Exossomos (Exo) originados de células dendríticas (DCs) carregam moléculas associadas à apresentação antigênica. Neste trabalho procurou-se estabelecer se Exo de DCs seriam capazes de conferir imunogenicidade às células tumorais. Os Exo isolados de culturas de DCs expressavam as moléculas HLA-ABC, HLA-DR, CD86, CD11c, CD81, CD54 e CD18. Estes foram então adicionados às células da linhagem humana de adenocarcinoma mamário, SK-BR-3, as quais passaram a expressar as moléculas HLA-DR, CD86 e CD11c. As células tumorais modificadas pelos Exo induziram a produção de IL-6 e IL-10, detectados no sobrenadante das co-culturas destas com linfócitos T. Estas células tumorais também induziram aumento do número de linfócitos produtores de IFN-<font face=\"Symbol\">g, pré-sensibilizados contra antígenos tumorais, e aumento da expressão de SOCS3 nestes. Em conclusão, nossos resultados mostram que, Exo de DCs alteram o fenótipo de células tumorais, modificando sua interação com linfócitos T, sem induzir nas mesmas capacidade de ativar respostas proliferativas ou citotóxicas de linfócitos T in vitro. / Exosomes (Exo) originated from dendritic cells (DCs) contain molecules involved in antigen presentation. The present work sought to determine if Exo from DCs would be able to transfer immunogenicity to tumor cells. Exo isolated from DCs cultures carried HLA-ABC, HLA-DR, CD86, CD11c, CD81, CD54 and CD18. These Exo were added to cultures of the human breast adenocarcinoma cell line, SK-BR-3, which gained expression of HLA-DR, CD86 and CD11c. Tumor cells modified by Exo induced IL-6 and IL-10 production, detected in the supernatant of their co-cultures with T lymphocytes. These tumor cells also induced an increase in the frequency of IFN-<font face=\"Symbol\">g-producing T lymphocytes, pre-sensitized against tumor antigens, and an increased expression of SOCS3. In conclusion, our results show that, Exo from DCs affect the phenotype of tumor cells, modifying their interaction with T lymphocytes, without inducing the ability to activate cytotoxic or T cell proliferative responses in vitro.

A identificação e caracterização das células reguladoras (Treg) trouxeram uma nova perspectiva para a indução da tolerância imunológica nos transplantes e um aumento na sobrevida dos enxertos. Uma vez que as células-tronco mesenquimais derivadas de tecido adiposo (ADSC) possuem a capacidade de suprimir uma resposta imune, nos questionamos se as ADSC poderiam melhorar a sobrevida de um enxerto em camundongos C57BL/6, associada à indução de Treg. Nossos resultados mostram que o tratamento com as ADSC aumentou a média de sobrevida do enxerto, com uma melhora na morfologia do tecido transplantado, um aumento na população de linfócitos Treg e na expressão de IFN-g e IL-10, alem de uma inibição da expressão de IL-17 e na proliferação de células T CD4+. Nossos achados sugerem que as ADSC suprimem a resposta imune ao enxerto por meio da indução de Treg, a qual inibe a participação das células Th17, com uma melhora no enxerto. Estes dados ajudam a desenvolver novos aspectos na estratégia terapêutica e possivelmente o uso futuro dessas células na prática clínica. / The identification and characterization de regulatory T cells that can control immune responsiveness to alloantigens opened up opportunities for new therapies in transplantation. After exposure to alloantigens in vivo, antigen-specific immunoregulatory activity is enriched in a population of CD4+ T cells that express high levels of CD25 and Foxp3. Adipose tissue contains one type of mesenchymal stem cells (ADSC) with capacity of suppressing immune response. In this work we propose the correlation of the ADSC cells in ameliorating skin graft survival, and if is associated with Foxp3 expression. Graft survival was enhanced in animals that received ASCs from the donor. Interesting, in the animals treated with ASCs from CBA presented a higher expression of Foxp3+ cells in the lymph-nodes. In treated mice Foxp3 expression was increased and IL-17 were increased in allogeneic group, suggesting a potent inflammatory response inside the graft. So far, these data suggest the ADSCs increase TReg population cells, inhibit IL-17 expression and prolonging skin graft survival.

