Immunology of Celiac Disease and the Small Intestine Dig Dis 2015;33:115–121 DOI: 10.1159/000369512
Small Bowel, Celiac Disease and Adaptive Immunity Ludvig M. Sollid a Rasmus Iversen a Øyvind Steinsbø a Shuo-Wang Qiao a Elin Bergseng a Siri Dørum a M. Fleur du Pré a Jorunn Stamnaes a Asbjørn Christophersen a Inês Cardoso a Kathrin Hnida a Xi Chen a Omri Snir a Knut E.A. Lundin a, b a
Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital – Rikshospitalet, and b Department of Gastroenterology, Oslo University Hospital – Rikshospitalet, Oslo, Norway
Abstract 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
© 2015 S. Karger AG, Basel 0257–2753/15/0332–0115$39.50/0 E-Mail [email protected] www.karger.com/ddi
(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. © 2015 S. Karger AG, Basel
The Gut and Its Immune System
The small bowel is essential for digestion and breakdown of food and absorption of nutrients. Only a single layer of epithelium is lining and protecting the gut. Along with nutrients, pathogenic bacteria and viruses are constantly hitting this epithelial surface layer. Bacteria and viruses that do not cause disease and which are in a symbiotic relationship with the host moreover colonize the Ludvig M. Sollid Centre for Immune Regulation and Department of Immunology University of Oslo and Oslo University Hospital – Rikshospitalet NO–0372 Oslo (Norway) E-Mail l.m.sollid @ medisin.uio.no
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Key Words Adaptive immunity · B cells · Celiac disease · Gluten · MHC genes · T cells
Celiac Disease and Involvement of the Adaptive Immune System
Celiac disease is an acquired disorder caused by an inappropriate immune response to cereal gluten proteins of wheat, barley and rye with clear signs of involvement of the adaptive immune system. It is a multifactorial disorder with influence of genes and environment. Gluten is obviously an important environmental factor as the disease goes in remission when gluten is eliminated from the diet. MHC is the single most important genetic factor which may account for as much as half of the genetic risk. The non-MHC genes have been extensively mapped by genome-wide association studies [1, 2], and most recently by the so-called ImmunoChip analysis [3]. Altogether 40 loci (including the MHC locus) have been identified with evidence of 54 independent signal genes. Many of these contain genes coding for molecules thought to be implicated in adaptive immunity, in particular the function of T cells and B cells. The culprit MHC genes implicated in celiac disease encode for HLA-DQ2.5 (DQA1*05, DQB1*02), HLA-DQ8 (DQA1*03, DQB1*03: 02) and HLA-DQ2.2 (DQA1*02:01, DQB1*02:02). One or more of these MHC allotypes are carried by all celiac disease patients. However, many healthy individuals also carry one of these MHC allotypes, so MHC is a necessary but not sufficient factor in celiac disease development. The risks for celiac disease associated with the various MHC allotypes are widely different. DQ2.5 confers the highest risk, whereas DQ2.2 and DQ8 confer much lower risks [4]. HLA-DQ 116
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molecules predispose to celiac disease by presenting gluten peptides to CD4 T cells [5]. Celiac disease patients have expansions of CD4 T-cell clones reactive to gluten peptides. Once these clonal expansions have taken place, a strong immune response to gluten will follow after an exposure to gluten, likely explaining why celiac disease patients have a lifelong intolerance to gluten. T cells recognize gluten peptides that are posttranslationally modified [6, 7]. The modification, the conversion of glutamine to glutamate, is called deamidation, and is mediated by the enzyme transglutaminase 2 (TG2). The TG2 enzyme targets in a sequence-specific manner certain glutamine residues [8, 9], and either crosslinks the glutamine with a primary amine which could be a lysine residue of another polypeptide, or converts the glutamine to glutamate by a reaction with water. In addition to T cells reactive to gluten, celiac disease patients have antibodies reactive to gluten as well as antibodies reactive to TG2 [10]. The anti-gluten antibodies react particularly well with gluten peptides that are deamidated [11]. Both anti-TG2 and anti-gluten antibodies disappear in subjects with celiac disease upon commencement of a gluten-free diet and reappear upon gluten challenge. Serologic examination plays an increasingly important role in the diagnostic workup of celiac disease [12]. The anti-TG2 antibodies and probably also the antideamidated gluten antibodies are HLA dependent in the sense that they are only formed in individuals who are HLA-DQ2 or HLA-DQ8 [13].
Immune Responses Initiated by Gluten Exposure: Priming and Effector Sites
Antibodies are produced by plasma cells, which differentiate from B cells after recognition of antigen by surface immunoglobulin which serves as the B-cell receptor (BCR). The intestinal mucosa of celiac disease patients is characterized by massive plasmacytosis. There is an increase in density of jejunal plasma cells producing IgA, IgM and IgG by 2.4, 4.6 and 6.5 times, respectively, when compared to healthy controls [14]. As IgA-producing plasma cells are dominating and IgG-producing plasma cells are few in the intestine, the increase in absolute numbers of plasma cells is by far the biggest for IgA, while there are still few IgG-producing plasma cells in the celiac lesion. The gluten-reactive CD4 T cells and plasma cells in the lamina propria are effector cells in the immune response (fig. 1). The mucosa can hence be considered an effector Sollid et al.
