Single-cell suspensions from duodenal biopsies or small intestinal resection were prepared as described (71 (link)) and blocked with FcR Blocking Reagent (Miltenyi Biotec) and stained with the following antibodies specific for CD markers; CD3-FITC (OKT3, Biolegend), CD11c-APC (S-HCl-3, BD Biosciences), CD14-APC (HCD14, Biolegend), CD19-PE-Cy7 (HIB19, Biolegend), CD27-BV421 (O232, Biolegend), CD38-APC-Cy7 (HIT2, Biolegend) and CD45-BV510 (H130, Biolegend), all at 1:20. Alternatively, CD3-Pacific Blue (OKT3, Biolegend), CD11c-APC (S-HCl-3, BD Biosciences), CD14-APC (HCD14, Biolegend), CD19-PerCP-Cy5.5 (HIB19, Biolegend), CD27-Pe-Cy7 (LG-7F9, Biolegend), CD38-APC-Cy7 (HIT2, Biolegend) and CD45-BV510 (H130, Biolegend), all at 1.5:50 were used. Peptide presentation was analyzed using mIgG2b 107, mIgG2b 3.C11 or mIgG2b OMV (all in-house generated, 10 μg/ml), followed by detection using anti-mIgG2b conjugated to PE (Biolegend, 1 μg/ml). Propidium iodide or LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen) were used for exclusion of dead cells and samples were immediately acquired on LSR Fortessa cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).
Frick R., Høydahl L.S., Petersen J., du Pré M.F., Kumari S., Berntsen G., Dewan A.E., Jeliazkov J.R., Gunnarsen K.S., Frigstad T., Vik E.S., Llerena C., Lundin K.E., Yaqub S., Jahnsen J., Gray J.J., Rossjohn J., Sollid L.M., Sandlie I, & Løset G.Å. (2021). A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T-cell activation. Science immunology, 6(62), eabg4925.
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