Cytotoxicity-related Gene Expression and Chromatin Accessibility Define a Subset of CD4+ T Cells that Mark Progression to Type 1 Diabetes

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of at-risk children, associated with progression to islet autoimmunity. We analysed gene expression by RNAseq in CD4⁺ and CD8⁺ T cells, NK cells and B cells, and chromatin accessibility by ATACseq in CD4⁺ T cells, in five genetically at-risk children with islet autoantibodies who progressed to diabetes over a median of 3 years (‘Progressors’) compared to five children matched for sex, age and HLA-DR who had not progressed (‘Non-progressors). In Progressors, differentially expressed genes (DEGs) were largely confined to CD4⁺ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 Progressors and 11 Non-progressors. Flow cytometry confirmed progression was associated with expansion of CD4⁺cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4⁺cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4⁺T cells play a role in promoting progression to type 1 diabetes.

Cytotoxicity-related Gene Expression and Chromatin Accessibility Define a Subset of CD4+ T Cells that Mark Progression to Type 1 Diabetes

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of at-risk children, associated with progression to islet autoimmunity. We analysed gene expression by RNAseq in CD4⁺ and CD8⁺ T cells, NK cells and B cells, and chromatin accessibility by ATACseq in CD4⁺ T cells, in five genetically at-risk children with islet autoantibodies who progressed to diabetes over a median of 3 years (‘Progressors’) compared to five children matched for sex, age and HLA-DR who had not progressed (‘Non-progressors). In Progressors, differentially expressed genes (DEGs) were largely confined to CD4⁺ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 Progressors and 11 Non-progressors. Flow cytometry confirmed progression was associated with expansion of CD4⁺cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4⁺cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4⁺T cells play a role in promoting progression to type 1 diabetes.

Cytotoxicity-related gene expression and chromatin accessibility define a subset of CD4+ T cells that mark progression to type 1 diabetes

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of at-risk children, associated with progression to islet autoimmunity. We analysed gene expression by RNAseq in CD4+ and CD8+ T cells, NK cells and B cells, and chromatin accessibility by ATACseq in CD4+ T cells, in five genetically at-risk children with islet autoantibodies who progressed to diabetes over a median of 3 years (‘Progressors’) compared to five children matched for sex, age and HLA-DR who had not progressed (‘Non-progressors). In Progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 Progressors and 11 Non-progressors. Flow cytometry confirmed progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ T cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes.

Relative Frequency of Islet Autoimmunity in Children and Adolescents with Autoimmune Thyroid Disease

The present study aims to investigate islet autoimmunity and susceptibility to type 1 diabetes (T1D) in children/adolescents with autoimmune thyroid disease (AITD), and family members of AITD patients with islet autoimmunity. Islet-cell cytoplasmic, glutamic-acid decarboxylase and tyrosine-phosphatase autoantibodies were measured in 161 AITD patients [(127 with autoimmune thyroiditis (AT); 34 with Graves’ disease (GD)], 20 family members of AITD patients with islet autoimmunity, and 155 age-matched controls. Islet autoimmunity was found in 10.6% of AITD patients, significantly more frequent than in controls (1.9%; p=0.002). Higher prevalence of islet autoantibodies was found in females with AITD (p=0.011) but not in males (p=0.16) as well as in AT (p=0.013) but not GD patients (p=0.19), compared to corresponding controls. Two or three islet autoantibodies were found concurrently in six AITD patients with islet autoimmunity. They all developed T1D and had significantly higher islet autoantibodies titers (p=0.01) compared to AITD patients with single islet autoantibodies but normal glucose metabolism. T1D was found in 3.7% of AITD patients compared to 0.2% in age-matched, general Croatian population. Islet autoantibodies were found in 5/20 family members of AITD patients with islet autoimmunity, among which two developed T1D. None of the controls was positive to more than one islet autoantibodies or developed T1D. Conclusion: Children/adolescents with AITD (particularly females and patients with AT) represent a risk group for islet autoimmunity and T1D, as well as family members of AITD patients with positive islet autoantibodies, but last observation must be examined in a larger number of patients.

