Co-expression of miR17-92 in CAR-T-cells confers resistance to suppressive effects of TMZ. CAR-T-cells and those co-transduced with miR-17-92 were co-cultured with aAPCs expressing EGFRvIII in the presence of the indicated concentrations of TMZ. (A-C) Open bars and closed bars represent results from CAR-T-cells (without miR-17-72) and miR-17-92 co-transduced CAR-T-cells, respectively. (A) IFN-γ produced by the transduced T cells during the last 24 h of 96 h co-culture. * indicates P < 0.05 for comparison of the two cell types at the given condition. TMZ significantly suppressed IFN-γ production in each of the two cell types (P < 0.05 by one-way ANOVA). (B) Relative proliferation levels between the groups were evaluated by WST1 assay following the 3-day co-culture. (C and D) Apoptotic death of CAR-T-cells evaluated by Annexin-V and PI. (C) Mean fluorescent intensity for Annexin-V on CAR-T-cells exposed to TMZ. Values indicate mean ± SD in triplicate wells. (* indicates P < 0.05) (D) Flow-cytometric histograms for Annexin-V+ and/or PI+ in one of the three experiments (all had similar results).