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Résumé : L’IL-7 et l’IL-15 sont des cytokines impliquées dans l’homéostasie des lymphocytes T CD8 naïfs et mémoires respectivement. Lors d’une réponse immunitaire, certaines cytokines pro-inflammatoires, comme l’IL-6 et l’IL-21, sont produites par les cellules du système immunitaire inné. Nous avons observé que certaines cytokines de ces deux groupes (homéostasie et pro-inflammatoires), peuvent avoir un effet synergique sur la fonction des lymphocytes T CD8. Spécifiquement, l’incubation des lymphocytes T CD8 naïfs avec l’IL-6 ou l’IL-21, en présence d’IL-7 ou d’IL-15 cause une forte prolifération qui est indépendante de l’antigène. De plus, la combinaison d’IL-15 avec l’IL-6 ou l’IL-21 entraîne une prolifération préférentielle des lymphocytes T mémoires, tandis que la combinaison avec l’IL-7 entraîne une prolifération des lymphocytes T naïfs. La stimulation des lymphocytes T CD8 avec l’IL-6 ou l’IL-21, en présence d’IL-7 ou d’IL-15, entraîne une augmentation de la phosphorylation en tyrosine de STAT5 ainsi qu’une augmentation de liaison à l’ADN. Nous avons étudié l’effet d’une pré-stimulation des cellules T CD8 naïves par les cytokines synergiques sur leur réponse subséquente à un antigène. Nous avons observé qu’une pré-stimulation avec l’IL-6 ou l’IL-21, en présence d’IL-7 ou d’IL-15, même pour une courte durée de 24 heures, augmente leur sensibilité aux antigènes, entraînant une robuste prolifération et une forte augmentation de cytotoxité spécifique à l’antigène gp33. Nous avons observé que les cytokines pro-inflammatoires en combinaison avec l’IL-7 induisent une augmentation accrue de la prolifération chez les lymphocytes T CD8 exprimant un TCR transgénique de forte affinité (P14), ainsi que les cellules exprimant un TCR de faible affinité (H-Y). De plus, la combinaison synergique de cytokines entraîne une forte expression du récepteur de l’IL-2R[gamma] (CD132), ainsi qu’une augmentation de la production d’IL-2 après stimulation antigénique. Une forte augmentation de l’expression de CD8 et de CD45, ainsi qu’une diminution drastique de l’expression de CD5 peut expliquer l’augmentation de l’avidité fonctionnelle du TCR suite à une stimulation avec les combinaisons de cytokines synergiques. La stimulation des lymphocytes T CD8 avec les combinaisons de cytokines, induit une augmentation de la phosphorylation de LAT ainsi qu‘AKT. Cependant, la stimulation subséquente du CD3 n’entraîne pas d’augmentation de la phosphorylation de LAT ainsi qu’AKT chez les lymphocytes T CD8 pré-stimulés avec les combinaisons de cytokines. Nous avons aussi observé que les lymphocytes T CD8 stimulés avec les combinaisons de cytokines augmentent l’expression de CD62L, ce qui peut favoriser leur migration vers les ganglions lymphatiques. En conclusion, la production de cytokines pro-inflammatoires (IL-6, IL-15, IL-21) par les cellules du système immunitaire inné lors d’une infection ou d’une inflammation, ainsi que la présence constitutive d’IL-7, peuvent stimuler la prolifération et l’activation des lymphocytes T CD8 de façon non spécifique à l’antigène. Cette stimulation entraîne une augmentation de l’avidité fonctionnelle de leur TCR causant ainsi une forte prolifération ainsi que l’acquisition de fonctions effectrices spécifiques. Cette liaison entre le système immunitaire inné et adaptatif, médiée par les cytokines pro-inflammatoires et les cytokines homéostatiques joue un rôle très important dans l’élimination des pathogènes ainsi que dans le développement de maladies auto-immunitaires. / Abstract : Homeostasis of naive and memory CD8[superscript +] T lymphocytes is dependent on two cytokines IL-7 and IL-15, respectively. During an immune response to an infection, cells of the innate immune system produce several pro-inflammatory cytokines. We have observed that these two groups of cytokines, namely proinflammatory and homeostatic, can have a synergistic effect on CD8 T lymphocytes. Specifically, incubation of naive CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 induced strong proliferation in an antigen independent manner. While the combination of IL-6 or IL-21 with IL-15 induced strong proliferation of memory CD8 T cells, naïve CD8 T cells responded better to the combination with IL-7. These stimulatory cytokine combinations elicited strong STAT5 phosphorylation and it’s binding to DNA in CD8 T cells. We investigated the effect of priming CD8 T cells with the synergistic combination of IL-6 or IL-21 and IL-7 on their subsequent response to antigen. We observed that cytokine priming for only 24 hours enhanced their sensitivity to antigen, resulting in strong proliferation, effectors functions and cytotoxicity. These effects were observed with CD8 T cells expressing transgenic TCR with strong (P14) or weak (H-Y) affinity towards cognate peptide antigens. Priming CD8 T cells with the synergistic combination of cytokines increased the expression of IL-2 receptor