This dissertation addresses the role for Janus Kinase 3 (Jak3) in CD4+ T cell homeostasis and function. Jak3 is a protein tyrosine kinase whose activity is essential for signals mediated by the γc dependent cytokines IL-2, -4, -7, -9, -15, and -21. Previous data have demonstrated that peripheral CD4+ T cells from Jak3-deficient mice have a memory phenotype and are functionally impaired in both proliferative and IL-2 responses in vitro. Interestingly, Jak3/γc activity has been previously shown to play a role in the prevention of T cell anergy. These studies were initiated to more precisely define the role for Jak3/γc cytokines in the prevention of T cell anergy and the maintenance of functional CD4+ T cell responses. We began to address this question by assessing global gene expression changes between wild type and Jak3-/- CD4+ T cells. These data indicate that Jak3-/- CD4+ T cells have an increase in gene expression levels of inhibitory surface receptors as well as immunosuppressive cytokines. Further analyses confirmed that Jak3-deficient T cells express high levels of PD-1, secrete a Trl-type cytokine profile following direct ex vivo activation, and suppress the proliferation of wild type T cells in vitro. These characteristics indicate that CD4+ Jak3-/- T cells share properties with regulatory T cell subsets that have an important role in peripheral tolerance and the prevention of autoimmunity. We next addressed whether these regulatory characteristics were T cell intrinsic or rather the result of expanding in a Jak3-deficient microenvironment characterized by a number of immune abnormalities and a disrupted splenic architecture. Jak3-/- CD4+ T cells proliferate in vivoin a lymphopenic environment and selectively acquire regulatory T cell characteristics in the absence of any additional activation signals. While the precise mechanism by which Jak3-deficient T cells acquire these characteristics remains unclear, our data indicate that one important component is a T cell-intrinsic requirement for Jak3 signaling. These findings indicate several interesting aspects of T cell biology. First, these studies, demonstrate that the homeostatic proliferation of CD4+ T cells is not dependent on signaling via γc-dependent cytokine receptors. And, second, that the weak activation signals normally associated with homeostatic expansion are sufficient to drive Jak3-/- T cells into a non-conventional differentiation program. Previous data indicate that, for wild type T cells, signaling through both the TCR as well as γc-dependent cytokine receptors promote the homeostatic proliferation of T cells in lymphopenic hosts. Since Jak3-/- T cells are unable to receive these cytokine signals, their proliferation is likely to be wholly dependent on TCR signaling. As a consequence of this TCR signaling, Jak3-/- T cells proliferate, but in addition, are induced to up regulate PD-1 and to selectively activate the IL-10 locus while shutting off the production of IL-2. Since this fate does not occur for wild type T cells in a comparable environment, it is likely that the unique differentiation pathway taken by Jak3-/- T cells reflects the effects of TCR signaling in the absence of γc-dependent cytokine signaling. Interestingly, wild type T cells undergoing homeostatic expansion in lymphopenic hosts show many common patterns of gene expression to freshly-purified unmanipulated Jak3-/- T cells. For instance, micro array analysis of gene expression in wild type CD4+ T cells after lymphopenia induced homeostatic expansion show a similar pattern of upregulation in surface markers (PD-1 and LAG-3), and cytokine signaling molecules (IL-10 and IFN-γ cytokine, receptors, and inducible gene targets) to that of Jak3-/- CD4+ T cells immediately ex vivo. These data suggest that the process of homeostatic proliferation normally induces immune attenuation and peripheral tolerance mechanisms, but that full differentiation into a regulatory T cell phenotype is prevented by γc-dependent cytokine signals. Taken together these data suggest that Jak3 plays an important role in tempering typical immune attenuation mechanisms employed to maintain T cell homeostasis and peripheral tolerance.

