HLA class II alleles in Norwegian patients with coexisting type 1 diabetes and celiac disease.

BACKGROUND: Type 1 diabetes (T1D) and celiac disease (CeD) are 2 distinct diseases, but there is an increased risk of developing CeD for T1D patients. Both diseases are associated with HLA-class II alleles, such as DQB1 *02:01 and DQB1 *03:02; however, their risk contribution vary between the diseases. MATERIALS AND METHODS: We genotyped HLA-DRB1 and - DQB1 in 215 patients with coexisting T1D and CeD identified from a T1D cohort, and compared them to patients with T1D (N = 487) and CeD (N = 327), as well as healthy controls (N = 368). RESULTS: The patients with coexisting T1D and CeD had an intermediate carrier frequency (72.8%) of the DRB1 *03:01- DQB1 *02:01- DQA1 *05:01 haplotype compared to T1D (64.1%) and CeD (88.7%) patients. The DRB1 *03:01- DQB1 *02:01- DQA1 *05:01/ DRB1 *04- DQB1 *03:02- DQA1 *03 haplotype combination, encoding DQ2.5 and DQ8 molecules, was equally frequent among patients with both T1D and CeD (52.6%) and T1D patients (46.8%) but significantly lower in CeD patients (9.5%). CONCLUSION: Overall, the patients with coexisting T1D and CeD had an HLA profile more similar to T1D patients than CeD patients.

The human intestinal B-cell response.

The intestinal immune system is chronically challenged by a huge plethora of antigens derived from the lumen. B-cell responses in organized gut-associated lymphoid tissues and regional lymph nodes that are driven chronically by gut antigens generate the largest population of antibody-producing cells in the body: the gut lamina propria plasma cells. Although animal studies have provided insights into mechanisms that underpin this dynamic process, some very fundamental differences in this system appear to exist between species. Importantly, this prevents extrapolation from mice to humans to inform translational research questions. Therefore, in this review we will describe the structures and mechanisms involved in the propagation, dissemination, and regulation of this immense plasma cell population in man. Uniquely, we will seek our evidence exclusively from studies of human cells and tissues.

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

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

Responsive population dynamics and wide seeding into the duodenal lamina propria of transglutaminase-2-specific plasma cells in celiac disease.

A hallmark of celiac disease is autoantibodies to transglutaminase 2 (TG2). By visualizing TG2-specific antibodies by antigen staining of affected gut tissue, we identified TG2-specific plasma cells in the lamina propria as well as antibodies in the subepithelial layer, inside the epithelium, and at the brush border. The frequency of TG2-specific plasma cells were found not to correlate with serum antibody titers, suggesting that antibody production at other sites may contribute to serum antibody levels. Upon commencement of a gluten-free diet, the frequency of TG2-specific plasma cells in the lesion dropped dramatically within 6 months, yet some cells remained. The frequency of TG2-specific plasma cells in the celiac lesion is thus dynamically regulated in response to gluten exposure. Laser microdissection of plasma cell patches, followed by antibody gene sequencing, demonstrated that clonal cells were seeded in distinct areas of the mucosa. This was confirmed by immunoglobulin heavy chain repertoire analysis of plasma cells isolated from individual biopsies of two untreated patients, both for TG2-specific and non-TG2-specific cells. Our results shed new light on the processes underlying the B-cell response in celiac disease, and the approach of staining for antigen-specific antibodies should be applicable to other antibody-mediated diseases.

Celiac disease: Autoimmunity in response to food antigen.

