The function of tissue transglutaminase in celiac disease

Celiac disease is a chronic small bowel disorder caused by an abnormal immune response to an array of epitopes of wheat gluten and related proteins of rye and barley in genetically susceptible individuals who express the HLA-DQ2/-DQ8 haplotype. Gluten peptides are efficiently presented by celiac disease-specific HLA-DQ2- and HLA-DQ8-positive antigen presenting cells to CD4(+) T-cells that, once activated, drive a T helper cell type 1 response leading to the development of the typical celiac lesion-villous atrophy, crypt hyperplasia and intraepithelial and lamina propria infiltration of inflammatory cells. Tissue transglutaminase (tTG) is a calcium dependent ubiquitous enzyme which catalyses posttranslational modification of proteins and is released from cells during inflammation. tTG is suggested to exert at least two crucial roles in celiac disease: as a deamidating enzyme, that can enhance the immunostimulatory effect of gluten, and as a target autoantigen in the immune response. Since glutamine-rich gliadin peptides are excellent substrates for tTG, and the resulting deamidated and thus negatively charged peptides have much higher affinity for the HLA-DQ2 and HLA-DQ8 molecules, the action of tTG is believed to be a key step in the pathogenesis of celiac disease. This review is focused on the function of tTG in celiac disease, although it also deals with novel advances in tTG-based therapies.     

Pathomechanisms in celiac disease

Celiac disease is a complex autoimmune disease which is characterized by a strong genetic association (HLA-DQ2 or -DQ8), gluten as nutritional etiological factor, and the enzyme tissue transglutaminase as endomysial autoantigen. Patients develop highly predictive IgA autoantibodies to tTG. Certain gluten peptides are presented by the disease-associated HLA-DQ2/DQ8 molecules leading to stimulation of gluten-specific T cells. This immune response which is driven in the lamina propria causes the mucosal transformation characteristic for celiac disease. Increased intestinal expression of tTG in patients with CD appears to play an important role in the pathogenesis of CD. Thus, modification of gluten peptides by tTG, especially deamidation of certain glutamine residues, can enhance their binding to HLA-DQ2 or -DQ8 and potentiate T cell stimulation. Furthermore, tTG-catalyzed cross-linking and consequent haptenization of gluten with extracellular matrix proteins allows for storage and extended availability of gluten in the mucosa. New therapeutic approaches aim at proteolytic destruction of immunodominant gliadin peptides that are resistant to intestinal enzymes by bacterial prolyl endopeptidases, the inhibition of tTG activity with highly specific enzyme inhibitors or at HLA-DQ2/DQ8 blocking peptide analogues.     

Analysis of the binding of gluten T-cell epitopes to various human leukocyte antigen class II molecules

Celiac disease is a prevalent disorder of the small intestine that is caused by an inflammatory reaction to dietary gluten in genetically susceptible individuals. More than 90% of patients express the HLA-DQ2 molecule, whereas DQ8 is carried by most of the remaining patients. DQ2- and DQ8-mediated presentation of gluten peptides to CD4+ T cells is a key event in the pathogenesis of the disease. The association of celiac disease with these human leukocyte antigen (HLA) molecules is explained by a preferential binding of gluten peptides to these HLA molecules, although the actual data on this in the literature are scarce. The objective of this study was to test this hypothesis. A panel of peptides representing DQ2-restricted gluten T-cell epitopes was tested for binding to various HLA class II molecules using various experimental approaches. The results demonstrate that the gluten T-cell epitopes mainly bind to the DQ2 molecule.     

The immune recognition of gluten in coeliac disease

Coeliac disease, the most common intestinal disorder of western populations, is an autoimmune enteropathy caused by an abnormal immune response to dietary gluten peptides that occurs in genetically susceptible individuals carrying the HLA-DQ2 or -DQ8 haplotype. Despite the recent progresses in understanding the molecular mechanisms of mucosal lesions, it remains unknown how increased amounts of gluten peptides can enter the intestinal mucosa to initiate the inflammatory cascade. Current knowledge indicates that different gluten peptides are involved in the disease process in a different manner, some fragments being 'toxic' and others 'immunogenic'. Those defined as 'toxic' are able to induce mucosal damage either when added in culture to duodenal endoscopic biopsy or when administered in vivo, while those defined as 'immunogenic' are able to specifically stimulate HLA-DQ2- or DQ8-restricted T cell clones isolated from jejunal mucosa or peripheral blood of coeliac patients. These peptides are able to trigger two immunological pathways: one is thought to be a rapid effect on the epithelium that involves the innate immune response and the other represents the adaptive immune response involving CD4+ T cells in the lamina propria that recognize gluten epitopes processed and presented by antigen presenting cells. These findings are the subject of the present review.     

