An optimised whole blood interleukin‐2 release assay is more sensitive than interferon‐γ ELIS pot for detecting and quantifying gluten‐specific CD 4 + T‐cell responses in coeliac disease

Figures

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  Figure 1. IL‐2 WBA and IFN‐γ ELISpot responses across concentrations and days in treated CD. IL‐2 fold change (peptide/PBS) from WBA stimulation with PBS or 0.2, 1 or 5 μg mL−1 of peptide (left) and IFN‐γ SFU (per million PBMC) from ELISpot stimulation with PBS or 1, 5 or 25 μg mL−1 of peptide (right) are shown for α‐gliadin (a, b), ω‐gliadin (c, d), and an equimolar pool of α‐gliadin and ω‐gliadin (e, f) in treated coeliac disease (CD) participants (n = 10) at baseline (Day 1) and Days 6–8 post gluten challenge. Median and 95% confidence intervals are shown. Dashed lines indicate calculated cutoffs. Friedman tests were performed to compare peptide concentrations; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 2. Comparison of IL‐2, IFN‐γ and IL‐10 WBA responses in treated CD and NCGS. IL‐2 (green), IFN‐γ (blue) and IL‐10 (purple) fold change from WBA stimulation with 5 μg mL−1 of α‐gliadin, ω‐gliadin, and α‐gliadin and ω‐gliadin at Day 1 (left) and Day 6 (right) in treated coeliac disease (CD) participants (top, n = 10, a, b) and non‐coeliac gluten sensitive (NCGS, c, d) participants (bottom, n = 3). Dashed lines indicate a twofold cutoff. Median and 95% confidence intervals are shown. Friedman tests were performed to compare cytokine responses; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 3. IL‐2 WBA reflects epitope hierarchy and immune modulation. IL‐2 fold change (peptide/PBS) from WBA stimulation with 5 μg mL−1 of peptide (left) and IFN‐γ SFU (per million PBMC) from ELISpot stimulation with 25 μg mL−1 of peptide (right) in treated coeliac disease (CD) participants (n = 10) at baseline (circle) and Day 6 (triangle) post gluten challenge (a, b). Responses to stimulation with PBS, α‐gliadin, ω‐gliadin, α‐gliadin and ω‐gliadin, γ‐gliadin and hor3a are shown. Responses to stimulation with α‐gliadin (blue) or ω‐gliadin (purple) alone (−, circle), with anti‐HLA‐DQ (+DQ, square), or with anti‐HLA‐DR (+DR, triangle) are shown (c, d, n = 4/5 for ELISpot). Dashed lines indicate calculated cut‐offs. Median and 95% confidence intervals are shown. Friedman tests were performed to compare response to different stimulation conditions; *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 4. Oral gluten challenge serum IL‐2 response and symptoms. Heatmap of patient‐reported symptom scores after single bolus gluten challenge are shown for 10 treated coeliac disease (CD) and three non‐coeliac gluten sensitive (NCGS) participants (a). Maximum reported severity scores are shown, ranging from 0 (no symptoms; white) to 10 (severe symptoms; red). Serum IL‐2 fold change (b) and concentration (c, 4 h compared to 0 h) are shown. Participants that vomited following gluten challenge are indicated in red. A 2‐fold cut‐off is indicated by the dashed line. IL‐2 concentrations below zero were set at 0.001 to allow graphing on a log10 scale. Median and 95% confidence intervals are shown. Mann–Whitney T tests were performed; **P < 0.01.