically the regulatory activities of memory B cells. GA exerts two major actions on the memory B cells. It generates an improved capacity of inhibition of the T cell proliferative response, whose mechanism is associated with a cellular contact involving HLA-DR molecules. In addition, GA supports a more effective control of the Th1 polarization which is most likely associated with its capacity to induce the production of IL-10 in these B cells. Finaly, GA modifies the memory B cell phenotype since the expression CD5, IL-21R, or PD-1 is significantly increased, all molecules involved in the suppressive function and the IL-10 production. In conclusion, our results show for the first time that amplification of Breg function and additionally its restoration when it is defective in patients, can be perfectly achieved in vitro. Currently, while the development of new process supporting the expansion of Bregs in the mouse model exist, the challenge is to extrapolate such methods in human. Through the control of their regulatory potential, regulatory B cells could be the targets of novel therapeutic approach in autoimmune diseases. This study might open up new horizons in this field.

Student Number : 9708297R - MSc (Med) dissertation - School of Medicine - Faculty of Health Sciences / B chronic lymphocytic leukaemia (B-CLL) is a monoclonal haematopoietic disorder with expansion of small lymphocytes of B-cells. B-CLL cells accumulate in blood, bone marrow, lymph nodes and spleen, resulting in enlargement of these organs and decreased bone marrow function. B-CLL is the most common leukaemia, with an annual incidence of 1.8 to 3.0 per 100 000 population in the United States. It is characterised by the accumulation of long-lived monoclonal CD5+ B lymphocytes. In vivo normal B-lymphocytes derive growth factors through interactions with T-cells and monocytes. In culture however, survival and growth of activated B-cells depends on the availability of external factors such as interleukins. B-CLL cells populations are unable to survive in culture long enough to respond to the addition of growth factors. Such factors are important for the proliferation and survival of many cell types and in the absence of cytokines, these cells die as a result of apoptosis. Chronic lymphocytic leukaemia cells are influenced in vitro by a number of exogenously added cytokines that include IFN- α, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-15, TGF- β and TNF- α. The aim of this study was to investigate the effect of cytokines e.g., IFN, IL-2, IL-6, IL7 and IL-10 on the proliferation and survival of B-CLL cells and furthermore to compare the induction and reduction of spontaneous and induced apoptosis in vitro. Patients with B-CLL were recruited from three centres. Thirty blood samples were collected, separated using Ficoll Hypaque Gradient and purified by rosetting with AET treated SRBC. The proliferation and survival of B-CLL cells were studied in vitro in response to GM-CSF, IFN, IL-2, IL-6, IL7 and IL-10,. The survival and apoptosis of B-CLL cells in cultures with or without interleukins and other growth factors were studied under microscopic examinations and DNA agarose gel electrophoresis. It was observed in B-CLL cells cultures that IFN and IL-2 enhanced proliferation significantly. IL6, IL-7 and GM-CSF also enhanced proliferation of B-CLL cells but not to the greater extent than IL2 and IFN. IL-10 inhibited proliferation of B-CLL cells when compared to controls. In a long-term (5-day) culture, survival of B-CLL cells was greatly enhanced by IFN and followed by IL-2. Therefore it appeared that IFN and IL-2 are the two most potent growth factors tested in this study to promote B-CLL cells proliferation and survival. The combination of these mitogens did not further enhanced proliferation. IL-6 and GM-CSF enhanced proliferation and survival of B-CLL cells. IL-7 promoted proliferation but had no effect on survival of B-CLL cells in-vitro. IL-10 enhanced apoptosis and did not promote survival of B-CLL cells in-vitro. IFN and IL2 are survival and promoting growth factors for B-CLL cells in culture. In contrast, IL-10 has demonstrated to induce apoptotic cell death of B-CLL cells. In conclusion B-CLL cells proliferated equally well with IFN and IL-2. IL-6, IL-7 and GM-CSF had a much lower proliferation and survival effect with noticeable antiapototic activity when compared to IFN and IL-2. IL-7 was found not to promote survival of B-CLL cells and IL-10 enhanced cell death by apoptosis.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 177-206). Also available on the Internet.

