Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-18T21:46:22Z No. of bitstreams: 1 Fernanda Oliveira Novais Avaliacao do papel....pdf: 2271339 bytes, checksum: eee5aa084065678cf55651296f356919 (MD5) / Made available in DSpace on 2012-07-18T21:46:22Z (GMT). No. of bitstreams: 1 Fernanda Oliveira Novais Avaliacao do papel....pdf: 2271339 bytes, checksum: eee5aa084065678cf55651296f356919 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Linfócitos T CD8+ são células do sistema imune adaptativo capazes de induzir a morte de células infectadas através de mecanismos citotóxicos. No modelo de infecção intradérmica por Leishmania revelou-se que os linfócitos T CD8+ são responsáveis tanto pela indução de patogênese bem como pela imunidade contra a infecção primária por L. major. Até o momento, o papel dos linfócitos T CD8+ não foi estudado na infecção experimental por L. braziliensis. Neste estudo, investigamos o recrutamento dos linfócitos T CD8+ para o sítio de infecção e determinamos a sua função. Cinco semanas após a infecção intradérmica por L. braziliensis, camundongos BALB/c apresentaram um aumento na porcentagem de linfócitos T CD8+ presentes na orelha infectada e estes produziram, principalmente, IFN-g e Granzima B. Já no linfonodo de drenagem, estas células não produzem granzima mas, sim, IFN-g e TNF-a. Utilizando o mesmo modelo de infecção, camundongos BALB/c ou C57BL/6 depletados de linfócitos T CD8+ ou camundongos deficientes em b2-microglobulina ou em CD8 apresentaram redução no tamanho da lesão ao longo da infecção e menor carga parasitária cinco semanas após a infecção. A depleção de linfócitos T CD8+ não induziu qualquer alteração no recrutamento e produção de IFN-g, TNF-a e IL-10 pelos linfócitos T CD4+ no sítio de infecção ou no linfonodo de drenagem. Além disso, a capacidade proliferativa ou a produção de citocinas específicas in vitro após estímulo com células dendríticas infectadas por L. braziliensis não sofreram alteração. A ausência de linfócitos T CD8+ após a 8 infecção por L. braziliensis também não alterou o recrutamento de monócitos inflamatórios nem a sua diferenciação em células dendríticas. Por último, a análise histológica e de citometria de fluxo mostrou aumento no recrutamento de neutrófilos para o sítio de infecção e este resultado pode ser correlacionado com o controle da doença. Para confirmar o envolvimento dos linfócitos T CD8+ no desenvolvimento de lesão por L. braziliensis, transferimos linfócitos T CD8+ de camundongos naïve ou imunes, bem como linfócitos T CD4+ somente ou linfócitos T CD8+ e T CD4+ para camundongos RAG-/-. Neste contexto, a transferência de linfócitos T CD8+ naïve ou imunes induziu uma intensa patologia no sítio de infecção bem como a disseminação de parasitas para outros sítios, como a orelha não infectada, pata e nariz. Camundongos RAG-/- controle e aqueles que receberam linfócitos T CD8+ naïve ou imunes apresentam a mesma quantidade de parasitas no sítio de infecção, embora o aspecto da lesão tenha sido muito diferente. A transferência de linfócitos T CD4+ foi capaz de controlar a carga parasitária nestes animais e o mesmo foi observado após transferência de linfócitos T CD4+ e T CD8+ em conjunto. Nestes animais não observamos lesões em outros sítios, indicando que os linfócitos T CD8+ contribuem para a disseminação dos parasitas. Por último, transferimos linfócitos T CD8+ provenientes de camundongos selvagens ou deficientes de IFN-g e perforina e observamos que, na ausência de perforina, a patologia e a disseminação parasitária são controladas. Portanto, este estudo sugere envolvimento dos linfócitos T CD8+ na patogênese induzida por L. braziliensis devido ao seu potencial citotóxico e, em paralelo, inibindo o recrutamento de neutrófilos para o sítio de infecção. / CD8+ T lymphocytes are part of the adaptive immune system and are considered cytotoxic because of their ability to induce death in infected cells. Using the intradermal model of Leishmania infection, it has been shown that CD8+ T cells play an essential role in both pathogenesis and immunity to primary infection with L. major in the skin. So far, the role of these lymphocytes in the experimental model of infection using L. braziliensis has not been evaluated. In this study we determined the recruitment and function of these cells upon infection with L. braziliensis. Five weeks after infection, the frequency of CD8+ T cells was increased in the dermal site and these cells produced mainly IFN-g and granzyme B in infected mice. In the draining lymph nodes, these cells produced high levels of IFN-g and TNF-a, but not granzyme B. Using the same intradermal model of infection, we analysed the