Senescent regulatory T cells retain potent suppressive function in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is an aging-associated malignancy characterized by a highly immunosuppressive, immune-rich tumor microenvironment and variable response to immune checkpoint blockade. Regulatory T cells (Tregs) are dominant orchestrators of immune suppression in ccRCC, yet the functional consequences of cellular senescence in these cells remain unknown and are widely presumed to be impairing. Single-cell RNA sequencing of treatment-naïve human ccRCC tumors revealed a distinct tumor-infiltrating Treg (TI-Treg) subpopulation co-expressing classic senescence markers (p16^INK4a, p21), senescence-associated beta-galactosidase activity (SA-beta-gal), BCL-xL, and robust immunosuppressive effector programs. Contrary to the prevailing view that senescence attenuates Treg function, we hypothesized that senescent TI-Tregs retain potent suppressive capacity. To functionally characterize these senescent TI-Treg subsets, we used a novel activity-based fluorescent senescence probe that labels SA-beta-gal and employed p16-tdTomato reporter mice for the isolation of viable senescent TI-Tregs. Additionally, using an in vitro co-culture system in which naïve Tregs are driven into senescence by ccRCC cell-derived factors, suppression assays demonstrated that senescent Tregs maintain strong inhibitory activity against CD4 + T-cell proliferation, comparable to non-senescent Tregs. Concomitant RNA sequencing confirmed persistence of canonical immunosuppressive pathways and acquisition of a senescence-reinforced suppressive transcriptome. These findings challenge the assumptions that senescence Tregs are dysfunctional in cancer and establish senescent Tregs as critical contributors to immunosuppression in aging-associated ccRCC. This work unravels senescent TI-Tregs as therapeutic targets/vulnerabilities for their selective elimination to restore antitumor immunity.