Submitted by MARIANA MIZIARA DE ABREU TEODORO null (marimiziara@gmail.com) on 2016-03-21T18:48:38Z No. of bitstreams: 1 Dissertação Mariana Miziara de Abreu Teodoro.pdf: 1597455 bytes, checksum: f121a89794f14989756c7e7c5208127a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-22T20:37:43Z (GMT) No. of bitstreams: 1 teodoro_mma_me_bot.pdf: 1597455 bytes, checksum: f121a89794f14989756c7e7c5208127a (MD5) / Made available in DSpace on 2016-03-22T20:37:43Z (GMT). No. of bitstreams: 1 teodoro_mma_me_bot.pdf: 1597455 bytes, checksum: f121a89794f14989756c7e7c5208127a (MD5) Previous issue date: 2016-03-01 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Doença de Chagas (DC), cujo agente etiológico é o protozoário Trypanosoma cruzi (T. cruzi), tornou-se nos últimos anos um grave problema de saúde pública, mesmo em países não-endêmicos. Um importante desafio para os pesquisadores da doença de Chagas é estabelecer os eventos que levam alguns pacientes a desenvolver a doença crônica assintomática, ou forma indeterminada, enquanto outros evoluem para uma doença severa, com lesões em tecidos cardíacos ou gastrointestinais. Alguns estudos têm sugerido que a ausência de sintomas clínicos em indivíduos com a forma crônica indeterminada está associada a um controle eficaz da resposta imune efetora contra o parasita, que evita uma resposta inflamatória excessiva, o que resultaria em lesão tecidual. Não existem estudos com o objetivo de confirmar este mecanismo em pacientes com a forma digestiva da DC, especialmente em relação ao controle modulador de mecanismos efetores envolvendo células Th17, por células Treg. No presente estudo, nós avaliamos em pacientes com a forma digestiva da DC e em pacientes com a forma indeterminada ou assintomática a taxa de ativação Th17/Treg, através da avaliação da freqüência de células Th17 e Treg por citometria de fluxo; da expressão do mRNA para Rorγt e Foxp3, fatores de transcrição envolvidos na diferenciação dessas células, respectivamente; bem como os níveis de IL-17a e IL-10 em sobrenadantes de cultura de células mononucleares de sangue periférico (PBMC’s). Foi demonstrado no presente estudo, que em pacientes com a forma digestiva da DC há um desequilíbrio na taxa de ativação de células Th17/Treg, favorecendo o perfil Th17, enquanto que em pacientes com a forma indeterminada, este desequilíbrio favorece o perfil Treg. Os nossos resultados apoiam a hipótese de que o desequilíbrio na taxa de ativação Th17/Treg observado no grupo de pacientes digestivos, favorecendo Th17 em detrimento de Treg, geraria um ambiente não regulador, que resulta em uma resposta inflamatória exacerbada, responsável pelas lesões dos tecidos gastrointestinais apresentadas por estes pacientes. / Chagas disease (CD) whose etiological agent is the protozoan Trypanosoma cruzi (T. cruzi) in recent years became a serious problem of public health even in nonendemic countries. One important challenge for the researchers of Chagas disease is to establish the events that lead some patients to develop asymptomatic or indeterminate chronic disease, while others undergo severe disease with lesions in cardiac or gastrointestinal tissues. Some studies have suggested that absence of clinical symptoms in individuals with the indeterminate chronic form is associated to a fine control of effector immune responses against the parasite that avoid a perpetuated inflammatory process which results in tissue injury. There are no studies aiming to confirm this mechanism in patients with digestive form of the disease, particularly in relation to the modulatory control by Treg cells of effector mechanisms involving Th17. Here, we studied in patients with the digestive form of CD and in those with the indeterminate or asymptomatic form of the disease, the ratio Th17/Treg activation by evaluating the frequency of Th17 and Tregs cells by flow cytometry, mRNA expression for Rorγt and Foxp3, the transcription factors involved in the differentiation of these cells respectively as well as the levels of IL-17a and IL-10 in peripheral blood mononuclear cells culture supernatants. We showed that in patients with digestive form of the disease there is an imbalance in the ratio Th17/Treg cells activation in favor of Th17, while in patients with the indeterminate form, this imbalance favour Treg activation. Our findings support the hypothesis that in contrast with indeterminate patients a non regulatory environment observed in patients with digestive CD, which results in an exacerbated inflammatory response exerted by Th17, due to lower Treg activation, may be responsible for tissue lesions in these patients. / FAPESP: 2013/02223-7

