ODNs directly bind KIR3DL2-Fc and down-regulate the expression of KIR3DL2 in freshly isolated NK cells. (A) Plates coated with KIR3DL2*003-, KIR3DL2*008-, or KIR2DL1-Fc soluble receptors were incubated with biotinylated sequence of ODN B or ODN C followed by streptavidin horseradish peroxidase (HRP)–conjugated second reagent or with anti–human IgG HRP-conjugated mAb. Arbitrary units (AU) on y-axis represent the ratio between optical density (OD) values obtained by analyzing ODN/KIR-Fc interaction and OD values obtained by testing binding of anti–human IgG HRP to the corresponding KIR-Fc molecules. Results are presented as medians of 3 to 8 independent experiments, each performed in duplicate. IQRs are reported. (B) Binding of ODN C to plates coated with KIR3DL2*003-Fc was tested in the absence or presence of dextran sulfate (DS) at the indicated concentrations. As control, we measured binding of an anti-KIR3DL2 mAb to KIR3DL2*003-Fc under the same experimental condition. AUs on y-axis represent the ratio between OD values obtained analyzing ODN/KIR3DL2-Fc interaction or mAb/KIR3DL2-Fc interaction and OD values obtained by testing the binding of anti–human IgG HRP to KIR3DL2-Fc–coated plates. A representative experiment of 3 independent ones is reported. (C) Binding of biotinylated sequences of ODN B or ODN C to plates coated with the indicated KIR-Fc molecules was determined and represented as described in panel A. Bars represent medians of 3 to 8 independent experiments performed in duplicate; IQRs are reported. (D) Freshly isolated NK cells were cultured for 20 hours either in the absence or in the presence of ODN C, and then assessed by double cytofluorimetric analysis for CD56 and KIR3DL2 (Q66 mAb) or KIR3DL1 (Z27 mAb) or KIR3DL2 and KIR3DL1 (AZ158 mAb) expression. AZ158^dull and AZ158^bright NK cells correspond to KIR3DL2⁺ and KIR3DL1⁺ NK cells, respectively.³²  Mean fluorescence intensities (MFIs) are reported. Results are representative of 3 independent experiments.