outcome of infection in the absence of CD8+ T lymphocytes using both antibody depletion in BALB/c and C57BL/6 mice and mice deficient in b2- microglobulin and CD8. In all groups, the absence of CD8+ T cells was correlated with better control of lesion development and parasite load in both depleted BALB/c and in b2-microglobulin deficient mice. In the absence of CD8+ T cells, CD4+ T lymphocytes were recruited to the same extension and produced same levels of IFN-g, TNF-a and IL-10 both in the infected ear and in draining lymph nodes when compared to infected mice that were not depleted. Also, there was no change in the proliferative potential and in IFN-g production by these cells after re-stimulation with infected dendritic cells. Analysis of inflammatory monocyte recruitment and differentiation of these cells into dendritic cells were similar in both depleted and non-depleted mice. On the other hand, histological and flow cytometric analyses showed increased neutrophil recruitment to the site of infection and this can be correlated with disease control. To confirm the role of CD8+ T cells in the lesion development of L. braziliensis infected mice, we then transferred CD8+ T cells from naïve or immune mice, as well as CD4+ T cells alone or together with T CD8+ to RAG deficient mice. The transfer of CD8+ T cells from immune or naïve mice into RAG recipients induced an intense pathology upon infection with L. braziliensis in the infection site, but also in uninfected tissues such as the uninfected ear, nose and footpad. Evaluation of parasite numbers in the infected ear showed that RAG deficient mice without T cells and those transferred with CD8+ T cells from naïve or immune mice had similar number of parasites although the pathology was very different. The transfer of CD4+ T cells alone or in association with CD8+ T cells induced parasite control in the infection site. In these mice, we could not detect lesions in other sites and we concluded that the transfer of CD8+ T cells alone induces parasite dissemination in RAG deficient mice. Finally, the transfer of CD8+ T cells from perforin deficient mice led to control in lesion development and in parasite dissemination. In this study we can conclude that CD8+ T cells are involved in the pathogenesis of L. braziliensis due to their cytotoxic potential and by inhibiting neutrophil recruitment to the infection site.

Abstract : CD8 + T cells can be programmed in their naïve state with pro-inflammatory cytokines such as IL-15 and IL-21. IL-15 induces the proliferation of CD8 + T cells and allows the generation of memory cells. IL-21 programs CD8 + T cells to become more cytotoxic while retaining a memory type phenotype. In the laboratory, we studied the effect of these two cytokines in different contexts by using the mouse model MHC-I-restricted Pmel-1 transgenic TCR specific to the melanoma-derived gp10025-33 antigen, which is also expressed by normal melanocytes. First, we elucidated the effect of IL-15 on CD8 + T cells in the Pmel-1 transgenic model lacking the protein Suppressor of cytokine signaling 1 (SOCS1). SOCS1 is a critical regulator of T cell homeostasis. We have found that these mice have CD8 + T-cells expressing surface proteins characteristic of memory T cells (CD44, Ly6C, CD122 and CD62L). However, these cells decrease the expression of the TCR and increase that of CD5, indicative of TCR activation in vivo. When stimulated in vitro, these cells displayed a highly cytotoxic phenotype but very low proliferation. Adoptive cell transfer studies in Rag1 - / - mice showed that these cells can undergo homeostatic proliferation under lymphopenic conditions. This proliferation was characterized by severe inflammatory lesions in the skin, extremities and eyes. This study demonstrates the importance of IL-15 and SOCS1 in the regulation of self-reactive cells that can be activated under lymphopenic conditions and can cause autoimmune diseases. Second, we studied the synergistic effect of IL-15 and IL-21 in native CD8+ T cells for cancer immunotherapy. We used the mouse melanoma model B16-F10 (B16) which expresses very weakly MHC-I molecules. In parallel, we studied the effect of NOD-like receptor CARD domain containing 5 (NLRC5) overexpression, the trans-activator of MHC-I genes, in B16 cells in order to increase their immunogenicity and restore anti-tumor immunity. We generated stable lines of B16 cells expressing NLRC5 (B16-5); the co-stimulatory molecule of T cells, CD80 (B16-CD80), or both (B16-5 / 80). The over-expressing NLRC5 cells positively regulated the MHC-I and LMP2, LMP7 and TAP1 genes. B16-5 cells efficiently presented gp10025-33 to CD8+ Pmel-1 T