Celiac disease (CD) is an increasingly common disease of the small intestine that occurs in genetically susceptible subjects by ingestion of cereal gluten proteins. Gluten is highly abundant in the modern diet and well tolerated by most individuals. In CD, however, an erroneous but highly specific, adaptive immune response is mounted toward certain parts of the gluten proteome. The resulting intestinal destruction is reversible and resolved upon removal of gluten from the diet. Post-translational modification (deamidation) of gluten peptides by transglutaminase 2 (TG2) is essential for the peptides to act as HLA-DQ-restricted T-cell antigens. Characteristically, deamidated gluten and the self-protein TG2 both become targets of highly disease specific B-cell responses. These antibodies share several peculiar characteristics despite being directed against vastly different antigens, which suggests a common mechanism of development. Importantly, no clear function has been ascribed to the antibodies and their contribution to disease may relate to their function as antigen receptors of the B cells rather than as soluble immunoglobulins. Adaptive immunity against gluten and TG2 appears not to be sufficient for establishment of the disease lesion, and it has been suggested that stress responses in the intestinal epithelium are essential for the development of full-blown disease and tissue damage. In this review we will summarize current concepts of the immune pathology of CD with particular focus on recent advances in our understanding of disease specific B-cell responses.

Coeliac disease-associated polymorphisms influence thymic gene expression.

Significant associations between coeliac disease (CD) and single nucleotide polymorphisms (SNPs) distributed over 40 genetic regions have been established. The majority of these SNPs are non-coding and 20 SNPs were, by expression quantitative trait loci (eQTL) analysis, found to harbour cis regulatory potential in peripheral blood mononuclear cells (PBMC). Almost all regions contain genes with an immunological relevant function, of which many act in the same biological pathways. One such pathway is T-cell development in the thymus, a pathway previously not explored in CD pathogenesis. The aim of our study was to explore the regulatory potential of the CD-associated SNPs (n=50) by eQTL analysis in thymic tissue from 42 subjects. In total, 43 nominal significant (P<0.05) eQTLs were found within 24 CD-associated chromosomal regions, corresponding to 27 expression-altering SNPs (eSNPs) and 40 probes (eProbes) that represents 39 unique genes (eGenes). Nine significant probe-SNP pairs (corresponding to 8 eSNPs and 7 eGenes) overlapped with previous findings in PBMC (rs12727642-PARK7, rs296547-DDX59, rs917997-IL18RAP, rs842647-AHSA2, rs13003464-AHSA2, rs6974491-ELMO1, rs2074404-NSF (two independent probes) and rs2298428-UBE2L3). When compared across more tissues, we found that 14 eQTLs could represent potentially novel thymus-specific eQTLs. This implies that CD risk polymorphisms could affect gene regulation in thymus.

Plasmacytoid dendritic cells are scarcely represented in the human gut mucosa and are not recruited to the celiac lesion.

Celiac disease (CD) is a chronic small intestinal inflammation precipitated by gluten ingestion. According to case reports, interferon (IFN)-α administration may induce development of overt CD. Plasmacytoid dendritic cells (PDCs) were thought to be the source of IFN-α and promote a T helper type 1 response leading to lesion formation. Surprisingly and contradicting to earlier findings, PDCs were described as the main antigen-presenting cells (APCs) in human duodenal mucosa and particularly in CD. Here we show that when assessed by flow cytometry and in situ staining, PDCs represent < 1% of APCs in both normal duodenal mucosa and the celiac lesion. Low levels of IFN-α were detected in the celiac lesion assessed by western blot, reverse transcriptase (RT)-PCR, and immunohistochemistry. In four cell populations sorted from the celiac lesion (based on their expression of HLA-DR and CD45), we found that equally low levels of mRNA for IFN-α were distributed among these cell populations. Together, these results suggest that relatively small amount of IFN-α, produced by a variety of cell types, is present in the celiac mucosa. IFN-λ, a type III IFN important in intestinal antiviral defense, was produced mainly by APCs, but its expression was not increased in the celiac lesion.

Novel therapies for coeliac disease.

