Th Cell Subset Profiling Protocol

The spleen and MLNs were aseptically separated, and single-cell suspensions were prepared as described previously [14 (link)–16 (link)]. Single-cell suspensions from the spleen and MLNs were stimulated with a brefeldin/monensin mixture [Brefeldin A; Multisciences (Lianke) Biotech, Hangzhou, China] and a phorbol-12-myristate-13-acetate/ionomycin mixture [Multisciences (Lianke) Biotech] for 5 h. Fluorescein isothiocyanate (FITC)-anti-mouse-CD4 (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) was used for surface staining. APC-anti-mouse-IFN-γ (eBioscience, Thermo Fisher Scientific), APC-anti-mouse-IL-4 (BioLegend, San Diego, CA, USA) and PE-anti-mouse-IL-17A (Biolegend) were utilized for intracellular staining after the membrane was ruptured by fixation/permeabilization (BD Biosciences, Franklin Lakes, NJ, USA) to analyze Th1, Th2 and Th17 cells as described previously [16 (link), 19 (link)]. Mononuclear cells were stained with FITC-anti-mouse-CD4, APC-anti-mouse-CD25 and PE-anti-mouse Foxp3 by using a mouse regulatory T cell staining kit (eBioscience) to evaluate regulatory T (Treg) cells. All samples were analyzed using a BD FACSCcanto flow cytometer (BD Biosciences), and data were analyzed with Flowjo v10.0.7 software (Tree Star, Ashland, OR, USA).