Celiac Disease: An Immunological Jigsaw

Immunity

Review Celiac Disease: An Immunological Jigsaw Bertrand Meresse,1,2,* Georgia Malamut,1,2,3 and Nadine Cerf-Bensussan1,2,* 1INSERM,

U989, 75015 Paris, France Paris Descartes, Paris Sorbonne Centre, Institut IMAGINE, 75015 Paris, France 3AP-HP, Department of Gastroenterology, Ho ˆ pital Europe´en Georges Pompidou, 75015 Paris, France *Correspondence: [email protected] (B.M.), [email protected] (N.C.-B.) DOI 10.1016/j.immuni.2012.06.006 2Universite ´

Celiac disease (CD) is a chronic enteropathy induced by dietary gluten in genetically predisposed people. The keystone of CD pathogenesis is an adaptive immune response orchestrated by the interplay between gluten and MHC class II HLA-DQ2 and DQ8 molecules. Yet, other factors that impair immunoregulatory mechanisms and/or activate the large population of intestinal intraepithelial lymphocytes (IEL) are indispensable for driving tissue damage. Herein, we summarize our current understanding of the mechanisms and consequences of the undesirable immune response initiated by gluten peptides. We show that CD is a model disease to decipher the role of MHC class II molecules in human immunopathology, to analyze the mechanisms that link tolerance to food proteins and autoimmunity, and to investigate how chronic activation of IEL can lead to T cell lymphomagenesis. Introduction Celiac disease (CD) is defined as a chronic small intestinal immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals (Ludvigsson et al., 2012). The term celiac (‘‘hollow’’ in Greek) referred to its digestive origin and was coined by the Greek physician Aretaeus, who describes the disease circa 100 AD in Roma. The link with the diet was definitively established in the 1950s, when W. Dicke, a Dutch pediatrician, demonstrated the causal role of grain storage proteins from wheat, barley, and rye (collectively called gluten), and proposed to treat patients by a long-life gluten-free diet (GFD) (Van de Kamer et al., 1953). Our present view of CD pathogenesis derives from several landmark findings. Major steps have been the recognition of HLA-DQ2 and DQ8 molecules as the main genetic predisposing factor and the demonstration of their role in orchestrating an adaptive immune response against gluten peptides (reviewed in Sollid, 2002). It is however now clear that this response, although necessary, is not sufficient and that other factors that impair immunoregulatory mechanisms and/or activate the large population of intestinal intraepithelial lymphocytes (IELs) are indispensable for driving tissue damage. Herein, we consider whether and how genetic and immunological studies can be integrated into a comprehensive plot and discuss possible clues for filling remaining gaps in our understanding of this complex disorder. Lessons from the Bedside The high prevalence and broad clinical spectrum of CD has been progressively uncovered since the 1970s after the demonstration that CD is specifically associated with serum autoantibodies against a tissue antigen (Seah et al., 1971), later identified with the ubiquitous enzyme tissue transglutaminase 2 (TG2) (Dieterich et al., 1997). TG2 antibodies are detected in 0.3%– 2% of individuals in Europe and in the US, and comparable prevalence rates are observed in a growing number of countries in which gluten is an important component of the diet (Aggarwal et al., 2012; Lionetti and Catassi, 2011). Positive serology is more particularly frequent in first-degree relatives, a finding consistent

with the importance of genetic predisposing factors (see below) and in patients with autoimmune diseases notably with type 1 diabetes (T1D) and thyroiditis (Aggarwal et al., 2012). Indeed, there is a strong link between CD and autoimmunity given that 5%–10% of patients with T1D develop CD, whereas, 15%– 20% of CD patients have or will develop autoimmune diseases (Cosnes et al., 2008). In individuals with positive serology, the diagnosis of CD is confirmed by duodenal biopsies that demonstrate the typical small intestinal lesions combining villous flattening, crypt hyperplasia, and increased numbers of IELs. Histological lesions and symptoms are however very variable and three situations are now defined (Ludvigsson et al., 2012). The first is symptomatic CD that can develop at all ages of life, with two peaks, in early childhood and in young women. Most symptoms can be ascribed to impaired absorption of nutrients. Several extraintestinal manifestations are less well understood and may be the consequence of the abnormal immune response initiated by gluten peptides (see below). A second condition, defined as silent CD, refers to the many individuals with positive serology and typical intestinal histology but no symptoms. The risk is the development of complications, notably autoimmune (Cosnes et al., 2008) or more rarely malignant (Elfstro¨m et al., 2011). Finally, a smaller group of asymptomatic individuals have serum TG2 antibodies but normal intestinal histology. Because some but not all can later develop epithelial lesions, this third situation is defined as latent or potential CD. In potential CD, there are often greater numbers of IELs and more particularly of TCRgd+ IELs, and TG2 antibodies can be detected in situ (Koskinen et al., 2008; Ludvigsson et al., 2012). Latent CD illustrates that the intestinal immune response revealed by the production of antibodies does not necessarily lead to intestinal tissue damage and may be restrained by regulatory mechanisms. Most CD patients are relieved by a life-long GFD, which interrupts the intestinal immune response triggered by gluten and allows recovery of a normal villous architecture. However, a few patients develop resistance to GFD. In so-called type I refractory celiac disease (RCD), immunological findings in the Immunity 36, June 29, 2012 ª2012 Elsevier Inc. 907

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Review Figure 1. Human Leukocyte Antigen Class II Associations with Celiac Disease and Type 1 Diabetes The majority of CD patients express the HLADQ2.5 heterodimer encoded by the HLA-DQA1*05 (a-chain) and HLA-DQB1*02 (b-chain) alleles. These two alleles are carried either in cis on the DR3-DQ2.5 haplotype or in trans in individuals who are DR5DQ7 and DR7DQ2 heterozygous. HLA-DQ8 that is encoded by the DR4DQ8 haplotype confers a lesser risk of CD. In individuals who are DR3DQ2.5 and DR4DQ8 heterozygous, transdimers DQ8.5 can form and confer high susceptibility to TID.

intestine, and notably the phenotype of IELs, resemble those observed in uncomplicated active CD. One plausible hypothesis is that the immunological reaction initiated by gluten has evolved toward autoimmunity. Accordingly, symptoms improve under immunosuppressive treatments. In contrast, in type II RCD, the normal population of CD3+ IELs is progressively replaced by clonal IELs with an abnormal phenotype and RCDII is now considered as an intraepithelial lymphoma. RCDII is the frequent first step toward the development of overt T lymphomas, the rare but most severe complication of CD (Malamut et al., 2009). Overall, clinical observations in CD reveal a striking overlap between CD and bona fide autoimmune diseases. Severity and age at onset of symptomatic CD are highly variable and might be influenced by life events that alter local immune regulation. IEL hyperplasia, a hallmark of CD and the origin of T lymphomas, points to severe impairment of IEL homeostasis in CD. The Keystone of CD: The Interplay between Gluten and MHC Class II HLA-DQ2 or -DQ8 Molecules Following studies highlighting a strong association between CD and the HLA complex, it was established in the late 1980s 908 Immunity 36, June 29, 2012 ª2012 Elsevier Inc.

that genetic susceptibility to CD is determined mainly by the HLA-DQ locus (Sollid et al., 1989). The strongest association is observed with HLA-DQ2.5 (Fallang et al., 2009). Thus, more than 90% of CD patients possess one or two copies of HLA-DQ2.5 encoded by HLA-DQA1*05 (for the a chain) and HLA-DQB1*02 (for the b chain). In individuals who carry the DR3DQ2 haplotype, this molecule is encoded by DQA1 and DQB1 alleles located on the same chromosome (cis configuration) whereas in individuals who are DR5DQ7 or DR7DQ2 heterozygous, it is encoded by alleles located on opposite chromosomes (trans configuration) (reviewed in Sollid et al., 2012; Figure 1). The resulting HLA-DQ2.5 heterodimers differ only by two amino acids, which do not influence their functional properties. In contrast, the highly homologous HLA-DQ2.2 molecule confers a much lesser risk due to the replacement of a tyrosine by a phenylalanine residue in DQa at position 22 that is important for peptide binding (Bodd et al., 2012; Fallang et al., 2009). Most of the remaining patients carry DR4DQ8 haplotypes and express a DQ8 molecule encoded by DQA1*03DQB1*03:02 (Sollid et al., 2012). Strikingly, individuals who are heterozygous for DQ2.5 (DQA1*05:01 and DQB1*02:01) or DQ8 (DQA1*03:01 and DQB1*03:02) are also predisposed to T1D with an almost five-fold higher risk than those who are homozygous for either of the DQ variants (Concannon et al., 2009). This high risk was recently associated with the formation of HLA-DQ8 transdimers between the a-chain of HLADQ2 (DQA1*05:01) and the b-chain of HLA-DQ8 (DQB1*03:02) or of HLA-DQ2 transdimers between the a-chain of HLA-DQ8 (DQA1*03:01) and the b-chain of HLA-DQ2 (DQB1*02:01) (Tollefsen et al., 2012; van Lummel et al., 2012) (Figure 1). In CD, the link between the main environmental factor, gluten, and the main genetic predisposing factor, the HLA-DQ2.5 molecule, was first disclosed by a seminal work demonstrating the presence of HLA-DQ2-restricted gluten-specific CD4+ T cells in the intestinal mucosal of CD patients (Lundin et al., 1993). The molecular bases of the interactions between gluten-derived peptides and HLA-DQ2.5/8 molecules are now well established

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Review Figure 2. Activation of Gluten-Specific CD4+ T Cell Responses by HLA-DQ2.5 Molecule Transglutaminase 2 (TG2) binds to and deamidates glutamine residues in Q-X-P sequences of gluten peptides into glutamic acid, introducing a negative charge that can interact with a positively charged lysine residue in position 6 of the peptide pocket of HLA-DQ2.5, resulting in enhanced peptide avidity for HLA-DQ2.5. HLA-DQ2.5-gluten peptide complexes expressed on antigen-presenting cells (APCs) can prime gluten-specific CD4 T cells. As for other dietary antigens, priming may occur in Peyer’s patches or in mesenteric lymph nodes after migration of CD103+ dendritic cells loaded with gluten peptides in lamina propria (Worbs et al., 2006). Unusual priming outside the gut has however been reported in HLA-DQ2 mice (Du Pre´ et al., 2011). Priming is followed by selection and clonal expansion of T cells diplaying high-avidity TCR (Qiao et al., 2011).

and depend on the physicochemical properties of the two partners. HLA-DQ2.5 and HLA-DQ8 have positively charged pockets, which preferentially bind peptides with negatively charged residues at anchor positions P4, P6, and P7 for HLADQ2 and P1, P4, and P9 for HLA-DQ8 (reviewed in Abadie et al., 2011). One common feature of gluten proteins immunogenic for CD patients is their high content in glutamine (Q) and proline (P) residues assembled in related repetitive sequences. Proline-rich peptides are particularly resistant to proteolysis by digestive enzymes, so that large immunogenic peptides remain undigested in the intestinal lumen and may come into contact with intestinal immune cells (Shan et al., 2002). Peptides containing glutamine (Q)-proline (P) rich motifs (notably the Q-X-P sequence) are excellent substrates for TG2. This multifunctional

enzyme is abundantly expressed in many tissues and with both intra- and extracellular localization and can convert neutral glutamine residues within Q-X-P sequences into negatively charged glutamate residues, thereby strongly increasing peptide avidity for HLA-DQ2.5 (reviewed in Abadie et al., 2011; Figure 2) or for HLA-DQ8 (Henderson et al., 2007). Finally, proline residues introduce structural constraints that are compatible with peptide binding to HLA-DQ2/8 molecules but not to other MHC class II molecules (Bergseng et al., 2005). As a consequence, HLA-DQ2/8+ dendritic cells can electively activate gluten-specific CD4+ T cells (Figure 2). Several gluten epitopes preferentially recognized in the context of either HLA-DQ2.5 or HLA-DQ8 have been defined (Sollid et al., 2012). Such epitopes may in turn drive the selection of T cells harboring a T cell receptor (TCR) of high avidity. Thus, a recent study showed clonal expansion, convergent recombination, and semipublic response of T cells recognizing the dominant gluten epitope DQ2.5-glia-a2. Reactive T cells preferentially used a TCR Vb6.7 chain with a conserved nongermline-encoded arginine residue in the CDR3b loop. This positively charged residue probably interacts with the negatively charged deamidated residue of DQ2.5-glia-a2 that binds to the HLA-DQ2.5 pocket in position P4 (Qiao et al., 2011). Further supporting the importance of HLA-DQ-restricted anti-gluten CD4+ T cell response in CD onset, correlations have been established between the richness in motifs predicted to be substrates for TG2 and to bind HLA-DQ molecules and the ‘‘toxicity’’ of individual cereal proteins (Vader et al., 2002). Conversely, that HLA-DQ8 and HLA-DQ2.2 bind a lesser number of gluten peptides than HLA-DQ2.5 and/or with a lesser avidity is Immunity 36, June 29, 2012 ª2012 Elsevier Inc. 909

