La parodontite est une pathologie immunoinflammatoire, entrainant une destruction du parodonte, suite à l’interaction entre un biofilm dysbiotique et une réponse immunitaire dérégulée. Parodontite et polyarthrite rhumatoïde présentent des mécanismes immunopathogéniques communs. Ainsi, les lymphocytes B (LB) ont un rôle majeur dans l’étiopathogénie de ces deux affections. Nous avons mené deux études cliniques observationnelles sur des cohortes de patients atteints de parodontite et traités pour leur polyarthrite rhumatoïde par deux immunothérapies touchant les LB : un anti-IL-6R(Tolicizumab) et un anti-CD20 (Rituximab). L’utilisation d’un anti-IL-6R a montré une diminution du TNF-α dans le fluide gingival après 6 mois de traitement, sans modification clinique de la parodontite. Par contre, l’anti-CD20, responsable d’une déplétion en LB, a permis une amélioration des paramètres cliniques parodontaux. Ensuite, nous avons étudié le phénotype des LB dans le sang et la gencive de patients atteints de parodontites sévères. Nos résultats montrent une augmentation des LB effecteurs "mémoires switchés" et une diminution des LB1 CD11b+ régulateurs. Notre travail a permis de mieux comprendre la distribution et le rôle des LB dans la réponse immunitaire dans les parodontites, aussi bien au niveau local que systémique et la discussion ouvre de nombreuses pistes de recherche et de nouvelles stratégies thérapeutiques. / Periodontitis is an immunoinflammatory pathologie, leading to periodontal destruction, following the interaction between a dysbiotic biofilm and a deregulated immune response. Periodontitis and rheumatoid arthritis exhibit common immunopathogenic mechanisms. Thus, B cells have a major role in the etiopathogenesis of these two diseases. We conducted two observational clinical studies in cohorts of patients with periodontitis and treated for rheumatoid arthritis with two immunotherapies for B cells: an anti-IL-6R (Tolicizumab) and an anti-CD20 (Rituximab). The use of anti-IL-6R showed a decrease in TNF-α in gingival crevicular fluid after 6 months of treatment, without clinical change in the periodontitis.On the other hand, the anti-CD20, responsible for a depletion in B cells, allowed an improvement of the periodontal clinical parameters. Then we studied the B cells phenotype in the blood and gingiva of patients with severe periodontitis. Our results show an increase in effector "switched memory" B cells and a decrease in regulator CD11b+ B1 cells.Our work has provided a better understanding of the distribution and role of B cells in the immune response in periodontitis, both locally and systemically, and the discussion opens many research approaches and new therapeutic strategies.

L'élimination des cellules tumorales par le système immunitaire repose sur la capacité des cellules dendritiques à correctement présenter l'antigène aux cellules effectrices. Nous avons donc développé une stratégie de vaccination thérapeutique basée sur des lipopolyplexes à ARNm (LPRs) : l'ARNm codant pour l'antigène est associé à un complexe de polylysine histidylée, et incorporé dans un liposome trimannosylé afin de mieux cibler les cellules dendritiques. En effet, les cellules dendritiques expriment largement les lectines de type C, qui sont des récepteurs reconnaissant principalement des résidus mannose. Dans le travail présenté ici, nous montrons que les LPR trimannosylés sont capables de fixer les cellules dendritiques humaines et murines. De manière très intéressante, nous montrons aussi que les LPR trimannosylés injectés chez la souris induisent le recrutement et l'activation de cellules dendritiques dans le ganglion drainant. De plus, quand les LPR trimannosylés vectorisent un ARNm codant pour l'antigène tumoral E7, ils sont capables d'induire une réponse T spécifique d'E7 au niveau systémique. Enfin, les LPRs trimannosylés permettent une réponse thérapeutique lorsqu'ils sont utilisés dans trois différents modèles de tumeurs : le modèle TC1 de carcinome exprimant E7, le modèle B16 de mélanome exprimant MART1 et le modèle EG7 de lymphome exprimant OVA. Cette stratégie apparaît donc prometteuse comme thérapie innovante anti-cancer. / Elimination of cancer cells requires an efficient cytotoxic immune response. In order to obtain such a response, antigens need to be uptaken by dendritic cells (DCs) and correctly presented to effector cells. We developed a strategy based on RNA lipopolyplexes (LPRs): antigenic mRNA is associated with a histidine-polylysine polyplexe and incorporated in a trimannosylated liposome to better target dendritic cells (DCs) in vivo, as DCs express several C-Type lectin receptors that preferentially bind to mannose. Here, we report that trimannosylated LPRs are efficient to target both human and murine DCs. Interestingly, in vivo experiments reveal that trimannosylated LPRs not only target DCs but also induce their recruitment and activation in draining lymph nodes. Furthermore, when combined with mRNA encoding E7 oncoprotein from HPV16, trimannnosylated LPRs trigger specific T-cell response against E7. Finally, when used as therapeutic vaccines in three different tumors models, LPRs promote curative therapeutic responses in E7-expressing TC1 tumor, in OVA-expressing EG7 lymphoma and in MART-1-expressing B16 melanoma, when combined with E7, OVA or MART-1 mRNA, respectively. Altogether, these results comfort us to considerate the use of this strategy for anti-cancer vaccine therapies.

