Coeliac disease: the paradox of diagnosing a food hypersensitivity disorder with autoantibodies.

Serum antibodies to the autoantigen transglutaminase 2 (TG2) are increasingly harnessed to diagnose coeliac disease. Diagnostic guidelines for children give recommendation for a no-biopsy-based diagnosis through detection of high amounts of IgA anti-TG2 antibodies in serum with confirmation of positivity in a separate blood sample by characteristic autoantibody-staining of tissue. While measurement of IgA anti-TG2 also is important in the diagnostic workup of adults, the adult guidelines still mandate examination of gut biopsies. This requirement might well change in the future, as might the necessity for confirming autoantibody positivity by tissue staining. The key role of autoantibody serology for diagnosis of coeliac disease is paradoxical. Coeliac disease was considered, and still can be considered, a food intolerance disorder where autoantibodies at face value are out of place. The immunological mechanisms underlying the formation of autoantibodies in response to gluten exposure have been dissected. This review presents the current insights demonstrating that the autoantibodies in coeliac disease are intimately integrated in the maladapted immune response to gluten.

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.

A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation.

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.

Generation of an HLA-DQ2.5 Knock-In Mouse.

The human MHC class II molecule HLA-DQ2.5 is implicated in multiple autoimmune disorders, including celiac disease, type 1 diabetes, and systemic lupus erythematosus. The pathogenic contribution of HLA-DQ2.5 in many of these disorders is not fully understood. There is thus a need for an HLA-DQ2.5 humanized mouse model with physiological expression of this MHC molecule that can be integrated into disease models. In this article, we report the generation of an HLA-DQ2.5 knock-in mouse strain on a C57BL/6 background in which sequences encoding the extracellular moieties of mouse MHC class II H2-IAa and H2-IAb1 have been replaced with those of HLA-DQA1*05:01 and HLA-DQB1*02:01 In heterozygous knock-in mice, the expression of HLA-DQ2.5 is superimposable with the expression of H2-IA. This was not the case in a regular untargeted HLA-DQ2.5 transgenic mouse. HLA-DQ2.5 in the knock-in animals is functional for T cell development and for Ag presentation to HLA-DQ2.5-restricted and gluten-specific T cells. Because C57BL/6 mice do not express H2-IEa, the only functional MHC class II molecule in homozygous HLA-DQ2.5 knock-in mice is the knock-in gene product. This alleviates the need for crossing with MHC class II knockout mice to study the isolated function of the MHC transgene. Our novel mouse strain provides an important tool to study the involvement of HLA-DQ2.5 in models of diseases with association to this HLA allotype.

Characterization of T-cell receptor transgenic mice recognizing immunodominant HLA-DQ2.5-restricted gluten epitopes.

We created a TCR transgenic mouse with CD4+ T cells recognizing the immunodominant DQ2.5-glia-ω2 gluten epitope. We show that these cells respond to deamidated gluten feed in vivo and compare them to previously published α2- and γ1-specific mice. These mice may help enlighten key aspects of celiac disease pathogenesis.

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.

A TCRα framework-centered codon shapes a biased T cell repertoire through direct MHC and CDR3ß interactions.

Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3ß loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3ß loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3ß. This allowed prediction of expanded DQ2.5-glia-α2-reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues.

Stereotyped antibody responses target posttranslationally modified gluten in celiac disease.

The role of B cells and posttranslational modifications in pathogenesis of organ-specific immune diseases is increasingly envisioned but remains poorly understood, particularly in human disorders. In celiac disease, transglutaminase 2-modified (TG2-modified; deamidated) gluten peptides drive disease-specific T cell and B cell responses, and antibodies to deamidated gluten peptides are excellent diagnostic markers. Here, we substantiate by high-throughput sequencing of IGHV genes that antibodies to a disease-specific, deamidated, and immunodominant B cell epitope of gluten (PLQPEQPFP) have biased and stereotyped usage of IGHV3-23 and IGHV3-15 gene segments with modest somatic mutations. X-ray crystal structures of 2 prototype IGHV3-15/IGKV4-1 and IGHV3-23/IGLV4-69 antibodies reveal peptide interaction mainly via germline-encoded residues. In-depth mutational analysis showed restricted selection and substitution patterns at positions involved in antigen binding. While the IGHV3-15/IGKV4-1 antibody interacts with Glu5 and Gln6, the IGHV3-23/IGLV4-69 antibody interacts with Gln3, Pro4, Pro7, and Phe8 - residues involved in substrate recognition by TG2. Hence, both antibodies, despite different interaction with the epitope, recognize signatures of TG2 processing that facilitates B cell presentation of deamidated gluten peptides to T cells, thereby providing a molecular framework for the generation of these clinically important antibodies. The study provides essential insight into the pathogenic mechanism of celiac disease.

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.

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.

T-cell and B-cell immunity in celiac disease.