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Review thought to explain the lesser risk of CD associated with these molecules (Bodd et al., 2012; Fallang et al., 2009). Finally, disease susceptibility has been associated with a dosage effect and DR3DQ2 homozygous individuals who express more at-risk molecules on antigen-presenting cells (APCs) have the highest risk for CD (Vader et al., 2003). Interestingly, recent observations might explain the high risk of HLA-DQ2-DQ8 heterozygous individuals for TID. Indeed the HLA-DQ8 transdimer can not only present a significant repertoire of gluten peptides but it can also bind with high avidity a much larger repertoire of peptides derived from the diabetogenic proteins GAD65, IA-2 and preproinsulin than the cis HLA-DQ8 dimer (Kooy-Winkelaar et al., 2011; van Lummel et al., 2012). Overall these data show how the main genetic risk factor for CD, the HLA-class II molecules DQ2 and DQ8, can orchestrate the activation of lamina propria CD4+ T cells against dietary gluten. They also provide evidence of the strong similarities between the mechanisms governing the recognition of exogenous gluten in CD and of pancreatic autoantigens in T1D. Loss of Tolerance and Tissue Damage in CD: A Need for Additional Environmental and Genetic Factors Only a small fraction of individuals with at-risk HLA and who are exposed to gluten develop CD, indicating that both factors, although necessary, are not sufficient for developing CD. This common observation was recently extended into a worldwide comparison of the prevalence of CD between countries depending on the frequencies of DR3DQ2 and DR4DQ8 haplotypes and on the amount of wheat consumption. Many but not all countries could be fitted into a correlation curve, and CD prevalence was notably much higher in Finland and Mexico or, on the contrary, much lower in Russia and Tunisia than expected (Abadie et al., 2011). Humanized mouse models further support the notion that an adaptive CD4+ T cell response to gluten is not sufficient to recapitulate CD and induce intestinal tissue damage. No enteropathy has been observed in mice expressing HLA-DQ8 and human CD4 (Black et al., 2002), nor in mice transgenic for HLA-DQ2.5 and a gluten-specific T cell receptor upon gluten feeding (de Kauwe et al., 2009; Du Pre´ et al., 2011). In these models, gluten-responsive T cells produced tumor-growth factor b (TGF-b) or Interleukin-10 (IL-10), reflecting the induction of immunoregulatory mechanisms (Black et al., 2002; Du Pre´ et al., 2011). Crossing HLA-DQ8 mice on a NOD background to promote autoimmunity resulted in the appearance of TG2 autoantibodies and of a skin disease reminiscent of dermatitis herpetiformis, but the mice did not develop intestinal lesions (Marietta et al., 2004). Therefore, complementary genetic and or environmental factors must influence CD penetrance and are likely necessary to break intestinal tolerance to gluten and induce intestinal tissue damage. Genetic Clues to Celiac Disease from Outside the HLA Locus In CD, the sibling recurrence risk of 10% and the concordance of 75%–80% between monozygotic twins stress the importance of genetic predisposition. Comparison between siblings also suggests that HLA contributes for no more than 35% of the genetic risk (Bevan et al., 1999; Nistico` et al., 2006). Twentysix non-HLA genomic loci significantly associated with CD, 910 Immunity 36, June 29, 2012 ª2012 Elsevier Inc.

most of which containing immune genes were first identified by genome wide associations studies (GWAS) (Dubois et al., 2010; Hunt et al., 2008). Dense genotyping using a custom Immunochip designed for 183 non-HLA risk loci associated with immune-mediated diseases then demonstrated 57 independent CD association signals in 39 separate non-HLA loci (Trynka et al., 2011). Twenty-nine of the total 54 independent non-HLA signals could be assigned to a single gene. Overall, these signals might explain 13.7% of the genetic variance of CD in individuals of European ancestry (Trynka et al., 2011). Yet, most identified variants are common allelic variants (detected in >5% of individuals in the general population) and each confers only small positive or negative risk with odd ratios (OR) between 0.7 and 1.5. Lower frequency variants were identified at four separate loci (RGS1, CD28-CTLA4-ICOS, SOCS1PRM1-PRM2, and PTPN2) by dense genotyping but OR did not exceed 1.7. One exome sequencing study in a CD family with six affected members also failed in identifying a rare causal variant (Szperl et al., 2011). Despite the now large number of loci associated with CD, causative mutations thus remain to be identified. A meta-analysis of genome-wide data sets for eQTLs (expression quantitative trait loci) mapping has suggested that
50% of CD-associated SNPs might affect the expression of nearby genes (Dubois et al., 2010). Using dense genotyping, the same authors further suggested that several signals are localized either around the transcription start site of specific genes (such as RUNX3, RGS1, ETS1, TAGAP, and ZFP36L1) or in the 30 untranslated region (IRF4, PTPRK, and ICOSLG). They notably observed that one variant present in the 30 UTR of the PTPRK locus is located in a predicted binding site for a microRNA (hsa-miR-1910) (Trynka et al., 2011). Strikingly, only very few CD-associated variants were found within coding sequences. One such interesting variant is a nonsynonymous SNPs in SH2B3 that is associated with other autoimmune (T1D, rhumatoid arthritis) and metabolic disorders. This gene encodes the adaptor protein LNK that influences a variety of signaling pathways, notably those mediated by Janus kinases and receptor tyrosine kinases (Devallie`re and Charreau, 2011). Overall, these data raise the question of the biological significance of the observed associations and of their exact contribution to CD pathogenesis. One interesting hypothesis is that CD-associated immune genes have been positively selected as conferring a benefit to the carriers, notably to resist against infectious diseases. Two studies have used the test of integrated haplotype similarity to detect signature of positive selection for SNPs tagging CD-associated genomic regions. Only three CD-associated regions showed evidence of positive selection: IL-18RAP, IL12A, and SH2B3 (Abadie et al., 2011; Zhernakova et al., 2010). Given that the selective sweep on SH2B3 might have occurred between 1,200 and 1,700 years ago, a possible link was suggested with the Justinian plague in 481–482 AD (Zhernakova et al., 2010). Given the lack of identified causal mutation and the low change in risk conferred by each variant, their individual role in CD pathogenesis remains uneasy to anticipate. A recent comprehensive review has compared variants identified in CD and in other immune-mediated disorders and used functional annotation databases to cluster variants into functional pathways

