Fig. 3: In vitro tumor targeting efficacy and mechanisms of Allo15CAR33-NKT cells. | Nature Communications

Fig. 3: In vitro tumor targeting efficacy and mechanisms of Allo15CAR33-NKT cells.

From: Allogeneic CD33-directed CAR-NKT cells for the treatment of bone marrow-resident myeloid malignancies

Fig. 3

a–e Studying the in vitro antitumor efficacy of Allo15CAR33-NKT cells against human AML cell lines. CAR33-T cells and non-CAR33-engineered PBMC-T cells were included as therapeutic cell controls. a Experimental design. b Schematics showing the indicated human AML cell lines. THP1-FG, THP1 cell line engineered to overexpress the firefly luciferase and green fluorescence protein dual reporters (FG); KG1-FG, KG1 cell line engineered to overexpress FG; HL60-FG, HL60 cell line engineered to overexpress FG; THP1-FGCD33-/-, THP1-FG cell line further engineered to knockout the CD33 gene; THP1-FGCD1d-/-, THP1-FG cell line further engineered to knockout the CD1d gene; THP1-FGCD33/CD1d-/-, THP1-FG cell line further engineered to knockout the CD33 and CD1d genes. c FACS detection of CD33 and CD1d expressions on the indicated AML cells. d Heatmap showing the NKR ligand expressions on the indicated AML cells. The number represents the percentage of NKR ligand-positive tumor cells out of the total tumor cells. Three independent tumor cell samples were analyzed, and the average numbers are presented. e Tumor cell killing data at 24 h (n = 4 from four different cell product donors). f, g Studying the tumor cell killing mechanisms of Allo15CAR33-NKT cells mediated by NKRs (i.e., NKG2D and DNAM-1). f Experimental design. g Tumor cell killing data at 24 h (E:T ratio = 10:1; n = 4 from four different cell product donors). h Diagram showing the CAR/TCR/NKR triple tumor-targeting mechanisms of Allo15CAR33-NKT cells, and the CAR single tumor-targeting mechanism of CAR33-T cells. GrzB, Granzyme B. Created in BioRender. FANG, Y. (2025) https://BioRender.com/j50y057. i, j Studying the expression of effector molecules of Allo15CAR33-NKT cells. i FACS detection of surface CD69 as well as intracellular Perforin and Granzyme B in Allo15CAR33-NKT cells. j Quantification of (i) (n = 3 from three different cell product donors). Representative of 3 experiments. Data are presented as the mean ± SEM. ns not significant, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, by one-way ANOVA (g, j), or two-way ANOVA (e). Source data and exact p values are provided as a Source Data file.

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