Type 1 Diabetes In Non-obese Diabetic Mice Research Articles

Immunopeptidome mining reveals a novel ERS-induced target in T1D.

Autoreactive CD8+ T cells play a key role in type 1 diabetes (T1D), but the antigen spectrum that activates autoreactive CD8+ T cells remains unclear. Endoplasmic reticulum stress (ERS) has been implicated in β-cell autoantigen generation. Here, we analyzed the major histocompatibility complex class I (MHC-I)-associated immunopeptidome (MIP) of islet β-cells under steady and ERS conditions and found that ERS reshaped the MIP of β-cells and promoted the MHC-I presentation of a panel of conventional self-peptides. Among them, OTUB258-66 showed immunodominance, and the corresponding autoreactive CD8+ T cells were diabetogenic in nonobese diabetic (NOD) mice. High glucose intake upregulated pancreatic OTUB2 expression and amplified the OTUB258-66-specific CD8+ T-cell response in NOD mice. Repeated OTUB258-66 administration significantly reduced the incidence of T1D in NOD mice. Interestingly, peripheral blood mononuclear cells (PBMCs) from patients with T1D, but not from healthy controls, showed a positive IFN-γ response to human OTUB2 peptides. This study provides not only a new explanation for the role of ERS in promoting β-cell-targeted autoimmunity but also a potential target for the prevention and treatment of T1D. The data are available via ProteomeXchange with the identifier PXD041227.

WITHDRAWN: Combined therapy of adenovirus vector mediated IGF-1 gene with anti-CD20 mAbs exerts potential beneficial role on type 1 diabetes in nonobese diabetic mice

To assess the protective effects of combined treatment with anti-CD20 monoclonal antibody (mAb) and adenovirus mediated mouse insulin-like growth factor 1 (Adv-mIGF-1) gene on type 1 diabetes (T1D) in nonobese diabetic (NOD) mice at early stage. To simultaneously restore the proportion of Th cells and block the interaction of B cells, NOD model mice were assigned to four groups which received PBS, Adv-mIGF-1 gene and anti-CD20 mAbs alone or combination, respectively. After 16 weeks of therapeutic intervention, blood samples and pancreatic tissues of mice were measured via the methods of ELISA, RT-PCR, western blotting, H&E staining, TUNEL and immunohistochemistry assays. Chronic combination intervention with Adv-mIGF-1 gene and anti-CD20 mAbs reduced the T1D-related morbidity, promoted the secretion of insulin, controlled the blood glucose levels (BGLs) and alleviated insulitis of experimental mice. In addition, current combination intervention also protected the pancreatic β cells via suppressing the expression of Fas and TNF-α, inhibiting Caspase-3/8 related apoptotic pathway, and activating the Bcl-2-related antiapoptotic pathway. Furthermore, current combination therapy also increased the expression levels of PDX-1 and CK-19 genes, and finally accelerated the proliferation and differentiation of pancreatic β-cells. In addition, combination therapy could also ameliorate the pathological characteristics of diabetic nephropathy in NOD mice. Combination treatment with Adv-mIGF-1 gene and anti-CD20 mAbs may exert a potential beneficial role on T1D in NOD mice.

Axenic Caenorhabditis elegans antigen protects against development of type-1 diabetes in NOD mice

Studies in humans and animals have demonstrated that infection with helminths (parasitic worms) is protective against a range of hyperinflammatory diseases. A number of factors limit translation into clinical use, including: potential contamination of helminths obtained from infected humans or animals, lack of batch to batch stability, and potential pathological risks derived from live worm infections. To overcome these limitations we tested whether an antigen homogenate of the non-pathogenic nematode Caenorhabditis elegans confers protection against type 1 diabetes mellitus (T1D) using the Non Obese Diabetic (NOD) mouse model.Our study demonstrates that twice weekly intraperitoneal injections of axenically cultured C. elegans antigen (aCeAg) confers substantial protection against type 1 diabetes in NOD mice. Whereas 80% of control mice (PBS-injected) developed clinical disease, only 10% of aCeAg-treated mice became diabetic. Additionally, aCeAg treated mice had significantly greater numbers of insulin-producing pancreatic islets and greater numbers of islets negative for lymphocyte infiltration. Immunological changes observed in aCeAg treated mice included increases in total IgE and total IgG1, consistent with induction of a type 2 immune response similar to that typically seen in parasitic worm infection. Although evidence suggests that helminth infections induce strong immunoregulatory signals, we did not observe significant changes in regulatory T cell numbers or in production of the regulatory cytokines TGFβ and IL-10. The lack of a regulatory response may be due to our time point of observation, or perhaps the mechanism of aCeAg efficacy may differ from that of helminth infection.Discovery that antigens obtained from a non-parasitic environmental nematode replicate the protective phenotype induced by parasitic worm infections may accelerate our ability to develop nematode-derived therapies for allergy and autoimmune diseases.

