Stephen N. McAdam

Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association?

Endomysial antibodies are a hallmark of coeliac disease. The existence of autoantibodies whose titres fluctuate with ingestion of gliadin is enigmatic. Gliadin seems to drive this antibody secretion as endomysial antibodies are produced in biopsy samples cultured with a peptic/tryptic digest of gliadin.1 The phenomenon of endomysial antibodies has been explained by molecular mimicry between gliadin and the endomysial antigen, or unmasking of cryptic epitopes in the endomysial antigen upon exposure to gliadin.1 2Recently, Dieterich and colleagues identified tissue transglutaminase (tTG) as the antigen for endomysial antibodies.3 Their data indicate that tTG forms complexes with gliadin, and they hypothesise that neoepitopes in the complex between gliadin and tTG initiate an immune response that is finally directed against gliadin and tTG.3 4 Based on the observation that tTG and gliadin form complexes, we would like to propose an alternative mechanism for the production of antibodies to tTG.nnThe prevailing view of B cell tolerance states that tolerance to soluble self-antigens, in contrast …

T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase

Celiac disease is an HLA‐DQ2‐associated disorder characterized by intestinal T cell responses to ingested wheat gliadins. Initial studies used gliadin that had been subjected to non‐enzymatic deamidation during pepsinu2009/u2009trypsin digestion to enrich for the gliadin‐specific T cells in small intestinal celiac biopsies. These T cells recognized synthetic gliadin peptides only after their deamidation in vitro by purified tissue transglutaminase (tTG). However, as these studies used a deamidated antigen for re‐stimulation prior to testing for antigen specificity, this raised the possibility that T cells specific for native epitopes had not been expanded in vitro and had thus been overlooked. To address this possibility and to look for more direct evidence that endogenous tTG mediates deamidation of gluten in the celiac lesions, we have here used a minimally deamidated chymotrypsin‐digest of gliadin to challenge biopsies and then investigated the specificity of the T cell lines derived from them. Interestingly, these T cell lines only barely responded to the chymotrypsin‐digested gliadins, but efficiently recognized the in vitro tTG‐treated variants of the same gliadins. Moreover, the addition of a tTG‐inhibitor during the gliadin challenge often resulted in T cell lines with abolished or reduced responses to deamidated gliadin. These data demonstrate that DQ2‐restricted T cells within adult celiac lesions predominantly recognize deamidated gliadin epitopes that are formed in situ by endogenous tTG.

HLA binding and T cell recognition of a tissue transglutaminase‐modified gliadin epitope

DQ2 confers susceptibility to celiac disease (CD) and intestinal CD4+ T cells of DQ2+ CD patients preferentially recognize deamidated gliadin peptides. This modification can be mediated by tissue transglutaminase (tTG). We have investigated what role the tTG‐modified residues play in DQ2 binding and T cell presentation using a model γ‐gliadin peptide (residues 134u2009–u2009153). Treatment of this peptide with tTG resulted in deamidation of Gln residues at positions 140, 148 and 150. Two of these residues act as DQ2 anchors at position P7 (148) and P9 (150) and increased the affinity of the modified peptide for DQ2 50‐fold. Testing of a mutant DQ2 molecule demonstrated that the Lys residue at β71 of DQ2 is important for binding of the deamidated peptide. A variant DQ2 molecule (with the same β‐chain but different α‐chain) that does not confer susceptibility to CD was capable of presenting the gliadin peptide, but not pepsin/trypsin‐digested gliadin, equally well to a T cell. This suggests that processing events might be involved in the preferential presentation of the gliadin peptide by the DQ2 molecule. Substitution of Gln with Glu in some positions not targeted by tTG, but in positions likely to be deamidated via non‐enzymatic mechanisms, disrupted T cell recognition. This provides additional evidence that tTG is responsible for modification of gliadin in vivo.

Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease

BACKGROUND/AIMS Coeliac disease is a chronic intestinal disorder most probably caused by an abnormal immune reaction to wheat gliadin. The identification of the HLA-DQ2 and HLA-DQ8 as the molecules responsible for the HLA association in coeliac disease strongly implicates a role for CD4 T cells in disease pathogenesis. Indeed, CD4 T cells specific for gliadin have been isolated from the small intestine of patients with coeliac disease. However, identification of T cell epitopes within gliadin has been hampered by the heterogeneous nature of the gliadin antigen. To aid the characterisation of gliadin T cell epitopes, multiple recombinant gliadins have been produced from a commercial Nordic wheat cultivar. METHODS The α-gliadin and γ-gliadin genes were amplified by polymerase chain reaction from cDNA and genomic DNA, cloned into a pET expression vector, and sequenced. Genes encoding mature gliadins were expressed inEscherichia coli and tested for recognition by T cells. RESULTS In total, 16 α-gliadin genes with complete open reading frames were sequenced. These genes encoded 11 distinct gliadin proteins, only one of which was found in the Swiss-Prot database. Expression of these gliadin genes produced a panel of recombinant α-gliadin proteins of purity suitable for use as an antigen for T cell stimulation. CONCLUSION This study provides an insight into the complexity of the gliadin antigen present in a wheat strain and has defined a panel of pure gliadin antigens that should prove invaluable for the future mapping of epitopes recognised by intestinal T cells in coeliac disease.

Staining of Celiac Disease-Relevant T Cells by Peptide-DQ2 Multimers

Gluten-specific T cells in the small intestinal mucosa are thought to play a central role in the pathogenesis of celiac disease (CD). The vast majority of these T cells recognize gluten peptides when presented by HLA-DQ2 (DQA1*05/DQB1*02), a molecule which immunogenetic studies have identified as conferring susceptibility to CD. We have previously identified and characterized three DQ2-restricted gluten epitopes that are recognized by intestinal T cells isolated from CD patients, two of which are immunodominant. Because almost all of the gluten epitopes are restricted by DQ2, and because we have detailed knowledge of several of these epitopes, we chose to develop peptide-DQ2 tetramers as a reagent to further investigate the role of these T cells in CD. In the present study, stable soluble DQ2 was produced such that it contained leucine zipper dimerization motif and a covalently coupled peptide. We have made four different peptide-DQ2 staining reagents, three containing the gluten epitopes and one containing a DQ2-binding self-peptide that provides a negative control for staining. We show in this study that peptide-DQ2 when adhered to plastic specifically stimulates T cell clones and that multimers comprising these molecules specifically stain peptide-specific T cell clones and lines. Interestingly, T cell activation caused severe reduction in staining intensities obtained with the multimers and an Ab to the TCR. The problem of TCR down-modulation must be taken into consideration when using class II multimers to stain T cells that may have been recently activated in vivo.

Studies of gliadin-specific T-cells in celiac disease.

Celiac disease is an immune-mediated disorder that primarily affects the small intestinal mucosa. It is one of the few human disorders of which it is possible, and ethically acceptable, to obtain samples from the disease-affected tissue. This chapter describes how small intestinal biopsy specimens are utilized for studies of cell-mediated immune responses in celiac disease. The focus is mainly on practical procedures for isolation, growth under sterile conditions, and subsequent analyses of gliadin-specific T-cells derived from the small biopsy specimens. This chapter also provides guidelines for the preparation of different gliadin antigens suitable for T-cell analysis. Note that most of the T-cell assays described necessitate serological and/or genomic HLA typing of the celiac disease patients from whom the T-cells are derived.

Genes and environment in celiac disease

Celiac disease is an intestinal disorder that develops as a result of interplay between genetic and environmental factors. HLA genes along with non-HLA genes predispose to the disease. Linkage studies have failed to identify chromosomal regions other than the HLA region which have major effects, indicating the existence of multiple non-HLA predisposing genes with modest effects. Association studies have shown that CTLA4 or a closely located gene is one of these genes. The primary HLA association in the majority of celiac disease patients is with DQ2 (DQA1*05/DQB1*02) and in the minority of patients with DQ8 (DQA1*0301/DQB1*0302). Gluten reactive CD4+ T cells can be isolated from small intestinal biopsies of celiac patients but not from controls. DQ2 or DQ8, but not other HLA molecules carried by patients, present peptides to these T cells. A number of distinct T cell gluten epitopes exist, most of them posttranslationally modified by deamidation. DQ2 and DQ8 bind the epitopes such that the glutamic acid residues created by deamidation are accommodated in pockets that have a preference for negatively charged side chains. There is evidence that deamidation in vivo is mediated by the enzyme tissue transglutaminase (tTG). Overall, the results point to control of the immune response to gluten by intestinal T cells restricted by the DQ2 or DQ8 molecules. This is likely to be a critical checkpoint for the development of celiac disease and could explain the dominant genetic role of HLA in this disorder. The products of the other predisposing genes may participate in pathway(s)that lead(s) to lesion formation. The minor genetic effects of the non-HLA genes could indicate a lack of critical checkpoints along these pathways, or that there are several pathways leading to the lesion formation.