gamma (CD132) and augmented the production of IL-2 when stimulated with antigen. These cells also expressed elevated levels of CD8 and CD45, as well as down modulate CD5, and these events may underlie the increased TCR avidity. Stimulation of CD8 T cells with the synergistic combination of cytokines induced phosphorylation of LAT and AKT. However, subsequent TCR stimulation did not further increase these phosphorylation events. We have observed that C D8 T cells primed with the synergistic combinations of cytokines up regulated CD62L, which could promote their migration through lymph nodes. In conclusion, inflammatory cytokines such as (IL-6, IL-15, IL-21) secreted by cells of the innate immune system during an infection or non-infectious inflammation, and basal levels of the homeostatic cytokine IL-7 can act in synergy with inflammatory cytokines to activate CD8 T lymphocytes in an antigen independent manner. This stimulation also results in an increase in the functional avidity of their TCR, as indicated by strong antigen responsiveness with increased proliferation and display of effectors functions. This connection between the innate and adaptive system mediated by inflammatory cytokines may play an important role in pathogen clearance and possibly in the development of autoimmune diseases.

B-lymphocytes have traditionally been thought to contribute to immunity and autoimmune disease through terminal differentiation into plasma cells that secrete antibody. However, studies in mice and recent clinical studies have demonstrated that genetically altered B-cell function and B-cell-targeted therapies can significantly affect autoimmune diseases that were predominantly thought to be T-cell-mediated. B-cell depletion in mouse models of disease has also led to the identification of alternative B-cell effector functions that regulate normal immune responses and autoimmune disease. This review highlights multiple B-cell effector mechanisms, including the promotion of cellular immunity, the negative regulation of immune responses, and the production of pathogenic antibodies. / Dissertation

Objectifs : Les lymphocytes T régulateurs de par leur rôle primordial dans l'homéostasie de la réponse immune sont des cellules idéales pour une immunothérapie antigène-spécifique dans les maladies auto-immunes. Les lymphocytes T régulateurs de type 1 ou Tr1 sont caractérisées par une forte sécrétion d'IL-10, cytokine qui joue un rôle déterminant dans leur capacité à supprimer des réponses immunes pathologiques dans différents contextes. L'objectif de ma thèse est d'évaluer le potentiel thérapeutique de cellules Tr1 spécifiques du collagène de type II (col-Treg) dans deux modèles de polyarthrite rhumatoïde (PR) chez la souris. Méthode : Les clones Col-Treg ne possèdent pas de marqueurs membranaires spécifiques mais sont caractérisés par un profil cytokinique particulier (IL10highIL4negIFN-γint) et par leur capacité de suppression in vitro. Tout comme les Tregs naturels, ils expriment une quantité importante de GITR, de CD39 et de Granzyme B. Une simple injection de cellules Col-Treg réduit l'incidence et les symptômes cliniques de l'arthrite à la fois de manière préventive et curative, avec un impact significatif sur les anticorps anti-collagène de type II. En outre, l'injection de Tr1 antigène spécifique in vivo diminue de manière significative la prolifération des cellules T antigène spécifique. Conclusion : Nos résultats démontrent le potentiel thérapeutique des cellules Col-Treg dans deux modèles d'arthrite expérimentale prouvant que les cellules Col-Treg représente une nouvelle approche thérapeutique de choix pour le traitement des patients atteint de polyarthrite et réfractaires aux traitements actuels. / Objectives : Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for therapies to suppress inflammation in an antigen-specific manner. Type 1 Treg cells (Tr1) are defined by their capacity to produce high levels of IL10, which contributes to their ability to suppress pathological immune responses in several settings. The aim of my PhD was to evaluate the therapeutic potential of collagen type II-specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice. Methods : Col-Treg clones were isolated and expanded from Collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibodies- and collagen-induced arthritis models. The in vivo suppressive mechanism on effector T cell proliferation was also investigated. Results : Col-Treg clones are characterized by a cytokine profile (IL10highIL4negIFN-γint) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and Granzyme B. Single infusion of Col-Treg cells reduced incidence and clinical symptoms of arthritis both in preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific type 1 Treg cells decreases significantly the proliferation of antigen-specific effector T cells in vivo. Conclusion : Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients refractory to current treatments.

