302 - Fluorescence Tracking of Tumor-Draining Lymph Node-Derived CD8 T Cell Responses Following Tumor Radiation
Presenter(s)
D. J. Friedman1, G. Kramer1, A. F. Alice1, J. Baird1, S. Ramin1, T. Medler1, C. Grassberger2, M. R. Crittenden3,4, M. Gough3, and K. H. Young4; 1Earle A. Chiles Research Institute, Portland, OR, 2Department of Radiation Oncology, University of Washington/Fred Hutchinson Cancer Center, Seattle, WA, 3Earle A. Chiles Research Institute, Providence Cancer Institute, Portland, OR, 4The Oregon Clinic, Portland, OR
Purpose/Objective(s): Radiation rapidly depletes intratumoral CD8 T cells yet tumors refill within days. We hypothesized that CD8 T cells recruited from the tumor-draining lymph node (TdLN) repopulate irradiated tumors and undergo antigen-independent, type I interferon (IFN) driven activation that enhances intratumoral retention.
Materials/Methods: Using Kaede photoconvertible mice, we tracked TdLN-derived cells entering tumors after CT-guided irradiation (single 2–12 Gy; or fractionated 2 Gyx3 or 6 Gyx3). Phenotypes were profiled by flow cytometry and scRNA-seq. Mechanism was tested in Ifnar1-/- or Sting-/- hosts and with cGAS-deficient cancer cells. Antigen-specific trafficking was evaluated with SIY/OVA models. A compartmental ordinary differential equation (ODE) model quantified cell survival and tumor–TdLN flow/retention.
Results: TdLN-derived effector CD8 T cells preferentially infiltrated tumors compared to non-draining lymph nodes, and radiation did not alter the magnitude of TdLN-to-tumor recruitment within the first 24 hours. By day 3 post-RT, recruited CD8 T cells demonstrated robust CD69 upregulation despite unchanged antigen-specific infiltration. CD69 induction required type I IFN signaling and cancer-cell intrinsic cGAS, whereas STING in host immune cells was dispensable, indicating tumor-cell sensing of DNA damage initiates the IFN-dependent activation program. scRNA-seq revealed a selective expansion of stem-like and proliferating CD8 subsets in irradiated tumors, consistent with preferential activation of non-exhausted cells. Dose escalation and fractionation induced proportional CD69 upregulation when matched for biologically effective dose, demonstrating that early T-cell activation integrates radiation dose rather than fraction structure. Kinetic studies showed a pronounced CD8 T-cell nadir at days 1–3 post-RT and full restoration by days 5–8. Mathematical modeling showed that cell kill alone could not account for the observed dynamics, and that reduced lymphocyte egress from tumors, consistent with CD69-mediated S1P1R suppression, was required. This identifies radiation-induced retention as a major factor shaping refilling kinetics.
Conclusion: Radiation triggers a rapid, type I IFN–dependent activation of newly infiltrating CD8 T cells originating from the TdLN, independent of antigen recognition. This early program enhances T-cell retention within the tumor and precedes radiation-driven antigen-specific expansion in the TdLN. These findings identify a narrow early post-RT window in which IFN signaling dominates T-cell behavior, highlight BED as a key regulator of early immune activation, and suggest that preserving TdLN function may be critical for optimizing T-cell–mediated tumor control. These insights have implications for sequencing radiation with checkpoint blockade, designing fractionation schemes that favor immune engagement, and avoiding collateral TdLN irradiation when systemic immunity is desired.