Les thérapies anti-tumorales se sont considérablement améliorées au cours de la dernière décennie. Toutefois, les traitements utilisés actuellement rencontrent d'importantes limitations, notamment dans le cas de cancers métastatiques, révélant l'urgence de développer de nouvelles approches. Ainsi, l'immunothérapie par transfert adoptif de cellules T représente une approche innovante particulièrement prometteuse. Son principe s'appuie sur l'injection de cellules T autologues spécifiques d'antigènes tumoraux, préalablement manipulées et amplifiées ex vivo, chez des patients rendus lymphopéniques par chimiothérapie et/ou radiothérapie. Toutefois, même si l'état lymphopénique est induit par ces 2 protocoles de conditionnements, leurs effets sur l'environnement de l'hôte ainsi que sur le devenir des cellules T greffées étaient, jusqu'à nos travaux, mal connus. Par le biais de modèles murins, nous avons pu démontrer que le devenir des cellules T diffère après transfert dans des souris irradiées ou traitées par chimiothérapie (Bu/Cy). Ainsi, après transfert dans des animaux irradiés, on observe une prolifération préférentielle des cellules T CD8, dépendante de l'IL-7, est observée alors qu'un transfert chez des souris traitées Bu/Cy se traduit par une prolifération rapide, indépendante de l'IL-7, des cellules T CD4. De plus, ces comportements sont associés à d'importantes modifications de l'environnement généré chez l'hôte. Plus spécifiquement, nous avons démontré, dans les organes lymphoïdes secondaires, que la localisation et la représentation des différentes sous-populations de cellules dendritiques présentes étaient différentiellement modulées par le type de conditionnement utilisé. Par ailleurs, l'élimination spécifique des cellules CD11c+ chez des souris traitées Bu/Cy était accompagnée d'une inhibition importante de la prolifération rapide des cellules T CD4 greffées. L'ensemble de nos travaux montrent que les traitements lymphopéniques génèrent des environnements distincts capables de moduler le devenir des cellules T greffées.Durant ma thèse, nous avons également abordé de façon originale un aspect novateur de l'environnement en étudiant le rôle potentiel des nutriments comme régulateurs métaboliques des fonctions effectrices des cellules T. La glutamine est l'acide aminé le plus abondant du plasma, pouvant contribuer aux besoins bionénergétiques et biosynthétiques des cellules T en prolifération. Nous avons démontré dans nos travaux qu'une carence en glutamine lors de l'activation de cellules T CD4 par leur TCR entrainait un délai dans l'activation de la voie mTOR, une réduction de la production intracellulaire d'ATP aux temps précoces et se traduisait par une diminution de la prolifération. De plus, ces conditions étaient associées à une augmentation de la conversion de cellules CD4 T naïves, via TGFβ, en cellules régulatrices Foxp3+ , y compris en condition de polarization Th1. Par contre, la carence en glutamine n'a pas inhibé la différenciation Th2. Les cellules T Foxp3+ ainsi générées en condition limitante de glutamine présentaient in vivo des fonctions suppressives aussi efficaces que celles des cellules régulatrices nTregs. En effet, elles ont la capacité de bloquer l'induction de la colite provoquée par la greffe de cellules T effectrices dans des souris Rag2-/- . Nos travaux démontrent ainsi que l'environnement métabolique peut être un régulateur clé de la différenciation des cellules T CD4. L'ensemble de mes travaux de thèse ont mis en évidence de nouveaux paramètres capables de potentiellement modifier la survie et la réactivité des cellules T greffées. / Anti-tumor therapies have improved significantly over the decade. However, the currently used treatments have important limitations, notably for metastatic cancers, and the development of new approaches is therefore a high priority. Adoptive T cell therapy (ACT) represents an innovative strategy that has shown much promise. This therapy is based on the infusion of tumor-specific T cells, which have been manipulated and expanded ex vivo, into patients who have been rendered lymphopenic by chemotherapy and/or irradiation. It is interesting to note that while lymphodepletion is attained by the vast majority of conditioning regimens, the effects of these protocols on the host environment and potentially, on the destiny of adoptively-transferred T cells had not been elucidated prior to the studies which we initiated. Using a murine model, we found that the fate of adoptively-transferred T cells differs markedly in mice rendered lymphopenic by sub-lethal irradiation as compared to a busulfan/cyclophosphamide (Bu/Cy) chemotherapy regimen. Irradiation-mediated lymphopenia resulted in a skewed IL-7-dependent proliferation of donor CD8+ T cells, whereas Bu/Cy treatment led to an increased IL-7-independent, rapid CD4+ T cell proliferation. These alterations in T cell proliferation were associated with striking changes in the host microenvironment. More specifically, we demonstrated that the proportion and localization of different dendritic cell (DC) subsets in lymphoid organs were differentially affected by the type of conditioning. Furthermore, we found that these DC controlled the rapid donor CD4+ T cell division detected in Bu/Cy-treated mice as depletion of CD11c+ DC inhibited this proliferation. Altogether, our studies demonstrate that lymphopenic regimens generate distinct host environments which modulate the fate of adoptively-transferred T cells. Durind my PhD, we also investigated an original and novel aspect of the microenvironement by studying the potential role of nutrients as metabolic regulators of T cell effector function. Glutamine is the most abundant amino acid in the plasma and contributes to the bioenergetic and biosynthetic requirements of proliferating T cells. Here, we demonstrated that activation of CD4+ T cells under glutamine-deprived conditions results in a delayed mTOR activation with reduced early ATP production and decreased proliferation. Moreover, these conditions resulted in the conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs). This de novo Treg differentiation occurred even under Th1-polarizing conditions and was TGFβ-dependent. Interestingly, glutamine deprivation did not inhibit Th2 differentiation. Importantly, these converted Foxp3+ T cells showed enhanced in vivo persistence and were highly suppressive, completely protecting Rag-deficient mice from the development of autoimmune inflammatory bowel disease as efficiently as natural-occuring Tregs. Thus, our data reveal the external metabolic environment to be a key regulator of a CD4 T lymphocyte's differentiation. Altogether, the data generated during my PhD provide new insights into the identification of parameters that can potentially alter the survival and reactivity of adoptively-transferred T cells.

