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Recent evidence suggests a role for B cells in the pathogenesis of young-onset type 1 diabetes (T1D), wherein rapid progression occurs. However, little is known regarding the specificity, phenotype, and function of B cells in young-onset T1D. We performed a cross-sectional analysis comparing insulin-reactive to tetanus-reactive B cells in the blood of T1D and controls using mass cytometry. Unsupervised clustering revealed the existence of a highly activated B cell subset we term BND2 that falls within the previously defined anergic BND subset. We found a specific increase in the frequency of insulin-reactive BND2 cells in the blood of young-onset T1D donors, which was further enriched in the pancreatic lymph nodes of T1D donors. The frequency of insulin-binding BND2 cells correlated with anti-insulin autoantibody levels. We demonstrate BND2 cells are pre-plasma cells and can likely act as APCs to T cells. These findings identify an antigen-specific B cell subset that may play a role in the rapid progression of young-onset T1D.

The greatest change in the treatment of people living with type 1 diabetes in the last decade has been the explosion of technology assisting in all aspects of diabetes therapy, from glucose monitoring to insulin delivery and decision making. Through screening of 835 peer-reviewed articles followed by systematic review of 70 of them (focusing on randomized trials and extension studies with ≥50 participants from the past 10 years), we conclude that novel technologies, ranging from continuous glucose monitoring systems, insulin pumps and decision support tools to the most advanced hybrid closed loop systems, improve important measures like HbA1c, time in range, and glycemic variability, while reducing hypoglycemia risk. Several studies included person-reported outcomes, allowing assessment of the burden or benefit of the technology in the lives of those with type 1 diabetes, demonstrating positive results or, at a minimum, no increase in self-care burden compared with standard care. Important limitations of the trials to date are their small size, the scarcity of pre-planned or powered analyses in sub-populations such as children, racial/ethnic minorities, people with advanced complications, and variations in baseline glycemic levels. In addition, confounders including education with device initiation, concomitant behavioral modifications, and frequent contact with the healthcare team are rarely described in enough detail to assess their impact. Our review highlights the potential of technology in the treatment of people living with type 1 diabetes and provides suggestions for optimization of outcomes and areas of further study for precision medicine-directed technology use in type 1 diabetes.Preface (Lay Abstract)We reviewed literature of the last decade to evaluate the impact of technology on the treatment of people living with type 1 diabetes. Screening of 835 articles and in-depth review of 70 showed that novel technologies, ranging from continuous glucose monitoring systems, insulin pumps and decision support tools to the most advanced hybrid closed loop systems, improve important measures like HbA1c and time in range, while reducing hypoglycemia risk. Of importance, several studies showed a positive impact on person-reported outcomes, like quality of life or, at a minimum, no increase in self-care burden compared with standard care.

Objective Previous studies showed that inhibiting lymphocyte costimulation reduces declining beta cell function in individuals newly-diagnosed with type 1 diabetes. We tested whether abatacept would delay or prevent progression of type 1 diabetes from normal (NGT) to abnormal (AGT) glucose tolerance or to diabetes and the effects of treatment on immune and metabolic responses. Research Design and Methods We conducted a phase-2, randomized, placebo-controlled, double-masked trial of abatacept in antibody-positive participants with NGT who received monthly abatacept/placebo infusions for 12 months. The endpoint was AGT or diabetes, assessed by oral glucose-tolerance tests. Results 101 participants received abatacept and 111 placebo. Eighty-one (35 abatacept/46 placebo) met the endpoint of AGT or type 1 diabetes diagnosis (HR=0.702 (0.452, 1.09)(p=0.11) The C-peptide responses to OGTTs were higher in the abatacept arm (p Conclusions Although abatacept treatment for one year did not significantly delay progression to glucose intolerance in at-risk individuals, it impacted immune cell subsets and preserved insulin secretion suggesting that costimulation blockade may modify progression of type 1 diabetes.

Context Participants with stage 1 or 2 type 1 diabetes (T1D) qualify for prevention trials, but factors involved in screening for such trials are largely unknown. Objective To identify factors associated with screening for T1D prevention trials. Methods This study included TrialNet Pathway to Prevention participants who were eligible for a prevention trial: oral insulin (TN-07, TN-20), teplizumab (TN-10), abatacept (TN-18), and oral hydroxychloroquine (TN-22). Univariate and multivariate logistic regression models were used to examine participant, site, and study factors at the time of prevention trial accrual. Results Screening rates for trials were: 50% for TN-07 (584 screened/1172 eligible), 9% for TN-10 (106/1249), 24% for TN-18 (313/1285), 17% for TN-20 (113/667), and 28% for TN-22 (371/1336). Younger age and male sex were associated with higher screening rates for prevention trials overall and for oral therapies. Participants with an offspring with T1D showed lower rates of screening for all trials and oral drug trials compared with participants with other first-degree relatives as probands. Site factors, including larger monitoring volume and US site vs international site, were associated with higher prevention trial screening rates. Conclusions Clear differences exist between participants who screen for prevention trials and those who do not screen and between the research sites involved in prevention trial screening. Participant age, sex, and relationship to proband are significantly associated with prevention trial screening in addition to key site factors. Identifying these factors can facilitate strategic recruitment planning to support rapid and successful enrollment into prevention trials.

Objective Electrochemiluminescence (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 human leukocyte antigen (HLA) haplotypes and genotypes with 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 1 RBA diabetes-related Ab [glutamic acid decarboxylase autoantibodies (GADA) or insulin autoantibodies (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 Ps < 0.05), while ECL and RBA-GADA z-score titers were higher in participants with HLA-DR3 haplotypes only (both Ps < 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.

Previously, we demonstrated low-dose anti-thymocyte globulin (ATG) and granulocyte colony stimulating factor (GCSF) immunotherapy preserved C-peptide for two years in a pilot study of subjects with established type 1 diabetes (n=25). Herein, we evaluated the long-term outcomes of ATG/GCSF in study participants with five years of available follow-up data (n=15). The primary endpoint was area under the curve (AUC) C-peptide during a two-hour mixed-meal tolerance test (MMTT). After five years, there were no statistically significant differences in AUC C-peptide when comparing subjects who received ATG/GCSF versus placebo (p = 0.41). A modeling framework based on mean trajectories in C-peptide AUC over five years, accounting for differing trends between groups, was applied to re-categorize responders (n=9) and non-responders (n=7). ATG/GCSF reponders demonstrated nearly unchanged HbA1c over five years [mean (95% CI) adjusted change = 0.29% (-0.69%, 1.27%)], but the study was not powered for comparisons against non-responders 1.75% (-0.57%, 4.06%) and placebo 1.44% (0.21%, 2.66%). These data underscore the importance of long-term follow up in previous and ongoing phase 2 trials of low-dose ATG in recent-onset type 1 diabetes.