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Figure 3. Hypothetical Role of Non-HLA CD-Associated Genes in Immune Responses Non-HLA loci associated with CD are enriched in genes predicted to control T cell activation (TCR signaling, antigen presentation, and recruitment differentiation into effector cells) and B cell help. Loci specific for CD are in red. Those shared with other autoimmune diseases (including T1D) are in black. Hypothetical groups of genes that may participate in T cell activation, T cell cytotoxicity, IL-21 production, and IgA response, are shown. The possible interplay between IL-21 and IL-15 in tissue damage is suggested (see also Figure 4 and text).

(Abadie et al., 2011). Strikingly, many loci are shared between CD and autoimmune diseases, and notably 12 overlapping loci were noted with T1D. CD-associated loci appeared more particularly enriched in genes predicted to control chemokine receptor activity, cytokine binding and production, and T and NK cell activation. In Figure 3, we have analyzed how genes associated with CD variants can be integrated into putative immune pathways, on the basis of the role(s) assigned to the corresponding genes in the literature. The obtained scheme illustrates the prominent association of CD with genes controlling T cell activation and recruitment. The association with the IL2IL21 locus, shared with T1D, is particularly interesting. Indeed, IL-21 is strongly increased in intestinal biopsies from active CD (Fina et al., 2008) and is produced in response to gluten peptides by specific CD4+ T cells from CD patients (Bodd et al., 2010). Moreover, CD-associated variants are also found in the IRF4 gene, a transcription factor that is activated in response to TCR stimulation and controls IL-21 production by CD4+ T cells

(Biswas et al., 2010). The potential importance of IL-21 in CD and associated autoimmunity is further suggested by a recent study showing how IL-21-producing CD4+ T cells that express the gut CCR9 homing receptor can target the pancreas of NOD mice and promote CD8-dependent tissue damage (McGuire et al., 2011). CD association with the TLR7-TLR8 region needs to be confirmed but is of potential interest given that the celiac risk variant correlated with changes in TLR8 expression in whole peripheral blood (Dubois et al., 2010). TLR7 and TLR8 are innate receptors for single-stranded viral RNA. They have been associated with susceptibility to hepatitis C (Wang et al., 2011a), which may play a favoring role in CD (see below). The genetic link with innate antiviral responses seems however less strong in CD than in T1D, in which a combination of GWAS and eQTL data has revealed an association with a whole network of antiviral innate immune genes (Foxman and Iwasaki, 2011). In conclusion, the search of genetic factors outside the HLA locus has not yet identified a rare causative variant. Rather, Immunity 36, June 29, 2012 ª2012 Elsevier Inc. 911

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Review a variable combination of common variants also often associated with autoimmune diseases may tune the immune system toward too energetic T cell immune responses. Clues for Infectious Cofactors in CD The role of infectious cofactors is a long-lasting hypothesis in CD that is supported by some epidemiological data. In a Swedish population-based register study comparing perinatal data in controls and in z3,400 infants who were born between the years 1987–1997 and developed CD, one main risk factor for contracting CD was exposure to neonatal infections (odds ratio [OR] = 1.52) (Sandberg-Bennich et al., 2002). Of note the period of study largely coincides with the so-called Swedish CD epidemic (1985–1996) during which CD incidence underwent a 4-fold increase. This epidemic is however usually ascribed to changes in infant feeding habits and notably to the delayed introduction of large amounts of gluten without the protection by breast feeding (Ivarsson, 2005). Two other observations are consistent with the triggering role of a virus. In children, early CD onset has been correlated with serological evidence of repeated rotavirus infection (Stene et al., 2006). In adults, recurrent observations report the development of CD in patients treated with IFN-a for hepatitis C (Bourlie`re et al., 2001). Despite strong suspicion that gastrointestinal or hepatic viruses may participate in CD onset, there has been however no formal demonstration of this (reviewed in Plot and Amital, 2009). In a recent study, viral RNA from enteroviruses was often detected in the intestinal mucosa of T1D but no significant difference was observed between controls and CD patients (Oikarinen et al., 2012), arguing against the hypothesis of a persistent enterovirus as incriminated in T1D or in Crohn’s disease (Foxman and Iwasaki, 2011). The role of transient or repeated intestinal infections such as by rotavirus(es) is however attractive. In mice, rotavirus induced Toll-like receptor 3 (TLR3)dependent production of CCL5 chemokine and of type I IFN-l and recruitment of cytotoxic IELs (Pott et al., 2012). Activation of TLR3 by poly-IC has also been associated in mice with the activation of TG2 and with the production of IL-15 (Dafik et al., 2012) and may thus simultaneously promote the activation of CD4+ LPL and of CD8+ IELs (see below). Recent studies have also considered a possible role for the microbiota. Not surprisingly, some changes in the composition of the fecal and duodenal microbiota have been reported (Nistal et al., 2012). The most original finding is the detection of rodshaped bacteria closely associated with the duodenal mucosa in biopsies of children born during the Swedish CD epidemic but not in more recent cases of CD. The pathogenic role of these bacteria, which belong to three distinc genders, remains unclear (Ou et al., 2009). Bacterial adhesion may be favored by gluteninduced inflammation and induce the activation of some Th17+ cells. Indeed a moderate induction of IL-17 is observed in duodenal biopsies in active CD but IL-17, in contrast with IL-21 and IFN-g, is not produced by gluten-specific CD4+ T cells (Bodd et al., 2010). Overall, the search for additional genetic and environment risk factors has provided interesting hypotheses but no decisive insight into the mechanisms, which convert immune tolerance to gluten into inflammation and tissue damage. We discuss below how to integrate other immunological features of CD into a comprehensive plot. 912 Immunity 36, June 29, 2012 ª2012 Elsevier Inc.

