Abstract PR008: Single cell transcriptomic investigation of tumor-infiltrating CD8+ T cells in renal cell carcinoma (RCC) resistant to immune checkpoint inhibition (ICI)

Abstract The most successful treatments for advanced RCC have been ICI-based combination therapies. However, most patients with advanced RCC have disease progression despite ICI treatment, suggesting analysis of immunological differences in patients that respond to or resist ICI therapy. CD8+ T cells display tremendous phenotypic diversity and play a critical role in anti-tumor immunity, but it is unclear what drives different CD8+ T cell phenotypes in the RCC tumor microenvironment (TME) and how individual phenotypes impact ICI response or resistance. 70 tumor samples from 63 RCC patients were collected before (n = 48) or after (n = 22) therapies (VEGFi, n = 9; ICI monotherapy, n = 20; ICI combination, n = 26; others, n = 15). 11 samples were collected from patients without tumors. RCC variants included 59 clear cell and 11 non-clear cell samples. 18 were labeled as clinical benefit and 11 as no-clinical benefit. Single-cell RNA sequencing (10x Genomics) was performed on these samples to generate a transcriptome of the RCC TME. Graph-based clustering identified cell type populations, which were annotated with known lineage genes. Non-negative matrix factorization (NMF) identified gene programs within exhausted CD8+ T cells (Tex). NicheNet predicted ligand-target interactions between macrophages and Tex cells. Within CD8+ T cells, Tex cells were identified through expression of TOX, PDCD1 (PD-1), and HAVCR2 (TIM-3). NMF generated 4 programs within Tex cells, expressing markers for immediate early genes, exhaustion/activation, tissue residency, and stress response. The tissue residency program associated with resistance to ICI therapy (p = 0. 05) ; the stress program was increased in ICI response (p 0. 002). NicheNet identified TGFb (TGFB1), produced by macrophages in non-responding tumors, as the ligand with the highest regulatory potential that generated tissue residency in Tex cells. It identified CD69 and IL7R as notable downstream genes which were associated with NCB via differential gene expression (p 0. 001). Expression of TGFb receptor (TGFBR2) was higher (p = 0. 01) in Tex cells from nonresponsive tumors. Naïve CD8 T cells from healthy donor peripheral blood mononuclear cells were cultured with stimulation under hypoxia (1% O2) or normoxia, and with/without TGFb. CD69/CD103 positive tissue-resident-like T cells were higher with TGFb under hypoxia (p = 0. 02). In hypoxic conditions (mimicking the RCC TME), TGFb led to more PD-1 expression on CD8+ T cells (p = 0. 012). Through scRNA-seq analysis, we identify an ICI-resistance circuit whereby tumor-associated macrophages produce TGF-beta, which leads to a tissue residency gene program in Tex cells associated with non-response to immunotherapy. Experimental validation studies demonstrate that TGFb and hypoxia are sufficient for induction of tissue residency and PD-1 expression on CD8+ T cells. This study provides a framework for using scRNA-seq to identify mechanisms of ICI resistance in RCC and nominates the TGFb axis as a targetable pathway to improve CD8 T cell-mediated anti-tumor immunity. Citation Format: Rishabh Rout, Soki Kashima, Miya Hugaboom, Zhaochen Ye, Nicholas Schindler, Anasuya Dighe, Maxine Sun, Gwo-Shu Mary Lee, Wenxin Xu, Sabina Signoretti, Bradley McGregor, Rana R. McKay, Toni Choueiri, David Braun. Single cell transcriptomic investigation of tumor-infiltrating CD8+ T cells in renal cell carcinoma (RCC) resistant to immune checkpoint inhibition (ICI) abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₂): Abstract nr PR008.