Esse trabalho objetivou avaliar o perfil genômico de pacientes HIV+ submetidos à imunoterapia com células dendríticas (DC). Os resultados obtidos mostraram que as DC utilizadas na imunoterapia podem ser diferentes em termos de perfil de expressão gênica entre os pacientes selecionados para o ensaio clínico e essas diferenças podem afetar a qualidade do produto administrado ao paciente. Nossos dados não correlacionaram diferenças na ativação das DC com esse perfil de expressão gênica distinto, possivelmente pelo número limitado de amostras disponíveis ou pela escassez de marcadores de ativação adequados. O único paciente que respondeu à imunoterapia mostrou, em leucócitos circulantes, um perfil de expressão específico e relacionado com um melhor controle da carga viral plasmática e com uma resposta imune de tipo Th1 quando comparado com os indivíduos que não responderam ao tratamento. A genotipagem revelou que esse paciente também carrega polimorfismos relacionados a menor progressão à AIDS o que também pode estar relacionado com a boa resposta à imunoterapia. / Aim of this project was the evaluation of genomic profile of HIV+ patients submitted to dendritic cell (DC)-based immunotherapy. Results showed that expression profile of DC used in immunotherapy could be different among patients selected for immunotherapy, and that those differences could affect the quality of final vaccine product, even if our data did not evidence significant difference in DC activation state with regard to expression profile, maybe due to limited size of available samples or to the lack of appropriate DC activation markers. The expression profile of peripheral blood leukocytes from the only good responder of immunotherapy trial was associated with better control of plasma viral load and a Th1 immune response compared to weak or transient responders. Genotyping analysis revealed that the good responder carries polymorphisms associated with a less progression toward AIDS, suggesting that also the genetic background could affect the response to immunotherapy.

Paracoccidioidomicose (PCM) é uma micose sistêmica causada por fungos dimórficos do gênero Paracoccidioides - P. brasiliensis ou P. lutzii. A alta incidência da PCM no Brasil, somada à gravidade que o quadro clínico pode assumir e ainda à ausência de um antifúngico de baixa toxicidade para tratamento de curta duração, motivaram a avaliação de adjuvantes e caracterização dos componentes antigênicos de P. brasiliensis em um modelo imunoterapêutico de PCM. Assim, inicialmente, avaliou-se o efeito de monofosforil lipídeo A (MPLA) de Salmonella minnesota, PAM3CSK4, hidróxido de alumínio e AS04 em camundongos cronicamente infectados com P. brasiliensis. Quando camundongos da linhagem BALB/c foram infectados pela via intratraqueal com 3 × 105 leveduras de P. brasiliensis, tratados no dia 20 após a infecção com um dos adjuvantes e analisados 30 dias após a administração do tratamento, observou-se que MPLA, ao contrário dos outros adjuvantes, induziu (1) uma redução significativa no número de unidades formadoras de colônias (UFC), (2) uma expressiva diminuição no número de granulomas e células fúngicas no tecido pulmonar e (3) uma resposta imunológica protetora (Th1) quando comparado aos animais controle tratados com PBS. Quanto ao rastreamento de antígenos de P. brasiliensis candidatos a agentes terapêuticos, evidenciou-se que as frações proteicas da preparação de exoantígenos (ExoAg) eram as mais promissoras. Dentre essas frações, a que continha o ExoAg majoritário diidrolipoil desidrogenase induziu maior porcentagem de proliferação de linfócitos T CD3+ esplênicos de camundongos infectados e tratados com MPLA. O gene de diidrolipoil desidrogenase foi clonado em vetor de expressão e a proteína recombinante produzida, purificada e utilizada no protocolo terapêutico da PCM experimental. Surpreendentemente, a administração terapêutica de diidrolipoil desidrogenase recombinante induziu uma redução significativa na contagem de UFC dos animais infectados cronicamente com P. brasiliensis, mesmo na ausência de adjuvantes, o que não foi observado quando foi administrada a preparação de ExoAg nos animais. A localização subcelular de diidrolipoil desidrogenase, por microscopia eletrônica de transmissão, utilizando anticorpos policlonais de camundongos contra essa proteína, mostrou que a mesma está localizada na mitocôndria e também no citoplasma. A análise da expressão gênica diferencial de diidrolipoil desidrogenase em P. brasiliensis mostrou que essa proteína é significativamente mais expressa em leveduras em comparação a hifas e formas de transição. Em conclusão, nossos resultados mostram os efeitos benéficos de MPLA na PCM experimental e sugerem que diidrolipoil desidrogenase é um alvo terapêutico potencial para o tratamento da PCM. / Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides - P. brasiliensis and P. lutzii. The high incidence of PCM in Brazil, added to the severity of the disease and the absence of an antifungal that brings together low toxicity and short-term treatment, led us to evaluate adjuvants and characterize antigenic components of P. brasiliensis that could be used as immunotherapy in an experimental model of PCM. In this work, firstly, we evaluated monophosphoryl lipid A (MPLA) from Salmonella minnesota, PAM3CSK4, aluminum hydroxide and AS04 in mice chronically infected with P. brasiliensis. When mice were infected intratracheally with 3 × 105 P. brasiliensis yeasts, treated with one of the adjuvants or vehicle on day 20 postinfection, and analyzed 30 days after the treatment, we observed that MPLA, unlike other adjuvants, induced (1) a significant reduction in the number of colony forming units (CFU) in the lungs of the mice, (2) an expressive decrease of granulomas and yeast cells in lung tissue, and (3) a more protective immune response (Th1) when compared with the control mice. Concerning to detection of antigens for PCM therapy, we noticed that among the fractions of a preparation of exoantigens (ExoAg), the one containing the protein identified as dihydrolipoyl dehydrogenase induced a high percentage of proliferation of splenic CD3+ T lymphocytes from mice infected and treated with MPLA. The gene of dihydrolipoyl dehydrogenase was cloned into expression plasmid vector, and the recombinant protein produced, purified and used in the therapeutic protocol of experimental PCM. Surprisingly, the therapeutic administration of recombinant dihydrolipoyl dehydrogenase, but not ExoAg preparation, induced a significant reduction in fungal load in the animals chronically infected with P. brasiliensis, even in the absence of adjuvants. Immunogold labeling and transmission electron microscopy revealed that the dihydrolipoyl dehydrogenase is localized in mitochondria and cytoplasm of P. brasiliensis. The differential expression of dihydrolipoyl dehydrogenase gene in P. brasiliensis showed that yeast had an expression more significant than hyphae, with an intermediate level of gene expression in the transitional forms. In conclusion, our results show that MPLA and dihydrolipoyl dehydrogenase are potential therapeutic targets for the treatment of PCM due to their beneficial effects in a therapy model of PCM.

