In vivo expression of perforin by CD8+ lymphocytes in autoimmune disease. Studies on spontaneous and adoptively transferred diabetes in nonobese diabetic mice

A potent cytolytic pore-forming protein (perforin or cytolysin) has previously been found to be associated with the cytoplasmic granules of CTL and NK cells. Inasmuch as all previous studies on perforin have been conducted with cultured CTL and NK cell lines, it is not clear whether perforin may play a role in the cytotoxicity mediated by CTL that have been primed in vivo. In this study, we investigated the presence of perforin in pancreata from nonobese diabetic (NOD) mice, which have been studied as a model of autoimmune, insulin-dependent (type I) diabetes mellitus. Whereas adult NOD mice spontaneously develop diabetes, it is possible to induce diabetes in young, irradiated NOD mice by adoptive transfer of splenocytes obtained from diabetic donors. By means of immunohistochemical analysis, we were able to detect perforin Ag in a small subpopulation of CD8+/Thy-1+/asialo GM1-/CD4- lymphocytes in the pancreatic islets of animals undergoing both spontaneous and adoptive transfer-mediated insulitis. Perforin+/CD8+ lymphocytes were found in small clusters and were observed to display the morphology of large granular lymphocytes. These observations show for the first time the presence of perforin-containing CD8+ lymphocytes in tissues of animals undergoing autoimmune disease.     

Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

Activating Fc gamma receptors participate in the development of autoimmune diabetes in NOD mice

Type 1 diabetes mellitus (T1D) in humans is an organ-specific autoimmune disease in which pancreatic islet beta cells are ruptured by autoreactive T cells. NOD mice, the most commonly used animal model of T1D, show early infiltration of leukocytes in the islets (insulitis), resulting in islet destruction and diabetes later. NOD mice produce various islet beta cell-specific autoantibodies, although it remains a subject of debate regarding whether these autoantibodies contribute to the development of T1D. Fc gammaRs are multipotent molecules that play important roles in Ab-mediated regulatory as well as effector functions in autoimmune diseases. To investigate the possible role of Fc gammaRs in NOD mice, we generated several Fc gammaR-less NOD lines, namely FcR common gamma-chain (Fc Rgamma)-deficient (NOD.gamma(-/-)), Fc gammaRIII-deficient (NOD.III(-/-)), Fc gammaRIIB-deficient (NOD.IIB(-/-)), and both Fc Rgamma and Fc gammaRIIB-deficient NOD (NOD.null) mice. In this study, we show significant protection from diabetes in NOD.gamma(-/-), NOD.III(-/-), and NOD.null, but not in NOD.IIB(-/-) mice even with grossly comparable production of autoantibodies among them. Insulitis in NOD.gamma(-/-) mice was also alleviated. Adoptive transfer of bone marrow-derived dendritic cells or NK cells from NOD mice rendered NOD.gamma(-/-) animals more susceptible to diabetes, suggesting a possible scenario in which activating Fc gammaRs on dendritic cells enhance autoantigen presentation leading to the activation of autoreactive T cells, and Fc gammaRIII on NK cells trigger Ab-dependent effector functions and inflammation. These findings highlight the critical roles of activating Fc gammaRs in the development of T1D, and indicate that Fc gammaRs are novel targets for therapies for T1D.     

Intranasal vaccination with proinsulin DNA induces regulatory CD4+ T cells that prevent experimental autoimmune diabetes

Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T(reg)) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ T(reg) that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+ CD25+ T(reg), CD4+ T(reg) induced by proinsulin DNA were both CD25+ and CD25- and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T(reg) and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T(reg), but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.     

