Nonobese Diabetic Background Research Articles

CD4(+) T cells from glutamic acid decarboxylase (GAD)65-specific T cell receptor transgenic mice are not diabetogenic and can delay diabetes transfer.

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

Flexibility of TCR Repertoire and Permissiveness of HLA-DR3 Molecules in Experimental Autoimmune Thyroiditis in Nonobese Diabetic Mice

Experimental autoimmune thyroiditis (EAT) is inducible in genetially susceptible mice by immunization with mouse thyroglobulin (mTg). With susceptibility linked to MHC class II, EAT is useful in studying human leukocyte antigen (HLA) associations with Hashimoto's thyroiditis. In non-obese diabetic (NOD) mice, ∼10% thyroiditis incidence occurs with aging. This potential was exploited to examine the T cell repertoire and HLA association in EAT. Similar to B10.K-Vβcmice with TCRBV genes reduced by ∼70%, mTg-immunized NOD-Vβcmice developed thyroiditis comparable to controls, indicating plasticity of the TCR repertoire for pathogenic epitopes. HLA association was evaluated by introducing HLA-DRA/DRB1*0301 (DR3) transgene into class II-negative NOD mice (Ab0/NOD). Previously, this HLA-DR3 transgene rendered EAT-resistant B10.M and Ab0mice susceptible to both mTg- and hTg-induced EAT. These results are now confirmed. mTg-induced thyroiditis in DR3+Ab0/NOD mice was comparable to that in NOD and DR3−NOD mice, and the proliferative response was stronger. By comparison, NOD mice were only moderately susceptible to hTg-induced EAT. However, thyroiditis was more severe in DR3+Ab0/NOD than in DR3−NOD mice, with no difference in proliferative response to hTg harbouring heterologous epitopes. The confirmed permissiveness of HLA-DR3 molecules on an NOD background for EAT induction by both mTg and hTg supports the importance of this class II gene implicated in some patient studies.

Congenic mapping and functional analysis of a second component of the MHC-linked diabetogenic gene (Idd16)

By using a congenic non-obese diabetic (NOD) mouse strain that possesses a recombinant majorhistocompatibility complex (MHC) from a diabetes-resistant sister strain, the CTS mouse, we havepreviously mapped a second component of the MHC linked susceptibility gene (Idd16) to the <11.8-centiMorgan (cM) segment of chromosome 17 adjacent to, but distinct from class II A and E genes(Idd1). To further localise and characterise Idd16, CTS-derived non-diabetogenic genetic interval,identified in our previous study, was transferred onto the NOD background, and a new recombinantwith recombination breakpoint at 1.3-cM proximal to the previous congenic chromosome was obtainedamong 49 intercrosses of heterozygous mice. The frequency of type 1 diabetes in mice possessing thenew recombinant chromosome was lower than that in the parental NOD strain, as in the case of theprevious congenic strain. The tumor necrosis factor a gene (Tnf), a strong candidate gene for type 1diabetes, is located within the newly localized Idd16 interval. Although our previous study indicatedthat the sequences of exons and exon-intron junctions of Tnf in the NOD mouse were identical to thosein the control mouse, defective expression of TNFα may well contribute to Idd16 effect. We thereforedetermined plasma TNFα levels in NOD and CTS mice. TNFa levels under basal conditions and afterstimulation with interferon gamma (IFN-gamma) and lipopolysaccharide (LPS), however, werecomparable among NOD, CTS and control mice. These data suggest that Idd16 is located in the <10.5-cM segment of chromosome 17 adjacent to Idd1, and that defective expression of TNFa may not beresponsible for Idd16 effect, at least under the condition used in the present study.

Both CD4+T Cells and CD8+T Cells Are Required for Iodine Accelerated Thyroiditis in NOD Mice

The nonobese diabetic (NOD) mouse, a spontaneous animal model for insulin-dependent diabetes mellitus, displays a tendency in common with human diabetic populations to develop autoimmune thyroiditis although incidence and severity of thyroid lesions vary widely among different colonies around the world. A congenic strain of NOD mice bearing I-Akon a NOD background (NOD-H2h4) has recently been derived and displays a much greater tendency to develop thyroiditis and autoantibodies to mouse thyroglobulin (MTg) although it is free of diabetes. Both thyroid infiltrates and autoantibody formation are accelerated and enhanced in NOD-H2h4mice by increased iodine intake. The effect of increased iodine intake on NOD mice themselves has not been directly investigated although a recent study of these animals given high or low doses of iodine showed no follicular destruction unless the mice were first rendered goitrous by iodine deprivation. We found that dietary iodine increased both the incidence and the severity of thyroid lesions in our NOD mice although autoantibodies to MTg were absent. NOD background genes appear to be essential for the development of these lesions, which were maximal after 4 weeks of iodine administration and showed no significant regression when the iodine was stopped. Furthermore, our studies show for the first time that both CD4+and CD8+T cells are necessary for the development of this accelerated but essentially spontaneous murine thyroid disease.

