Protect Non-obese Diabetic Mice Research Articles

Regulatory CD4+ T cells redirected against pathogenic CD8+ T cells protect NOD mice from development of autoimmune diabetes.

In this study, we report a novel therapeutic approach redirecting antigen-specific CD4+ T cells recognizing a hybrid insulin peptide (BDC2.5 T cell receptor (TCR) transgenic CD4+ T cells) to attract and suppress islet-specific CD8+ T cells T cells in the non-obese diabetic (NOD) mouse model, and prevent the development of autoimmune diabetes. Purified BDC2.5 CD4+ T cells were induced to differentiate into regulatory T cells (Tregs). The Tregs were then electroporated with mRNA encoding chimeric human β2 microglobulin (hβ2m) covalently linked to insulin B chain amino acids 15-23 (designated INS-eTreg) or islet-specific glucose-6-phosphatase related protein (IGRP) peptide 206-214 (designated IGRP-eTreg). Immunoregulatory functions of these engineered regulatory T cells (eTregs) were tested by in vitro assays and in vivo co-transfer experiments with β-cell-antigen-specific CD8+ T cells in NOD.Scid mice or by adoptive transfer into young, pre-diabetic NOD mice. These eTregs were phenotyped by flow cytometry, and shown to have high expression of FoxP3, as well as other markers of Treg function, including IL-10. They suppressed polyclonal CD4+ T cells and antigen-specific CD8+ T cells (recognizing insulin or IGRP), decreasing proliferation and increasing exhaustion and regulatory markers in vitro. In vivo, eTregs reduced diabetes development in co-transfer experiments with pathogenic antigen-specific CD8+ T cells (INS-CD8+ or IGRP-CD8+ cells) into NOD.Scid mice. Finally, when the eTreg were injected into young NOD mice, they reduced insulitis and prevented spontaneous diabetes in the recipient mice. Our results suggest a novel therapeutic strategy to protect NOD mice by targeting antigen-specific cytotoxic CD8+ T cells, using redirected antigen-specific CD4+ Treg cells, to suppress autoimmune diabetes. This may suggest an innovative therapy for protection of people at risk of development of type 1 diabetes.

β-Cell Cre Expression and Reduced Ins1 Gene Dosage Protect Mice From Type 1 Diabetes.

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type-specific analysis of gene function in preclinical models. In the type 1 diabetes (T1D) research field, multiple lines of nonobese diabetic (NOD) mice have been engineered to express Cre recombinase in pancreatic β cells using insulin promoter fragments, but tissue promiscuity remains a concern. Constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mice on the C57/bl6-J background have high β-cell specificity with no reported off-target effects. We explored whether NOD:Ins1Cre mice could be used to investigate β-cell gene deletion in T1D disease modeling. We studied wild-type (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null (Ins1Cre/Neo) littermates on a NOD background. Female Ins1Neo/WT mice exhibited significant protection from diabetes, with further near-complete protection in Ins1Cre/WT mice. The effects of combined neomycin and Cre knockin in Ins1Neo/Cre mice were not additive to the Cre knockin alone. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced insulitis at age 12 weeks. Collectively, these data confirm previous reports that loss of Ins1 alleles protects NOD mice from diabetes development and demonstrates, for the first time, that Cre itself may have additional protective effects. This has important implications for the experimental design and interpretation of preclinical T1D studies using β-cell-selective Cre in NOD mice.

Role of type l IFN in lacrimal gland-protective Treg dysfunction in nonobese diabetic mouse model of Sjögren’s disease