cells and induced their proliferation. This proliferation was very robust when Pmel-1 naive cells were pre-stimulated with IL-15 and IL-21 prior to activation. In the presence of CD80, B16-5 cells stimulate Pmel-1 cells even without the addition of gp100, indicating that NLRC5 facilitates the treatment and presentation of endogenous tumor antigens. During subcutaneous implantation, B16-5 cells showed a significant reduction in tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that tumor cells expressing NLRC5 generated an anti-tumor immunity. This response is dependent on CD8 + T cells since in mice depleted of these cells, B16-5 cells formed large subcutaneous and pulmonary tumors. Finally, immunization with irradiated B16-5 cells allowed anti-tumor protection during challenge of parental B16 cells. Collectively, our results indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity. / Les cellules T CD8+ peuvent être programmées à leur état naïf avec des cytokines pro-inflammatoires telles que l’IL-15 et l’IL-21. IL-15 induit la prolifération de cellules T CD8+ et permet la génération de cellules T CD8+ mémoire. IL-21 programme les cellules T CD8+ à devenir plus cytotoxiques tout en conservant un phénotype de type mémoire. Dans le laboratoire, nous avons étudié l’effet de ces deux cytokines dans différent contextes en utilisant le modèle de souris transgénique Pmel-1 qui possède des récepteurs de cellules T (TCR) spécifiques envers le peptide gp10025-33, exprimé par des cellules de mélanome et aussi par des mélanocytes. Premièrement, nous avons élucidé l’effet de l’IL-15 sur les cellules T CD8+ dans le modèle transgénique Pmel-1 déficient dans la protéine Suppressor of cytokine signaling 1 (SOCS1). SOCS1 est un régulateur critique de l’homéostasie de lymphocytes T. Nous avons trouvé que ces souris ont de cellules T CD8+ exprimant des protéines de surface caractéristique de cellules T mémoire (CD44, Ly6C, CD122 et CD62L). Cependant, ces cellules diminuent l’expression du TCR et augmentent celle de CD5, ce qui témoigne d’une activation du TCR in vivo. Lorsque stimulées in vitro, ces cellules montrent un phénotype hautement cytotoxique mais une prolifération très faible. Lorsque nous avons fait des études de transfert cellulaire adoptive dans de souris Rag1-/-, ces cellules ont proliféré de façon importante causant des lésions inflammatoires sévères dans la peau, les extrémités et les yeux. Cette étude démontre l’importance de l’IL-15 et de SOCS1 dans la régulation de cellules auto-réactives qui peuvent être activées sous des conditions lymphopéniques et qui peuvent causer de maladies auto-immunitaires. Deuxièmement, nous avons étudié l’effet synergique d’IL-15 et d’IL-21 dans les cellules T CD8+ naïves pour l’immunothérapie du cancer. Nous avons utilisé le modèle B16-F10 (B16) de mélanome de souris qui exprime très faiblement des molécules de CMH-I. En parallèle, nous avons étudié l’effet de la surexpression de NOD-like receptor CARD domain containing 5 (NLRC5), le trans-activateur de gènes de CMH-I dans les cellules B16, dans le but d’augmenter leur immunogénicité et de restaurer l’immunité anti-tumorale. Nous avons généré des lignées stables de cellules B16 exprimant NLRC5 (B16-5); la molécule co-stimulatrice de cellules T, CD80 (B16-CD80), ou les deux (B16-5 / 80). Les cellules sur-exprimant NLRC5 ont régulé positivement de manière constitutive les gènes MHC-I et LMP2, LMP7 et TAP1. Les cellules B16-5 ont efficacement présenté gp10025-33 aux cellules T CD8+ Pmel-1 et ont induit leur prolifération. Cette prolifération a été très robuste lorsque les cellules naïves Pmel-1 étaient pré-stimulées avec IL-15 et IL-21 avant leur activation. En présence de CD80, les cellules B16-5 stimulent les cellules Pmel-1 même sans l'addition de gp100, ce qui indique que NLRC5 facilite l’apprêtement et la présentation des antigènes tumoraux endogènes. Lors de l'implantation sous-cutanée, les cellules B16-5 ont montré une réduction significative de la croissance tumorale chez des hôtes C57BL/6, mais pas chez des hôtes immuno-déficients, ce qui indique que les cellules tumorales exprimant NLRC5 ont provoqué une immunité anti-tumorale. Cette réponse est dépendante de cellules T CD8+ puisque chez les souris déplétées de ces dernières, les cellules B16-5 ont formé de grandes tumeurs sous-cutanées et pulmonaires. Enfin, l'immunisation avec des cellules B16-5 irradiées a permis une protection anti-tumorale lors de la réimplantation des cellules B16 parentales. Collectivement, nos résultats indiquent que NLRC5 pourrait être exploité pour restaurer l'immunogénicité tumorale et pour stimuler l’immunité anti-tumorale protectrice.

