T-Cell Response to Gluten in Patients With HLA-DQ2.2 Reveals Requirement of Peptide-MHC Stability in Celiac Disease

doi:10.1053/j.gastro.2011.11.021

Gastroenterology

Volume 142, Issue 3, March 2012, Pages 552-561

https://doi.org/10.1053/j.gastro.2011.11.021 Get rights and content

Background & Aims

Celiac disease is a diet-induced, T cell–mediated enteropathy. The HLA variant DQ2.5 increases risk of the disease, and the homologous DQ2.2 confers a lower level of risk. As many as 5% of patients with celiac disease carry DQ2.2 without any other risk alleles. Epitopes commonly recognized by T cells of patients with HLA-DQ2.5 bind stably to DQ2.5 but unstably to DQ2.2. We investigated the response to gluten in patients with HLA-DQ2.2.

Methods

We generated intestinal T-cell lines and clones from 7 patients with HLA-DQ2.2 (but not DQ2.5) and characterized the responses of the cells to gluten. The epitope off-rate was evaluated by gel filtration and T cell–based assays. Peptide binding to DQ2.2 was studied with peptide substitutes and DQ2 mutants.

Results

Patients with DQ2.2 and no other risk alleles had gluten-reactive T cells that did not respond to the common DQ2.5-restricted T-cell epitopes. Instead, many of the T cells responded to a distinct epitope that was not recognized by those from patients with HLA-DQ2.5. This immunodominant epitope bound stably to DQ2.2. A serine residue at P3 was required for the stable binding. The effect of this residue related to a polymorphism at DQα22 that was previously shown to determine stable binding of peptides to DQ2.5.

Conclusions

High levels of kinetic stability of peptide–major histocompatibility complexes are required to generate T-cell responses to gluten in celiac disease; the lower risk from DQ2.2 relates to constraints imposed on gluten peptides to stably bind this HLA molecule. These observations increase our understanding of the role of the major histocompatibility complex in determining T-cell responses in patients with celiac disease and are important for peptide-based vaccination strategies.

Section snippets

Subjects

Seven subjects who expressed HLA-DQ2.2 but not DQ2.5 or DQ8 were included in the study (see Table 1). They obtained their diagnosis of celiac disease according to the American Gastroenterological Association guidelines,¹⁶ except one patient who had normal gut histology but highly positive anti-transglutaminase 2 (TG2) serology. Biopsy specimens were taken as part of routine clinical follow-up or patients volunteering for endoscopy for research purposes. Serologic typing, genomic typing, or both

DQ2.2 Patients Have Gluten-Reactive CD4⁺ T Cells in the Intestine

To evaluate whether patients with celiac disease who carry DQ2.2 but not DQ2.5 or DQ8 (hereafter referred to as DQ2.2 patients with celiac disease) have gluten-reactive T cells in their small intestine, we generated intestinal TCLs from 7 DQ2.2 patients with celiac disease and found gluten-reactive TCLs in 4 of these (Table 1).

Identification of a T-Cell Epitope Recognized by DQ2.2 Patients With Celiac Disease

From the TCLs generated, we established a total of 27 gluten-reactive TCCs. The TCCs were in an initial screen tested for reactivity against a panel of epitopes most

Discussion

The motivation behind this study was to understand whether and how patients with celiac disease who express DQ2.2, but not DQ2.5 or DQ8, make a T-cell response to gluten. The results underscore the notion that high kinetic stability of peptide-MHC is a key factor for establishment of antigluten T-cell responses and the development of celiac disease.

