Clinical Features and HLA Genetics Differ in Children at Type 1 Diabetes Onset by Hispanic Ethnicity.

CONTEXT: Type 1 diabetes incidence continues to increase in children, especially among Hispanic Whites (HW). OBJECTIVE: We investigated the clinical, immunologic, and genetic characteristics of HW and Non-Hispanic White (NHW) children that presented at type 1 diabetes diagnosis. METHODS: In this single-center, observational study, children who were diagnosed with type 1 diabetes (<20 years old) and tested for islet autoantibodies within 1 year of diagnosis were included in the study and divided into two groups by Hispanic ethnicity. RESULTS: Of 1297 children, 398 HW children presented with a younger age at diabetes onset (10.2 ± 3.9 vs. 11.1 ± 4.1 years, p<0.001) and more diabetic ketoacidosis (62.4% vs. 51.9%, p<0.001) compared to NHW children (n=899). There was no difference in sex, A1c levels, or the number and prevalence of islet autoantibodies between the two cohorts. A subset of our cohort was HLA typed as specific alleles confer strong genetic risk for type 1 diabetes (e.g., HLA-DR4 and DQ8). Among 637 HLA-typed children, HW children had a significantly higher prevalence of the DR4-DQ8 haplotype compared to NHW children (79.1% vs. 60.1%, p<0.001), and this frequency was much higher than a reference Hispanic population (OR = 6.5, 95% CI 4.6-9.3). CONCLUSIONS: Hispanic White children developing type 1 diabetes have a high prevalence of HLA DR4-DQ8, which can be utilized to select individuals for immune monitoring with islet autoantibodies to lessen diabetic ketoacidosis and potentially prevent diabetes onset.

Tracking DNA-based antigen-specific T cell receptors during progression to type 1 diabetes.

T cells targeting self-proteins are important mediators in autoimmune diseases. T cells express unique cell-surface receptors (TCRs) that recognize peptides presented by major histocompatibility molecules. TCRs have been identified from blood and pancreatic islets of individuals with type 1 diabetes (T1D). Here, we tracked ~1700 known antigen-specific TCR sequences, islet antigen or viral reactive, in bulk TCRß sequencing from longitudinal blood DNA samples in at-risk cases who progressed to T1D, age/sex/human leukocyte antigen-matched controls, and a new-onset T1D cohort. Shared and frequent antigen-specific TCRß sequences were identified in all three cohorts, and viral sequences were present across all ages. Islet sequences had different patterns of accumulation based upon antigen specificity in the at-risk cases. Furthermore, 73 islet-antigen TCRß sequences were present in higher frequencies and numbers in T1D samples relative to controls. The total number of these disease-associated TCRß sequences inversely correlated with age at clinical diagnosis, indicating the potential to use disease-relevant TCR sequences as biomarkers in autoimmune disorders.

Temporal development of T cell receptor repertoires during childhood in health and disease.

T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next-generation sequencing technologies. However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. Here, we deep sequenced the TCR-ß chain repertoires from longitudinal peripheral blood DNA samples at 4 time points beginning early in life (median age of 1.4 years) from children who progressed to T1D (n = 29) and age/sex-matched islet autoantibody-negative controls (n = 25). From 53 million TCR-ß sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independently of disease. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and, separately, those specific for insulin and its precursor, preproinsulin. Insulin-reactive TCR-ß sequences were more common and frequent in number as the disease progressed in those who developed T1D compared with genetically at risk nondiabetic children, and this was not the case for influenza-reactive sequences. As an independent validation, we sequenced and analyzed TCR-ß repertoires from a cohort of new-onset T1D patients (n = 143), identifying the same preproinsulin-reactive TCRs. These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders.

T-cell responses to hybrid insulin peptides prior to type 1 diabetes development.

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell-mediated destruction of insulin-producing ß-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Failed Genetic Protection: Type 1 Diabetes in the Presence of HLA-DQB1*06:02.

