PTCy-treated chimeras have equally suppressive tTregs that are differentially demethylated compared with controls. (A-B) In vitro suppression. Sorted Foxp3^– Tcons (CD4⁺ and CD8⁺; 1 × 10⁵) were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured with irradiated CD3^– splenocytes (1 × 10⁵) plus anti-CD3 (1 μg/mL) alone or with titrating numbers of sorted Tregs (CD4⁺Foxp3^RFP+) for 96 hours. CFSE dilution was measured by flow cytometry. The overlay depicts proliferation of Tcons alone or cocultured with posttransplant (day +28) pTregs, posttransplant (day +28) tTregs, or pretransplant Tregs in a 2:1 ratio. (C) Treg-specific demethylated region (TSDR) methylation profiles. CD4⁺ Foxp3^– Tcons, pTregs, and tTregs of different lymphoid organs from PBS- (pooled from 3 organs) and PTCy-treated alloBMT recipients (day +28) were sorted and genomic DNA isolated for bisulfite sequencing and pyrosequencing of the TSDR. Residue numbers denote individual CpG motifs in reference to the transcription initiation site of Foxp3. Bar colors represent 0% to 100% methylated regions. Data are representative of 2 independent experiments with 3 to 5 chimeras per group.