A Novel Liposome Formulation Carrying Both an Insulin Peptide and a Ligand for Invariant Natural Killer T Cells Induces Accumulation of Regulatory T Cells to Islets in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic β cells by autoantigen-reactive diabetogenic cells. Antigen-specific therapies using islet autoantigens for restoring immune tolerance have emerged as promising approaches for the treatment of T1D but have been unsuccessful in humans. Herein, we report that RGI-3100-iB, a novel liposomal formulation carrying both α-galactosylceramide (α-GalCer), which is a representative ligand for invariant natural killer T (iNKT) cells, and insulin B chain 9–23 peptide, which is an epitope for CD4+ T cells, could induce the accumulation of regulatory T cells (Tregs) in islets in a peptide-dependent manner, followed by the remarkable prevention of diabetes onset in nonobese diabetic (NOD) mice. While multiple administrations of a monotherapy using either α-GalCer or insulin B peptide in a liposomal formulation was confirmed to delay/prevent T1D in NOD mice, RGI-3100-iB synergistically enhanced the prevention effect of each monotherapy and alleviated insulitis in NOD mice. Immunopathological analysis showed that Foxp3+ Tregs accumulated in the islets in RGI-3100-iB-treated mice. Cotransfer of diabetogenic T cells and splenocytes of NOD mice treated with RGI-3100-iB, but not liposomal α-GalCer encapsulating an unrelated peptide, to NOD-SCID mice resulted in the prevention of diabetes and elevation of Foxp3 mRNA expression in the islets. These data indicate that the migration of insulin B-peptide-specific Tregs to islet of NOD mice that are involved in the suppression of pathogenic T cells related to diabetes onset and progression could be enhanced by the administration of liposomes containing α-GalCer and insulin B peptide.

Low Methoxyl Pectin Protects against Autoimmune Diabetes and Associated Caecal Dysfunction.

This study aims to examine the protective effects of specific low-methoxyl pectin (LMP) on the development of type 1 diabetes (T1D). Female non-obese diabetic (NOD) mice are weaned onto either control or 5% LMP supplemented diets for up to 22 weeks of age. T1D incidence, gut barrier function, and pancreatic-gut immune responses are analyzed. LMP supplementation significantly dampened the onset of T1D in NOD mice. LMP supplementation induces caecal homeostasis, as indicated by the increasing SCFAs production, higher expression of tight junction proteins claudin 1, zonula occludens-2 in caecum. Furthermore, LMP-mediated caecal homeostasis impacts gut-pancreatic immunity, as evidenced by increased regulatory T cell population, modulated inflammatory cytokine expression, and suppressed NOD like receptor protein 3 (NLRP3) inflammasome activation in both caecum and pancreas. The data demonstrate that LMP limits T1D development by inducing caecal homeostasis to shape pancreatic immune environment, providing a scientific basis for using LMP as a novel functional supplementation to intervene T1D.

Artesunate prevents type 1 diabetes in NOD mice mainly by inducing protective IL-4-producing T cells and regulatory T cells.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the immune-mediated destruction of insulin-producing β cells. Recent studies showed that in addition to malaria, artemisinin and its derivative, artesunate (AS), could alleviate several autoimmune diseases. However, whether AS has a role in the prevention or treatment of T1D is still unknown. Therefore, in this study we administrated AS or DMSO in the drinking water of nonobese diabetic (NOD) mice, a mouse model of T1D. We found that AS administration significantly prevented the incidence of T1D. The frequency of IL-4-producing CD4+ single-positive T cells and CD8+ T cells was significantly elevated, and IFN-γ-producing T cells were reduced in the spleen and pancreatic lymph nodes. In the pancreas, the skewing to IL-4-producing T cells was also observed. In addition, more regulatory T cells were found in the pancreas. mRNA levels of proinflammatory cytokines, including TNF-α and IL-6, were decreased. In addition, AS administration promoted the functional maturity of β cells in vitro. Our findings demonstrate that AS administration can prevent T1D in NOD mice mainly by reducing autoimmune T cells and increasing protective T cells. Our data constitute the first functional study of AS in T1D, which may provide a new rationale for future translational studies.-Li, Z., Shi, X., Liu, J., Shao, F., Huang, G., Zhou, Z., Zheng, P. Artesunate prevents type 1 diabetes in NOD mice mainly by inducing protective IL-4-producing T cells and regulatory T cells.

