The CD3×CD123 DART binds the CD3/TCR complex and human CD123 and mediates cell association and T-cell activation and expansion in vitro. (A) Expression of GFP and CD123 in K562 and A20 cell lines transduced with GFP alone or GFP-CD123. (B) K562^GFP-CD123 and (C) A20^GFP-CD123 cells were incubated at a 1:1 ratio with VPD450-labeled Jurkat cells in the presence of the indicated DARTs (10 ng/mL). K562^GFP and A20^GFP cell lines were used as controls. Association is measured by flow cytometry as the percentage of GFP⁺VPD450⁺ events divided by the total number of GFP⁺ and VPD450⁺ cells. Each bar in the summary graphs represents the average of 3 separate experiments, where samples were analyzed in duplicate in each experiment. (D-E) Human T cells (4 × 10⁴ cells/well) were cultured with IL-2 (10 IU/mL) and irradiated K562^GFP or K562^GFP-CD123 cells (100 Gy) at a 1:1 ratio in the presence or absence of the indicated DARTs for 5 days. (D) Assay of T-cell activation after exposure to DARTs. An increase in CD25 expression by flow cytometry demonstrates activation of T cells in the presence of CD3×CD123 DART compared with control DARTs. (E) Assay of T-cell proliferation after exposure to DARTs. An increase in the number of CD3⁺ cells demonstrates proliferation of T cells in the presence of K562^GFP-CD123 cells and CD3×CD123 DART compared with control DARTs and K562^GFP cells. One representative example is shown out of two experiments with different donors. The data are shown as means ± standard deviations, where each point was measured in triplicate. *P < .05, **P < .01, ***P < .001.