Immobilized anti-Ly49A or anti-Ly49G mAbs inhibit NKG2D receptor-mediated IFN-γ and GM-CSF production by NK cells. ELISA plates (Immunolon; Nunc, Rochester, NY) were coated with 5 μg/mL anti-NKG2D mAb in the presence or absence of titrated concentrations of either anti-Ly49A (A1), anti-Ly49G2 (4D11) alone, or both together. Independently, appropriate isotype antibody controls were also titrated in similar concentrations to define the specificity of the inhibition. NK cells (10⁵ per well) derived from B6 were added to the mAb-coated plates and incubated for 18 to 20 hours before harvesting the culture supernatants. Culture supernatants were assayed for the effect of inhibitory Ly49 mAbs on NKG2D-mediated IFN-γ (A) or GM-CSF (B) productions. In the top panels of A and B, indicates anti-NKG2D mAb A10 alone; □, isotype control, IgG1; ▪, anti-Ly49A; ○, isotype control, IgG2; and •, anti-Ly49G2. In the bottom bar graphs, □ indicates A10 alone, and , anti-Ly49A and anti-Ly49G2 combined with indicated mAb concentrations.