Selective expansion of TCF7-expressing tumor-reactive T cell subpopulations during ovarian tumor-infiltrating T cell production ex vivo.

Tumor-infiltrating lymphocyte (TIL) therapy was recently approved for melanoma patients; however, the dynamic changes in T cell subpopulations during TIL production remain poorly understood. Here, we analyzed epithelial ovarian cancer samples at various stages of ex vivo TIL culture using paired single-cell RNA and TCR sequencing. We also assessed the expansion potential and tumor reactivity of the identified TIL subpopulations. Single-cell transcriptomic analysis revealed that CD8 TILs exhibited reduced cellular diversity following ex vivo expansion, selectively expanding stem-like TCF7 precursors of exhausted T cells (Tpex) and effector-like tissue-resident memory (Trm) cells. TCR clonotype analysis showed that Tpex cells accumulated through self-renewal, while Trm cells primarily originated from TCF7GZMK early effector memory cells in tumors. Additionally, TCR tracing identified preferential activation and reprogramming of CD4 T follicular helper (Tfh)-like cells, especially TCF7 ones. All three TCF7 subpopulations showed robust expansion potential and tumor reactivity in vitro. Notably, CCR7CD200 T cells, enriched for TCF-1CD8 Tpex and CD4 Tfh-like cells in the tumor microenvironment, exhibited self-renewal during in vitro expansion and demonstrated tumor reactivity both in vivo and in vitro. These findings highlight the selective expansion of tumor-reactive TCF7 T cells during TIL culture and suggest that CCR7 and CD200 serve as important surface markers for generating stem-like, tumor-reactive cells, potentially improving TIL therapy in cancers.