M. Fleur du Pré

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.

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.

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.

CD62LnegCD38+ 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-β (TGF-β) 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 CD62LnegCD38+ during both tolerance induction and abrogation of intestinal homeostasis. This mucosal CD62LnegCD38+ T-cell phenotype was efficiently induced by RA and TGF-β in mice, whereas for human CD4+ T cells RA alone was sufficient. The CD4+CD62LnegCD38+ 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 β7 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+CD62LnegCD38+. The total percentage of circulating CD62LnegCD38+ 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 CD62LnegCD38+ cells interleukin-10 (IL-10) and interferon-γ (IFN-γ) transcripts were readily detected in circulating mucosal T cells.CONCLUSIONS:By selecting for CD62LnegCD38+ 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.

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.

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.

Changes in Natural Foxp3+Treg but Not Mucosally-Imprinted CD62LnegCD38+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 CD62LnegCD38+. Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L+Foxp3+ Treg or mucosally-imprinted CD62LnegCD38+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 CD62LnegCD38+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.

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.

A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β 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 CDR3β 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 CDR3β 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 CDR3β. 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.