A key step in the development of an adaptive immune response is the activation of naive T cells by dendritic cells (DCs). DCs sample antigens in the periphery and migrate to the lymphoid organs were they provide different signals to T cells: they present antigenic peptides in the context of MHC molecules, express costimulatory or coinhibitory ligands and produce cytokines that influence T cell fate. The integration of these signals will either induce tolerance or lead to the activation and expansion of effector T cells which will mediate the immune response.<p>The costimulatory CD70/CD27 pathway plays important roles in the development of pro-inflammatory Th1 and CTL responses. CD70 expression on DCs has also been described as a molecular switch from tolerance to immunity. Accordingly, its activity is tightly regulated in vivo. The aim of this work was to investigate the mechanisms controlling the expression of CD70 on dendritic cells and CD27 on T lymphocytes.<p>First, we described a cell-extrinsic mechanism of inhibition exerted on DCs by regulatory T cells (Tregs). Indeed, Tregs controlled Th1 priming in vivo and in vitro by downregulating CD70 on DCs. This control involved a transfer of the CD27 receptor to DCs, possibly via the production of CD27-bearing microvesicles by T cells at the immunological synapse. Acquisition of CD27 by DCs induced the internalization of both CD27 and CD70 and probably their lysosomal degradation. As a consequence, DCs were impaired in their ability to efficiently prime Th1 cells. Second, we analyzed CD70 and CD27 expression in the periphery and provided evidence for a cell-intrinsic control of CD27 expression by ectodomain shedding in the gastrointestinal tract.<p>While they efficiently clear infections, inflammatory responses can also be deleterious to the organism. By restraining CD70 expression on DCs, Tregs would promote tolerance and limit inflammation. Interestingly, tolerance is particularly important in the intestines, which are in constant contact with dietary antigens and the commensal microbiota. Accordingly, we propose that a second layer of control of CD27-driven costimulation takes place in the gut :by shedding CD27, T cells would be desensitized for any potential CD70-dependent costimulation.<p>To further investigate the physiological significance of the mechanisms described above, the immune response will be monitored in animals specifically lacking CD27 expression in the Treg population or expressing a nonsheddable CD27 receptor.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21451号 / 医博第4418号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 椛島 健治, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Une résidence à long terme de lymphocytes T (LTs) au sein de la plupart des tissus non lymphoïdes a été récemment décrite, notamment à la suite d’infections. Ces cellules confèreraient à l’hôte une meilleure protection en cas de réinfection. À l'aide de deux approches expérimentales différentes, l'injection d'anticorps bloquant l’entrée des LTs dans les ganglions lymphatiques (LNs) et la génération de parabioses par chirurgie, nous avons pu mettre en évidence, à l’état basal, la résidence d’une proportion significative des LTs αβ mémoires CD4+, des LTs αβ régulateurs CD4+ et d’une sous-population des LTs γδ dans les organes lymphoïdes secondaires. Les LTs CD4+ régulateurs et mémoires résidents ont en commun de nombreuses caractéristiques phénotypiques et fonctionnelles, et partagent avec leurs homologues issus de tissus non lymphoïdes une signature transcriptionnelle commune de résidence. Les LTs γδ résidents, quant à eux, arborent des caractéristiques phénotypiques et fonctionnelles proches de celles des cellules du système immunitaire inné. Si le microbiote semble jouer un rôle important dans la résidence des LTs αβ CD4+ des plaques de Peyer (PPs), son rôle ne semble pas être prépondérant dans la résidence de ces cellules au sein des LNs. Comme dans de nombreux tissus non lymphoïdes, la sous-expression de S1PR1 pourrait en partie expliquer la résidence des LTs αβ CD4+. Par contre, les LTs γδ seraient, eux, retenus dans les tissus lymphoïdes de par des interactions étroites avec les macrophages. Enfin, la résidence des LTs αβ augmente avec l'âge au point que la majorité des LTs CD4+ régulateurs et mémoires des LNs et des PPs sont en fait résidents chez des souris âgées. Nos résultats montrent que la résidence des cellules T n'est pas seulement une caractéristique des tissus non lymphoïdes mais qu’elle peut être étendue aux organes lymphoïdes secondaires. Le rôle respectif de ces différentes populations de LTs devra être