Les rythmes circadiens contrôlent de nombreux aspects de la physiologie chez les mammifères. Parmi ces processus physiologiques, les horloges circadiennes contrôlent entre autres la réponse immunitaire innée et adaptative. Depuis des décennies, de nombreuses études ont commencé à couvrir ce sujet. Cependant, le contrôle circadien de la réponse adaptative reste peu étudié. Dans le cadre de ce projet de recherche de doctorat, nous avons exploré le rôle des rythmes circadiens dans la réponse des lymphocytes T CD8 à la présentation antigénique par des cellules dendritiques. Des travaux du laboratoire publiés par Erin E. Fortier et al. ont mis en évidence une différence jour/nuit dans l’expansion des lymphocytes T CD8 dont le récepteur T (TCR) est spécifique au complexe KbOVA exprimé par les cellules dendritiques ainsi que dans le nombre de lymphocytes T CD8 CD44hi IFN+ spécifiques pour l’antigène de l’ovalbumine (OVA)1. En effet, la réponse des lymphocytes T CD8 est plus importante après une vaccination faite en milieu de jour (zeitgeber time (ZT) 6) par rapport à une vaccination faite en milieu de nuit (ZT18). Cependant, ces travaux de recherche n’ont pas démontré le rôle des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Mes travaux de recherche de doctorat ont dans un premier temps confirmé l’implication des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Nous avons ensuite démontré la contribution des horloges circadiennes des cellules dendritiques ainsi que le rôle essentiel des horloges circadiennes des lymphocytes T CD8 dans ce rythme de réponse. De plus, nous avons montré que ce rythme avait un impact sur la capacité des lymphocytes T CD8 à contrôler une infection bactérienne (Listeria monocytogenes). En effet, les variations jour/nuit de la charge bactérienne dans la rate et le foie des souris de type sauvage étaient abolies dans les souris déficientes pour le gène des horloges circadiennes Bmal1 dans les lymphocytes T CD8. Dans un second temps, nous avons mis en évidence suite à l’analyse du transcriptome des lymphocytes T CD8 de souris naïves collectés toutes les 4 heures sur 48 heures que ces cellules sont plus enclines à être activées le jour et à l’opposé plus enclines à être inhibées la nuit. Ces résultats corrèlent avec le rythme de réponse des lymphocytes T CD8 à la vaccination. Dans un dernier temps, nous avons confirmé que le rythme de réponse des lymphocytes T CD8 à la présentation antigénique agissait de manière précoce dans l’activation de ces cellules. Pour cela nous avons irradié des souris de type sauvage et nous les avons ensuite reconstituées avec une moelle osseuse contenant 1% de précurseurs de cellules OT-I (lymphocytes T CD8 spécifiques au complexe KbOVA, exprimant la chaîne du TCR V5. Après vaccination de ces souris en milieu de jour subjectif (circadian time (CT) 6) ou en milieu de nuit subjective (CT18), nous avons observé un rythme de la réponse des lymphocytes CD8 pour différents marqueurs impliqués dans la réponse précoce des lymphocytes T CD8, telles que CD69, CD5, IRF4, et la phosphorylation de S6 (marqueur de l’activité de mTOR) et de AKT. L’ensemble des recherches de mon doctorat ont permis de mettre en évidence un tout nouveau mécanisme impliquant les horloges circadiennes dans la réponse des lymphocytes T CD8 en réponse à une vaccination. Une meilleure compréhension du fonctionnement des horloges circadiennes dans la réponse immunitaire permettra de mettre en place des nouveaux traitements personnalisés en fonction du type d’infection et du type de maladie, délivré à un certain moment de la journée dans le but d’améliorer l’efficacité tout en réduisant les effets secondaires. / Circadian rhythms control various aspects of the physiology in mammals. Among these processes, circadian clocks control the innate and the adaptive immune responses. Since few decades, numerous studies started to uncover the role of the circadian system in the immune response. However, the circadian control of the adaptive immune response remains poorly studied. My PhD work focused on the circadian control of the CD8 T cell response to vaccination by dendritic cells. Erin E. Fortier et al. published in The Journal of Immunology that wild type mice vaccinated with antigen presenting cells loaded with the OVA peptide present a day/night variation of the CD8 T cell response after a vaccination done during the middle of the day (zeitgeber time (ZT) 6) compared to a vaccination done during the middle of the night (ZT18)1. Indeed, the proportion of CD8 KbOVA+ cells and the proportion of CD8 CD44hi IFN+ T cells were higher during the middle of the day than the middle of the night. However, this work showed a diurnal but not a circadian rhythm, that remained to be confirmed. The first part of my PhD research confirmed a role of the circadian system in the rhythm of the CD8 T cell response to vaccination. We showed a contribution of the dendritic cell clock as well as an essential role of the CD8 T cell clock. Moreover, this rhythm impacts the ability to control an infectious challenge as shown by a circadian variation in bacterial load (Listeria monocytogenes) in wild type but not in mice lacking clock in mature CD8 T cells. The second part of my research focused on the analysis of the transcriptome of CD8 T cells from naive mice collected every 4 hours over 48 hours. We showed that CD8 T cells are more prone to be activated during the day and at the opposite are more prone to be inhibited during the night. These results are correlated with the rhythm of the CD8 T cell response to vaccination. Finally, during the third part of my PhD, we confirmed that the rhythm of the CD8 T cell response to antigen presentation was acting at the early stage of the CD8 T cell activation. We used wild type mice reconstituted with bone marrow cells containing 1% of OT-I precursor cells (CD8 T cells restricted for the KbOVA complex, expressing the receptor chain of the TCR for the OVA peptide, V5) and vaccinate these mice during the middle of the subjective day (CT6) or during the middle of the subjective night (CT18). At the early stage of the CD8 T cell response to vaccination, we showed a higher expression of several activation markers after a vaccination done during the middle of the day than during the middle of the night, such as CD5, CD69, IRF4 and the phosphorylation of S6 (marker of the mTOR activity) and AKT. Altogether, my PhD work highlights a new mechanism involving the circadian system in the control of the immune response. A better understanding of how circadian clocks act on the immune response will allow implementing new treatment strategies in order to increase their efficacy as well as to decrease side effects.