AIMS: To test whether a long-acting GLP-1 receptor agonist would improve glucose control in patients with type 1 diabetes (T1D) and to determine whether the presence of residual beta cell function would affect the response. In addition, we sought to determine whether the drug would affect beta cell function. METHODS: We performed a randomized placebo-controlled trial of exenatide extended release (ER) in participants with T1D with and without detectable levels of C-peptide. Seventy-nine participants were randomized to exenatide ER 2 mcg weekly, or placebo, stratified by the presence or absence of detectable C-peptide levels. The primary outcome was the difference in glycated haemoglobin (HbA1c) levels at 24 weeks. Participants were followed for another 6 months off study drug. RESULTS: At week 24, the time of the primary outcome, the least squares (LS) mean HbA1c level was 7.76% (95% confidence interval [CI] 7.42, 8.10) in the exenatide ER group versus 8.0% (95% CI 7.64, 8.35) in the placebo group (P = 0.08). At week 12 the LS mean HbA1c levels were 7.71% (95% CI 7.37, 8.05) in the exenatide ER group versus 8.05% (95% CI 7.7, 8.4) in the placebo group (P = 0.01). The improvement at week 12 was driven mainly by those with detectable levels of C-peptide. Those treated with exenatide ER lost weight at 12 and 24 weeks compared to those treated with placebo (P

Purpose of review Although type 1 diabetes (T1D) is characterized by destruction of the pancreatic beta cells by self-reactive T cells, it has become increasingly evident that B cells also play a major role in disease development, likely functioning as antigen-presenting cells. Here we review the biology of islet antigen-reactive B cells and their participation in autoimmune diabetes. Recent findings Relative to late onset, individuals who develop T1D at an early age display increased accumulation of insulin-reactive B cells in islets. This B-cell signature is also associated with rapid progression of disease and responsiveness to B-cell depletion therapy. Also suggestive of B-cell participation in disease is loss of anergy in high-affinity insulin-reactive B cells. Importantly, loss of anergy is seen in patient's healthy first-degree relatives carrying certain T1D risk alleles, suggesting a role early in disease development. Summary Recent studies indicate that islet-reactive B cells may play a pathogenic role very early in T1D development in young patients, and suggest utility of therapies that target these cells.

The clinical diagnosis of new-onset type 1 diabetes has, for many years, been considered relatively straightforward. Recently, however, there is increasing awareness that within this single clinical phenotype exists considerable heterogeneity: disease onset spans the complete age range; genetic susceptibility is complex; rates of progression differ markedly, as does insulin secretory capacity; and complication rates, glycemic control, and therapeutic intervention efficacy vary widely. Mechanistic and immunopathological studies typically show considerable patchiness across subjects, undermining conclusions regarding disease pathways. Without better understanding, type 1 diabetes heterogeneity represents a major barrier both to deciphering pathogenesis and to the translational effort of designing, conducting, and interpreting clinical trials of disease-modifying agents. This realization comes during a period of unprecedented change in clinical medicine, with increasing emphasis on greater individualization and precision. For complex disorders such as type 1 diabetes, the option of maintaining the “single disease” approach appears untenable, as does the notion of individualizing each single patient’s care, obliging us to conceptualize type 1 diabetes less in terms of phenotypes (observable characteristics) and more in terms of disease endotypes (underlying biological mechanisms). Here, we provide our view on an approach to dissect heterogeneity in type 1 diabetes. Using lessons from other diseases and the data gathered to date, we aim to delineate a roadmap through which the field can incorporate the endotype concept into laboratory and clinical practice. We predict that such an effort will accelerate the implementation of precision medicine and has the potential for impact on our approach to translational research, trial design, and clinical management.

Type 1 diabetes is the prototypical CD4 T cell–mediated autoimmune disease. Its genetic linkage to a single polymorphism at position 57 of the HLA class II DQβ chain makes it unique to study the molecular link between HLA and disease. However, investigating this relationship has been limited by a series of anatomical barriers, the small size and dispersion of the insulin-producing organ, and the scarcity of appropriate techniques and reagents to interrogate antigen-specific CD4 T cells both in man and rodent models. Over the past few years, single-cell technologies, paired with new biostatistical methods, have changed this landscape. Using these tools, we have identified the first molecular link between MHC class II and the onset of type 1 diabetes. The translation of these observations to man is within reach using similar approaches and the lessons learned from rodent models.

BackgroundType 1 diabetes is a chronic autoimmune disease that leads to destruction of insulin-producing beta cells and dependence on exogenous insulin for survival. Some interventions have delayed the loss of insulin production in patients with type 1 diabetes, but interventions that might affect clinical progression before diagnosis are needed.MethodsWe conducted a phase 2, randomized, placebo-controlled, double-blind trial of teplizumab (an Fc receptor-nonbinding anti-CD3 monoclonal antibody) involving relatives of patients with type 1 diabetes who did not have diabetes but were at high risk for development of clinical disease. Patients were randomly assigned to a single 14-day course of teplizumab or placebo, and follow-up for progression to clinical type 1 diabetes was performed with the use of oral glucose-tolerance tests at 6-month intervals.ResultsA total of 76 participants (55 [72%] of whom were ≤18 years of age) underwent randomization - 44 to the teplizumab group and 32 to the placebo group. The median time to the diagnosis of type 1 diabetes was 48.4 months in the teplizumab group and 24.4 months in the placebo group; the disease was diagnosed in 19 (43%) of the participants who received teplizumab and in 23 (72%) of those who received placebo. The hazard ratio for the diagnosis of type 1 diabetes (teplizumab vs. placebo) was 0.41 (95% confidence interval, 0.22 to 0.78; P = 0.006 by adjusted Cox proportional-hazards model). The annualized rates of diagnosis of diabetes were 14.9% per year in the teplizumab group and 35.9% per year in the placebo group. There were expected adverse events of rash and transient lymphopenia. KLRG1+TIGIT+CD8+ T cells were more common in the teplizumab group than in the placebo group. Among the participants who were HLA-DR3-negative, HLA-DR4-positive, or anti-zinc transporter 8 antibody-negative, fewer participants in the teplizumab group than in the placebo group had diabetes diagnosed.ConclusionsTeplizumab delayed progression to clinical type 1 diabetes in high-risk participants. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01030861.).

We recently established that hybrid insulin peptides (HIPs) are present in human islets and that T cells reactive to HIPs are found in the residual islets of organ donors with type 1 diabetes (T1D). Here, we investigate whether HIP-reactive T cells are indicative of ongoing autoimmunity in patients with T1D. We used interferon-γ enzyme-linked immune absorbent spot analyses on peripheral blood mononuclear cells (PBMCs) to determine whether patients with new-onset T1D or control subjects displayed T-cell reactivity to a panel of 16 HIPs. We observed that nearly one-half of the patients responded to one or more HIPs. Responses to four HIPs were significantly elevated in patients with T1D but not in control subjects. To characterize the T cells reactive to HIPs, we used a carboxyfluorescein succinimidyl ester–based assay to clone T cells from PBMCs. We isolated six nonredundant, antigen-specific T-cell clones, most of which reacting to their target HIPs in the low nanomolar range. One T-cell clone was isolated from the same patient on two different blood draws, indicating persistence of this T-cell clone in the peripheral blood. This work suggests that HIPs are important target antigens in human subjects with T1D and may play a critical role in disease.

