Les cellules T régulatrices (Treg) jouent un rôle important dans différentes infections chroniques comme le VIH. Lors de l’infection chronique par le VIH, une augmentation du nombre de ces cellules limite la réponse des cellules T effectrices spécifiques du virus mais permet également le contrôle de la très forte activation du système immunitaire. La régulation de l’activité suppressive des Treg constitue une voie importante pour le développement d’un vaccin, l’efficacité de la surveillance tumorale et l’auto-immunité. Dans ce travail, nous avons étudié différents mécanismes de régulation des Treg chez les patients infectés par le VIH et sujets sains. Le but de mon travail a été d’étudier les mécanismes impliqués dans les fonctions suppressives des Treg et de leur régulation. Nous nous sommes particulièrement intéressés au rôle de Notch sur la sensibilité des cellules T effectrices à la fonction suppressive des Treg, en montrant que l’activation de Notch rendait les cellules T effectrices plus sensibles à l’action suppressive des Treg, et ceci même en présence d’un très faible nombre de Treg. Nous avons démontré que certains ligands de Notch, tels que DL-4 et Jagged-1 mais non DL-1, régulent l’effet inhibiteur des Treg sur les cellules T effectrices par une surexpression de TGFβ-RII et à la phosphorylation de Smad3. Le rôle important de l’enzyme CD39 dans la fonction suppressive des Treg a été décrit, mais peu d’études ont démontré son rôle dans l’infection par le VIH. Nous avons montré que les Treg de patients infectés par le VIH experiment plus fortement le CD39, et que leur effet suppresseur était inhibé en utilisant un anticorps monoclonal anti-CD39 et maintenu en utilisant un agoniste de l’adénosine. Nous avons également montré que le polymorphisme du gène CD39, associé à une plus faible expression de CD39, était corrélé à une plus lente progression de la maladie. Nos résultats montrent non seulement les mécanismes impliqués dans l’activité suppressive des Treg lors de l’infection par le VIH, mais constituent une approche intéressante pour en modifier les fonctions. Enfin, nous avons recherché l’effet de l’IL-7 sur le phénotype des Treg et l’expression de molécules impliquées dans leur fonction suppressive. Nos résultats montrent deux effets synergique de l’IL-7 sur les Treg mémoires: diminution de l’expression de CD39 à leur surface induisant une diminution de leur fonction suppressive, et surexpression du récepteur à l’ATP induisant un phénotype Th17. L’administration d’IL-7 in vivo chez des patients infectés par le VIH a confirmé la modification de phénotype des Treg en Th17 et notamment une surexpression du facteur de transcription spécifique des Th17, RORγt. En conclusion, notre travail apporte de nouvelles connaissances sur les mécanismes impliqués dans les fonctions inhibitrices des Treg et comment moduler ces fonctions. Ceci pourrait avoir un impact clinique direct, soit dans le traitement de maladies associées à un dysfonctionnement des Treg (infections chroniques virales, sclérose multiple, diabète de type 1, arthrite rhumatoide et lupus érythémateux), soit dans les stratégies vaccinales. / T regulatory cells (Treg cells) play an important role during various chronic infections like HIV. Increase in Treg cell number during chronic phase limit the HIV specific T effector cell response but may control exaggerated activation of the immune system. Modulation of regulatory T cell (Treg cells) suppression has important implications for vaccine development, the effectiveness of tumor surveillance, and the emergence of autoimmunity. Here we studied various mechanisms of Treg cells modulation in HIV+ patients and healthy subjects. The aim of my work was to decipher some aspects of the mechanisms involved in Treg cell mediated suppressive effects and the modulation of Treg cell suppressive effects in healthy subjects and HIV- infected patients.We have extended knowledge on the role of Notch in Treg/Effectors T cells cross talk. Here we focused our interest on the role of Notch pathway on the sensitivity of effectors T cells to Treg cell-mediated suppression, showing that Notch activation may significantly increase the sensitivity of effector cells to Treg cells even at a low ratio. We demonstrated that Notch ligands DL-4 and Jagged-1, but not DL-1 modulate significantly the suppressive effect of Treg cells on effectors T cells through an up regulation of TGF-RII expression and the phosphorylated form of Smad3 protein.A critical role of CD39 has been described for Treg cells in general but only a few studies have analyzed its role in HIV infection. We showed an increase in CD39 expression on Treg cells in a cohort of HIV- infected patients. Treg cells inhibitory effects were relieved by CD39 down modulation using an antiCD39 monoclonal antibody and this suppressive effect was reproduced on effector CD8+ T cells by an adenosine agonist. We found that CD39 gene polymorphism associated with a lower CD39 expression correlated with slower progression to AIDS. Our results shows not only the mechanism by which Treg cell suppression occurs during HIV infection but also provide an attractive approach to modify Treg cell functions.Finally, we have investigated the role of IL-7 on the phenotype of Treg cells and expression of molecules involved in the suppressive functions of these cells. Our results show that IL-7 exerts two synergistic effects on memory Treg cells. It decreases their suppressive effect by decreasing CD39 expression and increases ATP receptor leading to a switch towards a Th17 phenotype. In vivo administration of IL-7 tipped the balance towards a higher expression of RORγT in PBMCs from HIV infected patients.In conclusion our study bring new findings in the mechanisms involved in Treg cell mediated suppression and the way to modulate these cells which could have direct clinical impact either in the treatment of diseases associated with Treg cell dysregulation (chronic viral infections, autoimmune disorders like multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and systemic lupus erthematosus) or during vaccination.

