A imunodeficiência comum variável (ICV) é uma imunodeficiência primária de origem heterogênea, definida como uma diminuição de pelo menos dois isótipos de imunoglobulinas, a falta de resposta anticórpica a imunizações e a exclusão de outras causas primárias de hipogamaglobulinemia. A ausência de níveis adequados de anticorpos em pacientes com ICV resulta em infecções bacterianas recorrentes, mais freqüentes no trato respiratório e digestivo, que podem levar a seqüelas sinusais e pulmonares. Nos últimos 6 anos iniciou-se a descoberta de genes relacionados à causa de doenças com o fenótipo de ICV, como os genes de TACI, BAFF-R, CD19 e ICOS. Dentre as alterações imunológicas, podemos também relatar deficiência de células B de memória (CD19+IgM-IgD-CD27+), levando a distúrbio de comutação isotípica e redução da secreção de imunoglobulinas. Atualmente tal característica vem sendo utilizada para classificar a ICV. No decorrer do presente trabalho pudemos observar que pacientes com ICV apresentam alterações na expressão de CD27 não somente em células B, mas também em células T, além de resposta linfoproliferativa ao estímulo de PHA reduzida. O CD27 consiste em uma molécula da família TNF presente constitutivamente em células T e após ativação em células B. Sua atuação na resposta imune está relacionada com a proliferação e co-ativação de células T específicas que atuam na interação T-B, na resposta de células B dependente de T. Dessa forma deficiências na via de CD27 podem resultar em defeitos nos mecanismos de comutação isotípica e de diferenciação de células B do centro germinativo, assim como de células de memória. Essas características podem ser observadas em modelos murinos de deficiências de CD27/CD70. Nossos achados permitem que uma nova janela se abra para o estudo da ICV. A avaliação dos distúrbios associados a defeitos de sinalização de CD27/CD70 em humanos pode se tornar uma nova ferramenta para a compreensão de uma deficiência tão pouco esclarecida. Tal enfoque pode eventualmente contribuir para o desenvolvimento de novos tratamentos, atuando diretamente na molécula em questão. Além disso, sugerimos também a utilização da fenotipagem das moléculas CD27 em linfócitos B e T, além da IgM e IgD de membrana para a caracterização da ICV, mais a análise da molécula CD154 para exclusão de outras imunodeficiências / Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder of heterogeneous origin, defined by a decrease of at least two immunoglobulin isotypes, lack of antibody response to immunization and the exclusion of other causes of primary hypogammaglobulinemia. The absence of adequate levels of antibodies in patients with CVID results in recurrent bacterial infections, most frequently in the respiratory and digestive tract, which can lead to sinusal and lung sequels. Over the past six years the discovery of genes related to the phenotype of CVID began, such as the genes of TACI, BAFF-R, CD19 and ICOS. Among the immunological changes, there is impairment of memory B cells (CD19+/IgM-IgD-CD27+), leading to disturbance of isotypic switching and reduced secretion of immunogobulins. Currently this feature has been used to classify CVID. During the present study we observed that patients with CVID present changes in the expression of CD27 not only in B cells, but also in T cells, and reduced lymphoproliferative response to PHA. CD27 molecule is a member of the TNF family present constitutively in T cells, and after activation in B cells. Its importance in the immune response is related to the proliferation and co-activation of specific T cells that act in T-B interaction, in the T cell dependent B cells response. Thus disturbances in the CD27 pathway can result in defects in isotypic switch and differentiation of germinal center B cells, as well as memory cells. These characteristics can be observed in murine models of CD27/CD70 deficiency. Our findings allow a new approach for the study of CVID. The evaluation of defects in CD27/CD70 signaling in humans might become a new tool for understanding an incompletely understood disease. Such an approach may contribute to the development of new treatments, acting directly on the molecule in question. In addition, we also suggest the use of phenotyping of CD27 molecules on B and T lymphocytes, in addition to membrane IgM and IgD to characterize CVID, associated to the analysis of the molecule CD154 to exclude other immunodeficiencies

INTRODUÇÃO: A infecção por HIV-1 é um grave problema de saúde pública causando elevada taxa de morbidade e mortalidade. Entretanto, alguns indivíduos são considerados resistentes à infecção por HIV-1, mesmo após repetidas exposições ao vírus. Vários fatores imunológicos e genéticos podem estar associados a resistência à infecção, como ativação de componentes da imunidade inata e também devido ao baixo perfil de ativação das células T. É possível que nos indivíduos expostos e não infectados por HIV-1 (ENI) ocorra uma importante atuação das células T secretoras de IL-17 e IL-22, e também as células T reguladoras, pois são necessárias para a manutenção e homeostase das mucosas associadas ao intestino (GALT). OBJETIVO: Avaliar o fenótipo e a função de células TCD4+ e TCD8+ em casais sorodiscordante ao HIV-1, compostos por indivíduos ENI e os parceiros