ISCOMATRIX facilitated cross-presentation of HLA-A2, HLA-B7, and HLA-Cw3-restricted epitopes from NY-ESO-1 by MoDCs and CD1c⁺ DCs. Immature HLA-A2, HLA-B7, and HLA-Cw3 triple-positive MoDCs (A-C) or triple-positive CD1c⁺ DCs (D) or singly positive CD1c⁺ DCs (E-F) were plated in triplicate in 96-well, round-bottom plates and either unpulsed or pulsed with 0.2 to 10 μg/mL of NY-ESO-1 protein added separately with ISCOMATRIX adjuvant or with NY-ESO-1/ISCOMATRIX vaccine for 18 hours. After being washed, the DCs were divided and cocultured with HLA-A2 or HLA-B7 or HLA-Cw3-restricted NY-ESO-1-specific CTL lines (A-D) or HLA-A2 or HLA-B7 clones (E-F) 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 are mean ± SD of triplicate wells, and one of 3 experiments is shown. *P < .01 vs NY-ESO-1 added separately with ISCOMATRIX adjuvant to the cultures.