A three-arm, randomized, double-masked, placebo-controlled phase 2b trial performed by the Type 1 Diabetes TrialNet Study Group previously demonstrated that low-dose anti-thymocyte globulin (ATG) (2.5 mg/kg) preserved β-cell function and reduced HbA1c for 1 year in new-onset type 1 diabetes. Subjects (N = 89) were randomized to 1) ATG and pegylated granulocyte colony-stimulating factor (GCSF), 2) ATG alone, or 3) placebo. Herein, we report 2-year area under the curve (AUC) C-peptide and HbA1c, prespecified secondary end points, and potential immunologic correlates. The 2-year mean mixed-meal tolerance test–stimulated AUC C-peptide, analyzed by ANCOVA adjusting for baseline C-peptide, age, and sex (n = 82) with significance defined as one-sided P < 0.025, was significantly higher in subjects treated with ATG versus placebo (P = 0.00005) but not ATG/GCSF versus placebo (P = 0.032). HbA1c was significantly reduced at 2 years in subjects treated with ATG (P = 0.011) and ATG/GCSF (P = 0.022) versus placebo. Flow cytometry analyses demonstrated reduced circulating CD4:CD8 ratio, increased regulatory T-cell:conventional CD4 T-cell ratios, and increased PD-1+CD4+ T cells following low-dose ATG and ATG/GCSF. Low-dose ATG partially preserved β-cell function and reduced HbA1c 2 years after therapy in new-onset type 1 diabetes. Future studies should determine whether low-dose ATG might prevent or delay the onset of type 1 diabetes.

PURPOSE OF REVIEW: To review the recent findings that small ‘drug-like’ compounds block disease-specific HLA molecules in type 1 diabetes (T1D). RECENT FINDINGS: The predominant genetic risk for developing T1D, the immune mediated form of diabetes, is conferred through human leukocyte antigen (HLA) genes. One such gene, termed HLA-DQ8, is present in 50–60% of patients with T1D and those at-risk. DQ8 presents disease-relevant peptides to T cells, which mediate tissue specific destruction of pancreatic islets. Using a structure-based approach to evaluate the ‘druggability’ of the DQ8 molecule, methyldopa, a clinically well-established oral anti-hypertensive agent, was discovered to bind DQ8. Methyldopa blocked the activation of DQ8 specific T cells responding to self-antigens such as insulin but not influenza. In a proof-of-concept clinical trial (NCT01883804), methyldopa was administered to recent-onset T1D patients with the DQ8 gene that confirmed the mechanism of action and diminished inflammatory T cell responses towards insulin. SUMMARY: Methyldopa blocks the diabetes-specific function of HLA-DQ8, which represents a personalized medicine approach to treat the underlying autoimmunity in T1D. Clinical trials are warranted and underway to evaluate methyldopa in potentially preserving residual beta-cell function in those with new-onset and at-risk for T1D.

Background: The TrialNet Immune Effects of Oral Insulin (OI) in Relatives at Risk for Type 1 Diabetes Mellitus (TN20) study (2-arm, randomized, open-label clinical trial for Stages 1-2 T1D ) observed, in a subset of participants (pts), a decline in insulin autoantibody (Ab) titers associated with an increase in islet-specific CD4+ T cell frequencies in Arm A (OI 67.5 mg/d) but not Arm B (500 mg/2 wks). Here, we compared the 2 arms to assess whether metabolic effects also occur in the 67.5 mg/d treatment arm. Methods: High-risk Ab+ pts (mean age 6.4±3.1 yrs; range 3-16 yrs) were randomized, treated for 6 months, and assessed at 0, 6 and 12 months. We compared the 2 arms for changes of mean glucose and C-peptide response curves (GCRCs) during OGTTs in pts with Diabetes Prevention Trial Risk Score (DPTRS) ≥6.75 (a subset where a metabolic effect of OI has been shown). Results: GCRC shapes and centroids (central point of GCRC shape) in each arm are shown in the Figure. The C-peptide/glucose ratio of centroid coordinates changed little in Arm A but decreased in Arm B (1.03±8.71 vs. -4.39±7.01; p Conclusions: The better metabolic outcome of 67.5 mg/d of OI than 500 mg/2 wks is consistent with the prior finding that immunologic change is associated with 67.5 mg/d, suggesting a linkage of immune and metabolic effects. Disclosure M. J. Redondo: Advisory Panel; Self; Provention Bio, Inc. A. Lernmark: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc. J. Sosenko: None. E. K. Sims: None. D. D. Cuthbertson: None. E. A. James: Consultant; Self; Provention Bio, Inc., Research Support; Self; Bristol-Myers Squibb Company, Janssen Pharmaceuticals, Inc., Novartis AG, Sanofi. S. Long: None. W. Kwok: None. L. Yu: None. A. W. Michels: Stock/Shareholder; Self; IM Therapeutics. Funding National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; JDRF

Background: Type 1 diabetes (T1D) genetic risk score (GRS) and islet autoantibody (AAb) specificity and titers influence T1D risk; however, these factors collected at baseline in cross sectional studies have not been effectively incorporated in the current models for prediction of progression along pre-clinical stages of T1D. Our aim was to develop an updated model of T1D prediction for research and clinical practice. Methods: AAb-positive relatives of individuals with T1D (n=1,233, 52% female, 88% European ancestry, age mean 15 yrs [SD 12.2]) from the TrialNet Pathway to Prevention were followed for a median of 3.1 years (IQR 1.1 -7.1). T1D developed in 498/1,233 (40.4%). T1D GRS, AAb number, AAb combination, AAb titer, age and sex were evaluated on the combined T1D prediction model, estimating its performance using 3-fold cross-validation (10x repeated). Results: Machine learning (survival random forest) and traditional methods (Cox models, extended Cox model) performed equivalently as measured by 5 yr horizon time-dependent AUC ROC (respectively 0.74, 0.74, 0.72), on calibration by integrated Brier score (respectively 0.15, 0.15, 0.16), and by calibration plots. AAb combinations with specific variables predicted T1D. In multivariate analysis, T1D GRS was a significant predictor of T1D in individuals positive for GADA, IAA or both. For any autoantibody combination including IA2A, the IA2A titer was predictive while the T1D GRS was not. Variable importance analyses showed that T1D was predicted by IA2A titer level, followed by T1D GRS and combinations of specific AAbs. Conclusion: We modeled individual estimates of T1D risk with T1D GRS, AAb characteristics, age, and sex. T1D prediction improved by adding T1D GRS and AAb characteristics to other variables, and was similar using machine learning or traditional models. T1D GRS was a significant predictor only in the absence of IA2A. In the presence of IA2A, IA2A titer was the most influential factor. Disclosure L. A. Ferrat: None. M. J. Redondo: Advisory Panel; Self; Provention Bio, Inc. A. Steck: None. H. M. Parikh: None. L. You: None. S. Onengut-gumuscu: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc. S. S. Rich: None. J. Krischer: None. R. A. Oram: Consultant; Self; Janssen Research & Development, LLC. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK121843-01); JDRF (3-SRA-2019-827-S-B)

The TrialNet OI prevention trial (TN07) with T1D as an endpoint showed no effect. However, in a smaller trial run in parallel with TN07 (termed secondary stratum 1), in which all participants had low first-phase insulin release, a statistically significant delay in T1D onset was found. Since trials with T1D as an endpoint take years to complete, we aimed to determine if combination C-peptide and glucose markers could identify a therapeutic benefit after 12 months of OI in this high-risk population. All participants were at-risk relatives with multiple autoantibodies including mIAA and normal glucose tolerance (n=40 with full data). The Figure shows changes in glucose and C-peptide response curves and their centroids (central points). Glucose rose and C-peptide declined in the placebo group compared to a minimal glucose rise with an increase of C-peptide in the OI group. Change from baseline to 1 year in the centroid ratio (C-peptide/glucose) differed significantly between groups (p=0.037 after adjustments for age, BMI, and baseline C-peptide and glucose). The AUC C-peptide/AUC glucose ratio and Index60 did not differ (both p=0.108). In conclusion, the findings support a positive effect of OI on combined glucose and C-peptide endpoints in high-risk individuals. These measures appear to detect differences in a shorter time than the standard T1D endpoint. Disclosure L. M. Jacobsen: None. M. A. Atkinson: None. J. Krischer: None. D. Schatz: Advisory Panel; Self; Abbott, Medtronic. J. Sosenko: None. D. D. Cuthbertson: None. E. K. Sims: None. H. M. Ismail: None. L. Dimeglio: Advisory Panel; Self; MannKind Corporation. T. M. Triolo: None. M. J. Redondo: Advisory Panel; Self; Provention Bio, Inc. M. Lundgren: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc.