infectados por HIV-1. MÉTODOS: Os casais sorodiscordantes ao HIV-1, consistiam de 23 indivíduos expostos não-infectados (ENI), 14 mulheres e 9 homens, com mediana de 41 anos e 21 parceiros infectados por HIV-1 (HIV), 20 homens e 1 mulher com mediana de 41 anos. Os controles saudáveis foram 24 indivíduos (14 mulheres e 10 homens) com mediana de 37 anos. Os casais sorodiscordantes foram compostos por 16 heterossexuais e 7 homossexuais, com tempo de relacionamento de 13 anos. As frequências de células Th17, Th22 e Tc22, as células T polifuncionais foram analisadas em células mononucleares (CMNs) do sangue periférico, estimulados com peptídeos da região Gag do HIV-1 e da enterotoxina B do Staphylococcus aureus (SEB), a frequência de células T reguladoras, o perfil fenotípico de exaustão/diferenciação e a expressão da integrina alfa4?7 e CCR9 em células T, foram realizados por citometria de fluxo. RESULTADOS: No grupo HIV, as células T CD4+ e CD8+ do sangue periférico mostrou maior frequência de CD95 e PD-1 e baixa expressão de CD127 comparado ao grupo ENI e controle. A frequência de células Th17 em CMNs aumentou nos grupos ENI e HIV-1 na condição sem estímulo, contudo, após estímulo com os peptídeos da região p24 da Gag do HIV-1 induziu resposta somente no grupo HIV-1. O grupo ENI mostrou resposta antígeno-especifica somente para IL-22. Além disto, avaliando as células Tc22 e Th22, foi verificado aumento da resposta aos peptídeos da Gag e também ao SEB, nos grupos HIV e ENI. A presença de células T polifuncionais antígeno-especificas, secretoras de 5-4 citocinas, foi detectada apenas em células T CD38+ no grupo HIV, enquanto os indivíduos ENI mostraram resposta polifuncional por células T CD38- somente ao estímulo policlonal por SEB. Uma diminuição do número absoluto de células T reguladoras (CD4+CD25+CD127low/-Foxp3+) foi detectada no grupo HIV comparado ao ENI e controle, com maior expressão de moléculas HLA-DR e CD95. Além disto, foi detectado diminuição na frequência de células TCD8+ ?4?7+ no grupo ENI e de células TCD4+ alfa4beta7+ nos grupos ENI e HIV. Houve uma correlação positiva entre as células Tc22 e Th22 com as células TCD8+ e TCD4+ que expressam alfa4beta7, no grupo ENI e HIV-1. CONCLUSÃO: Os indivíduos ENI são capazes de desenvolver resposta antígeno-específicas relacionadas com a IL-22, que possui importante função na imunidade de mucosas. Além disto, mostram presença de células T polifuncionais com baixo perfil de ativação a estímulo policlonal. Os dados evidenciam que os indivíduos ENI, mostram indução de células Tc22, aumento de expressão de moléculas de migração para o intestino e equilíbrio entre as células efetoras e Treg, que em conjunto, devem exercer importante papel para a resistência à infecção por HIV-1 / INTRODUCTION: The HIV-1 infection is a major public health problem causing high morbidity and mortality. However, some individuals are considered resistant to HIV-1 infection even after repeated HIV-1 exposures. Several immunologic and genetic factors could be associated with the resistance to infection, such as activation of innate immunity components and due to the low profile of T-cell activation. It is possible that in HIV-1 exposed uninfected individuals (EU) occurs an important activity of the T cells secreting IL-17 and IL-22, including regulatory T cells, which are necessary to maintenance of homeostasis of gut-associated lymphoid tissue (GALT). AIM: To evaluate the phenotype and function of CD4+ and CD8+ T cells in HIV-1-serodiscordant couples, composed by the EU individuals and the infected HIV-1 partners. METHODS: The HIV-1-serodiscordant couples consisted of 23 EU individuals, 14 women and 9 men, with a median age of 41 years and 21 partners infected by HIV-1, 20 men and 1 woman, with a median of 41 years. Healthy controls consisted of 24 individuals (14 women and 10 men) with a median age of 37 years. The serodiscordant couples were composed by 16 homosexuals and 7 heterosexuals, reporting a median relationship duration of 13 years with a single partner. The frequency of Th17, Th22 and Tc22 cells, the polyfunctional T cells were assessed in mononuclear cells (MNCs) from peripheral blood, stimulated with the peptides from the gag region of HIV-1 and enterotoxin B from Staphylococcus aureus (SEB), the frequency of regulatory T cells and the exhaustion/differentiation phenotypic profile and expression of integrin alfa4beta7 and CCR9 in T cells were assessed by flow cytometry. RESULTS: In HIV group, CD4+ and CD8+ T cells from peripheral blood showed a higher frequency of PD-1, and CD95 and low expression of CD127 compared to ENI and control groups. The frequency of Th17 cells in MNCs increased in ENI and HIV-1 groups in the unstimulated conditions, however, upon stimulation with p24 peptides of HIV-1 Gag induced response only in HIV-1 group. The ENI group showed antigen-specific response only for IL-22. Moreover, evaluating the Tc22 and Th22 cells, it was found increased response to Gag peptides and also for SEB in both, HIV and ENI groups. The presence of polyfunctional antigen-specific T cells secreting 5-4 cytokines, was only detected in CD38+ T cells from HIV group, while ENI individuals showed polyfunctional CD38- T cells response only with the polyclonal stimulus with