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
- Author(s)
- Hardy, MY; Goel, G; Russell, AK; Chen Yi Mei, SLG; Brown, GJE; Wang, S; Szymczak, E; Zhang, R; Goldstein, KE; Neff, KM; Williams, LJ; Truitt, KE; Dzuris, JL; Tye-Din, JA; Anderson, RP;
- Journal Title
- Frontiers in Immunology
- 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.
- Publisher
- Frontiers Media
- Keywords
- Il-2; T cells; coeliac disease; cytokine release assay; cytokines; diagnosis
- Research Division(s)
- Immunology
- Publisher's Version
- https://doi.org/10.3389/fimmu.2021.661622
- Open Access at Publisher's Site
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- Refer to copyright notice on published article.
Creation Date: 2021-06-10 11:47:03
Last Modified: 2021-06-10 11:59:50