T regulatory (TR) cells suppress T-cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also has a pivotal role in regulation of T-cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating TR-cell functions that inhibit T-cell responses during virus-associated malignancy, TR cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4+ CD25+ and CD8+CD25 + TR cells, as well as high levels of PD-1 expressions on these TR cells were found in the peripheral blood of subjects with HCV-L compared with those from non-HCV-L or HCV alone or HS. TR cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of TR cells in peripheral blood mononuclear cells from HCV-L improved T-cell proliferation. Additionally, the suppressed T-cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in TR-cell number and the ability of TR to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating TR cells that suppress T-cell functions in the setting of HCV-associated B-cell lymphoma.

Interleukin-27 (p28/EBI3) is an immunomodulatory cytokine expressed by activated antigen presenting cells. Although first discovered to be involved in Th1 cell differentiation, further studies demonstrated the immunosuppressive functions of IL-27 including inhibition of Th2 and Th17 differentiation, development of a tolerogenic phenotype in dendritic cells (DC), and promoting type 1 regulatory T cells (Tr1). The anti-inflammatory effects of IL-27 have been demonstrated in vivo in murine models of parasitic infections and autoimmune diseases. Despite the prevalence of studies detailing the induction of IL-27 expression and the role of IL-27 in Tr1 differentiation, little is known about factors that negatively regulate IL-27 expression and Tr1 differentiation. Prostaglandin E2 (PGE2), a lipid mediator abundant at inflammatory sites, was shown to act as a proinflammatory agent in models of inflammatory/autoimmune diseases primarily by promoting CD4 Th1/Th17 differentiation. Here we describe a novel proinflammatory mechanism for PGE2 through the inhibition of IL-27 production in conventional dendritic cells (cDC) and the inhibition of Tr1 differentiation. PGE2 inhibits IL-27 production in bone marrow-derived DC and macrophages, as well as in splenic cDC, through EP2/EP4 receptors, induction of cAMP, and downregulation of IRF1 expression and binding to the p28 IL-27 ISRE site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFN-β, STAT1 or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, EPAC, PI3K, or MAPKs. We observed similar inhibition of p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. In addition to the inhibition of IL-27 production in APCs, PGE2 also directly affects Tr1 differentiation by reducing IL-27-induced CD4+CD49b+LAG-3+Foxp3- Tr1 cells and IL-10 production. The inhibitory effect is mediated by EP4 and induction of cAMP in differentiating CD4 T cells. IL-27-induced Tr1 differentiation and function depends primarily on the sustained expression of c-Maf in addition to AhR and Blimp-1. PGE2 significantly reduced expression of c-Maf without affecting AhR and only marginally reducing Egr-2/Blimp-1 expression. The effects of PGE2 on Tr1 cells are independent of STAT1/STAT3 signaling and of IL-21 signaling. In addition, the effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. The effects of PGE2 on both IL-27 production and IL-27-induced Tr1 differentiation represent novel proinflammatory mechanisms of PGE2. / Microbiology and Immunology

Les cellules T régulatrices (Treg) jouent un rôle important dans différentes infections chroniques comme le VIH. Lors de l’infection chronique par le VIH, une augmentation du nombre de ces cellules limite la réponse des cellules T effectrices spécifiques du virus mais permet également le contrôle de la très forte activation du système immunitaire. La régulation de l’activité suppressive des Treg constitue une voie importante pour le développement d’un vaccin, l’efficacité de la surveillance tumorale et l’auto-immunité. Dans ce travail, nous avons étudié différents mécanismes de régulation des Treg chez les patients infectés par le VIH et sujets sains. Le but de mon travail a été d’étudier les mécanismes impliqués dans les fonctions suppressives des Treg et de leur régulation. Nous nous sommes particulièrement intéressés au rôle de Notch sur la sensibilité des cellules T effectrices à la fonction suppressive des Treg, en montrant que l’activation de Notch rendait les cellules T effectrices plus sensibles à l’action suppressive des Treg, et ceci même en présence d’un très faible nombre de Treg. Nous avons démontré que certains ligands de Notch, tels que DL-4 et Jagged-1 mais non DL-1, régulent l’effet inhibiteur des Treg sur les cellules T effectrices par une surexpression de TGFβ-RII et à la phosphorylation de Smad3. Le