SEB. A decreased absolute number of regulatory T cells (CD4 + CD25 + CD127low /-Foxp3 +) was detected in HIV group compared to the EU and control groups, with higher expression of HLA-DR and CD95 molecules. In addition, it was detected decreased frequency of CD8+ alfa4beta7 + T cells in the ENI group and CD4+ alfa4beta7+ T cells in both, ENI and HIV groups. There was a positive correlation between Tc22 and Th22 cells with the CD8+ and CD4+ T cells expressing alfa4beta7, in the ENI and HIV-1 groups. CONCLUSION: The EU individuals are able to develop antigen-specific response related to IL-22, which has an important function in the mucosal immunity. In addition, showed presence of polyfunctional T cells with low activation profile to polyclonal stimuli. The data show that the EU individuals, showed induction of Tc22 cells, increased expression of homing molecules into the intestine and balance between effector cells and Treg cells, which together, must play an important role in the HIV-1 resistance

A hepatite C é uma doença que leva à inflamação do fígado, sendo causada pelo vírus da hepatite C (VHC). Estima-se que existam 130 a 170 milhões de casos de infecção crônica pelo VHC por todo o mundo. Apesar do VHC ser principalmente hepatotrópico, foi evidenciada a detecção de RNA viral em células mononucleares do sangue periférico (PBMC). Evidências clínicas e experimentais têm demonstrado um importante tropismo do VHC por células do sistema imune, em especial por PBMCs. Apesar deste interessante achado, a importância da infecção do sistema imune na história natural da doença não é totalmente conhecida. É possível observar em alguns estudos que, em pacientes que desenvolvem infecção aguda, observa-se uma vigorosa resposta mediada por células T, específica ao VHC, mediada, por células T CD4+ e T CD8. Esta resposta é detectada na fase inicial da doença, e prolonga-se durante vários anos após a resolução do vírus. Pelo contrário, os pacientes que desenvolvem infecção crônica, apresentam, normalmente, respostas T fracas e / ou de curta duração, bem como defeitos nas funções efetoras de células T específicas. As respostas mediadas por células T com este perfil resultam, normalmente, em um baixo controlo da viremia e na sua persistência. Outra importante questão que permanece incerta até o presente momento é como a infecção das células imunes pelo VHC altera a sua função, principalmente no que concerne às PBMCs. Informações referentes às PBMCs e o VHC são pouco estudadas e referidas na literatura internacional, embora sejam de fundamental importância para a compreensão do seu peso na história natural da infecção. OBJETIVO: Baseado nessas incertezas o presente estudo tentará contribuir para a elucidação da influência do parasitismo por VHC na função de células TCD4+ e TCD8+ em pacientes naïve com indicação de tratamento com IFNpeg+RBV e comparar os dados obtidos antes, durante e pós-tratamento com os valores encontrados dos controles negativos, avaliando a influência do tratamento sobre a função dessas células. Verificar a presença do RNA-VHC nas PBMCs por RT-PCR em Tempo Real. Verificar a influência de fatores do vírus e do hospedeiro, tais como genótipo, carga viral, idade, gênero, raça, polimorfismo IL28B e biópsia hepática sobre as células parasitadas pelo VHC e sua produção de citocinas; Determinar a contagem e a função de linfócitos T CD4+ e CD8+ no sangue periférico desses pacientes; Determinar a influência do parasitismo de linfócitos pelo VHC sobre a função dos linfócitos TCD4+ e TCD8+. METODOLOGIA: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios, atendidos no Ambulatório de Hepatites da Divisão de Clínica de Doenças Infecciosas e Parasitárias do HC FMUSP. A contagem de células da subpopulação de linfócitos T CD4+ e CD8+ periféricas foram realizadas por citometria de fluxo FACSCanto II (BD) por meio do software MultiSet(BD). Foi analisada a presença/ausência do RNA-VHC em PBMC por PCR em tempo real no sistema TaqMan, no termociclador Applied Biosystems StepOne(TM). A função dos linfócitos TCD4+ e TCD8+ foram avaliadas através da técnica ELISPOT utilizanso o Kit Human IFN-gamma/IL-4 Dual-Color FluoroSpot. A contagem dos spots foi realizada em um leitor automatizado CTL-ImmunoSpot® S6 FluoroSpot Line. As PBMCs foram fracionadas e depletadas, utilizando-se o Kit Dynabeads FlowComp Human CD4 (Invitrogen Life Technologies) segundo as instruções do fabricante. RESULTADOS: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios. O genótipo 1 foi o mais prevalente 61,5%. O RNA do VHC foi detectado nas PBMCS em 88,4% dos pacientes. XVII Nossos resultados mostraram um aumento de células CD4 e CD8 parasitadas pelo VHC antes do tratamento, com valores estatisticamente significantes quando comparadas aos controles normais apenas no caso das CD4. Ocorreu, porém, um nítido prejuízo na produção de interleucinas por estas células parasitadas, particularmente a produção de IFN- y, com valores altamente significantes (0,009). Na semana 12, podemos ver o aumento de células CD4 no pré-tratamento, porém com diminuição na semana 12 e no follow up; entretanto a produção de IL-4 pelas células CD4 aumenta na semana 12 e cai novamente no seguimento; com as células CD8 ocorre leve queda na semana 12 porém uma tentativa de recuperação