Treg Depletion Assessment by Flow Cytometry

Treg depletion was assessed as previously described [11 (link), 13 (link), 16 (link)]. In brief, blood samples were obtained at baseline, 4 and 8 weeks after the first KW-0761 treatment, and every 4 weeks during the continuous treatment until the point of study discontinuation. PBMCs were isolated from heparinized blood by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Fairfield, CT). Cells were stored in liquid N2 until used. Treg depletion was evaluated by flow cytometry. After thawing, PBMCs were incubated with mAb at 4°C for 20 minutes. Cells were stained with anti-CD4-PerCP (clone SK3; BD Biosciences, San Jose, CA), anti-CD25-APC (clone 2A3; BD Biosciences), and anti-CD45RA-FITC (clone ALB11; Beckman Coulter, Brea, CA) mAbs. The intracellular staining of FoxP3 was performed with anti-FoxP3-PE (clone PCH101; eBioscience) mAb and a FoxP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA) according to the manufacturer’s instructions. After the incubation, cells were washed and analyzed by FACS Calibur (BD Biosciences). CD45RA⁺ FoxP3^lo resting/naïve Tregs, CD45RA^-FoxP3^hi activated/effector Tregs (eTregs), and CD45RA^-FoxP3^lo non-Tregs were analyzed as previously described [7 (link)].