rôle important de l’enzyme CD39 dans la fonction suppressive des Treg a été décrit, mais peu d’études ont démontré son rôle dans l’infection par le VIH. Nous avons montré que les Treg de patients infectés par le VIH experiment plus fortement le CD39, et que leur effet suppresseur était inhibé en utilisant un anticorps monoclonal anti-CD39 et maintenu en utilisant un agoniste de l’adénosine. Nous avons également montré que le polymorphisme du gène CD39, associé à une plus faible expression de CD39, était corrélé à une plus lente progression de la maladie. Nos résultats montrent non seulement les mécanismes impliqués dans l’activité suppressive des Treg lors de l’infection par le VIH, mais constituent une approche intéressante pour en modifier les fonctions. Enfin, nous avons recherché l’effet de l’IL-7 sur le phénotype des Treg et l’expression de molécules impliquées dans leur fonction suppressive. Nos résultats montrent deux effets synergique de l’IL-7 sur les Treg mémoires: diminution de l’expression de CD39 à leur surface induisant une diminution de leur fonction suppressive, et surexpression du récepteur à l’ATP induisant un phénotype Th17. L’administration d’IL-7 in vivo chez des patients infectés par le VIH a confirmé la modification de phénotype des Treg en Th17 et notamment une surexpression du facteur de transcription spécifique des Th17, RORγt. En conclusion, notre travail apporte de nouvelles connaissances sur les mécanismes impliqués dans les fonctions inhibitrices des Treg et comment moduler ces fonctions. Ceci pourrait avoir un impact clinique direct, soit dans le traitement de maladies associées à un dysfonctionnement des Treg (infections chroniques virales, sclérose multiple, diabète de type 1, arthrite rhumatoide et lupus érythémateux), soit dans les stratégies vaccinales. / T regulatory cells (Treg cells) play an important role during various chronic infections like HIV. Increase in Treg cell number during chronic phase limit the HIV specific T effector cell response but may control exaggerated activation of the immune system. Modulation of regulatory T cell (Treg cells) suppression has important implications for vaccine development, the effectiveness of tumor surveillance, and the emergence of autoimmunity. Here we studied various mechanisms of Treg cells modulation in HIV+ patients and healthy subjects. The aim of my work was to decipher some aspects of the mechanisms involved in Treg cell mediated suppressive effects and the modulation of Treg cell suppressive effects in healthy subjects and HIV- infected patients.We have extended knowledge on the role of Notch in Treg/Effectors T cells cross talk. Here we focused our interest on the role of Notch pathway on the sensitivity of effectors T cells to Treg cell-mediated suppression, showing that Notch activation may significantly increase the sensitivity of effector cells to Treg cells even at a low ratio. We demonstrated that Notch ligands DL-4 and Jagged-1, but not DL-1 modulate significantly the suppressive effect of Treg cells on effectors T cells through an up regulation of TGF-RII expression and the phosphorylated form of Smad3 protein.A critical role of CD39 has been described for Treg cells in general but only a few studies have analyzed its role in HIV infection. We showed an increase in CD39 expression on Treg cells in a cohort of HIV- infected patients. Treg cells inhibitory effects were relieved by CD39 down modulation using an antiCD39 monoclonal antibody and this suppressive effect was reproduced on effector CD8+ T cells by an adenosine agonist. We found that CD39 gene polymorphism associated with a lower CD39 expression correlated with slower progression to AIDS. Our results shows not only the mechanism by which Treg cell suppression occurs during HIV infection but also provide an attractive approach to modify Treg cell functions.Finally, we have investigated the role of IL-7 on the phenotype of Treg cells and expression of molecules involved in the suppressive functions of these cells. Our results show that IL-7 exerts two synergistic effects on memory Treg cells. It decreases their suppressive effect by decreasing CD39 expression and increases ATP receptor leading to a switch towards a Th17 phenotype. In vivo administration of IL-7 tipped the balance towards a higher expression of RORγT in PBMCs from HIV infected patients.In conclusion our study bring new findings in the mechanisms involved in Treg cell mediated suppression and the way to modulate these cells which could have direct clinical impact either in the treatment of diseases associated with Treg cell dysregulation (chronic viral infections, autoimmune disorders like multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and systemic lupus erthematosus) or during vaccination.