Coeliac disease is a widespread, lifelong disorder for which dietary control represents the only accepted form of therapy. There is an unmet need for nondietary therapies to treat this condition. Most ongoing and emerging drug-discovery programmes are based on the understanding that coeliac disease is caused by an inappropriate T-cell-mediated immune response to dietary gluten proteins. Recent genome-wide association studies lend further support to this pathogenic model. The central role of human leucocyte antigen genes has been validated, and a number of new risk loci have been identified, most of which are related to the biology of T cells and antigen-presenting cells. Here, we review the status of potential nondietary therapies under consideration for coeliac disease. We conclude that future development of novel therapies will be aided considerably by the identification of new, preferably noninvasive, surrogate markers for coeliac disease activity.

Density of CD163+ CD11c+ dendritic cells increases and CD103+ dendritic cells decreases in the coeliac lesion.

Coeliac disease is a chronic inflammation of the intestinal mucosa controlled by gluten-specific T cells restricted by disease-associated HLA-DQ molecules. We have previously reported that mucosal CD11c(+) dendritic cells (DCs) are responsible for activation of gluten-reactive T cells within the coeliac lesion. In mice, intestinal CD11c(+) DCs comprise several functionally distinct subsets. Here, we report that HLA-DQ(+) antigen-presenting cells (APCs) in normal human duodenal mucosa can be divided into four subsets with striking similarities to those described in mice: CD163(+) CD11c(-) macrophages (74%), and CD11c(+) cells expressing either CD163 (7%), CD103 (11%) or CD1c (13%). CD103(+) and CD1c(+) DCs belonged to partly overlapping populations, whereas CD163(+) CD11c(+) APCs appeared to be a distinct population. In the coeliac lesion, we found increased density of CD163(+) CD11c(+) APCs, whereas the density of CD103(+) and CD1c(+) DCs was decreased, suggesting that distinct subpopulations of APCs in coeliac disease may exert different functions in the pathogenesis.

Interleukin-15 induces interleukin-17 production by synovial T cell lines from patients with rheumatoid arthritis.

IL-17-producing T cells (Th17 cells) are believed to contribute to local inflammation and joint damage in rheumatoid arthritis (RA). Limited data exist on Th17 cells located within the inflamed synovial tissue (ST) of patients with RA. Here, we aimed to generate polyclonal T cell lines (TCLs) from the RA ST and assess their cytokine production, including the effects of exogenous IL-15 on IL-17 production in vitro. For five patients with RA, polyclonal TCLs were established from ST obtained by joint surgery. Synovial TCLs were expanded and stimulated by anti-CD3/CD28 microbeads and exogenous cytokines. Cytokine production was assessed by culture supernatant analyses and intracellular flow cytometry, and TCLs were sorted based on their surface expression of CCR6. In addition to IL-17, we detected IL-6, IL-10, IFN-γ and TNF-α in the synovial TCL culture supernatants. Exogenous IL-15 increased the production of IL-17 as well as the other cytokines except IFN-γ. For IL-17, this effect was more pronounced after prolonged culture times. Intracellular flow cytometry confirmed the presence of IL-17+ and IL-17+ IFN-γ+ CD4+ T cells in the TCLs. IL-17+ and IL-17+ IFN-γ+ T cells were enriched in the CD4+ CCR6+ population. In conclusion, Th17 cells can be detected after polyclonal expansion and stimulation of RA synovial TCLs generated by joint surgery. The Th17 cells from the RA ST were enriched in the CD4+ CCR6+ population, and they were sensitive to exogenous IL-15. Th17 cells present within the synovial compartment may contribute to the RA pathogenesis and local joint damage.

HLA-DQ2-restricted gluten-reactive T cells produce IL-21 but not IL-17 or IL-22.

We have analyzed the production of the effector cytokines interleukin (IL)-17, IL-21, and IL-22 in gluten-reactive CD4(+) T cells of celiac disease patients, either cultured from small intestinal biopsies or isolated from peripheral blood after an oral gluten challenge. Combining intracellular cytokine staining with DQ2-α-II gliadin peptide tetramer staining of intestinal polyclonal T-cell lines, we found that gluten-specific T cells produced interferon-γ (IFN-γ) and IL-21, but not IL-17 or IL-22, even if other T cells of the same lines produced these cytokines. Similarly, in DQ2-α-II-specific T cells in peripheral blood of gluten-challenged patients, very few stained for intracellular IL-17, whereas many cells stained for IFN-γ. We conclude that gluten-reactive T cells produce IL-21 and IFN-γ, but not IL-17. Their production of IL-21 suggests a role for this cytokine in the pathogenesis of celiac disease.

