Les lymphocytes Th17 forment une population de cellules T CD4+ pro-inflammatoires, impliqués non seulement dans l'élimination d'agents pathogènes, mais ayant aussi un rôle délétère dans l'induction de maladies inflammatoires chroniques. Ils expriment spécifiquement le récepteur de chimiokines CCR6, qui a pour ligand le CCL20 mais aussi les β-defensine-1, 2 et 3, peptides ayant une activité antimicrobienne. Les cellules souches mésenchymateuses (CSMs) représentent une population cellulaire hétérogène exerçant diverses propriétés immunomodulatrices.Les résultats obtenus dans ce travail de thèse montrent que l'environnement inflammatoire contribue à augmenter l'adhésion des lymphocytes Th17 aux CSMs, et qu'elle est régulée par l'interaction du CCR6 avec ses ligands ; que les CSMs exercent, en partie via la sécrétion de PGE2, des effets anti-inflammatoires en faisant acquérir un phénotype régulateur aux lymphocytes Th17 différenciés, soulignant ainsi la plasticité de ces derniers.De plus, nous avons montré que les lymphocytes Th17 activés par l'antigène produisent du CCL20 et induisent, via la production de l'IL-17 et de l'IL-22, la sécrétion d'hBD-2, mais pas celle des hBD-1 et 3, par des kératinocytes épidermiques humains et de la peau reconstituée; que le CCL20, ainsi que la hBD-2, induisent l'arrêt de ces cellules sur l'endothélium enflammé in vitro en conditions de cisaillement. Finalement, l'activation spécifique d'antigène des lymphocytes Th17 entraîne une perte de l'expression de CCR6, ce qui provoque ainsi un état transitoire de non réponse à une nouvelle stimulation de ces cellules avec les ligands de CCR6, permettant leur migration ultérieure hors du tissu enflammé. / Th17 cells form a population CD4+ T cells with strong pro-inflammatory properties that are not only involved in the clearance of pathogens, but also play a deleterious role of in the pathogenesis of inflammatory disease. Th17 cells specifically express CCR6, a chemokine receptor that binds to its unique chemokine ligand, CCL20, as well as to human β-defensin (hBD)-1, 2 and 3, peptides with anti-microbial activity. Mesenchymal stem cells (MSC) represent a heterogenous population that exert broad immunomodulatory effects.The results from the studies carried out during this thesis show that the inflammatory environment contributes to increased adhesion of Th17 cells to MSCs, which is mediated via the interaction of CCR6 with its ligands, and that MSCs exert, in part via the secretion of PGE2, anti-inflammatory effects through the induction of a T regulatory cell phenotype in fully differentiated tissue-infiltrating Th17 cells, thereby underscoring the plasticity of the latter cells.Furthermore, the results show that antigen-activated Th17 cells produce CCL20 and induce, via the production of both IL-17 and IL-22, the secretion of hBD-2, but not 1 and 3, by normal human epidermal keratinocytes and reconstituted skin, and that CCL20, as well as hBD-2, induce arrest of these cells onto inflamed endothelium in vitro under conditions of shear stress. Finally, antigen-specific activation of Th17 cells also causes a loss of CCR6 expression from their cell surface and thus results in a transitory state of non-responsiveness to further stimulation of these cells with CCR6 ligands, which is likely to permit their subsequent migration out of inflamed tissue.

