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

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

Detailed Description
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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.

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

Conditions
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Type1diabetes

Intervention / Treatment
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* Diagnostic Test: MAIT cells analysis
* Diagnostic Test: MAIT cytokines production analysis
* Diagnostic Test: Lactulose/Mannitol Test
* Procedure: UGI Endoscopy
* Diagnostic Test: Coxsackie virus B infection status
* Diagnostic Test: Coxsackie virus B infection status


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: 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 Criteria: 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

Ages Eligible for Study
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Minimum Age: 12 Months
Maximum Age: 15 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| recent onset patients<br>patients with recently diagnosed type 1 diabetes | Diagnostic Test: MAIT cells analysis<br>* 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.<br>Diagnostic Test: MAIT cytokines production analysis<br>* 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.<br>Diagnostic Test: Lactulose/Mannitol Test<br>* 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.<br>Procedure: UGI Endoscopy<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>|
| at risk patients<br>patients with a high genetic risk type 1 diabetes | Diagnostic Test: MAIT cells analysis<br>* 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.<br>Diagnostic Test: MAIT cytokines production analysis<br>* 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.<br>Diagnostic Test: Lactulose/Mannitol Test<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>|
| control subjects<br>control patients (no risk of type 1 diabetes and no diagnosed type 1 diabetes) | Diagnostic Test: MAIT cells analysis<br>* 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.<br>Diagnostic Test: MAIT cytokines production analysis<br>* 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.<br>Diagnostic Test: Lactulose/Mannitol Test<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>|
| control subjects for endoscopy<br>patients without type 1 diabetes requiring UGI endoscopy for any medical reason | Diagnostic Test: MAIT cells analysis<br>* 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.<br>Diagnostic Test: MAIT cytokines production analysis<br>* 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.<br>Procedure: UGI Endoscopy<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>Diagnostic Test: Coxsackie virus B infection status<br>* 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.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| blood MAIT cells frequency | percentage of MAIT cells in the peripheral blood | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| MAIT cells membrane markers analysis | CD25 positive cells in percentage, CD69 positive cells in percentage, CD44 positive cells in percentage, PD1 positive cells in percentage , KLRG1 positive cells in percentage, TIM3 positive cells in percentage | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| Cytokines production in the cytoplasm of the MAIT | Antibodies against IL- 2 , IFN-y, TNF-α, IL-2, IL-10, IL-13, IL-17 and granzyme B expressed in Cells % | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| MAIT cell ligands measurements | Percentage of MAIT cells activated in vitro when cultivated in presence of subject stool sample | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| Presence of CVB in the stools | CVB specific ARN detection in subjects stools samples by qPCR | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| Blood immunoglobulins against CVB infection statu | Specific antibodies (Ig G) against CVB measurements in patients blood sample | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| Measurement of intestinal gut mucosal permeability | Assessing the permeability of the intestine using the Lactulose-Mannitol test. Concentration of lactulose and manitol in subjects urine samples in mg/dl | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| Evaluation of gut mucosal integrity | qPCR for the expression of key epithelial molecules and cytokines on gut samples | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) | 
| 16 S sequencing in the Subjects stools | The 16S rRNA gene sequencing will be used for the classification and identification of microbes species in subjects stools. Each species will be identified and quantified as a percentage of the total number of identified species in the stools. | Sample collected the day of enrollment (Control group and at risk patients) or up to 7 days after diagnosis and day of enrollment (recent onset group) |