α cd3ε

Small intestinal and colonic lamina propria mononuclear cells were isolated as previously described66 (link). Briefly, Peyer’s Patches were removed, and whole small intestine and colon were opened longitudinally, cut into pieces and incubated for 30 minutes in cold calcium- and magnesium-free PBS containing 30 mM EDTA, washed extensively, and incubated for several rounds in iDMEM (Life Technologies) containing collagenase D (0.7 mg/ml; Roche) and Liberase TL (2.5 mg/ml; Roche) at 37 °C. The remaining intestinal fragments were collected and pressed through 100μm filters (BD Biosciences), mixed with the collected supernatants. Mononuclear cells were separated by a 40/80% (wt/vol) Percoll (Sigma-Aldrich) density gradient and washed prior to staining for flow cytometry.
For cytokine quantification, either single-cell suspensions or isolated CD4⁺ T cells from mLNs by a negative magnetic separation (MACS) using CD4⁺ T cell isolation kit (Miltenyi Biotech) were cultured on plate bound α-CD3ε (clone: 145-2C11) and α-CD28 (clone: 37.51) (eBioscience) (both at 2.5 μg/ml) for 72 hours.

After flow sorting, cells were cultured in 96-well plates (Corning, Corning, NY) at 50,000 cells per type per well in advanced RPMI (Gibco/Fisher Scientific, Waltham, MA) supplemented with 5% Australian-produced heat-inactivated fetal bovine serum, 55μM β-mercaptoethanol, 100U/mL and 100μg/mL Penicillin/Streptomycin, and 0.2mM L-glutamine (Gibco/Fisher Scientific, Waltham, MA) at 5% CO₂, 37°C. For activating conditions, wells were coated with αCD3ε (eBioscience, San Diego, CA) at 2.5μg/mL in PBS then incubated at 37°C for 4 hours followed by two washes with PBS before receiving cells. For fixation studies, indicated cell types were resuspended in 2% paraformaldehyde in PBS for 10 minutes on ice. Cells were washed twice in PBS before being co-cultured with live CD4⁺FoxP3⁺ cells. All co-culture experiments were performed at 1:1 cell ratios, except for the experiments in Fig 1C, which were performed by maintaining constant Tconv cell numbers (50k) and altering the number of Tregs relative to the 50k Tconv, as indicated.