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Background Head and neck squamous cell carcinoma (HNSCC) accounts for 4-7% of all cancers globally with about 50% 5-year survival rate for advanced cases. However, HNSCC presents therapeutic challenges due to its immunosuppressive tumor microenvironment, high recurrence rates, and limited efficacy of current immunotherapies. Adoptive cell therapy (ACT) using Tumor-infiltrating lymphocyte (TIL) therapy has shown promise in solid tumors, but challenges such as T cell exhaustion, limited availability, and complex ex vivo expansion protocols remain barriers to widespread clinical adoption. Sentinel lymph nodes (SLNs), as primary immune sites for tumor antigen presentation, represent a physiologically relevant reservoir of tumor-primed T cells. We hypothesized that SLN-derived TILs (SLN-TILs) exhibit enhanced therapeutic features compared to non-sentinel lymph node (NSLN)- and tumor-derived TILs, and that tumor-directed radiation (tdRT) could further prime this response in HNSCC models.Methods Fresh Tumors, NSLNs and SLNs were collected from HPV-negative HNSCC mouse models (donors). In this model, 4MOSC1 cells are orthotopically injected into the buccal mucosa of C57BL/6 mice. Phenotypic profiling was performed using ELISA and multiplex IHC (mIHC), assessing CD4+/CD8+ populations, activation, tumor reactivity and exhaustion of CD4+/CD8+ cells. Tumors, NSLNs and SLNs were separately digested, and single cells were collected and used for CD45+ cell isolation. Subsequently, CD45+ cells were expanded using an IL-2 supplemented media. CD45+ cells from different sources later were injected retro-orbitally into the retrobulbar venous sinus of recipient mice. In a subset of recipient mice, low dose tdRT (4 Gy) was administered to recipients prior to CD45+ cell transfer to further enhance SLN-TILs effect (tdRT_SLN group). Lastly, Tumor growth and survival of each group (tdRT_SLN, SLN, NSLN, and Tumor) were measured ( figure 1A).Results SLN-derived TILs displayed a favorable activation (CD137+)/exhaustion (PD-1, TIM-3) balance compared to conventional Tumor-TILs. CD137 upregulation and higher IFN-γ release confirmed stronger functional responses in SLN-TILs. NSLN-TILs exhibited intermediate features. Adoptive transfer of SLN-TILs (SLN group) resulted in significantly robust tumor regression compared to control (vehicle). In SLN-TILs combined with tdRT (tdRT_SLN group) complete tumor regression was observed for 70% of the individuals. tdRT_SLN mice remained tumor-free for the entirety of experiment. tdRT_SLN also showed highest survival ( figure 1B-C). Data was analyzed using two-way ANOVA via Prism 10.Conclusions SLN-TILs are highly functional, less exhausted, and better suited for adoptive transfer than Tumor- or NSLN-TILs in HNSCC characterized by higher overall survival and tumor regression thanks to increased expression of activation marker CD137, release of INFγ and reduced exhaustion profiles.Acknowledgements We would like to thank Gleiberman Head and Neck Cancer Center and Iris and Matthew Strauss Chancellor’s Endowed Chair for Head and Neck Surgery for funding this research.Ethics Approval The animal study was approved by Institutional Animal Care and Use Committee of UCSD (#S16200), University of California San Diego. The study was conducted in accordance with the local legislation and institutional requirements.Abstract 376 Figure 1A. Schematic shows the experiment design; B. Survival curve; C. Tumor growth curve (p<0.05 is significant)