Anti il 5 pe

The following stimuli were used for PBMC stimulation: native AgB at 10 ug/mL, (produced at the Istituto Superiore di Sanità, as previously reported in [9 (link)]), costimulatory molecules anti-CD28 and anti-CD49d monoclonal antibodies (mAb) at 1ug/mL each (BD Bioscence, San Jose, USA), staphylococcal enterotoxin B (SEB) at 200 ng/mL (Sigma, St. Louis, MO, USA).
The fluorescently conjugated mAb used in this study were: AQUA DYE- AmCyan (Invitrogen Life Technology, Monza, IT), anti-CD4 peridinin chlorophyllprotein (PerCP)-Cy5.5-conjugated (Miltenyi Biotec S.r.l., BO, Italy), anti-CD3 allophycocyanin (APC)H7-conjugated (Miltenyi), anti-TNF-α phycoerythrin (PE)-Cy7-conjugated (eBiosceience, San Diego, CA, USA), anti- IFN-γ Horizon V450-conjugated (BD Biosciences), anti-IL-2 fluorescein isothiocyanate (FITC)-conjugated (BD Biosciences), anti-IL-4 PE (BD Biosciences), anti-IL-5 PE (Biolegend, San Diego, CA, USA), anti-IL-13 PE (Biolegend), anti-IL-10 allophycocyanin (APC)-conjugated (BD Biosciences).

Intracellular staining of FoxP3, IFNγ, IL-5, IL-17, and IL-10 was performed in CD4⁺ T cells from HD purified by negative selection using CD4⁺CD45RO⁺ microbeads kit following the protocol of manufacturer (Miltenyi Biotec). Memory T CD4⁺ cells were sorted in FACSAria III obtaining more than 98% purity. Cells were recovered overnight with RPMI complete media and the next day 1 × 10⁶ memory CD4⁺ T cells were challenged with ANDV-GP VLP in presence of 2.5 μL of anti-CD3/CD28 beads (ThermoFisher Scientific), also, an anti-CD3/CD28 condition was included and without stimuli was used as control (mock). After 3 days, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin (Sigma Aldrich) in presence of 1 μL brefeldin A (Biolegend) for 5 h. Later, cells were stained with fixable viability stain AF780 and fixed/permeabilized with transcription factor buffer set following manufacturer instructions (BD Biosciences). Intracellular staining was performed using the following antibodies: anti-IFNγ-FITC (Biolegend), anti-IL-5-PE (BioLegend), anti-FoxP3-eFluor450 or anti-IL-10-PE, and anti-IL-17-FITC (eBioscience).