Les maladies glomérulaires sont une des principales causes d'insuffisance rénale terminale de nos jours et constituent un problème de santé publique. Le concept classique dans les glomérulopathies accorde à l'agression systémique immune, toxique ou hypertensive le rôle principal dans la formation des lésions glomérulaires. L'hypothèse développée dans ce manuscrit est que épithélium et endothélium, deux composants principaux du parenchyme rénal sont des acteurs majeurs dans la formation des lésions glomérulaires. Trois modèles expérimentaux de glomérulopathie (inflammatoire, toxique et hypertensive) chez la souris nous ont permis d'étudier une voie de signalisation épithéliale γC/JAK/STAT classiquement décrite dans les cellules immunitaires et le système de réponse à l'hypoxie endothéliale afin d'étayer cette hypothèse. Après avoir discuté le rôle principal classiquement attribué aux lymphocytes T dans le modèle anti-membrane basale glomérulaire, un modèle animal de glomérulonéphrite inflammatoire, nous avons démontré le rôle protecteur de la chaîne γ commune (γC) glomérulaire et de sa protéine d'aval STAT5 dans le podocyte au cours du modèle anti-MBG et de la néphropathie à l'adriamycine. Enfin, nous avons étudié le rôle protecteur de EPAS1 (HIF-2α), une sous-unité régulatrice du complexe HIF, dans l'endothélium au cours des lésions glomérulaires hypertensives. Au total, ce travail met en évidence le rôle majeur de l'épithélium et l'endothélium rénal, étroitement liés, dans la formation des lésions glomérulaires. Le parenchyme rénal représente un acteur à part entière dans la physiopathologie de ces lésions comme le montrent les travaux sur les systèmes γC/STAT5 et HIF. / Glomerular diseases are a leading cause of kidney failure and represent a public health problem. Classically, systemic effectors such as the immune system, drug toxicity or hypertension are thought to be the main drivers of glomerular diseases. The hypothesis developed in this manuscript is that epithelium and endothelium, the two main components of the renal parenchyma, are major players in the formation of glomerular lesions. Three experimental models of glomerular disease (inflammatory, toxic and hypertensive) in mice allowed us to study epithelial γC / JAK / STAT signaling classically described in immune cells and the endothelial hypoxia inducible system in order to support this hypothesis. After discussing the main role traditionally assigned to T cells in the anti- glomerular basement membrane model, an animal model of inflammatory glomerulonephritis, we demonstrated the protective role of the glomerular interleukin common γ chain (γC) receptor and its dependent podocyte-specific STAT5 during the anti-GBM model and adriamycin nephropathy. We then showed the protective role of endothelial EPAS1 (HIF-2α), a regulatory subunit of HIF complex in focal segmental glomerulosclerosis (FSGS) induced by angiotensin II. In total, this work highlights the important role of the closely linked renal epithelium and endothelium in the formation of glomerular lesions using three experimental models of glomerular diseases. The renal parenchyma is a full player in the pathophysiology of these lesions as shown by the works studying γC / JAK / STAT and HIF systems.

L’échappement des tumeurs à la surveillance du système immunitaire est une des raisons pour lesquelles le cancer parvient à se développer. Un des objectifs de notre équipe consiste à étudier les lymphocytes T CD4+ et leurs rôles dans un contexte de cancer. Mon travail de thèse a eu pour objectif de déterminer si les résultats murins obtenus au laboratoire étaient transposables à l’Homme, et surtout dans le cancer du sein. Nous avons tout d’abord mis en évidence que les lymphocytes Th17 infiltrant les cancers du sein inhibent les fonctions effectrices des lymphocytes T cytotoxiques de manière dépendante des ectonucléotidases. Enfin, nous avons montré qu’une infiltration tumorale riche en cellules Th17 est associée à un moins bon pronostic clinique des patientes atteintes de cancer du sein. Nous avons ensuite eu pour objectif de tenter de limiter la différenciation de ces cellules Th17. Nous avons démontré que l’activation de SIRT1 diminue l’acétylation de STAT3 ce qui perturbe le programme de différenciation de ces cellules. L’activation de SIRT1 in vivo limite l’expansion des Th17 et conduit à un ralentissement de la croissance tumorale. Ce concept fut validé chez l’Homme et ouvre donc la possibilité d’association d’agonistes de SIRT1 avec la chimiothérapie. Le dernier projet porte sur les lymphocytes Th9 et leur rôle pronostique dans le cancer du sein. Nous avons mis en évidence que les propriétés effectrices des lymphocytes Th9 humains pouvaient être augmentées par l’IFNα via l’activation du facteur de transcription IRF1. Nous avons enfin démontré que l’infiltration tumorale de lymphocytes Th9 était associée au meilleur pronostic des patientes. / Tumor escape to immune system surveillance is one of the reasons why human cancer achieves to grow. In my research team, we aim to study CD4 T cell populations and their functions in the context of cancer. My work was precisely to determine if the results obtained in mice could be transposable in humans, in the context of breast cancer. . We first unraveled that tumor infiltrating Th17 cells could inhibit effector and cytotoxic functions of Th1 and CD8 T cells in an ectonucleotidase-dependent manner. Finally, we showed that high tumor infiltration in IL-17+ cells were significantly associated with a worse clinical prognosis for breast cancer patients. Then, we aim to prevent Th17 cell differentiation. We first established that SIRT1 activation reduces STAT3 acetylation thus limiting Th17 cell differentiation. Activation of SIRT1 limits in vivo Th17 cell expansion and leads to the decrease of tumor growth. This concept has been validated in humans and gives the possibility to associate SIRT1 agonists with chemotherapy. The last project concerns the role and the prognosis impact of Th9 lymphocytes in breast cancer. We highlighted that the effector properties of human Th9 cells could be increased by IFNα via IRF1 activation. Finally, we attested that the tumor infiltration of Th9 lymphocytes was associated with a better prognosis for breast cancer patients.

