Single-cell Immune Response to Controlled Gluten Ingestion in Pediatric Celiac Disease (CELLiomicS)

Single-cell Study of the Systemic Immune Response to Controlled Gluten Intake in Pediatric Celiac Disease

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.

Study Overview

• Status: Recruiting
• Conditions: Autoimmune Diseases; Celiac Disease; Gluten Sensitivity
• Intervention / Treatment: Dietary supplement: Placebo; Procedure: Collection of blood, stool and urine samples on Day 1 and Day 8.; Dietary supplement: Gluten 50 mg; Dietary supplement: Gluten 5 g

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 51
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Rafael Martín Masot, MD, PhD; Phone Number: +34 951290000; Email: rafammgr@uma.es
• Study Contact Backup: Name: Lara María Bossini Castillo, MD, PhD; Phone Number: +34 663148283; Email: lbossinicastillo@ugr.es

Study Locations

• Spain
  • Málaga, Spain, 29010
    • Recruiting
    • Hospital Regional Universitario de Malaga
    • Contact: Rafael Martín Masot, MD, PhD; Phone Number: +34 951 290 000; Email: rafammgr@uma.es
    • Contact: Lara María Bossini Castillo, MD, PhD; Email: lbossinicastillo@ugr.es
    • Principal Investigator: Rafael Martín Masot, MD, PhD
    • Principal Investigator: Lara María Bossini Castillo, MD, PhD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child
• Accepts Healthy Volunteers: No

Description

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.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Quadruple

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Placebo Comparator: Placebo Group; Participants receive a placebo preparation once daily on Days 1, 2, and 3. Blood samples are collected on Day 1 (baseline) and Day 8 (post-intervention). Participants and evaluators remain blinded to allocation.	Dietary supplement: Placebo; Placebo preparation identical in appearance and administration schedule to gluten doses. Administered once daily on Days 1, 2, and 3. Used as the comparator. Participants and evaluators remain blinded to allocation.; Procedure: Collection of blood, stool and urine samples on Day 1 and Day 8.; In addition to blood samples, stool and urine samples will be collected for complementary analyses (including fecal gluten immunogenic peptides (GIP) and exploratory metabolomic assays)
Experimental: Low-Dose Gluten (50 mg); Participants receive 50 mg of gluten once daily on Days 1, 2, and 3, simulating accidental low-level exposure. Blood samples are collected on Day 1 (baseline) and Day 8 (post-intervention). Participants and evaluators remain blinded to allocation.	Procedure: Collection of blood, stool and urine samples on Day 1 and Day 8.; In addition to blood samples, stool and urine samples will be collected for complementary analyses (including fecal gluten immunogenic peptides (GIP) and exploratory metabolomic assays); Dietary supplement: Gluten 50 mg; Participants receive 50 mg of gluten once daily on Days 1, 2, and 3. This dose simulates accidental low-level gluten exposure in children with celiac disease. Participants and evaluators remain blinded to allocation.
Experimental: High-Dose Gluten (5 g); Participants receive 5 g of gluten once daily on Days 1, 2, and 3, simulating a dietary transgression. Blood samples are collected on Day 1 (baseline) and Day 8 (post-intervention). Participants and evaluators remain blinded to allocation.	Procedure: Collection of blood, stool and urine samples on Day 1 and Day 8.; In addition to blood samples, stool and urine samples will be collected for complementary analyses (including fecal gluten immunogenic peptides (GIP) and exploratory metabolomic assays); Dietary supplement: Gluten 5 g; Participants receive 5 g of gluten once daily on Days 1, 2, and 3. This dose simulates a dietary transgression in children with celiac disease. Participants and evaluators remain blinded to allocation.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in peripheral blood immune cell gene expression after controlled gluten ingestion	Change from baseline (Day 1) to post-intervention (Day 8) in gene expression profiles of peripheral blood immune cells measured by single-cell RNA sequencing (scRNA-seq).	Day 1 to Day 8
Change in peripheral blood T-cell receptor repertoire after controlled gluten ingestion	Change from baseline (Day 1) to post-intervention (Day 8) in T-cell receptor (TCR) diversity and clonality in peripheral blood T cells measured by TCR sequencing	Day 1 to Day 8