Submitted by REGINALDO KELLER FERNANDES null (regiskeller@msn.com) on 2017-04-12T12:10:23Z No. of bitstreams: 1 Tese Dr definitiva.pdf: 5601863 bytes, checksum: 50eec58640e31a0c12ce54dde368d5a3 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-17T20:28:15Z (GMT) No. of bitstreams: 1 fernandes_rk_dr_bot.pdf: 5601863 bytes, checksum: 50eec58640e31a0c12ce54dde368d5a3 (MD5) / Made available in DSpace on 2017-04-17T20:28:15Z (GMT). No. of bitstreams: 1 fernandes_rk_dr_bot.pdf: 5601863 bytes, checksum: 50eec58640e31a0c12ce54dde368d5a3 (MD5) Previous issue date: 2017-02-24 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A paracoccidioidomicose (PCM) é uma micose sistêmica endêmica na América Latina, principalmente no Brasil, Argentina e Venezuela, e que promove um importante impacto na saúde pública. Seu agente etiológico é um fungo termodimórfico pertencente ao gênero Paracoccidioides que compreende o Paracocidioides brasiliensis (Pb) e suas espécies crípticas S1, PS2, PS3 e Paracoccidioides lutzii. As consequências da interação do fungo com as células da resposta imune inata, tais como as células dendríticas (DC), destacando a capacidade destas células para instruir a resposta imune adaptativa, não são totalmente compreendidas. Em estudo anterior, descobrimos que DCs não maturam em resposta ao desafio com Pb. Esta falha foi associada à inibição de PGE2 nas DCs pelo fungo, uma vez que este eicosanóide é um fator importante para a maturação dessas células. Na tentativa de melhor entender este processo e suas conseqüências para a instrução da resposta adaptativa CD4, nós buscamos analisar o perfil transcricional de DCs em resposta ao Pb assim como o de linfócitos CD4+ cocultivados com DCs sensibilizados com o fungo. Para estas análises, nós utilizamos a metodologia de RNA-seq, que permitiu o sequenciamento de alto rendimento e a quantificação sistemática de expressão gênica. Após as análises, os genes que foram regulados positivamente ou negativamente em ambas as células (DCs e CD4) foram listados e as funções das proteínas codificadas por eles, identificadas. A análise geral das proteínas codificadas por esses genes, como diversas citocinas e quimiocinas, mediadores inflamatórios, bem como fatores de transcrição envolvidos na diferenciação de populações de linfócitos, permitiram determinar o perfil de resposta adaptativa que foi diferenciado após Interação de DCs com células CD4, bem como alguns mecanismos que levaram a este perfil. / FAPESP: 2013/14733-0

Intestinal inflammatory disorders such as Crohn’s disease contribute significantly to human mortality and morbidity. Although the cells and molecules involved in suppression of intestinal inflammation have been extensively documented in mouse models, a full understanding of how these work together in the healthy and diseased gut remains elusive. It is known, however, that tight regulation over TH17 cells and regulatory T cells (Treg) is required to maintain immune homeostasis in the intestine. Activation of the cytokine transforming growth factor-β (TGFβ), which is secreted by immune cells as an inactive complex, plays a crucial role in the induction of both Treg and TH17 cells. Recent work has shown that the αvβ8 integrin is required for activation of TGFβ by murine dendritic cells (DC). Murine integrin αvβ8 is thus of fundamental importance for Treg and TH17 induction and, subsequently, for intestinal immune homeostasis. However, little is known about the signals controlling the expression of integrin αvβ8 on intestinal DC. Furthermore, whether this system is also important for regulation of the human system is entirely unknown. Here, expression of integrin αvβ8 is shown on human intestinal CD1c+CD103+SIRPα+ DC and CD1c+CD103-SIRPα+ DC, but not on CD141+CD103+SIRPα- DC. Expression of integrin αvβ8 is increased on DC from Crohn’s disease patients, and on DC from non-IBD donors treated with LPS. Both LPS and a number of probiotic bacteria were also able to induce integrin αvβ8 expression on human monocyte-derived DC (moDC), which suggested that the microbiota may play a role in the regulation of human integrin αvβ8. Importantly, we have also shown that TGFβ is activated by integrin αvβ8 on human moDC, and that integrin αvβ8 promotes expression of forkhead box P3 (FOXP3) in naïve human T cells in vitro. Together, these data suggest that integrin αvβ8-mediated activation of TGFβ by DC may play an important role in the regulation of human T cell responses in the human intestine, and that this pathway may be perturbed during intestinal inflammation.