NOD Model Research Articles

CD4+CD25+Foxp3+ T regulatory cell stimulation by high dose gp96 is costimulation and APC independent (131.3)

Abstract The immunogenicity of heat shock protein-peptide complexes (HSPs) against tumors and pathogens is initiated when the HSP engages its receptors, CD91 and maybe others, on antigen presenting cells (APCs). This allows APCs to obtain antigen in the form of peptides, and acquire signals that indicate for them to mature, express co-stimulation and migrate (1). However, high doses of HSPs tend to negate ongoing T cell responses (2), an effect shown to involve generation of a CD4+ suppressor response (3). This immune suppression has been demonstrated to protect mice from autoimmune conditions including experimental autoimmune encephalomyelitis (EAE) and the NOD model of diabetes (3). Here we provide evidence that highly enriched CD4+CD25+Foxp3+ T regulatory cells proliferate up to 3 fold more in the presence of the HSP gp96 and IL-2 compared to IL-2 alone. LPS or albumin were used as controls and did not support proliferation. This effect is APC and co-stimulation independent and may occur via CD91 which is expressed on T regulatory cells or by another receptor. Overall CD4+CD25+Foxp3+ T regulatory cells are stimulated directly by HSPs and may be the mechanism by which high doses of HSPs induce a suppressor response. This study was supported by NIH grant CA84479-02 and by a research agreement with Antigenics Inc.

CD8 + T lymphocytes specific for glutamic acid decarboxylase 90–98 epitope mediate diabetes in NOD SCID mouse

During the past decade, glutamic acid decarboxylase (GAD) has been considered a crucial β-cell autoantigen involved in type 1 diabetes in the NOD mouse and human. Recently, the etiological role of GAD has remained controversy. In the NOD mouse, some previous studies argued in favor of a regulatory role for GAD-specific CD4 + T cells, and no diabetogenic CD8 + T cells specific for GAD have been identified so far, discrediting the importance of GAD in β-cell injury. Here, we identified, in the NOD model, a relevant GAD CD8 + T cell epitope (GAD 90–98) using immunization with a plasmid encoding GAD, a protocol relying on in vivo processing of peptides from the autoantigenic protein. In pancreatic lymph nodes of naïve female NOD mice, CD8 + T lymphocytes recognizing GAD 90–98 peptide were detected during the initial phase of invasive insulitis (between 4 and 8 weeks of age), suggesting an important role for these cells in the first stage of the disease. GAD 90–98 specific CD8 + lymphocytes lysed efficiently islet cells in vitro and transferred diabetes into NOD SCID mice (100%). Finally, diabetes was accelerated greatly in 3-week-old female NOD mice injected i.p. with GAD 90–98, strengthening the role of GAD-specific CTLs in diabetes pathogenesis.

Sex-Specific Effect of Insulin-Dependent Diabetes 4 on Regulation of Diabetes Pathogenesis in the Nonobese Diabetic Mouse

Many human autoimmune diseases are more frequent in females than males, and their clinical severity is affected by sex hormone levels. A strong female bias is also observed in the NOD mouse model of type I diabetes (T1D). In both NOD mice and humans, T1D displays complex polygenic inheritance and T cell-mediated autoimmune pathogenesis. The identities of many of the insulin-dependent diabetes (Idd) loci, their influence on specific stages of autoimmune pathogenesis, and sex-specific effects of Idd loci in the NOD model are not well understood. To address these questions, we analyzed cyclophosphamide-accelerated T1D (CY-T1D) that causes disease with high and similar frequencies in male and female NOD mice, but not in diabetes-resistant animals, including the nonobese diabetes-resistant (NOR) strain. In this study we show by genetic linkage analysis of (NOD x NOR) x NOD backcross mice that progression to severe islet inflammation after CY treatment was controlled by the Idd4 and Idd9 loci. Congenic strains on both the NOD and NOR backgrounds confirmed the roles of Idd4 and Idd9 in CY-T1D susceptibility and revealed the contribution of a third locus, Idd5. Importantly, we show that the three loci acted at distinct stages of islet inflammation and disease progression. Among these three loci, Idd4 alleles alone displayed striking sex-specific behavior in CY-accelerated disease. Additional studies will be required to address the question of whether a sex-specific effect of Idd4, observed in this study, is also present in the spontaneous model of the disease with striking female bias.

