Effects of proteasome inhibition on ISCOMATRIX facilitated HLA-A2-restricted cross-presentation of NY-ESO-1 or Melan-A by MoDCs. Immature MoDCs (1 × 105) were either untreated or treated with 0.2 to 0.8μM lactacystin for 45 minutes before and throughout the 18-hour pulse with 10 μg/mL NY-ESO-1/ISCOMATRIX vaccine or 10 μg/mL NY-ESO-1 (A) or Melan-A protein (B) together with 10 μg/mL ISCOMATRIX adjuvant in 96-well, round-bottom plates. (C) MoDCs were first washed 3 times to remove serum before being treated with butabindide oxalate for 45 minutes before and throughout the 18-hour antigen pulse. In parallel, untreated or inhibitor-treated MoDCs were pulsed with cognate peptide for 30 minutes and then washed thoroughly before the 4-hour coculture with CTL lines to control for nonspecific effects of the inhibitors. A standard ICS was then performed, and IFN-γ levels assessed by flow cytometry. Data are mean ± SD of 3 separate donors. *P < .01 vs no addition of lactacystin or butabindide oxalate.
