Figure 2.

Peptide-specific precursor CTLs were activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex. PBMCs were isolated from patient 1 suffering from colon cancer (Table1) who had been vaccinated with survivin-2B_80-88 peptide in our clinical study. The patient's PBMCs were shown to contain the survivin-2B-specific CD8⁺ T cells. PBMCs were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B_80-88 (400 μg/mL), Hsp90 (400 μg/mL), survivin-2B_75-93 precursor peptide (400 μg/mL), and Hsp90 (400 μg/mL)–survivin-2B_75-93 precursor peptide (400 μg/mL) complex in AIM V medium containing 10% human serum and interleukin-2 (50 U/mL) for 7 days. The stimulated PBMCs were stained with HIV tetramer (tet) or survivin-2B tetramer at 37°C for 20 min. Then a phycoerythrin-Cy5-conjugated anti-CD8 antibody was added at 4°C for 30 min. Cells were washed twice with PBS. After washing, cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry using FACSCalibur and CellQuest software. CD8⁺ living cells were gated, and cells labeled with survivin-2B tetramer were referred to as tetramer-positive cells. The frequency of CTL precursors was calculated as the number of tetramer-positive cells divided by the number of CD8⁺ cells. Data are shown as means + SEM of three independent experiments. *P < 0.01.