FIG. 2.

Identification of HLA-A*0201-restricted epitopes of HPV-16 E5 protein by vaccination of HLA-A*0201 transgenic mice with peptide plus CpG ODN 1826. Four- to 6-week-old HLA-A*0201 transgenic mice were immunized three times each with T2 cell-binding peptides plus CpG ODN 1826 (12) at 1-week intervals, with five mice in each test group. Five days after the last vaccination, splenocytes were harvested and stimulated with each indicated peptide; then, intracellular-cytokine staining with flow cytometry was performed to determine the number of CD8⁺ IFN-γ⁺ double-positive cells. (A) splenocytes from vaccinated mice were stimulated in vitro with the indicated peptide and stained with CD8 and IFN-γ antibodies. The results of one representative assay from three identical independent experiments are shown. The percentage of CD8⁺ and IFN-γ⁺ double-positive cells in the gated T-cell populations are shown in the upper corners of the plots. (B) Summary of the three independent experiments. The data represent the means and standard errors of three experiments. The x-axis values were calculated as follows: increase of E5-specific splenocytes = (number of vaccinated splenocytes stimulated with indicated peptide)/(number of vaccinated splenocytes stimulated with irrelevant peptide) × 100%. (C) The T2 cell-binding activities of the wild-type peptide E5 63-71 and the mutant E5 63-71 M. The synthesized wild-type E5 63-71 peptide sequence is YIIFVYIPL, and that of the mutant E5 63-71 is YGIFVYIPG. The measurement of T2 cell binding is described in the legend to Fig. 1B. (D) Splenocytes from vaccinated mice were stimulated in vitro with the E5 63-71 or E5 63-71 M peptide and stained with CD8 and IFN-γ antibodies as described for panel A. (E) Summary of the three independent experiments.