Le Fas Ligand (FasL) est le déclencheur naturel de l’apoptose induite par le récepteur Fas. Il est produit sous la forme d’une protéine membranaire homotrimérique, qui peut être clivée par une métalloprotéase pour engendrer une forme soluble (sFasL). Cette forme conserve sa structure homotrimérique, mais son activité pro-apoptotique est très faible car elle n’atteint pas un degré de polymérisation suffisant. Au laboratoire, une molécule sFasL hautement polymérique a été obtenue en fusionnant un domaine de type immunoglobuline au domaine extracellulaire du FasL. Ce domaine permet de polymériser le sFasL sous forme hexamérique et dodécamérique. Cette molécule appelée pFasL possède une activité anti-tumorale in vitro et in vivo dans un modèle de greffe de cellules humaines tumorales à des souris immunodéficientes. Dans cette étude, nous nous sommes focalisés sur l'amélioration de pFasL, avec deux objectifs complémentaires. Tout d'abord, nous avons fusionné un récepteur à l’antigène de lymphocytes Tγδ (TCRγδ) au pFasL (TCR-pFasL) de façon à orienter son activité vers les cellules tumorales carcinomateuses exprimant l’antigène spécifique de ce TCR, qui est l’EPCR (Endothelial Protein C Receptor). Ensuite, nous souhaitions améliorer la production et l’activité spécifique de pFasL et son dérivé TCR-pFasL. La stratégie de ciblage dépendante du TCR n’a pas été validée dans cette étude, mais nous avons pu décrire une approche originale pour améliorer la production et/ou l’activité cytotoxique de ces chimères en co-exprimant celles-ci avec du sFasL non apoptotique. Cette approche a été validée avec deux autres chimères obtenues à partir de pFasL, l’une contenant la molécule de présentation antigénique HLA-A2 et la seconde le ligand CD80 du récepteur CD28, pour lequel le ciblage fonctionne. Notre étude ouvre ainsi des perspectives pour appliquer ce protocole à une grande variété de protéines de fusion dérivées du sFasL pour des applications diverses, en recherche et en thérapie. / Fas ligand (FasL) is the natural trigger for apoptosis induced by the Fas receptor. FasL exists as an homotrimer on the cell surface, which can be cleaved by a metalloprotease to generate a soluble form (sFasL). The sFasL form retains its homotrimeric structure, but displays almost no pro-apoptotic activity because it does not reach a sufficient degree of polymerisation. In the laboratory, a highly polymeric molecule sFasL was obtained by fusing an immunoglobulin-like domain to the extracellular domain of FasL. This domain polymerises the molecule through disulphide bonds, leading to hexameric and dodecameric compounds. This molecule, called pFasL, possesses an anti-tumor activity in vitro but also in vivo in a model of human tumor cell transplanted to immunodeficient mice. In this study, we focused on improving pFasL with two complementary objectives. Firstly, we fused to pFasL an antigen receptor of γδ T-cells (TCRγδ, leading to TCR-pFasL) to direct its activity towards carcinoma cells expressing this TCR specific antigen, which is the EPCR (Endothelial Protein C Receptor). Then, we wanted to improve the production and specific activity of pFasL and its derivative TCR-pFasL. In this study, the TCR dependent targeting strategy has not been validated, but we have described a novel approach to improve the production and/or cytotoxic activity of these chimeras by coexpressing them with non-apoptotic sFasL. This strategy has been validated with two other chimeras obtained from pFasL, one containing the antigen-presenting molecule HLA-A2 and the second one CD80, the ligand for the CD28 receptor. For this latter one, the cell targeting activity proved efficient. Our study opens up opportunities to improve and develop a wide variety of sFasL-derived fusion proteins for various applications in research and therapy.