Les traitements par immunothérapie adoptive actuels sont compromis par une faible infiltration et expansion des LT CD8 injectés. Les travaux antérieurs de l'équipe ont montré une synergie entre les signaux du TCR et du récepteur à l'IL-2 pour la différenciation en Lymphocytes T (LT) effecteurs compétents, un effet qui peut être mimé par une forme constitutivement active du facteur de transcription STAT5 (STAT5CA). Mon projet de thèse à viser à exprimer ce STAT5 actif dans des LT CD8 anti-tumoraux dans le but d'augmenter leur réactivité. Nous démontrons que STAT5CA soutient l'expression de gènes contrôlant la migration, la survie, la composition des granules cytolytiques, la sécrétion de cytokines de type Tc1 (IFNy/TNFα) et leur potentiel de réponse secondaire. Les LT modifiés par STAT5CA sur-expriment les facteurs de transcription T-bet et Eomes qui sont associés respectivement à la différenciation en LT effecteur mémoire et central mémoire. Le potentiel cytolytique et migratoire des LT modifiés par STAT5CA rend ces cellules plus efficaces que des LT contrôlent pour induire une régression tumorale dans des modèles de mélanome transplanté ou induit (modèle TiRP). Ces résultats suggèrent une résistance accrue aux mécanismes d'immunosuppression intra-tumoraux. Contrairement aux LT contrôles, les LT modifiés par STAT5CA sous-expriment l'IL-6Rα et le TGFβRII et ont une moindre sensibilité à l'action immunosuppressive des cytokines IL-6 et TGFβ1. / Adoptive therapies are compromised by poor infiltration and expansion of injected CD8 T cells. Previous work in our team has demonstrated that signals from TCR and IL-2 receptor are acting in synergy to promote the differentiation of CD8 T cells. Moreover, this IL-2 effect can be mimicked by a constitutive active form of STAT5 (STAT5CA). During my PhD, I expressed this active STAT5 in anti-tumor CD8 T cells in order to enhance their activity. We demonstrated that STAT5CA sustains gene expression involved in migration, survival, cytolytic granules composition, Tc1 cytokine secretion (IFNy/TNFα) and secondary response potential. T cells transduced with STAT5CA up-regulate expression of the transcription factors T-bet and Eomes which are involved respectively in effector or central memory T cell differentiation. Cytolytic and migratory properties of STAT5CA T cells contribute to their capacities to induce regression of both transplanted and induced (TiRP model) melanomas, while control T cells were inefficient. Those results suggest that STAT5CA T cells are less sensitive to tumor-derived immunosuppression. Compared to control T cells, STAT5CA T cells show a down-regulation of IL-6Rα and TGFβRII which correlate with their decreased sensitivity to IL-6 and TGF1 derived immunosuppression. Moreover, STAT5CA T cells infiltrate lung, liver and pancreas which open possible treatments for highly frequent tumors that are not efficiently cured.

L’allergie de type I, une réaction de type hypersensibilité immédiate (HIS), a considérablement augmenté au cours des dernières décades dans les pays industrialisés. Le rôle des lymphocytes T régulateurs (Treg) dans le contrôle des maladies allergiques a été récemment apprécié. Cependant, il n’existe actuellement aucune connaissance précise si et comment les CD4+ Treg peuvent réguler l’allergie de type I. Le but du projet était d’étudier la contribution de cellules Treg dans le contrôle de l’allergie de type I chez la souris et chez l’homme. En utilisant un modèle murin d’HSI aux protéines de lait de vache, ß-lactoglobuline(BLG), nous avons montré que l’absence de Treg augmentait les réponses des cytokines Th1et Th2 et des anticorps IgE, IgG1 et IgG2a spécifique de la BLG. De plus, l’absence de Treg a augmenté la sévérité de l’anaphylaxie chez les souris sensibilisées lors de l’épreuve orale avec la BLG, avec une augmentation concomitante de la protéase des mastocytes muqueux 1(mMCP-1) dans le sérum. La contribution des cellules Treg dans un modèle murin d’anaphylaxie systémique passive (PSA) a été également étudiée. Une anaphylaxie plus sévère a été observée chez les souris déficientes en CHM de classe II ainsi que chez des souris B6 traitées avec un anti-CD4. En revanche, la déplétion sélective des Foxp3+ Treg chez des souris Tg DEREG n’a pas d’effet sur l’anaphylaxie.La capacité des Treg à contrôler la dégranulation des basophiles a été étudiée chez l’homme. Les données préliminaires ont montré que les Treg ont