A2-CAR CD8⁺ Tregs are specifically activated through the CAR. (A-B) A2-CAR and Her2-CAR CD8⁺ Tregs were cultured with allogeneic APCs expressing or not HLA-A*02 at a ratio of 1:1 and then analyzed for activation markers. (A) CAR-Tregs were cocultured with APCs for 5 to 20 minutes and fixed and stained for phosphorylated Zap70, as revealed by mean fluorescence intensity. Time 0 corresponds to no contact with APCs. n = 5 for each group. (Left) Mean ± SEM. (Right) Representative staining. Two-way RM ANOVA test and Bonferroni posttest, *P < .05; **P < .01. (B) Cells were cocultured for 24 hours in the presence of brefeldin A for the last 4 hours, and analyzed by flow cytometry for activation markers and cytokines expression. Unstimulated Tregs (Tregs with no APCs) are shown as basal control. Percentage of CAR CD8⁺ Tregs expressing the marker is shown. n = 3-7 for each group. Mean ± SEM are represented. Mann-Whitney U test, *P < .05; ****P < .0001.