Apesar dos avanços sobre a efetiva participação das células T reguladoras (Treg) na resposta imune antitumoral, ainda existem vários pontos que precisam ser esclarecidos. Visto que, os fatores que controlam a migração destas células para o microambiente tumoral ainda não estão totalmente definidos, o esclarecimento dos mecanismos de migração de células Treg no contexto do câncer poderia fornecer novos alvos para o desenvolvimento de terapias mais específicas. Diversos modelos de estudo demonstraram que o recrutamento preferencial de células Treg ao invés de outros tipos de células T pode ser explicado pela expressão diferencial de receptores de quimiocinas como o CCR5. Assim, é de extrema importância estabelecer qual é o papel de CCR5 na migração de células Treg em tumores induzidos quimicamente e seu envolvimento no desenvolvimento tumoral. Baseado no exposto, o presente estudo analisou o envolvimento de CCR5 na migração de células Treg e a sua correlação com o desenvolvimento de carcinoma espinocelular (CEC) induzido quimicamente. Os resultados obtidos demonstraram que camundongos geneticamente deficentes de CCR5 (CCR5KO) apresentaram baixo número de células Treg nas lesões e foram menos suscetíveis ao desenvolvimento de carcinoma espinocelular. Na fase de progressão tumoral verificou-se o desenvolvimento de CEC in situ por animais CCR5KO em combinação com a maior infiltração leucocitária, enquanto camundongos do grupo controle (WTCEC) apresentaram lesões de CEC bem diferenciado associado à elevada frequência de células Treg no microambiente tumoral e menor infiltração leucocitária. Interessantemente, a transferência adotiva de células Treg CCR5+ para animais CCR5KO (CCR5CEC Treg) resultou no acúmulo destas células no microambiente tumoral, elevado nível de CCL4, CCL17 e CCL22, e aumento da suscetibilidade desses animais à carcinogênese química. Verificou-se o desenvolvimento de CEC indiferenciado por animais CCR5CEC Treg e este foi associado à elevada frequência de macrófagos, células mielóides e dendríticas, linfócitos CD19+, T CD4+, T CD8+ e células Treg na fase de progressão tumoral. Outro aspecto relevante de nosso estudo foi à observação de que a transferência adotiva de células T CD4+CD25-CCR5+ para animais CCR5KO (CCR5CEC CD4+) induziu o desenvolvimento de CEC moderadamente diferenciado com características intermediárias as lesões observadas em animais WTCEC e CCR5CEC Treg. A transferência adotiva de células T CD8+CCR5+ para animais CCR5KO (CCR5CEC CD8+) promoveu o aparecimento precoce de papilomas e inibiu a progressão de papilomas para o CEC. A menor suscetibilidade à carcinogênese química de animais CCR5CEC CD8+ foi associada ao alto número de macrófagos, células mielóides, linfócitos B e T CD8+, células NK detectado nas lesões destes animais. Dessa forma, os resultados descritos estabelecem que a quimiotaxia de células Treg para o microambiente tumoral é dependente de CCR5 e estas células regulam aspectos críticos desta doença, sugerindo que o bloqueio da migração de células Treg CCR5+ seria uma importante estratégia imunoterapêutica no combate deste tipo de câncer. / Considering the advances on the effective participation of regulatory T cells (Treg) in the antitumor immune response, there are still several points that need to be clarified. The mechanisms that control the Treg cells migration to the tumor microenvironment are not completely defined, for these reason, establish these mechanisms could provide new targets for the development of more specific therapies. Several study models have demonstrated that preferential recruitment of Treg cells rather than other types of T cells can be explained by the differential expression of chemokine receptors such as CCR5. Thus, the present study examined the involvement of CCR5 in the migration of Treg cells and their correlation with the development of squamous cell carcinoma (SCC) chemically induced. The results showed that CCR5 knockout mice (CCR5KO) showed a low number of Treg cells in the lesions and these animals were less susceptible to the development of squamous cell carcinoma. SCC in situ was developed in CCR5KO mice and associated with high leukocytes infiltration, whereas the development SCC well differentiated in the control group (WTSCC) was associated with a high number of Treg cells and lower leukocyte infiltration in the tumor microenvironment. Interestingly, adoptive transfer of CCR5+Treg cells to CCR5KO mice (CCR5SCC Treg) resulted in the accumulation of these cells, high levels of CCL4, CCL17 and CCL22 in the tumor microenvironment and increased susceptibility to chemical carcinogenesis. CCR5SCCTreg mice developed SCC undifferentiated associated with a higher incidence of macrophages, myeloid and dendritic cells, CD19+, CD4+ T, CD8+ T lymphocytes, and Treg cells in the stage of tumor progression. Another relevant aspect of our study was the observation that adoptive transfer of CD4+CD25-CCR5+ T cells to CCR5KO animals (CCR5SCC CD4+) induced the development of SCC moderately differentiated with intermediate features observed in the WTSCC and CCR5SCC Treg mice. The adoptive transfer of CD8+CCR5+ T cells to CCR5KO mice (CCR5SCC CD8+) promoted the early incidence of papillomas and inhibited the progression to SCC. Reduced susceptibility to skin carcinogenesis in CCR5SCC CD8+ mice was associated with high frequency of macrophages, myeloid cells, B lymphocytes, CD8+ T lymphocyte and NK cells. In this study we showed that the migration of Treg cells to the tumor microenvironment is CCR5 dependent and that it regulates critical aspects of tumor development. The development of drugs that blocks CCR5+ Treg cells migration could be an important immunotherapeutic strategy to control this type of cancer.