Abstract Sjogren’s disease is a chronic autoimmune disease of the lacrimal and salivary glands characterized by foci of inflammation in the tissue composed of aggregations of lymphocytes. Male non-obese diabetic (NOD) mice spontaneously develop autoimmunity of the lacrimal glands. Tregs from these mice are dysfunctional. Disruption of the type I interferon (IFN) signaling pathway protects NOD mice from developing lacrimal gland disease. Whether type l IFN signaling drives lacrimal gland protective Treg dysfunction is not known. Here we investigated the role of type l IFN in modulating Treg phenotype and function. Through adoptive transfer studies comparing cells from wild-type or IFNAR1-deficient donor mice, we found that recipients of IFNAR1-deficient cells had fewer foci of inflammation. IFNAR1 expression was correlated with a population of CD44loCD62Lhi Tregs. Co-adoptive transfer of both WT and IFNAR1-deficient cells determined that this altered Treg phenotype was due to intrinsic type l IFN signaling. In vitro studies suggest that Treg phenotype can be shaped by IFNα in a dose dependent manner. Ongoing in vivo studies will interrogate the impact of IFNAR1 expression on Treg function. Type l IFN signaling plays a role in shaping Treg phenotype and may antagonize Treg function. These studies will help define the role of type l IFN signaling in early pathogenesis of Sjögren’s disease and may provide insight into other systemic autoimmune diseases. Supported by grants from NIH (R01EY027731-01A1)

High-fat diet prevents the development of autoimmune diabetes in NOD mice.

AimsType 1 diabetes (T1D) has a strong genetic predisposition and requires an environmental trigger to initiate the beta‐cell autoimmune destruction. The rate of childhood obesity has risen in parallel to the proportion of T1D, suggesting high‐fat diet (HFD)/obesity as potential environmental triggers for autoimmune diabetes. To explore this, non‐obese diabetic (NOD) mice were subjected to HFD and monitored for the development of diabetes, insulitis and beta‐cell stress.Materials and MethodsFour‐week‐old female NOD mice were placed on HFD (HFD‐NOD) or standard chow‐diet. Blood glucose was monitored weekly up to 40 weeks of age, and glucose‐ and insulin‐tolerance tests performed at 4, 10 and 15 weeks. Pancreata and islets were analysed for insulin secretion, beta‐cell mass, inflammation, insulitis and endoplasmic reticulum stress markers. Immune cell levels were measured in islets and spleens. Stool microbiome was analysed at age 4, 8 and 25 weeks.ResultsAt early ages, HFD‐NOD mice showed a significant increase in body weight, glucose intolerance and insulin resistance; but paradoxically, they were protected from developing diabetes. This was accompanied by increased insulin secretion and beta‐cell mass, decreased insulitis, increased splenic T‐regulatory cells and altered stool microbiome.ConclusionsThis study shows that HFD protects NOD mice from autoimmune diabetes and preserves beta‐cell mass and function through alterations in gut microbiome, increased T‐regulatory cells and decreased insulitis. Further studies into the exact mechanism of HFD‐mediated prevention of diabetes in NOD mice could potentially lead to interventions to prevent or delay T1D development in humans.

Replacing murine insulin 1 with human insulin protects NOD mice from diabetes.

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

Norovirus Changes Susceptibility to Type 1 Diabetes by Altering Intestinal Microbiota and Immune Cell Functions.

Environmental factors contribute to Type 1 diabetes (T1D) susceptibility. The gut microbiome, which includes bacteria, viruses, and fungi, contributes to this environmental influence, and can induce immunological changes. The gut viral component of the microbiome, related to T1D has mostly focused on coxsackieviruses and rotavirus. The role of norovirus, another common enteric virus, in susceptibility to T1D was hitherto unknown. Norovirus is highly infectious and encountered by many children. We studied the mouse norovirus 4 (MNV4), related to human noroviruses, in the Non-obese diabetic (NOD) mouse model, to determine its role in influencing susceptibility to T1D. We infected MNV-free NOD mice with MNV4 by exposing the mice to MNV4-positive bedding from an endemically-infected mouse colony to mimic a natural infection. Control MNV-free NOD mice were exposed to MNV-free bedding from the same colony. Interestingly, MNV4 infection protected NOD mice from the development of T1D and was associated with an expansion of Tregs and reduced proinflammatory T cells. We also found MNV4 significantly modified the gut commensal bacteria composition, promoting increased α-diversity and Firmicutes/Bacteroidetes ratio. To elucidate whether T1D protection was directly related to MNV4, or indirectly through modulating gut microbiota, we colonized germ-free (GF) NOD mice with the MNV4-containing or non-MNV4-containing viral filtrate, isolated from filtered fecal material. We found that MNV4 induced significant changes in mucosal immunity, including altered Tuft cell markers, cytokine secretion, antiviral immune signaling markers, and the concentration of mucosal antibodies. Systemically, MNV4-infection altered the immune cells including B cell subsets, macrophages and T cells, and especially induced an increase in Treg number and function. Furthermore, in vitro primary exposure of the norovirus filtrate to naïve splenocytes identified significant increases in the proportion of activated and CTLA4-expressing Tregs. Our data provide novel knowledge that norovirus can protect NOD mice from T1D development by inducing the expansion of Tregs and reducing inflammatory T cells. Our study also highlights the importance of distinguishing the mucosal immunity mediated by bacteria from that by enteric viruses.

