Investigating mechanisms of resistance to immune checkpoint inhibitors (ICIs) in renal cell carcinoma (RCC).

4545 Background: Though RCC often exhibits substantial CD8 T-cell infiltration, CD8 abundance does not predict clinical benefit from ICIs. We sought to elucidate tumor microenvironment features that uncouple CD8 presence from ICI response and to evaluate strategies to overcome resistance mechanisms. Methods: We performed single-cell RNA sequencing on 70 RCC tumors (59 clear-cell; 11 non–clear-cell), including samples obtained during systemic therapy (ICI-based therapy, n = 46; others, n = 24). Best response to ICI-based therapy was assessed according to RECIST: 18 responders (R; CR/PR) and 11 non-responders (NR; PD). Tumor-reactive TCRs were inferred using VDJdive. Non-negative matrix factorization identified gene programs associated with NR. Bulk RNA-seq data from landmark clinical trials (HCRN, JAVELIN Renal 101, CheckMate 9ER, COSMIC-313) were analyzed using Scissor (Sun et al. Nat Biotechnol 2022) to map PFS-associated bulk signals to single-cell states in our reference dataset, with summarized enrichment as an observed-to-expected (O/E) ratio. Ligand–receptor (NicheNet) and pathway analyses implicated upstream drivers. Functional validation was performed using primary human T cells cultured under normoxia or hypoxia, with or without TGFβ1. A TGFβ activation assay was used to evaluate rescue of T-cell function by the clinical-stage latent TGFβ1-selective inhibitor SRK-181. Results: We profiled 443,337 high-quality cells including 86,865 CD8 T cells. In NR tumors, a subset with high tumor-specific T-cell abundance (5/11, 45.5%) was enriched for an exhausted CD8 state with a tissue-resident memory program (Tex-rm; p < 0.01). Across multiple ICI-treated clinical trial cohorts, we identified Tex-rm cells as most associated with shorter PFS (O/E 1.34–1.82). Ligand–receptor and pathway analyses implicated enhanced TGFβ signaling in Tex-rm compared with other Tex. In vitro, hypoxia plus TGFβ1 increased a Tex-rm–like phenotype (CD69⁺CD103⁺PD-1⁺TIM-3⁺) relative to controls (p < 0.05 for each). TGFβ inhibited T-cell IFNγ production, while treatment with the TGFβ inhibitor SRK-181 restored it (p < 0.05). Conclusions: We identified a Tex-rm program associated with ICI intrinsic resistance in RCC despite high abundance of tumor-specific T cells. Functional data support a role for TGFβ signaling in promoting this dysfunctional state, suggesting that inhibiting TGFβ1 may help restore T-cell function and overcome ICI resistance in some non-responders.