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gut surface. To be well functioning, the body should thus respond to and expel harmful bacteria and viruses, but at the same time not respond to foodstuff or to symbiotic bacteria and viruses. This is a formidable challenge to the immune system, and it is no wonder that the gut immune system is large and intricate. The immune system can broadly be separated into innate and adaptive immunity. Whereas innate immunity is based on nonspecific and generic recognition, adaptive immunity is based on specific recognition by antigen receptors of B and T lymphocytes. Another hallmark of adaptive immunity is clonal expansion of specific cells, and usually there is also establishment of memory. The same division of innate and adaptive immunity also applies to the immune system of the gut. The involvement of the adaptive immune system in the pathogenesis is more obvious for some gut diseases than others.
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site for the anti-gluten immune response. The induction of the immune response, however, likely takes place in organized lymphoid tissue, be that mesenteric lymph nodes or Peyer’s patches. In these structures, naïve T cells will be primed to become effector T cells that circulate via blood to seed into the mucosa. Likewise, B cells will be primed to become plasma blasts that circulate via blood and seed into the mucosa to end-differentiate into plasma cells. In this priming of B cells, CD4 T cells are usually instrumental in providing cognate help. This happens either in germinal centers or extrafollicularly. Binding of antigen by the surface immunoglobulin BCR will lead to increased uptake of this antigen into the endosomes for antigenic processing. As a consequence, by mass action,
there is high likelihood that MHC class II molecules of the B cell will display peptide fragments for which the BCR is specific. It is currently not known how gluten peptides access these organized lymphoid structures, nor is it known how TG2, which seems to be mainly catalytically inactive in situ [15], is active at these sites. A particularly interesting question in relation to antiTG2 antibodies is what dictates the formation of autoantibodies in response to exposure to a foreign antigen. As a first step to analyze this, we have established a panel of recombinant monoclonal antibodies from single TG2specific plasma cells of the celiac lesion, and compared these with monoclonal antibodies established from gluten-specific plasma cells.
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adaptive immune system in the pathogenesis of celiac disease. Two key players in the disease are CD4+ T cells specific for deamidated gluten peptides and plasma cells specific for TG2 or gluten. The CD4+ T cells recognize gluten peptides in the context of the disease-associated HLA-DQ2.5 or HLA-DQ8 molecules. The immune reaction is initiated in organized lymphoid tissue (mesenteric lymph nodes or Peyer’s patches), which serves as a priming site for the immune responses. Primed cells (T cells and plasma blasts) migrate via blood to seed into the lamina propria to become effector cells. The enzyme TG2 is the target of autoantibodies, and it is also responsible for creating deamidated gluten peptides.
Antigenpresenting cell
IgA and IgM plasma cells have surface expression of immunoglobulins. This allows selection of antigen-specific cells by use of labeled antigen. We used biotinylated TG2 multimerized on fluorophore-conjugated streptavidin to stain and flow sort TG2-specific plasma cells [16]. We further found that on average 10% of plasma cells in the celiac lesion are specific for TG2, and that the percentage of anti-TG2-positive cells goes down in patients who are on a gluten-free diet. We PCR amplified and sequenced the VH and VL genes of single plasma cells and inserted them into expression vectors for expression as human IgG1 in HEK cells. We found that there is a restricted repertoire of immunoglobulin heavy and light chains among TG2-specific antibodies with overusage of the heavy-chain gene segments IGVH5-51, IGVH3 and IGVH4. Many of the cells also use IGVK1 light-chain gene segments. The antibodies display limited somatic hypermutation, yet the mutations introduced do affect binding affinity indicative of affinity maturation and that T-cell help is important for the formation of the antibodies. The epitopes recognized by the antibodies are conformational. Competitive ELISA experiments indicate that the antibodies recognize at least four different epitopes which are spatially related [17]. The epitopes correspond broadly to the IGVH gene segments used by the antibodies. The localization of the epitopes has been mapped by hydrogen-deuterium exchange, by analysis of interference with TG2 binding to fibronectin and by single amino acid mutation analysis [35]. The so-called epitope 1 used by IGVH5-51 antibodies involves residues Glu8 and Lys30; epitope 2 used by IGVH3 antibodies involves Arg19, and epitope 3 used by IGVH4 antibodies involves Glu94. Epitope 1 and epitope 4 overlap with the fibronectin-binding site of TG2. In parallel with anti-TG2 antibodies, the celiac disease patients make antibodies to gluten. We established gluten-specific monoclonal antibodies by two distinct approaches [18]. In one approach, VH and VL genes were established from cultures of intestinal biopsy single-cell suspensions which by ELISA were shown to contain antibodies reactive with deamidated complex gluten antigen. In the second approach, VH and VL genes were established from single plasma cells of biopsies sorted by flow cytometry using synthetic gluten peptides known to harbor epitopes recognized by serum antibodies of celiac disease patients. A panel of 38 antibodies was established. Typically, the antibodies bind gluten 118
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peptides related to T-cell epitopes and many have higher reactivity to deamidated peptides. There is restricted VH and VL combination and usage among the antibodies, with many antibodies carrying the IGVH323/IGVL4-69 and IGVH3-15/IGVK4-1 combinations. These gluten-specific antibodies also have limited somatic hypermutations.
