PD-1 topographically defines distinct T cell subpopulations in urothelial cell carcinoma of the bladder and predicts patient survival

Introduction: The pre-existing tumor-infiltrating T cell landscape in urothelial cell carcinoma of the bladder (UCB) may obtain prognostic significance and guide treatment decisions, particularly regarding immunotherapy. However, the current studies typically lead to inconsistent conclusions due to the extreme heterogeneity of T cells in cancer. Herein, we investigated the heterogeneity, distribution and clinical significance of tumor-infiltrating T cells based on PD-1 expression, their spatial organization, and the balance between subsets in a series of UCB patients.

Methods: Flow cytometry for PD-1, CD4, and CD8 was performed in 6 UBC patients and 5 healthy donors. A series of 155 UBC patients with tissue slides were stained for triple color immunofluorescence. Stromal and intratumoral regions of the cancer tissue were respectively evaluated. Features derived from triple staining were analyzed for their correlations with clinical characteristics and patient prognosis.

Results: Flow cytometric analysis showed PD-1⁺ T cells were more frequently accumulated at the tumor site than in blood (p < 0.001). The proportion of PD-1⁺ T cells within CD4⁺ and/or CD8⁺ T cells is higher in the intratumoral region, as compared with the stroma by immunofluorescence evaluation (all p < 0.001, n = 155). Moreover, a high proportion of PD-1⁺ T cells within T cells in the intratumoral region, but not in the stroma, was predictive of a poorer overall survival (p = 0.0075) and recurrence-free survival (p = 0.0062), and was positively associated with aggressive clinical features (all p < 0.05). However, a low CD4/CD8 ratio among the PD-1⁺ T cells in the tumor stroma, but not in the intratumoral region, was significantly associated with shorter overall survival (p = 0.0164) and recurrence-free survival (p = 0.0016), which emerged as an independent predictor in multivariate analysis for UCB patients.

Conclusions: Taken together, our results emphasize that PD-1 expression in T cell subsets, based on their topographic micro-localizations, provides valuable prognostic information for UCB patients.