The adaptive immune response in celiac disease

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with a new suggested nomenclature [54] is given in Table 1. DQ2-gluten ...... (4):395–402. 7. Dubois PCA, Trynka G, Franke L, Hunt KA, Romanos J, Curtotti.
Semin Immunopathol DOI 10.1007/s00281-012-0314-z

REVIEW

The adaptive immune response in celiac disease Shuo-Wang Qiao & Rasmus Iversen & Melinda Ráki & Ludvig M. Sollid

Received: 5 March 2012 / Accepted: 10 April 2012 # Springer-Verlag 2012

Abstract 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.

Keywords Celiac disease . Gluten . T cells . B cells . Antigen-presenting cells

This article is published as part of the Special Issue on Celiac Disease [34:5]. S.-W. Qiao (*) : R. Iversen : M. Ráki : L. M. Sollid (*) Department of Immunology and Centre for Immune Regulation, University of Oslo, Oslo University Hospital—Rikshospitalet, 0027 Oslo, Norway e-mail: [email protected] e-mail: [email protected] M. Ráki Department of Pathology, Oslo University Hospital—Rikshospitalet, 0027 Oslo, Norway

Introduction Celiac disease is caused by an inappropriate immune response to gluten which is the cohesive mass that remains when dough is washed to remove starch. Traditionally and strictly speaking, gluten is a name of wheat proteins only, but gluten is now increasingly used as a term to denote proteins of wheat, barley, rye, and oat that are rich in proline (Pro) and glutamine (Gln) residues. Gluten proteins of wheat, barley, and rye can elicit celiac disease (CD) [1], whereas oat appears safe for most CD patients [2]. Of the wheat gluten proteins, both gliadins (alcohol soluble, divided into α, γ, and ω subtypes) and glutenins (alcohol insoluble) are harmful [1]. Notably, gluten proteins are very good substrates for the enzyme transglutaminase 2 (TG2). This enzyme can convert Gln residues to glutamate (Glu) in a process known as deamidation. The disease-causing immune response in CD is thus not to regular gluten antigen, but rather to posttranslationally modified gluten. The enzyme that mediates this posttranslational modification is the very same enzyme that the autoantibodies in CD are directed against. This is hardly coincidental, and although the mechanism for the generation of antibodies to TG2 is still unclear, it is very likely that it is related to the specificity of TG2 and the enzyme’s liking of gluten peptides as substrates. In this way, the adaptive immune response to the foreign antigen gluten induces a parallel immune response to the self-antigen TG2. The immune response in celiac disease thus has two Janus faces: on the one side, it shows a hypersensitivity reaction to edible antigen, thus being a food intolerance disorder, and on the other side, it shows an autoimmune reaction with antibodies to a self-antigen with unprecedented disease specificities and sensitivities. This all happens in individuals with a certain genetic predisposition. Which genes that confer this predisposition

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represent a key to understand this fascinating Janusfaced immune response.

Predisposing genes Human leukocyte antigen (HLA) genes Both genetic and environmental factors contribute to the development of CD. The importance of genes is signified by familial clustering and by high concordance in twins. The probandwise concordance rates in monozygotic and dizygotic twins are about 80 and 20 %, respectively [3, 4]. Genome-wide association studies (GWAS) have demonstrated that CD is a polygenic disorder with involvement of many genes, perhaps several hundreds, and that the HLA locus encoding human major histocompatibility complex (MHC) molecules is by far the most important genetic factor contributing about 40 % of the genetic variance of the disease [5–8]. The primary HLA association for CD is conferred by class II HLA-DQ genes. Approximately 90 % of the CD patients express the HLA-DQ2.5 molecule (also simply known as HLA-DQ2), encoded by DQA1*05/DQB1*02 genes, and the majority of the remaining patients express HLA-DQ8, encoded by DQA1*03/DQB1*03:02 genes [9, 10]. The relative risk of CD in individuals expressing DQ2.5 is estimated to be around 30–40 [9, 11], among one of the highest for autoimmune diseases [11]. When the genes encoding the α- and the β-chains of the HLA-DQ2.5 heterodimer are carried on the same chromosome (in cis position), they are most often found as part of the highly conserved A1-B8-DR3-DQ2 haplotype. This particular haplotype is associated with several autoimmune diseases such as type 1 diabetes and myasthenia gravis and is thus sometimes referred to as the “autoimmune haplotype” [11]. Patients that are DR5-DQ7/DR7-DQ2 heterozygous also express the HLA-DQ2.5 molecules, but then the two encoding genes are carried on different chromosomes (in trans position). The relevant DQA1 allele (DQA1*05) is encoded by the DR5DQ7 haplotype and the relevant DQB1 allele (DQB1*02) is encoded by the DR7-DQ2 haplotype. Gluten peptides show preferred binding to DQ2.5 molecules compared to other HLA class II molecules [12]. Peptide binding studies have shown that contact between DQ2.5 and the side chains of the peptides, so-called anchor residue and pocket interactions, takes place at the P1, P4, P6, P7, and P9 positions [13–16]. These studies and the crystal structure of the DQ2.5 molecule bound with the DQ2.5-glia-α1a peptide [17] reveal features that explain why DQ2.5 is especially suitable for the binding and presentation of gluten peptides. Peptide main chain amide groups normally participate in several hydrogen bonds to HLA class II residues along the

binding site, but as Pro has a ring structure and lacks the amide hydrogen, it cannot engage in such hydrogen bonds. The multiple Pro residues in the gluten epitopes are spaced throughout the binding site so that there is minimal interference with these main chain hydrogen bonds [18]. Furthermore, the lysine (Lys) residue at β71 in DQ2.5 creates a unique positive electrostatic region between the P4 and the P6/P7 pockets [17], thus favoring binding of negatively charged residues, such as Glu residues, in these pockets. Although one copy of DQ2.5 is sufficient to predispose for CD, epidemiological evidence suggests a gene dosage effect. Individuals carrying either DR3-DQ2 or DR7-DQ2 haplotypes on the other chromosome, in addition to the first DR3-DQ2, have higher risks for CD than those with a solitary copy of DR3-DQ2 [19, 20]. The common denominator of the DR3-DQ2 and DR7-DQ2 haplotypes is their shared DQβ-chain, which seemingly causes the gene dosage effect. Interestingly, the DR7-DQ2 haplotype alone does not confer much risk for CD, despite that the encoded DQ2.2 (DQA1*02:01/B1*02) molecule in its membrane distal domains differs by only 10 amino acids in the αchain from the related DQ2.5 molecule. However, a closer dissection of haplotypes carried by a few CD patients that are neither DQ2.5 nor DQ8 does reveal a disease association of the DQ2.2 molecule. In the largest such study so far, 41 of 61 non-DQ2.5-non-DQ8 CD patients were DQ2.2 positive, of which 11 of the 41 patients were DQ2.2 homozygous [21]. The peptide binding specificities of DQ2.5 and DQ2.2 are very similar [16, 22], but DQ2.2 does seem to have an additional binding pocket at P3 with a preference for serine (Ser), threonine (Thr), and aspartate (Asp) and with Pro being disfavored at this position [16]. Indeed, many gluten-derived peptides can bind DQ2.2 molecules and be presented by DQ2.2 antigen-presenting cells (APCs). Gluten-reactive CD4+ T cells generated from DQ2.5+ CD patients can often recognize these gluten–DQ2.2 complexes [23]. The observation that Pro is disfavored in the P3 binding pocket of DQ2.2 was linked to the differences in gluten epitope presentation and CD association of these two related molecules [24] but was later shown not to be the deciding factor [23]. A more recent study suggests that it is rather the longer peptide off-rate from DQ2.5 compared to DQ2.2 that explains the much higher disease risk associated with DQ2.5 [25]. A phenylalanine (Phe) (in DQ2.2) to tyrosine (Tyr) (in DQ2.5) polymorphism in DQα22 results in improved ability of the DQ2.5 molecule to retain its peptide cargo and thus protracted gluten presentation of DQ2.5+ APCs relative to that of DQ2.2+ cells. The dissociation half-life of the CLIP1 peptide and the gluten-derived DQ2.5-glia-γ1 peptide, for instance, is 14–30 times longer from DQ2.5 than DQ2.2. This means that these peptides will remain bound to DQ2.5 molecules for days, whereas they will dissociate from DQ2.2 molecules within hours [25]. Studies of CD8+

