Immunotherapies, specifically checkpoint inhibitors blocking PD ligand-1 (PD-L1) and its inhibitory receptor PD-1, lead to durable responses in a minority of patients with platinum-refractory and platinum-naïve bladder transitional cell carcinoma (TCC), and improved survival in the platinum-refractory setting. However, the features that predict responses, and the immune populations in TCC that are responsible for benefit with anti-PD-1/PD-L1, remain obscure. This proposal addresses the novel hypothesis that anti-PD-L1 modulates the activity of regulatory T cells (Tregs), which we and others have described in TCC, and addresses the following Specific Aims: 1. Does anti-PD-L1 therapy reduce the abundance of Tregs, including in specific spatial microenvironments associated with TCC tumors? 2. Does anti-PD-L1 therapy change the antigenic repertoire of Tregs in TCC? These Aims will be addressed using samples, specifically pre-treatment biopsies and immunotherapy-treated cystectomy specimens, from patients with localized TCC treated with neoadjuvant atezolizumab (anti-PD-L1) before cystectomy as part of an ongoing clinical trial, as well as patients from a companion cohort of non-immunotherapy-treated TCC patients (standard of care). Aim 1 addresses effects of anti-PD-L1 on Treg number and distribution via flow cytometric and immunohistochemical comparison of atezolizumab-treated cystectomies and either paired pre-treatment biopsies or standard of care cystectomies. Aim 2 uses T cell receptor (TCR) sequencing of Tregs from atezolizumab-treated and standard of care cystectomies as well as paired pre-treatment biopsies, to understand how anti-PD-L1 modulates Treg TCR repertoire diversity as well as the frequency of specific Treg clonotypes. This will reveal novel roles for Tregs in responsiveness to anti-PD-L1 in TCC, leading to avenues for therapeutic targeting of Tregs or enhancement of immune responses to specific tumor antigens that are suppressed by Tregs.