Les événements biochimiques de l'activation T ont été décrits depuis longtemps et sont bien connus. A l'échelle moléculaire la liaison du Récepteur des Cellules T (TCR) présent à la surface du lymphocyte T avec un peptide (du soi ou non soi), ce dernier étant chargé sur le Complexe Majeur d'Histocompatibilité (MHC), présent à la surface des Cellules Présentatrices d'Antigène (CPA) conduit à l'initiation de la réponse immunitaire. La réponse des lymphocytes T est extrêmement spécifique, sensible, robuste et semble présenter des caractéristiques de mécano-transduction. En effet, il a été démontré récemment que le TCR agirait comme un mécanosenseur alors que le lymphocyte T peut sentir la mécanique de son environnement à une échelle cellulaire. Cependant, la majorité de ces études ont été réalisées en opposant un lymphocyte T à un substrat inerte limitant la compréhension sur la contribution éventuelle de chaque partenaire cellulaire car la présence de l'APC peut induire des changements dans l'organisation du lymphocyte T.Le but de cette thèse a été de mettre en place un suivi de l’activation des lymphocytes T pat une APC modèle, sous force grâce à l’utilisation de la microscopie à force atomique. Il a mené à plusieurs développements méthodologiques originaux validés de manière expérimentale avec un système cellulaire modèle (hybridomes murins vs. Cellules COS modifiées pour être des APC pouvant être modifiées à souhait grâce à l’expression d’une grande variété de molécules impliquées dans la réponse immunitaire). / The biochemical events of T activation have been described for a long time and are well known. At the molecular level, the binding of the T-cell receptor (TCR) present on the surface of the T-cell with a peptide (self or non-self) loaded on the Major Histocompatibility Complex (MHC), present on the surface of the Antigen Presenting Cells (APC), leads to the initiation of the immune response. In addition, the T cell response is extremely specific, sensitive, robust and appears to have mechano-transduction characteristics. Indeed, it has recently been demonstrated that the TCR would act as a mechanosensor while the T lymphocyte can feel the mechanics of its environment on a cellular scale. However, the majority of these studies were performed by opposing/facing a T cell to an inert substrate (e.g., bead, functionnlized glass slides molecularly decorated or not), limiting the understanding of the possible contribution of each cell partner because the presence of APC can induce changes in the organization of the T cell. The aim of this thesis was to set up a follow-up of the activation, under force, between a model APC and a T lymphocyte. It has led to several original methodological developments experimentally validated with a model cell system (mouse hybridomas vs. COS cells modified to be complexifiable APCs).

A persistência de células T de memória funcionais é importante para garantir uma imunidade protetora na infecção pelo Vírus da Imunodeficiência Humana (HIV). As células T de memória têm sido subdivididas em memória central (TCM), memória efetora (TEM) e memória efetora altamente diferenciada (TEMRA) com base na expressão de moléculas de superfície como CCR7 e CD45RA, e na capacidade de produzir citocinas e proliferar. Recentemente, identificamos 18 peptídeos derivados de seqüências do consenso B do HIV-1, ligadores de múltiplas moléculas HLA-DR e amplamente reconhecidos por linfócitos T de sangue periférico de pacientes infectados pelo HIV. Diante disso e considerando a importância das células T de memória na manutenção da resposta imune específica, nosso objetivo foi caracterizar fenotípica e funcionalmente as subpopulações de células T de memória de indivíduos infectados pelo HIV envolvidas no reconhecimento in vitro desses epitopos. Foram incluídos 14 indivíduos controles sadios e 61 pacientes HIV+ com contagem de linfócitos T CD4+ maior que 250 células/mm3. Os pacientes HIV+ foram divididos em seis diferentes grupos clínicos de acordo com o estágio da infecção, carga viral (CV) plasmática e uso de terapia anti-retroviral (ART): não progressores por longo tempo (LTNP), avirêmicos em uso de ART (AV-ART), virêmicos em uso de ART (VI-ART), virêmicos sem uso de ART (VI sem ART), virêmicos recéminfectados sem uso de ART (VI-RI) e controladores. Células mononucleares do sangue periférico dos indivíduos do estudo foram estimuladas com o conjunto de peptídeos do HIV-1 e com um conjunto de peptídeos do Citomegalovírus (CMV). A freqüência de células de memória produtoras de IFN- e IL-2 e a proliferação celular antígeno-específica foram detectadas por citometria de fluxo de multiparâmetros. Nossos resultados mostraram que o conjunto de peptídeos do HIV-1 foi capaz de ativar subpopulações funcionais de memória TCM, TEM e TEMRA secretoras de IFN- e IL-2 em 100% dos pacientes HIV+ dos diferentes grupos clínicos. O conjunto de peptídeos do HIV-1 também induziu proliferação das subpopulações de linfócitos T de memória. As freqüências de TEMRA CD4+IFN-+, TEMRA CD4+IFN-+ total, TCM CD8+IFN-+, TCM CD8+IFN-+ total, TEM CD8+IFN-+, TEM CD8+IFN-+ total e TEMRA CD8+IFN-+ correlacionaram-se negativamente com a carga viral do HIV em pacientes virêmicos. Esses dados sugerem que essas subpopulações de memória funcionais são importantes no controle da viremia. Comparando as respostas HIV e CMVespecíficas observamos freqüências mais elevadas de células T de memória produtoras de IL-2, IFN-/IL-2 e IFN- em respostas ao pool de peptídeos do HIV. Esses dados sugerem que esse conjunto de peptídeos derivados de seqüências do HIV-1 ativa respostas polifuncionais de subpopulações de linfócitos T de memória. Nossos resultados mostraram que o conjunto de peptídeos do HIV-1 foi capaz de estimular diferentes subpopulações distintas de linfócitos T de memória produtores de IFN-, IFN-,/IL-2 e IL-2 de indivíduos em diferentes estágios da infecção pelo HIV e sugerem o envolvimento de subpopulações de memória funcionais no controle da viremia. Estes achados fortalecem a possibilidade de uso desses peptídeos em uma formulação vacinal bem-sucedida em humanos / The persistence of functional memory T cell is important to ensure a protective immunity to Human Immunodeficiency Virus (HIV) infection. Memory T cells have been subdivided into central memory (TCM), effector memory (TEM) and highly