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T-cell responses in animal models have demonstrated that a few-fold difference in peptide off-rate can result in 30,000fold differences in the in vivo antigenicity [26]. Thus, in DQ2.2 individuals, intestinal APCs bound with gluten peptides will in most cases lose their antigenic cargo before reaching the draining lymph nodes and therefore no presentation to naïve CD4+ T cells can take place. On the molecular level, the Tyr residue in DQα22 of the DQ2.5 molecule appears to form a hydrogen bond to the peptide main chain in pocket P3, whereas the Phe residue at the same position in DQ2.2 cannot. Interestingly, the only DQ2.2-restricted T-cell epitope identified so far contains a Ser in the P3 position (Table 1). The hydroxyl group of the Ser side chain could potentially substitute for the hydroxyl group that is removed by the Tyr to Phe substitution thereby enabling hydrogen bond interaction between the peptide and HLA at this site [27]. These findings suggest that the kinetic stability of complexes between peptide and HLA molecule is of importance for the association of HLA with disease. Non-HLA genes GWAS studies have revealed association to a number of non-HLA gene regions and the majority of these contain immune-related genes [5–8]. In fact, genes implicated in T-cell and B-cell function are overrepresented, thus testifying to the role of the adaptive immune system in the pathogenesis of CD [28]. The relative contribution of each of these genes is minute compared to the HLA genes, with odds ratios usually below 1.5. The collective effect of the 39 non-HLA loci described so far is estimated to contribute about 14 % of the genetic variance [8]. Many of the nonHLA polymorphisms seem to act by influencing gene expression [7, 8]. Furthermore, many non-HLA CD risk loci are shared with other immune-related diseases, in particular type 1 diabetes and rheumatoid arthritis [29–32]. A shared genetic background among these diseases points to common pathogenic pathways [33].

CD4+ T-cell responses to gluten The strong association to HLA class II genes reveals the importance of CD4+ T-cell responses in CD pathogenesis. In the active celiac lesion, there is an increased infiltration of T cells in the epithelium [34] and increased activation of T cells in the lamina propria [35]. The infiltration of T cells in the lamina propria of the active celiac lesion is dominated by CD4+ memory T cells (CD45RO+) bearing the α/β T-cell receptor (TCR) [36]. In vitro gluten stimulation of intestinal biopsies from treated CD patients leads to upregulation of the activation marker CD25 (α-chain of the IL-2

Table 1 List of celiac disease relevant T-cell epitopes recognized by CD4+ T cells of celiac disease patients Epitope

Peptide binding register: P1-P9 1

2

3

4

5

6

7

8

9

DQ2.5-restricted epitopes DQ2.5-glia-α1a

P

F

P

Q

P

E

L

P

Y

DQ2.5-glia-α1b DQ2.5-glia-α2

P P

Y Q

P P

Q E

P L

E P

L Y

P P

Y Q Q Q

DQ2.5-glia-α3

F

R

P

E

Q

P

Y

P

DQ2.5-glia-γ1 DQ2.5-glia-γ2 DQ2.5-glia-γ3 DQ2.5-glia-γ4a DQ2.5-glia-γ4b

P I Q

Q Q Q

Q P P

S E E

F Q Q

P P P

E A Y

Q Q P

S P

Q Q

P P

E E

Q Q

E E

F F

P P

Q Q

DQ2.5-glia-γ4c DQ2.5-glia-γ4d DQ2.5-glia-γ5

Q

Q

P

E

Q

P

F

P

P Q

Q Q

P P

E F

Q P

P E

F Q

C P

Q Q Q

DQ2.5-glia-ω1 DQ2.5-glia-ω2 DQ2.5-glut-L1

P P P

F Q F

DQ2.5-glut-L2 DQ2.5-hor-1

F P

S F

P P S Q P

Q E E Q Q

P Q Q Q P

E P E E E

Q F Q S Q

P P P P P

F W V F F

DQ2.5-hor-2 DQ2.5-hor-3 DQ2.5-sec-1

P P P

Q I F

P P P

E E Q

Q Q P

P P E

F Q Q

P P P

Q Y F

DQ2.5-sec-2 DQ2.5-ave-1a

P P

Q Y

P P

E E

Q Q

P E

F E

P P

Q F

DQ2.5-ave-1b P Y DQ2.2-restricted epitopes DQ2.2-glut-L1 P F

P

E

Q

E

Q

P

F

S

E

Q

E

Q

P

V

G

S

F

Q

P

S

Q

E

Q Q G

P P Y

Q Q Y

Q Q P

P P T

F Y S

P P P

Q E Q

DQ8-restricted epitopes DQ8-glia-α1 E DQ8-glia-γ1a DQ8-glia-γ1b DQ8-glut-H1

E E Q

L Q

Only the nine amino acid core sequences found within the HLA class II binding grooves are shown. In most cases peptides longer than nine amino acids are needed in order to elicit responses in CD4+ T cells from CD patients. Glu residues (E) formed by TG2-mediated deamidation that are important for recognition by T cells are shown in bold. Additional Gln residues (Q) also targeted by TG2 are underlined. Adapted from [54]

receptor), a phenomenon that is not observed in controls [37]. After in vitro gluten challenge, CD4+ TCRαβ+ T cells recognizing gluten can be readily isolated from the biopsy specimens of CD patients, either in active disease or on gluten-free diet, but not from controls [38, 39]. Notably, these disease-specific T cells recognize gluten in an HLADQ2.5- or HLA-DQ8-restricted manner, strongly indicating