La dernière décennie a connu une révolution dans le traitement du cancer en s'éloignant des médicaments conventionnels qui ciblent directement la tumeur (comme les chimiothérapies et les thérapies moléculaires ciblées) au profit des immunothérapies, et en particulier les inhibiteurs des "checkpoint" immunitaires. Ces immunothérapies libèrent sélectivement le système immunitaire de l'hôte contre la tumeur et ont démontré une rémission durable sans précédent chez des patients atteints de cancers que l'on croyait incurables, comme le mélanome métastatique, le carcinome rénal métastatique et les stades avancés du cancer du poumon non à petites cellules. Cependant, plus de la moitié des patients ne répondent pas à ces traitements, et sont donc contraints à recevoir d’autres traitements potentiellement toxiques et coûteux. Face aux résultats prometteurs des immunothérapies anti‐PD1/PD‐L1, la recherche de nouvelles cibles/marqueurs moléculaires permettant d'améliorer l'efficacité et de permettre un traitement personnalisé s'est intensifiée. Nos travaux portent sur l’étude de la protéine Sequestosome 1 (SQSTM1/p62) qui est un substrat de l'autophagie sélective et une plateforme de signalisation impliquée dans l’agressivité tumorale. Nous mettons en évidence l’implication de SQSTM1/p62 dans l'inflammation tumorale et dans l’évasion immunitaire et ceci grâce à la stabilisation du messager de PD‐L1. Nous observons que la surexpression tumorale de SQSTM1/p62 caractérise les tumeurs infiltrées et immunosuppressives qui répondent le mieux aux anti‐PD1/PD‐L1. Ainsi, nous proposons SQSTM1/p62 en tant que biomarqueur potentiel et cible thérapeutique pour améliorer la stratification des patients susceptibles de répondre aux immunothérapies. / The past decade has witnessed a new revolution in cancer treatment by shifting away from the conventional drugs that directly target the tumor (such as chemotherapies and molecular targeted therapies) towards immune‐based therapies, and in particular the so‐called immune checkpoint inhibitors. These immunotherapies selectively release the host immune system against the tumor and have shown unprecedented durable remission for patients with cancers that were thought incurable such as metastatic melanoma, metastatic renal cell carcinoma and late stages of non‐small cell lung cancer. However, more than half of the patients fail to respond to these treatments and are therefore forced to receive other potentially toxic and costly treatments. In this era of promising anti‐PD1/PD‐L1 immunotherapies, the quest for reliable molecular markers/therapeutic targets that would enhance the effectiveness and allow a personalized adaptation of the treatments has intensified. In our work we focus on the scaffold protein and autophagy adaptor Sequestosome 1 (SQSTM1/p62), and we show that it is required for tumor inflammation and immune evasion. We find that SQSTM1/p62 tumor overexpression characterizes infiltrated and immunosuppressive tumors and predicts for response to anti‐PD1/PD‐L1 therapies. Thus, we propose SQSTM1/p62 as a potential biomarker and a therapeutic target to improve the stratification of patients to immunotherapies.

abstract: Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies. / Dissertation/Thesis / Masters Thesis Biology 2020