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disease that affects millions of people worldwide. Although the exact cause of MS is unknown, it is clear that CD4+ T helper cells play a significant role, namely T helper 1 (Th1) and T helper 17 (Th17) cells. The Notch family of proteins plays a role in the development and differentiation of T helper cells. Previous data has shown that inhibition of Notch impairs the ability of T helper cell differentiation. Additionally specific inhibition of certain Notch members inhibits specific T helper cell differentiation, for example the inhibition of Notch 1 inhibits Th1 and iTreg polarization [Samon et al., 2008]. However, the effects of the other Notch family members on CD4+ T cells are not fully studied. We propose that Notch 3 plays an extensive role in the regulation of Th1, Th2, Th17, and iTreg polarizations. In addition, we propose that Notch 3 regulates function of T helper cell function in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Data in this thesis show that Notch 3 plays a significant role in the polarization of Th1, Th17 and iTreg polarization [Karlsson et al., 2011]. We present evidence that the heterozygous and homozygous Notch 3 knockout exhibits a significant decrease in polarization toward Th1, Th17 and iTreg cell fates. Exopolysaccharide (EPS) is a compound that has been previously shown to play a protective role in other inflammatory diseases. EPS has been shown to produce anti-inflammatory macrophages. We propose that a similar anti-inflammatory effect might be possible in EAE. We found that EPS had a significant effect on EAE induction, decreasing the onset and peak disease score. EPS also reduced the concentration of IFN-γ, IL17A, and GM-CSF in the supernatants of the splenocytes after restimulation with MOG. Further experimental data is needed to prove the effects of EPS on EAE and the method by which EPS function. These data indicate that Notch 3 could be crucial in regards to EAE due to the effects on Th1 and Th17 which are instrumental in EAE induction [Raphael et al., 2015].

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24329号 / 生博第488号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 杉田 昌彦, 教授 垣塚 彰 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Asthma is defined as reversible airflow obstruction and an estimated 1-in-3 Canadians will be diagnosed over their lifetime. Many clinical phenotypes of asthma exist, but allergic asthma is the most common presentation. Despite effective therapies, approximately 65% of Canadian asthmatics have poorly controlled disease. Thus, there remains pressing need to develop disease modifying therapies. Allergen-specific immunotherapy (SIT) is a disease-modifying therapy for allergic disease that consists of repeatedly administering doses of allergen, to an allergic individual; over 100 years of clinical use, SIT has been demonstrated to reduce symptoms of disease both during and after cessation of therapy. Widespread clinical uptake of SIT has been limited by the risk of developing anaphylaxis as a response to therapy. Peptide immunotherapy is a derivation of SIT, that attempts to retain the disease-modifying benefits, while lessening the risk of anaphylaxis, by treating subjects with allergen-derived T-cell peptide epitopes. Peptide immunotherapy has been demonstrated to reduce symptoms of allergic disease in treated subjects; however, it remains unknown how administration of a single (or several) T-cell epitopes can modulate immune responses to entire complex allergens. Additionally, how genetic diversity in peptide epitope presentation effects the development of immune tolerance is unknown. In this thesis, we sought to characterize these mechanisms of peptide immunotherapy; the hypothesis was, “The induction of immunosuppression by peptide immunotherapy involves the infectious spread of tolerance beyond the treatment epitope, and is dependent upon treatment peptide dose and affinity to MHC”. Through the definition of these mechanistic traits we hoped to expedite and inform the design of future peptide based therapeutics. The studies presented within this thesis examine the topic of immune modulation of allergic disease in mouse models, and have focused upon broadly pertinent characteristics of immune modulation, such as the number, dose and affinity of immunomodulatory epitopes. / Thesis / Doctor of Philosophy (PhD) / Asthma is a disease of the airways that can cause difficulties in breathing. In some people, asthma develops because their immune system reacts in an uncontrolled manner to common environmental proteins, called allergens. Whole allergen immunotherapy is a treatment for asthma, where asthmatic people are injected with doses of allergen until their immune system no longer responds to (or ‘tolerates’) the allergen. In some people, injection of allergen can lead to a life-threatening immune response known as ‘anaphylaxis’. Peptide-immunotherapy is a form of whole allergen immunotherapy where people are given small fragments of the allergen (a ‘peptide’) rather than the whole allergen. The benefit of peptide immunotherapy is that the treatment peptides are too small to cause anaphylaxis, but remain large enough to teach the immune system. In this thesis, we examined how treatment with small peptides teaches the immune system to tolerate the larger and more complex whole allergen.