Os linfócitos T CD4+ orquestram a resposta imune adaptativa, auxiliando os macrófagos, os linfócitos T CD8+ e os linfócitos B na resposta mais eficiente frente a um antígeno. As etapas que caracterizam uma resposta imune adaptativa são: apresentação do antígeno, ativação e diferenciação dos linfócitos T, expansão clonal e morte celular para retorno à homeostasia via AICD. Este tipo de morte ocorre via FAS/FASL. Tendo em vista que o mecanismo pelo qual a PGE2 inibe a expressão de fasl ainda não é conhecido, o presente trabalho tem como objetivo compreender o mecanismo molecular de atuação da PGE2 sobre os linfócitos T CD4+ na inibição gênica do FASL, tendo como hipótese o envolvimento do repressor transcricional ICER. Foi verificado que PGE2 10-8 M é capaz de proteger as DO11.10 da AICD e que, nesta concentração, ela induz a expressão de icer. Este repressor apresenta uma expressão transiente e observa-se seu aumento concomitantemente à inibição da expressão de fasl. Sendo assim, sugere-se a participação de ICER na via de inibição do fasl pela PGE2. / CD4+ T lymphocytes orchestrate the adaptive immune response, helping the macrophages, CD8+ T lymphocytes and B lymphocytes to reach an efficient antigen-specific immune response. The adaptive immune response is characterized by different phases: antigen, T lymphocyte activation and differentiation, clonal expansion and, finally, clonal cell death to return to homeostasis via AICD. This cell death occurs via FAS/FASL. Since the mechanism by which PGE2 inhibits fasl expression is not known, our aim is to understand the molecular mechanism responsible for PGE2 inhibition of fasl in CD4+ lymphocytes. Our hypothesis is that the transcriptional repressor ICER, which binds to CRE sites on gene promoters, is involved in this process. We verified that PGE2 10-8 M protects DO11.10 cells from AICD, and in that concentration, PGE2 increases icer expression. This repressor has a transient expression and we observed that its expression is increased in the same time that fasl expression is inhibited. Thus, we suggest the involvement of ICER in fasl inhibition pathway by PGE2.

La voie Fas-FasL est la voie majeure d’apoptose dont le rôle est indispensable pour l’homéostasie des cellules hématopoïétiques et la tolérance périphérique. Mon projet de thèse consiste à étudier le rôle de FasL dans la réponse anti tumorale, notamment le rôle de son expression sur les cellules myéloïdes, en l’occurrence les macrophages et les cellules myéloïdes suppressives.Les souris Fasl KO sont caractérisées par une accumulation des différentes populations de cellules hématopoïétiques dans les organes lymphoïdes périphériques. Cependant, elles ne développent pas de tumeurs spontanées. De façon intéressante, nos résultats montrent que lors qu’elles sont transplantées par les cellules tumorales, leur survie est significativement diminuée par rapport aux souris contrôles (Fasl fl/fl), ce qui suggère un rôle de FasL dans la réponse anti-tumorale. Une caractérisation fine de la répartition des cellules myéloïdes chez les souris Fasl KO porteuses de tumeur, montre une répartition différentielle des cellules Gr1+, par une accumulation des M-MDSC, dans la rate de ces souris. En plus, un enrichissement de l’infiltrat tumoral par les macrophages TAM chez les souris Fasl KO a été observé. Ces macrophages, indépendamment de génotype exècrent une forte activité d’arginase et iNOS et une inhibition de la prolifération des cellules T in vitro. Ainsi, la mortalité plus importante chez les souris Fasl KO pourrait, en partie, être associée à cet enrichissement des TAM dans l’infiltrat des souris déficientes en FasL.Afin de déterminer si cette accumulation des cellules myéloïdes immunosuppressives déficientes en FasL est spécifique d’un environnement tumoral ou le reflet d’un état inflammatoire, nous avons examiné le phénotype des macrophages dans un