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.

A200 HUMANIZED CELIAC-PRONE EPITHELIUM IN VITRO EXPRESS MHC-II AND CO-STIMULATORY MOLECULES NECESSARY FOR GLUTEN PEPTIDE PRESENTATION

Background The role intestinal epithelial cells (IECs) play in the breakdown of tolerance to gluten at an early stage in celiac disease (CeD) is unclear. Epithelial stress is a feature of CeD, and although the triggers are largely unknown, it is accompanied by expression of several markers that could be involved in initiation of inflammatory responses. IECs have been shown to express MHC class II (MHC-II) molecules and participate in antigen presentation in several models. Whether IECs can participate in gluten peptide presentation, the major environmental trigger in celiac disease, is unknown. To study this, a model expressing human MHC-II, HLA DQ8 or HLA-DQ2, would be required. Aims To develop organoid monolayers from transgenic mice expressing human celiac risk genes: HLA-DQ8 and -DQ2. To investigate conditions leading to the induction of epithelial MHC-II and its main co-stimulatory molecules, CD80, CD86 and CD40, that could enable early gluten peptide presentation. Methods In order to show pathophysiological significance of the model, we used two approaches, either induction of inflammation in vivo through gluten sensitization, or direct stimulation of the monolayers using pro-inflammatory cytokines relevant in CeD, such as IFNγ. Mice were sensitized with Pepsin-Trypsin digested gliadin and cholera toxin (CT) once a week for 3 weeks, followed by a challenge phase in which they only received gliadin. Control mice received CT only. We then developed organoid monolayers from the duodenum followed by stimulation with 10 ng/ml IFNγ. Finally, markers necessary for gluten peptide presentation, the expression of MHC-II and its co-stimulatory molecules, were evaluated using flow cytometry. Results Both in vivo gluten sensitization and in vitro stimulation of the organoid derived monolayer with IFNγ induced a proinflammatory response, that independently primed the epithelium to express MHC-II molecules (p =0.02 and <0.0001, respectively). When in vivo sensitization and in vitro IFNγ stimulation were combined, epithelial MHC-II expression was further upregulated (p <0.0001). Lastly, only the combination of gluten sensitization and in vitro IFNγ induced expression of MHC-II co-stimulatory molecules, which are necessary for antigen presentation. Conclusions Our findings support that gluten induced-inflammation in vivo as well as independent stimuli that release IFNγ enhance the capacity of the IECs to express MHC-II molecules. However, co-stimulatory molecules are only expressed by the epithelium when both gluten tolerance is broken by in vivo sensitization and the organoid monolayers is further exposed to IFNγ. The results support the hypothesis that the epithelium participates in gluten peptide presentation and that this pathway is stimulated by both gluten-dependent and independent inflammation. Funding Agencies CIHRSupported by CIHR and a Farncombe Family Grant to EFV and TFD.

Differential Physiological Responses Elicited by Ancient and Heritage Wheat Cultivars Compared to Modern Ones

Although ancient, heritage, and modern wheat varieties appear rather similar from a nutritional point of view, having a similar gluten content and a comparable toxicity linked to their undigested gluten peptide, whenever the role of ancient end heritage wheat grains has been investigated in animal studies or in clinical trials, more anti-inflammatory effects have been associated with the older wheat varieties. This review provides a critical overview of existing data on the differential physiological responses that could be elicited in the human body by ancient and heritage grains compared to modern ones. The methodology used was that of analyzing the results of relevant studies conducted from 2010 through PubMed search, by using as keywords “ancient or heritage wheat”, “immune wheat” (protein or peptides), and immune gluten (protein or peptides). Our conclusion is that, even if we do not know exactly which molecular mechanisms are involved, ancient and heritage wheat varieties have different anti-inflammatory and antioxidant proprieties with respect to modern cultivars. It is, therefore, reasonable to assume that the health proprieties attributed to older cultivars could be related to wheat components which have positive roles in the modulation of intestinal inflammation and/or permeability.

Comprehensive Detection of Isopeptides between Human Tissue Transglutaminase and Gluten Peptides

Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in CD pathogenesis, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent Nε-(γ-glutamyl)-lysine isopeptide crosslinks resulting in TG2–gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we used different proteomics software tools that enable the comprehensive identification of isopeptides. Thus, 34 different isopeptides involving 20 TG2 lysine residues were identified in a model system, only six of which were previously known. Additionally, 36 isopeptides of TG2-TG2 multimers were detected. Experiments with different TG2-gluten peptide molar ratios revealed the most preferred lysine residues involved in isopeptide crosslinking. Expanding the model system to three gluten peptides with more glutamine residues allowed the localization of the preferred glutamine crosslinking sites. These new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.