INTRODUÇÃO: As infecções causadas por bactérias ou vírus são frequentes em pacientes com imunodeficiência comum variável (ICV) devido à deficiência de anticorpos e associação com alteração da função das células T. OBJETIVOS: Avaliar o efeito da ativação de receptores Toll-like (TLR) utilizando ligantes de TLRs em células T monofuncionais ou polifuncionais em pacientes com ICV. MÉTODOS: Foram selecionados 16 pacientes com ICV do Ambulatório de Manifestações Dermatológicas das Imunodeficiências Primárias ADEE3003 HC-FMUSP e 16 controles saudáveis. Os métodos utilizados de citometria de fluxo foram: a) análise em sangue periférico de linfócitos B, linfócitos T quanto ao perfil de ativação/maturação, linfócitos T foliculares (Tfh) e células T reguladoras (Treg); b) dosagem de citocinas e quimiocinas em amostras de soro e em sobrenadante de culturas de células mononucleares do sangue periférico (CMNs) estimuladas com agonistas de TLRs; c) avaliação das células TCD4+ mono e polifuncionais secretoras de IL-17a, IL-22, TNF, IFN- e IL-10, e expressão de marcador de ativação crônica de CD38 estimuladas por agonistas de TLR2, TLR3 e TLR7/8 e estímulos policlonais como enterotoxina B de Staphylococcus aureus (SEB) e acetato miristato de forbol e ionomicina (PMA/IONO); d) células Th22 e Tc22 estimuladas com TLR e SEB. RESULTADOS: Na ICV, os linfócitos B do sangue periférico mostram diminuída frequência, sendo em maior frequência de linfócitos B naïve (CD19+IgD+CD27-), e ausência de células B de memória. Além disto, um aumento na expressão do marcador de exaustão PD-1 foi observado nas células TCD4+ de memória efetora (CD45RA-CCR7-) e na expressão de CD38, em células TCD8+ terminalmente diferenciadas (CD45RA + CCR7-). Em contraste, houve diminuição na frequência de células T reguladoras naïve nos pacientes com ICV. Nos indivíduos com ICV foi observado aumento na frequência de células TCD4+ TNF+ sob estímulo TLR2 e TLR7/8 comparado ao grupo controle, enquanto que sob estímulo com PMA/IONO houve menor frequência de células TCD4+ e TCD8+ secretoras de IFN-y IL-17a, IL-22 ou TNF. Já em células TCD8+ houve importante redução na ativação via TLR3 na resposta de IL-22, IFN-y e IL-17a. Contudo, os estímulos com TLR7/8 e SEB foram capazes de aumentar a frequência de células Th22 e Tc22 nos pacientes com ICV. Em geral, as células TCD4+, que secretam simultaneamente 4 a 5 citocinas induzidas por TLR foram preservadas em ICV. Embora as células TCD4+ polifuncionais secretoras de 3 citocinas, foram capazes de responder a estímulos via TLR2 e TLR7/8, as células TCD8+ não responderam para qualquer estímulo via TLRs. Além disso, as células T que expressam CD38 mostraram menor polifuncionalidade aos estímulos via TLRs e PMA/IONO. O perfil inflamatório nos pacientes com ICV foi observado pela elevação sérica de IL-6, CCL-2, CCL-5, CXCL8, CXCL-9, CXCL-10. Alteração na resposta aos agonistas de TLRs em ICV pode ser observada com a ativação dos agonistas de TLRs em CMNs, que mostrou maior produção de TNF e diminuição de CCL2 e CXCL8 após ativação via TLR4. Em contraste, o agonista de TLR7/8, teve ação oposta induzindo CXCL10 e reduzindo os níveis de CXCL9. Chama atenção no ICV, à reduzida secreção de IFN-alfa induzida por TLR7/8, que não foi observada com a ativação via TLR9. CONCLUSÕES: Até o momento, os achados em ICV mostram alterações nas células T, seja quanto à baixa frequência de células T reguladoras naïve e a reduzida resposta efetora, em especial das células TCD8+. Contudo, enfatiza o potencial de adjuvante dos agonistas de TLR7/8 na ativação das células T / INTRODUCTION: Infections caused by bacteria or viruses are common in patients with Common Variable Immunodeficiency (CVID), due to antibody deficiency and association with altered function of T cells. OBJECTIVES: To evaluate the effect of Toll-like receptors (TLR) activation using TLR agonists on the monofunctional or polyfunctional T cells in patients with CVID. METHODS: We selected 16 patients with ICV from the Dermatologic Manifestations of Primary Immunodeficiencies Clinic ADEE3003 HC-FMUSP and 16 healthy controls. The methods used for flow cytometry were: a) analysis of peripheral blood B lymphocytes, T lymphocytes were assessed by the activation/maturation profile, follicular T cells (Tfh) and regulatory T cells (Treg); b)evaluation of cytokines and chemokines serum levels and in supernatants of mononuclear cell cultures from peripheral blood (PBMC) stimulated with TLR agonists; c) evaluation of mono and polyfunctional CD4+ T cells secreting IL-17a, IL-22, TNF, IFN-y and IL-10, and expression chronic activation marker of CD38 stimulated by agonists of TLR2, TLR3 and TLR7/8 and polyclonal stimuli such as Staphylococcus aureus enterotoxin B (SEB) and