Il 17 af488

Intracellular cytokine assays were performed as previously described [14 (link),26 (link)]. Briefly 0.5 mL fresh sodium heparin anticoagulated blood was incubated with SEB or antigens (as above) for 2 h at 37 °C, before the addition of 10 µg/mL brefeldin A (Sigma-Aldrich), and incubated for a further 4 h, before addition of 2 mM EDTA. Aliquots were stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7, CD8-APC-Cy7 and CD73-PE, lysed with FACSLyse (BD Biosciences), permeabilized with FACSPerm2 (BD Biosciences), and then incubated with anti-IL-2-APC (BD Biosciences), IFN-γ-Pacific Blue (BioLegend, San Diego, CA, USA) or –FITC (BD Biosciences) and IL-17-AF488 (BioLegend). Cells were then analyzed on an LSR II, as described above.
CD25+CD134+ (“OX40”) assays were performed as previously described [33 (link)]. Briefly, 0.25 mL IMDM was added to 0.25 mL fresh Na Hep anticoagulated blood and incubated with SEB or antigens (as above) for 40–48 h at 37 °C. Aliquots were stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7, CD25-APC, CD134-FITC and CD73-PE, lysed with Optilyse C, and then analyzed on an LSR II, as described above.

After asphyxiation by carbon dioxide in rats, 20 mg of mucosa was weighed and added to 5 ml of DMEM solution (Biowest, France) containing 5 mg of collagenase type II (Sigma-Aldrich, USA) and 50 µg of DNAase (Sigma-Aldrich, USA) and digested at 37 °C for 1.5 h. The resulting suspension was filtered and centrifuged at 450 g for 5 min. Nasal mucosal cells were obtained by filtration and centrifugation at 450 g for 5 min and resuspended in appropriate PBS. The nasal mucosal cell suspension was divided into several portions, some of which were incubated with IL4-PE (Biolegend, USA), IL5-APC (Biolegend, USA), IL17-AF488 (Biolegend, USA), and IFNγ-PE (Biolegend, USA) at 4 °C for 60 min in the darkness. Another portion of mucosa was incubated with CD4-BV650 (Biolegend, USA) and IL4-PE (Biolegend, USA) or IFNγ-PE (Biolegend, USA) for 60 min at 4 °C in the dark. After rinsing with PBS and resuspension, mucosal samples were transferred to a flow cytomete (FACS Celesta, USA) and analyzed with FlowJo software (version 10.6.2, Treestar, USA).

BAL cells and PBMCs were isolated and stimulated at 1 × 10⁶ cells/mL with Ag85A, ESAT-6/CFP-10 peptides (2 µg/mL each), and PPD (20 µg/mL); unstimulated cells and SEB-stimulated cells were used as negative and positive controls, respectively. Brefeldin A (Sigma) was added to the cells 2 h after stimulation, and cells were incubated overnight at 37 °C and 5% CO₂.
Harvested BAL cells and PBMCs were stained with the live/dead red viability marker (Thermo Fisher) for the exclusion of dead cells, followed by surface staining with CD4-Pacific Blue (Biolegend, San Diego, CA, USA), CD14, and CD19 on ECD (Beckman Coulter, Brea, CA, USA). Cells were then permeabilised and stained with CD3-AF700 (Ebioscience, San Diego, CA, USA), CD8-APC/H7 (BD), IFN-γ-PECY7 (Ebioscience) and TNF-α-AF-647 (Biolegend), IL-2 PE (Beckman Coulter), and IL-17-AF488 (Biolegend).
Cytokine-producing CD4+ and CD8+ T-cells were gated on CD3+, CD14−, CD19− single T cells. Data were analysed using Flowjo (BD) and are presented as background-subtracted antigen-specific responses.