Characterization of the immunophenotype and tumor specificity of CD8+ T-cells in chromophobe renal cell carcinoma (ChRCC) and renal oncocytic neoplasms.
Authors: Labaki Chris , Hobeika Charbel , Hou Yue , Zhang Long , Bi Kevin , Bakouny Ziad , Camp Sabrina Yvonne , Priolo Carmen , Khabibullin Damir , Schindler Nicholas R , Alchoueiry Michel , Denize Thomas , Saliby Renee Maria , Matar Sayed , Signoretti Sabina , Van Allen Eliezer Mendel , Shukla Sachet A , Henske Elizabeth , Choueiri Toni K. , Braun David A. 
Published: June 2023

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Background: ChRCC is an uncommon kidney cancer variant that has a poor prognosis in the metastatic setting, with limited response to current standard-of-care immune checkpoint inhibitors (ICIs) used for other RCC histologies. We evaluated the tumor-immune microenvironment of ChRCC and other related oncocytic neoplasms to better understand the immunophenotype of these tumors. Methods: We performed paired single-cell RNA sequencing (scRNA-seq), single-cell T-cell receptor sequencing (scTCR-seq), and CD45 immunohistochemistry (IHC) of ChRCC, renal oncocytoma (RO) and low-grade oncocytic tumor (LOT) tumor and matched normal samples. Bulk RNA-sequencing (RNA-seq) data of clear cell RCC (ccRCC), papillary RCC (pRCC) and ChRCC were additionally analyzed using The Cancer Genome Atlas (TCGA) kidney cancer cohorts. T cell antigenic specificities from scTCR-seq were inferred using a comprehensive database of annotated T-cell receptor sequences (VDJdb). Single-cell transcriptomic signatures were used to infer the tumor specificity (Oliveira G, Nature, 2021 and Lowery FJ, Science, 2022) and viral specificity (Oliveira G, Nature, 2021) of CD8+ T-cells from ChRCC, as compared to those from ccRCC (Braun DA, Cancer Cell, 2021). Results: ChRCC and other oncocytic tumors had a lower infiltration of CD45+ immune cells as compared to ccRCC (p<0.01). Single-cell analysis was performed on 46,817 cells from 5 tumors (ChRCC: n=3, RO: n=1 and LOT: n=1) and 4 normal samples. Across all tumors, CD8+ T cell clusters displayed a lower expression of immune checkpoints (i.e. PDCD1 [PD-1], CTLA4, LAG3, HAVCR2 [TIM-3], and TIGIT) as compared to ccRCC. This was further validated in a bulk RNA-seq analysis using TCGA data, with a significantly lower expression of all immune checkpoints in ChRCC compared to both ccRCC (p<0.01) and papillary RCC (pRCC; p<0.01). Analysis of the T cell receptor repertoire (scTCR-seq) of ChRCC, RO and LOT samples did not show any pattern of clonal expansion, and a higher proportion of T cells in ChRCC were inferred to have a viral specificity, as compared to ccRCC (0.79 vs. 0.1%, respectively). CD8+ T cells from ChRCC (vs. ccRCC) displayed a significantly lower expression of two signatures of tumor specificity (p<0.01), and a higher expression of the viral-specific signature (p<0.01). Conclusions: In ChRCC, there is low infiltration by CD45+ immune cells. Although infiltrating CD8+ T cells have a predominantly non-exhausted immune phenotype, they likely lack anti-tumor specificity (i.e. are “bystander” T cells). These findings may help to understand the molecular basis for the lack of response to immunotherapy recently identified among patients with advanced ChRCC, and support future therapeutic strategies to increase infiltration of tumor-specific T cells into the tumor microenvironment.