Roles of IgA Antibodies to Gluten Peptides and Tissue Transglutaminases in and Outside the Gut Serum IgA specific for gluten peptides and for the autoantigen TG2 are hallmarks of active CD. They are enriched in polymeric IgA and produced by intestinal plasma cells, the density of which is markedly increased in CD LP (Colombel et al., 1990; Di Niro et al., 2012). A recent study showed that 10% of IgA plasma cells isolated from the intestinal LP of untreated CD produced TG2 antibodies. Despite their extensive characterization through the derivation of a large panel of monoclonal antibodies from single anti-TG2 lamina propria plasma cells (Di Niro et al., 2012), the mechanism that explains their specific production in active CD and their disappearance after a strict GFD remains unclear. Hapten-like recognition of TG2 crosslinked to gluten has been suggested. Indeed, in the presence of acyl acceptors bearing a lysine side chain, modification of glutamine residues by TG2 results in the formation of isopeptide bounds and protein crosslinking and TG2 is known to interact electively with glutenderived peptides. This hypothesis is also compatible with the observation that TG2 antibodies do not inhibit the crosslinking activity of TG2. It remains however to be substantiated (Di Niro et al., 2012). Other pending questions are the exact roles of TG2 and TG2 antibodies in CD pathogenesis. TG2 is a multifunctional enzyme that has been involved in a variety of physiological functions, including matrix assembly and tissue repair, receptor signaling, proliferation, cell motility, and endocytosis. A role of TG2 in CD beyond its established function in deamidation and presentation of gluten peptides to the adaptive immune system is thus suspected although not clearly delineated. In vitro studies have also suggested that TG2 IgA autoantibodies might modulate enterocyte proliferation and differentiation as well as epithelial barrier function (reviewed in Klo¨ck et al., 2012). Yet, the presence of TG2 IgA in the mucosa of latent CD indicates that they are not sufficient to induce intestinal tissue damage (Koskinen et al., 2008). Transglutaminase antibodies may however play a central role in some extraintestinal manifestations of autoimmunity, notably in dermatitis herpetiformis (DH), a gluten-dependent blistering skin disease. Patients with DH have serum antibodies reactive against TG2 but also against TG3, the epidermal version of TG2, due either to cross-reactivity or epitope spreading (Sa´rdy et al., 2002). DH skin lesions do not contain T cells but dermal deposits of IgA and TG3 that are associated with neutrophil infiltrates in blistering areas. Formation of dermal IgA and TG3 deposits are recapitulated in human skin grafted into SCID mice injected with goat-anti-human TG3, or serum from CD or DH patients (Zone et al., 2011). Moreover, a glutendependent blistering disease was observed in gluten-fed HLA-DQ8xNOD mice, which developed circulating TG2 antibodies, dermal IgA deposits, and neutrophil infiltrates (Marietta et al., 2004; Figure 4). Finally, a possible pathogenic role of antigliadin secretory IgA (SIgA) has recently been suggested because of its binding to CD71. CD71 is best known as a high avidity receptor for transferrin. In humans, it is also a receptor of weak affinity for polymeric IgA (Moura et al., 2001). In active CD, this receptor is strongly upregulated in small intestinal enterocytes and becomes expressed at the apical surface, allowing the binding

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Figure 4. Hypothetic Scheme of Immune Responses in CD As shown on the left, the B cell response. IgA-producing B cells primed in gut-associated lymphoid tissue (GALT, center) migrate into LP and differentiate into plasma cells, which produce dimeric IgA released into the intestinal lumen as secretory IgA. In active CD, SIgA-gluten complexes formed in the intestinal lumen can bind the CD71 receptor up-regulated at the apical surface of enterocytes, resulting in their rapid retro-transport into LP. Binding of SIgA to CD71 may also activate signal transduction into epithelial cells. These mechanisms may exacerbate immune responses. Intestinal dimeric IgA can be released into blood and participate in extra-intestinal autoimmunity, notably in dermatitis herpertiformis (see text). As shown on the right, the T cell response. In active CD, IL-15, produced by enterocytes and LP dendritic cells and macrophages, favors the activation of IFN-g-producing and cytotoxic CD8+ IELs harboring NK cell receptors. Gluten-specific CD4+ T cells may participate in IEL activation via cross-priming or via the production of IL-21 that synergizes with IL-15 to activate cytotoxic CD8+ T cells. In the presence of IL-15, CD8-T IELs can induce epithelial lesions via the interactions of their NK receptors NKG2D and NKG2C with their respective ligands MICA and HLA-E upregulated on enterocytes. IL-15 and IL-21 may also cooperate and enable autoreactive CD8+ T cells to respond to weakly agonistic TCR self-ligands (bottom right). IL-15 then favors accumulation of activated CD8+ T cells by stimulating their survival and inhibiting their responses to immunoregulatory mechanisms, further increasing the risk of developing T-dependent autoimmune diseases (T1D), thyroiditis, and perhaps type I refractory celiac disease (RCDI). Finally, IL-15 can promote the emergence of unusual IEL-derived T lymphomas sharing characteristics of both NK and T cells and able to drive epithelial lesions. Onset of lymphoma is revealed by refractoriness to the gluten-free-diet. In a majority of patients, the lymphoma is first intraepithelial (type II refractory celiac disease [RCDII]) and can secondarily transform into overt enteropathy-associated type T lymphoma (EATL).