Les basophiles sont les granulocytes les plus rares. Ils sont impliqués dans la polarisation des réponses immunitaires de type Th2, dans la différenciation des lymphocytes B et dans la protection contre les infections helminthiques. Les basophiles sont impliqués dans la modulation des réponses immunitaires, en particulier dans les maladies auto-immunes et inflammatoires. Des études récentes ont montré que les basophiles murins sont cellules présentatrices d’antigène (CPA) et induisent des réponses Th2 et IgE contre les allergènes et les infections helminthiques.Par conséquent, Nous avons exploré les fonctions des basophiles humains, en particulier comme CPA professionnelles. Les résultats montrent que les basophiles, contrairement aux cellules dendritiques et monocytes, n’expriment pas HLA-DR et les marqueurs de co-stimulations CD80 et CD86. De plus, la stimulation des basophiles par divers allergènes, comme des ligands de TLR et IgE, n’induit pas des changements dans l’expression de ces marqueurs. Enfin, nos résultats montrent que les basophiles ne favorise pas les réponses immunitaire de type Th2 ou Th17. Ainsi,notre étude montre que les basophiles humains circulant ne possèdent pas des fonctions de CPA professionnelles. Des plus, les basophiles sont impliqués dans la pathogenèse de maladies auto-immunes et inflammatoires dépendantes des réponses Th2 et médiées par les lymphocytes B. Puisque la dérégulation des basophiles joue un rôle important dans le développement des réponses immunitaires dans différentes conditions pathologiques, nous avons exploré les mécanismes de régulations qui modulent les fonctions les basophiles. En particulier, nous avons étudié le rôle suppresseur des lymphocytes T régulateurs (Tregs) CD4+CD25+FoxP3, des cellules clés dans la maintenance de l’homéostasie immune, sur les fonctions des basophiles. Nos résultats montrent que les fonctions des basophiles, contrairement à la majorité des cellules immunes, ne sont pas régulées par les Tregs. Bien au contraire, nos résultats montrent que les lymphocytes T favorisent l’activation des basophiles. En résumé, nous avons exploré de nouveaux mécanismes cellulaires et moléculaires impliqués dans la régulation des fonctions des basophiles humains. Ces résultats nous permettent de mieux comprendre le rôle des basophiles dans les conditions inflammatoires et dans le développement de nouvelles stratégies thérapeutiques. / Basophils are the rare granulocytes and play an important role in the polarization of Th2 responses, differentiation of B cells and protection against helminths. Basophils have a major influence on immune responses and various roles of these cells in autoimmune and inflammatory diseases are emerging. Recent reports showed that murine basophils function as antigen presenting cells (APCs) to induce Th2 and IgE responses to allergens and helminths. Therefore, I explored whether human basophils possess the features of APCs. I found that unlike dendritic cells (DCs) and monocytes, steady-state circulating human basophils did not express HLA-DR and co-stimulatory molecules CD80 and CD86. Basophils remained negative for these molecules following stimulation with various allergens, toll-like receptor ligands and IgE cross-linking.Unlike DCs, basophils did not promote Th2 and Th17 responses. Together, these results demonstrate the inability of circulating human basophils to function as professional APC. Further, basophils were also reported to be implicated in the pathogenesis of Th2 –associated and B cell-mediated autoimmune and inflammatory diseases. Considering the impact of dysregulated function of basophils on the outcome immune responses in various pathological conditions, it was essential to investigate the regulatory mechanisms by which basophil functions are kept in check. As CD4+CD25+FoxP3+ regulatory T cells (Tregs) are critical for the maintenance of immune homeostasis, I sought to investigate the interaction of Tregs with human basophils and its repercussion on basophil functions. My results indicated that unlike other immune cells that aresusceptible to Treg-mediated suppression, basophils are refractory to regulatory mechanism of Tregs. On the contrary I found that T cells could promote activation of basophils. My results thus provided an insight on cellular and molecular basis of regulation of human basophil functions. These data will have a repercussion in understanding role of basophils ininflammatory conditions and in designing therapeutic strategies.