phorbol myristate acetate and ionomycin (PMA / IONO); d) analysis of Tc22 and Th22 cells stimulated with TLR and SEB. RESULTS: In the CVID, the peripheral blood B cells show decreased frequency, being higher frequency of naïve B cells (IgD+ CD19+ CD27-) and lack of memory B cells. Moreover, an increased expression of PD-1, an exhaustion marker, was detected in the CD4+ T cell effector memory (CD45RA- CCR7-) and expression of CD38 on CD8+ T terminally differentiated cells (CD45RA+ CCR7-). In contrast, a decreased frequency of naïve regulatory T cells was detected in the patients with CVID. In CVID patients it was observed increased frequency of T CD4+ TNF+ cells upon TLR2 and TLR7/8 agonists compared to the control group, while under stimulation with PMA /IONO there was a lower frequency of CD4+ and CD8+T cells secreting IFN-y, IL-17a, IL-22 or TNF. The CD8+T cells showed a significant reduction of in the IL-22 response, IFN-? and IL-17a induced by TLR3 activation. However, stimulation with TLR7/8 and SEB were able to increase the frequency of Th22 and TC22 cells in the patients with CVID. In CVID patients it was observed increased frequency of T CD4+ TNF+ cells upon TLR2 and TLR7/8 agonists compared to the control group, while under stimulation with PMA /IONO there was a lower frequency of CD4+ and CD8+ T cells secreting IFN-y, IL-17a, IL-22 or TNF. The CD8+ T cells showed a significant reduction of in the IL-22 response, IFN-y and IL-17a induced by TLR3 activation. However, stimulation with TLR7/8 and SEB were able to increase the frequency of Th22 and TC22 cells in the patients with CVID. In general, CD4+ T cells that secrete simultaneously 4 to 5 cytokines induced by TLR were preserved in CVID. Although polyfunctional CD4+ T cells secreting 3 cytokines were able to respond to TLR2 and TLR7/8 agonists, the CD8+ T cells did not respond to any stimuli. In addition, T cells expressing CD38, showed lower polyfunctionality to the stimuli via TLRs and PMA/IONO. Furthermore, the inflammatory status in the patients with CVID was observed by the increased serum levels of IL-6, CCL-2, CCL-5, CXCL8, CXCL-9, CXCL-10. In contrast, the agonist of TLR7/8 had opposite action inducing CXCL10 and reducing the CXCL9 levels. Noteworthy in CVID, that the reduced secretion of IFN-alfa induced by TLR7/8 was not observed with TLR9 activation. CONCLUSIONS: To date, the CVID findings shows alterations in the T cells, as the low frequency of naïve regulatory T cells and reduced effector response, mainly of CD8+ T cells. However, it emphasizes the adjuvant potential of the TLR7/8 agonist in the T cells activation

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

Acredita-se que as células dendríticas (DCs) sejam as mais eficientes na ativação de linfócitos T (LT) naive. Com a possibilidade de geração in vitro de DCs, muitos protocolos explorando este potencial vêm sendo desenvolvidos, principalmente em abordagens imunoterapêuticas para o câncer. Todavia, em situações fisiológicas a apresentação antigênica dificilmente ocorre por um tipo celular único. Deste modo, este trabalho investigou os padrões de reposta de LT induzidos por DCs maduras (mDCs) em combinação com diferentes APCs, incluindo linfócitos B, monócitos, macrófagos e DCs imaturas. Os padrões de resposta gerados pelos LT foram analisados por citometria de fluxo, ELISA e BIOPLEX. A estimulação dos LT pelas mDCs isoladas apresentaram o maior poder de estimulação, seguidas pelas outras APCs. Todas as combinações diminuiram a resposta induzida pelas mDCs, principalmente de LT CD4+. Deste modo, os dados confirmam o efeito das interações de APCs na estimulação de LT provendo ferramentas para o refinamento de abordagens imunoterapêuticas. / Dendritic cells (DC) are the main APC able to activate T cells (TC). Since they may be generated in vitro, many protocols based on their immunostimulatory potential are currently underway, mainly in immunotherapeutic approaches for cancer. In physiological conditions, however, other APC may participate in antigen presentation, thus influencing the immune response pattern developed. Therefore, the aim of this study was to evaluate the TC response patterns induced in vitro by mature DC (mDC) combined with different APC, including B cells, monocytes, macrophages and immature DC. The response patterns were analyzed flow cytometry, ELISA and Multiplex flow immunoassay. The TC stimulation by isolated mDC presented the highest capacity of stimulation, followed by the other APC. All combinations decreased the response induced by mDC, mainly CD4+ T cells. These data confirm the effects of APC interactions upon TC stimulation and may provide a tool for fine-tuning of immune response induction in immunotherapeutic approaches.

