Crosstalk Between Mucosal-Associated Invariant T (MAIT) Cells and the Gut Microbiota and Mucosa in the Development of Type 1 Diabetes in Children

Summary

To investigate in a prospective way changes in Mucosal-Associated Invariant T (MAIT) cells frequency, phenotype and function in link with the gut microbiota, gut integrity and the presence of Coxsackie virus B in two cohorts of pediatric patients: patients with a high genetic risk of type 1 diabetes and pediatric patients with recently diagnosed T1D by comparison with control subjects

Tasks:

1. To measure blood MAIT cells frequency, phenotype and function in the three cohorts
2. To analyze gut microbiota and the presence of Coxsackie B enterovirus (CVB) and their impact on MAIT cell function
3. To evaluate gut integrity and analyze the gut mucosa
4. To integrate all the data obtained with T1D development and evolution

Conditions

• Type1diabetes

Interventions

DIAGNOSTIC_TEST

MAIT cells analysis

For FACS analysis, MAIT cells will be identified as CD3+ CD4- CD161high Vα7.2+ T cells. Surface markers will be analyzed to determine their activation status (CD25, CD69, CD44), their exhaustion (PD1, KLRG1, TIM3), their migration capacity (CCR6), and their proliferation and survival will be analyzed by Ki67 and BCL2 expression.

DIAGNOSTIC_TEST

MAIT cytokines production analysis

Cytokine production will be assessed after PMA-ionomycin activation, followed by intracytoplasmic staining with antibodies against IL-2, IFN-γ TNF-α, IL-2, IL-4, IL-10, IL-13, IL-17 and granzyme B. To determine the capacity of MAIT cells to response to TCR stimulation (exhaustion), in vitro stimulation will be performed in the presence of specific bacterial ligands. Activation marker expression will be analyzed by FACS and cytokines released in the supernatant by Cytometry based assay.

DIAGNOSTIC_TEST

Lactulose/Mannitol Test

After an overnight fast, the patients will drink 50 ml solution of 5 g lactulose and 2 g mannitol. Urine will be collected during before and 5 hours later.

PROCEDURE

UGI Endoscopy

Duodenal biopsy collection and analysis. Biopsies will be analyzed by qPCR for the expression of key epithelial molecules such as the fucosyl transferase 2 (fut2), tight junction proteins (occludin, claudin4), antimicrobial peptides (Reg3, LL37) and mucus component (mucin 2). Immune cells function will also be assessed by q-PCR for key cytokines/molecules such as IL-23, IL-17, IL-22, Foxp3, IL-10, TGFb and CVB.

DIAGNOSTIC_TEST

Coxsackie virus B infection status

Serology against CVB and CVB specific-qPCR measurement in gut microbiota samples will be performed in all patients of the three cohorts, and analysis of the gut mucosa by qPCR and immunochemistry with anti-CVB VP1 protein mAb will be performed on a subset of patients.

DIAGNOSTIC_TEST

Coxsackie virus B infection status

Stool samples are collected and directly kept under anaerobic condition. Within an hour the samples are processed: one fraction is aliquoted and frozen at -80°C and another fraction is used to prepared fecal supernatant for the bioassay of MAIT ligands, aliquoted and frozen. Bioassay to measure the presence of MAIT cell ligands by bioassays using WT3 cell line as well as plate bound MR117. 16S sequencing of all samples and according to the results obtained with the bioassay and 16S, 10 samples will be selected for metagenomic analysis.

Sponsors & Collaborators

• Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement

  collaborator OTHER

• Tampere University

  collaborator OTHER

• Assistance Publique - Hôpitaux de Paris

  collaborator OTHER

• Institut National de la Santé Et de la Recherche Médicale, France

  lead OTHER_GOV

Eligibility

Min Age

12 Months

Max Age

15 Years

Sex

ALL

Healthy Volunteers

Yes

Timeline & Regulatory

Start

2022-01-01

Primary Completion

2026-08-11

Completion

2027-08-14

Countries

• France

Study Locations