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

ConditionType1diabetes

Trial statusRecruiting

Trial phaseNot listed

Trial typeObservational

Biological sexAll

Age12-15

SponsorInstitut National de la Santé Et de la Recherche Médicale, France

About this trial

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

Eligibility criteria

This trial accepts healthy volunteers

Qualifiers

age > 12 months and < 15 years

recently diagnosed type 1 diabetes according ISPAD criteria

age > 12 months and < 15 years

siblings of type 1 diabetic patient

Disqualifiers

no health care insurance

parents or tutors unable to sign the consent

personal history of autoimmune disease and/or inflammatory disease except from T1D for RD and CE groups

use of corticosteroids during the month before inclusion

Trial population

Study population: patients recently diagnosed with type 1 diabetes and patients with a high risk of type 1 diabetes

Trial design

Design model

Case-control

Time perspective

Prospective

Treatments tested in this trial

MAIT cells analysis
Diagnostic test
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.
MAIT cytokines production analysis
Diagnostic test
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.
Lactulose/Mannitol Test
Diagnostic 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.
UGI Endoscopy
Procedure/Surgery
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.
Coxsackie virus B infection status
Diagnostic test
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.
Coxsackie virus B infection status
Diagnostic test
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.

Treatment groups

180 Participants

are divided into 4 treatment groups

Group A: recent onset patients5 interventions

Group B: at risk patients4 interventions

Group C: control subjects4 interventions

Group D: control subjects for endoscopy4 interventions

Trial outcomes