INTRODUÇÃO: A variante hipocromiante da micose fungoide - MF - (MFh) apresenta características peculiares, como a predileção por indivíduos jovens e melanodérmicos e curso clínico crônico. Estudos especulam a patogênese da hipocromia comparando-a à do vitiligo. No Brasil, faltam dados que permitam conhecer sua importância na saúde pública. O presente trabalho visou avaliar a epidemiologia, a histopatologia e a imunofenotipagem de uma amostra de pacientes com diagnóstico de MFh e propor hipóteses dos mecanismos patogênicos da hipocromia, além de comparar pacientes portadores de lesões hipocrômicas exclusivas com aqueles portadores de outras formas de MF com lesões hipocrômicas concomitantes. MÉTODOS: Foram selecionados pacientes do Ambulatório de Linfomas Cutâneos do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e classificados em três grupos: A (21 portadores apenas de lesões hipocrômicas); B (15 portadores de outras formas de MF com lesões hipocrômicas concomitantes) e C (8 pacientes com diagnóstico de MF clássica, estes apenas para avaliações histológica e imuno-histoquímica). Foram obtidos dados clinicoepidemiológicos e realizadas análises histológica e imuno-histoquímica de biópsias das lesões e de pele normal, como controle. Para o estudo imuno-histoquímico foram utilizados os marcadores para imunofenotipagem da neoplasia, Melan-A, tirosinase, SCF, CD117 e MITF. RESULTADOS: Do total de pacientes acompanhados naquele ambulatório, os pacientes com MF portadores de lesões hipocrômicas corresponderam a 16%. As medianas das idades de início da doença e dos tempos de história foram de, no grupo A 25 anos e 8 anos; no grupo B, 29 anos e 13 anos, respectivamente; houve predomínio de indivíduos melanodérmicos , acometimento do sexo feminino e a maioria dos pacientes encontrava-se em estágios iniciais da doença em ambos os grupos. A avaliação histológica revelou achados semelhantes, como epidermotropismo de linfócitos atípicos e infiltrado dérmico linfomonocitário nas lesões hipocrômicas e não-hipocrômicas. O imunofenótipo CD8+ do infiltrado neoplásico epidérmico foi mais frequente no grupo A, ao passo que os grupos B e C apresentaram mais casos com imunofenótipo CD4+. A avaliação da função melanocítica das lesões hipocrômicas do grupo A revelou diminuição significativa da imunomarcação dos melanócitos por todos marcadores em comparação à pele normal e às lesões do grupo C. Em relação ao grupo B, não houve diferenças para as lesões hipocrômicas, não-hipocrômicas e pele normal, quando avaliadas dentro do próprio grupo (exceto para Melan A). A expressão de SCF pelos queratinócitos foi irregular sobretudo nas lesões hipocrômicas. DISCUSSÃO: Os pacientes com lesões hipocrômicas apresentaram características semelhantes (idade precoce, predomínio do sexo feminino, doença indolente). Mostrou-se que indivíduos melanodérmicos tem maior chance de apresentar lesões hipocrômicas. Além da redução de melanócitos e do receptor melanocítico CD117 em relação à pele normal já demonstradas previamente, mostrou-se, como no vitiligo, a redução da expressão do MITF, fator vital para a função e sobrevida do melanócito. Além disso, também se explicitou desbalanço da produção de citocinas melanogênicas pelos queratinócitos. CONCLUSÃO: A presença de lesões hipocrômicas pode ser considerada um marcador de bom prognóstico na MF. Diferentes mecanismos, como ação celular citotóxica e a alteração do microambiente da unidade epidérmica, colaboram para hipocromia das lesões da MFh / INTRODUCTION: The hypopigmented variant of mycosis fungoides - MF - (MFh) presents specific characteristics, such as a predilection for young and melanodermic individuals, and chronic clinical course. Studies speculate the pathogenesis of the hypopigmentation comparing it to vitiligo\'s. In Brazil, the lack of data prevents the knowledge of its importance in public health. This study aimed to evaluate the epidemiology, the histopathology and the immunophenotyping of a sample of patients diagnosed with MFh and to propose hypotheses of the pathogenic mechanisms of hypopigmentation, in addition to comparing exclusive hypopigmented lesion-bearer patients with those bearing other types of MF with concomitant hypopigmented lesions. METHODS: Patients were selected from the Cutaneous Lymphoma Clinic, from Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo and classified in three groups: A (21 hypopigmented only lesion- bearers); B (15 bearers of other types of MF with concomitant hypopigmented lesion) and C (8 patients diagnosed with classical MF, being those only for histology and immunohistochemistry evaluations). Clinical- epidemiological data were obtained and histology and immunohistochemistry analyses of lesion biopsies and normal skin, as a control, were made. For the immunohistochemistry study, the markers for immunophenotyping the neoplasm, Melan-A, tyrosinase, SCF, CD117 and MITF were used. RESULTS: Of the total number of patients treated at that clinic, the MF patients bearing hypopigmented lesions were 16%. The medians of the age of disease onset and the medical history time were 25 years and 8 years in group A; 29 years and 13 years in group B, respectively; there were a predominance of melanodermic individuals, involvement of the female sex, and the majority of the patients were in early stages of the disease in both groups. The histological evaluation revealed similar findings, such as epidermotropism of atypical lymphocytes and lympho-monocytic dermal infiltrate in hypopigmented and non-hypopigmented lesions. The CD8+ immunophenotype of the epidermal neoplastic infiltrate was more frequent in group A, while groups B and C showed more cases of CD4+ immunophenotype. The evaluation of the melanocytic function of the hypopigmented lesions in group A revealed a significant decrease of immunostaining of the melanocytes by all markers when compared to normal skin and group C lesions. Regarding group B, there were no differences to hypopigmented and non-hypopigmented lesions and normal skin, when evaluated within the group itself (except for Melan A). The SCF expression by the keratinocytes was irregular especially in hypopigmented lesions. DISCUSSION: Patients with hypopigmented lesions showed similar characteristics (early age, female sex predominance, indolent disease). It has been showed that melanodermic subjects are more likely to have hypopigmented lesions. In addition to the previously-showed reduction of melanocytes and CD117 melanocytic receptor related to normal skin, it has been showed, as in vitiligo, the reduction of MITF expression, a vital factor for the function and survival of the melanocyte. Besides that, it has been also made explicit a production imbalance of melanogenic cytokines by the keratinocytes. CONCLUSION: The presence of hypopigmented lesions can be considered a marker of good prognosis in MF. Different mechanisms, such as cytotoxic cellular action and the change of the microenvironment of the epidermal unit, collaborate for the hypopigmentation of the lesions of MFh