A hanseníase é uma doença crônica causada por Mycobacterium leprae e apresenta diversas formas clínicas. O entendimento da interação parasita-hospedeiro na hanseníase evidenciou que ocorre a persistência assintomática do patógeno, caracterizando um estado de latência. Os fatores mais importantes relacionados com a permanência do patógeno são: a patogenicidade do agente infeccioso e o perfil da resposta imune, no qual os eventos de migração celular, produção de citocinas, as células efetoras e reguladoras são extremamente relevantes. As células T reguladoras (Treg) desempenham papel central na regulação da resposta imune em infecções crônicas o que favorece a persistência do patógeno. A importância de células T reguladores na hanseníase ainda é pouco conhecida. Neste trabalho investigou-se a presença de células T reguladoras em lesões e sangue periférico de indivíduos com hanseníase. Inicialmente avaliou-se a proliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) de pacientes com hanseníase. Os resultados evidenciaram que não há diferenças quanto à proliferação de células T e produção de IFN-γ e TNF-α por células desses pacientes, mas a produção de IL-4 e IL-5 foi detectada apenas entre os pacientes com hanseníase virchoviana. Em relação à presença de células T reguladoras, os resultados evidenciaram aumento no número de linfócitos T CD4+CD25+FoxP3+ no sangue periférico de pacientes com hanseníase virchoviana. As células T reguladoras dos pacientes com hanseníase apresentaram elevada expressão de moléculas co-inibitórias PD-1, CTLA-4, GITR e ICOS. De modo relevante, as células T CD4+CD25+ isolados de pacientes com hanseníase virchoviana apresentaram maior atividade supressora quando comparado às células isoladas de pacientes com hanseníase tuberculóide. As células T CD4+CD25+ de pacientes com hanseníase virchoviana inibiram a proliferação de PBMC alogênico e a produção de IFN-γ e TNF-α. Os resultados demonstraram também que nas amostras de lesão de pele de pacientes com hanseníase virchoviana há acúmulo de células CD25+ produtoras de IL-10 e TGF-β, enquanto que estas células não foram detectadas nas lesões de pacientes com hanseníase tuberculóide. Dessa forma, os resultados descritos indicam que pacientes com hanseníase virchoviana apresentam aumento no número de células T reguladoras circulantes e no infiltrado inflamatório, e estas células apresentaram maior atividade supressora. O acúmulo de células T reguladoras no sítio da infecção pode ser correlacionado com o controle da resposta imune e conseqüente persistência de M. leprae. / Leprosy is caused by Mycobacterium leprae and its clinical features depend on the host immune background. The understanding of parasite-host interactions in leprosy have highlighted asymptomatic persistence of the pathogen, which indicates that this infection becomes latent. The most important factors related to the permanence of pathogens are: the pathogenicity of the infectious agents; the profile of the immune response developed by the host whose events of cellular migration, cytokines production, and the effector and regulatory cells are extremely relevant. The regulatory T cells (Treg) seem to play a central role in the regulation of the immune response in chronic infections, which favors the persistence of the pathogen. Herein, we analyzed the relation between tuberculoid and lepromatous leprosy with the presence and function of T regulatory cells from peripheral blood mononuclear cells (PBMC) and skin lesions from these patients. First, the proliferation and cytokine production of PBMC isolated from leprosy patients were analyzed. We did not observe any difference in the proliferation ability or IFN-γ and TNF-α release; however, the production of IL-4 and IL-5 was detected only in patients with lepromatous leprosy. Furthermore, T CD4+CD25+FoxP3+ cells were detected in the PBMC of patients with leprosy and these cells from lepromatous patients showed high expression of co-inhibitory molecules such as PD-1, GITR, CTLA-4 and ICOS. T CD4+CD25+cells isolated from patients with lepromatous leprosy were significantly more suppressive than the cells obtained from tuberculoid patients. In addition, TCD4+CD25+ cells isolated from patients with lepromatous leprosy inhibited allogeneic PBMC proliferation and their production of IFN-γ and TNF-α. The results also demonstrated that IL- 10 and TGF-ß were co-expressed with CD25+ cells at the inflammatory infiltrate of skin lesions from lepromatous patients, but similar results were not detected among tuberculoid patients. Thus, these results indicate that lepromatous leprosy patients have an enhanced presence of Treg cells with a suppressive ability in the blood and in the inflammatory infiltrate. The accumulation of Treg cells at the infection sites might be associated to the control of immune response and consequently to Mycobacterium leprae presistence.

