Microbiota-dependent proteolysis of gluten subverts diet-mediated protection against type 1 diabetes.

Diet and commensals can affect the development of autoimmune diseases like type 1 diabetes (T1D). However, whether dietary interventions are microbe-mediated was unclear. We found that a diet based on hydrolyzed casein (HC) as a protein source protects non-obese diabetic (NOD) mice in conventional and germ-free (GF) conditions via improvement in the physiology of insulin-producing cells to reduce autoimmune activation. The addition of gluten (a cereal protein complex associated with celiac disease) facilitates autoimmunity dependent on microbial proteolysis of gluten: T1D develops in GF animals monocolonized with Enterococcus faecalis harboring secreted gluten-digesting proteases but not in mice colonized with protease deficient bacteria. Gluten digestion by E. faecalis generates T cell-activating peptides and promotes innate immunity by enhancing macrophage reactivity to lipopolysaccharide (LPS). Gnotobiotic NOD Toll4-negative mice monocolonized with E. faecalis on an HC + gluten diet are resistant to T1D. These findings provide insights into strategies to develop dietary interventions to help protect humans against autoimmunity.

Identification of gluten T cell epitopes driving celiac disease.

CD4+ T cells specific for cereal gluten proteins are key players in celiac disease (CeD) pathogenesis. While several CeD-relevant gluten T cell epitopes have been identified, epitopes recognized by a substantial proportion of gluten-reactive T cells remain unknown. The identification of such CeD-driving gluten epitopes is important for the food industry and in clinical settings. Here, we have combined the knowledge of a distinct phenotype of gluten-reactive T cells and key features of known gluten epitopes for the discovery of unknown epitopes. We tested 42 wheat gluten-reactive T cell clones, isolated on the basis of their distinct phenotype and with no reactivity to known epitopes, against a panel of synthetic peptides bioinformatically identified from a wheat gluten protein database. We were able to assign reactivity to 10 T cell clones and identified a 9-nucleotide oligomer core region of five previously uncharacterized gliadin/glutenin epitopes. This work represents an advance in the effort to identify CeD-driving gluten epitopes.

Focused B cell response to recurring gluten motif with implications for epitope spreading in celiac disease.

Antibodies to deamidated gluten peptides are accurate diagnostic markers of celiac disease. However, binding of patient antibodies to all possible gluten epitopes has not previously been investigated. Here, we assess serum antibody specificity across the gluten proteome by use of high-density peptide arrays. We confirm the importance of deamidation for antibody binding, and we show that the response is remarkably focused on the known epitope QPEQPFP (where E results from deamidation of Q). In addition, we describe an epitope in native (non-deamidated) gluten, QQPEQII (where E is gene encoded), which is associated with both B cell and T cell reactivity. Antibodies to this native epitope are cross-reactive with the major deamidated epitope due to recognition of the shared PEQ motif. Since cross-reactive B cells can present peptides to different gluten-specific T cells, we propose that such B cells play a role in epitope spreading by engaging T cells with multiple specificities.

Phenotype-Based Isolation of Antigen-Specific CD4+ T Cells in Autoimmunity: A Study of Celiac Disease.

The pathogenic immune response in celiac disease (CeD) is orchestrated by phenotypically distinct CD4+ T cells that recognize gluten epitopes in the context of disease-associated HLA-DQ allotypes. Cells with the same distinct phenotype, but with elusive specificities, are increased across multiple autoimmune conditions. Here, whether sorting of T cells based on their distinct phenotype (Tphe cells) yields gluten-reactive cells in CeD is tested. The method's efficiency is benchmarked by parallel isolation of gluten-reactive T cells (Ttet cells), using HLA-DQ:gluten peptide tetramers. From gut biopsies of 12 untreated HLA-DQ2.5+ CeD patients, Ttet+ /Tphe+ , Ttet- /Tphe+ , and Ttet- /Tphe- cells are sorted for single-cell T-cell receptor (TCR)-sequencing (n = 8) and T-cell clone (TCC)-generation (n = 5). The generated TCCs are TCR sequenced and tested for their reactivity against deamidated gluten. Gluten-reactivity is observed in 91.2% of Ttet+ /Tphe+ TCCs, 65.3% of Ttet- /Tphe+ TCCs and 0% of Ttet- /Tphe- TCCs. TCR sequencing reveals clonal expansion and sequence sharing across patients, features reflecting antigen-driven responses. The feasibility to isolate antigen-specific CD4+ T cells by the sole use of phenotypic markers in CeD outlines a potential avenue for characterizing disease-driving CD4+ T cells in autoimmune conditions.