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 β-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.

Type 1 diabetes results from autoimmune-mediated destruction of β cells. The tyrosine kinase inhibitor imatinib might affect relevant immunological and metabolic pathways, and preclinical studies show that it reverses and prevents diabetes. Our aim was to evaluate the safety and efficacy of imatinib in preserving β-cell function in patients with recent-onset type 1 diabetes.We did a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Patients with recent-onset type 1 diabetes (100 days from diagnosis), aged 18-45 years, positive for at least one type of diabetes-associated autoantibody, and with a peak stimulated C-peptide of greater than 0·2 nmol LPatients were screened and enrolled between Feb 12, 2014, and May 19, 2016. 45 patients were assigned to receive imatinib and 22 to receive placebo. After withdrawals, 43 participants in the imatinib group and 21 in the placebo group were included in the primary ITT analysis at 12 months. The study met its primary endpoint: the adjusted mean difference in 2-h C-peptide AUC at 12 months for imatinib versus placebo treatment was 0·095 (90% CI -0·003 to 0·191; p=0·048, one-tailed test). This effect was not sustained out to 24 months. During the 24-month follow-up, 32 (71%) of 45 participants who received imatinib had a grade 2 severity or worse adverse event, compared with 13 (59%) of 22 participants who received placebo. The most common adverse events (grade 2 severity or worse) that differed between the groups were gastrointestinal issues (six [13%] participants in the imatinib group, primarily nausea, and none in the placebo group) and additional laboratory investigations (ten [22%] participants in the imatinib group and two [9%] in the placebo group). Per the trial protocol, 17 (38%) participants in the imatinib group required a temporary modification in drug dosing and six (13%) permanently discontinued imatinib due to adverse events; five (23%) participants in the placebo group had temporary modifications in dosing and none had a permanent discontinuation due to adverse events.A 26-week course of imatinib preserved β-cell function at 12 months in adults with recent-onset type 1 diabetes. Imatinib might offer a novel means to alter the course of type 1 diabetes. Future considerations are defining ideal dose and duration of therapy, safety and efficacy in children, combination use with a complimentary drug, and ability of imatinib to delay or prevent progression to diabetes in an at-risk population; however, careful monitoring for possible toxicities is required.Juvenile Research Diabetes Foundation.

Aims/hypothesis Accurate prediction of disease progression in individuals with pre-symptomatic type 1 diabetes has potential to prevent ketoacidosis and accelerate development of disease-modifying therapies. Current tools for predicting risk require multiple blood samples taken during an OGTT. Our aim was to develop and validate a simpler tool based on a single blood draw. Methods Models to predict disease progression using a single OGTT time point (0, 30, 60, 90 or 120 min) were developed using TrialNet data collected from relatives with type 1 diabetes and validated in independent populations at high genetic risk of type 1 diabetes (TrialNet, Diabetes Prevention Trial–Type 1, The Environmental Determinants of Diabetes in the Young [1]) and in a general population of Bavarian children who participated in Fr1da. Results Cox proportional hazards models combining plasma glucose, C-peptide, sex, age, BMI, HbA1c and insulinoma antigen-2 autoantibody status predicted disease progression in all populations. In TrialNet, the AUC for receiver operating characteristic curves for models named M60, M90 and M120, based on sampling at 60, 90 and 120 min, was 0.760, 0.761 and 0.745, respectively. These were not significantly different from the AUC of 0.760 for the gold standard Diabetes Prevention Trial Risk Score, which requires five OGTT blood samples. In TEDDY, where only 120 min blood sampling had been performed, the M120 AUC was 0.865. In Fr1da, the M120 AUC of 0.742 was significantly greater than the M60 AUC of 0.615. Conclusions/interpretation Prediction models based on a single OGTT blood draw accurately predict disease progression from stage 1 or 2 to stage 3 type 1 diabetes. The operational simplicity of M120, its validity across different at-risk populations and the requirement for 120 min sampling to stage type 1 diabetes suggest M120 could be readily applied to decrease the cost and complexity of risk stratification. Graphical abstract

Background: Type 1 diabetes results from autoimmune-mediated destruction of beta cells. The tyrosine kinase inhibitor imatinib may impact relevant immunologic and metabolic pathways, and preclinical studies show that it reverses and prevents diabetes. Our goal was to evaluate the safety and efficacy of imatinib in preserving beta cell function in subjects with recent-onset type 1 diabetes. Methods: We conducted a phase 2, randomised, placebo-controlled, blinded clinical trial. Patients with recent-onset type 1 diabetes, aged 18-45 years, and with peak C-peptide ≥ 0.2 nmoles/L on mixed meal tolerance test (MMTT) were enrolled from 9 sites in the USA and Australia. Participants were randomly assigned 2:1 to receive either 400 mg imatinib or matching placebo for 26 weeks, respectively, using a computer-generated blocked randomisation scheme stratified by study site. The primary endpoint was the change in 2-hour area under the curve (AUC) C-peptide response to MMTT from baseline to 12 months, with further observation out to 24 months. Analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01781975. Findings: Patients were screened and enrolled into the trial between February 12, 2014 and May 19, 2016. 45 patients were assigned to imatinib, 22 to placebo. The study met its primary endpoint: the 12-mo C-peptide AUC mean was 0·583 (95% CI: 0·519, 0·650) nmoles/L in the imatinib group versus 0·489 (95% CI: 0·403, 0·579) nmoles/L for the placebo group, adjusted for baseline C-peptide, age, and gender (p = 0·048, 1-tail test). In secondary analyses, compared to the placebo group, imatinib-treated subjects required less exogenous insulin and trended towards lower HbA 1c , with higher beta cell glucose sensitivity, lower proinsulin/C-peptide ratios, improved insulin sensitivity, and higher serum adiponectin levels. No significant changes were noted in autoantibody titers and immune cell phenotyping. Interpretation: A 26-wk course of imatinib preserves beta cell function at 12 months in adults with recent onset type 1 diabetes. Although no overt effects on immune responses were detected, imatinib may have novel metabolic effects on both beta cell function and insulin sensitivity. Trial Registration: This study is registered with ClinicalTrials.gov, number NCT01781975. Funding: Juvenile Research Diabetes Foundation Declaration of Interests: SG has served on advisory boards for Avotres, Provention Bio, and Tolerion, and participated in clinical trials with Intrexon, Janssen, Provention Bio, and Tolerion. JB has consulted for Vertex Pharmaceuticals, Inc, and participated in clinical trials for Avotres, Caladrius, Janssen, and Tolerion. PG has served on advisory boards for Caladrius, Bristol Myers Squibb, and Lilly, received grant support from Caladrius, Novo Nordisk and Pfizer, and is co-founder and chief medica officer for ImmunoMolecular Therapeutics, Inc. RM has a patent application for DNA methylation in inflammatory disease. SW reports serving on advisory boards for Boehringer Ingelheim Pharmaceuticals, Inc. and Medtronic, and a DSMB for the National Institutes of Health. All other authors report no potential conflicts of interest. Ethics Approval Statement: This phase 2, randomised, placebo-controlled, double-blinded clinical trial was conducted according to the Declaration of Helsinki and in accordance with good clinical practice guidelines, performed under an investigational new drug application (IND 117644) and was approved by independent institutional review boards at each participating center.