no follow up; sua produção de IFN-y cai na semana 12 e no follow up para números estatisticamente significantes. É a chamada \"exaustão\" de função destas células já descrita in vitro por alguns autores. Estes dados poderão ser muito úteis nas futuras observações desses pacientes que serão tratados com esquemas de DAAs sem interferon alfa. CONCLUSÃO: Em conclusão, os resultados desta pesquisa confirmam a importante influência do parasitismo das células CD4 e CD8 em suas funções / Hepatitis C is a disease that causes inflammation of the liver, caused by hepatitis C virus (HCV). It is estimated that there are 130 million to 170 million cases of chronic HCV infection worldwide. Although HCV is primarily hepatotropic was evidenced by detection of viral RNA in peripheral blood mononuclear cells (PBMC). Clinical and experimental evidence have demonstrated an important tropism of HCV by immune system cells, in particular by PBMC. Despite this interesting finding, the importance of infection of the immune system in the natural history of the disease is not fully known. It can be observed in some studies in patients who develop acute infection a vigorous response is observed mediated by T cells specific to HCV mediated by CD4 + and CD8. This response is detected at the initial stage of the disease, and extends for years after the resolution of the virus. Conversely, patients who develop chronic infection, are typically in the low T responses and / or short-term as well as defects in the effector functions of specific T cells. The responses mediated by T cells with the profile usually result in a low control of viremia and its persistence. Another important issue that remains unclear to date is how the infection of immune cells by HCV alters its function, especially with regard to the PBMCs. Information relating to the PBMCs and HCV are little studied and reported in the international literature, although they are of fundamental importance for the understanding of its weight in the natural history of infection. OBJECTIVE: Based on these uncertainties the present study attempts to contribute to the elucidation of the influence of parasitism by HCV on CD4 T cell function + and CD8 + in naïve patients with treatment indication with IFNpeg + RBV and compare the data obtained before, during and after treatment with the values found negative controls, assessing the influence of treatment on the function of these cells. Verify the presence of HCV-RNA in PBMC by RT-PCR in real time. Check the influence of virus and host factors such as genotype, viral load, age, gender, race, IL28B polymorphism and liver biopsy on the parasitized cells with HCV and their cytokine production; Determine count and function of lymphocytes T CD4 + and CD8 + peripheral blood of patients; To determine the influence of parasitism lymphocytes HCV on the function of CD4 + and CD8 + T lymphocytes. METHODS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls, treated at the Hepatitis Clinic of the Division of Clinical Infectious and Parasitic Diseases of the FMUSP. The lymphocyte subpopulation of T-cell count CD4 + and CD8 + peripheral been made by FACSCanto II flow cytometer (BD) through the multiset software (BD). the presence / absence of HCV-RNA in PBMC by real-time PCR was analyzed in the TaqMan system, Applied Biosystems thermal cycler StepOne (TM). The role of CD4 + T lymphocytes and CD8 + were assessed by ELISPOT technique utilizanso the Human Kit IFN-gamma / IL-4 Dual-Color FluoroSpot. The counting of the spots was carried out in an automated reader CTL-ImmunoSpot® S6 FluoroSpot Line. And fractionated PBMCs were depleted using Dynabeads kit is the Human CD4 FlowComp (Invitrogen Life Technologies) according to manufacturer\'s instructions. RESULTS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls. Genotype 1 was the most prevalent 61.5%. The HCV RNA was detected in PBMC in 88.4% of patients. Our results showed an increase of CD4 and CD8 cells parasitized with HCV prior to treatment, with statistically significant amounts compared to controls only if CD4. There was however, a marked impairment in production of interleukins for these parasitized cells, particularly the production of IFN- y, with highly significant values (0.009). At week 12, we can see the increase in CD4 cells before treatment, but with decreased at week 12 and at follow up; However IL-4 production by CD4 cells increased at week 12 and again falls in the following; with the CD8 cells is slightly lower at week 12 but a recovery attempt at follow up; their IFN-y production drops at week 12 and follow up to statistically significant numbers. It is called \"exhaust\" function of these cells in vitro been described by some authors. This data is very useful in future observations of these patients are treated with AADs regimens without interferon alfa. CONCLUSION: In conclusion, the results confirm the important influence of the parasitism of CD4 and CD8 cells in their functions

