Isolation and Expansion of Human Tregs

Human primary cell isolation and culture were performed as described previously [57 (link),58 (link)]. Briefly, PBMCs were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) from buffy coats of healthy male blood donors at the Shanghai Blood Center. nTregs were separated with a FACSAria II cell sorter (BD Biosciences, USA) using the monoclonal antibodies anti-CD4-FITC, anti-CD25-PE, and anti-CD127- PE-Cy7. Naïve T cells were gated from a population of CD4+ CD25- effector T cells and separated with anti-CD45RA-Percp-CY5.5 (all from BD Biosciences, USA). Induced Tregs (iTregs) were induced from naïve T cells with recombinant TGF-β (5 ng/ml) and IL-2 (100 U/ml). The purified human nTregs and iTregs were expanded with anti-CD3/CD28 beads (Invitrogen, USA) and 500 U/ml IL-2 (R&D, USA). Both cell types were cultured with X-VIVO 15 medium (Lonza, Cologne, Germany) supplemented with 10% heat-inactivated human AB serum (Irvine Scientific, USA), 1% GlutaMax, and 1% NaPyr (Both from GIBCO, Life Technologies, USA). Cell fraction purity was determined using intracellular FOXP3 staining with FOXP3-PE-A (eBioscience, USA), following the manufacturer’s instructions. FACS data were then analyzed using FlowJo software (Tree Star, USA).