modèle d’inflammation induite par le thioglycollate. Les résultats montrent que les macrophages CD11b+F480+, recrutés sur le site de l’inflammation, lorsqu’elles sont déficientes en FasL, sur-expriment les gènes anti-inflammatoires comme IL-10, Arg1, CCL17. La caractérisation plus fine de cette population de macrophages a montré que la population responsable de ce phénotype suppressive est F480+CD115+IL-4R+. Chez les souris Fasl KO, le pourcentage des macrophages F480+CD115+IL-4R+ est significativement augmenté en comparaison avec les souris contrôles. L’analyse fonctionnelle de cette population CD115+ a montré que ces cellules, inhibent la prolifération et la production d’IFN- des cellules T activées. Ces caractéristiques fonctionnelles sont en faveur d’un phénotype anti-inflammatoire de ces macrophages, qui lorsqu’ils sont déficients en FasL, leur recrutement sur le site de l’inflammation est plus important.L’ensemble de ces résultats suggère que l’expression de FasL sur les cellules myéloïdes pourrait jouer un rôle dans leur polarisation vers un phénotype pro inflammatoire. Ainsi, ce travail pourrait apporter de nouvelles approches de levée de l’immunosuppression pour une immunothérapie efficace. / Fas-FasL pathway is the major pathway of apoptosis, the role of which is essential for the homeostasis of hematopoietic cells and peripheral tolerance. The project of my dissertation is to investigate the role of FasL in anti-tumor response, particulary the role of its expression on myeloid cells i.e., macrophages and myeloid derived suppressor cells. Fasl KO mice are characterized by an accumulation of different populations of hematopoietic cells in the peripheral lymphoid organs. However, they do not develop spontaneous tumors. Interestingly, our results show that when they are transplanted by tumor cells, their survival rate is significantly reduced in comparison to control mice, suggesting the implication of FasL in the anti-tumor response. Detailed characterization of the distribution of myeloid cells in Fasl KO mice injected with tumor cells shows a differential distribution of the sub populations of Gr1+ cells, an M-MDSC accumulation in the spleen. Furthermore, the enrichment of tumor infiltrate by suppressive macrophages in Fasl KO mice was observed. These macrophages, regardless of genotype, have a high arginase and iNOS activity and they inhibit the proliferation of T cells in vitro. Thus, the higher mortality in Fasl KO mice could in part be explained by the enrichment of tumor infiltrate by TAM in mice that are FasL deficient.To determine whether the accumulation of FasL deficient immunosuppressive myeloid cells is specific to a tumor environment or is the reflection of an inflammatory condition, we examined the phenotype of macrophages in an experimental inflammation induced by thioglycollate. The results show that CD11b + F480 + macrophages, which are recruited to the site of inflammation, when they are FasL deficient, they upregulate anti-inflammatory genes such as IL-10, Arg1, CCL17. More detailed characterization of this population of macrophages shows that the population responsible for the suppressor phenotype is F480+CD115+IL4R+. In Fasl KO mice, the percentage of F480+CD115+IL4R+ macrophages is significantly increased compared with control mice. Functional analysis of CD115+ population shows that they inhibit proliferation of activated T cells and their IFN-g production. These functional characteristics favor an anti-inflammatory phenotype of these macrophages, suggesting that when deficient in FasL, their recruitment to the site of inflammation is more important.Taken together, these results suggest that the expression of FasL on myeloid cells plays a significant role in their bias towards a pro inflammatory phenotype pointing toward a new class of approaches to raising immunosuppression for more effective immunotherapy.

