CI/NKG2^- NK cells in B6 mice respond poorly to cross-linking of stimulatory receptors. (A-C) Splenocytes from poly(I:C)-treated β2m^-/- or B6 mice were cultured for 5 hours on plates coated with increasing concentrations of the anti-NKG2D mAb MI-6 (A), the anti–NKR-P1C mAb PK136 (B), the anti-Ly49D mAb SED85 (C), or control rat IgG (A-C) in the presence of brefeldin A before staining and analysis. NK cells were gated as NK1.1⁺CD3^- cells except in panel B, where they were gated as DX5⁺CD3^- cells. Results are depicted as percentage ± SEM (n = 3 mice) of IFN-γ⁺ cells in each subset. In panels B and C, data were normalized based on percentage of NKR-P1C⁺ or Ly49D⁺ NK cells, respectively, in each subset, as determined before stimulation. CI/NKG2^- NK cells produced significantly less IFN-γ than their CI/NKG2⁺ counterparts after anti-NKG2D (P ≤ .03 for all doses), anti–NKR-P1C (P ≤ .04 for all but the lowest dose), or anti-Ly49D stimulation (P ≤ .03 for all but the lowest dose). (D) NK cells were stimulated with the indicated dilutions of a mixture of ionomycin and PMA for 5 hours in the presence of brefeldin A before analysis. The highest concentrations (dilution factor = 1) were 2.5 μg/mL ionomycin and 250 ng/mL PMA.