Genetic variation in the loci encoding immunoglobulin genes (IGH, IGK and IGL) affects the repertoire of B-cell receptors (BCRs). Such effects were previously demonstrated for total peripheral blood B cells, but so far have not been investigated at scale for isolated naïve B cells. As B cells are implicated in the pathogenesis of autoimmune diseases, genetically encoded features of the naïve BCR repertoire may affect disease risk, for instance in celiac disease (CeD) which is hallmarked by stereotyped disease-specific antibodies that recognize antigen in their germline configuration. Here we have characterized the BCR repertoire of naïve B cells in 102 individuals with CeD and 102 control subjects by undertaking gene usage quantitative trait loci analyses based on repertoire sequencing and single nucleotide polymorphism genotyping. Variants within each of the loci had significant effects on the naïve BCR repertoires, with the usage of 80% of IGH genes, 54% of IGK genes and 84% of IGL genes being significantly affected by gene polymorphisms. Effects of genetic polymorphisms on BCR usage were observed for genes implicated in stereotypic responses previously associated with CeD, yet no strong evidence for CeD predisposing effects of polymorphisms within the IGH, IGK and IGL loci was uncovered.

Polymorphisms of human leukocyte antigen (HLA) genes confer risks for human diseases. Predisposing effects related to T cell receptor (TCR) recognition of peptide-HLA can be selection of TCR repertoire and/or preferential presentation of disease-driving epitopes. In celiac disease (CeD), HLA-DQ2.5 predisposes by presenting gluten peptides to CD4+ T cells that typically employ stereotyped TCRs. Here, we analyzed whether genetic variants within the HLA and TR loci shape the naive TCR repertoire. We sequenced the αβ TCR repertoires of naive CD4+ T cells of 103 CeD subjects and 103 controls and performed gene usage quantitative trait loci analyses. The naive CD4+ TCR repertoire was significantly affected by TRA, TRB, and in particular HLA polymorphisms. The presence of HLA-DQ2.5 influenced the TCR repertoire, resulting in significant enrichment of TCR genes being involved in recognition of gluten epitopes in the repertoires of CeD subjects versus controls. HLA thus affects disease risk by selection of a disease-relevant TCR repertoire.

BACKGROUND:Coeliac disease (CeD) affects 1-2% of the western population. Diagnosis is based on serology and duodenal biopsy, but serology-based diagnosis in adults has been approved in Europe. OBJECTIVE:To evaluate the diagnostic performance of IgA anti-transglutaminase 2 (IgA-TG2) and IgG anti-deamidated gliadin peptides (IgG-DGP), and their combinations, compared with biopsy in a real-world secondary care setting. METHODS:Adult patients referred to secondary care endoscopy service at Oslo University Hospital for suspected CeD were invited to participate. CeD diagnosis followed European and Norwegian guidelines, requiring positive serology and mucosal damage. RESULTS:Among 312 evaluable patients, 215 were diagnosed with CeD between 2018 and 2024. Analysis of IgA-TG2 above threshold (>4 U/ml) showed 93% specificity and 94% sensitivity, while IgG-DGP (>20 U/ml) showed 88% specificity and 83% sensitivity. In ROC analyses, the AUC values were 0.98 and 0.95, respectively. Higher threshold (2x, 3x, 5x and 10x ULN) of IgA-TG2 increased specificity (99% to 100%) but lowered sensitivity (82% to 49%). Using IgG-DGP did not increase specificity but detected six missed CeD cases by IgA-TG2. Forty-two percent (n = 92) of cases could be diagnosed with a no-biopsy approach with 10x ULN IgA-TG2 at referral with 100% specificity. CONCLUSION:Serology correlates strongly with histological changes, supporting diagnosis without gastroscopy. A 10x ULN threshold shows excellent specificity at the expense of sensitivity, thus lower thresholds may be preferable due to diminishing gains in specificity. IgG-DGP serves as a valuable complementary marker, that improves sensitivity and helps identify patients with weak IgA-TG2 responses.

