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AIMS: We examined the effect of growth hormone (GH) counter-regulation on carbohydrate metabolism in individuals with life-long diminished insulin secretion (DIS).METHODS: Adults homozygous for the E180 splice site mutation of GHR [Laron syndrome (LS)], adults with a gain-of-function mutation in CDKN1c [Guevara-Rosenbloom syndrome (GRS)], and controls were evaluated for body composition, leptin, total and high molecular weight (HMW) adiponectin, insulin-like growth factor (IGF) axis molecules, and a 5-hour oral glucose tolerance test (OGTT), with measurements of glucose, insulin, glucagon, ghrelin, pancreatic polypeptide, gastric inhibitory peptide, glucagon-like peptide-1, peptide YY, and islet amyloid polypeptide (IAPP).RESULTS: Both syndromic cohorts displayed DIS during OGTT. LS subjects had higher serum concentrations of total and HMW adiponectin, and lower levels of IGF-I, IGF-II, and IGF-Binding Protein-3 than individuals in other study groups. Furthermore, they displayed normal glycemic responses during OGTT with the lowest IAPP secretion. In contrast, individuals with GRS had higher levels of protein glycation, deficient glucose control during OGTT, and increased secretion of IAPP.CONCLUSIONS: A distinct metabolic phenotype depending on GH counter-regulatory status, associates with diabetes development and excess glucose-induced IAPP secretion.

Type 1 diabetes (T1D) presents with two therapeutic challenges: the need to correct underlying autoimmunity and restore β-cell mass. We harnessed the unique capacity of regulatory T cells (Tregs) and the T cell receptor (TCR) to direct tolerance induction along with tissue-localized delivery of therapeutic agents to restore endogenous β-cell function. Specifically, we designed a combinatorial therapy involving biomaterials-based poly(lactic-co-glycolic acid) nanoparticles co-loaded with the Treg growth factor, IL-2, and the β-cell regenerative agent, harmine (a tyrosine-regulated kinase 1A [DYRK1A] inhibitor), conjugated to the surface of Tregs. We observed continuous elution of IL-2 and harmine from nanoparticles for at least 7 days in vitro. When conjugated to primary human Tregs, IL-2 nanoparticles provided sufficient IL-2 receptor signaling to support STAT5 phosphorylation for sustained phenotypic stability and viability in culture. Inclusion of poly-L-lysine (PLL) during nanoparticle-cell coupling dramatically increased conjugation efficiency, providing sufficient IL-2 to support in vitro proliferation of IL-2-dependent CTLL-2 cells and primary murine Tregs. In 12-week-old female non-obese diabetic mice, adoptive transfer of IL-2/harmine nanoparticle-conjugated NOD.BDC2.5 Tregs, which express an islet antigen-specific TCR, significantly prevented diabetes demonstrating preserved in vivo viability. These data provide the preclinical basis to develop a biomaterials-optimized cellular therapy to restore immune tolerance and promote β-cell proliferation in T1D through receptor-targeted drug delivery within pancreatic islets.

OBJECTIVE Women are broadly underrepresented in scientific leadership positions and their accomplishments are not provided equal recognition compared with those of men, but the imbalance in the field of diabetes is unknown. Hence, we analyzed multiple aspects of historical and present-day female representation in the diabetes field. RESEARCH DESIGN AND METHODS We quantified gender representation at annual American Diabetes Association (ADA) meetings; editorial board service positions for ADA and the European Association for the Study of Diabetes (EASD) journals; principal investigators for ADA, JDRF, and National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases P30 grant funding; and ADA, JDRF, and EASD award recipients. RESULTS There are many women in the field of diabetes: registration for the ADA Scientific Sessions has been 43% female since 2016, and for over five decades, women comprised 83% of ADA Presidents of Health Care and Education. Yet, only 9% of ADA Presidents of Medicine and Science have been women. Women were well represented on editorial boards for journals focused on diabetes education (Diabetes Spectrum, 89% female) and primary care (Clinical Diabetes, 49% female) but not for the more academically targeted Diabetes Care (34% female), Diabetes (21% female), and Diabetologia (30% female). Only one-third of ADA Pathway to Stop Diabetes and JDRF grants have been awarded to women, and females only lead 2 of 18 (11%) of the P30-supported Diabetes Research Centers. Finally, only 2–12% of major ADA, JDRF, and EASD awards were given to women, without significant change over time. CONCLUSIONS Despite increasing recognition of gender imbalance in research and medicine, many disparities in the field of diabetes persist. We call for decreasing barriers for advancement of female investigators and creating environments that promote their retention and equitable recognition for their contributions to the field.

Emerging data suggest that type 1 diabetes affects not only the β-cell-containing islets of Langerhans, but also the surrounding exocrine compartment. Using digital pathology, machine learning algorithms were applied to high-resolution, whole-slide images of human pancreata to determine whether the tissue composition in individuals with or at risk for type 1 diabetes differs from those without diabetes. Transplant-grade pancreata from organ donors were evaluated from 16 nondiabetic autoantibody-negative controls, 8 nondiabetic autoantibody-positive subjects with increased type 1 diabetes risk, and 19 persons with type 1 diabetes (0 to 12 years' duration). HALO image analysis algorithms were implemented to compare architecture of the main pancreatic duct as well as cell size, density, and area of acinar, endocrine, ductal, and other nonendocrine, nonexocrine tissues. Type 1 diabetes was found to affect exocrine area, acinar cell density, and size, whereas the type of difference correlated with the presence or absence of insulin-positive cells remaining in the pancreas. These changes were not observed before disease onset, as indicated by modeling cross-sectional data from pancreata of autoantibody-positive subjects and those diagnosed with type 1 diabetes. These data provide novel insights into anatomic differences in type 1 diabetes pancreata and demonstrate that machine learning can be adapted for the evaluation of disease processes from cross-sectional data sets.

