KIR2DL2 signaling blocks activating responses. (A) Control NKL cells (left) and NKL cells expressing KIR2DL2 (right) were stimulated with α-NK and either wild-type (WT) HLA-Cw3 or HLA-Cw3 containing the ILT2-binding mutation (IBM) as indicated. Both HLA-Cw3 proteins contained the importin-α peptide with p8 Ala. IFN-γ secretion was quantified by ELISA. Two independent experiments are shown for each cell type. Although maximal IFN-γ secretion varied from day to day, the relative differences in cytokine production between different stimulus conditions were consistent. All other figures presented in this paper used HLA-Cw3 containing the ILT2-binding mutation. (B–D) NKL cells expressing wild-type KIR2DL2 (B–D) or KIR2DL2(mut) (D) were added to plastic wells containing immobilized α-NK and the indicated HLA-Cw3 proteins. (B) IFN-γ secretion from NKL cells expressing KIR2DL2, measured by ELISA. Two independent experiments are shown. (C) Representative degranulation responses measured by surface expression of CD107a. Unstim., unstimulated. As with IFN-γ secretion, maximal degranulation responses were quite variable. However, the relative differences between stimulus conditions were consistent. (D) Dose–response curves showing induced degranulation from NKL cells expressing either wild-type KIR2DL2 or KIR2DL2(mut) (both Tyr 302 and Tyr 332 mutated to Phe) as a function of the concentration of HLA-Cw3(Ala) used during protein immobilization. In A and B, error bars represent SEM between replicates, with n = 3. All data are representative of at least two independent experiments. P-values were calculated using Student’s t test.