A WHOLE BLOOD INTERLEUKIN-2 RELEASE ASSAY OFFERS HIGHER SENSITIVITY TO DETECT GLUTEN-SPECIFIC CD4+ T CELLS THAN INTERFERON-γ ELISPOT IN CELIAC DISEASE AND ACCURATELY REFLECTS GLUTEN PEPTIDE IMMUNOGENICITY

Introduction: Celiac disease (CeD) is caused by proinflammatory gluten-specific CD4+ T cells that reside in the small intestine and circulation at low frequency. Approaches to detect these T cells have relied on short-term gluten challenge to expand the cells in blood followed by detection using interferon (IFN)-γ ELISpot or tetramers specific for immunodominant epitopes, both of which are highly sensitive but technically demanding. In vivo or in vitro interleukin (IL)-2 release to gluten is a novel marker of gluten-specific T cells and may offer a simpler approach to detecting these T cells and assessing their specificity.

Aim: Assess the performance of IL-2 release in an in vitro whole blood assay (WBA) before and after in vivo expansion of gluten-specific T cells by oral gluten-challenge and compare these readouts to existing T cell assessment tools based on IFN-γ ELISpot and tetramers.

Methods: Adults with treated CeD underwent a single-dose gluten challenge (10g vital wheat gluten). IL-2 WBA and IFN-γ ELISpot (using peripheral blood mononuclear cells) were performed at baseline and day 6 after gluten challenge. For the WBA, fresh whole blood was incubated with gluten peptides with assessment of IL-2 release (S-plex, MesoScale Discovery) after 24 hours. A series of peptides encompassing epitopes of varying immunogenicity were assessed in the WBA and IFN-γ ELISpot. The effect of pan HLA-DQ blocking using SPVL3 was also assessed.

Results: Ten adults with treated HLA-DQ2.5+ CeD following a gluten-free diet were recruited. At baseline, significant responses to peptides encompassing the immunodominant α-gliadin or ω-gliadin epitopes were seen in 70% by IL-2 WBA and 10% by IFN-g ELISpot. Six days post challenge, significant responses to these peptides were increased to 90% by IL-2 WBA and 40% by IFN-g ELISpot. HLA-DQ blocking significantly reduced the WBA response by a mean of 30-fold (Wilcoxon matched-pairs test, p=0.002). IL-2 WBA responses enabled ranking of peptide immunogenicity (mean IL-2 stimulation after gluten challenge by 5mg/ml of α-gliadin 15pg/ml; ω-gliadin 9.2 pg/ml; γ-gliadin 2 pg/ml; Hor3a 1.3 pg/ml; negative control 0.3 pg/ml). The single patient with entirely negative functional T cell assays to gluten is receiving work-up to confirm their CeD diagnosis. Tetramer data is pending.

Conclusions: The IL-2 WBA is more sensitive than IFN-γ ELISpot in detecting the gluten-specific T cell before and after gluten challenge in CeD. It can measure the functional blockade of disease-associated HLA and can accurately reflect the dominance hierarchies of immunogenic gluten peptides concordant with published data. These findings indicate the IL-2 WBA offers considerable advantages over IFN-γ ELISpot for detection and tracking of gluten-specific T cells and may better support clinical trial immunomonitoring and T cell epitope mapping and discovery.