O controle de respostas imunes patológicas (autoimunidade, alergia, rejeição de transplantes) tem sido um dos principais objetivos dos imunologistas. Apesar dos avanços recentes, a maioria dos tratamentos atuais ainda procura diminuir a imunidade e inflamação em vez de restabelecer o estado saudável da tolerância imunológica. Sepse é uma doença desencadeada pela presença de bactérias e/ou produtos bacterianos como lipopolissacarídeos (LPS), componente principal da membrana externa de bactérias gram-negativas, ativando a resposta imune do hospedeiro. A caracterização do perfil de linfócitos na resposta à tolerância ao LPS são de extrema importância para a contribuição do estudo da imunodepressão na sepse. O objetivo deste estudo foi investigar se a comprovada redução de mortalidade previamente vista em modelo de sepse animal através tolerância ao LPS, pode ser associada com o aumento da população de linfócitos T CD4+ regulatórios e Th17. Camundongos machos C57/6, receberam por via subcutânea ( s.c.) injecções de LPS ( 1mg/kg ) durante 5 dias , seguido por perfuração e ligadura cecal (CLP ) . Citocinas e linfócitos marcados foram medidos durante, após a tolerância e o desafio CLP. Ambos os subtipos de células T analisados Treg e Th17 , mostrou aumento destas células no baço durante e após a tolerância. Este estudo demonstrou que a mortalidade reduzida depois de tolerância previamente constatada pode ser associada com o aumento da população de células T regulatórias e Th17 devido a imunorregulação do hiperinflamação e recrutamento de neutrófilos / The control of pathological immune responses (autoimmunity, allergy, transplant rejection) has been a major goal of immunologists. Despite recent advances, most current treatments still seeks to reduce immunity and inflammation rather than restore the healthy state of immune tolerance. Sepsis is a disease triggered by the presence of bacteria and / or bacterial products like lipopolysaccharide (LPS), the main component of the outer membrane of gram-negative bacteria, activating the immune response of the host. The characterization of lymphocyte profile in response to LPS tolerance is extremely important for the study of immunosuppression in sepsis contribution. The aim of this study was to investigate whether the proven reduction in mortality seen previously in animal sepsis model by tolerance to LPS, can be associated with the increase in population of CD4 + regulatory and Th17. Mice C57 / 6 mice received subcutaneous (s.c.) injection of LPS (1mg / kg) for 5 days, followed by cecal ligation and puncture (CLP). Cytokines and marked lymphocytes were measured during after tolerance and CLP challenge. Both subtypes of T cells Treg and Th17 analyzed showed an increase of these cells in the spleen during and after tolerance. This study demonstrated that reduced mortality after previously seen tolerance may be associated with increasing the population of regulatory T cells and Th17 because immunoregulation of the hiperinflamação and neutrophil recruitment

A paracoccidioidomicose (PCM), doença sistêmica de caráter granulomatoso, é causada pelo fungo termodimórfico Paracoccidioides brasiliensis (Pb). A gp43 secretada pelo Pb, possui um trecho específico de 15 aminoácidos designado como (P10), é reconhecido por linfócitos T. No presente trabalho, avaliamos a ativação da resposta imune e o efeito aditivo da imunização com o peptídeo P10, em camundongos induzidos à imunossupressão com dexametasona. Os resultados indicam um efeito aditivo da imunização com P10 e o tratamento com as drogas em camundongos BALB/c imunossuprimidos e infectados; associado a redução da carga fúngica no pulmão, baço e fígado desses animais, detectamos aumento de citocinas proinflamatorias, no homogenato de pulmão e no sobrenadante de cultura celular. Animais imunossuprimidos e imunizados com P10 apresentaram um aumento significativo na produção de Óxido Nítrico (NO). A eficiencia na resposta levou a um aumento na sobrevida de 100%. Estes resultados sugerem que o P10, representa uma alternativa promisoria na geração de uma vacina anti-PCM. / Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis (Pb). The gp43 secreted by Pb has a 15-mer peptide designated as P10 that is recognized by T lymphocytes. In the present work we evaluated the activation of immune response and the additive effect of P10 immunization in BALB/c mice induzed to immunossupression with Dexamethasone. Results indicate an additive effect of P10 immunization and the treatment with drugs in BALB/c mice immunosuppressed and infected; associated with a significant reduction in fungal burden in the lung, spleen and liver of these animals. Also, we detected an increase of proinflammatory cytokines such as IFN-<font face=\"symbol\">g, TNF-<font face=\"symbol\">a e IL-12, in lung homogenates and cell culture supernatant. Immunosuppressed animals that were immunized with P10 showed an increase in the Nitric Oxide production. The efficiency of response led to a 100% survival in animals immunized with P10 and treated with antifungal drugs. Our results suggest that P10 represents a promising alternative of anti-PCM vaccine generation.

Neste trabalho, investigamos os mecanismos da resposta imune estimulados por PAS-1 para o desencadeamento do seu efeito modulatório na inflamação alérgica pulmonar. Para tanto, camundongos C57BL/6 selvagens, IL-12-/-, IFN-<font face=\"Symbol\">g-/- e IL-10-/- foram imunizados e desafiados com OVA ou PAS-1 ou OVA + PAS-1. Ainda, camundongos C57BL/6 imunizados e desafiados com OVA receberam transferência adotiva de células CD19+, B220+, CD3+, CD4+, CD8+, CD4+CD25-, CD4+CD25+ PAS-1-primadas. Nossos resultados demonstraram que o efeito imunomodulatório de PAS-1 é mediado por IFN-<font face=\"Symbol\">g e IL-10, mas não por IL-12, e que somente camundongos que receberam células CD8+ ou CD4+CD25+ não desenvolveram inflamação pulmonar e produziram, respectivamente, IFN-<font face=\"Symbol\">g e IL-10/TGF-ß. Em conjunto, estes resultados demonstraram que o efeito imunomodulatório de PAS-1 é devido à estimulação de células T CD8+ produtoras de IFN-<font face=\"Symbol\">g e por CD4+CD25+ secretoras de IL-10 e TGF-ß. / In this work, we investigate the immune response mechanisms triggered by PAS-1 to promote its immunomodulatory effect in the lung allergic inflammation. For that, wild type, IL-12-/-, IFN-<font face=\"Symbol\">g-/- and IL-10-/- C57BL/6 mice were immunized and challenged with OVA or PAS-1 or OVA + PAS-1. Moreover, wild type C57BL/6 mice were adoptively transferred with PAS-1-primed CD19+, B220+, CD3+, CD4+, CD8+, CD4+CD25-, and CD4+CD25+ lymphocytes. Our results demonstrated that the immunomodulatory effect induced by PAS-1 is mediated by IFN-<font face=\"Symbol\">g and IL-10, but not by IL-12, and only mice which received CD8+ or CD4+CD25+ T cells did not present lung allergic inflammation and produced IFN-<font face=\"Symbol\">g and IL-10/TGF-ß, respectively. Taken together, these results demonstrated that the imunomodulatory effect induced by PAS-1 is due to stimulating CD8+ T cells, which secrete IFN-<font face=\"Symbol\">g, and CD4+CD25+ T cells, which secrete IL-10 and TGF-ß.

