HLA-A*02⁺ recognition by A2-CAR CD8⁺ Tregs induces a fine-tuned activation of ECs. (A-B) HLA-A*02⁺ (red, blue, green, and pink lines) or HLA-A*02⁻ (purple line) ECs were labeled with Fura-2 probe calcium flux reporting marker and cocultured with A2-CAR (red and purple lines) or Her2-CAR CD8⁺ Tregs (blue line) or CD8⁺ Teffs (green line) for 15 minutes under microscopic observation (time-lapse assay) to measure calcium flux in ECs. Positive control of activation was obtained by adding 10 µg/mL anti-HLA-A*02 mAbs to ECs culture (pink line). (A) Activation score of ECs over coculture duration with T cells was calculated by doing the ratio of free calcium (340 nm emission of Fura-2) divided by bound calcium (380 nm emission of Fura-2). Mean ± SEM are represented. Two-way RM ANOVA test, *P < .05; **P < .01. (B) Representative photos after 5 minutes of coculture. Resting cells are colored in blue and turn to red when activated. (C) A2-CAR (red) or Her2-CAR CD8⁺ Tregs (blue) were cultured with HLA-A*02⁺ ECs for 6 hours and analyzed by quantitative reverse transcription PCR for expression of genes associated to status of ECs or adhesion or cell recruitment. Tumor necrosis factor α (purple) and IFN-γ (green) were added as controls for EC activation. Gene expression was normalized to basal expression in unstimulated ECs. Mean ± SEM is shown. Mann-Whitney U test, *P < .05; **P < .01.