A hanseníase é geralmente agravada pelo aparecimento de reações, que são quadros inflamatórios de difícil tratamento e a principal causa de seqüelas. Nossa hipótese é de que deficiência e/ou perda da função das células T reguladoras (Tregs) podem estar envolvidas no desenvolvimento das reações. Além da avaliação da frequência das Tregs circulantes em pacientes com reação tipo 1 (R1) e reação tipo 2 (R2), também foi avaliada a frequência in situ de FoxP3, IL-17, IL-6 e TGFbeta. Pacientes com R2 apresentaram expressiva diminuição na frequência das Tregs circulantes e in situ em comparação com pacientes com R1 e com os controles. Paralelamente a diminuição das Tregs nas R2 foi observado aumento da expressão de IL-17 in situ e diminuição da expressão de TGFbeta. Biópsias obtidas de pacientes com R1 e R2 antes do episódio reacional mostraram números de células FoxP3+ e IL-17+ similares entre os dois grupos. Entretanto, nas biópsias obtidas durante a reação foi observado diminuição de Tregs e aumento de células IL-17+ em pacientes com R2, enquanto que pacientes com R1 apresentaram o oposto: aumento de Tregs e diminuição de células IL-17+. Além disso, foi observada diminuição da expansão das Tregs frente ao estímulo in vitro com Mycobacterium leprae e uma tendência a baixa expressão de FoxP3 e da molécula imunossupressora CTLA-4 em Tregs de pacientes com R2. Nossos resultados sugerem que nas R2, a diminuição na frequência de Tregs possa estar favorecendo o desenvolvimento de uma resposta Th17, a qual é característica deste tipo de reação. Adicionalmente, com a finalidade de obter um número suficiente de Tregs para realização de ensaios funcionais com estas células, uma vez que se trata de uma subpopulação com baixa freqüência no sangue periférico ( < 10%), foram estabelecidos e avaliados três protocolos distintos para expansão in vitro de Tregs: protocolo Rapamicina, protocolo TGFbeta e protocolo Vitamina D3. Todos os protocolos foram capazes de induzir expansão de Tregs viáveis nos grupos estudados (paucibacilares e multibacilares sem reação, R1 e R2). Em todos os grupos estudados as Tregs expandidas apresentaram capacidade de suprimir a proliferação de linfócitos TCD4+ e TCD8+. Apesar dos três protocolos testados apresentarem capacidade de expandir Tregs in vitro, selecionamos para ensaios futuros os protocolos Rapamicina e TGFbeta por apresentarem melhor custo-benefício. A expansão in vitro será utilizada para estudos funcionais das Tregs buscando melhor entendimento do envolvimento desta subpopulação na patogenia das reações hansênicas / Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of FoxP3 and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-beta expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-beta expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3 + (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule CTLA-4 in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction. In addition, aiming to provide sufficient number of Tregs to perform functional assays with these cells, as they correspond to a subtle subpopulation among the peripheral blood mononuclear cells ( < 10%), we established and analyzed three different protocols for in vitro Tregs: a Rapamycin protocol, a TGFbeta protocol and a Vitamina D3 protocol. All three protocols were able to induce the expansion of viable in the four types of patients of the study (paucibacillary and multibacillary patients without reaction, and R1 and R2 patients). In these four groups the tregs were able to suppress the proliferative response of TCD4+ e TCD8+ lymphocytes. Although the three protocols resulted in expansion of Tregs, we selected two of them, Rapamycin and TGFbeta for further assays since they showed better cost-benefit. The in vitro expansion will be used to perform functional assays of the Tregs aiming at a better understanding of the involvement of this subpopulation in the pathogenesis of the leprosy reaction episodes