The increasing incidence of type 2 diabetes and of health consequences related to complications from inadequate glycemic control is of major concern, even in the midst of a worldwide pandemic. Desp...

Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-gamma ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-gamma ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells in 3 longitudinal samples collected 6 months apart. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative response - but not inflammatory - is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, and our data may indicate that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity. Disclosure R.L. Baker: None. T. Delong: None. M. Rewers: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. K.M. Haskins: None. Funding American Diabetes Association (1-15-ACE-14 to T.D.); JDRF (2-SRA-2017-511-S-B); National Institutes of Health (R21AI133059-01A1)

Electrochemiluminescence (ECL) assays are high-affinity autoantibodies (Abs) that are more specific than radiobinding assay (RBA) Abs for the prediction of type 1 diabetes (T1D) in natural history studies screening for risk and progression to T1D. We analyzed 603 subjects from the TrialNet Pathway to Prevention Study who were positive for at least one RBA diabetes related Ab and for whom ECL and HLA data were available. We sought to characterize ECL-GADA and ECL-IAA positivity and levels (z-scores) by HLA genotype and haplotypes. Mean age at initial visit was 19.4 ± 13.7 years; 345 (57.2%) were female and 104 (17.2%) carried the high-risk HLA- DR3/4*0302 genotype. At initial visit 425 (70%) of subjects were positive for either ECL-GADA or ECL-IAA, with 207 (34.3%) ECL-IAA positive subjects and 367 (60.9%) ECL-GADA positive subjects. For subjects with high-risk HLA-DR3/4 genotype (n=104), 80 (76.9%) were positive for ECL-GADA, while 42 (40.4%) were positive for ECL-IAA. ECL and RBA-GADA z-scores were higher in subjects with HLA-DR3 haplotypes, while ECL-IAA (but not RBA-IAA) z-scores were associated with HLA-DR4 haplotypes. Associations of ECL and RIA Abs with high-risk HLA haplotypes are shown in the Table. In conclusion, ECL and RBA-GADA positivity are associated with both HLA-DR3 and DR4 haplotypes, whereas only ECL-IAA (but not RBA-IAA) positivity and levels are associated with HLA-DR4 haplotype. Disclosure T.M. Triolo: None. L. Pyle: None. L. Yu: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. A. Steck: None. Funding American Diabetes Association (1-14-CD-17 to A.S.); National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; JDRF

Context Concordance for persistent islet autoimmunity (IA) and type 1 diabetes in monozygotic twins after probands are diagnosed is variable (30%-70%). Risk for development of IA in dizygotic twins is thought to be similar to nontwin siblings. Little is known in regard to the development of celiac autoimmunity (CDA) in twins of subjects with type 1 diabetes. Objective Our aim was to investigate the development of IA and CDA in cotwins of probands with type 1 diabetes. Methods Since 1995, the Twin Family Study has followed 336 twins (168 twin probands with type 1 diabetes and 168 cotwins) for a median of 14 years (interquartile range:10-18 years). Cotwins were followed for the development of IA, type 1 diabetes, and CDA. Results In monozygotic cotwins, cumulative incidence by age 20 was 14% for IA and 10% for CDA. Development of IA and CDA by age 20 was 9% and 12% in dizygotic cotwins, respectively. While the numbers are small, IA by age 30 years was 26% in monozygotic and 39% in dizygotic twins. In proportional hazards models, the proband’s younger age at diagnosis, but not sex or human leukocyte antigen were associated with time to IA and CDA in cotwins. Conclusion CDA risk by age 20 in cotwins was 10% to 12%. With long-term follow-up, cumulative incidence for IA is high in dizygotic twins, similar to monozygotic twins, suggesting a role of possible early environmental factors shared by type 1 diabetes discordant cotwins.

OBJECTIVE Dysbiosis of the gut microbiota has been linked to disease pathogenesis in type 1 diabetes, yet the functional consequences to the host of this dysbiosis are unknown. We investigated the functional interactions between the microbiota and the host associated with type 1 diabetes disease risk. RESEARCH DESIGN AND METHODS We performed a cross-sectional analysis of stool samples from subjects with recent-onset type 1 diabetes (n = 33), islet autoantibody–positive subjects (n = 17), low-risk autoantibody-negative subjects (n = 29), and healthy subjects (n = 22). Metaproteomic analysis was used to identify gut- and pancreas-derived host and microbial proteins, and these data were integrated with sequencing-based microbiota profiling. RESULTS Both human (host-derived) proteins and microbial-derived proteins could be used to differentiate new-onset and islet autoantibody–positive subjects from low-risk subjects. Significant alterations were identified in the prevalence of host proteins associated with exocrine pancreas output, inflammation, and mucosal function. Integrative analysis showed that microbial taxa associated with host proteins involved in maintaining function of the mucous barrier, microvilli adhesion, and exocrine pancreas were depleted in patients with new-onset type 1 diabetes. CONCLUSIONS These data support that patients with type 1 diabetes have increased intestinal inflammation and decreased barrier function. They also confirmed that pancreatic exocrine dysfunction occurs in new-onset type 1 diabetes and show for the first time that this dysfunction is present in high-risk individuals before disease onset. The data identify a unique type 1 diabetes–associated signature in stool that may be useful as a means to monitor disease progression or response to therapies aimed at restoring a healthy microbiota.

Very recently one of the largest type 1 diabetes prevention trials using daily administration of oral insulin or placebo was completed. After 9 years of study enrollment and follow-up, the randomized controlled trial failed to delay the onset of clinical type 1 diabetes, which was the primary end point. The unfortunate outcome follows the previous large-scale trial, the Diabetes Prevention Trial–Type 1 (DPT-1), which again failed to delay diabetes onset with oral insulin or low-dose subcutaneous insulin injections in a randomized controlled trial with relatives at risk for type 1 diabetes. These sobering results raise the important question, “Where does the type 1 diabetes prevention field move next?” In this Perspective, we advocate for a paradigm shift in which smaller mechanistic trials are conducted to define immune mechanisms and potentially identify treatment responders. The stage is set for these interventions in individuals at risk for type 1 diabetes as Type 1 Diabetes TrialNet has identified thousands of relatives with islet autoantibodies and general population screening for type 1 diabetes risk is under way. Mechanistic trials will allow for better trial design and patient selection based upon molecular markers prior to large randomized controlled trials, moving toward a personalized medicine approach for the prevention of type 1 diabetes.

Autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), is the most common autoimmune disorder in the United States, affecting over 20 million people. At the time of diagnosis, both HD and GD are characterized by the accumulation of B and T lymphocytes in the thyroid gland and production of autoantibodies targeting the thyroid, indicating that a breach in tolerance of autoreactive lymphocytes has occurred. However, few studies have sought to understand the underlying pathogenesis of AITD that ultimately leads to production of autoantibodies and loss of thyroid function. In this study, we analyzed the phenotype of thyroid antigen-reactive B cells in the peripheral blood of recent onset and long standing AITD patients. We found that in recent onset patients thyroid antigen-reactive B cells in blood no longer appear anergic, rather they express CD86, a marker of activation. This likely reflects activation of these cells leading to their production of autoantibodies. Hence, this study reports the early loss of anergy in thyroid antigen-reactive B cells, an event that contributes to development of AITD.

