BCG Induces PD-1 Upregulation on Circulating CD8⁺ T Cells and IL-6 and IL-8 Secretion In Vitro

Backgrounds: Bacillus Calmette-Guérin (BCG) remains the most effective adjuvant therapy for non-muscle-invasive bladder cancer (NMIBC); however, the immunological underpinnings of its efficacy remain incompletely understood. This study aims to elucidate the dual immunomodulatory roles of BCG by integrating systemic and tumor-intrinsic analyses, through determining the systemic effects of BCG instillation on immune checkpoint expression and direct inflammatory response in a previously established in vitro tumor model. Methods: We investigated systemic and tumor-intrinsic immune responses to BCG. Flow cytometry was used to evaluate immune checkpoint expression on circulating lymphocyte subsets in NMIBC patients (n = 7) at various stages of BCG therapy. In parallel, an in vitro model of PD-L1 modulation using breast cancer cell lines (MDA-MB-231 and MCF-7) was stimulated with BCG and TLR agonists, and the secretion of IL-6 and IL-8 was assessed using an ELISA. Results: Peripheral immune profiling revealed stable lymphocyte frequencies, but a significant increase in PD-1 expression on CD8⁺ T cells following BCG exposure (p = 0.0068), with no significant modulation in CTLA-4 levels. In vitro, MDA-MB-231 cells exhibited robust IL-6 secretion upon high-dose BCG stimulation (p = 0.0277), whereas MCF-7 cells showed increased IL-8 release (p < 0.0001). Other TLR agonists had limited effects. Conclusions: BCG induces dual immunomodulation, characterized by PD-1 upregulation in systemic CD8⁺ T cells and the release of pro-inflammatory cytokines from epithelial tumor cells. These findings support the potential of combining BCG with immune checkpoint inhibitors and underscore IL-6 and IL-8 as candidate biomarkers of tumor-intrinsic responsiveness.