<p>  </p> <p>Cancer is well known as a disease involving genetic disorders, which make them distinguishable from normal tissue by the altered molecular signatures. Theoretically, malignant cells can be recognized and attacked by innate and adaptive immune system as “non-self” species, and the idea to take advantage of host immunity to treat cancer has been discussed for over a century. Through the multi-disciplinary research efforts from immunology, cancer biology, cell engineering <em>etc.</em>, cancer immunotherapy has been successfully translated from benchside to bedside. While the clinical application of immunotherapeutic regimens has achieved extraordinary success including the unprecedented long-term survival of metastatic melanoma patients, we must take it seriously that only a small proportion (about 20% on average) of patients benefit from immunotherapy, and many develop secondary progression after the initial response. Advancements have been made in biomarker development to identify the group the patients who may benefit from immunotherapy, yet the accuracy and adaptability remain to be improved. In general, the performance of immunotherapy is hardly satisfactory as the current situation.</p> <p><br></p> <p>The effect out of T cell-mediated immune response is mediated by plenty pairs of receptor-ligand interactions in the immune synapse between T cells and target cells. Despite the T cell receptor-mediated first signal and CD28-mediated second signal, a huge collection of co-signals molecules serves unneglectable roles to keep the T-cell immune response fine-tuned under appropriate threshold. Inadequate co-signaling transduction result in with immune deficiency or autoimmunity depending on the type of signal (stimulatory or inhibitory). 4-1BB is a significant co-receptor which is mainly expressed on T cells and delivers activation signal to drive T cell proliferation and cytotoxicity. 4-1BB is targetable for cancer treatment and can be used as a tumor-reactive T cell marker as well. Hence, it is of substantial importance to understand how co-signaling molecules, such as 4-1BB, are regulated under specific physiological or pathological conditions.</p> <p><br></p> <p>Proteins are regulated at multiple levels, including transcription, translation, localization, and interaction with other biomolecules (covalently or non-covalently). Post-translational modification (PTM) constitutes a critical type of mechanism that elicit multidimensional effects to the biophysical properties of target proteins. Herein, I sought to elaborate how 4-1BB, an TNFRSF family co-stimulatory receptor, is possessed and regulated by PTMs, particularly ubiquitination and <em>N</em>-glycosylation. In the first part of this study, I confirmed that 4-1BB is degraded through ubiquitination-proteasome pathway and identified FBXL20 to be the E3 ligase subunit mediating 4-1BB polyubiquitination. While conducting the first section, I noticed that 4-1BB is heavily <em>N</em>-glycosylated and thereby dissected the biological significance of this modification which made up the second part of this study. I experimentally characterized that 4-1BB necessitates its <em>N</em>-glycans to be efficaciously transported to cell membrane through the secretory pathway. Plus, the glycosylated 4-1BB has short half-life. Without the spatial hindrance established by <em>N</em>-linked carbohydrate moieties, the exposed C121 residue of 4-1BB can be used to forms stable multimer which intracellular retention and stabilization of 4-1BB.</p> <p><br></p> <p>This study uncovered the post-translational mechanisms of action of 4-1BB regulation for the first time. More fundamentally, we provided a blueprint to study the post-translational regulation network of immune receptors which may be applied for future investigations in other targets. Our ultimate hope is to be able to grasp the dynamic of key immune regulators in the context of microenvironment and based on which pair the right therapeutics with the correct populations.</p>