Co-delivery of siAlox15 and sunitinib for reversing the new-onset of type 1 diabetes in non-obese diabetic mice

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by proinflammatory T cell infiltration associated cytokine secretion and reactive oxygen species (ROS) production. In this study, our objective was to determine whether co-delivery of sunitinib, a member of tyrosine kinase inhibitor (TKI) family, and siRNA against Alox15 (siAlox15) could reverse the new-onset of T1D in non-obese diabetic (NOD)/ShiLtJ female mice which spontaneously develop diabetes. A cationic copolymer methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (mPEG-b-PCC-g-DC-g-TEPA) was used to encapsulate sunitinib with 8.32-9.39% loading efficacy and form complex with siAlox15 at the N/P ratio of 16:1. These micellar formulations exhibited sustained release of sunitinib for around 50% at 24 h and protected siAlox15 from serum degradation for at least 10 h. Further, both sunitinib and siAlox15 inhibited human peripheral blood mononuclear cell (PBMC) proliferation and activation by downregulating the phosphorylation of Akt, platelet-derived growth factor receptor (PDGFR) at protein levels and Alox15 in mRNA levels, respectively. Besides, siAlox15 treatment reversed the cytokine induced ROS upregulation in rat INS-1E β cells. Finally, systemic administration of these micelles carrying sunitinib and siAlox15 efficiently reversed T1D in NOD mice as indicated by decreased blood glucose concentration to below 250 mg/dL. The mice showed fast response to high glucose which was determined by intraperitoneal glucose tolerance test and promoted serum insulin production. Tissue staining indicated that sunitinib and siAlox15 combination could inhibit T cell infiltration to mouse islets, thereby promoting islet survival and function. In summary, our results demonstrated that combination therapy of sunitinib and siAlox15 could be a novel, reliable and safe approach for patients who were recently diagnosed with T1D.

Diabetes progression and alterations in gut bacterial translocation: prevention by diet supplementation with human milk in NOD mice

Impaired intestinal barrier function occurs before type 1 diabetes (T1D) onset with a possible contribution of microbial translocation. Breastfeeding is associated with enhanced mucosal intestinal integrity and T1D protection. Our aim was to study the potential of human milk (HM) to prevent diabetes onset and modulate the translocation of gut bacteria susceptible to breastfeeding or associated to diabetes onset. We show that HM intake can prevent T1D in nonobese diabetic mice independently of bifidobacteria colonization. Prior to diabetes onset, HM mice harbored splenic bacterial counts and plasma lipopolysaccharides level similar to control mice but exhibited a reduced expansion of Anaerotruncus sp. in pancreas and Lactobacillus johnsonii and Barnesiella in Peyer's patches (PP). Surprisingly, pancreas and PP bacterial expansion did not correlate with their own gut localization but with ileal Escherichia coli and cecal HM-susceptible bacteria (the promoted L. murinus and Bacteroides vulgatus, and the repressed B. fragilis and E. coli), respectively. Besides, higher colonic B. vulgatus counts induced by HM intake were associated with low islet infiltration and pancreatic E. coli expansion. On another hand, splenic dendritic cells (DCs) were identified as negative covariate of PP Barnesiella, suggesting a possible HM contribution to preserving splenic DCs through the reduction of Barnesiella translocation. Fecal B. vulgatus also negatively correlated with PP Barnesiella expansion, indicating that the mouse coprophagic behavior likely added to HM effect. Our findings provide evidence that HM has a multilevel impact and cooperates with some gut bacteria for controlling bacterial translocation at the earliest stage of insulitis.

Dietary Cows' Milk Protein A1 Beta-Casein Increases the Incidence of T1D in NOD Mice.