Os linfócitos T CD4+ orquestram a resposta imune adaptativa, auxiliando os macrófagos, os linfócitos T CD8+ e os linfócitos B na resposta mais eficiente frente a um antígeno. As etapas que caracterizam uma resposta imune adaptativa são: apresentação do antígeno, ativação e diferenciação dos linfócitos T, expansão clonal e morte celular para retorno à homeostasia via AICD. Este tipo de morte ocorre via FAS/FASL. Tendo em vista que o mecanismo pelo qual a PGE2 inibe a expressão de fasl ainda não é conhecido, o presente trabalho tem como objetivo compreender o mecanismo molecular de atuação da PGE2 sobre os linfócitos T CD4+ na inibição gênica do FASL, tendo como hipótese o envolvimento do repressor transcricional ICER. Foi verificado que PGE2 10-8 M é capaz de proteger as DO11.10 da AICD e que, nesta concentração, ela induz a expressão de icer. Este repressor apresenta uma expressão transiente e observa-se seu aumento concomitantemente à inibição da expressão de fasl. Sendo assim, sugere-se a participação de ICER na via de inibição do fasl pela PGE2. / CD4+ T lymphocytes orchestrate the adaptive immune response, helping the macrophages, CD8+ T lymphocytes and B lymphocytes to reach an efficient antigen-specific immune response. The adaptive immune response is characterized by different phases: antigen, T lymphocyte activation and differentiation, clonal expansion and, finally, clonal cell death to return to homeostasis via AICD. This cell death occurs via FAS/FASL. Since the mechanism by which PGE2 inhibits fasl expression is not known, our aim is to understand the molecular mechanism responsible for PGE2 inhibition of fasl in CD4+ lymphocytes. Our hypothesis is that the transcriptional repressor ICER, which binds to CRE sites on gene promoters, is involved in this process. We verified that PGE2 10-8 M protects DO11.10 cells from AICD, and in that concentration, PGE2 increases icer expression. This repressor has a transient expression and we observed that its expression is increased in the same time that fasl expression is inhibited. Thus, we suggest the involvement of ICER in fasl inhibition pathway by PGE2.

The mechanisms responsible for myocardial dysfunction in the setting of sepsis remain undefined. Fas ligation with its cognate ligand (FasL) induces apoptosis and activates cellular inflammatory responses associated with tissue injury. We determined whether interruption of Fas/FasL interaction by cardiac-specific expression of soluble Fas (sFas), a competitive inhibitor of FasL, would improve myocardial dysfunction and inflammation in a lipopolysacharide (LPS)-induced mouse model of sepsis. Wild-type (WT) and sFas transgenic mice were injected intraperitoneally with 10 mg/kg LPS or with an equivalent volume of saline. At 18 h after LPS administration, echocardiographic evaluation revealed a significant decrease in left ventricular fractional shortening in the WT mice, whereas the fractional shortening was preserved in the sFas mice. Activation of nuclear factor-kappa B (NF-κB) and the increase in the transcript levels of proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 resulting from LPS treatment were attenuated in the myocardium of sFas mice. sFas expression also inhibited LPS-induced upregulation of Toll-like receptor 4 (TLR-4) and inducible nitric oxide synthase (iNOS), and formation of peroxynitrite in the myocardium. LPS-induced increase in caspase-3/7 activity and apoptotic cell death were suppressed in sFas mice compared with WT mice. LPS-induced lung injury and increase in lung water content were also significantly reduced in sFas mice. These data indicate that neutralization of FasL by expression of sFas significantly preserves cardiac function and reduces inflammatory responses in the heart, suggesting that Fas/FasL signaling pathway is important in mediating the deleterious effects of LPS on myocardial function.

Tumor-distal immune suppression promotes tumor growth by preventing the recruitment of leukocytes to the tumor-proximal microenvironment. Tumor necrosis factor (TNF)-a is both secreted by and expressed on the cell-surface (mTNF-a) of macrophages. When stimulated with LPS, tumor-burdened host (TBH) macrophages secrete more TNF-a than normal host (NH) macrophages. In this study, I showed that mTNF-a is elevated both in freshly isolated and stimulated TBH macrophages. Additionally, I analyzed the expression of Fas and FasL on freshly isolated and LPS-stimulated macrophages and found no differences between TBH and NH macrophages. Fas and Fas ligand (FasL) cell-surface expression was analyzed on NH and TBH T-cells. While no difference was observed in freshly isolated cells, cell-surface expression of both proteins remained higher in TBH T-cells than NH T-cells after mitogenic stimulation. Fas and FasL analysis was also extended to the MethKDE fibrosarcoma and I found that these tumor cells express high levels of FasL. Because past observations show increased TNF-a mRNA expression in TBH macrophages relative to NH macrophages, I hypothesized that NF-kB activation may be increased as well. NF-kB is a transcription factor whose activation is required for TNF-a transcription. I observed increased NF-kB activation in both splenic and peritoneal TBH macrophages. Interestingly, electrophoretic mobility shift analysis (EMSA) suggests that different species of NF-kB were found in each distinct population of macrophages. Together, these data demonstrate that cell-surface tumoricidal molecules and NF-kB are dysregulated in the tumor-burdened host. / Master of Science

