PD-1 and LAG-3 Dominate Checkpoint Receptor–Mediated T-cell Inhibition in Renal Cell Carcinoma

Drugs targeting the programmed cell death protein 1 (PD-1) pathway are approved as therapies for an increasing number of cancer entities, including renal cell carcinoma. Despite a significant increase in overall survival, most treated patients do not show durable clinical responses. A combination of checkpoint inhibitors could provide a promising improvement. The aim of the study was to determine the most promising checkpoint blockade combination for renal cell carcinoma patients. Tumor-infiltrating lymphocytes (TIL) and autologous peripheral blood mononuclear cells (PBMC) were isolated from patients undergoing surgery for primary tumors. Cells were stained for multicolor flow cytometry to determine the (co)expression of five inhibitory receptors (iR), PD-1, LAG-3, Tim-3, BTLA, and CTLA-4, on T-cell populations. The function of these TILs was assessed by intracellular cytokine staining after in vitro stimulation in the presence or absence of PD-1 ± LAG-3 or Tim-3-specific antibodies. Although the percentage of iR+ T cells was low in PBMCs, both CD4+ and CD8+ T cells showed increased frequencies of PD-1+, LAG-3+, and Tim-3+ cells on TILs. The most frequent iR combination was PD-1 and LAG-3 on both CD4+ and CD8+ TILs. Blockade of PD-1 resulted in significant LAG-3, but not Tim-3, upregulation. The dual blockade of PD-1 and LAG-3, but not PD-1 and Tim-3, led to increased IFNγ release upon in vitro stimulation. Together, these data suggest that dual blockade of PD-1 and LAG-3 is a promising checkpoint blockade combination for renal cell carcinoma.