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Doctoral Thesis
DOI
https://doi.org/10.11606/T.5.2020.tde-29012021-100912
Document
Author
Full name
Bruna Tereso Santillo
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Sumida, Telma Miyuki Oshiro (President)
Rigato, Paula Ordonhez
Sato, Maria Notomi
Silva, Laís Teodoro da
Title in Portuguese
Análise fenotípica e funcional de células dendríticas polarizadas para o tipo alfa-1 (alfa-DC1) derivadas de monócitos de indivíduos infectados pelo HIV-1
Keywords in Portuguese
Células dendríticas
HIV
Imunoterapia
Interferon Gama
Interferon tipo I
Abstract in Portuguese
Resumo: A imunoterapia baseada em células dendríticas derivadas de monócitos (MoDCs) constitui uma estratégia promissora para o manejo de indivíduos infectados pelo HIV podendo atuar como um tratamento complementar para pacientes que fazem uso de terapia antirretroviral. Protocolos para obtenção de MoDCs convencionais, aqui denominadas IL4-DCs, utilizam como mediadores da diferenciação celular as citocinas IL-4 e GM-CSF que originam DCs imaturas com características mielóides. O estímulo das DCs imaturas com citocinas pró-inflamatórias TNF-alfa, IL-1beta e IL-6 ativa as MoDCs tornando-as aptas a apresentar antígenos e iniciar uma resposta imune específica. As IL4-DCs tem demonstrado capacidade de induzir resposta específica de linfócitos T, mas ainda demonstram baixo potencial de migrar para os linfonodos e produzir IL-12p70, essencial para estimular linfócitos Th1. Um protocolo alternativo tem proposto a utilização de interferons dos tipos I e II, além de agonistas de TLR para suplementar o estímulo para ativação de MoDCs. As MoDCs assim estimuladas são denominadas células dendríticas polarizantes para o tipo alfa-1 (alfa-DC1) e apresentam capacidade de produzir níveis elevados de IL-12p70 levando a uma potente resposta Th1. O potencial terapêutico de alfa-DC1 foi demonstrado inicialmente em protocolos de imunoterapia para pacientes de tumores e mais recentemente foi desenvolvido protocolo para indivíduos infectados pelo HIV. Há alguns anos nosso grupo de pesquisa está envolvido com protocolo clínico de imunoterapia baseada em IL4-DCs pulsadas com vírus quimicamente inativado. Neste contexto, com o objetivo de buscar alternativas para aperfeiçoar a imunoterapia, a nossa proposta foi avaliar in vitro aspectos fenotípicos e funcionais das alfa-DC1 pulsadas com HIV quimicamente inativado com potencial de aplicação na imunoterapia. Para tanto, foram avaliadas alfa-DC1 e IL4-DCs de pacientes e controle quanto à morfologia, expressão de moléculas de superfície, produção de IL-12p70, capacidade de estimular a produção de IFN-y e expressão de CD107a em linfócitos T autólogos e capacidade de estimular a proliferação de linfócitos T alogênicos. Os resultados revelaram que as alfa-DC1 apresentam maior expressão de CD40, CCR7 e CCR5, quando comparadas às IL4-DCs. Os resultados dos ensaios funcionais demonstraram que as alfa-DC1 tendem a produzir maiores níveis de IL-12p70 e que ambas alfa-DC1 e IL4-DCs foram capazes de estimular linfócitos T autólogos de pacientes a produzirem IFN-y. Ainda, ambas MoDCs foram capazes de induzir a proliferação de linfócitos T alogênicos de pacientes e HIV-. Embora as alfa-DC1 e IL4-DCs constituam populações distintas de MoDCs, demonstrando potencial de produção de IL-12p70 distintos, observamos que a capacidade de reconhecer e apresentar antígenos virais e estimular resposta de linfócitos T foi semelhante entre estas duas populações
Title in English
Phenotypic and functional analysis of alpha-type1 polarized dendritic cells (alpha-DC1) derived from monocytes of HIV-1 infected subjects
Keywords in English
Dendritic cells
HIV
Immunotherapy
Interferon type I
Interferon-Gamma
Abstract in English
Abstract: Immunotherapy based on monocyte-derived dendritic cells (MoDCs) has been shown to be a promising tool for treating HIV-infected individuals, and can act as a complementary treatment for HIV-infected individuals in use of antiretroviral therapy. Protocols for obtaining conventional MoDCs, here called IL4-DCs, use as mediators of cell differentiation the IL-4 and GM-CSF cytokines that originate immature DCs with myeloid characteristics. Stimulation with TNF-alpha, IL-1beta and IL-6 pro-inflammatory cytokines activates MoDCs making them able to present antigens and initiate a specific immune response. IL4-DCs have shown able to induce specific-T cell response, but still unable to migrate to lymph nodes and increase IL-12p70 production, essential to stimulate Th1 cells. An alternative protocol has proposed the use of type I and II interferons, in addition to TLR agonists to supplement the activation stimulus of MoDCs. These MoDCs are called alpha-type-1 polarized DCs (alpha-DC1), which have an exuberant capacity to produce high levels of IL-12p70 cytokine and to induce a potent Th1 response, consequently. The therapeutic potential of alpha-DC1 was first demonstrated in immunotherapy protocols for tumor patients and more recently a protocol has been developed for HIV-infected individuals. For some years, our research group has been involved in a clinical immunotherapy protocol based on IL4-DCs stimulated with chemically-inactivated virus. In this context, the aim of this study was to investigate alternatives to improve immunotherapy with proposing to evaluate in vitro phenotypic and functional aspects of alpha-DC1 pulsed with chemically inactivated HIV prospecting as a potential application in HIV immunotherapy. For that, alphaDC1 and IL4-DCs of HIV+ and HIV- subjects were evaluated for morphology, surface molecule expression, IL-12p70 production, ability to stimulate IFN-y production and CD107a expression in autologous T lymphocytes and ability to stimulate the proliferation of allogeneic T lymphocytes. The results revealed that alpha-DC1. The results of functional assays demonstrate that alpha-DC1 tends to produce higher levels of IL-12p70 and that both alpha-DC1 and IL4-DCs were able to stimulate IFN-y producing autologous T lymphocytes of HIV+ subjects. Also, both of MoDCs were able to induce allogeneic T lymphocytes proliferation of HIV+ and HIV- subjects. Although alpha-DC1 and IL4-DCs are distinct MoDCs populations that demonstrated different potential to release IL-12p70, we observed that the ability to recognize and present viral antigens and stimulate T lymphocyte response was similar between these two populations
 
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Publishing Date
2021-02-02
 
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