Alcohol represents the third leading cause of preventable death in the United States. Yet, despite its prevalent role in impeding human health, there is much to understand about how it elicits its effects on the body and how the body and brain change when an individual becomes physiologically dependent upon alcohol. The work presented herein represents an effort to elucidate the acute and chronic effects of alcohol on the nervous system. We investigate two specific protein pathways and their role in alcohol's effects on the body. The first begins with brain-derived neurotrophic factor (BDNF), which acts on TrkB, and ends with KCC2. We demonstrate that BDNF expression is increased in the VTA during withdrawal from chronic but not acute alcohol exposure and that this increase persists for at least seven days. Concomitantly, we demonstrate that the activation of GABAA channels on produces less inhibition of VTA GABA neurons in mice treated with chronic intermittent ethanol exposure than in alcohol naïve mice. This effect likewise persisted for at least seven days. We illustrate that BDNF has no apparent direct effect on VTA GABA neuron firing rate. The second pathway begins with the T cell marker CD5 and ends with the anti-inflammatory cytokine, IL-10. We demonstrate that in a genetic CD5 knockout (CD5 KO) mouse model both alcohol consumption as well as the sedative properties of alcohol are reduced. Since CD+ B cells secrete more IL-10 than CD5- B cells, we also demonstrate the effects of IL-10 on VTA neurons. We show that IL-10 has direct effects on VTA dopamine (DA) neurons by increasing their firing activity. We relatedly illustrate that IL-10 produces an increase in DA release in the nucleus accumbens (NAc). However, contrary to our hypotheses, we show that IL-10 produces conditioned place aversion rather than conditioned place preference in a place conditioning paradigm, suggesting that IL-10 might mediate pain-induced secretions of DA. Collectively, these results suggest two potential therapeutic targets to reduce alcohol consumption that need further validation. They also suggest a novel mechanism for the sedative effects of alcohol at moderate and high doses.

Alcohol represents the third leading cause of preventable death in the United States. Yet, despite its prevalent role in impeding human health, there is much to understand about how it elicits its effects on the body and how the body and brain change when an individual becomes physiologically dependent upon alcohol. The work presented herein represents an effort to elucidate the acute and chronic effects of alcohol on the nervous system. We investigate two specific protein pathways and their role in alcohol’s effects on the body. The first begins with brain-derived neurotrophic factor (BDNF), which acts on TrkB, and ends with KCC2. We demonstrate that BDNF expression is increased in the VTA during withdrawal from chronic but not acute alcohol exposure and that this increase persists for at least seven days. Concomitantly, we demonstrate that the activation of GABAA channels on produces less inhibition of VTA GABA neurons in mice treated with chronic intermittent ethanol exposure than in alcohol naïve mice. This effect likewise persisted for at least seven days. We illustrate that BDNF has no apparent direct effect on VTA GABA neuron firing rate. The second pathway begins with the T cell marker CD5 and ends with the anti-inflammatory cytokine, IL-10. We demonstrate that in a genetic CD5 knockout (CD5 KO) mouse model both alcohol consumption as well as the sedative properties of alcohol are reduced. Since CD+ B cells secrete more IL-10 than CD5- B cells, we also demonstrate the effects of IL-10 on VTA neurons. We show that IL-10 has direct effects on VTA dopamine (DA) neurons by increasing their firing activity. We relatedly illustrate that IL-10 produces an increase in DA release in the nucleus accumbens (NAc). However, contrary to our hypotheses, we show that IL-10 produces conditioned place aversion rather than conditioned place preference in a place conditioning paradigm, suggesting that IL-10 might mediate pain-induced secretions of DA. Collectively, these results suggest two potential therapeutic targets to reduce alcohol consumption that need further validation. They also suggest a novel mechanism for the sedative effects of alcohol at moderate and high doses.

