Autoimmune-prone Nonobese Diabetic Mice Research Articles

In autoimmune-prone NOD mice, altered DC subsets induce aberrant activation of CD4 T cells, yet DCIR2+ DCs can still induce tolerance. (BA15P.218)

Abstract Stimulation of CD4+ T cells by antigen targeted in vivo to dendritic cells (DCs) in the absence of inflammation usually leads to tolerance. However, DCs are altered in autoimmune-prone mice such as non-obese diabetic (NOD) mice, a model for type 1 diabetes. We observed altered DC responses in NOD mice to innate immune stimuli such as CpG, which may alter the ability of these DCs to induce tolerance. In addition, determining which antigen presenting cell populations are capable of inducing tolerance in the context of chronic autoimmunity is critical for antigen-specific treatments for autoimmune disease. We recently reported that in NOD mice, DEC-205+ DCs induce effector responses, not tolerance, in autoreactive CD4+ T cells. We now show that DCIR2+ DCs, distinct from DEC-205+ DCs, can be tolerogenic in NOD mice. Beta cell antigen targeted via chimeric antibody to DCIR2+ DCs delays disease induction in a transfer model of type 1 diabetes. DCIR2+ DC stimulation induces partial deletion of autoreactive CD4+ T cells and remaining cells are unable to produce IFN-γ. These disparate T cell outcomes after targeting antigen to DEC-205+ or DCIR2+ DCs are underpinned by differential gene expression in antigen-specific T cells early after stimulation. In conclusion, we have characterized defects in DCs in NOD mice, and the resulting altered T cell responses with the goal of identifying ways to induce tolerance even in the context of autoimmunity.

Premature CD4+ T Cell Aging and Its Contribution to Lymphopenia-Induced Proliferation of Memory Cells in Autoimmune-Prone Non-Obese Diabetic Mice

Lymphopenia-induced proliferation (LIP), a mechanism to maintain a constant number of T cells in circulation, occurs in both normal aging and autoimmune disease. The incidence of most autoimmune diseases increases with age, and premature CD4+ T cell aging has been reported in several autoimmune diseases. In this study, we tested the hypothesis that premature CD4+ T cell aging can cause autoimmune disease by examining whether premature CD4+ T cell aging exists and causes LIP in our mouse model. Non-obese diabetic (NOD) mice were used because, in addition to Treg defects, the LIP of T cells has been shown to plays a causative role in the development of insulin-dependent diabetes mellitus (IDDM) in these mice. We found that with advancing age, NOD mice exhibited an accelerated decrease in the number of CD4+ T cells due to the loss of naïve cells. This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio. In addition, both the percentage of CD28+ cells in CD4+ T cells and IL-2 production decreased, while the percentage of FAS+CD44+ increased, suggesting that NOD mice exhibit premature CD4+ T cell aging. This process preferentially contributed to LIP of memory cells. Therefore, our results suggest that premature CD4+ T cell aging underlies the development of IDDM in NOD mice. Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4+ T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.

Characterization of antigen-specific CD4+CD25+ regulatory T-cells expanded with IL-2/IL-2mAb complexes in NOD mice (115.9)

Abstract Type-1 diabetes is an autoimmune disease caused by the destruction of insulin-producing pancreatic beta cells by auto-reactive T-cells. It results from a failure of the immune system to maintain tolerance to self-antigens. CD4+CD25+ regulatory T-cells represent one major mechanism by which auto-reactive T-cells are controlled in the periphery. Deficiency in this cell population number or function could tilt the regulatory cell-pathogenic cell balance and lead to type-1 diabetes development. In this study, we describe a new method to expand antigen-specific CD4+ CD25+ regulatory T-cells from autoimmune-prone non-obese diabetic mice. CD4+ CD25+ regulatory T-cells were expanded in vivo using a combination of IL-2, anti-IL-2 antibody, BDC 2.5 peptide, and rapamycin. The expanded CD4+ CD25+ regulatory T-cells expressed a classical phenotype, displayed suppressive functions in vivo, and preliminary data suggest that these cells can delay diabetes development in NOD mice. This new method of peripheral tolerance induction could be used in humans to promote tolerance to islet cell transplants without relying on immunosuppressive drugs.