Extracellular lysosome‐associated membrane protein‐1 (LAMP‐1) mediates autoimmune disease progression in the NOD model of type 1 diabetes

Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.

SYSTEMIC TGF-BETA1 GENE THERAPY INDUCES CD4+CD25+ FOXP3+ REGULATORY T CELLS, RESTORES SELF-TOLERANCE AND FACILITATES ISLET REGENERATION IN OVERTLY DIABETIC NOD MICE

O251* Aims: Type 1 diabetes results from autoaggressive T cell mediated destruction of insulin producing beta cells of the pancreas. Recent data support the idea that restoration of self-tolerance may facilitate islet cell regeneration/recovery. In view of the potent immunoregulatory activity of transforming growth factor beta1 (TGF-β1), we explored the hypothesis that systemic TGF-β1 therapy restores self-tolerance, facilitates islet regeneration and cures autoimmune diabetes. Methods: We designed and constructed a replication deficient adenovirus (Ad) vector encoding the active form of human TGF-β1 and tested the hypothesis that systemic TGF-β1 gene therapy restores self-tolerance and facilitates islet regeneration in the NOD model. Overtly diabetic NOD mice received intravenous injection of Ad.AH.TGF-β1 at a dose of 5x1010 particle units and seven to fourteen days after the injection, the mice were transplanted with 500-600 syngeneic islets under the kidney capsule. Results: Hyperglycemia occurred by day 17 in all untreated NOD mice whereas 50% of syngeneic grafts functioned until day 60 in the NOD mice treated with Ad.AH.TGF-β1. Analysis of retrieved islet grafts showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Staining for IFN-γ was negative whereas TGF-β1 and IL-10, and PD-L1 and PD-L2 all showed heightened expression. Having demonstrated that Ad.AH.TGF-β1 protects syngeneic islet grafts, we investigated whether systemic TGF-β1 gene therapy is associated with islet regeneration in the native pancreas. Thus, we removed the long-term functioning syngeneic islet grafts and examined whether the mice remained normoglycemic; we also removed the endogenous pancreas and examined the status of native islets. Normoglycemia was observed in 66% of the mice despite removal of the syngeneic islet grafts suggesting recovery of endogenous islet function upon treatment with Ad.AH.TGF-β1. Examination of the native pancreas revealed scattered islets, among pancreatic exocrine tissues, with intact islet structures (panel A). There were few intra-islet cellular infiltrates. However, similar to what was found in the syngeneic islet graft, there were well-demarcated lymphocytic infiltrates on the periphery of the islets without invasion of the islets themselves (Panel A. Peri-insulitis but not insulitis). Moreover, the intact islets stained strongly for insulin (Panel B) providing definitive evidence for functional restoration of endogenous beta cells.FigureConclusions: Our data demonstrate that systemic TGF-beta1 gene restores self-tolerance and facilitates islet regeneration in previously diabetic NOD mice. Our studies support the idea that in-vivo expansion of CD4+CD25+ Foxp3+ regulatory T cells is an effective strategy for constraining established islet directed autoimmunity and curing type 1 diabetes.

Cure of prediabetic mice by viral infections involves lymphocyte recruitment along an IP-10 gradient.

Viruses can cause but can also prevent autoimmune disease. This dualism has certainly hampered attempts to establish a causal relationship between viral infections and type 1 diabetes (T1D). To develop a better mechanistic understanding of how viruses can influence the development of autoimmune disease, we exposed prediabetic mice to various viral infections. We used the well-established NOD and transgenic RIP-LCMV models of autoimmune diabetes. In both cases, infection with the lymphocytic choriomeningitis virus (LCMV) completely abrogated the diabetic process. Interestingly, such therapeutic viral infections resulted in a rapid recruitment of T lymphocytes from the islet infiltrate to the pancreatic draining lymph node, where increased apoptosis was occurring. In both models this was associated with a selective and extensive expression of the chemokine IP-10 (CXCL10), which predominantly attracts activated T lymphocytes, in the pancreatic draining lymph node, and in RIP-LCMV mice it depended on the viral antigenic load. In RIP-LCMV mice, blockade of TNF-alpha or IFN-gamma in vivo abolished the prevention of T1D. Thus, virally induced proinflammatory cytokines and chemokines can influence the ongoing autoaggressive process beneficially at the preclinical stage, if produced at the correct location, time, and levels.

