Anti 1 a 1 e pecy7 m5 114

Cervical draining lymph nodes (dLNs) were harvested and single-cell suspensions were prepared. To avoid non-specific staining, cells were blocked with an anti-FcR blocking antibody (eBioscience, San Diego, CA, USA). Mature DCs were stained with anti-CD11c Alexa 488 (N418, BioLegend), anti-CD45 PE (30-F11, eBioscience) and anti-I-A/I-E PeCy7 (M5/114.15.2, BioLegend). Tregs were stained with anti-CD4 FITC (RM4-5), anti-CD25 PE (PC61) and anti-Foxp3 PECy7 (FJK-16s) (BioLegend, San Diego, CA, USA). For intracellular IFN-γ and IL-17 staining, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml inomycin (Sigma-Aldrich, St. Louis, MO, USA) for 12 hours at 37°C and 5% CO₂ in the presence of GolgiStop (0.7μl per 100 μl cell culture media; BD Biosciences, San Jose, CA) to inhibit cytokine secretion. Cells were then stained with anti-CD4 FITC, anti-IFN-γ APC (XMG1.2) and anti-IL-17 PECy7 (TC11-18H10.1) (Biolegend). All antibodies and their matched isotype controls and fixation and permeabilization buffers were purchased from eBioscience. Stained cells were examined using the LSRII Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo software X 10.0.7. (FlowJo LLC, Ashland, OR, USA).