Les myosites sont des maladies auto-immunes, caractérisées par des atteintes musculaires et extra musculaires. Le diagnostic des myosites peut être difficile et nécessite de l’expertise, afin d’éviter l’administration de thérapie inapproprié. Les mécanismes impliqués au cours des myosites sont peu connus. Notre but était de décrire le profil immunitaire des patients, afin d’identifier des biomarqueurs. Nous avons utilisé un panel de 36 marqueurs pour caractériser les PBMC issus de patients actifs (MIs, SAS anti-Jo1, myopathies anti-SRP et anti-HMGCR) et de sujets sains par cytométrie de masse combiné au « barcoding ». Tout d’abord, nous avons mis au point une procédure technique pour la détection simultanée de cibles extracellulaires et intracellulaires. En utilisant différents outils bio-informatiques, nous avons isolé une fréquence de lymphocytes CD8+T-bet+ > 51.5% comme étant un biomarqueur spécifique de la MIs en comparaison aux autres myosites, avec une sensibilité de 94,74% et une spécificité de 88,46%. De plus, nous avons identifié un profil immunitaire CD8+T-bet+ CD57- activé, potentiellement capable de prolifération et de maintien de mécanismes auto-immuns chez les patients atteints de MIs. Chez les patients anti-Jo1, nous avons observé une dérégulation de l’homéostasie des lymphocytes B, caractérisée par une diminution des lymphocytes B mémoires circulants. La présence de ces derniers dans le muscle des patients suggère qu’ils se nichent dans le muscle afin d’éviter l’action des immunosuppresseurs. Ces travaux ont permis l’identification de biomarqueurs et de phénotypes cellulaires potentiellement impliqués au cours de la MIs et du SAS anti-Jo1. / Myositis is an autoimmune disease characterized by muscular and extra-muscular disorders. In the early stages of the disease, the diagnosis of myositis can be misleading and requires expertise, in order to avoid the administration of inappropriate treatment. The mechanisms involved in these diseases are poorly understood. Our aim was to describe the immune profile specific to each patient group, in order to identify biomarkers that may be useful for diagnosis and management of patients. We used a panel of 36 markers by mass cytometry to characterize PBMCs derived from active patients (sIBM, anti-Jo1 ASyS, anti-SRP and anti-HMGCR myopathies) and healthy subjects. First, we developed and optimized a technical procedure for the simultaneous detection of extracellular and intracellular targets by mass cytometry. Using different bioinformatics tools, we isolated a frequency of CD8 +T-bet + cells > 51.5% as a specific biomarker for sIBM compared to other myositis, with a sensitivity of 94.74% and a specificity of 88. , 46%. In addition, we identified an activated CD8 + T-bet + CD57- immune profile, potentially capable of proliferation and the maintenance of autoimmune mechanisms in patients with sIBM. In anti-Jo1 patients, we observed a dysregulation of B cell homeostasis, characterized by a decrease in circulating memory B cells. The presence of the latter in the muscle of patients suggests that they nest in the muscle to avoid immunosuppressants. This work allowed the identification of a biomarker that could enhance the diagnosis of MIs compared to other myositis and the identification of cells potentially involved during sIBM and anti-Jo1 ASyS.

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.

Cancer is the second most common cause of death in the world, and the number of people with the disease increases each year. The therapy of the disease currently stands on four pillars; surgery, chemotherapy, radiotherapy, and immunotherapy. Through the past few years, immunotherapy has become the fastest developing treatment modality. However, despite its unprecedented efficacy in some patients, the majority of patients still does not respond to the therapy. Therefore, there is a need to investigate the mechanisms that make immunotherapy inefficient. Cell-based cancer immunotherapy is the treatment modality which uses live ex vivo-produced tumor-targeting immune cells to treat cancer. One of the mechanisms that may compromise its therapeutic efficacy is the expression of inhibitory molecules on the surface of the produced immune cells. Tim-3 is the inhibitory molecule which attracts attention in recent years. Tim-3 expression in the tumor cells and the tumor-infiltrating immune cells is often associated with worse prognosis and more aggressive forms of the disease. However, its role in the in vitro or ex vivo-produced immune cells is difficult to predict. In this work, an in vitro study model which is based on in vitro-produced antigen-specific CD8+ T cells with high expression of Tim-3 has been...

