{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T16:12:59Z","timestamp":1790784779743,"version":"4.1.0"},"reference-count":28,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,5,19]],"date-time":"2021-05-19T00:00:00Z","timestamp":1621382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>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 (\u201cRESET CeD\u201d) 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-\u03b3 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 \u03bcg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-\u03b3 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-\u03b3 before and after gluten challenge. IL-2 and IFN-\u03b3 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-\u03b3 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-\u03b3 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.<\/jats:p>","DOI":"10.3389\/fimmu.2021.661622","type":"journal-article","created":{"date-parts":[[2021,5,19]],"date-time":"2021-05-19T14:00:21Z","timestamp":1621432821000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":34,"title":["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"],"prefix":"10.3389","volume":"12","author":[{"given":"Melinda Y.","family":"Hardy","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gautam","family":"Goel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy K.","family":"Russell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Swee Lin G.","family":"Chen Yi Mei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregor J. 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