Single-cell gene expression profiling reveals heterogeneity within Treg subpopulations. The 3 Treg subpopulations were sorted from adult peripheral blood of healthy donors. Each population was stimulated with CD3/CD28 coated beads for 24 hours then sorted as single cells into 96-well plates. Expression of the indicated mRNA transcript was assessed by single-cell quantitative RT-PCR using microfluidic arrays (Fluidigm). (A) The heat map representation of single-cell gene expression profiling was obtained by 2-dimensional hierarchical clustering analysis using Euclidean distance and average linkage using Qlucore Omics Explorer software. Each row corresponds to the expression level (Ct value) of a single gene and each column represents a single cell (black squares: naive conventional CD4⁺ T cells; green squares: RA⁺DR⁻ Tregs; blue squares: RA⁻DR⁻ Tregs; and red squares: RA⁻DR⁺ Tregs). (B) Dot plots were obtained by analyzing Ct values in GraphPad Prism software. Expression of transcripts for Treg markers FOXP3, CTLA4, FAS, IL2RA (CD25), IKZF2 (Helios), LRRC32 (GARP), TNFRSF18 (GITR), IL1R2, and chemokine receptors CCR8, CCR6, CCR4 were plotted for single RA⁻DR⁺ Tregs (red triangles), single RA⁻DR⁻ Tregs (blue triangles), single RA⁺DR⁻ Tregs (green squares), and CD4⁺CD25⁻CD45RA⁺ Tconvs (black dots). The expression of the housekeeping gene B2M was also analyzed in each T-cell subpopulation. One representative experiment of 3 from independent donors is shown.