Association of high-affinity autoantibodies with type 1 diabetes high-risk HLA haplotypes

Objective ECL assays are high-affinity autoantibody (Ab) tests that are more specific than Abs detected by traditional radiobinding assays (RBA) for risk screening and prediction of progression to type 1 diabetes. We sought to characterize the association of high-risk HLA haplotypes and genotypes with electrochemiluminescence (ECL) positivity and levels in relatives of individuals with type 1 diabetes. Methods We analyzed 602 participants from the TrialNet Pathway to Prevention Study who were positive for at least one RBA diabetes related Ab (GADA or IAA) and for whom ECL and HLA data were available. ECL and RBA Ab levels were converted to SD units away from mean (Z-scores) for analyses. Results Mean age at initial visit was 19.4+13.7 years; 344 (57.1%) were female and 104 (17.3%) carried the high-risk HLA- DR3/4*0302 genotype. At initial visit 424/602 (70.4%) participants were positive for either ECL-GADA or ECL-IAA, and 178/602 (29.6%) were ECL negative. ECL and RBA-GADA positivity were associated with both HLA-DR3 and DR4 haplotypes (all p<0.05), while ECL and RBA-GADA z-score titers were higher in participants with HLA-DR3 haplotypes only (both p<0.001). ECL-IAA (but not RBA-IAA) positivity was associated with the HLA-DR4 haplotype (p<0.05). Conclusions ECL-GADA positivity is associated with the HLA-DR3 and HLA-DR4 haplotypes and levels are associated with the HLA-DR3 haplotype. ECL-IAA positivity is associated with HLA-DR4 haplotype. These studies further contribute to the understanding of genetic risk and islet autoimmunity endotypes in type 1 diabetes.

Dynamic changes in immune gene co-expression networks predict development of type 1 diabetes

Significant progress has been made in elucidating genetic risk factors influencing Type 1 diabetes (T1D); however, features other than genetic variants that initiate and/or accelerate islet autoimmunity that lead to the development of clinical T1D remain largely unknown. We hypothesized that genetic and environmental risk factors can both contribute to T1D through dynamic alterations of molecular interactions in physiologic networks. To test this hypothesis, we utilized longitudinal blood transcriptomic profiles in The Environmental Determinants of Diabetes in the Young (TEDDY) study to generate gene co-expression networks. In network modules that contain immune response genes associated with T1D, we observed highly dynamic differences in module connectivity in the 600 days (~ 2 years) preceding clinical diagnosis of T1D. Our results suggest that gene co-expression is highly plastic and that connectivity differences in T1D-associated immune system genes influence the timing and development of clinical disease.

Telomere Length is Not a Main factor for the Development of Islet Autoimmunity and Type 1 Diabetes in the TEDDY Study

The Environmental Determinants of Diabetes in the Young (TEDDY) study enrolled 8,676 children, 3-4 months of age, born with HLA-susceptibility genotypes for islet autoimmunity (IA) and type 1 diabetes (T1D). Whole-genome sequencing (WGS) was performed in 1,119 children in a nested case-control study design. Telomere length was estimated from WGS data using five tools: Computel, Telseq, Telomerecat, qMotif and Motif_counter. The estimated median telomere length was 5.10 kb (IQR: 4.52 kb – 5.68 kb) using Computel. The age when the blood sample was drawn had a significant negative correlation with telomere length (P = 0.003). European children, particularly those from Finland (P = 0.041) and from Sweden (P = 0.001), had shorter telomeres than children from the U.S.A. Paternal age (P = 0.019) was positively associated with telomere length. First degree relative status, presence of gestational diabetes in the mother, and maternal age did not have a significant impact on estimated telomere length. HLA-DR4/4 or HLA-DR4/X children had significantly longer telomeres compared to children with HLA-DR3/3 or HLA-DR3/9 haplogenotypes (P = 0.008). Estimated telomere length was not significantly different with respect to any IA (P = 0.377), IAA-first (P = 0.248), GADA-first (P = 0.248) or T1D (P = 0.861). These results suggest that telomere length has no major impact on the risk for IA, the first step to develop T1D. Nevertheless, telomere length was shorter in the T1D high prevalence populations, Finland and Sweden.

