Anti cd3ε clone 2c11

Colonic-LP CD45⁺CD3⁺CD4⁺CD44^low cells were sorted and seeded into 96-well plates for stimulation with plate-bound anti-CD3ε (clone 2C11, 5 µg/ml, eBioscience) and soluble anti-CD28 (clone 37.51, 2 µg/ml, eBioscience) antibodies alone or in combination with recombinant IL-12 (100 ng/ml, Peprotech) in complete RPMI medium and were incubated for 8 h at 37^°C. After incubation, supernatants were collected for cytokine ELISA detection.
For STAT4 staining, CD4⁺ T cells were incubated at 37^°C for 5 h in complete RPMI medium alone or with recombinant IL-12 (100 ng/ml, Peprotech) and then fixed (4% PFA) for 30 min at room temperature. The cells were then rinsed three times in PBS for 5 min and incubated in a blocking buffer (0.1% Saponin in 1% BSA–PBS) for 30 min at room temperature. The cells were then incubated for 2 h at room temperature with purified anti-STAT4 (15A1B41, Biolegend) and IgG2b isotype (RTK4530, Biolegend) antibodies diluted in a blocking buffer, followed by two washes in a blocking buffer for 5 min and incubated overnight at 4^°C. The following day, the cells were incubated for 1 h with a PE-conjugated secondary antibody (Thermo Scientific), then washed and mounted with Prolong gold DAPI antifade reagent (Molecular Probes).

Sorted splenic-naïve CD4⁺ T cells (CD45RB^highCD44^lowCD62L⁺) from Cnb1^fl/fl mice were stained with CellTrace™ Violet (Molecular Probes) and cocultured with sorted splenic dendritic cells (CD11c^highMHCII⁺) at a 5:1 ratio in the presence or absence of sorted Treg cells (CD45RB^lowCD25⁺GiTR⁺) from Cnb1^fl/fl or Cnb1^CD4 mice and plate-bound anti-CD3ε (clone 2C11, 2 µg/ml, eBioscience) and anti-CD28 (clone 37.51, 2 µg/ml, eBioscience) antibodies, and IL-2 (200 U/ml). After 4 days, proliferation of the naïve CD4⁺ T cells was measured by dye dilution. Data were analyzed using FlowJo (TreeStar Inc.).

Mononuclear cells isolated from colonic-LP were stimulated with PMA (1 µg/ml, Sigma) and ionomycin (1 µg/ml) for 6.5 h in vitro in the presence of Brefeldin A (10 µg/ml, Sigma) for the final 5 h. The cells were then harvested and surface-labeled with anti-CD4 antibody before fixation/permeabilization using BD Cytofix/Cytoperm™ kits (BD Bioscience). Intracellular staining was performed for 30 min at 4^°C using anti-IL-2 (JES5H4), IL-17 (TC11-18H10.1), IL-10 (JES5-16E3), IL-4 (BVD6-24G2), and TGFβ1 (TW7-16B4) antibodies, all from Biolegend.
For apoptosis experiments with sorted cells, DAPI⁻CD45⁺CD4⁺ T cells from colonic LP were sorted and seeded into 96-well plates for stimulation with plate-bound anti-CD3ε (clone 2C11, 5 µg/ml, eBioscience) and soluble anti-CD28 (clone 37.51, 2 µg/ml, eBioscience) antibodies in complete RPMI medium and incubated for 24 h at 37^°C. After incubation, cell death was quantified by Annexin V and propidium iodide staining (BD Bioscience).