Effects of proteasome inhibition on ISCOMATRIX facilitated cross-presentation of HLA-A2, HLA-B7, and HLA-Cw3-restricted epitopes from NY-ESO-1 by MoDCs. Immature HLA-A2, HLA-B7, and HLA-Cw3 triple-positive MoDCs (1 × 105) were plated in triplicate in 96-well, round-bottom plates and untreated or treated with 1 or 2μM lactacystin for 45 minutes before and throughout the 18-hour pulse with 10 μg/mL of the NY-ESO-1/ISCOMATRIX vaccine or NY-ESO-1 protein added separately with ISCOMATRIX adjuvant or with NY-ESO-1/ICs. After being washed, MoDCs were divided and cocultured with HLA-A2 or HLA-B7 or HLA-Cw3-restricted NY-ESO-1-specific CTL lines for 4 hours in the presence of 10 μg/mL BFA. A standard ICS was then performed, and IFN-γ levels were assessed by flow cytometry. Data represent the mean ± SD of triplicate wells, and one of 3 experiments is shown. *P < .01 vs no addition of lactacystin.