Myeloid-derived suppressor cells prevent type 1 diabetes in murine models

Effective immunotherapy for type 1 diabetes (T1D) relies on active induction of peripheral tolerance. Myeloid-derived suppressor cells (MDSCs) play a critical role in suppressing immune responses in various pathologic settings via multiple mechanisms, including expansion of regulatory T cells (Tregs). In this study, we investigated whether MDSCs could act as APCs to induce expansion of Ag-specific Tregs, suppress T cell proliferation, and prevent autoimmune T1D development. We found that MDSC-mediated expansion of Tregs and T cell suppression required MHC-dependent Ag presentation. A murine T1D model was established in INS-HA/RAG(-/-) mice in which animals received CD4-HA-TCR transgenic T cells via adoptive transfer. We found a significant reduction in the incidence of diabetes in recipients receiving MDSC plus HA, but not OVA peptide, leading to 75% diabetes-free mice among the treated animals. To test further whether MDSCs could prevent diabetes onset in NOD mice, nondiabetic NOD/SCID mice were injected with inflammatory T cells from diabetic NOD mice. MDSCs significantly prevented diabetes onset, and 60% of MDSC-treated mice remained diabetes free. The pancreata of treated mice showed significantly lower levels of lymphocyte infiltration in islet and less insulitis compared with that of the control groups. The protective effects of MDSCs might be mediated by inducing anergy in autoreactive T cells and the development of CD4(+)CD25(+)Foxp3(+) Tregs. Thist study demonstrates a remarkable capacity of transferred MDSCs to downregulate Ag-specific autoimmune responses and prevent diabetes onset, suggesting that MDSCs possess great potential as a novel cell-based tolerogenic therapy in the control of T1D and other autoimmune diseases.     

IL-10-deficiency unmasks unique immune system defects and reveals differential regulation of organ-specific autoimmunity in non-obese diabetic mice

Interleukin (IL)-10 is a potent anti-inflammatory cytokine and ablation of IL-10 exacerbates Th1-type autoimmune diseases. Even though type 1 diabetes (T1D) in NOD mice is believed to be Th1-mediated, the incidence and severity of T1D is unaltered in IL-10-deficient NOD mice raised under pathogen-free conditions. We describe for the first time, the outcome of IL-10 deficiency on islet and other organ-specific autoimmunity in NOD mice raised in a conventional facility. IL-10-deficient NOD mice under such conditions were protected from spontaneous as well as cyclophosphamide-induced diabetes, but were susceptible to diabetes induced by adoptive transfer of splenocytes from spontaneously diabetic NOD mice. Whereas the incidence of rectal prolapse was very high in this NOD.IL-10(-/-) mouse colony, IL-10-deficient C57Bl/6 mice raised under similar conditions seldom developed rectal prolapse. While injection of complete Freund's adjuvant (CFA) significantly reduced insulitis, it did not ameliorate colitis in IL-10-deficient NOD mice indicating differential regulation of organ-specific autoimmunity by CFA. Phenotypic characterization of IL-10(-/-) mice revealed a significant increase in splenic macrophage numbers in NOD but not on the B6 background. This was accompanied by a heightened systemic inflammatory cytokine response and mortality following in vivo challenge with a toll-like receptor 9 agonist, CpG-containing DNA.     

Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.     

TNF receptor 1-dependent beta cell toxicity as an effector pathway in autoimmune diabetes

Autoimmune diabetes is characterized by a chronic progressive inflammatory autoimmune reaction that ultimately causes the selective elimination of pancreatic beta cells. To address the question of whether the cell death-inducing cytokines TNF and lymphotoxin alpha are involved in this process, we generated nonobese diabetic (NOD) mice that are deficient for TNF receptor 1 (TNFR1 or TNFRp55). Insulitis developed in these mice similarly to that in normal control NOD mice, but progression to diabetes was completely abrogated. Since this was probably due to the complex immunomodulatory effects of TNF and lymphotoxin alpha signaled via TNFR1 on lymphohemopoietic cells, adoptive transfer experiments with spleen cells from diabetic NOD mice were conducted. It was found that the absence of TNFR1 in recipients delayed diabetes induced by normal control and precluded diabetes induced by perforin-deficient spleen cells. In a CD8+ T cell-mediated model of diabetes, however, diabetes induced by adoptive transfer of TCR transgenic lymphocytic choriomeningitis virus glycoprotein-specific CD8+ T cells was not delayed by the absence of TNFR1 in recipient mice. Together with the described expression patterns of perforin and TNF in the mononuclear islet infiltrates of NOD mice, these results indicate that two diabetogenic effector mechanisms are delivered by distinct cell populations: CD8+ T cells lyse beta cells via perforin-dependent cytotoxicity, whereas CD4+ T cells, macrophages, and dendritic cells contribute to diabetes development via TNFR1-dependent beta cell toxicity.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

