Combinatorial immunotherapy drives exhaustion in tumor antigen-specific CD8+ T cells within the mouse renal tumor...

Immunotherapies have greatly improved outcomes for patients with renal cell carcinoma (RCC), yet response rates remain suboptimal and the factors promoting therapy resistance versus sensitivity are incompletely understood. Currently, no preclinical model of orthotopic renal cancer exists that permits evaluation of tumor antigen-specific (TAS) CD8+ tumor-infiltrating lymphocytes (TILs). To address this deficiency, we developed a mouse renal cancer model that permits tracking of adoptively transferred TAS CD8+ T cells. Renca-LUC tumor cells were transduced to express tumor ERK (tERK), a model antigen expressing a one-amino-acid change from wild-type ERK, resulting in recognition by tERK/H-2Kd-specific DUC Thy1.1 TCR transgenic CD8+ T cells. Renca-tERK-LUC challenge into DUC Thy1.1 mice results in rapid tumor clearance. To assess intratumoral TAS T-cell responses, we adoptively transferred limited numbers of DUC Thy1.1 T cells into mice with established renal tumors, followed by clinically relevant anti-PD-1 + anti-VEGFR-2 combinatorial immunotherapy. We used standard flow cytometry gating as well as unbiased Leiden clustering of stained cells to assess TIL phenotypes and prevalence. Therapy responders showed intratumoral gene expression changes reflective of those seen in human RCC responders to therapy, and had increased frequencies of activated and exhausted CD8+ TILs, activated CD4+ TILs, and NKp46+ natural killer cells. Examination of CD8+ TILs revealed unique aspects of the TAS response characterized by reduced phenotypic cluster heterogeneity and heightened levels of PD-1+CD39+CD44+CD8+ TILs versus endogenous CD8+ TILs from the same tumors. Future studies using this model should yield insights into the biology of TAS CD8+ TILs in renal tumors and facilitate the development of novel immunotherapies for patients with RCC.