Soluble FAS ligand is not required for pancreatic islet inflammation or beta-cell destruction in non-obese diabetic mice

CD8+ T cells play a central role in beta-cell destruction in type 1 diabetes. CD8+ T cells use two main effector pathways to kill target cells, perforin plus granzymes and FAS ligand (FASL). We and others have established that in non-obese diabetic (NOD) mice, perforin is the dominant effector molecule by which autoreactive CD8+ T cells kill beta cells. However, blocking FASL pharmacologically was shown to protect NOD mice from diabetes, indicating that FASL may have some role. FASL can engage with its receptor FAS on target cells either as membrane bound or soluble FASL. It has been shown that membrane-bound FASL is required to stimulate FAS-induced apoptosis in target cells, whereas excessive soluble FASL can induce NF-κB-dependent gene expression and inflammation. Because islet inflammation is a feature of autoimmune diabetes, we tested whether soluble FASL could be important in disease pathogenesis independent of its cell death function. We generated NOD mice deficient in soluble FASL, while maintaining expression of membrane-bound FASL due to a mutation in the FASL sequence required for cleavage by metalloproteinase. NOD mice lacking soluble FASL had normal numbers of lymphocytes in their spleen and thymus. Soluble FASL deficient NOD mice had similar islet inflammation as wild-type NOD mice and were not protected from diabetes. Our data indicate that soluble FASL is not required in development of autoimmune diabetes.

Glycated Whey Proteins Protect NOD Mice against Type 1 Diabetes by Increasing Anti-Inflammatory Responses and Decreasing Autoreactivity to Self-Antigens.

Our previous studies suggested that early glycation products (EGPs) generated in the first step of Maillard reaction/glycation were anti-inflammatory. The objectives of the present study were to determine the effects of EGPs derived from the whey protein isolate-glucose system on type 1 diabetes (T1D), and the underlying immunological mechanisms. In non-obese diabetic (NOD) mice, EGPs at the physiological dose of 600 mg/kg/day increased glucose metabolism, decreased non-fasting blood glucose levels and T1D incidence, decreased insulin resistance, and decreased the pancreatic immune infiltration. The protective effects were accompanied with decreases in CD4-CD8+ thymocytes, CD8+ T cells and serum insulin autoantibody levels, and increases in splenic CD4+CD25+ T cells, macrophage M2/M1 ratio and serum IL-10 level. However, similar treatment with EGPs produced minimal effect on the multiple low-dose streptozotocin-induced hyperglycemia. In conclusion, EGPs protected NOD mice against T1D via increasing anti-inflammatory immune responses and decreasing autoreactivity to self-antigens.

Complex dietary polysaccharide modulates gut immune function and microbiota, and promotes protection from autoimmune diabetes.