TCRpower: quantifying the detection power of T-cell receptor sequencing with a novel computational pipeline calibrated by spike-in sequences.

T-cell receptor (TCR) sequencing has enabled the development of innovative diagnostic tests for cancers, autoimmune diseases and other applications. However, the rarity of many T-cell clonotypes presents a detection challenge, which may lead to misdiagnosis if diagnostically relevant TCRs remain undetected. To address this issue, we developed TCRpower, a novel computational pipeline for quantifying the statistical detection power of TCR sequencing methods. TCRpower calculates the probability of detecting a TCR sequence as a function of several key parameters: in-vivo TCR frequency, T-cell sample count, read sequencing depth and read cutoff. To calibrate TCRpower, we selected unique TCRs of 45 T-cell clones (TCCs) as spike-in TCRs. We sequenced the spike-in TCRs from TCCs, together with TCRs from peripheral blood, using a 5' RACE protocol. The 45 spike-in TCRs covered a wide range of sample frequencies, ranging from 5 per 100 to 1 per 1 million. The resulting spike-in TCR read counts and ground truth frequencies allowed us to calibrate TCRpower. In our TCR sequencing data, we observed a consistent linear relationship between sample and sequencing read frequencies. We were also able to reliably detect spike-in TCRs with frequencies as low as one per million. By implementing an optimized read cutoff, we eliminated most of the falsely detected sequences in our data (TCR α-chain 99.0% and TCR ß-chain 92.4%), thereby improving diagnostic specificity. TCRpower is publicly available and can be used to optimize future TCR sequencing experiments, and thereby enable reliable detection of disease-relevant TCRs for diagnostic applications.

Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease.

Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4+-T-cell receptor beta variable (TRBV) 29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4- (TRAV9-2+) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4+-TRBV29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2+-TRBV7-3+ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4+ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2+ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3ß loop of the TCR and Arg70ß from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.

Pathogenic T Cells in Celiac Disease Change Phenotype on Gluten Challenge: Implications for T-Cell-Directed Therapies.

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 α4ß7, 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.

Frequency of Gluten-Reactive T Cells in Active Celiac Lesions Estimated by Direct Cell Cloning.

Chronic inflammation of the small intestine in celiac disease is driven by activation of CD4+ T cells that recognize gluten peptides presented by disease-associated HLA-DQ molecules. We have performed direct cell cloning of duodenal biopsies from five untreated and one refractory celiac disease patients, and three non-celiac disease control subjects in order to assess, in an unbiased fashion, the frequency of gluten-reactive T cells in the disease-affected tissue as well as the antigen fine specificity of the responding T cells. From the biopsies of active disease lesions of five patients, 19 T-cell clones were found to be gluten-reactive out of total 1,379 clones tested. This gave an average of 1.4% (range 0.7% - 1.9%) of gluten-reactive T cells in lamina propria of active celiac lesions. Interestingly, also the patient with refractory celiac disease had gluten-reactive T cell clones in the lamina propria (5/273; 1.8%). In comparison, we found no gluten-reactive T cells in any of the total 984 T-cell clones screened from biopsies from three disease control donors. Around two thirds of the gluten-reactive clones were reactive to a panel of peptides representing known gluten T-cell epitopes, of which two thirds were reactive to the immunodominant DQ2.5-glia-α1/DQ2.5-glia-α2 and DQ2.5-glia-ω1/DQ2.5-glia-ω2 epitopes. This study shows that gluten-reactive T cells in the inflamed duodenal tissue are prevalent in the active disease lesion, and that many of these T cells are reactive to T-cell epitopes that are not yet characterized. Knowledge of the prevalence and epitope specificity of gluten-specific T cells is a prerequisite for therapeutic efforts that target disease-specific T cells in celiac disease.