Molecular profile of the T cell receptors of regulatory and effector CD4+ T cells recognizing overlapping determinants on glutamic acid decarboxylase (524–543)

Glutamic acid decarboxylase (GAD65) is one of the autoantigens that initiates pathogenic T cell responses against insulin-secreting pancreatic beta cells in Type 1 diabetes (T1D). Previously it was shown that spontaneously arising pathogenic T cell responses in the NOD mouse model are confined to GAD 530–543 (p530). However, regulatory T cell subpopulations, which can prevent diabetes, can also be generated, for example, by immunization with GAD 524–538 (p524) or GAD 524–543. Interestingly, two functionally distinct subpopulations of T cells which recognize overlapping determinants of GAD 524–543, p524 and p530, utilize distinct TCR Vβ families, Vβ4 for pathogenic, and Vβ12 for regulatory T cells. We characterized T cell receptors (TCRs) from each subpopulation of T cells and visualized p524-specific TCR/p524/I-A g7 and p530-specific TCR/p530/I-A g7 complexes via molecular modeling to help us understand, at a molecular level, the in vivo expansion of p524- or p530-specific T cells in the NOD model of T1D. The absolute restriction in Vβ usage but not Vα usage and conserved CDR3β lengths for both T cell subpopulations demonstrates that the beta chains are main contributors in shaping both p524/I-A g7 and p530/I-A g7 restricted TCRs. However, only Vβ4+ T cells but not Vβ12+ T cells contain a common motif (DWG) in CDR3β and may involve all of CDR1β, CDR2β, and CDR3β in the recognition of the C-terminus of p530. These observations imply that the spontaneously arising p530-restricted TCRs may be selected under stringent structural frameworks to bind p530/I-A g7 with high affinity. Thus, the pathogenic p530-specific T cells may arise from a small pool of autoreactive T cells upon breaking tolerance.

Distinct Characteristics and Features of Allogeneic Chimerism in the NOD Mouse Model of Autoimmune Diabetes

The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.

Combining mouse congenic strains and microarray gene expression analyses to study a complex trait: the NOD model of type 1 diabetes.

Combining congenic mapping with microarray expression profiling offers an opportunity to establish functional links between genotype and phenotype for complex traits such as type 1 diabetes (T1D). We used high-density oligonucleotide arrays to measure the relative expression levels of >39,000 genes and ESTs in the NOD mouse (a murine model of T1D and other autoimmune conditions), four NOD-derived diabetes-resistant congenic strains, and two nondiabetic control strains. We developed a simple, yet general, method for measuring differential expression that provides an objective assessment of significance and used it to identify >400 gene expression differences and eight new candidates for the Idd9.1 locus. We also discovered a potential early biomarker for autoimmune hemolytic anemia that is based on different levels of erythrocyte-specific transcripts in the spleen. Overall, however, our results suggest that the dramatic disease protection conferred by six Idd loci (Idd3, Idd5.1, Idd5.2, Idd9.1, Idd9.2, and Idd9.3) cannot be rationalized in terms of global effects on the noninduced immune system. They also illustrate the degree to which regulatory systems appear to be robust to genetic variation. These observations have important implications for the design of future microarray-based studies in T1D and, more generally, for studies that aim to combine genome-wide expression profiling and congenic mapping.

Mucosal administration of IL-10 enhances oral tolerance in autoimmune encephalomyelitis and diabetes.

IL-10 is an immunoregulatory cytokine that can modulate immune processes, inhibiting the expression of inflammatory T(h)1 type responses as well as affecting antigen-presenting cell function. In addition, IL-10 has been shown to be active at mucosal surfaces. In the present study, we examined the role of IL-10 on orally and nasally induced tolerance. Treatment of (PL/J x SJL)F(1) mice with low-dose oral myelin basic protein (MBP) (0.5 mg) and simultaneous oral IL-10 given 3 times reduced the severity and incidence of experimental autoimmune encephalomyelitis (EAE), whereas administration of oral IL-10 alone or MBP alone given in these doses had no effect. Lymphocytes from mice treated orally with MBP and IL-10 proliferated less, and produced decreased amounts of IFN-gamma and IL-2 and increased amounts of IL-10 and transforming growth factor-beta upon in vitro stimulation with MBP. Nasal administration of antigen and IL-10 reduced proliferative responses and IFN-gamma production, increased IL-10 production, and enhanced protection from EAE. In addition, oral IL-10 combined with oral myelin oligodendrocyte glycoprotein (MOG) 35-55 reduced relapses in MOG-induced EAE in the NOD mouse, as well as enhanced the protective effect of oral insulin in the NOD model of diabetes. These results demonstrate that IL-10 is biologically active at mucosal surfaces and can act synergistically to enhance the tolerogenic effects of mucosally administered antigen.