A asma afeta aproximadamente 300 milhões de pessoas no mundo e é a maior causa de internação hospitalar em crianças nos países desenvolvidos. Essa doença é incurável e por vezes refratária ao tratamento em um número significativo de pacientes. As taxas de prevalência de tabagismo entre pacientes asmáticos são semelhantes aos da população em geral e o impacto da fumaça de cigarro nestes pacientes ainda é clinicamente controverso. O objetivo deste trabalho foi traçar o perfil inflamatório e analisar o papel das células dendríticas sobre a imunomodulação provocada pela exposição à fumaça do cigarro na inflamação alérgica pulmonar induzida previamente por ovalbumina (OVA) em um modelo murino. Primeiramente avaliamos in vivo a ação da fumaça de cigarro na inflamação pulmonar alérgica crônica avaliando a responsividade brônquica, o remodelamento pulmonar, a produção de anticorpos antígeno-específicos, o perfil de células inflamatórias pulmonares e sistêmicas e a produção de citocinas inflamatórias e moduladoras. Em seguida, realizamos estudo in vitro do perfil de maturação, migração e inflamatório de células dendríticas expostas a OVA e/ou a extrato de fumaça de cigarro. Nosso estudo mostrou que a sensibilização e desafios inalatórios com OVA levaram à inflamação pulmonar de característica Th2 com aumento de responsividade brônquica, remodelamento, altos níveis de IgE e de citocinas pró-inflamatórias como IL-4, IL-5 e IL-13. A exposição à fumaça de cigarro, surpreendentemente, levou a uma redução dos níveis de IL-4, IL-5 e IL-13, e simultaneamente reduziu os níveis de citocinas anti-inflamatórias como IL- 10 e TGF-beta em animais sensibilizados e desafiados com antígeno. Foi observada nestes animais, uma redução no número de eosinófilos no lavado broncoalveolar e aumento no número de neutrófilos no pulmão. A combinação da inflamação alérgica com exposição à fumaça de cigarro levou a um aumento do recrutamento e ativação de células dendríticas linfoides nos linfonodos mediastinais, que mostrou relação direta com aumento do influxo de células T CD8+ e ativação das mesmas no pulmão. A inflamação alérgica juntamente com a exposição à fumaça de cigarro, levou a uma redução no recrutamento de células dendríticas plasmocitoides além de reduzir o recrutamento de células T regulatórias. In vitro, mostramos que o extrato de fumaça de cigarro combinado ao antígeno aumenta a capacidade migratória e fagocítica do antígeno pelas BMDCs. No entanto, houve redução da expressão gênica para IL-13 neste mesmo grupo. Concluímos que neste modelo de inflamação pulmonar alérgica crônica combinada com a exposição à fumaça de cigarro leva a uma descaracterização do perfil inflamatório característico da resposta Th2 com a redução do recrutamento de eosinófilos, redução dos níveis de IL-4, IL-5 e IL-13 aliados a um aumento do número de neutrófilos, o que pode estar relacionado ao aumento do recrutamento e ativação de células dendríticas linfoides bem como de células T CD8+ e redução local de células dendríticas plasmocitoides. Mostramos ainda que a fumaça de cigarro juntamente com o antígeno leva as células dendríticas a aumentarem sua capacidade fagocítica porém, reduzir sua capacidade pró-inflamatória pela expressão gênica reduzida de IL-13 / Asthma affects approximately 300 million people worldwide and it is the major cause of hospitalization among children in developed countries. This disease is often refractory to treatment in a high number of patients. The prevalence rates of smoking among asthmatic patients are similar to the general population and the impact of cigarette smoke is also clinically controversial. The main goal of this study is to outline, in a murine model, the inflammatory profile and to analyze the role of dendritic cells on the immunomodulation caused by exposure to cigarette smoke in ovalbumin (OVA)-induced pulmonary allergic inflammation. First, we evaluated in vivo the action of cigarette smoke on chronic allergic pulmonary inflammation, evaluating the bronchial responsiveness, pulmonary remodeling, the production of antigen-specific antibodies, pulmonary and systemic inflammatory cell profile and the production of inflammatory and modulating cytokines. Next, we performed an in vitro study of the maturation, migration and inflammatory profile of dendritic cells exposed to OVA and/or cigarette smoke extract. Our study showed that sensitization and challenge with OVA led to Th2-type lung inflammation with increased bronchial responsiveness, remodeling, high levels of IgE and proinflammatory cytokines such as IL-4, IL-5 and IL-13. Exposure to cigarette smoke has surprisingly led to a reduction in levels of IL-4, IL-5 and IL-13, and simultaneously reduced levels of anti-inflammatory cytokines such as IL-10 and TGF-beta in animals sensitized and challenged with the antigen. We also observed a reduction in the number of eosinophils in bronchoalveolar lavage fluid and an increase in the number of neutrophils in the lung of these animals. The combination of allergic inflammation with exposure to cigarette smoke led to increased recruitment and activation of lymphoid dendritic cells in the mediastinal lymph nodes, which showed to be direct related with increased activation and influx of CD8+ T cells in lung. Allergic inflammation combined with cigarette smoke led to a decrease of plasmacytoid dendritic cells a well as regulatory T cells. In vitro, we showed that cigarette smoke extract combined with antigen increased migratory and phagocytic capacity of BMDCs. However, there was a reduction of IL-13 gene expression in this same group. We conclude that in this model of chronic pulmonary allergic inflammation combined with exposure to cigarette smoke leads to a mischaracterization of the characteristic inflammatory profile of the Th2 response with the reduction of eosinophil recruitment, reduction of levels of IL-4, IL-5 and IL-13 allied to increased number of neutrophils, which is related to increased recruitment and activation of lymphoid dendritic cells as well as CD8+ T cells and local decrement of plasmacytoid dendritic cells. We further show that cigarette smoke combined with antigen increases dendritic cell phagocytic capacity however, reduces its pro-inflammatory capacity by the reduced gene expression of IL-13