A sepse é descrita como uma disfunção orgânica ameaçadora à vida secundária à resposta desregulada do organismo a uma infecção, e é responsável por uma alta e crescente taxa de internação hospitalar no Brasil e no mundo. Apesar da diminuição da mortalidade ao longo do tempo, efeitos da doença a longo prazo como infecções secundárias e mortes tardias têm sido observadas. A sepse caracteriza-se pela liberação de mediadores inflamatórios e posterior imunossupressão compensatória, e o distúrbio na homeostase pode causar danos às células e órgãos levando a um estado mais grave da doença. Os linfócitos T reguladores (Tregs) são células responsáveis pela tolerância periférica e modulação de processos inflamatórios e são essenciais na imunossupressão observada na sepse. Este trabalho teve por objetivo estudar a relação entre a atividade imunológica (quantificação de Tregs, monócitos e citocinas inflamatórias) e a susceptibilidade a novas infecções e mortalidade em pacientes sépticos admitidos na Unidade de Emergência do HC-FMRP-USP. Foram incluídos 33 pacientes sépticos e 34 controles saudáveis, entre outubro de 2014 e novembro de 2015. Os pacientes foram acompanhados durante a internação na admissão (D0), D2, D7, D14, D21 e D28, e 12 pacientes retornaram para avaliação pelo menos três meses após a alta. Os pacientes tiveram porcentagens de Treg em linfócitos CD4+ elevados em relação aos controles nos momentos D2 e retorno (p = 0,0004), sendo que no D2 pacientes que desenvolveram complicação infecciosa tiveram porcentagens significativamente menores que os que não desenvolveram complicação (p = 0,0015). Além disso, porcentagens altas de Treg na admissão (D0) correlacionaram-se inversamente com o tempo de internação dos pacientes sobreviventes (p = 0,0271). Valores absolutos de Treg nos pacientes estiveram significativamente elevados no retorno em relação à admissão (p = 0,0074). Pacientes no retorno tiveram maior porcentagem de monócitos CD163+ em relação aos controles (p = 0,036). Pacientes na admissão tiveram menor porcentagem demonócitos HLA-DR+ em relação aos controles e aos pacientes no retorno (p = 0,0057). Pacientes tiveram valores de IL-6, IL-8, IL-10 e ST2 superiores em relação aos controles em diversos momentos da internação e retorno (p < 0,0001, p < 0,0001, p = 0,0030 e p < 0,0001, respectivamente). Valores de ST2 na admissão correlacionaram-se com o escore SOFA (p = 0,0252), tempo de internação (p = 0,0105), presença de complicação infecciosa (p = 0,0356) e mortalidade (p = 0,0114) dos pacientes. Valores de IL-8 na admissão também se correlacionaram com a presença de complicação infecciosa (p = 0,0387). Podemos inferir, portanto, que apesar de uma inflamação exacerbada já na admissão dos pacientes, evidenciada por altos valores de citocinas inflamatórias, há um aumento posterior de células Treg, imunorreguladoras, no D2, e o mesmo foi benéfico em relação à recuperação mais rápida dos pacientes e instalação de uma nova infecção. Porém, a imunossupressão continua sendo exibida mesmo após a alta dos pacientes, como podemos observar com a alta porcentagem de Tregs e monócitos CD163+ e valores de IL-10 nos pacientes em retorno em relação aos controles. Além disso, muitos dos pacientes sobreviventes à primeira internação foram internados novamente, e alguns deles foram a óbito no ano seguinte. / Sepsis is defined by a life-threatening organ dysfunction caused by a dysregulated host response to infection, and it is responsible for a high and increasing rate of hospital admission in Brazil and worldwide. Despite the decrease in mortality over time, long-term disease effects such as secondary infections and late deaths have been observed. Sepsis is characterized by the release of inflammatory mediators and subsequent compensatory immunosuppression, and the homeostasis disorder can cause damage to cells and organs leading to a more severe disease state. Regulatory T lymphocytes (Tregs) are cells responsible for peripheral toleran ce and modulation of inflammatory processes and are essential in the immunosuppression observed in sepsis. This study aimed to investigate the relationship between immunological activity (quantification of Tregs, monocytes and inflammatory cytokines) and susceptibility to new infections and mortality in