Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin

Abstract

Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease.

Figure 1

HPLC purification of the gluten digest combined with a T cell bioassay and mass spectral analysis. (A) rp-HPLC profile of the pepsin/trypsin digest of gluten after three rounds of purification. (B) Stimulatory capacity of the collected rp-HPLC fractions shown in A. T cells (10⁴) of TCC S2 were stimulated with 10⁵ PBMCs (3,000 rad) from an HLA-DQ2/DQ8-positive donor in the presence of 2 μl of each rp-HPLC fraction (total fraction size: 100 μl). Values show mean cpm (×10⁻³) of duplicate cultures. SD is <10%. (C) Matrix-associated laser desorption ionization mass spectrometry profile (Lasermat) of the bioactive fraction 54 (see A and B).

Figure 2

Partial product ion mass spectrum of the 35-mer gliadin peptide (residues 198–232). The spectrum displays two series of peaks, the so-called b-ions and y-ions, that allow the sequence to be elucidated. A part of the y-ion series consists of the masses 857.5, 985.5, 1,084.5, and 1,171.6, which correspond to the partial sequence Glutamine/Lysine (Q/K),Valine (V), Serine (S). This information, together with the total mass of the 35-mer peptide (3,897 Da), was put in the program peptidesearch. This led to several hits, but only one gliadine-related peptide was found, gda09 198–232. In a similar fashion, the peptide sequences of the 2,479- and 2,679-Da peaks (Fig. 1C) were elucidated. These peaks were found to represent gliadin residues 3–24 (sequence VPVPQLQPQNPSQQQPQEQVPL) and glutenin residues 50–72 (sequence SQQQQPPFSQQQPPFSQQELPIL).

Figure 3

Truncation analysis of gliadin peptide 198–222. T cells (10⁴) (TCC S2) were stimulated with 10⁵ PBMCs (3,000 rad) from an HLA-DQ2/DQ8-positive donor in the presence of 7.5 μg/ml the indicated peptide sequences. Amino acids are shown as single-letter codes. (A) Results of a set of 18-mer peptides covering the gliadin 198–222 sequence. (B) Results of a set of truncated variants of the central portion of gliadin 198–222. Values show mean cpm (×10⁻³) of truplicate cultures. SD is <10%.