Research-based immunotherapy trials seeking to prevent or reverse a number of autoimmune diseases, including type 1 diabetes, have seen near universal suspension due to the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diabetes and hyperglycemia are now appreciated as significant risk factors for COVID-19 morbidity and mortality; however, the vast majority of studies have reported on adults. Recent data in children and adolescents with type 1 diabetes suggest no increased risk of COVID-19. Even with immense appreciation for COVID-19 morbidity and mortality, we believe compelling arguments exist to carefully and thoughtfully resume certain type 1 diabetes phase 2–3 immunotherapy trials. In this Perspective, we consider the experience of trials that never halted or have resumed in the oncology and rheumatology fields, and advocate for staged type 1 diabetes immunotherapy trial resumption. With this, we present recommendations to achieve equipoise and mitigate risks for SARS-CoV-2 infection in the weeks surrounding infusion. Given the fact that the COVID-19 pandemic is expected to persist for some time, it is in the best interest of our patients that we find ways to safely move our field forward.

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.

Exocrine pancreas abnormalities are increasingly recognized as features of type 1 diabetes. We previously reported reduced serum trypsinogen levels and in a separate study, smaller pancreata at and prior to disease onset. We hypothesized that three pancreas enzymes (amylase, lipase and trypsinogen) might serve as serological biomarkers of pancreas volume and risk for type 1 diabetes. Amylase, lipase, and trypsinogen were measured from two independent cohorts, together comprising 800 serum samples from single-autoantibody positive (1AAb+) and multiple-AAb+ (≥2AAb+) subjects, individuals with recent-onset or established type 1 diabetes, their AAb negative (AAb-) first-degree relatives, and AAb- controls. Lipase and trypsinogen were significantly reduced in ≥2AAb+, recent-onset, and established type 1 diabetes subjects versus controls and 1AAb+, while amylase was reduced only in established type 1 diabetes. Logistic regression models demonstrated trypsinogen plus lipase (AUROC=81.4%) performed equivalently to all three enzymes (AUROC=81.4%) in categorizing ≥2AAb+ versus 1AAb+ subjects. For Cohort 2 (n=246), linear regression demonstrated lipase and trypsinogen levels could individually and collectively serve as indicators of BMI-normalized relative pancreas volume (RPVBMI, PBMI and may improve disease staging in pre-type 1 diabetes.

Co-stimulation serves as a critical checkpoint for T cell development and activation, and several genetic variants affecting co-stimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism in CD226 (rs763361; G307S) has been shown to increase susceptibility to type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. CD226 competes with the co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) to bind CD155 to amplify TCR signaling. We previously found that Cd226 knockout protected non-obese diabetic (NOD) mice from disease, but the impact of CD226 signaling on individual immune subsets remained unclear. We focused on regulatory T cells (Tregs) as a population of interest, as prior reports demonstrated that human CD226+ Tregs exhibit reduced FOXP3+Helios+ purity and suppressive function following expansion. Hence, we hypothesized that global deletion of Cd226 would increase Treg stability and accordingly, Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Indeed, crossing the NOD.Cd226-/- and NOD.Foxp3-GFP-Cre.R26-loxP-STOP-loxP-YFP Treg-fate tracking strains resulted in increased Treg induction and decreased FoxP3-deficient “ex-Tregs” in the pancreatic lymph nodes. We generated a Treg-conditional knockout (TregΔCd226) strain and found that female TregΔCd226 mice had decreased insulitis and diabetes incidence compared to TregWT mice. Additionally, we observed increased TIGIT expression on Tregs and conventional CD4+ T cells within the pancreas of TregΔCd226 versus TregWT mice. These findings demonstrate that an imbalance of CD226/TIGIT signaling may contribute to Treg destabilization in the NOD mouse and highlight the potential for therapeutic targeting of this pathway to prevent or reverse autoimmunity.

IL-2 has been proposed to restore tolerance via regulatory T cell (Treg) expansion in autoimmunity, yet off-target effects necessitate identification of a combinatorial approach. We recently reported reduced levels of immunoregulatory insulin-like growth factor-1 (IGF1) during type 1 diabetes (T1D) progression. Thus, we hypothesized that IGF1 would synergize with IL-2 to expand Tregs. We observed IGF1R was elevated on murine memory and human naïve Treg subsets. IL-2 and IGF1 promoted murine PI3K/Akt and human STAT5 signaling in Tregs. IL-2 and IGF1 treatment expanded Tregs beyond either agent alone in NOD mice. Incubation of naïve human CD4+T cells with IL-2 and IGF1 enhanced Treg proliferationin vitro, without the need for T cell receptor ligation. This synergism was attributed to increased high-affinity IL-2Rα expression on naïve Tregs, in contrast to intermediate-affinity IL-2Rβ and IL-2Rγ subunit enhancement on naïve conventional T cells (Tconv). We then demonstrated that IGF1 and IL-2 or the IL2Rγ-chain-dependent cytokine, IL-7, can be used to induce proliferation of genetically-engineered naïve Treg or Tconv cells, respectively. These data support the potential use of IGF1 in combination with common γ-chain cytokines to drive T cell expansions bothin vitroandin vivofor cellular therapeutics and genetic modifications.

