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.