The contribution of cows’ milk containing beta-casein protein A1 variant to the development of type 1 diabetes (T1D) has been controversial for decades. Despite epidemiological data demonstrating a relationship between A1 beta-casein consumption and T1D incidence, direct evidence is limited. We demonstrate that early life exposure to A1 beta-casein through the diet can modify progression to diabetes in non-obese diabetic (NOD) mice, with the effect apparent in later generations. Adult NOD mice from the F0 generation and all subsequent generations (F1 to F4) were fed either A1 or A2 beta-casein supplemented diets. Diabetes incidence in F0–F2 generations was similar in both cohorts of mice. However, diabetes incidence doubled in the F3 generation NOD mice fed an A1 beta-casein supplemented diet. In F4 NOD mice, subclinical insulitis and altered glucose handling was evident as early as 10 weeks of age in A1 fed mice only. A significant decrease in the proportion of non-conventional regulatory T cell subset defined as CD4+CD25−FoxP3+ was evident in the F4 generation of A1 fed mice. This feeding intervention study demonstrates that dietary A1 beta-casein may affect glucose homeostasis and T1D progression, although this effect takes generations to manifest.

Abnormal T-Cell Development in the Thymus of Non-obese Diabetic Mice: Possible Relationship With the Pathogenesis of Type 1 Autoimmune Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells in the pancreas, by direct interactions with autoreactive pancreas infiltrating T lymphocytes (PILs). One of the most important animal models for this disease is the non-obese diabetic (NOD) mouse. Alterations in the NOD mouse thymus during the pathogenesis of the disease have been reported. From the initial migratory disturbances to the accumulation of mature thymocytes, including regulatory Foxp3+ T cells, important mechanisms seem to regulate the repertoire of T cells that leave the thymus to settle in peripheral lymphoid organs. A significant modulation of the expression of extracellular matrix and soluble chemoattractant molecules, in addition to integrins and chemokine receptors, may contribute to the progressive accumulation of mature thymocytes and consequent formation of giant perivascular spaces (PVS) that are observed in the NOD mouse thymus. Comparative large-scale transcriptional expression and network analyses involving mRNAs and miRNAs of thymocytes, peripheral T CD3+ cells and PILs provided evidence that in PILs chemokine receptors and mRNAs are post-transcriptionally regulated by miR-202-3p resulting in decreased activity of these molecules during the onset of T1D in NOD mice. In this review, we discuss the abnormal T-cell development in NOD mice in the context of intrathymic expression of different migration-related molecules, peptides belonging to the family of insulin and insulin-like growth factors as well as the participation of miRNAs as post-transcriptional regulators and their possible influence on the onset of aggressive autoimmunity during the pathogenesis of T1D.

Anti-TCRβ mAb in Combination With Neurogenin3 Gene Therapy Reverses Established Overt Type 1 Diabetes in Female NOD Mice.

Insulin-producing β cells in patients with type 1 diabetes (T1D) are destroyed by T lymphocytes. We investigated whether targeting the T-cell receptor (TCR) with a monoclonal antibody (mAb) abrogates T-cell response against residual and newly formed islets in overtly diabetic nonobese diabetic (NOD) mice. NOD mice with blood glucose levels of 250 to 350 mg/dL or 350 to 450 mg/dL were considered as new-onset or established overt diabetes, respectively. These diabetic NOD mice were transiently treated with an anti-TCR β chain (TCRβ) mAb, H57-597, for 5 days. Two weeks later, some NOD mice with established overt diabetes further received hepatic gene therapy using the islet-lineage determining gene Neurogenin3 (Ngn3), in combination with the islet growth factor gene betacellulin (Btc). We found that anti-TCRβ mAb (50 µg/d) reversed >80% new-onset diabetes in NOD mice for >14 weeks by reducing the number of effector T cells in the pancreas. However, anti-TCRβ mAb therapy alone reversed only ∼20% established overt diabetes in these mice. Among those overtly diabetic NOD mice whose diabetes was resistant to anti-TCRβ mAb treatment, ∼60% no longer had diabetes when they also received Ngn3-Btc hepatic gene transfer 2 weeks after initial anti-TCRβ mAb treatment. This combination of Ngn3-Btc gene therapy and anti-TCRβ mAb treatment induced the sustained formation of periportal insulin-producing cells in the liver of overtly diabetic mice. Therefore, directly targeting TCRβ with a mAb potently reverses new-onset T1D in NOD mice and protects residual and newly formed gene therapy-induced hepatic neo-islets from T-cell‒mediated destruction in mice with established overt diabetes.

