This study investigates how the immune system of children with celiac disease responds to controlled, small amounts of gluten. Children on a strict gluten-free diet are randomly assigned to receive either placebo, 50 mg of gluten, or 5 g of gluten once daily for three days, simulating real-life accidental exposure or dietary transgression. Blood samples are collected on Day 1 (before gluten intake) and Day 8 (five days after the last dose). Stool and urine samples are also collected for complementary analyses.

Using single-cell ribonucleic acid (RNA) sequencing, T-cell receptor sequencing, microRNA profiling, and exploratory metabolomics, the study aims to characterize changes in immune cell populations and gene expression after gluten exposure. The objective is to determine whether even very small amounts of gluten induce measurable systemic immune responses and whether these responses differ according to the dose administered. Understanding these mechanisms may support the development of new biomarkers and improve clinical management of pediatric celiac disease.

Celiac disease is a chronic, immune-mediated enteropathy triggered by gluten ingestion in genetically predisposed individuals. In pediatric patients, strict adherence to a gluten-free diet (GFD) leads to mucosal recovery and clinical improvement; however, the systemic immunological landscape during long-term GFD remains incompletely understood. Accidental gluten exposure is common in daily life and may trigger subclinical immune activation even in the absence of overt symptoms. The biological consequences of such low-dose exposures, particularly at the single-cell level, are not fully characterized in children.

CELLiomicS (Single-cell Immune Response to Controlled Gluten Ingestion in Pediatric Celiac Disease) is a randomized, controlled, parallel-group clinical study designed to characterize systemic immune responses to controlled gluten ingestion in pediatric patients with established celiac disease. Eligible participants are children aged 8-14 years who have followed a strict GFD for at least 18 months before enrollment. The minimum estimated sample size was 45 participants with biopsy- or serology-confirmed celiac disease. Anticipating dropout rate or non-analyzable data of approximately 10-15%, the study planned to recruit a total of 51 participants.

Participants are randomized into three intervention groups: placebo, 50 mg gluten, or 5 g gluten. The selected gluten doses represent two clinically relevant scenarios: (1) accidental low-level exposure (50 mg), and (2) dietary transgression (5 g), both below the threshold typically required to cause serological relapse. Gluten or placebo is administered once daily on Days 1, 2, and 3. Blood samples are obtained on Day 1 (baseline) and Day 8 (five days after the final dose). Stool and urine samples are also collected at the same time points for complementary analyses, including fecal gluten immunogenic peptides (GIP) and exploratory metabolomics.

Peripheral blood mononuclear cells are processed for high-dimensional immune profiling using single-cell RNA sequencing and T-cell receptor (TCR) sequencing (10x Genomics). These analyses enable characterization of immune cell composition, transcriptional activity, and clonal T-cell dynamics before and after gluten challenge. In addition, exosomal microRNA expression is assessed as approved by the ethics committee amendment SICEIA-2025-001434, and untargeted metabolomic profiling of blood, stool, and urine samples is performed to explore systemic metabolic signatures associated with gluten exposure.

The primary objective is to identify gluten-induced transcriptional and cellular changes in peripheral immune cells between baseline (Day 1) and post-intervention (Day 8). Secondary objectives include: (1) assessing dose-dependent immune activation; (2) identifying immune cell subsets and gene expression programs associated with different gluten quantities; (3) characterizing TCR repertoire alterations following gluten exposure; (4) evaluating inter-individual variability in immune responses; (5) analyzing exosomal microRNA changes; and (6) identifying metabolomic signatures associated with gluten ingestion.

By providing a high-resolution view of systemic immune activation following controlled gluten ingestion, this study aims to deepen understanding of the biological impact of real-world gluten exposure in pediatric celiac disease and to support the development of novel biomarkers for dietary monitoring and personalized clinical management.

Inclusion Criteria:

• Age 8 to 14 years at study entry.
• Diagnosis of celiac disease according to ESPGHAN 2020 criteria.
• At least 18 months on a strict gluten-free diet (GFD).
• Adequate adherence to the GFD, demonstrated by negative fecal gluten immunogenic peptides (GIP) prior to inclusion.
• Asymptomatic from a gastrointestinal perspective in the preceding weeks.
• Ability to swallow the gluten/placebo preparation.
• Written informed consent from parents/legal guardians and assent from the child.

Exclusion Criteria:

• Obesity defined as BMI ≥ 95th percentile according to WHO criteria.
• Diagnosed inflammatory bowel disease or diabetes mellitus.
• Acute infectious illness at the time of inclusion.
• Chronic hepatic, pulmonary, renal, or rheumatologic disease.
• History of severe acute reactions to accidental gluten ingestion.
• Use of oral corticosteroids or immunosuppressive therapy in the previous 3 months.
• Any condition that, in the opinion of the investigators, may contraindicate participation or compromise study integrity.

Individual participant data (IPD) will not be shared because the dataset contains highly sensitive genomic, transcriptomic, and immunological data from pediatric participants. Data cannot be sufficiently anonymized to ensure participant privacy under GDPR regulations. Only aggregated results will be made publicly available.

Quadruple-blind (participant, care provider, investigator, outcomes assessor). Only three designated team members not involved in routine clinical assessment or outcome evaluation have access to the randomization list in order to prepare the gluten/placebo doses and monitor safety. Data entry and statistical analyses are performed using a blinded database.