of intraluminal SIgA complexed to gliadin peptides and their subsequent retrotransport into lamina propria via the recycling endocytic pathway (Matysiak-Budnik et al., 2008; Lebreton et al., 2012). In active CD, SIgA thus fail to play a normal protective role in excluding dietary antigens, but rather behaves as a Trojan horse facilitating the entrance of intact peptides into the mucosa (Figure 4, left). Our recent data indicate that TG2 participates in this process as necessary for the endocytosis of CD71 (Lebreton et al., 2012). How this mechanism contributes to CD pathogenesis remains to be evaluated. IgA-deficient patients are prone to develop CD, indicating that SIgA are dispensable

for CD. However, inadvertent transepithelial passage of gluten peptides may be facilitated in the latter patients by the lack of an efficient SIgA barrier (Wang et al., 2011b). In immunoproficient patients with active CD, CD71-mediated retrotranscytosis of SIgA-gliadin complexes may thus also sustain the immune response by enhancing epithelial permeability to immunogenic peptides. Of note, the binding of polymeric IgA to CD71 can activate signal transduction into erythropoietic precursors (Coulon et al., 2011). It will be interesting to determine whether binding of SIgA to CD71 might also induce a signaling cascade in enterocytes. Immunity 36, June 29, 2012 ª2012 Elsevier Inc. 913

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Review Intraepithelial Lymphocytes: Role in Intestinal Damage and T cell Lymphomagenesis IELs form a large population of lymphocytes that are thought to act as sentinels protecting the epithelial barrier. In CD, however, their chronic activation leads to epithelial damage and, in a small subset of patients, to T cell lymphomagenesis. The mechanisms that switch on their abnormal activation and drive their transformation are only partially elucidated. Upregulation of IL15 observed in the intestine of patients with active CD and RCDII probably plays an important role although the mechanism that drives IL-15 expression remains poorly understood (Di Sabatino et al., 2006; Hu¨e et al., 2004; Mention et al., 2004; Meresse et al., 2004). A first puzzling observation in CD is the increase in TCRgd+ IELs. The role of the latter cells remains largely enigmatic. The current consensus assumes that TCRgd+ IELs recognize tissue-specific stress signals and might help maintain epithelium integrity by exerting cytotoxicity or releasing soluble factors. This protective function may be preserved in CD. Indeed an increase in TCRgd+ IELs is observed at all stages of CD, except in severe cases of RCD II where they tend to disappear (reviewed in Meresse and Cerf-Bensussan, 2009). Their early and persistent increase in CD may reflect chronic epithelial stress favored by exposure to gluten. An alternative but nonexclusive hypothesis may be IL-15-driven accumulation. Thus, mouse TCRgd+ IELs strictly depend on survival signals delivered by enterocytederived IL-15 (Schluns et al., 2004). Whether TCRgd+ IELs can turn into epithelial aggressors in active CD is not excluded but not yet documented. In contrast, the second and main subset of human IELs, consisting in CD8+TCRab+ cells (CD8-T IELs), is now held responsible for intestinal tissue damage (Figure 4, right). In active CD, CD8-T IELs undergo a marked expansion that is associated with enhanced intraepithelial expression of IFN-g (Olaussen et al., 2002), perforin, and granzymes and with epithelial apoptosis (Di Sabatino et al., 2006; Oberhuber et al., 1996). Because all changes subside after GFD, one first possible hypothesis is that CD8-T IELs are activated through cross-presentation of gluten peptides. This mechanism is suggested by data from Gianfrani and coworkers who have identified a peptide mapping to the 123–132 position of A-gliadin (QLIPCMDVVL), which can be recognized in the context of HLA-A*0201 by CD8+ T lymphocytes isolated from CD mucosa. This peptide stimulates their in vitro production of IFN-g and their cytotoxicity against the intestinal epithelial cell line Caco-2 (Mazzarella et al., 2008). A second nonexclusive hypothesis involves the coordinated activation of CD8-T IELs by IL-15 and by NK receptors interacting with their epithelial ligands. CD8-T IELs express several NK receptors, notably CD94 and the activating NK receptor NKG2D. Both receptors are upregulated in CD, probably in response to IL-15 (Jabri et al., 2000; Meresse et al., 2004). Moreover, whereas CD94 associates with NKG2A in control IELs, forming heterodimers that induce inhibitory signals, NKG2A is downregulated in active CD, and CD94 associates on a substantial fraction of IELs with a second partner, NKG2C, that recruits the DAP12 adaptor and transduces activating signals (Meresse et al., 2006). Accordingly, CD8-T IELs from active CD can in vitro kill targets expressing MICA and HLA-E, the two nonclassical MHC class I molecules that are the respective 914 Immunity 36, June 29, 2012 ª2012 Elsevier Inc.

