abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2017

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-03-10T13:11:37Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Rafael de Freitas e Silva.pdf: 13510018 bytes, checksum: 080b7ee10e7a8be555cb7a7ca29c10fb (MD5) / Made available in DSpace on 2017-03-10T13:11:37Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Rafael de Freitas e Silva.pdf: 13510018 bytes, checksum: 080b7ee10e7a8be555cb7a7ca29c10fb (MD5) Previous issue date: 2016-09-06 / As leishmanioses são doenças causadas por protozoários do gênero Leishmania e estão presentes em 98 países e territórios e possuem incidência anual de 2 milhões de casos. A Leishmania (Viannia) braziliensis (L.V. braziliensis) é uma das principais espécies causadoras da leishmaniose cutânea (LC) no Brasil. Apesar disso, ainda não há uma vacina segura e eficaz para ser utilizada em seres humanos. Nesse sentido, o objetivo deste trabalho foi identificar no proteoma predito de L.V. braziliensis, potenciais epítopos de linfócitos T e avaliá-los por meio de ensaios imunológicos. No primeiro capítulo, o proteoma predito de L. braziliensis foi comparado ao de outras espécies e analisado quanto a presença de epítopos. Nessa etapa foram encontrados epítopos derivados de mais de 8 mil proteínas conservadas entre diferentes espécies de Leishmania. Os epítopos foram clusterizados e então utilizados para etapa de docagem molecular com estruturas de MHC I e MHC II depositadas no Protein Data Bank. A docagem molecular resultou em epítopos peptídicos de 15 aminoácidos com alta afinidade de ligação às moléculas de MHC I e MHC II. Os 10 melhores resultados foram então sintetizados e avaliados, in vitro, quanto à capacidade de estimular a proliferação de células mononucleares do sangue periférico (PBMC) de indivíduos com LC após o tratamento (PT). Os resultados indicaram que 50% das moléculas testadas apresentaram capacidade de estimular, significativamente (p<0,05), a proliferação celular quando comparado às células de indivíduos saudáveis que não vivem em região endêmica para LC. No segundo capítulo, os peptídeos foram avaliados quanto à capacidade de estimular a proliferação de PBMC de indivíduos com LC em sua fase ativa (AD) e indivíduos moradores de área endêmica para LC resistentes à infecção (RT). Em paralelo, quantificou-se a expressão do fator de transcrição T-bet em PBMC de indivíduos PT, e citocinas dos perfis Th1, Th2 e Th17 foram mensuradas no sobrenadante de cultura das células de indivíduos PT e AD. Os resultados demonstraram altos níveis de proliferação nas células do grupo RT para todos os peptídeos testados. Além disso, níveis significativos de Tbet foram observados em linfócitos T CD4+ e CD8+ após estímulo com seis peptídeos. Níveis significativos de IFN-γ, TNF e IL-6 foram observados no sobrenadante das células do grupo PT com quatro dos peptídeos testados. Altos níveis dessas citocinas também foram encontrados no sobrenadante do grupo AD. No terceiro capítulo, avaliou-se o efeito dos peptídeos sobre células dendríticas de medula (BMDC) murinas, produção de citocinas de sobrenadante, e células dendríticas esplênicas murinas após estímulo com os peptídeos. Verificou-se altos níveis de MHC II e CD40 em uma subpopulação de BMDC estimuladas com as moléculas e altos níves de TNF e IL-6 após 48h de estímulo. Para as células esplênicas, foram observados altos níveis de subpopulações celulares expressando CD11b+, IL-12p70+, CD205+ e CD11b+ após estímulo com o peptídeo que teve o melhor resultado in silico. Por fim, os resultados indicam o grande potencial imunogênico que os epítopos identificados apresentam, o que dá suporte ao desenvolvimento futuro de abordagens vacinais. / The leishmaniasis are diseases caused by protozoans from the genus Leishmania which are present in 98 countries and territories, with an annual incidence of 2 million cases. Among the other species, Leishmania (Viannia) braziliensis is the main specie implicated with cutaneous leishmaniasis (CL) in Brazil. Besides that, there is no safe and effective vaccine against leishmaniasis to be applied in humans. In this context, the aim of this work was to identify in the predicted proteome of L. braziliensis potential CD4+ and CD8+ T cell epitopes and evaluate them by immunological assays. In the first chapter, the predicted proteome of L. braziliensis was compared with other species and analyzed for the presence of epitopes. In this step, epitopes from more than 8,000 conserved proteins were found among other species of Leishmania. The epitopes were clustered and then used for the molecular docking with MHC I and MHC II structures deposited in the Protein Data Bank. This approach resulted in 15 aminoacids peptide epitopes with high binding affinity for MHC I and MHC II. The 10 best results were synthesized and evaluated in vitro for their capacity to stimulate the proliferation of peripheral blood mononuclear cells (PBMC) of individuals with CL post treatment (PT). The results have shown that 50% of the tested molecules had the capacity to stimulate, significantly (p<0.05), cell proliferation when compared with cells of healthy individuals living in non-endemic regions. For the second chapter, the peptides were evaluated for their capacity to stimulate the proliferation of PBMC from CL individuals with active disease (AD) and of individuals resistant to infection (RT) living in endemic region. In parallel, the T-bet transcription factor expression was quantified in PBMC of PT individuals, and cytokines from the Th1, Th2 and Th17 profiles were measured in culture supernatant of PT and AD groups. High levels of cell proliferation in the RT group were demonstrated for all peptides tested. Moreover, significant levels of T-bet in CD4+ and CD8+ T cells were verified after stimulation with six peptides. For IFN-γ, TNF and IL-6, significant levels were detected in the supernatant of cultures from the PT group with four peptides tested. High levels of these same cytokines were also present in the supernatant of AD group. In the third chapter, the peptide effects over murine bone marrow dendritic cells (BMDC), the production of cytokines in the supernatant and murine spleen dendritic cell subsets were evaluated after peptide stimuli. High levels of MHC II and CD40 were verified for stimulated BMDC and high levels of TNF and IL-6 after 48h of stimuli. For spleen cells, high levels of cells expressing CD11b+, IL-12p70+, CD205+ e CD11b+ were observed after stimulation with the peptide which showed the best in silico result. In conclusion, the results indicate the great immunogenic potential of the identified peptides and support the further development of vaccine approaches using those molecules.