Cytexpert experiment based software

After 72 h of polarization, cell culture supernatants were assayed by ELISA to measure the levels of mouse IFN-γ, IL-4, IL-9, and IL-17A (BioLegend) according to the manufacturer’s instructions. For intracellular staining, cells were cultured for 3 days, restimulated for 1 additional day, and then stimulated for 4 h at 37 °C in RPMI-1640 medium containing phorbol 12-myristate 13-acetate (50 ng ml⁻¹, Sigma-Aldrich, St Louis, MO, USA) and ionomycin (1 μg ml⁻¹, Sigma-Aldrich). After staining for surface markers, the cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm Kit, Thermo Fisher Scientific) and then stained for intracellular products. The following monoclonal antibodies were used for the flow cytometric analyses: fixable viability dye eFluor^TM 450- or phycoerythrin-conjugated anti-CD4 and allophycocyanin-conjugated anti-IFN-γ, anti-IL-4, anti-IL-9, anti-IL-17A, or anti-Foxp3. All events were acquired on a BeckmanCoulter DxFLEX flow cytometer equipped with CytExpert experiment-based software (BeckmanCoulter, Inc.), and data were analyzed using FlowJo software (TreeStar). Gating strategies were presented in Supplementary Fig. 9.

Intracellular staining was performed as described previously (40 (link)). Splenocytes and peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: PE-Cy 7-CD4 (#201516; BioLegend, San Diego, CA, USA), FITC-CD25 (#202103; BioLegend), PE-Foxp3 (#12-5773-82; eBioscience, San Diego, CA, USA), FITC-IFN-γ (#507804; BioLegend), PE-Cy5.5-IL-17A (#45-7177-82, eBioscience), and PE-IL-10 (#555088, BD Biosciences, Franklin Lakes, NJ, USA). Events were analyzed using FlowJo software (Tree Star, Ashland, OR, USA) and CytExpert experiment-based software (Beckman Coulter, Inc, Bera, CA, USA.).

According to the manufacturer’s instructions, intranuclear staining was carried out with fixation/permeabilization buffer solution (00-5123-43 and 00-5223-56, eBioscience, San Diego, CA, USA). For intracellular staining, cells were stimulated for 4 h at 37 °C in medium containing PMA (P1585, 50 ng ml⁻¹), ionomycin (I3909, 1 μg ml⁻¹) (both from Sigma–Aldrich), and brefeldin A solution (00-4506-51, eBioscience) and were then subjected to an intracellular staining protocol (00-8222-49, eBioscience). Stained cells were analyzed on a Beckman Coulter DxFLEX flow cytometer equipped with CytExpert experiment-based software (Beckman Coulter, Inc., 250 South Kraemer Boulevard Brea, CA, USA), and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The gating strategies are presented in Supplementary Fig. 11.