Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (T_regs) and CD141⁺ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141⁺ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10^-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ T_regs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of T_regs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of T_regs with matched specificity for therapeutic intervention.

Made available in DSpace on 2015-07-22T20:50:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Melanomas se destacam entre os tumores sólidos por apresentar alto potencial de malignidade e incidência crescente, especialmente entre indivíduos jovens. Identificação de marcadores moleculares em melanomas é de enorme interesse para uso clínico e para estudos relacionados ao seu desenvolvimento. A linhagem de melanoma murino B16 tem sido amplamente empregada visando melhor compreensão do processo metastático. Objetivo: identificar moléculas na superfície de células B16, empregando anticorpos monoclonais, que apresentem função biológica importante para essas células, bem como investigar expressão de moléculas reconhecidas pelos mesmos mAbs em melanoma humano. Métodos: camundongos C57Bl/6 foram imunizados com células B16 irradiadas para produção de híbridos produtores de mAb. Resultados: após fusão foi obtido mAb da classe IgM, designado G12F2, que reconheceu uma única banda de aproximadamente 230 kDa em extrato total de células B16. A molécula era expressa na superfície celular e não por células não tumorigênicas, como fibroblastos ou melanócitos melan-a. Células não tumorigênicas, derivadas de melan-a, também não a expressaram ao passo que células tumorigênicas, de mesma origem, expressaram-na em grande quantidade. Variante menos metastática da linhagem B16 expressou menor quantidade desta molécula quando comparado à variante mais metastática. A neutralização da molécula de 230 kDa com mAb G12F2 inibiu proliferação, migração e invasão por células B16 in vitro. Também nestas condições, G12F2 promoveu atividade citolítica contra células B16, mediada por complemento. Por outro lado, adição in vitro de mAb G12F2 em nada alterou adesão das células B16 à fibronectina e laminina, ou adesão célula-célula. In vivo, o tratamento com mAb G12F2 inibiu crescimento do nódulo tumoral e formação de metástases pulmonares. Quando testado contra extrato de tumores de origem humana, como carcinoma e melanoma, mAb G12F2 reconheceu banda de 75 e 67 kDa, respectivamente. Por fim, foi demonstrado que mAb inibiu proliferação de células de melanoma humano in vitro. Conclusões: a molécula de 230 KDa parece ter importância durante crescimento do melanoma murino; identificação de molécula homóloga em melanoma humano fornecerá subsídios para diagnóstico e protocolos visando imunoterapia. / TEDE / BV UNIFESP: Teses e dissertações

Meduloblastoma (MB) e Tumor Teratóide Rabdóide Atipico (TTRA) são tumores malignos do sistema nervoso central (SNC) que ocorrem na infância. Embora tenha havido um aumento na sobrevida, recorrência e metástases são frequentes e as opções terapêuticas poucas e tóxicas para crianças. Uma alternativa à terapia tradicional é a imunoterapia, que pode contornar os efeitos tóxicos associados à radioterapia e quimioterapia. De uma forma geral a imunoterapia depende da presença de antígenos associados a tumor (TAA) e/ou processamento de antígenos expressos pelo HLA classe I. No entanto, TAA para MB ou ATRT não estão bem definidos e tecidos neuronais tem a expressão de HLA classe I muito baixa. As células Natural Killer (NK) não dependem de TAA para citólise e são particularmente ativas na ausência de moléculas do HLA classe I tendo, portanto, potencial para o tratamento dessas doenças. Atualmente as barreiras para a aplicação clínica de células NK são quantitativas e qualitativas. Com o intuito de quebrar a barreira quantitativa foi criada uma plataforma tecnológica para a expansão das NK ex vivo. Essa plataforma consiste no co-cultivo de células mononucleares de sangue periférico com células apresentadoras de antígeno artificiais (aAPC), que expressam Interleucina 21 ligada à membrana. Com essa tecnologia foi possível expandir células NK na ordem de 40.000 vezes. Também foi demonstrada a persistência das células NK expandidas in vitro por 3 semanas após infusão em cérebro murino e que essas células expressam altos níveis de citocinas antitumorais e estimulantes do sistema imune: Interferon gama e Fator de Necrose Tumoral alfa. Finalmente, foi demonstrada a atividade citolítica das células NK tanto in vitro, contra um painel de células de MB e ATRT, quanto in vivo em um modelo murino de MB. Os resultados obtidos fornecem a primeira evidência pré-clínica que provê suporte ao uso de células NK expandidas usando essa plataforma tecnológica no combate a cânceres de cérebro pediátricos. Com base nesses dados, nosso grupo no MD Anderson Cancer Center iniciou um ensaio clínico fase I para testar a segurança e eficácia de administração locorregional de células NK expandidas usando aAPC para o tratamento de tumores pediátricos da fossa posterior do cérebro. / Medulloblastoma (MBs) and Atypical Teratoid/Rhabdoid Tumor (ATRT) are malignant pediatric brain tumors. Although survival has improved, recurrence and metastasis are frequent with few therapeutic options for these children. Immunotherapy is an alternative to traditional therapies that may circumvent the potential toxicities associated with traditional chemotherapy and radiation approaches. Many immune based therapies rely on the presence of tumor associated antigens (TAAs) and/or antigen processing and class I human leukocyte antigen (HLA I) expression. However, TAAs for MB are not well defined and neuronal tissues have very low HLA I expression. Natural killer (NK) cells do not rely on TAA for cytolysis and therefore have potential for the treatment of these diseases. The current barriers to clinical application of NK therapy are quantitative and qualitative. To overcome quantitative barriers, we have worked with a platform technology for the ex vivo expansion of NK cells through co-culture of peripheral blood mononuclear cells with artificial antigen presenting cells expressing membrane-bound IL-21 (mbIL21) to promote a 40,000-fold expansion of NK cells. We also demonstrate prolonged life-span of ex vivo expanded NK cells and persistence for up to 3 weeks post-infusion in the murine brain. These cells express high levels of immune stimulatory and anti-tumor cytokines - interferon gamma and TNF-alpha following activation. Finally we demonstrate NK cytolytic activity in vitro against a panel of primary and established ATRT and MB cells and in vivo following locoregional delivery in a mouse orthotopic model of MB. Our data provide the first pre-clinical evidence supporting the use of mbIL21 expanded NK cells against pediatric brain tumors. Based on this data we have initiated a novel Phase I clinical trial at MD Anderson Cancer Center to assess the safety and efficacy of locoregional delivery of mbIL21 expanded NK cells for the treatment of posterior fossa pediatric brain tumors.

