CXCR4-overexpressing CD33.CAR-CIKs have enhanced in vivo BM homing ability. Data are presented as individual values and the mean ± SD. (A) Percentage of migration of CXCR4-overexpressing CD33.CAR⁺-CIKs in response to mouse BM supernatant (n = 5 independent experiments using CAR-CIKs generated from 1 donor and supernatant samples from 5 different mice; ∗P = .027 using paired t test). (B) Scheme of CD33.CAR-CIKs homing model using NSG mice inoculated via tail vein injection with 10⁷ CD33.CAR⁺-, CD33.CAR⁺-CXCR4^wt–, or CD33.CAR⁺-CXCR4^mut–CIKs. Mice were euthanized 7, 10, or 14 days after infusion, and hCD45⁺CD3⁺ T cells were enumerated in the BM, PB, and spleen via flow cytometry. (C) Representative flow cytometry plots of hCD45⁺ cell engraftment within mice BM at 7 days after infusion. (D-E) Summary of the percentages and absolute numbers of hCD45⁺ in (D) the BM, and (E) the PB and spleen of mice at 7 days after infusion (n = 12 mice in the CD33.CAR⁺- and CD33.CAR⁺-CXCR4^mut–CIKs groups, n = 11 mice in the CD33.CAR⁺-CXCR4^wt–CIKs group. Three independent experiments using CAR-CIKs generated from 3 different donors. For percentage of hCD45⁺ in the BM: ∗∗∗P = .0006 and ∗∗P = .0038. For the absolute number of hCD45⁺ in the BM: ∗∗∗∗P < .0001, ∗∗P = .0035, and ∗P = .0369. For the absolute number of hCD45⁺ in the spleen: ∗P = .0393. A mixed-effect model was used). (F-G) Summary of the percentages and absolute numbers of hCD45⁺ in the BM of mice euthanized 10 days (F) or 14 days (G) after infusion (n = 12 mice in the CD33.CAR⁺-CIKs group, n = 10 mice in the CD33.CAR⁺-CXCR4^wt–CIKs group, and n = 13 mice in the CD33.CAR⁺-CXCR4^mut–CIKs group for experiments at 10 days; ∗∗∗∗P < .0001. n = 11 mice in the CD33.CAR⁺-CIKs group, n = 8 mice in the CD33.CAR⁺CXCR4^wt-CIKs group, and n = 10 mice in the CD33.CAR⁺-CXCR4^mut–CIKs group for experiments at 14 days; ∗∗∗∗P < .0001, ∗∗P = .0029, and ∗P = .0425. Three independent experiments using CAR-CIKs generated from 3 different donors. A mixed-effect model was used).