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).