The CD3×CD123 DART induces T-cell activation, expansion, and redirected killing of blasts in primary AML samples. PBMCs (2 × 10⁵ cells/well in 96-well plate) from primary AML samples (n = 6) were incubated with DARTs at 0.1 ng/mL in the absence of exogenous cytokines for 6 days. (A) Representative flow cytometry analyses reveal an increase in the relative percentage of CD45^hi lymphocytes compared with CD45^dim blasts in response to CD3×CD123 DART. (B) Representative flow cytometry of CD25 expression in T cells after exposure to DARTs. (C) T-cell number and (D) CD25 expression in primary patient samples. (E) AML blast percentage after DART exposure in primary patient samples. (F) Correlation between the relative percentage of surviving blasts after exposure to CD3×CD123 DART for 6 days and baseline expression of CD123 on AML blasts. The percentage of AML cells expressing CD123 and the relative mean fluorescence intensity (RMFI) of CD123 is shown. (G) Dose-response relationship in total number of surviving blasts after exposure to CD3×CD123 DART. PBMCs (2 × 10⁵ cells/well in 96-well plate) from AML patient 5 were incubated with PBS (No DART) or DARTs in the absence of exogenous cytokines for 6 days. Error bars represent the SD of duplicate or triplicate cultures in a single experiment. (H) Mononuclear cells from primary AML patient 1 were treated in duplicate for 6 days with DARTs 0.1 to 10 ng/mL in complete medium followed by incubation of viable cells in methylcellulose-based medium. Colonies were counted after 7 to 14 culture days. Data are representative of 2 patient samples. *P < .05, **P < .01, ***P < .001.