IL-12, IL-23, and IL-27 regulation of ITAM receptors. (A) IL-23 and IL-27 synergy of IFN-γ induction by NK receptors. Enriched NK cells were evaluated for synergy with media, IL-12, IL-23, and IL-27 by addition into the assay for 4 hours after precoating with anti-Ly49D (4E5). Values are representative of 3 experiments. (B) Reversal of the dominant inhibitory signal by IL-12. Highly enriched Ly49G2⁺ NK cells were expanded for 4 days with IL-2 after selection by antibody-coated magnetic beads. Cells were depleted of CD3, CD19, and CD24, then selected into Ly49G2⁺ subsets. Cells were greater than 95% Ly49G2⁺, 52% Ly49D⁺, 88% NKG2D⁺, and greater than 95% NK1.1⁺. NK cells were precoated with NKR antibodies, then cross-linked for 4 hours with or without IL-12. Values are representative of 2 experiments. (C) In vivo evaluation of NKG2D synergy with IL-12. B6 mice were injected intrasplenically with either Baf3- or Rae1γ-expressing Baf3 cells (5 × 10⁵ cells). After 15 minutes, spleens were surgically removed. After 1 hour, mice were injected intraperitoneally with 10 ng IL-12 protein, and serum was collected for indicated times to 48 hours. Serum was evaluated for cytokine production using the CBA T_H1/T_H2 kit (Becton Dickinson). Values represent mean ± SE, with 5 mice per group.