LLC-B é uma neoplasia hematológica derivada de uma população de linfócitos B maduros CD5+ localizados na zona do manto dos folículos linfóides e é a mais comum das doenças linfoproliferativas. É uma doença clinicamente heterogênea na qual certos pacientes apresentam quadros indolentes que durante muitos anos podem ser controlados com pouco ou nenhum tratamento. Relatos da literatura sugerem que os linfócitos T na LLC-B podem ser incapazes de iniciar, manter e concluir uma resposta imune para a célula B maligna e outros antígenos, e podem estar diretamente envolvidos na manutenção do tumor. Os linfócitos são ativados, proliferam e polarizam sua resposta para padrões pro-inflamatórios ou antiinflamatórios, aumentando sua população e tornando-se capazes para realizar suas funções efetoras. Embora o processo de ativação dos linfócitos Th seja indispensável para a defesa do hospedeiro, é necessário que haja um equilíbrio homeostático, onde as células auto-reativas ou recorrentemente ativadas sejam eliminadas. A esse último mecanismo de manutenção do equilíbrio imunológico, dá-se o nome de Tolerância Periférica, sendo que o processo de morte celular induzida por ativação (AICD) constitui um dos principais mecanismos para sua manutenção. Assim, neste estudo também foram analisados membros do grupo de receptores de membrana da superfamília dos receptores de fatores de necrose tumoral (tumor necrosis factor receptor, TNFR). Esta família TNFR inclui diversos receptores, entre eles o FAS (CD95) e seu ligante FASL. O objetivo central deste trabalho é investigar alterações no compartimentos de linfócitos T como, Th1, Th2, Th17 e Treg, bem como membros da via extrínseca de morte, FAS e FASL nos linfócitos T CD4+. Os resultados mostraram que o número absoluto dos linfócitos T CD4+, CD8+ e Th1 é heterogêneo, sendo que alguns pacientes apresentaram aumento e outros diminuição destas células quando comparados com o grupo controle do estudo. Em relação a expressão de FAS e FASL os resultados também apresentaram heterogeneidade. Sendo assim foi analisado cada paciente e comparados com os fatores de prognósticos e dados clínicos em cada caso. Ainda há muito para ser investigado, mas este trabalho tem como perspectivas buscar melhor entendimento da participação dos linfócitos T CD4+ nas LLC-B, expandindo as possibilidade de tratamento e busca de novos alvos terapêuticos. / B-CLL is a hematologic malignancy derived from a mature population of CD5+ Blymphocytes located in the mantle zone of the lymphoid follicles and is the most common lymphoproliferative disorders. It is a clinically heterogeneous disorder in which patients have certain frames idle for many years that can be controlled with little or no treatment. Literature reports suggest that T lymphocytes in B-CLL may be unable to initiate, sustain and complete an immune response to the malignant B cell and other antigens, and may be directly involved in tumor maintenance. The lymphocytes are activated, proliferate and polarize their response patterns to pro-inflammatory or anti-inflammatory, increasing its population and becoming able to perform their effector functions. Although the process of Th lymphocyte activation is essential for host defense, there must be a homeostatic balance, where autoreactive cells are eliminated or recurrently activated. The latter mechanism of maintenance of immune balance, gives the name of Peripheral Tolerance, and the process of activation-induced cell death (AICD) is a major mechanism for its maintenance. In this study group members of the superfamily of membrane receptors of tumor necrosis factor (tumor necrosis factor receptor, TNFR) receptors were also analyzed. The TNFR family includes many receptors, including FAS (CD95) and its ligand FasL. The central objective of this study is to investigate changes in T lymphocyte compartments as Th1, Th2, Th17 and Treg as well as members of the extrinsic death pathway, FAS and FASL in CD4+ lymphocytes. The results showed that the absolute number of CD4+, CD8+ and Th1 lymphocytes is heterogeneous, with some patients showed an increase and others decrease of these cells when compared with the control group of the study. Regarding the expression of FAS and FASL results also showed heterogeneity. Thus each patient was analyzed and compared to the prognostic factors and to clinical data in each case. Much remains to be investigated, but this work has the prospects look better understanding of the role of the B-CLL CD4+ T lymphocytes, expanding the possibilities of treatment and the search for new therapeutic targets.