Non-obese Diabetic (NOD) Mice are Genetically Susceptible to Experimental Autoimmune Prostatitis (EAP)

Rodents develop inflammatory, non-infectious, prostatitis upon autoimmuniz-ation with male accessory gland (MAG) extracts in complete Freund’s adjuvant (CFA). Although there appears to be differences among strains, with respect to susceptibility to induction, specific details are not known about the genetic bases of such differences. Because NOD mice have inherited a genetic predisposition to autoimmune lesions affecting, apart from the islets of Langerhans, a large array of secretory glands such as salivary glands, thyroid, parathyroids and adrenal cortex, we selected this strain to assess the influence of inherited genes upon experimentally-induced autoimmune prostatitis (EAP). Indeed, MAG extracts injected into young NOD males in association with CFA cause a severe inflammatory reaction in the prostate, accompanied by a humoral and T cell-mediated response. NOD mice develop a more aggressive form of EAP than Wistar rats, the strain of reference used to establish the model. In NOD mice, disease begins earlier, affects 100% of the animals, does not require boosting and leads to florid infiltrates circumscribed to lateral and dorsal prostatic lobes. Immune mice develop a T cell-mediated response to MAG assessed byin vitroproliferation and accompanied by the release of IFN-γ, whereas IL-4 is not detectable in the same culture super-natants. To assess the influence of the NOD background genes upon EAP susceptibility, we tested C57BL/6.H2g7mice in parallel. NOD mice are considerably more susceptible to EAP induction than congenic C57BL/6.H2g7mice. Both strains demonstrate a detectable humoral and cell-mediated response against MAG, but the histopathological manifestations are considerably more dramatic in NOD than in the C57BL/6.H2g7strain. Our results thus support the notion that NOD mice have background genes which favour severe autoimmune manifestations, irrespective of the target tissue.

Evidence for the presence of insulin-dependent diabetes-associated alleles on the distal part of mouse chromosome 6.

Type 1 diabetes (IDDM) is a complex disorder with multifactorial and polygenic etiology. A genome-wide screen performed in a BC1 cohort of a cross between the nonobese diabetic (NOD) mouse with the diabetes-resistant feral strain PWK detected a major locus contributing to diabetes development on the distal part of chromosome 6. Unlike the majority of other Idd loci identified in intraspecific crosses, susceptibility is associated with the presence of the PWK allele. Genetic linkage analysis of congenic lines segregating PWK chromosome 6 segments in a NOD background confirmed the presence of the Idd locus within this region. The genetic interval defined by analysis of congenic animals showed a peak of significant linkage (P = 0.0005) centered on an approximately 9-cM region lying between D6Mit11 and D6Mit25 genetic markers within distal mouse chromosome 6. [Genetic markers polymorphic between the NOD and PWK strains are available as a supplement at http://www.genome.org]

Development of an I-Ag7-expressing Antigen-presenting Cell Line: Intrinsic Molecular Defect in Compact I-Ag7Dimer Generation

Insulin-dependent diabetes mellitus (IDDM) results from chronic, T-cell dependent, autoimmune destruction of the insulin-producing β-cells in the Langerhans’ islets of the pancreas. Non-obese diabetic (NOD) mice spontaneously develop IDDM that resembles human type I diabetes. The susceptibility to diabetes in the NOD strain is a complex polygenic trait that determines a phenotype of immune alterations. The unique MHC class II molecule expressed by NOD mice (I-Ag7) plays a major role in the development of disease. Recently, it has been reported that I-Ag7molecules generate a lower proportion of compact αβheterodimers, compared to other haplotypes. However, it is not clear whether this reflects an intrinsic defect of this molecule to bind peptide stably or is the result of abnormal processing and/or peptide loading into the I-Ag7molecule. Our aim was to develop and characterize a suitable antigen-presenting cell (APC) that expressed I-Ag7in the context of a non-diabetes-prone antigen processing and presentation machinery. Here, we report the generation of a mouse DAP.3 fibroblast cell line (DAP.3Ag7) that constitutively expresses high levels of I-Ag7. Using DAP.3 cells transfected with I-Ag7or I-Ak, we show that the expression of compact dimers in the same cell type is proportionally less for I-Ag7molecules than for I-Akmolecules, implying an intrinsic defect of the I-Ag7molecule as the cause for the low generation of compact dimers. However, DAP.3Ag7cells are able to process and present antigen, as indicated by I-Ag7-dependent IL-2 production by a GAD67-specific NDO T-cell hybridoma after stimulation with GAD and live, but not fixed, DAP.3Ag7cells. The IL-2 response to GAD when presented by DAP.3Ag7was significantly higher than the response to GAD presented by NOD splenocytes. Based on these data, we conclude that the low generations of compact dimers is an intrinsic feature of I-Ag7molecules and not affected by other genes in the NOD background. The DAP.3Ag7cell line should be a valuable tool with which to dissect the role of the I-Ag7molecule in antigen presentation and T-cell activation in NOD mice, which clearly contributes to the development of IDDM.