Crypt hyperplasia is a key feature of celiac disease (CeD) and several other small intestinal inflammatory conditions. Analysis of the gut epithelial crypt zone by mass spectrometry-based tissue proteomics revealed a strong IFN-γ signal in active CeD. This signal, hallmarked by increased expression of MHC molecules, was paralleled by diminished expression of proteins associated with fatty acid metabolism. Crypt hyperplasia and the same proteomic changes were observed in WT mice administered IFN-γ. In mice with conditional KO of the IFN-γ receptor in gut epithelial cells, these signature morphological and proteomic changes were not induced with IFN-γ administration. IFN-γ was thus a driver of crypt hyperplasia in CeD by acting directly on crypt epithelial cells. The results are relevant to other enteropathies with involvement of IFN-γ.

Coeliac disease is an autoimmune disorder driven by gluten and managed through a strict gluten-free diet. We tested teriflunomide, a drug known for its ability to alter T-cell activity, in a phase 2a trial of 15 patients with coeliac disease. Participants received teriflunomide or placebo including a loading dose, followed by a 3-day oral gluten challenge to monitor T-cell responses. The main aim was to observe attenuation of gluten-specific T-cell activation markers after gluten exposure. We found no significant differences in T-cell activity between groups, concluding that teriflunomide is ineffective as a non-dietary treatment for coeliac disease.

BACKGROUND & AIMS:The treatment of celiac disease (CeD) with gluten-free diet (GFD) normalizes gut inflammation and disease-specific antibodies. CeD patients have HLA-restricted, gluten-specific T cells persisting in the blood and gut even after decades of GFD, which are reactivated and disease driving upon gluten exposure. Our aim was to examine the transition of activated gluten-specific T cells into a pool of persisting memory T cells concurrent with normalization of clinically relevant biomarkers during the first year of treatment. METHODS:We followed 17 CeD patients during their initial GFD year, leading to disease remission. We assessed activation and frequency of gluten-specific CD4+ blood and gut T cells with HLA-DQ2.5:gluten tetramers and flow cytometry, disease-specific serology, histology, and symptom scores. We assessed gluten-specific blood T cells within the first 3 weeks of GFD in 6 patients and serology in an additional 9 patients. RESULTS:Gluten-specific CD4+ T cells peaked in blood at day 14 while up-regulating Bcl-2 and down-regulating Ki-67 and then decreased in frequency within 10 weeks of GFD. CD38, ICOS, HLA-DR, and Ki-67 decreased in gluten-specific cells within 3 days. PD-1, CD39, and OX40 expression persisted even after 12 months. IgA-transglutaminase 2 decreased significantly within 4 weeks. CONCLUSIONS:GFD induces rapid changes in the phenotype and number of gluten-specific CD4+ blood T cells, including a peak of nonproliferating, nonapoptotic cells at day 14. Subsequent alterations in T-cell phenotype associate with the quiescent but chronic nature of treated CeD. The rapid changes affecting gluten-specific T cells and disease-specific antibodies offer opportunities for clinical trials aiming at developing nondietary treatments for patients with newly diagnosed CeD.

Summary: Autoantibodies against the enzyme transglutaminase 2 (TG2) are characteristic of celiac disease (CeD), and TG2-specific immunoglobulin (Ig) A plasma cells are abundant in gut biopsies of patients. Here, we describe the corresponding population of autoreactive B cells in blood. Circulating TG2-specific IgA cells are present in untreated patients on a gluten-containing diet but not in controls. They are clonally related to TG2-specific small intestinal plasma cells, and they express gut-homing molecules, indicating that they are plasma cell precursors. Unlike other IgA-switched cells, the TG2-specific cells are negative for CD27, placing them in the double-negative (IgD−CD27−) category. They have a plasmablast or activated memory B cell phenotype, and they harbor fewer variable region mutations than other IgA cells. Based on their similarity to naive B cells, we propose that autoreactive IgA cells in CeD are generated mainly through chronic recruitment of naive B cells via an extrafollicular response involving gluten-specific CD4+ T cells.