The Tec kinase ITK is activated by TCR stimulation and also required for TCR downstream signaling. Previous studies have reported differential roles of ITK and another Tec family kinase RLK in CD4+ TH differentiation and effector function. However, these findings are confounded by the complex T cell developmental defects in Itk-/- mice. Furthermore, the function of ITK in tissue-resident T cells in the intestine and anti-viral immune response to a persistent infection has not been studied previously. In addition to T cells, recent studies have indicated an expression of ITK in ILC2, but not in other ILC subsets. Yet, the role of ITK in ILC2 has not been characterized. Here, I have examined the role of ITK and RLK in CD4+ TH subsets using a small molecule inhibitor PRN694. I found that PRN694 impaired TH1 differentiation in vitro, and PRN694 administration prevented TH1-mediated colitis progression in vivo. In an MHV68 infection model, Itk-/- mice failed to control viral replication in the intestine, while gut-homing of CD8+ T cells was greatly impaired. Finally, I found that ILC2 number was markedly reduced in the intestine of Itk-/- mice. Gut-specific defect of Itk-/- ILC2 is associated with a low availability of IL-2 in the intestine of Itk-/- mice. Collectively, these data suggest that ITK is important in T cell migration to the intestine and ILC2 homeostasis in the intestine, thereby contributing to the protective response to a latent virus and intestinal tissue homeostasis.

La reconnaissance des cellules tumorales par le système immunitaire, appelée immunosurveillance, permet le contrôle de lacroissance des tumeurs voir dans certains cas leur élimination. Cependant, de nombreuses études d’exploration du système immunitaire dans le contrôle de la réponse antitumorale ont mis en évidence des mécanismes complexes d’échappement à cette immunosurveillance,avec par exemple pour les lymphocytes TCD8+ (LT CD8+) principal acteur de cette réponse, un défaut de migration, de reconnaissance des cellules tumorales ou d’activation au sein de la tumeur. Cette inhibition de fonction des LTCD8+, peut être liée à un phénomène appelé exhaustion, lié à l’expression à leur surface de molécules de costimulation inhibitrices telles que PD1, TIM-3, Lag3, CTLA-4. L’interaction de ces récepteurs avec leurs ligands engendre une perte de contrôle de la réponse antitumorale, favorisant alors la progression tumorale. Afin de lever ce phénomène d’exhaustion induit sur les LT CD8+ et restaurer la réponse antitumorale, plusieurs stratégies de traitements, visant à inhiber ces « checkpoints inhibiteurs » ont étédéveloppées. L’effet clé de la ciclosporine-A(CsA) repose sur la modulation de l’activité des lymphocytes T, ce qui explique son rôle dans la prévention du rejet de greffe. Cependant, il reste à déterminer si la CsA exerce d'autres effets sur le système immunitaire.Les évidences scientifiques montrent un effet paradoxal de la faible dosage de cyclosporine-A (fd-CsA). Ces résultats nous ont permis d’émettre l’hypothèse d’un rôle de la fd-CsA dans la modulation de la réponse antitumorale des LT CD8+, Nous avons pu observer à l’inverse, qu’à faible dose de ciclosporine-A, soit une dose équivalente entre 10 et 30 ng/mL, l’expression de PD1 était significativement diminuée à la surface des LT CD8+ activé. En revanche, à cette faible dose, aucun effet significatif sur la diminution d’expression du marqueur d’activation CD69 n’a été observé. Des expériences effectuées in vivo dans le modèle murin de mélanome B16F10 et MCA nous ont par ailleurs permis de montrer une réduction de la croissance tumorale chez les souris traitées par fd-CsA par rapport aux souris non traitées. En utilisant un modèle murin de fibrosarcome MCA, nous avons montré que nous restaurions in vivo une réponse immunitaire antitumorale et qu’un traitement de ces souris par fd-CsA en combinaison avec l’anti PDL-1 permettait une guérison après traitement, alors que l’anti PDL-1 seul n’avait pas d’effet. Cet effet novateur de la fd-CsA permet donc de visualiser de nouvelles stratégies thérapeutiques dans la réponse antitumorale qui pourraient bénéficier aux futurs patientsdiagnostiqués d’un cancer. / The recognition of tumor cells by the immune system, called immunosurveillance, allows the control of tumor growth or in some cases their elimination. However, numerous studies of the immune system in the control of the antitumor response have revealed complex mechanisms by means of which this immunosurveillance can be evaded. Examples of this occurrence are CD8+ T lymphocytes, the main element of this response are defective migration, tumor cell non-recognition or non-activation within the tumor. This inhibition of CD8+ T cell function may be related to a phenomenon called exhaustion, which may be a result of the expression on their surface of inhibitory costimulatory molecules such as PD1, TIM-3, Lag3, CTLA-4. The interaction of these receptors with their ligands causes a loss ofcontrol of the antitumor response, thus promoting tumor progression. To overcome this phenomenon of exhaustion induced in CD8+ T cells and restore the antitumor response, several treatment strategies aimed at inhibiting these "inhibitory checkpoints" have been developed. The key effect of cyclosporin-A (CsA) is modulation of T-cell activity, which explains its role in the prevention of transplantrejection. However, it remains to be determined whether CsA has other effects on the immune system. Preliminary results have allowed us to demonstrate the paradoxical effect of cyclosporin-A (CsA) in the antitumor response. Thus, these unexpected results have allowed us to hypothesize a role for Ld-CsA in modulating the antitumor response of CD8+ Tcells. We observed that Ld-CsA at equivalent dose between 10 and 30 ng/mL, significantly decreased PD1 expression at the activated CD8+ T cell surface. Furthermore, at this lowdose, no significant effect was observed on the expression of the CD69 activation marker. We have also shown that Ld-CsA increases the vaccine response in vivo. In vivo experiments with the murine B16F10 melanoma model and MCA fibrosarcoma have also shown areduction in tumor growth in mice treated with d-CsA compared to untreated mice. More recently, using a mouse model of MCA-OVAfibrosarcoma, we have shown that we can restore the in-vivo antitumor immune response and that the treatment of these mice by Ld-CsAin combination with the anti PDL-1, allowed tumor regression, whereas anti PDL-1 alone had no effect. This novel effect of Ld-CsA allows us therefore to visualize new therapeutic strategies for the antitumor response which may benefit future patients diagnosed with cancer.

