Anti foxp3 pe clone pch101

CD4+ T lymphocytes were purified from PBMCs isolated from three anonymous blood donors (with ages ranging from 18 to 65 years) by negative selection using the Human CD4+ T Cell Enrichment Kit (#19052, Stemcell Technologies) according to the manufacturers' instructions. Purification was over 90% as assessed by staining with 5 ng/μl of anti-CD4-FITC (clone RPA-T4, eBioscience) and flow cytometric analysis (FACSCalibur).
Isolated CD4+ T lymphocytes (2 × 10⁵) were resuspended in RPMI medium (control) or preincubated for 24 hours with 50% of AMSC-derived CM and SF, then seeded in triplicate into 96-well plates with prebound 0.5 μg/ml anti-CD3 (clone OKT3, eBiosciences), 0.5 μg/ml anti-CD28 (clone CD28.6, eBiosciences), and recombinant IL-2 at a concentration of 250 U/ml (Peprotech). After 3 days, in vitro-stimulated CD4+ T cells were stained with 5 ng/μl of anti-CD25-APC (clone BC96, eBiosciences) and 5 ng/μl of anti-FoxP3-PE (clone PCH101, eBioscience) and T reg proliferation was tested with flow cytometry (FACSCalibur, Becton Dickinson). The data were analysed using the Flowing Software.

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)].

Cryopreserved expanded Tregs and Tconvs were thawed and immunostained with anti-CD3-PE-Cy7, anti-CD4-qdot655 (Invitrogen), anti-CD25-PE-Cy5 (eBiosciences), anti-CD39-FITC (eBioscience), anti-CD45RA-horizon v450 (BD Pharmingen), anti-FOXP3-PE (clone PCH101, eBiosciences), anti-CTLA4-APC (BD Pharmingen), anti-HELIOS-FITC (Biolegend). For intracellular staining, the eBioscience FOXP3 staining buffer kit was used. Dead cells were eliminated using the LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Invitrogen). Flow-cytometry data were acquired on a LSR Fortessa (BD Biosciences).