Diabetes In NOD Mice Research Articles

Long-term drench of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 protects against type 1 diabetes of NOD mice via stimulating GLP-1 secretion.

Type 1 diabetes is an autoimmune disease that results in the specific destruction of insulin-producing beta cells in the pancreas. The aim of this study was to investigate the mechanism of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) against type 1 diabetes. Long-term drench of XG5 EPS delayed the onset of autoimmune diabetes and had fewer islets with high-grade infiltration (an insulitis score of 3 or 4) than untreated NOD mice. Oral administration of 50 mg kg-1 d-1 XG5 EPS increased the insulin and glucagon-like peptide-1 (GLP-1) levels of serum, stimulated GLP-1 secretion and upregulated gcg mRNA expression of colon in NOD mice. Moreover, oral administration of 50 mg kg-1 d-1 XG5 EPS significantly increased total short-chain fatty acids levels in the colon contents, especially those of acetic acid and butyric acid. In NCI-H716 cells, 500 and 1000 μmol L-1 sodium butyrate promoted the secretion of GLP-1 and upregulated the mRNA expression of gcg and PC3, while XG5 EPS and sodium acetate did not stimulate the GLP-1 secretion. Therefore, long-term drench of XG5 EPS delayed the onset of autoimmune diabetes, which may be directly correlated with the increase of butyrate in the colon of NOD mice. Long-term drench of 50 mg kg-1 d-1 XG5 EPS promoted the expression and secretion of GLP-1 by increasing the production of butyric acid, thereby delaying T1D onset in NOD mice. © 2021 Society of Chemical Industry.

Coreceptor therapy has distinct short- and long-term tolerogenic effects intrinsic to autoreactive effector T cells.

Immunotherapies are needed in the clinic that effectively suppress β cell autoimmunity and reestablish long-term self-tolerance in type 1 diabetes. We previously demonstrated that nondepleting anti-CD4 (αCD4) and αCD8α antibodies establish rapid and indefinite remission in recent-onset diabetic NOD mice. Diabetes reversal by coreceptor therapy (CoRT) is induced by suppression of pathogenic effector T cells (Teffs) and the selective egress of T cells from the pancreatic lymph nodes and islets that remain free of infiltration in the long term. Here, we defined CoRT-induced events regulating early Teff function and pancreatic residency, and long-term tolerance. TCR-driven gene expression controlling autoreactive Teff expansion and proinflammatory activity was suppressed by CoRT, and islet T cell egress was dependent on sphingosine-1 phosphate. In both murine and human T cells, CoRT upregulated the Foxo1 transcriptional axis, which in turn was required for suppression and efficient pancreatic egress of Teffs. Interestingly, long-term tolerance induced in late-preclinical NOD mice was marked by reseeding of the pancreas by a reduced CD8+ Teff pool exhibiting an exhausted phenotype. Notably, PD-1 blockade, which rescues exhausted Teffs, resulted in diabetes onset in protected animals. These findings demonstrate that CoRT has distinct intrinsic effects on Teffs that impact events early in induction and later in maintenance of self-tolerance.

Protective effect and mechanism of Schistosoma japonicum soluble egg antigen against type 1 diabetes in NOD mice

Protective effect and mechanism of Schistosoma japonicum soluble egg antigen against type 1 diabetes in NOD mice

Mouse IL-2/CD25 Fusion Protein Induces Regulatory T Cell Expansion and Immune Suppression in Preclinical Models of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with an IL-2-deficient state, with regulatory T cells (Tregs) showing diminished immune regulatory capacity. A low dose of IL-2 has shown encouraging clinical benefits in SLE patients; however, its clinical utility is limited because of the requirement of daily injections and the observation of increase in proinflammatory cytokines and in non-Tregs. We recently showed that a fusion protein of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, was effective in treating diabetes in NOD mice by selectively inducing Treg expansion. In this report, we show that mouse IL-2 (mIL-2)/CD25 at doses up to 0.5 mg/kg twice a week induced a robust Treg expansion without showing signs of increase in the numbers of NK, CD4+Foxp3-, or CD8+ T cells or significant increase in proinflammatory cytokines. In both NZB × NZW and MRL/lpr mice, mIL-2/CD25 at 0.2-0.4 mg/kg twice a week demonstrated efficacy in inducing Treg expansion, CD25 upregulation, and inhibiting lupus nephritis based on the levels of proteinuria, autoantibody titers, and kidney histology scores. mIL-2/CD25 was effective even when treatment was initiated at the time when NZB × NZW mice already showed signs of advanced disease. Furthermore, we show coadministration of prednisolone, which SLE patients commonly take, did not interfere with the ability of mIL-2/CD25 to expand Tregs. The prednisolone and mIL-2/CD25 combination treatment results in improvements in most of the efficacy readouts relative to either monotherapy alone. Taken together, our results support further evaluation of IL-2/CD25 in the clinic for treating immune-mediated diseases such as SLE.

