Crosstalk Between Mucosal-Associated Invariant T (MAIT) Cells and the Gut Microbiota and Mucosa in the Development of Type 1 Diabetes in Children
Crosstalk Between Mucosal-Associated Invariant T Cells and the Gut Microbiota and Mucosa in the Development of Type 1 Diabetes in Children
Institut National de la Santé Et de la Recherche Médicale, France
180 participants
Jan 1, 2022
OBSERVATIONAL
Conditions
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
Eligibility
Inclusion Criteria14
- Recent onset group
- age \> 12 months and \< 15 years
- recently diagnosed type 1 diabetes according ISPAD criteria
- At risk subjects:
- age \> 12 months and \< 15 years
- siblings of type 1 diabetic patient
- HLA DR3 and DR4 positive
- Control subjects:
- age \> 12 months and \< 15 years
- no HLA associated with high risk type 1 diabetes
- no antibodies against pancreas antigenes
- Control subjects for UGI endoscopy:
- age \> 12 months and \< 15 years
- suspicion of coeliac disease or gastritis
Exclusion Criteria12
- For all groups:
- 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
- pregnant subjects
- medical contraindication of anesthetic topics
- For control subjects for UGI endoscopy control and Recent onset-endoscopy group:
- age below 8 years for Recent onset-endoscopy group
- age below 4 for UGI endoscopy control group
- cardiac or respiratory insufficiency, cardiac rhythm disorders, coagulation disease, patients treated with anticoagulant or antiaggregant drug
- history of allergy to anesthetic drug
Interventions
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.
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.
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.
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.
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.
Locations(2)
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NCT05054361