Characterization of peripheral regulatory CD4+ T cells that prevent diabetes onset in nonobese diabetic mice

The period that precedes onset of insulin-dependent diabetes mellitus corresponds to an active dynamic state in which pathogenic autoreactive T cells are kept from destroying beta cells by regulatory T cells. In prediabetic nonobese diabetic (NOD) mice, CD4+ splenocytes were shown to prevent diabetes transfer in immunodeficient NOD recipients. We now demonstrate that regulatory splenocytes belong to the CD4+ CD62Lhigh T cell subset that comprises a vast majority of naive cells producing low levels of IL-2 and IFN-gamma and no IL-4 and IL-10 upon in vitro stimulation. Consistently, the inhibition of diabetes transfer was not mediated by IL-4 and IL-10. Regulatory cells homed to the pancreas and modified the migration of diabetogenic to the islets, which resulted in a decreased insulitis severity. The efficiency of CD62L+ T cells was dose dependent, independent of sex and disease prevalence. Protection mechanisms did not involve the CD62L molecule, an observation that may relate to the fact that CD4+ CD62Lhigh lymph node cells were less potent than their splenic counterparts. Regulatory T cells were detectable after weaning and persist until disease onset, sustaining the notion that diabetes is a late and abrupt event. Thus, the CD62L molecule appears as a unique marker that can discriminate diabetogenic (previously shown to be CD62L-) from regulatory T cells. The phenotypic and functional characteristics of protective CD4+ CD62L+ cells suggest they are different from Th2-, Tr1-, and NK T-type cells, reported to be implicated in the control of diabetes in NOD mice, and may represent a new immunoregulatory population.     

NK3-like NK cells are involved in protective effect of polyinosinic-polycytidylic acid on type 1 diabetes in nonobese diabetic mice

Type 1 diabetes in NOD mice is characterized by the uncontrolled Th1 immune responses and deficiency of regulatory or suppressor cells. Previous study has shown that NOD mice treated with polyinosinic-polycytidylic acid (poly(I:C)) have a markedly reduced incidence of diabetes, but the underlying mechanisms remain unclear. In this study, we report that the prevention of diabetes by poly(I:C) is associated with the formation of Th2-enriched environment in spleen and pancreas. We further show that the prevention of diabetes and the formation of Th2-enriched environment depend on the presence of NK cells. Long-term poly(I:C)-treated NK cells exhibit a NK3-like phenotype, and are involved in the induction of Th2 bias of spleen cells in response to islet autoantigens via TGF-beta-dependent manner. Therefore, NK cells mediate the protective effect of poly(I:C) possibly through the promotion of Th2 bias of immune responses. These findings suggest that NK cells can participate in the regulation of autoimmune diabetes.     

NKT cells are required for complete Freund's adjuvant-mediated protection from autoimmune diabetes

Autoimmune diabetes in NOD mice can be prevented by application of Ags derived from Mycobacterium tuberculosis in the form of bacillus Calmette-Guérin or CFA. Disease protection by CFA is associated with a reduction in the numbers of pathogenic β-cell specific, self-reactive CTLs, a phenomenon dependent on the presence and function of NK cells. However, the mechanisms by which NK cells are activated and recruited by heat-killed M. tuberculosis within CFA are unclear. In this study, we report that CFA-mediated NK cell activation and mobilization is dependent on CD1d expression. The administration of M. tuberculosis from CFA results in rapid NKT cell activation and IFN-γ secretion both in vitro and in vivo. CFA-induced NKT cell activation is intact in MyD88(-/-) mice suggesting that the mechanism is independent of TLR signaling. Furthermore, CD1d expression was found to be essential for both M. tuberculosis-triggered NKT cell activation and CFA-mediated protection of NOD mice from diabetes. Collectively, these findings reveal hitherto previously unidentified roles for NKT cells in the adjuvant-promoting effects of CFA on innate and adaptive immunity.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585)

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.     

