A2-CAR CD8⁺ Treg therapy prevents HLA-A*02⁺ PBMCs from inducing xenogeneic GVHD. (A) Schematic depicting the model of GVHD xenogeneic reaction induced by 1.5 × 10⁷ IV injected human PBMCs toward 1.5 Gy irradiated mouse tissues. PBMCs used were from a HLA-A*02⁺ healthy volunteer (HV#1) and were co-injected or not with CAR CD8⁺ Tregs generated from a HLA-A*02⁻ healthy volunteer (HV#2) at PBMCs:Tregs ratios of 1:1 or 3:1. (B-C) Percentage of mouse survival (B) and percentage of mouse body weight followed-up over time (C) after cell transfer in mice injected with PBMCs alone (green line, n = 4) or cotreated with A2-CAR CD8⁺ Tregs at a ratio of 1:1 (red line, n = 4) or 3:1 (orange line, n = 3), or with Her2-CAR CD8⁺ Tregs at a ratio of 1:1 (blue line, n = 4). (B) Log Rank (Mantel Cox) test, **P < .01. (C) One line represents 1 mouse. Two-way RM ANOVA test, ***P < .001; ****P < .0001. (D) Percentage of human cells in mice blood from day 0 to day 100. n = 3 for each group and n = 2 for PBMCs alone. One line represents 1 mouse. (E) Histological analysis of organ integrity by HPS coloration at day 15 for mice injected with PBMCs and day 100 for mice injected with PBMCs:A2-CAR Tregs at a ratio of 3:1 and 1:1. Arrows show immune cell infiltrate, stars hepatocyte necrosis.