Selective activation of naïve B cells with unique epitope specificity shapes autoantibody formation in celiac disease.

Many antibody responses induced by infection, vaccination or autoimmunity show signs of convergence across individuals with epitope-dependent selection of particular variable region gene segments and complementarity determining region 3 properties. However, not much is known about the relationship between antigen-specific effector cells and antigen-specific precursors present in the naïve B-cell repertoire. Here, we sought to address this relationship in the context of celiac disease, where there is a stereotyped autoantibody response against the enzyme transglutaminase 2 (TG2). By generating TG2-specific monoclonal antibodies from both duodenal plasma cells and circulating naïve B cells, we demonstrate a discord between the naïve TG2-specific repertoire and the cells that are selected for autoantibody production. Hence, the naïve repertoire does not fully reflect the epitope preference and gene usage observed for memory B cells and plasma cells. Instead, distinct naïve B cells that target particular TG2 epitopes appear to be selectively activated at the expense of TG2-binding B cells targeting other epitopes.

Biopsy Proteome Scoring to Determine Mucosal Remodeling in Celiac Disease.

BACKGROUND & AIMS: Histologic evaluation of gut biopsies is a cornerstone for diagnosis and management of celiac disease (CeD). Despite its wide use, the method depends on proper biopsy orientation, and it suffers from interobserver variability. Biopsy proteome measurement reporting on the tissue state can be obtained by mass spectrometry analysis of formalin-fixed paraffin-embedded tissue. Here we aimed to transform biopsy proteome data into numerical scores that give observer-independent measures of mucosal remodeling in CeD. METHODS: A pipeline using glass-mounted formalin-fixed paraffin-embedded sections for mass spectrometry-based proteome analysis was established. Proteome data were converted to numerical scores using 2 complementary approaches: a rank-based enrichment score and a score based on machine learning using logistic regression. The 2 scoring approaches were compared with each other and with histology analyzing 18 patients with CeD with biopsies collected before and after treatment with a gluten-free diet as well as biopsies from patients with CeD with varying degree of remission (n = 22). Biopsies from individuals without CeD (n = 32) were also analyzed. RESULTS: The method yielded reliable proteome scoring of both unstained and H&E-stained glass-mounted sections. The scores of the 2 approaches were highly correlated, reflecting that both approaches pick up proteome changes in the same biological pathways. The proteome scores correlated with villus height-to-crypt depth ratio. Thus, the method is able to score biopsies with poor orientation. CONCLUSIONS: Biopsy proteome scores give reliable observer and orientation-independent measures of mucosal remodeling in CeD. The proteomic method can readily be implemented by nonexpert laboratories in parallel to histology assessment and easily scaled for clinical trial settings.

Autoantibody binding and unique enzyme-substrate intermediate conformation of human transglutaminase 3.

Transglutaminase 3 (TG3), the autoantigen of dermatitis herpetiformis (DH), is a calcium dependent enzyme that targets glutamine residues in polypeptides for either transamidation or deamidation modifications. To become catalytically active TG3 requires proteolytic cleavage between the core domain and two C-terminal ß-barrels (C1C2). Here, we report four X-ray crystal structures representing inactive and active conformations of human TG3 in complex with a TG3-specific Fab fragment of a DH patient derived antibody. We demonstrate that cleaved TG3, upon binding of a substrate-mimicking inhibitor, undergoes a large conformational change as a ß-sheet in the catalytic core domain moves and C1C2 detaches. The unique enzyme-substrate conformation of TG3 without C1C2 is recognized by DH autoantibodies. The findings support a model where B-cell receptors of TG3-specific B cells bind and internalize TG3-gluten enzyme-substrate complexes thereby facilitating gluten-antigen presentation, T-cell help and autoantibody production.

Separate Gut Plasma Cell Populations Produce Auto-Antibodies against Transglutaminase 2 and Transglutaminase 3 in Dermatitis Herpetiformis.