760-P: Allogeneic "Neo-Islets," Organoids Composed of Cultured Pancreatic Islet and Mesenchymal Stromal Cells from Diverse Donors, Afford a Marked Increase in the Global Availability of a Curative, Novel, Anti-Rejection-Drug-Independent Biotherapy of T1DM

The scarcity of suitable pancreas/islet donors, the life-long need for potentially toxic anti-rejection drugs for transplants and certain biotherapies, and failure of some immune-isolating encapsulation devices collectively warrant the urgent development of novel therapies for T1DM that significantly expand the donor pool without reliance on anti-rejection drugs. Allogeneic, i.p.-administered “Neo-Islets” (NIs) are potentially such a therapy. We previously reported that the use of NIs reestablished permanent normoglycemia in autoimmune diabetic NOD mice without immunosuppression. Our FDA-guided pilot study (INAD 012-0776) in insulin dependent, spontaneously diabetic pet dogs showed similar, durable responses, also without immunosuppression. Scoring systems have improved allotransplantation outcomes while their use may actually waste potentially valuable cells for novel diabetes therapies such as NIs. In preparation for a Phase 1 clinical trial with NIs, we examined whether human “non-clinical grade” islets from diverse, nondiabetic human and canine donors (n=6-8 each) could be processed to generate fully functioning NIs. Islet gene expression profiles (insulin, others) in both species closely correlate with population doublings and GSIS responses. Human-NIs establish normoglycemia in STZ diabetic NOD/SCID mice. Based on these results, an estimated 600 therapeutic doses can be generated from a single islet donor. Conclusion: human-NIs created from diverse, “non-clinical grade” donors have the potential to greatly expand patient access to this curative therapy, facilitated by biotherapy-specific adjustments in the current scoring systems. Disclosure C. Westenfelder: Consultant; Self; SymbioCellTech. S. S. Chowdhury: Employee; Self; SymbioCellTech. A. Gooch: Employee; Self; SymbioCellTech.

1239-P: Evidence of Altered Alpha- and Beta-Cell Lysosomes prior to Onset of Type 1 Diabetes

Autophagic degradation is a dynamic recycling process that contributes to cellular homeostasis and mitigation of cellular stress. While there are multiple forms of -phagic degradation processes, a common endpoint is the final degradation step in the lumen of the acidic lysosomes. Post-mitotic cells, including beta cells, can accumulate tertiary lysosomes called telolysosomes which contain oxidized proteins and lipids that are not completely digested. We recently found that autophagy is impaired in the beta cells of individuals with T1D and also in islets of diabetic NOD mice. We also observed a significant increase in the number of autophagosomes, and telolysosomes in the beta cells of autoantibody positive organ donors when compared to nondiabetic organ donors. We now also find a 1.5-fold increase in the number of telolysosomes in the alpha cells of autoantibody positive donors when compared to nondiabetic organ donors (p=0.03). This was mirrored by our previous observation of globally increased lysosome numbers across the islets of pre-diabetic NOD mice, suggesting that these lysosomes may be dysfunctional. To test this, we performed a lysosomal enzyme activity assay to analyze the lysosomal cysteine protease Cathepsin H (a T1D susceptibility locus). Interestingly, we found that the activity of Cathepsin H was increased by ~2-fold in islets of pre-diabetic NOD mice when compared to NOR controls (p=0.02). Taken together, these data suggest a compensatory hyperactivation of the lysosomal enzyme Cathepsin H in the islets of pre-diabetic NOD mice, potentially as a mechanism to rid the cell of degradation-resistant oxidized proteins and lipids whose accumulation in lysosomes might lead to the presence of telolysosomes. Disclosure C. Muralidharan: None. J. Crowder: None. A. K. Linnemann: None. Funding Network for Pancreatic Organ Donors with Diabetes (nPOD; RRID:SCR_014641); JDRF (nPOD: 5-SRA-2018-557-Q-R); The Leona M. and Harry B. Helmsley Charitable Trust (2018PG-T1D053)

Erratum. Microencapsulated G3C Hybridoma Cell Graft Delays the Onset of Spontaneous Diabetes in NOD Mice by an Expansion of Gitr+ Treg Cells. Diabetes 2020;69:965-980.

