Human ML NK cells are effectively transduced with CAR lentiviral vectors. (A) Schematic representation of the lentiviral cassette encoding the αCD19 CAR. P2A indicates the ribosomal P2A skip site. Transmembrane (TM)/hinge: CD8α; costimulatory domain (D1): CD137; and stimulatory domain (D2): CD3ζ. ITAMs indicated in light blue. (B) Schema of in vitro experiments. Purified NK cells were activated with IL-12, IL-15, and IL-18 or were control treated for 16 hours, washed, and transduced with CAR lentivirus for 2 days. After differentiating for 1 week, NK cell phenotype and functionality were assessed. (C) Summary of data of viral transduction efficiency between low-dose cNK cells (blue open triangle) and ML NK cells (green triangle; n = 17-23 donors from 15 independent experiments; each symbol represents 1 donor; line indicates mean ± SEM). Data were compared using paired Student t test. (D) Representative bivariate flow plots showing expression of GFP and sCD19 staining on 19-CAR-ML NK cells. (E) Representative overlaid viSNE plot of cNK (blue), ML (orange), and CAR-ML NK cells (green). (F) Representative viSNE density plot of cNK, ML NK, and CAR-ML NK cells. (G) Linear regression model of phenotypic markers of 19-CAR-ML NK cells (x-axis) and ML NK cells (y-axis). hCD19, human CD19; LTR, long terminal repeat; ns, no significance; scFv, single-chain variable fragment.