Diagnosis of celiac disease on a gluten-free diet: a multicenter prospective quasi-experimental clinical study

Abstract

Background: Diagnosing celiac disease (CD) in individuals adhering to a gluten-free diet (GFD) presents significant challenges. Current guidelines recommend a gluten challenge (GC) lasting at least 6-8 weeks, which has several limitations. Our aim was to compare four approaches previously proposed for diagnosing CD on a GFD: IL-2 serum levels, gut-homing CD8⁺ T cells, % TCRγδ⁺ intraepithelial lymphocytes (IELs), and UBE2L3 gene expression. Additionally, we evaluated the CD8⁺ T-cell-based method with a 3-day GC against the standard GC protocol.

Methods: We conducted a multicenter prospective quasi-experimental clinical study. Two subsets of individuals were considered: (1) 20 patients with CD previously diagnosed and 15 non-CD controls, to evaluate the first aim; (2) 41 individuals with uncertain diagnosis who were on a GFD and required GC following current clinical guidelines, to assess the second aim. All participants underwent a 3-day GC (10 g gluten/day).

Results: Among CD patients and non-CD controls, the sensitivity and specificity of IL-2, gut-homing CD8⁺ T cells, and UBE2L3 were 82.4% and 83.3%, 88.2% and 100%, and 52.9% and 100%, respectively. The percentage of TCRγδ⁺ IELs showed 88.2% sensitivity. In the uncertain diagnosis group, a CD8⁺ T-cell positive response was observed in 8 of the 41 subjects.

Conclusions: The percentage of TCRγδ⁺ IELs and the analysis of IL-2 levels and gut-homing CD8⁺ T cells are promising diagnostic methods for CD on a GFD. Notably, our results suggest that the CD8⁺ T-cell assay may provide a consistent and reliable alternative to the extended GC, eliminating the need for invasive procedures to obtain duodenal samples and prolonged gluten ingestion. However, further research with larger cohorts are necessary to validate these findings and establish their definitive clinical utility.

Keywords: CD8+ T cells; Celiac disease; Gluten challenge; Gluten-free diet.

Fig. 1

Design of the two studies presented. Number and type of participants, along with the timing of sample collection and the type of analysis performed are included. ¹In patients with CD and SGRS. ²In patients with CD. 4 h, 4 hours; anti-TG2, anti-transglutaminase type 2; CD, celiac disease; GC, gluten challenge; GIP, gluten immunogenic peptides; IELs, intraepithelial lymphocytes; SGRS, suspected gluten-related functional bowel disease symptoms

Fig. 2

Baseline UBE2L3 gene expression data. UBE2L3 Ct exon 4/Ct exon 5 ratio in subjects from each study group. The dotted red line indicates the cut-off for CD diagnosis. Red dots represent subjects with positive fecal GIP. Centerlines in the box represent the median; box limits indicate the 25th and 75th percentiles (IQR)

Fig. 3

Baseline data of the percentage of TCRγδ⁺ intraepitelial lymphocytes (IELs) in patients with celiac disease (CD). The dotted red line indicates the cut-off for CD diagnosis. Red dots represent subjects with positive fecal GIP

Fig. 4

Serum IL-2 data. IL-2 concentration baseline and after 4 h of gluten challenge is shown in subjects from each study group: a patients with celiac disease (CD), and b non-CD controls. The dotted red line indicates the cut-off for CD diagnosis. Lines connect results from individual patients. Centerlines in the box show the median; box limits indicate the 25th and 75th percentiles (IQR). Red dots represent subjects with positive fecal GIP

Fig. 5

Gut-homing CD8⁺ T-cell responses. Percentage of activated gut-homing CD8⁺ T cells baseline and on day 6 after starting the 3-day gluten challenge is shown in subjects from each study group: a patients with celiac disease, b patients with suspected gluten-related functional bowel disease symptoms (SGRS), c healthy controls, and d individuals with uncertain diagnosis. In patients with a response at day 6 exceeding the cut-off (dotted red line), the percentage of CD8⁺ CD103⁺ β7^hi CD38⁺ CCR9⁺ cells/total CD8⁺ cells is also shown. Note that including CCR9 eliminates the two false positives (green dots) in the group of uncertain diagnosis. Red dots represent subjects with positive fecal GIP