A bispecific antibody targeting HLA-DQ2.5-gluten peptides potently blocks gluten-specific T cells induced by gluten ingestion in patients with celiac disease.

The gluten-free diet for celiac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complexes of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52). OBJECTIVE: To assess the ability of DONQ52 to inhibit CeD patient-derived T-cell responses to the most immunogenic gluten peptides that encompass immunodominant T cell epitopes. METHODS: We employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T-cell responses. HLA-DQ2.5+ CeD patients consumed food containing wheat, barley, or rye for 3 days with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon (IFN)-γ enzyme-linked immunosorbent spot assay (ELISpot) testing responses to gluten peptides encompassing a series of immunodominant T cell epitopes. The inhibitory effect of DONQ52 (4 or 40 µg/mL) was assessed and compared to pan-HLA-DQ blockade (SPVL3 antibody). RESULTS: In HLA-DQ2.5+ CeD patients, DONQ52 reduced T cell responses to all wheat gluten peptides to an equivalent or more effective degree than pan-HLA-DQ antibody blockade. It reduced T cell responses to a cocktail of the most immunodominant wheat epitopes by a median of 87% (IQR 72-92). Notably, DONQ52 also substantially reduced T-cell responses to dominant barley hordein and rye secalin derived peptides. DONQ52 had no effect on T-cell responses to non-gluten antigens. CONCLUSION: DONQ52 can significantly block HLA-DQ2.5-restricted T cell responses to the most highly immunogenic gluten peptides in CeD. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against multiple gluten peptide:HLA-DQ2.5 complexes. This work provides proof-of-concept multi-specific antibody blockade has the potential to meaningfully inhibit pathogenic gluten-specific T-cell responses in CeD and supports ongoing therapeutic development.

Past, Present, and Future of Noninvasive Tests to Assess Gluten Exposure, Celiac Disease Activity, and End-Organ Damage.

Although many biomarkers have been proposed, and several are in widespread clinical use, there is no single readout or combination of readouts that correlates tightly with gluten exposure, disease activity, or end-organ damage in treated patients with celiac disease. Challenges to developing and evaluating better biomarkers include significant interindividual variability-related to immune amplification of gluten exposure and how effects of immune activation are manifest. Furthermore, the current "gold standard" for assessment of end-organ damage, small intestinal biopsy, is itself highly imperfect, such that a marker that is a better reflection of the "ground truth" may indeed appear to perform poorly. The goal of this review was to analyze past and present efforts to establish robust noninvasive tools for monitoring treated patients with celiac disease and to highlight emerging tools that may prove to be useful in clinical practice.

Generation of circulating autoreactive pre-plasma cells fueled by naive B cells in celiac disease.

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-Free Diet Induces Rapid Changes in Phenotype and Survival Properties of Gluten-Specific T Cells in Celiac 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.

Advances in Nonresponsive and Refractory Celiac Disease.

Nonresponsive celiac disease (CeD) is relatively common. It is generally attributed to persistent gluten exposure and resolves after correction of diet errors. However, other complications of CeD and disorders clinically mimicking CeD need to be excluded. Novel therapies are being evaluated to facilitate mucosal recovery, which might benefit patients with nonresponsive CeD. Refractory CeD (RCeD) is rare and is divided into 2 types. The etiology of type I RCeD is unclear. A switch to gluten-independent autoimmunity is suspected in some patients. In contrast, type II RCeD represents a low-grade intraepithelial lymphoma. Type I RCeD remains a diagnosis of exclusion, requiring ruling out gluten intake and other nonmalignant causes of villous atrophy. Diagnosis of type II RCeD relies on the demonstration of a clonal population of neoplastic intraepithelial lymphocytes with an atypical immunophenotype. Type I RCeD and type II RCeD generally respond to open-capsule budesonide, but the latter has a dismal prognosis due to severe malnutrition and frequent progression to enteropathy-associated T-cell lymphoma; more efficient therapy is needed.

Gut tissue-resident memory T cells in coeliac disease.