INTRODUÇÃO: O aumento na expectativa de vida da população justifica o interesse em compreender melhor o processo de envelhecimento. Uma projeção recente do Instituto Brasileiro de Geografia e Estatística prevê que a população de idosos vai triplicar até 2050. Esse aumento trará para o sistema de saúde do Brasil um desafio. O aumento do número de doenças, o comprometimento imunológico, a diminuição da resposta às vacinas são evidenciados na população idosa. A associação entre encurtamento do telômero e senescência celular tem sido estabelecida laboratorialmente. Neste estudo avaliamos comparativamente três grupos de indivíduos idosos quanto à atividade física na categoria de alto desempenho, moderado desempenho e sedentário. Comparamos o comprimento do telômeros em linfócitos T entre os grupos citados. Avaliamos a cognição, a qualidade de vida e incidência de depressão dos idosos dos grupos citados. MÉTODOS: Os pacientes foram selecionados no ambulatório do Instituto de Ortopedia e Traumatologia da Universidade de São Paulo. Avaliamos as características clínicas, funcionais, qualidade de vida, depressão, cognição, miniavaliação nutricional, imunidade e medida do telômeros em linfócitos T. RESULTADOS: Foi detectada correlação estatística significante entre a medida do telômeros em linfócitos e CD8CD28- e a atividade física. Foi, também, detectada diferença na mini avaliação mental e consumo de O2. CONCLUSÕES: Comprovou-se diferença estatística entre a medida do telômeros em linfócitos e CD8CD28- bem como na cognição e a atividade física / INTRODUCTION: The increase in population life expectancy justifies the interest in better understanding the aging process. A recent projection of the Brazilian Institute of Geography and Statistics predicts that the elderly population will triple by 2050. This increase will bring a challenge to the healthcare system in Brazil. The increase in the number of diseases, the immunological impairment, and the decreased response to vaccines are evident, especially in the elderly population. The association between telomere shortening and cellular senescence has been established in a laboratory setting. In this study we compared three groups of elderly individuals regarding their physical activity in the categories of high performance, moderate performance and sedentary. We compared telomere length in T cells between the aforementioned groups. We evaluated the cognition and incidence of depression in the elderly of the aforementioned groups. METHODS: Patients were selected from the clinic of the Institute of Orthopedics and Traumatology, University of São Paulo. We evaluated the clinical characteristics, functional characteristics, quality of life, depression, cognition, nutritional mini-assessment, immunity and telomere length in T cells. RESULTS: A statistically significant correlation was found between telomere length in CD8CD28- T cells and physical activity. Also, differences in the mental mini assessment and the consumption of O2 were detected. CONCLUSION: A statistically significant correlation was proven between the length of the telomeres in CD8CD28- T cells and cognition, as well as in physical activity

by Li Pok Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 94-114). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of contents --- p.XII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Human Th lymphocytes and their immunopathogenic roles --- p.1 / Chapter 1.1.1 --- Characteristics of Th lymphocytes --- p.1 / Chapter 1.1.2 --- Migration and activation --- p.1 / Chapter 1.1.3 --- Th cell differentiation --- p.2 / Chapter 1.1.4 --- Pathological roles --- p.4 / Chapter 1.2 --- Cytokines as modulator in Th lymphocyte activation --- p.6 / Chapter 1.2.1 --- IL-18 --- p.6 / Chapter 1.2.2 --- IL-25 --- p.7 / Chapter 1.3 --- Surface marker expression in Th lymphocytes --- p.8 / Chapter 1.3.1 --- Adhesion molecules --- p.8 / Chapter 1.3.2 --- Cytokine and chemokine receptors --- p.9 / Chapter 1.3.3 --- Costimulatory molecules --- p.11 / Chapter 1.4 --- Cytokine and chemokine release from Th lymphocytes / Chapter 1.4.1 --- Thl cytokines --- p.13 / Chapter 1.4.2 --- Th2 cytokines --- p.14 / Chapter 1.4.3 --- Chemokines --- p.15 / Chapter 1.5 --- Intracellular signaling pathways in Th lymphocytes --- p.19 / Chapter 1.5.1 --- p38 MAPK pathway --- p.19 / Chapter 