New genetic models and mesenchymal cells as tools to ameliorate anti-tumor immunity

Resume Le but final de ce projet est d'utiliser des cellules T ou des cellules souches mesenchymateuses modifiees genetiquement afin de surexprimer localement les deux chemokines CXCL13 et CCL2 ensemble ou chacune separement a l'interieur d'une tumeur solide. CXCL13 est suppose induire des structures lymphoides ectopiques. Un niveau eleve de CCL2 est presume initier une inflammation aigue. La combinaison des deux effets amene a un nouveau modele d'etude des mecanismes regulateur de la tolerance peripherique et de l'immunite tumorale. Les connaissances acquises grâce a ce modele pourraient permettre le developpement ou l'amelioration des therapies immunes du cancer. Le but premier de ce travail a ete l'etablissement d'un modele genetique de la souris permettant d'exprimer specifiquement dans la tumeur les deux chemokines d'interet a des niveaux eleves. Pour accomplir cette tâche, qui est en fait une therapie genetique de tumeurs solides, deux types de cellules porteuses potentielles ont ete evaluees. Des cellules CD8+ T et des cellules mesenchymateuses de la moelle osseuse transferees dans des receveurs portant une tumeur. Si on pouvait repondre aux besoins de la therapie genetique, independamment de la therapie immune envisagee, on possederait la un outil precieux pour bien d'autres approches therapeutiques. Plusieurs lignees de souris transgeniques ont ete generees comme source de cellules CD8+ T modifiees afin d'exprimer les chemokines d'interet. Dans une approche doublement transgenique les proprietes de deux promoteurs specifiques de cellules T ont ete combinees en utilisant la technologie Cre-loxP. Le promoteur de granzyme B confere une dependance d'activation et le promoteur distal de lck assure une forte expression constitutive des que les cellules CD8+ T ont ete activees. Les transgenes construits ont montre une bonne performance in vivo et des souris qui expriment CCL2 dans des cellules CD8+ T activees ont ete obtenues. Ces cellules peuvent maintenant etre utilisees avec differents protocoles pour transferer des cellules T cytotoxiques (CTL) dans des receveurs porteur d'une tumeur, permettant ainsi d'evaluer leur capacite en tant que porteuse de chemokine d'infiltrer la tumeur. L'etablissement de souris transgeniques, qui expriment pareillement CXCL13 est prevu dans un avenir proche. L'evaluation de cellules mesenchymateuses de la moelle osseuse a demontre que ces cellules se greffent efficacement dans le stroma tumoral suite a la co-injection avec des cellules tumorales. Cela represente un outil precieux pour la recherche, vu qu'il permet d'introduire des cellules manipulees dans un modele tumoral. Les resultats confirment partiellement d'autres resultats rapportes dans un modele ameliore. Cependant, l'efficacite et la specificite suggerees de la migration systemique de cellules mesenchymateuses de la moelle osseuse dans une tumeur n'ont pas ete observees dans notre modele, ce qui indique, que ces cellules ne se pretent pas a une utilisation therapeutique. Un autre resultat majeur de ce travail est l'etablissement de cultures de cellules mesenchymateuses de la moelle osseuse in vitro conditionnees par des tumeurs, ce qui a permis a ces cellules de s'etendre plus rapidement en gardant leur capacite de migration et de greffe. Cela offre un autre outil precieux, vu que la culture in vitro est un pas necessaire pour une manipulation therapeutique. Abstract The ultimate aim of the presented project is to use genetically modified T cells or mesenchymal stem cells to locally overexpress the two chemokines CXCL13 and CCL2 together or each one alone inside a solid tumor. CXCL13 is supposed to induce ectopic lymphoid structures and a high level of CCL2 is intended to trigger acute inflamation. The combination of these two effects represents a new model for studying mechanisms that regulate peripheral tolerance and tumor immunity. Gained insights may help developing or improving immunotherapy of cancer. The primary goal of the executed work was the establishment of a genetic mouse model that allows tumor-specific expression of high levels of the two chemokines of interest. For accomplishing this task, which represents gene therapy of solid tumors, two types of potentially useful carrier cells were evaluated. CD8+ T cells and mesenchymal bone marrow cells to be used in adoptive cell transfers into tumor-bearing mice. Irrespectively of the envisaged immunotherapy, satisfaction of so far unmet needs of gene therapy would be a highly valuable tool that may be employed by many other therapeutic approaches, too. Several transgenic mouse lines were generated as a source of CD8+ T cells modified to express the chemokines of interest. In a double transgenic approach the properties of two T cell-specific promoters were combined using Cre-loxP technology. The granzyme B promoter confers activation-dependency and the lck distal promoter assures strong constitutive expression once the CD8+ T cell has been activated. The constructed transgenes showed a good performance in vivo and mice expressing CCL2 in activated CD8+ T cells were obtained. These cells can now be used with different protocols for adoptively transferring cytotoxic T cells (CTL) into tumor-bearing recipients, thus allowing to study their capacity as tumor-infiltrating chemokine carrier. The establishment of transgenic mice likewisely expressing CXCL13 is expected in the near future. In addition, T cells from generated single transgenic mice that have high expression of an EGFP reporter in both CD4+ and CD8+ cells can be easily traced in vivo when setting up adoptive transfer conditions. The evaluation of mesenchymal bone marrow cells demonstrated that these cells can efficiently engraft into tumor stroma upon local coinjection with tumor cells. This represents a valuable tool for research purposes as it allows to introduce manipulated stromal cells into a tumor model. Therefore, the established engraftment model is suited for studying the envisaged immunotherapy. These results confirm to some extend previously reported results in an improved model, however, the suggested systemic tumor homing efficiency and specificity of mesenchymal bone marrow cells was not observed in our model indicating that these cells may not be suited for therapeutic use. Another major result of the presented work is the establishment oftumor-conditioned in vitro culture of mesenchymal bone marrow cells, which allowed to more rapidly expand these cells while maintaining their tumor homing and engrafting capacities. This offers another valuable tool as in vitro culture is a necessary step for therapeutic manipulations.