Transduction of melanoma cells with the aim to induce avidine expression on tumor cell surface was studied. Subsequently the method enabling quantification of binding of ligands to the cell surface was developed.

The aim of this thesis was to study cancer immunotherapy based on combination of TLR7 signalling and activation of phagocytosis of cancer cells. This thesis was focused on the possibility of enhancing its effect. The possibilities of removing immunosuppressive barriers (esp. MDSCs and adenosine) were studied, using a combination with chemotherapy or enzyme - adenosine deaminase and purine nucleoside phosphorylase. The effect of enzymes was studied in both in vivo and in vitro experiments. As another way of optimizing the therapy used so far, the modulation of the therapeutic mixture using lower molecular weight substances was tried.

Oomycete Pythium insisiosum is the etiologic agent of pythiosis, chronic and granulomatous disease, which affects humans and animals in tropical and subtropical areas of the world. Due to ergosterol absence in the plasmatic membrane of this microorganism, treatments based on antifungal agents have been ineffective. Immunotherapy has emerged showing promising results. In this context, the improvement of the immunotherapy was the main goal of this study, whose first purpose was to characterize the immunodominant antigens from this species (P. insidosum ATCC 58637 and P. insidiosum 210-LAPEMI) through sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE). Some proteins showing 15 kDa to 120 kDa were detected, and bands were transferred to nitrocellulose membranes in order to develop the western blot tests. The second purpose was to characterize the humoral response to pythiosis in equine, in bovine and in experimental pythiosis in rabbits, considering the no treated disease, the disease cured by immunotherapy, the disease treated with immunotherapy but not cured, and the disease cured spontaneously. To encompass all these cases we have included sera from: a) horses cured by immunotherapy; b) horses with pythiosis but not responsive to immunotherapy; c) horses with pythiosis but not treated; d) bovines with pythiosis and showing spontaneous cure; e) rabbits with experimental pythiosis but not treated; and f) rabbits with experimental pythiosis treated with immunotherapy. All specimens recognized three immunodominant proteins: 74, 33 and 32 kDa. The sera from horses cured by immunotherapy (a) and bovine sera (d) recognized another immunodominant protein, 55 kDa, which was weakly positive or negative in the other groups. Thus, the 74, 33 and 32 kDa immunodominant proteins suggest a relevant function in the humoral response to pythiosis because they were recognized by horses, rabbits and bovines. In addition, the 55 kDa antigen, which is being reported here for the first time, is likely to be involved in the cure mechanisms stimulated by immunotherapy. / O oomiceto Pythium insidiosum é o agente etiológico da pitiose, doença granulomatosa crônica que afeta animais domésticos (eqüinos, bovinos, etc) e o homem, em regiões tropicais e subtropicais de todo mundo. A ausência de ergosterol na membrana plasmática deste microorganismo torna a antifungicoterapia muito limitada, donde a imunoterapia (a base de hifas rompidas) tem emergido com resultados promissores. A identificação de antígenos imunodominantes é fundamental para a consolidação dos imunoterápicos utilizados no tratamento da pitiose. Objetivando-se o refinamento destes imunoterápicos, o presente estudo teve como proposta inicial, caracterizar os antígenos imunodominantes desta espécie (P. insidosum ATCC 58637 e P. insidiosum 210-LAPEMI) através de eletroforese em gel de poliacrilamida (SDS-PAGE). Diversas proteínas de 120 a 15 kDa foram detectadas e, a seguir, eletrotransferidas para membranas de nitrocelulose para realização de western blot. O segundo objetivo foi caracterizar a resposta humoral à doença em eqüinos, bovinos e na infecção experimental em coelhos, considerando-se a doença curada pela imunoterapia, doença tratada pela imunoterapia e não curada, doença não tratada, e na doença evidenciando cura espontânea. Para tanto, utilizou-se o soro de: a) eqüinos doentes e curados pela imunoterapia; b) eqüinos doentes e sem resposta a imunoterapia; c) eqüinos doentes e não tratados; d) bovinos doentes e com cura espontânea; e) coelhos infectados e tratados pela imunoterapia; f) coelhos infectados e não tratados. Todos os soros reconheceram três proteínas imunodominantes: 74, 33 e 32 kDa. O soro dos eqüinos doentes curados pela imunoterapia (a) e o soro dos bovinos (d) reconheceram também outra proteína imunodominante de 55 kDa, a qual, foi fracamente reconhecida ou ausente nos demais grupos. Assim, comprova-se que as proteínas de 74, 33 e 32 kDa desempenham relevante papel na resposta imune à pitiose, pois foram imunogênicas nas três espécies avaliadas. Em adição, sugere-se que a proteína de 55 kDa possa estar envolvida no mecanismo de cura promovido pela imunoterapia; ressalta-se que esta proteína é aqui pela primeira vez relatada.

