2.12. Dendritic Cell (DC) Vaccine

DCs were prepared as described previously [15]. Briefly, bone marrow cells from femurs and tibias were cultured in RPMI-1640 supplemented with 10% FBS, 10 mM HEPES (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), MEM Non-Essential Amino Acids Solution (Nacalai Tesque), 5 mM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 ng/mL GM-CSF (PeproTech) for 8 days. DCs were stimulated with 1 μg/mL lipopolysaccharide (FUJIFILM Wako Pure Chemical Corporation) for 16 h and pulsed with peptides at 1 μg/mL for 2 h. For induction of neoantigen-specific CD8⁺ T cells, 1 × 10⁶ neoepitope peptide-pulsed DCs were subcutaneously injected into the flank of mice twice biweekly. Two weeks after the second vaccination, the splenocytes were stimulated with the corresponding peptides and IFN-γ production was determined by intracellular cytokine staining. For evaluation of anti-tumor effects of neoepitope peptide-pulsed DC vaccines, mice were inoculated with 5 × 10⁶ YTN16 cells on day 0 into the right flank. Neoepitope peptide-pulsed DCs were injected into the left flank on day 5. Tumor growth was monitored every 2 to 3 days.