Gluten-specific CD4+ T cells are central players in the pathogenesis of celiac disease (CeD), an inflammatory disease driven by exposure to dietary gluten proteins. Patients with CeD are treated with a lifelong gluten-free diet. When gluten is reintroduced to the diet of patients in remission, there is a wave of activated (CD38+) gluten-specific CD4+ T cells in the blood that peaks on days 6‒8 after the first gluten exposure. This increase in the frequency of activated gluten-specific CD4+ effector-memory T cells (TEM) in the blood can be detected by IFN-γ enzyme-linked immunospot assay (Anderson et al., 2000; Tye-Din et al., 2010) or by HLA-DQ:gluten tetramers (Ráki et al., 2007; Sarna et al., 2018b; Zühlke et al., 2019).

The notion that CD4+ T cells play a central role in coeliac disease (CeD) became strong with the observation that gluten-reactive T cells selectively recognize gluten antigen in the context of the CeD-associated HLA class II allotypes. Today a whole body of evidence points to these gluten-specific CD4+ T cells as essential drivers of CeD pathology. The interaction of T cells with B cells seems to be particularly important as this leads to the formation of CeD-associated antibodies as well as to clonal expansion of T cells. Here we will discuss this and other key aspects of the CeD pathogenesis.

Gluten‐specific CD4 + T cells being drivers of celiac disease (CeD) are obvious targets for immunotherapy. Little is known about how cell markers harnessed for T‐cell‐directed therapy can change with time and upon activation in CeD and other autoimmune conditions. In‐depth characterization of gluten‐specific CD4 + T cells and CeD‐associated (CD38 + and CD103 + ) CD8 + and γδ + T cells in blood of treated CeD patients undergoing a 3 day gluten challenge is reported. The phenotypic profile of gluten‐specific cells changes profoundly with gluten exposure and the cells adopt the profile of gluten‐specific cells in untreated disease (CD147 + , CD70 + , programmed cell death protein 1 (PD‐1) + , inducible T‐cell costimulator (ICOS) + , CD28 + , CD95 + , CD38 + , and CD161 + ), yet with some markers being unique for day 6 cells (C‐X‐C chemokine receptor type 6 (CXCR6), CD132, and CD147) and with integrin α 4 β 7, C‐C motif chemokine receptor 9 (CCR9), and CXCR3 being expressed stably at baseline and day 6. Among gluten‐specific CD4 + T cells, 52% are CXCR5 + at baseline, perhaps indicative of germinal‐center reactions, while on day 6 all are CXCR5 − . Strikingly, the phenotypic profile of gluten‐specific CD4 + T cells on day 6 largely overlaps with that of CeD‐associated (CD38 + and CD103 + ) CD8 + and γδ + T cells. The antigen‐induced shift in phenotype of CD4 + T cells being shared with other disease‐associated T cells is relevant for development of T‐cell‐directed therapies.

Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen.

We compared the αβ T-cell receptor repertoires of CD8+ αβ intraepithelial lymphocytes from celiac disease patients and healthy subjects by single-cell sequencing. We demonstrate that the repertoires of untreated celiac disease patients were more polyclonal and more diverse than what was observed in both treated patients and healthy subjects.

Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3β, and paired CDR3α:CDR3β motif candidates. Among these, a previously known conserved CDR3β R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3β motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRβ sequences of which 145, 102 and 78 belonged to TCRα, TCRβ and paired TCRαβ sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent.

The semi-public T-cell response towards the gluten epitope DQ2.5-glia-alpha 2 uses a prototypic TCR encoded by the germline segments TRAV26-1 and TRBV7-2. Through mutagenesis experiments, we show that a TRAV26-1encoded recognition motif contacts the MHC beta-chain and the TCR CDR3 beta loop underpinning this conserved T-cell response restricted to the prototypic TCRs.

Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ+ IL-2+ TNF-α+) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders.