Anti-B7–H3 mAbs T-1A5, HEK5-1B3, and 58B1 enhance NK cell-mediated apoptosis in AML. (A) Representative images obtained at 0, 9, and 16 hours show apoptosis in OCI-AML3 cells treated with anti-B7–H3 antibodies and/or NK cells. OCI-AML3 cells were labeled with CytoLight red and annexin V-green. The overlap (yellow) represents apoptotic AML cells. The images were acquired by an Incucyte live-cell imager. (B) Time kinetics showing apoptosis in OCI-AML3 cells treated with anti-B7–H3 mAbs T-1A5, HEK5-1B3, and 58B1 or control IgG1 and NK cells measured each hour for 16 hours. (C-D) Bar graph shows the percentage of annexin V binding (green staining) at 16 hours in OCI-AML3 (C) and U937 (D) cells treated with anti-B7–H3 mAbs (1 μg/mL) and NK cells at a 2:1 NK:target ratio. Data are plotted as mean values with error bars representing standard error (Student unpaired t test). (E) Similar experiments were performed in primary AML cells (patient1) expressing B7-H3. (F) The bar graph shows the percentage of annexin V binding (green staining) at 8 and 24 hours in AML patients 1 and 2, respectively. *P < .05, **P < .0001, ***P < .0001.