MART-1 mRNA–loaded CD40-B and DCs generate functional antitumor T cells. (A) CD40-B and DCs from healthy donors were electroporated with MART-1 mRNA or GFP mRNA and used to stimulate autologous PBLs weekly for 3 weeks. T-cell cultures were then analyzed with tetramers as in Figure 2, but using HLA-A2/MART-1 tetramer. (B) T-cell cultures stimulated with MART-1 mRNA–loaded CD40-B were assayed by IFN-γ ELISPOT using either T2 cells (left panel) pulsed with tax peptide or MART-1 peptide (shown compared to T cells only) or mRNA electroporated autologous DC (right panel) using GFP mRNA or MART-1 mRNA (shown compared with T cells only). Bars represent one standard deviation. P values shown are for comparisons between starred experiments. (C) T-cell cultures stimulated with CD40-B loaded with MART-1 mRNA or GFP mRNA were assayed for lysis of tumor cells. Left panel: closed symbols indicate MART-1+ Malme-3M cells; open symbols, MART-1+ SK-MEL-113 cells, shown for MART-1 T cells (circles) and GFP T cells (squares). Malme-3M tumor targets matched donors only at HLA-A2; SK-MEL-113 cells and donors were HLA mismatched. Right panel: ▴ MART-1^– SW-480 carcinoma cells electroporated with MART-1 mRNA. ▵ SW-480 cells electroporated with GFP mRNA, shown for MART-1 T cells. Similar results were obtained with 2 donors, except for experiments in panel B, which were performed for 1 donor.