In the legend for Fig. 3 B of the above-cited article, the term “splenoctyes” was inadvertently used to describe the …

Insulin Receptor-Expressing T Cells Appear in Individuals at Risk for Type 1 Diabetes and Can Move into the Pancreas in C57BL/6 Transgenic Mice.

Insulin receptor (IR) expression on the T cell surface can indicate an activated state; however, the IR is also chemotactic, enabling T cells with high IR expression to physically move toward insulin. In humans with type 1 diabetes (T1D) and the NOD mouse model, a T cell-mediated autoimmune destruction of insulin-producing pancreatic β cells occurs. In previous work, when purified IR+ and IR- T cells were sorted from diabetic NOD mice and transferred into irradiated nondiabetic NOD mice, only those that received IR+ T cells developed insulitis and diabetes. In this study, peripheral blood samples from individuals with T1D (new onset to 14 y of duration), relatives at high-risk for T1D, defined by positivity for islet autoantibodies, and healthy controls were examined for frequency of IR+ T cells. High-risk individuals had significantly higher numbers of IR+ T cells as compared with those with T1D (p < 0.01) and controls (p < 0.001); however, the percentage of IR+ T cells in circulation did not differ significantly between T1D and control subjects. With the hypothesis that IR+ T cells traffic to the pancreas in T1D, we developed a (to our knowledge) novel mouse model exhibiting a FLAG-tagged mouse IR on T cells on the C57BL/6 background, which is not susceptible to developing T1D. Interestingly, these C57BL/6-CD3FLAGmIR/mfm mice showed evidence of increased IR+ T cell trafficking into the islets compared with C57BL/6 controls (p < 0.001). This transgenic animal model provides a (to our knowledge) novel platform for investigating the influence of IR expression on T cell trafficking and the development of insulitis.

Natural Protection From Type 1 Diabetes in NOD Mice Is Characterized by a Unique Pancreatic Islet Phenotype.

The NOD mouse develops spontaneous type 1 diabetes, with some features of disease that are very similar to the human disease. However, a proportion of NOD mice are naturally protected from developing diabetes, and currently, studies characterizing this cohort are very limited. Here, using both immunofluorescence and multiparameter flow cytometry, we focus on the pancreatic islet morphology and immune infiltrate observed in naturally protected NOD mice. We show that naturally protected NOD mice are characterized by an increased frequency of insulin-containing, smaller-sized, pancreatic islets. Although mice remain diabetes free, florid immune infiltrate remains. However, this immune infiltrate is skewed toward a regulatory phenotype in both T- and B-cell compartments. Pancreatic islets have an increased frequency of IL-10-producing B cells and associated cell surface markers. Resident memory CD69+CD8+ T cells show a significant shift toward reduced CD103 expression, while CD4+ T cells have increased FoxP3+CTLA4+ expression. These data indicate that naturally protected NOD mice have a unique islet signature and provide new insight into regulatory mechanisms within pancreatic islets.

A Versatile Microencapsulation Platform for Hyaluronic Acid and Polyethylene Glycol.

Cell microencapsulation is a rapidly expanding field with broad potential for stem cell therapies and tissue engineering research. Traditional alginate microspheres suffer from poor biocompatibility, and microencapsulation of more advanced hydrogels is challenging due to their slower gelation rates. We have developed a novel, noncytotoxic, nonemulsion-based method to produce hydrogel microspheres compatible with a wide variety of materials, called core-shell spherification (CSS). Fabrication of microspheres by CSS derived from two slow-hardening hydrogels, hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), was characterized. HA microspheres were manufactured with two different crosslinking methods: thiolation and methacrylation. Microspheres of methacrylated HA (MeHA) had the greatest swelling ratio, the largest average diameter, and the lowest diffusion barrier. In contrast, PEGDA microspheres had the smallest diameters, the lowest swelling ratio, and the highest diffusion barrier, while microspheres of thiolated HA had characteristics that were in between the other two groups. To test the ability of the hydrogels to protect cells, while promoting function, diabetic NOD mice received intraperitoneal injections of PEGDA or MeHA microencapsulated canine islets. PEGDA microspheres reversed diabetes for the length of the study (up to 16 weeks). In contrast, islets encapsulated in MeHA microspheres at the same dose restored normoglycemia, but only transiently (3–4 weeks). Nonencapsulated canine islet transplanted at the same dose did not restore normoglycemia for any length of time. In conclusion, CSS provides a nontoxic microencapsulation procedure compatible with various hydrogel types.Impact statementCore-shell spherification, described here for the first time, is a versatile method of coating cells to protect them following transplantation. Before the invention of this technique, only instantaneously hardening hydrogels, like alginate, could be used to encapsulate cells. With this new technology, biocompatible hydrogels can now be used for encapsulation without harsh emulsion chemicals. The technique involves a temporary shell of alginate around the slow-hardening gel that provides time for crosslinking to occur. Subsequently, the alginate shell is easily removed, leaving the cells in a protective microsphere with a higher surface area for diffusion than large encapsulating devices.

