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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.
Hardy, MY, Goel, G, Russell, AK, Chen Yi Mei, SLG, Brown, GJE, Wang, S, Szymczak, E, Zhang, R, Goldstein, KE, Neff, KM, et al
Frontiers in immunology. 2021;:661622
Abstract
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.
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Serum cytokines elevated during gluten-mediated cytokine release in coeliac disease.
Goel, G, Daveson, AJM, Hooi, CE, Tye-Din, JA, Wang, S, Szymczak, E, Williams, LJ, Dzuris, JL, Neff, KM, Truitt, KE, et al
Clinical and experimental immunology. 2020;(1):68-78
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Abstract
Cytokines have been extensively studied in coeliac disease, but cytokine release related to exposure to gluten and associated symptoms has only recently been described. Prominent, early elevations in serum interleukin (IL)-2 after gluten support a central role for T cell activation in the clinical reactions to gluten in coeliac disease. The aim of this study was to establish a quantitative hierarchy of serum cytokines and their relation to symptoms in patients with coeliac disease during gluten-mediated cytokine release reactions. Sera were analyzed from coeliac disease patients on a gluten free-diet (n = 25) and from a parallel cohort of healthy volunteers (n = 25) who underwent an unmasked gluten challenge. Sera were collected at baseline and 2, 4 and 6 h after consuming 10 g vital wheat gluten flour; 187 cytokines were assessed. Confirmatory analyses were performed by high-sensitivity electrochemiluminescence immunoassay. Cytokine elevations were correlated with symptoms. Cytokine release following gluten challenge in coeliac disease patients included significant elevations of IL-2, chemokine (C-C motif) ligand 20 (CCL20), IL-6, chemokine (C-X-C motif) ligand (CXCL)9, CXCL8, interferon (IFN)-γ, IL-10, IL-22, IL-17A, tumour necrosis factor (TNF)-α, CCL2 and amphiregulin. IL-2 and IL-17A were earliest to rise. Peak levels of cytokines were generally at 4 h. IL-2 increased most (median 57-fold), then CCL20 (median 10-fold). Cytokine changes were strongly correlated with one another, and the most severely symptomatic patients had the highest elevations. Early elevations of IL-2, IL-17A, IL-22 and IFN-γ after gluten in patients with coeliac disease implicates rapidly activated T cells as their probable source. Cytokine release after gluten could aid in monitoring experimental treatments and support diagnosis.
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Prospective longitudinal study: use of faecal gluten immunogenic peptides to monitor children diagnosed with coeliac disease during transition to a gluten-free diet.
Comino, I, Segura, V, Ortigosa, L, Espín, B, Castillejo, G, Garrote, JA, Sierra, C, Millán, A, Ribes-Koninckx, C, Román, E, et al
Alimentary pharmacology & therapeutics. 2019;(12):1484-1492
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Abstract
BACKGROUND Treatment for coeliac disease is a lifelong strict gluten-free diet. Although guidelines recommend regular follow-up with dietary interviews and coeliac serology, these methods may be inaccurate. AIM: To evaluate the usefulness of faecal gluten immunogenic peptides to support the diagnosis and to determine the adherence to the gluten-free diet in coeliac children. METHODS Multicentre prospective observational study including 64 coeliac children. Faecal gluten peptides, and tissue transglutaminase and deamidated gliadin peptide antibodies were analyzed at diagnosis, and 6, 12 and 24 months thereafter. Gluten consumption was estimated from gluten peptide levels. RESULTS Most children (97%) had detectable gluten peptides at diagnosis. On a gluten-free diet, the rate of gluten peptides increased from 13% at 6 months to 25% at 24 months. Mean estimated gluten exposure dropped from 5543 mg/d at diagnosis to 144 mg/d at 6 months, then increased to 606 mg/d by 24 months. In contrast, deamidated gliadin peptide antibodies normalised and only 20% had elevated tissue transglutaminase antibody by 24 months. The elevation of tissue transglutaminase antibody was more prolonged in patients with detectable gluten peptides (P < 0.05). Nevertheless, absolute levels of tissue transglutaminase antibody had low sensitivity to identify patients with detectable gluten peptides (P > 0.1). Dietitian assessment was only moderately correlated with gluten peptide detection (κ = 0.5). CONCLUSIONS Faecal gluten peptides testing may guide treatment of coeliac disease prior to diagnosis and during the assessment diet adherence. Further studies could determine if early identification of gluten exposure reduces the need for expensive/invasive investigations for non-responsive coeliac disease. ClinicalTrials.gov Number: NCT02711397.