Mechanisms for Formation of Anti-TG2 Antibodies
It is striking that TG2 which is essential to create deamidated gluten epitopes recognized by CD4 T cells is also the target of highly celiac disease-specific TG2 autoantibodies. This is unlikely to be coincidental, and suggests a causal link between the two phenomena. T-cell help to TG2-specific B cells by gluten-specific T cells is a model that would explain a causal link [19]. It would also explain why the TG2 antibodies are only formed in HLADQ2- or HLA-DQ8-expressing subjects and only when the subjects consume gluten. Gluten peptides are good substrates for TG2. When incubating gluten peptides with TG2, covalent complexes of gluten and TG2 are formed. Such complexes can be bound by TG2-specific B cells, which upon internalization and processing can present gluten epitopes in the context of HLA-DQ2 or HLA-DQ8 to T cells. The T cells recognize deamidated peptides, and one can envisage three ways deamidated peptides are generated from such complexes. These are: (a) The covalent bond is the thioester bond between TG2active site cysteine and peptide glutamine in the enzyme substrate intermediate. Upon reaction of the enzyme substrate intermediate with water, the deamidated peptide is formed. (b) The covalent bond is the isopeptide bond between a lysine residue of TG2 and a glutamine residue of the peptide. Isopeptidase activity of TG2 could regenerate the enzyme substrate intermediate, which again could react with water for formation of a deamidated peptide. (c) The peptide could at least contain two glutamine residues targeted by TG2, one could be deamidated and one could be isopeptide bonded to TG2. Upon internalization in B cells, endoproteases in endosomes would cleave the peptide and release the deamidated epitope. If TG2-specific B cells would be able to present gluten peptides to T cells, the B cells would get their help, but at the same time the T cells would become activated, clonally expand and likely generate memory T cells. Interaction between TG2specific B cells and gluten-specific T cells could thus be an important amplification loop for the anti-gluten T-cell response. Sollid et al.
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TG2 and Gluten-Specific IgA Antibodies Produced by Plasma Cells of the Celiac Lesion
B cells specific for gluten antigen could receive cognate help from gluten-specific T cells. The observation that anti-gluten antibodies bind peptides related to T-cell epitopes speaks in favor of this mechanism. B cells which bind and internalize deamidated gluten epitopes would load HLA-DQ2 or HLA-DQ8 molecules with deamidated epitopes and would hence be particularly well suited to receive help from T cells recognizing deamidated gluten antigen. This may explain why anti-deamidated gliadin antibodies and anti-TG2 antibodies both are excellent markers of disease with high clinical diagnostic performance as they both reflect the action of gluten-specific T cells recognizing deamidated peptides.
Selection of T-Cell Epitopes
Which parts of gluten proteins are recognized by T cells is the result of several processes. T-cell epitopes are localized to proline-rich regions of gluten proteins [20]. This is so partly because proline-rich sequences are resistant to proteolytic degradation by digestive enzymes [21]. It is also reflecting that proline is part of the recognition sequence of TG2. Typically, TG2 is targeting glutamine residues in the sequence QXP where Q is glutamine, X is any amino acid and P is proline [8, 9]. The central role of TG2 in selection of T-cell epitopes is signified by the finding that among the peptides being targeted by TG2 in a very complex gluten digest mixture, peptides harboring T-cell epitopes were heavily overrepresented [22]. Finally, binding of peptides to MHC is a selective force. The importance of this is reflected by the observation that T cells of celiac patients which recognize gluten peptides in the context of the disease predisposing HLA molecules DQ2.5, DQ2.2 and DQ8 by and large recognize different cohorts of gluten peptides.
T-Cell Epitopes Presented by DQ2.5, DQ2.2 and DQ8
sentation of DQ2.5-restricted T-cell epitopes revealed presentation of most epitopes [24, 25]. These observations were in apparent contrast to the huge difference in risk to celiac disease conferred by the two HLA-DQ2 variants. Later, it was found that incubating the antigen-presenting cells after pulsing with the antigen epitopes before the addition of T cells to the culture revealed striking differences between DQ2.5 and DQ2.2 antigen-presenting cells [26]. The gluten epitopes were only being effectively presented by DQ2.5-expressing cells speaking to stable binding of gluten epitopes as prerequisite for establishing the in vivo T-cell response. This led to the prediction that DQ2.2 would present other gluten epitopes which bind stably to DQ2.2. Recent research has revealed that this indeed is the case [26]. Using HLA molecules as affinity matrix for isolation of DQ2.2-binding peptides of gluten digests, three DQ2.2 epitopes were identified [27], one of which was previously characterized as an immunodominant epitope [28]. Characteristically, these epitopes all have serine at position P3. This suggests that Ser at P3 is a unique characteristic of DQ2.2-restricted epitopes. Moreover, peptide binding experiments indicated that Ser and Thr are anchor residues for binding of peptides to DQ2.2 but not to DQ2.5 [28, 29]. This notion was further established by elution of endogenous peptides from B-lymphoblastoid cell lines [36]. This analysis revealed big differences in the repertoires of peptides eluted from DQ2.2 compared to DQ2.5, with a minority of peptides being presented by both HLADQ molecules. Characterization of the binding motifs by neural network analysis of the eluted peptides revealed a preference of serine, threonine and aspartate at P3 by DQ2.2, which was not found for DQ2.5. Thus, DQ2.5 and DQ2.2 appear to have similar peptide-binding motifs, with DQ2.2 having an additional anchor requirement at P3. Gluten peptides that shall bind stably to DQ2.2 will need to fulfill this extra requirement. Hence, fewer peptides in the gluten proteome will be able to bind stably to DQ2.2 than DQ2.5, and this likely explains the differential celiac disease risks of the two HLA-DQ2 variants.
