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GLUTEN-SPECIFIC CD4+ T CELL DETECTION USING A WHOLE BLOOD INTERLEUKIN-2 RELEASE ASSAY IS SENSITIVE AND SPECIFIC FOR CELIAC DISEASE
Date
May 19, 2024
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Background: Celiac disease (CeD) is prevalent but frequently undiagnosed. Central to its pathogenesis are proinflammatory HLA-DQ-restricted gluten-specific CD4+ T cells in the intestine and circulation. CeD diagnosis relies on celiac serology and duodenal histology, but these are inaccurate in patients not regularly consuming gluten. Immune studies employing tetramers can detect gluten-specific T cells in treated CeD with high sensitivity but are impractical for clinical use. In vivo or in vitro interleukin (IL)-2 release to gluten is a novel marker of gluten-specific T cells and may offer a simple approach to CeD diagnosis.
Aim: Assess the diagnostic performance of IL-2 release in a prototype “gluten challenge in-tube” ex vivo whole blood assay (WBA) and in vivo following oral gluten-challenge in a “real life” community study comprising people with and without CeD.
Methods: Adults with treated CeD (gluten-free diet with negative CeD serology), active CeD (gluten-containing diet), non-celiac gluten sensitivity (NCGS) and healthy controls had blood collected for WBA. Fresh whole blood was incubated for 24 hours with gluten peptides encompassing immunodominant T cell epitopes and IL-2 release assessed (S-plex, MesoScale Discovery). The WBA was repeated weekly in the active CeD cohort for 4 weeks after commencing a gluten-free diet and was performed at two timepoints in a subset of treated CeD participants to assess reproducibility. A subset of treated CeD participants also undertook single-dose gluten challenge (10g vital wheat gluten) and serum IL-2 assessment 4 hours later.
Results: The IL-2 WBA was assessed in 75 treated CeD (70 HLA-DQ2.5 +/- DQ2.2 or DQ8; 3 HLA-DQ8; 1 HLA-DQ7; 1 HLA-DQ2.2), 6 active CeD (all HLA-DQ2.5), 21 NCGS (38% HLA-DQ2.5) and 52 healthy controls (37% HLA-DQ2.5). The WBA had a sensitivity of 83% (68/81) and specificity of 93% (68/73) to detect CeD with an AUC of 0.92 (Fig. 1). Excluding CeD patients on immunosuppressive medication and those with the HLA-DQ8 genotype increased sensitivity to 86% (64/74) and gave an AUC of 0.95. The WBA inter-assay consistency was high (R2=0.88, p=0.002). IL-2 WBA levels in active CeD temporarily increased after commencing a gluten-free diet (Fig. 2). Four hour IL-2 responses after in vivo gluten-challenge in treated CeD (n=15) concorded with IL-2 WBA results.
Conclusions: The IL-2 WBA is sensitive and specific for CeD and has high reproducibility. It can detect CeD in patients adhering to a gluten-free diet with negative CeD serology. CeD patients with negative IL-2 WBA and controls with positive IL-2 WBA are undergoing clinical work-up to review their diagnoses. Correlation of the WBA with gluten-specific T cell phenotype and tetramer frequency is needed and is underway. The IL-2 WBA is a promising non-invasive immune diagnostic for CeD that offers considerable advantages over current approaches.
Aim: Assess the diagnostic performance of IL-2 release in a prototype “gluten challenge in-tube” ex vivo whole blood assay (WBA) and in vivo following oral gluten-challenge in a “real life” community study comprising people with and without CeD.
Methods: Adults with treated CeD (gluten-free diet with negative CeD serology), active CeD (gluten-containing diet), non-celiac gluten sensitivity (NCGS) and healthy controls had blood collected for WBA. Fresh whole blood was incubated for 24 hours with gluten peptides encompassing immunodominant T cell epitopes and IL-2 release assessed (S-plex, MesoScale Discovery). The WBA was repeated weekly in the active CeD cohort for 4 weeks after commencing a gluten-free diet and was performed at two timepoints in a subset of treated CeD participants to assess reproducibility. A subset of treated CeD participants also undertook single-dose gluten challenge (10g vital wheat gluten) and serum IL-2 assessment 4 hours later.
Results: The IL-2 WBA was assessed in 75 treated CeD (70 HLA-DQ2.5 +/- DQ2.2 or DQ8; 3 HLA-DQ8; 1 HLA-DQ7; 1 HLA-DQ2.2), 6 active CeD (all HLA-DQ2.5), 21 NCGS (38% HLA-DQ2.5) and 52 healthy controls (37% HLA-DQ2.5). The WBA had a sensitivity of 83% (68/81) and specificity of 93% (68/73) to detect CeD with an AUC of 0.92 (Fig. 1). Excluding CeD patients on immunosuppressive medication and those with the HLA-DQ8 genotype increased sensitivity to 86% (64/74) and gave an AUC of 0.95. The WBA inter-assay consistency was high (R2=0.88, p=0.002). IL-2 WBA levels in active CeD temporarily increased after commencing a gluten-free diet (Fig. 2). Four hour IL-2 responses after in vivo gluten-challenge in treated CeD (n=15) concorded with IL-2 WBA results.
Conclusions: The IL-2 WBA is sensitive and specific for CeD and has high reproducibility. It can detect CeD in patients adhering to a gluten-free diet with negative CeD serology. CeD patients with negative IL-2 WBA and controls with positive IL-2 WBA are undergoing clinical work-up to review their diagnoses. Correlation of the WBA with gluten-specific T cell phenotype and tetramer frequency is needed and is underway. The IL-2 WBA is a promising non-invasive immune diagnostic for CeD that offers considerable advantages over current approaches.
Figure 1. IL-2 WBA response in treated CeD, active CeD, healthy and NCGS cohorts.
Figure 2. IL-2 WBA response in active CeD whilst eating gluten (Day 0; D0), and weekly for 4 weeks after starting a gluten-free diet (W1-W4).
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