A biased view toward celiac disease

Celiac disease is an autoimmune-like disorder that is triggered by dietary gluten and has a strong genetic association with the human leukocyte antigen locus, specifically, HLA-DQ2.5/DQ8. Here, Dahai-Koirala et al. apply ex vivo single-cell sequencing of TCRs from celiac disease patients, and show that biased T-cell receptor usage underpins the response to two gluten epitopes, which has implications for disease pathogenesis, diagnosis, and treatment.     

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5+-associated coeliac disease

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell-mediated disease diagnosed by the presence of gluten-dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten-free diet (GFD) mobilizes gluten-reactive T cells measurable by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA-DQ2·5. We aimed to develop whole blood assays to detect gluten-specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA-DQ2·5⁺), GFD donors confirmed not to have CD (n = 6 HLA-DQ2·5⁺, 11 HLA-DQ2·5⁻) and donors with CD not following GFD (n = 4, all HLA-DQ2·5⁺). Plasma IFN-γ and IFN-γ inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN-γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN-γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA-DQ2·5⁺ CD patients; the whole blood IP-10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten-reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.     

Immunomodulatory Strategies for Celiac Disease

Celiac disease (CD) is the most common food-sensitive enteropathy in humans and is caused by the lack of immune tolerance (oral tolerance) to gluten. The identification of gluten-specific T cells in the lamina propria of celiacs and the strong association with HLA-DQ2 and -DQ8 genes support a central role of CD4⁺ T cells in CD pathogenesis. Studies focused on the modulation of autoimmunity in different experimental models highlighted possible immune therapeutic protocols useful also for the management of CD. On the basis of these observations, a series of strategies have been designed: some of them are based on the identification of immunogenic epitopes and their suppression via enzymatic treatment or by using peptide analogues; others rely on the delivery of unmodified antigen through the nasal route or coadministered with downregulatory cytokines. studies are generally early stage but encouraging in paving a way for an alternative treatment for celiac disease.     

TCR sequencing of single cells reactive to DQ2.5-glia-α2 and DQ2.5-glia-ω2 reveals clonal expansion and epitope-specific V-gene usage

CD4+ T cells recognizing dietary gluten epitopes in the context of disease-associated human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 molecules are the key players in celiac disease pathogenesis. Here, we conducted a large-scale single-cell paired T-cell receptor (TCR) sequencing study to characterize the TCR repertoire for two homologous immunodominant gluten epitopes, DQ2.5-glia-α2 and DQ2.5-glia-ω2, in blood of celiac disease patients after oral gluten challenge. Despite sequence similarity of the epitopes, the TCR repertoires are unique but shared several overall features. We demonstrate that clonally expanded T cells dominate the T-cell responses to both epitopes. Moreover, we find V-gene bias of TRAV26, TRAV4, and TRBV7 in DQ2.5-glia-α2 reactive TCRs, while DQ2.5-glia-ω2 TCRs displayed significant bias toward TRAV4 and TRBV4. The knowledge that antigen-specific TCR repertoire in chronic inflammatory diseases tends to be dominated by a few expanded clones that use the same TCR V-gene segments across patients is important information for HLA-associated diseases where the antigen is unknown.     

Celiac disease: an immunological jigsaw

Celiac disease (CD) is a chronic enteropathy induced by dietary gluten in genetically predisposed people. The keystone of CD pathogenesis is an adaptive immune response orchestrated by the interplay between gluten and MHC class II HLA-DQ2 and DQ8 molecules. Yet, other factors that impair immunoregulatory mechanisms and/or activate the large population of intestinal intraepithelial lymphocytes (IEL) are indispensable for driving tissue damage. Herein, we summarize our current understanding of the mechanisms and consequences of the undesirable immune response initiated by gluten peptides. We show that CD is a model disease to decipher the role of MHC class II molecules in human immunopathology, to analyze the mechanisms that link tolerance to food proteins and autoimmunity, and to investigate how chronic activation of IEL can lead to T?cell lymphomagenesis.     

Surface expression of transglutaminase 2 by dendritic cells and its potential role for uptake and presentation of gluten peptides to T cells

Celiac disease is a chronic small intestinal inflammation driven by gluten-reactive T cells of the intestinal mucosa. These T cells are HLA-DQ2 or -DQ8 restricted, and predominantly recognize gluten peptides that are deamidated by the enzyme transglutaminase 2 (TG2). Our recent results strongly suggest that duodenal CD11c(+) dendritic cells (DC) are directly involved in T cell activation in the celiac lesion. The aim of this study was to investigate whether surface-associated TG2 could be involved in receptor-mediated endocytosis of gluten peptides, a process that may contribute to the preferential recognition of deamidated peptides. We found that both monocyte-derived DC and local CD11c(+) DC in the duodenal mucosa expressed cell surface-associated TG2. As phenotypic characterization of CD11c(+) DC in the celiac lesion suggests that these cells may be derived from circulating monocytes, we used monocyte-derived DC in functional in vitro studies. Using a functional T cell assay, we obtained evidence that cell surface-associated TG2 is endocytosed by monocyte-derived DC. However, we were unable to obtain evidence for a role of surface TG2 in the loading and subsequent generation of deamidated gluten peptides in these cells.     

