Antigen-presenting activity in mice immunized with mRNA-transfected cells loaded with α-GalCer. (A) We examined CD1d RNA expression levels in B16 cells, NIH3T3 cells, and murine bone marrow–derived DCs (mBM-DCs) as well as B16 and NIH3T3 cells after retrovirus-mediated transfer of a murine CD1d gene (CD1d^hi-B16, CD1d ^hi-NIH3T3) by real-time PCR. (B) Because the retroviral vector contained both murine CD1d and GFP genes, the stable CD1d^hi cell lines were selected to a purity of more than 98% on a FACSVantage cell sorter (left panel). The CD1d expression of parental cell lines or CD1d transfectants was analyzed by FACS (right panel). (C) After a 4-hour transduction, the expression of OVA protein was measured from cell lysates by ELISA. (D) The antigen-presenting activity was evaluated by coculturing OVA-transgeneic CD8⁺ T cells (OT-I cells) with each type of transfectant for 48 hours. The supernatants were collected and IFN-γ secretion was measured by ELISA. In this experiment, the B16 cell line was pretreated with IFN-γ for 12 hours to enhance MHC class I expression before coculture. (E) C57BL/6 mice were given 2 × 10⁶ OT-I cells and then immunized 24 hours later with OVA mRNA-transfectants loaded with or without α-GalCer. Absolute numbers of OT-I cells in the spleen were measured 3 days later. α-GalCer–loaded CD1d^hi-B16 transfected with OVA mRNA (CD1d^hi-B16/Gal-ova) was administered to mice as a positive control. Data are representative of 2 independent experiments with 2 mice per group. Data are means plus or minus SEM of 4 mice per group. *P < .025 (CD1d^hi-NIH3T3-ova vs CD1d^hi-NIH3T3/Gal-ova, CD1d^hi-B16-ova vs CD1d^hi-B16/Gal-ova).