differentiated effector memory (TEMRA) based on the expression of surface molecules such as CCR7 and CD45RA, and the ability to produce cytokines and proliferate. Recently, we identified 18 peptides derived from B consensus sequences of HIV-1 that bind to multiple HLA-DR molecules and are widely recognized by peripheral blood T lymphocytes from HIV-infected patients. Given this and considering the importance of memory T cells in the maintenance of specific immune response, our objective was to characterize phenotypic and functionally memory T cell subsets from HIV-infected individuals involved in the recognition of these epitopes in vitro. The study included 14 healthy control subjects and 61 HIV+ patients with CD4+ lymphocytes counts higher than 250 cells/mm3. The HIV+ patients were divided into six different clinical groups according to the stage of infection, plasma viral load (VL) and antiretroviral therapy use (ART): long-term non-progressors (LTNP), aviremic under ART (AV-ART), viremic under ART (VI-ART), viremic without using ART (VI without ART), recently infected viremic without using ART (VI-RI) and controllers. Peripheral blood mononuclear cells from study subjects were stimulated with HIV-1 peptide pool and with a cytomegalovirus (CMV) peptide pool. The frequencies of IFN- and IL-2 producing memory cells and antigenspecific cell proliferation were detected by multiparametric flow cytometry. Our results showed that the HIV-1 set of peptides was able to activate TCM, TEM and TEMRA functional memory subsets that secrete IFN- and IL-2 in 100% of the HIV patients from the different clinical groups. The HIV-1 set of peptides also induced memory T lymphocyte subsets proliferation. TEMRA CD4+IFN-+, total TEMRA CD4+IFN-+, TCM CD8+IFN-+, total TCM CD8+IFN-+, total TEM CD8+IFN-+, TEM CD8+IFN-+ and TEMRA CD8+IFN- + frequencies negatively correlated with HIV viral load in viremic patients. These data suggest that these functional memory subsets are important to control the viremia. When comparing the HIV and CMV-specific responses we observed higher frequencies of IL-2, IFN-/IL-2 and IFN- producing memory T cells in response to HIV peptide pool. These data suggest that this set of HIV sequence derived peptides activates polyfunctional response of memory T lymphocyte subsets. Our results showed that the HIV-1 peptide set was able to stimulate different IFN-, IFN-/IL-2 e IL-2 producing memory T lymphocytes from individuals in different stages of HIV infection and suggest the involvement of functional memory subsets in the control of viremia. These findings strengthen the possibility of using these peptides in a successful vaccine formulation in humans

Em 1993, a Organização Mundial da Saúde declarou a tuberculose (TB) como uma emergência global devido à sua relevância epidemiológica e a necessidade de seu controle. Atualmente, a TB ainda é considerada um problema de saúde pública e requer o desenvolvimento de vacinas e terapias que sejam mais eficazes na sua prevenção e tratamento. Nesse sentido, o Laboratório de Vacinas Gênicas da Faculdade de Medicina de Ribeirão Preto estuda há mais de dez anos a eficácia da vacina gênica DNAhsp65 na profilaxia e terapia da TB. Com o intuito de complementar o conhecimento existente sobre os mecanismos imunes desencadeados pela vacina DNAhsp65, assim como sua associação às drogas convencionais utilizadas no tratamento da TB, objetivou-se neste trabalho a caracterização de células natural killer (NK), T natural killer (NKT), e T ?? na imunoterapia da tuberculose experimental com a vacina DNAhsp65, no tratamento com as drogas rifampicina (RIF) e isoniazida (INH), e na associação DNAhsp65-drogas. Inicialmente, camundongos BALB/c foram infectados com Mycobacterium tuberculosis (Mtb) cepa H37Rv no dia 0 e nos dias 1, 7, 15, 30 e 70 após a infecção, foi promovida a eutanásia dos animais infectados ou não (controle) para análise das células T não convencionais no pulmão por citometria de fluxo. No dia 30 após a infecção, os animais infectados receberam os diferentes tratamentos: vacina DNAhsp65, vetor pVAX1, drogas RIF e INH, ou as drogas em associação à vacina. Dez dias após o fim dos tratamentos, foi promovida a eutanásia dos animais para análise das populações celulares no pulmão e linfonodo por citometria de fluxo, imunohistoquímica e PCR em tempo real. Os animais somente infectados com Mtb apresentaram aumento significativo no número das células NK (CD3-CD49b+), NKT (CD3+CD49b+) e T ?? (CD3+??+) logo na primeira semana após a infecção, e esta diferença em relação aos animais controle permaneceu em até 70 dias após a infecção. Entre as células NK presentes no pulmão, observou-se predominância da subpopulação CD11bhighCD27low em todos os animais. Nos animais infectados, verificou-se aumento significativo das subpopulações de NK: CD11bhighCD27high e CD11blowCD27high, nos dias 7 e 15 e somente no dia 15 após a infecção, respectivamente. Entre a população de células T ?? presentes no pulmão, houve predomínio do fenótipo CD27- em animais controles e infectados nos diferentes tempos experimentais. Quanto aos animais infectados com Mtb e tratados com DNAhsp65, verificou-se aumento significativo de células T ?? produtoras de IFN-? e IL-17 no pulmão, e apesar de não ter sido observada diferença na freqüência de células NK e NKT neste grupo, as células NK apresentavam maior expressão da molécula FasL relacionada à morte celular induzida por apoptose. Nos grupos drogas e DNAhsp65-drogas observou-se aumento da freqüência de células T ?? no pulmão, assim como aumento de células NK produtoras de IL-10 e que expressavam o marcador de ativação CD69. Os resultados deste trabalho mostram mais uma vez a eficácia da vacina DNAhsp65 e da associação DNAhsp65- drogas no tratamento de animais infectados com Mtb e sugerem que células T não convencionais como as células NK, NKT e T ?? podem participar na modulação da resposta immune na TB. Estes achados devem ser levados em consideração no desenho de novas estratégias terapêuticas e também profiláticas para a TB. / In 1993, the World Health Organization declared tuberculosis (TB) as a global emergence due to its epidemical relevance and the need to improve its control. Nowadays, TB still remains a public health problem and requires the development of more effective vaccines and therapies. In this sense, the