Impaired Activated/Memory Regulatory T Cell Clonal Expansion Instigates Diabetes in NOD Mice.

Regulatory T cell (Treg) insufficiency licenses the destruction of insulin-producing pancreatic β-cells by autoreactive effector T cells (Teffs), causing spontaneous autoimmune diabetes in NOD mice. We investigated the contribution to diabetes of the T-cell receptor (TCR) repertoires of naive regulatory T cells (nTregs), activated/memory Tregs (amTregs), and CD4+ Teffs from prediabetic NOD mice and normal C57BL/6 (B6) mice. NOD mice amTreg and Teff repertoire diversity was unexpectedly higher than that of B6 mice. This was due to the presence of highly expanded clonotypes in B6 amTregs and Teffs that were largely lost in their NOD counterparts. Interleukin-2 (IL-2) administration to NOD mice restored such amTreg clonotype expansions and prevented diabetes development. In contrast, IL-2 administration only led to few or no clonotype expansions in nTregs and Teffs, respectively. Noteworthily, IL-2-expanded amTreg and nTreg clonotypes were markedly enriched in islet-antigen specific TCRs. Altogether, our results highlight the link between a reduced clonotype expansion within the activated Treg repertoire and the development of an autoimmune disease. They also indicate that the repertoire of amTregs is amenable to rejuvenation by IL-2.

Preventive effect of fermented brown rice and rice bran on spontaneous type 1 diabetes in NOD female mice

Abstract Consumption of brown rice and rice bran fermented with Aspergillus oryzae (FBRA) suppresses spontaneously occurring diabetes in female NOD mouse. While control diet-fed mice showed glucosuria and hyperglycemia at around 20 week of age and the ratio reached to 57% at 30 weeks of age, the ratio did not increase in the 0.5% FBRA-containing diet-fed group. The FBRA-fed group at 30 weeks of age kept higher ratio of intact islets and showed significantly lower insulitis score compared to the control diet group, with dose-dependency from 0.25% to 0.5% dietary concentration of FBRA. The percentage of diabetic mice was significantly lower at 24 weeks of age as compared to the control group (p = 0.01, log rank test). These results indicate that the suppressive effects of dietary administration of 0.5% FBRA in delaying the spontaneous onset of diabetes in NOD mice is probably achieved by maintaining the number of intact islets.

GABAB-Receptor Agonist-Based Immunotherapy for Type 1 Diabetes in NOD Mice.

Some immune system cells express type A and/or type B γ-aminobutyric acid receptors (GABAA-Rs and/or GABAB-Rs). Treatment with GABA, which activates both GABAA-Rs and GABAB-Rs), and/or a GABAA-R-specific agonist inhibits disease progression in mouse models of type 1 diabetes (T1D), multiple sclerosis, rheumatoid arthritis, and COVID-19. Little is known about the clinical potential of specifically modulating GABAB-Rs. Here, we tested lesogaberan, a peripherally restricted GABAB-R agonist, as an interventive therapy in diabetic NOD mice. Lesogaberan treatment temporarily restored normoglycemia in most newly diabetic NOD mice. Combined treatment with a suboptimal dose of lesogaberan and proinsulin/alum immunization in newly diabetic NOD mice or a low-dose anti-CD3 in severely hyperglycemic NOD mice greatly increased T1D remission rates relative to each monotherapy. Mice receiving combined lesogaberan and anti-CD3 displayed improved glucose tolerance and, unlike mice that received anti-CD3 alone, had some islets with many insulin+ cells, suggesting that lesogaberan helped to rapidly inhibit β-cell destruction. Hence, GABAB-R-specific agonists may provide adjunct therapies for T1D. Finally, the analysis of microarray and RNA-Seq databases suggested that the expression of GABAB-Rs and GABAA-Rs, as well as GABA production/secretion-related genes, may be a more common feature of immune cells than currently recognized.

