Analytical and functional approaches to assess the immunogenicity of gluten proteins

Gluten proteins are the causative agents of celiac disease (CD), a lifelong and worldwide spread food intolerance, characterized by an autoimmune enteropathy. Gluten is a complex mixture of high homologous water-insoluble proteins, characterized by a high content of glutamine and proline amino acids that confers a marked resistance to degradation by gastrointestinal proteases. As a consequence of that, large peptides are released in the gut lumen with the potential to activate inflammatory T cells, in CD predisposed individuals. To date, several strategies aimed to detoxify gluten proteins or to develop immunomodulatory drugs to recover immune tolerance to gluten are under investigation. This review overviews the state of art of both analytical and functional methods currently used to assess the immunogenicity potential of gluten proteins from different cereal sources, including native raw seed flours and complex food products, as well as drug-treated samples. The analytical design to assess the content and profile of gluten immunogenic peptides, described herein, is based on the oral-gastro-intestinal digestion (INFOGEST model) followed by extensive characterization of residual gluten peptides by proteomic and immunochemical analyses. These approaches include liquid chromatography–high-resolution mass spectrometry (LC-MS/MS) and R5/G12 competitive ELISA. Functional studies to assess the immune stimulatory capabilities of digested gluten peptides are based on gut mucosa T cells or peripheral blood cells obtained from CD volunteers after a short oral gluten challenge

Consistency in Polyclonal T-cell Responses to Gluten between Children and Adults with Celiac Disease

BACKGROUND & AIMS: Developing antigen-specific approaches for diagnosis and treatment of celiac disease requires a detailed understanding of the specificity of T cells for gluten. The existing paradigm is that T-cell lines and clones from children differ from those of adults in the hierarchy and diversity of peptide recognition. We aimed to characterize the T-cell response to gluten in children vs adults with celiac disease. METHODS: Forty-one children with biopsy-proven celiac disease (median age, 9 years old; 17 male), who had been on strict gluten-free diets for at least 3 months, were given a 3-day challenge with wheat; blood samples were collected and gluten-specific T cells were measured. We analyzed responses of T cells from these children and from 4 adults with celiac disease to a peptide library and measured T-cell receptor bias. We isolated T-cell clones that recognized dominant peptides and assessed whether gluten peptide recognition was similar between T-cell clones from children and adults. RESULTS: We detected gluten-specific responses by T cells from 30 of the children with celiac disease (73%). T cells from the children recognized the same peptides that were immunogenic to adults with celiac disease; deamidation of peptides increased these responses. Age and time since diagnosis did not affect the magnitude of T-cell responses to dominant peptides. T-cell clones specific for dominant α- or ω-gliadin peptides from children with celiac disease had comparable levels of reactivity to wheat, rye, and barley peptides as T-cell clones from adults with celiac disease. The α-gliadin-specific T cells from children had biases in T-cell receptor usage similar to those in adults. CONCLUSIONS: T cells from children with celiac disease recognize similar gluten peptides as T cells from adults with celiac disease. The findings indicate that peptide-based diagnostics and therapeutics for adults may also be used for children. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR

Recent Progress and Recommendations on Celiac Disease From the Working Group on Prolamin Analysis and Toxicity

Celiac disease (CD) affects a growing number of individuals worldwide. To elucidate the causes for this increase, future multidisciplinary collaboration is key to understanding the interactions between immunoreactive components in gluten-containing cereals and the human gastrointestinal tract and immune system and to devise strategies for CD prevention and treatment beyond the gluten-free diet. During the last meetings, the Working Group on Prolamin Analysis and Toxicity (Prolamin Working Group, PWG) discussed recent progress in the field together with key stakeholders from celiac disease societies, academia, industry and regulatory bodies. Based on the current state of knowledge, this perspective from the PWG members provides recommendations regarding clinical, analytical and legal aspects of CD. The selected key topics that require future multidisciplinary collaborative efforts in the clinical field are to collect robust data on the increasing prevalence of CD, to evaluate what is special about gluten-specific T cells, to study their kinetics and transcriptomics and to put some attention to the identification of the environmental agents that facilitate the breaking of tolerance to gluten. In the field of gluten analysis, the key topics are the precise assessment of gluten immunoreactive components in wheat, rye and barley to understand how these are affected by genetic and environmental factors, the comparison of different methods for compliance monitoring of gluten-free products and the development of improved reference materials for gluten analysis

IL-10-producing regulatory cells impact on celiac disease evolution

Celiac Disease (CD) is a T-cell mediated disorder caused by immune response to gluten, although the mechanisms underlying CD progression are still elusive. We analyzed immune cell composition, plasma cytokines, and gliadin-specific T-cell responses in patients with positive serology and normal intestinal mucosa (potential-CD) or villous atrophy (acute-CD), and after gluten-free diet (GFD). We found: an inflammatory signature and the presence of circulating gliadin-specific IFN-γ+ T cells in CD patients regardless of mucosal damage; an increased frequency of IL-10-secreting dendritic cells (DC-10) in the gut and of circulating gliadin-specific IL-10-secreting T cells in potential-CD; IL-10 inhibition increased IFN-γ secretion by gliadin-specific intestinal T cells from acute- and potential-CD. On GFD, inflammatory cytokines normalized, while IL-10-producing T cells accumulated in the gut. We show that IL-10-producing cells are fundamental in controlling pathological T-cell responses to gluten: DC-10 protect the intestinal mucosa from damage and represent a marker of potential-CD

Tristetraprolin/ZFP36 Regulates the Turnover of Autoimmune-Associated HLA-DQ mRNAs.