Development of insulitis and diabetes in B cell-deficient NOD mice.

Insulin-dependent diabetes mellitus (IDDM) is believed to be an autoimmune disease that results from autoimmune destruction of the insulin-secreting beta-cells of the pancreas. In addition to a lymphocytic infiltration (insulitis) of the islets, patients with IDDM have autoantibodies directed against the components of the islet cells. Several beta-cell proteins have been identified as candidate autoantigens. The non-obese diabetic (NOD) mouse is a murine model for spontaneous IDDM. It is generally accepted that IDDM in patients and NOD mice results from the T lymphocyte-mediated destruction of beta-cells. However, the direct role of B lymphocytes in the disease process has not yet been clarified. To test directly the role of B cells in IDDM, we have generated B cell-deficient NOD mice by backcrossing the microMT-/- B cell 'knockout mice' onto the NOD background. The mice had no evidence of functional B cells as determined by flow cytometry and antibody production. We show that two out of seven of these mice developed insulitis and diabetes. These results suggest that despite an absence of B cells some NOD mice can still develop insulitis and diabetes.

Local production of human IL-6 promotes insulitis but retards the onset of insulin-dependent diabetes mellitus in non-obese diabetic mice

We produced transgenic mice which overexpress human IL-6 in pancreatic beta cells of C57BL/6 x CBA and non-obese diabetic (NOD) mice. Transgenic C57BL/6 x CBA mice back-crossed onto a C57BL/6 background do not develop insulitis or diabetes. In contrast, NOD/F1 transgenic mice develop a lymphocytic infiltrate of pancreatic islets which is not seen in negative littermates. Immunohistochemistry reveals these cells to be predominantly CD4+, CD8+, B220+ cells. Despite the insulitis, these mice do not develop diabetes. Transgenic rat insulin promoter-IL-6 mice were therefore also made on an inbred NOD background. These mice showed no difference in the onset or extent of insulitis when compared with non-transgenic NOD mice and no difference was found in the phenotype of the infiltrating cells. However, transgenic NOD mice had lower average fasting glucose levels and delayed onset of diabetes compared with age and sex matched littermate negative NOD mice. As a consequence, transgenic NOD mice also had longer survival than littermate negative NOD mice. We conclude that the expression of IL-6 by beta cells does not cause insulitis or diabetes in C57BL/6 x CBA mice, but that the interaction of IL-6 and diabetes susceptibility genes causes insulitis in NOD/F1 mice. Since IL-6 delays the onset of diabetes and prolongs survival of NOD mice, it is possible that the protective effect is caused by local IL-6 action on the islets, by the infiltrating lymphocytes or both.

I-E+ nonobese diabetic mice develop insulitis and diabetes.

The development of type I diabetes in the nonobese diabetic (NOD) mouse is under the control of multiple genes, one or more of which is linked to the major histocompatibility complex (MHC). The MHC class II region has been implicated in disease development, with expression of an I-E transgene in NOD mice shown to provide protection from insulitis and diabetes. To examine the effect of expressing an I-E+ or I-E- non-NOD MHC on the NOD background, three I-E+ and three I-E- NOD MHC congenic strains (NOD.H-2i5, NOD.H-2k, and NOD.H-2h2, and NOD.H-2h4, NOD.H-2i7, and NOD.H-2b, respectively) were developed. Of these strains, both I-E+ NOD.H-2h2 and I-E- NOD.H-2h4 mice developed insulitis, but not diabetes. The remaining four congenic strains were free of insulitis and diabetes. These results indicate that in the absence of the NOD MHC, diabetes fails to develop. Each NOD MHC congenic strain was crossed with the NOD strain to produce I-E+ and I-E- F1 mice; these mice thus expressed one dose of the NOD MHC and one dose of a non-NOD MHC on the NOD background. While a single dose of a non-NOD MHC provided a large degree of disease protection to all of the F1 strains, a proportion of I-E+ and I-E- F1 mice aged 5-12 mo developed insulitis and cyclophosphamide-induced diabetes. When I-E+ F1 mice were aged 9-17 mo, spontaneous diabetes developed as well. These data are the first to demonstrate that I-E+ NOD mice develop diabetes, indicating that expression of I-E in NOD mice is not in itself sufficient to prevent insulitis or diabetes. In fact, I-E- F1 strains were no more protected from diabetes than I-E+ F1 strains, suggesting that other non-NOD MHC-linked genes are important in protection from disease. Finally, transfer of NOD bone marrow into irradiated I-E+ F1 recipients resulted in high incidences of diabetes, indicating that expression of non-NOD MHC products in the thymus, in the absence of expression in bone marrow-derived cells, is not sufficient to provide protection from diabetes.