ligands of NKG2D and CD94-NKG2C. Because MICA and HLA-E are upregulated on enterocytes in active CD, the latter cells may become in vivo the target of a cytolytic attack by IELs (Meresse et al., 2004; Meresse et al., 2006; Figure 4, right). If activation of CD94-NKG2C heterodimers seems sufficient to stimulate IFN-g production and cytotoxicity by CD8-T IELs, the exact role of NKG2D as a direct trigger or as a cosignal for the TCR remains controversial. In vitro evidence indicates that signals delivered by IL-15 may bypass a requirement for the TCR (Meresse et al., 2004). Signals delivered by NKG2D and IL-15 might also lower the activating threshold of the TCR (Hu¨e et al., 2004; Roberts et al., 2001) and thus foster the activation of gliadin-specific CD8+ T cells or of CD8+ T cells bearing TCR with a low affinity for self-antigens. That NKG2D signaling can trigger or exacerbate autoimmunity is supported by observations in the NOD model of TID. RAE-1, the mouse homolog of MICA was found upregulated in prediabetic pancreas islets, whereas NKG2D was expressed on autoreactive intrapancreatic CD8+ T cells, and progression to diabetes was prevented by a nondepleting anti-NKG2D antibody (Ogasawara et al., 2004). More recently, it was also shown that transgenic expression of RAE1ε in pancreas b-islet cells, although not sufficient to induce diabetes, could stimulate the recruitment of CD8+ T cells regardless of their antigen specificity and induce insulitis (Markiewicz et al., 2012). In CD, chronic upregulation of activating NKR on CD8-T IELs and of their ligands in intestinal and extraintestinal tissues may thus favor the emergence of autoreactivity in and outside the gut (Figure 4, right). Finally, gluten-driven chronic activation is also associated in a small subset of patients with the emergence of IEL-derived lymphomas (Cellier et al., 2000; Malamut et al., 2009; Spencer et al., 1988). Characterization of malignant IELs in RCDII patients indicates that, alike CD8-T IELs in active CD, they share characteristics of both T and NK cells: they contain clonal TCR rearrangements and all chains of CD3 but also express several NK markers, notably CD94, NKG2D, and NKP46. One major difference is the lack of membrane expression of TCR-CD3 complexes and generally of CD8 (Cellier et al., 1998; Tjon et al., 2008). In vitro, they exert a strong cytotoxicity against enterocyte lines that can be blocked by NKG2D antibody (Hu¨e et al., 2004). This functional property probably accounts for the severe epithelial lesions observed in RCDII. Consistent with data in mice showing that chronic upregulation of IL-15 drives the emergence of T leukemias or lymphomas with NK markers (Fehniger et al., 2001), we have observed that IL-15 plays a central role in RCDII (Figure 4, right). In RCDII, IL-15 is upregulated in enterocytes and drives the NK-like cytotoxicity of RCDII IELs (Hu¨e et al., 2004; Mention et al., 2003). Via Janus kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5), IL-15 stimulates expression of the antiapoptotic B cell lymphoma-extra large (Bcl-xL) protein, which rescues transformed RCDII IELs from apoptosis and allowed their massive accumulation and malignant progression (Malamut et al., 2010). Our most recent data further suggest that IL-15 drives the emergence of RCDII IELs from a T cell precursor reprogrammed into induced T to NK cells (N. Montcuquet et al., unpublished data). Overall these data illustrate how the interplay between IEL and enterocyte-derived IL-15 can orchestrate intestinal tissue

Immunity

Review damage and promote the onset of T cell lymphoma, a rare but most severe complication of CD. Which Clues for the Missing Pieces of the CD Jigsaw? No activation of IELs is observed in humanized gluten-fed HLA-DQ2 or -DQ8 mice, suggesting that IELs are not activated or that immunoregulatory mechanisms curb their activation (Black et al., 2002; de Kauwe et al., 2009). In contrast, mice overexpressing IL-15 in their gut epithelium develop a massive expansion of activated intestinal NK1.1+CD8+ T cells and a small intestinal enteropathy that progresses with age (Ohta et al., 2002). Therefore, why is gluten-specific activation of CD4+ T cells necessary in CD to drive CD8-T IEL activation that appears largely IL-15 dependent? Using OTII mice with CD4+T cells specific for OVA crossed with mice overexpressing IL-15 in the gut epithelium, we have observed that CD4+T cell activation by OVA feeding strongly accelerates the onset of the enteropathy (E. Ramiro et al. unpublished data). Help from CD4+ T cells may be necessary to license dendritic cells for CD8+ T cell cross-priming (Kurts et al., 2010). IL-15 might then stimulate the survival of activated CD8+ T cells (see above) and also interfere with two important immunoregulatory mechanisms. Indeed, by activating JNK, IL-15 can inhibit the Smad3 cascade of TGF-b in human T cells, notably CD8+ (Benahmed et al., 2007). Of note, mice lacking Smad3 develop intestinal inflammation and expansion of activated CD8+ T cells (Yang et al., 1999) and mice with T cells lacking a functional TGF-b receptor develop autoimmunity and expanded numbers of CD8+ T cells harboring activating NK receptors (Marie et al., 2006). In addition, by activating phosphoinositide 3 kinase, IL-15 can also block the response of effector T cells, notably CD8+ to the immunoregulatory effect of FOXP3+ regulatory T (Treg) cells (Ben Ahmed et al., 2009). Accordingly, lymphocytes from patients with active CD, and notably IELs, do not respond to the immunosuppressive effects of autologous or heterologous Treg cells (Hmida et al., 2012; Zanzi et al., 2011). Finally, IL-15 combined with retinoic acid may inhibit the generation of induced Treg cells, but it is unclear whether the later mechanism operates in CD (DePaolo et al., 2011). If evidence converges toward a role for IL-15 in IEL activation in CD, unsolved questions remain regarding the mechanisms involved in stimulating the expression of this cytokine in CD. IL-15 can be synthesized by many cell types and is generally expressed linked to the a-chain of its receptor (IL-15Ra), which trans-presents IL-15 to adjacent lymphocytes bearing the signaling module IL15Rb/gc. In humans, IL-15 regulation is thought to be mainly posttranscriptional, although the underlying mechanism(s) remain largely unknown (reviewed in Fehniger and Caligiuri, 2001). Consistent with this notion, increased expression of IL-15 protein but not of IL-15 mRNA has been observed in situ in active CD and in RCDII. IL-15 was detected in lamina propria cells and in enterocytes, notably at the cell surface of the latter cells (Di Sabatino et al., 2006; Mention et al., 2003). Using intestinal organ cultures, we and others have observed that gluten peptides and notably peptide 31-49 common to the N terminus of A-gliadin might upregulate intestinal synthesis of IL15 (Hu¨e et al., 2004; Maiuri et al., 2003). Whether and how this peptide, which is not presented by HLA-DQ2, might exert this inducing effect remains hotly debated, however. Other

