Fig. 1.

FOXP3 promoter RT-PCR assay design. Naïve T cells (CD4+CD45RA+CD62L+CD25−) were sorted from CD4 enriched buffy coats (a, red gate) and put in culture with IL-2 and CD3/CD28 stimulation in medium alone (b), in the presence of TGF-β (c) or both TGF-β and retinoic acid (d). At day 7, cells were sorted based on FOXP3 expression. FOXP3^high (green gates) and FOXP3^low samples (red gates), as well as baseline naïve tTreg controls (a, green gate) were pyrosequenced at the promoter and three conserved non-coding sequence regions of the FOXP3 gene (e). Promoter CpG sites at positions −126, −77 and −56 from transcription start site showed the greatest differential methylation in FOXP3^high TGF-induced T cells (iTregs) (indicated with arrows). These sites were selected to design primers and RT-PCR probes specific for their methylation status (f). Specificity of methylated and unmethylated RT-PCR primers and probes was validated using unmethylated (g) and methylated (h) plasmid standards dilutions at 31,250, 6250, 1250, 250 and 50 copy numbers per reaction