Figure S5 shows representative gating strategies for the co-culture assays with different timepoints (6h, 48h) and conditions (baseline, unstimulated, T-cell stimulated).
ARTICLE ABSTRACT
The successful use of expanded tumor-infiltrating lymphocytes (TILs) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T-cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n=58) from treatment-naïve RCC patients, “pre-rapidly expanded” TILs (pre-REP TIL, n=15) and “rapidly expanded” TILs (REP TILs, n=25) according to a clinical-grade TIL production protocol, with scRNA+TCRab-seq, TCRb-seq, and flow cytometry. REP TILs encompassed a greater abundance of CD4+ than CD8+ T-cells, with increased LAG-3 and low PD-1 expressions in both CD4+ and CD8+ T-cell compartments compared to the pre-REP TIL and tumor T-cells. The REP protocol preferentially expanded small clones of the CD4+ phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T-cell clones in the tumor do not expand during the expansion protocol. Additionally, by generating a catalog of RCC-associated TCR motifs from >1000 scRNA+TCRab-seq and TCRb-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs.
