Soluble anti human cd3

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].

Cells were stimulated with either: phorbol myristate acetate [PMA; 50 ng/mL or 100 ng/mL, Sigma Aldrich] and ionomycin [1 µg/mL; Sigma Aldrich] for 2-4hrs, soluble anti-human CD3 [1 µg/mL, UCHT1] and soluble anti-human CD28 [1 µg/mL, CD28.2, both BD Biosciences] overnight, or anti-human CD3 and recombinant human IL-12 [1ng/mL, Life Technologies] overnight. GolgiStop and GolgiPlug [both BD Biosciences] were added at the beginning of the PMA/ionomycin stimulation, or 4 h before the end of stimulation for the CD3/CD28 or CD3/IL-12 stimulation. Cells were stained with fixable viability dye and fixed for 1 h [Foxp3 staining buffer set, eBioscience]. Cells were then washed twice with 1× permeabilization buffer and stained for all relevant surface and intracellular markers for 1 h. The antibodies used were CD4-FITC, CD8-BV650, CD45-AF700, Fixable Viability Dye-eFluor-780, GM-CSF-PE-Dazzle, CD25-BV786, IL-10-PE-Cy7, IFNγ-PE-Dazzle, IL-22-PE-Cy7, IL-4-BV711 and IL-17A-eFluor450 [Supplementary Table 1A].