septic patients admitted to the HC-FMRP-USP Emergency Unit. Thirty-three septic patients and 34 healthy controls were included between October 2014 and November 2015. Patients were followed up at admission (D0) and during hospitalization at D2, D7, D14, D21 and D28, and 12 patients returned for evaluation at least three months after discharge. Patients had higher Treg percentages on CD4 + lymphocytes than had controls at D2 and on return (p = 0.0004), whereas in D2 patients who developed infectious complications had significantly lower percentages than those who did not develop complications (p = 0.0015). In addition, high percentages of Treg at admission (D0) correlated inversely with the length of hospital stay of surviving patients (p = 0.0271). Absolute values of Treg in patients were significantly elevated in the return relative to admission (p = 0.0074). Patients on return had a higher percentage of CD163 + monocytes than controls (p = 0.036). Patients on admission had a lower percentage of HLA-DR+ monocytes than controls and patients on return (p = 0.0057). Patients had higher IL-6, IL-8, IL-10 and ST2 levels than controls at various times ofhospitalization and return (p < 0.0001, p < 0.0001, p = 0.0030 and p < 0.0001, respectively). ST2 values at admission were correlated with SOFA score (p = 0.0252), length of hospital stay (p = 0.0105), presence of infectious complication (p = 0.0356) and mortality (p = 0.0114). IL-8 values at admission also correlated with the presence of infectious complication (p = 0.0387). We can infer, therefore, that despite exacerbated inflammation as soon as at the admission of the patients, demonstrated by high levels of inflammatory cytokines, there is a posterior increase of immunoregulatory Treg cells at D2, and that was beneficial in relation to the faster recovery of the patients and setting up of new infection. However, immunosuppression continues to appear even after discharge of patients, as can be observed with the high percentage of CD163 + monocytes, Tregs and L-10 levels in patients on return compared to controls. In addition, many of the patients surviving the first hospitalization were hospitalized again, and some of them died the following year.

Linfócitos T CD4+ (LTCD4) sofrem morte pelo reestímulo do TCR em um processo chamado de AICD (activation-induced cell death) no fim de uma resposta imune. Resultados prévios de nosso grupo de pesquisa mostraram que melatonina foi capaz de inibir o processo de AICD em LTCD4 em experimentos in vitro. Portanto, o objetivo deste trabalho é verificar se a melatonina é capaz de agir como estimulador, aumentando a resposta imune mediada pelos LTCD4 in vivo. Nós observamos que 3 e 9mg/Kg de melatonina aumentaram a resposta de DTH (Delayed-type Hypersensitivity), de forma diretamente proporcional à dose utilizada. O tratamento com melatonina estimulou um aumento da proliferação e do numero absoluto de LTCD4 específicos de antígeno. Em experimentos de diferenciação linfocitária in vitro, nós observamos que o tratamento com melatonina estimulou a produção de LTCD4 do perfil Th1 e Th2, no entanto inibiu a produção de linfócitos Th17. Em conclusão, nossos resultados sugerem um efeito estimulador da melatonina sobre a função de linfócitos T CD4+. / CD4+ T lymphocytes (LTCD4+) suffer cell death by a process known as activation-induced cell death (AICD) at the end of immune response. Previous results from our group showed that melatonin can inhibits AICD process in LTCD4 at in vitro experiments. Therefore, the aim of this work is verify if a melatonin can act as immune stimulator increasing a LTCD4-mediated immune response in vivo. We showed that 3 and 9 mg/Kg of exogenous melatonin added during immunization resulted in potentiation dose-dependent of Delayed-type hypersensitivity (DTH) response. The treatment with melatonin increased the absolute number LTCD4 antigen-specific, probably by increment of its proliferation. In experiment of T cell differentiation, we observed that the treatment with melatonin stimulated LTCD4 production of Th1 and Th2 profile however blocked the Th17 lymphocytes production. In conclusion, our results support the idea about a regulator role of melatonin on LTCD4 lymphocytes function for development of an immune response.