OBJETIVO: Avaliar a eficácia e segurança da imunoterapia tópica com imiquimode creme 5% no tratamento do carcinoma basocelular nodular periocular. MÉTODOS: Pacientes com carcinoma basocelular confirmado por biopsia e com contraindicação clínica para a cirurgia reconstrutiva devido ao alto risco ou que recusaram a cirurgia por razões estéticas ou fobia foram incluídos no estudo. O tratamento foi iniciado após treinamento do paciente e de acompanhante. A posologia foi de 5 vezes por semana por 8 a 16 semanas. Acompanhamento quinzenal foi realizado durante a vigência do tratamento com questionário, exame biomicroscópico, medida da acuidade visual e documentação fotográfica. As características clínicas das lesões foram mensuradas através do software ImageJ. Após 12 semanas do fim da terapia, uma nova biópsia na região da lesão foi guiada por fotografia. O seguimento dos pacientes foi semestral, após fim do tratamento, com biópsias anuais da região até o presente momento. RESULTADOS: 19 foram tratadas. A taxa de cura histológica foi de 89,5% após três meses do final do tratamento, e de 84,2% nos três anos de seguimento (39,5 meses). A taxa de cura histológica em três anos foi de 100% para lesões menores que 10 mm, e de 81,8% para lesões maiores que 10 mm. De uma forma geral, os efeitos colaterais da medicação foram mais frequentes durante as oito primeiras semanas de tratamento. Quanto menor foi a distância da lesão à margem palpebral, maior foi chance de o paciente desenvolver ectrópio no tratamento (p = 0,045). Assim, como quanto maior foi a inflamação, maior foi a chance de desenvolver ectrópio, dor e edema durante o tratamento (p = 0,017, p = 0,016 e p = 0,044, respectivamente). CONCLUSÕES: Imiquimode creme 5% mostrou-se eficaz para o tratamento alternativo do carcinoma basocelular periocular, principalmente em lesões menores que 10 mm. Em adição, demonstrou um interessante efeito neoadjuvante sobre as lesões maiores que 10 mm que não foram curadas. Mostrou-se um tratamento seguro; entretanto, um cuidado maior deve ser dado às lesões próximas à margem palpebral devido ao maior risco de complicações e desenvolvimento de ectrópio / Objective: to evaluate the efficacy and safety of topical administration of 5% imiquimod cream in the treatment of periocular nodular basal cell carcinoma (BCC). Methods: Patients with periocular nodular basal cell carcinoma confirmed by biopsy and clinical contraindication to reconstructive surgery due to high risk or who decline surgery for aesthetic reasons or phobia were included in the study. The medication was applied once a day, five days a week for 8-16 weeks. Treatment was initiated after provision of patient and caretaker training. During treatment, patients were followed up twice a month with questionnaires, biomicroscopic examinations, measurement of visual acuity and photographic documentation. The clinical characteristics of the tumors were registered with the software ImageJ. Twelve weeks after the end of treatment, an image-guided biopsy of the tumor site was performed. Patients have since been attending follow-up visits every six months, and biopsies of the region are performed annually. Results: 19 tumors were treated with imiquimod. The average histological cure rate was 89.5% after 3 months at the end of the treatment and 84.2% after 3 years of follow-up (39.5 months). The 3-year histological cure rate was 100% for smaller tumors and 81.8% for larger tumors (>10 mm). In general, drug-related side effects were more frequent during the first 8 weeks of treatment. The smaller the distance between tumor and lid margin, the greater the probability of developing ectropion during treatment (p=0.045). Likewise, the more severe the inflammation, the greater the probability of developing ectropion, pain and edema during treatment (p=0.017, p=0.016 and p=0.044 respectively). Conclusion: Topical administration of 5% imiquimod cream was found to be an efficacious and relatively safe alternative treatment for periocular BCC, especially for tumors smaller than 10 mm, with interesting neoadjuvant effects on uncured tumors larger than 10 mm. However, special care is required when treating tumors near the eyelid margin due to the risk of complications and development of ectropion

