Bladder cancer is the most expensive cancer per capita to treat in the US. Non-muscle invasive bladder cancer (NMIBC) which accounts for 70-75% of all newly diagnosed tumors and has only a single FDA approved first- line treatment option, Bacillus Calmette-Guérin (BCG). BCG has been the only approved first-line therapy for intermediate and high-risk NMIBC for more than 40 years. While BCG can induce durable responses, ~50% of patients have recurrence or progression of their disease. The current dosing of BCG is arbitrary with no data currently available to support the dosage, strain nor regimen. Furthermore, no clear mechanism of action behind BCG’s anti-neoplastic activity in NMIBC has been delineated. Better understanding of the immune response initiated by exposure to BCG and how such response is sustained over many repeated doses is essential to help improve its therapeutic action. Further, it may lead to discovery of predictive biomarkers and novel targets leading to advance care for patients, reduce the financial burden associated with the disease, and improve access to BCG. Our preliminary data have identified a significant Type-1 cytokine response to BCG in all patients inducing an activation and recruitment of IFN-g producing NKG2A+ natural killer (NK) cells and NKG2A+PD-1+ CD8 T cells to the tumor microenvironment (TME). Our preliminary analyses of post-BCG-treated specimens demonstrate IFN-g signaling as the most upregulated signature in BCG resistant specimens and strongly correlates with increased HLA-E and PD-L1 expression on the recurring tumors. Monalizumab (anti-NKG2A) is a novel therapeutic target and is currently in clinical trials across several cancer types. A role for NKG2A/HLA-E alone or in conjunction with PD-1/PD-L1 in regulating NK and CD8 T cells remains elusive with virtually nothing known in bladder cancer. Our hypothesis is that chronic activation from repeated exposures to BCG drives immune dysregulation followed by functional exhaustion in a significant proportion of patients, which then leads to disease recurrence or progression. The immune dysregulation in the TME is driven by NKG2A/PD-1 on CD8 T cells, NKG2A on NK cells and HLA-E and PD-L1 on tumor cells. To further validate our findings and better understand BCG mechanisms we will study the following aims: Aim 1: To analyze intratumoral NK cells and T cells and their interactions with tumors in response to BCG therapy. We will test the hypothesis that upregulated expression of HLA-E and PD-L1 on recurring tumors are directly in contact with infiltrating NKG2A+ NK and NKG2A+ PD-1+ CD8 T cells. Aim 2: To longitudinally assess blood and tumor phenotypes and functions in response to BCG therapy. We will test the hypothesis that repeated exposures to BCG drives prolonged activation and dysregulation of NKG2A+ NK and NKG2A+ PD-1+ CD8 T cells. Aim 3: To determine the effects of combination PD-L1 and NKG2A blockade on anti-tumor immunity. We will test the hypothesis th...