exploré. / In the last decade, numerous data have demonstrated the existence of T cells residing in non-lymphoid tissues, mostly after infectious diseases. These resident memory T cells may represent a first line of defense against pathogens at front-line sites of microbial exposure upon reinfection. Using two different experimental approaches such as the injection of integrin-neutralizing antibodies that inhibits the entry of circulating lymphocytes into lymph nodes and long-term parabiosis experiments, we have highlighted the long-term residence of a substantial proportion of regulatory and memory CD4 αβ T cells and γδ T cells within the secondary lymphoid organs of specific pathogen free mice. Resident γδ T cells display innate-like characteristics. Lymph node-resident regulatory and memory CD4 αβ T cells share many phenotypic and functional characteristics, including a core transcriptional profile, with their cell-counterparts from non-lymphoid tissues. Microbiota plays an important role in αβ T-cell residence in Peyer’s patches but only a small one if any in lymph nodes. Like in many non-lymphoid tissues, S1PR1 down-regulation may account forαβ T-cell residency within secondary lymphoid organs although other mechanisms may account for this especially in the case of lymph node memory CD4 T cells. Specific in vivo cell-depletion strategies have allowed us to demonstrate that macrophages are the main actors involved in the long-term retention of γδ T cells in secondary lymphoid organs. Strikingly, T-cell residence increases with age to the point that the majority of regulatory and memory CD4 αβ T cells from LNs and Peyer’s patches are in fact resident T cells in old mice. Altogether, our results show that T-cell residence is not only a hallmark of non-lymphoid tissues but can be extended to secondary lymphoid organs.

Au cours d’une réponse immunitaire primaire, les lymphocytes T CD8 mémoires émergent à partir d'un environnement de forte activation immunitaire. Les cellules régulatrices T CD4 FoxP3+ (LTregs) jouent un rôle clé de suppression de la réponse immunitaire. Nous montrons que les LTregs sont nécessaires pour la génération d’une réponse mémoire T CD8 fonctionnelle. En absence de LTregs lors du priming, les LT CD8 mémoires générées prolifèrent faiblement et ne parviennent pas à se différencier après une réactivation antigénique en effecteurs cytotoxiques secondaires fonctionnelles. Nous suggérons que les LTregs agissent tôt, lors de la phase d'expansion des LT CD8, en réduisant l’exposition des précurseurs mémoires T CD8 à l'interleukine-2. Ce nouveau rôle crucial des LTregs a des implications pour le développement optimal de vaccin.Chez les patients sous traitement antirétroviral efficace et prolongée (ART), le VIH peut persister dans un petit pool de cellules T CD4 mémoires quiescentes de longue durée de vie infectées par du virus latent intégré. Ce réservoir latent comprend différentes sous-populations mémoires. Nos résultats suggèrent une contraction progressive de la taille du réservoir latent autour d'un noyau formé de sous-populations T CD4 mémoires moins différenciées (centrales mémoires TCM et souches mémoires TSCM). Ce processus très lent semble dépendre de la taille initiale et du taux de décroissance qui diffère entre les sous-populations mémoires infectées de manière latente. Nos résultats suggèrent également une extrême stabilité du sous-réservoir TSCM, dont la taille est directement liée à l'exposition cumulée au virus plasmatique avant le début du traitement ART, soulignant l'importance d'une initiation précoce du traitement antirétroviral efficace. La présence de cette dynamique intrinsèque dans le réservoir latent peut avoir des implications pour la conception de stratégies optimales de purge thérapeutique contre le VIH. / During the primary immune response, CD8 memory emerges from an environment of strong immune activation. The FoxP3 regulatory CD4 T-cell subset (Treg) is known as a key suppressive component of the immune system. We report that Tregs are required for the generation of functional CD8 memory. In the absence of Tregs during priming, the resulting memory cells proliferate poorly and fail to differentiate into functional cytotoxic secondary effectors following antigen reactivation. We find that the Tregs act early, during the expansion phase of primary CD8 effectors, by fine tuning interleukin-2 exposure of CD8 memory precursors. This crucial new role of Tregs has implications for optimal vaccine development. In patients who are receiving prolonged antiretroviral treatment (ART), HIV can persist within a small pool of long-lived