Étude du rôle du TGF-β dans la différenciation mémoire des lymphocytes T et son implication potentielle pour l'immunothérapie adoptive

Over the last three decades, adoptive immunotherapy (IA) has emerged as a curative therapy for advanced hematologic malignancies and melanoma. However, its effectiveness remains limited to only a small number of patients. The efficacy of AI mainly relies on the ability of the transferred T cells to efficiently traffic and infiltrate the tumor, and to specifically recognize tumor antigens. The transferred T cells should also be able to overcome the immunosuppressive microenvironment established by the tumor to evade immune surveillance and to proliferate and persist long-term in the patient to provide protection against eventual relapse. Many efforts have been done to improve these T-cell properties in order to optimize the effectiveness of this therapy so it can benefit to a larger number of patient. In this line, Our work focuses on the improvement of two parameters in particular; (i) the ability of T lymphocytes (LTs) to engraft an persist long-term in-vivo, which is related to their differentiation state at the time of adoptive transfer (AT) and (ii) their ability to traffic and infiltrate the tumor. In the context of AI, several evidences indicate that T cell differentiation is inversely correlated with their anti-tumor efficacy. Indeed, the process of T cell differentiation results in a loss of their proliferative capacity, self-renewal ability and their multipotency, which reduces their persistence and their ability to induce long-term protection against a possible relapse. Thus, adoptive transfer of poorly differentiated LT, such as Stem cell memory T cells (Tscm) or Central Memory T cells (Tcm) improves the therapeutic efficacy of AI. In the first part of this thesis we have attempted to generate human memory LT ex-vivo to improve the efficiency of AI, using the TGF-β cytokine (Transforming growth factor beta). In T cell biology, TGF-β is mostly known for its immunosuppressive properties. However, we now know that TGF-β is much more than an immunosuppressive cytokine and that it exerts its pleiotropic effects in a context-dependent manner. In particular, recent studies have shown that TGF-β is necessary for the formation and maintenance of memory T cells. In concurrence with this, we demonstrated that exposure of human LTs to TGF-β during their ex-vivo activation phase promoted memory T cell differentiation, associated with phenotypic and transcriptional expression similar to that observed in central memory T cells. Interestingly, TGF-β inhibits the expression of the transcription factor BLIMP1, a master regulator of effector T cell differentiation and function, and inversely increases ID3 expression, a key factor in the generation and maintenance of memory T cells. Moreover, the exposure of LTs to TGF-β did not alter their proliferation or polyfunctionality and did not lead to the generation of regulatory T cells (Treg). On the other hand, adoptive transfer of LT exposed to TGF-β in immunodeficient mice induces a greater in-vivo reactivity which resulted in the faster onset of GVHD (Graft versus host disease) and improved persistence in-vivo. Finally, adoptive transfer of genetically engineered LT to express a chimeric antigen receptor (CAR) exposed to TGF-β in immunodeficient mice revealed improved toxicity and induced a more effective anti-tumor response. Our study revealed a new role for TGF-β in human T cell memory differentiation and demonstrated that TGF-β signaling could be targeted to generate Tcm ex-vivo in the context of AI. TGF-β has also been associated with the formation and maintenance of a newly identified LT population that is permanently resident in non-lymphoid tissues. These tissue-resident T cells (Trm) play a crucial role in protecting epithelial tissues against infectious and inflammatory diseases, as well as in controlling the anti-tumor response. Their presence into the tumors is associated with a good prognosis and a better response to immunotherapies. As a result, adoptive transfer of LT with Trm properties, which can infiltrate and be retained more effectively into the tumors, could improve the efficacy of AI. In the second part of this thesis we sought to determine whether a brief TGF-β exposure during ex-vivo T cell activation for AI may confer a tumor infiltration/retention phenotype to effector T cell within tumors in-vivo and hence improve AI efficacy. Using OT-I TcR transgenic model, we found that exogenous TGF-β favors the expression of the integrin CD103 and the chemokine receptor CXCR3 in ex-vivo stimulated OT-I-T cells in the presence of the cognate antigen. We also found that TGF-β-treated T cells exhibited strong migration to recombinant CXCL9, CXCL10 and CXCL11, the three CXCR3 ligands and to LLC-OVA (Lewis lung carcinoma expressing OVA peptide) supernatants. This migration was dependant on Giα proteins associated receptors in general, and more specifically on CXCR3. Furthermore, using in vivo competitive homing assays, we found that TGF-β improves T-cell trafficking in LLC-OVA tumors. Finally, adoptive transfer of TGF-β-treated T cells into LLC-OVA-bearing mice slightly but significantly delayed tumor growth at early time points, which correlated with higher intratumoral T cell infiltration and maintenance of CD103 expression in vivo. In order to improve the therapeutic efficacy of our strategy, it would be interesting to envisage a combination therapy associating the adoptive transfer of LT-OT-I treated with TGF-β with check-point inhibitors. The synergistic effect of these two therapies may enhance both T cell localization within tumor and citotoxicity thereby enhancing the anti-tumor response and improving the therapeutic effectiveness of AI. Our results have revealed that the use of TGF-β during the ex-vivo activation phase of LT promotes the induction a memory phenotype as well as a migratory and intra-tumor retention potential that can improve the efficacy of AI. We have provide the rational for clinical use of TGF-β for the generation of improved T cells for AI.

Keyphrases: TGF-β, T cell memory differentiation, Adoptive immunotherapy