Kinetic stability of peptide-MHC complexes has been shown to be decisive for antigen-presenting cells to successfully activate naïve T cells in the

Acknowledgments

The authors thank the patients with celiac disease who have donated biological material to the study, Jørgen Jahnsen (Oslo University Hospital-Aker) for providing biopsy specimens of the gut from one of the patients, Maria Stensland (Proteomics Core Facility, Oslo University Hospital) and Siri Dørum for mass spectrometry analysis and help with interpretation of data, Bjørg Simonsen for help with performing the competitive peptide binding assays, Lars Egil Fallang and Elin Bergseng for the

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The binary HLA-DQ2.2-glut-L1 and HLA-DQ2.5-glia-α2 structures revealed that the difference in epitope binding preference of HLA-DQ2.5 and HLA-DQ2.2 molecules was attributed to this single polymorphic residue at position 22 of HLA-DQ2 α-chain (Figure 3) [12]. This DQα22 polymorphism perturbs a crucial hydrogen (H)-bonding network with the gluten epitope main chain required for stability [6,31]. Since HLA-DQ2.2-Phe22α cannot contribute to the H-bond network, unlike HLA-DQ2.5-Tyr22α, the HLA-DQ2.2 has an additional selectivity constraint toward epitopes containing p3-Ser needed for compensatory H-bonds with nearby HLA residues (Figure 3) [6,12,31].
Celiac disease (CeD) is a human leukocyte antigen (HLA)-linked autoimmune-like disorder that is triggered by the ingestion of gluten or related storage proteins. The majority of CeD patients are HLA-DQ2.5⁺, with the remainder being either HLA-DQ8⁺ or HLA-DQ2.2⁺. Structural studies have shown how deamidation of gluten epitopes engenders binding to HLA-DQ2.5/8, which then triggers an aberrant CD4⁺ T cell response. HLA tetramer studies, combined with structural investigations, have demonstrated that repeated patterns of TCR usage underpins the immune response to some HLADQ2.5/8 restricted gluten epitopes, with distinct TCR motifs representing common landing pads atop the HLA–gluten complexes. Structural studies have provided insight into TCR specificity and cross-reactivity towards gluten epitopes, as well as cross-reactivity to bacterial homologues of gluten epitopes, suggesting that environmental factors may directly play a role in CeD pathogenesis. Collectively, structural immunology-based studies in the CeD axis may lead to new therapeutics/diagnostics to treat CeD, and also serve as an exemplar for other T cell mediated autoimmune diseases.
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These findings have been brought to attention in Table 1 sequences throughout bold (E) and underlined (Q) font in the respective residues. Unfortunately, there were not enough experimental data to cross-compare epitopes HLA-DQ binding affinity (Arentz-Hansen et al., 2000; Bodd et al., 2012; Dorum et al., 2014; Klock et al., 2012; Kooy-Winkelaar et al., 2011; Qiao et al., 2005; Stepniak et al., 2005) (Table S2). The affinity column (Table 1), listed for each epitope, represent the antigen affinity to bind the receptor.
A ranking of gluten T-cell epitopes triggering celiac disease (CD) for its potential application in the safety assessment of innovative food proteins is developed. This ranking takes into account clinical relevance and information derived from key steps involved in the CD pathogenic pathway: enzymatic digestion, epitope binding to HLA-DQ receptors of the antigen-presenting cells and activation of pro-inflammatory CD4 T-cells, which recognizes the HLA-DQ-epitope complex and initiates the inflammatory response. In silico chymotrypsin digestion was the most discriminatory tool for the ranking of gluten T-cell epitopes among all digestive enzymes studied, classifying 81% and 60% of epitopes binding HLA-DQ2.5 and HLA-DQ8 molecules, respectively, with a high risk. A positive relationship between the number of prolines and the risk of gluten T-cell epitopes was identified. HLA-binding data analysis revealed the additional role played by the flanking regions of the 9-mer epitopes whereas the integration of T-cell activation data into the ranking strategy was incomplete because it was difficult to combine results from different studies. The overall ranking proposed in decreasing order of immunological relevance was: α-gliadins > ω-gliadins > hordeins > γ-gliadins ∼ avenins ∼ secalins > glutenins. This novel approach can be considered as a first step to reshape the risk assessment strategy of innovative proteins and their potential to trigger CD.
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The notion that CD4+ T cells play a central role in coeliac disease (CeD) became strong with the observation that gluten-reactive T cells selectively recognize gluten antigen in the context of the CeD-associated HLA class II allotypes. Today a whole body of evidence points to these gluten-specific CD4+ T cells as essential drivers of CeD pathology. The interaction of T cells with B cells seems to be particularly important as this leads to the formation of CeD-associated antibodies as well as to clonal expansion of T cells. Here we will discuss this and other key aspects of the CeD pathogenesis.
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The Human Leukocyte Antigen (HLA) has a crucial role in the development and pathogenesis of coeliac disease (CD). The genes HLA-DQA1 and HLA-DQB1, both lying in this region and encoding the HLA-DQ heterodimer, are the main genetic predisposing factors to CD. Approximately 90% of CD patients carry the heterodimer HLA-DQ2.5, leaving only a small proportion of patients with lower risk heterodimers (HLA-DQ8, HLA-DQ2.2 or HLA-DQ7.5). These HLA-DQ molecules act as receptors present in the surface of antigen presenting cells and show high affinity for deamidated gluten peptides, which bind and present to CD4⁺ T cells. This triggers the immunological reaction that evolves into CD. Since specific HLA genetics is present in almost the totality of CD patients, HLA typing has a very high negative predictive value, and it can be used to support diagnosis in specific scenarios. HLA risk has been associated to different CD-related features, such as age at onset, clinical outcomes, antibody levels and grade of histological lesion; but further research is needed. HLA-DQ genotypes have been also suggested to modulate the composition of the gut microbiota.
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CD1 glycoproteins are a class of antigen presenting molecules that bind and present non-peptidic antigens (lipids and glycolipids) for immune recognition. CD1 polymorphisms, although limited, could have a critical role in antimicrobial, anticancer, and autoimmune responses and disease susceptibility. Ethnic differences and interactions between genetic and environmental factors make it attractive the study of these molecules in autoimmune inflammatory disorders, such as celiac disease (CD), in which a strong genetic predisposition (HLA-DQ2/DQ8) and pressure of environmental factors have a central role. CD1A, CD1D and CD1E polymorphisms in exon 2 were assessed in patients from Morocco affected by CD, using direct sequencing analysis, in order to investigate possible associations with the disease in a North African population. Differences in genotype and haplotype distribution of CD1E between celiac patients and controls were found: in particular, an increase of CD1E*02/02 homozygous (OR 2.93, CI 1.30-6.59, p = 0.007) and CD1A*02-E*02 estimated haplotypes in CD, compared with controls. Frequencies of CD1A and CD1D genotypes/alleles were not different between groups. CD1E*02/02, previously suggested as a potential immune protective genotype to malaria susceptibility, could be an additional gene involved in celiac risk in this geographic area.

