Figure 2.

BiTEs secreted from T cells specifically bind to target antigens and T cells

(A) Schematic vector maps of 806CAR/806BiTE/Hu08CAR/Hu08BiTE constructs. (B) Flow cytometric detection of T cell transduction by mCherry expression. (C) Conditioned media of T cells in each group tested by ELISA to evaluate the binding ability of secreted BiTEs to recombinant EGFR, EGFRvIII, and IL13Rα2 proteins. (D) The GSC line 5077 was lentivirally transduced to overexpress EGFRvIII or IL13Rα2 (red) and assessed via flow cytometry. Staining control was showed (blue). (E) Conditioned media of CAR/BiTE T cells was collected and co-cultured with UTD T cells and target cells. BiTE binding on T cells was detected by biotinylated protein L with secondary streptavidin coupled FITC after 16-h co-culture. The median fluorescence intensity was quantified on CD4- and CD8-positive T cells. (F) Flow based results of representative samples in (E). CD8 was stained to distinguish the CD4-positive and CD8-positive subgroups of T cells along the x axis. Statistically significant differences were calculated by one-way ANOVA with post hoc Tukey test. ∗∗∗∗p < 0.0001. Data are presented as means ± standard deviation.