Dermatitis herpetiformis (DH) is an inflammatory skin disorder often considered as an extra intestinal manifestation of celiac disease (CeD). Hallmarks of CeD and DH are auto-antibodies to transglutaminase 2 (TG2) and transglutaminase 3 (TG3), respectively. DH patients have auto-antibodies reactive with both transglutaminase enzymes. Here it is reported that in DH both gut plasma cells and serum auto-antibodies are specific for either TG2 or TG3 with no TG2-TG3 cross reactivity. By generating monoclonal antibodies from TG3-specific duodenal plasma cells of DH patients, three conformational epitope groups are defined. Both TG2-specific and TG3-specific gut plasma cells have few immunoglobulin (Ig) mutations, and the two transglutaminase-reactive populations show distinct selection of certain heavy and light chain V-genes. Mass spectrometry analysis of TG3-specific serum IgA corroborates preferential usage of IGHV2-5 in combination with IGKV4-1. Collectively, these results demonstrate parallel induction of anti-TG2 and anti-TG3 auto-antibody responses involving separate B-cell populations in DH patients.

Expression of transglutaminase 2 in human gut epithelial cells: Implications for coeliac disease.

BACKGROUND: Formation of complexes between transglutaminase 2 (TG2) and gluten can mechanistically explain why TG2 serves both as B-cell autoantigen and as an enzyme that creates deamidated gluten epitopes in coeliac disease (CeD). A model has been proposed where TG2 released from shed epithelial cells encounters high concentrations of dietary gluten peptides to form these TG2:gluten complexes. In this work we have characterised TG2 protein expression in gut epithelial cells in humans. METHODS: Western blot analysis, immunofluorescence staining and mass spectrometry in combination with laser capture microdissection to gain spatial resolution were used to characterise TG2 expression in the epithelial cell layer of healthy and coeliac disease affected duodenum. FINDINGS: TG2 is expressed in human duodenal epithelial cells, including cells in the apical region that are shed into the gut lumen. In untreated CeD the apical expression of TG2 is doubled. Enzymatically active TG2 is readily released from isolated human intestinal epithelial cells. CONCLUSION: Shed epithelial cells are a plausible source of pathogenic TG2 enzyme in CeD. Increased epithelial TG2 expression and increased epithelial shedding in active CeD may reinforce action of luminal TG2 in this condition.

Transglutaminase 2 affinity and enzyme-substrate intermediate stability as determining factors for T-cell responses to gluten peptides in celiac disease.

The adaptive immune response of celiac disease (CeD) involves presentation of gluten peptides to CD4+ T cells by transglutaminase 2 (TG2) specific B cells. This B-cell/T-cell crosstalk is facilitated by involvement of TG2:gluten peptide complexes that act principally in the form of enzyme-substrate intermediates. Here, we have addressed how gluten peptide affinity and complex stability in the presence of secondary substrates affect the uptake of TG2:gluten peptide complexes by TG2-specific B cells and the activation of gluten-specific T cells. We studied affinity of various gluten peptides for TG2 by biochemical assay, and monitored uptake of gluten peptides by TG2-specific B cells by flow cytometry. Crosstalk between TG2-specific B cells and gluten-specific T cells was assayed with transfectants expressing antigen receptors derived from CeD patients. We found that gluten peptides with high TG2 affinity showed better uptake by TG2-specific B cells. Uptake by B cells, and subsequent activation of T cells, was negatively affected by polyamines acting as secondary TG2 substrates. These results show that affinity between gluten peptide and TG2 governs the selection of T-cell epitopes via enhanced uptake of TG2:gluten complexes by TG2-specific B cells, and that exogenous polyamines can influence the CeD immune responses by disrupting TG2:gluten complexes.

Injection of prototypic celiac anti-transglutaminase 2 antibodies in mice does not cause enteropathy.

BACKGROUND: Celiac disease is an autoimmune enteropathy driven by dietary intake of gluten proteins. Typical histopathologic features are villous flattening, crypt hyperplasia and infiltration of inflammatory cells in the intestinal epithelium and lamina propria. The disease is hallmarked by the gluten-dependent production of autoantibodies targeting the enzyme transglutaminase 2 (TG2). While these antibodies are specific and sensitive diagnostic markers of the disease, a role in the development of the enteropathy has never been established. METHODS: We addressed this question by injecting murine antibodies harboring the variable domains of a prototypic celiac anti-TG2 immunoglobulin into TG2-sufficient and TG2-deficient mice evaluating for celiac enteropathy. RESULTS: We found no histopathologic abnormalities nor clinical signs of disease related to the injection of anti-TG2 IgG or IgA. CONCLUSIONS: Our findings do not support a direct role for secreted anti-TG2 antibodies in the development of the celiac enteropathy.

TG2-gluten complexes as antigens for gluten-specific and transglutaminase-2 specific B cells in celiac disease.

