Abstract
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.