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that T cells are the primary players in the pathogenesis of CD [10]. Gluten-specific CD4+ T cells isolated from CD biopsies display TH1 or TH0 cytokine profile, dominated by the production of interferon-γ (IFN-γ) [40]. Although polyclonal T-cell lines generated from CD biopsies can secrete IL-17 upon stimulation, intracellular cytokine staining combined with tetramer staining showed that gluten-specific T cells produced IFN-γ and IL-21, but not IL-17 or IL-22. IL-17 is produced by T cells other than the gluten-specific T cells of the same lines [41]. The posttranslational modification of Gln→Glu deamidation at specific sites is crucial for T-cell recognition for a majority of the gluten T-cell epitopes [42–45] (Table 1). Although Gln→Glu deamidation can occur chemically at low pH, such as the milieu in the stomach, a wealth of evidences suggest that the generation of deamidated T-cell epitopes in CD is a well-controlled enzymatic process mediated by TG2 [46–48]. In particular, the sequence specificity of TG2 matches the deamidation requirements by T cells, and TG2 specificity is shown to participate in the selection of gluten T-cell epitopes [45, 49]. When a complex mixture of glutenderived peptides was given to TG2 as substrates, the enzyme modifies a limited number of peptides with high efficiency [50]. A vast majority of these peptides contain wellcharacterized gluten T-cell epitopes showing that TG2 specificity is a major factor in shaping the gluten-specific T-cell response in CD. The negative charges introduced by the TG2-mediated deamidation increase the binding affinity of gluten peptides to HLA-DQ2.5 and HLA-DQ8, and deamidation is in many cases essential for T-cell recognition. Importantly, the deamidation decreases the off-rate of gluten peptides such that the dissociation of peptides from DQ2.5 molecules is slower [51]. Both HLA molecules prefer negatively charged amino acid residues in certain binding pockets (P4, P6, and P7 for DQ2; P1 and P9 for DQ8) [17, 52]. To date, more than 15 different gluten-derived T-cell epitopes have been reported in CD [23, 42–45, 47] and all contain a deamidated residue in at least one of these pocket positions. This also relates to epitopes of barley and rye gluten proteins [53]. Notably, some of these barley- and rye-derived epitopes elicit T-cell responses that are specific for these cereal proteins whereas other epitopes can stimulate T cells that are reactive across cereal species [53]. A list of the currently known epitopes with a new suggested nomenclature [54] is given in Table 1. DQ2-gluten tetramers based on biotinylated, soluble recombinant DQ2.5 molecules complexed with gluten epitope peptide have been generated. When multimerized with fluorescence-conjugated streptavidin, these tetramers can be used to detect epitope-specific CD4+ T cells in flow cytometry [55]. Direct sequencing of the TCR genes of tetramer-sorted gliadin-α2-reactive T cells reveals biased

usage of the TRBV7-2 gene segment and a highly conserved arginine (Arg) in the CDR3β loop [56]. The Arg interacts directly with the DQ2.5-glia-α2 antigen complex and is crucial for the TCR recognition. Evidence suggests that the archetypical TCR that has been selected in vivo by the gluten antigen recognizes soluble DQ2.5-glia-α2 antigen complex with Glu residue (deamidated sequence) more vigorously than the same complex with Gln residue (native sequence). Therefore, deamidation by TG2 not only increases DQ2.5-gluten complex stability on the surface of APCs by increasing peptide binding affinity, but also directly participates in the selection of the gluten-reactive TCR repertoire in vivo. Owing to its high Pro content, gliadin is remarkably resistant to luminal and brush-border proteolysis and large fragments remain intact after digestion. Therefore, in contrast to other dietary antigens that are broken down to smaller fragments, the larger fragments of the gluten antigen remain antigenic after luminal and brush-border proteolysis. The most illustrative peptide fragment is the 33mer produced by digestion of certain α-gliadin proteins. This 33mer fragment remains intact even after extended incubation with gastric, pancreatic, and intestinal brush-border membrane enzymes [57]. It contains six overlapping copies of two different DQ2.5-restricted T-cell epitopes and is recognized by T-cell lines from nearly all adult CD patients. The two epitopes contained within this 33mer peptide, the DQ2. 5-glia-α1 and DQ2.5-glia-α2 epitopes, are often referred to as the dominant T-cell epitopes in CD. Intriguingly, the 33mer peptide can bind DQ2.5 molecules directly on the surface of APCs and can thus be presented to T cells without further intracellular processing [58]. The gliadin-derived epitopes cluster in the Pro-rich regions of gliadin, a property that both confers relative proteolytic resistance and favors TG2mediated deamidation [44]. Where and when the necessary gluten deamidation takes place is still in question. TG2 is enzymatically active only in the presence of millimolar levels of Ca2+. Upon activation, the enzyme undergoes a dramatic conformational change in which the C-terminal residues are displaced by as much as 120 Å [59]. TG2 is a ubiquitous protein found both intracellularly and extracellularly. The intracellular Ca2+ concentration is low and tightly controlled. By contrast, the Ca2+ concentration is high in the extracellular environment, and thus, it has been assumed that extracellular TG2 is enzymatically active. However, a recent study reveals that the majority of extracellular TG2 is inactive, despite an environment conductive to enzyme activation [60]. Extracellular TG2 can be transiently activated by certain types of inflammation or injury signals [60]. The enzymatic activity of TG2 is found to be tightly regulated by the redox potential of the environment, mediated by a redox-sensitive cysteine triad consisting of Cys230, Cys370, and Cys371 [61]. TG2 is

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reversibly inactivated by oxidation, and Ca2+ can protect against oxidation and inactivation. In the current model of TG2 activation, TG2 is released into the extracellular matrix upon cell wounding and remains catalytically active for a short period of time but then becomes silenced through oxidation. A change to a reductive environment could rescue TG2 from an inactive state. Interestingly, the redox state of lymphoid tissue is modified by ongoing immune reactions. The thiol content is increased after antigen stimulation, particularly in mesenteric lymph nodes after intraperitoneal immunization [62]. An ongoing immune response, including those directed against infectious agents, may thus facilitate TG2 activity and an immune response to deamidated gluten. DQ2.5- or DQ8-restricted gluten-reactive CD4+ T cells can be cultured from the intestinal lesions of both CD patients in active disease or on gluten-free diet. It is difficult to estimate the exact prevalence of gluten-reactive T cells in the lamina propria due to lack of techniques to visualize and quantify these antigen-specific cells directly. In the peripheral blood, the frequency of gluten-reactive T cells is usually too low to be detected by conventional ELISpot or tetramer staining. In treated CD patients who are on gluten-free diet, a 3-day gluten challenge leads to synchronized activation of a large number of gluten-reactive memory T cells in the gut. Six days after initiation of the gluten challenge, activated gluten-reactive T cells in systemic circulation—presumably en route from mesenteric lymph nodes homing back to the intestinal tissues—can be detected in the peripheral blood by ELISpot or tetramer staining [63, 64]. In those studies, the frequency of T cells specific to the dominant gliadin-α1 or gliadin-α2 epitopes is estimated to be between 1:5,000 and 1:25,000 among peripheral blood mononuclear cells on day 6 after gluten challenge. The detection sensitivity of tetramer staining can be greatly enhanced by introducing an enrichment step using paramagnetic microbead-aided separation. With this method, gluten-reactive CD4+ T cells can be directly visualized in the peripheral blood of CD patients without the need of gluten challenge. With this method, the frequency of gliadin-α1 or gliadin-α2 tetramer-positive T cells is estimated to be 1:50,000 to 1:500,000 among peripheral blood mononuclear cells from CD patients (Christophersen et al., unpublished). Taken together, the lamina propria CD4+ T-cell response directed against deamidated gluten peptides presented by the disease associated HLA-DQ2.5 and DQ8 molecules is a key event in the pathogenesis of CD. The CD4+ T-cell response is directed against several different epitopes from different parts of the gluten protein. The selection of these epitopes is governed by a number of factors. The epitopes cluster in Pro-rich regions of the gluten protein because they are more proteolytic resistant, thus generating larger peptide fragments that are antigenic. Next, TG2 deamidates certain