été incapables de contrôler la dégranulation des basophiles chez les donneurs sains. En revanche, une légère mais reproductible down-regulation de la dégranulation/activation des basophiles a été observée dans l’allergie induite par les médicaments chez l’homme. Dans l’ensemble, notre travail met en évidence un nouveau rôle encore non identifié des Treg dans le contrôle de l’HSI et souligne que chez la souris et l’homme, les Treg peuvent dans certaines conditions, limiter la sévérité de l’HSI en agissant sur les mastocytes et/ou basophiles effecteurs / Type I allergy, an immediate type hypersensitivity reaction (ITH), has dramatically increased during the past decades affecting up to 30% of the population in industrialized countries. The role of regulatory T cells (Treg) in the control of allergic diseases has been recently appreciated. However, there is currently no precise knowledge of whether and howCD4+ Treg can regulate type I allergy. The aim of this project was to investigate the contribution of Treg in the control of type I allergy. Using a murine model of ITH to cow’s milk protein the ß-lactoglobulin (BLG), we showed that lack of Treg increased BLG-specific Th2 and Th1 cytokine response and BLG-specific IgE, IgG1 and IgG2a antibodies.Furthermore, absence of Treg enhanced the severity of the anaphylaxis with concomitant increase in serum mucosal mast cell protease 1 (mMCP-1) in sensitized mice upon oral challenge with BLG. Furthermore, we investigated the contribution of Treg in a murine model of passive systemic anaphylaxis (PSA). More sever anaphylaxis was observed in both MHCclass II KO mice and after anti-CD4 mAb treatment. Alternatively, selective depletion of Foxp3+ Treg in DEREG Tg mice did not show changes in anaphylaxis.The capacity of Treg to control basophils degranulation was investigated in humans. Preliminary data showed that Treg were unable to control basophils degranulation in healthy donors. In contrast, a slight butreproducible downregulation of basophils activation/ degranulation was observed in allergic individuals. Taken together, our work points out to a novel as yet unidentified role of Treg in the control of ITH and emphasizes that both mouse and human Treg can, in certain conditions, limit the severity of ITH by acting on mast cells and/or basophils effectors of ITH

Les lymphocytes T Vγ9Vδ2 ont été étudiées depuis les années quatre-vingt pour leurs puissantes propriétés anti-infectieuses, démontrées aussi bien in vitro que dans des modèles animaux et confirmées par de nombreuses observations cliniques.L'implication de ces cellules dans l’immunité anti-infectieuse réside dans leur reconnaissance d’une famille de molécules produites par des pathogènes intracellulaires appelées phosphoantigènes dont l’activateur naturel le plus puissant à ce jour est le HDMAPP : (E)-1-hydroxy-2-méthylbut-2-ényl 4-diphosphate.Après avoir défini et synthétisé un synthon clé, sur lequel nous avons couplé des groupements pyrophosphonate et pyrophosphoramidate, nous avons réalisé des bioisostères de la molécule HDMAPP. Nous avons également synthétisé des isomères géométriques, analogue de position et isomères cis, des dérivés carbonylés, acide et ester. La bioactivité de ces molécules a été testée in vitro et pour les plus actives in vivo. Les résultats obtenus indiquent que l'utilisation de composés bioisostères de HDMAPP peuvent représenter de nouvelles pistes prometteuses pour l'immunothérapie. / Vγ9Vδ2 T lymphocytes have been studied since the early eighties for their potent anti-infectious properties, attested both in vitro and in animal models and supported by many clinical observations. The involvement of Vγ9Vδ2 T cells in anti-infectious immunity lies in their recognition of an original family of molecules produced by intracellular pathogens so-called phosphoantigens and whose most potent natural activator to date is the HDMAPP: (E)-1-hydroxy-2-methylbut -2-enyl diphosphate.Having defined and synthesized a key intermediate on which we have linked pyrophosphonate and pyrophosphoramidate moieties, we have made bioisosters of the molecule HDMAPP. We also synthesized geometric isomers, analogue of position and cis isomers, carbonyl derivatives, acid and ester. The bioactivity of these molecules was tested in vitro and for the most active in vivo. The results indicate that the use of bioisosters compounds of HDMAPP may represent promising new leads for immunotherapy.

