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Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed amethod of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

The use of a standardized outcome metric enhances clinical trial interpretation and cross-trial comparison. If a disease course is predictable, comparing modeled predictions with outcome data affords the precision and confidence needed to accelerate precision medicine. We demonstrate this approach in type 1 diabetes (T1D) trials aiming to preserve endogenous insulin secretion measured by C-peptide. C-peptide is predictable given an individual’s age and baseline value; quantitative response (QR) adjusts for these variables and represents the difference between the observed and predicted outcome. Validated across 13 trials, the QR metric reduces each trial’s variance and increases statistical power. As smaller studies are especially subject to random sampling variability, using QR as the outcome introduces alternative interpretations of previous clinical trial results. QR can provide model-based estimates that quantify whether individuals or groups did better or worse than expected. QR also provides a purer metric to associate with biomarker measurements. Using data from more than 1300 participants, we demonstrate the value of QR in advancing disease-modifying therapy in T1D. QR applies to any disease where outcome is predictable by pre-specified baseline covariates, rendering it useful for defining responders to therapy, comparing therapeutic efficacy, and understanding causal pathways in disease. The use of a standardized outcome metric enhances clinical trial interpretation and cross-trial comparison. Here, the authors show the implementation of such a metric using type 1 diabetes trial data, reassess and compare results from these trials, and extend its use to define response to therapy.

The immunopathogenesis of type 1 diabetes mellitus is associated with T-cell autoimmunity. To be fully active, immune T cells need a co-stimulatory signal in addition to the main antigen-driven signal. Abatacept modulates co-stimulation and prevents full T-cell activation. We evaluated the effect of abatacept in recent-onset type 1 diabetes. In this multicentre, double-blind, randomised controlled trial, patients aged 6–45 years recently diagnosed with type 1 diabetes were randomly assigned (2:1) to receive abatacept (10 mg/kg, maximum 1000 mg per dose) or placebo infusions intravenously on days 1, 14, 28, and monthly for a total of 27 infusions over 2 years. Computer-generated permuted block randomisation was used, with a block size of 3 and stratified by participating site. Neither patients nor research personnel were aware of treatment assignments. The primary outcome was baseline-adjusted geometric mean 2-h area-under-the-curve (AUC) serum C-peptide concentration after a mixed-meal tolerance test at 2 years' follow-up. Analysis was by intention to treat for all patients for whom data were available. This trial is registered at ClinicalTrials.gov, NCT00505375. 112 patients were assigned to treatment groups (77 abatacept, 35 placebo). Adjusted C-peptide AUC was 59% (95% CI 6·1–112) higher at 2 years with abatacept (n=73, 0·378 nmol/L) than with placebo (n=30, 0·238 nmol/L; p=0·0029). The difference between groups was present throughout the trial, with an estimated 9·6 months' delay (95% CI 3·47–15·6) in C-peptide reduction with abatacept. There were few infusion-related adverse events (36 reactions occurred in 17 [22%] patients on abatacept and 11 reactions in six [17%] on placebo). There was no increase in infections (32 [42%] patients on abatacept vs 15 [43%] on placebo) or neutropenia (seven [9%] vs five [14%]). Co-stimulation modulation with abatacept slowed reduction in β-cell function over 2 years. The beneficial effect suggests that T-cell activation still occurs around the time of clinical diagnosis of type 1 diabetes. Yet, despite continued administration of abatacept over 24 months, the decrease in β-cell function with abatacept was parallel to that with placebo after 6 months of treatment, causing us to speculate that T-cell activation lessens with time. Further observation will establish whether the beneficial effect continues after cessation of abatacept infusions. US National Institutes of Health.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 μL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1μL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.

Studies of new therapies to preserve insulin secretion in early type 1 diabetes require several years to recruit eligible subjects and to see a treatment effect; thus, there is interest in alternative study designs to speed this process. Most people with longstanding type 1 diabetes no longer secrete insulin. However, studies from pancreata of those with longstanding T1D show that beta cells staining for insulin can persist for decades after diagnosis, and this is paralleled in work showing proinsulin secretion in individuals with longstanding disease; collectively this suggests that there is a reserve of alive but “sleeping” beta cells. Here, we designed a novel clinical trial platform to test whether a short course of therapy with an agent known to have effects in type 1 diabetes with residual endogenous insulin could transiently induce insulin secretion in those who no longer produce insulin. A therapy that transiently “wakes up” sleeping beta cells might be tested next in a fully powered trial in those with endogenous insulin secretion. In this three-arm non-randomized pilot study, we tested three therapies known to impact disease: two beta-cell supportive agents, liraglutide and verapamil, and an immunomodulatory agent, golimumab. The golimumab treated arm was not fully enrolled due to uncertainties about immunotherapy during the COVID-19 pandemic. Participants had mixed-meal tolerance test (MMTT)-stimulated C-peptide below the quantitation limit (<0.02 ng/mL) at enrollment and received 8 to 12 weeks of therapy. At the completion of therapy, none of the individuals achieved the primary outcome of MMTT-stimulated C-peptide ≥ 0.02 ng/mL. An exploratory outcome of the verapamil arm was MRI-assessed pancreas size, diffusion, and longitudinal relaxation time, which showed repeatability of these measures but no treatment effect. The liraglutide and golimumab arms were registered on clinicaltrials.gov under accession number NCT03632759 and the verapamil arm under accession number NCT05847413. Trail registration: Protocols are registered in ClinicalTrials.gov under accession numbers NCT03632759 and NCT05847413 .

