Mode of antigen delivery and DC activation influence cross-presentation and MHC II presentation efficiency. MoDCs from an HLA-A2⁺ donor were pulsed with NY-ESO-1 protein, NY-ESO-1/IC (mAb ES121), or NY-ESO-1/IMX at the indicated concentrations (referring to antigen content) and cultured overnight in media containing GM-CSF and IL-4 in the absence or presence of CD40L, LPS, or R-848, or a combination of TNF-α, IL-1β, IL-6, and PGE₂ (TIIP). Antigen-pulsed MoDCs were cocultured with NY-ESO-1–specific T cells and induction of IFN-γ production by CD8⁺ T cells (A-B) or CD4⁺ T cells (C) was quantified by intracellular cytokine staining assay (ICS). (A,C) ○ indicates GM-CSF + IL-4 only; ▪, CD40L. Data in panels A and C are representative of 9 experiments using MoDCs from different donors. ^*P < .01. In panel B, CD8⁺ T-cell IFN-γ induced by MoDCs cultured with GM-CSF and IL-4 only was normalized to 100%. Data are mean plus or minus standard deviation (SD) of 6 to 10 experiments.