RT Journal Article SR Electronic T1 1021 A pan-cancer signature identifies tumor-reacting CD8+ TILs and reveals their functional heterogeneity JF Journal for ImmunoTherapy of Cancer JO J Immunother Cancer FD BMJ Publishing Group Ltd SP A1064 OP A1064 DO 10.1136/jitc-2022-SITC2022.1021 VO 10 IS Suppl 2 A1 Chamberlain, Christopher A1 Draghi, Arianna A1 Khan, Shawez A1 Harbst, Katja A1 Presti, Mario A1 Schina, Aimilia A1 Præst Jensen, Agnete Witness A1 Rasmussen, Anne-Christine Kiel A1 Lorentzen, Torben A1 Schou, Jakob Vasehus A1 Jönsson, Göran A1 Svane, Inge A1 Donia, Marco YR 2022 UL http://jitc.bmj.com/content/10/Suppl_2/A1064.abstract AB Background Identification of CD8+ tumor-reactive tumor-infiltrating lymphocytes (TILs) is a key focus for understanding TIL:tumor interactions in the tumor microenvironment and improving adoptive cell therapies. Current methods concentrate on the detection of TILs endowed with tumor-specific T cell receptors (TCRs) and largely overlook their functional profiles. In addition, analyses of actively tumor-reacting TIL function are often analyte limited and may not reflect the heterogeneity of TIL responses to stimulation. Therefore, we set out to identify tumor-reacting CD8+ TILs and thoroughly characterize their functional profiles at the single-cell level pan-cancer.Methods Minimally in vitro cultured TILs and matching autologous tumor cell lines (TCLs) were derived from metastatic melanoma (n=6), renal cell carcinoma (n=3), sarcoma (n=2), colorectal cancer (n=1), and ovarian cancer (n=1) patient samples. Bulk TILs were cocultured with their wildtype or a major histocompatibility complex I and II deficient control TCL, sorted for CD8+ (fluorescence-activated cell sorting), and then underwent single-cell RNA sequencing (scRNAseq, 10xGenomics 5’ v2/3’ v3.1) to form a discovery dataset. Publicly available scRNAseq datasets (n=25, 12 histologies) of fresh tumor biopsies were obtained from online databases (Gene Expression Omnibus, Genome Sequence Archive, European Genome-phenome Archive) or the authors directly and merged into a singular validation dataset. Cell Ranger and Seurat were used for data processing and analysis.Results Distinct clusters of tumor-reacting CD8+ TILs could be easily bioinformatically identified using known reactivity markers. A tumor-reacting CD8+ TIL signature (TR8S) derived via comparison to non-reacting cells clearly differentiated tumor-reacting and non-reacting CD8+ bulk TILs for all samples and histologies. Deeper characterization of TR8Shi cells revealed diverse sub-populations with distinct and heterogeneous functional profiles, many of which could be re-identified in the validation dataset. In particular, an XCL1hiXCL2hi cluster and a separate CCL3hiCCL4hi cluster dominated the reacting population. The majority of tumor-reacting CD8+ TIL sub-populations were shared across tumor histologies.Conclusions Our results indicate that the TR8S is an effective pan-cancer tool for identifying and characterizing actively tumor-reacting CD8+ TILs. Furthermore, our data highlight the complexity of the actively tumor-reacting CD8+ TIL population and that deeper investigation of their functional abilities is warranted. Specifically, the prevalence of distinct clusters may have predictive/prognostic strength and could direct future immunotherapies.Acknowledgements The authors sincerely thank all the patients who consented to the use of their samples for this study. The following funding sources are thanked for their continuous support: Lundbeck Foundation (grants R233-2016-3728, R286-2018-991, R307-2018-3636), The Danish Cancer Society (grants R180-A11339, R184-A11806, R204-A12535), Independent Research Fund Denmark (8045-00067B), Danish National Board of Health (4-1612-236/8, Empowering Cancer Immunotherapy in Denmark), and Herlev and Gentofte Hospital Research Council (Clinician-Scientist grant to MD). All physicians, surgeons, and technicians involved in patient enrolment and sample procurement are thanked for their efforts. The authors of the public scRNAseq data used are thanked for sharing the data either directly or via public databases.Ethics Approval Fresh tumour material from patients with cancer was acquired via surgical resection or needle biopsy in the context of standard treatment or enrolment in clinical trials (Ethical approval references: H-20070020, H-19076238, Data Protection approval P-2021-303) at the National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev, Denmark. All procedures were performed in compliance with the clinical protocols approved by the Ethics Committee of the Capital Region of Denmark and national regulations for biomedical research.