Anti cd45ro ecd

Flow cytometry was performed as described previously [27] (link). Briefly, antigen-specific phenotypes and cytokine secretion profiles were assessed using a qualified polychromatic flow cytometry (PFC) panel. PBMC were co-incubated with peptide pools matched to the GRIN/ENV insert, 1 µg/ml SEB (Sigma-Aldrich, St. Louis, MO, USA) or mock stimuli, CD107a PECy5, BD Golgistop (Becton Dickinson, San Jose, CA, USA) and Brefeldin A (Sigma-Aldrich, Poole Dorset, UK) for 6 hours at 37°C. Cells were stained for viability with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen, Eugene, OR, USA), and then surface stained by anti-CD4 QD605, anti-CD8 pacific orange, anti-CD19 pacific blue (Invitrogen, Paisley, UK), anti-CD27 APC-H7, anti-CD14 pacific blue, anti-CD57 FITC, anti-B7 integrin PE (Becton Dickinson, San Jose, CA), and anti-CD45RO ECD (Beckman Coulter, High Wycombe, UK). Finally cells were stained intracellularly with anti-CD3 QD655 (Invitrogen, Paisley, UK), anti-IFNγ PE Cy7, anti-TNF-α A700 and anti-IL-2 APC (Becton Dickinson, San Jose, CA, USA) washed and acquired on the same day. At least 750,000 events were acquired on a custom-built BD LSR II cytometer. Data were analyzed and presented using FlowJo (version 8.8 Treestar).

Treg cells were stained for 30 min with anti-CD4 APC, anti-CD25-PECy7, anti-CD38-FITC, anti-CD45RO-ECD and anti-HLA-DR-PECy5 (Beckman Coulter, Barcelona, Spain). Fixation and permeabilization for intracellular staining was done with the Anti-Human Foxp3 Staining Set (eBiosciences, San Diego, CA, USA), and cells were stained with anti-Foxp3-PE (eBiosciences) or anti-p24 protein (KC57-FITC; Beckman Coulter). Data acquisition was performed in a Beckman Coulter GALLIOS cytometer. The functionality of the Treg cells was studied measuring the capacity of the Treg to suppress the activation of effector cells. Allogenic PBMC were stimulated with anti-CD3/anti-CD28 coated beads (0.5 beads for 10⁵ cells) (Gibco, Van Allen Way, Carlsbad, California) and incubated for 7 hours. Then these cells were co-cultured with previously expanded and treated Treg cells in a ratio of 1:1 in X-VIVO medium supplemented with 10% of AB human serum. The analysis was performed by using the Regulatory T–Cell Function Kit (BD Biosciences, San Jose, CA, US), according to manufacturer´s instructions.