Fig. 2

MSLN-CAR T cells target MUC16-overexpressing cells for cytotoxic killing. A Structure of the pLGNe-MUC16 expression plasmid used for overexpression assays. B The level of MUC16 protein overexpression in SKOV3 cell line (left) was evaluated by Western blot (light), with Tubulin was used as a loading control. Lane 1 = molecular marker; Lanes 2 = SKOV3 cell lysate; Lanes 3 = MUC16 overexpression in SKOV3 cell lysate. B and C consist of three technical replicates. The pictures represent one example of three technical replicates. C Flow cytometry was used to detect MUC16 expression on the surface of viable, SKOV3 cells. D Cytotoxicity of MSLN-CAR T cells following their co-culture with MUC16-overexpressing cells as tested with a luciferase-based assay. CAR T cells were incubated with luciferase-expressing target cells for 20 h at an effector to target (E:T) ratio of 2:1. E, F ELISA-based quantification of IFN-γ (E) and TNF-α (F) released in response to coculture with Mock or MSLN-CAR T cells at an E:T ratio of 2:1. G, H CAR T cells were co-incubated with target cells expressing luciferase at varying effector to target (E:T) ratios for 20 h. D–H consist of three biologic replicates and three technical replicates. Statistics: two-tailed one-way ANOVA. The results are presented as the mean volume ± SD; ns = not significant; *P < 0.05, **P < 0.01 vs CD19-CAR or NTD