L’infection par le VIH induit une activation immunitaire chronique qui est suspectée d’être un des moteurs de la pathogénèse du SIDA. L’identification des mécanismes de contrôle de cette activation immunitaire, ainsi qu’une meilleure compréhension du contrôle spontané de l’infection chez certains patients, sont des étapes essentielles vers la conception de thérapies innovantes. Nous avons utilisé le modèle d’infection du macaque cynomolgus (Macaca fascicularis) par le virus de l’immunodéficience simienne (SIV) pour étudier ces deux points fondamentaux de la physiopathologie de l’infection.Il est proposé que l’induction précoce de l’activation immunitaire chronique soit une conséquence de la surexpression des gènes induits par les interférons (ISG) en réponse aux interférons de type I (IFN-I). Ces IFN-I ( et ) sont notamment produits par les cellules dendritiques plasmacytoïdes (pDC) en réponse aux virus. Notre premier objectif a été d’étudier la dynamique de production des IFN-I et celle des pDC durant l’infection, en parallèle de celle de l’activation immunitaire chronique et de la virémie. Nos résultats montrent que les pDC sont activées dans les tissus et qu’elles sont responsables de la production d’IFN-I observée transitoirement au cours de la primo-infection. La dynamique des pDC (apoptose, activation, renouvellement) entraîne un épuisement de la capacité des pDC à produire de l’IFN-I, qui pourrait rendre compte à la fois de l’altération fonctionnelle des pDC et de l’arrêt de la production d’IFN-I. De manière surprenante, ce contrôle de la production d’IFN-I n’est pas suivi d’un contrôle de la surexpression des ISG, ce qui suggère l’existence d’autres mécanismes inducteurs des ISG et souligne l’origine multifactorielle de l’activation immunitaire chronique.Dans une seconde étude, nous avons analysé l’impact d’une déplétion in vivo des lymphocytes exprimant le CD8 chez des animaux contrôlant spontanément la réplication virale sur le long terme. Quatre des cinq animaux de l’étude n’expriment pas de complexe majeur d’histocompatibilité (CMH) précédemment associé au contrôle, et aucun d’entre eux ne présente de forte réponse LT CD8. La déplétion transitoire des cellules CD8 entraîne chez quatre des contrôleurs une augmentation transitoire de la virémie qui se stabilise ensuite à des valeurs similaires aux niveaux pré-déplétion lors du retour des cellules CD8+. Un de ces animaux contrôle sa virémie avant la restauration des LT CD8. Chez le cinquième animal, la déplétion des CD8 n’a pas été accompagnée d’une élévation de virémie. Globalement, le contrôle de la virémie après l’élévation transitoire n’a pas été accompagné d’une augmentation de leur fonction antivirale. En revanche, une expansion et une activation des LT CD4 consécutive à la déplétion des CD8 ont été remarquées et corrélées positivement avec la virémie plasmatique. Ces résultats suggèrent que les réponses LT CD8 ne sont pas les principales responsables du contrôle à long terme de la virémie chez ces animaux. Dans notre modèle, d’autres mécanismes, tels qu’un réservoir de virus limité ou un meilleur contrôle de l’activation immunitaire, semblent participer à ce phénotype de contrôle.En conclusion, ces résultats éclairent la contribution des pDC, des IFN-I et des LT CD8 dans la physiopathologie du VIH, et permettent de proposer un nouveau modèle d’étude des mécanismes immunologiques précoces mis en place chez les patients contrôleurs de la virémie à long terme. / HIV infection induces a chronic immune activation, which is suspected to be a driving force in the pathogenesis of AIDS. Identifying control mechanisms of this immune activation, and a better understanding of the spontaneous control observed in some patients, are essential steps towards the development of innovative therapies. We used the model of cynomolgus macaques (Macaca fascicularis) infected by the simian immunodeficiency virus (SIV) to study these two fundamental fields of HIV pathogenesis.It is proposed that early induction of chronic immune activation is a consequence of an interferon-induced genes (ISG) overexpression in response to type I interferons (IFN-I). These I IFN (α and β) are preferentially produced by plasmacytoid dendritic cells (pDC) in response to the virus. Our first objective was to study the dynamics of pDC and IFN-I production during infection, together with chronic immune activation and viremia analysis. Our results indicate that pDCs are activated in tissues and are responsible for the transient IFN-I production observed during primary infection. The dynamics of pDCs (apoptosis, activation, renewal) induces an impaired IFN-I production by pDC, which could account both the functional defect of pDCs and the arrest of IFN-I production. Surprisingly, control of IFN-I production is not followed by down-regulation of ISG, which suggests the existence of other mechanisms that induce ISG and emphasizes the multifactorial origin of chronic immune activation.In a second study, we analyzed the impact of a in vivo CD8 T cells depletion in animals which spontaneously control viral replication in the long term. Importantly, four of the five animals in the study do not express major histocompatibility complex (MHC) previously associated with control, and none of them display strong CD8 response. The transient depletion of CD8 cells results in four controllers in a transient increase in viremia, which then stabilizes at values similar to pre-depletion levels when CD8+ cells come back. One of these animals controls their viremia before the restoration of CD8. In the fifth animal, CD8 depletion was not followed by a rise in viremia. Overall, the control of viremia after the transient increase was not associated to an increase of the antiviral function of CD8 T cells. In contrast, CD4 T cells expansion and activation were noticed and positively correlated to plasma viremia. These results suggest that CD8 responses are not the main cause of long-term control of viremia in these animals. In our model, other mechanisms, such as smaller reservoir or better control of immune activation, seem to be involved in this controller phenotype.In conclusion, these results shed light on the contribution of pDCs, IFN-I and CD8 T cells in the pathogenesis of HIV, and allow us to propose a new model for studying early immunological mechanisms in HIV controllers.

