Evaluating Responses to Gluten Challenge: A Randomized, Double-Blind, 2-Dose Gluten Challenge Trial

Abstract

Background & aims: Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology.

Methods: In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence.

Results: All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose-response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot, gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2-restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or near significantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure.

Conclusions: Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796.

Keywords: Biomarkers; Celiac Disease; Gluten Challenge; T Cells.

Figure 1.

Change in Vh:Cd and IEL count following gluten challenge. Change in duodenal mucosal biopsy Vh:Cd (A) and IEL count (B) after 3 g or 10 g gluten. Boxplots represent medians and quartiles, and lines between data points connect results from individual patients.. P values calculated using paired one-sided Student t-test for Vh:Cd and one-sided Wilcoxon signed-rank test for IEL count. GC, gluten challenge; IEL, intraepithelial lymphocyte; Vh:Cd, villous height:crypt depth ratio.

Figure 2.

Characterization of small intestine damage by VCE. Small intestine changes detected by VCE in patients receiving 3 g or 10 g gluten. Lines between data points connect results from individual patients P values calculated using one-sided Wilcoxon signed-rank test. GC, gluten challenge; VCE, video capsule endoscopy.

Figure 3.

Change in gluten-specific T cells. Change in gluten-specific T cells measured by IFN-γ ELISpot (A) or HLA-DQ2-gluten tetramer-positive, CD38+ β7+ EM CD4 T cells (B). Lines between data points connect data from individual patients Day 1 and day 6 results were compared, and P values calculated using one-sided Wilcoxon signed-rank test. One patient (3 g gluten) lacked a day 6 sample for ELISpot analysis. CD, cluster of differentiation; ELISpot, enzyme-linked immune absorbent spot; EM, effector memory; GC, gluten challenge; HLA, human leukocyte antigen; IFN-γ, interferon gamma.

Figure 4.

Change in gut-homing CD8+ T cells. Gut-homing EM CD8+ T cells, express α4, β7 and are CD38 bright. Boxplots represent means and the lines between data points connect results from individual patients (n = 12) Day 1 pre-dose and day 6 results were compared, and P values calculated by one-sided Wilcoxon signed-rank test. Two patients (one receiving 3 g and one receiving 10 g of gluten) lacked day 6 samples and were not included in this analysis. CD, cluster of differentiation; EM, effector memory; GC, gluten challenge.

Figure 5.

Change in plasma IL-2. Boxplots represent means. Lines between data points connect data from individual patients. Day 1 pre-dose and post-dose results were compared, and P values calculated by one-sided Wilcoxon signed-rank test. GC, gluten challenge; IL, interleukin; LLOQ, lower limit of quantification.

Figure 6.

Data integration. Biomarker correlations. Maximum biomarker response was compared with both doses pooled and at each gluten dose by Spearman correlation. Vh:Cd change was inverted before calculating correlation because decreasing Vh:Cd signifies increasing severity, while for other markers increasing score reflects increasing severity. CD, cluster of differentiation; CDSD, Celiac Disease Symptom Diary; ELISpot, enzyme-linked immune absorbent spot; IEL, intraepithelial lymphocyte; IL, interleukin; LP, lamina propria; VCE, video capsule endoscopy; Vh:Cd, villous height:crypt depth ratio.