Certain HLA class II genes increase the risk for type 1 diabetes (T1D) development while others provide protection from disease development. HLA class II alleles encode MHC proteins on antigen-presenting cells, which function to present peptides and activate CD4 T cells. The DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype provides dominant protection across all stages of T1D and is a common haplotype found in Caucasians. However, it is present in <1% of people with T1D. Knowing which metabolic, immunologic, and genetic features are unique to individuals who fail genetic protection and develop T1D is important for defining the underlying mechanisms of DQB1*06:02-mediated protection. We describe a T1D cohort with DQB1*06:02 (n = 50) and compare them to individuals with T1D and without DQB1*06:02 (n = 2,759) who were identified over the last 26 years at the Barbara Davis Center for Diabetes. The age at diagnosis was similar between the cohorts and normally distributed throughout childhood and early adulthood. The average hemoglobin A1c was 10.8 ± 2.8% (95 ± 7 mmol/mol) at diagnosis in those DQB1*06:02 positive. The majority of T1D DQB1*06:02 + individuals were positive for one or more islet autoantibodies; however, there was a greater proportion who were islet autoantibody negative compared with those T1D DQB1*06:02 - individuals. Interestingly, DQB1*03:02, which confers significant T1D risk, was present in only those DQB1*06:02 + individuals with islet autoantibodies. This is one of the largest studies examining patients presenting with clinical T1D in the presence of DQB1*06:02, which provides a population to study the mechanisms of failed genetic protection against T1D.

Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes.

Major histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes (T1D), the DQ8 molecule is common, confers significant disease risk, and is involved in disease pathogenesis. We hypothesized that blocking DQ8 antigen presentation would provide therapeutic benefit by preventing recognition of self-peptides by pathogenic T cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the MHC antigen-binding cleft. A limited number of the predicted compounds inhibited DQ8 antigen presentation in vitro, with 1 compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. An existing drug with a similar structure, methyldopa, specifically blocked DQ8 in patients with recent-onset T1D and reduced inflammatory T cell responses to insulin, highlighting the relevance of blocking disease-specific MHC class II antigen presentation to treat autoimmunity.

Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes.

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing ß-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Islet Autoantibody Measurements from Dried Blood Spots on Filter Paper Strongly Correlate to Serum Levels.

Type 1 diabetes (T1D) is increasing in incidence and predictable with measurement of serum islet autoantibodies (iAb) years prior to clinical disease onset. Identifying iAb positive individuals reduces diabetic ketoacidosis and identifies individuals for T1D prevention trials. However, large scale screening for iAb remains challenging as assays have varying sensitivities and specificities, insulin autoantibodies remain difficult to measure and venipuncture is generally required to obtain serum. We developed an approach to reliably measure all four major iAb, including insulin autoantibodies, from dried blood spots (DBS) on filter-paper. By spiking iAb positive serum into iAb negative whole blood in a dose titration, we optimized the conditions for autoantibody elution from filter paper as measured by fluid phase radioimmunoassays. After assessing stability of measuring iAb from DBS over time, we then screened iAb from DBS and the corresponding serum in new-onset T1D (n = 52), and controls (n = 72) which included first-degree relatives of T1D patients. iAb measured from eluted DBS in new-onset T1D strongly correlated with serum measurements (R2 = 0.96 for mIAA, GADA = 0.94, IA-2A = 0.85, ZnT8A = 0.82, p<0.01 for each autoantibody). There were no false positives in control subjects, and 5/6 with previously unknown iAb positivity in sera were detected using DBS. With further validation, measuring iAb from DBS can be a reliable method to screen for T1D risk.

The effect of season on inflammatory response in captive baboons.

INTRODUCTION: Highly seasonal animals demonstrate predictable changes in immune function that coincide with changes in photoperiod. Little is known about the effect of season on immune response in baboons. The objective of this study was to determine the effect of season on inflammatory response in baboons. MATERIALS AND METHODS: Peripheral blood mononuclear cell cytokine response following immune stimulation and serum markers of inflammation were assessed during each season in two groups of young male baboons: one housed under natural light and one in a controlled environment of 12 hours light:12 hours dark. RESULTS: A seasonal immune rhythm was evident in both groups, with a greater TNF-α and IL-6 response to stimulation and serum CRP concentration in June and September compared with December. CONCLUSIONS: Season is an important experimental confounder, and therefore, time of year should be controlled when designing studies and analyzing data from immune studies in baboons.

Age-associated alteration in innate immune response in captive baboons.

Baboons are an ideal model for studies of human inflammatory response due to their physiological and immunological similarities to people; however; little is known about how age affects immune function in the baboon. We sought to determine if baboons show age-related innate immune changes similar to that described in people. Age was correlated with increased serum C-reactive protein and interleukin-6 or, however, no change in interleukin-10 concentration was observed (n = 120 baboons). Cytokine release from unstimulated peripheral blood mononuclear cells as well as following immune (lipopolysaccharide) stimulation increased with age. When whole blood was assayed, both lipopolysaccharide stimulated and unstimulated samples showed an age-related increase in interleukin-6 response, although the unstimulated cytokine response was reduced compared with that observed in peripheral blood mononuclear cells. Tumor necrosis factor-α response was not related to age. Cytokine response in lipopolysaccharide-stimulated whole blood was negatively correlated with serum DHEA-S concentration and positively correlated with TGF-ß concentration.