Oligonucleotide IMT504 Improves Glucose Metabolism and Controls Immune Cell Mediators in Female Diabetic NOD Mice.

Type 1 diabetes occurs as a consequence of progressive autoimmune destruction of beta cells. A potential treatment for this disease should address the immune attack on beta cells and their preservation/regeneration. The objective of this study was to elucidate whether the immunomodulatory synthetic oligonucleotide IMT504 was able to ameliorate diabetes in NOD mice and to provide further understanding of its mechanism of action. We found that IMT504 restores glucose homeostasis in a diabetes mouse model similar to human type 1 diabetes, by regulating expression of immune modulatory factors and improving beta cell function. IMT504 treatment markedly improved fasting glycemia, insulinemia, and homeostatic model assessment of beta cell function (HOMA-Beta cell) index. Moreover, this treatment increased islet number and decreased apoptosis, insulitis, and CD45+ pancreas-infiltrating leukocytes. In a long-term treatment, we observed improvement of glucose metabolism up to 9 days after IMT504 cessation and increased survival after 15 days of the last IMT504 injection. We postulate that interleukin (IL)-12B (p40), possibly acting as a homodimer, and Galectin-3 (Gal-3) may function as mediators of this immunomodulatory action. Overall, these results validate the therapeutic activity of IMT504 as a promising drug for type 1 diabetes and suggest possible downstream mediators of its immunomodulatory effect.

Targeting transcriptional coregulator OCA-B/Pou2af1 blocks activated autoreactive T cells in the pancreas and type 1 diabetes.

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

InsB9-23 Gene Transfer to Hepatocyte-Based Combined Therapy Abrogates Recurrence of Type 1 Diabetes After Islet Transplantation.

The induction of antigen (Ag)-specific tolerance represents a therapeutic option for autoimmune diabetes. We demonstrated that administration of a lentiviral vector enabling expression of insulin B chain 9-23 (InsB9-23) (LV.InsB) in hepatocytes arrests β-cell destruction in prediabetic NOD mice by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). LV.InsB in combination with a suboptimal dose of anti-CD3 monoclonal antibody (combined therapy [CT], 1 × 5 μg [CT5]) reverts diabetes and prevents recurrence of autoimmunity after islet transplantation in ∼50% of NOD mice. We investigated whether CT optimization could lead to abrogation of recurrence of autoimmunity. Therefore, alloislets were transplanted after optimized CT tolerogenic conditioning (1 × 25 μg [CT25]). Diabetic NOD mice conditioned with CT25 when glycemia was <500 mg/dL remained normoglycemic for 100 days after alloislet transplantation and displayed reduced insulitis, but independently from the graft. Accordingly, cured mice showed T-cell unresponsiveness to InsB9-23 stimulation and increased Treg frequency in islet infiltration and pancreatic lymph nodes. Additional studies revealed a complex mechanism of Ag-specific immune regulation driven by CT25, in which both Tregs and PDL1 costimulation cooperate to control diabetogenic cells, while transplanted islets play a crucial role, although transient, recruiting diabetogenic cells. Therefore, CT25 before alloislet transplantation represents an Ag-specific immunotherapy to resolve autoimmune diabetes in the presence of residual endogenous β-cell mass.

CD70 Inversely Regulates Regulatory T Cells and Invariant NKT Cells and Modulates Type 1 Diabetes in NOD Mice

The CD27-CD70 costimulatory pathway is essential for the full activation of T cells, but some studies show that blocking this pathway exacerbates certain autoimmune disorders. In this study, we report on the impact of CD27-CD70 signaling on disease progression in the NOD mouse model of type 1 diabetes (T1D). Specifically, our data demonstrate that CD70 ablation alters thymocyte selection and increases circulating T cell levels. CD27 signaling was particularly important for the thymic development and peripheral homeostasis of Foxp3+Helios+ regulatory T cells, which likely accounts for our finding that CD70-deficient NOD mice develop more-aggressive T1D onset. Interestingly, we found that CD27 signaling suppresses the thymic development and effector functions of T1D-protective invariant NKT cells. Thus, rather than providing costimulatory signals, the CD27-CD70 axis may represent a coinhibitory pathway for this immunoregulatory T cell population. Moreover, we showed that a CD27 agonist Ab reversed the effects of CD70 ablation, indicating that the phenotypes observed in CD70-deficient mice were likely due to a lack of CD27 signaling. Collectively, our results demonstrate that the CD27-CD70 costimulatory pathway regulates the differentiation program of multiple T cell subsets involved in T1D development and may be subject to therapeutic targeting.