Les traitements par immunothérapie adoptive actuels sont compromis par une faible infiltration et expansion des LT CD8 injectés. Les travaux antérieurs de l'équipe ont montré une synergie entre les signaux du TCR et du récepteur à l'IL-2 pour la différenciation en Lymphocytes T (LT) effecteurs compétents, un effet qui peut être mimé par une forme constitutivement active du facteur de transcription STAT5 (STAT5CA). Mon projet de thèse à viser à exprimer ce STAT5 actif dans des LT CD8 anti-tumoraux dans le but d'augmenter leur réactivité. Nous démontrons que STAT5CA soutient l'expression de gènes contrôlant la migration, la survie, la composition des granules cytolytiques, la sécrétion de cytokines de type Tc1 (IFNy/TNF&#945;) et leur potentiel de réponse secondaire. Les LT modifiés par STAT5CA sur-expriment les facteurs de transcription T-bet et Eomes qui sont associés respectivement à la différenciation en LT effecteur mémoire et central mémoire. Le potentiel cytolytique et migratoire des LT modifiés par STAT5CA rend ces cellules plus efficaces que des LT contrôlent pour induire une régression tumorale dans des modèles de mélanome transplanté ou induit (modèle TiRP). Ces résultats suggèrent une résistance accrue aux mécanismes d'immunosuppression intra-tumoraux. Contrairement aux LT contrôles, les LT modifiés par STAT5CA sous-expriment l'IL-6R&#945; et le TGF&#946;RII et ont une moindre sensibilité à l'action immunosuppressive des cytokines IL-6 et TGF&#946;1. / Adoptive therapies are compromised by poor infiltration and expansion of injected CD8 T cells. Previous work in our team has demonstrated that signals from TCR and IL-2 receptor are acting in synergy to promote the differentiation of CD8 T cells. Moreover, this IL-2 effect can be mimicked by a constitutive active form of STAT5 (STAT5CA). During my PhD, I expressed this active STAT5 in anti-tumor CD8 T cells in order to enhance their activity. We demonstrated that STAT5CA sustains gene expression involved in migration, survival, cytolytic granules composition, Tc1 cytokine secretion (IFNy/TNF&#945;) and secondary response potential. T cells transduced with STAT5CA up-regulate expression of the transcription factors T-bet and Eomes which are involved respectively in effector or central memory T cell differentiation. Cytolytic and migratory properties of STAT5CA T cells contribute to their capacities to induce regression of both transplanted and induced (TiRP model) melanomas, while control T cells were inefficient. Those results suggest that STAT5CA T cells are less sensitive to tumor-derived immunosuppression. Compared to control T cells, STAT5CA T cells show a down-regulation of IL-6R&#945; and TGF&#946;RII which correlate with their decreased sensitivity to IL-6 and TGF&#61538;1 derived immunosuppression. Moreover, STAT5CA T cells infiltrate lung, liver and pancreas which open possible treatments for highly frequent tumors that are not efficiently cured.

No description available.

L'activation de TLR9 par injection directe de nucléotides CpG non méthylés dans une tumeur peut induire une réponse immunitaire thérapeutique, mais les lymphocytes T régulateurs (Tregs) inhibent ensuite la réponse immunitaire antitumorale et limitent ainsi le pouvoir des stratégies d'immunothérapies contre le cancer.Chez des souris porteuses de tumeurs, nous avons constaté que les Tregs dans la tumeur expriment préférentiellement les marqueurs cellulaires de surface CTLA-4 et OX40. Nous montrons que la co-injection intratumorale d'anti-CTLA-4 et anti-OX40 avec du CpG en intra-tumoral aboutit à l’élimination des Tregs infiltrant la tumeur. Cette immunomodulation in situ, réalisée avec de faibles doses d'anticorps dans une tumeur unique, génère une réponse immunitaire antitumorale systémique capable d’éradiquer la maladie disséminée chez la souris. De plus, cette modalité de traitement est efficace contre des lésions de lymphome du SNC avec métastases leptoméningées, des sites qui sont généralement considérés comme des sanctuaires de cellules tumorales pour les traitements systémiques conventionnels.Ces résultats démontrent que les effecteurs immunitaires anti-tumoraux activés par immunomodulation locale peuventt éradiquer des cellules tumorales siègeant dans des sites éloignés. Nous proposons que, plutôt que d'utiliser des anticorps monoclonaux pour cibler les cellules cancéreuses par voie systémique, des anticorps monoclonaux pourraient être utilisés pour cibler les cellules immunitaires infiltrant la tumeur localement, provoquant ainsi une réponse immunitaire systémique. / Activation of TLR9 by direct injection of unmethylated CpG nucleotides into a tumor can induce a therapeutic immune response; however, regulatory T-cells (Tregs) eventually inhibit the antitumor immune response and thereby limit the power of cancer immunotherapies. In tumor-bearing mice, we found that Tregs within the tumor preferentially express the cell surface markers CTLA-4 and OX40. We show that intratumoral coinjection of anti–CTLA-4 and anti-OX40 together with CpG depleted tumor-infiltrating Tregs. This in situ immunomodulation, which was performed with low doses of antibodies in a single tumor, generated a systemic antitumor immune response that eradicated disseminated disease in mice. Further, this treatment modality was effective against established CNS lymphoma with leptomeningeal metastases, sites that are usually considered to be tumor cell sanctuaries in the context of conventional systemic therapy. These results demonstrate that antitumor immune effectors elicited by local immunomodulation can eradicate tumor cells at distant sites. We propose that, rather than using mAbs to target cancer cells systemically, mAbs could be used to target the tumor infiltrative immune cells locally, thereby eliciting a systemic immune response.