Les cellules dendritiques (CD) jouent un rôle majeur dans l'initiation, la régulation et le maintien des réponses immunes contre les pathogènes. Le ciblage d'antigènes (Ag) liés à des anticorps monoclonaux (AcM) dirigés contre des récepteurs spécifiques de CD est une approche vaccinale prometteuse induisant une réponse immunitaire chez l'animal. Cependant, certaines protéines de fusion AcM-Ag ne peuvent pas être produites. J'ai donc développé des AcM fusionnés à un domaine dockérine et des Ag fusionnés à un domaine cohésine, permettant ainsi l'assemblage non-covalent de complexes AcM-Ag. Ainsi, des complexes non-covalents anti-CD40-Ag ou anti-Langerine-Ag du virus influenza ont induit l'expansion in vitro de lymphocytes T CD4+ et CD8+ spécifiques, et la production d'anticorps chez la souris. De même, le ciblage de ces Ag via DCIR, récepteur exprimé sur différentes populations de CD humaines, ont induit l'expansion de lymphocytes T CD8+ mémoires in vitro. Enfin, j'ai montré que l'addition de peptides glycosylés flexibles facilite la production d'AcM anti-CD40 fusionnés à 5 peptides du VIH. Ces peptides conservés et immunogènes sont dérivés des protéines Gag, Nef et Pol. Ce prototype vaccinal, testé in vitro, sur des cellules de patients séropositifs, induit l'expansion d'un vaste répertoire de lymphocytes T CD4+ et CD8+ spécifiques et multifonctionnels. Ces cellules T CD8+ cytotoxiques sont capables de supprimer la réplication du VIH in vitro. En conclusion, ce travail a permis de développer plusieurs prototypes de vaccins ciblant les CD et a démontré leur potentiel à induire des réponses immunes efficaces, justifiant leur application à visée préventive et thérapeutique. / Dendritic cells (DCs), as the most potent antigen-presenting cells, have a pivotal role in the initiation, regulation and maintenance of immune responses against cancers and pathogens. Targeting antigens (Ag) directly to DCs via anti-DC receptor monoclonal antibody-antigen (mAb-Ag) fusion proteins is a promising approach to vaccine development and has been shown to induce potent immunity in animal models. Thus, I developed mAbs fused to a dockerin domain and antigens fused to a cohesin domain, enabling non-covalent assembly of mAb-Ag complexes particularly when direct mAb-Ag fusions could not be produced. Delivery of influenza Ags to CD40 and Langerin via these non-covalent complexes, respectively expanded Ag-specific CD4+ and CD8+ T cells in vitro and elicited Ag-specific antibody responses in mice. Similarly, targeting influenza Ags in vitro with such complexes to DCIR on various human DC subsets expanded Ag-specific memory CD8+ T cells. Finally, I found that flexible glycosylated peptide linkers enabled production of an anti-CD40 mAb fused to a string of 5 conserved T cell epitope- rich regions of HIV Gag, Nef and Pol. In vitro, this prototype vaccine expanded a broad repertoire of Ag- specific and multifunctional CD4+ and CD8+ T cells from HIV-infected patients. These cytotoxic expanded CD8+ T cells were effective in suppressing in vitro HIV replication. In conclusion, our work facilitated the development of prototype DC-targeting vaccines and demonstrated their potential to induce effective immune responses, supporting their use for preventive and therapeutic applications.

L'objectif de notre travail est de comprendre la contribution relative des voies de signalisation précoces du TCR et de CD28 dans l'activation des lymphocytes T naïfs. Notre étude a d'abord montré que dans les cellules T CD4+ naïves, la stimulation du TCR augmente de manière significative la liaison en deux dimensions (2D) de CD28 avec ses ligands B7, et ceci dépend à la fois du domaine cytoplasmique de CD28 et de l'activité des src kinases. Par la suite, notre analyse biochimique a démontré que l'engagement du TCR par son ligand (CMHp) potentialise la phosphorylation de CD28 stimulée par son ligand B7. En outre, la stimulation conjointe du TCR et de CD28 augmente fortement la phosphorylation des protéines de signalisation proximales telles que les molécules Vav-1 et PLCγ-1. Nous avons également examiné la mobilisation des ions calcique (Ca++). Nous avons trouvé que l'engagement du TCR ou de CD28 seul est capable de déclencher une élévation de la concentration intracellulaire d'ions Ca++ dans des cellules T naïves. Cette élévation qui se caractérise par de fortes fluctuations de la concentration calcique impliquerait principalement 2 types de canaux calciques de la membrane plasmique. De façon attendue, une stimulation conjointe des lymphocytes par le TCR et CD28 augmente l'amplitude moyenne de la réponse calcique. Nos données ont révélé que seule une stimulation conjointe, et non individuelle, du