Comprehensive Analysis of CDR3 Sequences in Gluten-Specific T-Cell Receptors Reveals a Dominant R-Motif and Several New Minor Motifs.

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, CDR3ß, and paired CDR3α:CDR3ß motif candidates. Among these, a previously known conserved CDR3ß 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:CDR3ß 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 TCRß sequences of which 145, 102 and 78 belonged to TCRα, TCRß and paired TCRαß 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.

Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease.

Gut intraepithelial γδ and CD8+ αß T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αß 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+ αß T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αß 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+ αß T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αß 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+ αß T-cell clonotypes that may not be specific for the gluten antigen.

A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease.

The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4+ T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5-mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.

A TRAV26-1-encoded recognition motif focuses the biased T cell response in celiac disease.

The semi-public T-cell response towards the gluten epitope DQ2.5-glia-α2 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 ß-chain and the TCR CDR3ß loop underpinning this conserved T-cell response restricted to the prototypic TCRs.

On the immune response to barley in celiac disease: Biased and public T-cell receptor usage to a barley unique and immunodominant gluten epitope.

Celiac disease (CeD) is driven by CD4+  T-cell responses to dietary gluten proteins of wheat, barley, and rye when deamidated gluten epitopes are presented by certain disease-associated HLA-DQ allotypes. About 90% of the CeD patients express HLA-DQ2.5. In such patients, five gluten epitopes dominate the anti-gluten T-cell response; two epitopes unique to wheat, two epitopes present in wheat, barley, and rye and one epitope unique to barley. Despite presence of barley in commonly consumed food and beverages and hence being a prominent source of gluten, knowledge about T-cell responses elicited by barley in CeD is scarce. Therefore, in this study, we explored T-cell response toward the barley unique epitope DQ2.5-hor-3 (PIPEQPQPY) by undertaking HLA-DQ:gluten peptide tetramer staining, single-cell T-cell receptor (TCR) αß sequencing, T-cell cloning, and T-cell proliferation studies. We demonstrate that majority of the CeD patients generate T-cell response to DQ2.5-hor-3, and this response is characterized by clonal expansion, preferential TCR V-gene usage and public TCR features thus echoing findings previously made for wheat gluten epitopes. The knowledge that biased and public TCRs underpin the T-cell response to all the immunodominant gluten epitopes in CeD suggests that such T cells are promising diagnostic and therapeutic targets.

CD38 expression on gluten-specific T cells is a robust marker of gluten re-exposure in coeliac disease.

Background: Increasing efforts are being put into new treatment options for coeliac disease (CeD), a chronic disorder of the small intestine induced by gluten. Interleukin-2 (IL-2) and gluten-specific CD4 + T cells increase in the blood after four hours and six days, respectively, following a gluten challenge in CeD patients. These responses are unique to CeD and are not seen in controls. We aimed to evaluate different markers reflecting a recall response to gluten exposure that may be used to monitor therapy. Methods: CeD patients on a gluten-free diet underwent a one- (n = 6) or three-day (n = 7) oral gluten challenges. We collected blood samples at several time points between baseline and day 8, and monitored gluten-specific CD4 + T cells for their frequency and CD38 expression using HLA-DQ:gluten tetramers. We assessed the IL-2 concentration in plasma four hours after the first gluten intake. Results: The frequency of gut-homing, tetramer-binding, CD4 + effector memory T (tetramer + ß7 + TEM) cells and the IL-2 concentration measured shortly after the first dose of gluten increased significantly after the one- and three-day gluten challenges, but large interindividual differences were exhibited. The frequency of tetramer + ß7 + TEM plateaued between days 6 and 8 and was lower after the one-day challenge. We observed a consistent increase in CD38 expression on tetramer + ß7 + TEM cells and did not find a significant difference between the one- and three-day challenges. Conclusions: The optimal time points for monitoring therapy response in CeD after a three-day oral gluten challenge is four hours for plasma IL-2 or six to eight days for the frequency of tetramer + ß7 + TEM cells, but both these parameters involved large interindividual differences. In contrast, CD38 expression on tetramer + ß7 + TEM cells increased uniformly and irrespectively of the length of gluten challenge, suggesting that this parameter is more suited for monitoring drug efficacy in clinical trials for CeD.

