CD4+CD25high (purity >99%) cells were isolated by cell sorting fr

CD4+CD25high (purity >99%) cells were isolated by cell sorting from Buffy coats from the National Blood Service. Treg (CD4+CD25highCD127low; typical purity >98%) and effector T cells (CD4+CD25-CD127+; purity >99%) were cell sorted from cones obtained from the Gefitinib datasheet National Blood Service. Human CD4+ T cells (1 × 106 cells/mL) were stimulated with plate-bound anti-CD3 (1 μg/mL; OKT-3) in RPMI containing 50 U/mL recombinant hIL-2 (Eurocetus), 10 ng/mL hIL-4

(NBS), and calcitriol (1α25VitD3; BIOMOL Research Labs) as indicated, for 7 day cycles. In some experiments, 5 ng/mL IL-10 (R&D), 5 μg/mL anti-TGF-β (clone 1D11; R&D), 5 μg/mL anti-IL-10R (clone 3F9-2; BD-Pharmingen), or the appropriate isotype control antibody were added, as indicated. Note cells used for proliferation analysis were stained at day 0 with 5 mM CellTrace™ Violet (Invitrogen), according to manufacturers’ instructions. Murine CD4+ T cells were FACS sorted on a MoFlo cytometer (Beckman Coulter) for CD4+ (purity >99%), CD4+CD44lowCD25− (Foxp3GFP−; purity >99%), or CD4+Foxp3GFP+ (purity >97%) from CD4-enriched spleen cells. Cells were stimulated in flat-bottom 96-well plates (0.25 × 106 cells/mL) with plate-bound anti-CD3 (145-2C11) at 2.5 mg/mL in cRPMI medium [45] containing 5 ng/mL recombinant mIL-2 (Insight Biotechnology) for 7 days. Cells were fed with IL-2 on day 3. Where YAP-TEAD Inhibitor 1 cost indicated, 1α25VitD3, 5 ng/mL recombinant hTGF-b1 (Insight

Biotechnology), and 10 nM all trans RA (Sigma-Aldrich) were added to T-cell cultures. CD4+ T-cell lines were generated as described above. CD4+CD45RA+ naïve T cells were labeled with 2 μM CFSE (Molecular Probes, Eugene) and co-cultured with the autologous line at the ratios indicated, with 0.1 μg/mL plate-bound anti-CD3 and 1 μg/mL anti-CD28 (clone 15E8; Sanquin). In some experiments, anti-IL-10R or IgG control was added to the co-culture. On day 5, cells were stained with propidium iodide (PI; Sigma-Aldrich) for dead cell exclusion and 30,000 CFSE positive viable responder cells were acquired on a FACSCaliber flow cytometer (Becton Dickinson). Human IL-10+ cells

were identified using a commercially available IL-10 Secretion Assay Detection Kit (Miltenyi Biotec). Foxp3 (clone PCH101) expression was determined by cell staining using the Foxp3 staining buffer set from Ebiosciences. Quadrant next markers were set according to the matched isotype control antibody staining. Antibodies used for cell surface phenotyping (BD Biosciences) were PD-1 (clone MIH4), CTLA-4 (clone BN13), CD62L (clone DREG-56), CD25 (clone M-A251), GITR (clone 110416), and CD38 (clone HIT2). Expression of Foxp3 in murine CD4+ T cells was determined by excluding dead cells with LIVE/DEAD Fixable Red Dead Cell Staining Kit (Invitrogen) and intracellular staining for Foxp3 with staining buffer set from eBiosciences. Samples were acquired on LSR II (BD) flow cytometer. RNA was extracted from cell pellets using RNeasy Mini kit (Qiagen).

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