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Background Tumor-infiltrating CD8 + T lymphocytes (TIL) are key determinants of response to immune checkpoint blockade (ICB) cancer immunotherapy and adoptive T cell therapy,1 2 but the identity of the subpopulations of TIL critical for tumor eradication remains incompletely understood.Methods To examine the cytolytic capacity of TIL, we generated a cohort of paired TIL and melanoma cell lines ( n = 10) from patients with advanced melanoma. To nominate cancer cell-intrinsic genes and pathways critical for elimination by TIL, we performed a genome-scale, in vitro loss-of-function CRISPR screen using Cas9-expressing patient-derived melanoma cells co-cultured with autologous TIL. To determine if enrichment of LTB+CD8 + T cells was associated with clinical response to lifileucel, we examined scRNA-seq data from TIL products (n = 34) for patients with advanced melanoma. To identify the origin of LTB + CD8 + TIL in TIL resection specimens, we performed matched scRNA-seq and TCR-seq analysis from baseline tumor digests and TIL products (n = 7).Results We confirmed that pMHC-TCR interactions were dispensable for in vitro TIL-mediated cancer cell lysis using anti-MHC-I neutralizing antibodies and CRISPR deletion of B2M and TRAC/TRBC in melanoma cells and TILs, respectively. The lymphotoxin β receptor (LTBR) and interferon (IFN) sensing (JAK1, JAK2, STAT1, IFNGR1, IFNGR2) pathways were nominated as key determinants of class I MHC-independent, TIL-mediated cancer cell killing from our CRISPR screen. Validation studies confirmed that dual LTbR and IFN sensing pathway activation is necessary and sufficient for cancer cell lysis, and that tumoricidal CD8+ TIL express high baseline lymphotoxin β (LTB) and upregulate lymphotoxin a (LTA) upon coculture with cancer cells. Using scRNA-seq and TCR-seq data from patients treated with lifileucel,3 we confirmed that tumoricidal TIL express high levels of LTB, are enriched in TIL products of melanoma patients responsive to lifileucel, and are expanded from putative neoantigen-reactive CD8+ T cells lacking LTB expression in resected tumors.Conclusions Here we identify and characterize a novel subset of CD8 + TIL capable of eliminating cancer cells independently of canonical pMHC-TCR interactions. Our findings demonstrate that putative neoantigen-reactive T cells in resected tumors undergo transcriptional reprogramming during in vitro expansion, giving rise to tumoricidal LTB+CD8+ TILs, endowing them with the capacity to eliminate cancer cells independent of canonical pMHC-TCR interactions. These results not only provide mechanistic insights on the role of LTBR/IFN signaling in class I MHC-independent, TIL-mediated cancer cell killing, but also may inform novel strategies to enrich specific TIL subsets during expansion or augment specific TIL functions.Acknowledgements This work was supported by NIH 1R37CA283560-01 (R.W.J.), and the MGH ECOR Fund for Medical Discovery (FMD) Research Fellowship Award (H.X.). Additional support provided by a generous gift from Robert and Marie McInnes. We acknowledge funding provided by the Krantz Family Center for Cancer Research Breakthrough Award (M.S-F., G.M.B, R.W.J.), the National Institute of Health Director’s New Innovator Award (DP2CA247831; A.C.V.), the Massachusetts Life Sciences Center Research Infrastructure Program in support of the Mass General Cancer Center Tumor Cartography Center, and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.References Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 2018;175(4):998-1013.Krishna S, et al. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 2020;370:1328–1334.Chesney J, et al. Efficacy and safety of lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, in patients with advanced melanoma after progression on immune checkpoint inhibitors and targeted therapies: pooled analysis of consecutive cohorts of the C-144-01 study. J Immunother Cancer 2022;10.Ethics Approval Melanoma tumor explants were collected and analyzed according to Dana-Farber/Harvard Cancer Center (DF/HCC) IRB-approved protocol 11-181. A cohort of patients treated at Massachusetts General Hospital was processed, isolated, and in vitro expanded for generating patient-derived cell lines and TIL. These studies were conducted according to the Declaration of Helsinki and approved by the DF/HCC IRB. Specimens and TIL products were collected as part of the C-144-01 study (ClinicalTrials.gov ID: NCT02360579). The study was approved by the Institutional Review Board (IRB) at each site and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines of the International Conference on Harmonization. All patients provided written informed consent.