(A) Reverse-phase HPLC of a pepsin-trypsin digest of avenin. Peptides in fraction 25, obtained from gel filtration, were eluted by an acetonitrile gradient (straight line), and 41 fractions were collected. Fractions recognized by T-cell lines in subsequent experiments are indicated by the numbers above the peaks.

(B) T-cell recognition of fractions obtained by reverse-phase HPLC. All 41 fractions obtained in (A) were tested for recognition by the intestinal T-cell line 422.2.4 (derived from an oat-intolerant celiac disease patient). The fractions were incubated with DR3-DQ2 homozygous antigen-presenting cells overnight before the T-cell line was added. Specific T-cell responses were measured by ³H-thymidine incorporation. Pepsin-trypsin-digested avenins, both TG2-treated and untreated, were used as control antigens.

(C) Sequences of the peptides in the stimulatory fractions from reverse-phase HPLC identified by tandem mass spectrometry and overlapping synthetic peptides used for T-cell assays. For better comparison, the amino acid sequence of the avenin precursor protein JQ1047 (gi82331) is taken as a consensus sequence, and deviating residues are underlined.