Lag 3 pe

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].

The following anti-human monoclonal antibodies (mAb) were used for flow cytometry staining: CD8-FITC, CD137-PE, CD39-PE-Cy7, CD4-AF700, LAG3-PE, PD-1-PE, PD-L1-PE (all eBioscience. Waltham, USA), BLTA-BV421, GITR-BV421, OX40-PE-Cy5, TIM3-PB (all Biolegend, London, UK), and CD45-AMCyan (BD Bioscience, San Jose, CA, USA). All antibodies were pre-titrated using stimulated and non-stimulated PBL to determine optimal staining dilutions. For IHC, PD-L1 (E1L3N) XP Rabbit mAb was purchased from Cell Signaling Technology, Danvers, MA, USA.

CAR expression was determined with a biotin-conjugated goat anti-murine Fab SP-longer Spacer immunoglobulin G (IgG) (1:40) (Jackson Immunoresearch, Philadelphia, PA, USA) and antigen-presenting cell (APC)-conjugated streptavidin (1:350) (Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed with 2 mL of FACS buffer (PBS + 2% BSA + 2 mM EDTA) between each step. For human T cell phenotyping, the following monoclonal antibodies (mAbs) were used: CD45RA-phycoerythrin (PE)/fluorescein isothiocyanate (FITC) (HI-100, 1:400), CD62L-PE-Cy7 (DREG56, 1:400), CD3-PerCP-Cy5/APC-780 (OKT3, 1:400), PD1-Allophycocyanin (APC) (MIH4, 1:200), LAG-3-PE (3DS223H, 1:200), TIM-3-APC-Cy7 (F38-2E2, 1:200), and CD95-APC (Fas/ApoI, DX2, 1:100), all from eBioscience (Thermo Fisher Scientific). Tonic signaling was determined by intracellular staining with pCD3z-PE (Tyr142, 3ZBR4S, 1:100) and Fix & Perm Kit (Nordic MuBio, Susteren, the Netherlands). Samples were acquired on a FACSCantoII cytometer (BD Dickinson, NJ, USA). FlowJo software (TreeStar, BD Biosciences, USA) was used for data analysis.