TCR et de CD28 augmente significativement le temps de résidence du Ca++ libres fluctuants par rapport aux cellules non stimulées. Par conséquent, cette augmentation du temps de résidence caractérise spécifiquement la réponse calcique induite par TCR et CD28. / In this work, we aimed at determining the relationship between and specific contribution of TCR and CD28 early signaling pathways in naïve CD4+ T cell activation. Our data showed that in naïve CD4+ T cells, TCR stimulation significantly increased the 2D binding of CD28 to its B7 ligands and this increase depended on both cytoplasmic tail of CD28 and activity of src kinases. Our biochemical analysis then demonstrated that TCR engagement with its ligand pMHC strongly enhanced the CD28 tyrosine phosphorylation triggered by B7. Moreover, the conjoint stimulation of TCR and CD28 markedly augmented activation of proximal signaling molecules such like Vav-1 and PLCγ-1 compared to the stimulation with each receptor alone. We next went to examine the calcium ion (Ca2+) mobilization. We found that in naïve CD4+ T cells, engagement with ligand of TCR or CD28 alone was able to trigger rise of the fluctuating cytosolic-free Ca2+ level. Unexpectedly, such rises implicated predominantly the involvements of two different types of calcium channels: Cav and CRAC channels. The conjoint stimulation with both TCR and CD28 enabled the augment of average amplitude of the calcium response. Through the time series analysis, our data unveiled that the conjoint, but not separate, TCR and CD28 stimulation in naïve CD4+ T cells significantly increased the fluctuating cytosolic-free Ca2+ dwell time relative to that found in unstimulated cells. The increase of the cytosolic-free Ca2+ dwell time therefore uniquely characterized the calcium response triggered by TCR and CD28 and presumably corresponded to a fundamental feature for the high efficiency of T cell activation induction.

Les lymphocytes T CD8 sont des acteurs clés de l'immunité adaptative, impliqués dans la lutte contre les pathogènes intracellulaires et les cellules tumorales. En outre, les lymphocytes T CD8 sont capables, lors d’une réponse primaire, de se différencier en cellules mémoires capables de se maintenir dans l’organisme et de le protéger efficacement contre une nouvelle rencontre avec ces dangers. Ceci est permis grâce à leurs fonctions effectrices améliorées, leur plus grand potentiel prolifératif et leur capacité à migrer dans les tissus non-lymphoïdes, des propriétés toutes trois régulées au niveau moléculaire par le cytosquelette d'actine.            Récemment, notre équipe a montré que pleckstrin, un gène impliqué dans la régulation du cytosquelette d'actine, était fortement exprimé dans les lymphocytes T CD8 mémoires (générés suite à une infection virale), par comparaison avec des cellules T CD8 naïves. Grâce à des expériences d'infection par le virus de la Vaccine in vivo, nous avons pu mettre en évidence que l'absence de pleckstrin n'altère pas les fonctions effectrices des lymphocytes T CD8 (i. e. cytotoxicité et production de cytokines). En revanche, pleckstrin semble nécessaire à la génération des lymphocytes T CD8 mémoires et à leur localisation au sein des tissus infectés.            Ces travaux de recherche apportent ainsi un regard nouveau sur les mécanismes cellulaires et moléculaires impliqués dans la mise en place d'un compartiment T CD8 mémoire au sein des tissus non-lymphoïdes. / CD8 T cells are key players of adaptive immunity, and are involved in the elimination of intracellular pathogens and cancer cells. During a primary immune response, memory CD8 T cells are generated, and are able to maintain long-term and protect efficiently the organism against a secondary encounter with these threats. Indeed, memory CD8 T cells mediate durable protection through enhanced effector functions, increased proliferative capacity and distinct migratory behaviors, three processes that are tightly regulated by actin cytoskeleton.            Recently, the laboratory has shown that pleckstrin, a gene involved in actin cytoskeleton, is highly up-regulated in pathogen-induced memory CD8 T cells compared to naïve cells. Using pleckstrin deficient CD8 T cells, we show in an in vivo model of Vaccinia virus infection that pleckstrin deficiency does not affect global CD8 functions, in terms of cytokine production and cytotoxicity. However, pleckstrin is required for the optimal generation and localization of memory CD8 T cells within infected tissues.            As a whole, this work gives new insight into the molecular and cellular mechanisms that allow the establishment of a memory CD8 T cell compartment within non-lymphoid tissues.