Made available in DSpace on 2014-08-20T14:37:58Z (GMT). No. of bitstreams: 1 tese_tatiana_antunes.pdf: 3388546 bytes, checksum: 6c26eaf4ab24c11f046c7e7b2f9f2566 (MD5) Previous issue date: 2010-03-10 / Sporotrichosis is a subcutaneous mycosis of zoonotic character, cosmopolitan development subacute or chronic whose agent the dimorphic fungus Sporothrix schenckii affecting man and various species of animals, especially the domestic cat, being considered of interest to Public Healt. Considering the difficulties in the therapeutic treatment of ringworm in this animal species, including toxicity and the development of resistance to antifungal agents traditionally used to treat disease the study aimed evaluate the immunogenic capacity of yeast cells of S. schenckii inactivated both in immunoprophylaxis and immunotherapy of sporotrichosis. We used 160 Wistar albin rats (Rattus norvegicus) at 70 days (80 in immunoprophylaxis and 80 in immunotheraphy) were divided into four groups that were immunized three times every 15 days. The groups were divided as follows: G1 (control, mineral oil), G2 (Ag + incomplete freund adjuvant), G3 (Ag + Freund's complete adjuvant) and G4 (Ag + incomplete freund adjuvant + propolis). Preparation of the vaccine used was an isolate of S. schenckii from yeast in the form of a case of cutaneous sporotrichosis of domestic cat. The fungus was grown in medium liquid (Brain-Heart broth), incubated for 10 days at 370C and kept under constant agitation to obtain the yeast form. The culture was filtered through a double layer of sterile gauze, centrifuged, washed twice with buffered saline (PBS), homogenized and standardized in 108 cells of S. schenckii / ml. The cells were inactivated with 0.02% thimerosal and then emulsified with mineral oil, and vaccines packaged in sterilized sealed and kept at a temperature of 40C throughout the experimental period. Was used a dose of 0.1 ml/animal intramuscularly. Rats that received the vaccine as immunoprophylaxis after the three doses were challenged, and inoculated subcutaneously with 2X103 cells / ml of S. schenckii in the right footpad and evaluated for 10 days. Animals treated with immunotherapy were inoculated with the agent after 14 days and received three doses of immunogen. After the experimental period all were euthanized and necropsy to mycological examination, histopathology and counting colony forming units. The results were: immunoprophylaxis in the pathological changes showed statistically significant differences (P <0.05) in the vaccinated groups (G2, G3 and G4) compared to the control group (G1) to the point of inoculation, with no statistical difference the evaluation of internal organs. Immunotherapy clinical evaluation at the injection showed that there was no statistical difference among the four experimental groups, but at the end of the experiment the groups G1, G2, G3 and G4 had respectively 8.3%, 58.3%, 41.7 % and 50% of injuries in the process of regression and healing. In relation to injuries in other body areas only in the last week of the experiment, the G2 and G3 differ statistically (P <0.05) in the control group (G1). Pathological changes were found in the internal organs of the four experimental groups, but with a greater number of lesions in group CONT. Both in immunoprophylaxis and immunotherapy the retroisolation of the agent and count of colony forming units showed that there was growth of S. schenckii in the four experimental groups, but with less frequency and quantification of CFU at the point of inoculation and internal organs of the groups G2, G3 and G4. In histopathologic evaluation to determine the presence of granulomas and pyogranulomas focal and multifocal in the point of inoculation and internal organs of the four groups, but the animals that received immunoprophylactic changes were more restricted to the point of inoculation. The results indicate that the three vaccine formulations (AIF ACF and AIFP) used as immunoprophylactic and immunotherapy have not been effective for the remission of the lesions of experimental cutaneous sporotrichosis. / Esporotricose é uma micose subcutânea de caráter zoonótico, cosmopolita de evolução subaguda ou crônica que tem como agente etiológico o fungo dimórfico Sporothrix schenckii afetando o homem e várias espécies de animais, principalmente o felino doméstico, sendo considerada de interesse para a Saúde Pública. Considerando as dificuldades terapêuticas no tratamento da micose nessa espécie animal, incluindo toxicidade e o desenvolvimento de resistência aos antifúngicos tradicionalmente utilizados na terapia da enfermidade o estudo objetivou avaliar a capacidade imunogênica de células leveduriformes de S. schenckii inativadas tanto na imunoprofilaxia como na imunoterapia da esporotricose. Foram utilizados 160 ratos albinos wistar (Rattus norvergicus) com 70 dias (80 na imunoprofilaxia e 80 na imunoterapia) sendo divididos em quatro grupos que receberam três doses do imunógeno a cada 14 dias. Os grupos foram divididos em: G1 (controle- óleo mineral), G2 (Ag+ adjuvante incompleto de freund), G3 (Ag + adjuvante completo de freund) e G4 (Ag + adjuvante incompleto de freund + própolis). Para preparação da vacina foi utilizado um isolado de S. schenckii na forma leveduriforme proveniente de um caso de esporotricose cutânea de felino doméstico. O fungo foi cultivado em meio líquido (Brain-Heart broth), incubado durante 10 dias a 37oC e mantido sob agitação constante para obtenção da forma leveduriforme. A cultura foi filtrada em dupla camada de gaze estéril, centrifugada, lavada duas vezes com solução salina tamponada (PBS), homogeneizada e padronizada em 108 células de