A hallmark of celiac disease is the gluten-dependent production of antibodies specific for deamidated gluten peptides (DGP) and the enzyme transglutaminase 2 (TG2). Both types of antibodies are believed to result from B cells receiving help from gluten-specific CD4+ T cells and differentiating into antibody-producing plasma cells. We have here studied the collaboration between DGP- and TG2-specific B cells with gluten-specific CD4+ T cells using transgenic mice expressing celiac patient-derived T-cell and B-cell receptors, as well as between B-cell transfectants and patient-derived gluten-specific T-cell clones. We show that multivalent TG2-gluten complexes are efficient antigens for both TG2-specific and DGP-specific B cells and allow both types of B cells to receive help from gluten-specific T cells of many different specificities.

Insights from tissue "omics" analysis on intestinal remodeling in celiac disease.

Celiac disease (CeD) is a prevalent intestinal disorder that only develops in genetically susceptible individuals when they mount a harmful CD4+ T-cell response towards gluten peptides. Intake of gluten leads to inflammation and remodeling of the small intestine with symptoms such as nausea and diarrhea. The only current treatment is a lifelong gluten free diet. The immunological basis for CeD is well characterized but the mechanisms that drive intestinal remodeling are still poorly understood. Transcriptome or proteome analysis of intestinal biopsies gives a global snapshot of all processes that occur in the tissue, including alterations in the epithelial cell layer. This paper will introduce concepts of intestinal remodeling, recapitulate the current understanding of CeD pathogenesis and discuss findings from relevant tissue "omics" studies. On the basis of this review, I give perspectives on what tissue "omics" studies can tell us about disease pathogenesis with a particular focus on the gluten induced intestinal remodeling.

In Well-Treated Celiac Patients Low-Level Mucosal Inflammation Predicts Response to 14-day Gluten Challenge.

In celiac disease (CeD), gluten activates adaptive immune cells that cause damage to the small intestinal mucosa. Histological evaluation of intestinal biopsies allows for grading of disease severity. CeD can effectively be treated with a life-long gluten-free diet. Gluten challenge of treated CeD patients is used to confirm diagnosis and to test drug efficacy in clinical trials, but patients respond with different magnitudes to the same gluten challenge. In this study of 19 well-treated CeD patients, proteome analysis of total tissue or isolated epithelial cell compartment from formalin-fixed paraffin embedded biopsies collected before and after 14-day gluten challenge demonstrates that patients with strong mucosal response to challenge have signs of ongoing tissue inflammation already before challenge. This low-level tissue inflammation at baseline is paralleled by increased gluten specific CD4+ T-cell frequencies in the gut and presence of a low-level blood inflammatory profile. Thus, apparently well-treated CeD is frequently not entirely quiescent, with presence of low-grade inflammation and antigluten immunity in the gut mucosa. Histology assessment alone appears insufficient to judge full recovery and gut mucosal healing of CeD patients. The findings raise a concern whether a seemingly proper gluten-free diet is able to curb gut inflammation in all CeD patients.

B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2.

Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2, we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient-derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2-/- mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2-/- mice with no signs of clonal deletion, receptor editing, or B cell anergy. The autoreactive B cells appeared ignorant to their antigen, and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten-TG2 complexes, and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help.

Efficient T cell-B cell collaboration guides autoantibody epitope bias and onset of celiac disease.

B cells play important roles in autoimmune diseases through autoantibody production, cytokine secretion, or antigen presentation to T cells. In most cases, the contribution of B cells as antigen-presenting cells is not well understood. We have studied the autoantibody response against the enzyme transglutaminase 2 (TG2) in celiac disease patients by generating recombinant antibodies from single gut plasma cells reactive with discrete antigen domains and by undertaking proteomic analysis of anti-TG2 serum antibodies. The majority of the cells recognized epitopes in the N-terminal domain of TG2. Antibodies recognizing C-terminal epitopes interfered with TG2 cross-linking activity, and B cells specific for C-terminal epitopes were inefficient at taking up TG2-gluten complexes for presentation to gluten-specific T cells. The bias toward N-terminal epitopes hence reflects efficient T-B collaboration. Production of antibodies against N-terminal epitopes coincided with clinical onset of disease, suggesting that TG2-reactive B cells with certain epitope specificities could be the main antigen-presenting cells for pathogenic, gluten-specific T cells. The link between B cell epitopes, antigen presentation, and disease onset provides insight into the pathogenic mechanisms of a T cell-mediated autoimmune condition.

Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry.

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography-mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa.

Dissecting the interaction between transglutaminase 2 and fibronectin.

In the extracellular environment, the enzyme transglutaminase 2 (TG2) is involved in cell-matrix interactions through association with the extracellular matrix protein, fibronectin (FN). The 45 kDa gelatin-binding domain of FN (45FN) is responsible for the binding to TG2. Previous studies have demonstrated that the FN-binding site of TG2 is located in the N-terminal domain of the enzyme although with conflicting results regarding the specific residues involved. Here we have mapped the FN interaction site of human TG2 by use of hydrogen/deuterium exchange coupled with mass spectrometry, and we confirm that the FN-binding site is located in the N-terminal domain of TG2. Furthermore, by combination of site-directed mutagenesis and surface plasmon resonance analysis we have identified the TG2 residues K30, R116 and H134 as crucial to maintain the high affinity interaction with FN. Mutation of all three residues simultaneously reduced binding to 45FN by more than 2000-fold. We also identified residues in the catalytic core domain of TG2 that contributed to FN binding, hence extending the binding interface between TG2 and FN. This study provides new insights into the high affinity interaction between TG2 and FN.

Epitope-dependent Functional Effects of Celiac Disease Autoantibodies on Transglutaminase 2.

Transglutaminase 2 (TG2) is a Ca2+-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2. Binding of the mAbs induced allosteric changes in TG2. Thus, we aimed to determine whether these mAbs could influence enzymatic activity through modulation of TG2 susceptibility to oxidative inactivation and Ca2+ affinity. All tested epitope 1 mAbs, as well as 679-14-D04, which recognizes a previously uncharacterized epitope, prevented oxidative inactivation and increased Ca2+ sensitivity of TG2. We have identified crucial residues for binding of 679-14-D04 located within a Ca2+ binding site. Epitope 1 mAbs and 679-14-D04, although recognizing separate epitopes, behaved similarly when assessing their effect on TG2 conformation, suggesting that the shared effects on TG2 function can be explained by induction of the same conformational changes. None of the mAbs targeting other epitopes showed these effects, but epitope 2 mAbs reduced the rate of TG2-catalyzed reactions. Collectively, these effects could be relevant to the pathogenesis of CD. In A20 B cells transduced with TG2-specific B-cell receptor, epitope 2-expressing cells had poorer uptake of TG2-gluten complexes and were less efficient in gluten epitope presentation to T cells than cells expressing an epitope 1 receptor. Thus, the ability of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients.

Transglutaminase 2 strongly binds to an extracellular matrix component other than fibronectin via its second C-terminal beta-barrel domain.

Transglutaminase 2 (TG2) is a ubiquitous crosslinking enzyme present in both intra- and extracellular in many cell types and tissues. TG2 is upregulated upon cellular stress or injury, and extracellular TG2 is implicated in several human diseases, including celiac disease. However, incomplete knowledge about extracellular TG2 biology limits our understanding of how TG2 is involved in disease. Here, we demonstrate that binding of TG2 to the ECM of small intestinal tissue sections is the sum of binding to fibronectin (FN) via its N-terminal domain and binding to an abundant, novel extracellular matrix (ECM) interaction partner via its second C-terminal beta-barrel domain. The latter interaction dominates and gives rise to the characteristic reticular staining pattern of extracellular TG2. Of relevance for celiac disease, we show that self-multimerized TG2 does not efficiently deposit in the intestinal ECM, and TG2 complexes may thus become free-floating antigens in tissues in contrast to monomeric TG2 that would readily become sequestered by the ECM. Upon injection of monoclonal antibody targeting the FN-binding site, we observe antibody deposition on extracellular TG2 in cryosections, suggesting that the FN-binding site of TG2 is exposed in vivo. This would explain how and why celiac autoantibodies recognizing the FN-binding site of TG2 can bind TG2 in vitro, in situ as well as in vivo.

Enhanced B-Cell Receptor Recognition of the Autoantigen Transglutaminase 2 by Efficient Catalytic Self-Multimerization.