While adenovirus (Ad) vectors have been shown to elicit potent antigen-specific T cell responses, the kinetics and nature of antigen-specific mucosa! and systemic T-cell responses has rarely been examined, especially following mucosal administration of Ad-based vectors. In the present studies, the phenotypic and functional characterization of antigen-specific CD8+ T cell responses following intrarectal (i.r.) vaccination with an Ad vector expressing Gallus gallus ovalbumin (OVA) was conducted. The frequencies of OVA-specific CD8+ T cells was maximal at 2 weeks post-vaccination in all tissues examined and then declined, demonstrating normal expansion and contraction kinetics. CD8+ T cells induced in the course of immunization exhibited phenotypic characteristics of effector memory T cells including up-regulation of the cell surface molecules CD43, CD44 and a low level of expression of CD127 at both local and systemic sites. While the discordance between the number of tetramer-reactive and cytokine-producing OVA-specific CD8+ T cells was observed, CD8+ T cells appeared to be fully functional in vivo. Upon secondary antigen exposure, the CD8+ T cell population expanded dramatically, particularly at the mucosa! surfaces. In addition, the CD8+ T cell response generated in the course of i.r. priming protected mice from intravaginal (i. vag.) vaccinia virus one month after immunization, thus underscoring the importance of inducing a tissue-resident effector memory T cell subset for protection against pathogens at mucosal surfaces. In developing future vaccines for mucosal diseases, the induction of a tissue-resident effector memory T cell subset should be one of the immunization objectives. Lentiviral vectors represent an attractive mode of genetic vaccination. Most commonly used, vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentiviral vectors do not efficiently infect epithelial cells from the apical side, and, therefore, are not suitable as mucosa! vaccines. In the present studies, Ebola Zaïre strain glycoprotein (EboZ)-pseudotyped lentiviral vectors, which have been previously used to deliver transgene to the lung epithelium, were delivered i.r. and evaluated as a mucosal booster vaccine. Rectal delivery of EboZ-pseudotyped lentiviral vectors expressing β-galactosidase (β-gal) had resulted in low, but detectable levels of β-gal expression 2 weeks after administration. When delivered on its own, EboZ-pseudotyped lentivirus did not prime detectable antigen-specific immune response. However, when delivered i.r. 30 days after i.r. Adβ-gal immunization, a significant enlargement (boost) of β-gal-specific CD8+ T cell responses, especially in the colonic lamina propria (LP), was observed as compared to the delivery of EboZ-pseudotyped vector encoding different transgenes or VSVG-pseudotyped lentivirus expressing β-gal. When these animals were i. vag. challenged with vaccinia virus expressing β-gal, a dramatic expansion of β-gal-specific CD8+ T cells, especially in the vaginal tract, was observed. In addition, this prime and boost strategy protected the mice from i. vag. vaccinia virus challenge. Therefore, i.r. Ad-based priming followed by i.r. EboZ-pseudotyped lentiviral boosting was an effective strategy for eliciting protective mucosal CD8+ T cell responses. / Thesis / Doctor of Philosophy (PhD)

T Zellen können in zwei Hauptpopulationen mit unterschiedlichen Aufgaben unterschieden werden. CD4+ T Zellen exprimieren im Zuge ihrer Aktivierung CD40L, welches ein zentraler kostimulatorischer Rezeptor zur Induktion von B-Zell basierter humoraler Immunität, APC Aktivierung und einer effizienten Effektor CD8+ T Zell Entwicklung ist („Helfer-Funktion“). Im Gegensatz dazu sind die zytotoxischen CD8+ T Zellen dazu vorbestimmt, infizierte oder maligne Zellen direkt abzutöten. Jedoch wurde eine Fraktion von CD8+ T Zellen identifiziert, die nach Aktivierung CD40L exprimiert. Bisher ist nicht verstanden, wie in solchen CD8+ T Zellen a) die CD40L Expression reguliert ist, b) wann und wie die Fähigkeit CD40L zu exprimieren implementiert wird und c) was die Folgen für das Immunsystem sind. In dieser Arbeit konnten wir zeigen, dass sowohl in