S. schenckii/ ml. As células foram inativadas com thimerosal a 0,02% e posteriormente emulsificadas com óleo mineral, sendo as vacinas acondicionadas em frascos estéreis fechados e mantidas a temperatura de 40C durante todo o período experimental. Foi utilizada uma dose de 0,1 ml/animal por via intramuscular. Os ratos que receberem a vacina como imunoprofilaxia após as três doses foram desafiados, sendo inoculados por via subcutânea com 2X103 células/ml de S. schenckii e avaliados durante 10 dias. Os animais tratados com imunoterápico foram inoculados com o agente e após 14 dias receberam três doses do imunógeno. Após o período experimental todos foram eutanasiados e feita necropsia para realização de análise micológica, histopatológica e contagem das Unidades Formadoras de Colônias. Os resultados obtidos foram: na imunoprofilaxia as alterações anatomopatológicas demonstraram diferença estatísticamente significativa (P<0,05) dos grupos vacinados (G2, G3 e G4) quando comparados ao grupo controle (G1) em relação ao ponto de inoculação, não havendo diferença estatística na avaliação dos órgãos internos. Na imunoterapia a avaliação clínica no ponto de inoculação evidenciou que não houve diferença estatística entre os quatro grupos experimentais, porém ao final do experimento os grupos G1, G2, G3 e G4 apresentavam respectivamente 8,3%, 58,3%, 41,7% e 50% das lesões em processo de regressão e cicatrização. Em relação a lesões em outras áreas corpóreas somente na última semana do experimento os grupos G2 e G3 diferiram estatisticamente (P<0,05) do grupo controle (G1). Alterações anatomopatológicas foram encontradas nos órgãos internos dos quatro grupos experimentais, porém com um maior numero de lesões nos animais do grupo CONT. Tanto na imunoprofilaxia quanto na imunoterapia o retroisolamento do agente e Contagem das Unidades Formadoras de colônias demonstraram que houve crescimento do S. schenckii nos quatro grupos experimentais, porém com menor freqüência e quantificação de UFCs no ponto de inoculação e órgãos internos dos grupos G2, G3 e G4. Na avaliação histopatológica foi verificada a presença de granulomas e piogranulomas focais e multifocais no ponto de inoculação e órgãos internos dos quatro grupos, porém os animais que receberam imunoprofilático as alterações ficaram mais restritas ao ponto de inoculação. Os resultados permitem concluir que as três formulações de vacinas (AIF, ACF e AIFP) utilizadas como imunoprofilático e imunoterápico não foram eficazes para a remissão completa das lesões de esporotricose cutânea experimental.

Le transfert adoptif de cellules T exprimant un récepteur chimérique reconnaissant un antigène (CAR), est un traitement qui génère des réponses impressionnantes dans les cancers hématologiques mais est beaucoup moins efficace pour le traitement de tumeurs solides. Les tumeurs solides modulent leur microenvironnement induisant des formes multiples d’immunosuppression qui inhibent l’efficacité des fonctions effectrices des cellules T ayant infiltrées la tumeur. Au cours de ma thèse, j’ai évalué le potentiel de deux stratégies pour améliorer les réponses anti-tumorales des cellules T CAR. La première se focalise sur l’étude du rôle potentiel des cellules immunes non T, exprimant un CAR sur la stimulation des fonctions et de la persistance de cellules T CAR+ dans le microenvironnement tumoral. Afin d’étudier la fonction des cellules CAR non T, nous avons généré un modèle de souris transgénique (vav-CAR) dans lequel les cellules immunes expriment un CAR reconnaissant l’antigène tumoral Her2 (ErbB2). Comme attendu, les cellules T CAR+ possèdent des fonctions anti-tumorales, mais nous avons aussi mis en évidence que les macrophages et les cellules NK exprimant le CAR montraient une réponse cytokinique, cytotoxique et phagocytiques spécifiques de l’antigène. De plus, en utilisant le modèle vav-CAR, nous avons démontré le potentiel des cellules immunes CAR+ dans le rejet des tumeurs et cela indépendamment des cellules T CD8+. Les cellules T CD4+ sont essentielles puisque leur élimination réduit considérablement les réponses anti-tumorales dans notre modèle vav-CAR. Il a été démontré que certaines sous-populations de cellules T auxiliaires participent aux réponses anti-tumorales avec les cellules Th1 et Th17 démontrant une efficacité plus robuste que les autres sous-populations. Notre deuxième stratégie s’est focalisée sur l’étude de l’impact du métabolisme au cours de la polarisation des cellules T CD4+ et plus particulièrement lors de la différenciation des cellules T CAR+ en cellules Th1. En effet, l’activation et différenciation des cellules T sont fortement associées à une augmentation des besoins métaboliques. Dans le microenvironnement tumoral, en raison de la forte demande en ressources de la tumeur, la déprivation en nutriments ainsi générée peut limiter l’accès aux nutriments d’autres types cellulaires et ainsi altérer le devenir et les fonctions des cellules immunes greffés