CXCR4^wt-overexpressing CD33.CAR-CIKs have superior antileukemic activity against KG-1 cells in the BM. Data are shown as mean values ± SD. (A) Scheme of the AML model using NSG mice inoculated, via tail vein injection, with 5 × 10⁶ KG-1 cells and 14 days later with CD33.CAR⁺-, CD33.CAR⁺-CXCR4^wt–, or CD33.CAR⁺-CXCR4^mut–CIKs (10⁷ cells per mouse). Mice were euthanized 25 days after CD33.CAR-CIKs infusion and hCD45⁺CD33⁺ leukemia cells were enumerated in the BM, PB, and spleen via flow cytometry. (B) Representative flow cytometry plots of hCD33⁺ cell engraftment in the BM of mice untreated (UNTR) or treated. (C-D) Summary of the percentages and absolute numbers of hCD33⁺ in (C) the BM (for percentage: ∗P = .0106 for CD33.CAR⁺- vs CD33.CAR⁺-CXCR4^wt–CIKs; and ∗P = .0336 for CD33.CAR⁺-CXCR4^wt– vs CD33.CAR⁺-CXCR4^mut–CIKs; for absolute number: ∗P = .0156 for CD33.CAR⁺- vs CD33.CAR⁺-CXCR4^wt–CIKs; and ∗P = .0418 for CD33.CAR⁺-CXCR4^wt– vs CD33.CAR⁺-CXCR4^mut–CIKs, using a mixed-effect model), and (D) the spleen and PB (for percentage in the spleen: ∗∗∗P = .0003 and ∗∗P = .0086; for absolute number in the spleen: ∗∗P = .00107 and ∗P = .033, using a mixed-effect model), of mice at 25 days (n = 11 mice per group. Three independent experiments using CAR-CIKs generated from 3 different donors). (E) In a second experimental setting, using the same model shown in panel B, femoral BM aspiration was performed on mice starting from day 14 after CD33.CAR-CIK injection until survival, and percentage of hCD33⁺ leukemia cells in the BM was analyzed via flow cytometry. (F) Summary of percentage of hCD33⁺ cells in the BM, n = 4 mice per group from 4 independent experiments using CAR-CIKs generated from 4 different donors. (G) Kaplan-Meier survival curves of the same mice in panel F. Comparisons of survival curves were determined using log-rank test. (H) Percentage of residual migration of conjugates formed by CD33.CAR⁺-, CD33.CAR⁺-CXCR4^wt–, or CD33.CAR⁺-CXCR4^mut–CIKs with CD33⁺ KG-1 (stable conjugates) relative to CD33⁻ KG-1 cells (control). The conjugates were allowed to migrate toward CXCL12 in a transwell filter allowing the migration of single cells only (n = 7 independent experiments using CAR-CIKs generated from 7 different donors; ∗∗∗P = .0006 and ∗∗P = .00101, using t test).