Context We recently demonstrated increased cellular proliferation in the pancreatic ductal gland (PDG) compartment of organ donors with type 1 diabetes, suggesting that PDGs may harbor progenitor cells capable of pancreatic regeneration. Objective We evaluated the impact of diabetes and pancreatic inflammation on PDG and interlobular duct (ILD) cellular proliferation and profiles. Methods Endocrine hormone expression (insulin, glucagon, somatostatin, pancreatic polypeptide) and proliferating Ki67+ cells were localized within the PDG and ILD compartments by multicolor immunohistochemistry in cross-sections from the head, body, and tail regions of pancreata from those with (n = 31) or without type 1 diabetes (n = 43). Whole-slide scanned images were analyzed using digital pathology. Results Type 1 diabetes donors with insulitis or histologically identified pancreatitis had increased cellular replication in the ILD and PDG compartments. Interestingly, while cellular proliferation within the pancreatic ductal tree was significantly increased in type 1 diabetes (PDG mean = 3.36%, SEM = 1.06; ILD mean = 2.78%, SEM = 0.97) vs nondiabetes(ND) subjects without pancreatic inflammation (PDG mean = 1.18%, SEM = 0.42; ILD mean = 0.74%, SEM = 0.15, P Conclusion These data suggest that increased pancreatic ductal cell replication is associated with sustained pancreatic inflammation; however, as replicating cells were hormone-negative, PDGs do not appear to represent a compelling endogenous source of hormone-positive endocrine cells.

Women are broadly underrepresented in scientific leadership positions and their accomplishments are not provided equal recognition compared with those of men, but the imbalance in the field of diabetes is unknown. Hence, we analyzed multiple aspects of historical and present-day female representation in the diabetes field.We quantified gender representation at annual American Diabetes Association (ADA) meetings; editorial board service positions for ADA and the European Association for the Study of Diabetes (EASD) journals; principal investigators for ADA, JDRF, and National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases P30 grant funding; and ADA, JDRF, and EASD award recipients. There are many women in the field of diabetes: registration for the ADA Scientific Sessions has been 43% female since 2016, and for over five decades, women comprised 83% of ADA Presidents of Health Care and Education. Yet, only 9% of ADA Presidents of Medicine and Science have been women. Women were well represented on editorial boards for journals focused on diabetes education (Diabetes Spectrum, 89% female) and primary care (Clinical Diabetes, 49% female) but not for the more academically targeted Diabetes Care (34% female), Diabetes (21% female), and Diabetologia (30% female). Only one-third of ADA Pathway to Stop Diabetes and JDRF grants have been awarded to women, and females only lead 2 of 18 (11%) of the P30-supported Diabetes Research Centers. Finally, only 2-12% of major ADA, JDRF, and EASD awards were given to women, without significant change over time. Despite increasing recognition of gender imbalance in research and medicine, many disparities in the field of diabetes persist. We call for decreasing barriers for advancement of female investigators and creating environments that promote their retention and equitable recognition for their contributions to the field.

Regulatory T cell (Treg) adoptive cell therapy (ACT) represents an emerging strategy for restoring immune tolerance in autoimmune diseases. Tregs are commonly purified using a CD4

SummaryThe spleen and lymph node represent important hubs for both innate and adaptive immunity1,2. Herein, we map immune, endothelial, and neuronal cell networks within these tissues from “normal”/non-diseased organ donors, collected through the NIH Human BioMolecular Atlas Program (HuBMAP)3, using highly multiplexed CODEX (CO-Detection by indEXing) imaging and 3D light sheet microscopy of cleared tissues. Building on prior reports4–6, we observed the lymph node subcapsular sinus expressing podoplanin, smooth muscle actin, and LYVE1. In the spleen, LYVE1 was expressed by littoral cells lining venous sinusoids, whereas podoplanin was restricted to arteries and trabeculae. 3D visualization of perivascular innervation revealed a subset of axonal processes expressing choline acetyl transferase in both tissues, in contrast with prior literature on human spleen7. We further report our novel observations regarding the distinct localization of GAP43 and β3-tubulin within the vascular anatomy of both lymph node and spleen, with Coronin-1A+ cells forming a dense cluster around β3-tubulin positive GAP43 low/negative segments of large vessels in spleen. These data provide an unprecedented 2D and 3D visualization of cellular networks within secondary lymphoid tissues, laying the groundwork for future disease-specific and system-wide studies of neural regulation of immunity in human lymphatics.

Type 1 diabetes (T1D) has a multifactorial autoimmune etiology, involving environmental prompts and polygenic predisposition. We hypothesized that pancreata from individuals with and at risk for T1D would exhibit dysregulated expression of genes associated with monogenic forms of diabetes caused by nonredundant single-gene mutations. Using a "monogenetic transcriptomic strategy," we measured the expression of these genes in human T1D, autoantibody-positive (autoantibody+), and control pancreas tissues with real-time quantitative PCR in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Gene and protein expression was visualized in situ with use of immunofluorescence, RNAscope, and confocal microscopy. Two dozen monogenic diabetes genes showed altered expression in human pancreata from individuals with T1D versus unaffected control subjects. Six of these genes also saw dysregulation in pancreata from autoantibody+ individuals at increased risk for T1D. As a subset of these genes are related to cellular stress responses, we measured integrated stress response (ISR) genes and identified 20 with altered expression in T1D pancreata, including three of the four eIF2α-dependent kinases. Equally intriguing, we observed significant repression of the three arms of the ISR in autoantibody+ pancreata. Collectively, these efforts suggest monogenic diabetes and ISR genes are dysregulated early in the T1D disease process and likely contribute to the disorder's pathogenesis., Diabetes, 70 (8)

