Abstract A36: Single-cell characterization of tumor-infiltrating T cells from renal cell carcinoma
Authors: Xu Yuexin , Morales Alicia J. , Towlerton Andrea M.H. , Warren Edus H. , Tykodi Scott S. 
Published: March 2020

Abstract
Purpose: T-cell infiltration of renal cell carcinoma (RCC) is commonly viewed as evidence for antitumor immunity that may be harnessed by T cell-directed immunotherapies. We hypothesize that bona fide tumor-reactive T cells constitute only a fraction of tumor-infiltrating lymphocytes (TIL) that may be composed largely of nonspecific passenger cells. This study deployed TRB-CDR3 repertoire characterization and targeted single-cell RNAseq of RCC versus normal tissue and peripheral blood mononuclear cells (PBMC)-derived T cells to analyze phenotypes that may associate with tumor-antigen specificity.
Experimental Procedures: RCC tumor, normal adjacent kidney tissue (NAT), skin, and PBMC were collected from 9 RCC patients at the time of nephrectomy surgery. Tissue samples were digested to single-cell suspensions and CD45+CD8+CD3+ T cells were sorted. Sequencing of TCR-β CDR3 regions (TRB-CDR3) was carried out on polyclonal CD8+ T-cell populations. Targeted single-cell RNAseq was then applied to sorted CD8+ T cells from the same samples focusing on TRB-CDR3 and TRA-CDR3 regions and 25 T-cell phenotype genes. Full-length T-cell receptor (TCR) genes from selected, clonally expanded, tumor-associated CD8+ T cells were cloned into lentiviral expression vectors for re-expression in CD8+ T cells from healthy donors to facilitate analysis of TCR specificity.
Results: TCR-β CDR3 sequencing demonstrated the frequency of clonally expanded TRB was higher in tumors (mean 45%; range 27%-65%) compared with PBMCs (mean 14%; range 4%-22%) or NATs (mean 15%; range 2%-26%) for all 9 cases. Expanded T-cell clonotypes present in the tumor and not detected in NAT or PBMC samples were present in every case. Dimensionality reduction of targeted single-cell RNAseq analysis identified phenotypic differences between clonally expanded and nonexpanded T cells in 3 of 9 tumors. Overall, IFN-γ, perforin, and granzymeB were upregulated in clonally expanded T cells, while IL-2, IL-13, and CTLA-4 were upregulated in nonexpanded cells. A total of 116 TRB-CDR3 and TRA-CDR3 sequence pairs were identified from clonally expanded RCC TIL, among which 49 were only found in tumor tissue. Based on effector gene expression, as well as the availability of an autologous RCC tumor line, a total of 12 expanded TRAB pairs were cloned into lentiviral expression vectors. Analysis of TCR specificity for autologous tumor as well as HLA-matched RCC cell lines will be updated.
Conclusions: T-cell clonal expansions associated with the microenvironment of RCC tumors and may represent antigen engagement. Phenotypic differences in effector gene expression between clonally expanded and nonexpanded CD8+ T cells from RCC TIL are heterogeneous between tumors. The association of such phenotypic differences with clinical outcomes is a priority for further investigation. Evaluation of alternate 10x platform and analysis of tumor antigen recognition by TCRs from clonally expanded T cells with cytotoxic phenotype unique to the RCC microenvironment is ongoing.
Citation Format: Yuexin Xu, Alicia J. Morales, Andrea M.H. Towlerton, Edus H. Warren, Scott S. Tykodi. Single-cell characterization of tumor-infiltrating T cells from renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A36.