Regulatory T cell–specific demethylation region DNA methylation analysis was performed as previously described17 using genomic DNA isolated from freshly sorted or expanded Treg cells using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). A minimum of 60-ng bisulfite-treated (EpiTect; Qiagen) genomic DNA was used in a real-time polymerase chain reaction (PCR) to quantify the Foxp3 TSDR. Real-time PCR was performed in a final reaction volume of 20 μL containing 10-μL FastStart universal probe master (Roche Diagnostics, Mannheim, Germany), 50-ng/μL lamda DNA (New England Biolabs, Frankfurt, Germany), 5-pmol/μL methylation or nonmethylation-specific probe, 30-pmol/μL methylation or nonmethylation-specific primers, and 60-ng bisulfite-treated DNA or a respective amount of plasmid standard. The samples were analyzed in triplicates on an ABI 7500 cycler and reported as % T cells with demethylated TSDR region.