CD4⁺T-cell subsets: phenotype, function, and DNA methylation. (A) Naive and rTreg from male blood donors were sorted (top panel) and activated with soluble anti-CD3 and -28 antibodies (100 and 200 ng/mL, respectively) and autologous irradiated CD4⁺ cell-depleted peripheral blood mononuclear cells for 4 days, supplemented with 20 U/mL IL-2 for an additional 2 days. Act-Naive and Act-rTreg were sorted as proliferating CTV^dim CD25⁺ cells (middle panel). FOXP3 expression was examined by intracellular staining (bottom panel). (B) Sorted CTV^dim CD25⁺ cells were incubated with Naive CD4⁺ T cells (5 × 10⁴) at 1:1, 1:2, 1:4, 1:8, and 1:16 ratios with anti-CD3 antibody (100 ng/mL), with or without IL-2 (20 U/mL) for 5 days. (C) Multidimensional scaling (MDS) analysis of the normalized, filtered data. The MDS plot is analogous to a principal components analysis plot. The axes represent the major sources of variation in the data based on the top 1000 genes with the largest standard deviations between samples; dimension 1 represents the largest source of variation, dimension 2 represents the next largest orthogonal source of variation, followed by dimension 3, etc. The left-hand MDS plot shows that the largest source of variation between samples was the difference in baseline methylation between the donors. Examination of further sources of variation reveals that methylation differences between cell types and activation status are also present, as shown in the right-hand MDS plot. Each sample is labeled with the donor ID and colored by cell type.