A erliquiose monocitotrópica canina é caracterizada como uma infecção persistente, podendo evoluir para doença fatal. Mecanismos imunopatogênicos estão implicados no desenvolvimento da doença, porém não é completamente compreendido o papel da resposta imune celular nas infecções causadas por Ehrlichia canis transmitida pelo carrapato Rhipicephalus sanguineus. A infecção do vetor ocorre somente durante o repasto em cães riquetsêmicos, porém não é reconhecido o nível de riquetsemia infectante durante o curso da infecção nos cães. O objetivo desse estudo foi verificar os níveis de riquetsemia relativa capazes de infectar o vetor (R. sanguineus) durante o curso da erliquiose canina; e avaliar a resposta imune celular induzida por exposição de cães a carrapatos infectados com Ehrlichia canis. Um cão foi utilizado para produção do inoculo e infecção de ninfas de R. sanguineus. Subseqüentemente, os carrapatos adultos infectados, oriundos das ninfas que se alimentaram nos cães infectados, foram utilizados para infecção dos cães dos grupos I (n=3) e II (n=3). Cães do grupo III (n=3) foram inoculados com sangue infectado com E. canis, por via intravenosa. Os grupos IV (n=3) e V (n=3) foram compostos por cães não infectados. Os grupos II e IV foram reinfestados periodicamente com ninfas não infectadas de R. sanguineus. Foram observadas, alterações clínicas como febre, apatia e disorexia nos cães infectados, durante a fase aguda da infecção, porém de forma mais intensa e precoce nos cães infectados por inoculação intravenosa (grupo III). As alterações hematológicas mais evidentes foram redução do número de plaquetas, leve redução na série vermelha e branca. Todos os animais infectados soroconverteram aos 14 dias após inoculação ou infestação com carrapatos infectados (dpi), mantendo títulos entre 10240 e 81920. Os níveis de riquetsemia foram variáveis durante o curso da infecção, persistindo até 364 dpi, porém mais constante na fase aguda da doença. Um pequeno número de carrapatos alimentados nos cães infectados apresentou amplificação de DNA de E. canis, porém foram demonstrados até 308 dpi. Foi observada uma redução na relação CD4:CD8 nos animais infectados, no 28° dpi, mantendo-se de forma mais branda até 252 dpi. Uma maior proporção de linfócitos T CD4+ produtores de IFN-γ e IL-4 foi observada no tempo zero de cães que não adquiriram infecção após infestação com carrapatos infectados. Os níveis séricos de citocinas mostraram de forma mais evidente a elevação de IL-10 e TNF-α em um cão que desenvolveu doença fatal, podendo indicar a participação de mecanismos imunes na apresentação clínica e na persistência do agente no organismo hospedeiro. / The canine monocytic ehrlichiosis is characterized as a persistent infection, which can develop fatal disease. Immunopathogenic mechanisms are implicated in the development of the disease, however it is not completely understood the role of cellular immune in the infection caused by Ehrlichia canis transmitted by the tick Rhipicephalus sanguineus. The vector becomes infected only while feeding on rickettsemic dogs, however the level of rickettsemia is not recognized during the course of the infection in the dogs. The objective of this study was to verify the level of relative rickettsemia capable to infect the vector (R. sanguineus) during the course of the canine ehrlichiosis; and to evaluate the cellular immune induced in dogs exposed to Ehrlichia canis-infected ticks. A dog was used for production of the inoculum and infection of nymphs of R. sanguineus. Infected adult ticks that had fed as nymphs on the infected dog were used to feed on and transmit the infection to dogs of groups I (n=3) and II (n=3). Group III dogs (n=3) were intravenously inoculated with E. canis-infected dogs. Group IV (n=3) and V (n=3) dogs were the control groups, never exposed to E. canis. Dogs of groups II and IV were reinfested periodically with uninfected R. sanguineus nymphs. Clinical alterations such as fever, apathy and dysorexia were observed in the infected dogs during the acute phase of the infection, however in a more intense and precocious form in the dogs infected by intravenous inoculation (group III). The more evident hematological alterations were reduction of the number of platelets, mild reduction in the red and white cells series. All infected animals soroconverted by 14 days after inoculation or infestation by infected ticks (dpi), showing titers between 10240 and 81920 during the study. The rickettsemia levels were variable during the course of the infection, persisting up to 364 dpi, however more constant in the acute phase of the disease. A small number of ticks that fed on infected dogs presented amplification of E. canis DNA, however they were demonstrated up to 308 dpi. A reduction in the relationship CD4:CD8 in the infected animals was observed in 28th dpi, keeping in a mild form up to 252 dpi. A larger proportion of lymphocytes T CD4+ producing IFN-γ and IL-4 it was observed in the zero time of dogs that did not acquire infection after infestation with infected ticks. The cytokine seric levels showed the elevation in a more evident form of IL-10 and TNF-α in a dog that developed fatal disease, what could indicate the participation of immune mechanisms in the clinical presentation and in the persistence of the agent in the organism host.