1.5.2 --- ERK pathway --- p.20 / Chapter 1.5.3 --- JNK pathway --- p.20 / Chapter 1.5.4 --- NF- k B pathway --- p.21 / Chapter 1.6 --- Pharmacological intervention of signaling pathways --- p.22 / Chapter 1.7 --- Aims and scope of the study --- p.24 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Blood samples --- p.26 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.26 / Chapter 2.1.3 --- Antibodies for costimulation of Th cells --- p.28 / Chapter 2.1.4 --- Recombinant human cytokines --- p.28 / Chapter 2.1.5 --- "Signaling pathway inhibitors: SB203580, PD98035, SP600125 and BAY117082" --- p.28 / Chapter 2.1.6 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.29 / Chapter 2.1.7 --- Reagents and buffers for the purification of human Th lymphocytes --- p.31 / Chapter 2.1.8 --- Reagents and buffers for protein array --- p.32 / Chapter 2.1.9 --- Reagents and buffers for Thl/2 cytokine and chemokine detection --- p.32 / Chapter 2.1.10 --- Reagents and buffers for protein extraction --- p.32 / Chapter 2.1.11 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis --- p.33 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.35 / Chapter 2.1.13 --- Reagents and buffers for non-radioactive electromobility shift assay (EMSA) --- p.37 / Chapter 2.1.14 --- Reagents and buffers for cell viability and proliferation assay --- p.39 / Chapter 2.1.15 --- Reagent kit for endotoxin level assay --- p.39 / Chapter 2.1.16 --- Other reagent kits --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Purification of human Th lymphocytes and cell culture --- p.41 / Chapter 2.2.2 --- Measurement of total and allergen-specific IgE concentrations --- p.41 / Chapter 2.2.3 --- Immunophenotyping of cells by flow cytometry --- p.42 / Chapter 2.2.4 --- Protein array --- p.42 / Chapter 2.2.5 --- Quantitative analysis of cytokines and chemokines by flow cytometry --- p.43 / Chapter 2.2.6 --- Quantitative analysis of IFN-γ by ELISA --- p.43 / Chapter 2.2.7 --- SDS-PAGE --- p.44 / Chapter 2.2.8 --- Western blot analysis --- p.44 / Chapter 2.2.9 --- EMSA / gel shift assay --- p.45 / Chapter 2.2.10 --- MTT assay --- p.46 / Chapter 2.2.11 --- Cell proliferation assay --- p.46 / Chapter 2.2.12 --- Endotoxin level assay --- p.47 / Chapter 2.2.13 --- Statistical analysis --- p.47 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of IL-18 and IL-25 on the induction of Thl/2 cytokine and chemokine release from costimulated Th lymphocytes --- p.48 / Chapter 3.1.1 --- IL-18 and IL-25 could up-regulate the protein expression of cytokines and chemokines --- p.48 / Chapter 3.1.2 --- IL-18 but not IL-25 induced the release of IFN-γ and TNF-α --- p.48 / Chapter 3.1.3 --- "IL-18 and IL-25 induced the release of IL-5, IL-6 and IL-10" --- p.49 / Chapter 3.1.4 --- "IL-18 induced the release of IP-10, MIG, RANTES, MlP-lα and IL-8" --- p.49 / Chapter 3.1.5 --- "IL-25 induced the release of IP-10, MIG and RANTES" --- p.49 / Chapter 3.1.6 --- IL-18 and IL-25 did not enhance the proliferation of costimulated Th cells --- p.49 / Chapter 3.2 --- "Effects of IL-18 and IL-25 on the activation of p38 MAPK, ERK, JNK and NF- k B" --- p.58 / Chapter 3.2.1 --- "Costimulation with or without IL-18 and IL-25 could activate p38 MAPK, ERK and JNK" --- p.58 / Chapter 3.2.2 --- Costimulation with or without IL-18 and IL-25 could induce NF- k B activity --- p.58 / Chapter 3.3 --- Effects of inhibitors on the IL-18 and IL-25-induced release of Thl/2 cytokines and chemokines --- p.63 / Chapter 3.3.1 --- "Optimal dosage of SB203580, PD98035, SP600125 and BAY117082" --- p.63 / Chapter 3.3.2 --- "SB203580, PD98035 and BAY 117082 but not SP600125 suppressed the IL-18 and IL-25-induced release of Thl/2 cytokines" --- p.63 / Chapter 3.3.3 --- SP600125 suppressed the IL-18 and IL-25-induced release of chemokines --- p.64 / Chapter 3.4 --- Effects of inhibitors on the cell surface expression of IL-18 and IL-25 receptors --- p.72 / Chapter 3.4.1 --- "SB203580, PD98035, BAY 117082 but not SP600125 could suppress IL-18 receptor on costimulated Th cells" --- p.72 / Chapter 3.4.2 --- "SB203580, SP600125, PD98035 and BAY 117082 could not suppress IL-25 receptor on costimulated Th cells" --- p.72 / Chapter 3.5 --- Effects of costimulation on the expression of cell surface markers on Th lymphocytes --- p.75 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effects of IL-18 and IL-25 on the release of Th1/2 cytokines and chemokines --- p.80 / Chapter 4.2 --- "Regulation of Thl/2 cytokines and chemokines through intracellular p38 MAPK, ERK, JNKand NF-kB" --- p.83 / Chapter 4.3 --- Effects of costimulation on different surface markers in Th cells --- p.87 / Chapter 4.4 --- Concluding remarks and future perspectives --- p.90 / References --- p.94