INTRODUÇÃO: A Síndrome de Sézary (SS) é um linfoma cutâneo de células T (LCCT), caracterizado por eritrodermia, linfadenopatia generalizada e presença de células tumorais na pele, linfonodos e sangue periférico. Os linfócitos TCD8+ têm papel fundamental na resposta imune antitumoral, entretanto, há escassos estudos evidenciando seu perfil fenotípico e funcional. Considerando que a resposta imunológica do paciente com SS está suprimida, estratégias para potencializar a imunidade inata e adaptativa com agonistas de receptores Toll-like (TLRs) têm sido exploradas. OBJETIVO: Caracterizar o perfil de marcadores de ativação/inibição das células TCD8+, seus estágios de diferenciação, capacidade de resposta a IL-7/IL-15 e ao agonista de TLR7/TLR8 de pacientes com SS. METODOLOGIA: Foram selecionados 15 pacientes com SS (7 homens e 8 mulheres) com 48-85 anos do Ambulatório de Linfomas Cutâneos, do HC-FMUSP, e um grupo de controle com 24 indivíduos sadios. A análise de marcadores de ativação/inibição e diferenciação celular em células TCD4/TCD8+ do sangue periférico foi realizada por citometria de fluxo. A expressão de marcadores extracelulares e citocinas intracelulares em células mononucleadas do sangue periférico (CMN) após estimulação com o agonista de TLR7/TLR8 foi analisada por citometria de fluxo. Além disto, o efeito de IL-7 e IL-5 em células T foi avaliado pela fosforilação de STAT5, na capacidade de proliferação mitogênica e expressão de BCL-2 em CMNs, como também pelos níveis séricos de IL-7 por citometria de fluxo. RESULTADOS: Os pacientes com SS mostram perfil fenotípico de ativação crônica nos linfócitos TCD8+ periféricos, decorrente do elevadopercentual de células TCD8+ CD38+, redução percentual de TCD8+ CD127+ (IL-7R) e da população naive. Além disso, ocorreu aumento de expressão de PD-1 na população naive de células TCD8+. O marcador de ativação, CD26, até então apenas relacionado com linfócitos TCD4, foi detectado em reduzida percentagem de linfócitos TCD8. A resposta para IL-7/IL-15 parece estar funcionalmente presente tanto nos linfócitos TCD4 quanto nos linfócitos TCD8. Contudo, foi encontrado um perfil diferenciado e heterogêneo de fosforilação de STAT5 assim como de expressão de BCL-2 nos linfócitos TCD8+ de pacientes com SS. O nível sérico de IL-7 reduzido dos pacientes com SS foi inversamente correlacionado com o número absoluto de linfócitos TCD4+. CONCLUSÃO: Os linfócitos TCD8+ dos pacientes com SS encontram-se reduzidos em números absolutos, e possuem um perfil alterado de diferenciação celular e expressão de marcadores extracelulares. A redução percentual da população de TCD8+ naive associada com a presença de moléculas de ativação crônica mostra um perfil de imunosenescência. As células TCD8+ exibem baixa capacidade de resposta aos ligantes de TLR intracelulares, provavelmente devido ao perfil de ativação crônica. Além disso, há resposta parcial dos linfócitos TCD8+ às citocinas ligantes do receptor yc. Nossos resultados evidenciam alterações em linfócitos TCD8+ que debilitam a resposta imune antitumoral e que pode contribuir com a patogênese da síndrome de Sézary / INTRODUCTION: Sézary syndrome (SS) is a cutaneous T cell lymphoma (CTCL), characterized by erythroderma, generalized lymphadenopathy and the presence of tumor cells in the skin, lymph nodes and peripheral blood. The TCD8+ lymphocytes play a key role in anti-tumor immune response, whereas, there are few studies showing its phenotypic and functional profile in SS. Considering that the immune response of SS patient is suppressed, strategies to enhancing the innate and adaptive immunity by Toll-like receptors (TLRs) agonists have been explored. OBJECTIVE: To characterize the profile of activation/inhibition markers of CD8+ T cells, their stages of differentiation, ability of response to IL-7/IL-15 and TLR7/TLR8 agonist of patients with SS. METHODOLOGY: Fifteen SS patients were enrolled (7 men and 8 woman) with 48-85 years from the Clinic of Cutaneous Lymphomas, HC-FMUSP, and a control group of 24 healthy individuals. Analysis of activation/inhibition markers and cellular differentiation in CD4/CD8 T cells from peripheral blood were assessed by flow cytometry. The expression of extracellular markers and intracellular cytokines in mononuclear cells in the peripheral blood (CMN) were evaluated by flow cytometry. Moreover, the effect of IL-7 and IL-15 stimulation in T cells was assessed by the STAT5 phosphorylation, proliferative mitogenic capacity, BCL-2 expression in CMNs as well as serum IL-7 levels by flow cytometry. RESULTS: Patients with SS show a phenotypic CD8 T peripheral lymphocytes profile of chronic activation, due to the high percentage of CD8+CD38+ T cells, reduced percentage of CD8+CD127+ (IL-7R) and naïve population. Furthermore, it was observed an increased PD-1 expression in the naïve CD8+ T cells. The activation marker CD26, previously only associated with CD4 T lymphocyte, was detected at decreased percentage in CD8 T lymphocytes. The TLR7/TLR8 agonist did not affect the IFN-? and TNF secretion of CD8 T lymphocytes of SS patients, in contrast to