Øyvind Molberg

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. The prevailing view of B cell tolerance states that tolerance to soluble self-antigens, in contrast …

The Molecular Basis for Oat Intolerance in Patients with Celiac Disease

ABSTRACT Background Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance. Methods and Findings We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine→glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation. Conclusions We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable.

HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

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 pepsin / trypsin 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.

Rituximab treatment of the anti-synthetase syndrome—a retrospective case series

OBJECTIVE Interstitial lung disease (ILD) is the major determinant of morbidity and mortality in the anti-synthetase syndrome (ASS). Here we have retrospectively assessed 11 ASS patients with ILD treated with the anti-CD20 mAB rituximab at our tertiary referral hospital. METHODS Data on clinical and laboratory parameters, lung imaging by high-resolution CT thorax and pulmonary function tests were collected from patient examinations done up to 6 months before rituximab was initiated, and at 3 and 6 months post-treatment. RESULTS All the 11 ASS patients had severe and progressive ILD and most of them had previously failed on cyclophosphamide and/or other immuno-modulating agents. Rituximab appeared to stabilize and/or improve the ILD in 7 of 11 ASS patients during the first 6 months after treatment. The rituximab treatment appeared to decrease the serum level of anti-Jo-1 antibodies, but the decrease was most often modest. One patient developed a fatal infection 3 months after the last infusion with rituximab. In the other ASS patients, the treatment was well tolerated. CONCLUSIONS This retrospective case series indicates a short-term beneficial effect of rituximab in ASS. Prospective, controlled studies are needed to validate this finding and further assess safety issues.

Intestinal T-cell responses to high-molecular-weight glutenins in celiac disease ☆

BACKGROUND & AIMS The chronic, small intestinal inflammation that defines celiac disease is initiated by a HLA-DQ2 restricted T-cell response to ingested gluten peptides after their in vivo deamidation by tissue transglutaminase (TG2). To date, celiac disease can only be treated by a lifelong abstinence from foods that contain wheat, rye, or barley; better therapeutic options are hence needed. An attractive target would be to identify nontoxic wheat cultivars or components thereof with intact baking qualities. Because these qualities are mainly determined by the high molecular weight (HMW) glutenin proteins of gluten, it is critical to know if these proteins are toxic or, more specifically, if they will trigger the activation of T cells in the celiac lesion. METHODS Different, highly purified HMW glutenins were isolated from wheat cultivars or expressed as recombinant proteins. The proteins were first tested for recognition by a large panel of gluten-specific T-cell lines established from celiac lesions and then applied during ex vivo challenges of celiac biopsies to allow for a direct identification of HMW specific T cells. RESULTS Intestinal T-cell responses to TG2-deamidated HMW glutenins but not the corresponding native proteins were detectable in 9 of the 22 adult and childhood celiac disease patients tested. CONCLUSIONS T cells within celiac lesions frequently recognize deamidated HMW glutenin proteins. This finding questions the possibility of implementing these proteins in novel food items destined to be nontoxic for celiac disease patients.

Refining the Rules of Gliadin T Cell Epitope Binding to the Disease-Associated DQ2 Molecule in Celiac Disease: Importance of Proline Spacing and Glutamine Deamidation

Celiac disease is driven by intestinal T cells responsive to proline-rich gluten peptides that often harbor glutamate residues formed by tissue transglutaminase-mediated glutamine conversion. The disease is strongly associated with the HLA variant DQ2.5 (DQA1*05, DQB1*02), and intestinal gluten-reactive T cells from DQ2.5-positive patients are uniquely restricted by this HLA molecule. In this study, we describe the mapping of two novel T cell epitopes of γ-gliadin and the experimental identification of the DQ2.5 binding register of these and three other γ-gliadin epitopes. The new data extend the knowledge base for understanding the binding of gluten peptides to DQ2.5. The alignment of the experimentally determined binding registers of nine gluten epitopes reveal positioning of proline residues in positions P1, P3, P6, and P8 but never in positions P2, P4, P7, and P9. Glutamate residues formed by tissue transglutaminase-mediated deamidation are found in position P1, P4, P6, P7, or P9, but only deamidations in positions P4 and P6, and rarely in P7, seem to be crucial for T cell recognition. The majority of these nine epitopes are recognized by celiac lesion T cells when presented by the related but nonassociated DQ2.2 (DQA1*0201, DQB1*02) molecule. Interestingly, the DQ2.2 presentation for most epitopes is less efficient than presentation by the DQ2.5 molecule, and this is particularly prominent for the α-gliadin epitopes. Contrary to previous findings, our data do not show selective presentation of DQ2.5 over DQ2.2 for gluten epitopes that carry proline residues at the P3 position.

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

The authors have analysed gliadin specific, CD4+ T cells isolated from small intestinal biopsies of 23 adult coeliac disease patients (20 on a gluten‐free diet and three untreated) and nine control patients. The biopsies were stimulated ex vivo with a peptic/tryptic digest of gliadin for 24 h, and activated T cells were positively selected with paramagnetic beads coated with an antibody against the interleukin‐2 receptor. The T cells were expanded and tested for gliadin reactivity and HLA restriction. Gliadin specific, polyclonal T cell lines were recovered from biopsies of all 23 patients. Inhibition studies of T cell lines from 21 patients with anti‐HLA monoclonal antibodies indicated predominant presentation of the gliadin antigen by HLA‐DQ2 in T cell lines from 11 patients (lines from seven patients with complete MoAb inhibition, the remaining with incomplete inhibition) and incomplete inhibition by HLA‐DR3 in lines from three patients. Nine gliadin specific T cell clones from six patients were established; all of these were HLA‐DQ2 restricted. Gliadin specific T cells were not found in biopsies from the non‐coeliac controls. Our findings demonstrate that gliadin reactive T cells are commonly found in the intestinal mucosa of CD patients and they support the notion that the majority of T cells recognize gliadin peptide(s) when presented by the disease associated DQ2 molecules.

Antigen Presentation to Celiac Lesion-Derived T Cells of a 33-Mer Gliadin Peptide Naturally Formed by Gastrointestinal Digestion

Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gluten proteins. A peptide fragment of 33 residues (α2-gliadin 56–88) produced by normal gastrointestinal proteolysis contains six partly overlapping copies of three T cell epitopes and is a remarkably potent T cell stimulator after deamidation by tissue transglutaminase (TG2). This 33-mer is rich in proline residues and adopts the type II polyproline helical conformation in solution. In this study we report that after deamidation, the 33-mer bound with higher affinity to DQ2 compared with other monovalent peptides harboring gliadin epitopes. We found that the TG2-treated 33-mer was presented equally effectively by live and glutaraldehyde-fixed, EBV-transformed B cells. The TG2-treated 33-mer was also effectively presented by glutaraldehyde-fixed dendritic cells, albeit live dendritic cells were the most effective APCs. A strikingly increased T cell stimulatory potency of the 33-mer compared with a 12-mer peptide was also seen with fixed APCs. The 33-mer showed binding maximum to DQ2 at pH 6.3, higher than maxima found for other high affinity DQ2 binders. The 33-mer is thus a potent T cell stimulator that does not require further processing within APC for T cell presentation and that binds to DQ2 with a pH profile that promotes extracellular binding.

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 134 – 153). 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.