Figure 2.

Tetramer staining before the first vaccination and after the fourth vaccination in the second protocol. FITC-labeled HLA-A*2402-HIV peptide (RYLRDQQLL) tetramer and PE-labeled HLA-A*2402-Survivin-2B80-88 peptide tetramer were uesd. For flow cytometric analysis, PBMCs, which were stimulated in vitro, were stained with the tetramers at 37°C for 20 min, followed by staining with FITC- or PerCP-conjugated anti-CD8 mAb (Beckton Dickinson Biosciences) at 4°C for 30 min. Cells were washed twice with PBS before fixation in 1% formaldehyde. Flow cytometric analysis was performed using FACSCalibur and CellQuest software (BD Biosciences). The frequency of CTL precursors was calculated as the number of tetramer-positive cells divided by the number of CD8-positive cells. The peptide-specific CTL frequency is indicated as the percentage of tetramer-positive CTL cells among CD8-positive T cells before the first vaccination and after the fourth vaccination. The peptide-specific CTL frequency after the fourth vaccination (B) was compared with that before the first vaccination (A). In the second protocol with the peptide mixed with IFA, the peptide-specific CTL frequency was increased in all 4 patients (100%).