the control group. The response to IL-7/IL-15 appears to be functional in both CD4 and CD8 T lymphocytes. However, it was founded a differentiated and heterogeneous profile of STAT5 phosphorylation and Bcl-2 expression in the CD8 T lymphocytes in SS patients. The reduced IL-7 serum of patients with SS was inversely correlated with the absolute number of CD4 T lymphocytes. CONCLUSION: CD8 T lymphocytes of patients with SS are reduced in absolute numbers, and show an altered cellular differentiation profile and extracellular markers expression. The reduced percentage of CD8 naïve population associated with chronic activation of molecules reveals an immunosenescence profile. The CD8 T cells exhibit low ability to ligands of intracellular TLR receptors, probably due to chronic activation profile. In addition, there are partial response of CD8 T lymphocytes to the cytokine receptor ?c. Our results show disturbance in CD8 T lymphocytes that may impair the anti-tumor response contributing to the pathogenesis of Sézary syndrome

INTRODUÇÃO: A variante hipocromiante da micose fungoide - MF - (MFh) apresenta características peculiares, como a predileção por indivíduos jovens e melanodérmicos e curso clínico crônico. Estudos especulam a patogênese da hipocromia comparando-a à do vitiligo. No Brasil, faltam dados que permitam conhecer sua importância na saúde pública. O presente trabalho visou avaliar a epidemiologia, a histopatologia e a imunofenotipagem de uma amostra de pacientes com diagnóstico de MFh e propor hipóteses dos mecanismos patogênicos da hipocromia, além de comparar pacientes portadores de lesões hipocrômicas exclusivas com aqueles portadores de outras formas de MF com lesões hipocrômicas concomitantes. MÉTODOS: Foram selecionados pacientes do Ambulatório de Linfomas Cutâneos do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e classificados em três grupos: A (21 portadores apenas de lesões hipocrômicas); B (15 portadores de outras formas de MF com lesões hipocrômicas concomitantes) e C (8 pacientes com diagnóstico de MF clássica, estes apenas para avaliações histológica e imuno-histoquímica). Foram obtidos dados clinicoepidemiológicos e realizadas análises histológica e imuno-histoquímica de biópsias das lesões e de pele normal, como controle. Para o estudo imuno-histoquímico foram utilizados os marcadores para imunofenotipagem da neoplasia, Melan-A, tirosinase, SCF, CD117 e MITF. RESULTADOS: Do total de pacientes acompanhados naquele ambulatório, os pacientes com MF portadores de lesões hipocrômicas corresponderam a 16%. As medianas das idades de início da doença e dos tempos de história foram de, no grupo A 25 anos e 8 anos; no grupo B, 29 anos e 13 anos, respectivamente; houve predomínio de indivíduos melanodérmicos , acometimento do sexo feminino e a maioria dos pacientes encontrava-se em estágios iniciais da doença em ambos os grupos. A avaliação histológica revelou achados semelhantes, como epidermotropismo de linfócitos atípicos e infiltrado dérmico linfomonocitário nas lesões hipocrômicas e não-hipocrômicas. O imunofenótipo CD8+ do infiltrado neoplásico epidérmico foi mais frequente no grupo A, ao passo que os grupos B e C apresentaram mais casos com imunofenótipo CD4+. A avaliação da função melanocítica das lesões hipocrômicas do grupo A revelou diminuição significativa da imunomarcação dos melanócitos por todos marcadores em comparação à pele normal e às lesões do grupo C. Em relação ao grupo B, não houve diferenças para as lesões hipocrômicas, não-hipocrômicas e pele normal, quando avaliadas dentro do próprio grupo (exceto para Melan A). A expressão de SCF pelos queratinócitos foi irregular sobretudo nas lesões hipocrômicas. DISCUSSÃO: Os pacientes com lesões hipocrômicas apresentaram características semelhantes (idade precoce, predomínio do sexo feminino, doença indolente). Mostrou-se que indivíduos melanodérmicos tem maior chance de apresentar lesões hipocrômicas. Além da redução de melanócitos e do receptor melanocítico CD117 em relação à pele normal já demonstradas previamente, mostrou-se, como no vitiligo, a redução da expressão do MITF, fator vital para a função e sobrevida do melanócito. Além disso, também se explicitou desbalanço da produção de citocinas melanogênicas pelos queratinócitos. CONCLUSÃO: A presença de lesões hipocrômicas pode ser considerada um marcador de bom prognóstico na MF. Diferentes mecanismos, como ação celular citotóxica e a alteração do microambiente da unidade epidérmica, colaboram para hipocromia das lesões da MFh / INTRODUCTION: The hypopigmented variant of mycosis fungoides - MF - (MFh) presents specific characteristics, such as a predilection for young and melanodermic individuals, and chronic clinical course. Studies speculate the pathogenesis of the hypopigmentation comparing it to vitiligo\'s. In Brazil, the lack of data prevents the knowledge of its importance in public health. This study aimed to evaluate the epidemiology, the histopathology and the immunophenotyping of a sample of patients diagnosed with MFh and to propose hypotheses of the pathogenic mechanisms of