Islet Autoantibody Type-Specific Titer Thresholds Improve Stratification of Risk of Progression to Type 1 Diabetes in Children

OBJECTIVE: To utilize islet autoantibody titers to improve the estimation of future type 1 diabetes risk in children. <p>RESEARCH DESIGN AND METHODS: Prospective cohort studies in Finland, Germany, Sweden and the US followed 24,662 children at increased genetic or familial risk to develop islet autoimmunity and diabetes. For 1,604 children with confirmed positivity, titers of autoantibodies against insulin (IAA), glutamic acid decarboxylase (GADA) and insulinoma-associated antigen-2 (IA-2A) were harmonized for diabetes risk analyses.</p> <p>RESULTS: Survival analysis from time of confirmed positivity revealed markedly different 5-year diabetes risks associated with IAA (n=909), GADA (n=1076) or IA-2A (n=714), when stratified by quartiles of titer, ranging from 19% (GADA 1^st quartile) to 60% (IA-2A 4^th quartile). The minimum titer associated with a maximum difference in 5-year risk differed for each autoantibody, corresponding to the 58.6^th, 52.4^th and 10.2^nd percentile of children specifically positive for each of IAA, GADA and IA-2A, respectively. Using these autoantibody type-specific titer thresholds in the 1,481 children with all autoantibodies tested, the 5-year risk conferred by single (n=954) and multiple (n=527) autoantibodies could be stratified from 6% to 75% (p<0.0001). The thresholds effectively identified children with 50% or higher 5-year risk when considering age-specific autoantibody screening (57-65% positive predictive value and 56-74% sensitivity for ages 1-5 years). Multivariable analysis confirmed the significance of associations between the three autoantibody titers and diabetes risk, informing a childhood risk surveillance strategy.</p> <p>CONCLUSIONS: This study defined islet autoantibody type-specific titer thresholds that significantly improved type 1 diabetes risk stratification in children.</p>

Islet Autoantibody Type-Specific Titer Thresholds Improve Stratification of Risk of Progression to Type 1 Diabetes in Children

OBJECTIVE: To utilize islet autoantibody titers to improve the estimation of future type 1 diabetes risk in children. <p>RESEARCH DESIGN AND METHODS: Prospective cohort studies in Finland, Germany, Sweden and the US followed 24,662 children at increased genetic or familial risk to develop islet autoimmunity and diabetes. For 1,604 children with confirmed positivity, titers of autoantibodies against insulin (IAA), glutamic acid decarboxylase (GADA) and insulinoma-associated antigen-2 (IA-2A) were harmonized for diabetes risk analyses.</p> <p>RESULTS: Survival analysis from time of confirmed positivity revealed markedly different 5-year diabetes risks associated with IAA (n=909), GADA (n=1076) or IA-2A (n=714), when stratified by quartiles of titer, ranging from 19% (GADA 1^st quartile) to 60% (IA-2A 4^th quartile). The minimum titer associated with a maximum difference in 5-year risk differed for each autoantibody, corresponding to the 58.6^th, 52.4^th and 10.2^nd percentile of children specifically positive for each of IAA, GADA and IA-2A, respectively. Using these autoantibody type-specific titer thresholds in the 1,481 children with all autoantibodies tested, the 5-year risk conferred by single (n=954) and multiple (n=527) autoantibodies could be stratified from 6% to 75% (p<0.0001). The thresholds effectively identified children with 50% or higher 5-year risk when considering age-specific autoantibody screening (57-65% positive predictive value and 56-74% sensitivity for ages 1-5 years). Multivariable analysis confirmed the significance of associations between the three autoantibody titers and diabetes risk, informing a childhood risk surveillance strategy.</p> <p>CONCLUSIONS: This study defined islet autoantibody type-specific titer thresholds that significantly improved type 1 diabetes risk stratification in children.</p>