Celiac disease is an inflammatory disorder with leukocyte infiltration and changes of tissue architecture of the small intestine. The condition develops in genetically susceptible individuals as the result of an inappropriate immune response to gluten proteins of wheat, barley and rye. The clinical manifestations and the histological changes normalize when gluten is eliminated from the diet. CD4(+) T cells that recognize gluten peptides bound to predisposing HLA-DQ molecules play a key role in the pathogenesis. These T cells recognize better gluten peptides that are deamidated, and this posttranslational modification is mediated by the enzyme transglutaminase 2 (TG2). Another hallmark of celiac disease is the production of antibodies to gluten as well as to TG2. A role for B cells in celiac disease pathogenesis is receiving increased recognition. This review will discuss the main discoveries in the field of T-cell and B-cell biology of celiac disease.

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.

Changes in natural Foxp3(+)Treg but not mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+)Treg in the circulation of celiac disease patients.

BACKGROUND: Celiac disease (CD) is an intestinal inflammation driven by gluten-reactive CD4(+) T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg) differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62L(neg)CD38(+). Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L(+)Foxp3(+) Treg or mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+) Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD. METHODS: Cell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients. RESULTS: In children, the percentages of peripheral blood CD4(+)Foxp3(+) Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L(+)Foxp3(+) Treg, but normal mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+) Treg frequencies were observed. CONCLUSIONS: Our data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3(+) Treg explains exuberant effector responses in CD. Changes in natural Foxp3(+) Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients.

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.

Tolerance to ingested deamidated gliadin in mice is maintained by splenic, type 1 regulatory T cells.

BACKGROUND & AIMS: Patients with celiac disease have permanent intolerance to gluten. Because of the high frequency of this disorder (approximately 1 in 100 individuals), we investigated whether oral tolerance to gluten differs from that to other food proteins. METHODS: Using transgenic mice that express human HLA-DQ2 and a gliadin-specific, humanized T-cell receptor, we compared gluten-specific T-cell responses with tolerogenic mucosal T-cell responses to the model food protein ovalbumin. RESULTS: Consistent with previous findings, the ovalbumin-specific response occurred in the mesenteric lymph nodes and induced Foxp3(+) regulatory T cells. In contrast, ingestion of deamidated gliadin induced T-cell proliferation predominantly in the spleen but little in mesenteric lymph nodes. The gliadin-reactive T cells had an effector-like phenotype and secreted large amounts of interferon gamma but also secreted interleukin-10. Despite their effector-like phenotype, gliadin-reactive T cells had regulatory functions, because transfer of the cells suppressed a gliadin-induced, delayed-type hypersensitivity response. CONCLUSIONS: Ingestion of deamidated gliadin induces differentiation of tolerogenic, type 1 regulatory T cells in spleens of HLA-DQ2 transgenic mice. These data indicate that under homeostatic conditions, the T-cell response to deamidated gliadin is tolerance, which is not conditioned by the mucosal immune system but instead requires interleukin-10 induction by antigen presentation in the spleen.

CD62L(neg)CD38⁺ expression on circulating CD4⁺ T cells identifies mucosally differentiated cells in protein fed mice and in human celiac disease patients and controls.

OBJECTIVES: The aim of this study was to identify new markers of mucosal T cells to monitor ongoing intestinal immune responses in peripheral blood. METHODS: Expression of cell-surface markers was studied in mice on ovalbumin (OVA)-specific T cells in the gut-draining mesenteric lymph nodes (MLN) after OVA feed. The effect of the local mucosal mediators retinoic acid (RA) and transforming growth factor-ß (TGF-ß) on the induction of a mucosal phenotype was determined in in vitro T-cell differentiation assays with murine and human T cells. Tetramer stainings were performed to study gluten-specific T cells in the circulation of patients with celiac disease, a chronic small-intestinal inflammation. RESULTS: In mice, proliferating T cells in MLN were CD62L(neg)CD38(+) during both tolerance induction and abrogation of intestinal homeostasis. This mucosal CD62L(neg)CD38(+) T-cell phenotype was efficiently induced by RA and TGF-ß in mice, whereas for human CD4(+) T cells RA alone was sufficient. The CD4(+)CD62L(neg)CD38(+) T-cell phenotype could be used to identify T cells with mucosal origin in human peripheral blood, as expression of the gut-homing chemokine receptor CCR9 and ß(7) integrin were highly enriched in this subset whereas expression of cutaneous leukocyte-associated antigen was almost absent. Tetramer staining revealed that gluten-specific T cells appearing in blood of treated celiac disease patients after oral gluten challenge were predominantly CD4(+)CD62L(neg)CD38(+). The total percentage of circulating CD62L(neg)CD38(+) of CD4 T cells was not an indicator of intestinal inflammation as percentages did not differ between pediatric celiac disease patients, inflammatory bowel disease patients and respective controls. However, the phenotypic selection of mucosal T cells allowed cytokine profiling as upon restimulation of CD62L(neg)CD38(+) cells interleukin-10 (IL-10) and interferon-γ (IFN-γ) transcripts were readily detected in circulating mucosal T cells. CONCLUSIONS: By selecting for CD62L(neg)CD38(+) expression that comprises 5-10% of the cells within the total CD4(+) T-cell pool we are able to highly enrich for effector T cells with specificity for mucosal antigens. This is of pivotal importance for functional studies as this purification enhances the sensitivity of cytokine detection and cellular activation.