mechanisms may participate in IL-15 upregulation. IL-15 may be retained at the surface of intestinal cells as a consequence of the increased expression of IL-15Ra observed at the mRNA and protein levels in CD mucosa (Bernardo et al., 2008). Synthesis of both IL-15Ra and IL-15 might also be stimulated by IFN-a. This cytokine is upregulated in CD intestine (Di Sabatino et al., 2007) and a recent study in transgenic mouse expressing emerald-green fluorescence protein under IL-15 promoter showed its inducing effect on IL-15 production (Colpitts et al., 2012). Of note, it has been suggested that IL-15 is not increased in all CD patients (Abadie et al., 2011). Because of IL-15 binding to IL-15Ra and to a generally low level of expression, precise quantitation of IL-15 is difficult and its upregulation might be difficult to demonstrate. However, it is also possible that other signals present in the CD mucosa might synergize with IL-15 and stimulate IEL activation even at low concentrations of IL-15. Such signals may notably be delivered by IL-21 that is produced by gluten-specific CD4+ T cells in active CD (Bodd et al., 2010; Fina et al., 2008). This cytokine exerts overlapping effects with IL-15 on IFN-g production, cytotoxicity, proliferation, and survival of NK and CD8+ T cells. Strikingly, IL-21 alone has only very moderate effects and this cytokine seems to act mainly on CD8+ T cells via its strong synergistic effects with IL-15 (Zeng et al., 2005), a conclusion supported by our unpublished data in human IELs. IL-21 released by CD4+ T cells and enterocytederived IL-15 might thus work in concert to stimulate activation and expansion of CD8+ T cells in CD (Figure 4, right). Consistent with this hypothesis, prior stimulation with both cytokines but not with either cytokine alone enabled autoreactive CD8+ T cells to respond to weakly agonistic TCR self-ligands and to induce tissue damage when adoptively transferred into a murine model of autoimmune diabetes. Moreover, no induction of diabetes was observed in the absence of endogenous production of IL-15, indicating that IL-21 effect depended in vivo on IL-15 (Ramanathan et al., 2011). The low amount of IL-15 produced by enterocytes at steady state may be sufficient to initiate the cooperation that may be amplified upon upregulation of IL-15. Interestingly, IL-21 is not upregulated in latent CD, suggesting that switching on IL-21 production is an important step toward overt CD and tissue damage (Sperandeo et al., 2011). The mechanism initiating the production of IL-21 in CD is however unknown. The critical role of IL-21 in the generation of efficient and sustained antiviral CD8+ T cell responses (Yi et al., 2010) points again to the possible cofactor role of viral infections in CD. It will also be interesting to define whether CD-associated gene polymorphisms in IL2-IL21 and IRF4 loci might influence IL-21 synthesis. In conclusion, more work is needed to delineate precisely how the HLA-DQ-driven gluten-specific CD4+ T cell response that is mandatory for CD onset can, in concert with IL-15, favor IEL activation, which is essential for tissue damage. The mechanisms underlying the excessive production of IL-15 remain also elusive. Because of its synergistic effects with IL-15, IL-21 may be one important actor of the dialog between CD4+ and CD8+ T cells. Concluding Remarks and Perspectives Two thousand years after the first description of CD, functional and genetic approaches progressively converge into a scheme in which immune responses triggered by dietary gluten share Immunity 36, June 29, 2012 ª2012 Elsevier Inc. 915

Immunity

Review broad similarities with responses conferring protection against viruses but also causing tissue damage in autoimmune diseases. As suspected in chronic viral infections, chronic exposure to gluten may lead to the onset of autoimmunity, either due to cross-reactivity of antibodies or of T cells generated in the gut in response to gluten or to impaired regulatory control of CD8+ T cells and emergence of autoreactive T cells. The role of MHC class II is now well understood, but much more work is needed to elucidate how a combination of predisposing traits and environmental cofactors may control the onset and the highly variable presentation and severity of CD. How can we translate present knowledge into help for CD patients? Sixty years after W. Dicke’s discovery that treating CD patients with a gluten-free diet is efficacious and without danger, this remains the gold-standard treatment for uncomplicated CD. Yet, GFD is a social burden for many patients and several approaches are being considered to alleviate the diet, including tolerogenic vaccines, which may perhaps reverse overt CD to latent CD, inhibitors of TG2 able to impair gluten presentation, or proteases able to digest gluten within the intestinal lumen (Sollid and Khosla, 2011). The most urgent unmet need is however an efficient treatment for patients who develop refractory celiac disease, notably those developing RCDII. The important role of IL-15 in the pathogenesis of RCDII suggests that blocking IL-15 or its signaling pathway might help to control the disease and prevent the evolution toward aggressive T lymphomas (Malamut et al., 2010). ACKNOWLEDGMENTS The authors acknowledge funding from INSERM, Ligue Contre le Cancer, Fondation Pour la Recherche Me´dicale, Agence Nationale pour la Recherche, Association pour la Recherche contre le Cancer, Association franc¸aise des Patients Intole´rants au Gluten, and Fondation Princesse Grace. They thank all members of INSERM U989 for sharing work and helpful discussions and J.C. Weill for critical review of the manuscript. REFERENCES Abadie, V., Sollid, L.M., Barreiro, L.B., and Jabri, B. (2011). Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu. Rev. Immunol. 29, 493–525. Aggarwal, S., Lebwohl, B., and Green, P.H. (2012). Screening for celiac disease in average-risk and high-risk populations. Therap Adv Gastroenterol 5, 37–47. Ben Ahmed, M.B., Belhadj Hmida, N., Moes, N., Buyse, S., Abdeladhim, M., Louzir, H., and Cerf-Bensussan, N. (2009). IL-15 renders conventional lymphocytes resistant to suppressive functions of regulatory T cells through activation of the phosphatidylinositol 3-kinase pathway. J. Immunol. 182, 6763–6770. Benahmed, M., Meresse, B., Arnulf, B., Barbe, U., Mention, J.J., Verkarre, V., Allez, M., Cellier, C., Hermine, O., and Cerf-Bensussan, N. (2007). Inhibition of TGF-beta signaling by IL-15: a new role for IL-15 in the loss of immune homeostasis in celiac disease. Gastroenterology 132, 994–1008. Bergseng, E., Xia, J., Kim, C.Y., Khosla, C., and Sollid, L.M. (2005). Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the celiac disease-associated HLA-DQ2 molecule. J. Biol. Chem. 280, 21791– 21796. Bernardo, D., Garrote, J.A., Allegretti, Y., Leo´n, A., Go´mez, E., Bermejo-Martin, J.F., Calvo, C., Riestra, S., Ferna´ndez-Salazar, L., Blanco-Quiro´s, A., et al. (2008). Higher constitutive IL15R alpha expression and lower IL-15 response threshold in coeliac disease patients. Clin. Exp. Immunol. 154, 64–73. Bevan, S., Popat, S., Braegger, C.P., Busch, A., O’Donoghue, D., Falth-Magnusson, K., Ferguson, A., Godkin, A., Hogberg, L., Holmes, G., et al. (1999).

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