Anti cd127 pe

Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation over Ficoll-Hypaque (GE, Fairfield, CT, USA). CD14⁺ monocytes were isolated by positive selection (CD14 beads, Miltenyi Biotec, Auburn, CA, USA) and immature monocyte-derived DCs were generated by culturing the isolated monocytes for 4 days in complete medium (RPMI 1640, supplemented with 10% of heat-inactivated fetal calf-serum, HEPES, Glutamine) with 500 U/ml rhIL-4 and 1,000 U/ml rhGM-CSF (both from PeproTech, Inc., Rocky Hill, NJ, USA). Complete medium, including cytokines, was replaced at day 3. After 4 days of differentiation, cells had the morphology of immature DCs (CD14^low and HLA-DR^hi expression; no adherence to the culture plate). Resting autologous CD4⁺ T cells were purified by negative selection using the Miltenyi CD4 separation kit (Auburn, CA, USA), according to the manufacturer’s instructions. Purified CD4⁺ T cells were then stained with anti-CD8-FITC, anti-CD25-APC (BD Pharmingen; San Diego, CA, USA), and anti-CD127-PE (Beckman Coulter, Fullerton, CA, USA), to separate Treg and Tcon by cell sorting (FACSAria, BD). The purity of Treg (CD8^negCD25^hiCD127^low) and Tcon (CD8^negCD25^lowCD127^hi) cells was evaluated post-sorting by CD4 and FOXP3 staining (clone PCH101, e-Bioscience; San Diego, CA, USA). Purity of the sorted populations was superior or equal to 90% (data not shown).

Fasting heparinized peripheral blood was centrifuged at 2000 rpm, 10 min, 22°C to collect the plasma. Aliquots of plasma were frozen at -80°C. The remaining blood was diluted with PBS 1:2. Peripheral blood mononuclear cells (PBMC) were separated by centrifugation through Ficoll-Hypaque (GE, Fairfield, CT). Aliquots of PBMC were frozen in FBS + 10% DMSO and stored in liquid nitrogen. CD4+ T cells were purified from the cryopreserved PBMC by negative selection using the Miltenyi CD4 negative separation kit (Auburn, CA), per the manufacturer’s instructions. In some experiments, healthy control Treg were used. To isolate bulk Treg, CD4+ T cells were stained with live dead aqua (Thermofisher, Waltham, MA), anti-CD8-FITC, anti-CD25-APC (BD Pharmingen, San Diego, CA), anti-CD127-PE (Beckman Coulter, Fullerton, CA), and sorted using a FACS Aria (BD). Purity of the sorted Treg was >90%, as determined by post-sorting analysis of FOXP3 expression.