The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2+, and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to α-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide–MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.

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In patients with celiac disease, inflammatory T cell responses to HLA-DQ2-bound gluten peptides are thought to cause disease. Two types of HLA-DQ2 molecules exist, termed HLA-DQ2.5 and HLA-DQ2.2. Whereas HLA-DQ2.5 predisposes to celiac disease, HLA-DQ2.2 does not. We now provide evidence that the diseaseassociated HLA-DQ2.5 molecule presents a large repertoire of gluten peptides, whereas the non-disease-associated HLA-DQ2.2 molecule can present only a subset of these. C eliac disease (CD) is the most common food-induced enteropathy in humans. Patients display a permanent intolerance toward the gluten proteins in wheat. About 95% of CD patients are HLA-DQ2 ϩ (DQA1*0501, DQB1*0201, termed HLA-DQ2.5 hereafter). HLA-DQ molecules bind and present peptides to antigen-specific T cells. It is now commonly accepted that HLA-DQ2.5 can bind and present gluten peptides and that these HLA-DQ-peptide complexes induce inflammatory T cell responses, causing disease. The HLA-DQ2.5 molecule preferentially binds peptides with negatively charged amino acids at anchor positions (1, 2). Whereas gluten peptides contain few negative charges, these charges can be introduced by the enzyme tissue transglutaminase (tTG) that selectively deamidates glutamine residues in gluten peptides (3-5).A second HLA-DQ2 molecule ex ists (DQA1*0201, DQB1*0202, termed HLA-DQ2.2 hereafter) with peptidebinding properties that are virtually identical with the properties of HLA-DQ2.5 (6). In fact, HLA DQ2.2 has been shown to bind and present a gluten peptide to T cells (7). Yet, HLA-DQ2.2 does not predispose for CD unless it is expressed together with HLA-DQ2.5 (8). Individuals homozygous for the diseaseassociated HLA-DQ2.5 genotype or HLA-DQ2.2/2.5 heterozygous have the highest risk for developing CD. In contrast, HLA-DQ2.5/non-DQ2.2 heterozygous individuals have an only slightly increased risk (9-11). To determine the mechanism underlying these observations we have now compared the gluten-specific T cell response in the context of the HLA-DQ2.5 and HLA-DQ2.2 molecules. Materials and MethodsSynthetic Peptides. Peptides were synthesized by standard fluorenylmethoxycarbonyl (Fmoc) chemistry on a SyroII peptide synthesizer (MultiSynTech, Witten, Germany). The integrity of the peptides was checked by RP-HPLC and mass spectrometry. The gluten epitopes were synthesized with glutamic acid residues at positions that are deamidated by tTG.

Gluten ingestion causes coeliac disease in susceptible individuals. Gluten is a heterogeneous mixture of glutenin and gliadin, the latter of which is considered responsible for disease induction. By combining high‐performance liquid chromatography purification steps of gluten with a T cell bioassay and mass spectral analyses, we have identified a glutenin peptide (glt04 707 – 742) that activates T cells from the small intestine of a coeliac disease patient and results in the secretion of large amounts of IFN‐γ. The minimal T cell stimulatory core of the peptide (residues 724 – 734) is repetitively present in glutenin molecules. Moreover, it was observed that a large number of naturally occurring variants of this peptide are recognized by the T cells. These data suggest that the large heterogeneity of glutenin proteins dramatically increases the number of available T cell epitopes. Together, the results provide new insight into the nature of the gluten antigens that lead to coeliac disease and suggest that glutenin, next to gliadin‐derived antigens, may be involved in the disease process.

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