Expression of a functional CAR by CD8⁺ Tregs does not affect their Treg-like phenotype and does not confer cytotoxic activity against HLA-A*02⁺ cells. (A) Cultured (14 days) A2-CAR (in red) and Her2-CAR (in blue) CD8⁺ Tregs and nontransduced CD8⁺ Tregs (NT, in green) were stimulated with PMA/ionomycin (except for CD25 analysis) in the presence of brefeldin A for 4 hours and analyzed for expression of Tregs-associated markers. Shown is the percentage of CD8⁺ Tregs expressing each marker. Mann-Whitney U test, *P < .05; **P < .01. (B) A2-CAR (red and purple lines, n = 6) and Her2-CAR (blue line, n = 4) CD8⁺ Tregs or A2-CAR CD8⁺ Teffs (green line) were cultured with ECs expressing (red, blue, and green lines) or not (purple line) HLA-A*02 in a range of CAR T cells:ECs ratios for 3 hours of culture, and ECs were analyzed for caspase 3 activation by flow cytometry. Percentage of specific EC death was calculated by the subtraction of the staining of ECs in absence of Tregs. Mean ± SEM is represented. Two-way RM test, **P < .01; ***P < .001. (C) A total of 1.5 × 10⁷  A2-CAR CD8⁺ Tregs or CD8⁺ Teffs were generated from HLA-A*02⁻ PBMCs. Irradiated HLA-A*02 transgenic NSG mice received either A2-CAR CD8⁺ Tregs or A2-CAR CD8⁺ Teffs alone to assess in vivo cytotoxicity as reflected by mice body weight loss (mean ± SEM). (D) Anatomopathological analysis of lesions in liver, lung, and gut performed at day 100 in HLA-A*02 transgenic NSG mice injected with A2-CAR CD8⁺ Tregs in comparison with A2-CAR CD8⁺ Teffs-injected mice analyzed at time of euthanasia. Arrows and stars show immune cell infiltrate and tissue injury.