AIMS/HYPOTHESIS: Normal cellular prion protein (PrP(C)) is a conserved mammalian glycoprotein found on the outer plasma membrane leaflet through a glycophosphatidylinositol anchor. Although PrP(C) is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. The misfolded pathogenic isoform PrP(Sc) (the scrapie form of PrP) is a causative agent of neurodegenerative prion diseases. The aim of this study is to evaluate PrP(C) localisation, expression and trafficking in pancreases from organ donors with and without type 1 diabetes and to infer PrP(C) function through studies on interacting protein partners. METHODS: In order to evaluate localisation and trafficking of PrP(C) in the human pancreas, 12 non-diabetic, 12 type 1 diabetic and 12 autoantibody-positive organ donor tissue samples were analysed using immunofluorescence analysis. Furthermore, total RNA was isolated from 29 non-diabetic, 29 type 1 diabetic and 24 autoantibody-positive donors to estimate PrP(C) expression in the human pancreas. Additionally, we performed PrP(C)-specific immunoblot analysis on total pancreatic protein from non-diabetic and type 1 diabetic organ donors to test whether changes in PrP(C) mRNA levels leads to a concomitant increase in PrP(C) protein levels in human pancreases. RESULTS: In non-diabetic and type 1 diabetic pancreases (the latter displaying both insulin-positive [INS(+)] and -negative [INS(−)] islets), we found PrP(C) in islets co-registering with beta cells in all INS(+) islets and, strikingly, unexpected activation of PrP(C) in alpha cells within diabetic INS(−) islets. We found PrP(C) localised to the plasma membrane and endoplasmic reticulum (ER) but not the Golgi, defining two cellular pools and an unconventional protein trafficking mechanism bypassing the Golgi. We demonstrate PrP(C) co-registration with established protein partners, neural cell adhesion molecule 1 (NCAM1) and stress-inducible phosphoprotein 1 (STI1; encoded by STIP1) on the plasma membrane and ER, respectively, linking PrP(C) function with cyto-protection, signalling, differentiation and morphogenesis. We demonstrate that both PRNP (encoding PrP(C)) and STIP1 gene expression are dramatically altered in type 1 diabetic and autoantibody-positive pancreases. CONCLUSIONS/INTERPRETATION: As the first study to address PrP(C) expression in non-diabetic and type 1 diabetic human pancreas, we provide new insights for PrP(C) in the pathogenesis of type 1 diabetes. We evaluated the cell-type specific expression of PrP(C) in the human pancreas and discovered possible connections with potential interacting proteins that we speculate might address mechanisms relevant to the role of PrP(C) in the human pancreas.

Within the human pancreas, exocrine and endocrine cells control secretion of digestive enzymes and production of hormones to maintain metabolic homeostasis, respectively. While the vast majority of type 1 diabetes research efforts have focused on endocrine function and autoimmunity, recent studies identified a series of unique features (e.g., reduced weight and volume, increased density of leukocytes) within the exocrine pancreas in this disease, but the mechanisms underlying these aberrancies are unknown. Therefore, we histologically assessed amylase, insulin, glucagon, lipase, and/or trypsinogen in 78 organ donor pancreata from birth through adulthood in control subjects and those at various stages of type 1 diabetes. While amylase-positive (AMY(+)) acinar cells were detectable in pancreata from all study groups, tissues from individuals >2 years of age contained clusters of acinar cells devoid of amylase (AMY(−)). A majority of these AMY(−) cell clusters localized proximal to islets (i.e., peri-islet). Additionally, most AMY(−) clusters were positive for the exocrine enzymes lipase and trypsinogen. Interestingly, type 1 diabetes pancreata displayed significant reductions in the frequency of these AMY(−) cell clusters. These results support a contribution of the islet-acinar axis in pancreatic development and underscore a potential role for the exocrine pancreas in the pathogenesis of type 1 diabetes.

The conceptual basis for a genetic predisposition underlying the risk for developing type 1 diabetes (T1D) predates modern human molecular genetics. Over half of the genetic risk has been attributed to the human leukocyte antigen (HLA) class II gene region and to the insulin (INS) gene locus - both thought to confer direction of autoreactivity and tissue specificity. Notwithstanding, questions still remain regarding the functional contributions of a vast array of minor polygenic risk variants scattered throughout the genome that likely influence disease heterogeneity and clinical outcomes. Herein, we summarize the available literature related to the T1D-associated coding variants defined at the time of this review, for the genes PTPN22, IFIH1, SH2B3, CD226, TYK2, FUT2, SIRPG, CTLA4, CTSH and UBASH3A. Data from genotype-selected human cohorts are summarized, and studies from the non-obese diabetic (NOD) mouse are presented to describe the functional impact of these variants in relation to innate and adaptive immunity as well as to β-cell fragility, with expression profiles in tissues and peripheral blood highlighted. The contribution of each variant to progression through T1D staging, including environmental interactions, are discussed with consideration of how their respective protein products may serve as attractive targets for precision medicine-based therapeutics to prevent or suspend the development of T1D.

We and others previously demonstrated that a type 1 diabetes genetic risk score (GRS) improves the ability to predict disease progression and onset in at-risk subjects with islet autoantibodies. Here, we hypothesized that GRS and islet autoantibodies, combined with age at onset and disease duration, could serve as markers of residual β-cell function following type 1 diabetes diagnosis. Generalized estimating equations were used to investigate whether GRS along with insulinoma-associated protein-2 autoantibody (IA–2A), zinc transporter 8 autoantibody (ZnT8A), and GAD autoantibody (GADA) titers were predictive of C-peptide detection in a largely cross-sectional cohort of 401 subjects with type 1 diabetes (median duration 4.5 years [range 0–60]). Indeed, a combined model with incorporation of disease duration, age at onset, GRS, and titers of IA–2A, ZnT8A, and GADA provided superior capacity to predict C-peptide detection (quasi-likelihood information criterion [QIC] = 334.6) compared with the capacity of disease duration, age at onset, and GRS as the sole parameters (QIC = 359.2). These findings support the need for longitudinal validation of our combinatorial model. The ability to project the rate and extent of decline in residual C-peptide production for individuals with type 1 diabetes could critically inform enrollment and benchmarking for clinical trials where investigators are seeking to preserve or restore endogenous β-cell function.

