Chef de file des inhibiteurs de tyrosines kinase, l’Imatinib Mesylate (IM) a révolutionné la prise en charge de la leucémie myéloïde chronique et des tumeurs stromales gastro-intestiales. En plus de son action directe sur les cellules tumorales, une partie de l’efficacité thérapeutique de l’IM a été attribuée à son aptitude à moduler la réponse immunitaire. Cette propriété soulève la possibilité que les résultats cliniques de l’IM pourraient être améliorés en le combinant efficacement à une immunothérapie. A cet effet, nous avons montré dans un modèle préclinique que l’interleukine-2 (IL-2) – adjuvant des cellules NK – augmente l’efficacité de l’IM. Nous avons également démontré une efficacité supérieure de l’IM en association au cyclophosphamide (CTX) du fait de l’inhibition des lymphocytes T régulateurs. Nous avons donc entrepris un essai clinique de phase 1 associant l’IM, l’IL-2 et le CTX chez des patients ayant une tumeur solide métastatique ou localement avancée. Les objectifs de cet essai sont (i) de déterminer la dose maximale tolérée d’IL-2 associée à l’IM et au CTX ; (ii) d’étudier les paramètres pharmacocinétiques de l’association ; (iii) d’évaluer l’efficacité de l’association et (iv) son effet sur les effecteurs de l’immunité. Au total, 17 patients ont été inclus dans cette étude. La DMT d’IL-2 associée à la dose fixe de 400 mg d’IM correspond à 6 MUI/j. A ce niveau de dose, tous les patients ont présenté au moins un effet indésirable imputable au traitement : principalement fièvre et frissons, augmentation des enzymes hépatiques, asthénie et nausée mais sans que ne soit observée de toxicité limitante. L’étude des paramètres pharmacocinétiques révèle une augmentation significative de l’exposition systémique à l’IM en fin de cycle et qui semble être imputable à l’IL-2. La pharmacocinétique de l’IL-2 n’est par contre pas modifiée par l’administration concomitante d’IM. Sur le plan des effecteurs de l’immunité, l’association IM, IL-2 et CTX diminue le taux de lymphocytes B, lymphocytes T (LT) CD4+ et LT CD8+ mais active les cellules NK puisqu’on observe une augmentation des marqueurs CD56bright, HLA-DR et TRAIL. De manière intéressante, la sous-population de cellules NK HLA-DR+ possède des capacités de dégranulation plus importante après exposition à cette association et son expansion est associée à une meilleure survie. Cette association pourrait donc s’avérer particulièrement intéressante dans le traitement de tumeurs sensibles d’une part à l’IM et d’autre part à la lyse par les cellules NK. Les GIST étant particulièrement sensibles à l’IM, nous avons étudié l’infiltrat tumoral présent au niveau de ses tumeurs. Nous avons ainsi pu mettre en évidence le rôle pronostique de l’infiltrat en LT et NK sur la survie sans progression des GIST. En vue d’une étude de phase 2, les GIST apparaissent donc être un modèle tumoral particulièrement pertinent pour évaluer les bénéfices de l’association IM, IL-2 et CTX. / Imatinib mesylate (IM) was the first tyrosine kinase inhibitor to be successfully used in clinical practice and its introduction has revolutionized the management of chronic myeloid leukemia and gastrointestinal stromal tumors. In addition to its direct effects on malignant cells, IM appears to exert immunological off-target effects that contribute to its anticancer effects. Thus, combining IM with immunotherapy might improve patients’ clinical outcome. Indeed, IM combined to Interleukin-2 (IL-2) - a cytokine that enhances natural killer (NK) cells functions - improved antitumor responses in preclinical models. We also observed synergistic effects of cyclophosphamide (CTX) and IM as a result of the inhibition of regulatory T lymphocytes. Based on the promising results of these preclinical studies, we developed a phase 1 clinical trial which combines metronomic CTX, IM and escalating doses of IL-2 in patients affected by refractory solid tumors. The goals of this study were (i) to determine the maximum tolerated dose of IL-2 combined with IM and CTX ; (ii) to study the pharmacokinetics of IM and IL-2 ; (iii) to evaluate clinical efficacy of the combined therapy and (iv) effects of the association on immune parameters. A total of 17 patients were enrolled in the study. The maximum tolerated dose of IL-2 combined with IM, given at a constant dose of 400 mg was determined to be 6 MIU/day. At this dose level, all patients experienced at least one treatment-related adverse event: fevers and chills, transaminase elevation, fatigue and nausea but no dose-limiting toxicities were observed. Pharmacokinetic studies revealed that the co-administration of IL-2 increases