Soluble NCR dimers recognize THP-1 cells and inhibit their killing and IFN-γ release by NK cells. (A) FACS analysis of THP-1 and K562-L cells stained with sNKp30 (shaded curve), sNKp44 (thin solid line), and sNKp46 (bold solid line) or anti-BirA1.4 mAb (dotted line) and with secondary FITC-labeled goat anti–mouse IgG. (B) Inhibition of NK cell–mediated killing of THP-1 cells by sNKp30, sNKp44, and sNKp46 dimers. A calcein release–based cytotoxicity assay was used to determine the lysis of THP-1 and K562-L cells by NK cells at the indicated effector-target ratios. The killing assay was performed in triplicate in the absence of sNCRs (▪) and in the presence of a mixture of sNKp30, sNKp44, and sNKp46 at 5 μg/mL (▨), 20 μg/mL (□), or 50 μg/mL (▦) of each dimer. Mean ± SEM values are shown. (C) Inhibition of IFN-γ release by NK cells cocultured with THP-1 cells, using anti-NKG2D mAb (20μg/mL) and sNKp30, sNKp44, and sNKp46 (10 μg/mL, each) or anti-BirA1.4 mAb (10μg/mL) in the indicated combinations. In the absence of blocking reagents (no mAbs), 100% IFN-γ release corresponded to 8.67 ± 3.5 ng/mL, as determined in 3 experiments.