hypopigmentation, in addition to comparing exclusive hypopigmented lesion-bearer patients with those bearing other types of MF with concomitant hypopigmented lesions. METHODS: Patients were selected from the Cutaneous Lymphoma Clinic, from Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo and classified in three groups: A (21 hypopigmented only lesion- bearers); B (15 bearers of other types of MF with concomitant hypopigmented lesion) and C (8 patients diagnosed with classical MF, being those only for histology and immunohistochemistry evaluations). Clinical- epidemiological data were obtained and histology and immunohistochemistry analyses of lesion biopsies and normal skin, as a control, were made. For the immunohistochemistry study, the markers for immunophenotyping the neoplasm, Melan-A, tyrosinase, SCF, CD117 and MITF were used. RESULTS: Of the total number of patients treated at that clinic, the MF patients bearing hypopigmented lesions were 16%. The medians of the age of disease onset and the medical history time were 25 years and 8 years in group A; 29 years and 13 years in group B, respectively; there were a predominance of melanodermic individuals, involvement of the female sex, and the majority of the patients were in early stages of the disease in both groups. The histological evaluation revealed similar findings, such as epidermotropism of atypical lymphocytes and lympho-monocytic dermal infiltrate in hypopigmented and non-hypopigmented lesions. The CD8+ immunophenotype of the epidermal neoplastic infiltrate was more frequent in group A, while groups B and C showed more cases of CD4+ immunophenotype. The evaluation of the melanocytic function of the hypopigmented lesions in group A revealed a significant decrease of immunostaining of the melanocytes by all markers when compared to normal skin and group C lesions. Regarding group B, there were no differences to hypopigmented and non-hypopigmented lesions and normal skin, when evaluated within the group itself (except for Melan A). The SCF expression by the keratinocytes was irregular especially in hypopigmented lesions. DISCUSSION: Patients with hypopigmented lesions showed similar characteristics (early age, female sex predominance, indolent disease). It has been showed that melanodermic subjects are more likely to have hypopigmented lesions. In addition to the previously-showed reduction of melanocytes and CD117 melanocytic receptor related to normal skin, it has been showed, as in vitiligo, the reduction of MITF expression, a vital factor for the function and survival of the melanocyte. Besides that, it has been also made explicit a production imbalance of melanogenic cytokines by the keratinocytes. CONCLUSION: The presence of hypopigmented lesions can be considered a marker of good prognosis in MF. Different mechanisms, such as cytotoxic cellular action and the change of the microenvironment of the epidermal unit, collaborate for the hypopigmentation of the lesions of MFh

Modelos murinos de infecção revelaram que as células T CD8+ (LTCs) são essenciais no controle de Trypanosoma cruzi, agente etiológico da Doença de Chagas. No entanto, durante a doença, os LTCs são notavelmente alterados e sub-ótimos, estabelecendo a fase crônica. Nos últimos anos, vários trabalhos sob a Doença de Chagas visam melhorar o desempenho dos LTCs. Apesar que os mecanismos citotóxicos, como perforina/granzima e interações Fas/FasL, pelos LTCs são bem caraterizados, a regulação de ambos não são completamente entendidos. Porém, nós investigamos se alguns parâmetros podem estar envolvidos na regulação in vivo das respostas efetoras dos LTCs pela vacina terapêutica de Trypanosoma cruzi baseado em Adenovírus humano tipo 5. Nós encontramos que a carga viral e o tempo após imunização têm um impacto na comutação do fenótipo citotóxico dos LTCs. Nossa descoberta evidencia os papeis não redundantes da perforina e FasL e a importância destes parâmetros na citotoxicidade FasL-independente, eventos envolvidos na busca uma efetiva vacina contra a Doença de Chagas. / Models of infection revealed that CD8+ T cells (CTLs) are essential for control of Trypanosoma cruzi, etiologic agent of Chagas disease. However, during disease, the CTLs are remarkably delayed and suboptimal establishing the chronic phase. In the latest years, several works about Chagas disease aim to enhance the performance of CTLs. Although the effector mechanisms of target cells elimination as perforin/granzymes and Fas/FasL interactions by CTLs are well characterized, the regulation of both are non-completed understood. Here, we investigated if some parameters can be involved in the in vivo regulation of CTL effector responses by a human type 5 Adenovirus-based Trypanosoma cruzi therapeutic vaccine. We found that, the viral load and time post-immunization have an impact in the CTL switching killing phenotype. Our findings shows the evidence of the nonredundant roles of the perforin and FasL and the importance of those parameters in the FasL-independent cytotoxicity, events related with the searching for an effective vaccine against Chagas disease.