Helper T cells (Th) are a vital component of the immune system responsible for directing other immune cells to eliminate pathogens and cancer. Specifically, Th cells facilitate B cell and cytotoxic T cell (Tc) activation and recruitment and enhance their function against cancer and infectious diseases. Th cells are a valuable resource for improving Tc responses in cancer treatment and have become a focus of immunotherapeutic research. While it is increasingly clear that helper T cells serve an important role, the details about which entities produce an effective Th cell response remain unclear. CD5 is a T cell co-receptor that negatively regulates T cell activation and helps fine-tune the TCR repertoire by altering TCR signaling during the selection process in the thymus. This work discusses the role of the co-receptor CD5 in influencing Th cell metabolism, as well as the study of two T cells called LLO118 and LLO56 that have different CD5 expression levels, and their functional response to altered peptides. Antibodies have revolutionized the world of cancer research and accelerated the development of therapies that trigger the immune system to target disease. In recent years, many antibody-based immunotherapies have emerged as effective candidates for combating cancer due to their refined specificity and ability to target a variety of epitopes. However, many therapies, such as those that target CD19 on B cell cancers, are also present on healthy cells, destroying both cancerous and healthy cells alike. Thymidine kinase 1 (TK1) is an enzyme involved in the DNA salvage pathway that converts thymidine into the nucleotide thymine. Recently, TK1 has been shown to be overexpressed on the surface of many cancers such as acute lymphoblastic leukemia. Importantly, TK1 is not expressed on the surface of healthy cells, making it an ideal cancer-specific antigen that can be targeted for cancer treatment. This work discusses our efforts to characterize TK1-specific single-chain antibodies from a yeast display library. According to the World Health Organization, genomics is defined as the study of all genes and their related functions. In contrast to genetics, genomics analyzes the entire DNA makeup of an organism rather than a single gene. In the past 20 years, the cost of genomic sequencing has decreased dramatically, making it affordable and accessible. A key area that must be addressed with genomic testing involves education about their promise, challenges, potential consequences, and ethical considerations. Genomic testing provides a powerful opportunity to educate everyone on scientific and ethical issues to increase understanding on the subject. This work discusses the influence of personal genomics in society and focuses on the importance, benefits, and consequences of genomics education in the classroom, clinic, and the public.

According to the centers for disease control and prevention (CDC) and the world healthorganization (WHO), heart disease and immune related diseases such as diabetes and cancer areamong the leading causes of death around the world. Thus, the regulation of the function ofimmune cell plays a key role in health and disease. Calcium (Ca2+) ions play a critical role inimmune cell activation, function and in a robust immune response. Defects in Ca2+ signalinginfluences the development of cardiac disease, Alzheimer disease, immune cell metabolism,muscle dysfunction, and cancer. Each immune cell is unique in its activation and function,making it relevant to understand how activation of each type of immune cell is regulated. Herewe describe the role of the nBMP2 protein in macrophage activation and function and the role ofthe CD5 co-receptor in helper T cell activation and function.The nuclear bone morphogenetic protein 2 (nBMP2) is the nuclear variant of the bonemorphogenetic protein 2 (BMP2), a growth factor important in heart development, neurogenesis,bone, cartilage and muscle development. To better understand the function of nBMP2, transgenicnBMP2 mutant mice were generated. These mice have a slow muscle relaxation and cognitivedeficit caused in part by abnormal Ca2+ mobilization. Mutant nBMP2 mice also have an impairedsecondary immune response to systemic bacterial challenge. Here we have further characterizedmacrophage activation and function from mutant nBMP2 mice before and after bacterialinfection. We describe how nBMP2 influences the Ca2+ mobilization response and phagocytosisin macrophages, revealing a novel role of the nBMP2 protein in immune cell regulation.CD5 is a surface marker on T cells, thymocytes, and the B1 subset of B cells. CD5 isknown to play an important role during thymic development of T cells. CD5 functions as anegative regulator of T cell receptor (TCR) signaling and fine tunes the TCR signaling response.Here we describe our characterization of CD5 regulation of Ca2+ signaling in naïve helper Tcells. We also outline our findings examining how CD5-induced changes in helper T cellactivation influence other biological processes such as immune cell metabolism, the diversity ofthe gut microbiome, and cognitive function and behavior. Thus, this work elucidates theinfluence of the CD5 co-receptor on the functional outcomes in multiple systems when CD5 isaltered.

