The pHI-Db minigene vaccine induces an H-2D^b–restricted, FLK₄₀₀–specific response. Groups of vaccinated C57BL/6 mice (n = 4) were killed 2 weeks after the last immunization. (A) ELISPOT assays were performed on splenocytes isolated by using either no stimulator or synthetic peptides FLK₉₄ (25 μg/mL), FLK₄₀₀ (10 μg/mL), or FLK₁₂₁₀ (25 μg/mL) as stimulators. (B-D) Isolated splenocytes were stimulated with FLK₄₀₀ (B), FLK₉₄ (C), or FLK₁₂₁₀ (D) peptides for 5 days. Thereafter cytotoxicity assays were performed with MS1 (♦) or RM9 (○) serving as target cells. Experiment was repeated twice with 2 μg/mL or 10 μg/mL stimulator peptides and with similar results. (E-G) Splenocytes isolated from pHI-Db–vaccinated mice were stimulated with FLK₄₀₀ (E), FLK₉₄ (F), or FLK₁₂₁₀ (G) peptides for 7 days, and restimulated twice weekly with irradiated FLK₄₀₀-loaded (E), FLK₉₄-loaded (F), or FLK₁₂₁₀-loaded (G) splenocytes from normal C57BL/6 mice. Thereafter cytotoxicity assays were performed with MS1 (♦) or RM9 (○) serving as target cells. Error bars indicate SD.