Despite the advances in systemic immunotherapy with immune checkpoint blockade, only a small fraction of patients with bladder cancer respond to this treatment. This highlights a need to better understand drug resistance and identify rational immunotherapeutic combinations. This thesis explores the immune checkpoint landscape of the bladder tumour microenvironment (TME) in three main groups of patients with bladder cancer: non-muscle invasive bladder cancer (NMIBC), muscle invasive bladder cancer (MIBC), and metastatic urothelial bladder cancer. The immune checkpoint landscape between NMIBC and MIBC in the bladder TME was very similar, but distinct to peripheral blood. Interestingly, I observed the presence of urine-derived lymphocytes (UDLs) in NMIBC as previously described, but also in MIBC. We identified UDLs as a readily accessible source of T cells in 32 patients with MIBC, and demonstrated that UDLs have a remarkably similar immunological T cell phenotype and TCR repertoire to tumour infiltrating lymphocytes (TILs) in patients with MIBC. Interestingly, patients with MIBC who were found to have high urinary lymphocytes, at the time of cystectomy, in particularly high expression of PD-1 (PD-1hi) on CD8+, had a reduced recurrence free survival. Furthermore, I developed a lab protocol for the in vitro expansion of cryopreserved UDLs. Expanded UDLs produced IFNg and TNFa when restimulated with autologous tumour samples. In addition, with collaborative efforts, we explored the antigenic specificity of T cells and observed the presence of neoantigen reactive T cells (NARTs) in the peripheral blood of a single patient. Finally, in nine patients with metastatic bladder cancer, I showed a poor prognostic association in patients with high UDL CD3+ count and an exhaustive phenotype of CD8+ PD-1hi UDLs as detected at the end of treatment, which could also be tracked longitudinally in metastatic patients undergoing systemic therapy. Taken together, the data suggest a role for UDLs as an immune biomarker to track response to systemic therapy. However, this requires a large prospective study to validate these findings. Crucially, the data above highlight the importance of incorporating UDL analysis in all future studies with a view to integrating into routine clinical practice in patients with bladder cancer