Establishment of a Regulatory Microenvironment by Lactococcus Expressing Colonization Factor Antigen I (CFA/I) Fimbriae Ameliorates Type 1 Diabetes (T1D) in Non-Obese Diabetic (NOD) Mice

Abstract Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae protect in murine models of multiple sclerosis and rheumatoid arthritis. Although CFA/I fimbriae initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection was found to be ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). Our recent findings show that oral dosing with a L. lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45% with a concomitant 8-fold increase in their splenic Foxp3+CD25+ Tregs. However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. We hypothesized that LL-CFA/I stimulates dendritic cells (DCs) to establish a regulatory microenvironment since DCs play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic. In this study, 4 wk-old NOD mice were orally dosed with LL-CFA/I and treated every 2 wks; control groups were given L. lactis vector or PBS. At 11 wks of age, frequency of insulitis was reduced by more than half, and insulin-specific T cells were reduced in the pancreatic lymph nodes (PaLNs; P < 0.05). To discern how this was mediated, groups of mice were examined at 3, 7, 14, and 21 days post-treatment for changes in DC and T cell phenotypes. As early as 3 days post-treatment, DCs exhibited significantly less expression of costimulatory molecules, CD40 and CD86, and stably maintained this phenotype for at least 7 wks. Splenic DCs showed reduced IL-6 production. These data show that orally dosing with LL-CFA/I ameliorates T1D in NOD mice by establishing a regulatory microenvironment via DCs. Work is supported by AI121745.

Targeting innate immunity to downmodulate adaptive immunity and reverse type 1 diabetes.

Type 1 diabetes (T1D) is characterized by specific destruction of pancreatic insulin-producing beta cells accompanied by evidence of beta-cell-directed autoimmunity such as autoreactive T cells and islet autoantibodies (IAAs). Currently, T1D cannot be prevented or reversed in humans. T1D is easy to prevent in the nonobese diabetic (NOD) spontaneous mouse model but reversing new-onset T1D in mice is more difficult. Since the discovery of the T-cell receptor in the 1980s and the subsequent identification of autoreactive T cells directed toward beta-cell antigens (eg, insulin, glutamic acid decarboxylase), the dream of antigen-specific immunotherapy has dominated the field with its promise of specificity and limited side effects. While such approaches have worked in the NOD mouse, however, dozens of human trials have failed. Broader immunosuppressive approaches (originally cyclosporine, subsequently anti-CD3 antibody) have shown partial successes (e.g., prolonged C peptide preservation) but no major therapeutic efficacy or disease reversal. Human prevention trials have failed, despite the ease of such approaches in the NOD mouse. In the past 50 years, the incidence of T1D has increased dramatically, and one explanation is the “hygiene hypothesis”, which suggests that decreased exposure of the innate immune system to environmental immune stimulants (e.g., bacterial products such as Toll-like receptor (TLR) 4-stimulating lipopolysaccharide [LPS]) dramatically affects the adaptive immune system and increases subsequent autoimmunity. We have tested the role of innate immunity in autoimmune T1D by treating acute-onset T1D in NOD mice with anti-TLR4/MD-2 agonistic antibodies and have shown a high rate of disease reversal. The TLR4 antibodies do not directly stimulate T cells but induce tolerogenic antigen-presenting cells (APCs) that mediate decreased adaptive T-cell responses. Here, we review our current knowledge and suggest future prospects for targeting innate immunity in T1D immunotherapy.

Insulin sensitizer prevents and ameliorates experimental type 1 diabetes.

Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune β-cell failure, whereas systemic resistance to insulin is considered the hallmark of insulin-independent type-2 diabetes (T2D). In contrast to this canonical dichotomy, insulin resistance appears to precede the overt diabetic stage of T1D and predict its progression, implying that insulin sensitizers may change the course of T1D. However, previous attempts to ameliorate T1D in animal models or patients by insulin sensitizers have largely failed. Sensitization to insulin by MEthyl-substituted long-chain DICArboxylic acid (MEDICA) analogs in T2D animal models surpasses that of current insulin sensitizers, thus prompting our interest in probing MEDICA in the T1D context. MEDICA efficacy in modulating the course of T1D was verified in streptozotocin (STZ) diabetic rats and autoimmune nonobese diabetic (NOD) mice. MEDICA treatment normalizes overt diabetes in STZ diabetic rats when added on to subtherapeutic insulin, and prevents/delays autoimmune T1D in NOD mice. MEDICA treatment does not improve β-cell insulin content or insulitis score, but its efficacy is accounted for by pronounced total body sensitization to insulin. In conclusion, potent insulin sensitizers may counteract genetic predisposition to autoimmune T1D and amplify subtherapeutic insulin into an effective therapeutic measure for the treatment of overt T1D.

Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice

Autoantigen-specific immunological tolerance represents a central objective for prevention of type 1 diabetes (T1D). Previous studies demonstrated mucosal antigen administration results in expansion of Foxp3+ and LAP+ regulatory T cells (Tregs), suggesting oral delivery of self-antigens might represent an effective means for modulating autoimmune disease. Early preclinical experiments using the non-obese diabetic (NOD) mouse model reported mucosal administration of T1D-related autoantigens [proinsulin or glutamic acid decarboxylase 65 (GAD)] delayed T1D onset, but published data are conflicting regarding dose, treatment duration, requirement for combinatorial agents, and extent of efficacy. Recently, dogma was challenged in a report demonstrating oral insulin does not prevent T1D in NOD mice, possibly due to antigen digestion prior to mucosal immune exposure. We used transplastomic plants expressing proinsulin and GAD to protect the autoantigens from degradation in an oral vaccine and tested the optimal combination, dose, and treatment duration for the prevention of T1D in NOD mice. Our data suggest oral autoantigen therapy alone does not effectively influence disease incidence or result in antigen-specific tolerance assessed by IL-10 measurement and Treg frequency. A more aggressive approach involving tolerogenic cytokine administration and/or lymphocyte depletion prior to oral antigen-specific immunotherapy will likely be required to impart durable therapeutic efficacy.

Oral insulin (human, murine, or porcine) does not prevent diabetes in the non-obese diabetic mouse

Studies have shown oral insulin prevents type 1 diabetes (T1D) in mouse models, however human trials were inconclusive. We tested the ability of different insulins to prevent T1D in non-obese diabetic mice. Mice received oral insulin or PBS twice weekly and disease was monitored. Contrary to previous studies, no insulin tested showed significant ability to prevent T1D, nor did testing of linked suppression in a delayed type hypersensitivity model have reproducible effect. To investigate delivery of antigen within the GI tract, blue dye was fed to mice. Dye traveled 5–8cm from stomach to small intestine within 10s, suggesting orally administered antigen may not get digested in the stomach in mice. Insulin incubated with jejunum extracts was instantly digested. Thus, in humans large doses of insulin may be required to achieve tolerance as antigen may be more vulnerable to digestion in the stomach even before reaching the small intestine.

PD-1 pathway-mediated regulation of islet-specific CD4+ T cell subsets in autoimmune diabetes.

Type 1 diabetes (T1D) is a CD4+ T cell-driven autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells. Clinical evidence and studies in non-obese diabetic (NOD) mice suggest that insulin is a major autoantigen. With this in mind, we developed insulin B10-23:IAg7 tetramer reagents to track insulin-specific CD4+ T cells in mice and interrogated the role of Programmed death-1 (PD-1) for peripheral tolerance. PD-1 is a T cell inhibitory receptor necessary to maintain tolerance and prevent T1D in NOD mice. PD-1 pathway inhibitors are increasingly used in the clinic for treating malignancies, and while many patients benefit, some develop adverse autoimmune events, including T1D. We therefore sought to understand the role of PD-1 in maintaining islet-specific tolerance in diabetes-resistant strains. B6.g7 mice express the same MHC Class II allele as NOD mice, have predominantly naïve insulin-specific CD4+ T cells in the periphery, and remain diabetes-free even after PD-1 pathway blockade. Here, we examined the trafficking potential of insulin-specific CD4+ T cells in NOD and B6.g7 mice with or without anti-PD-L1 treatment, and found that PD-L1 blockade preferentially increased the number of CD44highCXCR3+ insulin-specific cells in NOD but not B6.g7 mice. Additionally, we investigated whether pancreatic islets in NOD and B6.g7 mice expressed CXCL10, a lymphocyte homing chemokine and ligand for CXCR3. Anti-PD-L1 treated and control NOD mice had detectable CXCL10 expression in the islets, while B6.g7 islets did not. These data suggest that islet tolerance may be in part attributed to the pancreatic environment and in the absence of pancreas inflammation, chemotactic cytokines may be missing. This, together with our previous data showing that PD-1 pathway blockade preferentially affects effector but not anergic self-specific T cells has implications for the use of checkpoint blockade in treating tumor patients. Our work suggests that determining tumor- and self-specific CD4+ T cell activation status (naïve, effector or anergic) prior to initiation of immunotherapy would likely help to stratify individuals who would benefit from this therapy versus those who might have adverse effects or incomplete tumor control.