Les anticorps monoclonaux (AcM) ont été utilisés pour traiter des cancers dès le début des années 1980, en particulier lors du travail pionnier de l’équipe de Ronald Levy dans le traitement des lymphomes. Ces traitements ont pendant longtemps été considérés comme une sérothérapie passive à effet immédiat et à court terme. Cependant, au cours de ces dernières années, le concept d’un effet « vaccinal » des anticorps à usage thérapeutique en oncologie a peu à peu vu le jour du fait de réponses cliniques à long terme observées chez certains patients et de différentes études précliniques. En 2010, notre équipe a démontré que des souris immunocompétentes injectées avec les cellules tumorales EL4-huCD20 et traitées avec un AcM anti-huCD20 générait une réponse immunitaire anti-tumorale à long-terme par le biais de mécanismes dépendants de la région constante de l’anticorps et de lymphocytes T CD4+. Mon travail de thèse a donc porté sur l’analyse des mécanismes cellulaires et moléculaires par lesquels le traitement par un AcM anti-CD20 génère une immunité cellulaire adaptative anti-tumorale. J’ai ainsi pu montrer que le traitement des souris avec l’AcM anti-CD20 conduit à une expansion de lymphocytes Th1 producteurs d’IFN-γ, à l’apparition de lymphocytes T CD4+ effecteurs mémoires spécifiques des cellules tumorales CD20+, et au blocage de l’expansion de lymphocytes Tregs induite par les cellules tumorales. Le rôle central dans la protection anti-tumorale et la genèse d’une réponse adaptative anti-tumorale joué par l’axe IL-12/IFN-γ et leurs principales sources cellulaires, cellules dendritiques (DCs) et cellules NK, a été démontré par des expériences de neutralisation de ces cytokines, qui provoque une importante diminution du nombre de Th1 spléniques, de déplétion des cellules NK, ainsi que par des analyses phénotypiques qui ont permis d’identifier des DCs activées par le traitement - comme le montre l’expression accrue des molécules de classe II du CMH et de co-stimulation CD80 et CD86 - comme une importante source cellulaire de l’IL-12. Enfin, nous avons pu montrer qu’un variant de l’IL-2, liant préférentiellement le récepteur de l’IL-2By et faiblement le récepteur de l’IL-2aBy exprimé majoritairement par les Tregs, permettait l’obtention d’une protection anti-tumorale accrue d’animaux porteurs de tumeurs et traités par l’AcM anti-CD20. En conclusion, nous avons démontré qu’un contexte immunitaire pro-tumoral façonné par la présence d’une tumeur en développement peut être inversé par le traitement par un anticorps anti-tumoral, aboutissant à un contexte anti-tumoral. Qu’une telle réponse immunitaire adaptative cellulaire puisse être observée chez des patients atteints de lymphomes, traités par un anticorps anti-CD20, reste encore à être déterminé. / Monoclonal antibodies have been used to treat cancers since the early 1980s, in particular with the pioneer work of Ronald Levy for the treatment of lymphomas. Those treatments have been considered for a long time as a passive serotherapy with immediate and short term actions. Yet, recently, the idea of a vaccine effect of therapeutic antibodies in oncology have appeared, after preclinical studies and clinic observations suggesting a long term immune response in patients. In 2010, our team demonstrated that immunocompetent mice injected with EL4-huCD20 tumor cells and treated with anti-huCD20 monoclonal antibody generated a long term anti-tumor immune response linked with mechanisms dependent on constant part of antibodies and CD4+ T cells. My PhD work was based on the analysis of cellular and molecular mechanisms by which the treatment by an anti-CD20 mAb generates a cellar adaptive anti-tumor immunity. I could show that the treatment of mice with anti-CD20 antibody lead to the expansion of Th1 lymphocytes IFN-γ producers, to the apparition of effector memory CD4+ T cells specific for CD20 antigen, and to the blockade of the expansion of Treg cells induced by tumor cells. The key role of an adaptive anti-tumor immune response played by IL-12/IFN- γ and their main cellular sources, dendritic cells and NK cells, in the anti-tumor protection and genesis, has been demonstrated by experiments of cytokine neutralization, provoking an important decrease of splenic Th1 number, by NK depletion and by phenotypic analysis that allowed the identification of DCs activated by the treatment – as it is shown by the increased expression of MHC-II and CD80 and CD86 costimulation molecules, - as an important cellular source of IL-12. Finally, we could show that a variant of IL-2, binding preferentially IL-2By with a lower affinity for the IL-2aBy receptor mainly expressed by Tregs, could induce an increased anti-tumor protection of tumor-bearing animals treated with anti-CD20 mAb. In conclusion, we have demonstrated that a pro-tumor immune contexture affected by a growing tumor can be modified by an anti-tumor antibody leading to an anti-tumor contexture. That such cellular adaptive immune response could be observed in lymphoma patients treated with anti-CD20 still need to be determined.