A eliminação de células-alvo por linfócitos T citotóxicos (CTLs) exerce um papel importante na imunidade protetora contra patógenos e células tumorais, o que pode ser desencadeada pela ação de perforina e granzima, ou pelas interações Fas-FasL. A interação Fas-FasL promove a apoptose pela formação de DISC, que é composto pela cauda citoplasmática de Fas, a proteína adaptadora FADD e a pro-caspase-8. Nesse complexo, a caspase-8 é ativada e induz a clivagem das caspases efetoras -3, -6 e -7, e/ou BID - membro da família Bcl-2, desencadeando o sinal apoptótico. Devido à sua relevância na sinalização apoptótica, a caspase-8 é alvo de inúmeros estudos. Uma correlação entre mutações no gene CASP8 de células tumorais e alta atividade citolítica foi demonstrada, sugerindo que a deficiência em caspase-8 pode representar um mecanismo de evasão imune. Nesse projeto, nosso objetivo foi avaliar o efeito da deficiência em caspase-8 na eliminação de células-alvo por linfócitos T CD8&#43. Para isso, nós estabelecemos uma técnica de citotoxicidade in vitro utilizando blastos de camundongos OTI como células efetoras e células RMA como alvo. Nesse ensaio, as células-alvo foram marcadas e pulsadas com diferentes concentrações de peptídeo OVA e colocadas em co-cultura a diferentes razões efetora: alvo (E:A) com linfócitos OTI, estimulados por 7 dias com ConA e rIL-2. A eliminação específica foi avaliada após 16 horas de co-cultura. Como controle para avaliar a especificidade ao MHC de classe I, utilizamos as células RMA-S, uma vez que expressam níveis reduzidos desta molécula na superfície. Os resultados demonstraram que o ensaio estabelecido é eficiente e antígeno-específico. Foi também observado que a eliminação das células RMA-S é menor em comparação com as células RMA, sendo essa diferença melhor discriminada em condições com números limitados de células efetoras. Para avaliar o efeito da caspase-8, células RMA caspase-8-suficientes e -deficientes, obtidas pela técnica CRISPR-Cas9, foram utilizadas como alvo nesse ensaio. Os resultados demonstram que, em condições com menor número de células efetoras, as células caspase-8-deficientes são significativamente mais resistentes à eliminação por linfócitos T CD8&#43, em comparação com as células caspase-8-suficientes. Portanto, nossos dados corroboram com a ideia de que a deficiência em caspase-8 poderia representar um mecanismo de evasão imune, principalmente na eliminação por linfócitos T CD8&#43. / The killing of target cells by cytotoxic T lymphocytes (CTLs) plays a major role in protective immunity to pathogens and tumor cells, which can be triggered by either the action of perforin and granzymes or FAS-FASL interactions. The Fas-FasL interaction leads to apoptosis through the formation of DISC, which is composed by the cytoplasmic tail of Fas, the adaptor protein FADD and the pro-caspase-8. At the complex, caspase-8 become activated and transduce the apoptotic signal by cleaving the effector caspases-3, -6 and -7 and/or the Bcl-2 family member Bid. Due to its role on apoptotic signaling, caspase-8 is the target of several studies. Recently, it has been demonstrated a correlation between mutations in CASP8 in tumors from patients with high cytolytic activity, suggesting that deficiency in caspase-8 may represent an immune evasion mechanism. Our project aimed to evaluate the effect of caspase-8 deficiency on the elimination of target cells by CD8&#43 T lymphocytes. In order to assess this elimination, we have established an in vitro cytotoxic assay using as effector, blasts from OTI mice, and as target, RMA cells. In this assay, the target cells were labeled, pulsed with OVA peptide at different concentrations and then co-cultured with OTI lymphocytes, which were stimulated with ConA and rIL-2, at different effector: target (E: T) ratios. The specific elimination was evaluated after 16 hours of co-culture. As a control to assess the specificity to MHCI of our assay, RMA-S cells were used because they have reduced levels of MHCI at surface. The results have shown that OTI blasts have effector capacity and that our established assay is antigen-specific. In addition, we observed that RMA-S elimination is reduced in comparison to RMA cells. The difference between RMA and RMA-S is better detected in conditions with limited number of effector cells. In order to assess the effect of caspase-8 in the resistance to elimination by CD8&#43 T cells, caspase-8-sufficient and deficient RMA cells (obtained by CRISPR-Cas9 technique) were used as targets in this assay. Our results demonstrate that caspase-8-deficient cells are more resistant to elimination than wild-type counterparts. This difference is better detected at conditions with reduced number of effector cells. Thus, our data suggests the deficiency in caspase-8 results in lower elimination by CD8&#43 T lymphocytes, suggesting this might represent an immune evasion mechanism.