resting memory CD4 T cells infected with integrated latent virus. This latent reservoir involves distinct memory subsets. We provide results that suggest a progressive reduction of the size of the blood latent reservoir around a core of less-differentiated memory subsets (central memory and stem cell-like memory).This process appears to be driven by the differences in initial sizes and decay rates between latently infected memory subsets. Our results also suggest an extreme stability of the TSCM sub-reservoir, the size of which is directly related to cumulative plasma virus exposure before the onset of ART, stressing the importance of early initiation of effective ART. The presence of these intrinsic dynamics within the latent reservoir may have implications for the design of optimal HIV therapeutic purging strategies.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Les cytokines jouent un rôle essentiel dans la réponse immunitaire ; elles modulent les propriétés et la survie de nombreuses cellules immunitaires. Les lymphocytes T (LT) sont régulés par un large spectre de cytokines. Néanmoins certaines sous-populations de LT restent peu définies à ce jour tout comme l’impact de cytokines sur ces cellules en conditions physiologiques et pathologiques. Une population de LT caractérisée par la double expression des corécepteurs CD4 et CD8 a été identifiée en périphérie chez des donneurs sains et sa fréquence est augmentée chez les patients atteints de certaines pathologies. Cependant, peu d’études ont déterminé le phénotype et les fonctions de ces cellules. Nous avons caractérisé le phénotype ex vivo des LT CD4+CD8+ issus de sujets sains et étudié la réponse de ces cellules à plusieurs cytokines importantes dans l’homéostasie et l’activation des lymphocytes. Notre étude révèle que les LT CD4+CD8+ forment une population hétérogène présentant de nombreuses caractéristiques des cellules mémoires. De plus, notre étude montre que ces cellules ont une capacité accrue de produire des cytokines et enzymes lytiques comparée aux LT CD4 et CD8. Finalement, nous avons observé qu’une plus grande proportion de LT CD4+CD8+ répond aux cytokines IL-2, IL-15 et IL-7 comparée aux LT CD4 et CD8 simples positifs. Plusieurs études ont révélé que certaines cytokines participent à la pathobiologie de maladies auto-immunes notamment la sclérose en plaques (SEP). La SEP est une maladie inflammatoire chronique du système nerveux central (SNC) caractérisée par une destruction de la gaine de myéline et une perte axonale à l’origine des symptômes cliniques. L’impact des médiateurs immunitaires dans la destruction et/ou la réparation reste néanmoins à éclaircir. Plusieurs études suggèrent que l’interleukine-27 (IL-27) joue un rôle central dans la SEP : l’IL-27 diminue la sévérité de la maladie dans un modèle murin de la SEP ; la réponse des patients à certaines thérapies corrèle avec des niveaux périphériques élevés d’IL-27. Notre étude avait pour but de déterminer comment l’IL-27 module les LT des patients atteints de SEP. Nous avons observé des niveaux d’IL-27 augmentés à la fois en périphérie et dans le SNC des patients SEP. Nous avons mis en évidence que les effets induits par l’IL-27 sont altérés dans les LT des patients SEP ; notamment l’induction de PD-L1 et la réponse à l’IL-12 sont plus faibles comparées aux LT de sujets sains. La voie de signalisation STAT3 est induite plus fortement en réponse à l’IL 27 dans les cellules de patients SEP en comparaison à celles de sujets sains. Finalement, nous avons démontré que la forme soluble du récepteur à l’IL-27 est présente en plus grande quantité dans le sérum des patients SEP et ces niveaux sont suffisants pour bloquer l’effet de l’IL-27 sur ses cellules cibles. En conclusion, nos résultats permettent de mieux comprendre comment certaines cytokines participent à la régulation des populations lymphocytaires. En condition pathologique, la modulation de ces cytokines pourrait s’avérer efficace dans un but thérapeutique afin de promouvoir et/ou inhiber certaines réponses immunitaires notamment dans la SEP. / Cytokines play an essential role in modulating the immune response. They act on cells from both innate and adaptive immunity by modulating their phenotype and influencing their survival. T cells are highly regulated by a broad spectrum of cytokines. Nevertheless, some subsets of T cells remain ill-defined and a better understanding of cytokine-mediated responses in physiological and pathological conditions requires further investigation. A T cell population characterized by the dual expression of CD4 and CD8 co-receptors has been identified in the periphery