IL-17 Producing T Cells in Celiac Disease: Angels or Devils?

Celiac disease (CD) is a very common chronic condition in human beings, affecting approximately one in 100 individuals. It is an autoimmune disease with a defined environmental trigger, the gluten contained in dietary cereals, occurring in genetically susceptible individuals. The disease has a very strong HLA association. More than 90% of CD patients have HLA-DQ2, and almost all of the remaining celiac population possesses HLA-DQ8 molecules. Th17 cells seem to participate in the disease pathogenesis producing and secreting either proinflammatory or anti-inflammatory cytokines.     

On the immune response to barley in celiac disease: Biased and public T-cell receptor usage to a barley unique and immunodominant gluten epitope

Celiac disease (CeD) is driven by CD4⁺ T-cell responses to dietary gluten proteins of wheat, barley, and rye when deamidated gluten epitopes are presented by certain disease-associated HLA-DQ allotypes. About 90% of the CeD patients express HLA-DQ2.5. In such patients, five gluten epitopes dominate the anti-gluten T-cell response; two epitopes unique to wheat, two epitopes present in wheat, barley, and rye and one epitope unique to barley. Despite presence of barley in commonly consumed food and beverages and hence being a prominent source of gluten, knowledge about T-cell responses elicited by barley in CeD is scarce. Therefore, in this study, we explored T-cell response toward the barley unique epitope DQ2.5-hor-3 (PIPEQPQPY) by undertaking HLA-DQ:gluten peptide tetramer staining, single-cell T-cell receptor (TCR) αβ sequencing, T-cell cloning, and T-cell proliferation studies. We demonstrate that majority of the CeD patients generate T-cell response to DQ2.5-hor-3, and this response is characterized by clonal expansion, preferential TCR V-gene usage and public TCR features thus echoing findings previously made for wheat gluten epitopes. The knowledge that biased and public TCRs underpin the T-cell response to all the immunodominant gluten epitopes in CeD suggests that such T cells are promising diagnostic and therapeutic targets.     

Celiac disease--sandwiched between innate and adaptive immunity

Celiac disease (CD) patients are intolerant to gluten, proteins in wheat, and related cereals. Virtually all patients are human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 positive and several studies have demonstrated that CD4 T cells specific for (modified) gluten peptides bound to these HLA-DQ molecules are found in patients but not in control subjects. These T cell responses are therefore thought to be responsible for disease development. Many immunogenic gluten peptides which may relate to the disease-inducing properties of gluten have now been identified. In addition, gluten can stimulate IL-15 production that ultimately leads to NKG2D-mediated epithelial cell killing. However, CD develops in only a minority of HLA-DQ2 and HLA-DQ8 individuals. This may be attributed to the default setting of the intestinal immune system: induction and maintenance of tolerance to dietary components and commensal flora. Although at present it is unknown why tolerance in CD is not established or broken, both environmental and genetic factors have been implicated. There is strong evidence for the existence of genes or gene variants on chromosomes 5, 6, and 19 that predispose to CD. In addition, type I interferons have been implicated in development of several autoimmune disorders, including CD. Thus, viral infection and/or tissue damage in the intestine may cause inflammation and induce protective Th1-mediated immunity leading to loss of tolerance for gluten. Once tolerance is broken, a broad gluten-reactive T cell repertoire may develop through determinant spreading. This may be a critical step toward full-blown disease.     

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.     

A structural and immunological basis for the role of human leukocyte antigen DQ8 in celiac disease

The risk of celiac disease is strongly associated with human leukocyte antigen (HLA) DQ2 and to a lesser extent with HLA DQ8. Although the pathogenesis of HLA-DQ2-mediated celiac disease is established, the underlying basis for HLA-DQ8-mediated celiac disease remains unclear. We showed that T helper 1 (Th1) responses in HLA-DQ8-associated celiac pathology were indeed HLA DQ8 restricted and that multiple, mostly deamidated peptides derived from protease-sensitive sites of gliadin were recognized. This pattern of reactivity contrasted with the more absolute deamidation dependence and relative protease resistance of the dominant gliadin peptide in DQ2-mediated disease. We provided a structural basis for the selection of HLA-DQ8-restricted, deamidated gliadin peptides. The data established that the molecular mechanisms underlying HLA-DQ8-mediated celiac disease differed markedly from the HLA-DQ2-mediated form of the disease. Accordingly, nondietary therapeutic interventions in celiac disease might need to be tailored to the genotype of the individual.     