Conserved Usage of Celiac Disease-Relevant BCRs and T-Cell Receptors
Gluten peptides recognized by T cells in the context of DQ2.5 have deamidations at positions P4, P6 and occasionally P7, whereas gluten peptides recognized in the context of DQ8 have deamidations at position P1 or P4 [23]. This different distribution of glutamate residues reflects the different peptide-binding motifs of DQ2.5 and DQ8. Early experiments using DQ2.2-expressing cells for pre-
T-cell receptors (TCRs) and BCRs have enormous diversity due to receptor rearrangement with recombination of variable (V), diversity (D) and joining (J) gene segments during T-cell and B-cell development. Despite this diversity, similar and clonally dominant BCRs and TCRs are observed across celiac disease patients. This is the case for
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Mechanisms for Formation of Anti-Gluten Antibodies
anti-TG2 antibodies predominantly using the IGVH5-51, IGVH3 or IGVH4 heavy-chain gene segments together with IGVK1 light-chain gene segment [16]. It is the case for anti-gluten antibodies with many antibodies using the IGVH3-23/IGVL4-69 and IGVH3-15/IGVK4-1 combinations [18]. It is also the case for TCRs recognizing certain peptide-MHC complexes. It is particularly prominent for TCRs recognizing the DQ2.5-glia-α2 epitope with overusage and preferred pairing of the TRAV26-1 and TRBV7-2 gene segments [30–33]. In addition to the overusage of the TRAV26-1/TRBV7-2 gene segments, these TCRs have a conserved arginine residue in their complementarity-determining region (CDR) 3β loop [30]. Recently resolved crystal structures of peptide-MHC-TCR complexes revealed this CDR3β arginine in the TCR serves as a lynchpin in the recognition of the DQ2.5-glia-α2 epitope, yet the arginine is not making any direct contact with the glutamate at the P4 in the epitope [33]. Similar preferred usage and pairing seem to be the case for TCRs recognizing the DQ8-glia-α1 epitope with overusage of the TRAV26-2 and TRBV9 gene segments [34] as well as for TCRs recognizing DQ2.5-glia-α1 with overusage of TRBV20-1, TRBV29-1 and TRAV4 gene segments [33]. Whether similar types of biased usage and chain pairing occur in TCRs recognizing other gluten T-cell epitopes remains to be established. Further work should shed light on why there is this conserved antigen receptor usage among presumably disease-relevant BCRs and TCRs in celiac disease. It is possible that the basis lies in a competition for which antigen receptors are best at recognizing certain structures, and that this selection follows the same rules in different subjects. Another interesting avenue to pursue in future studies is whether the conserved receptor usage that seems to be a hallmark of the disease can be harnessed in DNA-based diagnostic approaches.
Conclusions
The adaptive immune system appears to be central in the pathogenesis of celiac disease. Current understanding advocates that gluten-specific CD4 T cells orchestrate an immune response to gluten which also involves an antibody response to the self-antigen TG2. Interactions between gluten-reactive CD4 cells and B cells appear to be key steps in the pathogenesis giving rise to antibodies to gluten (including deamidated gluten fragments) and to TG2. These antibodies serve as excellent surrogate markers for celiac disease. The interaction between T cells and B cells not only gives rise to antibodies, but also amplifies the anti-gluten T-cell response. Interference with the distinct steps in the immune reactions that lead to gluten hypersensitivity could control celiac disease and would represent strategies for novel therapies of this prevalent disorder.
Acknowledgements The work in the authors’ laboratory is supported by grants from the Research Council of Norway (partially through its Centres of Excellence funding scheme, grant No. 179573/V40), the European Commission (grant Nos. ERC-2010-Ad-268541, FP7-People2011-ITN-289964), the University of Oslo, the South-East Norway Regional Health Authority as well as by a Research Prize of the United European Gastroenterology.
Disclosure Statement The authors declare no conflicts of interest.