Therapeutic and Diagnostic Implications of T Cell Scarring in Celiac Disease and Beyond.

Few therapeutic and diagnostic tools specifically aim at T cells in autoimmune disorders, but are T cells a narrow target in these diseases? Lessons may be learned from celiac disease (CeD), one of the few autoimmune disorders where the T cell driving antigens are known, i.e. dietary gluten proteins. T cell clonotypes specific to gluten are expanded, persist for decades and express a distinct phenotype in CeD patients. Cells with this phenotype are increased also in other autoimmune conditions. Accordingly, disease-specific CD4+ T cells form an immunological scar in CeD and probably other autoimmune disorders. We discuss approaches how such T cells may be targeted for better treatment and diagnosis via their antigen specificity or via their expression of characteristic phenotypic markers.

Distinct phenotype of CD4+ T cells driving celiac disease identified in multiple autoimmune conditions.

Combining HLA-DQ-gluten tetramers with mass cytometry and RNA sequencing analysis, we find that gluten-specific CD4+ T cells in the blood and intestines of patients with celiac disease display a surprisingly rare phenotype. Cells with this phenotype are also elevated in patients with systemic sclerosis and systemic lupus erythematosus, suggesting a way to characterize CD4+ T cells specific for disease-driving antigens in multiple autoimmune conditions.

Discriminative T-cell receptor recognition of highly homologous HLA-DQ2-bound gluten epitopes.

Celiac disease (CeD) provides an opportunity to study the specificity underlying human T-cell responses to an array of similar epitopes presented by the same human leukocyte antigen II (HLA-II) molecule. Here, we investigated T-cell responses to the two immunodominant and highly homologous HLA-DQ2.5-restricted gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). Using HLA-DQ2.5-DQ2.5-glia-α1a and HLA-DQ2.5-DQ2.5-glia-ω1 tetramers and single-cell αß T-cell receptor (TCR) sequencing, we observed that despite similarity in biased variable-gene usage in the TCR repertoire responding to these nearly identical peptide-HLA-II complexes, most of the T cells are specific for either of the two epitopes. To understand the molecular basis of this exquisite fine specificity, we undertook Ala substitution assays revealing that the p7 residue (Leu/Gln) is critical for specific epitope recognition by both DQ2.5-glia-α1a- and DQ2.5-glia-ω1-reactive T-cell clones. We determined high-resolution binary crystal structures of HLA-DQ2.5 bound to DQ2.5-glia-α1a (2.0 Å) and DQ2.5-glia-ω1 (2.6 Å). These structures disclosed that differences around the p7 residue subtly alter the neighboring substructure and electrostatic properties of the HLA-DQ2.5-peptide complex, providing the fine specificity underlying the responses against these two highly homologous gluten epitopes. This study underscores the ability of TCRs to recognize subtle differences in the peptide-HLA-II landscape in a human disease setting.

Disease-driving CD4+ T cell clonotypes persist for decades in celiac disease.

Little is known about the repertoire dynamics and persistence of pathogenic T cells in HLA-associated disorders. In celiac disease, a disorder with a strong association with certain HLA-DQ allotypes, presumed pathogenic T cells can be visualized and isolated with HLA-DQ:gluten tetramers, thereby enabling further characterization. Single and bulk populations of HLA-DQ:gluten tetramer-sorted CD4+ T cells were analyzed by high-throughput DNA sequencing of rearranged TCR-α and -ß genes. Blood and gut biopsy samples from 21 celiac disease patients, taken at various stages of disease and in intervals of weeks to decades apart, were examined. Persistence of the same clonotypes was seen in both compartments over decades, with up to 53% overlap between samples obtained 16 to 28 years apart. Further, we observed that the recall response following oral gluten challenge was dominated by preexisting CD4+ T cell clonotypes. Public features were frequent among gluten-specific T cells, as 10% of TCR-α, TCR-ß, or paired TCR-αß amino acid sequences of total 1813 TCRs generated from 17 patients were observed in 2 or more patients. In established celiac disease, the T cell clonotypes that recognize gluten are persistent for decades, making up fixed repertoires that prevalently exhibit public features. These T cells represent an attractive therapeutic target.