Introdução: O timo é um órgão linfóide especializado responsável por criar um microambiente propício para a diferenciação e maturação de células T. A quantificação dos níveis de TRECs (círculos excisados durante o rearranjo do TCR) vem sendo utilizada para avaliação e quantificação da função tímica em células do sangue periférico. Estudos que tenham realizado a quantificação dos níveis de TREC em crianças e adolescentes saudáveis brasileiros são escassos na literatura. Objetivo: No presente estudo, avaliamos os níveis de TREC em células mononucleares do sangue periférico associado à análise da expressão de linfócitos T CD3, CD4, CD8, de linfócitos T ativados co-expressando CD38 e HLA-DR e linfócitos T reguladores co-expressando CD25 e Foxp3 em crianças e adolescentes saudáveis em diferentes faixas etárias. Material e métodos: A quantificação dos níveis de sjTREC de DNA genômico em células mononucleares de sangue periférico foi realizada pelo método de PCR quantitativo em tempo real. A concentração de TREC foi expressa em número de cópias de TREC/?g de DNA. A análise da expressão dos marcadores CD3, CD4, CD8, CD38, HLA-DR, CD25 e Foxp3 foi realizada por citometria de fluxo. Resultados: Foram avaliadas 95 crianças e adolescentes, 46 do sexo feminino e 49 do sexo masculino com idades entre 1 e 18 anos. A média do número de cópias de TREC foi de 8,9 ± 3,6 x 104 TRECs/?g DNA. Não encontramos diferença significativa nos valores de TREC entre o sexo feminino e masculino (8,2 ± 3,3 x 104 TRECs/?g DNA vs 9,5 ± 3,9 x 104 TRECs/?g DNA, respectivamente, p = 0,085). Houve uma correlação inversa e significativa entre idade e os níveis de TREC (r = -0,846; p < 0,001), refletindo a já conhecida queda da função tímica com a idade. A expressão de CD3, CD4 e CD8 variou de 45 a 62%, 60 a 65% e 14 a 26%, respectivamente. Não houve correlação significativa entre a proporção de CD3, CD4 e CD8 e a idade dos indivíduos avaliados. Houve uma correlação inversa fraca entre os níveis de linfócitos T ativados expressando CD4+CD38+HLA-DR+ e idade (r = -0,286; p = 0,023), porém não encontramos correlação entre linfócitos T CD8+CD38+HLA-DR+ e idade (r = -0,229; p = 0,072). Houve uma correlação inversa entre os valores de linfócitos T expressando CD4+CD25+Foxp3+ e idade (r = -0,467; p = 0,04). Adicionalmente, encontramos correlação positiva entre a expressão de linfócitos T reguladores e o número de cópias de TREC/?g DNA (r = 0,529; p = 0,02). Conclusão: No presente estudo encontramos uma queda da função tímica com a idade, avaliada pela quantificação dos níveis de TREC em sangue periférico, que se correlacionaram positivamente com a proporção de células T reguladoras em crianças e adolescentes saudáveis. / Introduction: The thymus is a specialized lymphoid organ that is responsible for providing an exclusive microenvironment for T cell maturation and differentiation. The quantification of TREC (T cell receptor excision circle) has been widely used to evaluate and quantify thymic function in peripheral blood cells. Studies that evaluated TREC levels in Brazilian healthy children and adolescents are scarce in the literature. Objective: In the present study, we evaluated the TREC levels in peripheral mononuclear cells associated with the analysis of CD3, CD4, CD8 T cell markers, and activated T cells coexpressing CD38 and HLA-DR, and regulatory T cells co-expressing CD25 and Foxp3 in healthy children and adolescents in different age groups. Material and Methods: The quantification of sjTREC levels in genomic DNA of peripheral blood mononuclear cells (PBMC) was performed