Celiac disease T cell clone response to CD3/CD28 stimulation

To identify the CD4+ T cell cytokines responsible for the proliferation of the Lin-IEL lines CD4+ T cell clone L10, which recognises DQ2-glia-α1, one of the immunodominant T cell epitopes in celiac disease, was stimulated for 3 hours in IMDM with plate-bound CD3/CD28-specific (2.5 µg/ml each) or control antibodies coated onto 6-well non-tissue culture treated plates. Three independent biological replicates were performed, each time including 6 million Ficoll-purified live cells per condition. RNA was purified from these cells using the RNAeasy mini kit (Qiagen, Venlo, the Netherlands). cDNA was amplified using the Applause WT-Amp system (NuGEN technologies, Bemmel, the Netherlands) and biotin-labelled with the Encore Biotin Module (NuGEN). Human Gene 1.0 ST arrays (Affymetrix, High Wycombe, UK) were employed to quantify global gene expression. CD4+ T clone L10 cells, isolated from a duodenal biopsy of a celiac disease patient (Petersen et al., 2014, Nat. Struct. Biol. 21:480-488) were stimulated for 3 hours in IMDM with plate-bound CD3/CD28-specific (2.5 µg/ml each) or control antibodies. Three independent biological replicates were performed, each time including 6 million Ficoll-purified live cells per condition.