Mice were transplanted as in Figure 2. For C, allografts were made using Foxp3-DTR-GFP–expressing B6C3F1 male donor mice. (A) At day +7 in the B6C3F1→B6D2F1 HCT model, Treg percentages were stable to slightly decreased after PTCy treatment. (B) However, at day +21, mice treated with 25 mg/kg PTCy had significantly higher percentages of CD4⁺CD25⁺Foxp3⁺ Tregs in all tissue compartments. This increase was blunted after 100 mg/kg PTCy, a dose associated with worse GVHD histopathologically. (C) At day +21, CD25⁺Foxp3(GFP)⁺ CD4⁺ T cells pooled from the liver, blood, peripheral lymph nodes, and spleens of mice (n = 3) treated with 25 mg/kg/d PTCy on days +3 and +4 were largely demethylated within the TSDR of the Foxp3 gene, suggesting that the CD4⁺CD25⁺Foxp3⁺ T cell population expanded after PTCy was primarily composed of natural Tregs. In contrast, CD25^–Foxp3(GFP)^– CD4⁺ T cells at day +21 from these same mice were nearly all methylated. The numbers indicate the CpG site location relative to the transcriptional start site. (D–E) The percentages of alloantigen-specific (Vβ6⁺) Tregs in the liver increased after PTCy at both days (D) +7 and (E) +21. Of note, all samples in the day +7 100 mg/kg PTCy group had Treg numbers of less than 100 cells and would therefore normally be excluded, but are included here for illustrative purposes. Combined results from (A, D) 3 (n = 6/group) or (B, E) 2 (n = 8/group except 100 mg/kg PTCy; n = 5) independent experiments are shown. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, 1-way ANOVA with Holm-Šidák post hoc test using the allogeneic (Allo) TCD BM and splenocyte, vehicle group (labeled Allo, vehicle for A, D and BM, splen, vehicle for B, E) as control.