Fig. 1: DNA methylation separates skin T_reg and blood CCR8⁺ T_reg cells from blood CD45RA⁺ T_reg cells.

a, Representative flow cytometry plot showing CD45RA⁻CCR8⁺ T_reg cells among CD4⁺CD127⁻CD25⁺ T_reg cells in human blood, fat and skin (left) and percentage of CD45RA⁻CCR8⁺ T_reg cells among CD4⁺CD127⁻CD25⁺ T_reg cells in human blood compared to skin (right; n = 6 healthy female donors; FACS Donors 1–6; median age: 46 years; range: 31–61 years). The P value was determined by two-tailed Wilcoxon signed-rank test. b, Sort layout showing isolation of blood CD45RA⁺ T_reg, blood CD45RA⁺ T_conv, blood CCR8⁺ T_reg, skin T_conv and skin T_reg cells in one healthy female donor (FACS Donor 7; age unknown). Not all gates are shown. c, Principal component analysis of DNA methylation in blood CD45RA⁺ T_reg cells (RA⁺ T_reg cells), blood CD45RA⁺ T_conv cells (RA⁺ T_conv cells), blood CCR8⁺ T_reg cells, skin T_conv cells and skin T_reg cells isolated from blood and skin from nine healthy female donors (Tissue Donors 7, 10 and 11 and Blood Donors 3–8). d, Methylation level by genomic position and corresponding differences with respect to blood CD45RA⁺ T_conv cells in blood CD45RA⁺ T_reg, blood CCR8⁺ T_reg, skin T_conv and skin T_reg cells in human donors as in c. Numbers in brackets indicate the average methylation level on autosomes and chromosome X. e, Schematic showing the extraction of cell-type signatures based on the largest numeric methylation gap between any two cell types (red arrows), which was required to be at least 0.15 and at least 1.5 times as large as the second-to-largest methylation gap (blue arrows) and resulted in the selection of signature regions; RA, CD45RA. f, DNA methylation in regions belonging to selected cell-type signatures as in e. Rows correspond to signature regions, and columns correspond to donors (n = 3 donors per cell type as in c). Numbers on the right indicate the number of regions per signature category. Signature nomenclature is based on the cell types that were hypomethylated in the corresponding signature regions. g, Distribution of cell-type signature regions from the signatures in e in genomic intervals defined by genes (top) and CpG islands (bottom). Numbers at the top indicate the number of regions per signature category. Data are representative of three or more independent experiments with three or more individual donors; TSS, transcription start site.