Anti cd25 pecy7

Cell surface markers activation was assessed by culturing PBMCs in 96-wells plates and stimulating them with 0.4% DMSO, a 1 µg/mL SARS-CoV-2 peptide pool, 100 nM Ang II, or both a 1 µg/mL SARS-CoV-2 peptide pool and 100 nM Ang II for 24 and 48 h, separately. Supernatants were collected post-stimulation and stored at −20 °C for ELISA. The pellets were washed and stained with the cell surface markers antibodies anti-CD4-PerCP5.5 (clone RM4-5; Invitrogen, Waltham, MA, USA), anti-CD8a-APC-eFluor 780 (clone RPA-T8; Invitrogen, Waltham, MA, USA), anti-CD25-PECY7 (Beckman Coulter, Brea, CA, USA), anti-CD38-E flour 450 (clone HIT2; eBioscience, Waltham, MA, USA), anti-CD69-FITC (clone H1.2F3; Invitrogen, Waltham, MA, USA), anti-CD127-PE (clone eBioRDR5; Invitrogen), anti-CD154-APC (clone 24-31; BioLegend, San Diego, CA, USA), and anti-CD8-FITC (Beckman Coulter, Brea, CA, USA) for 1 h on ice, before the cells were washed and acquired using a BD FACS Canto II Flow Cytometer (BD Biosciences, San Jose, CA, USA) as previously described [56 (link)]. The BD FACSDiva software was used to analyze the results. The gating strategy (Figure S4) was performed by BD LSRFortessa.

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.