by real time quantitative PCR. TREC concentration was expressed as the number of copies of TREC/?g DNA. The analysis of CD3, CD4, CD8, CD38, HLA-DR, CD25 and Foxp3 expression was performed using flow cytometry methodology. Results: Ninety-five healthy children and adolescents were analyzed, 46 girls and 49 boys. The mean TREC count in PBMC in all individuals was 8.9 ± 3.6 x 104 TRECs/?g DNA. There was no significant difference in the number of TRECs/?g DNA between female and male gender (8.2 ± 3.3 x 104 TRECs/?g DNA vs 9.5 ± 3.9 x 104 TRECs/?g DNA, p = 0.085). There was an inverse correlation between age and TREC counts in PBMC in the 95 individuals (r = -0.846, p < 0.001), reflecting the well-known decrease of thymic function that occurs with age. The expression of CD3, CD4 and CD8 surface markers ranged between 45-62%, 60-65% and 14- 26%, respectively. There was no significant correlation between CD3, CD4 and CD8 proportion and age. There was a weak correlation between activated T cells expressing CD4+CD38+HLA-DR+ and age (r = -0.286; p = 0.023), however there was no significant correlation between T cells expressing CD8+CD38+HLA-DR+ and age (r = -0.229; p = 0.072). There was an inverse correlation between T cells expressing CD4+CD25+Foxp3+ and age (r = -0.467; p = 0.04). Additionally, we also found a positive correlation between CD4+CD25+Foxp3+ values and numbers of TREC/?g DNA in all studied individuals (r = 0.529, p = 0.02). Conclusion: In the present study we found a decrease in the thymic function with age, accessed by the quantification of the TREC level in peripheral blood, which was positively associated with the proportion of regulatory T cells.

Chez des patients allergiques, il est possible de retrouver dans leur sérum desanticorps spécifiques du médicament (IgE) et dans leur sang des lymphocytes T spécifiquesdu médicament. La présence de LT spécifiques du médicament chez des patients allergiquessuggère la présentation du médicament par des cellules présentatrices d’antigène telles queles cellules dendritiques.Nous nous sommes alors intéressés à mieux comprendre l’implication deslymphocytes T et des cellules dendritiques dans le développement des allergies auxantibiotiques comme la pénicilline G (ou Benzyl-Pénicilline) ou le sulfaméthoxazole.Ce travail de thèse a permis: i) de démontrer la présence de lymphocytes Tspécifiques de la pénicilline G dans le sang périphérique de donneurs non allergiques à unefréquence mesurable, ii) de développer deux approches expérimentales et de modélisationpour l’identification des épitopes potentiellement présentés aux lymphocytes T et iii)d’étudier l’effet des médicaments sur les cellules dendritiques.Les perspectives de ce travail sont de mieux comprendre les mécanismes impliquésdans les allergies médicamenteuses au niveau des lymphocytes T et des cellules dendritiqueset de développer des tests de prédiction du « potentiel allergique » des médicaments, afinde mieux prédire les allergies médicamenteuses lors du développement des médicaments. / In allergic patients, antibodies like IgE or T-lymphocytes specific for the drugimplicated can be detected and measured. Presence of T-lymphocytes specific for the drugin allergic patients suggested the presentation of the culprit drug to T-lymphocytes byantigen presenting cells like dendritic cells.We were interested in better understanding the implication of T-lymphocytes anddendritic cells in the development of antibiotics allergies such as penicillin G (or Benzyl-Penicillin) or sulfamethoxazole.The results obtained in this work allowed us: i) to demonstrate the presence ofbenzyl-penicillin-specific T cells in the peripheral blood of non-allergic donors, at adetectable frequency; ii) to develop two approaches: one experimental and one usingmodelisation for the identification of epitopes potentially presented to T-lymphocytes andiii) to study the effect of drugs on DC.The perspectives of this research work are to better understand the mechanismsimplicated in drug allergies and to develop predictive tests for “drug allergic potential", inorder to be able to better predict drug allergies during drug development.