Figure 3.

P4-10 CAR T cells specifically respond to GFRα4-expressing cells in vitro

(A) NFAT-GFP reporter Jurkat cells expressing no CAR (NTD), 19bbz, or the P4-10bbz CAR were co-cultured with the indicated stimuli overnight, followed by assessment of GFP expression in the Jurkat cells by flow cytometry (∗p < 0.05, ANOVA, Dunnett’s multiple comparisons test compared to media control). A representative of 3 independent experiments is shown. (B) Primary human T cells expressing no CAR (NTD), 19bbz, or P4-10bbz were co-cultured for approximately 18 h with ⁵¹Cr-loaded TT cells or K562 cells expressing the indicated antigens, at the indicated effector:target ratios. Following co-culture, supernatant was assessed for released radioactivity. Percentage of the lysis was calculated according to the formula: 100 × (experimental − spontaneous)/(maximum − spontaneous), where spontaneous represents radioactivity released from cultures of target cells alone and maximum represents radioactivity released by target cells lysed with 5% SDS. Error bars represent 1 standard deviation of three technical replicates, and results are representative of at least 3 independent experiments. (C) Primary human T cells expressing no CAR (NTD), 19bbz, or P4-10bbz were co-cultured for 24 h with media, Nalm6 cells, TT cells, or beads coated with anti-CD3/anti-CD28 antibodies. IL-2 and IFNγ in co-culture supernatants were measured by ELISA. All error bars indicate 1 standard deviation of three technical replicates, and results are representative of at least 3 independent experiments.