Fluorescence tracking reveals CD8 T cell recruitment from lymph nodes to irradiated tumors and early activation via type I interferon 2260446

Abstract Introduction Radiation therapy is a potent local cancer treatment but depletes intratumoral CD8 T cells. Despite this, tumors rapidly refill with T cells post-treatment. The source and activation mechanisms of these repopulating T cells remain unclear. Understanding early recruitment and differentiation is critical to optimizing radiation-immunotherapy combinations. Methods We used Kaede photoconvertible mice to track CD8 T cells migrating from tumor-draining lymph nodes (TdLN) to irradiated tumors. Tumors were implanted subcutaneously and treated with CT-guided radiation (2—12Gy) using the SARRP. Photoconversion was performed by targeted 405nm light exposure to TdLN prior to radiation. CD8 T cell activation and phenotype were assessed by flow cytometry and scRNA-seq. Mechanistic studies used IFNAR1-/-, Sting-/-, and cGAS-/- mice, and adoptive transfer of antigen-specific 2C T cells. Fractionation effects and IFN signaling were evaluated using knockout models and RNA-seq datasets. Results TdLN-derived CD8 T cells preferentially migrated to irradiated tumors and rapidly upregulated CD69 independent of antigen specificity. Activation was mediated by type I IFN signaling triggered by cGAS in irradiated cancer cells. CD69 expression was dose-dependent and enriched in progenitor exhausted T cells. scRNA-seq revealed a shift toward stem-like and proliferating subsets post-radiation. Antigen-specific CD8 T cells also migrated and acquired exhaustion markers upon tumor entry, but their recruitment was not increased by radiation. Fractionated radiation (e.g., 2Gy ×3) induced similar CD69 upregulation at higher single doses. Conclusion Radiation induces early, non-specific activation of newly recruited CD8 T cells via type I IFN, shaping the tumor immune environment before antigen-specific expansion. These findings highlight the role of TdLN-derived T cells and suggest that radiation timing and dose critically influence immune synergy. Funding Source National Cancer Institute R01CA182311, R01CA244142, R01CA208644, 1R01CA293806-01A1, and the Providence Foundation Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)