Variable effects of transgenic c-Maf on autoimmune diabetes.

Autoimmune diabetes is associated with T helper 1 polarization, but protection from disease can be provided by the application of T helper 2 (Th2) cytokines. To test whether genetic manipulation of T-cells can provide protective Th2 responses, we developed transgenic mice in which T-cells express the interleukin-4-specific transcription factor c-Maf. When crossed with a transgenic model that combines a class II restricted T-cell receptor specific for influenza hemagglutinin with islet beta-cell expression of hemagglutinin, the c-Maf transgene provided significant protection from spontaneous autoimmunity but not from adoptively transferred diabetes. In a second transgenic model in which islet cells express the lymphocytic choriomeningitis virus nucleoprotein, the virus infection triggers autoimmune diabetes within a few weeks involving both CD4 and CD8 T-cells; here too transgenic c-Maf provided significant protection. Surprisingly, when the c-Maf transgene was backcrossed with the NOD model of spontaneous disease, no protection was evident. Thus, transgenic c-Maf can strongly influence autoimmune disease development in some models, but additional factors, such as background genetic differences, can influence the potency of its effect.

A noncytolytic IL-10/Fc fusion protein prevents diabetes, blocks autoimmunity, and promotes suppressor phenomena in NOD mice.

We have been successful in our efforts to develop a long lived noncytolytic murine IL-10/Fc fusion protein. In the nonobese diabetic mouse (NOD) model, administration of IL-10/Fc from 5 to 25 wk of age completely prevented the occurrence of diabetes. Moreover, these mice remained disease-free long after cessation of IL-10/Fc therapy. Immunohistochemistry studies show that IL-10/Fc treatment inhibits expression of TNF-alpha, proinflammatory cytokine, as well as Th1-type cytokines, IL-2 and IFN-gamma, but promotes expression of IL-4 and IL-10, Th2-type cytokines, by islet-infiltrating leukocytes. In an adoptive transfer model of diabetes in NOD mice, we found that: 1) IL-10/Fc treated hosts bear leukocytes that block expression of diabetes and 2) these leukocytes persisted even 8 wk after cessation of IL-10/Fc treatment. The potent antidiabetogenic effects provided by IL-10/Fc in the NOD model, together with its apparent lack of systemic toxicity, are notable.

β-Cell Destruction May Be a Late Consequence of the Autoimmune Process in Nonobese Diabetic Mice

The NOD mouse is an animal model of IDDM that shows many of the characteristics of human IDDM. It has been proposed that beta-cell destruction in IDDM progresses over time in a linear manner. Recently, we and others have demonstrated that T helper type 1 (Th1) cells have pathogenic roles in the NOD model and proposed that cytokine balances change as the disease progresses. However, it has not been demonstrated how or when the cytokine balances change or how the beta-cell destruction progresses. We have recently demonstrated that the cytokine profiles of CD45RB(low) CD4+ cells correlate either with their pathogenic or with their protective roles in the NOD mouse. To further analyze this apparent correlation between the shift in cytokine level and IDDM, we examined the anti-CD3-induced cytokine profiles of this subset from NOD mice of various ages compared with that from age-matched I-Ak transgenic NOD and BALB/c mice as controls. A significantly higher ratio of anti-CD3-induced interferon-gamma/interleukin-4 was found in diabetic NOD mice (P < 0.0001) but not in age-matched nondiabetic NOD mice. This cytokine ratio did not change significantly until the onset of diabetes in NOD mice. Based upon these results, we propose that IDDM in the NOD mouse progresses as a predominant inflammatory beta-cell dysfunction without actual beta-cell destruction until late in the disease process. This supports the possibility that late-stage immunotherapy may preserve islet beta-cell mass.

Pathogenic and protective roles of CD45RB(low) CD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse.