Apesar dos avanços sobre a efetiva participação das células T reguladoras (Treg) na resposta imune antitumoral, ainda existem vários pontos que precisam ser esclarecidos. Visto que, os fatores que controlam a migração destas células para o microambiente tumoral ainda não estão totalmente definidos, o esclarecimento dos mecanismos de migração de células Treg no contexto do câncer poderia fornecer novos alvos para o desenvolvimento de terapias mais específicas. Diversos modelos de estudo demonstraram que o recrutamento preferencial de células Treg ao invés de outros tipos de células T pode ser explicado pela expressão diferencial de receptores de quimiocinas como o CCR5. Assim, é de extrema importância estabelecer qual é o papel de CCR5 na migração de células Treg em tumores induzidos quimicamente e seu envolvimento no desenvolvimento tumoral. Baseado no exposto, o presente estudo analisou o envolvimento de CCR5 na migração de células Treg e a sua correlação com o desenvolvimento de carcinoma espinocelular (CEC) induzido quimicamente. Os resultados obtidos demonstraram que camundongos geneticamente deficentes de CCR5 (CCR5KO) apresentaram baixo número de células Treg nas lesões e foram menos suscetíveis ao desenvolvimento de carcinoma espinocelular. Na fase de progressão tumoral verificou-se o desenvolvimento de CEC in situ por animais CCR5KO em combinação com a maior infiltração leucocitária, enquanto camundongos do grupo controle (WTCEC) apresentaram lesões de CEC bem diferenciado associado à elevada frequência de células Treg no microambiente tumoral e menor infiltração leucocitária. Interessantemente, a transferência adotiva de células Treg CCR5+ para animais CCR5KO (CCR5CEC Treg) resultou no acúmulo destas células no microambiente tumoral, elevado nível de CCL4, CCL17 e CCL22, e aumento da suscetibilidade desses animais à carcinogênese química. Verificou-se o desenvolvimento de CEC indiferenciado por animais CCR5CEC Treg e este foi associado à elevada frequência de macrófagos, células mielóides e dendríticas, linfócitos CD19+, T CD4+, T CD8+ e células Treg na fase de progressão tumoral. Outro aspecto relevante de nosso estudo foi à observação de que a transferência adotiva de células T CD4+CD25-CCR5+ para animais CCR5KO (CCR5CEC CD4+) induziu o desenvolvimento de CEC moderadamente diferenciado com características intermediárias as lesões observadas em animais WTCEC e CCR5CEC Treg. A transferência adotiva de células T CD8+CCR5+ para animais CCR5KO (CCR5CEC CD8+) promoveu o aparecimento precoce de papilomas e inibiu a progressão de papilomas para o CEC. A menor suscetibilidade à carcinogênese química de animais CCR5CEC CD8+ foi associada ao alto número de macrófagos, células mielóides, linfócitos B e T CD8+, células NK detectado nas lesões destes animais. Dessa forma, os resultados descritos estabelecem que a quimiotaxia de células Treg para o microambiente tumoral é dependente de CCR5 e estas células regulam aspectos críticos desta doença, sugerindo que o bloqueio da migração de células Treg CCR5+ seria uma importante estratégia imunoterapêutica no combate deste tipo de câncer. / Considering the advances on the effective participation of regulatory T cells (Treg) in the antitumor immune response, there are still several points that need to be clarified. The mechanisms that control the Treg cells migration to the tumor microenvironment are not completely defined, for these reason, establish these mechanisms could provide new targets for the development of more specific therapies. Several study models have demonstrated that preferential recruitment of Treg cells rather than other types of T cells can be explained by the differential expression of chemokine receptors such as CCR5. Thus, the present study examined the involvement of CCR5 in the migration of Treg cells and their correlation with the development of squamous cell carcinoma (SCC) chemically induced. The results showed that CCR5 knockout mice (CCR5KO) showed a low number of Treg cells in the lesions and these animals were less susceptible to the development of squamous cell carcinoma. SCC in situ was developed in CCR5KO mice and associated with high leukocytes infiltration, whereas the development SCC well differentiated in the control group (WTSCC) was associated with a high number of Treg cells and lower leukocyte infiltration in the tumor microenvironment. Interestingly, adoptive transfer of CCR5+Treg cells to CCR5KO mice (CCR5SCC Treg) resulted in the accumulation of these cells, high levels of CCL4, CCL17 and CCL22 in the tumor microenvironment and increased susceptibility to chemical carcinogenesis. CCR5SCCTreg mice developed SCC undifferentiated associated with a higher incidence of macrophages, myeloid and dendritic cells, CD19+, CD4+ T, CD8+ T lymphocytes, and Treg cells in the stage of tumor progression. Another relevant aspect of our study was the observation that adoptive transfer of CD4+CD25-CCR5+ T cells to CCR5KO animals (CCR5SCC CD4+) induced the development of SCC moderately differentiated with intermediate features observed in the WTSCC and CCR5SCC Treg mice. The adoptive transfer of CD8+CCR5+ T cells to CCR5KO mice (CCR5SCC CD8+) promoted the early incidence of papillomas and inhibited the progression to SCC. Reduced susceptibility to skin carcinogenesis in CCR5SCC CD8+ mice was associated with high frequency of macrophages, myeloid cells, B lymphocytes, CD8+ T lymphocyte and NK cells. In this study we showed that the migration of Treg cells to the tumor microenvironment is CCR5 dependent and that it regulates critical aspects of tumor development. The development of drugs that blocks CCR5+ Treg cells migration could be an important immunotherapeutic strategy to control this type of cancer.