173. Insulin B9-23 LV-Driven Expression in Hepatocytes Combined With Suboptimal Dose of Anti-CD3 mAb Cures Type 1 Diabetes in NOD Mice

Type 1 diabetes (T1D) is an autoimmune disease resulting in complete destruction of insulin-producing pancreatic β cells. In T1D in human and in the non-obese diabetic (NOD) mouse, the spontaneous murine model of T1D, auto-reactive T cells target islet-associated antigens. Induction of antigen (Ag)-specific tolerance could cure Type 1 Diabetes (T1D) but it has not been achieved yet. We previously showed that lentiviral vector (LV)-mediated gene expression in hepatocytes induces active tolerance toward the encoded-Ag. Systemic administration of a single dose of Integrase competent (IC) or integrase defective (ID) LV.ET.InsB9-23.142T, enabling stable and transient expression of InsB9-23 in hepatocytes, respectively, arrests β cell destruction in NOD mice at advanced pre-diabetic stage by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). In the present study we tested the efficacy of hepatocytes-directed LV.ET. InsB9-23.142T gene transfer in protecting from disease progression at later stages and in reversing T1D.Treatment with LV.ET.InsB9-23.142T in NOD mice with glucose levels ranging from 200mg/mL to 250mg/mL blocked T1D progression in only 27% of the mice. Co-expression of the late auto-Ags-derived epitopes GAD206-220 and IGRP195-214 in hepatocytes did not improve the efficacy of LV.ET.InsB9-23.142T treatment. LV.ET.InsB9-23.142T treatment in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL did not result in reversion to normoglycemic levels in any of the treated mice.We next combined InsB9-23 gene transfer with anti-CD3 monoclonal antibody (mAb) treatment. Treatment with anti-CD3 mAb at optimal doses is able per se to reverse T1D in NOD mice. Therefore, we tested decreasing doses of anti-CD3 mAb in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL to identify the sub-optimal dose unable to revert T1D. We found that a single administration of anti-CD3 mAb at 5μg instead of 10μg results was not effective. This sub-optimal dose of anti-CD3 mAb (1X 5μg) was administered together with LV.ET.InsB9-23.142T to NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL. Results showed T1D reversal in 75% of ICLV-treated and 40% of the IDLV-treated mice. These data indicate that the LV.ET. InsB9-23.142T treatment combined with sub-optimal anti-CD3 mAb treatment is able to reverse overt diabetes.

Type 1 diabetes in NOD mice unaffected by mast cell deficiency.

Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4(+) and CD8(+) T cells, in the regulation of T-cell and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization, all of which could significantly impact the immune response against self-antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell-deficient nonobese diabetic (NOD) mice, NOD.Cpa3(Cre/+) (Heidelberg) and NOD.Kit(W-sh/W-sh) (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process nonessential and excluding them as potential therapeutic targets.

PH of Drinking Water Influences the Composition of Gut Microbiome and Type 1 Diabetes Incidence

Nonobese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D), progression of which is similar to that in humans, and therefore are widely used as a model for understanding the immunological basis of this disease. The incidence of T1D in NOD mice is influenced by the degree of cleanliness of the mouse colony and the gut microflora. In this report, we show that the T1D incidence and rate of disease progression are profoundly influenced by the pH of drinking water, which also affects the composition and diversity of commensal bacteria in the gut. Female NOD mice that were maintained on acidic pH water (AW) developed insulitis and hyperglycemia rapidly compared with those on neutral pH water (NW). Interestingly, forced dysbiosis by segmented filamentous bacteria (SFB)-positive fecal transfer significantly suppressed the insulitis and T1D incidence in mice that were on AW but not in those on NW. The 16S rDNA–targeted pyrosequencing revealed a significant change in the composition and diversity of gut flora when the pH of drinking water was altered. Importantly, autoantigen-specific T-cell frequencies in the periphery and proinflammatory cytokine response in the intestinal mucosa are significantly higher in AW-recipient mice compared with their NW counterparts. These observations suggest that pH of drinking water affects the composition of gut microflora, leading to an altered autoimmune response and T1D incidence in NOD mice.