KD of B7-H3 expression induced NK cell-mediated apoptosis. (A-C) Time kinetics graph of B7-H3 KD cells and scrambled shRNA MV4-11 (A), U937 (B), and OCI-AML3 (C) AML cells cultured with rhB7-H3 protein in the presence or absence of NK cells at a 4:1 NK:target cell ratio. Leukemic cells were labeled with CytoLight red and annexin V green reagents. Apoptosis of leukemic cells was assessed via annexin V green staining every hour for up to 18 hours. (D-F) Bar graphs showing the percentage of annexin V positive cells (green staining) in B7-H3 KD MV4-11 at 18 hours (D), U937 at 18 hours (E), and OCI-AML3 at 14 hours (F) AML cells and scrambled shRNA control cells treated with rB7-H3 protein and NK cells at a 2:1 ratio. Data are plotted as mean values with error bars representing standard error (Student unpaired t test). (G-I) B7-H3 expression in AML patient samples and controls using our 3 anti-B7–H3 mAbs. PBMCs from AML patients and controls were stained with primary anti-B7–H3 mAbs T-1A5, HEK5-1B3, and 58B1 and secondary conjugated goat anti-mouse IgG (Alexa Fluor 647). DAPI was used to exclude dead cells, and B7-H3 expression was measured by flow cytometry (Wilcoxon rank-sum test). (J) Overlay plots showing cell-surface staining of B7-H3 in AML cell lines OCI-AML3, U937, MV4-11, and THP-1 using our anti-B7–H3 mAbs T-1A5, HEK5-1B3, and 58B1 measured by flow cytometry. MFI: mean fluorescence intensity. ***P < .0001.