Figure 3.

CAR-Mediated Activation of NF-κB and NFAT Reporter Genes

(A) Schematic representation of CAR-modified reporter cells. scFv, single-chain fragment variable; S, spacer; TM, transmembrane domain. (B) CAR expression of transduced reporter cells. ROR1-CAR was directly stained with AF647-labeled ROR1 protein, and CD19-CAR was directly detected by its N-terminal c-Myc tag. UTD, untransduced. (C) Target antigen expression of transduced BW5147 stimulator cells in comparison to native BW5147 cells. Numeric values represent the MFI of transduced cells minus the MFI of native cells. (D) MFI of ECFP and EGFP after stimulation of ROR1-CAR and CD19-CAR reporter cells with stimulator cells at a 2.5:1 ratio for 24 hr. (E) ECFP and EGFP expression kinetics of stimulated ROR1-CAR reporter cells. Data represent MFI ± SD. (F) NF-κB and NFAT activation of ROR1-CAR and CD19-CAR reporter cells co-cultured with human K562 cells expressing either ROR1 or CD19 or JeKo-1 cells expressing both antigens. The activation in percent ± SD was calculated by normalizing CAR stimulation to the positive control (stimulation with BW-OKT3). (G) MFI of ECFP and EGFP after a 24-hr stimulation of ROR1-CAR reporter cells with plate-bound ROR1 protein. Statistical significance for (E) (n = 4) and (F) (n = 3) was determined using two-way ANOVA with Holm-Sidak post hoc test; ns, not significant; **p < 0.01, ***p < 0.001, and ****p < 0.0001.