Lymphotoxin-driven cancer cell eradication by tumoricidal CD8 + TIL

Abstract 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, LTBlo CD8+ T cells in resected tumors. Significance We have uncovered a previously unrecognized mechanism of TIL-mediated tumor eradication, providing mechanistic insights into the role of LTBR/IFN signaling in TIL-mediated cancer cell killing, and potentially offering insights into novel strategies to isolate, enrich, and expand tumoricidal TIL or augment specific TIL functions to enhance tumor control. Competing Interest Statement R.W.J. is a member of the advisory board for and has a financial interest in Xsphera Biosciences Inc., a company focused on using ex vivo profiling technology to deliver functional, precision immune-oncology solutions for patients, providers, and drug development companies. R.W.J. has received honoraria from Incyte (invited speaker), G1 Therapeutics (advisory board), Bioxcel Therapeutics (invited speaker). R.W.J. has ownership interest in U.S. patents US20200399573A9 and US20210363595A1. R.W.J.'s interests were reviewed by Mass General Brigham and MUSC in accordance with their conflict-of-interest policies. R.J.S. has served in a consulting/advisor role for Marengo, Merck, Novartis, Pfizer, Replimune and has received grant funding from Merck. A.C.V. has been a paid consultant to Bristol Myers Squibb and AbbVie, and has a financial interest in 10X Genomics. 10X Genomics company designs and manufactures gene sequencing technology for use in research, and such technology is being used in this research; these interests were reviewed by The Massachusetts General Hospital and Mass General Brigham in accordance with their institutional policies. N.P.S has been a paid consultant to Hera Biotech. G.M.B. has sponsored research agreements through her institution with: Olink Proteomics, Teiko Bio, InterVenn Biosciences, Palleon Pharmaceuticals. She served on advisory boards for: Iovance, Merck, Nektar Therapeutics, Novartis, and Ankyra Therapeutics. She consults for: Merck, InterVenn Biosciences, Iovance, and Ankyra Therapeutics. She holds equity in Ankyra Therapeutics. D.B.K. is a scientific advisor for Immunitrack, a wholly owned subsidiary of Eli Lilly and Company and Breakbio. D.B.K. owns equity in Affimed N.V., Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Celldex Therapeutics, Editas Medicine, Immunitybio, Lexicon Pharmaceuticals. Moderna, Sana biotechnologies. Summit Therapeutics. BeiGene, a Chinese biotech company, supported unrelated SARS COV-2 research at TIGL. K.T.F. has/had served on the Board of Directors of Clovis Oncology, Strata Oncology, Checkmate Pharmaceuticals, Kinnate Pharmaceuticals and Scorpion Therapeutics; Scientific Advisory Boards of PIC Therapeutics, Apricity, Fog Pharma, Tvardi, xCures, Monopteros, Vibliome, ALX Oncology, Karkinos, Soley Therapeutics, Alterome, IntrECate, and PreDICTA; and as consultant to Novartis, Genentech, Takeda, and Transcode Therapeutics; and received research funding from Novartis. Footnotes correcting issues for symbols were incorrectly reformatted