the systemic exposure of patients to IM. In contrast, the pharmacokinetics of IL-2 was not modified by IM. The combined therapy markedly reduced the absolute numbers of B lymphocytes, CD4+ T cells and CD8+ T cells in a IL-2 dose-dependant manner. The NK cell compartment was activated, exhibiting a significant upregulation of CD56bright, HLA-DR and TRAIL. Interinstingly, the abundance of HLA-DR+ NK cells after one course of combined therapy positively correlated with both progression free- and overall survival. Thus, it could be of interest to evaluate this immunotherapeutic regimen in a tumor model sensitive to IM and to lysis by NK cells and evaluate whether the adjunction of IL-2 can boost the efficacy of IM. GIST are particularly sensitive to IM, thus we performed a retrospective study of the immune infiltrates and their prognostic value in these tumors. We found that both LT and NK cell infiltrates were independent prognostic factors for progression-free survival. For a phase 2 clinical trial, gastrointestinal stromal tumors appear to be a particularly relevant to evaluate the benefits of the association IM, IL-2 and CTX.

Desde o início da pandemia de HIV/aids, a imunoterapia vem sendo utilizada como alternativa terapêutica numa tentativa de estimular uma resposta do sistema imunológico contra o agente agressor. Esta abordagem tem sido considerada promissora para obtenção do controle da infecção em longo prazo. A administração de células apresentadoras de antígeno, em especial células dendríticas, é fundamentada pelos conceitos de imunoterapia passiva e de terapia celular ativa (vacina terapêutica) além da perspectiva da eliminação dos chamados reservatórios virais, unindo assim estas diversas estratégias de intervenção. Dentre os diferentes antígenos do HIV utilizados para pulsar as células dendríticas, alguns dos melhores resultados foram obtidos com a inativação química do vírus, preservando a integridade da estrutura da sua superfície.Há alguns anos nosso grupo vem trabalhando com terapia celular baseada em células dendríticas derivadas de monócitos autólogos, pulsadas com HIV inativado quimicamente, seguindo o protocolo descrito por Lu e colaboradores em 2004. Aqui, nós apresentamos os resultados de um ensaio clínico de fase I/II que teve como objetivos avaliar a tolerância, segurança e impacto imunovirológico, de diferentes formulações do produto em pacientes cronicamente infectados pelo HIV, sem uso de antirretrovirais. Os participantes foram alocados em três braços para receber composições distintas: 3x107 células dendríticas sem pulso adicional de HIV inativado (Braço A), 3x106 células dendríticas com pulso adicional de HIV inativado (Braço B) ou 3x107 células dendríticas com pulso adicional de HIV inativado (Braço C). O número de participantes no braço A evoluiu com uma considerável diminuição ao longo do período de observação do estudo, prejudicando as avaliações. As análises dos outros braços mostraram que as preparações foram seguras, não se observando eventos adversos relacionados à intervenção. Os resultados sugeriram um aumento na carga viral plasmática associados a uma redução das sub-populações de linfócitos TCD4+ e TCD8+ nos pacientes do braço C além de uma redução na quantidade de linfócitos T reguladores nos indivíduos do braço B / Since the beginning of the HIV / aids pandemic, immunotherapy is being used as an alternative therapy in attempt to stimulate an immune response against the pathogenic agent. This approach has been considered as promising for achieving the control of infection in the long term. Administration of antigen presenting cells, particularly dendritic cells is based on the concepts of passive immunotherapy and active cellular therapy (therapeutic vaccine), with the perspective of eliminating the so-called viral reservoirs, thus joining different intervention strategies. From different antigens tested for loading DCs, some of the best results were obtained with chemically inactivated virus, which preserves its surface proteins.Our group has been working with cellular therapy based on dendritic cells derived from autologous monocytes pulsed with chemicallyinactivatedHIV, following the protocol described by Lu et al in 2004.Here, we present the results of a phase I / II clinical trial aimed to evaluate tolerance, safety and immunovirological impact of different product formulations in chronically HIV-infected individuals, naïve for antiretroviral treatment.Participants were allocated to receive: 3x107 un-pulsed DCs (Arm A), 3x106 HIV-pulsed DCs (Arm B) or 3x107 HIV-pulsed DCs (Arm C). The number of participants in the arm A evolved with a considerable decrease over the study, so any considerations about effect of DCs without antigen overload were difficult to carry out. Outcomes in other arms showedthat they were safe, with no adverse events related to the products. The results suggested an increase in plasma viral load and decline in CD4+ and CD8+ T cells subpopulations after intervention in arm C. Additionally, we observed a decrease in percentage of regulatory T cells in arm B patients

