Age-associated mucosal and systemic immunomodulation by Bacillus Calmette-Guérin immunotherapy in Non-Muscle Invasive Bladder Cancer

Abstract

Non-muscle invasive bladder cancer (NMIBC) constitutes approximately 75% of bladder cancer cases. Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy remains the standard of care for high-risk NMIBC, however clinical outcomes remain suboptimal. This limited efficacy is largely attributed to an incomplete understanding of mucosal and systemic immune responses triggered by repeated BCG administration. Building on prior findings linking high intra-tumoral B cell density with poor BCG response, this study investigates the role of aging and chronic inflammation-induced mucosal immune dysfunction in promoting pre-treatment immune exhaustion and resistance to BCG immunotherapy. Using human tumor specimens and murine models, we showed these factors promote the formation of tertiary lymphoid structures (TLSs) enriched with exhausted immune cells. Immunophenotyping of peri-tumoral TLSs in pre-BCG tumor sections revealed increased expression of immune exhaustion-related markers in non-responders. Given the enrichment of B cells in TLSs and their established role in mucosal immunity, we next examined B cell subsets as potential modulators of the tumor-immune microenvironment (TIME). In a carcinogen-exposed aging murine model, atypical B cells (ABCs)-a subset of exhausted B cells-emerged as key contributors to local and systemic immunosuppression, correlating with disease progression following BCG treatment. B cell depletion prior to BCG in carcinogen-exposed mice promoted urothelial recovery and attenuated tumor development. To translate these finding clinically, longitudinal immune profiling of NMIBC patients receiving BCG was performed systemically and locally. Non-responders exhibited high frequency of circulating and tumor-infiltrating ABCs, alongside increased systemic IL10 and CCL23 with repeated BCG. Spatial immune-phenotyping revealed localization of ABCs within peri-tumoral TLSs in non-responders, implicating them as biomarkers of immune exhaustion. Together, these data underscore the complex interplay between systemic and mucosal immunity in determining BCG response. In patients with a pre-treatment immunosuppressive phenotype, repeated BCG administration fails to reconstitute effective immunity and instead augments immune exhaustion, as evidenced by-but not limited to-ABC expansion. This study provides the first evidence linking ABC-mediated mucosal immune exhaustion to BCG failure, identifying ABCs as biomarkers and drivers of therapeutic resistance. These findings offer a novel therapeutic axis and support the need for pre-treatment immune profiling to enable better patient stratification and early intervention in high-risk NMIBC.