of healthy donors and its frequency is notably increased in several pathologies. However, very few studies have determined the phenotype and functions of these cells under physiological conditions. We performed an ex vivo phenotypic characterization of CD4+CD8+ T cells from healthy subjects and analyzed the response of these cells to several important cytokines implicated in homeostasis and activation of immune cells. Our study reveals that CD4+CD8+ T cells are heterogeneous and exhibit many characteristics of memory cells. In addition, our study shows that these cells have an enhanced ability to produce cytokines and lytic enzymes compared to CD4 and CD8 T cells. Finally, we found that a higher proportion of CD4+CD8+ T cells respond to IL-2, IL-15 and IL-7 cytokines compared to CD4 and CD8 counterparts. Numerous studies showed that lymphocyte populations and some cytokines are involved in the development of several autoimmune diseases, including multiple sclerosis (MS). MS is a chronic inflammatory disease of the central nervous system (CNS) characterized by the destruction of the myelin sheath, axonal loss and oligodendrocytic death leading to clinical symptoms. The immune mediators involved in the destruction and/or repair remain to be clarified. Several studies suggest that interleukin-27 (IL-27) plays a central role in MS: IL-27 dampens the severity of the disease in a MS mouse model and in MS patients the response to some therapies correlates with higher levels of peripheral IL-27. The purpose of our study was to determine how IL-27 modulates T cells in MS patients. We observed that IL-27 levels are increased in both periphery and CNS of MS patients. We found that IL-27-mediated effects are impaired in T cells from MS patients in particular PD-L1 induction and IL-12 response that are lower compared to T cells from healthy subjects. IL-27-triggered STAT3 signaling pathway is also enhanced in cells from MS patients compared to healthy subjects. Finally, our study showed that the soluble form of the receptor of IL-27 is present in greater levels in the serum of MS patients and the measured doses are sufficient to block the capacity of IL-27 to act on its target cells. To conclude, our results provide a better understanding of how certain cytokines participate in the regulation of lymphocyte populations. Under pathological conditions, modulation of these cytokines may be effective for therapeutic applications in order to promote and/or inhibit immune responses, particularly in MS.

Paracoccidioides brasiliensis é um fungo dimórfico patogênico agente etiológico da paracoccidioidomicose (PCM), uma micose endêmica no Brasil. A busca por alternativas para reduzir o tempo de tratamento da PCM levou ao desenvolvimento de uma vacina de DNA contendo a sequência do peptídeo P10 de P. brasiliensis. Neste trabalho, avaliamos a geração de células T de memória e células T reguladoras em camundongos imunizados com esta vacina de DNA antes e após o desafio com o fungo, através da análise de seus esplenócitos e linfócitos pulmonares por citometria de fluxo. Os resultados mostram um aumento no percentual de células T reguladoras e de memória no baço e pulmões dos animais imunizados antes e depois de 30, 60 e 120 dias do desafio em comparação com os grupos controle e não imunizado. Outro experimento revelou que o modelo experimental da PCM in vivo é capaz de induzir a expressão de ROR&#947t. Este estudo mostra que nossa vacina de DNA contra a PCM gera células com fenótipo de reguladoras e de memória, caracterizando seu potencial para o tratamento desta micose. / Paracoccidioides brasiliensis is a dimorphic fungal pathogen that is the etiological agent of paracoccidioidomycosis (PCM), a mycosis endemic in Brazil. The search for new alternatives to reduce the duration of PCM treatment led to the development of a DNA vaccine encoding the peptide P10 of P. brasiliensis. Presently, we evaluated the generation of memory and regulatory T cells in mice immunized with this DNA vaccine, before and after the challenge with the fungus by analizing their splenocytes and pulmonary lymphocytes by flow cytometry. The results show an increase in the percentage of regulatory and memory T cells on spleens and lungs of immunized mice before and after 30, 60 and 120 days of challenge compared with the control and untreated groups. Another experiment revealed that the PCM in vivo infection model is capable of inducing ROR&#947t expression. This study demonstrates that our DNA vaccine against PCM generates cells with a regulatory and memory phenotype, which shows its potencial in the treatment of this mycosis.