Wu Xiujuan. / "May 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 294-314). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Le chondrosarcome (CHS) est caractérisé par une grande chimio et radiorésistance ; il y a un besoin urgent de nouvelles stratégies thérapeutiques pour cette tumeur. Parmi celles-ci, certaines approches d'immunothérapie pourraient être d'un grand intérêt. Nous étudions actuellement l'implication du système immunitaire dans la progression du CHS et la réponse thérapeutique à la fois sur des échantillons humains et dans le modèle de chondrosarcome de rat (SRC).Dans le CHS humain et de rat, des infiltrats immunitaires composés de lymphocytes et macrophages ont été identifiés dans la zone péritumorale. L’infiltration immunitaire est en corrélation avec l’évolution de la tumeur (grade, envahissement et taille). L'expression de PD1 et PDL1 ont été détectée dans les infiltrats immunitaires et cellules tumorales du CHS chez l’homme et le rat. Le niveau d'expression PD-L1 en corrélation avec la survie des patients et le taux de rechute. Dans le model SRC, la déplétion sélective de lymphocytes T a entrainé une accélération de la progression tumorale, tandis que la déplétion de macrophages l’a ralenti. Les splénocytes isolés de rats porteurs de CHS ont montré une cytotoxicité spécifique dirigée contre les cellules de chondrosarcome (27%), qui a diminué de manière significative avec des rats appauvrie en CD3 (11%). La voie de signalisation PI3K/mTOR ne peut pas être associée à une immunothérapie car elle induit une action immunosuppressive in vivo.L'environnement immunitaire contribue à la progression du CHS à la fois chez l’homme et chez le rat, ce qui suggère que une approche immunomodulatrice avec des anticorps bloquant PDL1 pourrait être testée pour le CHS / Chondrosarcoma is highly resistant to chemotherapy and radiation and there is an urgent need in developing new therapeutic strategies for this malignancy; among these, some immunotherapy approaches could be of great interest. We are currently investigating the immune system implication in chondrosarcoma progression and therapeutic response both on human samples and in rat chondrosarcoma model (SRC). In human and rat chondrosarcoma, immune infiltrates composed of lymphocytes and macrophages were identified in the peritumoral area. Immune infiltrates composition was found correlated with tumors characteristics and evolution (grade, invasiveness and size). Expression of PD-1 and PD-L1 was detected in CHS immune infiltrates, both in human and rat (and on tumor cells). PD-L1 expression level correlated with patients survival and relapse rate. In SRC, T lymphocytes depletion resulted in an accelerated tumor progression, while CD163+ macrophages depletion slowed down tumor progression. Splenocytes isolated from CHS bearing SRC showed a specific cytotoxicity directed against chondrosarcoma cells (27%), which significantly decreased in CD3 depleted SRC (11%). The immune environment contributes to CHS progression in both human and animal models, this associated with expression of immune checkpoint PD1/PDL1 suggest that immunomodulatory approaches with PD-L1 blocking antibody could be applied in CHS; this approach is currently being tested in SRC

Imunização terapêutica utilizando células dendríticas derivadas de monócitos (MoDCs) pulsadas com antígenos de HIV constitui um meio promissor de potencializar a resposta imune específica anti-HIV em pacientes infectados. Neste contexto, é importante ressaltar que células dendríticas além de estimular a resposta imune específica, podem ser capazes de promover a tolerância periférica em linfócitos T CD4+ e T CD8+ ao induzir deleção, anergia ou através da expansão de células T reguladoras (T regs). Experimentos in vitro foram conduzidos para avaliar a capacidade de MoDCs pulsadas com HIV autólogo inativado em induzir apoptose celular, respostas celulares específicas e a geração de T regs. Os pacientes avaliados neste estudo foram indivíduos infectados pelo HIV, sem uso de tratamento antirretroviral (n = 14) com número de células T CD4+ acima de 350 células/L. MoDCs foram geradas a partir