DNA vaccine encoding full-length FLK-1 induces FLK₄₀₀-specific responses. (A) ELISPOT assays performed with freshly isolated splenocytes from pFLK-1–vaccinated mice and stimulator with FLK₉₄, FLK₄₀₀, FLK₁₂₁₀, or no peptide. (B) Splenocytes isolated from pFLK-1–vaccinated mice were first stimulated in vitro for 5 days with peptides indicated by “primary stimulators,” then restimulated in ELISPOT assays. Stimulators used in such ELISPOT assays are unloaded, FLK₉₄-loaded, FLK₄₀₀-loaded, or FLK₁₂₁₀-loaded splenocytes from normal C57BL/6 mice. Splenocytes from pFLK-1 (C), pHI-Db (D), pCMV (E), and pHI (F) groups of mice were stimulated with irradiated MS1 cells for 5 days, and cytotoxicity assays were performed against unloaded (▵) or FLK₄₀₀-loaded (▪) EO771 target cells. ^*P < .02 compared to unloaded EO771 target cells. The killing of FLK₉₄-loaded or FLK₁₂₁₀-loaded EO771 cells was indistinguishable from that of unloaded EO771 cells (data not shown). This experiment was repeated once with similar results (data not shown). Error bars indicate SD.