Concerns that infection with SARS-CoV-2, the etiological agent of COVID-19, may cause new-onset diabetes persist amidst an evolving research landscape, and precise risk assessment is hampered by at times conflicting evidence. Here, leveraging comprehensive single-cell analyses of in vitro SARS-CoV-2-infected human pancreatic islets, we demonstrate that productive infection is strictly dependent on the SARS-CoV-2 entry receptor ACE2 and targets all pancreatic cell types. Importantly, the infection remains highly circumscribed, largely non-cytopathic, and despite high viral burden in infected subsets, promotes only modest cellular perturbations and inflammatory responses. Similar experimental outcomes are also observed after islet infection with endemic coronaviruses. Thus, the limits of pancreatic SARS-CoV-2 infection, even under in vitro conditions of enhanced virus exposure, do not support the proposition that in vivo targeting of beta cells by SARS-CoV-2 precipitates new-onset diabetes. If restricted pancreatic damage accrued by COVID-19 increases cumulative diabetes risk, however, remains to be evaluated. Funding: These efforts were supported by JDRF 3-PDF-2018-575-A-N (V.v.d.H.); NIH/NIDDK R01DK12392, NIH/NIAID P01AI042288 and NIH/NIAID U54AI142766-S1 (M.A.A.); NIH/NIAID Center of Excellence for Influenza Research and Response/Center for Research for Influenza Pathogenesis and Transmission contract # 75N93019R00028, NIH/NIAID U19AI135972 (supplement), Defense Advanced Research Projects Agency HR0011-19-2-0020, JPB Foundation, and Open Philanthropy Project # 2020-215611 (5384), Anonymous (A.G.-S.); NIH/NIAID R01AI151029 and NIA/NIAID U01AI150748 (B.R.R.); NIH/NIDDK R01DK130425 (M.S.); and NIH/NIAID R01AI134971, NIH/NIDDK U01DK123716, NIH/NIDDK U01DK104162, NIH/NIDDK P30DK020541 and NIH/NIDDK R01DK130425 (D.H.). Funding: The AG-S laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines and Merck, outside of the reported work. Declaration of Interests: AG-S has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories and Pfizer, outside of the reported work. AG-S is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. All other authors declare no conflict of interest. Ethics Approval Statement: Our study is considered “not human subjects research” since all donor islet preparations were provided as de-identified tissue specimens by a commercial purveyor

Type 1 diabetes (T1D) pathophysiology, while incompletely understood, has in part been attributed to aberrant presentation of self-antigen plus proinflammatory co-stimulation by professional antigen-presenting cells (APCs). Therapies targeting dendritic cells (DCs) offer an avenue to restore antigen-specific tolerance by promoting presentation of self-antigen in an anti-inflammatory or suppressive context. Here, we describe a subcutaneously administered, dual-sized biodegradable microparticle (MP) platform that includes phagocytosable (~1 μm) and non-phagocytosable (~30 μm) MPs to deliver pro-tolerogenic factors both intra- and extracellularly, as well as the T1D-associated autoantigen, insulin, to DCs for amelioration of autoimmunity. This MP platform resulted in increased recruitment of DCs, suppressive skewing of DC phenotype with diminished expression of CD86 and MHC-II, increased regulatory T cell (Treg) frequency, and upregulated expression of the checkpoint inhibitor programmed cell death protein 1 (PD-1) on T cells. When administered concomitantly with anti-CD3 antibody, which provides transient T cell depletion while preserving Treg populations, in 12-week-old non-obese diabetic (NOD) mice, regulatory immune populations persisted out to 20 weeks of age; however, combination anti-CD3 + dual sized MP (dMP) therapy failed to synergistically inhibit diabetes onset.

SUMMARYDiabetes is associated with increased mortality from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of the key molecule for SARS-CoV-2 infection of cells, angiotensin-converting enzyme-2 (ACE2). Specifically, we analyzed five public scRNAseq pancreas datasets and performed fluorescencein situhybridization, Western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) patients. Thesein silicoandex vivoanalyses demonstrated pancreatic expression of ACE2 is prominent in pancreatic ductal epithelium and the microvasculature, with rare endocrine cell expression of this molecule. Pancreata from COVID-19 patients demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression primarily limited to ducts. SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, appears an unlikely central pathogenic feature of COVID-19 as it relates to diabetes.

