Single-cell analysis of cytokine-producing Tregs. (A) IL-17A– and IFN-γ–producing Tregs and Tconvs were isolated using a cytokine capture assay on sorted populations of CD4⁺CD25^highHLADR⁻CD45RA⁻ (RA⁻DR⁻ Tregs) Tregs and CD4⁺CD25⁻CD45RA⁻HLADR⁻ Tconvs (RA⁻DR⁻ Tconv). Single IFN-γ– and IL-17A–producing cells were sorted into 96 cells plates and single-cell gene expression profiling was performed using 48.48 Dynamic Arrays (18 cycles preamplification). The heat map represents 2-dimensional hierarchical clustering using Euclidean distance and average linkage. (B) mRNA expression of Treg markers (FOXP3, CTLA4, Helios), Th1 and Th17 transcription factors (TBX21, RORC2), and cytokines (IFN-γ, IL17A, IL10, IL26, IL22, IL4) in single RA⁻DR⁻ Tregs and RA⁻DR⁻ Tconvs. A representative experiment performed with 4 independent donors is shown.