Peptide p60

hPBMCs, CD4⁺, or CD3⁺ T lymphocytes (5 × 10⁵ cells/well) were treated with phytohemagglutinin (PHA, 12.5 ug/ml, Sigma Aldrich, St. Louis, MO, USA) to stimulate T lymphocyte proliferation in the absence or presence of PGRN and incubated in 48-well culture plates (Corning, NY, USA) for 5 days. EdU (5-ethynyl-2-deoxyuridine, 10 μM, Invitrogen, Carlsbad, CA, USA) was added to the culture plates 18 hr before harvest. Based on a click reaction, EdU were reacted with azide, which is coupled to Alexa Flour 488 (Invitrogen, Carlsbad, CA, USA) dye. After harvesting the cells, the extent of cell proliferation was measured by flow cytometry (Beckman Coulter, Miami, FL, USA). Data were analyzed with FlowJo software (FlowJo, Ashland, OR, USA). Human TGF-β RI Kinase Inhibitor VI (SB431542, Selleckchem, Houston, TX, USA) or Treg inhibitor (Peptide P60, Abbiotec, San Diego, CA, USA) was used to block the formation of iTregs, and recombinant human TGF-β 1 protein (R&D System, Minneapolis, MN, USA) was used to induce the formation of iTregs.