Laboratory of Genetic Vaccines from the School of Medicine of Ribeirão Preto studies, for more than ten years, the efficiency of the genetic vaccine DNAhsp65 in TB prophylaxis and therapy. In order to complement the knowledge about the immune mechanisms triggered by DNAhsp65 vaccine and by its association with conventional drugs used in TB, our aim in this work was to characterize natural killer (NK), natural killer T (NKT) and gamma-delta (??) T cells in the immunotherapy of experimental tuberculosis with the DNAhsp65 vaccine, in the treatment with rifampicin and isoniazid drugs and in the association DNAhsp65-drugs. Initially, BALB/c mice were infected with Mtb strain H37Rv on day 0, and on days 1, 7, 15, 30 and 70 after infection, infected animals or not (control) were euthanized for lung cell analysis by flow cytometry. On day 30 after infection, infected animals received the following treatment: DNAhsp65 vaccine, pVAX1 vector, rifampicin and isoniazid drugs, or drugs in association with DNAhsp65. Ten days after the end of treatment, animals were euthanized for lung and lymph node cell analysis by flow cytometry, immunohistochemistry and real time PCR. Infected animals showed a significant increase of NK (CD3-CD49b+), NKT (CD3+CD49b+) and ?? (CD3+??+) T cells in the first week of infection and this difference compared to control animals remained until 70 days after infection. Within the lung NK cell population, we observed a predominance of CD11bhighCD27low phenotype in all animals. In infected animals, we verified a significant increase of the following NK cell subpopulations: CD11bhighCD27high and CD11blowCD27high on days 7 and 15, and only on day 15 after infection, respectively. Within the lung ?? T cell population, there was a predominance of CD27- ?? T cell in control and infected animals in the different experimental times. In infected animals and subsequently vaccinated with DNAhsp65, we verified a significant increase in ?? T cells producing IFN-? and IL-17 in the lungs. Although we have not seen any differences in NK and NKT cells in this group, NK cells showed higher expression of FasL molecule related to induced cell death by apoptosis. In DNAhsp65-drugs and drugs groups, we observed an increase in lung ?? T cells frequency, as well as increase in NK cells producing IL-10 and expressing CD69, an activation marker. Our results confirm the effectiveness of DNAhsp65 vaccine and its association with drugs in Mtb infected animals and suggest a modulation in the immune response through unconventional T cells such as NK, NKT and ?? T cells. These findings should be taken into consideration in the design of new therapeutic and prophylactic strategies for TB.

Embora faltem evidências diretas da relação causal entre a vacinação recente e o desenvolvimento da anemia hemolítica imunomediada (AHIM) em cães, pode ser identificada uma associação temporal entre elas. Constituem-se em objetivos deste trabalho avaliar: a presença de imunoglobulinas (IgG e IgM) e complemento (C3>) na superfície eritrocitária e o potencial do estímulo mitogênico de membranas eritrocitárias autólogas sobre os linfócitos periféricos de cães nos momentos pré-vacinal (imediatamente antes da vacinação com vacinas polivalente e antirrábica) e pós-vacinal (28 a 38 dias após a imunização). Vinte e um cães adultos e hígidos, machos e fêmeas, foram submetidos à anamnese, exame físico e avaliações laboratoriais nos dois momentos do estudo. O teste da antiglobulina direta (n=15) foi realizado com o reagente de Coombs polivalente, nas diluições de 1:2 a 1:8. A detecção de imunoglobulinas (IgG, IgM) e complemento (C3) na superfície de eritrócitos por citometria de fluxo (n=21) foi realizada utilizando anticorpos anti-IgG de cão produzido em ovelha cadeia pesada, anti-IgM produzido em cabra e anti-C3 de cão produzido em cabra, todos conjugados com fluoresceína de isotiocianato (FITC). As células mononucleares do sangue periférico foram isoladas por gradiende, marcadas com CFSE e estimuladas com Concanavalina A (ConA) e com membranas eritrocitárias autólogas em duas concentrações (ME1 e ME2). Foi utilizado o Índice de Proliferação (IP) como indicador da proliferação celular, obtido pela divisão das intensidades de fluorescência obtidas por citometria de fluxo das amostras basal e estimulada. As comparações das variáveis \"hemácias marcadas com anti-Ig/C3\" e \"IP de linfócitos\" foram realizadas utilizando-se o t-Student para amostras pareadas. As comparações dos IP de linfócitos frente aos diferentes antígenos (Con A, ME1 e ME2) foram realizadas por meio da ANOVA com medidas repetidas. Quando houve diferença significante entre os índices, foram realizadas comparações múltiplas (teste de Bonferroni). Foi considerado um nível de significância de 5%. Observou-se que os cães se encontravam em boas condições de saúde, nos dois momentos do estudo, com as variáveis hematológicas e bioquímicas mantidas próximas entre si e resultado do teste da antiglobulina direta negativo (n=21). A porcentagem de hemácias marcadas com IgG e IgM nos momentos pré-vacinal (1,06±0,49% e 1,42±1,59%) e pós-vacinal (0,83±0,56% e 1,35±1,71%) não foi alterada, com p=0,261 e p=0,699, respectivamente. A porcentagem de hemácias com C3 na superfície no momento pós-vacinação (0,40±0,38%) foi, em média, menor do que no momento pré-vacinação (0,71±0,33%), com p=0,019. Os índices de proliferação obtidos com a ConA, ME1 e ME2 no momento pré-vacinal (2,15±0,83; 1,03±0,07; 1,05±0,11) e pós- vacinal (2,13±0,58; 1,02±0,05; 1,02±0,05) não se modificaram, com p=0,935; p=0,845 e p=0,222, respectivamente. Em ambos os momentos, os índices de proliferação celular observados com o uso de ConA foram, em media, maiores do que os índices com ME1 e ME2 (p<0,001). A baixa porcentagem de hemácias com IgG, IgM ou C3 na superfície e a ausência de resposta proliferativa dos linfócitos quando estimulados com membranas eritrocitárias, indicam que, neste experimento, não houve nenhuma evidência de que o estímulo vacinal pudesse estar relacionado ao desenvolvimento da AHIM. / Despite the lack of evidence regarding a causal link between recent vaccination and development of immune mediated hemolytic anemia (IMHA) in dogs, a temporal association between them has been identified in some cases. The aim of this study was to evaluate: the presence of immunoglobulins (IgG and IgM) and complement (C3) on the surface of red blood cells and the potential for mitogenic stimulation of peripheral lymphocytes against autologous red blood cell membranes on dogs at pre-vaccination (immediately prior to vaccination with polyvalent and anti-rabies vaccines) and post-vaccination (after 28 and 38 days after vaccination). Twenty-one healthy adult dogs (both males and females) were subjected to physical examination and complementary laboratory exams in the aforementioned two instances of the study (i.e. pre-vaccination and post-vaccination). Direct antiglobulin test (n=15) was performed using the polyvalent Coombs reagent in 1:2 to 1:8 dilutions. Immunoglobulins (IgG, IgM) detection and identification of complement (C3) on the surface of red blood cells were done by flow cytometry (n=21) using antibodies anti-dog IgG heavy chain produced in sheep, anti-dog IgM produced in goat and anti-dog C3 produced in goat - all in conjunction with fluorescein isothiocyanate (FITC). The peripheral blood mononuclear cells were isolated by gradient, labeled with CFSE and stimulated with concanavalin A (Con A) and autologous erythrocyte membranes in two concentrations (EM1 and EM2). The Proliferation Index (PI), used as an indicator of cell proliferation, was obtained by dividing the fluorescence intensities of basal and stimulated samples, both obtained by flow cytometry. A comparison was made between the variables \"labeled red blood cells with anti-Ig/C3\" and \"PI lymphocyte\" using the paired Student\'s t-test. Comparisons of PI lymphocytes to different antigens (Con A, EM1 and EM2) were performed using ANOVA of repeated measures. Whenever significant differences between the indices were found, multiple comparisons (Bonferroni test) were then performed. A 5% significance level was considered. At the two instances of the study, dogs were presented in good health status with both hematological and biochemical variables kept close together and negative results for direct antiglobulin test (n=21). The percentage of red blood cells labeled with IgG and IgM in the pre-vaccination (1.06±0.49% and 1.42±1.59%) and post-vaccination (0.83±0.56% and 1.35±1.71%) were kept similar, with p=0.261 and p=0.699, respectively. The percentage of erythrocytes with C3 on the surface at the time post-vaccination (0.40±0.38%) was on average lower than in the pre-vaccination (0.71±0.33%), p=0.019. The proliferation index obtained with ConA, ME1 and ME2 in the pre-vaccination (2.15±0.83, 1.03±0.07, 1.05±0.11) and post-vaccination (2.13±0.58, 1.02±0.05, 1.02±0.05) did not differ significantly, with p=0.935, p=0.845 and p=0.222, respectively. In both instances, rates of cell proliferation observed with the use of ConA were, on average, higher than the rates with ME1 and ME2 (p<0.001). The low percentage of erythrocytes with IgG, IgM or C3 on the surface and the absence of lymphocyte proliferative response when stimulated with erythrocyte membranes, indicate that, at least in this experiment, there is no evidence of any association between vaccine stimulation and development of IMHA in dogs.

O timo é um órgão de grande importância imunológica durante a vida fetal e no período neonatal, para que o indivíduo se torne imunocompetente. É considerado, anatomicamente, o maior órgão com alta atividade linfopoiética no indivíduo jovem. O timo dos gatos apresenta duas porções, torácica e cervical, onde cada uma delas apresenta um lobo direito e esquerdo em sua maioria. A maior contribuição vascular origina-se da artéria torácica interna esquerda e do tronco braquiocefálico. Possui coloração rósea-pálido, localizado em região de mediastino cranial, entre os pulmões e na base do coração a porção torácica, e a porção cervical estende-se além das costelas em sentido cranial localizada ventralmente a traquéia. Foram utilizados 12 fetos de gatos domésticos, sem raça definida, machos e fêmeas, divididos em três grupos. Os timos foram processados para o estudo da microscopia de luz, e as análises estereológicas foram realizadas utilizando o método disector físico associado com o princípio de ConnEuler. As variações de volume, comprimento, espessura e largura de maneira geral apresentaram aumento conforme o desenvolvimento dos animais, com diferenças entre os sexos. As medidas estereológicas relativas a densidade numérica vascular (Nv(vasc)) apresentam-se maiores nas fêmea, ocorrendo uma diminuição gradativa e o número total de vasos no órgão (N(vasc)) apresentou valores maiores nos machos com uma diminuição gradual. A estimação da densidade do comprimento do vaso (Lv) e da densidade de superfície de área (Sv) apresentaram diminuição aos 45 dias de idade, e a densidade do comprimento do vaso (Lv) apresentou valor maior nos machos de 35 e 55 dias, enquanto que na densidade de superfície de área (Sv) os valores variaram entre os sexos. / During the phoetal life and neonatal period, the thymus has a great importance to became an individual healthy. Anatomically is the largest organ with high limphopoietic activity in young individual. The cat thymus presents two portions, thoracic and cervical, where each one of them presents a right and left lobe at the most. The major contribution initiates from the inner thoracic artery and from braquicephalicus trunk. The organ presents pink-pale color, located in region of cranial mediastine, between lungs and at the base of the heart; the thoracic portion and the cervical extend beyond the ribs located ventrally to the trachea. For this study were used twelve fetus of the mongrel domestic cats, males and females, divided into three groups. The thymus were processed for the light-microscopy, and the stereological analyses were done using the physical disector method associated with the ConnEuler principle. The volume of the organ, lenght, thickness and wide increased gradually with the development, with diferences between sex. The stereological variables related to vascular number density (Nv(vasc)) were greater in females, decreasing gradually and the vascullar total number (N(vasc)) were greater in males decreasing gradually. The lenght density (Lv) and the surface area density showed decreasing at forty-five days old, and the lenght density were greater in males ate thirty-five and fifty-five days old, while the surface area density the values were varied between sex.

Une réponse immunitaire efficace contre un large spectre de pathogènes ou contre des cellules tumorales nécessite l’activation des lymphocytes T CD4+. L’engagement du récepteur T par un peptide antigénique apprêté sur les produits de classe-II du complexe majeur d’histocompatibilité (peptide-CMH, pCMH) portés par une cellules présentatrices d’antigène (CPAg), conduit à de nombreux remaniements du lymphocyte T. Il s’établit notamment à l’interface entre le lymphocyte T et la CPAg, une structure spécialisée qui est la synapse immunologique (ou synapse immune). La synapse est le siège d’événements de signalisation intenses où diverses molécules de signalisation nécessaires l’amplification et la diversification du signal provenant du TCR sont recrutées. Ces évènements de signalisation sont régulés par l’adaptateur transmembranaire LAT (« Linker for Activation of T cells ») qui est présent à la membrane plasmique ainsi que dans des compartiments intracellulaires. La fraction intracellulaire de LAT est recrutée à la synapse immune et il est proposé que ces compartiments vésiculaires participent à la signalisation lymphocytaire T. L’objectif de ce travail de thèse a été de déterminer les voies de trafic intracellulaire nécessaires au recrutement de la fraction intracellulaire de LAT à la synapse immunologique et de comprendre le rôle de ce transport dans l’activation et la réponse lymphocytaire T. Par des approches d’extinction de l’expression de différentes molécules de transport intracellulaires dans les cellules T Jurkat ou des lymphocytes T CD4+ primaires humains ou par l’utilisation de souris Knock-Out (KO), nous avons mis en évidence plusieurs voies de transport impliquées dans le transport de LAT. Nous avons ainsi mis en évidence que le recrutement de LAT à la synapse immunologique nécessite une voie de sécrétion dépendante de la protéine SNARE vésiculaire VAMP7. L’analyse plus avant du transport de LAT a par ailleurs permis de montrer que LAT est présente dans des compartiments de recyclage alors que VAMP7 est principalement localisée dans l’appareil de Golgi. L’étude de la petite GTPase Rab6 et de la protéine t-SNARE syntaxine-16 qui sont impliquées dans des voies de transport rétrograde entre les endosomes de recyclage et l’appareil de Golgi, a permis de dévoiler que cette voie de transport est requise dans le recrutement de LAT à la synapse immunologique, ainsi qu’à la réponse lymphocytaire T in vitro, ex vivo et in vivo. Enfin, le rôle de la protéine de transport intraflagellaire IFT20, qui a déjà été mis en cause dans le transport du TCR, a été analysé chez la souris et a également montré des défauts de recrutement de LAT à la synapse et dans l’activation lymphocytaire T ex vivo et in vivo. Nos résultats mettent ainsi en évidence que la régulation du transport intracellulaire dans les lymphocytes T joue un rôle crucial dans l’activation lymphocytaire T. Nous proposons ainsi un modèle dans lequel LAT est constitutivement internalisé depuis la membrane plasmique et poursuit une voie de recyclage dépendante de l’appareil de Golgi qui contient la machinerie de sécrétion associé à VAMP7. Cette voie de transport intracellulaire, conditionnerait la resécrétion polarisée de LAT à la synapse immunologique dans les conditions d’activation et une réponse lymphocytaire T robuste. / The immune response against a broad spectrum of pathogens or against tumor cells requires the CD4 + T lymphocytes activation. The triggering of T Cell Receptor (TCR) by peptide-MHC through antigen-presenting cells (APC) leads to numerous T-cell remodeling and the establishment of a specialized structure at the interface between the T lymphocyte and the APC: the immunological synapse. The synapse is the site of intense signaling events where various signaling molecules are recruited in order to amplify and diversify the signal initiated by the TCR. These signaling events are regulated by the transmembrane adapter LAT ("Linker for activation of T cells") which is present at the plasma membrane as well as in intracellular compartments. The intracellular fraction of LAT is recruited at the immune synapse and it is proposed that these vesicular compartments participate in T lymphocyte signaling. The objective of this thesis work was to determine the intracellular trafficking pathways required for the recruitment of the intracellular pool of LAT to the immunological synapse and understand the role of this transport in T cell activation and response. By silencing the expression of different intracellular transport molecules in Jurkat T cells or primary human CD4 + T lymphocytes, or by using Knock-Out (KO) mice, we have highlighted several trafficking pathways involved in the transport of LAT to the immune synapse. We have demonstrated that the recruitment of LAT to TCR activation sites requires a secretion pathway dependent on the vesicular SNARE protein VAMP7. Further analysis of the transport of LAT showed that LAT is present in recycling compartments whereas VAMP7 is mainly located in the Golgi apparatus. The study of the small GTPase Rab6 and the t-SNARE syntaxin-16 protein that are involved in the retrograde transport pathways between the recycling endosomes and the Golgi apparatus, demonstrated that this route of transport is required for the recruitment of LAT to the immunological synapse and for T lymphocyte response in vitro, ex vivo and in vivo as well. Finally, the role of intraflagellar transport protein IFT20, which has already been implicated in the transport of TCR, was analyzed in mice and also showed defects in LAT recruitment to synapse and T lymphocyte activation ex vivo and in vivo. Our results thus show that the regulation of intracellular transport plays a crucial role in T lymphocyte activation. We thus propose a model in which LAT is constitutively internalized from the plasma membrane and pursues a Golgi-dependent recycling pathway that contains the secretion machinery associated with VAMP7. This intracellular transport pathway would thus allow the polarized LAT re-secretion to the immunological synapse under activation conditions and a robust T lymphocyte response.

