De novo priming of CD8⁺ T cells with RNA-pulsed DCs. (A) Coexpression of HLA-A2 and tyrosinase proteins was detected by flow cytometry in DCs of an HLA-A2⁺ donor transfected with 24 μg tyrosinase ivt-RNA (left histograms) and DCs of an HLA-A2⁻ donor transfected with 48 μg HLA-A2 and 24 μg tyrosinase ivt-RNA (right histograms). Stained samples are represented by filled curves and corresponding controls by empty curves. Data are representative of 9 independent experiments, demonstrating that all DCs used for T-cell priming coexpressed both proteins. (B) Columns represent the amount of IFN-γ (picograms per milliliter) secreted by a tyrosinase-independent HLA-A2 allo-reactive T-cell clone (JB4) and an HLA-A2–restricted tyrosinase peptide–specific T-cell clone (Tyr-F8) after coincubation with RNA-pulsed DCs, 10 hours after electroporation. IFN-γ was quantified in culture supernatants by ELISA. Mean values and mean deviations represent duplicates. n.d. indicates not detectable. (C) DCs of an HLA-A2⁻ donor were transfected with HLA-A2 (48 μg) and tyrosinase (24 μg) or melan-A (48 μg) ivt-RNA alone and in combination and used for coincubation with the HLA-A2 allo-reactive CTL clone (JB4), 2 HLA-A2–restricted tyrosinase peptide–specific CTL clones (Tyr-F8 and IVS-B), and an HLA-A2–restricted melan-A peptide–specific CTL clone (A42) 10 hours after electroporation. IFN-γ was quantified in culture supernatants by ELISA and presented as picograms per milliliter. Mean values and mean deviations represent duplicates. n.d. indicates not detectable.