Enhancement of ADCC activity by YB-AHM and PBMCs pretreated with Len.

(A) RPMI 8226, U266, and OPM-2 MM cell lines, and the control HEL leukemic cell line, were incubated with PBMCs from a healthy donor for 4 hours at an E/T ratio of 10 in the presence AHM (○) or YB-AHM (♦) at various concentrations as indicated. The viability of target cells was analyzed by a flow cytometric PKH26 assay. ADCC activity was determined by percentages of 7AAD⁺ dead cells in PKH26-labeled target MM cells. (B) RPMI 8226 cells were incubated with PBMCs from a healthy donor for 4 hours in the presence of 0.1 µg/mL of AHM (○) or YB-AHM (♦) or absence (▪) at various E/T ratios as indicated. (C) PBMCs from 3 normal donors were cultured alone or in the presence of Len (3 µM) for 48 hours. These PBMCs were added to PKH26-labeled RPMI 8226 cells at an E/T ratio of 10 in the presence or absence of AHM (0.1 µg/mL) or YB-AHM (0.1 µg/mL) for 4 hours. (D) PKH26-labeled RPMI 8226 cells were incubated in triplicate with Len-treated or untreated PBMCs from a patient with MM at an E/T ratio of 10 in the presence or absence of YB-AHM (0.1 µg/mL) for 4 hours. Representative flow cytometric result is shown (left). PKH26-labeled RPMI 8226 cells were distributed in red squares. (E) PKH26-labeled RPMI 8226 cells were incubated with Len-treated or untreated PBMCs from 3 patients with MM at an E/T ratio of 10 in the presence or absence of YB-AHM (0.1 µg/mL) for 4 hours. Data presented are mean ±SD (*, p<0.05; **, p<0.01).