CART-mediated transfection of anti-CD19 CAR generates cytotoxic human CAR NK cells. Isolated primary resting human NK cells were transfected with an mRNA encoding an hCAR or mCAR. The cells were incubated for a total of 18 hours before being cocultured with wild-type CD19⁺ Nalm6 (filled circles) or CD19KO Nalm6 (open circles) target cells for 6 hours followed by flow cytometric analysis. (A) hCAR expression by NK cells in the absence of target cells, as detected by fluorescein isothiocyanate–conjugated soluble human CD19. (B) Percentage of dead CD19⁺ wild-type Nalm6 or CD19KO Nalm6 cells after a 20:1 effector/target (E/T) ratio coculture with transfected NK cells.⁶¹  (C) CD107a, IFN-γ, and TNFα expression after 1:3 E/T ratio coculture with Nalm6 cells. For all panels, n = 8 donors. *P < .05 by Wilcoxon matched-pairs signed rank test with Bonferroni correction for multiple testing.