IL-15–expressing CAR NK cells exhibit robust antigen-specific cytotoxicity and IL-15 secretion against Raji cells. (A) Schematic representation of the lentiviral vectors encoding anti-CD70 CAR (70-IL-15) and anti-CD19 CAR (19-IL-15). (B) Percentage transduced NK cells expressing anti-CD70 CAR or anti-CD19 CAR 2 days after transduction (n = 5). Data show mean ± standard deviation (SD), representative of 3 independent experiments. (C) Correlation analysis between IL-15 production and percentage of CAR⁺ NK cells, 48 hours after anti-CD70 CAR or anti-CD19 CAR lentiviral transduction. Simple line regression was used to fit the correlation between IL-15 concentration and the percentage of CAR⁺ cells. (D) Proliferation of anti-CD70 CAR, anti-CD19 CAR NK cells, or non-NT-NK cells after stimulation with irradiated K562-mbIL21-mb41BBL feeder cells for 15 days. Data were representative of mean ± SD; n = 5. (E) Flow cytometry–based cytotoxicity assay of indicated CAR NK cells against Raji, CD19 KO Raji, or CD70 KO Raji. Bars in green indicate the percentage of NT-NK or CAR NK cells, and bars in blue indicate target tumor cells in the cocultured system. Data were representative of mean ± SD of 3 independent experiments; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. Two-way analysis of variance (ANOVA) and Tukey tests were used for P values. (F) The indicated Raji lymphoma cells were cocultured with the indicated CAR-transduced NK cells at a ratio of 1:1 for 5 hours, respectively, and then the percentage of CD107a⁺ cells in each group were detected by flow cytometry. ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. P values were determined by 1-way ANOVA test. (G) Enzyme-linked immunosorbent assay detection of IL-15 in the supernatants after the indicated Raji cells were cocultured with the indicated CAR-transduced NK cells at a ratio of 1:2 for 24 hours or 48 hours. Data show mean ± SD, and P values were calculated by 1-way ANOVA and Tukey test; ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. ns, no significance.