infiltrant la tumeur. En conséquence, modifier les cellules immunes CAR+ afin qu’elles puissent résister à la compétition métabolique du microenvironnement tumoral pourrait leur permettre de conserver leurs fonctions effectrices. En étudiant l’impact de la déprivation en nutriments sur la différenciation des cellules T, nous avons trouvé que des concentrations limitantes en glutamine, l’acide aminé le plus abondant du plasma, inhibaient le potentiel des cellules T à se différencier vers la voie Th1 associée à la production d’IFNγ. Au contraire, cette condition favorisait la conversion de cellules T CD4 naïves en cellules régulatrices Foxp3+ ayant des fonctions suppressives (Tregs). De plus, nous avons montré que la présence d’un seul métabolite dérivé de la glutamine, l’α-ketoglutarate (αKG), suffisait à augmenter les fonctions effectrices anti-tumorales de plusieurs sous-types de cellules T auxiliaires CAR+, augmentant la production d’IFNγ et diminuant l’expression de FOXP3. Ainsi, durant ma thèse, j’ai développé un modèle murin vav-CAR, générant un outil permettant d’étudier et manipuler les fonctions de multiples populations de cellules immunitaires exprimant un CAR. Ce modèle permettra de promouvoir l’utilisation de cellules immunes optimisées exprimant un CAR dans le cadre d’immunothérapies dirigées contre des tumeurs solides. De plus, en utilisant ce modèle, nous avons identifié un métabolite de la glutamine, qui orchestre les réponses immunitaires au moyen d’une reprogrammation métabolique des cellules T CD4. / The adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) as a treatment for cancer has achieved impressive responses in haematological malignancies, but has been less successful in the treatment of solid tumors. The tumor microenvironment of solid tumors presents multiple forms of immunosuppression, inhibiting the efficient effector function of infiltrating anti-tumor T cells. During my PhD, we assessed the potential of two strategies to enhance the anti-tumor function of CAR T cells. The first focuses on the potential of other CAR-expressing immune subsets to stimulate CAR T cell function and persistence in the tumor microenvironment. To elucidate the function of CAR-expressing non-T lymphocytes, we generated a transgenic mouse model (vav-CAR) in which immune cells express a CAR against the Her2 (ErbB2) tumor antigen. As expected, CAR T cells harboured anti-tumor function but we also found that CAR-modified macrophages and natural killer cells (NKs) exhibited significant antigen specific cytokine secretion, cytotoxicity and phagocytosis. Moreover, using the vav-CAR model, we demonstrated the potential of CAR immune cells to mediate tumor rejection independently of CD8+ T cells. CD4+ T cells were critical for this response as their deletion severely abrogated the anti-tumor responses in our vav-CAR model. Distinct T helper subsets have been shown to participate to anti-tumor responses, with Th1 and Th17 cells demonstrating a more robust efficacy as compared to other T helper subsets. Our second strategy was focused on the impact of metabolism in the polarisation of CD4+ T cells, in particular the differentiation of CAR T cells to Th1 lineage. T cell activation and polarisation is highly associated with increased metabolic needs. Given that nutrient deprivation in the tumor microenvironment, due to a high demand of the tumor for resources, can limit the nutrients available for other cell types, the fate and function of adoptively transferred immune cells may be altered upon entering the tumor. Therefore, modifying CAR immune cells to resist metabolic suppression in the tumor microenvironment may help retain their effector functions. Upon assessing the effects of nutrient deprivation on T cell differentiation, we found that limiting concentrations of glutamine, the most abundant amino acid in the plasma, inhibited the potential of T cells to undergo Th1 differentiation with associated IFNγ secretion. Rather, this condition resulted in the conversion of naïve CD4+ T cells into suppressive Foxp3+ regulatory T cells (Tregs). Furthermore, we determined that a single glutamine-derived metabolite, α-ketoglutarate (αKG), enhanced the anti-tumor effector functions of multiple CAR T helper subsets, increasing the production of IFNγ and reducing FOXP3 expression.Thus, during my PhD, I generated a vav-CAR model, providing a platform in which the function of multiple CAR-bearing immune subsets can be studied and manipulated. This model will promote the utilisation of optimized CAR-bearing immune cells in adoptive immunotherapy for solid tumors. Furthermore, using the CAR model, we have identified a glutamine metabolite that orchestrates immune responses through the metabolic reprogramming of CD4 T cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis, an important disease in humans and animals that is prevalent in tropical and subtropical areas. In Brazil it was first reported in 1974, then several