Immunotherapy has long been recognized as a promising therapy to fight cancer due to its specificity, its long term effects towing to immunological memory and its targeting of the immune system rather than of the tumor itself. However, control of tumor growth by immunotherapy is hindered by a number of obstacles including the activation of various immunosuppressive mechanisms and the lack of specific tumor antigen, which can strongly activate anti-tumor T cells. Immunotherapy of cancer has gained wide recognition in recent years with the characterization of immune checkpoints involved in the dampening of effective tumor antigen-specific T cell functions. The targeting of these immune checkpoints led to significant and impressive clinical successes. In addition, cancer mutations theoretically represent ideal targets for cancer immunotherapy as they combine a favorable safety due to the lack of their expression in healthy tissues and their capability of high immunogenicity as they are not affected by central tolerance mechanisms. Although, neoantigens are mostly patient-specific, recent advances in next-generation sequencing and computational prediction allowed the rapid and affordable characterization of genetic alterations in tumor and the identification of resulting neoepitopes, paving the road to patient-tailored immunotherapy. Moreover, several studies highlighted that neoepitope-specific T cells may be a key player in the efficacy of immune checkpoint blockade. More investigations are thus needed to understand whether mutational burden, presence of neoepitope-specific T cells and immune checkpoint expression are linked to cancer clinical outcome, particularly in bladder cancer.Bladder cancer remains a public health concern due to its prevalence, high risk of recurrence and associated cost of management. Although Bacillus Calmette-Guérin (BCG) instillations for urothelial cancer therapy is the most successful immunotherapy on a large scale and considered as a standard treatment for this indication, repeated BCG treatments are associated with significant toxicity and failure, underlying the necessity for alternative or complementary immunotherapy and for better understanding of T cell responses generated within bladder mucosa. Thus, the aim of this grant proposal is two-fold:1 - Evaluate in bladder cancer patients the spontaneous and treatment-mediated anti-neoantigen CD8+ T cell repertoire.2 - Comprehensively characterize the expression profile of immune checkpoints by CD8+ neoepitope-specific T cells.Experimental design and methods: Sequencing of non-muscle invasive (NMIBC) and muscle invasive bladder cancer tumors followed by bioinformatics analysis will be performed in order to establish putative neoepitopes. Pool of peptides will be then screened for recognition by autologous peripheral mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL), but also by T cells isolated from urine of NMIBC patients undergoing BCG treatment. Specificity and class-I HLA restriction will be confirmed by neoepitope peptide-MHC multimer complex labeling. Finally, functional characterization (cytokine secretion, proliferation and cytotoxicity) and V repertoire analysis will be performed. In parallel, we will analyze in-depth the expression pattern of 37 immune checkpoint inhibitors by neoepitope-specific T cells using Chipcytometry in PBMC and frozen section of bladder tumors.Overall, we believe that in depth study of neoantigen-specific T cells and immune checkpoints will provide valuable clues to the development of new therapeutic strategies for bladder cancer patients.