Plasma cytokine quantification
The plasma levels of IL-5, IL-13, IL-6, IL-17A and TNF⍺ were quantified using Quanterix Single Molecule-Arrays (SiMoA) analyser using the Simoa™️ IL-5 Advantage Kit, Simoa™️ IL-13 Advantage Kit and Simoa™️ Cytokine 3-Plex B Advantage Kit respectively following the manufacturer’s protocol.
Detection of cytokine secretion by flow cytometry
Frozen cells were thawed using RPMI 1640 Medium (Gibco), supplemented with 10% FBS (BioWhittaker) and washed with homemade 1X MACS buffer (1X PBS + 0.5% BSA + 2mM EDTA). Cells were then pre-stained for 15 minutes at 37oC with 5% CO2 using the following antibodies: anti-CD294 (clone BM16, BD Pharmingen) and anti-CD45RB (clone MEM-55, ImmunoTools). Cells were washed with 1X MACS buffer and then stimulated for 4 hours at 37oC with 5% CO2 with 20ng/ml Phorbol myristate acetate (PMA) (Sigma-Aldrich), 1µM Ionomycin calcium salt (Sigma-Aldrich) and GolgiPlug (BD Biosciences).
After 4 hours of stimulation, cells were washed with 1X PBS (Gibco) and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) for 10 minutes at room temperature. Cells were then washed with 1X MACS buffer and stained for 15 minutes at 4oC using the following antibodies: anti-CD161 (clone HP-3G10, eBioscience). Intracellular staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s instructions. Cells were fixed with Fixation/Permeabilization solution (BD Biosciences) for 20 minutes at 4oC. Cells were washed and kept in 1X MACS buffer overnight at 4oC. On the next day, cells were permeabilized with 1X Perm/Wash buffer for 15 minutes at 4oC and stained with the following antibodies:  anti-CD3 (clone UCHT1, BD Pharmingen), anti-CD4 (clone RPA-T4, BD Pharmingen) and anti-IL-5 (clone TRFK5, BioLegend). After 30 minutes, cells were washed with 1X Perm/Wash buffer and resuspended in 1X MACS buffer. Samples were acquired using BD LSR II flow cytometer and analysed using FlowJo v10 (BD).