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.

Although B cells reactive with islet autoantigens are silenced by tolerance mechanisms in healthy individuals, they can become activated and contribute to the development of type 1 diabetes. We previously demonstrated that high-affinity insulin-binding B cells (IBCs) occur exclusively in the anergic (BND) compartment in peripheral blood of healthy subjects. Consistent with their activation early in disease development, high-affinity IBCs are absent from the BND compartment of some first-degree relatives (FDRs) as well as all patients with autoantibody-positive prediabetes and new-onset type 1 diabetes, a time when they are found in pancreatic islets. Loss of BND IBCs is associated with a loss of the entire BND B-cell compartment consistent with provocation by an environmental trigger or predisposing genetic factors. To investigate potential mechanisms operative in subversion of B-cell tolerance, we explored associations between HLA and non-HLA type 1 diabetes–associated risk allele genotypes and loss of BNDs in FDRs. We found that high-risk HLA alleles and a subset of non-HLA risk alleles (i.e., PTPN2 [rs1893217], INS [rs689], and IKZF3 [rs2872507]), relevant to B- and T-cell development and function are associated with loss of anergy. Hence, the results suggest a role for risk-conferring alleles in perturbation of B-cell anergy during development of type 1 diabetes.

Background: Electrochemiluminescence (ECL) assays have shown promise for enhancing the prediction of type 1 diabetes (T1D) with autoantibodies. We thus studied relatives of T1D patients to determine whether ECL assays can be used to refine risk assessments for T1D among individuals either positive for single GADA or single mIAA autoantibodies. Subjects and Methods: TrialNet Pathway to Prevention (PTP) study participants with either GADA or mIAA single autoantibodies were tested for ECL positivity during their participation in the TrialNet PTP study. Those ECL positive (ECL+) were compared with those ECL negative (ECL−) for conversion to multiple autoantibodies, 6-month glycemic progression (PS6M), and the progression to T1D. Results: The progression to multiple autoantibodies was significantly higher for those GADA/ECL+ (n = 107) than those GADA/ECL− (n = 78) (P = 0.001) and for those mIAA/ECL+ (n = 24) than those mIAA/ECL− (n = 63) (P

BACKGROUND. The cytokine IL-7 is critical for T cell development and function. We performed a Phase Ib study in patients with type 1 diabetes (T1D) to evaluate how blockade of IL-7 would affect immune cells and relevant clinical responses. METHODS. Thirty-seven subjects with T1D received s.c. RN168, a monoclonal antibody that blocks the IL -7 receptor α (IL7Rα) in a dose-escalating study. RESULTS. Between 90% and 100% IL-7R occupancy and near-complete inhibition of pSTAT5 was observed at doses of RN168 1 mg/kg every other week (Q2wk) and greater. There was a significant decline in CD4(+) and CD8(+) effector and central memory T cells and CD4(+) naive cells, but there were fewer effects on CD8(+) naive T cells. The ratios of Tregs to CD4(+) or CD8(+) effector and central memory T cells versus baseline were increased. RNA sequencing analysis showed downmodulation of genes associated with activation, survival, and differentiation of T cells. Expression of the antiapoptotic protein Bcl-2 was reduced. The majority of treatment-emergent adverse events (TEAEs) were mild and not treatment related. Four subjects became anti–EBV IgG(+) after RN168, and 2 had symptoms of active infection. The immunologic response to tetanus toxoid was preserved at doses of 1 and 3 mg/kg Q2wk but reduced at higher doses. CONCLUSIONS. This trial shows that, at dosages of 1–3 mg/kg, RN168 selectively inhibits the survival and activity of memory T cells while preserving naive T cells and Tregs. These immunologic effects may serve to eliminate pathologic T cells in autoimmune diseases. TRIAL REGISTRATION. NCT02038764. FUNDING. Pfizer Inc.

Context The incidence of type 1 diabetes (T1D) is increasing worldwide. The quest to understand T1D etiology and how to predict diabetes is ongoing; and, in many ways, those goals intertwine. Although genetic components associate with T1D, not all individuals with T1D have those components, and T1D does not develop in all subjects with those components. Objective More robust methods for prediction of T1D are needed. We investigated if high CD4+CD40+ T-cell (Th40) levels can be used as a biomarker. Methods Th40 levels were assessed along with other parameters in blood collected from prediabetic subjects in TrialNet. Results In prediabetic subjects stratified according to Th40 cell level, patterns paralleled those seen between control subjects and those with T1D. Cytokine patterns were significantly different between those with high Th-40 levels (Th40-high) and those with low levels, and a CD4/CD8 double-positive population was more represented in Th40-high groups. Subjects experiencing impaired glucose tolerance had a significantly higher Th40 level than did control subjects. HLA DR4/DR4 and DQ8/DQ8 were more likely found among Th40-high subjects. Interestingly, HLA DR4/DR4 subjects were significantly older compared with all other subjects, suggesting that this haplotype, together with a high Th40 level, may represent someone in whom T1D will develop after age 30 years, which is reported for 42% of T1D cases. Conclusion Considering the differences found in relation to prediabetic Th40 cell level, it may be possible to devise methods that more accurately predict who will proceed toward diabetes and, possibly, indicate prediabetic stage.

Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies.TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits.After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time.Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects.

Aims/hypothesis Type 1 diabetes results from T cell mediated destruction of beta cells. We conducted a trial of antithymocyte globulin (ATG) in new-onset type 1 diabetes (the Study of Thymoglobulin to ARrest T1D [START] trial). Our goal was to evaluate the longer-term safety and efficacy of ATG in preserving islet function at 2 years. Methods A multicentre, randomised, double-blind, placebo-controlled trial of 6.5 mg/kg ATG (Thymoglobulin) vs placebo in patients with new-onset type 1 diabetes was conducted at seven university medical centres and one Children’s Hospital in the USA. The site-stratified randomisation scheme was computer generated at the data coordinating centre using permuted-blocks of size 3 or 6. Eligible participants were between the ages of 12 and 35, and enrolled within 100 days from diagnosis. Subjects were randomised to 6.5 mg/kg ATG (thymoglobulin) vs placebo in a 2:1 ratio. Participants were blinded, and the study design included two sequential patient-care teams: an unblinded study-drug administration team (for the first 8 weeks), and a blinded diabetes management team (for the remainder of the study). Endpoints assessed at 24 months included meal-stimulated C-peptide AUC, safety and immunological responses. Results Fifty-eight patients were enrolled; at 2 years, 35 assigned to ATG and 16 to placebo completed the study. The pre-specified endpoints were not met. In post hoc analyses, older patients (age 22–35 years) in the ATG group had significantly greater C-peptide AUCs at 24 months than placebo patients. Using complete preservation of baseline C-peptide at 24 months as threshold, nine of 35 ATG-treated participants (vs 2/16 placebo participants) were classified as responders; nine of 11 responders (67%) were older. All participants reported at least one adverse event (AE), with 1,148 events in the 38 ATG participants vs 415 in the 20 placebo participants; a comparable number of infections were noted in the ATG and placebo groups, with no opportunistic infections nor difficulty clearing infections in either group. Circulating T cell subsets depleted by ATG partially reconstituted, but regulatory, naive and central memory subsets remained significantly depleted at 24 months. Beta cell autoantibodies did not change over the 24 months in the ATG-treated or placebo participants. At 12 months, ATG-treated participants had similar humoral immune responses to tetanus and HepA vaccines as placebo-treated participants, and no increased infections. Conclusions/interpretation A brief course of ATG substantially depleted T cell subsets, including regulatory cells, but did not preserve islet function 24 months later in the majority of patients with new-onset type 1 diabetes. ATG preserved C-peptide secretion in older participants, which may warrant further study. Trial registration: ClinicalTrials.gov NCT00515099 Public data repository: START datasets are available in TrialShare www.itntrialshare.org Funding: National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH). The trial was conducted by the Immune Tolerance Network (ITN).