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8 Vader LW, de Ru A, van Der WY, Kooy YM, Benckhuijsen W, Mearin ML, Drijfhout JW, van Veelen P, Koning F: Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 2002;195:643–649. 9 Fleckenstein B, Molberg, Qiao SW, Schmid DG, Von Der MF, Elgstoen K, Jung G, Sollid LM: Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 2002;277:34109–34116. 10 Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D: Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3: 797– 801. 11 Osman AA, Gunnel T, Dietl A, Uhlig HH, Amin M, Fleckenstein B, Richter T, Mothes T: B cell epitopes of gliadin. Clin Exp Immunol 2000;121:248–254. 12 Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, Troncone R, Giersiepen K, Branski D, Catassi C, Lelgeman M, Maki M, Ribes-Koninckx C, Ventura A, Zimmer KP; ESPGHAN Working Group on Coeliac Disease Diagnosis; ESPGHAN Gastroenterology Committee; European Society for Pediatric Gastroenterology, Hepatology, and Nutrition: European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 2012;54:136–160. 13 Bjorck S, Brundin C, Lorinc E, Lynch KF, Agardh D: Screening detects a high proportion of celiac disease in young HLA-genotyped children. J Pediatr Gastroenterol Nutr 2010;50:49–53. 14 Baklien K, Brandtzaeg P, Fausa O: Immunoglobulins in jejunal mucosa and serum from patients with adult coeliac disease. Scand J Gastroenterol 1977;12:149–159. 15 Siegel M, Strnad P, Watts RE, Choi K, Jabri B, Omary MB, Khosla C: Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury. PLoS One 2008;3:e1861. 16 Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, Huang M, Iversen R, du Pre MF, Qiao SW, Lundin KE, Wilson PC, Sollid LM: High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med 2012;18:441–445. 17 Iversen R, Di Niro R, Stamnaes J, Lundin KE, Wilson PC, Sollid LM: Transglutaminase 2-specific autoantibodies in celiac disease tar-
Small Bowel, Celiac Disease and Adaptive Immunity Ludvig M. Sollid a Rasmus Iversen a Øyvind Steinsbø a Shuo-Wang Qiao a Elin Bergseng a Siri Dørum a M. Fleur du Pré a Jorunn Stamnaes a Asbjørn Christophersen a Inês Cardoso a Kathrin Hnida a Xi Chen a Omri Snir a Knut E.A. Lundin a, b a
Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital – Rikshospitalet, and b Department of Gastroenterology, Oslo University Hospital – Rikshospitalet, Oslo, Norway
Abstract 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
© 2015 S. Karger AG, Basel 0257–2753/15/0332–0115$39.50/0 E-Mail [email protected] www.karger.com/ddi
(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. © 2015 S. Karger AG, Basel
The Gut and Its Immune System
The small bowel is essential for digestion and breakdown of food and absorption of nutrients. Only a single layer of epithelium is lining and protecting the gut. Along with nutrients, pathogenic bacteria and viruses are constantly hitting this epithelial surface layer. Bacteria and viruses that do not cause disease and which are in a symbiotic relationship with the host moreover colonize the Ludvig M. Sollid Centre for Immune Regulation and Department of Immunology University of Oslo and Oslo University Hospital – Rikshospitalet NO–0372 Oslo (Norway) E-Mail l.m.sollid @ medisin.uio.no
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Key Words Adaptive immunity · B cells · Celiac disease · Gluten · MHC genes · T cells
Celiac Disease and Involvement of the Adaptive Immune System
Celiac disease is an acquired disorder caused by an inappropriate immune response to cereal gluten proteins of wheat, barley and rye with clear signs of involvement of the adaptive immune system. It is a multifactorial disorder with influence of genes and environment. Gluten is obviously an important environmental factor as the disease goes in remission when gluten is eliminated from the diet. MHC is the single most important genetic factor which may account for as much as half of the genetic risk. The non-MHC genes have been extensively mapped by genome-wide association studies [1, 2], and most recently by the so-called ImmunoChip analysis [3]. Altogether 40 loci (including the MHC locus) have been identified with evidence of 54 independent signal genes. Many of these contain genes coding for molecules thought to be implicated in adaptive immunity, in particular the function of T cells and B cells. The culprit MHC genes implicated in celiac disease encode for HLA-DQ2.5 (DQA1*05, DQB1*02), HLA-DQ8 (DQA1*03, DQB1*03: 02) and HLA-DQ2.2 (DQA1*02:01, DQB1*02:02). One or more of these MHC allotypes are carried by all celiac disease patients. However, many healthy individuals also carry one of these MHC allotypes, so MHC is a necessary but not sufficient factor in celiac disease development. The risks for celiac disease associated with the various MHC allotypes are widely different. DQ2.5 confers the highest risk, whereas DQ2.2 and DQ8 confer much lower risks [4]. HLA-DQ 116
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molecules predispose to celiac disease by presenting gluten peptides to CD4 T cells [5]. Celiac disease patients have expansions of CD4 T-cell clones reactive to gluten peptides. Once these clonal expansions have taken place, a strong immune response to gluten will follow after an exposure to gluten, likely explaining why celiac disease patients have a lifelong intolerance to gluten. T cells recognize gluten peptides that are posttranslationally modified [6, 7]. The modification, the conversion of glutamine to glutamate, is called deamidation, and is mediated by the enzyme transglutaminase 2 (TG2). The TG2 enzyme targets in a sequence-specific manner certain glutamine residues [8, 9], and either crosslinks the glutamine with a primary amine which could be a lysine residue of another polypeptide, or converts the glutamine to glutamate by a reaction with water. In addition to T cells reactive to gluten, celiac disease patients have antibodies reactive to gluten as well as antibodies reactive to TG2 [10]. The anti-gluten antibodies react particularly well with gluten peptides that are deamidated [11]. Both anti-TG2 and anti-gluten antibodies disappear in subjects with celiac disease upon commencement of a gluten-free diet and reappear upon gluten challenge. Serologic examination plays an increasingly important role in the diagnostic workup of celiac disease [12]. The anti-TG2 antibodies and probably also the antideamidated gluten antibodies are HLA dependent in the sense that they are only formed in individuals who are HLA-DQ2 or HLA-DQ8 [13].