This mini-review describes observations of the 1990ies with culturing of gluten-specific and astrovirus-specific CD4+ T cells from duodenal biopsies from subjects who presumably had a long time between the exposure to gluten or astrovirus antigens and the sampling of the biopsy. In these studies, it was also observed that antigen-specific CD4+ T cells migrated out of the gut biopsies during overnight culture. The findings are suggestive of memory T cells in tissue which are resident, but which also can be mobilised on antigen stimulation. Of note, these findings were made years before the term tissue-resident memory T cells was invoked. Since that time, many observations have accumulated on these gut T cells, particularly the gluten-specific T cells, and we have insight into the turnover of CD4+ T cells in the gut lamina propria. These data make it evident that human antigen-specific CD4+ T cells that can be cultured from gut biopsies indeed are bone fide tissue-resident memory T cells.

Celiac Disease in Children.

Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. In patients with suspected celiac disease, measurement of serum IgA antibodies to tissue transglutaminase-2 has a high sensitivity and specificity and is the first screening test that should be ordered. The diagnosis of celiac disease is based on the presence of mucosal damage in small intestinal biopsies in patients having circulating celiac disease-specific antibodies. Celiac disease management includes lifelong adherence to a gluten-free diet and continuous long-term follow-up.

Determination of Urinary Gluten Immunogenic Peptides to Assess Adherence to the Gluten-Free Diet: A Randomized, Double-Blind, Controlled Study.

INTRODUCTION: The adherence to a gluten-free diet (GFD) is a trending topic in the management of celiac disease. The aim of our study was to evaluate the diagnostic performance of urinary gluten immunogenic peptides (GIP) determination to detect gluten contamination of the GFD. METHODS: In study A, 25 healthy adults on a standard GFD performed 6 gluten challenges (0, 10, 50, 100, 500, and 1,000 mg) with quantification of urinary GIP before (T0) and during the following 24 hours. In study B, 12 participants on a gluten contamination elimination diet underwent urinary GIP determination at T0 and after challenge with 5 or 10 mg gluten. Urine GIP concentration was determined by an immunochromatographic assay. RESULTS: In study A, 51 of 150 baseline urine samples were GIP+ on GFD and 7 of 17 were GIP+ after the zero-gluten challenge, whereas only 55 of 81 were GIP+ after the 10-1,000 mg gluten challenges. There was no significant change in the 24-hour urinary GIP when increasing gluten from 10 to 1,000 mg. In study B, 24 of 24 baseline urine samples were GIP-, whereas 8 of 24 were GIP+ after 5 or 10 mg of gluten. DISCUSSION: Traces of gluten in the standard GFD may cause positivity of urinary GIP determination, whereas a false negativity is common after a gluten intake of 10-1,000 mg. Owing to the impossibility of standardizing the test in normal conditions, it seems unlikely that urinary GIP determination may represent a reliable tool to assess the compliance to the GFD of patients with celiac disease or other gluten-related disorders.

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.

Molecular and Structural Parallels between Gluten Pathogenic Peptides and Bacterial-Derived Proteins by Bioinformatics Analysis.

Gluten-related disorders (GRDs) are a group of diseases that involve the activation of the immune system triggered by the ingestion of gluten, with a worldwide prevalence of 5%. Among them, Celiac disease (CeD) is a T-cell-mediated autoimmune disease causing a plethora of symptoms from diarrhea and malabsorption to lymphoma. Even though GRDs have been intensively studied, the environmental triggers promoting the diverse reactions to gluten proteins in susceptible individuals remain elusive. It has been proposed that pathogens could act as disease-causing environmental triggers of CeD by molecular mimicry mechanisms. Additionally, it could also be possible that unrecognized molecular, structural, and physical parallels between gluten and pathogens have a relevant role. Herein, we report sequence, structural and physical similarities of the two most relevant gluten peptides, the 33-mer and p31-43 gliadin peptides, with bacterial pathogens using bioinformatics going beyond the molecular mimicry hypothesis. First, a stringent BLASTp search using the two gliadin peptides identified high sequence similarity regions within pathogen-derived proteins, e.g., extracellular proteins from Streptococcus pneumoniae and Granulicatella sp. Second, molecular dynamics calculations of an updated α-2-gliadin model revealed close spatial localization and solvent-exposure of the 33-mer and p31-43 peptide, which was compared with the pathogen-related proteins by homology models and localization predictors. We found putative functions of the identified pathogen-derived sequence by identifying T-cell epitopes and SH3/WW-binding domains. Finally, shape and size parallels between the pathogens and the superstructures of gliadin peptides gave rise to novel hypotheses about activation of innate immunity and dysbiosis. Based on our structural findings and the similarities with the bacterial pathogens, evidence emerges that these pathologically relevant gluten-derived peptides could behave as non-replicating pathogens opening new research questions in the interface of innate immunity, microbiome, and food research.