Gene editing of the human T cell receptor locus enables optimized in vitro testing of autoreactive T cell function in type 1 diabetes. The generation of antigen specific T cells and subsequent implementation as “living drugs” has garnered great interest in the immunotherapy space. One of the main caveats to this approach is the potential for heterologous chain pairing between the α- and β-chains of the endogenous and de-novo T cell receptors (TCRs). The consequences of chain mispairing can range from reduced antigen specific functionality to off-target autoimmune reactivity. The latter is an important consideration when designing therapies for autoimmune and inflammatory diseases, specifically with regards to the introduction of regulatory T cells (Treg) adoptive cell therapy. To date, infusions of polyclonal Tregs have demonstrated safety but have failed to reverse established type 1 diabetes (T1D). This demonstrates the need for the generation of safe and effective antigen-specific Tregs capable of recognizing β-cells in T1D. We have previously shown the ability to generate GAD-specific Tregs which maintain lineage stability and suppressive phenotype. To extend this work, we deleted the endogenous TCR α-chain gene (TRAC) via CRISPR/cas9 in CD8+T cells. We achieved >90% editing efficiency through ribonucleoprotein (RNP) complex mediated single guide RNA delivery. Moreover, we found TRAC-KO CD8+ T cells displayed increased peptide MHC-dextramer staining, CD69 and CD25 upregulation after stimulation with cognate antigen, and increased antigen-specific killing of β-cell lines when compared to mock guide treated T cells. We believe this workflow can be applied to generate antigen specific Tregs, which we expect to provide increased suppressive capacity. Hence, we present a TCR-gene editing protocol in primary human T cells that results in efficient deletion of the endogenous TCR α-chain and robust enhancement of antigen-specific T cell function. Disclosure L.D. Peters: None. A.L. Posgai: None. T.M. Brusko: Board Member; Self; OneVax, LLC. Consultant; Self; Caladrius Biosciences, Inc., Sanofi-Aventis. Speaker’s Bureau; Self; CSL Behring. Funding National Institutes of Health (P01AI42288, R01DK106191, T325T32DK108736-03HIRN, UC4 P0063595)

AIMS Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. METHODS We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n = 111) or non-type 1 (n = 42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulin-containing islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). RESULTS Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P = 0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median

IL-12 and IL-18 synergize to promote T(H)1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We investigated the influence of these cytokines on immune cells involved in human T1D progression: natural killer (NK) cells, regulatory T cells (Tregs), and cytotoxic T lymphocytes (CTL). NK cells from T1D patients exhibited higher surface CD226 versus controls and lower CD25 compared to first-degree relatives and controls. Changes in NK cell phenotype towards terminal differentiation were associated with cytomegalovirus (CMV) seropositivity, while possession of IL18RAP, IFIH1, and IL2RA T1D-risk variants impacted NK cell activation as evaluated by immuno-expression quantitative trait loci (eQTL) analyses. IL-12 and IL-18 stimulated NK cells from healthy donors exhibited enhanced specific killing of myelogenous K562 target cells. Moreover, activated NK cell increased expression of NKG2A, NKG2D, CD226, TIGIT and CD25, which enabled competition for IL-2 upon co-culture with Tregs, resulting in Treg downregulation of FOXP3, production of IFNγ, and loss of suppressive function. We generated islet-autoreactive CTL “avatars”, which upon exposure to IL-12 and IL-18, upregulated IFNγ and Granzyme-B leading to increased lymphocytotoxicity of a human β-cell line in vitro. These results support a model for T1D pathogenesis wherein IL-12 and IL-18 synergistically enhance CTL and NK cell cytotoxic activity and disrupt immunoregulation by Tregs.

It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from β cells, reflect their recent demise, and can be used to assess β cell damage in type 1 diabetes. Herein we describe an ultrasensitive assay for detection of a β cell–specific DNA methylation signature, by simultaneous assessment of 6 DNA methylation markers, that identifies β cell DNA in mixtures containing as little as 0.03% β cell DNA (less than 1 β cell genome equivalent). Based on this assay, plasma from nondiabetic individuals (N = 218, aged 4–78 years) contained on average only 1 β cell genome equivalent/mL. As expected, cell-free DNA (cfDNA) from β cells was significantly elevated in islet transplant recipients shortly after transplantation. We also detected β cell cfDNA in a patient with KATP congenital hyperinsulinism, in which substantial β cell turnover is thought to occur. Strikingly, in contrast to previous reports, we observed no elevation of β cell–derived cfDNA in autoantibody-positive subjects at risk for type 1 diabetes (N = 32), individuals with recent-onset type 1 diabetes (4 months, N = 38). We discuss the utility of sensitive β cell cfDNA analysis and potential explanations for the lack of a β cell cfDNA signal in type 1 diabetes.

Diabetes is associated with increased mortality from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of the key molecule for SARS-CoV-2 infection of cells, angiotensin-converting enzyme-2 (ACE2). Specifically, we analyzed five public scRNAseq pancreas datasets and performed fluorescence in situ hybridization, Western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) patients. These in silico and ex vivo analyses demonstrated pancreatic expression of ACE2 is prominent in pancreatic ductal epithelium and the microvasculature, with rare endocrine cell expression of this molecule. Pancreata from COVID-19 patients demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression primarily limited to ducts. SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, appears an unlikely central pathogenic feature of COVID-19 as it relates to diabetes. Funding: These efforts were supported by NIH P01 AI42288 and UC4 DK108132 (MAA), JDRF (MAA), NIH R01 DK122160 (MCT), NIH R01 AI134971 (DH), NIH P30 DK020541 (D.H.), JDRF 3-PDF-2018-575-A-N (VvdH), R01 DK093954 (CEM); VA Merit Award I01BX001733 (CEM), Imaging Core of NIH/NIDDK P30 DK097512 (CEM), gifts from the Sigma Beta Sorority, the Ball Brothers Foundation, and the George and Frances Ball Foundation (CEM), the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641) (5-SRA-2018-557-Q-R) and The Leona M. & Harry B. Helmsley Charitable Trust (2018PG-T1D053). Conflict of Interest: The authors declare no relevant conflicts of interest exist. Ethical Approval: Transplant-quality pancreas, duodenum, and kidney were recovered by JDRF nPOD (www.jdrfnpod.com) from 36 COVID-19 negative organ donors without diabetes (Table S2) according to established protocols and procedures (Campbell-Thompson et al., 2012), as approved by the University of Florida Institutional Review Board (201400486), the United Network for Organ Sharing (UNOS), and according to federal guidelines with informed consent obtained from each donor’s legal representative.

Interest in adoptive T-cell therapies has been ignited by the recent clinical success of genetically-modified T cells in the cancer immunotherapy space. In addition to immune targeting for malignancies, this approach is now being explored for the establishment of immune tolerance with regulatory T cells (Tregs). Herein, we will summarize the basic science and clinical results emanating from trials directed at inducing durable immune regulation through administration of Tregs. We will discuss some of the current challenges facing the field in terms of maximizing cell purity, stability and expansion capacity, while also achieving feasibility and GMP production. Indeed, recent advances in methodologies for Treg isolation, expansion, and optimal source materials represent important strides toward these considerations. Finally, we will review the emerging genetic and biomaterial-based approaches on the horizon for directing Treg specificity to augment tissue-targeting and regenerative medicine.

Insulin-like growth factors (IGFs), specifically IGF1 and IGF2, promote glucose metabolism, with their availability regulated by IGF-binding proteins (IGFBPs). We hypothesized that IGF1 and IGF2 levels, or their bioavailability, are reduced during type 1 diabetes development. Total serum IGF1, IGF2, and IGFBP1–7 levels were measured in an age-matched, cross-sectional cohort at varying stages of progression to type 1 diabetes. IGF1 and IGF2 levels were significantly lower in autoantibody (AAb)+ compared with AAb− relatives of subjects with type 1 diabetes. Most high-affinity IGFBPs were unchanged in individuals with pre–type 1 diabetes, suggesting that total IGF levels may reflect bioactivity. We also measured serum IGFs from a cohort of fasted subjects with type 1 diabetes. IGF1 levels significantly decreased with disease duration, in parallel with declining β-cell function. Additionally, plasma IGF levels were assessed in an AAb+ cohort monthly for a year. IGF1 and IGF2 showed longitudinal stability in single AAb+ subjects, but IGF1 levels decreased over time in subjects with multiple AAb and those who progressed to type 1 diabetes, particularly postdiagnosis. In sum, IGFs are dysregulated both before and after the clinical diagnosis of type 1 diabetes and may serve as novel biomarkers to improve disease prediction.

The human T lymphocyte compartment is highly dynamic over the course of a lifetime. Of the many changes, perhaps most notable is the transition from a predominantly naïve T cell state at birth to the acquisition of antigen-experienced memory and effector subsets following environmental exposures. These phenotypic changes, including the induction of T cell exhaustion and senescence, have the potential to negatively impact efficacy of adoptive T cell therapies (ACT). When considering ACT with CD4+CD25+CD127–/lo regulatory T cells (Tregs) for the induction of immune tolerance, we previously reported ex vivo expanded umbilical cord blood (CB) Tregs remained more naïve, suppressed responder T cells equivalently, and exhibited a more diverse T cell receptor (TCR) repertoire compared to expanded adult peripheral blood (APB) Tregs. Herein, we hypothesized that upon further characterization, we would observe increased lineage heterogeneity and phenotypic diversity in APB Tregs that might negatively impact lineage stability, engraftment capacity, and the potential for Tregs to home to sites of tissue inflammation following ACT. We compared the phenotypic profiles of human Tregs isolated from CB versus the more traditional source, APB. We conducted analysis of fresh and ex vivo expanded Treg subsets at both the single cell (scRNA-seq and flow cytometry) and bulk (microarray and cytokine profiling) levels. Single cell transcriptional profiles of pre-expansion APB Tregs highlighted a cluster of cells that showed increased expression of genes associated with effector and pro-inflammatory phenotypes (CCL5, GZMK, CXCR3, LYAR, and NKG7) with low expression of Treg markers (FOXP3 and IKZF2). CB Tregs were more diverse in TCR repertoire and homogenous in phenotype, and contained fewer effector-like cells in contrast with APB Tregs. Interestingly, expression of canonical Treg markers, such as FOXP3, TIGIT, and IKZF2, were increased in CB CD4+CD127+ conventional T cells (Tconv) compared to APB Tconv, post-expansion, implying perinatal T cells may adopt a default regulatory program. Collectively, these data identify surface markers (namely CXCR3) that could be depleted to improve purity and stability of APB Tregs, and support the use of expanded CB Tregs as a potentially optimal ACT modality for the treatment of autoimmune and inflammatory diseases.

Summary Recent studies in Type 1 Diabetes (T1D) support an emerging model of disease pathogenesis that involves intrinsic β-cell fragility combined with defects in both innate and adaptive immune cell regulation. This combination of defects induces systematic changes leading to organ-level atrophy and dysfunction of both the endocrine and exocrine portions of the pancreas, ultimately culminating in insulin deficiency and β-cell destruction. In this review, we discuss the animal model data and human tissue studies that have informed our current understanding of the cross-talk that occurs between β-cells, the resident stroma, and immune cells that potentiate T1D. Specifically, we will review the cellular and molecular signatures emerging from studies on tissues derived from organ procurement programs, focusing on in situ defects occurring within the T1D islet microenvironment, many of which are not yet detectable by standard peripheral blood biomarkers. In addition to improved access to organ donor tissues, various methodological advances, including immune receptor repertoire sequencing and single-cell molecular profiling, are poised to improve our understanding of antigen-specific autoimmunity during disease development. Collectively, the knowledge gains from these studies at the islet–immune interface are enhancing our understanding of T1D heterogeneity, likely to be an essential component for instructing future efforts to develop targeted interventions to restore immune tolerance and preserve β-cell mass and function.

