Clone pch101

Peripheral blood mononuclear cells (PBMCs) from patients and healthy volunteers were stained with CD4-FITC, clone (RPA-T4); CD8-PerCP, clone (SK1); CD25-APC, clone (BC96); and CD127-PE, clone (hIL7R-M21) fluorochrome-conjugated monoclonal antibodies (mAb). Intracellular staining for FoxP3 (clone-PCH101) was performed after cell surface staining by fixation and permeabilization as per manufacturer protocol (eBiosciences, San Diego, CA). The antibodies for cell surface staining and isotype controls were from BD Biosciences, eBiosciences, and from BioLegend. (San Diego, CA). Data were collected using FACSCalibur (BD Biosciences) and analyzed by BD Cell Quest software (Becton-Dickinson, Mountain View, CA) or FCS De Nova software (Thornhill, Ontario, Canada).

Immunohistochemical staining of 4 μm sections was conducted according to standard avidin-biotin-peroxidase protocols using monoclonal antibodies against IDO (clone 10.1, 1/200, Millipore, Billerica, USA), Foxp3 (clone PCH101, 1/50, eBioscience, San Diego, USA) and CD8 (clone CD8/144B, RTU, Dako). For antigen retrieval, slides were boiled (97° C) for 20 min (PT Link, Dako). A mouse linker (Dako) was added to the protocol in order to amplify the signal of the primary mouse anti-IDO and anti-Foxp3. Antibody detection was visualized using 3-amino-9-ethylcarbazole (AEC) for IDO and diaminobenzidene (DAB) for Foxp3 and CD8 detection. Sections were counterstained with hematoxylin.

Fresh PBMC (ex-vivo) from MS patients and healthy controls were stained with CD4 PerCP-Cy5.5 (Biolegend), CD25 BV421 (Biolegend), CD39 FITC (Miltenyi Biotech), followed by fixation, permeabilization and intracellular staining with FoxP3 PE (clone 236A/E7, eBioscience; clone 150D/E4, eBioscience; clone 259D/C7, Becton Dickinson; clone PCH101, eBioscience). Live/Dead Fixable Aqua Dead cell stain (Invitrogen) was added to the cocktail of surface mAbs. Cells were acquired on a Beckman Coulter CyAn flow cytometer and analyzed using FlowJo 9.9.5 software. For the experiments on the Treg phenotype fresh PBMCs from MS and HD donors were stained with CD4APC-e780 (eBioscience), CD25 BV421 (Biolegend), CD45RA PE-Cy7 (Beckman Coulter), CD39 FITC (Miltenyi Biotech), FoxP3 PE (150D/E4, eBioscience), PD-1 BV650 (Becton Dickinson), CD127 APC-Alexa647 (Miltenyi Biotech), Live/dead cell stain. Cells were acquired on a Beckman Coulter CytoFLEX, and analyzed using Flowjo 10. Statistical analysis was performed using unpaired Student’s T test.