A hallmark of the gluten-driven enteropathy celiac disease is autoantibody production towards the enzyme transglutaminase 2 (TG2) that catalyzes the formation of covalent protein-protein cross-links. Activation of TG2-specific B cells likely involves gluten-specific CD4 T cells as production of the antibodies is dependent on disease-associated HLA-DQ allotypes and dietary intake of gluten. IgA plasma cells producing TG2 antibodies with few mutations are abundant in the celiac gut lesion. These plasma cells and serum antibodies to TG2 drop rapidly after initiation of a gluten-free diet, suggestive of extrafollicular responses or germinal center reactions of short duration. High antigen avidity is known to promote such responses, and is also important for breakage of self-tolerance. We here inquired whether TG2 avidity could be a feature relevant to celiac disease. Using recombinant enzyme we show by dynamic light scattering and gel electrophoresis that TG2 efficiently utilizes itself as a substrate due to conformation-dependent homotypic association, which involves the C-terminal domains of the enzyme. This leads to the formation of covalently linked TG2 multimers. The presence of exogenous substrate such as gluten peptide does not inhibit TG2 self-cross-linking, but rather results in formation of TG2-TG2-gluten complexes. The celiac disease autoantibody epitopes, clustered in the N-terminal part of TG2, are conserved in the TG2-multimers as determined by mass spectrometry and immunoprecipitation analysis. TG2 multimers are superior to TG2 monomer in activating A20 B cells transduced with TG2-specific B-cell receptor, and uptake of TG2-TG2-gluten multimers leads to efficient activation of gluten-specific T cells. Efficient catalytic self-multimerization of TG2 and generation of multivalent TG2 antigen decorated with gluten peptides suggest a mechanism by which self-reactive B cells are activated to give abundant numbers of plasma cells in celiac disease. Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway.

Small bowel, celiac disease and adaptive immunity.

BACKGROUND: Celiac disease is a multifactorial and polygenic disease with autoimmune features. The disease is caused by an inappropriate immune response to gluten. Elimination of gluten from the diet leads to disease remission, which is the basis for today's treatment of the disease. There is an unmet need for new alternative treatments. KEY MESSAGES: Genetic findings point to adaptive immunity playing a key role in the pathogenesis of celiac disease. MHC is by far the single most important genetic factor in the disease. In addition, a number of non-MHC genes, the majority of which have functions related to T cells and B cells, also contribute to the genetic predisposition, but each of them has modest effect. The primary MHC association is with HLA-DQ2 and HLA-DQ8. These HLA molecules present gluten epitopes to CD4+ T cells which can be considered to be the master regulators of the immune reactions that lead to the disease. The epitopes which the T cells recognize are usually deamidated, and this deamidation is mediated by the enzyme transglutaminase 2 (TG2). Celiac disease patients have disease-specific antibodies. In addition to antibodies to gluten, these include autoantibodies to TG2. Antibodies to deamidated gluten are nearly as specific for celiac disease as the anti-TG2 antibodies. Both types of antibodies appear only to be produced in subjects who are HLA-DQ2 or HLA-DQ8 when they are consuming gluten. CONCLUSION: It is hardly coincidental that TG2 is implicated in T-cell epitope formation and at the same time a target for autoantibodies. Understanding this connection is one of the major challenges for obtaining a complete understanding of how gluten causes tissue destruction and remodeling of the mucosa in the small bowel.

Transglutaminase 2 interactions with extracellular matrix proteins as probed with celiac disease autoantibodies.

Transglutaminases have been implicated in various human diseases. A prominent example is the involvement of transglutaminase 2 (TG2) in the gluten-sensitive enteropathy celiac disease, where the enzyme is both the target of autoantibodies and responsible for the generation of immunogenic gluten epitopes. Here, we aimed to characterize the microenvironment of TG2 in the extracellular matrix (ECM) in order to gain insights into the antigenic structures that are recognized by autoantibodies in celiac disease. A panel of TG2-specific mAbs established from gut plasma cells of celiac disease patients was employed as probes to characterize the interactions between TG2 and ECM constituents. With immunofluorescence staining, microplate protein-binding and surface plasmon resonance assays, we found that the main epitope (epitope 1) recognized by TG2-specific gut plasma cells overlaps with the fibronectin (FN)-binding site of TG2. Furthermore, we found that the same TG2 amino acids that are involved in binding of epitope 1 mAbs are also important for efficient binding of FN. Notably, epitope 1 mAbs recognize TG2 in tissue sections, suggesting that some TG2 in the extracellular matrix has interaction partners in addition to FN. We demonstrate that collagen VI is a strong candidate, on the basis of its tissue expression pattern and ability to bind TG2. Collagen VI may thus serve as a matrix for deposition of TG2 in a context that can also be recognized by epitope 1-targeting autoantibodies.