Antigen-reactive T-cell enrichment
Identification and enrichment of antigen-reactive T-cells was performed by applying the recently described ARTE technique 7. Briefly, 0.5 –1 x107 PBMC were cultured in RPMI1640 supplemented with 5% human AB-serum and stimulated for 6 h with 1 µg/mL CD40 (Miltenyi Biotec, Bergisch Gladbach, Germany) in the presence or absence of the pepsin-trypsin digested 33-mer gliadin peptide (200 µg/mL) (Sigma- Aldrich), OVA-peptide (Invitrogen) or soybean or peanut extract (200 µg/mL) (Greer Laboratories). For the last 2 h, 1 µg/mL brefeldin A (Sigma- Aldrich) was added. Cells were indirectly labeled with anti-CD154-biotin antibody, followed by anti-biotin MicroBeads (CD154 MicroBead-Kit, Miltenyi Biotec) and magnetically enriched using MS columns (Miltenyi Biotec).