Les cellules dendritiques (DC) sont des cellules présentatrices d’antigènes qui jouent un rôle central dans l’immunité en initiant et en orientant les réponses T primaires. Parmi elles, les cellules dendritiques plasmacytoïdes (pDC) sont des cellules clé de la réponse anti-virale et sont également impliquées dans l’induction de tolérance. Dans le cancer du sein, la présence de pDC et de lymphocytes T régulateurs (Treg) dans les tumeurs sont des facteurs de mauvais pronostic. Nous avons développé un modèle murin de tumeur mammaire dans lequel l’échappement des tumeurs à l’immuno-surveillance semble associé à leur infiltration par des pDC et des Treg reproduisant ainsi la pathologie humaine. Les DC myéloïdes associées aux tumeurs (TAmDC) et les TApDC sont immatures et conservent leur capacité à internaliser des antigènes in vivo. De façon importante, les TApDC sont spécifiquement altérées pour leur capacité à produire des cytokines et chimiokines inflammatoires en réponse aux ligands du Toll Like Receptor (TLR)-9 mais conservent une réponse normale aux ligands du TLR7. En revanche, les TAmDC produisent spontanément de grandes quantités d’IL-10 mais ne sécrètent pas d’IL-12. Enfin, les deux sous populations sont bloquées pour leur capacité à induire l’activation des lymphocytes T CD4+. En parallèle, les pDC normales sont capables d’internaliser des corps apoptotiques de cellules tumorales à condition que celles-ci soient infectées par un virus. Ces résultats suggèrent que les pDC et les mDC sont différemment altérées dans l’environnement tumoral et fournissent de nouvelles pistes pour des stratégies d’immunothérapie dans le cancer du sein / Dendritic cells (DC) as professional antigen presenting cells, are key regulators of adaptive immune responses. Among them, plasmacytoid dendritic cells (pDC) are professional interferon producing cells critical for antiviral immunity but are also implicated in tolerance induction. In breast cancer, we previously reported that the pDC and regulatory T cells (Treg) infiltration in primary tumors represent independent prognostic factors associated with poor outcome. The current study was designed to understand this negative impact of tumor-associated pDC (TApDC). For this purpose, we have developed a murine mammary tumor model that closely mimic the human pathology, including TApDC and Treg infiltration associated with immune escape. We showed that tumor-associated myeloid DC (TAmDC) and TApDC are immature and preserve their ability to internalize antigens in vivo. Most importantly, TApDC are specifically altered for cytokine production in response to Toll-Like Receptor (TLR)-9 ligands while preserving unaltered response to TLR7 ligands. TAmDC spontaneously mature and produce large amounts of IL-10, but are unable to secrete IL-12. Finally both subsets are inhibited in their ability to activate CD4+ T cells. Alternatively, we showed that normal pDC are able to uptake apoptotic bodies from virally infected tumor cells. These results suggest that pDC and mDC are differentially altered in the tumor microenvironment. They further provide critical insights for developing new therapeutic strategies using TLR7 ligands to target TApDC activation in breast cancer