Les cellules T ɑβ conventionnelles expriment des récepteurs antigéniques qui peuvent reconnaître et répondre à une grande variété d’agents pathogènes. En parallèle, des mécanismes cruciaux sont en place pour empêcher les cellules T de réagir aux auto-antigènes afin de prévenir le développement d’auto-immunité. Dans le but d’assurer la génération d’un réservoir de cellules T fonctionnelles, diverses et tolérantes au soi, les récepteurs des cellules T (TCR) ɑβ appropriés sont sélectionnés dans le thymus en fonction de la quantité et de la qualité des interactions avec les peptides du soi présentés par le complexe majeur d’histocompatibilité (CMH) sur les cellules présentatrice d’antigènes (CPA). Chez les nouveau-nés, des mécanismes intrinsèques et extrinsèques aux cellules influencent les interactions entre le TCR et le complexe CMH-peptide du soi, résultant en un répertoire de cellules T qui possèdent des propriétés distinctes par rapport à leurs homologues adultes. Par ailleurs, les souris diabétiques non-obèses (NOD), qui ont des cellules T auto-réactives qui attaquent les cellules β du pancréas, responsables de la production d’insuline, sont porteuses de polymorphismes génétiques qui peuvent influencer la sélection thymique. Ainsi, nous avons émis l’hypothèse que des facteurs intrinsèques et extrinsèques aux cellules modulent la sélection thymique tout au long de la vie et peuvent ultimement contribuer à la fonction et au dysfonctionnement de cellules T effectrices. La force globale de la signalisation TCR perçue lors du développement des lymphocytes T peut être mesurée en évaluant le niveau d’expression de différentes molécules, telles que CD5. Nous avons découvert que, tant chez la souris que l’humain, le répertoire des cellules T néonatales est composé de cellules T exprimant des niveaux plus élevés de CD5 que ceux des adultes. Cette augmentation des niveaux d’expression de CD5 n’est pas due à des défauts de tolérance centrale. En fait, nous avons plutôt démontré que les thymocytes exprimant un TCR de faible affinité pour les antigènes du soi ne sont pas sélectionnés efficacement chez les nouveau-nés et donc ne font pas partie du répertoire des cellules T néonatales, alors que ces thymocytes se développent adéquatement chez les adultes. Cette modification dans les seuils de sélection thymique biaise le niveau basal d’auto-réactivité du répertoire de cellules T néonatales et pourrait expliquer en partie les différences de réponse aux infections observées entre les nouveau-nés et les adultes. En comparant les niveaux de CD5 sur les thymocytes et les cellules T périphériques de souris NOD, prédisposées au diabète, avec ceux de souris C57BL/6 (B6), résistantes au développement du diabète auto-immun, nous avons découvert que les populations de cellules T des souris NOD ne perçoivent pas nécessairement des signaux TCR plus forts lorsqu’ils interagissent avec des antigènes du soi. Au contraire, une plus grande proportion de cellules T CD4+ et régulatrices avec un plus faible niveau de CD5 se différencient chez les souris NOD. Ce phénotype est fortement dépendant du locus du CMH des souris NOD. En revanche, les niveaux de CD5 sur les cellules T CD8+ périphériques des souris NOD sont plus élevés que ceux des souris B6, en raison d’un biais de survie intrinsèque aux cellules. Ces différences en niveau d’expression de CD5 sur le répertoire de cellules T des souris NOD ont des conséquences fonctionnelles directes et pourraient contribuer au développement ou à la progression du diabète auto-immun. Enfin, nous avons évalué si la sélection thymique était modulée par une molécule de co-signalisation, le co-stimulateur inductible de cellules T (ICOS). ICOS appartient à la famille des molécules de co-signalisation de type CD28 et se lie au ligand de ICOS (ICOSL). Alors que d’autres molécules de co-signalisation ont été démontré comme étant impliquées dans la tolérance centrale, le rôle joué par ICOS n’est pas clair. Nous avons démontré que ICOSL est exprimé par une variété de CPA thymiques importantes dans l’induction de la tolérance centrale et que ICOS est exprimé à la hausse durant la sélection thymique, en fonction de la force du signal TCR perçue par les thymocytes. Nous fournissons également, pour la première fois, une preuve que la voie ICOS-ICOSL pourrait avoir un rôle dans la régulation fine de la sélection négative. En conclusion, les résultats présentés dans cette thèse démontrent que la sélection thymique être altérés dans le contexte de l’ontogénie et du diabète auto-immun, conduisant au développement de cellules T avec une auto-réactivité basale relativement plus élevée ou plus faible en comparaison aux animaux adultes en bonne santé. Ces modulations pourraient avoir des conséquences importantes sur la fonction immunitaire et doivent être considérées pour le développement de futurs vaccins ou approches thérapeutiques chez ces populations. / Conventional ɑβ T cells express antigen receptors that can recognize and respond to a wide range of foreign pathogens. In parallel, critical mechanisms are in place to prevent T cells from reacting to self-antigens causing autoimmunity. To ensure the generation of a functional, diverse yet self-tolerant T cell pool, appropriate ɑβ T cell receptors (TCR) are selected in the thymus based on the quality and