de células mononucleares de sangue periférico e em seguida foram pulsadas com vírus autólogo inativado por Aldrithiol-2, tratadas com estímulo para maturação e então cultivadas com linfócitos autólogos. A apoptose de linfócitos T e MoDCs e a frequência de células efetoras e reguladoras foram avaliadas por citometria de fluxo. Os resultados obtidos mostraram que não houve diferença nos níveis de apoptose de células T CD4+, T CD8+ ou MoDCs entre os grupos pulsadas e não pulsadas com HIV inativado. Foi observado que tanto MoDCs pulsadas quanto aquelas não pulsadas com o vírus autólogo inativado foram capazes de induzir células T CD4+ secretoras de IFN-, enquanto que apenas MoDCs pulsadas levou a um aumento no percentual de células T CD8+ efetoras. Pacientes com contagem de células T CD4+ acima de 500 células/L apresentaram um percentual maior de células T CD4+ secretoras de IFN após estimulo de MoDCs pulsadas. Esta diferença não foi observada em células T CD8 +. T regs também foram induzidas in vitro após cocultivo com MoDCs. Níveis basais mais elevados de T regs foram encontrados em pacientes com carga viral plasmática baixa. Em conjunto, os resultados indicam que MoDCs pulsadas com HIV-1 são capazes de induzir linfócitos T efetores, mas também aumentam a frequência de T regs in vitro. Além disto, pacientes com maior contagem de células T CD4 + foram capazes de responder de forma mais eficiente ao estímulo com MoDCs pulsadas. Viremia persistente na infecção crônica pelo HIV pode estar associada significativamente à perda de T reg / Therapeutic immunization using inactivated autologous HIVpulsed dendritic cells (DCs) is a promising strategy to enhance specific anti-HIV immune responses in infected patients. In this context, it is important to note that DC besides stimulate a specific immune response, may be able to promote tolerance in peripheral CD4 + and CD8 + T cells inducing deletion, anergy or through expansion of regulatory T cells (T reg). In vitro experiments were conducted to evaluate the capacity of autologous HIVstimulated DC to induce apoptosis, effector cellular T cell responses and T reg generation. For these purposes, we used peripheral blood from HAARTnaïve HIVinfected patients (n=14) with CD4+ T cell counts above 350 cell/L for generation of monocytederived DC (MoDC). MoDC were pulsed with aldrithiol-2 (AT-2)-inactivated autologous virus and matured. MoDC were then cocultured with autologous lymphocytes and the apoptosis, production of IFN- and T reg cell frequency were evaluated by flow cytometry. There was no difference in the rate of apoptosis of CD4, CD8 T cells or MoDC between the groups pulsed and not pulsed with inactivated HIV. MoDC pulsed or not with inactivated autologous virus induced IFN- +CD4+ T cells, whereas only pulsed MoDC were able to increase effector CD8+ T cells percentage along the time culture. Patients with CD4+ T cell counts above 500 had an increased percentage of CD4 + T cells secreting IFN- upon DC pulsed with HIV. This difference was not observed in CD8 + T cells. Interestingly, T reg were also induced in vitro after MoDC cocultivation. Higher baseline T reg counts were found in patients with lower plasma viral loads. These results show that MoDC pulsed with HIV-1 are able to induce effector lymphocyte but also elevate the frequency of T reg in vitro. Patients with higher CD4+ T cell counts are able to respond more efficiently to the stimulation with pulsed MoDC, and persistent viremia in chronic HIV infection is associated with significant loss of T reg

O melanoma é um dos tipos de câncer de pele cuja frequência tem crescido nos últimos anos e apresentado elevada taxa de mortalidade, apesar de ter reduzida prevalência. Mesmo havendo um considerável avanço nas propostas terapêuticas nos últimos anos, ainda se vê necessário o desenvolvimento de novas abordagens, sendo a terapia gênica uma promissora possibilidade para tal. Utilizando vetores adenovirais com promotor responsivo à p53 (PGTx beta) para a transferência gênica de p19Arf (proteína supressora de tumor) e interferon-beta (citocina imunomodulatória) em células de melanoma murino com o gene Trp53 selvagem, o nosso grupo demonstrou previamente que a combinação dos dois genes, mas não o tratamento individual, promove efeito citotóxico sinérgico com a liberação de marcadores de morte imunogênica, in vitro; e significativa redução da progressão tumoral acompanhada de uma forte resposta imunológica de linfócitos T CD4+ e CD8+, células NK e neutrófilos contra desafios tumorais, in vivo. Porém, como a translação para modelos de melanomas humanos ainda estava em estágio