ISCOMATRIX facilitated HLA-A2-restricted cross-presentation of NY-ESO-1 or Melan-A by MoDCs and CD1c⁺ DCs. Immature MoDCs (1 × 10⁵) or freshly isolated CD1c⁺ DCs (1 × 10⁵) were either unpulsed or pulsed 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 the absence or presence of 100 ng/mL IFN-γ, 1800 U/mL IFN-α, 2 μg/mL CD40L, 100 ng/mL LPS, 2.5 μg/mL CpG-A (2216), 1 μg/mL R848, or 12.5 μg/mL Poly I:C in 96-well, round-bottom plates. After 18 hours, DCs were washed and cocultured with HLA-A2-restricted NY-ESO-1 or Melan-A-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. DCs pulsed with cognate peptide for 30 minutes before the 4-hour coculture with CTL lines served as positive controls. Data are mean ± SD of 3 separate donors. (A) *P < .01 vs unpulsed CD1c⁺ DCs and vs CD1c⁺ DCs pulsed with NY-ESO-1 and ISCOMATRIX adjuvant separately with or without additional stimulation. (B) *P < .01 vs unpulsed MoDCs.