ULBP2-BB4 increases the suspectibility of MM cell lines to NK cell–mediated cytotoxicity. (A) NK cells from healthy donors were incubated overnight with IL-2 (10 U/mL; black line, marked with an arrow) or IL-2 + IL-15 (100 ng/mL; green line, marked with an arrow), and the expression of the NKG2D receptor was analyzed with a mouse anti-NKG2D antibody and a secondary FITC-coupled goat anti–mouse IgG. Note that NKG2D receptor expression was induced using IL-2/IL-15, whereas IL-2 alone revealed only minor effects. One representative polyclonal NK cell population is shown. (B-D) The IL-2/IL-15–stimulated NK cells were used at the indicated effector-target ratios for killing assays with U-266 (B), RPMI-8226 (C), and 293T (D) tumor cells in the presence of ULBP2-BB4 (5 μg/mL; •) or the single-chain BB4 (2.5 μg/mL; ○). For blocking experiments (▵) the NK cells were preincubated with 10 μg/mL anti-NKG2D antibody. Measurements were performed in triplicates and 1 of at least 2 independent experiments is indicated. The increase of cell lysis through ULBP2-BB4 was significant at all effector-target ratios for U-266 and RPMI-8226 cells, whereas no increase was observed for 293T cells. Error bars indicate SD (n=3).