abstract: Combination therapy has shown to improve success for cancer treatment. Oncolytic virotherapy is cancer treatment that uses engineered viruses to specifically infect and kill cancer cells, without harming healthy cells. Immunotherapy boosts the body's natural defenses towards cancer. The combination of oncolytic virotherapy and immunotherapy is explored through deterministic systems of nonlinear differential equations, constructed to match experimental data for murine melanoma. Mathematical analysis was done in order to gain insight on the relationship between cancer, viruses and immune response. One extension of the model focuses on clinical needs, with the underlying goal to seek optimal treatment regimens; for both frequency and dose quantity. The models in this work were first used to estimate parameters from preclinical experimental data, to identify biologically realistic parameter values. Insight gained from the mathematical analysis in the first model, allowed for numerical analysis to explore optimal treatment regimens of combination oncolytic virotherapy and dendritic vaccinations. Permutations accounting for treatment scheduled were done to find regimens that reduce tumor size. Observations from the produced data lead to in silico exploration of immune-viral interactions. Results suggest under optimal settings, combination treatment works better than monotherapy of either type. The most optimal result suggests treatment over a longer period of time, with fractioned doses, while reducing the total dendritic vaccination quantity, and maintaining the maximum virotherapy used in the experimental work. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics for the Life and Social Sciences 2016

Pythiosis is a granulomatous, life-threatening disease of humans and animals, which is caused by the oomycete Pythium insidiosum. The treatment of the infections caused by this pathogen is complicated because of its cell wall and cytoplasmic membrane characteristics. Therefore, immunotherapy obtained from P. insidiosum culture extracts has emerged as an alternative treatment for equine pythiosis. Notwithstanding, the mechanisms by which immunotherapy work are not yet fully understood. The aims of this study were: a) to evaluate the immunological parameters in immunotherapy by measuring interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels; b) to evaluate the biochemical parameters (hepatic function) in the horses of the experiment. To address this issue, 10 horses were divided in two groups: control (n=5), composed of animals without pythiosis; and infected (n=5), composed of animals with the disease. Animals from both groups received four doses of the immunotherapeutic product PitiumVac®. A significant increase in IL-2 and IFN- γ levels was observed after 42 days of immunotherapy in the infected group, and the animals were considered cured. The immunotherapy also improved the hepatic function of the animals, decreasing alanine aminotransferase and alkaline phosphatase levels. This may be a result of the interaction of different immunotherapeutic antigen present in the immune system, causing an increase in the production of these cytokines and establishing a predominance of TH1. / A pitiose é uma doença infecciosa, com risco de morte, que acomete animais e humanos, caracterizada por lesões granulomatosas, tendo como agente etiológico o oomiceto Pythium insidiosum. O tratamento de infecções causadas por P. insidiosum em animais e humanos é complicado pelas características do agente, sobretudo a composição de sua parede celular e membrana plasmática. A imunoterapia surgiu como uma alternativa para o tratamento da pitiose equina, com a produção de imunoterápico a partir de culturas do próprio agente. Apesar dos estudos sobre a doença e a imunoterapia, ainda não há um completo conhecimento dos mecanismos envolvidos. . Os objetivos deste trabalho foram: a) analisar os parâmetros imunológicos envolvidos na resposta frente à imunoterapia por meio da quantificação da interleucina 2 (IL2) e interferon gama (IFN-γ); b) analisar os parâmetros bioquímicos (função hepática) dos equinos envolvidos no experimento. Para tal foram utilizados 10 equinos, divididos em 2 grupos: controle n=5( animais hígidos sem a doença) e infectado n=5( com pitiose clínica). Todos os 10 animais dos 2 grupos receberam 4 doses do imunoterápico PitiumVac a cada 14 dias. Os parâmetros imunológico dos animais demostraram uma diferença estatística significativa no grupo infectado após o tratamento com o imunoterápico PitiumVac®. Após 42 dias de imunoterapia foi observado um aumentando de forma significativa os níveis séricos de IL-2 e IFN-γ no grupo dos animais infectados, culminando com a cura clínica destes animais. Após a imunoterapia foi constatada uma melhora na função hepática, com a diminuição da alanina aminotransferase (ALT) e fosfatase alcalina (FA). Este resultado pode ser decorrente da interação distinta do antígeno presente no imunoterápico com o sistema imunológico, provocando o aumento na produção destas citocinas e estabelecendo um predomínio de resposta TH1.

Made available in DSpace on 2014-10-09T12:54:59Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:12Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP