CD40-B and DCs loaded with FluMP mRNA induce functional, specific T cells in healthy donors. (A) CD40-B and DCs from healthy donors were electroporated with FluMP mRNA or GFP mRNA and cocultured with autologous PBL for 7 days. T-cell cultures were then harvested and stained with anti-CD8, anti-CD14, anti-CD4, and HLA-A2/FluMP or HLA-A2/tax tetramers. Cells were gated on CD8⁺CD4^–CD14^– mononuclear cells, and the percentage of CD8⁺ tetramer+ cells is indicated. (B) Day 7 T-cell cultures stimulated with FluMP mRNA-loaded CD40-B (right and left panels) or DCs (middle panel) were assayed by IFN-γ ELISPOT using either T2 cells (left and middle panels) pulsed with tax peptide or FluMP peptide (shown compared with T cells only) or mRNA electroporated DC (right panel) using GFP mRNA or FluMP 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 were stimulated once with CD40-B loaded with FluMP mRNA (upper left and lower left panels), once with DCs loaded with FluMP mRNA (upper right panel), or once (lower left) or 3 times (lower right) with CD40-B loaded with GFP mRNA and were assayed for lysis of peptide-loaded T2 cells (• FluMP peptide; ○ tax peptide) or mRNA-loaded SW-480 carcinoma cells (lower panels: ▴ or , FluMP mRNA–loaded targets; ▵ or ⋄, GFP mRNA–loaded targets; ▴ or ▵, FluMP mRNA–stimulated T cells; or ⋄, GFP mRNA–stimulated T cells). SW-480 cells are HLA-A2⁺ and MHC class II–. Similar results were obtained with 3 donors.