gluten peptides with high efficiency based on the sequence specificity of TG2. Deamidation increases affinity and kinetic binding stability of gluten peptides to DQ2.5 and DQ8 through the introduction of negative charges that are preferred by these HLA class II proteins. In addition, at least for some epitopes, the deamidation also enhances the TCR recognition directly. Lastly, luminally processed, TG2deamidated, Pro-rich gluten peptides are docked into the binding grooves of DQ2.5 and DQ8 molecules such that Pro residues are placed in positions where the lack of hydrogen bond network is least penalized, and the negatively charged Glu residues are found in pockets where these are favored. Contrary to DQ2.2, the DQ2.5 molecule binds gluten epitope peptides with relatively long half-life such that APCs that have picked up the antigen cargo in the intestinal tissue can travel to the mesenteric lymph nodes where antigen presentation and priming of naïve CD4+ T cells can take place. After activation, gluten-reactive CD4+ T cells home back to lamina propria after a brief transit in the general circulation. In the tissue, primed CD4+ T cells are either re-activated by local resident APCs presenting gluten peptides in patients that consume gluten, or remain dormant as memory T cells in patients that are on gluten-free diets. Activated glutenreactive CD4+ T cells contribute to celiac pathology by secretion of proinflammatory cytokines such as IFN-γ and most probably by helping the activation, isotype switch, and plasma cell differentiation of gluten-reactive as well as TG2-reactive B cells.

B-cell immunity to gluten and TG2 There is increased humoral activity in CD. The density of plasma cells in the active celiac lesion is increased two- to threefold [65], and the disease is hallmarked by a variety of specific serum antibodies targeting both self and non-self molecules [66]. Assessment of antibodies in serum has been harnessed for diagnostic purposes for over 40 years. For a long time, the prevailing test was measurement of IgA antibodies to native wheat gluten or gliadin [67, 68]. However, this test is less used in clinical practice today as such antibodies are found in patients suffering from other conditions, including psoriasis [69] and rheumatoid arthritis [70], and as other serological tests have higher diagnostic value [71]. A test measuring antibodies to deamidated gliadin peptides was developed based on the central role of TG2-mediated deamidation in generating gluten-derived T-cell epitopes [72]. The antibody epitopes reflect the substrate specificity of TG2, and in many cases, they are overlapping with known T-cell epitopes [73]. Such tests have been found to have very strong performance [74]; however, the most sensitive and specific tests are the ones based on detection of serum autoantibodies [75].

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Different types of serum autoantibodies targeting connective tissue fibers have been known as markers for CD for more than 50 years. The so-called reticulin antibodies were described first [76, 77]. Later, the endomysium antibodies reactive with structures surrounding smooth muscles fibers in monkey esophagus [78] or human umbilical cord [79] were described. In 1997, the major target autoantigen of the endomysium antibodies was identified as TG2 [80], and it was later shown that the reticulin antibodies are also directed against this protein [81]. The discovery of TG2 as the main autoantigen in CD has led to the development of enzymelinked immunosorbent assays that can rapidly detect TG2specific serum autoantibodies with close to 100 % sensitivity and specificity [71]. Both IgA and IgG class TG2specific autoantibodies can be detected in serum but the IgA test has been found to be more sensitive. However, in the case of IgA deficiency, the IgG test becomes an important diagnostic tool [82]. As a result of the strong performance of serological tests, the European Society for Gastroenterology and Hepatology in its new guidelines allow the diagnosis of CD in children without duodenal biopsy. The diagnosis can be made in HLA-DQ2.5 or DQ8-positive subjects without a biopsy if IgA anti-TG2 is 10 times above the upper limit of normal subjects and if the presence of IgA endomysial antibodies is documented in an independent serum sample [83]. The strong connection between CD and the production of antiTG2 autoantibodies is underlined by the observation that deposits of TG2-specific IgA can be found in the intestinal mucosa even in rare patients where serum autoantibodies are undetectable [84]. In IgA-deficient patients, these deposits are made up of IgM [85]. It thus appears that TG2-specific autoantibodies are produced by all CD patients and the response is directly implicated in the mechanisms that drive the disease. Autoantibody deposits have in some cases been detected years before any intestinal damage was observed and the production of TG2-specific autoantibodies is therefore considered an early marker for developing CD [86]. The antibodies are produced locally by plasma cells residing in the intestinal mucosa and end up in the serum through “spillover” into the circulation. Anti-TG2 antibodies may also be produced elsewhere like in the spleen and bone marrow. This particularly relates to IgG production. Autoantibodies with other specificities than TG2 have also been described in CD, but these do not display the same high sensitivity and specificity as the TG2-specific antibodies [87]. Identified autoantigens include abundant proteins like actin and various types of collagens as well as other members of the transglutaminase family: TG3, TG6, and Factor XIII. Interestingly, complexes between IgA and TG3 have been found in the skin of patients with dermatitis herpetiformis [88], and autoantibodies targeting the neuronal enzyme TG6 have been associated with ataxia

[89]. Thus, there seems to be a connection between these types of autoantibodies and the development of extraintestinal manifestations of CD. The finding of gluten-specific antibodies in CD patients is to be expected given the CD4+ T-cell response to gluten. However, the formation of autoantibodies to TG2 is unexpected as well as their gluten dependence. The antibodies disappear from the circulation if gluten is removed from the diet and they reappear if a gluten-containing diet is reintroduced [90]. The dependence of an autoimmune response on the exposure to a foreign protein antigen (gluten) makes CD unique among autoimmune conditions and an interesting model for studying mechanisms of autoimmunity. The basis for the link between the ingestion of gluten and the activation of autoreactive B cells with concomitant plasma cell differentiation and production of antibodies, however, is not clearly understood. As will be discussed in the following sections, different scenarios explaining the gluten-dependent production of autoantibodies have been suggested but they are not easily investigated experimentally. Recent data providing detailed information about autoreactive plasma cells in the CD lesion, however, give important hints to understand the mechanisms underlying this autoimmune response. Thus, one central question is how the TG2-specific antibodies are produced. Another is what role they play in the disease.

Biological role of TG2-specific autoantibodies The production of TG2-specific autoantibodies appears to be tightly connected to the development of CD, but despite their central role in diagnosis, the biological significance of the antibodies is uncertain. Thus, it has been debated whether the anti-TG2 response plays any part in the pathogenesis of CD. Experiments in which cell cultures have been treated with patient serum antibodies suggest that anti-TG2 autoantibodies can have various biological effects. These include induction of proliferation [91] and inhibition of differentiation [92] in epithelial cells: effects that fit clinical features observed in patient intestinal biopsies. CD autoantibodies have also been found to induce monocyte activation and increase epithelial cell permeability [93]. In accordance with the latter effect, it has been suggested that TG2-specific antibodies can facilitate transport of gliadin peptides across the intestinal epithelium, thereby helping to pave the way for an anti-gliadin immune response [94]. In addition, it has been reported that anti-TG2 autoantibodies can induce apoptosis in neuronal cells [95] and trophoblasts [96], supposedly contributing to neurological and pregnancy-related complications in CD, respectively. Finally, inhibition of angiogenesis [97] and increased blood vessel permeability [98] have been observed in vitro after treatment of endothelial cells with TG2-specific serum antibodies.

