CD64 expression on susceptible AML cells trigger DNTs to produce TNFα. (A-B) DNTs and AML cells were cultured alone or together for 2 hours. The intracellular expression of TNFα was measured on DNTs (CD3⁺CD33^–) by flow cytometry. The bar graph shows the percentage expression (left), and the flow plot shows a representative histogram with MFI values (right) (A). The level of TNFα in the supernatants of cocultures (left) and AML or DNT cell alone groups (right) were determined by enzyme-linked immunosorbent assay (ELISA) (B). The experiments were performed in triplicates. The data shown are representative of 2 independent experiments. (C) CD64 expression by OCI-AML3^AAVS control (orange), OCI-AML3^CD64KO (dark blue), KG-1a (red), and MV4-11 (blue) cells. Representative histogram shows expression measured by flow cytometry with MFI values. (D-E) OCI-AML3^AAVS control, OCI-AML3^CD64KO, and KG-1a cells were cocultured with DNTs for 2 hours. Specific killing of AML cells was measured using flow cytometry (D). The level of TNFα from the coculture supernatants was determined by ELISA (E). The experiment was performed in triplicates, and the data are representative of 2 independent experiments. (F) OCI-AML3^CD64KO cells were untreated or pretreated with rTNFα (100 ηg/mL), washed with PBS, then cocultured with DNTs for 24 hours. Specific killing of AML cells by DNT was determined by flow cytometry. Data shown are representative of 2 independent experiments done in triplicates. Student t test and 1-way/2-way ANOVA were used. ∗∗P < .01; ∗∗∗P < .001. n.d., not detected; ns, nonsignificant.