Cd4 rm4 5

Spleen and lymph node samples were forced through 100 μm filters to prepare single-cell suspensions. Dead cells were removed by gradient centrifugation with lymphocyte M (Cedarlane, Burlington, NC, USA) and live cells were stained with surface antibodies for FACS analysis. To isolate lymphocytes from colon, less than 0.1 cm colon pieces were cut and digested at 37°C with 3 mg mL⁻¹ dispase II (Thermo Fisher Scientific, Waltham, MA, USA), 1 mg mL⁻¹ collagenase D (Roche) and 0.1 mg mL⁻¹ DNase I (Roche) for 1 h until no visible pieces were present. The digested tissue was passed through a 100 μm filter, followed by centrifugation on a Percoll gradient.
For intracellular staining, the cells were first stained with antibodies for surface marker expression, then permeabilized and stained with antibodies for intracellular protein for 1 h using Foxp3 Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA). Data were collected in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD, Franklin Lakes, NJ, USA). Antibodies to the following markers were used: CD4 (RM4–5, Thermo Fisher Scientific), CD3 (145–2C11, Biolegend, Dedham, MA, USA), TCRβ (H57–597, BD Biosciences), RORγt (AFKJS-9, Thermo Fisher Scientific), CD25 (PC61, BD Biosciences), Foxp3(FJK-16s, Thermo Fisher Scientific).