Objective Biomarkers are needed to characterize heterogeneity within populations at risk for type 1 diabetes. The ratio of proinsulin to C-peptide (PI:C ratio), has been proposed as a biomarker of beta cell dysfunction and is associated with progression to type 1 diabetes. However, relationships between PI:C ratios and autoantibody type and number have not been examined. We sought to characterize PI:C ratios in multiple islet autoantibody positive, single autoantibody positive and autoantibody negative relatives of individuals with type 1 diabetes. Methods We measured PI:C ratios and autoantibodies with both electrochemiluminescence (ECL) assays (ECL-IAA, ECL-GADA and ECL-IA2A) and radiobinding (RBA) assays (mIAA, GADA, IA2A and ZnT8A) in 98 relatives of individuals with type 1 diabetes followed in the TrialNet Pathway to Prevention Study at the Barbara Davis Center for a mean of 7.4 ​± ​4.1 years. Of these subjects, eight progressed to T1D, 31 were multiple autoantibody (Ab) positive, 37 were single Ab positive and 22 were Ab negative (by RBA). Results In cross-sectional analyses, there were no significant differences in PI:C ratios between type 1 diabetes and/or multiple Ab positive subjects (4.16 ​± ​4.06) compared to single Ab positive subjects (4.08 ​± ​4.34) and negative Ab subjects (3.72 ​± ​3.78) (p ​= ​0.92) overall or after adjusting for age, sex and BMI. Higher PI:C ratios were associated with mIAA titers (p ​= ​0.03) and showed an association with ECL-IA2A titers (p ​= ​0.09), but not with ECL-IAA, GADA, ECL-GADA, IA2A nor ZnT8A titers. In mixed-effects longitudinal models, the trajectories of PI:C ratio over time were significantly different between the Ab negative and multiple Ab positive/type 1 diabetes groups, after adjusting for sex, age, and BMI (p ​= ​0.04). Conclusions PI:C ratio trajectories increase over time in subjects who have multiple Ab or develop type 1 diabetes and may be a helpful biomarker to further characterize and stratify risk of progression to type 1 diabetes over time., Highlights • Higher PI:C ratios are associated with mIAA titers. • Trajectories of PI:C increase overtime in multiple antibody positive subjects. • PI:C could be a useful biomarker to trend in those at risk for type 1 diabetes.

The long-term effects of successful immune therapies for treatment of type 1 diabetes have not been well studied. The Autoimmunity-Blocking Antibody for Tolerance (AbATE) trial evaluated teplizumab, an Fc receptor non-binding humanised anti-CD3 monoclonal antibody in individuals with new-onset type 1 diabetes, and ended in 2011. Clinical drug-treated responders showed an increased frequency of ‘partially exhausted’ CD8+ T cells. We studied the clinical, immunological and metabolic status of participants after an average follow-up of 7 years. Participants with detectable C-peptide at year 2 of AbATE returned for follow-up. C-peptide responses were assessed by 4 h mixed-meal tolerance test. Autoantibodies and HbA1c levels were measured and average daily insulin use was obtained from patient logs. Peripheral blood mononuclear cells were analysed by flow cytometry and cytokine release. Fifty-six per cent of the original participants returned. Three of the original control group who did not return had lost all detectable C-peptide by the end of the 2 year trial. The C-peptide responses to a mixed-meal tolerance test were similar overall in the drug vs control group of participants but were significantly improved, with less loss of C-peptide, in drug-treated responders identified at 1 year. However, the improvements in C-peptide response were not associated with lower HbA1c levels or insulin use. Drug-treated responders showed a significantly increased frequency of programmed cell death protein 1-positive central memory and anergic CD8+ T cells at follow-up. These findings suggest there is reduced decline in C-peptide and persistent immunological responses up to 7 years after diagnosis of diabetes in individuals who respond to teplizumab. ClinicalTrials.gov NCT02067923; the protocol is available at www.immunetolerance.org (ITN027AI).

OBJECTIVE A pilot study suggested that combination therapy with low-dose anti-thymocyte globulin (ATG) and pegylated granulocyte colony-stimulating factor (GCSF) preserves C-peptide in established type 1 diabetes (T1D) (duration 4 months to 2 years). We hypothesized that 1) low-dose ATG/GCSF or 2) low-dose ATG alone would slow the decline of β-cell function in patients with new-onset T1D (duration RESEARCH DESIGN AND METHODS A three-arm, randomized, double-masked, placebo-controlled trial was performed by the Type 1 Diabetes TrialNet Study Group in 89 subjects: 29 subjects randomized to ATG (2.5 mg/kg intravenously) followed by pegylated GCSF (6 mg subcutaneously every 2 weeks for 6 doses), 29 to ATG alone (2.5 mg/kg), and 31 to placebo. The primary end point was mean area under the curve (AUC) C-peptide during a 2-h mixed-meal tolerance test 1 year after initiation of therapy. Significance was defined as one-sided P value < 0.025. RESULTS The 1-year mean AUC C-peptide was significantly higher in subjects treated with ATG (0.646 nmol/L) versus placebo (0.406 nmol/L) (P = 0.0003) but not in those treated with ATG/GCSF (0.528 nmol/L) versus placebo (P = 0.031). HbA1c was significantly reduced at 1 year in subjects treated with ATG and ATG/GCSF, P = 0.002 and 0.011, respectively. CONCLUSIONS Low-dose ATG slowed decline of C-peptide and reduced HbA1c in new-onset T1D. Addition of GCSF did not enhance C-peptide preservation afforded by low-dose ATG. Future studies should be considered to determine whether low-dose ATG alone or in combination with other agents may prevent or delay the onset of the disease.

It has been reported that 2.5%–30% of human peripheral CD27(–) B cells are autoreactive and anergic based on unresponsiveness to antigen receptor (BCR) stimulation and autoreactivity of cloned and expressed BCR. The molecular mechanisms that maintain this unresponsiveness are unknown. Here, we showed that in humans anergy is maintained by elevated expression of PTEN, a phosphatidylinositol 3,4,5P-3-phosphatase. Upregulation of PTEN was associated with reduced expression of microRNAs that control its expression. Pharmacologic inhibition of PTEN lead to significant restoration of responsiveness. Consistent with a role in conferring risk of autoimmunity, B cells from type 1 diabetics and autoimmune thyroid disease patients expressed reduced PTEN. Unexpectedly, in healthy individuals PTEN expression was elevated in on average 40% of CD27(–) B cells, with levels gradually decreasing as IgM levels increase. Our findings suggest that a much higher proportion of the peripheral repertoire is autoreactive than previously thought and that B cells upregulate PTEN in a manner that is proportional to the recognition of autoantigens of increasing avidity, thus tuning BCR signaling to prevent development of autoimmunity while providing a reservoir of cells that can be readily activated to respond when needed.