Combination of Anti-CD4 with Anti-LFA-1 and Anti-CD154 Monoclonal Antibodies Promotes Long-Term Survival and Function of Neonatal Porcine Islet Xenografts in Spontaneously Diabetic NOD Mice

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.

In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.

The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

Long-range histone acetylation of the Ifng gene is an essential feature of T cell differentiation.

Histone acetylation of promoters precedes activation of many genes. In addition, long-range histone acetylation patterns can be established over many kilobases of the chromatin of linked families of genes that are under common transcriptional control. It is not known whether establishment of long-range histone acetylation patterns is limited to gene families or is a common feature of many genes. The Ifng gene is not known to be a member of a gene family but exhibits complex strain-, cell lineage-, and stimulus-dependent regulation. For example, stimulation of naive T cells through their antigen receptor does not initiate Ifng gene transcription. However, stimulation of naive T cells through their antigen and IL-12 receptors initiates differentiation programs that yield effector cells with 100-fold greater rates of transcription of the Ifng gene after stimulation through the antigen receptor. Here, we demonstrate that these differentiation programs establish long-range histone hyperacetylation patterns that extend at least 50 kb in both upstream and downstream directions of the Ifng gene. Establishment of these histone acetylation patterns and Ifng gene expression is relatively IL-12-independent in T cells from autoimmune-prone nonobese diabetic mice. These results indicate that gene expression programs that mediate T cell differentiation are regulated by long-range histone acetylation patterns and that defective control of these patterns may contribute to development of autoimmunity.

Central role of defective apoptosis in autoimmunity.

Lymphocyte development, selection and education represent tightly controlled immune processes that normally prevent autoimmunity. Lymphocyte development likely induces cellular selection through apoptosis to remove potentially autoreactive cells. Dysregulated apoptosis, both interrupted as well as accelerated apoptosis, are now demonstrated as central defects in diverse murine autoimmune disease. In murine models of autoimmune lupus, mutations in cell death receptor Fas (CD95) and its ligand, FasL (CD95 L), have been identified. These errors create a lymphoid system resistant to apoptosis. In contrast, select lymphoid subpopulations of maturing autoimmune prone non-obese diabetic mice have identifiable and pathogenic T cells with both in vivo and in vitro heightened apoptosis after drug interventions. In part, these defects are due to faulty activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) that normally protect against apoptotic death. The genetic basis of interrupted NF-kappaB in pathogenic memory T cells in diabetes is attributable to a developmentally controlled gene defect in an essential subunit of the proteasome. No specific gene in most common forms of human autoimmune disease has yet been identified. Functional assays from diverse laboratories repeatedly demonstrate heightened apoptosis in multiple cellular signaling pathways for cell death, suggesting a common theme in disease causality.

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice.

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

Two-step development of Hashimoto-like thyroiditis in genetically autoimmune prone non-obese diabetic mice: effects of iodine-induced cell necrosis.

The administration of a high iodide dose (HID; 10 micrograms/day) to goitrous mice is known to induce thyroid cell necrosis and inflammation, which, in most strains, is transient. In this study, we analyzed the effects of iodide in autoimmune prone non-obese diabetic (NOD) mice. Control NOD mice fed a standard diet (MID; 1 microgram I/day) or HID did not spontaneously develop thyroiditis. In NOD mice previously made goitrous, HID provoked thyroid cell necrosis and diffuse inflammation within 4 days. Inflammatory cells consisted of MHC-class II+ antigen-presenting cells, CD4+ T helper cells and CD8+ T suppressor/cytotoxic cells. After 96 days of treatment with HID, thyroiditis similar to Hashimoto's disease was obtained in 100% of the animals, with destruction of thyroid follicles, large clusters of T and B cells, and antithyroid antibodies in the plasma. When treating goitrous mice with MID, no cell necrosis was observed and no autoimmune thyroiditis was obtained. The early iodide-induced cell necrosis and inflammation may thus be considered as an important factor in the induction and persistence of autoimmune thyroiditis in individuals carrying a genetic susceptibility to autoimmune disease.