HLA class II genes encode highly polymorphic heterodimeric proteins functioning to present antigens to T cells and stimulate a specific immune response. Many HLA genes are strongly associated with autoimmune diseases as they stimulate self-antigen specific CD4+ T cells driving pathogenic responses against host tissues or organs. High expression of HLA class II risk genes is associated with autoimmune diseases, influencing the strength of the CD4+ T-mediated autoimmune response. The expression of HLA class II genes is regulated at both transcriptional and post-transcriptional levels. Protein components of the RNP complex binding the 3'UTR and affecting mRNA processing have previously been identified. Following on from this, the regulation of HLA-DQ2.5 risk genes, the main susceptibility genetic factor for celiac disease (CD), was investigated. The DQ2.5 molecule, encoded by HLA-DQA1*05 and HLA-DQB1*02 alleles, presents the antigenic gluten peptides to CD4+ T lymphocytes, activating the autoimmune response. The zinc-finger protein Tristetraprolin (TTP) or ZFP36 was identified to be a component of the RNP complex and has been described as a factor modulating mRNA stability. The 3'UTR of CD-associated HLA-DQA1*05 and HLA-DQB1*02 mRNAs do not contain canonical TTP binding consensus sequences, therefore an in silico approach focusing on mRNA secondary structure accessibility and stability was undertaken. Key structural differences specific to the CD-associated mRNAs were uncovered, allowing them to strongly interact with TTP through their 3'UTR, conferring a rapid turnover, in contrast to lower affinity binding to HLA non-CD associated mRNA

Statement of the Prolamin Working Group on the Determination of Gluten in Fermented Foods Containing Partially Hydrolyzed Gluten

On August 12, 2020, the U.S. Food and Drug Administration (FDA) has finalized a rule related to gluten-free labeling for foods containing fermented, hydrolyzed ingredients. The FDA believes that there is no scientifically valid analytical method e ective for determining gluten in fermented or hydrolyzed foods. In the absence of an analytical method, the FDA has decided to evaluate gluten-free claims on these foods based only on evidence that the food or ingredient used is gluten-free before fermentation or hydrolysis. For example, barley-based beers from which gluten is removed during brewing using special filtration, adsorption and/or enzymatic treatment are therefore excluded from bearing a gluten-free label. The Prolamin Working Group (PWG) acknowledges that the FDA rule is a regulatory act and might have to take into consideration several aspects other than scientific evidence, including risk assessment. Nevertheless, the PWG thinks that science has to be the most important driver for regulatory acts in risk management.Fil: Scherf, Katharina Anne. Karlsruher Institut Für Technologie; AlemaniaFil: Catassi, Carlo. Università Politecnica Delle Marche; ItaliaFil: Chirdo, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Ciclitira, Paul J.. University of East Anglia; Reino UnidoFil: Feighery, Conleth Francis. Universidad de Dublin; IrlandaFil: Gianfrani, Carmen. Institute of Biochemistry and Cell Biology; ItaliaFil: Koning, Frits. Leiden University; Países BajosFil: Lundin, Knut E. A.. University of Oslo; NoruegaFil: Masci, Stefania. No especifíca;Fil: Schuppan, Detlef. No especifíca;Fil: Smulders, Marinus J. M.. Wageningen University and Research; Países BajosFil: Tranquet, Olivier. No especifíca;Fil: Troncone, Riccardo. University Federico II; ItaliaFil: Koehler, Peter. No especifíca

Statement of the Prolamin Working Group on the Determination of Gluten in Fermented Foods Containing Partially Hydrolyzed Gluten

On August 12, 2020, the U.S. Food and Drug Administration (FDA) has finalized a rule related to gluten-free labeling for foods containing fermented, hydrolyzed ingredients. The FDA believes that there is no scientifically valid analytical method effective for determining gluten in fermented or hydrolyzed foods. In the absence of an analytical method, the FDA has decided to evaluate gluten-free claims on these foods based only on evidence that the food or ingredient used is gluten-free before fermentation or hydrolysis. For example, barley-based beers from which gluten is removed during brewing using special filtration, adsorption and/or enzymatic treatment are therefore excluded from bearing a gluten-free label

Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases

Tolerogenic dendritic cells play a critical role in promoting antigen-specific tolerance via dampening of T cell responses, induction of pathogenic T cell exhaustion and antigen-specific regulatory T cells. Here we efficiently generate tolerogenic dendritic cells by genetic engineering of monocytes with lentiviral vectors co-encoding for immunodominant antigen-derived peptides and IL-10. These transduced dendritic cells (designated DCIL-10/Ag) secrete IL-10 and efficiently downregulate antigen-specific CD4+ and CD8+ T cell responses from healthy subjects and celiac disease patients in vitro. In addition, DCIL-10/Ag induce antigen-specific CD49b+LAG-3+ T cells, which display the T regulatory type 1 (Tr1) cell gene signature. Administration of DCIL-10/Ag resulted in the induction of antigen-specific Tr1 cells in chimeric transplanted mice and the prevention of type 1 diabetes in pre-clinical disease models. Subsequent transfer of these antigen-specific T cells completely prevented type 1 diabetes development. Collectively these data indicate that DCIL-10/Ag represent a platform to induce stable antigen-specific tolerance to control T-cell mediated diseases