A neuroimunologia é o ramo da imunologia que estuda a relação entre sistema imune e o sistema nervoso. Muitos estudos têm demonstrado a capacidade direta de neurotransmissores em modular a resposta imune, assim como de citocinas em influenciar funções cognitivas. Neste contexto, o glutamato possui papel de destaque, por se tratar do neurotransmissor excitatório mais importante e mais abundante no sistema nervoso central dos mamíferos. Sua função é exercida através de dois tipos de receptores principais: i) os receptores ionotrópicos (iGluR) e ii) os receptores metabotrópicos (mGluR). A descoberta da expressão de receptores de glutamato em células do sistema imune tem despertado interesse científico, levantando questões acerca de sua expressão e função. No presente trabalho, avaliamos parâmetros como viabilidade celular, linfoproliferação e ativação de MAP quinase pelo receptor NMDAR esplenócitos totais e linfócitos cultivados in vitro. Nossos resultados demonstram que linfócitos em repouso e ativados apresentam diferentes perfis de expressão do receptor NMDAR. O uso do antagonista deste receptor, o MK801, foi capaz de reduzir a proliferação de linfócitos T CD4 e T CD8 estimulados com anti-CD3 em cultura de esplenócitos. Tal redução pode ser explicada por um aumento na taxa de morte celular, o que foi avaliado através de marcação com anexina-V, indicador de apoptose, ou 7-AAD, indicador de necrose. Para entendermos um pouco a respeito da sinalização do receptor NMDAR no sistema imune, avaliamos a fosforilação da MAP quinase ERK 1,2 em linfócitos T CD4 ativados na presença do agonista (NMDA) ou do antagonista (MK801) do receptor. Observamos um aumento na ativação desta quinase na presença de NMDA, o que é revertido na presença do MK801. Ao avaliar o papel do receptor NMDAR in vivo, verificamos uma redução significativa na gravidade da encefalomielite experimental auto-imune em animais tratados com MK801. Mais interessante, esta redução se correlaciona também com uma redução na fosforilação de ERK 1,2 em esplenócitos totais obtidos ao dia 7 pós-imunização. Em resumo, nossos dados sugerem que o receptor NMDA possui o papel de ativador de vias intracelulares importantes, como as da MAP quinase ERK 1,2; e que o seu bloqueio resulta em morte celular in vitro. Logo, isso indica a importância do glutamato como modulador da intensidade da resposta e viabilidade de linfócitos T CD4 e T CD8 in vitro e in vivo. Sendo assim, nossos resultados contribuem para um melhor entendimento dos fenômenos de imunoregulação, especialmente aqueles no campo da neuroimunologia ou neuroimunomodulação. / Neuroimmunology is a field within immunology which studies the relationship between the nervous system and the immune system. Several studies have demonstrated the direct ability of neurotransmitters in modulating the immune response, as for cytokines in influencing cognitive functions. In this context, glutamate stands out for being the most important and abundant neurotransmitter in the mammal central nervous system. Its role is exerted through two main types of receptor: i) ionotropic receptors (iGluR) and ii) metabotropic receptors (mGluR). The discovery of glutamate receptor expression in immune cells has led to scientific interest, raising issues concerning its expression and function. In the present study, we evaluated parameters such as cell viability, lymphoproliferation, and activation of the MAP quinase pathway by the NMDA receptor on total splenocytes and lymphocytes cultured in vitro. Our results demonstrate that naive and activated lymphocytes present different profiles of NMDA receptor expression. The use of MK801, an antagonist for this receptor, was able to reduce the T CD4 and T CD8 lymphocyte proliferation stimulated with anti-CD3 in splenocyte culture. Such reduction may be explained by the increase of the cellular death rate, evaluated by annexin-V staining, indicator of apoptosis or 7-AAD, indicator of necrosis. With the intent of understanding part of the NMDA receptor signaling in the immune system, we evaluated the ERK 1,2 MAP quinase phosphorylation in T CD4 lymphocytes activated in the presence of the agonist (NMDA) or the antagonist (MK801) of the receptor. We observed an increase in this quinase activation in the presence of NMDA, which is reversed by the MK801. When evaluating the role of the NMDA receptor in vivo, we verified a significant reduction in the degree of experimental auto-immune encephalomyelitis in animals treated with MK801. More interesting, this reduction also correlates to a reduction on the phosphorilation of ERK 1,2 in total splenocytes obtained at the seventh day post-immunization. In sum, our data suggest that the NMDA receptor has the role of activating important intracellular pathways, such as the MAP quinases ERK 1,2; and that its blockage results in cellular death in vitro. As so, this indicates the importance of glutamate as a modulator of the intensity of response and the viability of T CD4 e T CD8 lymphocytes in vitro e in vivo. Thus, our result contribute for a better understanding of the immunoregulation phenomena, especially those in the neuroimmunology ou neuroimmunomodulation field.

