Anti-CD3 Induced Mouse Intestinal Immunity

8–10-week-old female C57BL/6 mice were injected with 20 µg anti-CD3 monoclonal antibody (Clone: 145-2C11) at 0 and 48 h^{17 (link)}. Mice were dosed every 12 h by oral gavage with 100 mg/kg vismodegib (LC laboratories), prepared in MCT from a fresh vial for each experiment (0.5% methylcellulose, 0.2% Tween 80), or carrier control, with four to five mice per group. Mice were harvested at 52 h, at which point serum was collected and small intestines were harvested in ice-cold PBS. Small intestines were cleaned using ice-cold PBS, cut into roughly 5 mm pieces and collected in complete IMDM. Tissue pieces were rotated for 30 min at room temperature to release intraepithelial lymphocytes (IELs). Cells were strained on a 70 µm filter and IELs were collected from the interface of a 40–80% Percoll gradient (GE Healthcare, cat no. 17089101). IELs were restimulated for 4 h with PMA and Ionomycin in the presence of Monensin and subjected to viability, surface, and intracellular flow cytometric staining (for details see section on flow cytometric methods). Serum IL-17a concentrations were determined using the ELISA MAX™ Deluxe Set Mouse IL-17A (Biolegend, cat no. 432504) according to the manufacturer’s instructions.