cases this disease has been reported throughout the country. The failure in the antifungal therapy combined with cases unresponsive to immunotherapy existing, lead to molecular studies in order to investigate possible genetic variations among Brazilian isolates, and this may be able to contribute on the research for the improvement of immunotherapy available. Thus, this study aimed to evaluate the genetic diversity of thirty P. insidiosum isolates from different regions of Brazil, and compare all of them with isolates from Thailand, one isolate from Central America and another isolate from North America, by sequencing and the analysis of genomic DNA regions corresponding to cytochrome c oxidase (COX II) and ITS1, 5.8S rRNA and ITS2 rDNA (ITS). The analyses of nucleotide sequences of both regions were carried out, individually and in combination, using the following methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data demonstrated all of P. insidiosum isolates as monophyletic in relation to the other Pythium species. Analises of COX II gene sequences subdivided P. insidiosum isolates into three groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian isolates. The molecular analyses performed in this study suggest an evolutionary proximity among all of American isolates, including the Brazilian, from Costa Rica and from the United States, that were grouped in a single group. COX II gene network analysis showed signs of a recent expansion of P. insidosum isolates, probably originated from the Asiatic to America continent. By analysis of COX II gene demonstrated the highest levels of genetic variability among P. insidiosum isolates studied in here, additionally, the highest levels of phylogenetic information were shown, when it was compared to the ITS region analysis. Nevertheless, both genetic markers selected for this study proved to be entirely congruent in the phylogenetic relations among Brazilian isolates of P. insidiosum, since clustered all of them into a single monophyletic group which did not shown to have genetic variability. The results indicate that the strain used in the production of the immunotherapic - Pitium-Vac ® - is representative of the Brazilian isolates of P. insidiosum. / O Pythium insidiosum é um oomiceto aquático causador da pitiose, uma importante enfermidade em humanos e animais, sendo prevalente em áreas tropicais e subtropicais. No Brasil foi relatada pela primeira vez em 1974. Desde então vários casos desta enfermidade tem sido descritos por todo o país. Os insucessos de terapias antifúngicas aliados aos casos não responsivos à imunoterapia existente impulsionam estudos moleculares a fim de investigar possíveis variações genéticas entre os isolados brasileiros de forma a contribuir nas pesquisas para a melhoria do imunoterápico disponível. Assim, o presente estudo teve por objetivo avaliar a diversidade genética de 30 isolados de P. insidiosum provenientes de diferentes regiões do Brasil, bem como compará-los com isolados tailandeses, um isolado da América Central e outro isolado da América do Norte através do sequenciamento e das análises das regiões do DNA genômico correspondentes à citocromo c oxidase (COX II) e ITS1, 5.8S rRNA e ITS2 do rDNA (ITS). As análises das sequências de nucleotídeos de ambas as regiões foram realizadas, individualmente e em combinação, utilizando as seguintes metodologias: máxima parcimônia (Maximum parsimony, MP); Neighbor-joining (NJ); máxima verossimilhança (Maximum likelihood, ML); e análise Bayesiana (Bayesian analysis, BA). Os dados demonstraram que todos os isolados de P. insidiosum são monofiléticos em relação às outras espécies de Pythium. A análise das sequências do gene COX II subdividiu os isolados de P. insidiosum em três grupos, cuja disposição demonstra os isolados tailandeses como parafilético em relação aos isolados brasileiros. As análises moleculares realizadas neste estudo sugerem uma proximidade evolutiva entre todos os isolados americanos, incluindo os brasileiros, da Costa Rica e dos Estados Unidos, os quais foram agrupados juntos em um único grupo. Na análise de network do gene COX II os resultados apresentaram sinais de uma recente expansão dos isolados de P. insidosum para a América, provavelmente oriundos do continente asiático. Pela análise do gene COX II foi possível evidenciar os maiores níveis de variabilidade genética entre os isolados de P. insidiosum avaliados, além disso, foram demonstrados os maiores níveis de informação filogenética, quando comparada à análise da região ITS. Contudo, os dois marcadores genéticos selecionados para este estudo revelaram-se inteiramente congruentes nas relações filogenéticas entre os isolados brasileiros de P. insidiosum, reunindo-os em um único grupo monofilético o qual não demonstrou haver variabilidade genética. Os resultados obtidos indicam que a cepa utilizada na produção do imunoterápico Pitium-Vac ® é representativa dos isolados brasileiros de P. insidiosum.