Em 1993, a Organização Mundial da Saúde declarou a tuberculose (TB) como uma emergência global devido à sua relevância epidemiológica e a necessidade de seu controle. Atualmente, a TB ainda é considerada um problema de saúde pública e requer o desenvolvimento de vacinas e terapias que sejam mais eficazes na sua prevenção e tratamento. Nesse sentido, o Laboratório de Vacinas Gênicas da Faculdade de Medicina de Ribeirão Preto estuda há mais de dez anos a eficácia da vacina gênica DNAhsp65 na profilaxia e terapia da TB. Com o intuito de complementar o conhecimento existente sobre os mecanismos imunes desencadeados pela vacina DNAhsp65, assim como sua associação às drogas convencionais utilizadas no tratamento da TB, objetivou-se neste trabalho a caracterização de células natural killer (NK), T natural killer (NKT), e T ?? na imunoterapia da tuberculose experimental com a vacina DNAhsp65, no tratamento com as drogas rifampicina (RIF) e isoniazida (INH), e na associação DNAhsp65-drogas. Inicialmente, camundongos BALB/c foram infectados com Mycobacterium tuberculosis (Mtb) cepa H37Rv no dia 0 e nos dias 1, 7, 15, 30 e 70 após a infecção, foi promovida a eutanásia dos animais infectados ou não (controle) para análise das células T não convencionais no pulmão por citometria de fluxo. No dia 30 após a infecção, os animais infectados receberam os diferentes tratamentos: vacina DNAhsp65, vetor pVAX1, drogas RIF e INH, ou as drogas em associação à vacina. Dez dias após o fim dos tratamentos, foi promovida a eutanásia dos animais para análise das populações celulares no pulmão e linfonodo por citometria de fluxo, imunohistoquímica e PCR em tempo real. Os animais somente infectados com Mtb apresentaram aumento significativo no número das células NK (CD3-CD49b+), NKT (CD3+CD49b+) e T ?? (CD3+??+) logo na primeira semana após a infecção, e esta diferença em relação aos animais controle permaneceu em até 70 dias após a infecção. Entre as células NK presentes no pulmão, observou-se predominância da subpopulação CD11bhighCD27low em todos os animais. Nos animais infectados, verificou-se aumento significativo das subpopulações de NK: CD11bhighCD27high e CD11blowCD27high, nos dias 7 e 15 e somente no dia 15 após a infecção, respectivamente. Entre a população de células T ?? presentes no pulmão, houve predomínio do fenótipo CD27- em animais controles e infectados nos diferentes tempos experimentais. Quanto aos animais infectados com Mtb e tratados com DNAhsp65, verificou-se aumento significativo de células T ?? produtoras de IFN-? e IL-17 no pulmão, e apesar de não ter sido observada diferença na freqüência de células NK e NKT neste grupo, as células NK apresentavam maior expressão da molécula FasL relacionada à morte celular induzida por apoptose. Nos grupos drogas e DNAhsp65-drogas observou-se aumento da freqüência de células T ?? no pulmão, assim como aumento de células NK produtoras de IL-10 e que expressavam o marcador de ativação CD69. Os resultados deste trabalho mostram mais uma vez a eficácia da vacina DNAhsp65 e da associação DNAhsp65- drogas no tratamento de animais infectados com Mtb e sugerem que células T não convencionais como as células NK, NKT e T ?? podem participar na modulação da resposta immune na TB. Estes achados devem ser levados em consideração no desenho de novas estratégias terapêuticas e também profiláticas para a TB. / In 1993, the World Health Organization declared tuberculosis (TB) as a global emergence due to its epidemical relevance and the need to improve its control. Nowadays, TB still remains a public health problem and requires the development of more effective vaccines and therapies. In this sense, the Laboratory of Genetic Vaccines from the School of Medicine of Ribeirão Preto studies, for more than ten years, the efficiency of the genetic vaccine DNAhsp65 in TB prophylaxis and therapy. In order to complement the knowledge about the immune mechanisms triggered by DNAhsp65 vaccine and by its association with conventional drugs used in TB, our aim in this work was to characterize natural killer (NK), natural killer T (NKT) and gamma-delta (??) T cells in the immunotherapy of experimental tuberculosis with the DNAhsp65 vaccine, in the treatment with rifampicin and isoniazid drugs and in the association DNAhsp65-drugs. Initially, BALB/c mice were infected with Mtb strain H37Rv on day 0, and on days 1, 7, 15, 30 and 70 after infection, infected animals or not (control) were euthanized for lung cell analysis by flow cytometry. On day 30 after infection, infected animals received the following treatment: DNAhsp65 vaccine, pVAX1 vector, rifampicin and isoniazid drugs, or drugs in association with DNAhsp65. Ten days after the end of treatment, animals were euthanized for lung and lymph node cell analysis by flow cytometry, immunohistochemistry and real time PCR. Infected animals showed a significant increase of NK (CD3-CD49b+), NKT (CD3+CD49b+) and ?? (CD3+??+) T cells in the first week of infection and this difference compared to control animals remained until 70 days after infection. Within the lung NK cell population, we observed a predominance of CD11bhighCD27low phenotype in all animals. In infected animals, we verified a significant increase of the following NK cell subpopulations: CD11bhighCD27high and CD11blowCD27high on days 7 and 15, and only on day 15 after infection, respectively. Within the lung ?? T cell population, there was a predominance of CD27- ?? T cell in control and infected animals in the different experimental times. In infected animals and subsequently vaccinated with DNAhsp65, we verified a significant increase in ?? T cells producing IFN-? and IL-17 in the lungs. Although we have not seen any differences in NK and NKT cells in this group, NK cells showed higher expression of FasL molecule related to induced cell death by apoptosis. In DNAhsp65-drugs and drugs groups, we observed an increase in lung ?? T cells frequency, as well as increase in NK cells producing IL-10 and expressing CD69, an activation marker. Our results confirm the effectiveness of DNAhsp65 vaccine and its association with drugs in Mtb infected animals and suggest a modulation in the immune response through unconventional T cells such as NK, NKT and ?? T cells. These findings should be taken into consideration in the design of new therapeutic and prophylactic strategies for TB.