Figure 6.

BiTEs’ activation significantly up-regulated checkpoints and T cell effector phenotypes

(A) Flow panel of EGFR (left) and IL13Rα2 (right) expression (red) on glioma line D270 with staining control (blue). (B) Flow cytometry determined T cell proliferation assay with CFSE staining was performed on UTD T cells, 806CAR T cells, 806BiTE T cells, Hu08CAR T cells, and Hu08BiTE T cells on day 4 (upper panel) and day 7 (lower panel) coculturing with D270 cell line. The median fluorescence intensity was quantified on CD4-positive and CD8-positive subgroups of T cells. (C) The expression of checkpoints (PD-1, CTLA-4, and TIM-3) on the T cells was determined by flow cytometry after overnight co-culturing of CAR/BiTE T cells with D270 cell line. Flow based results of representative samples were illustrated, CD8 was stained to distinguish the CD4-positive and CD8-positive subgroups of T cells along the x axis. The median fluorescence intensity was quantified and compared between CAR T cells and BiTE T cells. (D) The expression of CD45RA, CCR7, and CD62L on the T cells was determined by flow cytometry after 4 days co-culturing of CAR/BiTE T cells with D270 cell line. Flow-based results of representative samples were illustrated. The median fluorescence intensity was quantified and compared between CAR T cells and BiTE T cells on CD4-positive and CD8-positive subgroups. Statistically significant differences were calculated by one-way ANOVA with post hoc Tukey test. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are presented as means ± standard deviation.