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These effects could contribute to the disorganization of the small intestinal vascular network that is seen in the CD lesion. Despite the reported effects of anti-TG2 autoantibodies in cell culture systems, in vivo studies have so far not been able to confirm that TG2-specific antibodies play a pathogenic role in CD. Thus, immunization of mice with human TG2 did not induce morphological changes in the small intestine [99]; neither did adenovirus-mediated expression of human TG2-specific single chain antibody fragments in mice [100]. However, injection of the same type of antibody fragments has later been shown to cause ataxia in mice, suggesting that TG2-specific autoantibodies might play a role in extraintestinal manifestations associated with CD [101]. When it comes to the direct effect of TG2-specific autoantibodies on the enzymatic activity of TG2, contradicting observations have been reported. Serum antibodies from CD patients have been observed to inhibit the transamidation activity of TG2 [102], but the degree of inhibition and its potential significance in vivo has later been questioned [103]. Other studies have reported increased enzymatic activity upon incubation of TG2 with serum-derived antibodies both in solution [104] and in a cell culture system [98]. Furthermore, a large panel of human monoclonal antibodies obtained from TG2-specific plasma cells in the CD intestinal lesion was recently shown not to affect the transamidation and deamidation activities of the enzyme significantly [105]. Epitopes recognized by TG2-specific autoantibodies So far, attempts to map the antigenic epitopes of TG2 have yielded somewhat divergent results. According to one binding study using TG2 fragments of varying lengths based on the structural domains of the enzyme, both the N- and C-terminal parts of TG2 harbor epitopes recognized by serum autoantibodies [106]. In a similar study, it was concluded that the catalytic central region of TG2 is the main antigenic target [107], while a third study found both the N-terminal and central parts of TG2 to be important for autoantibody binding [108]. The epitopes recognized by TG2-specific autoantibodies are known to be conformational as disruption of the threedimensional structure of the enzyme abolishes antibody binding. This complicates binding experiments using TG2 fragments as antigen since interactions between individual domains will be lost in truncated protein variants. Thus, the effects seen by deleting one domain may not imply antibody binding to that specific region alone. This line of thought is supported by a recent report in which site-directed mutagenesis was used to identify a single major epitope made up of residues from different TG2 domains at the intersection between the N-terminal, C-terminal, and central region [109]. The binding of antibodies to this type of epitope is likely to be affected by

the conformation of TG2 and can explain why the open, active conformation of the enzyme has been demonstrated to be the better antigen in diagnostic tests [110]. In another study, it was reported that mutation of the catalytic triad in the central region of TG2 causes a strong decrease in anti-TG2 IgA binding [111]. This led the authors to suggest that the autoantibody response is mainly directed against the region surrounding the active site. This conclusion, however, is now questioned by accumulating evidence claiming that the autoantibodies do not inhibit the enzymatic activity of TG2. Our own data obtained from crosscompetitive binding studies with plasma cell-derived monoclonal antibodies suggest that at least five main TG2 epitopes exist and that the binding to these is affected by the conformation of the enzyme with most antibodies displaying stronger affinity to the open than the closed state (Di Niro et al., unpublished). Further studies need to be carried out to determine which amino acid residues make up these different epitopes. Human monoclonal antibodies specific for TG2 In 2001, Marzari and co-workers constructed phage-display libraries of single-chain antibody variable regions obtained from IgA-producing lymphocytes isolated from the blood or intestinal biopsies of CD patients [112]. In the samples derived from intestinal biopsies but not from blood, they were able to select TG2-specific antibody fragments, indicating that anti-TG2 serum IgA is produced locally in the intestinal mucosa. Analysis of the TG2-specific gene repertoire revealed a preference for VH5 family gene segment usage among IgA-producing cells in CD patients. Interestingly, this preference was not found in TG2-specific antibody fragments obtained from a naïve B-cell library, suggesting that VH5 family selection is not explained by intrinsic TG2 affinity associated with this particular heavy chain variable region. The VH5 family preference was recently confirmed in a comprehensive study of TG2-specific plasma cells in the intestinal mucosa of CD patients [105]. Here, it was shown that high numbers of TG2-specific plasma cells could be detected in intestinal biopsies from CD patients but not from healthy controls. These cells were sorted and subjected to single-cell PCR, thereby allowing the production of TG2specific monoclonal antibodies. This approach has the advantage that it produces antibodies with the correct pairing of heavy and light chains whereas the phage-display method results in antibody fragments with random pairing of heavy and light chain variable regions. In accordance with the phage-display study, it was found that almost half of the generated TG2-specific antibodies belonged to the VH5 family. Furthermore, the amount of somatic mutations in the variable region gene segments was determined and

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compared to other B-cell subsets, clearly showing a reduced number of mutations in TG2-specific plasma cells. The mutations that had accumulated, though, were shown to have a positive effect on the affinity of the antibodies as reversion of mutations back to the germline sequence resulted in lower TG2 affinity for all but one of the antibodies tested. This suggests that TG2-specific B cells have been through a T-cell-dependent selection process in a germinal center reaction. For some reasons, however, this reaction appears somewhat limited so that only few mutations accumulate. Models to explain anti-TG2 B-cell response When TG2 was first discovered as the main autoantigen in CD, the authors speculated that anti-TG2 antibodies could be the result of gluten-induced cellular damage with concomitant release of intracellular TG2 leading to increased crosslinking of dietary gliadin to other molecules, including TG2 itself, and the possible formation of antigenic neoepitopes [80]. Shortly after, a model based on the hapten-carrier principle was proposed stating that gliadin peptides could serve as a carrier when bound in a complex with TG2 [113]. This would allow TG2-specific B cells to take up TG2–gliadin complexes through B-cell receptor (BCR)-mediated endocytosis followed by endosomal processing and presentation of gliadin peptides on MHC class II molecules on the cell surface, ultimately resulting in TG2specific B cells receiving activation signals from gliadinspecific CD4+ T cells (Fig. 1). This model nicely explains why anti-TG2 autoantibodies disappear when gluten is removed from the diet. In addition, the strict HLA dependence of the antibody response strongly implies the involvement of T cells. Accordingly, in a recent screening study on Swedish children, TG2-specific autoantibodies were only detected in individuals having the specific HLA types associated with CD [114]. According to a different theory, TG2-specific autoantibodies can be a result of molecular mimicry as it has been reported that a subset of CD antibodies targeting deamidated gliadin peptides also recognizes TG2 [115]. Through crossreactivity, an initial antibody response against gliadin might therefore spread to include anti-TG2 antibodies. The ability of TG2 to efficiently crosslink gliadin peptides to itself has been demonstrated in vitro, thereby supporting the hapten-carrier mechanism [116]. In line with this model, the TG2-mediated crosslinking of gliadin peptides to other proteins could also explain the production of antibodies targeting other autoantigens than TG2. Hence, it has been shown that TG2 can crosslink gliadin peptides to collagens [117]. Recently, the hapten-carrier model was demonstrated more directly as it was shown that a retrovirally transduced murine B-cell line expressing a human TG2specific BCR and HLA-DQ2.5 can take up preformed TG2–

