Multiplexed single-cell pathology reveals the association of CD8 T-cell heterogeneity with prognostic outcomes in renal cell carcinoma

Despite the high sensitivity of renal cell carcinoma (RCC) to immunotherapy, RCC has been recognized as an unusual disease in which CD8⁺ T-cell infiltration into the tumor beds is related to a poor prognosis. To approach the inner landscape of immunobiology of RCC, we performed multiplexed seven-color immunohistochemistry (CD8, CD39, PD-1, Foxp3, PD-L1, and pan-cytokeratin AE1/AE3 with DAPI), which revealed the automated single-cell counts and calculations of individual cell-to-cell distances. In total, 186 subjects were included, in which CD39 was used as a marker for distinguishing tumor-specific (CD39⁺) and bystander (CD39⁻) T-cells. Our clear cell RCC cohort also revealed a poor prognosis if the tumor showed increased CD8⁺ T-cell infiltration. Intratumoral CD8⁺CD39⁺ T-cells as well as their exhausted CD8⁺CD39⁺PD-1⁺ T-cells in the central tumor areas enabled the subgrouping of patients according to malignancy. Analysis using specimens post-antiangiogenic treatment revealed a dramatic increase in proliferative Treg fraction Foxp3⁺PD-1⁺ cells, suggesting a potential mechanism of hyperprogressive disease after uses of anti-PD-1 antibody. Our cell-by-cell study platform provided spatial information on tumors, where bystander CD8⁺CD39⁻ T-cells were dominant in the invasive margin areas. We uncovered a potential interaction between CD8⁺CD39⁺PD-1⁺ T-cells and Foxp3⁺PD-1⁺ Treg cells due to cell-to-cell proximity, forming a spatial niche more specialized in immunosuppression under PD-1 blockade. A paradigm shift to the immunosuppressive environment was more obvious in metastatic lesions; rather the infiltration of Foxp3⁺ and Foxp3⁺PD-1⁺ Treg cells was more pronounced. With this multiplexed single-cell pathology technique, we revealed further insight into the immunobiological standing of RCC.