Fig. 1
Treg signature molecules confirm the quality of iTreg and control cells used for molecular profiling. a Human naïve CD4+ T cells were stimulated (‘stim.’) with anti-CD3/-CD28 antibodies and IL-2 for up to 6 days in serum-free medium. For iTreg generation, TGF-β1, rapamycin (Rapa), all-trans retinoic acid (ATRA), or butyrate were added. At indicated time points (h: hours, d: days), RNA and protein were extracted for RNA-Seq and proteomics. Unstimulated (‘unstim.’) nTregs (ex vivo CD25high cells) were used as a positive control, unstimulated naïve CD4+ T cells were used as the ‘time zero’ control, both harvested on the day of isolation. G01–G07: Treatment group abbreviations. b, c Cultures as in (a), except unstimulated cells cultured in medium (+ IL-2) for 6 days. On day 6, an aliquot was re-stimulated for 4 h with phorbol 12-myristate 13-acetate/ionomycin plus Brefeldin A, stained for the given surface and intracellular markers and analyzed by flow cytometry. b Histograms for FOXP3 are shown for individual molecular profiling donors. Pre-gating: live CD4 + CD25++ cells (filled histograms) or live CD4+ cells (open histograms). ‘isotype’: anti-FOXP3 antibody isotype staining for each sample colored as beneath. Grey dashed lines: gate to determine FOXP3+ cell fraction; corresponding values are displayed in (c). c Expression of flow cytometry markers, gated on viable CD4+ cells (except for last three columns depicting live cell fraction, and columns 4–6, which are gated on live CD4 + CD25++ cells). The percentage of positive cells for the given marker is indicated by the color scale, columns show individual donors (D1, D2, D3). Grey indicates samples not measured or not applicable markers. FOXP3 and IKZF4 (Eos) expression from RNA-Seq (d) and proteomics (e) data, respectively. Dots: individual donors (mean per donor for proteomics samples with technical replicates), lines: mean of n = 3 donors. Statistical analysis, see Methods and Additional file 3: Table S2