NSG mice were transplanted with HLA-A*02:01⁺ human skin and injected 6 weeks later with either PBS (n = 3), or HLA-A*02:01^neg PBMCs alone (n = 4) or with a 2:1 ratio of autologous H1k2 hA2-CAR Tregs (H1k2, n = 6) or ΔNGFR Tregs (n = 6). PBMCs/hA2-CAR Tregs were from 2 individual donors, tested in one experiment. Mice were monitored 3 times weekly and sacrificed 28 days after cell injection for mRNA and histology assessment. (A) Body weight was monitored 3 times weekly, and (B) the proportion of human CD45⁺ cells in the blood (left) and spleen (right) was measured on day 28. (C) Cumulative histological score of transplanted skin sections as determined by H&E staining. (D) Transplanted skin grafts were immunostained at experiment end point to quantify the amount of involucrin expression and proportion of Ki-67⁺ cells in the epidermis. Scale bars: 100 μm (top row), 20 μm (bottom row). (E) mRNA expression of the indicated genes within transplanted skin sections was determined by qPCR. (F) Transplanted skin grafts harvested at the experiment endpoint were immunostained to quantify the proportion of FOXP3⁺ cells within human CD45⁺ cells. Scale bars: 50 μm (top row), 20 μm (bottom row). (G) Transplanted skin grafts, intestine, lung, and liver sections were immunostained at the experiment end point to show the proportion of FOXP3⁺ cells within human CD45⁺ cells in each tissue. Scale bars: 100 μm. Each data point represents 1 mouse. Box-and-whisker plots show mean ± range. Statistical significance determined by 1-way ANOVA comparing PBMCs with PBMCs + NGFR or H1k2. For immunofluorescence quantifications in D and F, each data point represents the average cell number counted in 18–27 fields of view from 1 section/mouse. *P < 0.05.