The medium was find more enriched with 1% inactivated autologous plasma, and cell cultures were stimulated with 0·5 μg/ml of αCD3 monoclonal antibody (mAb) (clone Okt3; eBioscience, San Diego, CA). For analysis of cytokine production by nTreg, 6 × 104 nTreg were polyclonally stimulated (as described above) and cultured for 62 hr. To verify that isolated nTreg did not proliferate, which would have indicated contamination with other T helper cells, we stained nTreg with CFSE, co-cultured them with Tres and measured CFSE dilution in

nTreg using FACS, as described above. Culture supernatants were collected and the amounts of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α were assessed using the Bio-Plex™ Cytokine IWR-1 datasheet Assay (Bio-Rad, Munich, Germany) on the Bio-Plex™ Protein Array System (BioRad), following the manufacturer’s instructions. To analyse the nTreg-mediated suppression of cytokine secretion we calculated the suppression ratio as: supernatant cytokine concentration (assay without nTreg)/supernatant cytokine concentration

(assay with nTreg). To analyze possible differences in the suppressive activity of nTreg on the proliferation of Tres subpopulations, we investigated the percentage of IL-2-, IL-4-, IL-10-, IL-17A-, IFN-γ- and TNF-α-producing cells within the proliferated Tres in representative blood samples. All samples were collected at 08:30 hr, purified as described above and cultured for 62 hr before being restimulated with 5 ng/ml of phorbol myristate acetate (PMA; Sigma-Aldrich, Munich, Germany) and 500 ng/ml of ionomycin (Sigma-Aldrich) for 4 hr (IL-2, IFN-γ or TNF-α), 6 hr (IL-17A) or 8 hr (IL-4 or IL-10); 1 μg/ml

of brefeldin A (BD Biosciences) was added to the cells after 1 hr of restimulation. Cells were then stained with αCD4-mAb labelled with allophycocyanin (clone M-T466; Miltenyi Biotec) and co-stained with αIL-2-mAb (clone N7.48A; Miltenyi Biotec), αIL-4-mAb (clone 8D4-8; BD Pharmingen, Heidelberg, Germany), αIL-10-mAb (clone B-T10; Miltenyi Biotec), αIL-17A (clone eBio64DEC17; eBiosciences), αIFN-γ-mAb (clone 45–15; Miltenyi Biotec), or αTNF-α-mAb (clone MAb11; BD Sclareol Pharmigen) labelled with phycoerythrin or allophycocyanin. The percentage of cytokine-producing cells was determined by gating on the proliferated CD4+ CFSE-stained T cells (see Fig. 2a) applying the Cellquestpro®Software (BD Biosciences). The aim of our study was to characterize the diurnal activity of T-cell subsets. We therefore analyzed whether the expression of CD126 (IL-6R alpha chainl; BD Bioscience), CD25 (IL-2R alpha chain; Miltenyi Biotec), or FOXP3 (clone PCH101; eBioscience) on/in nTreg or Tres, changed over a diurnal cycle. Additionally, we assessed whether the isolated T-cell subsets contained the same amount of FOXP3−, CD45RA+ and CD25− T cells. The expression of these markers was analyzed using FACS.