Cd8 bv650

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].

Briefly, the small intestine was placed in Hank’s Balanced Salt Solution (HBSS) buffer supplemented with 10% fetal bovine serum. Peyers patches were excised and tissue was placed in digestion media containing 1 mM DTT, 1 mM EDTA in calcium/magnesium-free HBSS supplemented with 2% fetal calf serum and subsequently treated with Collagenase IV/Dnase digestion mix (0.5 mg/ml of collagenase IV and 200 μg/ml of Dnase). Lymphocytes were enriched using a 44/67% discontinuous Percoll (GE Lifesciences, Pittsburgh PA) gradient. Splenic lymphocytes were treated with ammonium chloride and washed. Cells were stimulated with phorbol 12-myristate 13-acetate and Ionomycin for 4 h at 37 °C in the presence of brefeldin A (GolgiPlug; BD Bioscience, San Jose, CA). Following stimulation, cells were stained with LIVE/DEAD Fixable Aqua (ThermoFisher Scientific, Waltham, MA), and the following antibody/fluorophore combinations APCC7-CD45, TCRβ-FITC, CD4-V500 (BD Bioscience), CD8-BV650, Foxp3-PECy7, IL17-PE, IFNγ-FITC (eBioscience, San Diego, CA), and fixed with fix/perm (eBioscience), were used according to the manufacturer’s instructions. Cells were acquired on an LSRII flow cytometer (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR); 100,000 events or greater were collected for each sample. Samples with yields less than 10,000 viable events were excluded from analysis.