Immune Responses Initiated by Gluten Exposure: Priming and Effector Sites
Antibodies are produced by plasma cells, which differentiate from B cells after recognition of antigen by surface immunoglobulin which serves as the B-cell receptor (BCR). The intestinal mucosa of celiac disease patients is characterized by massive plasmacytosis. There is an increase in density of jejunal plasma cells producing IgA, IgM and IgG by 2.4, 4.6 and 6.5 times, respectively, when compared to healthy controls [14]. As IgA-producing plasma cells are dominating and IgG-producing plasma cells are few in the intestine, the increase in absolute numbers of plasma cells is by far the biggest for IgA, while there are still few IgG-producing plasma cells in the celiac lesion. The gluten-reactive CD4 T cells and plasma cells in the lamina propria are effector cells in the immune response (fig. 1). The mucosa can hence be considered an effector Sollid et al.
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gut surface. To be well functioning, the body should thus respond to and expel harmful bacteria and viruses, but at the same time not respond to foodstuff or to symbiotic bacteria and viruses. This is a formidable challenge to the immune system, and it is no wonder that the gut immune system is large and intricate. The immune system can broadly be separated into innate and adaptive immunity. Whereas innate immunity is based on nonspecific and generic recognition, adaptive immunity is based on specific recognition by antigen receptors of B and T lymphocytes. Another hallmark of adaptive immunity is clonal expansion of specific cells, and usually there is also establishment of memory. The same division of innate and adaptive immunity also applies to the immune system of the gut. The involvement of the adaptive immune system in the pathogenesis is more obvious for some gut diseases than others.
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site for the anti-gluten immune response. The induction of the immune response, however, likely takes place in organized lymphoid tissue, be that mesenteric lymph nodes or Peyer’s patches. In these structures, naïve T cells will be primed to become effector T cells that circulate via blood to seed into the mucosa. Likewise, B cells will be primed to become plasma blasts that circulate via blood and seed into the mucosa to end-differentiate into plasma cells. In this priming of B cells, CD4 T cells are usually instrumental in providing cognate help. This happens either in germinal centers or extrafollicularly. Binding of antigen by the surface immunoglobulin BCR will lead to increased uptake of this antigen into the endosomes for antigenic processing. As a consequence, by mass action,
there is high likelihood that MHC class II molecules of the B cell will display peptide fragments for which the BCR is specific. It is currently not known how gluten peptides access these organized lymphoid structures, nor is it known how TG2, which seems to be mainly catalytically inactive in situ [15], is active at these sites. A particularly interesting question in relation to antiTG2 antibodies is what dictates the formation of autoantibodies in response to exposure to a foreign antigen. As a first step to analyze this, we have established a panel of recombinant monoclonal antibodies from single TG2specific plasma cells of the celiac lesion, and compared these with monoclonal antibodies established from gluten-specific plasma cells.
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adaptive immune system in the pathogenesis of celiac disease. Two key players in the disease are CD4+ T cells specific for deamidated gluten peptides and plasma cells specific for TG2 or gluten. The CD4+ T cells recognize gluten peptides in the context of the disease-associated HLA-DQ2.5 or HLA-DQ8 molecules. The immune reaction is initiated in organized lymphoid tissue (mesenteric lymph nodes or Peyer’s patches), which serves as a priming site for the immune responses. Primed cells (T cells and plasma blasts) migrate via blood to seed into the lamina propria to become effector cells. The enzyme TG2 is the target of autoantibodies, and it is also responsible for creating deamidated gluten peptides.
Antigenpresenting cell
IgA and IgM plasma cells have surface expression of immunoglobulins. This allows selection of antigen-specific cells by use of labeled antigen. We used biotinylated TG2 multimerized on fluorophore-conjugated streptavidin to stain and flow sort TG2-specific plasma cells [16]. We further found that on average 10% of plasma cells in the celiac lesion are specific for TG2, and that the percentage of anti-TG2-positive cells goes down in patients who are on a gluten-free diet. We PCR amplified and sequenced the VH and VL genes of single plasma cells and inserted them into expression vectors for expression as human IgG1 in HEK cells. We found that there is a restricted repertoire of immunoglobulin heavy and light chains among TG2-specific antibodies with overusage of the heavy-chain gene segments IGVH5-51, IGVH3 and IGVH4. Many of the cells also use IGVK1 light-chain gene segments. The antibodies display limited somatic hypermutation, yet the mutations introduced do affect binding affinity indicative of affinity maturation and that T-cell help is important for the formation of the antibodies. The epitopes recognized by the antibodies are conformational. Competitive ELISA experiments indicate that the antibodies recognize at least four different epitopes which are spatially related [17]. The epitopes correspond broadly to the IGVH gene segments used by the antibodies. The localization of the epitopes has been mapped by hydrogen-deuterium exchange, by analysis of interference with TG2 binding to fibronectin and by single amino acid mutation analysis [35]. The so-called epitope 1 used by IGVH5-51 antibodies involves residues Glu8 and Lys30; epitope 2 used by IGVH3 antibodies involves Arg19, and epitope 3 used by IGVH4 antibodies involves Glu94. Epitope 1 and epitope 4 overlap with the fibronectin-binding site of TG2. In parallel with anti-TG2 antibodies, the celiac disease patients make antibodies to gluten. We established gluten-specific monoclonal antibodies by two distinct approaches [18]. In one approach, VH and VL genes were established from cultures of intestinal biopsy single-cell suspensions which by ELISA were shown to contain antibodies reactive with deamidated complex gluten antigen. In the second approach, VH and VL genes were established from single plasma cells of biopsies sorted by flow cytometry using synthetic gluten peptides known to harbor epitopes recognized by serum antibodies of celiac disease patients. A panel of 38 antibodies was established. Typically, the antibodies bind gluten 118
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peptides related to T-cell epitopes and many have higher reactivity to deamidated peptides. There is restricted VH and VL combination and usage among the antibodies, with many antibodies carrying the IGVH323/IGVL4-69 and IGVH3-15/IGVK4-1 combinations. These gluten-specific antibodies also have limited somatic hypermutations.