A proteína Bim é uma potente molécula pró-apoptótica da família Bcl-2 que participa na indução da via intrínseca de apoptose. Pouco se sabe sobre o papel de Bim na resposta imune contra patógenos. Utilizando um modelo murino de infecção pelo T. cruzi, nós observamos um aumento da carga parasitaria e da mortalidade de animais Bim-/-. Macrófagos peritoneais isolados de camundongos Bim-/- no pico de sua parasitemia apresentaram diminuição da produção de NO e da sua atividade microbicida, provavelmente devido a uma deficiência no influxo da subpopulação SPM (small peritoneal macrophages). Ademais, neste mesma fase, esplenócitos apresentaram uma deficiência da produção de NO e das citocinas pró-inflamatórias IFN-γ e IL-6 e os animais Bim-/- apresentaram uma diminuição da citotoxicidade in vivo contra antígenos específicos ao T. cruzi. Em conjunto, nossos resultados sugerem um papel importante da proteína Bim no controle da replicação e eliminação do parasita na fase inicial da infecção. / Bim protein is a potent pro-apoptotic molecule of Bcl-2 family that participates in the induction of intrinsic apoptosis pathway. Little is known about its role on the immune response against pathogens. Using a murine model of T. cruzi infection, we observed an increased parasitemia and mortality in Bim-/- mice. Peritoneal macrophages isolated from these mice at the peak of parasitemia displayed decreased NO production and microbicidal activity, probably due to a defect in the influx of the small peritoneal macrophage (SPM) subpopulation. Moreover, we also observed a deficiency in nitric oxide production, pro-inflammatory cytokines IFN-γ and IL-6 secretion by splenocytes at this period of infection. Finally, Bim-/- mice has an impaired in vivo cytotoxic response against antigens from T. cruzi. Together, our results suggest an important role of Bim in the control of parasite replication and elimination in acute phase of T. cruzi infection.

Baixas doses de agentes desmetilantes do DNA (DNMTi) tem efeitos antitumorais por induzirem parada do ciclo celular e aumentarem a imunogenicidade se tornando mais visível para o sistema imune. Crescentes evidências sugerem que o tratamento com DNMTis podem aumentar a sinalização imune nas células tumorais por meio do mecanismo de mimetismo viral bem como aumento da expressão de antígenos associados ao tumor, destacando assim, o seu potencial clínico na combinação com outras terapias. Enquanto os efeitos dos DNMTis têm sido na maior parte investigados nas células tumorais, os efeitos sobre o sistema imune ainda permanecem desconhecido, especialmente sobre baixas doses. Assim sendo, nosso objetivo foi investigar os efeitos da administração de agentes desmetilantes do DNA sobre a função efetora dos linfócitos T CD8 e seu papel frente a resposta antitumoral. Para isso, injetamos células tumorais CT26 na concentração de 5x105 por animal no dia 0. Cada grupo de animais foi tratado ou não com 5-AZA-CdR (0,5 mg/kg). Os camundongos foram tratados diariamente intraperitonealmente do dia 5 ao dia 9. Como controle adicional, em um grupo de animais, foi utilizado anticorpo neutralizante para depletar células T CD8. O tratamento diminuiu o tamanho tumoral, aumentou o infiltrado de células T CD8 e aumentou a capacidade de produção de citocinas por essas células. Em adição, todos esses efeitos foram abolidos na ausência das células T CD8. Em nossos ensaios de estimulação ex vivo e de imunização com adenovírus, o tratamento gerou aumento da ativação celular, aumento da produção de citocinas, diminuição da proliferação e aumento de morte celular. Por fim em nossos ensaios de citotoxicidade, o tratamento aumentou a capacidade citotóxica dessas células. Nossos resultados mostram que o tratamento in vivo com DNMTis em tumores de camundongos levou ao aumento da infiltração de células T CD8 e que a diminuição do tamanho tumoral é dependente da ação das células T CD8. Uma vez que observamos a capacidade efetoras das células T CD8, o tratamento levou ao aumento da produção de citocinas e aumento da indução de morte celular de células alvo. Em resumo, nossos resultados demonstram que o tratamento com DNMTis aumenta a resposta antitumoral e aumentam o potencial citotóxico das células T CD8. / Low doses of the DNA methylation inhibitor (DNMTis) on cancer cells has durable antitumorigenic effects by inducing cell cycle arrest and immune-modulatory properties that increases their visibility by the immune system. Growing evidence suggest that DNMTis can upregulate immune signaling in cancer cells through viral mimicry and upregulation of tumour associated antigens, highlighting clinical potential to combine with immunotherapy. While the effects of DNMTis have been mostly studied in cancer cells, their effects on the immune system remains vastly unknown specially at clinically relevant low doses. Therefore, we aimed to investigate the effect of in vivo administration of DNA demethylating inhibitor on the effector function of CD8+ T cells and antitumor immune response. We injected 5x105 CT26 cells at day 0 in three group of mice (n=5 mice per group). Each group of mice were either mock treated or treated with Decitabine (5-AZA-CdR or DAC, at a dosage of 0,5 mg/kg). The mice were treated intraperitoneally daily from days 5 to 9. As a control, in one group of mice, it was used neutralizing antibody to deplete CD8+ T cells. The treatment significantly decreased tumor burden and in the absence of CD8+ T cells this effect was abolished. The treatment led to increase in CD8+ T cells infiltration into the tumor microenvironment and enhance secretion of effector cytokines. In addition, all these effects were abolished in the lack of CD8 T cells. Furthermore, using ex vivo and adenovirus immunization, the treatment led to enhance of cellular activation, decrease of proliferation and enhance of cell death. In addition, using cytotoxic assays, the treatment enhanced the killing capability of these cells. to characterize the CD8 T cells, we used ex vivo stimulation protocols. Remarkably, our results show that treatment in vivo with DNMTi in established murine tumors model led to increase CD8+ T cell infiltration and the decrease in tumor burden is dependent of CD8 T cells. Once the effector capacities of these CD8+ T cells were assessed, DNMTi treatment increased their effector cytokine production and increased killing of target cells. Altogether, our results show that treatment of CD8+ T cells enhance antitumor immune response and their cytolytic potential.