Abstract not available

Optimal CD4+ T cell activation is key to the generation of a productive immune response. Naïve circulating CD4+ T cells are quiescent under normal conditions and undergo activation only upon encounter of the T cell receptor (TCR) with Major Histocompatibility Complex (MHC)-encoded class II molecules on antigen presenting cells (APCs). Processed antigens (derived from pathogens, tumors or self tissue during autoimmunity) in complex with MHC class II are recognized by specific TCRs on CD4+ T cells. After this encounter, the highly complex and regulated process of CD4+ T cell activation results in the differentiation of naïve T cells into effectors and their clonal expansion. Apart from binding to its cognate peptide-MHC-II complex, several other factors define the extent and magnitude of T cell activation. This context is an important determinant of the nature of the subsequent T cell response. One of the factors involved is the strength of the signal (SOS) which is delivered to the cell upon ligation of the TCR to the MHC-peptide complex. The SOS, which can vary from weak to strong, is determined by the affinity/avidity of the TCR for the MHC-peptide complex, antigen concentrations, the duration of engagement, etc. Extreme weak or strong signals can lead to non-productive T cell responses with the former resulting mostly in anergy and the latter in cell death. Signals of optimal strength are the ones that translate into functional T cell responses. However, mechanisms by which signal strength information is translated into distinct T cell responses are still not very well understood. Binding of the TCR to the MHC-peptide complex triggers several signaling cascades and leads to generation of intracellular signaling intermediates, including Ca2+. Rise in intracellular Ca2+ levels is one of the first events to occur upon initiation of T cell activation. The initial increase is brought about due to release of Ca2+ from intracellular smooth endoplasmic reticulum stores. Once intracellular stores have been emptied, the increase is sustained by a process termed as capacitative Ca2+ entry, involving opening of Ca2+ channels in the plasma membrane known as Ca2+ release activated channels (CRACs). Consequently, Ca2+ flows from the extracellular milieu into the cell. A sustained Ca2+ increase is essential for activation of the transcription factor, NF-AT whose primary job is to initiate transcription of IL-2, a cytokine crucial for CD4+ T cell proliferation. The other intracellular signaling intermediates which are the focus of work presented in this study are reactive oxygen species (ROS). TCR signaling leads to generation of ROS, which may be either mitogenic or detrimental to T cell activation. Low levels of ROS, especially H2O2, inactivate phosphatases leading to activation of kinases and signaling pathways resulting in increased proliferation. However, high levels of ROS cause oxidative stress leading to reduced T cell activation, hyporesponsiveness and death. The experimental system used for this study consists of purified mouse lymph node CD4+ T cells. These cells were activated with varying strengths of the primary signal to better understand the roles of Ca2+ and ROS in modulating T cell activation and function. The signal strength was either varied by addition of different concentrations of ionomycin or thapsigargin, pharmacological agents that increase intracellular Ca2+ concentrations. Alternatively, signal through the surface TCR-CD3 complex was initiated using anti-CD3 in two modes: soluble (weak signal) or plate immobilized (strong signal). Increasing concentrations of ionomycin or thapsigargin or changing the mode of anti-CD3 from soluble to plate bound enhances IL-2 amounts, thereby converting a weak signal to a strong one. The work presented has been divided into three parts, each dealing with a distinct aspect of T cell activation. I. SOS and CTLA4-CD80/CD86 interactions: The binding of the TCR to its cognate MHC-peptide complex delivers the primary signal. However, this alone is not sufficient to drive T cell activation and an additional costimulatory signal emanating from the binding of CD28, a constitutively expressed receptor on T cells, to its ligands CD80 and CD86 is required. Another receptor that binds to CD80 and CD86 is CTLA-4 although it does so with a ~100 fold higher affinity. CTLA-4, unlike CD28, is expressed upon T cell activation and is considered to downregulate T cell activation. Its role as a negative regulator is highlighted by the phenotype of Ctla4 -/-mice which die of massive lymphoproliferation. However, there have also been reports of some plasticity in the effects mediated by CTLA-4. Previous work from our laboratory showed