Figure 2.
P4-10 CAR T cells specifically respond to GFRα4 protein in vitro
(A) Schematic of CAR construct inserted downstream of an EF1a-derived promoter within a 3rd-generation lentiviral vector plasmid. (B) Primary human T cells were activated using anti-CD3/anti-CD28-coated beads followed by transduction with lentiviral vectors encoding the indicated CAR genes or were left non-transduced (NTD). Seven days later, T cells were stained with biotinylated F(ab′)2-specifc goat anti-mouse or donkey anti-rabbit followed by streptavidin-AF647 and analysis by flow cytometry. (C) NFAT-GFP reporter Jurkat cells expressing no CAR (NTD), 19bbz, or the P4-10bbz CAR were cultured in wells that had been coated overnight with GFRα1, GFRα2, GFRα3, GFRα4, and OKT3 proteins. Following overnight culture, cells were analyzed by flow cytometry for GFP expression. A representative of 3 independent experiments is shown. (D and E) NFAT-GFP reporter Jurkat cells (D) or primary human T cells (E) expressing no CAR (NTD), or the P4-10bbz CAR were cultured in wells that had been coated overnight with OKT3 or GFRα4 (bound) or in media with soluble GFRα4 (soluble). Following overnight culture, cells were analyzed by flow cytometry for GFP expression (*p < 0.001 compared to media control, ANOVA, Dunnett’s multiple comparisons test). Representatives of 2 independent experiments each are shown. Error bars indicate 1 standard deviation of the mean.