Cytolytic assays and IFN-γELISPOT analyses. For induction of antigen-specific CTL, T cells were isolated via magnetic bead separation from HLA-A0201–positive healthy volunteers. DCs were matured for 24 hours as indicated, loaded with the HLA-A0201-restriced MART-1/MelanA epitope ELAGIGILTV (10μM), and then used as stimulators at a stimulator to responder ratio of 1:20. After 7 days, cells were restimulated with autologous, peptide-loaded DCs at a stimulator to responder ratio of 1:10. Seven days later, CD8⁺ T cells were isolated and analyzed for cytolytic activity in standard europium release assays using MART-peptide pulsed or control peptide pulsed T2 cells as targets. Simultaneously, IFN-γ secreting CD8⁺ T cells were enumerated after addition of MART-1 peptide (10μM) in ELISPOT assays and counts obtained with control peptide were subtracted. The data are shown as the mean ± SEM of triplicate cultures and are from 1 representative experiment of 2 performed. Cytotoxicity is expressed as LU/10⁷ cells at a reference lysis level of 20%.