that CTLA-4-CD80/CD86 interactions could either inhibit or stimulate T cell activation depending on the SOS. To identify the molecular mediators of the differential effects of CTLA-4, the role of Ca2+ and ROS was evaluated. During activation with phorbol myristate acetate (PMA) and low amounts of ionomycin, intracellular amounts of Ca2+ were greatly reduced upon blockade of CTLA-4-CD80/CD86 interactions. Further experiments demonstrated that CTLA4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of ionomycin or thapsigargin (strong SOS) but the opposite occurred with PMA and low amounts of ionomycin or thapsigargin (weak SOS). These results were confirmed by activating cells with anti-CD3 either in the soluble or plate bound form. Considerably higher amounts of intracellular Ca2+ were present in cells activated with plate bound anti-CD3 compared to those activated with soluble anti-CD3. These amounts, further augmented by CTLA-4-CD80/CD86 interactions, probably became toxic to cells as increased proliferation was observed, using reagents that blocked these interactions. The opposite, however, was seen in cells activated with soluble anti-CD3. Also, CTLA4-CD80/CD86 interactions enhanced the generation of ROS. Studies with catalase revealed that H2O2 is required for IL-2 production and cell cycle progression during activation with a weak SOS. However, the high amounts of ROS produced during activation with a strong SOS reduced cell cycle progression. Together, this study identifies intracellular Ca2+ and ROS to play important roles in the modulation of T cell responses by CTLA4-CD80/CD86 interactions. II. SOS and CD4 downregulation: This study was initiated to identify early T cell functional responses that would help predict the strength of the primary signal. Using the in vitro culture system of varying signal strengths, it was found that CD4 surface expression was controlled by signal strength. CD4 is a surface glycoprotein expressed on the TH subset along with the TCR. It performs two main functions: First, binding to MHC class II and strengthening the TCR-MHC interaction, i.e. functioning as a coreceptor. Second, due to its association with p56lck a src family tyrosine kinase, the presence of CD4 along with the TCR enhances signal transduction. Also, CD4 acts as a receptor for entry for the AIDS virus. It is known that CD4 is downregulated from the surface and degraded upon T cell activation by a protein kinase-C dependent process in human and mouse T cells. Experiments presented in this study showed increased CD4 downregulation with a strong signal. The roles of intracellular mediators were assessed and high intracellular Ca2+ amounts, but not PMA activation, was required for sustained CD4 downregulation. Also, increased H2O2 amounts in cells activated with a strong SOS inhibited CD4 downregulation. Most interestingly, the pattern of CD4 downregulation was different between peripheral T cells and thymocytes, suggesting a correlation with CD4+ T cell development. III. Modulation of CD4+ T cell activation by small molecule plant growth regulators: An important area of investigation in T cell biology is the identification of molecules that modulate T cell activation. Towards this end, the mechanisms by which small molecule plant growth regulators, naphthalene acetic acid (NAA), 2,4 dichlorophenoxyacetic acid (2,4D) and indole acetic acid (IAA), influence CD4+ T cell activation was studied. It is useful to recall that IAA is the natural auxin present in plants, NAA is a synthetic auxin and 2,4D is a herbicide. These compounds, but not structurally similar control molecules, increased the activation and IL-2 production in CD4+ T cells activated with either soluble anti-CD3 or a combination of PMA and ionomycin. An investigation into the mechanisms of action by these compounds revealed increased early generation of intracellular ROS and Ca2+. Interestingly, the nature of their effects was found to rely on the strength of the primary signal: IL-2 and proliferation were increased in cells activated with a weak signal, but lowered proliferation was observed in cells activated with a strong signal. Cells activated with strong signal posses high amounts of ROS and Ca2+ and further increase in their amounts by IAA, NAA and 2,4D resulted in growth suppression. However, augmentation of Ca2+ and ROS amounts in cells activated with a weak signal was mitogenic. The role of these compounds during in vivo T cell responses needs to be addressed. Taken together, results presented in this study emphasize the importance of the role of SOS in determining T cell responses. In addition, the roles of Ca2+ and ROS downstream of the primary signal in modulating CD4+ T cell activation were demonstrated.