MoDCs cross-present the HLA-A2-restricted epitopes from NY-ESO-1 and Melan-A in the presence of ISCOMATRIX adjuvant. Immature MoDCs (1 × 105) were either unpulsed or pulsed with 100 or 10 μg/mL of (A) NY-ESO-1 or (B) Melan-A protein alone or together with 10 μg/mL ISCOMATRIX adjuvant for 18 hours in 96-well, round-bottom plates. After being washed, MoDCs were 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 intracellular cytokine stain (ICS) was then performed, and IFN-γ levels were assessed by flow cytometry. MoDCs pulsed with cognate peptide for 30 minutes before the 4-hour co-culture were also included to assess maximal peptide-specific IFN-γ responses. (C-F) Immature MoDCs were pulsed with 10 μg/mL of NY-ESO-1 or Melan-A protein together with 10 μg/mL ISCOMATRIX adjuvant for 18 hours or pulsed with antigen or ISCOMATRIX for 30 minutes before being washed 3 times, and then chased with antigen or ISCOMATRIX at the times indicated (C-D) or with antigen together with ISCOMATRIX (E-F). Pulsed MoDCs were cultured for a further 18 hours before being cocultured with specific CTL lines as in panels A and B. Data are mean ± SD of 3 separate donors. **P < .02, *P < .01 vs no addition of ISCOMATRIX (A-B) and vs the protein chase (C-D).
