The median age was 5·1 years (range 4·0–6·1). All control children were tested negative for TGA at the time of sampling. The study was approved by the Ethics Committee of the Kuopio University Hospital and written informed consent was obtained from all parents/guardians and age-appropriate children

(>10 years of age). Purified tetanus toxoid (TT; National Institute of Health and Welfare, Helsinki, Finland) was used as an independent control antigen at a final concentration of 1 µg/ml and purified phytohaemagglutinin (PHA) as a mitogen control of cell functionality at 2 µg/ml (Remel, Crossways, Dorset, UK). gTG was prepared as follows. First, native gliadin from wheat powder (Sigma-Aldrich, find protocol St Louis, MO, USA) was dissolved in dimethyl sulphoxide (DMSO) and diluted with 4 mM CaCl2 dilution [CaCl2 dissolved to phosphate-buffered saline (PBS)] to a concentration of 4 mg/ml. TTG from guinea pig liver (Sigma-Aldrich) was dissolved in PBS to a concentration of

0·8 mg/ml. Deamidation of gliadin with TTG was accomplished by incubation of these two antigens in a final volume of 100 µl (25 µl gliadin dilution, 25 µl TTG dilution and 50 µl PBS) for 2 h at 37°C. Finally, 20 µl of this mixture per 1-ml culture medium was used to stimulate cells. Native gliadin alone was used at a final concentration of 10 µg/ml and TTG alone at 2 µg/ml. Peripheral blood mononuclear cells (PBMC) were also stimulated with 10 µg/ml of synthetic gTG peptides QLQPFPQPELPY (Q12Y) and PQPELPYPQPELPY RAD001 chemical structure (P14Y) (purity > 95%; GL Biochem, Shanghai, China) containing the earlier-reported immunodominant gliadin epitopes α-I and α-II, respectively [5]. Peripheral blood mononuclear cells (PBMC) were isolated from fresh venous blood by Ficoll Histopaque gradient centrifugation (Sigma-Aldrich), according to the manufacturer’s Non-specific serine/threonine protein kinase protocol. PBMCs were washed twice with PBS and labelled with CFSE (Invitrogen, Molecular Probes, Carlsbad, CA, USA). Briefly, PBMC at 107/ml were suspended in 1 µM CFSE in PBS and incubated for 10 min at 37°C. After incubation

cells were washed with culture medium (RPMI-1640 supplemented with 5% inactivated human AB serum (Sigma Aldrich), 2 mM l-glutamine, 20 µM 2-mercaptoethanol, 1 mM natrium pyruvate, non-essential amino acids, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 mM HEPES), reincubated for 30 min at +37°C and washed again to remove unbound CFSE. Finally cells were suspended in culture medium at 106/ml and stimulated with different antigens in a volume of 200 µl in 96-well round-bottomed plates (Costar, Corning Incorporated, Corning, NY, USA). Cells were maintained at 37°C and 5% CO2 incubator in six to eight equal wells per antigen and analysed on day 10 by flow cytometry [fluorescence activated cell sorter (FACS) Canto II; Becton Dickinson, Mountain View, CA, USA) using FACSDiva software (BD Pharmingen, San Jose, CA, USA).