Suppression of autoimmune diabetes by viral IL-10 gene transfer

Th1 cell activation and cytokine production shift the balance between Th1 and Th2, favoring the up-regulation of proinflammatory activity that leads to destruction of insulin-producing pancreatic beta cells in type 1 diabetes. Th2-type cytokines, such as IL-10, have immune regulatory function. Administration of IL-10, or IL-10 gene transfer, prevents autoimmune diabetes in nonobese diabetic (NOD) mice. However, constant administration of purified rIL-10 is not practical for long-term therapy to prevent diabetes. In this study, we transferred the BCRF-1 gene, an open reading frame in the Epstein-Barr viral genome with remarkable homology to mouse IL-10 (viral IL-10 or vIL-10), by an adeno-associated viral (AAV) vector to NOD mice to attain sustained vIL-10 gene expression. Like endogenous mouse IL-10, vIL-10 has potent immunoregulatory and immunosuppressive functions, but can be specifically distinguished from endogenous mouse IL-10 for monitoring of the transgene expression. A single systemic administration of AAV vIL-10 significantly reduced insulitis and prevented diabetes development in NOD mice. This protective effect correlated with sustained transgene expression and protein production. Moreover, splenocytes from the treated mice blocked diabetes transfer to NOD recipients, suggesting that vIL-10 induces an active suppression of autoimmunity. This study provides evidence to support the possibility of using vIL-10 gene therapy to prevent type 1 diabetes.     

Inhibition of autoimmune diabetes by Fas ligand: the paradox is solved

Previous reports that diabetogenic lymphocytes did not induce diabetes in nonobese diabetic (NOD)-lpr mice suggested the critical role of Fas-Fas ligand (FasL) interaction in pancreatic beta cell apoptosis. However, recent works demonstrated that FasL is not an effector molecule in islet beta cell death. We addressed why diabetes cannot be transferred to NOD-lpr mice despite the nonessential role of Fas in beta cell apoptosis. Lymphocytes from NOD-lpr mice were constitutively expressing FasL. A decrease in the number of FasL+ lymphocytes by neonatal thymectomy facilitated the development of insulitis. Cotransfer of FasL+ lymphocytes from NOD-lpr mice completely abrogated diabetes after adoptive transfer of lymphocytes from diabetic NOD mice. The inhibition of diabetes by cotransferred lymphocytes was reversed by anti-FasL Ab, indicating that FasL on abnormal lymphocytes from NOD-lpr mice was responsible for the inhibition of diabetes transfer. Pretreatment of lymphocytes with soluble FasL (sFasL) also inhibited diabetes transfer. sFasL treatment decreased the number of CD4+CD45RBlow cells and increased the number of propidium iodide-stained cells among CD4+CD45RBlow cells, suggesting that sFasL induces apoptosis on CD4+CD45RBlow "memory" cells. These results resolve the paradox between previous findings and suggest a new role for FasL in the treatment of autoimmune disorders. Our data also suggest that sFasL is involved in the deletion of potentially hazardous peripheral "memory" cells, contrary to previous reports that Fas on unmanipulated peripheral lymphocytes is nonfunctional.     