L’immunothérapie allergénique (ITA) est la seule thérapie capable d’agir sur l’étiologie des allergies. La compréhension des mécanismes d’action de ce traitement et la mise en évidence de biomarqueurs d’efficacité favoriserait l’optimisation de l’ITA. A l’aide des tétramères de classe II, nous avons suivi les lymphocytes T CD4 périphériques spécifiques d’allergènes, acteurs centraux de la réaction allergique, dans des conditions normales, pathologiques ou en cours d’ITA, afin d’établir un lien entre ces trois situations physiologiques. Nous avons mis en évidence des différences entre les réponses lymphocytaires T CD4 spécifiques d’allergènes saisonniers et perannuels, chez les individus sains et allergiques. Puis, lors de 2 études cliniques d’ITA sublinguale, l’une menée chez des adultes allergiques aux pollens de graminées traités pendant 4 mois et l’autre menée chez des enfants allergiques aux acariens traités pendant 1, nous avons respectivement observé une diminution des lymphocytes Th2A et une augmentation de la production d’IFN- γ liées au traitement. Toutefois, ces variations ne corrèlent pas avec l’efficacité clinique de l’ITA observée dans ces deux études. Les limites d’utilisation des tétramères de classe II nous ont amené à rechercher si l’expression de marqueurs d’activation membranaires pouvait remplacer un marquage « tétramère ». Alors qu’une corrélation insuffisante a été observée entre le marquage « tétramère » et l’expression des marqueurs d’activation testés, nous avons mis en évidence 3 populations cellulaires aux propriétés fonctionnelles diverses, soulignant l’hétérogénéité des réponses lymphocytaires spécifiques d’allergènes. De plus, la découverte des lymphocytes Th2A pourrait être une approche prometteuse pour le suivi des réponses lymphocytaires T CD4 spécifiques d’allergènes lors d’ITA à plus long terme. / Allergenic immunotherapy (AIT) is currently the only curative treatment for allergic disease. Whereas efficacy of this treatment is well established, its mechanisms of action are not clearly understood and predictive as well as surrogate biomarkers are needed to further support AIT development. We focused on allergen specific CD4 T cells, highly involved in allergic inflammation, and monitored their responses both in normal and pathologic conditions, or during AIT. Using MHC class II tetramers, we highlighted distinct patterns of polarization between seasonal and perennial allergen-specific CD4 T cells as well as between healthy and allergic individuals. Then, allergen-specific CD4 T cell responses were monitored during 2 double-blind placebo-controlled sublingual AIT clinical trials. After short term AIT (4 months), we observed a decrease of Th2A cells, a newly define subset, thought to contain most allergen-specific CD4+ T cells. IFN-γ production was increased after one year of treatment. However, these variations were not related to AIT clinical efficacy. We further compared the expression of various activation markers and MHC class II tetramer staining following in vitro stimulation in order to circumvent inherent limitation of tetramers. No correlation could be established between tetramer staining and the expression of multiple activation markers in allergen-stimulated CD4 T cells. Combining these methods helps understanding patient heterogeneity regarding CD4 T cell responses. Moreover, Th2A cells detection is likely a promising approach to identify allergen-specific CD4 T cell during long-term AIT.