Antigen processing of NY-ESO-1/IC and NY-ESO-1/IMX occurs via 2 distinct cytosolic pathways. MoDCs were incubated in the absence (□) or presence (▪) of inhibitors 30 to 45 minutes before pulsing with NY-ESO-1/IC or NY-ESO-1/IXM (each 10 μg/mL): (A) concanamycin B (20 nM); (B) lactacystin (10 μM); (C) epoxomicin (5 μM); and (D) brefeldin A (1 μM). After overnight culture, MoDCs were assessed for their ability to induce IFN-γ by CD8⁺ T cells. To control for unspecific effects, MoDCs were pulsed with peptide (0.3 μg/mL) for each inhibitor condition. CD8⁺ T-cell IFN-γ induced by DCs in the absence of inhibitors was normalized to 100%. Data are mean plus or minus SD of 4 to 6 experiments. (E) MoDCs were incubated in the absence or presence of vaccinia virus encoding either ICP-47 or OVA at 3 or 6 MOI before pulsing with NY-ESO-1 formulations (□, NY-ESO-1/IC; •, NY-ESO-1/IMX) or peptide (○). CD8⁺ T-cell IFN-γ induced by MoDCs in the absence of virus was normalized to 100%. Data are mean plus or minus SD of 2 experiments.