Fas is detectable on beta cells in accelerated,but not spontaneous,diabetes in nonobese diabetic mice

Fas (CD95) is a potential mechanism of pancreatic beta cell death in type 1 diabetes. beta cells do not constitutively express Fas but it is induced by cytokines. The hypothesis of this study is that Fas expression should be measurable on beta cells for them to be killed by this mechanism. We have previously reported that up to 5% of beta cells isolated from nonobese diabetic (NOD) mice are positive for Fas expression by flow cytometry using autofluorescence to identify beta cells. We have now found that these are not beta cells but contaminating dendritic cells, macrophages, and B lymphocytes. In contrast beta cells isolated from NODscid mice that are recipients of T lymphocytes from diabetic NOD mice express Fas 18-25 days after adoptive transfer but before development of diabetes. Fas expression on beta cells was also observed in BDC2.5, 8.3, and 4.1 TCR-transgenic models of diabetes in which diabetes occurs more rapidly than in unmodified NOD mice. In conclusion, Fas is observed on beta cells in models of diabetes in which rapid beta cell destruction occurs. Its expression is likely to reflect differences in the intraislet cytokine environment compared with the spontaneous model and may indicate a role for this pathway in beta cell destruction in rapidly progressive models.     

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.     

Improved outcomes in NOD mice treated with a novel Th2 cytokine-biasing NKT cell activator

Activation of CD1d-restricted invariant NKT (iNKT) cells by alpha-galactosylceramide (alphaGalCer) significantly suppresses development of diabetes in NOD mice. The mechanisms of this protective effect are complex, involving both Th1 and Th2 cytokines and a network of regulatory cells including tolerogenic dendritic cells. In the current study, we evaluated a newly described synthetic alphaGalCer analog (C20:2) that elicits a Th2-biased cytokine response for its impact on disease progression and immunopathology in NOD mice. Treatment of NOD mice with alphaGalCer C20:2 significantly delayed and reduced the incidence of diabetes. This was associated with significant suppression of the late progression of insulitis, reduced infiltration of islets by autoreactive CD8(+) T cells, and prevention of progressive disease-related changes in relative proportions of different subsets of dendritic cells in the draining pancreatic lymph nodes. Multiple favorable effects observed with alphaGalCer C20:2 were significantly more pronounced than those seen in direct comparisons with a closely related analog of alphaGalCer that stimulated a more mixed pattern of Th1 and Th2 cytokine secretion. Unlike a previously reported Th2-skewing murine iNKT cell agonist, the alphaGalCer C20:2 analog was strongly stimulatory for human iNKT cells and thus warrants further examination as a potential immunomodulatory agent for human disease.     

A mechanism for IL-10-mediated diabetes in the nonobese diabetic (NOD) mouse: ICAM-1 deficiency blocks accelerated diabetes

Neonatal islet-specific expression of IL-10 in nonobese diabetic (NOD) mice accelerates the onset of diabetes, whereas systemic treatment of young NOD mice with IL-10 prevents diabetes. The mechanism for acceleration of diabetes in IL-10-NOD mice is not known. Here we show, by adoptive transfers, that prediabetic or diabetic NOD splenocytes upon encountering IL-10 in the pancreatic islets readily promoted diabetes. This outcome suggests that the compartment of exposure, not the timing, confers proinflammatory effects on this molecule. Moreover, injection of IL-10-deficient NOD splenocytes into transgenic IL-10-NOD.scid/scid mice elicited accelerated disease, demonstrating that pancreatic IL-10 but not endogenous IL-10 is sufficient for the acceleration of diabetes. Immunohistochemical analysis revealed hyperexpression of ICAM-1 on the vascular endothelium of IL-10-NOD mice. The finding suggests that IL-10 may promote diabetes via an ICAM-1-dependent pathway. We found that introduction of ICAM-1 deficiency into IL-10-NOD mice as well as into NOD mice prevented accelerated insulitis and diabetes. Failure to develop insulitis and diabetes was preceded by the absence of GAD65-specific T cell responses. The data suggest that ICAM-1 plays a role in the formation of the "immunological synapse", thereby affecting the generation and/or expansion of islet-specific T cells. In addition, ICAM-1 also played a role in the effector phase of autoimmune diabetes because adoptive transfer of diabetogenic BDC2.5 T cells failed to elicit clinical disease in ICAM-1-deficient IL-10-NOD and NOD mice. These findings provide evidence that pancreatic IL-10 is sufficient to drive pathogenic autoimmune responses and accelerates diabetes via an ICAM-1-dependent pathway.