gliadin complexes and subsequently present peptides to gliadin-specific T cells [105]. These findings strongly support the hapten-carrier model as a valid explanation for the generation of TG2-specific autoantibodies. The model does not explain the low number of mutations found in TG2-specific plasma cells or the preference for the VH5 gene segment, though. However, the generated panel of human TG2specific monoclonal antibodies might provide some hints to explain these findings. Based on the observation that none of the antibodies inhibits enzymatic activity of TG2, it was speculated that crosslinking mediated by BCR-bound TG2 could be directly involved in B-cell activation. Interestingly, it was found that TG2 catalyzes the covalent crosslinking of antibodies of the IgD and IgM but not the IgA class [105]. As BCR crosslinking is known to stimulate B-cell activation, this could explain the activation of naïve cells, which express surface IgD and IgM, but the stimulatory effect would be lost when the B cell has switched to IgA expression. Isotype switching could therefore terminate the accumulation of somatic mutations at an early point in plasma cell differentiation. Pursuing this hypothesis, one can speculate that the VH5 preference is a result of VH5 antibodies being more prone to this type of crosslinking than antibodies belonging to other VH families. Thus, VH5 antibodies might tend to bind TG2 in a way that favors crosslinking. In support of this view, almost all VH5 antibodies recognize the same epitope on TG2 whereas antibodies of other VH families target other parts of the TG2 surface (Di Niro et al., unpublished). TG2 binding by VH5 antibodies could therefore orient the enzyme in a way that is optimal for crosslinking. Apart from providing an explanation for the gluten-dependent production of TG2specific autoantibodies, the hapten-carrier model hypothesizes that TG2-specific B cells can play a role as APCs for gliadinspecific T cells. Given the early presence of anti-TG2 autoantibodies before the appearance of histological changes in the gut [86], it is possible that an amplification of the gliadin-directed T-cell response by involvement of TG2-specific B cells is required for the immune response to reach a magnitude where it causes tissue destruction. Thus, although there is no clear evidence that anti-TG2 autoantibodies are pathogenic, TG2specific B cells might still be indirectly responsible for driving the pathogenesis.

Antigen-presenting cells in celiac disease The adaptive immune response is initiated by APCs, primarily dendritic cells (DCs) but also macrophages and B cell subsets, which present to T-cell antigenic fragments in complex with cell surface MHC class II molecules. This antigen priming usually takes place in organized lymphoid tissues. APCs travel from peripheral tissues to lymph nodes where they generate effector or tolerogenic T cells that

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Fig. 1 Model to explain the gluten-dependent production of TG2specific antibodies. 1 TG2–gliadin complexes are taken up by a TG2-specific B cell through BCR-mediated endocytosis. Uptake of complexes can happen if active BCR-bound TG2 binds gliadin peptides in the active site or gliadin peptides are crosslinked to TG2 through the enzyme’s transamidation activity. 2 After lysosomal

processing, gliadin peptides are presented on the B-cell surface bound to HLA-DQ2.5 (or HLA-DQ8) and recognized by a gliadin-specific CD4+ T cell. 3 After receiving activation help from the T cell, the B cell differentiates into a plasma cell and starts secreting TG2-specific autoantibodies

operate in the peripheral tissues. The re-activation of effector and tolerogenic T cells in peripheral tissues will again require interaction with APCs. The instruction that determines which type of cell the naïve T cell will differentiate into, in particular whether it shall become an effector or regulatory T cell, is provided by the APCs which sense relevant signals in the tissue. In the gut, by default, regulatory T cells are generated in order to maintain oral tolerance to food antigens [118]. Antigen presentation is a key initial step during the pathogenesis of CD both in mesenteric lymph nodes and the intestinal lamina propria. Direct assessment of human APCs in situ has however been difficult, since the draining mesenteric lymph nodes are hardly accessible. Most data on antigen presentation in CD come therefore from studies of APCs in intestinal biopsies, which we will discuss in light of functional studies in mice. In particular, we try to address which APCs drive priming and reactivation of the antigluten T-cell response, and where do the various APCs operate. In the healthy duodenum, macrophages and DCs constitute the main populations of APCs, whereas B cells are only found occasionally. The classical markers for macrophages and DCs are CD163 and CD11c, respectively. Roughly, 83 % of APCs, i.e., cells that express HLA-DQ, in normal human duodenum are CD163+ and approximately 24 % are CD11c+. In addition to the classical macrophages and DCs that are single positive for one of these two markers only, there is also a considerable cell population (about 7 % of all

HLA-DQ+APCs) with an intermediate phenotype (intermediate DCs) that express both markers [119, 120]. The same subpopulations are present in the celiac lesion, but the density and phenotype of APCs are characteristically altered as discussed below. Tissue macrophages Macrophages are tissue-resident cells that differentiate from circulating monocytes [121, 122]. These cells are long-lived with low turnover in the tissue. In the human skin, for example, only 46 % of macrophages were replaced by donor cells 100 days after sex-mismatched bone marrow transplantation [123]. Macrophages in the small intestine occupy a cellular zone beneath the enterocytes and they are also found scattered in the lamina propria. Human jejunal macrophages were reported to have a decreased capacity to produce inflammatory cytokines and do not express receptors typical for innate immune responses, such as the lipopolysaccharide (LPS) co-receptor CD14 or IgA and IgG receptors [124], suggesting that these cells do not mediate mucosal inflammation in the steady state. Indeed, when stimulated with LPS, an obligate inducer of innate immune response, intestinal macrophages did not release detectable levels of the inflammatory cytokines IL-1, IL-6, or TNF-α [124]. Still, human intestinal macrophages express the pattern recognition receptors TLR3-9, have potent phagocytic activity, and

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role in the pathogenesis of CD, for example as producers of cytokines.

effectively eliminate phagocytosed microbes [125, 126]. Similarly, macrophages residing in the murine lamina propria spontaneously produce IL-10 and were shown to be important in the maintenance of functional regulatory T cells (Tregs), suggesting a key regulatory function in intestinal homeostasis [127, 128]. Phenotypic markers of human and murine intestinal macrophages match fairly well. These cells express CD68, CD163, and DC-SIGN (Fig. 2). Expression of CD14, a classical monocyte marker, by these cells was observed in the normal human duodenum [120], but not in normal human jejunum [125]. This discrepancy may relate to differential expression in the proximal and distal small intestine, or alternatively to the different fine specificities of the anti-CD14 antibodies that were used in these studies. In the celiac lesion, macrophages that are marked by CD163+CD11c− phenotype have a decreased density, although many of them are activated and express CD25 [129]. The role of activated macrophages is elusive. The cells were not found to be effective presenters of gluten peptides to gluten reactive T-cell clones [119]. Nonetheless, activated macrophages may still play an important

In contrast to tissue macrophages, DCs are messenger cells with a rapid turnover. In patients receiving sex-mismatched bone marrow grafts, nearly all skin DCs were replaced within 40 days after transplantation [123]. The two main subgroups of DCs, myeloid and plasmacytoid DCs, are characterized respectively by their abilities to prime naïve T cells and to secrete large amounts of IFN-α in response to viral infections. Plasmacytoid DCs residing in the mesenteric lymph nodes of mice were, in addition, shown to induce mucosal T-cell-independent IgA synthesis through the cytokines APRIL and BAFF [130]. Plasmacytoid DCs probably do not migrate in intestinal or hepatic lymph, distinguishing these cells from myeloid DCs that carry intestinal antigens to mesenteric lymph nodes [131]. The immunophenotype of intestinal DCs is complex. In mice, DCs are divided into functionally and ontogenically distinct subpopulations based on the expression of CD11c,

