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Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1and Suv39h2enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1nor Suv39h2individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-?B and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.

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The gut microbiota can influence immune-cell function by the production of short-chain fatty acids. Mackay and colleagues show that diets enriched for acetate and butyrate protect non-obese diabetic mice from insulitis and diabetes progression.

Differentiation of naïve CD4+ T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4+ T cells is dependent on Ezh2. Naïve CD4+ T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor–mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ–producing CD4+ and CD8+ T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.

Differentiation of naïve CD4+T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10loci in naïve CD4+T cells is dependent on Ezh2. Naïve CD4+T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor–mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ–producing CD4+and CD8+T cells responding to Listeria monocytogenesinfection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.

Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4+ T cells (Naive). We detected 2315 differentially methylated cytosine–guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4+T cells (Naive). We detected 2315 differentially methylated cytosine–guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motifdomain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGITlocus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

Vaccination against self-antigens to avert pathological immunity to self - ‘negative vaccination’ - is the Holy Grail of autoimmune disease therapy. This approach depends on deletion or inactivation of pathogenic T cells, or induction of protective, ‘regulatory’ T cells. While effective in inbred rodent models, it is yet to be translated to humans. Reasons for this include its application only in end-stage disease, ignorance about antigen form, route of delivery and dose-schedule required for a bio-response, lack of meaningful and measurable bio-response markers, co-activation of pathogenic immunity and genetic heterogeneity.

Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1β, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings.

Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1β, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings.

The progressive increase in the incidence of both type 1 diabetes mellitus (T1DM) and T2DM, which is associated with changing environmental conditions, highlights overlapping clinical and pathogenetic features of these diabetes stereotypes. The article proposes that the common thread is a proinflammatory environment that activates innate immunological and inflammatory pathways, which lead to β-cell dysfunction in T2DM, insulin resistance in both T1DM and T2DM, and enhanced adaptive immunity that kills β cells in T1DM.

Type 1 diabetes (T1D) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing ? cells in the islets of the pancreas. An understanding of pathogenetic mechanisms paves the way for vaccination strategies to prevent T1D. Primary prevention by traditional ‘positive’ vaccination awaits clear evidence of a the role for infectious agents in triggering the disease process. Following initiation of disease, at-risk individuals with underlying islet inflammation can be identified by the presence of circulating autoantibodies to islet antigens. This pre-clinical phase of T1D, before insulin deficiency leads to symptoms of hyperglycemia, provides a window for secondary prevention. The therapeutic Holy Grail in autoimmune disease is ‘negative’ vaccination against autoantigens that drive immune-mediated pathology, to induce disease-specific immune tolerance. This can be achieved by administering autoantigen via a ‘tolerogenic’ (e.g. mucosal) route, cell (e.g. resting dendritic cell), mode (e.g. with blockade of co-stimulation molecules) or form (as an ‘altered peptide ligand’). These strategies to induce immune tolerance are effective in rodent models of autoimmune disease, but in application to humans the results have so far been disappointing. This review discusses the prospects of vaccination to prevent T1D, focusing on autoantigen-specific mucosal tolerance.

A dendritic cell (DC) imbalance with a marked deficiency in CD4−8+ DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4−8+ DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4−8+ DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tryosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.

T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR−/−). We also compared the action of GC on GITR+ and GITR− T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR−/− mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR−/− cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10−8 to 10−6 M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.

The Melbourne Prediabetes Family Study, a prospective study of first-degree relatives of people with type 1 diabetes (T1D), provided an opportunity to examine late-onset autoimmune diabetes within the context of a family history of T1D. We compared genetic, immunologic, and clinical features in relatives of people with T1D, who developed early- versus late-onset diabetes.

Cell lineage development is a finely tuned process of proliferation and differentiation, survival and apoptosis, that is regulated by numerous extracellular signals. Here we review some of the extracellular signals—including insoluble cell-cell and extracellular matrix-cell interactions, as well as soluble factors—that appear critical for pancreatic β-cell development. Knowledge of how these signals control the development of pancreatic endocrine stem/precursor cells into fully functional insulin-secreting βcells is a platform for the restoration of β-cell function and the cure therapy of type 1 diabetes.

ABSTRACTRotavirus infection in children at risk of developing type 1 diabetes has been temporally associated with development of pancreatic islet autoantibodies. In this study, nonobese diabetic mice were shown to be susceptible to rhesus rotavirus infection and pancreatic islets from nonobese diabetic mice, nonobese diabetes-resistant mice, fetal pigs, and macaque monkeys supported various degrees of rotavirus growth. Human rotaviruses replicated in monkey islets only. This islet susceptibility shows that rotavirus infection of the pancreas in vivo might be possible.

Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti‐CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both Th1 and Th2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti‐CD45RB antibody with co‐stimulation blockade. The primary effect of anti‐CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti‐CD45RB treatment led to reduced expression of LFA‐1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA‐1 may enhance co‐stimulation, and both LFA‐1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti‐CD45RB antibody is due to effects on both T cell priming and trafficking.

Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti-CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both T(h)1 and T(h)2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti-CD45RB antibody with co-stimulation blockade. The primary effect of anti-CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti-CD45RB treatment led to reduced expression of LFA-1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA-1 may enhance co-stimulation, and both LFA-1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti-CD45RB antibody is due to effects on both T cell priming and trafficking.

The innate immune system existed prior to the emergence of adaptive immunity in sharks and higher vertebrates. Homologues of many mammalian innate immune‐system elements such as the toll‐like receptors exist in species as distant as Drosophila. Selective pressure has led to the development of highly conserved, soluble, and cell‐surface receptors that recognize functionally essential molecules shared by microbial pathogens. It is thought that molecular patterns that exquisitely distinguish pathogenic cells from mammalian cells are recognized. Therefore, it would seem unlikely that innate immune‐system elements should recognize mammalian tissues. However, there is increasing evidence to suggest that this is the case and that innate immunity promotes rejection of transplanted mammalian tissues, particularly those from other species (xenografts). Evidence for innate recognition of mammalian grafts, the nature of this recognition, and the bi‐directional interactions between innate and adaptive immunity that contribute to graft rejection are discussed in this review, with the emphasis on nonvascular xenografts.

Extracellular signals that guide pancreas cell development are not well characterized. In an in vitro culture system of dissociated pancreas cells from the E15.5 mouse fetus we show that, in the presence of the extracellular matrix protein laminin-1, bone morphogenetic proteins (BMPs-4, -5 and -6)promote the development of cystic epithelial colonies. Transforming growth factor β1 (TGF-β1) and activin A antagonise this effect of BMP-6 and inhibit colony formation. Histological analysis revealed that the colonies are composed of E-cadherin-positive epithelial cells, which in localised areas are insulin positive. The colonies also contain occasional glucagon-positive cells, but no somatostatin- or α-amylase-positive cells. These findings indicate that members of the TGF-β superfamily regulate pancreas epithelial cell development and can promote the formation of islet-like structures in vitro.

Cytotoxic CD8 T lymphocytes (CTL) are effectors of pancreatic islet β-cell destruction in type 1 diabetes but, with the exception of a single report, CTL to islet antigen peptides have not been identified. We used autologous blood monocyte-derived dendritic cells to elicit HLA-A2-restricted CTL to a peptide, MVWESGCTV (aa 797–805), that is contiguous with a dominant CD4 T-cell epitope in the islet antigen tyrosine phosphatase IA-2. IA-2 peptide-specific CTL activity measured as 51Cr release from autologous lymphoblasts was detected in 2/6 islet antibody-positive relatives at high risk for type 1 diabetes but also in 2/6 closely HLA-matched controls. All subjects had CTL activity to an HLA-A2-restricted Epstein–Barr virus peptide. CTL to the IA-2 self-peptide were therefore not disease-specific, consistent with other evidence that autoreactive T cells are present in healthy individuals.

Background: The ability to manipulate the development of pancreatic insulin-producing βcells has implications for the treatment of type 1 diabetes. Previously, we found that laminin-1, a basement membrane trimeric glycoprotein, promotes β-cell differentiation. We have investigated the mechanism of this effect, using agents that block the receptors for laminin-1, α6integrin, and α-dystroglycan (α-DG). Materials and Methods: Dissociated cells from 13.5-day postcoitum (dpc) fetal mouse pancreas were cultured for 4 days with laminin-1, with and without monoclonal antibodies and other agents known to block integrins or α-DG. Fetuses fixed in Bouin’s solution or fetal pancreas cells fixed in 4% paraformaldehyde were processed for routine histology and for immunohistology to detect hormone expression and bromodeoxyuridine (BrdU) uptake. Results: Blocking the binding of laminin-1 to α6integrin with a monoclonal antibody, GoH3, abolished cell proliferation (BrdU uptake) and doubled the number of βcells. Inhibition of molecules involved in α6integrin signaling (phosphotidylinositol 3-kinase, F-actin, or mitogen-activated protein kinase) had a similar effect. Nevertheless, βcells appeared to develop normally in α6integrin-deficient fetuses. Blocking the binding of laminin-1 to α-DG with a monoclonal antibody, IIH6, dramatically decreased the number of βcells. Heparin, also known to inhibit laminin-1 binding to α-DG, had a similar effect. In the presence of heparin, the increase in βcells in response to blocking α6integrin with GoH3 was abolished. Conclusions: These findings reveal an interplay between α6integrin and α-DG to regulate laminin-1-induced β-cell development. Laminin-1 had a dominant effect via α-DG to promote cell survival and β-cell differentiation, which was modestly inhibited by α6signaling.

