Figure 1.

Ex vivo expansion of CD33.CAR-CIKs is associated with downregulation of CXCR4, and its expression can be stably increased using bicistronic SB transposon vectors without phenotypic alterations. Data are presented as individual values and the mean ± standard deviation (SD). (A-B) Summary of the percentage of CXCR4 expression on circulating CD3⁺ T cells and on CD33.CAR-CIKs at the end of ex vivo expansion. The analysis was performed on (A) all CD3⁺ cells (n = 16 different donors; ∗∗∗∗P < .0001 using paired t test) and on (B) CD4⁺ and CD8⁺ subpopulations (n = 8 different donors; ∗∗∗P = .0003 for CD4⁺; and ∗∗∗P = .00011 for CD8⁺, using t test). (C) Schematic representation of the vectors encoding CD33.CAR and CXCR4^wt or CXCR4^mut. Representative flow cytometry plots showing CD33.CAR and CXCR4 expression in CIKs transduced with CD33.CAR, CD33.CAR(2A)CXCR4^wt, or CD33.CAR(2A)CXCR4^mut transposon vectors. (D-E) Summaries of CXCR4 expression percentage in culture during time (D) and of CXCR4 mean fluorescence intensity (MFI) at the end of the culture (E) on CD33.CAR-, CD33.CAR-CXCR4^wt–, and CD33.CAR-CXCR4^mut–CIKs (n = 19 different donors; ∗∗∗∗P < .0001; ∗∗∗P = .0003; ∗∗P = .0012, using paired t test). (F) Summary of CD33.CAR expression percentage (left) and MFI (right) on CD33.CAR-, CD33.CAR-CXCR4^wt–, and CD33.CAR-CXCR4^mut–CIKs after ex vivo expansion (n = 19 different donors). (G) Percentages of CD4⁺, CD8⁺, CD56⁺ cells on CD3⁺ for CD33.CAR-, CD33.CAR-CXCR4^wt– and CD33.CAR-CXCR4^mut–CIKs after ex vivo expansion (n = 13 different donors; ∗P = .015 for CD4⁺; and ∗P = .031 for CD8⁺, using paired t test). (H) Different memory cell subsets (naive, central memory [CM], effector memory [EM], and effector memory reexpressing CD45RA [EMRA] cells) on CD3⁺ for CD33.CAR-, CD33.CAR-CXCR4^wt–, and CD33.CAR-CXCR4^mut–CIKs after ex vivo expansion (n = 13 different donors).