<p>This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy.</p><p>The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated <i>ex vivo</i>.</p><p>Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3⁺ to CD3⁺ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications.</p><p>Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. <i>In vitro</i> expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4⁺ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048).</p><p>In conclusion, human solid tumours are recognised in sentinel nodes and <i>in vitro</i> expanded sentinel node-acquired CD4⁺ T lymphocytes seem useful in the treatment of patients with disseminated cancer.</p>

This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy. The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated ex vivo. Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3+ to CD3+ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications. Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. In vitro expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4+ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048). In conclusion, human solid tumours are recognised in sentinel nodes and in vitro expanded sentinel node-acquired CD4+ T lymphocytes seem useful in the treatment of patients with disseminated cancer.

Background: Tobacco smoking is the main cause of chronic obstructive pulmonary disease, COPD, but the mechanisms by which cigarette smoke induces COPD are still elusive. T lymphocytes have been implicated in the pathogenesis of the disease, but their role in the airway inflammation in COPD is not fully understood. The aim of this thesis was therefore to address T lymphocyte subsets and their activation in the airways of subjects with COPD, in comparison to smokers with normal lung function (S) and never smokers (NS). Methods: Subjects with moderate to severe COPD were recruited along with controls. They were all non-atopic and clinically stable, without any exacerbation during at least three months prior to inclusion. Only medication with short-acting β2-agonists and/or anti-cholinergic drugs was permitted. All subjects underwent bronchoscopy with endobronchial mucosal biopsy sampling as well as bronchial wash, BW, and bronchoalveolar lavage, BAL, collection. Biopsies were immunohistochemically stained for inflammatory cells and markers. BW and BAL fluids were prepared for differential cell counts. Soluble markers were measured in BW and lymphocyte subsets were determined in BAL using flow cytometry. Results: In biopsies, an increase in epithelial CD3+ and CD8+ cells was found in COPD, compared to NS. In BAL fluid, CD8+ cells were enhanced, whereas CD4+ cells were reduced in subjects with COPD and S, compared to NS. Furthermore, CD4+ and CD8+ cells were more activated both in COPD and S, in terms of increased expression of CD25, CD69 and HLA-DR. NKG2D-expressing CD8+ T cells in BAL fluid were enhanced in both COPD and S. CD4+CD25bright cells were upregulated in COPD and S, suggesting the presence of regulatory T cells. Further analyses of T cell subsets with the more specific markers for regulatory T cells, FoxP3 and CD127, indicated a smoking-induced expansion of non-regulatory T cells, which tended to normalize after smoking cessation in COPD. Currently smoking subjects with COPD still expressed high proportions of activated non-regulatory CD4+ T cells. The data on FoxP3 expression further indicated that the increase in CD25 expression in COPD and S was not only associated with the expansion of regulatory T cells. As CD127 expression is reported to be inversely associated with FoxP3, the data indicate the expansion of a non-regulatory CD25+ population in smokers and patients with stable COPD. The immunohistochemical staining for the NKG2D ligands MICA and MICB on epithelial cells was unchanged. Conclusion: The results of this thesis suggest a role for CD4+ and CD8+ T-cells in clinically stable COPD, indicating that T-cells are of importance in the long-term inflammatory response in COPD. Regardless of current smoking habits, activated CD8+ T lymphocytes were found to be increased in BAL fluid from subjects with COPD, suggesting that changes in CD8+ T cells are associated with a persistent immune response and, thus, of importance in COPD pathogenesis. In contrast, the expansion of non-regulatory CD25+CD4+ cells in BAL fluid seemed to be preferentially smoke-related. In summary, the data indicate that, among airway T cells, changes in CD8+ cells seem to be highly associated with COPD pathogenesis, whereas changes in CD4+ cells appear to be related to cigarette smoke-induced responses. Further, a non regulatory population of helper T cells was identified in BAL fluid of COPD patients, which may contribute to the persistent cytotoxic T cell responses.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.