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Dose-dependent changes in immune gene expression after gluten exposure	Change from baseline (Day 1) to post-intervention (Day 8) in immune-related gene expression levels measured by single-cell RNA sequencing (scRNA-seq), comparing placebo, 50 mg gluten, and 5 g gluten groups.	Day 1 to Day 8
Dose-dependent changes in T-cell receptor repertoire clonality after gluten exposure	Change from baseline (Day 1) to post-intervention (Day 8) in T-cell receptor (TCR) clonality and diversity measured by TCR sequencing, comparing placebo, 50 mg gluten, and 5 g gluten groups.	Day 1 to Day 8
Inter-individual variability in immune gene expression response to gluten exposure across participants	Inter-individual variability in changes from baseline (Day 1) to post-intervention (Day 8) in immune gene expression profiles measured by single-cell RNA sequencing (scRNA-seq).	Day 1 to Day 8
Changes in serum exosomal microRNA expression after gluten exposure	Change from baseline (Day 1) to post-intervention (Day 8) in serum exosomal microRNA expression levels.	Day 1 to Day 8
Changes in metabolomic profiles after gluten exposure	Change from baseline (Day 1) to post-intervention (Day 8) in untargeted metabolomic profiles measured in blood, stool, and urine samples.	Day 1 to Day 8
Changes in peripheral blood immune cell type proportions after gluten exposure	Change from baseline (Day 1) to post-intervention (Day 8) in the relative proportions of immune cell types identified in peripheral blood by single-cell RNA sequencing (scRNA-seq).	Day 1 to Day 8

Collaborators and Investigators

• Sponsor: Fundación Pública Andaluza para la Investigación de Málaga en Biomedicina y Salud
• Collaborators: Andaluz Health Service; Fundación Pública Andaluza para la Investigación Biomédica Andalucía Oriental; Hospital Regional de Malaga; Biohealth Research Institute in Granada (ibs.GRANADA); Consorcio Centro de Investigación Biomédica en Red (CIBER); Universidad de Málaga
• Investigators: Principal Investigator: Rafael Martín Masot, MD, PhD, Hospital Regional Universitario de Málaga / IBIMA-BIONAND; Principal Investigator: Lara María Bossini Castillo, MD, PhD, IBS Granada

Study record dates

Study Major Dates

• Study Start (Actual): July 7, 2025
• Primary Completion (Estimated): April 1, 2026
• Study Completion (Estimated): November 1, 2026

Study Registration Dates

• First Submitted: December 17, 2025
• First Submitted That Met QC Criteria: January 15, 2026
• First Posted (Actual): January 23, 2026

Study Record Updates

• Last Update Posted (Actual): April 27, 2026
• Last Update Submitted That Met QC Criteria: April 22, 2026
• Last Verified: November 1, 2025

More Information

Terms related to this study

• Keywords: Pediatrics; Immune Response; Celiac Disease; Omics; Gluten-Free Diet; Single-Cell RNA Sequencing; Gluten Exposure
• Additional Relevant MeSH Terms: Metabolic Diseases; Intestinal Diseases; Immune System Diseases; Digestive System Diseases; Gastrointestinal Diseases; Malabsorption Syndromes; Nutritional and Metabolic Diseases; Autoimmune Diseases; Celiac Disease; Amino Acids, Peptides, and Proteins; Proteins; Digestive System Physiological Phenomena; Digestive System and Oral Physiological Phenomena; Plant Proteins; Prolamins; Grain Proteins; Seed Storage Proteins; Defecation; Glutens
• Other Study ID Numbers: PI-0070-2024

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: 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.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No