The adoptive transfer of splenocytes from diabetic NOD mice to NOD-scid/scid (NOD-scid) recipients results in diabetes. This model was used to test the effect of cotransfer of splenocyte subsets from young nondiabetic NOD mice. As shown previously in other NOD models, the CD4+ subset from young nondiabetic mice significantly delayed the onset of diabetes in splenocyte cotransfers (P < 0.001). The data presented here showed that the development of diabetes in NOD-scid recipients correlated with a rapid increase in peripheral CD45RB(low) CD4+ cells. However, the CD45RB(low) subset of CD4+ cells from young nondiabetic mice protected from diabetes transfer in this model. We therefore examined whether CD45RB(low) CD4+ cells from diabetic mice were pathogenic rather than protective. CD45RB(low) CD4+ splenocytes from diabetic NOD mice were transferred along with CD8+ splenocytes from diabetic mice into NOD-scid recipients, and all of the recipients became diabetic within 5 weeks posttransfer. In contrast, no recipients (0 of 10) of CD45RB(high) CD4+ cells along with CD8+ splenocytes from diabetic mice became diabetic within 5 weeks posttransfer (P < 0.001). A correlate for the difference between CD45RB(low) CD4+ cells from diabetic NOD mice and CD45RB(low) CD4+ cells from nondiabetic mice, which showed protective effect in splenocyte cotransfers, was found in cytokine production after stimulation with anti-CD3 antibodies in vitro. CD45RB(low) CD4+ cells from diabetic mice showed a significantly higher ratio (approximately fivefold) of gamma-interferon (IFN-gamma) to interleukin (IL)-4 when compared with CD45RB(low) CD4+ cells from nondiabetic mice (P < 0.001). In conclusion, the function of the CD45RB(low) population of CD4+ cells changes from a protective to a pathogenic one during the development of disease in the NOD mouse. This change in function correlates with cytokine production in vitro; increased IFN-gamma-to-IL-4 ratio is associated with pathogenic potential and occurs coincident with (or after) the onset of diabetes.

Effects of ethinylestradiol on the course of spontaneous autoimmune disease in NZB/W and NOD mice.

Sex hormones affect (auto)immune responses in various ways. Investigations of the effects of estrogens have produced contradictory results. We studied the effects of gender, gonadectomy and of (supra)physiological doses of (the orally active) ethinylestradiol (EE) in two spontaneous autoimmune disease models: the NZB/NZW F1 and NOD mice. In both models we confirmed the female preponderance and the aggravating effects of gonadectomy in males but not in females. The accelerated mortality found in NZB/W mice treated with supraphysiological doses of EE was not associated with increased proteinuria, increased IgG-type anti-DNA levels or increased mononuclear cell infiltrations in the submandibular gland. In contrast, we found a severe reduction in body weight and in the weights of various organs (indications of toxicity), and a decrease rather than an increase in proteinuria and in mononuclear cell infiltrations (indications for autoimmunity). Physiological doses of EE did not significantly affect disease symptoms. In the NOD model a near-physiological, non-toxic dose of EE did not cause consistent changes on immunological disease symptoms either. Therefore, we conclude that the sexual dichotomy in spontaneous autoimmune models is due to protective effects of androgens and that the mortality by estrogens is due to toxic effects rather than accelerated autoimmunity.

Pancreatic transplantation in the spontaneously diabetic rodent

Pancreatic islet transplantation experiments in diabetic BB rats have defined a formidable obstacle to the successful use of therapeutic islet replacement for IDDM: an anti-beta cell autoimmune response directed at the transplanted islet tissue. Importantly this response was subsequently documented to also occur in humans following pancreas transplantation and in NOD mice after islet grafting. The model of recurrent disease has proven to be very useful in probing the pathogenesis of autoimmune diabetes. The fact that the response is rapid and consistently reproducible, and allows easy manipulation of the target beta cells makes for an ideal investigative system. The model of recurrent diabetogenesis in BB rats has determined that islets relocated to either the renal subcapsule or abdominal testis site might be exempt from autoimmune damage. Moreover, pancreatic grafts may be less vulnerable to autoimmunity than isolated islets. Also of potential relevance to the clinical situation are the data from BB rat studies which suggest an MHC restricted autoimmune process. However, since other studies favour an non-MHC specific islet damage the question may ultimately need to be resolved by human studies. In both the NOD and BB models immunosuppressive agents have been identified which successfully eliminate the threat of autoimmune damage to transplanted beta cells. In the NOD the efficacy of anti-L3T4 mAb directly implicates T helper cells in the response. In the BB the effectiveness of mAb OX-8 administration just as clearly defines the participation of either CTL or NK like cells in beta cell damage. Use of either or both types of monoclonal antibody could ultimately find application in human islet or pancreas transplantation.