DNAM-1–mediated signal induces maturation and activation of CD8α DCs. (A) Spleen cells from C57BL /6 mice were stained with PE-conjugated anti-CD11c, FITC-conjugated anti-CD8, and biotin-conjugated anti–DNAM-1, followed by APC-conjugated streptavidin. DNAM-1 expressions on CD8α^-CD11c⁺ and CD8α⁺CD11c⁺ cells were analyzed by flow cytometry. (B) CD11c⁺ DCs were separated from splenocytes of C57BL /6 mice by positive selection using MACS. Purified DCs were stimulated for 4 days with plate-coated anti–DNAM-1 mAb or control IgG, and stained with anti-CD11c, anti-CD8, and either control immunoglobulin, anti-CD80, anti-CD86, or anti–MHC class II (I-Ab). Mean fluorescence intensity of CD80, CD86, and I-Ab expressions on CD8⁺CD11c⁺ or CD8^-CD11c⁺ DCs were determined by flow cytometry. Data are representative of several independent experiments. (C) CD11c⁺CD8α⁺ or CD11c⁺CD8α^- DCs were stimulated with plate-coated mAbs indicated for 4 days, pulsed with the OVA peptide, and then cocultured with CD4⁺ T cells from splenocytes of OT-II mice for 7 days. CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 for 48 hours and IFN-γ concentrations in the culture supernatant were determined by ELISA. Data are representative of several independent experiments.