Fig. 2 Putative relationship between antigen-presenting cell subsets in blood and in the small intestine (courtesy of Ann-Christin Røberg Beitnes). Arrows indicate migration to the tissue with putative ways of differentiation. Myeloid DCs enter the duodenum and some upregulate CD103. CD1c−CD103+ DC may derive from CD1c+ DC by downregulation of CD1c or from another DC subset in blood. Classical monocytes migrate to the tissue and probably differentiate into

intermediate monocyte-derived DCs and macrophages. It is uncertain whether macrophages can redifferentiate to intermediate DCs upon inflammation. The contribution of non-classical monocytes to APCs in the small intestine—especially to monocyte-derived DCs—is uncertain. Very few plasmacytoid DCs migrate to the small intestinal mucosa, both in steady state and celiac disease. Mo-DC monocyte-derived DCs

Classical dendritic cells

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CD103, and CX3CR1. In human duodenum, about 50 % of CD11c+ DCs express CD103 [120], but there are no reports on expression of CX3CR1. The αE integrin CD103 is believed to characterize DCs that migrate to the lymph nodes in order to activate T cells. Indeed, murine CD103+ DCs were shown to induce gut-homing receptors on responding T cells and differentiation of FoxP3+ regulatory T cells in vitro. Induction of regulatory T cells requires TGF-β and conversion of retinal to retinoic acid by CD103+ DCs, which were shown to express high levels of aldh1a2, the key enzyme metabolizing retinal acid to retinoic acid [132, 133]. The function of CD103 is unknown. Indeed, this molecule seems to be merely a marker for tolerogenic DC, as DCs isolated from mesenteric lymph nodes of wild-type and CD103 knock-out mice induce similar T-cell responses. Human CD103+ DCs are also readily detected in the lymph nodes and, similar to their murine counterparts, display a more mature phenotype than their CD103− counterparts, assessed by expression of maturation markers CD40 and CD83 [132]. In addition, human DCs expressing CD103 also preferentially induce Treg cells compared to their CD103− counterparts [134]. Together, these findings demonstrate a remarkable cross-species preservation of phenotypic and functional properties of CD103+ DCs in the mesenteric lymph nodes of mice and men. In the normal duodenal mucosa, a subset of CD11c+ DCs also expresses CD1c, a glycoprotein that mediates presentation of lipid antigens and is present on about 80 % of peripheral blood myeloid DCs (Fig. 2). Notably, expression of CD1c and CD103 overlaps but CD14 is virtually absent on CD103+ and CD1c+ dendritic cells (Fig. 2), supporting the hypothesis that these cells are not derived from monocytes [120], similar to murine CD103+ DCs that differentiate from committed DC precursors [135]. In the lamina propria of patients with CD, the density of classical myeloid DCs (Fig. 2) is reduced [120]. Lower tissue density may be secondary to inflammation-induced expansion of the tissue volume or may represent genuine cell depletion due to migration to lymph nodes, diminished recruitment, cell death, or a combination of these events. Notably, reduced density of myeloid DCs is already apparent after a 3-day gluten provocation, before any change in tissue architecture, suggesting that these cells are partially depleted from the lamina propria of celiac patients with active disease [136]. Since activated myeloid DCs of mice migrate to mesenteric lymph nodes, the decreased intestinal density of myeloid DCs in CD may be partly caused by migration of these cells to the lymph nodes. Intermediate dendritic cells In addition to the well-known resident macrophages and classical myeloid DCs, there is an additional subset of APCs in the normal duodenum [120]. These cells feature an

intermediate phenotype and express markers typical of both macrophages and DCs, CD163, and CD11c, respectively. Similar intermediate populations of APCs are also described in human skin [137] and colon [138, 139]. In mice, intermediate DCs appear to originate from monocytes rather than DC precursors and they are believed to be tissue-resident cells important in modifying local immune responses. In murine gut, these cells express the chemokine receptor CX3CR1, which they do not in the human intestine. Notably, murine monocyte-derived DCs can be distinguished from conventional DCs by the expression of the lectin-receptor DC-SIGN, and both cell types showed equal ability to activate T cells [140]. In the human healthy duodenal mucosa, about 65 % of CD163+CD11c+ APCs, i.e., the intermediate DCs, express the monocyte marker CD14, which may indicate that these cells stem from monocytes [120]. In addition, similar to murine monocyte-derived DCs, CD14+CD11c+ DCs in the duodenal mucosa also express DC-SIGN/CD209. Furthermore, CCR2, a chemokine receptor that was shown to mediate monocyte recruitment into injured tissues, is also expressed by these cells. In the celiac lesion, there are DCs that express CD11c and co-express the activation markers CD86 and/or DC-LAMP [119]. Such cells, which would be classical or intermediate DCs, were found to be effective stimulators of gluten-reactive T cells [119]. Intermediate cells of CD163+CD11c+ phenotype have a significantly increased density in the celiac lesion. Several lines of evidence suggest that these cells are directly involved in the immunopathology of CD. Indeed, unlike classical DCs and macrophages that show decreased tissue concentration, the density of these intermediate DCs increases after a 3-day gluten challenge of celiac patients. Furthermore, increased density of intermediate DCs precedes the typical inflammation-related architectural changes of the intestine as well as the surge of intraepithelial lymphocytes and eosinophils, which suggests that intermediate cells may take part in disease initiation [136]. Plasmacytoid dendritic cells In humans, plasmacytoid DCs (PDCs) typically reside in lymphoid tissues and rarely infiltrate inflamed tissues. Although there are reports on plasmacytoid DCs recruited to skin and airways [141–143], there is little evidence for recruitment to human intestinal lamina propria [144, 145]. The abundance of PDCs in the human duodenum is debated. Ráki et al. report that PDCs is a minor cell population in the normal duodenal mucosa, representing about 1 % of all APCs in the lamina propria with no increase in the celiac lesion (Ráki et al., submitted). This is in contrast to the findings of Di Sabatino et al. who reported that PDCs, defined by CD123 expression in flow cytometry, are the main APC in the small intestine [146]. However, cells other