The dietary supplement and prebiotic values of β-glucan-rich products have been widely recognized and dietary approaches for modulating autoimmunity have been increasingly explored, we assess the impact of oral administration of high-purity yeast β-glucan (YBG) on gut immune function, microbiota and type 1 diabetes (T1D) using mouse models. Oral administration of this non-digestible complex polysaccharide caused a dectin-1-dependent immune response involving increased expression of interleukin-10 (IL-10), retinaldehyde dehydrogenase (Raldh) and pro-inflammatory cytokines in the gut mucosa. YBG-exposed intestinal dendritic cells induced/expanded primarily Foxp3+ , IL-10+ and IL-17+ T cells, ex vivo. Importantly, prolonged oral administration of low-dose YBG at pre-diabetic stage suppressed insulitis and significantly delayed the appearance of T1D in non-obese diabetic (NOD) mice. Further, prolonged treatment with YBG showed increased Foxp3+ T-cell frequencies, and a significant change in the gut microbiota, particularly an increase in the abundance of Bacteroidetes and a decrease in the Firmicute members. Oral administration of YBG, together with Raldh-substrate and β-cell antigen, resulted in better protection of NOD mice from T1D. These observations suggest that YBG not only has a prebiotic property, but also an oral tolerogenic-adjuvant-like effect, and these features could be exploited for modulating autoimmunity in T1D.

Lymphocyte-Derived Exosomal MicroRNAs Promote Pancreatic β Cell Death and May Contribute to Type 1 Diabetes Development

Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the β cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to β cells favoring apoptosis. Inactivation of these miRNAs in recipient β cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved inchemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in β cells. The induction of these genes may promote the recruitment of immune cells and exacerbate β cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.

TRIF deficiency protects non-obese diabetic mice from type 1 diabetes by modulating the gut microbiota and dendritic cells

The incidence of type 1 diabetes (T1D) is determined by both genetic and environmental factors. In recent years, the gut microbiota have been identified to be an important environmental factor that could modify diabetes susceptibility. We have previously shown that Myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein downstream of most innate immune Toll-like receptor (TLR) signaling, is important for mediating diabetes susceptibility in the non-obese diabetic (NOD) mouse model of human T1D. Here we report the role of TIR-domain-containing adapter-inducing interferon-β (TRIF) in T1D development, as TRIF is an important adaptor protein downstream of TLR3 and TLR4 signaling. We found that TRIF-deficient (TRIF−/-) NOD mice were protected from development of diabetes, but only when housed with TRIF-deficient (TRIF−/-) NOD mice. When housed with TRIF-sufficient wild type (WT, i.e., TRIF+/+) NOD mice, the mice developed diabetes. We further investigated the gut microbiota as a potential cause for the altered diabetes development. Interestingly, TRIF−/−NOD mice had a different microbiota composition compared to WT NOD mice, only if they were housed with TRIF−/−NOD mice. However, the composition of gut microbiota in the TRIF−/−NOD mice was indistinguishable from WT NOD mice, if they were housed with WT NOD mice. The difference in the gut microbiota in TRIF−/−NOD mice, due to cohousing, accorded with the diabetes development in TRIF−/−NOD mice. Comparing the gut microbiota in TRIF−/- and WT NOD mice, we identified changes in percentage of Sutterella, Rikenella and Turicibacter species. Moreover, bacteria from WT NOD mice induced significantly stronger inflammatory immune responses in vitro compared to those from TRIF−/−NOD mice. Further immunological analysis revealed impaired function of dendritic cells and reduced T cell activation and proliferation in TRIF−/−NOD mice. Our data show that TRIF-deficiency protects NOD mice from diabetes development through alteration of the gut microbiota and reduced immune cell activation; however, that protection is over-ridden upon exposure to WT NOD bacteria. Therefore exposure to different microbiota can modify disease susceptibility determined by genetic factors related to innate immunity.

Transplantation of Fecal Microbiota Shaped by Diet.

Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with protection from disease. We reconstituted germ free (GF) NOD mice with fecal bacteria from protected NOD mice fed with high acetate- and butyrate-yielding diets, to test whether the transferred gut microbiota protect against the development of T1D. GF NOD mice that received a microbiota shaped by high acetate- but not butyrate-yielding diet showed a marked protection against diabetes. This fecal transplantation assay demonstrated the potential for a dietary technology to reshape the gut microbiota that enables specific bacteria to transfer protection against T1D.

