Lymphotoxin-driven cancer cell eradication by tumoricidal CD8 ⁺ TIL

Tumor-infiltrating lymphocyte (TIL) therapy is FDA-approved for patients with treatment-resistant advanced melanoma, but the TIL subpopulations critical for tumor eradication remains incompletely understood. Using patient-derived TIL-melanoma co-cultures, we identified and characterized a novel subset of CD8 ⁺ TIL, capable of class I HLA-independent cancer cell lysis. The lymphotoxin β receptor (LTβR) and interferon (IFN) sensing pathways were nominated as key determinants of TIL-mediated cancer cell killing from a whole-genome, loss-of-function CRISPR screen. Validation studies confirmed that dual LTβR and IFN sensing is necessary and sufficient for cancer cell lysis, and that expanded CD8 ⁺ TIL express high lymphotoxin β ( LTB ) and upregulate lymphotoxin α ( LTA ) upon coculture with cancer cells. Leveraging paired scRNA-seq and scTCR-seq data, we confirmed that enrichment of LTB ⁺ CD8 ⁺ T cells is associated with clinical response to TIL, and that LTB ⁺ CD8 ⁺ TIL are expanded from putative neoantigen-reactive, LTB ^lo CD8 ⁺ T cells in resected tumors.