CD4+ als auch in CD8+ T Zellen die CD40L Expression durch DNA-Methylierung regulatorischer Regionen des CD40LG Lokus reguliert wird. Die Demethylierung zentraler Elemente wird im Thymus implementiert, manifestiert sich mit der T-Zell Reifung und geht mit einer zunehmenden Stabilität der CD40L Expression einher. Erhöhte Expression von CD5 und NUR77 in CD40L+ CD8+ SP Thymozyten weisen auf eine positive Selektion mit hoher Affinität gegen Selbst-peptide während der Reifung im Thymus hin, welche das weitere Schicksal der CD40L exprimierenden CD8+ T Zellen beeinflusst. Naive CD40L+ CD8+ T Zellen besitzen ein anderes TCR Repertoire als CD40L- CD8+ T Zellen und reifen im Zuge ihrer Aktivierung bevorzugt zu Gedächtniszellen mit Zytokin- und Chemokinrezeptorprofilen von Tc2, Tc17 und Tc22 Zellen heran. Mit ihrem nicht-zytotoxischen Phänotyp und ihrer Genexpressionsignatur ähneln diese Zellen stark Helfer-CD4+ T Zellen und können von den klassisch zytotoxischen Tc1 und Tc17+1 Zellen durch ihre IL-6R und fehlende SLAMF7 Expression sowie der Expression von Markern die auf eine Fähigkeit in die Haut zu wandern schließen lassen, unterschieden werden. Zusammenfassend zeigen wir hier, dass naive CD8+ T Zellen von den frühsten Entwicklungsstadien im Thymus an nicht homogen sind und die Fähigkeiten über CD40L Expression eine Helferfunktion auszuüben beziehungsweise über die Sekretion zytolytischer Moleküle Zielzellen abzutöten unabhängig vom CD4+ or CD8+ T-Zell Status sind. Zellen mit Zytokin- und Genexpressionsignaturen, die mit denen der CD8+ Helfer-T Zellen übereinstimmen, wurden von uns und anderen in Geweben (Haut, Lunge) identifiziert und tragen zu den verschiedensten autoinflammatorischen Erkrankungen bei. Diese Arbeit insinuiert daher die Notwendigkeit einer grundlegenen Neubewertung der CD8+ T Zell Fähigkeiten und Funktionen in Immunantworten. / The T cell compartment consists of two major subsets with diverse assignments. CD4+ T cells express CD40L upon activation, a central co-stimulatory receptor to induce B cell mediated humoral immunity, activate APCs and prime efficient effector CD8+ T cell development (“helper function”). In contrast, cytotoxic CD8+ T cells are predetermined to kill infected or malignant cells directly. However, a fraction of CD8+ T cells expressing CD40L upon activation was identified. So far, it is not understood in CD8+ T cells a) how CD40L expression is regulated, b) when and how the ability of CD40L expression is implemented and c) what are the implications for the immune system. In this thesis, we found that CD40L expression is regulated by DNA-methylation of regulatory regions of the CD40LG locus in CD4+ as well as CD8+ T cells. The de-methylation of central elements is implemented in the thymus and increases with T cell maturation reflected by enhanced stability of CD40L expression. Elevated CD5 and NUR77 expression of CD40L+ CD8+ SP thymocytes suggests that high affine detection of self-peptides during positive selection in the thymus implements CD40L expression ability and predetermines the fate of the CD40L imprinted CD8+ T cells. CD40L+ naïve CD8+ T cells differ in their TCR repertoire from their CD40L- counterparts and preferentially mature into memory cell subsets with cytokine and chemokine receptor profiles of Tc2, Tc17 and Tc22 cells. With their non-cytotoxic phenotype and gene expression signatures, the CD40L+ memory CD8+ T cell subsets Tc2, Tc17 and Tc22 widely resemble helper CD4+ T cells and can be distinguished from classical cytotoxic Tc1 and Tc17+1 cells by their IL-6R and absent SLAMF7 expression and their skin migratory phenotype. Altogether, we demonstrate that from the earliest developmental stages in thymus onwards naive CD8+ T cells are not homogenous and the abilites to provide “CD40L based help” or “cytotoxicity mediated killing” are independent of the CD4+ or CD8+ T cell status. Cells with helper-type CD8+ T cell cytokine and gene-expression signatures were found at barrier sites (skin, lung) by us and others where they contribute to multiple autoinflammatory diseases. Therefore, this work insinuates the need to revisite CD8+ T cell capablities and function in immune responses.