Após a resposta imune, a expansão dos linfócitos T CD4 é seguida de uma fase de retração chamada Morte Celular induzida por Ativação (AICD), para que a homeostasia seja reestabelecida. Nosso grupo demonstrou que DCs estimuladas com LPS produzem PGE2 que inibe a expressão de FASL e bloqueia a AICD dos linfócitos T CD4. Nossa hipótese é que a apresentação de antígenos em contexto de infecção tenha um impacto na expressão de FASL e na sobrevivência das células T CD4, de maneira dependente de TLRPGE2. Para comprovar nossa hipótese nós estudamos a apresentação de OVA in vitro e in vivo. Observamos que a adição de LPS durante a apresentação de OVA aumenta a ativação e proliferação das células T CD4 específicas. O pré-tratamento dos camundongos com Indometacina, um inibidor da enzima COX, reduz a frequência das células específicas através do aumento na expressão de FASL e da apoptose, mas sem interferir com a proliferação. Nós sugerimos que a PGE2 produzida em resposta ao LPS regule a sobrevivência dos linfócitos T CD4 durante a persistência do estímulo antigênico. / After immune response, expansion of antigen-specific CD4 T cells is followed by a contraction phase due to Activation-Induced Cell Death (AICD) to reestablish homeostasis. Our group demonstrated that LPS stimulated-DCs produce PGE2 that protects CD4 T cells from AICD by preventing TCR/CD3-mediated FASL upregulation. Our hypothesis is that antigen presentation in the context of infection impacts on FASL expression and survival of CD4 T cells, dependently on TLR-mediated PGE2 release. To approach our hypothesis we studied OVA presentation in vitro and in vivo. We observed that the addition of LPS during OVA presentation increased specific CD4+ T cells activation and proliferation. Pretreatment of mice with indomethacin, an inhibitor of COX enzyme, reduces the frequency of specific T cells by increasing FASL expression and apoptosis, but did not interfere with proliferation. We suggest that PGE2 produced in response to LPS regulates the survival of CD4 T lymphocytes during persistent antigen stimulation.

Após a resposta imune, a expansão dos linfócitos T CD4 é seguida de uma fase de retração chamada Morte Celular induzida por Ativação (AICD), para que a homeostasia seja reestabelecida. Nosso grupo demonstrou que DCs estimuladas com LPS produzem PGE2 que inibe a expressão de FASL e bloqueia a AICD dos linfócitos T CD4. Nossa hipótese é que a apresentação de antígenos em contexto de infecção tenha um impacto na expressão de FASL e na sobrevivência das células T CD4, de maneira dependente de TLRPGE2. Para comprovar nossa hipótese nós estudamos a apresentação de OVA in vitro e in vivo. Observamos que a adição de LPS durante a apresentação de OVA aumenta a ativação e proliferação das células T CD4 específicas. O pré-tratamento dos camundongos com Indometacina, um inibidor da enzima COX, reduz a frequência das células específicas através do aumento na expressão de FASL e da apoptose, mas sem interferir com a proliferação. Nós sugerimos que a PGE2 produzida em resposta ao LPS regule a sobrevivência dos linfócitos T CD4 durante a persistência do estímulo antigênico. / After immune response, expansion of antigen-specific CD4 T cells is followed by a contraction phase due to Activation-Induced Cell Death (AICD) to reestablish homeostasis. Our group demonstrated that LPS stimulated-DCs produce PGE2 that protects CD4 T cells from AICD by preventing TCR/CD3-mediated FASL upregulation. Our hypothesis is that antigen presentation in the context of infection impacts on FASL expression and survival of CD4 T cells, dependently on TLR-mediated PGE2 release. To approach our hypothesis we studied OVA presentation in vitro and in vivo. We observed that the addition of LPS during OVA presentation increased specific CD4+ T cells activation and proliferation. Pretreatment of mice with indomethacin, an inhibitor of COX enzyme, reduces the frequency of specific T cells by increasing FASL expression and apoptosis, but did not interfere with proliferation. We suggest that PGE2 produced in response to LPS regulates the survival of CD4 T lymphocytes during persistent antigen stimulation.

