The transfer of CD8⁺iTregs prophylactically attenuates cGVHD pathogenicity. (A-B) Lethally irradiated BALB/c mice were transplanted with TCD-BM cells (5 × 10⁶ cells per mouse) and whole splenocytes from WT B6 donors (0.35 × 10⁶ cells per mouse). The recipients without iTreg transfer served as the cGVHD control group (WT SP). A total of 0.35 × 10⁶ Tregs was transferred per mouse on the day of BMT (day 0 [d0]). All recipient mice were monitored for survival (data not shown), body weight (A), and cGVHD clinical scores (B). The experiment was ended on day 50 to 55, and recipient thymus and spleen were analyzed. Representative FACS plot, percentages, and total number of CD4⁺CD8⁺ cells in thymus (C) and B-cell reconstitution in spleen (D) are shown on gated donor cells. Percentages of CD4⁺ IFN-γ⁺ cells (E) and Tfh cells (CD4⁺Foxp3⁻PD^hiCXCR5⁺) (F). (G) Line graphs (left panel) and mean fluorescent intensity (MFI; right panel) of CD86 expression. (H) Recipient skin was stained using Masson trichrome stain. (I) Collagen deposition in skin was calculated using the TT/CT ratio. Data are combined from 2 independent experiments (n = 5 or 6 per group). *P ≤ .05, **P ≤ .01, ***P ≤ .001, 1-way ANOVA.