DC CIK cells express both CAR and CCR efficiently and show potent and specific in vitro antileukemic activity against CD123⁺CD33⁺ targets. (A) Flow cytometric analysis of CD33 and CD123 expression on AML primary cells, KG-1 cell line, and on normal hCD34⁺ cells and endothelial TIME cell line. (B) CD33 and CD123 quantification on cell surface. The number of CD33 (n = 2 for KG-1, n = 4 for CD34⁺, n = 12 for AML blasts) and CD123 (n = 3 for KG-1, n = 5 for TIME, n = 4 for CD34⁺, n = 12 for AML blasts) molecules on the cell surface was quantified using a BD QuantiBRITE PE fluorescence quantitation kit. (C) DC IL-3z/CD33 vector scheme. Single CARs are included as controls. (i) single IL-3z CAR, (ii) single CD33 CAR with CD28-4-1BB in cis costimulation, and (iii) bicistronic DC IL-3z/CD33 CCR with a self-cleaving 2A peptide. (D) Representative dot plot of IL-3z CAR, CD33 CAR, and DC expression on CIK cells at the end of differentiation. Unmanipulated (NT) CIK cells were used as control. (E) Expression of IL-3 and scFv CD33 on the surface of IL-3z CAR (n = 4), CD33 CAR (n = 4), and DC CIK cells (n = 6) by flow cytometry at the end of differentiation. (F) Short-term (E:T ratio of 5:1; n = 4 in all groups) and (G) long-term (E:T ratio of 1:100; n = 4 in all groups) cytotoxicity and (H) cytokine release against CD123⁺/CD33⁺ KG-1, OCI-AML3, and THP-1 AML cell lines (n = 4 in all groups except for OCI-AML3 and MHH-CALL-4, n = 2). CD123⁻/CD33⁻ CHH-CALL4 B-ALL cell line has been included as control. Summary from 4 independent CAR CIK cell donors is shown in panels F, G, and H. Paired comparisons were performed using the Tukey test and adjusted for multiple comparisons. ns, not significant (P value >.05); ∗∗P value <.01, ∗∗∗P value <.001, ∗∗∗∗P value <.0001. B-ALL, B-cell acute lymphoblastic leukemia; LS, leader sequence; SP, spacer; TM, transmembrane domain.