Apc anti mouse il 4

Splenic CD4⁺ cells from approximately 28-day-old mice were obtained using the CD4⁺ T cell isolation kit (Miltenyi Biotec, 130-104-454) or by sorting with the BD FACSAria II cell sorter. CD4⁺ cells were processed using the Mouse Th1/Th2/Th17 (BD Biosciences, 560758) and Th17/Treg (BD Biosciences, 560767) phenotyping kits. Cells were cultured for 3–4 hours with or without 50 ng/mL PMA and 1 μg/mL ionomycin and then harvested for RNA-Seq or flow analysis. The following antibodies were used: PerCP-Cy5.5 anti–mouse CD4 (BD Biosciences, 550954), PE anti–mouse IL-17a (BD Biosciences, 559502), FITC anti–mouse IFN-γ (BD Biosciences, 554411), APC anti–mouse IL-4 (BD Biosciences, 554436), and Alexa Fluor 647 anti–mouse FOXP3 (BD Biosciences, 560402).

The extraction and purification of ATI were prepared as described by Zevallos, V. F [20 (link)]. Briefly, high-gluten wheat flour (Triticum aestivum L.) was extracted by using ammonium bicarbonate buffer, the supernatant was fractionally precipitated by using ammonium sulfate, and then the mixture was dialyzed, sterile filtrated, lyophilized and further purified by FPLC. Gliadin from wheat, bovine serum albumin (BSA), Poly:IC and DSS (MW: 40,000 Da) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Sodium salt of Caboxy Methyl Cellulose (CMC) with food grade was obtained from Jiangsu Tailida New Materials CO., Ltd. (Jiangsu Tailida, Nantong, China). Cholera Toxin (CT, B subunit) and tissue lysate were acquired from Absin (Absin, Shanghai, China). Mouse IL-1β, TNF-α, IL-4 and IL-13 enzyme-linked immunosorbent assay kits were purchased from Luminex (Univ-bio, Shanghai, China). APC anti-mouse IL-4, PE/Cyanine 7 anti-mouse CD4, FITC anti-mouse INF-γ and Fixable Viability Stain 510 were procured from BD Pharmingen (BD Biosciences, Piscataway, NJ, USA). Other analytical grade reagents were purchased from Xilong Scientific (Xilong Scientific, Guangdong, China).