This study investigates the role of CD4+ regulatory T (Treg) cells in the immunosuppressive microenvironment of prostate cancer (PCa) and its implications for prognosis and therapy prediction. The authors used single-cell and bulk RNA sequencing to analyze 56,924 cells from 14 samples with varying Gleason scores. They identified Treg cells and Th17 cells as key players in shaping the tumor microenvironment (TME). The ratio of Treg to Th17 cells was positively correlated with Gleason groups, indicating a poor prognosis. Multiplex immunofluorescence confirmed higher Treg cell infiltration in high-risk PCa samples. Trajectory analysis revealed distinct differentiation paths for Treg and Th17 cells, with Treg cells showing higher expression of genes involved in negative regulation of the immune system. Patients were classified into two phenotypes: rich-Treg PCa (TregR) and poor-Treg PCa (TregP), based on their Treg activity scores. TregR exhibited poorer prognosis, higher TGF-β expression, and mutations in TP53 and PIK3CA, which contributed to resistance to anti-PD-1 therapy and chemotherapy. The study suggests that combining immunotherapy with TGF-β inhibition may improve treatment outcomes in TregR PCa.This study investigates the role of CD4+ regulatory T (Treg) cells in the immunosuppressive microenvironment of prostate cancer (PCa) and its implications for prognosis and therapy prediction. The authors used single-cell and bulk RNA sequencing to analyze 56,924 cells from 14 samples with varying Gleason scores. They identified Treg cells and Th17 cells as key players in shaping the tumor microenvironment (TME). The ratio of Treg to Th17 cells was positively correlated with Gleason groups, indicating a poor prognosis. Multiplex immunofluorescence confirmed higher Treg cell infiltration in high-risk PCa samples. Trajectory analysis revealed distinct differentiation paths for Treg and Th17 cells, with Treg cells showing higher expression of genes involved in negative regulation of the immune system. Patients were classified into two phenotypes: rich-Treg PCa (TregR) and poor-Treg PCa (TregP), based on their Treg activity scores. TregR exhibited poorer prognosis, higher TGF-β expression, and mutations in TP53 and PIK3CA, which contributed to resistance to anti-PD-1 therapy and chemotherapy. The study suggests that combining immunotherapy with TGF-β inhibition may improve treatment outcomes in TregR PCa.