A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation.

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.

A Case Study of the Response of Immunogenic Gluten Peptides to Sourdough Proteolysis.

Celiac disease is activated by digestion-resistant gluten peptides that contain immunogenic epitopes. Sourdough fermentation is a potential strategy to reduce the concentration of these peptides within food. However, we currently know little about the effect of partial sourdough fermentation on immunogenic gluten. This study examined the effect of a single sourdough culture (representative of those that the public may consume) on the digestion of immunogenic gluten peptides. Sourdough bread was digested via the INFOGEST protocol. Throughout digestion, quantitative and discovery mass spectrometry were used to model the kinetic release profile of key immunogenic peptides and profile novel peptides, while ELISA probed the gluten's allergenicity. Macrostructural studies were also undertaken. Sourdough fermentation altered the protein structure, in vitro digestibility, and immunogenic peptide release profile. Interestingly, sourdough fermentation did not decrease the total immunogenic peptide concentration but altered the in vitro digestion profile of select immunogenic peptides. This work demonstrates that partial sourdough fermentation can alter immunogenic gluten digestion, and is the first study to examine the in vitro kinetic profile of immunogenic gluten peptides from sourdough bread.

A Sensitive Whole Blood Assay Detects Antigen-Stimulated Cytokine Release From CD4+ T Cells and Facilitates Immunomonitoring in a Phase 2 Clinical Trial of Nexvax2 in Coeliac Disease.

Improved blood tests assessing the functional status of rare gluten-specific CD4+ T cells are needed to effectively monitor experimental therapies for coeliac disease (CD). Our aim was to develop a simple, but highly sensitive cytokine release assay (CRA) for gluten-specific CD4+ T cells that did not require patients to undergo a prior gluten challenge, and would be practical in large, multi-centre clinical trials. We developed an enhanced CRA and used it in a phase 2 clinical trial ("RESET CeD") of Nexvax2, a peptide-based immunotherapy for CD. Two participants with treated CD were assessed in a pilot study prior to and six days after a 3-day gluten challenge. Dye-dilution proliferation in peripheral blood mononuclear cells (PBMC) was assessed, and IL-2, IFN-γ and IL-10 were measured by multiplex electrochemiluminescence immunoassay (ECL) after 24-hour gluten-peptide stimulation of whole blood or matched PBMC. Subsequently, gluten-specific CD4+ T cells in blood were assessed in a subgroup of the RESET CeD Study participants who received Nexvax2 (maintenance dose 900 µg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-γ and IL-10 release from PBMCs attributable to CD4+ T cells, but the PBMC CRA was substantially less sensitive than whole blood CRA. Only modest gluten peptide-stimulated IL-2 release could be detected without prior gluten challenge using PBMC. In contrast, whole blood CRA enabled detection of IL-2 and IFN-γ before and after gluten challenge. IL-2 and IFN-γ release in whole blood required more than 6 hours incubation. Delay in whole blood incubation of more than three hours from collection substantially reduced antigen-stimulated IL-2 and IFN-γ secretion. Nexvax2, but not placebo treatment in the RESET CeD Study was associated with significant reductions in gluten peptide-stimulated whole blood IL-2 and IFN-γ release, and CD4+ T cell proliferation. We conclude that using fresh whole blood instead of PBMC substantially enhances cytokine secretion stimulated by gluten peptides, and enables assessment of rare gluten-specific CD4+ T cells without requiring CD patients to undertake a gluten challenge. Whole blood assessment coupled with ultra-sensitive cytokine detection shows promise in the monitoring of rare antigen-specific T cells in clinical studies.

Efficient Hydrolysis of Gluten-Derived Celiac Disease-Triggering Immunogenic Peptides by a Bacterial Serine Protease from Burkholderia gladioli.