Les lymphocytes T sont caractérisés par l’expression d’un récepteur à l’antigène des cellules T (TCR), soit le preTCR, soit le γδ TCR et le αβ TCR clonotypique, associé à un complexe de transduction formé du CD3 et de la chaîne ζ. La signalisation du complexe TCR:CD3:ζ est cruciale pour le développement des cellules T et pour leur activation spécifique par l'antigène. Ces signaux déclenchent la prolifération, la différentation, les fonctions effectrices et l’apoptose des cellules T. Les évènements proximaux de la signalisation du TCR:CD3:ζ impliquent des protéines tyrosine kinases (PTK) des familles CSK, SRC, SYK et TEC dont les acteurs principaux sont CSK (C-terminal SRC kinase), LCK (lymphocyte-specific protein tyrosine kinase), ZAP-70 (ζ chain-associated protein tyrosine kinase of 70 kDa) et ITK (interleukin-2-inducible T cell kinase). Après stimulation du complexe TCR:CD3:ζ, les PTKs sont activées et déclenchent une cascade de phosphorylation sur tyrosine dont la phosphorylation des motifs activateurs ITAM (immunoreceptor tyrosine-based activation motif) du CD3 et de la chaîne ζ, et la phosphorylation des protéines adaptrices. Celles-ci conduisent à l’assemblage du signalosome LAT:SLP-76, lequel contrôle en grande partie la diversité des signaux associés au complex TCR:CD3:ζ, dont le flux calcique, l’activation de la cascade des MAP kinases, l’activation des facteurs de transcription NF-κB, NFAT et AP-1 ainsi que la réorganisation du cytosquellette d’actine, l’adhésion cellulaire et la motilité. Pendant les cinq dernières décennies, le système immunitaire a été analysé extensivement in vitro et à l’aide de modèles animaux comme la souris. Par ailleurs, l’étude des déficits immunitaires héréditaires chez l’homme a permis aussi des avancées importantes et novatrices dans la compréhension du système immunitaire humain que les modèles animaux ne permettaient pas d’appréhender. Dans ma thèse de doctorat je rapporte le premier cas de déficit humain en LCK de transmission autosomique récessive et l’identification de nouvelles mutations autosomiques récessives provoquant un défaut humain de ZAP-70 et un défaut humain d’ITK. Je rends compte des phénotypes cliniques et immunologiques associés à ces immunodéficiences et je caractérise les défauts biochimiques de la signalisation TCR:CD3:ζ associés à ces déficits. Enfin, je compare mes observations avec les modèles murins déficients Lck-/-, Zap-70-/- et Itk-/- déjà bien établis. / T lymphocytes express either a preTCR, or a clonotypic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs results in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, and adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility. Throughout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulate the subtleties found in men. In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.