We recently established, using the NOD mouse model, that hybrid insulin peptides (HIPs), formed spontaneously in islet beta-cells by fusion of insulin C-peptide fragments to peptides of Chromogranin A (ChgA) or Islet Amyloid Polypeptide (IAPP), are ligands for diabetogenic CD4 T cell clones. The goal of this study was to investigate whether HIP-reactive T cells were indicative of ongoing autoimmunity in human T1D patients. To investigate their relevance to human disease, we used IFN-gamma ELISPOT to determine whether HIP-reactive T cells with an inflammatory phenotype were present in PBMCs of new onset T1D patients. We observed that PBMCs from new onset T1D patients responded to 8 of 16 HIPs tested and that nearly half of the patients tested responded to one or more HIPs. We used a CFSE based assay to further characterize T cells reactive to HIP peptides. CD4+CFSEdimCD25+ T cells were sorted single into single wells using FACS and cloned. T cell clonality was confirmed using TCR deep sequencing and T cell reactivity to specific HIPs was confirmed by ELISA. A total of 6 T cell clones were isolated and show a variety of TCR usage. One T cell clone was isolated from the same patient on two different blood draws, indicating a high frequency of this T cell clone in the peripheral blood. While all the T cell clones isolated secreted IFN-gamma in response to HIPs, they also produced other inflammatory cytokines such as TNF-alpha and GM-CSF and reacted to HIPs in the low nanomolar range. Our findings provide new evidence that HIP-reactive T cells are critical players in the pathogenesis of T1D and indicate that HIP-reactive T cells present in the peripheral blood can potentially serve as a biomarker of disease in a subset of patients. Disclosure R.L. Baker: None. T. Delong: None. P. Gottlieb: Advisory Panel; Self; Bristol-Myers Squibb Company. Research Support; Self; Caladrius Biosciences, Inc.. Advisory Panel; Self; Eli Lilly and Company. Board Member; Self; ImmunoMolecular Therapeutics. Consultant; Self; Kamada. Research Support; Self; JDRF, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc., MacroGenics, Inc., Pfizer Inc.. Advisory Panel; Self; Viacyte, Inc.. Research Support; Self; GlaxoSmithKline plc., Janssen Pharmaceuticals, Inc.. K.M. Haskins: None.

Concordance for persistent islet autoimmunity (IA) and type 1 diabetes (T1D) in monozygotic twins (MZ) after one twin is diagnosed has been highly variable (30-70%), while risk for development of IA in dizygotic twins (DZ) is thought to be similar to non-twin siblings. Since 1995, the Twin Family Study at the Barbara Davis Center has followed 336 twins (168 twin probands diagnosed with T1D and 168 cotwins) with a median follow-up of 14 years (ICR:10-18 years). Zygosity testing confirmed a total of 80 MZ pairs and 88 DZ pairs. Cotwins were followed for the development of IA and celiac autoimmunity (CDA). In MZ cotwins, cumulative incidence for IA was 14% by age 20 and 63% by age 45, while development of CDA was 10% by 20 years. Development of IA and CDA by age 20 was 9.2% and 12.3% in DZ cotwins respectively, with development of IA reaching 70% by age 30 (Figure). In Cox proportional hazards models, only the proband’s age at diagnosis, but not sex, HLA-DR3 nor DR4 were associated with time to IA and CDA in cotwins. In MZ twins, younger age of the proband was associated with an increased hazard ratio (HR) for both IA and CDA, while younger age in DZ twins was associated with an increased HR for IA only. In conclusion, CDA risk does not appear to increase after age 15. With long term follow-up, cumulative incidence for IA is high in DZ twins, similar to MZ twins, suggesting a role of possible early environmental factors shared by T1D-discordant cotwins. Disclosure T.M. Triolo: None. A.R. Fouts: None. L. Pyle: None. L. Yu: None. P. Gottlieb: Advisory Panel; Self; Bristol-Myers Squibb Company. Research Support; Self; Caladrius Biosciences, Inc.. Advisory Panel; Self; Eli Lilly and Company. Board Member; Self; ImmunoMolecular Therapeutics. Consultant; Self; Kamada. Research Support; Self; JDRF, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc., MacroGenics, Inc., Pfizer Inc.. Advisory Panel; Self; Viacyte, Inc.. Research Support; Self; GlaxoSmithKline plc., Janssen Pharmaceuticals, Inc.. A. Steck: None.

Background Minnesota is home to the largest Somali population in USA, and pediatric diabetes teams are seeing increasing numbers of Somali children with diabetes. Objective To assess the immune basis of diabetes in Somali children in the Twin Cities, Minnesota. Methods A total of 31 Somali children ≤19 yr were treated for type 1 diabetes (T1D) at the University of Minnesota Masonic Children's Hospital and Children's Hospitals and Clinics of Minnesota underwent analysis of human leukocyte antigen (HLA) alleles (n = 30) and diabetes autoantibodies [glutamic acid decarboxylase (GAD65), islet antigen 2 (IA-2), zinc transporter 8 (ZnT8); n = 31]. HLA alleles were analyzed in 49 Somalis without diabetes (controls). Anti-transglutaminase autoantibodies (TGA) for celiac disease were also measured. Results In Somali children with T1D aged 13.5 ± 5 yr (35% female, disease duration 6.5 ± 3.6 yr), the most common HLA allele was DRB1*03:01 (93%, compared with 45% of Somali controls), followed by DRB1*13:02 (27%). There was a relatively low frequency of DR4 (13%). Controls showed a similar pattern. All 31 participants were positive for at least one diabetes autoantibody. Insulin antibodies were positive in 84% (all were on insulin). Excluding insulin antibodies, 23 (74%) subjects tested positive for at least one other diabetes autoantibody; 32% had 1 autoantibody, 32% had 2 autoantibodies, and 10% had 3 autoantibodies. GAD65 autoantibodies were found in 56% of subjects, IA-2 in 29%, and ZnT8 in 26%. Four (13%) were TGA positive. Conclusion The autoantibody and HLA profiles of Somali children with diabetes are consistent with autoimmune diabetes. Their HLA profile is unique with an exceptionally high prevalence of DRB1*03:01 allele and relative paucity of DR4 alleles compared with African Americans with T1D.

Insights from prospective, longitudinal studies of individuals at risk for developing type 1 diabetes have demonstrated that the disease is a continuum that progresses sequentially at variable but predictable rates through distinct identifiable stages prior to the onset of symptoms. Stage 1 is defined as the presence of β-cell autoimmunity as evidenced by the presence of two or more islet autoantibodies with normoglycemia and is presymptomatic, stage 2 as the presence of β-cell autoimmunity with dysglycemia and is presymptomatic, and stage 3 as onset of symptomatic disease. Adoption of this staging classification provides a standardized taxonomy for type 1 diabetes and will aid the development of therapies and the design of clinical trials to prevent symptomatic disease, promote precision medicine, and provide a framework for an optimized benefit/risk ratio that will impact regulatory approval, reimbursement, and adoption of interventions in the early stages of type 1 diabetes to prevent symptomatic disease.