FLT3 CAR NK-92 cells prolong the survival of mice that received human AML cell grafts but do not reject HSCs and their repopulation. (A) Bioluminescence imaging of NSG mice bearing human AML. NSG mice were inoculated with luciferase-expressing FLT3⁺ MOLM-13 AML cells via tail vein injection (day 0). Mice were injected on days 7 and 14 with 1 × 10⁷ unmodified NK-92 cells (NT), EV NK-92 cells, or FLT3 CAR NK-92 cells, followed by imaging of mice on day 20. (B) Mice bearing MOLM-13 cells treated with unmodified NK-92 cells, EV NK-92 cells, or FLT3 CAR NK-92 cells. Overall survival was estimated using Kaplan-Meier survival curves (n = 5 for each group). (C) Expression of FLT3 and CD123 on the surface of CD34⁺ HSCs and DCs including the plasmacytoid DC (pDC) and the conventional DC (cDC). (D) Scheme of the in vivo experiment assessing the impact of FLT3 CAR NK-92 on CD34⁺ HSC and differentiated cells. On day 1, 5 × 10⁵ human CD34⁺ HSCs were simultaneously IV injected into NSGS mice that express human IL-3, granulocyte-macrophage colony–stimulating factor, and stem cell factor. Four months later, mice were IV injected with either 5 × 10⁶ FLT3 CAR NK92 cells or 5 × 10⁶ EV NK92 cells weekly for 4 doses, and then mice were sacrificed on day 150 (n = 5 mice for each group). (E) At the time of euthanization, human CD34⁺ HSCs and their differentiation as measured based on the presence of mature lymphocytes and myeloid cells in the BM were assessed. Human CD45⁺ and CD34⁺ cells were counted as engrafted cells derived from human HSCs. A representative example of human CD45⁺CD34⁺, CD45⁺CD34⁻CD19⁺, CD45⁺CD34⁻CD11c⁺, and CD45⁺CD34⁻CD3⁺ from the BM is shown, defining human HSCs, B cells, DCs, and T cells, respectively. (F) Cumulative flow cytometric data are shown, demonstrating no difference in absolute cell number of total human CD45⁺ cells or the percentages of HSCs, B cells, DCs, or T cells as defined earlier, between mice infused with FLT3 CAR NK-92 cells and those with EV NK-92 cells. Data are presented as mean ± standard error of the mean (SEM). ∗∗P < .01. n.s., not significant.