La polyendocrinopathie auto-immune de type 1 (PEA1) est liée aux mutations du gène AIRE. En l’absence de AIRE se développe un défaut de tolérance immune centrale, à l’origine de pathologies auto-immunes multiples spécifiques d’organe. Notre objectif était d’évaluer l’effet d’une altération« exemplaire » du répertoire T sur les empreintes auto-réactives humorales. Les données cliniques etimmunologiques ont été recueillies chez des patients atteints de PEA1, qui ont bénéficié du séquençage du gène AIRE. Chez ces patients ont été analysés les profils d’auto-réactivité sérique IgGet IgM vis à vis des tissus pancréatique et surrénalien, en comparaison à des patients atteints d’autres endocrinopathies auto-immunes, et à des sujets sains. Les bandes antigéniques discriminantes ont été sélectionnées grâce à un test de Chi-2, et une approche immuno-protéomique a permis leur caractérisation moléculaire. Dix-neuf patients atteints de PEA1 ont pu être étudiés. Ils présentaient de1 à 10 manifestations cliniques liées à la maladie. Quatre mutations du gène AIRE différentes ont été identifiées, et la délétion 13-bp dans l’ exon 8 (c.967-979del13) s’est avérée la plus fréquente. L’étude en immuno-empreinte a permis d’identifier 6 antigènes préférentiellement reconnus par les patients atteints de PEA1. Leur caractérisation par approche immuno-protéomique a montré qu’il s’agissait à lafois d’antigènes tissus-spécifiques (lipase pancréatique reconnue à la fois par les IgM et les IgG,amylase pancréatique reconnue par les IgG, Regenerating Protein 1 alpha pancréatique ciblée par lesIgM) mais également ubiquitaires (péroxiredoxine-2 reconnue à la fois par les IgG et les IgM, HeatShock cognate 71kDa Protein ciblée par les IgM, aldose réductase reconnue par les IgG). Ainsi, une altération majeure du répertoire T auto-réactif, telle que celle liée aux mutations du gène AIRE, affecte de manière importante les réponses humorales auto-réactives dépendantes d’ IgG, mais également d’IgM. Ces modifications touchent à la fois des antigènes tissu-spécifiques et ubiquitaires, nous faisant évoquer un rôle au moins partiel de AIRE dans des phénomènes T-indépendants et /ou des altérations de l’immunité naturelle. / Autoimmune polyendocrine syndrome type 1 (APS1) is caused by mutations in the AIRE gene thatinduce central tolerance breakdown which results in tissue-specific autoimmune diseases. Ourobjective was to evaluate the effect of a well-defined T cell repertoire impairment on humoralsystemic self-reactive footprints. Clinical and immunological data were collected, and pathologicalmutations in the AIRE gene were identified by DNA sequencing. Comparative serum self-IgG andself-IgM reactivities, directed towards pancreatic and adrenal protein extracts, of APS1 patients,patients suffering from other autoimmune endocrinopathies and healthy subjects, were tested using Western blotting. Discriminant protein bands were selected using the Chi-square test and molecularcharacterization of these bands was conducted using a proteomic approach. Nineteen patients wereidentified with APS1. Clinical manifestations varied greatly, showing 1 to 10 components. Fourdifferent AIRE gene mutations were identified, and the 13-bp deletion in exon 8 (c.967-979del13) wasthe most prevalent. A singular distortion of seric self-IgG and self-IgM repertoires was noted in APS1patients. IgG and IgM antibodies recognized significantly one tissue-specific (pancreatictriacylglycerol lipase) and one ubiquitous antigens (peroxiredoxin-2). IgM recognized one tissuespecific (Pancreatic regenerating protein 1!) and one ubiquitous antigen (Heat Shock cognate 71kDaProtein). IgG also recognized one tissue-specific (pancreatic amylase) and one ubiquitous antigen(aldose reductase). As expected, a well-defined self-reactive T cell repertoire impairment affected thetissue-specific self-IgG repertoire but also self-IgM repertoire. Our study also reveals discriminant responses against ubiquitous antigens with IgG and IgM antibodies. Some common discriminantantigenic targets were found for IgG and IgM. All these data suggest that T cell-dependent but also T cell-independent mechanisms are involved in APS1. The potent involvement of complementary events related to potent dysfunction in the innate immune response is discussed.

Vitiligo is an autoimmune disease in which CD8+ T cells selectively destroy melanocytes, leading to a patchy, disfiguring depigmentation of the skin. Our group and others have highlighted the central role of IFN-γ-dependent chemokines in the progression of disease; however, IFN-γ is also reported to have pleiotropic effects on melanocyte biology. We examined whether IFN-γ has a direct role in melanocyte killing. We tested the T-cell effector functions IFN-γ, Fas ligand and perforin by deleting them from autoreactive T cells used to induce vitiligo in mice. We found that disease incidence, disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFN-γ deficient or Fas ligand deficient gp100-specific T cells; however, perforin was dispensable and led to increased disease scores and T cell accumulation. To determine how melanocytes are affected by IFN-γ signaling during vitiligo, we performed single-cell RNA-sequencing on suction blister biopsies obtained from vitiligo and healthy subjects. We discovered that integrin expression and TGFb2 signaling was decreased only in lesional melanocyte transcriptomes. Moreover, melanocytes appear to participate in their own demise by increasing HLA expression and recruiting effector cells through the chemotactic ligand CCL18. The loss of melanocyte retention factors may explain their clean disappearance from the skin during keratinocyte turnover. Taken together, we believe IFN-γ production by autoreactive T cells in the skin leads to clean loss of melanocytes by downregulation of melanocyte retention factors and by increasing their potential to be detected by effector cells during vitiligo.

