490 Galectin-9 Drives TIM-3+ Natural Killer Cell Dysfunction in Head and Neck Squamous Cell Carcinoma

Background Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that play a critical role in tumor immunosurveillance. Their activation states are regulated by an interplay of activating and inhibitory surface receptors, including T-cell immunoglobulin and mucin-domain containing molecule 3 (TIM-3), an immune checkpoint receptor (ICR) that is expressed on terminally-differentiated NK cells. The role TIM-3 plays in the context of NK cell-mediated anti-tumor response remains evasive, partly because TIM-3 has four known ligands: galectin-9, phosphatidylserine, HMGB1 and CEACAM-1. Methods In the context of head and neck squamous cell carcinoma (HNSCC), single-cell RNA sequencing and flow cytometry were implemented to study the prevalence, phenotypes and functional differences of TIM-3+ NK cells in patient tumors and blood. In vitro killing and proliferation assays were used to evaluate whether the four TIM-3 ligands differentially modulate TIM-3+ NK cell functions and whether abrogation of TIM-3/ligand interaction is a valid therapeutic strategy to enhance NK cell-mediated anti-tumor effector mechanisms. Finally, TCGA survival analysis and digital spatial profiling were employed to study the potential impact of etiology-associated differences on HNSCC patient survival. Results We demonstrate that TIM-3 is the dominant NK cell ICR, that it marks dysfunctional NK cells in tumors but not in circulation, and that galectin-9 is the only TIM-3 ligand that consistently suppresses NK cell cytotoxic and proliferative capacity. Galectin-9-induced effects on cytotoxicity can be abrogated using the clinical-grade anti-TIM-3 blocking antibody, MBG453. Clinically, high intratumoral TIM-3+ NK cell gene signature associates with worse outcome in HPV+, but not HPV- HNSCC patients. This may be due to higher intratumoral galectin-9 protein expression in HPV+ HNSCC lesions, as well as higher frequencies of circulating and tumor-infiltrating TIM-3+ NK cells in HPV+ patients. Conclusions Our data stress the importance of TIM-3 in the context of NK cells and suggest that targeting the TIM-3/galectin-9 pathway may be a cogent immunotherapeutic strategy to reinvigorate NK cell effector function in HPV+ HNSCC patients. Acknowledgements We thank Merida Serrano, Amy Cuda and Denise Kroll for assistance with patient sample procurement. This research utilized the Hillman Cancer Center Flow Cytometry Core Facility, supported in part by award P30 CA047904 (RLF). This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. Ethics Approval Peripheral blood and tumor tissues from treatment-naïve HNSCC patients were collected with their written consent in accordance with the Declaration of Helsinki, under the University of Pittsburgh Cancer Institute Review Board–approved protocol (99-069).