quantity of their interactions with self-peptides presented by the major histocompatibility complex (MHC) on thymic antigen presenting cells (APC). In neonates, cell-intrinsic and -extrinsic mechanisms influence TCR and self-peptide-MHC interactions resulting in a T cell pool that exhibits distinct functions as compared to their adult counterparts. Similarly, non-obese diabetic (NOD) mice, which contain autoreactive T cells that attack insulin-producing pancreatic β cells, carry genetic polymorphisms that can influence thymic selection. Therefore, we hypothesized that cell-intrinsic and -extrinsic factors modulate thymic selection throughout life and may ultimately contribute to the function and dysfunction of effector T cells. The overall perceived strength of TCR signaling during T cell development can be measured by several molecules, including CD5. We found that, in both mice and humans, the neonatal T cell pool is composed of T cells with higher CD5 levels than their adult counterparts. The increased CD5 levels are not due to defects in central tolerance. Instead, we demonstrated that thymic selection is altered in neonates. Thymocytes expressing a TCR with low affinity to self-antigen that develop in adults are not efficiently selected into the neonatal T cell pool. This shift in thymic selection thresholds skews the basal self-reactivity of the neonatal T cell repertoire and may explain, in part, differences in the neonatal versus adult response to infections. By comparing CD5 levels on thymocytes and peripheral T cells from diabetes-prone NOD mice with those from autoimmune-resistant C57BL/6 (B6) mice, we found that T cell populations in NOD mice do not necessarily perceive stronger TCR signals when interacting with self-antigens. Rather, NOD mice allow the differentiation of more CD4+ T cells and thymic Tregs with lower CD5 levels. This phenotype is strongly dependent on the NOD MHC locus. In contrast, CD5 levels on peripheral NOD CD8+ T cells are higher than those in the B6 mice that is likely due to a cell-intrinsic survival bias. These differences in CD5 levels in the NOD T cell pool have direct functional consequences and may contribute to the development or progression of autoimmune diabetes. Lastly, we investigated whether thymic selection is modulated by the co-signaling molecule, inducible T cell costimulator (ICOS). ICOS belongs to the CD28 family of co-signaling molecules and binds to the ICOS ligand (ICOSL). While other co-signaling molecules have been implicated in central tolerance, the role played by ICOS is unclear. We demonstrated that ICOSL is expressed by an array of thymic APCs important for central tolerance induction, and that ICOS is upregulated during thymic selection relative to the strength of TCR signaling the thymocytes perceive. We also provide, for the first time, evidence that the ICOS-ICOSL pathway may fine-tune negative selection. In conclusion, the results presented in this thesis demonstrate that thymic selection appears to be altered within the context of ontogeny and autoimmune diabetes, leading to the development of T cells with relatively higher or lower basal self-reactivity as compared to healthy adult animals. These modulations may have significant consequences on immune function and require careful consideration for future vaccination and therapeutic approaches within these populations.

Immunotherapy, especially the adoptive transfer of T cells and immune checkpoint blockade therapy, have revolutionized cancer therapy. In particular, utilizing antigen-specific T cells for adoptive cell therapy has enabled the development of specific and effective strategies. It has paved the way for developing more accurate and personalized cancer immunotherapies. Adoptive cell therapy (ACT) results depend on the characteristics of ex vivo expanded T cells, such as their differentiation and clonal diversity. However, ex vivo expanded specific T cells often express several inhibitory receptors involved in T-cell exhaustion and markers of terminal effector differentiation. Accordingly, we hypothesized that blocking one or several inhibitory receptors during the ex vivo expansion could improve the expansion and differentiation of antigen-specific T cells. Preconditioning the ACT products and combinatorial immunotherapy approaches are newly developed concepts in cancer therapy to optimize cancer immunotherapy for a larger group of patients. To study the development of antigen-specific T-cells in combination with checkpoint blockade, we have adopted a method that allows the expansion of rare antigen-specific T cell precursors from PBMCs via multiple stimulations, using antigen-pulsed dendritic cells. In the current study, we utilized our protocol to generate and expand antigen-specific CD8+ T cells targeting the oncogenic Epstein-Barr virus (EBV)-LMP2 and a tumor-associated antigen (TAA) from the Wilms Tumor 1 (WT1) protein. We employed two approaches to abolish the negative regulatory receptors, antibody-mediated blockade and deletion via CRISPR/Cas9. We evaluated the impact of checkpoint blockade on antigen-specific T cells development, proliferation, and function. Additionally, TCR clonality and transcriptomic changes were assessed by genomic studies, including single-cell RNA (scRNA) sequencing and T-cell