Description

Subjects: Multi study. Subjects will be included in the pediatric diabetes and endocrinology unit in Necker Sick children hospital and in the pediatric unit of ANTONY hospital.

• Recent-onset (RO) n=40: New onset patients will be included shortly after diagnosis.
• At risk (AR) cohort n=70: Routinely screened siblings of T1D patients previously tested positive for HLA DR3 and DR4 will be asked to be a part of the study.
• Control (C) cohort (n=50): Control subjects will be patients consulting at Necker Hospital for endocrine testing
• Control for Endoscopy (CE) n= 20: patients consulting at Necker Hospital or at Antony hospital for UGI endoscopy

  Analysis

MAIT cell 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. 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.

Gut microbiota analysis: 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.

Coxsackie virus B (CVB) infection status: Specific antibodies against coxsackie virus B 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 q-PCR and immunochemistry will be performed on a subset of patients.

Gut integrity: the investigators will assess the permeability of the intestine using the Lactulose-Mannitol test in a sub sample of RO, RD and AR groups (> 5 years of age, n=20). In brief 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 after ingestion.

Gut mucosa analysis: In a subset of patients (without celiac disease as determined by the dosage of antibodies against transglutaminase), duodenal biopsies will be obtained during an IUG endoscopy . Duodenal biopsy will be performed in RD subjects older than 8 years of age and in CE subjects older than 4 years of age. These 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.

Based on previous study, the sample size should allow statistical differences between AR, RO and C groups. The investigators anticipate to observe blood MAIT cell abnormalities in RO patients and some at risk children after seroconversion but before diabetes onset. This new data will strengthen our predictive model (see preliminary data). Since MAIT cells recognize bacterial ligand we hypothesize that MAIT cells alteration could occur in parallel with microbiota changes and/or CVB infection. The investigators anticipate observing gut mucosa abnormalities in RO children and the severity of these abnormalities may correlate with the level of MAIT cells defect and the presence of CVB infection. The investigators expect to demonstrate that MAIT cells represent a new biomarker of progression toward diabetes as well as a functional immune marker of the aggressiveness of the autoimmune disease. As such this study could determine the accurate therapeutic window for preventive strategies based on MAIT cells manipulation.