Il 17a pe cy7

The following antibodies (Abs) were used for short-term culture or surface marker and intracellular cytokine staining for flow cytometry (all Abs were from Biolegend): anti-CD3-PerCP-cy5.5 (Clone UCHT1), anti-CD8-APC-Cy7 (Clone SK1), anti-CD4-PE-Cy7 (Clone RPA-T4), anti-Vδ₁-APC (Clone REA173), anti-Vδ₂-PE (Clone B6), anti-Vγ₂-FITC (Clone 7A5), anti-TNF-α-PE (Clone MAb11), anti-IFN-γ-PE-Cy7 (Clone 4S.B3), anti-interleukin-17A (IL-17A)-PE-Cy7 (Clone BL168), anti-Granzyme A-PE (Clone CB9), anti-Perforin-APC (Clone dG9), anti-granulocyte macrophage colony-stimulating factor (GM-CSF)-APC (Clone BVD2-21C11). The isotype control mAbs were purchased from the related company, respectively. The reagents listed below were all commercial products: brefeldin A (GolgiPlug, BD Biosciences), Cytofix/Cytoperm, and Perm buffer (BD Biosciences). The isotype control mAbs were purchased from the related company, respectively.

T‐cell polyfunctionality was measured as previously described (Kim et al., 2017). Briefly, splenocytes were stimulated with 5 μg/mL cEDIII for 4 h in the presence of 5 μg/mL brefeldin A. Cells were then washed with PBS and stained with a viability dye (eFluor 780; 1:1000 dilution; Thermo Fisher, Hemel Hempstead, UK) alongside an Fc receptor blockade (TruStain, 1 μg/mL; Biolegend London, UK) for 20 min at 4 °C. Following this, cells were washed in flow cytometry buffer and then fixed in 100 μL BD Cytofix (Becton Dickinson, Oxford, UK) for 30 min at 4 °C. Cells were washed and then stained with the following antibodies at optimized concentrations for 45 min at 4 °C: CD3‐FITC, CD4‐PerCP‐Cy5.5, CD8‐Alexa Fluor 700, IFN‐γ‐PE Dazzle, IL‐2‐PE, IL‐17A‐PE‐Cy7 and TNF‐α‐APC (all from Biolegend). Fluorescence minus one and PMA/ionomycin‐stimulated cells were used to determine gating boundaries and serve as positive controls. Cells were then washed twice with permeabilization buffer and flow cytometry buffer and then acquired on a LSR II (Beckton Dickinson, Oxford, UK) instrument.

Cells were stimulated in U-bottom 96-well plates (0.5 × 10⁶ cells/0.2 ml media/well) for 5 h with 50 ng/ml phorbol myristate acetate (PMA), 1 µg/ml ionomycin, and 10 µg/ml brefeldin A. DNAse (1 U/ml) was added 15 min before the stimulation period ended. The cells were then washed, stained with a fixable viability dye (#L34975, Thermo Fisher Scientific, Buenos Aires, Argentina), washed, stained with CD4 (CD4 FITC #100406, Biolegend), and then fixed, permeabilized, and stained for intracellular cytokines (IFN-γ PE #505808, IL-4 BV421 #504120, IL-17A PE-Cy7 #506922, Biolegend) with the Cyto-Fast™ Fix/Perm Buffer Set (#426803, BioLegend) as per the manufacturer’s instructions.