Cd4 apc

Cell phenotype was assessed by flow cytometry (BD LSRFortessa; BD Biosciences) after incubation with the following fluorescein-labeled antibodies: CD45-allophycocyanin (APC)-eFluor780, CD3e-fluorescein isothiocyanate (FITC), CD4-APC, CD8a-phycoerythrin (PE)-cy7, CD25-PE-cy7, Foxp3-PE, IFN-γ-AlexaFluor488, IL-4-PE-cy7, CD107a-PE, granzymeB-peridinin chlorophyll (PerCP)-eFluor710, perforin-FITC, CD11c-PE-cy7, CD80-PerCP-eFluor710, CD83-FITC, B7-H4-PE, and CD31-PE. All antibodies were purchased from eBioscience (San Diego, CA, USA), except CD4-APC, CD8a-PE-cy7, and Foxp3-PE (BD Biosciences). Intracellular antigens were determined after incubation with ionomycin (500 ng/mL; Abcam, Cambridge, UK) and phorbol-12-myristate13-acetate (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h and monensin (2 μM; eBioscience) for an additional 4 h. Fixation and permeabilization were performed prior to antibody incubation, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Thymocyte cell suspensions were stained for 20 min at room temperature with CD4-APC, CD8a-PE, TCRb-BV421 and CD69-FITC (BD-Pharmingen). Single cells were sorted into 96-well plates containing lysis buffer using a FACSAria Fusion flow cytometer (BD Biosciences) and the gates depicted in Supplementary Fig. 1. Flow cytometry standard files were analysed with DIVA (BD Biosciences) and FlowJo v10 (TreeStar Inc) analysis software.
C57BL/6 (C57BL/6OlaHsd, Envigo, UK) and Mice lacking MHC class II expression^{41 (link)} (JAX stock #003584) were maintained separately under specific pathogen-free conditions under Project Licences issued by the Home Office, UK. OT-I Rag2^−/− and CD8.4 OT-I Rag2^−/− mice^{32 (link)} were maintained together at the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, in accordance with laws of the Czech Republic. Six-week-old male or female mice were killed by cervical dislocation, and thymocyte or lymph-node cell suspensions were stained with CD4-APC, TCRb-FITC, CD69-BV421 (Pharmingen) or CD4-Alexa Fluor 700, CD8a-PE or CD8a-BV421, TCRb-APC (Biolegend), and LIVE/DEAD NIR (ThermoFisher). Data acquisition was on a Cytek Aurora flow cytometer (Cytek Biosciences) and flow cytometry standard files were analysed with FlowJo v10 (TreeStar Inc) analysis software. Data were further analysed using GraphPad Prism v5.04 (GraphPad Software).

To assess the capacity of MSCs to induce the generation of T-regulatory (Treg) cells, PBMC-MSC cocultures have been set up. In short, 500 × 10³ PBMCs from five different donors obtained by Ficoll gradient centrifugation were plated in the presence of 50,000 allogeneic MSCs in RPMI 10% FBS with IL-2300 U/ml in a 24-multiwell flat-bottomed plate for 1 week of culture [36 (link)]. At day 7, PBMCs were harvested and analyzed by flow cytometry after incubation with the following conjugated monoclonal antibodies: CD25 FITC, CD127 PE, CD4 APC, CD3 PECy7 and CD45 APCCy7 (Becton Dickinson). Treg cell induction by MSCs was evaluated as the percentage of CD25^High/CD4⁺/CD127^Low/− PBMCs [37 (link)].