Effect of 5-azaC on the expression of FOXP3 and on the methylation pattern of FOXP3 promoter. (A) Regulatory T cells were identified as CD4⁺CD25⁺FoxP3⁺CD127⁻ cells. A representative dot plot of 6 cases analyzed is shown. (B) Mean fluorescence intensity for FoxP3 at 0 and after 7 and 14 days of culture; a significant increase in the mean fluorescence intensity for FoxP3 was observed along the 14 days of culture in the presence of 5-azaC compared with controls; (C) quantitative PCR showed a significant increase in FOXP3 expression in treated versus untreated T cells from day 7 to 14 of culture. (D) Effect of 5-azaC in the methylation status of the FOXP3 gene. (i) Schematic overview of the FOXP3 gene, including exons and position of its promoter CpG island. It is shown the position of the amplicons designed for methylation analysis. The bottom panel depicts a summary of the methylation levels measured by bisulfite sequencing of different samples indicated below. Each box represents 1 of the 4 amplicons studied with the average methylation rate according to the color code (scale is shown at the bottom: black indicates no methylation; gray, methylation). (ii) Graph corresponding to the change of methylation for one of the amplicons of the Foxp3 gene at different time points (4, 7, 11, and 14 days) and presence or absence of 5-azaC (100 and 0nM). (iii) Individual bisulfite sequencing of the 4 selected regions of the Foxp3 gene according to Baron et al.³²  Fifteen clones are shown. Methylated and nonmethylated CpG sites are represented as black and white squares, respectively. Percentage of methylation is shown at the right hand side of each section. Eight samples are represented corresponding to 4 time points (4, 7, 11, and 14 days) in the presence or absence of 5-azaC (100 and 0nM).