La composition des compartiments lymphocytaires T est soumise à de constantes modifications, sous l'effet conjugué de la production de nouvelles spécificités, de la sélection lors d'une rencontre avec un antigène et de la mort cellulaire. Face à ces flux de cellules entre les différents compartiments centraux et périphériques, les mécanismes de la régulation homéostatique assurent le fait que le système immunitaire se maintienne à l'équilibre. A l'image d'un écosystème, on peut alors observer que les lymphocytes T entrent en compétition les uns avec les autres pour des niches de sélection et des ressources en quantité limitée. Durant ce travail de thèse, réalisé chez la souris, nous avons analysé le comportement des lymphocytes T ab CD8+ face à un bouleversement de leur environnement, et nous avons travaillé sur les molécules de classe I du CMH comme modèle de ressource nécessaire à la survie des cellules T ab CD8+.<br />Dans un premier temps, nous avons étudié la sélection centrale et périphérique de cellules T ab CD8+ exprimant deux transgènes codant respectivement pour le TCR aHY, spécifique de l'antigène mâle H-Y, et le TCR P14, spécifique du peptide gp33-41 issu du virus de la chorioméningite lymphocytaire (LCMV). Ce modèle reproduit un phénomène courant dans le système immunitaire, puisqu'on trouve chez l'homme et la souris jusqu'à 30% de cellules exprimant deux TCR différents à leur surface. Nos résultats montrent que l'expression de deux TCR par les cellules T ab CD8+ leur permet d'échapper partiellement à la sélection négative dans le thymus, et de résister à la délétion clonale à la périphérie. Dans un second temps, nous avons étudié l'établissement d'une infection chronique par le LCMV dans des souris n'ayant pour lymphocytes que des cellules T exprimant le TCR P14 (souris MoP14). Nous avons pu observer que cette infection passe par la sélection de variants viraux spécifiquement mutés au niveau de l'épitope gp33-41, mais également par la modification du comportement des cellules T ab CD8+ des animaux. L'ensemble de ces données plaide pour un modèle d'adaptation des lymphocytes T ab CD8+ à leurs conditions environnementales.<br />Enfin, nous avons étendu ce travail à l'étude de l'influence des molécules de classe I du CMH sur la survie et la prolifération homéostatique des lymphocytes T ab CD8+, en utilisant une gamme de souris transgéniques pour le TCR. Nos résultats montrent une variété de comportements en relation avec la réactivité croisée supposée des différents TCR utilisés.

Plusieurs populations de cellules immunitaires possèdent une activité cytotoxique permettant l'élimination de cellules altérées. Cette fonction cellulaire est ainsi déterminante dans le contrôle des infections, des processus tumoraux, ou encore des maladies inflammatoires chroniques. Mon projet de thèse se concentre sur des aspects fondamentaux de l'activité lytique de deux populations de lymphocytes T cytotoxiques : les lymphocytes T CD8+ et les lymphocytes T CD4+ régulateurs de type 1. Pour cela, l'exploration des mécanismes de cette activité a été conduite au travers de deux modèles, pathologique et thérapeutique, à différentes échelles biologiques : au niveau de la population ou de la cellule individuelle, mais aussi différentes échelles d'organisations moléculaires : cellulaire et nanoscopique. Nous avons pu démontrer que l'activité de lyse de lymphocytes T CD8+ cytotoxiques face à un excès de cellules cibles est efficace sur des temps prolongés, reposant sur une capacité individuelle fortement hétérogène à effectuer une lyse multiple. L'importance de cette activité de lyse soutenue a été renforcée par l'identification d'un défaut lytique particulièrement prononcé sur le long- terme chez des lymphocytes T CD8+ issus de patients atteints du syndrome de Wiskott-Aldrich. Ce défaut est lié à une activation réduite de l'intégrine LFA-1 et un délai dans la délivrance du coup létal. De plus, la protéine WASP permet de restreindre LFA-1 de haute-affinité en nanoclusters denses ainsi que de permettre l'organisation en un ring de LFA-1 et la localisation des granules lytiques à l'intérieur de celui-ci. Par ailleurs, les lymphocytes T CD4+ régulateurs de type 1 développés dans le cadre d'une thérapie cellulaire (Ovasave(r)) démontrent une capacité de lyse envers les cellules myéloïdes, en complément d'une activité immunosuppressive sur les lymphocytes T conventionnels. Cette activité est mise en place sur du long-terme, jusqu'à atteindre une efficacité optimale, lié à un délai dans la délivrance du coup létal. De manière surprenante, malgré une spécificité pour l'ovalbumine, l'activité cytotoxique semble être indépendante de l'activation du TCR. En outre, la lyse est granzyme-dépendante mais perforine-indépendante. Ainsi ces lymphocytes T thérapeutiques manifestent une activité cytotoxique alternative. Pour conclure, mon projet de thèse a permis de caractériser une activité de lyse soutenue basée sur une capacité individuelle hétérogène. Cette habilité à soutenir une lyse sur du long-terme implique une stabilité de la synapse, où WASP joue notamment un rôle clé pour l'activation et l'organisation de LFA-1. Les lymphocytes T régulateurs thérapeutiques démontrent aussi une activité de lyse soutenue, cependant les acteurs moléculaires sont non conventionnels. De manière générale, une activité de lyse soutenue permettrait de calibrer une réponse cytotoxique prolongée en rapport à la taille de la population