Fig. 5: DNA methylation patterns reflect similarity of skin T_reg and blood CCR8⁺ T_reg cells.

a, Methylation (Tissue Donors 7, 10 and 11 and Blood Donors 3–8), chromatin accessibility (ATAC Donors 1, 2, 4 and 5) and gene expression (Tissue Donors 7, 8, 10 and 11 and Blood Donors 3–12) of features that are different between skin T_reg and blood CD45RA⁺ T_reg cells (blood RA⁺ T_reg cells). Annotation bars indicate blood CCR8⁺ T_reg cell positioning. b, Proportions of features placing blood CCR8⁺ T_reg cells closer to skin T_reg cells or blood CD45RA⁺ T_reg cells. Donors are as in a. c, Smoothed methylation (top left), raw methylation (top right), chromatin accessibility (bottom left) and gene expression (bottom right, mean ± s.d.) in skin T_reg, blood CCR8⁺ T_reg and blood CD45RA⁺ T_reg cells for regions where blood CCR8⁺ T_reg cells are closer to blood CD45RA⁺ T_reg cells than to skin T_reg cells regarding chromatin accessibility. Highlighted regions mark DMRs and differential peaks. P values were determined by two-tailed Wald test implemented in DESeq2 (n = 4, 5 and 5 donors for skin T_reg cells, blood CCR8⁺ T_reg cells and blood CD45RA⁺ T_reg cells, respectively) with a Benjamini–Hochberg correction. Donors are as in a. d, Enrichment of bZIP motifs in feature sets defined by blood CCR8⁺ T_reg cell positionings. P values were determined by one-tailed binomial test implemented in homer³¹ (n = 6,424 DMRs, 292,033 DMRs, 1,128 peaks and 2,064 peaks for columns 1–4) with a Benjamini–Hochberg correction. e, Methylation around genomic sites (±200 bases) for bZIP motifs overlapping with DMRs (mean across three biological replicates) in skin T_reg, blood CCR8⁺ T_reg and blood RA⁺ T_reg cells. Annotation bars indicate whether blood CCR8⁺ T_reg cells are closer to skin T_reg cells or blood CD45RA⁺ T_reg cells as in a. Donors are as in a. Heat maps are ordered as in d. f, Chromatin accessibility in skin T_reg, blood CCR8⁺ T_reg and blood CD45RA⁺ T_reg cells around genomic sites for a selected bZIP motif (see Fig. 4). Donors are as in a. g, Module scores (with cell numbers, median and bottom/top quartile) of genes upregulated in blood CD45RA⁺ T_reg or skin T_reg cells in TCR-matched blood CCR8⁺ T_reg, skin T_reg and blood CD45RA⁺ T_reg cells from two donors (TCR Donors 6 and 7). h,i, Concatenated flow cytometry plot showing CCR8 expression (h) and percentage of CCR8⁺ cells among Kaede-red⁺ T_reg cells and Kaede-green⁺ T_reg cells (i) in skin dLN T_reg cells of Kaede mice 6 days after skin photoconversion from Kaede-green to Kaede-red (data concatenated from seven mice; mean ± s.d.). The P value was determined by two-tailed Wilcoxon signed-rank test. Data are representative of two or more independent experiments with two or more individual donors.