Four novel coeliac disease regions replicated in an association study of a Swedish-Norwegian family cohort.

Recent genome-wide association studies have identified 1q31 (RGS1), 2q11-12 (IL18RAP), 3p21 (CCR1/CCR3/CCR2), 3q25-26 (IL12A/SCHIP1), 3q28 (LPP), 4q27 (IL2/IL21), 6q25 (TAGAP) and 12q24 (SH2B3) as susceptibility regions for coeliac disease (CD). We have earlier replicated association with the IL2/IL21 region. This study aimed at replicating the remaining regions in a family cohort using the transmission disequilibrium test, which is not prone to population stratification as a source of false-positive results. Nine single nucleotide polymorphisms (SNPs) within these regions were genotyped in 325 Swedish-Norwegian CD families. We found significant associations with the same alleles in the regions 1q31 (rs2816316; P(nc)=0.0060), 3p21 (rs6441961; P(nc)=0.0006), 3q25-26 (rs17810564; P(nc)=0.0316 and rs9811792; P(nc)=0.0434) and 3q28 (rs1464510; P(nc)=0.0037). Borderline, but non-significant, associations were found for rs917997 (IL18RAP), whereas no evidence for association could be obtained for rs13015714 (IL18RAP) or rs1738074 (TAGAP). The lack of replication of the latter SNPs could be because of limited power. rs3184504 (SH2B3) was not analysed because of assay failure. The most significantly associated region, 3p21 (CCR1/CCR3/CCR2), was further analysed by typing of 30 SNPs, with the aim of identifying the causal variant responsible for the initial association. Several SNPs showed association with CD, but none displayed associations stronger than rs6441961, nor did any of them add to the effect initially marked by rs6441961 in a conditional analysis. However, differential effects of rs6441961(*)C carrying haplotypes were indicated, and we thus cannot exclude the possibility that our inability to obtain evidence for multiple independent effects in the CCR1/CCR3/CCR2 gene region was related to a power issue.

Diagnosis and treatment of celiac disease.

The understanding of the pathogenesis of celiac disease has made huge advances in recent years. The disease is caused by an inappropriate immune response to dietary gluten proteins. This immune response is controlled by CD4(+) T cells in the lamina propria that recognize gluten peptides in the context of disease predisposing HLA-DQ2 and HLA-DQ8 molecules.(1, 2) These T cells are specific for proline- and glutamine-rich gluten peptides that are resistant to proteolysis and that have been become deamidated by the enzyme transglutaminase 2 (TG2). Strikingly, celiac disease patients produce antibodies to this same enzyme when exposed to dietary gluten. Here we discuss how the new insight in the pathogenesis has lead to development of new diagnostics and nourished research into novel treatments.

The monoclonal antibody 6B9 recognizes CD44 and not cell surface transglutaminase 2.

The multifunctional enzyme transglutaminase 2 (TG2) can be located intracellularly, in the extracellular matrix (ECM) and on the cell surface. Cell surface TG2 (csTG2) is poorly recognized both by most TG2-specific commercial antibodies and celiac disease-associated anti-TG2 autoantibodies. The recent characterization of a csTG2-specific monoclonal antibody (mAb), which did not recognize ECM-associated TG2, suggested major conformational differences between csTG2 and TG2 found in the ECM. Subsequent findings based on this antibody indicated ubiquitous abundance and novel roles of csTG2 in innate immune responses. We wished to identify the epitope of 6B9 so as to shed light on the disparate antibody binding properties of csTG2- and ECM-associated TG2. Surprisingly, and despite thorough effort, we were unable to isolate TG2 as the antigen of 6B9. We found that 6B9 does not react with recombinant human TG2. In immunoprecipitation experiments, 6B9 pulled down an 85 kDa protein which was identified as CD44 by mass spectrometry. Several flow cytometry experiments including the testing of CD44s transfectants indicated that CD44, and not csTG2, is the antigen of 6B9. We conclude that 6B9 does not recognize csTG2 but rather the cell surface glycoprotein CD44. Thus, recent knowledge of csTG2 gained through the use of 6B9 should be reevaluated.