Mechanisms for Formation of Anti-TG2 Antibodies
It is striking that TG2 which is essential to create deamidated gluten epitopes recognized by CD4 T cells is also the target of highly celiac disease-specific TG2 autoantibodies. This is unlikely to be coincidental, and suggests a causal link between the two phenomena. T-cell help to TG2-specific B cells by gluten-specific T cells is a model that would explain a causal link [19]. It would also explain why the TG2 antibodies are only formed in HLADQ2- or HLA-DQ8-expressing subjects and only when the subjects consume gluten. Gluten peptides are good substrates for TG2. When incubating gluten peptides with TG2, covalent complexes of gluten and TG2 are formed. Such complexes can be bound by TG2-specific B cells, which upon internalization and processing can present gluten epitopes in the context of HLA-DQ2 or HLA-DQ8 to T cells. The T cells recognize deamidated peptides, and one can envisage three ways deamidated peptides are generated from such complexes. These are: (a) The covalent bond is the thioester bond between TG2active site cysteine and peptide glutamine in the enzyme substrate intermediate. Upon reaction of the enzyme substrate intermediate with water, the deamidated peptide is formed. (b) The covalent bond is the isopeptide bond between a lysine residue of TG2 and a glutamine residue of the peptide. Isopeptidase activity of TG2 could regenerate the enzyme substrate intermediate, which again could react with water for formation of a deamidated peptide. (c) The peptide could at least contain two glutamine residues targeted by TG2, one could be deamidated and one could be isopeptide bonded to TG2. Upon internalization in B cells, endoproteases in endosomes would cleave the peptide and release the deamidated epitope. If TG2-specific B cells would be able to present gluten peptides to T cells, the B cells would get their help, but at the same time the T cells would become activated, clonally expand and likely generate memory T cells. Interaction between TG2specific B cells and gluten-specific T cells could thus be an important amplification loop for the anti-gluten T-cell response. Sollid et al.
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TG2 and Gluten-Specific IgA Antibodies Produced by Plasma Cells of the Celiac Lesion
B cells specific for gluten antigen could receive cognate help from gluten-specific T cells. The observation that anti-gluten antibodies bind peptides related to T-cell epitopes speaks in favor of this mechanism. B cells which bind and internalize deamidated gluten epitopes would load HLA-DQ2 or HLA-DQ8 molecules with deamidated epitopes and would hence be particularly well suited to receive help from T cells recognizing deamidated gluten antigen. This may explain why anti-deamidated gliadin antibodies and anti-TG2 antibodies both are excellent markers of disease with high clinical diagnostic performance as they both reflect the action of gluten-specific T cells recognizing deamidated peptides.
Selection of T-Cell Epitopes
Which parts of gluten proteins are recognized by T cells is the result of several processes. T-cell epitopes are localized to proline-rich regions of gluten proteins [20]. This is so partly because proline-rich sequences are resistant to proteolytic degradation by digestive enzymes [21]. It is also reflecting that proline is part of the recognition sequence of TG2. Typically, TG2 is targeting glutamine residues in the sequence QXP where Q is glutamine, X is any amino acid and P is proline [8, 9]. The central role of TG2 in selection of T-cell epitopes is signified by the finding that among the peptides being targeted by TG2 in a very complex gluten digest mixture, peptides harboring T-cell epitopes were heavily overrepresented [22]. Finally, binding of peptides to MHC is a selective force. The importance of this is reflected by the observation that T cells of celiac patients which recognize gluten peptides in the context of the disease predisposing HLA molecules DQ2.5, DQ2.2 and DQ8 by and large recognize different cohorts of gluten peptides.