Role of TGF-β in Self-Peptide Regulation of Autoimmunity

Transforming growth factor (TGF)-β has been implicated in regulation of the immune system, including autoimmunity. We have found that TGF-β is readily produced by T cells following immunization with self-peptide epitopes that downregulate autoimmune responses in type 1 diabetes (T1D) prone nonobese diabetic (NOD) mice. These include multiple peptide epitopes derived from the islet β-cell antigens GAD65 (GAD65 p202-221, GAD65 p217-236), GAD67 (GAD67 p210-229, GAD67 p225-244), IGRP (IGRP p123-145, IGRP p195-214) and insulin B-chain (Ins. B:9-23) that protected NOD mice from T1D. Immunization of NOD mice with the self-MHC class II I-Ag7 β-chain-derived peptide, I-Aβg7 p54-76 also induced large amounts of TGF-β and also protected these mice from diabetes development. These results indicate that peptides derived from disease related self-antigens and MHC class II molecules primarily induce TGF-β producing regulatory Th3 and Tr1-like cells. TGF-β produced by these cells could enhance the differentiation of induced regulatory iTreg and iTreg17 cells to prevent induction and progression of autoimmune diseases. We therefore suggest that peripheral immune tolerance could be induced and maintained by immunization with self-peptides that induce TGF-β producing T cells.

Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes

Chimeric major histocompatibility complex (MHC) molecules supplemented with Tcell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified Tcells against pathogenic CD8 Tcells. We have shown that β2 microglobulin (β2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15-23 (InsB15-23) to the N terminus of β2m/CD3-ζ, redirected polyclonal CD8 Tcells against pathogenic CD8 Tcells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/β2m/CD3-ζ genes to a reporter Tcell line and purified primary mouse CD8 Tcells. The peptide/β2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter Tcell line transfected with InsB15-23/β2m/CD3-ζ mRNA was activated by an InsB15-23-H-2Kd-specific CD8 Tcell hybrid only when the transfected Tcells expressed H-2Kd. Primary NOD CD8 Tcells expressing either InsB15-23/β2m/CD3-ζ or islet-specific glucose-6-phosphatasecatalytic subunit-related protein, amino acids 206-214 (IGRP206-214)/β2m/CD3-ζ killed their respective autoreactive CD8 Tcell targets invitro. Furthermore, transfer of primary CD8 Tcells transfected with InsB15-23/β2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 Tcells, offering a new approach for the treatment of type 1 diabetes.

Perinatal tolerance to proinsulin is sufficient to prevent autoimmune diabetes.

High-affinity self-reactive thymocytes are purged in the thymus, and residual self-reactive T cells, which are detectable in healthy subjects, are controlled by peripheral tolerance mechanisms. Breakdown in these mechanisms results in autoimmune disease, but antigen-specific therapy to augment natural mechanisms can prevent this. We aimed to determine when antigen-specific therapy is most effective. Islet autoantigens, proinsulin (PI), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were expressed in the antigen-presenting cells (APCs) of autoimmune diabetes-prone nonobese diabetic (NOD) mice in a temporally controlled manner. PI expression from gestation until weaning was sufficient to completely protect NOD mice from diabetes, insulitis, and development of insulin autoantibodies. Insulin-specific T cells were significantly diminished, were naive, and did not express IFN-γ when challenged. This long-lasting effect from a brief period of treatment suggests that autoreactive T cells are not produced subsequently. We tracked IGRP206-214-specific CD8+ T cells in NOD mice expressing IGRP in APCs. When IGRP was expressed only until weaning, IGRP206-214-specific CD8+ T cells were not detected later in life. Thus, anti-islet autoimmunity is determined during early life, and autoreactive T cells are not generated in later life. Bolstering tolerance to islet antigens in the perinatal period is sufficient to impart lasting protection from diabetes.

Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice

Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

A Bispecific Protein Capable of Engaging CTLA-4 and MHCII Protects Non-Obese Diabetic Mice from Autoimmune Diabetes

Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3+ regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9–12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4–13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.