inicial, ainda não haviam sido confirmamos se esses benefícios também seriam recapitulados. Observações inicias sugeriam que apenas a transferência gênica de interferon-beta seja suficiente para induzir morte celular em linhagens humanas portadoras de TP53 selvagem, sem ainda terem sido identificado o efeito da transferência de p14ARF e nem a necessidade de p53 endógeno para a resposta. Dessa forma, o presente projeto buscou avaliar os efeitos antitumorais provocados pela terapia gênica combinada de p14ARF e interferon-beta em modelos de melanoma humano utilizando linhagens com e sem a via da p53 integra. Para isso, foram utilizadas diferentes linhagens celulares com TP53 selvagem ou com distintas mutações e também foram construídos vetores adenovirais com o promotor constitutivo CMV, tornando assim possível a expressão dos transgenes de maneira independente do status do TP53 endógeno. O presente trabalho revelou que a transferência combinada do interferon-beta e p14ARF revelou vantagem quanto ao estímulo citotóxico e regulação negativa na dinâmica da população em ambas as linhagens UACC-62 e SK-Mel-29, independentemente do estado da via da p53. Na avaliação dos mecanismos de morte foi observado que ambas a linhagens apresentaram marcação positiva para marcadores da via da apoptose, porém com possível participação de outras modalidades de morte-celular, como a necrose, para a linhagem com o TP53 mutado (SK-Mel-29). Além disso, mostramos que os tratamentos potencialmente induzem vias de morte com caráter imunogênico pela secreção de ATP e exposição da calreticulina, sendo este último marcador mais significantemente observado mediante o tratamento combinado. Assim, recapitulamos o benefício observado em modelo murino para a transferência gênica do interferon-beta e p14ARF em modelo de melanoma humano, e investigamos marcadores importantes à translação da proposta terapêutica para o melanoma / Melanoma is one of the types of skin cancer whose frequency has grown in the last years and presents a high mortality rate, despite its low prevalence. Although there has been considerable progress in therapeutic proposals in recent years, it is still necessary to develop new approaches, being gene therapy a promising possibility for this. With the use of adenoviral vectors with a p53 responsive promoter (PGTx beta) for the gene transfer of p19Arf (tumor suppressor protein) and interferon-beta (immunomodulatory cytokine) in murine melanoma cells bearing wild-type Trp53 gene, our group previously demonstrated that the combination of the two genes, but not individual treatment, promotes a synergistic cytotoxic effect with the release of immunogenic death markers in vitro; and significant reduction of tumor progression with a strong immune response mediated by CD4+ and CD8+ T lymphocytes, NK cells and neutrophils in tumor challenges in vivo. However, as the translation for human melanoma models was still at an early stage, it still was not possible to confirm whether these benefits would also be recapitulated in a human model. Initial observations suggested that interferon-beta gene transfer is sufficient to induce cell death in wild-type TP53-bearing human melanoma cell lines, with the effect of p14ARF gene transfer and the role for endogenous p53 in this response yet to be investigated. Thus, the present work aimed to evaluate the antitumor effects induced upon the combined gene transfer of p14ARF and interferon-beta in human melanoma cell lines with and without a functional p53 pathway. For this, different cell lines bearing wild-type TP53 or with different mutations were used and adenoviral vectors with the constitutive CMV promoter were also constructed, making possible the expression of the transgenes independently of the endogenous TP53 status. The present work showed that the combined transfer of interferon-beta and p14ARF was advantageous in cytotoxic stimulation and negative regulation in population dynamics for both cell lines UACC-62 and SK-Mel-29, regardless of p53 pathway status. In the evaluation of the triggered cell death mechanisms it was observed that both cell lines presented positive markers of the apoptosis pathway, but with possible participation of other cell death mechanism, such as necrosis, for the mutated TP53 cell line SK-Mel-29. In addition, we showed that the treatments potentially induced cell death pathways with immunogenic features including the secretion of ATP and calreticulin exposure, being the latter marker more significantly presented after the combined treatment. Thus, we recapitulated the benefit observed in murine model for the gene transfer of interferon-beta and p14ARF in the model of human melanoma, and investigated important markers for the translation of the melanoma therapeutic proposal

