Volunteers provided written informed consent. The University of Chicago IRB (12623B) approved the study. PBMC were isolated by Ficoll-Paque density centrifugation. Cells were isolated from duodenal biopsies of patients with CeD on GFD via 1-hour mechanical disruption in RPMI containing 2 mM EDTA (46-034-CI, Corning) and 1.5 mM MgCl2 (BP214-500, Thermo Fisher Scientific) for acquisition of intraepithelial lymphocytes and subsequent 1-hour mechanical disruption in RPMI containing collagenase type IV (C5138-100, Sigma-Aldrich) (39) for acquisition of lamina propria lymphocytes. Cells staining positive for anti-CD3 (PE-Cy7, 300420, UCHT1, BioLegend), TCRαβ (αβ T cell receptor; BV421, 306722, IP26, BioLegend), and CD4 (PerCP/Cy5.5, 317428, OKT4, BioLegend) were purified by fluorescence-activated cell sorting (fig. S4). Five to 10,000 sorted cells were expanded with phytohemagglutinin-L (1 μg/ml; M5030, Calbiochem/EMD Millipore, Billerica, MA) and a mixture of irradiated heterologous PBMCs and Epstein Barr virus–transformed B lymphoblastoid cell lines in RPMI + 10% human serum AB (S40110, Atlanta Biologicals, Norcross, GA) and maintained with IL-2 (100 U/ml; 136, National Institutes of Health AIDS Reagent Program) (40). One round of cell expansion for 29 days was performed. Cells were 98.5 to 99.5% TCRαβ/CD4 double positive. For stimulation with anti-CD3 (555329, BD Bioscience, Franklin Lakes, NJ) and anti-CD28 (555726, BD Bioscience), 96-well flat-bottom plates (07-200-656, Thermo Fisher Scientific) were coated overnight (anti-CD3, 1.5 μg/ml; anti-CD28, 1 μg/ml) at 4°C. Cells were then plated 200 μl per well in RPMI + 10% human serum AB (S40110, Atlanta Biologicals, Norcross, GA) at a concentration of 1 × 106 cells/ml counted using a hemocytometer and incubated 24 hours at 37°C. Supernatants were harvested and frozen at −80°C until evaluation.

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