RT promotes priming of Ag-specific cells. (A) A total of 5 × 10⁵ B16-SIY tumor cells were subcutaneously injected into the lower back of C57BL/6 (n = 8-9 per group). Fourteen days after tumor challenge, mice received localized RT (20 Gy) on the tumors and were transferred intravenously with CFSE-labeled naive 2C cells. Four to 5 days after adoptive transfer, mice were killed for analysis of DLN and spleen. The degree of CFSE dilution via FACS was determined by gating on the 1B2⁺CD8⁺ lymphocyte population. The RT group has more proliferative T cells than the no RT group (***P < .001). (B) A total of 5 × 10⁵ B16-SIY tumor cells were subcutaneously injected into the lower back of C57BL/6 (n = 5 or 6 per group). Fourteen days after tumor challenge, mice received local RT (20 Gy) on the tumors and were killed 5 days later for tetramer⁺ cell analysis. DLN and spleen were harvested, collagenase digested, and then stained for FACS. Cells were gated on CD11c⁺ cells. Similar experiments were repeated twice. The RT group has more positive cells than the no-RT group (***P < .001). (C) A total of 2 × 10⁵ B16 tumor cells were subcutaneously injected into the lower back of C57BL/6 mice (n = 4-6 per group). Fourteen days after tumor challenge, mice received localized RT (20 Gy) on the tumors and were analyzed 48 hours later. DLN was isolated, collagenase-digested (1.5 mg/mL), and then stained for FACS. Cells were gated on CD11c⁺ cells. Mean ± SD for the no-RT group was 6.8 ± 4, and for the RT group 14.6 ± 2. Similar experiments were repeated at least twice.