Volunteers provided written informed consent. The Regional Committee for Medical and Health Research Ethics South East Norway (REK 2010/2720) approved the study. Intestinal TCCs were prepared from duodenal biopsies from CeD patients as previously described (36). To obtain peripheral blood TCCs, PBMC from CeD patients were stained with HLA-DQ2.5:gluten peptide tetramers (peptide-loaded major histocompatibility complex molecules, pMHC) labeled with phycoerythrin, sorted, and cloned by limited dilution according to previously reported methods (37). Six gut-derived and six blood-derived TCCs were considered to be clonal as judged by pMHC staining of >99% of cells. TCCs were seeded (1.5 million per well) into 48-well plates precoated with anti-CD3 (5 μg/ml; 300414, BioLegend, San Diego, CA) and incubated for 24 hours in 1.5 ml of RPMI 1640 supplemented with 10% heat-inactivated human serum and penicillin-streptomycin containing anti-CD28 (1 μg/ml; 302914, BioLegend). Medium (0.1 ml) was removed and centrifuged, and supernatants were frozen. In parallel, eight TCCs were also cultured for 24 hours with cognate peptide and monocyte-derived DCs derived from a HLA-DQ2.5+ donor not affected by CeD. DCs were prepared by incubating positively selected PBMC CD14+ cells (CD14 microbeads; Miltenyi Biotec Inc., San Diego, CA) with GM-CSF (1000 U/ml) and IL-4 (500 U/ml). Cell cultures were replenished on alternate days with half the medium replaced with RPMI supplemented with 10% fetal calf serum containing GM-CSF and IL-4. On day 6, lipopolysaccharide (150 ng/ml) was included with GM-CSF and IL-4 in the medium added. On day 7, mature DCs were collected, washed, counted, and resuspended in RPMI + 10% human serum (0.5 million/ml) and incubated overnight with PBS or matched cognate peptide, either 10 μM DQ2·5-glia-γ1 peptide (YQQLPQPEQPQQSFPEQERPF) or 2 μM α-gliadin-33mer peptide (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF). Peptide-pulsed DCs (0.4 million) and TCCs (1.2 million) were added to each 48 well in a total volume of 1.2 ml. Medium (0.1 ml) was removed and centrifuged, and supernatants were frozen. Six TCCs were incubated in wells coated with immobilized epitope-specific pMHC (38). TCCs (260,000) were incubated with plate-bound pMHC in nine replicate wells (225 μl per well). Thirty microliters of medium was removed from each of three wells and pooled, producing three pooled samples for each time point that were frozen.

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