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than PDCs may also express CD123. Among APCs, some macrophages express low levels of CD123 detectable by flow cytometry, and the CD123-bright cells detected in immunohistochemistry by Di Sabatino et al. could be capillary endothelial cells since these strongly express CD123. Notably, PDCs are scarce in the lamina propria of mice [147, 148]. Plasmacytoid DCs are large producers of IFN-α, and as treatment with IFN-α is reported to induce development of clinically overt CD, this has been taken to support roles for both IFN-α and PDCs in the pathogenesis of CD. However, scrutinizing the case reports on IFN-α-induced CD, it is hard to conclude whether IFN-α therapy initiated the disease or just aggravated the symptoms, since pre-treatment serum samples were either not available or they were scored positive for endomysial antibodies [149–152]. Furthermore, most cells can produce IFN-α, albeit at much lower levels than PDCs and production by such cells can account for the modest production of IFN-α in intestinal biopsies of untreated CD patients. If PDCs play a role in the initiation of CD, we believe that this is more likely to take place in mesenteric lymph nodes than in the lamina propria. B cells B cells are also professional APCs and efficiently present antigens taken up by receptor-mediated endocytosis via the BCR. There are very few B cells in the lamina propria of naïve or memory phenotypes, and most of the cells in the B-cell lineage in the lamina propria are plasmablasts or plasma cells with scarce expression of HLA class II molecules [153]. B cells are probably more important as APCs in the mesenteric lymph nodes where they may activate naïve and central memory T cells. Origin of small intestinal antigen-presenting cells Precursors of DCs have not yet been described in the human blood, but three distinct cell subsets may generate tissue DCs: monocytes, classical myeloid DCs, and plasmacytoid DCs (Fig. 2). These subsets can be distinguished by their distinct phenotypes. Classical peripheral blood monocytes express high levels of CD11c, CD163, and CD14. Myeloid DCs also express high levels of CD11c, whereas plasmacytoid DCs are CD11c negative (Fig. 2). The majority of myeloid DCs in blood express CD1c (Fig. 2). In peripheral blood, plasmacytoid DCs display a unique phenotype of CD11c−CD123+CD45RA+. Little is known, however, about the phenotypes of APCs in tissues and their correlates in the blood. The main reason for this is the technical difficulty to obtain sufficient material from tissues, the heterogeneity of resident cells, and differentiation of cells upon entering the tissues.

Subpopulations of intestinal APCs in CD may be replenished by peripheral monocytes. Because most circulating monocytes do not express HLA-DQ, the finding that the density of CD14+DQ−cells is increased in the celiac lesion supports the monocytic origin of CD163+CD11c+CD14+ cells which are the intermediate DCs that are mostly increased in active CD lesions [120]. Indeed, monocytes are known to be attracted by intestinal stroma-derived TGF-β, IL-8, and MCP-1 [121], which are upregulated in intestinal biopsies of CD patients, but not controls, after 24 h of rectal gluten challenge [154]. There is also evidence that gliadin peptides may induce phenotypic and functional maturation of monocyte-derived DCs and activate monocytes from CD patients [155, 156]. Roles of different antigen-presenting cells in celiac disease As the initiation of mucosal effector and tolerogenic T-cell responses takes place in organized lymphoid tissues, such as the mesenteric lymph nodes in the gut, antigen transport and DC migration are necessary for T-cell response. Interestingly, it seems that distinct subtypes of APCs migrate to the lymph nodes and activate naïve T cells or stimulate effector T cells locally in the mucosa. In mice, only CD103+ DCs express CCR7 and migrate to the lymph nodes, whereas CXCR3+ APCs, the intermediate subset of monocyte-derived DCs and macrophages, probably modulate local adaptive immune responses [135, 157]. It was proposed that CD103+ DCs might be inherently tolerogenic and a distinct population of CD103− APCs drive effector T-cell responses [158, 159]. Alternatively, CD103+ DCs may adapt to the altered environment and acquire the ability to produce proinflammatory cytokines and imprint gut-homing effector T cells. Supporting this latter hypothesis, in a murine model of inflammatory bowel disease, CD103+ DCs in the mesenteric lymph nodes had lost tolerogenic properties upon intestinal inflammation [160], although it was not formally proven in that study that these cells migrated from the lamina propria. Consistent with the idea that the function of DCs is conditioned by the microenvironment, it was recently shown that retinoic acid also may drive inflammation rather than tolerance depending on additional signals. In a recent study, Jabri and colleagues established a new mouse model of CD in which mice were engineered to constitutively express IL-15 in the intestinal mucosa and showed that combined with high IL-15 levels, retinoic acid acts on DCs to promote an inflammatory immune response and not tolerance [161]. Taken together, the function of mucosal DCs is conditioned by the local environment, especially intestinal epithelial cells. Human intestinal epithelial cells release thymic stromal lymphopoietin, which inhibits IL-12 production by DCs [162]. Enterocytes and cells of the lamina propria also release TGF-β

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and retinoic acid, factors that imprint DCs and macrophages to a tolerogenic state [134]. During inflammation, newly recruited cells that are not tolerized by the intestinal microenvironment may migrate to lymph nodes and initiate effector T-cell responses. It remains to be established which populations of mononuclear phagocytes influence differentiation of T cells towards the Th0/Th1 phenotype that is typical for glutenreactive CD4+ T cells in CD, and whether IL-12 production or other cytokines mediate this process. However, based on phenotypical observations of APCs in intestinal biopsies of CD patients shortly after gluten challenge, a scenario where the inflammatory response in lamina propria in CD is characterized by depletion of myeloid DCs and an influx of blood monocytes that differentiate into intermediate DCs can be envisaged. In the same scenario, the priming of naïve gluten T cells into effector T cells is done by myeloid DCs that travel from the tissue to mesenteric lymph nodes carrying gluten antigens as well as inflammatory signals, and the reactivation of memory T cells in the small intestinal mucosa is mainly mediated by intermediate DCs. B cells likely play an important role as APCs in the mesenteric lymph nodes for the amplification of gluten T-cell response. The TG2-specific B cells will preferentially stimulate T cells specific for deamidated gluten peptides. B cells specific for deamidated gluten peptides may receive T-cell help from the same T cells specific for deamidated peptides. This may explain why antibodies to deamidated gluten peptides are so good predictors of CD.

Perspectives With a known causative antigen that can be removed and the possibility to study diseased tissues in addition to peripheral blood, CD has proven to be a valuable model for studying autoimmune and chronic inflammatory diseases in humans. Understanding the pathogenesis of CD has given us insights that may be useful in unraveling the mechanism for autoimmune diseases in general. From the highly complex gluten protein, T-cell epitopes are generated as a result of luminal digestion, specific posttranslational modification by TG2 and binding to HLA-DQ2.5 or HLA-DQ8 molecules. Sustained binding to the HLA molecule is required so that the peptide–MHC complex can survive the journey from the tissue to the draining lymph node where presentation to naïve T cells may take place. The mechanisms explaining why naïve T cells responding to gluten develop into effector T cells and not regulatory T cells in patients with CD have not been worked out completely. Evidence supports the hapten-carrier model as an explanation to why the antibody response to TG2 is dependent on gluten exposure, but the final proof remains. This link between an autoimmune

response and exposure to an exogenous antigen may represent a mechanism for autoimmunity beyond CD. Posttranslational modification of exogenous antigen by an endogenous enzyme may be the link to anti-enzyme or anti-modified autoantigen antibodies found in several autoimmune diseases such as the anti-GAD65 antibodies in diabetes and anti-citrulline antibodies in rheumatic arthritis. Finally, we hope that one day our knowledge on CD mechanism will benefit CD patients in forms of novel therapeutics supplementing or even replacing the gluten-free diet. Acknowledgments The work in the authors’ laboratory is supported by grants from the Research Council of Norway, the South-Eastern Norway Regional Health Authority, the Norwegian ExtraFoundation for Health and Rehabilitation (EXTRA funds), the JDRF, and by various grants from the European Commission (MRTN-CT-2006036032, FOOD-CT-2006-36383, and ERC-2010-AdG-268541).

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