The mammalian pregnancy comprises a challenge to the maternal immune system since the fetus is semi-allogeneic and could thus be rejected. Pregnancy success is associated with the placenta that is not only essential for oxygen supply, nourishment and pregnancy hormones but also plays a role in the protection of the fetus against maternal immunologic attack. The aim of the current studies was to elucidate the role of human placenta as an immunomodulatory organ with a special focus on placental exosomes as vehicles for establishment of maternal tolerance to the fetus. We discovered that the syncytiotrophoblast in human normal pregnancy constitutively produces and secretes exosomes. Exosomes are 30-100 nanometer-sized membrane vesicles of endosomal origin that convey intercellular communication. Exosomes are produced and released through the endosomal compartment and reflect the type and the activation state of the cells that produce and secrete them. They carry cytosolic and membrane-bound proteins and nucleic acids and can influence and re-program recipient cells. Depending on their interactions with cells of the immune system they can be divided into immunostimulatory or immunosuppressive. We developed methods for isolation and culture of trophoblast and placental explants from human normal first trimester pregnancy and isolated exosomes from the culture supernatants.  These exosomes were characterized biochemically and functionally regarding mechanisms with potential importance in the establishment of maternal tolerance towards the fetus. The following aspects were studied: 1) exosomal modulation of the NKG2D receptor-ligand system, a major cytotoxic pathway for NK- and cytotoxic T cells and thus potentially dangerous to the fetus; 2) placental exosome-mediated apoptosis of activated immune effector cells; and 3) Foxp3-expressing T regulatory cells in human pregnant uterine mucosa, the decidua. Using immuno electron microscopy we show that human early syncytiotrophoblast constitutively expresses the stress-inducible NKG2D ligands MICA/B and ULBP1-5, and the apoptosis inducing molecules FasL and TRAIL. While MICA/B were expressed both on the cell surface and intracellularly on the limiting membrane of multivesicular bodies (MVB) and on exosomes, the ULBP1-5, FasL and TRAIL  were solely  processed through the MVB of the endosomal compartment and secreted on exosomes. The NKG2D ligand-expressing placental exosomes were able to internalize the cognate receptor from the cell surface of activated NK- and T cells thus down regulating their cytotoxic function. In our studies of apoptosis we found that placental exosomes carry the proapoptotic ligands FasL and TRAIL in their active form as a hexameric complex of two homotrimeric molecules, required for triggering of the apoptotic signaling pathways. This finding was supported by the ability of isolated placental FasL/TRAIL expressing exosomes to induce apoptosis in activated peripheral blood mononuclear cells (PBMC) and Jurkat T cells. Additionally, we studied Foxp3-expressing T regulatory (Treg) cells in paired human decidual and blood samples from pregnant women compared to non-pregnant controls. The CD4+CD25+Foxp3+ Treg cells were 10 fold enriched in the decidual mucosa compared to peripheral blood of pregnant women and non-pregnant controls. We discovered a pool of Foxp3-expressing, CD4+CD25- cells in human decidua, a phenotype consistent with naïve/precursor Foxp3+ Treg cells. These results suggest local enrichment of Treg cells in decidua of normal pregnancy. Furthermore, we have results indicating that the exosomes, isolated from placental explant cultures, carry PD-L1 and TGFβ on their surface, molecules known to promote induction of Treg cells. Taken together, our results provide evidence that placental exosomes are immunosuppressive and underline their role in the maternal immune modulation during pregnancy. The constitutive production and secretion of immunosuppressive placental exosomes create a protective exosomal gradient in the blood surrounding the feto-placental unit. This “cloud of immunosuppressive exosomes” conveys immunologic privilege to the developing fetus and thus contributes to the solution of the immunological challenge of mammalian pregnancy.

A hanseníase é uma doença crônica causada por Mycobacterium leprae e apresenta diversas formas clínicas. O entendimento da interação parasita-hospedeiro na hanseníase evidenciou que ocorre a persistência assintomática do patógeno, caracterizando um estado de latência. Os fatores mais importantes relacionados com a permanência do patógeno são: a patogenicidade do agente infeccioso e o perfil da resposta imune, no qual os eventos de migração celular, produção de citocinas, as células efetoras e reguladoras são extremamente relevantes. As células T reguladoras (Treg) desempenham papel central na regulação da resposta imune em infecções crônicas o que favorece a persistência do patógeno. A importância de células T reguladores na hanseníase ainda é pouco conhecida. Neste trabalho investigou-se a presença de células T reguladoras em lesões e sangue periférico de indivíduos com hanseníase. Inicialmente avaliou-se a proliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) de pacientes com hanseníase. Os resultados evidenciaram que não há diferenças quanto à proliferação de células T e produção de IFN-γ e TNF-α por células desses pacientes, mas a produção de IL-4 e IL-5 foi detectada apenas entre os pacientes com hanseníase virchoviana. Em relação à presença de células T reguladoras, os resultados evidenciaram aumento no número de linfócitos T CD4+CD25+FoxP3+ no sangue periférico de pacientes com hanseníase virchoviana. As células T reguladoras dos pacientes com hanseníase apresentaram elevada expressão de moléculas co-inibitórias PD-1, CTLA-4, GITR e ICOS. De modo relevante, as células T CD4+CD25+ isolados de pacientes com hanseníase virchoviana apresentaram maior