T-Cell Epitopes Presented by DQ2.5, DQ2.2 and DQ8
sentation of DQ2.5-restricted T-cell epitopes revealed presentation of most epitopes [24, 25]. These observations were in apparent contrast to the huge difference in risk to celiac disease conferred by the two HLA-DQ2 variants. Later, it was found that incubating the antigen-presenting cells after pulsing with the antigen epitopes before the addition of T cells to the culture revealed striking differences between DQ2.5 and DQ2.2 antigen-presenting cells [26]. The gluten epitopes were only being effectively presented by DQ2.5-expressing cells speaking to stable binding of gluten epitopes as prerequisite for establishing the in vivo T-cell response. This led to the prediction that DQ2.2 would present other gluten epitopes which bind stably to DQ2.2. Recent research has revealed that this indeed is the case [26]. Using HLA molecules as affinity matrix for isolation of DQ2.2-binding peptides of gluten digests, three DQ2.2 epitopes were identified [27], one of which was previously characterized as an immunodominant epitope [28]. Characteristically, these epitopes all have serine at position P3. This suggests that Ser at P3 is a unique characteristic of DQ2.2-restricted epitopes. Moreover, peptide binding experiments indicated that Ser and Thr are anchor residues for binding of peptides to DQ2.2 but not to DQ2.5 [28, 29]. This notion was further established by elution of endogenous peptides from B-lymphoblastoid cell lines [36]. This analysis revealed big differences in the repertoires of peptides eluted from DQ2.2 compared to DQ2.5, with a minority of peptides being presented by both HLADQ molecules. Characterization of the binding motifs by neural network analysis of the eluted peptides revealed a preference of serine, threonine and aspartate at P3 by DQ2.2, which was not found for DQ2.5. Thus, DQ2.5 and DQ2.2 appear to have similar peptide-binding motifs, with DQ2.2 having an additional anchor requirement at P3. Gluten peptides that shall bind stably to DQ2.2 will need to fulfill this extra requirement. Hence, fewer peptides in the gluten proteome will be able to bind stably to DQ2.2 than DQ2.5, and this likely explains the differential celiac disease risks of the two HLA-DQ2 variants.
Conserved Usage of Celiac Disease-Relevant BCRs and T-Cell Receptors
Gluten peptides recognized by T cells in the context of DQ2.5 have deamidations at positions P4, P6 and occasionally P7, whereas gluten peptides recognized in the context of DQ8 have deamidations at position P1 or P4 [23]. This different distribution of glutamate residues reflects the different peptide-binding motifs of DQ2.5 and DQ8. Early experiments using DQ2.2-expressing cells for pre-
T-cell receptors (TCRs) and BCRs have enormous diversity due to receptor rearrangement with recombination of variable (V), diversity (D) and joining (J) gene segments during T-cell and B-cell development. Despite this diversity, similar and clonally dominant BCRs and TCRs are observed across celiac disease patients. This is the case for
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Mechanisms for Formation of Anti-Gluten Antibodies
anti-TG2 antibodies predominantly using the IGVH5-51, IGVH3 or IGVH4 heavy-chain gene segments together with IGVK1 light-chain gene segment [16]. It is the case for anti-gluten antibodies with many antibodies using the IGVH3-23/IGVL4-69 and IGVH3-15/IGVK4-1 combinations [18]. It is also the case for TCRs recognizing certain peptide-MHC complexes. It is particularly prominent for TCRs recognizing the DQ2.5-glia-α2 epitope with overusage and preferred pairing of the TRAV26-1 and TRBV7-2 gene segments [30–33]. In addition to the overusage of the TRAV26-1/TRBV7-2 gene segments, these TCRs have a conserved arginine residue in their complementarity-determining region (CDR) 3β loop [30]. Recently resolved crystal structures of peptide-MHC-TCR complexes revealed this CDR3β arginine in the TCR serves as a lynchpin in the recognition of the DQ2.5-glia-α2 epitope, yet the arginine is not making any direct contact with the glutamate at the P4 in the epitope [33]. Similar preferred usage and pairing seem to be the case for TCRs recognizing the DQ8-glia-α1 epitope with overusage of the TRAV26-2 and TRBV9 gene segments [34] as well as for TCRs recognizing DQ2.5-glia-α1 with overusage of TRBV20-1, TRBV29-1 and TRAV4 gene segments [33]. Whether similar types of biased usage and chain pairing occur in TCRs recognizing other gluten T-cell epitopes remains to be established. Further work should shed light on why there is this conserved antigen receptor usage among presumably disease-relevant BCRs and TCRs in celiac disease. It is possible that the basis lies in a competition for which antigen receptors are best at recognizing certain structures, and that this selection follows the same rules in different subjects. Another interesting avenue to pursue in future studies is whether the conserved receptor usage that seems to be a hallmark of the disease can be harnessed in DNA-based diagnostic approaches.
Conclusions
The adaptive immune system appears to be central in the pathogenesis of celiac disease. Current understanding advocates that gluten-specific CD4 T cells orchestrate an immune response to gluten which also involves an antibody response to the self-antigen TG2. Interactions between gluten-reactive CD4 cells and B cells appear to be key steps in the pathogenesis giving rise to antibodies to gluten (including deamidated gluten fragments) and to TG2. These antibodies serve as excellent surrogate markers for celiac disease. The interaction between T cells and B cells not only gives rise to antibodies, but also amplifies the anti-gluten T-cell response. Interference with the distinct steps in the immune reactions that lead to gluten hypersensitivity could control celiac disease and would represent strategies for novel therapies of this prevalent disorder.
Acknowledgements The work in the authors’ laboratory is supported by grants from the Research Council of Norway (partially through its Centres of Excellence funding scheme, grant No. 179573/V40), the European Commission (grant Nos. ERC-2010-Ad-268541, FP7-People2011-ITN-289964), the University of Oslo, the South-East Norway Regional Health Authority as well as by a Research Prize of the United European Gastroenterology.
Disclosure Statement The authors declare no conflicts of interest.
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