Simple SummaryToday’s standard of treatment for advanced head and neck squamous cell carcinoma (HNSCC) is invasive surgical resection. Radiation co-therapy has received a great deal of attention for its ability to reduce tumor recurrence, common in these cancers. The aim of our study was to investigate the ability of tumor-targeted radiation therapy to generate tumor antigens capable of eliciting host immune response pathways. Using a murine model of HNSCC with tumor cells injected into immune-competent mice, we determined that antigens generated by the irradiation of tumor cells induced potent immune responses compared to other methods for tumor antigen preparations. This response was also found to display comparable sensitivity, but greater specificity for T cell activation compared to conventional anti-CD3 and CD28 antibodies. This information may be utilized to predict patient responses to radiation and immune therapy.AbstractHead and neck squamous cell carcinoma (HNSCC) is common and deadly, and there is a need for improved strategies to predict treatment responses. Ionizing radiation (IR) has been demonstrated to improve HNSCC outcomes, but its effects on immune responses are not well characterized. We determined the impact of IR on T cell immune responses ex vivo. Human and mouse HNSCC cells were exposed to IR ranging from 20 to 200 Gy to determine cell viability and the ability to stimulate T-cell-specific responses. Lymph node cells of LY2 and MOC2 tumor-bearing or non-tumor-bearing mice were re-stimulated with a tumor antigen derived from LY2 or MOC2 cells treated with 200 Gy IR, ultraviolet (UV) exposure, or freeze/thaw cycle treatments. T cell proliferation and cytokine production were compared to T cells restimulated with plate-bound CD3 and CD28 antibodies. Human and mouse HNSCC cells showed reduced viability in response to ionizing radiation in a dose-dependent manner, and induced expression of T cell chemotactic cytokines. Tumor antigens derived from IR-treated LY2 and MOC2 cells induced greater proliferation of lymph node cells from tumor-bearing mice and induced unique T cell cytokine expression profiles. Our results demonstrate that IR induces potent tumoral immune responses, and IR-generated tumor antigens can potentially serve as an indicator of antitumor immune responses to HNSCC in ex vivo T cell restimulation assays.