atividade supressora quando comparado às células isoladas de pacientes com hanseníase tuberculóide. As células T CD4+CD25+ de pacientes com hanseníase virchoviana inibiram a proliferação de PBMC alogênico e a produção de IFN-γ e TNF-α. Os resultados demonstraram também que nas amostras de lesão de pele de pacientes com hanseníase virchoviana há acúmulo de células CD25+ produtoras de IL-10 e TGF-β, enquanto que estas células não foram detectadas nas lesões de pacientes com hanseníase tuberculóide. Dessa forma, os resultados descritos indicam que pacientes com hanseníase virchoviana apresentam aumento no número de células T reguladoras circulantes e no infiltrado inflamatório, e estas células apresentaram maior atividade supressora. O acúmulo de células T reguladoras no sítio da infecção pode ser correlacionado com o controle da resposta imune e conseqüente persistência de M. leprae. / Leprosy is caused by Mycobacterium leprae and its clinical features depend on the host immune background. The understanding of parasite-host interactions in leprosy have highlighted asymptomatic persistence of the pathogen, which indicates that this infection becomes latent. The most important factors related to the permanence of pathogens are: the pathogenicity of the infectious agents; the profile of the immune response developed by the host whose events of cellular migration, cytokines production, and the effector and regulatory cells are extremely relevant. The regulatory T cells (Treg) seem to play a central role in the regulation of the immune response in chronic infections, which favors the persistence of the pathogen. Herein, we analyzed the relation between tuberculoid and lepromatous leprosy with the presence and function of T regulatory cells from peripheral blood mononuclear cells (PBMC) and skin lesions from these patients. First, the proliferation and cytokine production of PBMC isolated from leprosy patients were analyzed. We did not observe any difference in the proliferation ability or IFN-γ and TNF-α release; however, the production of IL-4 and IL-5 was detected only in patients with lepromatous leprosy. Furthermore, T CD4+CD25+FoxP3+ cells were detected in the PBMC of patients with leprosy and these cells from lepromatous patients showed high expression of co-inhibitory molecules such as PD-1, GITR, CTLA-4 and ICOS. T CD4+CD25+cells isolated from patients with lepromatous leprosy were significantly more suppressive than the cells obtained from tuberculoid patients. In addition, TCD4+CD25+ cells isolated from patients with lepromatous leprosy inhibited allogeneic PBMC proliferation and their production of IFN-γ and TNF-α. The results also demonstrated that IL- 10 and TGF-ß were co-expressed with CD25+ cells at the inflammatory infiltrate of skin lesions from lepromatous patients, but similar results were not detected among tuberculoid patients. Thus, these results indicate that lepromatous leprosy patients have an enhanced presence of Treg cells with a suppressive ability in the blood and in the inflammatory infiltrate. The accumulation of Treg cells at the infection sites might be associated to the control of immune response and consequently to Mycobacterium leprae presistence.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Histopathologic examination of the tumor microenvironment demonstrates the presence of a vast repertoire of infiltrating lymphocytes and antigen presenting cells (APC’s). Recent studies establish a strong correlation between the tumor microenvironment cell composition and prognostic value in terms of cell type, location and ratio, referred to as a tumor’s immunoscore. More specifically, the relationship between T regulatory (Treg) cell to T effector (Teff) cell percentage predominates as a mechanism of tumor immune evasion. Further investigation of the factors influencing the development of Treg and Teff cells is therefore warranted. Gammainterferon‐inducible lysosomal thiol reductase (GILT) acts to influence antigenic processing and presentation by MHC class II cells, ultimately impacting lymphocyte development. Evaluation of the role of GILT expression in MHC class II+ APC’s with respect to Treg and Teff cell development in primary melanoma lesions, to our knowledge, has not been reported. Therefore our investigation focuses on elucidating a plausible relationship between GILT presence and Treg to Teff cell ratio. The aim of our study is to examine a possible association between GILT expression in APC’s and Treg:Teff cell ratio. We hypothesized GILT expression in melanoma cells would result in a decreased Treg to Teff ratio or an enhanced T cell‐mediated response. Our study included 17 de‐identified primary melanoma specimens previously stained and scored for Treg, Teff, CD8, MHC class II and GILT. Scoring was performed through identification of four areas per specimen with highest Treg and Teff cell density. These four areas were then averaged with ± standard deviation (SD). With use of landmark association, these four areas were identified and scored for MHC class II and GILT in APC’s and tumor cells with consideration to presence/absence, intensity and frequency of staining. Statistical significance was not reached relative to our hypothesized relationship of a decreased Treg to Teff cell ratio in the presence of GILT+ MHC class II. Similarly, we did not reach statistical significance when comparing individual cell types to GILT, MHC class II and GILT + MHC class. In our study, we were unable reach statistical significance relative to our proposed correlation between MHC class II and GILT presence leading to a decreased Treg to Teff cell ratio or enhanced T‐cell mediated immune response. A major limitation of our study included the small sample size leading to a probable type II error, prompting the need for further investigation of the factors influencing the Treg to Teff cell ratio within the melanoma tumor microenvironment on a larger scale.