The tumour microenvironment comprises the non-cancerous cells present in the tumour mass (fibroblasts, endothelial, and immune cells), as well as signalling molecules and extracellular matrix. Tumour growth, invasion, metastasis, and response to therapy are influenced by the tumour microenvironment. Therefore, characterising the cellular and molecular components of the tumour microenvironment, and understanding how they influence tumour progression, represent a crucial aim for the success of cancer therapies. High-grade serous ovarian cancer provides an excellent opportunity to systematically study the tumour microenvironment due to its clinical presentation of advanced disseminated disease and debulking surgery being standard of care. This thesis first presents a case report of a long-term survivor (>10 years) of metastatic high-grade serous ovarian cancer who exhibited concomitant regression/progression of the metastatic lesions (5 samples). We found that progressing metastases were characterized by immune cell exclusion, whereas regressing metastases were infiltrated by CD8+ and CD4+ T cells. Through a T cell - neoepitope challenge assay we demonstrated that pre- dicted neoepitopes were recognised by the CD8+ T cells obtained from blood drawn from the patient, suggesting that regressing tumours were subjected to immune attack. Immune excluded tumours presented a higher expression of immunosuppressive Wnt signalling, while infiltrated tumours showed a higher expression of the T cell chemoattractant CXCL9 and evidence of immunoediting. These findings suggest that multiple distinct tumour immune microenvironments can co-exist within a single individual and may explain in part the hetero- geneous fates of metastatic lesions often observed in the clinic post-therapy. Second, this thesis explores the prevalence of intra-patient tumour microenvironment het- erogeneity in high-grade serous ovarian cancer at diagnosis (38 samples from 8 patients), as well as the effect of chemotherapy on the tumour microenvironment (80 paired samples from 40 patients). Whole transcriptome analysis and image-based quantification of T cells from treatment-naive tumours revealed highly prevalent variability in immune signalling and distinct immune microenvironments co-existing within the same individuals at diagnosis. ConsensusTME, a method that generates consensus immune and stromal cell gene signatures by intersecting state-of-the-art deconvolution methods that predict immune cell populations using bulk RNA data was developed. ConsensusTME improved accuracy and sensitivity of T cell and leukocyte deconvolutions in ovarian cancer samples. As previously observed in the case report, Wnt signalling expression positively correlated with immune cell exclusion. To evaluate the effect of chemotherapy on the tumour microenvironment, we compared site-matched and site-unmatched tumours before and after neoadjuvant chemotherapy. Site- matched samples showed increased cytotoxic immune activation and oligoclonal expansion of T cells after chemotherapy, unlike site-unmatched samples where heterogeneity could not be accounted for. In addition, low levels of immune activation pre-chemotherapy were found to be correlated with immune activation upon chemotherapy treatment. These results cor- roborate that the tumour-immune interface in advanced high-grade serous ovarian cancer is intrinsically heterogeneous, and that chemotherapy induces an immunogenic effect mediated by cytotoxic cells. Finally, the different deconvolution methods were benchmarked along with ConsensusTME in a pan-cancer setting by comparing deconvolution scores to DNA-based purity scores, leukocyte methylation data, and tumour infiltrating lymphocyte counts from image analysis. In so far as it has been benchmarked, unlike the other methods, ConsensusTME performs consistently among the top three methods across cancer-related benchmarks. Additionally, ConsensusTME provides a dynamic and evolvable framework that can integrate newer de- convolution tools and benchmark their performance against itself, thus generating an ever updated version. Overall, this thesis presents a systematic characterisation of the tumour microenvironment of high grade serous ovarian cancer in treatment-naive and chemotherapy treated samples, and puts forward the development of an integrative computational method for the systematic analysis of the tumour microenvironment of different tumour types using bulk RNA data.