cible, ainsi que le partage avec d'autres fonctions cellulaires comme la sécrétion de cytokines. / During different pathological conditions such as infections, tumoral processes or chronic inflammation diseases, altered cells are eliminated through a cytotoxic activity mediated by several immune cell populations. This cellular function is therefore crucial for carrying out the action of the immune system. My thesis project focuses on fundamental aspects of the lytic activity of two cytotoxic lymphocyte populations: CD8+ T cells and type-1 CD4+ regulatory T cells. To explore the mechanisms of this activity, this study has been driven on two cases, pathological and therapeutic models, at the population and single-cell levels and also at the cellular and nanoscopic scales of the molecular organisation. We have been able to demonstrate that the CD8+ T cell lysis activity against an excess of target cells is effective over prolonged periods, relying on a highly heterogeneous individual capacity to perform multiple lysis. The importance of this sustained cytotoxic activity was reinforced by the identification of a lytic defect, particularly pronounced on a long time period, of CD8+ T cells from Wiskott-Aldrich syndrome patients. This defect is related to a reduced activation of the LFA-1 integrin and delay in the lethal hit delivery. In addition, the WASP protein allows to restrict high affinity LFA-1 to dense nanoclusters as well as the assembly of LFA- 1 ring and the localization of the lytic granules inside this ring. Moreover, type-1 CD4+ regulatory T cells from a cellular therapy (Ovasave(r)) demonstrated a cytotoxic activity toward myeloid cells, additionally to an immunosuppressive activity on conventional T cells. This activity is implemented over long time periods, until reaching optimal efficiency, and is related to a delay in the lethal hit delivery. Surprisingly, despite a specificity for ovalbumin, the cytotoxic activity measured in absence of the antigen suggests a TCR independence. In addition, lysis is not mediated by perforin but is exclusively granzyme-dependent. Thus, these therapeutic T cells exhibit an alternative cytotoxic activity. To conclude, my thesis project permits to characterize a sustained lysis activity relying on a heterogeneous individual capacity. This ability to sustain a lytic activity involves stability of the synapse, where WASP plays a key role towards the activation and organization of LFA-1. The therapeutic regulatory T lymphocytes also demonstrated a sustained cytotoxic activity, however the molecular actors are unconventional. On the whole, sustained lytic activity would be key to the calibration of cytotoxic responses in relation to the size of the target population, as well as sharing with other cellular functions such as cytokine secretion.

Les données de la littérature ont récemment souligné l’importance du dialogue réciproque qui s’instaure entre les cellules Natural Killer (NK) et les cellules dendritiques (DC) au cours des phases précoces de la réponse immune pour l’initiation des réponses T spécifiques. La présentation croisée d’antigènes (Ag), processus qui permet aux DC de présenter des Ag exprimés par d’autres cellules aux lymphocytes T CD8+, est requise pour le développement d’une immunité cellulaire spécifique dans la plupart des infections par des pathogènes intracellulaires et des tumeurs. L’étude des mécanismes physiologiques impliqués dans la régulation de cette fonction suscite donc un intérêt majeur. Ce travail a permis de mettre en lumière une coopération entre les cellules NK et les DC humaines pour la présentation croisée d’un Ag exprimé par des cibles tumorales. Dans ce contexte, nous avons montré que la lyse de ces cibles par les cellules NK n’est pas nécessaire à la capture de leurs Ag par les DC. Au contraire, la sécrétion d’IFN-γ et de TNF-α par les cellules NK activées au contact des cibles tumorales joue un rôle prépondérant dans l’induction de la présentation croisée d’Ag. Ainsi, nous avons identifié une nouvelle fonction « helper » des cellules NK à l’interface entre l’immunité innée et adaptative. Le ciblage de cette fonction pourrait offrir de nouvelles perspectives en immunothérapies anti-tumorales dont le but ultime est le développement d’une immunité cellulaire spécifique / Recent reports have demonstrated the importance of the reciprocal crosstalk between natural killer (NK) cells and dendritic cells (DC) occurring during early phase of immune response for shaping downstream T cell immunity. Antigen (Ag) cross-presentation, a process by which DC present Ag from neighboring cells to CD8+ T lymphocytes is a prerequisite for the developpment of specific cellular immunity against most intracellular pathogens and tumors. A more detailed understanding of the mechanisms that regulate this specific DC function is thus a major challenge for immunologists. Here, we highlight the cooperation between NK and DC for tumor cell-derived Ag cross-presentation. In this context, we show that the NK cell-mediated lysis of target cells is not required for Ag capture by DC. In contrast, both IFN-γ and TNF-α produced by NK cells upon recognition of tumor cells play a critical role in the induction of Ag cross-presentation. These findings define a novel « helper» function of NK cells bridging innate and adaptive immunity. This novel function could be harnessed in cancer immunotherapy for inducing Ag-specific cellular immunity