receptor sequencing. Supporting our hypothesis, we observed that blocking both PD-L1 and TIM3 (not any of them alone) significantly enhanced LMP2 and WT1-specific T cell generation and expansion. Additionally, checkpoint blockade resulted in higher specific T cell function, including cytokine production and in vitro targeted cytotoxicity. Using scRNA-seq and TCR sequencing approaches, we first remarked that the specific T cells are highly oligoclonal and identified a few dominant shared clones between donors. Immune checkpoint blockade did not confer consistent transcriptional signatures but may have a clonotype and donor-specific impact on the expression of activation and exhaustion-related genes. Overall, immune checkpoint blockade did not markedly alter the clonal composition of the T-cell product. We also evaluated the impact of CD5 deletion in antigen-specific T cell priming and expansion as an inhibitory receptor and a part of the immune response synapse. However, in a human ACT setting, our data show that the CRISPR/Cas9 mediated CD5 deletion only has modest effects on antigen-specific T-cell generation. However, future combinations with the blockade of other immune checkpoint may be warranted. Conclusion We demonstrated that blocking PD-L1 and TIM3 during the ex vivo expansion improves antigen-specific T-cell yield. We show that blocking multiple checkpoints can synergistically optimize specific T-cell production without compromising the response's specificity. It is a rapidly implementable strategy to enhance the number and quality of ex vivo expanded antigen-specific T cells for immunotherapy. / Le transfert adoptif de cellules T et le traitement par le blocage des points de contrôle immunologiques ont révolutionné le traitement du cancer. En particulier, l'utilisation de cellules T antigène-spécifiques en thérapie cellulaire adoptive a facilité le développement d'immunothérapies anticancéreuses plus précises et personnalisées. Les résultats de la thérapie cellulaire adoptive (TCA) sont liés à la qualité des cellules T spécifiques expansées ex vivo, telles que leur état de différenciation et leur diversité clonale. Cependant, le pré-conditionnement des produits de thérapie cellulaire adoptive et les traitements d'immunothérapie combinatoire sont de nouveaux concepts en développement de la thérapie du cancer pour optimiser l'immunothérapie du cancer dans un plus grand groupe de patients. Nous avons formulé l’hypothèse que le blocage d'un ou plusieurs récepteurs inhibiteurs au cours de l'expansion ex vivo favorise une meilleure expansion et une meilleure fonction des cellules T destinées à la TCA. Pour étudier l’expansion et la différenciation de cellules T antigène-spécifiques lors d’un blocage des points de contrôle, nous avons adopté une méthode qui nous permet de stimuler et expanser de rares cellules T antigène-spécifiques à partir de cellules mononuclées du sang périphérique (PBMCs) via de multiples stimulations utilisant des cellules dendritiques chargées avec l’antigène d’intérêt. Nous avons utilisé deux approches pour supprimer l’activité des récepteurs régulateurs négatifs, les anticorps bloquants des points de contrôle et la délétion génique via CRISPR/Cas9. Nous démontrons que le blocage combiné de PD-L1 et TIM3 améliore considérablement la l'expansion de cellules T CD8+ spécifiques à des antigènes viraux et tumoraux. De plus, le blocage des points de contrôle a entraîné la génération de cellules T spécifiques fonctionelles tel que démontré par la production de cytokines et la cytotoxicité in vitro. En utilisant de séquençage de l'ARN en cellule unique (scRNA-seq) et de séquençage des récepteurs des lymphocytes T (TCP-seq), nous avons remarqué que les cellules T spécifiques sont très fortment oligoclonales. Nous avons également identifié quelques clones dominants partagés entre les donneurs. L’application de l’inhibition des points de controles ne confère pas de signatures transcriptionelles particulières mais pourrait affecter certains clones provenant de certains donneurs davantage que d’autres. De plus, le peu de changements dans la composition clonale des cellules expandues suggèrent que le blocage de ces points de contrôle immunologiques n’altère pas de façon significative le produit cellulaire obtenu. Des données récentes soutiennent également un rôle du CD5 dans la régulation de l'activation des cellules T naïves et leur état fonctionnel. Cependant, dans un contexte compatible avec la TCA, nos données montrent que la suppression de CD5 via CRISPR/Cas9 n'a que des effets modestes sur la génération de cellules T antigène-spécifiques. Par contre, la combinaison éventuelle avec l’inhibition de d’autres points de contrôle immunologiques pourrait être envisagée. En conclusion, nos travaux fournissent une nouvelle méthode pour générer des cellules T spécifiques pour la TCA et la caractérisation à plus haute résolution de cellules T spécifiques expansées ex vivo. Nous avons donc démontré que le blocage combiné de plusieurs points de contrôle peut optimiser de manière synergique la production de cellules T spécifiques sans compromettre la spécificité de la réponse. Il s'agit là d'une stratégie rapidement applicable pour améliorer le nombre et la qualité des cellules T antigène-spécifiques expansées ex vivo pour l'immunothérapie.