Leukemia is a hematological neoplasm with abnormal genetic mutation or chromosomal translocation in the myeloblast or lymphoblast, and characterized by accumulation of immature cells and malfunction of lymphocytes and myeloid-derived cells. The prognosis of treatment depends on genetic mutation, chromosomal aberration, disease progression and age of patients. Currently, bone marrow transplantation is a useful therapeutic strategy, but the success in therapy is limited by the bone marrow of donors and life-threatening events such as immune repulsion. Although chemotherapy improves leukemia treatment, long-term chemotherapy usually leads to the production of drug-resistant cancer cells. Thus, the development of new modality in overcoming drug-resistant should be beneficial for in leukemia therapy. In this thesis, Naja nigricollis toxin £^, piceatannol, caffeine, and Bungarus multicinctus protease inhibitor-like protein 1 (PILP-1) are employed to investigate the molecular mechanisms in regulating apoptosis and invasion of leukemic cell lines K562 and U937. Hopefully, the signaling pathways elicited by these treatments may be aid in identifying new targets in treating leukemia. Toxin £^ inducing cell death is found to evoke p38 MAPK-mediated Bcl-2 down-regulation, which facilitates mitochondria dysfunction, ROS generation and cytiochrome c release. Finally, activation of caspases leads to apoptotic death of toxin £^-treated cells. Piceatannol elicits Ca2+/p38£\ MAPK- mediated c-Jun and ATF-2 phosphorylation, leading to up-regulation of Fas/FasL protein expression and autocrine Fas-mediated death pathway activation. Caffeine treatment down-regulates MMP-2/-9 down-regulation via Ca2+/ROS-mediated inactivation of ERK/c-Fos and activation of p38 MAPK/c-Jun pathway. Consequently, caffeine treatment suppresses invasion of leukemia cells. PILP-1-induced ADAM17 down-regulation suppresses Lyn-mediated Akt phosphorylation, resulting in death of PILP-1-treated leukemia cells. Taken together, the results of the present study elucidate the signaling pathways responsible for apoptosis and invasion of leukemia cells. Moreover, these findings might suggest new targets in developing therapeutic strategy in treating leukemia.

Ma thèse porte sur l'étude des rôles régulateurs des GTPases Rho et de leurs effecteurs ROCK sur l'expression de ligands du système immunitaire, sur des cellules de mélanomes murins et humains ainsi que les conséquences sur le développement tumoral de modulateurs de la voie RhoA/ROCK. A l'heure actuelle, les traitements du mélanome métastatique ont une efficacité limitée, c'est pourquoi les nouvelles stratégies s'orientent vers l'immunothérapie notamment en recherchant de nouvelles molécules pharmacologiques capables d'amplifier les réponses immunes anti mélanome. Mon travail a porté sur l'étude de trois ligands de l'immunité modulés par la voie RhoA/ROCK : - Nous avons étudié la régulation du ligand MICA qui est exprimé sur des mélanomes humains, mais qui est reconnu par les cellules NK humaines et murines du système immun inné. En utilisant des statines, qui sont des inhibiteurs de l'activité des Rho, nous avons induit une surexpression membranaire de MICA sans toxicité cellulaire. Cette surexpression s'accompagne d'une sensibilisation des mélanomes à la lyse par les cellules NK. Elle induit également un ralentissement de leur croissance tumorale sous-cutanée en souris NMRI nu/nu et une diminution de l'implantation des métastases pulmonaires. Nous avons aussi montré que cette régulation de MICA induite par les statines ne dépendait pas de l'inhibition des GTPases Ras ou Rho mais de la voie de PPAR?. - Nous avons ensuite étudié la régulation de la molécule de costimulation CD70 par les GTPases Rho sur des mélanomes humains et son rôle dans ces tumeurs. Nous avons montré que les mélanomes primitifs expriment CD70, que cette expression diminue au cours de la maladie et que la GTPase RhoA et la voie des MAPK contrôlent positivement l'expression de CD70 sur nos lignées