Freshly isolated PBMCs from non-IBD controls were stimulated overnight with soluble anti-human CD3 [1 µg/mL] and soluble anti-human CD28 [1 µg/mL, both BD Biosciences] to induce LAG-3 surface expression. Cells were stained with the following antibodies: CD4-FITC, CD8a-BV650, CD45-AF700, LAG-3-PE, and 4’,6-Diamidino-2-Phenylindole, Dilactate [DAPI, Biolegend, Supplementary Table 1A ]. The PBMCs were sorted on a FACSAriaIII using a 70-µm nozzle and those T cells [CD4+ and CD8+] that were LAG-3+ or LAG-3− were sorted into separate collection tubes.
To determine cytokine expression, sorted LAG-3+ and LAG-3− cells were cultured for 3 h at 37°C in modified Dulbecco’s medium [MDM, Life Technologies] with 10% FCS and 25 mM HEPES [Life Technologies] with or without PMA [100 ng/mL]/ionomycin [1 µg/mL] to induce cytokine production and with GolgiPlug and GolgiStop [BD Biosciences] to prevent extracellular secretion of cytokines. Cells were stained with fixable viability dye eFluor-780 [eBioscience], then fixed using the fixation buffer for 1 h. The cells were washed twice with 1× permeabilization buffer and stained with the following antibodies: CD4-FITC, CD8-BV650, CD45-AF700, LAG-3-PE, GM-CSF-PE-Dazzle, Foxp3-BV421, CD25-BV786, IL-10-PE-Cy7, IFNγ-PE-Dazzle, IL-22-PE-Cy7, IL-4-BV711 and IL-17A-BV421 [Supplementary Table 1A ].
To determine cytokine expression, sorted LAG-3+ and LAG-3− cells were cultured for 3 h at 37°C in modified Dulbecco’s medium [MDM, Life Technologies] with 10% FCS and 25 mM HEPES [Life Technologies] with or without PMA [100 ng/mL]/ionomycin [1 µg/mL] to induce cytokine production and with GolgiPlug and GolgiStop [BD Biosciences] to prevent extracellular secretion of cytokines. Cells were stained with fixable viability dye eFluor-780 [eBioscience], then fixed using the fixation buffer for 1 h. The cells were washed twice with 1× permeabilization buffer and stained with the following antibodies: CD4-FITC, CD8-BV650, CD45-AF700, LAG-3-PE, GM-CSF-PE-Dazzle, Foxp3-BV421, CD25-BV786, IL-10-PE-Cy7, IFNγ-PE-Dazzle, IL-22-PE-Cy7, IL-4-BV711 and IL-17A-BV421 [