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by the Research Council of Norway (to L.M.S.) and Biomedical Research Council of Singapore (to C.-Y.K.). M.B. is a recipient of a doctoral fellowship from the Norwegian Foundation for Health and Rehabilitation (EXTRA fund).

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Original ResearchBasic and Translational—Alimentary TractT-Cell Response to Gluten in Patients With HLA-DQ2.2 Reveals Requirement of Peptide-MHC Stability in Celiac Disease

Background & Aims

Methods

Results

Conclusions

Section snippets

Subjects

DQ2.2 Patients Have Gluten-Reactive CD4+ T Cells in the Intestine

Identification of a T-Cell Epitope Recognized by DQ2.2 Patients With Celiac Disease

Discussion

Acknowledgments

Hum Immunol

Hum Immunol

Gastroenterology

Immunity

Trends Biochem Sci

Bioorg Med Chem

Gastroenterology

Curr Opin Immunol

Hum Immunol

Hum Immunol

Mucosal Immunol

Tissue-mediated control of immunopathology in coeliac disease

Nat Rev Immunol

Gliadin specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls

Scand J Immunol

Gliadin-specific, HLA-DQ(alfa1*0501,beta1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients

J Exp Med

Original Research
Basic and Translational—Alimentary Tract
T-Cell Response to Gluten in Patients With HLA-DQ2.2 Reveals Requirement of Peptide-MHC Stability in Celiac Disease

DQ2.2 Patients Have Gluten-Reactive CD4⁺ T Cells in the Intestine

Gliadin-specific, HLA-DQ(alfa10501,beta10201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients