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The induction of antigen (Ag)-specific tolerance and replacement of islet β-cells are major ongoing goals for the treatment of type 1 diabetes (T1D). Our group previously showed that a hybrid insulin peptide (2.5HIP) is a critical autoantigen for diabetogenic CD4+ T cells in the NOD mouse model. In this study, we investigated whether induction of Ag-specific tolerance using 2.5HIP-coupled tolerogenic nanoparticles (NPs) could protect diabetic NOD mice from disease recurrence upon syngeneic islet transplantation. Islet graft survival was significantly prolonged in mice treated with 2.5HIP NPs, but not NPs containing the insulin B chain peptide 9-23. Protection in 2.5HIP NP-treated mice was attributed both to the simultaneous induction of anergy in 2.5HIP-specific effector T cells and the expansion of Foxp3+ regulatory T cells specific for the same Ag. Notably, our results indicate that effector function of graft-infiltrating CD4+ and CD8+ T cells specific for other β-cell epitopes was significantly impaired, suggesting a novel mechanism of therapeutically induced linked suppression. This work establishes that tolerance induction with an HIP can delay recurrent autoimmunity in NOD mice, which could inform the development of an Ag-specific therapy for T1D.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab–treated CD11b+ cells showed significant upregulation of genes associated with bone marrow–derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab–induced CD11b+ cells, derived ex vivo from TLR4-Ab–treated mice, suppress T cells, and TLR4-Ab–conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab–induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal “second signal” for the activation/maturation of MDSCs that can reverse acute T1D.

Excess of chronic contact between microbial motifs and intestinal immune cells are known to trigger a low-grade inflammation involved in many pathologies such as obesity and diabetes.The important skewing of intestinal adaptive immunity in the context of diet-induced obesity (DIO) is well described but how dendritic cells (DCs) participate to these changes is still poorly documented. To address this question, transgenic mice with enhanced DCs lifespan and immunogenicity (DChBcl-2 mice), are challenged with a high fat diet.Those mice display resistance to DIO and metabolic alterations. The DIO resistant phenotype is associated with healthier parameters of intestinal barrier function and lower intestinal inflammation. DChBcl-2 DIO-resistant mice demonstrate a particular increase in tolerogenic DC numbers and function which is associated with strong intestinal IgA, Th17 and T regulatory immune responses.Microbiota composition and function analyses reveal that the DChBcl-2 mice microbiota is characterized by a lower immunogenicity and an enhanced butyrate production. Cohousing experiments and fecal microbial transplantations are sufficient to transfer the DIO resistance status to WT mice demonstrating that maintenance of DCs tolerogenic ability sustains a microbiota able to drive DIO resistance. DCs tolerogenic function is revealed as a new potent target in metabolic diseases management.

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.

The long-term success of pancreatic islet transplantation (Tx) as a cure for type 1 diabetes remains limited. Islet loss after Tx related to apoptosis, inflammation, and other factors continues to limit Tx efficacy. In this project, we demonstrate a novel approach aimed at protecting islets before Tx in nonhuman primates (NHPs) (baboons) by silencing a gene (caspase-3) responsible for induction of apoptosis. This was done using siRNA (siCas-3) conjugated to magnetic nanoparticles (MNs). In addition to serving as carriers for siCas-3, these nanoparticles also act as reporters for MRI, so islets labeled with MN-siCas-3 can be monitored in vivo after Tx. In vitro studies showed the antiapoptotic effect of MN-siCas-3 on islets in culture, resulting in minimal islet loss. For in vivo studies, donor baboon islets were labeled with MN-siCas-3 and infused into recipient diabetic subjects. A dramatic reduction in insulin requirements was observed in animals transplanted with even a marginal number of labeled islets compared with controls. By demonstrating the protective effect of MN-siCas-3 in the challenging NHP model, this study proposes a novel strategy to minimize the number of donor islets required from either cadaveric or living donors.

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2–based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.

T cells isolated from the pancreatic infiltrates of nonobese diabetic mice have been shown to recognize epitopes formed by the covalent cross-linking of proinsulin and secretory granule peptides. Formation of such hybrid insulin peptides (HIPs) was confirmed through mass spectrometry, and responses to HIPs were observed among the islet-infiltrating T cells of pancreatic organ donors and in the peripheral blood of individuals with type 1 diabetes (T1D). However, questions remain about the prevalence of HIP-specific T cells in humans, the sequences they recognize, and their role in disease. We identified six novel HIPs that are recognized in the context of DRB1*04:01, discovered by using a library of theoretical HIP sequences derived from insulin fragments covalently linked to one another or to fragments of secretory granule proteins or other islet-derived proteins. We demonstrate that T cells that recognize these HIPs are detectable in the peripheral blood of subjects with T1D and exhibit an effector memory phenotype. HIP-reactive T-cell clones produced Th1-associated cytokines and proliferated in response to human islet preparations. These results support the relevance of HIPs in human disease, further establishing a novel posttranslational modification that may contribute to the loss of peripheral tolerance in T1D.

β-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29–42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.

Type 1 diabetes is an autoimmune-mediated disease that culminates in the targeted destruction of insulin-producing β-cells. CD4 responses in NOD mice are dominated by insulin epitope B:9-23 (InsB9-23) specificity, and mutation of the key T-cell receptor (TCR) contact residue within the epitope prevents diabetes development. However, it is not clear how insulin self-antigen controls the selection of autoimmune and regulatory T cells (Tregs). Here we demonstrate that mutation of insulin epitope results in escape of highly pathogenic T cells. We observe an increase in antigen reactivity, clonality, and pathogenicity of insulin-specific T cells that develop in the absence of cognate antigen. Using a single TCR system, we demonstrate that Treg development is greatly diminished in mice with the Y16A mutant epitope. Collectively, these results suggest that the tyrosine residue at position 16 is necessary to constrain TCR reactivity for InsB9-23 by both limiting the development of pathogenic T cells and supporting the selection of Tregs.

Accumulating evidence supports a critical role for posttranslationally modified (PTM) islet neoantigens in type 1 diabetes. However, our understanding regarding thymic development and peripheral activation of PTM autoantigen-reactive T cells is still limited. Using HLA-DR4 humanized mice, we observed that deamidation of GAD65115–127 generates a more immunogenic epitope that recruits T cells with promiscuous recognition of both the deamidated and native epitopes and reduced frequency of regulatory T cells. Using humanized HLA/T-cell receptor (TCR) mice, we observed that TCRs reactive to the native or deamidated GAD65115–127 led to efficient development of CD4+ effector T cells; however, regulatory T-cell development was reduced in mice expressing the PTM-reactive TCR, which was partially restored with exogenous PTM peptide. Upon priming, both the native-specific and the deamidated-specific T cells accumulated in pancreatic islets, suggesting that both specificities can recognize endogenous GAD65 and contribute to anti–β-cell responses. Collectively, our observations in polyclonal and single TCR systems suggest that while effector T-cell responses can exhibit cross-reactivity between native and deamidated GAD65 epitopes, regulatory T-cell development is reduced in response to the deamidated epitope, pointing to regulatory T-cell development as a key mechanism for loss of tolerance to PTM antigenic targets.

Recognition of beta-cell antigens by autoreactive T cells is a critical step in the initiation of autoimmune Type1 diabetes (T1D). A complete protection from diabetes development in non-obese diabetic (NOD) mice harboring a point mutation in the insulin B-chain 9-23 epitope points to a dominant role of insulin in diabetogenesis. Generation of NOD mice lacking the Chromogranin A protein (NOD.ChgA-/-) completely nullified the autoreactivity of the BDC2.5 T cell and conferred protection from diabetes onset. These results raised the issue concerning the dominant antigen that drives the autoimmune process. Here we revisited the NOD.ChgA-/- mice and found that their lack of diabetes development may not be solely explained by the absence of Chromogranin A reactivity. NOD.ChgA-/- mice displayed reduced presentation of insulin peptides in the islets and periphery, which corresponded to impaired T cell priming. Diabetes development in these mice was restored by antibody treatment targeting regulatory T cells or inhibiting TGFb and PD-1 pathways. Therefore, the global deficiency of chromogranin A impairs recognition of the major diabetogenic antigen insulin, leading to broadly impaired autoimmune responses controlled by multiple regulatory mechanisms.

We identified autoantibodies (AAb) reacting with a variant IA-2 molecule (IA-2var) that has three amino acid substitutions (Cys27, Gly608, and Pro671) within the full-length molecule. We examined IA-2var AAb in first-degree relatives of type 1 diabetes (T1D) probands from the TrialNet Pathway to Prevention Study. The presence of IA-2var–specific AAb in relatives was associated with accelerated progression to T1D in those positive for AAb to GAD65 and/or insulin but negative in the standard test for IA-2 AAb. Furthermore, relatives with single islet AAb (by traditional assays) and carrying both IA-2var AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progress rapidly to onset of T1D. Molecular modeling of IA-2var predicts that the genomic variation that alters the three amino acids induces changes in the three-dimensional structure of the molecule, which may lead to epitope unmasking in the IA-2 extracellular domain. Our observations suggest that the presence of AAb to IA-2var would identify high-risk subjects who would benefit from participation in prevention trials who have one islet antibody by traditional testing and otherwise would be misclassified as “low risk” relatives.

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.

IntroductionTo evaluate the effects of efpeglenatide, a long-acting glucagon-like peptide-1 receptor agonist (GLP-1 RA), on gastric emptying, glucose metabolism, and islet beta-cell function versus liraglutide and placebo in people with type 2 diabetes.Research design and methodsThis phase Ib study (ClinicalTrials.gov identifier: NCT02059564) randomized participants (n=47) to three cohorts. Within the first two cohorts, participants were randomized to placebo, efpeglenatide 6 mg weekly (QW; first cohort), or efpeglenatide 16 mg monthly (QM; second cohort). The third cohort received liraglutide 1.8 mg daily (QD). Gastric emptying was assessed through the pharmacokinetic (PK) profile of acetaminophen at baseline and steady state. Glucose metabolism and beta-cell function were assessed based on mixed-meal tolerance testing and a graded glucose infusion procedure.ResultsTreatment duration was approximately 3 months for efpeglenatide 16 mg QM and 1 month for efpeglenatide 6 mg QW and liraglutide. At peak drug concentrations, efpeglenatide 6 mg QW was non-inferior to liraglutide 1.8 mg QD in delaying gastric emptying, as assessed by acetaminophen PK (lower bound of 90% CI for the efpeglenatide:liraglutide ratio >0.8 for area under the curve (AUC)0–120, AUC0–180, AUC0–360 and maximum concentration (Cmax)). Efpeglenatide 16 mg QM did not decrease the rate of gastric emptying to as great an extent as liraglutide (ie, non-inferiority was not shown). Compared with liraglutide, both efpeglenatide dosing regimens demonstrated comparable or more favorable glucometabolic effects and improved beta-cell function. All gastrointestinal adverse events reported with efpeglenatide were mild or moderate in severity and transient over treatment and follow-up.ConclusionsThe glucometabolic effects of efpeglenatide 6 mg QW and 16 mg QM were comparable to liraglutide. Additional studies are necessary to further examine these benefits of efpeglenatide.Trial registration numberNCT02059564.

IntroductionZinc transporter 8 autoantibodies (ZnT8A) are biomarkers of beta cell autoimmunity in type 1 diabetes that have become more widely available to clinicians in recent years. Robust control population-defined thresholds are essential to ensure high clinical specificity in islet autoantibody testing. We aimed to determine the optimal cut-offs for ZnT8A testing.Research design and methods97.5th and 99th centile cut-offs were determined using residual clinical sera from 1559 controls aged between 0 and 83 years with no history of diabetes and a hemoglobin A1c level of less than 6.0% (ResultsUsing the manufacturer’s limit of detection, 6 WHO U/mL, 16.2% of people in the control cohort had detectable levels of ZnT8A and those who had detectable ZnT8A were much more likely to be younger (pConclusionsAge-related thresholds are needed for ZnT8A testing. In those aged

NKG2D is implicated in autoimmune diabetes. However, the role of this receptor in diabetes pathogenesis is unclear owing to conflicting results with studies involving global inhibition of NKG2D signaling. We found that NKG2D and its ligands are present in human pancreata, with expression of NKG2D and its ligands increased in the islets of patients with type 1 diabetes. To directly assess the role of NKG2D in the pancreas, we generated NOD mice that express an NKG2D ligand in β-islet cells. Diabetes was reduced in these mice. The reduction corresponded with a decrease in the effector to central memory CD8(+) T-cell ratio. Further, NKG2D signaling during in vitro activation of both mouse and human CD8(+) T cells resulted in an increased number of central memory CD8(+) T cells and diabetes protection by central memory CD8(+) T cells in vivo. Taken together, these studies demonstrate that there is a protective role for central memory CD8(+) T cells in autoimmune diabetes and that this protection is enhanced with NKG2D signaling. These findings stress the importance of anatomical location when determining the role NKG2D signaling plays, as well as when developing therapeutic strategies targeting this pathway, in type 1 diabetes development.

In type 1 diabetes, autoimmune β-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8(+) T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8(+) T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8(+) T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8(+) T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Enteroviruses, including the Coxsackievirus Bs (CVB), have been implicated as causal agents in human type 1 diabetes. Immunization of at-risk individuals with a CVB vaccine provides an attractive strategy for elucidating the role of CVBs in the disease etiology. Previously, we have shown that an inactivated whole-virus vaccine covering all CVB serotypes (CVB1-6) is safe to administer and highly immunogenic in preclinical models, including nonhuman primates. Before initiating clinical trials with this type of vaccine, it was also important to address 1) whether the vaccine itself induces adverse immune reactions, including accelerating diabetes onset in a diabetes-prone host, and 2) whether the vaccine can prevent CVB-induced diabetes in a well-established disease model. Here, we present results from studies in which female NOD mice were left untreated, mock-vaccinated, or vaccinated with CVB1-6 vaccine and monitored for insulitis occurrence or diabetes development. We demonstrate that vaccination induces virus-neutralizing antibodies without altering insulitis scores or the onset of diabetes. We also show that NOD mice vaccinated with a CVB1 vaccine are protected from CVB-induced accelerated disease onset. Taken together, these studies show that CVB vaccines do not alter islet inflammation or accelerate disease progression in an animal model that spontaneously develops autoimmune type 1 diabetes. However, they can prevent CVB-mediated disease progression in the same model. acceptedVersion

Toll-like receptor 9 (TLR9) is highly expressed in B cells, and B cells are important in the pathogenesis of type 1 diabetes (T1D) development. However, the intrinsic effect of TLR9 in B cells on β-cell autoimmunity is not known. To fill this knowledge gap, we generated NOD mice with a B-cell–specific deficiency of TLR9 (TLR9fl/fl/CD19-Cre+ NOD). The B-cell–specific deletion of TLR9 resulted in near-complete protection from T1D development. Diabetes protection was accompanied by an increased proportion of interleukin-10 (IL-10)–producing B cells. We also found that TLR9-deficient B cells were hyporesponsive to both innate and adaptive immune stimuli. This suggested that TLR9 in B cells modulates T1D susceptibility in NOD mice by changing the frequency and function of IL-10–producing B cells. Molecular analysis revealed a network of TLR9 with matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and CD40, all of which are interconnected with IL-10. Our study has highlighted an important connection of an innate immune molecule in B cells to the immunopathogenesis of T1D. Thus, targeting the TLR9 pathway, specifically in B cells, may provide a novel therapeutic strategy for T1D treatment.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes. ispartof: DIABETES vol:70 issue:2 pages:516-528 ispartof: location:United States status: published

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at-risk for T1D, multiple studies have analyzed and reported alterations in follicular helper T cells (Tfh) in presymptomatic AAb-positive subjects and patients with T1D. Yet, it is still not clear whether the regulatory counterpart of Tfh cells, represented by follicular regulatory T cells (Tfr), is similarly altered. To address this question, we performed analyses in peripheral blood, spleen and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb-negative and -positive subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed between T1D, AAb-negative and AAb-positive adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared to non-diabetic controls. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.

In type 1 diabetes (T1D), a lifelong autoimmune disease, T cells infiltrate the islets and the exocrine pancreas in high numbers. CD8(+) T cells are the main cell type found in the insulitic lesion, and CD8(+) T cells reactive against β-cell antigens have been detected in peripheral blood and in the pancreas of patients with short- or long-term disease. In the Diabetes Virus Detection (DiViD) study, researchers collected pancreatic tissue, by pancreatic tail resection, from living patients with recent-onset T1D. These tissues have been extensively studied by the scientific community, but the autoreactive nature of the T-cell infiltrate has remained unexplored. Our objective was to determine the number and localization of these cells in pancreas samples obtained through the DiViD study. Here, we demonstrate the presence of high frequencies of CD8(+) T cells reactive against a highly relevant epitope derived from the preproinsulin signal peptide in pancreatic tissue samples from these donors. We also show the heterogeneity of islet distribution and CD8(+) T-cell infiltration. Our findings contribute to the current limited existing knowledge of T-cell reactivity in the pancreas of donors with recent-onset T1D and indicate that antigen-specific therapies directed toward preproinsulin could have high clinical impact.

ObjectiveHere we looked for possible mechanisms regulating the progression of type 1 diabetes mellitus (T1DM). In this disease, autoaggressive T cells (T conventional cells, Tconvs) not properly controlled by T regulatory cells (Tregs) destroy pancreatic islets.Research design and methodsWe compared the T-cell compartment of patients with newly diagnosed T1DM (NDT1DM) with long-duration T1DM (LDT1DM) ones. The third group consisted of patients with LDT1DM treated previously with polyclonal Tregs (LDT1DM with Tregs). We have also looked if the differences might be dependent on the antigen specificity of Tregs expanded for clinical use and autologous sentinel Tconvs.ResultsPatients with LDT1DM were characterized by T-cell immunosenescence-like changes and expansion of similar vβ/T-cell receptor (TCR) clones in Tconvs and Tregs. The treatment with Tregs was associated with some inhibition of these effects. Patients with LDT1DM possessed an increased percentage of various proinsulin-specific T cells but not GAD65-specific ones. The percentages of all antigen-specific subsets were higher in the expansion cultures than in the peripheral blood. The proliferation was more intense in proinsulin-specific Tconvs than in specific Tregs but the levels of some proinsulin-specific Tregs were exceptionally high at baseline and remained higher in the expanded clinical product than the levels of respective Tconvs in sentinel cultures.ConclusionsT1DM is associated with immunosenescence-like changes and reduced diversity of T-cell clones. Preferential expansion of the same TCR families in both Tconvs and Tregs suggests a common trigger/autoantigen responsible. Interestingly, the therapy with polyclonal Tregs was associated with some inhibition of these effects. Proinsulin-specific Tregs appeared to be dominant in the immune responses in patients with T1DM and probably associated with better control over respective autoimmune Tconvs.Trial registration numberEudraCT 2014-004319-35.

Type 1 diabetes (T1D) is an autoimmune disease of insulin-producing β-cells. Islet transplantation is a promising treatment for T1D, but long-term graft viability and function remain challenging. Oxidative stress plays a key role in the activation of alloreactive and autoreactive immunity toward the engrafted islets. Therefore, targeting these pathways by encapsulating islets with an antioxidant may delay immune-mediated rejection. Utilizing a layer-by-layer approach, we generated nanothin encapsulation materials containing tannic acid (TA), a polyphenolic compound with redox scavenging and anti-inflammatory effects, and poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer. We hypothesize that transplantation of PVPON/TA-encapsulated allogeneic C57BL/6 islets into diabetic NOD mice will prolong graft function and elicit localized immunosuppression. In the absence of systemic immunosuppression, diabetic recipients containing PVPON/TA-encapsulated islets maintained euglycemia and delayed graft rejection significantly longer than those receiving nonencapsulated islets. Transplantation of PVPON/TA-encapsulated islets was immunomodulatory because gene expression and flow cytometric analysis revealed significantly decreased immune cell infiltration, synthesis of reactive oxygen species, inflammatory chemokines, cytokines, CD8 T-cell effector responses, and concomitant increases in alternatively activated M2 macrophage and dendritic cell phenotypes. Our results provide evidence that reducing oxidative stress following allotransplantation of PVPON/TA-encapsulated islets can elicit localized immunosuppression and potentially delay graft destruction in future human islet transplantation studies.

Preserving endogenous insulin production is clinically advantageous and remains a vital unmet challenge in the treatment and reversal of type 1 diabetes. Although broad immunosuppression has had limited success in prolonging the so-called remission period, it comes at the cost of compromising beneficial immunity. Here, we used a novel strategy to specifically deplete the activated diabetogenic T cells that drive pathogenesis while preserving not only endogenous insulin production but also protective immunity. Effector T (Teff) cells, such as diabetogenic T cells, are naturally poised on the edge of apoptosis because of activation-induced DNA damage that stresses the p53 regulation of the cell cycle. We have found that using small molecular inhibitors that further potentiate p53 while inhibiting the G2/M cell cycle checkpoint control drives apoptosis of activated T cells in vivo. When delivered at the onset of disease, these inhibitors significantly reduce diabetogenic Teff cells, prolong remission, preserve functional islets, and protect islet allografts while leaving naive, memory, and regulatory T-cell populations functionally untouched. Thus, the targeted manipulation of p53 and cell cycle checkpoints represents a new therapeutic modality for the preservation of islet β-cells in new-onset type 1 diabetes or after islet transplant.

Autoimmune diseases such as type 1 diabetes (T1D) arise from unrestrained activation of effector lymphocytes that destroy target tissues. Many efforts have been made to eliminate these effector lymphocytes, but none has produced a long-term cure. An alternative to depletion therapy is to enhance endogenous immune regulation. Among these endogenous alternatives, naturally occurring Igs have been applied for inflammatory disorders but have lacked potency in antigen-specific autoimmunity. We hypothesized that naturally occurring polyclonal IgMs, which represent the majority of circulating, noninduced antibodies but are present only in low levels in therapeutic Ig preparations, possess the most potent capacity to restore immune homeostasis. Treatment of diabetes-prone NOD mice with purified IgM isolated from Swiss Webster (SW) mice (nIgMSW) reversed new-onset diabetes, eliminated autoreactive B lymphocytes, and enhanced regulatory T-cell (Treg) numbers both centrally and peripherally. Conversely, IgM from prediabetic NOD mice could not restore this endogenous regulation, which represents an unrecognized component of T1D pathogenesis. Of note, IgM derived from healthy human donors was similarly able to expand human CD4 Tregs in humanized mice and produced permanent diabetes protection in treated NOD mice. Overall, these studies demonstrate that a potent, endogenous regulatory mechanism, nIgM, is a promising option for reversing autoimmune T1D in humans.

The role of interferons, either pathogenic or protective, during autoimmune diabetes remains controversial. Herein, we examine the progression of diabetes in NOD mice lacking the type I (IFNAR) or type II (IFNGR) interferon receptor and, for the first time, in mice deficient in both receptors (double knockout [DKO]). All mice were bred, maintained, and monitored in a single specific pathogen-free facility with high female and low male diabetes incidence. Our expectation was that removal of interferon signaling would reduce autoimmune destruction. However, examination of diabetes incidence in the IFNAR- and IFNGR-deficient NOD mice showed a reduction in females and an increase in males. In DKO mice, diabetes occurred only in female mice, at decreased incidence and with delayed kinetics. These results show that interferons act as both positive and negative modulators of type 1 diabetes disease risk dependent on sex.

Transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) is a receptor for the TNF superfamily cytokines, B cell–activating factor (BAFF), and A proliferation–inducing ligand (APRIL). Here, we demonstrate that TACI-deficient mice subjected to high-fat diet (HFD) are protected from weight gain and dysregulated glucose homeostasis. Resistance to HFD-induced metabolic changes in TACI-deficient mice does not involve TACI-mediated adipogenesis. Instead, accumulation of M2 macrophages (Mϕs), eosinophils, and type 2 innate lymphoid cells in visceral adipose tissue (VAT) is implicated in the protection from obesity-induced assaults. In support of this hypothesis, adoptively transferred TACI-deficient peritoneal or adipose tissue Mϕs, but not B cells, can improve glucose metabolism in the obese host. Interestingly, the transferred TACI-deficient Mϕs not only home to host VAT but also trigger the accumulation of host M2 Mϕs and eosinophils in VAT. The increase in host M2 Mϕs in VAT is likely a result of eosinophil recruitment in response to eotaxin-2 produced by TACI-deficient Mϕs. Insulin signaling experiments revealed that IL-10 secreted by TACI-deficient Mϕs is responsible for maintaining adipocyte insulin sensitivity. Thus, the adoptive transfer experiments offer a model where TACI-deficient Mϕs accumulate in VAT and protect against metaflammation and obesity-associated dysregulation of glucose metabolism.

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.β2m(−/−) mice transgenically expressing human disease–associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8(+) T-cell responses mediated by these human class I variants. NOD.β2m(−/−)-A2.1 mice were previously used to identify β-cell autoantigens presented by this human class I variant to pathogenic CD8(+) T cells and for testing therapies to attenuate such effectors. However, NOD.β2m(−/−) mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-K(d) and H2-D(b) classical class I variants either individually or in tandem (cMHCI(−/−)). Ablation of the H2-A(g7) class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II(−/−)). NOD-cMHCI(−/−) mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8(+) T-cell development and T1D susceptibility to NOD-cMHCI(−/−) mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.

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.

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these “isletokines” in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ–induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet–specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti–IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell–induced inflammatory loss of graft function after islet cell transplantation.

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)–mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+) to determine the function of MLL1 in wound healing. Mll1f/fLyz2Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1f/fLyz2Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.

Certain HLA class II genes increase the risk for type 1 diabetes (T1D) development while others provide protection from disease development. HLA class II alleles encode MHC proteins on antigen-presenting cells, which function to present peptides and activate CD4 T cells. The DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype provides dominant protection across all stages of T1D and is a common haplotype found in Caucasians. However, it is present in

Lymph node stromal cells (LNSC) are essential for providing and maintaining peripheral self-tolerance of potentially autoreactive cells. In type 1 diabetes, proinsulin-specific CD8(+) T cells, escaping central and peripheral tolerance, contribute to β-cell destruction. Using G9Cα(−/−)CD8(+) T cells specific for proinsulin, we studied the mechanisms by which LNSC regulate low-avidity autoreactive cells in the NOD mouse model of type 1 diabetes. Whereas MHC-matched NOD-LNSC significantly reduced G9Cα(−/−)CD8(+) T-cell cytotoxicity and dendritic cell–induced proliferation, they failed to sufficiently regulate T cells stimulated by anti-CD3/CD28. In contrast, non-MHC–matched, control C57BL/6 mouse LNSC suppressed T-cell receptor engagement by anti-CD3/CD28 via MHC-independent mechanisms. This C57BL/6-LNSC suppression was maintained even after removal of the LNSC, demonstrating a direct effect of LNSC on T cells, modifying antigen sensitivity and effector function. Thus, our results suggest that a loss of NOD-LNSC MHC-independent suppressive mechanisms may contribute to diabetes development.

Insulin is a major autoantigen in type 1 diabetes, targeted by both CD8 and CD4 T-cells. We studied an insulin-reactive T-cell receptor (TCR) alpha-chain transgenic non-obese diabetic (NOD) mouse on a TCRCα and proinsulin2 (PI2)-deficient background, designated as A22Cα-/-PI2-/-NOD mice. These mice develop a low incidence of autoimmune diabetes. To test the role of gut microbiota on diabetes development in this model system, we treated the A22Cα-/-PI2-/-NOD mice with enrofloxacin, a broad-spectrum antibiotic. The treatment led to male mice developing accelerated diabetes. We found that enrofloxacin increased the frequency of the insulin-reactive CD8+ T-cells and activated the cells in the Peyer’s patches (PP) and pancreatic lymph nodes (PLNs), together with induction of immunological effects on the antigen-presenting cell populations. The composition of gut microbiota differed between the enrofloxacin-treated and untreated mice and also between the enrofloxacin-treated mice that developed diabetes, compared with those that remained normoglycemic. Our results provide evidence that the composition of the gut microbiota is important for determining the expansion and activation of insulin-reactive CD8+ T-cells.

Multiple studies of B- and T-cell compartments and their response to stimuli demonstrate alterations in established type 1 diabetes (T1D). Yet it is not known whether these alterations reflect immune mechanisms that initiate islet autoimmunity, promote disease progression, or are secondary to disease. To address these questions, we used samples from the TrialNet Pathway to Prevention study to investigate T-cell responses to interleukin (IL)-2 and regulatory T cell–mediated suppression, the composition of the B-cell compartment, and B-cell responses to B-cell receptor and IL-21 receptor engagement. These studies revealed stage-dependent T- and B-cell functional and immune phenotypes; namely, early features that differentiate autoantibody-positive at-risk first-degree relatives (FDRs) from autoantibody-negative FDRs and persisted through clinical diagnosis; late features that arose at or near T1D diagnosis; and dynamic features that were enhanced early and blunted at later disease stages, indicating evolving responses along the continuum of T1D. We further explored how these specific phenotypes are influenced by therapeutic interventions. Our integrated studies provide unique insights into stable and dynamic stage-specific immune states and define novel immune phenotypes of potential clinical relevance.

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic β-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4+ T cells compartmentalized within networks of CD45−gp38+CD31− fibroblastic reticular cell (FRC)–like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of β-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing β-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention.

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

Diabetes is prevalent among solid organ transplant recipients and is universal among islet transplant recipients. Whereas diabetes is often considered to result in an immune-compromised state, the impact of chronic hyperglycemia on host alloimmunity is not clear. Potential immune-modifying effects of obesity, autoimmunity, or diabetogenic agents like streptozotocin may confound understanding alloimmunity in experimental models of diabetes. Therefore, we sought to determine the role of chronic hyperglycemia due to insulinopenia on alloimmunity using the nonautoimmune, spontaneously diabetic H-2b–expressing C57BL/6 Ins2Akita mice (Akita). Akita mice harbor a mutated Ins2 allele that dominantly suppresses insulin secretion, resulting in lifelong diabetes. We used BALB/c donors (H-2d) to assess alloimmunization and islet transplantation outcomes in Akita recipients. Surprisingly, chronic hyperglycemia had little effect on primary T-cell reactivity after alloimmunization. Moreover, Akita mice readily rejected islet allografts, and chronic hyperglycemia had no impact on the magnitude or quality of intragraft T-cell responses. In contrast, allospecific IgM and IgG were significantly decreased in Akita mice after alloimmunization. Thus, whereas diabetes influences host immune defense, hyperglycemia itself does not cause generalized alloimmune impairment. Our data suggest that immune compromise in diabetes due to hyperglycemia may not apply to cellular rejection of transplants.

Overcoming the immune response to establish durable immune tolerance in type 1 diabetes remains a substantial challenge. The ongoing effector immune response involves numerous immune cell types but is ultimately orchestrated and sustained by the hematopoietic stem cell (HSC) niche. We therefore hypothesized that tolerance induction also requires these pluripotent precursors. In this study, we determined that the tolerance-inducing agent anti-CD45RB induces HSC mobilization in nonautoimmune B6 mice but not in diabetes-prone NOD mice. Ablation of HSCs impaired tolerance to allogeneic islet transplants in B6 recipients. Mobilization of HSCs resulted in part from decreasing osteoblast expression of HSC retention factors. Furthermore, HSC mobilization required a functioning sympathetic nervous system; sympathectomy prevented HSC mobilization and completely abrogated tolerance induction. NOD HSCs were held in their niche by excess expression of CXCR4, which, when blocked, led to HSC mobilization and prolonged islet allograft survival. Overall, these findings indicate that the HSC compartment plays an underrecognized role in the establishment and maintenance of immune tolerance, and this role is disrupted in diabetes-prone NOD mice. Understanding the stem cell response to immune therapies in ongoing human clinical studies may help identify and maximize the effect of immune interventions for type 1 diabetes.

While the autoimmune destruction of pancreatic ß-cells underlying type 1 diabetes (1D) development is ultimately mediated by T-cells in NOD mice and also likely humans, B-lymphocytes play an additional key pathogenic role. It appears expression of plasma membrane bound immunoglobulin (Ig) molecules that efficiently capture ß-cell antigens allows autoreactive B-lymphocytes bypassing normal tolerance induction processes to be the subset of antigen presenting cells most efficiently activating diabetogenic T-cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or not (hen egg lysozyme; HEL) expressed by ß-cells have proven useful in dissecting the developmental basis of diabetogenic B-lymphocytes. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B-lymphocytes in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin autoreactive B-lymphocytes infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach.

NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8(+) T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8(+) T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23-reactive TRBV19(+)CD8(+) T cells and causes diabetes; 2) insulin B15-23-reactive TRBV19(+)CD8(+) T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8(+) T-cell compartment; 4) a biased repertoire of insulin-reactive CD8(+) T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8(+) T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes.

Adipose tissue (AT) inflammation contributes to impaired insulin action, which is a major cause of type 2 diabetes. RBP4 is an adipocyte- and liver-derived protein with an important role in insulin resistance, metabolic syndrome, and AT inflammation. RBP4 elevation causes AT inflammation by activating innate immunity, which elicits an adaptive immune response. RBP4-overexpressing mice (RBP4-Ox) are insulin resistant and glucose intolerant and have increased AT macrophages and T-helper 1 cells. We show that high-fat diet–fed RBP4−/− mice have reduced AT inflammation and improved insulin sensitivity versus wild type. We also elucidate the mechanism for RBP4-induced macrophage antigen presentation and subsequent T-cell activation. In RBP4-Ox, AT macrophages display enhanced c-Jun N-terminal kinase, extracellular signal–related kinase, and p38 phosphorylation. Inhibition of these pathways and of NF-κB reduces activation of macrophages and CD4 T cells. MyD88 is an adaptor protein involved in proinflammatory signaling. In macrophages from MyD88−/− mice, RBP4 fails to stimulate secretion of tumor necrosis factor, IL-12, and IL-6 and CD4 T-cell activation. In vivo blockade of antigen presentation by treating RBP4-Ox mice with CTLA4-Ig, which blocks costimulation of T cells, is sufficient to reduce AT inflammation and improve insulin resistance. Thus, MyD88 and downstream mitogen-activated protein kinase and NF-κB pathways are necessary for RBP4-induced macrophage antigen presentation and subsequent T-cell activation. Also, blocking antigen presentation with CTLA4-Ig improves RBP4-induced insulin resistance and macrophage-induced T-cell activation.

Developing effective immune therapies for type 1 diabetes (T1D) remains an immense challenge. To date, results from most of the clinical trials have been largely disappointing, with only temporary improvement and limited efficacy (1,2). One of the major obstacles is the heterogeneous and multifactorial nature of the disease. Developing a personalized immune intervention protocol that can attenuate the specific anti-islet autoimmune responses in high-risk individuals or new-onset patients would be a more productive approach. It is well established that autoreactive T lymphocytes that recognize pancreatic β-cell antigens (Ags) (e.g., insulin, GAD65, and Znt8) play central roles in mediating the anti-islet autoimmunity in T1D (3–7). Recent studies demonstrated the presence of a variety of islet-infiltrating, β-cell Ag-reactive T cells in T1D patients (8). Characterizing these autoreactive T cells, including their frequencies, phenotypes, predominant Ag targets, cytokine production, and T-cell receptor specificities, in high-risk individuals or new-onset patients will help to monitor the disease progression and develop the right regimens for immune intervention. However, unlike the well-established autoantibody assays that can detect the presence of the autoantibodies against β-cell antigens with high sensitivity and specificity, detecting autoreactive T cells is more challenging (9). Tissue biopsy is clinically prohibitive and unjustifiable, as the procedure is dangerously invasive and the immune cell–infiltrated islets are likely distributed irregularly within the pancreas. Liquid biopsy is the primary tool to assess autoreactive T cells in T1D patients. Regrettably, autoreactive T cells are extremely rare and hard to detect in peripheral blood, even with recent advances of the highly sensitive tetramer- and multimer-based technologies (10,11). In addition, the limited amount of blood that can be sampled provides only …

Bacteroides fragilis (BF) is an integral component of the human colonic commensal microbiota. BF is also the most commonly isolated organism from clinical cases of intra-abdominal abscesses, suggesting its potential to induce proinflammatory responses upon accessing the systemic compartment. Hence, we examined the impact of mucosal and systemic exposures to BF on type 1 diabetes (T1D) incidence in NOD mice. The impact of intestinal exposure to BF under a chemically induced enhanced gut permeability condition, which permits microbial translocation, in T1D was also examined. While oral administration of heat-killed (HK) BF to prediabetic mice caused enhanced immune regulation and suppression of autoimmunity, resulting in delayed hyperglycemia, mice that received HK BF by intravenous injection showed rapid disease progression. Importantly, polysaccharide A–deficient BF failed to produce these opposing effects upon oral and systemic deliveries. Furthermore, BF-induced modulation of disease progression was observed in wild-type, but not TLR2-deficient, NOD mice. Interestingly, oral administration of BF under enhanced gut permeability conditions resulted in accelerated disease progression and rapid onset of hyperglycemia in NOD mice. Overall, these observations suggest that BF-like gut commensals can cause proinflammatory responses upon gaining access to the systemic compartment and contribute to T1D in at-risk subjects.

Optimal immune-based therapies for type 1 diabetes (T1D) should restore self-tolerance without inducing chronic immunosuppression. CD4(+)Foxp3(+) regulatory T cells (T(regs)) are a key cell population capable of facilitating durable immune tolerance. However, clinical trials with expanded T(regs) in T1D and solid-organ transplant recipients are limited by poor T(reg) engraftment without host manipulation. We showed that T(reg) engraftment and therapeutic benefit in nonautoimmune models required ablative host conditioning. Here, we evaluated T(reg) engraftment and therapeutic efficacy in the nonobese diabetic (NOD) mouse model of autoimmune diabetes using nonablative, combinatorial regimens involving the anti-CD3 (αCD3), cyclophosphamide (CyP), and IAC (IL-2/JES6–1) antibody complex. We demonstrate that αCD3 alone induced substantial T-cell depletion, impacting both conventional T cells (T(conv)) and T(regs), subsequently followed by more rapid rebound of T(regs). Despite robust depletion of host T(conv) and host T(regs), donor T(regs) failed to engraft even with interleukin-2 (IL-2) support. A single dose of CyP after αCD3 depleted rebounding host T(regs) and resulted in a 43-fold increase in donor T(reg) engraftment, yet polyclonal donor T(regs) failed to reverse diabetes. However, infusion of autoantigen-specific T(regs) after αCD3 alone resulted in robust T(reg) engraftment within the islets and induced remission in all mice. This novel combinatorial therapy promotes engraftment of autoantigen-specific donor T(regs) and controls islet autoimmunity without long-term immunosuppression.

Progression to clinical type 1 diabetes varies among children who develop β-cell autoantibodies. Differences in autoantibody patterns could relate to disease progression and etiology. Here we modeled complex longitudinal autoantibody profiles by using a novel wavelet-based algorithm. We identified clusters of similar profiles associated with various types of progression among 600 children from The Environmental Determinants of Diabetes in the Young (TEDDY) birth cohort study; these children developed persistent insulin autoantibodies (IAA), GAD autoantibodies (GADA), insulinoma-associated antigen 2 autoantibodies (IA-2A), or a combination of these, and they were followed up prospectively at 3- to 6-month intervals (median follow-up 6.5 years). Children who developed multiple autoantibody types (n = 370) were clustered, and progression from seroconversion to clinical diabetes within 5 years ranged between clusters from 6% (95% CI 0, 17.4) to 84% (59.2, 93.6). Children who seroconverted early in life (median age

ObjectiveImprovement in insulin alternatives is leading to a delayed presentation of microvascular and macrovascular complications of diabetes. The objective of this study was to evaluate the long-term outcomes of older (≥50 years) diabetic patients who receive a pancreas transplantation (PT).Research design and methodsWe retrospectively evaluated all 338 PTs performed at our center between 2000 and 2016 (mean follow-up 9.4±4.9 years). Recipient and graft survivals were estimated for up to 10 years after PT. Major adverse cardiovascular events (MACEs) before and after PT were included in the analysis.ResultsThirty-nine patients (12%) were ≥50 years old (52.7±2.3 years) at the day of PT, of which 29 received a simultaneous pancreas–kidney transplantation (SPK) and 10 a pancreas after kidney transplantation (PAK). SPK recipients were first transplants, whereas in the PAK up to 50% were pancreas re-transplantations. Recipient and pancreas graft survivals at 10 years were similar between the group 0.05). The prevalence of MACE prior to PT was similar between both groups (31% vs 29%). Following PT, older recipients presented inferior post-transplant MACE-free survival. In a multivariate regression model, diabetes vintage (HR 1.054, p=0.03) and pre-transplantation MACE (HR 1.98, p=0.011), but not recipient age (HR 1.45, p=0.339), were associated with post-transplant MACE.ConclusionsLong-term survival of older pancreas transplant recipients are similar to younger counterparts. Diabetes vintage, but not age, increased the risk of post-transplantation MACE. These results suggest pancreas transplantation is a valuable treatment alternative to older diabetic patients.