Elucidating the Role of Ezh2 in Tolerogenic Function of NOD Bone Marrow-Derived Dendritic Cells Expressing Constitutively Active Stat5b.

Tolerogenic dendritic cells (toDCs) are crucial to controlling the development of autoreactive T cell responses and the prevention of autoimmunity. We have reported that NOD.CD11cStat5b-CA transgenic mice expressing a constitutively active (CA) form of Stat5b under the control of a CD11c promoter are protected from diabetes and that Stat5b-CA-expressing DCs are tolerogenic and halt ongoing diabetes in NOD mice. However, the molecular mechanisms by which Stat5b-CA modulates DC tolerogenic function are not fully understood. Here, we used bone marrow-derived DCs (BMDCs) from NOD.CD11cStat5b-CA transgenic mice (Stat5b-CA.BMDCs) and found that Stat5b-CA.BMDCs displayed high levels of MHC class II, CD80, CD86, PD-L1, and PD-L2 and produced elevated amounts of TGFβ but low amounts of TNFα and IL-23. Stat5b-CA.BMDCs upregulated Irf4 and downregulated Irf8 genes and protein expression and promoted CD11c+CD11b+ DC2 subset differentiation. Interestingly, we found that the histone methyltransferase Ezh2 and Stat5b-CA bound gamma-interferon activated site (GAS) sequences in the Irf8 enhancer IRF8 transcription, whereas Stat5b but not Ezh2 bound GAS sequences in the Irf4 promoter to enhance IRF4 transcription. Injection of Stat5b-CA.BMDCs into prediabetic NOD mice halted progression of islet inflammation and protected against diabetes. Importantly, inhibition of Ezh2 in tolerogenic Stat5b-CA.BMDCs reduced their ability to prevent diabetes development in NOD recipient mice. Taken together, our data suggest that the active form of Stat5b induces tolerogenic DC function by modulating IRF4 and IRF8 expression through recruitment of Ezh2 and highlight the fundamental role of Ezh2 in Stat5b-mediated induction of tolerogenic DC function.

Persistent IL-2 Receptor Signaling by IL-2/CD25 Fusion Protein Controls Diabetes in NOD Mice by Multiple Mechanisms.

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.

Microencapsulated islet allografts in diabetic NOD mice and nonhuman primates.

Our goal was to assess the efficacy of encapsulated allogeneic islets transplanted in diabetic NOD mice and streptozotocin (STZ)-diabetic nonhuman primates (NHPs). Murine or NHP islets were microencapsulated and transplanted in non-immunosuppressed mice or NHPs given clinically-acceptable immunosuppressive regimens, respectively. Two NHPs were treated with autologous mesenchymal stem cells (MSCs) and peri-transplant oxygen therapy. Different transplant sites (intraperitoneal [i.p.], omental pouch, omental surface, and bursa omentalis) were tested in separate NHPs. Graft function was monitored by exogenous insulin requirements, fasting blood glucose levels, glucose tolerance tests, percent hemoglobin A1c (% HbA1c), and C-peptide levels. In vitro assessment of grafts included histology, immunohistochemistry, and viability staining; host immune responses were characterized by flow cytometry and cytokine/chemokine multiplex ELISAS. Microencapsulated islet allografts functioned long-term i.p. in diabetic NOD mice without immunosuppression, but for a relatively short time in immunosuppressed NHPs. In the NHPs, encapsulated allo-islets initially reduced hyperglycemia, decreased exogenous insulin requirements, elevated C-peptide levels, and lowered % HbA1c in plasma, but graft function diminished with time, regardless of transplant site. At necropsy, microcapsules were intact and non-fibrotic, but many islets exhibited volume loss, central necrosis and endogenous markers of hypoxia. Animals receiving supplemental oxygen and autologous MSCs showed improved graft function for a longer post-transplant period. In diabetic NHPs and mice, cell-free microcapsules did not elicit a fibrotic response. The evidence suggested that hypoxia was a major factor for damage to encapsulated islets in vivo. To achieve long-term function, new approaches must be developed to increase the oxygen supply to microencapsulated islets and/or identify donor insulin-secreting cells which can tolerate hypoxia.