Tuberculosis (TB), a transmissible disease caused by Mycobacterium tuberculosis (Mtb), is a global health threat. To design an effective vaccine, we need to better understand how different elements of our immune system collaborate to fight against Mtb. CD4 T cells are crucial in protective immunity to Mtb because they produce cytokines including interferon-γ. In contrast, CD8 T cells are thought to play a modest role. Whether CD4 T cells act as “helper” cells to promote optimal CD8 T cell responses during TB is unknown. We argue CD8 T cells’ role are likely underestimated because CD8 T cell functions are compromised without CD4 T cells. Here, using two independent models, I show that CD4 T cell help promotes CD8 T cell effector functions and prevents CD8 T cell exhaustion. I demonstrate CD4 and CD8 T cells synergistically enhance the survival of infected mice. Purified helped, but not helpless, CD8 T cells effectively restrict intracellular Mtb growth. Thus, CD4 T cell help is indispensable for generating protective CD8 T cell responses. In addition, I investigate the mechanisms of CD4 T cell help. Signals from CD4 T cells, and signals relayed by antigen presenting cells collectively shape CD8 T cell responses. We infer that vaccines aimed for eliciting both CD4 and CD8 T cells, in which CD8 T cells are properly helped by CD4 T cells, are more likely to be successful.

No description available.

T cells use their highly variable T cell receptor (TCR) to engage major histocompatibility molecules (MHC) presenting peptides on the surface of antigen presenting cells during an immune response. The TCR repertoire of developing T cells is shaped by thymic selection, resulting in a self-tolerant and foreign peptide specific naïve T cell population. However, naive T cells are alloreactive and generate immune responses towards foreign MHC alleles in clinical settings involving transplantation. While T cell immune responses towards foreign pathogens are peptide specific, the overall specificity of allo-responses is still debated. Under normal circumstances, immune system homeostasis and self-tolerance is maintained by specialized natural T regulatory cells (nTregs) that develop in the thymus. nTregs respond to self-peptide MHC they encountered in peripheral tissues with immune-suppressive activities. However, the identify of self-peptides that stimulate nTregs, specificity towards these self-peptides, and the method nTreg TCRs engage self-peptide MHC molecules is not clear. Here, we built a library of defined MHC-linked self-peptides eluted from the I-Ab MHC molecule to screen alloreactive T cells and self-reactive nTregs for activating self-peptides. We used this library to show that negative selection shapes the TCR repertoire’s specificity to self-peptides. We also provide evidence that alloreactive T cells have degenerate self and foreign peptide recognition if the foreign MHC allele is largely different from the host’s MHC allele. Finally, we identified a self-peptide that activates an nTreg, and present protein crystal structures that reveal its TCR engages self and foreign peptide MHC complexes via fairly conventional mechanisms.

The contributions of peptide antigen affinity for TCR in driving T cell memory is unclear. Effector CD4 T cells must recognize cognate antigen again at an effector checkpoint, 5-8 days post-infection, to generate an optimal memory population. In this thesis, we examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We used an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP- peptide variants that bind FluNP TCR with a broad range of avidity. Varying peptide avidity in vivo at the effector checkpoint revealed that higher affinity interactions yielded greater numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. These memory cells demonstrated enhanced protection against lethal IAV infection with a robust early day 5 secondary effector response in the lung. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors while IL-2R expression was unaffected. However, IL-2Ra expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory and can instruct future vaccine design.

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.

Ex vivo expansion of T lymphocytes is a central process in the generation of cellular therapies targeted at tumors and other disease-relevant structures,which currently cannot be reached by established pharmaceuticals. The influence of culture conditions on T cell functions is, however, incompletely understood. In clinical applications of ex vivo expanded T cells, so far, a relatively classical standard cell culture methodology has been established. The expanded cells have been characterized in both preclinical models and clinical studies mainly using a therapeutic endpoint, for example antitumor response and cytotoxic function against cellular targets, whereas the influence of manipulations of T cells ex vivo including transduction and culture expansion has been studied to a much lesser detail, or in many contexts remains unknown. This includes the circulation behavior of expanded T cells after intravenous application, their intracellular metabolism and signal transduction, and their cytoskeletal (re)organization or their adhesion, migration, and subsequent intra-tissue differentiation. This review aims to provide an overview of established T cell expansion methodologies and address unanswered questions relating in vivo interaction of ex vivo expanded T cells for cellular therapy.