B7-H4.Ig inhibits the development of Type 1 diabetes by regulating Th17 cells in NOD mice

Type 1 diabetes (T1D) is an autoimmune disease characterized by immunological destruction of insulin-producing pancreatic β-cells and subsequent hyperglycemia. The non-obese diabetic (NOD) mouse strain spontaneously develops a disease similar to human T1D and is commonly used as an animal model for studying this disease. We have previously shown that the administration of B7-H4-immunoglobulin fusion protein (B7-H4.Ig), a newly identified T-cell co-inhibitory signaling molecule, blocks the onset of diabetes in NOD mice. However, the mechanism(s) by which B7-H4 protects NOD mice from T1D is not fully understood. IL-17 is a pro-inflammatory cytokine, produced by Th17 cells, that activates T cells and other immune cells to produce a variety of cytokines and chemokines. Increasing evidence has shown that therapeutic agents targeting the IL-17 molecule or directly inhibiting IL-17-producing cells regulate autoimmune diabetes in NOD mice, suggesting that IL-17 is involved in the pathogenesis of this disease. In this study, we investigate whether B7-H4.Ig treatment inhibits the generation of Th17 cells which subsequently decreases IL-17 production and prevents the onset of T1D in NOD mice. Pre-diabetic female NOD mice were injected intraperitoneally with control mouse IgG or B7-H4.Ig starting at 4weeks of age for 12weeks. Our data showed that the frequency of Th17 cells in B7-H4.Ig-treated mice was significantly decreased. In addition, our data showed that B7-H4.Ig-treated mice had decreased levels of pro-inflammatory cytokines and Th17-associated cytokines, and an increased level of the potent Th17 inhibitor IFN-γ. To further investigate the effect of B7-H4.Ig on differentiation of Th17 cells, we co-cultured splenocytes with Th17-polarizing cytokines in the absence or presence of B7-H4.Ig. Our results indicated that splenocytes, under the Th17 driving conditions in the presence of B7-H4.Ig, had significantly decreased the numbers of Th17 cells compared to cells co-cultured in the absence of B7-H4.Ig. Together, this study suggests that blocking the generation of Th17 cells with the administration of B7-H4.Ig effectively inhibits the development of T1D in NOD mice.

Antigen-dependent immunotherapy of non-obese diabetic mice with immature dendritic cells

Immunotherapy can be used to induce immunological tolerance by a number of different protocols. During the last decade the ability to use tolerogenic dendritic cells (DCs) to prevent autoimmunity has received much attention. Many studies have attempted to use immature or semi-mature DCs to induce tolerance in the non-obese diabetic (NOD) mouse model of human type 1 diabetes. However, most studies to date have used protocols in which generation of DCs involved a culture step in fetal bovine serum (FBS)-supplemented medium which may affect tolerance induction in a non-specific fashion. Indeed, several studies have shown that DCs cultured in the presence of FBS will induce a powerful T helper type 2 (Th2) immune response towards FBS-related antigens which can suppress an ongoing immune response. Hence, this may interfere with diabetes development in the NOD mouse by induction of immune deviation rather than by antigen-specific tolerance. In order to test whether antigen-specific tolerance induction by DC therapy is feasible in the NOD mouse, we therefore generated immature DCs using autologous serum [normal mouse serum (NMS)-supplemented cultures] instead of FBS, and we show that these DCs can protect NOD mice from diabetes, if pulsed with insulin-peptide antigens before adoptive transfer. Our data therefore support that DC therapy is able to prevent diabetes in the NOD mouse in an antigen-specific manner.

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)-17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL-17 on the progress of adoptively transferred diabetes. IL-17-producing cells in non-obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co-transfer assays. Unexpectedly, we found that in vivo neutralization of IL-17 did not protect NOD-severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, gammadelta(+) T cells were dominated by IL-17-producing cells and were found to be the major source of IL-17. Interestingly, these IL-17-producing gammadelta T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up-regulating transforming growth factor (TGF)-beta production. Our data suggest that the presence of IL-17 did not increase the chance of the development of diabetes; gammadelta T cells protected NOD mice from diabetes in a TGF-beta-dependent manner, irrespective of their role as major IL-17 producers.