Submitted by Mayara Caroline Rosolem null (mayara_rosolem@yahoo.com.br) on 2017-12-13T13:13:58Z No. of bitstreams: 1 Tese_Mayara_Caroline_Rosolem.pdf: 2522128 bytes, checksum: af877102b33947d3515c6cd2c29b01af (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2017-12-13T13:40:57Z (GMT) No. of bitstreams: 1 rosolem_mc_dr_jabo.pdf: 2522128 bytes, checksum: af877102b33947d3515c6cd2c29b01af (MD5) / Made available in DSpace on 2017-12-13T13:40:57Z (GMT). No. of bitstreams: 1 rosolem_mc_dr_jabo.pdf: 2522128 bytes, checksum: af877102b33947d3515c6cd2c29b01af (MD5) Previous issue date: 2017-11-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os tumores mamários malignos possuem várias formas de evadir o sistema imune e, dentre elas está a modulação das células dendríticas (DCs), por interferência na sua maturação, resultando em apresentação de antígenos ineficiente aos linfócitos T e consequente indução da tolerância imunológica. As DCs moduladas pelo tumor recrutam muitos linfócitos T regulatórios (Tregs) para o microambiente tumoral, o que amplia os efeitos imunossupressores locais. A forte relação entre as DCs e os linfócitos Tregs no microambiente tumoral ainda foi pouco estudada, principalmente em cães. Por isso, este estudo teve o objetivo de avaliar a relação existente entre as DCs e os linfócitos Tregs em carcinomas mamários caninos do tipo simples de cadelas, por meio da técnica de imunohistoquímica. Foram utilizadas 10 amostras de glândula mamária sem tumor (G1) e 40 amostras de neoplasias mamárias (G2: adenomas; G3: carcinomas papilares; G4: carcinomas tubulares e G5: carcinomas sólidos), que foram submetidas a imunodetecção de linfócitos Treg (FOXP3+), linfócitos T CD4 e T CD8, MHC-II, DCs mieloides (imaturas e maduras), as citocinas TGF-β, IL-10, a enzima Indoleamine 2,3-dioxigenase (IDO) e dos receptores de quimiocina (CCR6 e CCR7). A maioria das células, citocinas e receptores imunológicos mostraram correlação positiva dentro da população tumoral e controles avaliados, principalmente sobre o efeito fixo “idade”, que teve alta correlação positiva com o tamanho tumoral e com CCR6. Quanto às correlações negativas, o anticorpo CD83 foi o único que não teve correlação significativa positiva com nenhuma outra variável. Observou-se que houve ocorreu relação entre as DCs mieloides imaturas/maduras e linfócitos Tregs, bem como TGF-β e IL-10, e a enzima IDO apresentaram marcante presença nas amostras malignas. A imunodetecção de CCR6 e CCR7 ocorreu principalmente nos tumores mais agressivos, onde CCR6 teve alta relação com as pacientes mais idosas e com tumores maiores, o que, no final de tudo pode refletir que o envelhecimento do sistema imunológico possa ser mais um fator pró-tumoral. / The malignant mammary tumors have several ways of evading the immune system, including the modulation of dendritic cells (DCs), by interfering with their maturation, resulting in inefficient presentation of antigens to T cells and consequent induction of immunological tolerance. Tumor-modulated DCs recruit many regulatory T cells (Tregs) into the tumor microenvironment, which amplifies local immunosuppressive effects. The strong relationship between DCs and Tregs cells into the tumor microenvironment was poorly studied, especially in dogs. Therefore, this study aimed to evaluate the relationship between DCs and Tregs cells in the simple type canine mammary carcinomas, using the immunohistochemical technique. Ten samples of mammary gland without tumor (G1) and 40 samples of mammary neoplasms (G2: adenomas, G3: papillary carcinomas, G4: tubular carcinomas and G5: solid carcinomas) were submitted to immunodetection of Treg cells (FOXP3 +), CD4 and CD8 T cells, MHC-II, myeloid DCs (immature and mature), cytokines TGF-β, IL10, Indoleamine 2,3-dioxygenase (IDO) and chemokine receptors (CCR6 and CCR7). Most of the cells, cytokines and immunological receptors showed positive correlation within the tumor population and controls evaluated, mainly on the fixed effect "age” that had high positive correlation with the tumor size and with CCR6. As for the negative correlations, the CD83 antibody was the only one that had no significant positive correlation with any other variable. It was observed that there was a relationship between immature / mature myeloid DCs and Tregs cells, as well as TGFβ and IL-10, and the IDO enzyme showed a marked presence in the malignant samples. Immunodetection of CCR6 and CCR7 occurred mainly in the most aggressive tumors, where CCR6 had a high relation with older patients and with larger tumors, which, in the end, may reflect that the aging of the immune system may be more of a pro- tumor. / FAPESP: 2012/09385-0