Association study of IL2/IL21 and FcgRIIa: significant association with the IL2/IL21 region in Scandinavian coeliac disease families.

The first genome-wide association study performed in a UK coeliac disease (CD) case-control cohort revealed association with a linkage disequilibrium block containing the KIAA1109/Tenr/IL2/IL21 genes. Also recently, an association with a non-synonymous polymorphism in FcgammaRIIa (CD32a) was reported in CD with an unusually strong P-value. We aimed to replicate the reported associations with the single nucleotide polymorphisms rs13119723 A>G and rs6822844 G>T in the KIAA1109/Tenr/IL2/IL21 region and rs1801274 G>A in the FcgammaRIIa gene in a family sample consisting of 325 Swedish/Norwegian families using the robust transmission disequilibrium test. The family sample used in this study included 100 families with two or more children affected by CD and 225 families with one affected child. We could confirm significant association between the polymorphisms rs13119723 A>G and rs6822844 G>T located in the KIAA1109/Tenr/IL2/IL21 region and CD (P-value 0.001 and 0.002, respectively). However, we found no association with the FcgammaRIIa rs1801274 G>A polymorphism (P-value=0.3). In conclusion, our results support the KIAA1109/Tenr/IL2/IL21 region as a true CD susceptibility region.

Fine mapping study in Scandinavian families suggests association between coeliac disease and haplotypes in chromosome region 5q32.

The previous genome-wide scan in Scandinavian families supported earlier evidence for linkage of a region on chromosome 5 (5q31-33) to coeliac disease. This study deals with further genetic mapping of an 18 cM region, spanning from marker GAh18A (131.87 Mb) to D5S640 (149.96 Mb). Linkage and association analyses were performed in a two-step approach. First, seven microsatellites were added. Strong evidence for linkage was obtained with a Zlr score of 3.96, P(nc) = 4 x 10(-5) at marker D5S436. The strongest association was with a haplotype consisting of the markers D5S2033 and D5S2490 (P(nc) < 0.001). In the second step, we added 17 microsatellites and 69 single nucleotide polymorphisms (SNPs) to the analysis. These markers were located close to or within candidate genes across the region of approximately 7 Mb beneath the linkage peak marked by D5S2017 and D5S812. A substantial increase of the linkage signal with a maximum Zlr score of 4.6 at marker rs1972644 (P(nc) = 2 x 10(-6)) was obtained and several SNPs showed association. Seven SNPs that individually showed the strongest association were genotyped in a second independent family sample set (225 trios). In the trio family sample as well as in the multiplex family sample, the strongest association was found with SNPs within the region flanked by the associated microsatellites D5S2033 and D5S2490 at 5q32.

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

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

Association analysis of MYO9B gene polymorphisms and inflammatory bowel disease in a Norwegian cohort.

Association between single nucleotide polymorphisms (SNPs) within the MYO9B gene and celiac disease was recently reported. The role of MYO9B in celiac disease was suggested to relate to an epithelial barrier defect. The region to which MYO9B localize is also linked with inflammatory bowel disease (IBD). For these reasons, we hypothesize that MYO9B could also be a susceptibility gene in IBD. To address this, we performed an association study of a Norwegian IBD cohort (149 patients with Crohn's disease, 308 patients with ulcerative colitis and 562 healthy controls) using SNPs, which tagged the celiac disease associated MYO9B haplotype. No association between these SNPs and IBD was observed. Our results failed to support the notion that MYO9B is a susceptibility gene in IBD.