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4 + T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism. Post translational modifications are involved in the regulation of a number of proteins and can be themselves modified by the cellular environment. Here the authors implicate citrullination of glucokinase in autoimmune diabetes

OBJECTIVE:--β-Cell function in type 1 diabetes clinical trials is commonly measured by C-peptide response to a secretagogue in either a mixed-meal tolerance test (MMTT) or a glucagon stimulation test (GST). The Type 1 Diabetes TrialNet Research Group and the European C-peptide Trial (ECPT) Study Group conducted parallel randomized studies to compare the sensitivity, reproducibility, and tolerability of these procedures. RESEARCH DESIGN AND METHODS--In randomized sequences, 148 TrialNet subjects completed 549 tests with up to 2 MMTT and 2 GST tests on separate days, and 118 ECPT subjects completed 348 tests (up to 3 each) with either two MMTTs or two GSTs. RESULTS:--Among individuals with up to 4 years' duration of type 1 diabetes, >85% had measurable stimulated C-peptide values. The MMTT stimulus produced significantly higher concentrations of C-peptide than the GST. Whereas both tests were highly reproducible, the MMTT was significantly more so (R² = 0.96 for peak C-peptide response). Overall, the majority of subjects preferred the MMTT, and there were few adverse events. Some older subjects preferred the shorter duration of the GST. Nausea was reported in the majority of GST studies, particularly in the young age-group. CONCLUSIONS:--The MMTT is preferred for the assessment of β-cell function in therapeutic trials in type 1 diabetes.

Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA⁹⁷ in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramere loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.

Glutamic acid decarboxylase (GAD) is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. In animal models of autoimmunity, treatment with a target antigen can modulate aggressive autoimmunity. We aimed to assess whether immunisation with GAD formulated with aluminum hydroxide (GAD-alum) would preserve insulin production in recent-onset type 1 diabetes. Patients aged 3–45 years who had been diagnosed with type 1 diabetes for less than 100 days were enrolled from 15 sites in the USA and Canada, and randomly assigned to receive one of three treatments: three injections of 20 μg GAD-alum, two injections of 20 μg GAD-alum and one of alum, or 3 injections of alum. Injections were given subcutaneously at baseline, 4 weeks later, and 8 weeks after the second injection. The randomisation sequence was computer generated at the TrialNet coordinating centre. Patients and study personnel were masked to treatment assignment. The primary outcome was the baseline-adjusted geometric mean area under the curve (AUC) of serum C-peptide during the first 2 h of a 4-h mixed meal tolerance test at 1 year. Secondary outcomes included changes in glycated haemoglobin A 1c (HbA 1c) and insulin dose, and safety. Analysis included all randomised patients with known measurements. This trial is registered with ClinicalTrials.gov, number NCT00529399. 145 patients were enrolled and treated with GAD-alum (n=48), GAD-alum plus alum (n=49), or alum (n=48). At 1 year, the 2-h AUC of C-peptide, adjusted for age, sex, and baseline C-peptide value, was 0·412 nmol/L (95% CI 0·349–0·478) in the GAD-alum group, 0·382 nmol/L (0·322–0·446) in the GAD-alum plus alum group, and 0·413 nmol/L (0·351–0·477) in the alum group. The ratio of the population mean of the adjusted geometric mean 2-h AUC of C-peptide was 0·998 (95% CI 0·779–1·22; p=0·98) for GAD-alum versus alum, and 0·926 (0·720–1·13; p=0·50) for GAD-alum plus alum versus alum. HbA 1c, insulin use, and the occurrence and severity of adverse events did not differ between groups. Antigen-based immunotherapy therapy with two or three doses of subcutaneous GAD-alum across 4–12 weeks does not alter the course of loss of insulin secretion during 1 year in patients with recently diagnosed type 1 diabetes. Although antigen-based therapy is a highly desirable treatment and is effective in animal models, translation to human autoimmune disease remains a challenge. US National Institutes of Health.

Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype. To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8). Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression. These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function.