Celiac disease is an autoimmune disorder triggered by toxic peptides derived from incompletely digested glutens in the stomach. Peptidases that can digest the toxic peptides may formulate an oral enzyme therapy to improve the patients' health condition. Bga1903 is a serine endopeptidase secreted by Burkholderia gladioli. The preproprotein of Bga1903 consists of an N-terminal signal peptide, a propeptide region, and an enzymatic domain that belongs to the S8 subfamily. Bga1903 could be secreted into the culture medium when it was expressed in E. coli. The purified Bga1903 is capable of hydrolyzing the gluten-derived toxic peptides, such as the 33- and 26-mer peptides, with the preference for the peptide bonds at the carbonyl site of glutamine (P1 position). The kinetic assay of Bga1903 toward the chromogenic substrate Z-HPQ-pNA at 37 °C, pH 7.0, suggests that the values of Km and kcat are 0.44 ± 0.1 mM and 17.8 ± 0.4 s-1, respectively. The addition of Bga1903 in the wort during the fermentation step of beer could help in making gluten-free beer. In summary, Bga1903 is usable to reduce the gluten content in processed foods and represents a good candidate for protein engineering/modification aimed to efficiently digest the gluten at the gastric condition.

Selection of Gut-Resistant Bacteria and Construction of Microbial Consortia for Improving Gluten Digestion under Simulated Gastrointestinal Conditions.

This work aimed to define the microbial consortia that are able to digest gluten into non-toxic and non-immunogenic peptides in the human gastrointestinal tract. METHODS: 131 out of 504 tested Bacillus and lactic acid bacteria, specifically Bacillus (64), lactobacilli (63), Pediococcus (1), and Weissella (3), showed strong gastrointestinal resistance and were selected for their PepN, PepI, PepX, PepO, and PepP activities toward synthetic substrates. Based on multivariate analysis, 24 strains were clearly distinct from the other tested strains based on having the highest enzymatic activities. As estimated by RP-HPLC and nano-ESI-MS/MS, 6 cytoplasmic extracts out of 24 selected strains showed the ability to hydrolyze immunogenic epitopes, specifically 57-68 of α9-gliadin, 62-75 of A-gliadin, 134-153 of γ-gliadin, and 57-89 (33-mer) of α2-gliadin. Live and lysed cells of selected strains were combined into different microbial consortia for hydrolyzing gluten under gastrointestinal conditions. Commercial proteolytic enzymes (Aspergillusoryzae E1, Aspergillusniger E2, Bacillussubtilis Veron HPP, and Veron PS proteases) were also added to each microbial consortium. Consortium activity was evaluated by ELISA tests, RP-HPLC-nano-ESI-MS/MS, and duodenal explants from celiac disease patients. RESULTS: two microbial consortia (Consortium 4: Lactiplantibacillus (Lp.) plantarum DSM33363 and DSM33364, Lacticaseibacillus (Lc.) paracasei DSM33373, Bacillussubtilis DSM33298, and Bacilluspumilus DSM33301; and Consortium 16: Lp. plantarum DSM33363 and DSM33364, Lc. paracasei DSM33373, Limosilactobacillusreuteri DSM33374, Bacillusmegaterium DSM33300, B.pumilus DSM33297 and DSM33355), containing commercial enzymes, were able to hydrolyze gluten to non-toxic and non-immunogenic peptides under gastrointestinal conditions. CONCLUSIONS: the results of this study provide evidence that selected microbial consortia could potentially improve the digestion of gluten in gluten-sensitive patients by hydrolyzing the immunogenic peptides during gastrointestinal digestion.

Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.

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.

Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n = 6; cohort 2, n = 12) and unaffected controls (cohort 3, n = 9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3 h, 6 h and 6 days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11 per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Gliadin Sequestration as a Novel Therapy for Celiac Disease: A Prospective Application for Polyphenols.

Celiac disease is an autoimmune disorder characterized by a heightened immune response to gluten proteins in the diet, leading to gastrointestinal symptoms and mucosal damage localized to the small intestine. Despite its prevalence, the only treatment currently available for celiac disease is complete avoidance of gluten proteins in the diet. Ongoing clinical trials have focused on targeting the immune response or gluten proteins through methods such as immunosuppression, enhanced protein degradation and protein sequestration. Recent studies suggest that polyphenols may elicit protective effects within the celiac disease milieu by disrupting the enzymatic hydrolysis of gluten proteins, sequestering gluten proteins from recognition by critical receptors in pathogenesis and exerting anti-inflammatory effects on the system as a whole. This review highlights mechanisms by which polyphenols can protect against celiac disease, takes a critical look at recent works and outlines future applications for this potential treatment method.

Evaluating Responses to Gluten Challenge: A Randomized, Double-Blind, 2-Dose Gluten Challenge Trial.

BACKGROUND & AIMS: Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology. METHODS: In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence. RESULTS: All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose-response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot, gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2-restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or near significantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure. CONCLUSIONS: Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796.