CART123 cells manifest multiple effector functions upon in vitro exposure to CD123-expressing targets. (A) Activation of CART123 cells by AML targets. CART123 cells were coincubated with normal bone marrow (NBM) cells, primary AML cells, or with CD123+ MOLM14 cells, and CD107a degranulation was measured via flow cytometry. CAR-expressing cells were identified by staining with goat anti-mouse F(ab′)2 reagent. CART123 cells alone and the CD123− Jurkat cell line were used as negative controls; P < .0001 (Student t test). (B) Antigen-specific cytokine production in response to CD123+ AML. CARs were detected using a goat anti-mouse F(ab′)2 (for CART123) or an anti-CAR19 idiotypic antibody (for CART19). Intracellular cytokines were interferon-γ, MIP1β, and tumor necrosis factor α. (C) Proliferation of CART123 in response to CD123+ primary AML. CART123 or CART19 cells were labeled with CFSE and exposed to primary AML cells for 96 hours. Proliferation was assessed by CFSE dilution; P < .001 (Student unpaired t test). (D) Cytotoxic targeting of primary AML blasts by CART123 after incubation for 16 hours at the indicated effector-to-target (E:T) ratios; CART19 cells were used as controls. Two representative examples are shown. (E) Cytokine profiling of CART123 (black) or CART19 (white) cells in response to 24-hour coincubation with MOLM14 cells. All results are representative of at least 2 experiments with similar results. IFN-γ, interferon-γ; IL-2, interleukin-2; GM-CSF, granulocyte macrophage colony-stimulating factor; TNF-α, tumor necrosis factor α.
