Rasmus Iversen

High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions

Celiac disease is an immune-mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2) are generated in response to the exogenous antigen gluten in individuals who express human leukocyte antigen HLA-DQ2 or HLA-DQ8 (ref. 3). We assessed in a comprehensive and nonbiased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire of ex vivo–isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are markedly expanded within the duodenal mucosa in individuals with active celiac disease. TG2-specific antibodies were of high affinity yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts, the TG2-specific ASCs had not recently proliferated and were not short-lived ex vivo. Altogether, these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favor massive generation of autoreactive B cells. TG2-specific antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naive IgD- and IgM-expressing B cells, thus possibly explaining why the antibody response to TG2 bears signs of a primary immune response despite the disease chronicity.Celiac disease (CD) is an immune mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2)1 are generated in response to the exogenous antigen gluten2 in individuals who are HLA-DQ2 or HLA-DQ83. We assessed in a comprehensive and non-biased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire on ex vivo isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are hugely expanded in patients with active CD, representing on average 10% of ASCs within the duodenal mucosa. Surprisingly, anti-TG2 antibodies were of high affinity and yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts4, the TG2-specific ASCs had neither recently proliferated nor were they short-lived ex vivo. Altogether these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favour massive generation of autoreactive B cells. Anti-TG2 antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM, but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naïve IgD/IgM-expressing B cells, thus possibly explaining why the anti-TG2 response bears signs of a primary immune response despite the disease chronicity.

The adaptive immune response in celiac disease

Compared to other human leukocyte antigen (HLA)-associated diseases such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, fundamental aspects of the pathogenesis in celiac disease are relatively well understood. This is mostly because the causative antigen in celiac disease—cereal gluten proteins—is known and the culprit HLA molecules are well defined. This has facilitated the dissection of the disease-relevant CD4+ T cells interacting with the disease-associated HLA molecules. In addition, celiac disease has distinct antibody responses to gluten and the autoantigen transglutaminase 2, which give strong handles to understand all sides of the adaptive immune response leading to disease. Here we review recent developments in the understanding of the role of T cells, B cells, and antigen-presenting cells in the pathogenic immune response of this instructive disorder.

Transglutaminase 2–Specific Autoantibodies in Celiac Disease Target Clustered, N-Terminal Epitopes Not Displayed on the Surface of Cells

The gluten-sensitive enteropathy celiac disease is tightly associated with the production of autoantibodies specific for the enzyme transglutaminase 2 (TG2). The mechanisms underlying the activation of autoreactive B cells, however, are not well defined. To gain more insight into this autoimmune response we have characterized the binding of TG2 by a panel of human mAbs generated by expression cloning of Ig genes from single plasma cells of the celiac disease lesion. The Abs were highly specific to TG2 and bound preferentially to the open, Ca2+-activated enzyme conformation. Epitope mapping revealed that they recognize few distinct conformational epitopes that cluster in the N-terminal half of the enzyme. Two of the epitopes were overlapping with the fibronectin binding site in TG2, and none of the epitopes was accessible when TG2 was in a cell surface–bound form. Based on our findings, we propose that the autoantibodies are generated against the soluble, catalytically active enzyme, whereas Abs reactive with cell surface–associated TG2 are absent from the response due to negative selection of B cells recognizing membrane-bound self-Ag. The findings give insight into the mechanisms controlling the formation of anti-TG2 autoantibodies in celiac disease.

Activity-regulating structural changes and autoantibody epitopes in transglutaminase 2 assessed by hydrogen/deuterium exchange

Significance The enzyme transglutaminase 2 (TG2) is the target of autoantibodies characteristic of the gluten-sensitive enteropathy celiac disease, and intact enzyme activity seems to be required for the disease-causing immune response. TG2 activity is regulated through conformational changes. Ca2+ binding is required for enzyme activity, whereas oxidation inactivates the enzyme. Using hydrogen/deuterium exchange monitored by mass spectrometry, we have studied differences between active and inactive forms of TG2 in solution and found that oxidation prevents Ca2+-induced structural changes. Further, we have characterized the TG2 binding of a panel of monoclonal autoantibodies derived from disease lesion plasma cells. Autoantibody binding affected the structure of TG2, and mapping of the targeted epitopes suggests a possible mechanism for the induction of the autoimmune response. The multifunctional enzyme transglutaminase 2 (TG2) is the target of autoantibodies in the gluten-sensitive enteropathy celiac disease. In addition, the enzyme is responsible for deamidation of gluten peptides, which are subsequently targeted by T cells. To understand the regulation of TG2 activity and the enzyme’s role as an autoantigen in celiac disease, we have addressed structural properties of TG2 in solution by using hydrogen/deuterium exchange monitored by mass spectrometry. We demonstrate that Ca2+ binding, which is necessary for TG2 activity, induces structural changes in the catalytic core domain of the enzyme. Cysteine oxidation was found to abolish these changes, suggesting a mechanism whereby disulfide bond formation inactivates the enzyme. Further, by using TG2-specific human monoclonal antibodies generated from intestinal plasma cells of celiac disease patients, we observed that binding of TG2 by autoantibodies can induce structural changes that could be relevant for the pathogenesis. Detailed mapping of two of the main epitopes targeted by celiac disease autoantibodies revealed that they are located adjacent to each other in the N-terminal part of the TG2 molecule.

Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease

Background: Knowledge of how celiac disease autoantibodies recognize transglutaminase 2 (TG2) is limited. Results: The interaction between TG2 and a celiac disease epitope 1 anti-TG2 antibody was studied by small angle x-ray scattering and mutational analysis. Conclusion: TG2 residues Arg-116 and His-134 are part of epitope 1. Significance: The study gives insights into key aspects of celiac disease. Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.

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.

Igs as Substrates for Transglutaminase 2: Implications for Autoantibody Production in Celiac Disease

Autoantibodies specific for the enzyme transglutaminase 2 (TG2) are a hallmark of the gluten-sensitive enteropathy celiac disease. Production of the Abs is strictly dependent on exposure to dietary gluten proteins, thus raising the question how a foreign Ag (gluten) can induce an autoimmune response. It has been suggested that TG2-reactive B cells are activated by gluten-reactive T cells following receptor-mediated uptake of TG2–gluten complexes. In this study, we propose a revised model that is based on the ability of the BCR to serve as a substrate to TG2 and become cross-linked to gluten-derived peptides. We show that TG2-specific IgD molecules are preferred in the reaction and that binding of TG2 via a common epitope targeted by cells using the IgH variable gene segment (IGHV)5–51 results in more efficient cross-linking. Based on these findings we hypothesize that IgD-expressing B cells using IGHV5–51 are preferentially activated, and we suggest that this property can explain the previously reported low number of somatic mutations as well as the overrepresentation of IGHV5–51 among TG2-specific plasma cells in the celiac lesion. The model also couples gluten peptide uptake by TG2-reactive B cells directly to peptide deamidation, which is necessary for the activation of gluten-reactive T cells. It thereby provides a link between gluten deamidation, T cell activation, and the production of TG2-specific Abs. These are all key events in the development of celiac disease, and by connecting them the model may explain why the same enzyme that catalyzes gluten deamidation is also an autoantigen, something that is hardly coincidental.

High-Throughput Single-Cell Analysis of B Cell Receptor Usage among Autoantigen-Specific Plasma Cells in Celiac Disease

Characterization of Ag-specific BCR repertoires is essential for understanding disease mechanisms involving humoral immunity. This is optimally done by interrogation of paired H chain V region (VH) and L chain V region (VL) sequences of individual and Ag-specific B cells. By applying single-cell high-throughput sequencing on gut lesion plasma cells (PCs), we have analyzed the transglutaminase 2 (TG2)-specific VH:VL autoantibody repertoire of celiac disease (CD) patients. Autoantibodies against TG2 are a hallmark of CD, and anti-TG2 IgA-producing gut PCs accumulate in patients upon gluten ingestion. Altogether, we analyzed paired VH and VL sequences of 1482 TG2-specific and 1421 non–TG2-specific gut PCs from 10 CD patients. Among TG2-specific PCs, we observed a striking bias in IGHV and IGKV/IGLV gene usage, as well as pairing preferences with a particular presence of the IGHV5-51:IGKV1-5 pair. Selective and biased VH:VL pairing was particularly evident among expanded clones. In general, TG2-specific PCs had lower numbers of mutations both in VH and VL genes than in non–TG2-specific PCs. TG2-specific PCs using IGHV5-51 had particularly few mutations. Importantly, VL segments paired with IGHV5-51 displayed proportionally low mutation numbers, suggesting that the low mutation rate among IGHV5-51 PCs is dictated by the BCR specificity. Finally, we observed selective amino acid changes in VH and VL and striking CDR3 length and J segment selection among TG2-specific IGHV5-51:IGKV1-5 pairs. Hence this study reveals features of a disease- and Ag-specific autoantibody repertoire with preferred VH:VL usage and pairings, limited mutations, clonal dominance, and selection of particular CDR3 sequences.

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.

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 todays 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.