Cytotoxicity and IFN-γ production of NK cells in mixed culture with CpG-pDCs is dependent on type I IFNs and GITR. (A-B) NK cells were cultured with CpG B (○), pDCs plus CpG B (•), or IFN-α (2000 U/mL, □) for 24 hours. Cytotoxic activity of NK cells against K562 or Daudi was evaluated by 6 hours of ⁵¹Cr-release assay at different E/T ratios. Data are represented as the mean percentage of specific lysis ± SD and are representative of 5 experiments. (C) NK cells were cultured alone, together, or separately with pDCs plus CpG B in the presence or absence of control Igs, anti-type I IFNs/IFNR (a cocktail of Abs to IFN-α, IFN-β, and IFN-α/β receptor), and/or anti-GITRL in transwell chamber plates (0.4-μm porous membrane). After 36 hours of culture, cytotoxic activity of NK cells against Daudi was evaluated by 6 hours of ⁵¹Cr-release assay at an E/T ratio of 10. Data are represented as the mean percentage of specific lysis ± SD and are representative of 5 experiments. (D) NK cells were cultured with pDCs, which were preactivated with CpG B for 36 hours in the presence or absence of isotype Igs, anti-type I IFNs/IFNR, and/or anti-GITRL. Following this culture, the cytotoxic activity of NK cells against Daudi was evaluated by 6 hours of ⁵¹Cr-release assay at an E/T ratio of 10. Data are represented as the mean percentage of specific lysis ± SD and are representative of 5 experiments. (E-F) NK cells were cultured for 36 hours together or separately with pDCs plus CpG B in transwell chamber plates in the presence of isotype Igs, anti-type I IFNs/IFNR, and/or anti-GITRL (E) or cultured for 36 hours with pDCs that were preactivated for 36 hours with CpG B, with or without IFN-α (2000 U/mL) in the presence of isotype Igs, anti-type I IFNs/IFNR, and/or anti-GITRL (F). IFN-γ in the supernatant of NK-pDC culture was determined by ELISA. The data shown are representative of 5 experiments.