Direct cloning and tetramer staining to measure the frequency of intestinal gluten‐reactive T cells in celiac disease

Knowledge of the frequency of disease‐driving CD4⁺ T cells in lesions of chronic human inflammatory diseases is limited. In celiac disease (CD), intestinal gluten‐reactive CD4⁺ T cells, which recognize gluten peptides only in the context of the disease‐associated HLA‐DQ molecules, are key pathogenic players. By cloning CD4⁺ T cells directly from intestinal biopsies of CD patients, we found that 0.5–1.8% of CD4⁺ T cells were gluten reactive. About half of the gluten‐reactive T cells were specific for either the immuno‐dominant DQ2.5‐glia‐α1a or DQ2.5‐glia‐α2 epitopes, suggesting that direct visualization of gluten‐specific T cells could be possible. Assessed by flow cytometry, tetramer‐positive T cells were present in 10/10 untreated CD patients with a frequency of 0.1–1.2% of CD4⁺ T cells. Gluten‐specific T cells were also detectable in most treated CD patients (7/10). Moreover, the frequency of gluten‐specific T cells correlated with the degree of histological damage in the gut mucosa as scored by Marsh‐grading, and also with serum IgA anti‐transglutaminase 2 antibody levels. Tetramer staining of gluten‐reactive T cells in biopsy material is a useful tool for future studies of such cells in CD and could also potentially serve as a diagnostic supplement in selected cases.     

Immunomodulatory strategies for celiac disease

Celiac disease (CD) is the most common food-sensitive enteropathy in humans and is caused by the lack of immune tolerance (oral tolerance) to gluten. The identification of gluten-specific T cells in the lamina propria of celiacs and the strong association with HLA-DQ2 and -DQ8 genes support a central role of CD4(+) T cells in CD pathogenesis. Studies focused on the modulation of autoimmunity in different experimental models highlighted possible immune therapeutic protocols useful also for the management of CD. On the basis of these observations, a series of strategies have been designed: some of them are based on the identification of immunogenic epitopes and their suppression via enzymatic treatment or by using peptide analogues; others rely on the delivery of unmodified antigen through the nasal route or coadministered with downregulatory cytokines. studies are generally early stage but encouraging in paving a way for an alternative treatment for celiac disease.     

Gliadin Peptides Activate Blood Monocytes from Patients with Celiac Disease

To elucidate the role of innate immune responses in celiac disease, we investigated the effect of gliadin on blood monocytes from patients with celiac disease. Gliadin induced substantial TNF-α and IL-8 production by monocytes from patients with active celiac disease, lower levels by monocytes from patients with inactive celiac disease, and even lower levels by monocytes from healthy donors. In healthy donor monocytes gliadin induced IL-8 from monocytes expressing HLA-DQ2 and increased monocyte expression of the costimulatory molecules CD80 and CD86, the dendritic cell marker CD83, and the activation marker CD40. Gliadin also increased DNA binding activity of NF-κB p50 and p65 subunits in monocytes from celiac patients, and NF-κB inhibitors reduced both DNA binding activity and cytokine production. Thus, gliadin activation of HLA-DQ2⁺ monocytes leading to chemokine and proinflammatory cytokine production may contribute to the host innate immune response in celiac disease.     

Celiac disease: quantity matters

Celiac disease (CD) is caused by uncontrolled immune responses to the gluten proteins in wheat and related cereals. Gluten is a complex mixture of gliadin and glutenin proteins and most modern wheat varieties contain up to 100 highly related, but distinct gluten proteins. Invariably, these gliadin and glutenin proteins contain immunogenic peptides, particularly so after the peptides have been modified by the enzyme tissue transglutaminase (TG2). This modification results in the conversion of glutamine residues in the gluten peptides into the negatively charged glutamic acid. This generates peptides that bind strongly to the disease predisposing HLA-DQ2.5 or -DQ8 molecules and this facilitates the induction of disease-inducing CD4 T cell responses, a hallmark of CD. It is well-known that the HLA-DQ genotype determines the risk of disease development. Moreover, the abundance of immunogenic peptides in the gluten proteins is likely linked to the observation that polyclonal T cell responses to multiple gluten peptides are usually found in patients with CD. However, not all patients respond to the same set of peptides. Here, I propose a model that integrates these observations and links them to the highly variable clinical spectrum of symptoms that are associated with CD. Moreover, I discuss whether it is feasible to alter wheat and/or gluten to make it suitable for consumption by CD patients.     

Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides

Celiac disease (CD) is a common CD4⁺ T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo.We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5⁺ CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye.Avenin-specific responses were observed in 6/73 HLA-DQ2.5⁺ CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability.Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.