Recent efforts addressing the pathogenesis of T1D have benefited greatly from access to transplant-quality human pancreas samples obtained from organ donors. While alcohol use is linked to pancreatitis clinically, the effect of alcohol and other illicit substances on pancreatic histopathology in T1D is not known. Hence, we determined the influence of alcohol, marijuana, tobacco, cocaine, and other controlled substances in T1D pancreata. In total, 140 T1D organ donor pancreata [median age at T1D onset=11 (range 0-36) years, duration=12 (0-84) years, 47% female] were recovered through the Network for Pancreatic Organ donors with Diabetes (nPOD) program. Paraffin sections were stained and reviewed for insulin positivity, insulitis (≥6 CD3+ cells adjacent/within the islet in ≥3 islets/section), amyloid staining, acute (acinar cell loss, mononuclear cell infiltrate, fat necrosis, and hemorrhage) and chronic pancreatitis (acinar cell loss, mononuclear cell infiltrate, and fibrosis) as well as chronic exocrine changes (acinar atrophy, fibrosis, fatty infiltration, or periductal fibrosis). Terminal hospital records were reviewed for a history of alcohol, tobacco, marijuana, and any illicit substance use. Of 140 T1D donors, 51% had a history of alcohol use, 40% tobacco use, 29% marijuana, and 20% other illicit substances. Urine drug screening on admission was available for 74 donors with 36 positive (49%). Alcohol use was associated with histopathological features of acute pancreatitis (p Disclosure B.S. Bruggeman: None. M. Campbell-Thompson: None. A.L. Posgai: None. M.A. Atkinson: None. D. Schatz: None. L.M. Jacobsen: None. Funding JDRF

With the conceptual advance some four decades ago that type 1 diabetes (T1D) represents an autoimmune disease, hope emerged that immune-based therapies would quickly evolve as a means to prevent and reverse the disorder. However, despite dozens of clinical trials seeking to achieve that purpose, the goal remains unfulfilled, at least in a pragmatic form. With the benefit of hindsight, multiple reasons are likely to account for this unfortunate situation, and several stand out: failure to appreciate disease heterogeneity; inappropriate utilization of and insight from rodent models of disease; inadequacies in addressing the immunologic and metabolic contributions to the disease; suboptimal trial designs; and lack of a clear understanding of the disorder’s pathogenesis. This review conveys how recent knowledge gains in these areas, combined with efforts related to disease staging and emerging mechanistic data from clinical trials, provide cautious optimism that an immune-based means to prevent the loss of β-cells in T1D will emerge into clinical practice.

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory Tcells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26× 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder Tcell proliferation. Immunosequencing of the Tcell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following invitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

The patient (nPOD 6310) was a 28-year-old female, 32 days postoperative from a cesarean delivery. Her past medical history was significant for chronic hypertension, severe preeclampsia, acute renal failure, and gestational diabetes mellitus (GDM). She was brought to the emergency department after collapsing, with total downtime of 20–30 min. On arrival, she was admitted to the intensive care unit and found to be in hypertensive crisis. Her hospital course was complicated by renal failure requiring four rounds of hemodialysis, hypertension requiring multiple agents for control, and a degree of hyperglycemia requiring insulin. Computed tomography of the head demonstrated anoxic encephalopathy with multiple infarcts within the brain. The patient was declared brain-dead and, with authorization obtained from next of kin, became an organ donor. The pancreas, along with blood, was recovered for research through the Network for Pancreatic Organ …

Diabetes is associated with increased mortality from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2), the key entry factor for SARS-CoV-2 infection. Specifically, we analyzed five public scRNA-seq pancreas datasets and performed fluorescence in situ hybridization, western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) cases. These in silico and ex vivo analyses demonstrated prominent expression of ACE2 in pancreatic ductal epithelium and microvasculature, but we found rare endocrine cell expression at the mRNA level. Pancreata from individuals with COVID-19 demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression that was primarily limited to ducts. These results suggest SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, is an unlikely central pathogenic feature of COVID-19-related diabetes., Graphical Abstract, Highlights • ACE2 mRNA and protein are expressed in human pancreatic ducts and microvasculature • ACE2 mRNA was rarely detected and at low levels in human pancreatic endocrine cells • Pancreatic ACE2 protein expression changes across the lifespan and correlates with BMI • SARS-CoV-2 NP was detected in ducts, but not endocrine cells, of COVID-19 pancreata, Kusmartseva et al. demonstrate preferential ACE2 expression in pancreatic microvascular and ductal structures, suggesting these constitute a more likely target than islet endocrine cells in SARS-CoV-2 infection. This notion was supported by detection of SARS-CoV-2 nucleocapsid protein in ductal epithelium, but not endocrine cells, of pancreata from individuals with COVID-19.

PURPOSE OF REVIEW: The immunosuppressive agent cyclosporine was first reported to lower daily insulin dose and improve glycemic control in patients with new-onset type 1 diabetes (T1D) in 1984. While renal toxicity limited cyclosporine’s extended use, this observation ignited collaborative efforts to identify immunotherapeutic agents capable of safely preserving β cells in patients with or at risk for T1D. RECENT FINDINGS: Advances in T1D prediction and early diagnosis, together with expanded knowledge of the disease mechanisms, have facilitated trials targeting specific immune cell subsets, autoantigens, and pathways. In addition, clinical responder and non-responder subsets have been defined through the use of metabolic and immunological readouts. SUMMARY: Herein, we review emerging T1D biomarkers within the context of recent and ongoing T1D immunotherapy trials. We also discuss responder/non-responder analyses in an effort to identify therapeutic mechanisms, define actionable pathways, and guide subject selection, drug dosing, and tailored combination drug therapy for future T1D trials.