The gold standard immunologic agent for bladder cancer treatment is bacillus Calmette-Guérin (BCG), a live attenuated Mycobacterium. Repeated BCG instillations into bladder significantly reduce recurrence rates and offer an alternative to complete bladder removal. Unfortunately, the relapses occur and when BCG therapy fails, there are few options available. Despite decades of use, the mechanism for BCG efficacy remain largely unknown. Although it is established that T cells are required, the importance of tumor- vs BCG-specific immune response is unknown. Importantly, understanding how BCG augments antigen-specific immunity is critical to developing strategies to avoid BCG failure. We hypothesize that antigen-specific immunity contributes to clinical activity of BCG in bladder cancer. This hypothesis will be tested in the following aims that incorporate mouse models and patient samples to explore mechanisms underlying immune and clinical responses. Aim 1. Test the hypothesis that tumor-specific immunity contributes to BCG efficacy in bladder cancer. We will test effects of BCG on orthotopic bladder tumor-bearing mice by adoptive transfer of naïve vs tumor-specific T cells that we have developed. The effect of BCG on tumor-specific T cells will be corroborated using patient PBMCs before and after BCG treatment. Aim 2. Test the hypothesis that BCG-specific immunity contributes to BCG efficacy in bladder cancer. Similar to Aim 1, we will test BCG-specific T cells in bladder tumor-bearing mice treated with BCG. We will also enrich and transfer human BCG-specific T cells into human-tumor-challenged immune-deficient mice followed by intravesical BCG treatment. Summary: These studies will provide mechanistic insights into BCG efficacy in treating bladder cancer, and help establishing a framework for rapid development of BCG combination strategies for individual patients.