de mélanome humain. De façon surprenante, nous avons aussi montré que CD70 possède une fonction non immunologique dans ces tumeurs. En effet, la trimérisation de CD70 favorise l'invasion tumorale et l'apparition de métastases en activant la voie de signalisation BRAF/MEK/ERK/RhoE et en inhibant les fibres de stress d'actine et des points focaux d'adhésion. - Enfin, nous nous sommes intéressés aux conséquences de la modulation de FasL sur le développement tumoral du mélanome murin B16F10. Des travaux précédents de l'équipe ont montré que la protéine RhoA et ses effecteurs ROCK régulent de façon négative l'expression de FasL à la membrane des cellules B16F10. Nous avons étudié le rôle in vivo de la surexpression de FasL induite par l'inhibition de ROCK par le H1152. Nous avons mis en évidence un ralentissement de la croissance tumorale in vivo chez les souris immunocompétentes. Ce contrôle du développement tumoral est dépendant de la voie Fas/FasL et de l'activité des lymphocytes TCD8+ et de l'IFN-?. De plus, l'inhibition de ROCK réduit le nombre de métastases pulmonaires sans intervention de la réponse immune adaptative. L'ensemble de mes travaux montre que le ciblage de la voie des GTPases Rho et de leurs effecteurs ROCK constitue une approche nouvelle pour amplifier les réponses immunes protectrices innées et adaptatives anti mélanome, suggérant que des inhibiteurs de cette voie pourraient être envisagés dans de nouveaux protocoles d'immunothérapie du mélanome / My thesis focuses on the study of the regulatory roles of Rho GTPases and their effectors ROCK on the expression of immune system ligands in murine and human melanoma cell lines and the impact on tumor development of modulators of the RhoA/ROCK pathway. Current therapies for metastatic melanoma have poor efficiency. It is the reason why new immunotherapeutic strategies are developed for to find new pharmacological molecules that could improve anti-melanoma immune responses. My work is based on the study of three immune ligands: - We studied the regulation by Rho GTPases of MICA ligand expression in human melanoma cell lines and their recognition by NK cells. Using statins, inhibitors of Rho GTPases activity, we have induced MICA over-expression without any cell toxicity. This MICA over-expression enhanced melanoma cells sensitivity to NK cells lysis, then reduced subcutaneous tumor growth in NMRI nu/nu mice and also decreased pulmonary metastases implantation. We also showed that statins-induced MICA over-expression was not linked to Ras or Rho GTPases inhibition but to PPAR? pathway. - Then, we studied the expression and the function of a co-stimulatory molecule, CD70, and its regulation by the Rho pathway in human melanomas. We demonstrated that the RhoA GTPase and MAPK pathway positively regulate CD70 expression in our melanoma cell lines. Surprisingly, we observed a non-immunological function of CD70 in melanoma. Indeed, CD70 trimerization enhanced melanomas invasion and metastatic capacities through an activation of BRAF/MEK/ERK/RhoE pathway, which inhibited stress fibers and focal adhesions. - Finally, we analyzed the consequences of FasL over-expression on B16F10 murine melanoma development in vivo. Our previous studies have showed that RhoA/ROCK pathway negatively regulates membrane FasL expression on B16F10 cells. We studied in vivo the role of this FasL over-expression induced by ROCK inhibitor H1152, on melanoma cells. We showed tumor growth shrinkage in immunocompetent mice, when B16F10 cells were pretreated with H1152. The Fas/FasL pathway and the activity of TCD8+ cells and IFN- ? induced this tumor slowing down. Moreover, ROCK inhibition induced a reduction of pulmonary metastases implantation independently of T lymphocytes response. Altogether, my work showed that targeting Rho GTPases/ROCK pathway could be interesting in order to improve innate and adaptative anti melanoma immune responses, suggesting that inhibitors of this pathway could be envisaged in new melanoma immunotherapy protocols