Malignant melanoma (MM) is the deadliest skin cancer with an ever-increasing incidence. New treatments have improved the prognosis for patients with advanced MM. Still, most patients do not respond, and the side effects can be severe, underlining the need for better therapies. The overall aim of this thesis was to evaluate new means to improve the treatment for patients with advanced MM. Immunostimulatory gene therapy (AdCD40L) was evaluated in a clinical study and BRAF-inhibitory treatment in rare cases of BRAF-mutated MM. Due to its immunogenicity, MM is an attractive target for immunostimulatory gene therapy. AdCD40L is an adenovirus carrying the human gene for CD40 ligand, which in different ways can stimulate the immune system to combat cancer. We conducted a Phase I/IIa study with AdCD40L in patients with metastatic MM having received established treatments. In cohort 1 (n=6), four weekly, intratumoural AdCD40L injections were given. In cohort 2 (n=9), low dose cyclophosphamide was added to increase the immune response. Since irradiation may act synergistically with immunotherapy, patients in cohort 3 (n=9) also received a single fraction of radiotherapy (8 Gy). This fraction was given towards the lesion selected for injections. The primary objectives were to assess the feasibility and safety of AdCD40L-treatment and secondarily its anti-tumour effects. Patients were thoroughly assessed for toxicity. The anti-tumour response was evaluated by imaging techniques (FDG-PET/CT, DW-MRI scans), tumour biopsies and blood tests. Plasma protein markers were measured with a multiplex platform. Another objective was to evaluate the potential of DW-MRI and FDG-PET/CT for prediction of AdCD40L treatment response, in terms of overall survival (OS). AdCD40L was well tolerated with mild transient reactions. Local and distant responses in PET/CT scans along with a significantly better 6-month survival in the cohorts that received cyclophosphamide conditioning were observed. Effector lymphocyte responses were elicited. All patients had an increased T effector/T regulatory-cell ratio and death receptors were significantly up-regulated post therapy. Inflammatory cytokines and other plasma proteins were altered in favourable ways by the AdCD40L treatment. The analyses support that the functional DWI parameters may be better early predictors of OS than the established metabolic and morphologic criteria of FDG-PET/CT and CT/MRI, respectively. In conclusion, the stimulation of the CD40 pathway to initiate anti-tumour immunity is a promising treatment alternative for MM patients. However, further studies with developed treatment schemes are warranted. In the first report ever on treatment of a pregnant patient with a BRAF-inhibitor, the therapy was initiated in the second trimester. The treatment with vemurafenib enabled prolonged gestation, hence reducing the risk of immaturity-related complications. Further, we report the first case worldwide of a patient with metastatic conjunctival melanoma who benefitted from treatment with vemurafenib. Additional studies are needed to assess the efficacy of BRAF -inhibitors in the different subtypes of ocular melanoma.

Le cancer colorectal est un problème de santé publique majeur pour lequel la recherche de nouveaux traitements est indispensable. Notre équipe a démontré l’efficacité d’une immunothérapie par un lipide A dans un modèle de cancer du colon chez le rat. Lorsque les rats sont porteurs de petites carcinomatoses, le lipide A induit la guérison de 95% des rats. L’étude des mécanismes d’action de cette immunothérapie nous a permis de montrer que l’effet antitumoral du lipide A est dépendante de la cytotoxicité induite par le granzyme B produit par les neutrophiles intratumoraux. En effet, nous avons montré que, dans le microenvironnement tumoral, les neutrophiles produisent du granzyme B et présentent un phénotype de type N2 protumorigène. Lorsque les rats sont traités par lipide A, il y a modification du phénotype des neutrophiles en type N1 antitumoral et libération du granzyme B qui induit l’apoptose des cellules tumorales. Lorsque les rats développent des tumeurs beaucoup plus volumineuses, l’efficacité du lipide A est diminuée et seul 40% des animaux sont guéris. L’injection préalable d’oxaliplatine permet alors de maintenir l’efficacité de l’immunothérapie par le lipide A. Nous avons montré que l’oxaliplatine induit la sénescence des cellules tumorales, générant ainsi un microenvironnement propice au recrutement au sein des tumeurs des neutrophiles, lesquels sont activables par l’immunothérapie subséquente. L’association de l’induction de la sénescence et de l’activation des cellules immunitaires par immunothérapie est une approche efficace et originale sur laquelle les recherches doivent se poursuivre. / Colorectal cancer is a major public health concern in France. Resistance to standard chemotherapy requires development of novel therapeutic approaches. In the past decades, our team showed the immunotherapeutic properties of lipid A in a model of colon cancer in rats. 95% of rats bearing small carcinomas were cured following treatment by lipid A. The study of mechanisms underlying this immunotherapy allowed us to show that the antitumor effect of lipid A was dependent on cytotoxicity induced by granzyme B produced by intratumoral neutrophils. Indeed, we have shown that, in the tumor microenvironment, neutrophils produced granzyme B and had a pro-tumorigenic N2 phenotype. When rats were treated with lipid A, neutrophils shifted to an antitumor N1 phenotype and released granzyme B, thus inducing apoptosis of tumor cells. In rats bearing advanced carcinoma, the effectiveness of lipid A was reduced and only 40% of animals were cured. An injection of oxaliplatin prior to lipid A treatment allowed sustaining the effectiveness of lipid A immunotherapy. In the present study, we showed that oxaliplatin injection induced tumor cell senescence. The microenvironment produced by senescent cells enabled then the recruitment of neutrophils within tumors, subsequently activated by lipid immunotherapy.Combining the induction of tumor cells senescence and activation of immune cells by an immunotherapeutic agent constitute an original and interesting therapeutic approach, but still studies must be carrying out to better understand underlying mechanisms.