Les môles hydatiformes sont une prolifération placentaire prétumorale pouvant évolueren tumeur alors traitée par chimiothérapie. Afin de réduire la mortalité et d’optimiser laprise en charge thérapeutique, l’objectif de cette thèse est d’identifier les gènespermettant de prédire la transformation en tumeur post môlaire et la chimiorésistance.Concernant la prédiction de la transformation, l’analyse de l’expression de gènescandidatssur tissu molaire décrit la relocalisation apicale de la Syncytine-1 en cas detransformation maligne, sans modification de transcription de ses récepteurs ni de deuxautres enveloppes rétrovirales placentaires. L’analyse sans à priori du transcriptome par3 méthodes différentes n’a pas permis d’identifier de gène différentiellement expriméselon la transformation. Cela suggère que la variabilité interindividuelle et les diverscritères utilisés pour le diagnostic de tumeur nuisent à l’identification de biomarqueursrobustes.Concernant la prédiction de la chimiorésistance, une approche transcriptomique largespectre sur tissu tumoral de choriocarcinome identifie une réduction de transcriptiond’HLA-G en cas de monochimiorésistance, confirmée au niveau protéique par immunohistochimie. L’analyse en réseaux de l’ensemble des gènes différentiellementexprimés suggère que la monochimiorésistance est associée à une altération de ladifférenciation des lymphocytes T alors que la polychimiorésistance est associée à unealtération de la prolifération des cellules sanguines.In fine, l’objectivation de l’expression trophoblastique du point de contrôle PD-L1 aconduit à évaluer l’efficacité d’un anti PD-L1 chez les patientes chimiorésistantes. Lesrésultats encourageants de cet essai et la possibilité de stratifier les patientes à l’aidedes marqueurs HLA-G et Syncytine-1 incitent à évaluer la place de l’anti PD-L1 associéà une monochimiothérapie en première ligne de traitement des tumeurstrophoblastiques. / Hydatidiform moles are a pretumoral placental proliferation which can turn into a tumorrequiring chemotherapy. In order to reduce mortality and propose an optimal therapeuticmanagement, the aim of this thesis is to identify genes which are predictive of postmolartumor transformation and chemoresistance.Concerning the prediction of transformation, the expression analysis of candidate-geneson molar tissue shows a relocalization of Syncytin-1 at the syncytiotrophoblast apicalborder in moles followed by malignant transformation, without modification oftranscription of its receptors and two other retroviral placental envelopes. A wholetranscriptomeapproach using 3 different microarrays-based methods did not identify anydifferentially expressed gene according to the post molar evolution. This may reflect thatinter-individual variability and the different criteria used for tumor diagnosis impede theidentification of robust biomarkers.Concerning the prediction of chemoresistance, a broad-spectrum transcriptomicapproach on choriocarcinoma tumor tissue identifies a down regulation of HLA-G in case of monochemoresistance, confirmed at the protein level by immunohistochemistry.Pathway analysis of the differentially expressed genes suggests thatmonochemoresistance is associated with impaired T-cell differentiation, whereaspolychemoresistance is associated with impaired proliferation of blood cells.Ultimately, the evidence of trophoblastic ubiquitous expression of the PD-L1 immunecheckpoint led us to the evaluation of the efficacy of PD-L1 blockade in chemoresistantpatients. The encouraging results of this trial and the possibility of stratifying patientswith HLA-G and Syncytin-1 markers encourages the assessment of PD-L1 blockadecombined with monochemotherapy as a first line treatment for trophoblastic tumors.

<p>The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (T_H) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. </p><p>To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65_495-503 peptide, a recombinant adenovirus coding for pp65, <i>in vitro</i> transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8⁺ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4⁺ T_H cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and T_H cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells <i>ex vivo</i> from virtually all stem cell donors for adoptive T cell transfer. </p><p>I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8⁺ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8⁺ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer. </p>

The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer. I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (&lt;0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.