018Z cells are efficiently killed by primary NK cells through interaction with NKG2D receptors. (A) IL-15 levels (enzyme-linked immunosorbent assay) in the medium of various ALL cell lines after overnight incubation at 37°C. (B) 018Z and REH cells were incubated for 5 hours with human primary NK cells at various effector to target (E:T) ratios. Specific killing of 018Z cells was significantly higher than that of REH cells for all E:T ratios. *P < .001; **P < .0001 (t test). (C) HLA-A/B/C expression on REH (black line) and 018Z cells (dashed line). (D) Binding of Fc-fusion proteins of NK receptors to 018Z cells. 018Z cells were incubated with various human Fc-fusion proteins of NK receptors followed by labeling with goat anti-hIgG (black line): (i) hNKG2D-Ig, (ii) mNKG2D-Ig, (iii) NKp44-Ig, (iv) hNKp46-Ig, (v) mNKp46-Ig, and (vi) NKp30-Ig. Nonspecific binding was detected by staining with human Fc-protein (filled gray). (vii) Expression of the hNKG2D ligands on 018Z cells. Purified mouse anti MICA (black line), MICB (dotted), and ULBP2 (dashed), followed by labeled goat anti-mIgG (filled gray). Results are representative of 3 independent experiments. (E-F) Killing assay of 018Z cells by IL-2-activated human primary NK cells with or without blocking antibodies for NKG2D and DNAM-1, respectively. Pretreatment of NK cells with blocking antibodies for NKG2D significantly reduced the killing of 018Z cells. Values are shown as means ± standard deviation of triplicates. *P < .00001 (t test, no mAb or isotype control compared with anti-hNKG2D).