Antibodies to glutamic decarboxylase (GADAb) are present in insulin-dependent diabetes (IDD) but their association with age and sex and their temporal profile in relation to disease onset have not been fully documented. We have examined the association between GADAb and islet cell antibodies (ICA), age and sex, and have cross-sectionally and longitudinally measured the levels of GADAb before and after diagnosis of IDD. GADAb were measured by allowing serum immunoglobulin prebound to protein A Sepharose to precipitate GAD enzymatic activity from a fetal pig brain extract. GADAb levels were above the normal range (mean+3SD of healthy controls, 460 nU/M1) in 19/44 (43%) at-risk subjects (ICA positive first degree relatives of persons with IDD), 35/108 (32%) recent-onset IDD subjects and 22/46 (47%) established IDD subjects. When analysed according to age and sex, GADAb levels were significantly higher (P<0.05) in post-pubertal females in at-risk, recent-onset and established IDD groups. There was a significant association between GADAb and ICA>20 in both first degree relatives (P<0.001) and recent-onset subjects (P<0.01) and GADAb were uncommon in the absence of ICA. Levels of GADAb were similar in at-risk, recent-onset and established IDD subjects and GADAb status remained stable in all but 2/41 at-risk subjects followed for 17 (mean, range 3-33) months. In conclusion, GADAb levels are strongly influenced by age, sex and ICA status, and generally remain stable in at-risk subjects and after the onset of clinical IDD. Copyright 1994, 1999 Academic Press

The purpose of the present study was to quantify beta cell proliferation in rabbits that had undergone subtotal beta cell ablation by labeling cell nuclei undergoing DNA synthesis with bromodeoxyuridine (BUdR). Ten New Zealand white rabbits were compared with five sham-operated controls. The beta cells in the head of the pancreas were destroyed by intravenous alloxan (200 mg/kg) while the tail of the pancreas was temporarily isolated from the systemic circulation by vascular occlusion clamps. On day 7, BUdR (70 mg/kg) was infused prior to killing to label newly synthesised DNA. Immunoperoxidase staining with anti-insulin sera and an anti-islet cell monoclonal antibody confirmed the absence of insulin-producing cells in the head of the pancreas. Beta cell proliferation within the islets of the tail of the pancreas was suggested by the appearance of cells undergoing mitosis and confirmed by BUdR labeling of cell nuclei detected with anti-BUdR monoclonal antibody. The mitotic index was significantly increased compared to control rabbits (p = 0.0235, Mann- Whitney U nonparametric test). This study demonstrates that the BUdR labeling can be used to document beta cell proliferation in vivo in the rabbit model after subtotal beta cell ablation.

Objectives: To determine the utility of various autoantibodies in predicting progression to clinical diabetes in first‐degree relatives of patients with type 1 diabetes mellitus. Participants: 3315 first‐degree relatives of patients with type 1 diabetes (1161 parents, 1206 siblings and 948 offspring) recruited through diabetes clinics, private endocrinologists, Diabetes Australia and the Juvenile Diabetes Foundation. Main outcome measures: Prevalence of islet cell antibodies (ICA) levels ≥20 JDFu, insulin autoantibodies (IAA) levels > 100nU/ml, and antibodies to glutamic acid decarboxylase (GADAb) and tyrosine phosphatase IA2 (IA2Ab); change in cell function over time; and development of clinical diabetes. Results: 2.6% of relatives had elevated ICA levels, 1.3% had elevated IAA levels and 0.3% had both. High ICA levels were significantly more frequent in siblings than in offspring or parents, and were more frequent in relatives younger than 20 years. GADAb were detected in 68% and IA2Ab in 57% of relatives with elevated ICA and/or IAA levels. Diabetes developed in 33 relatives (25 siblings, 2 offspring and 6 parents). Before diagnosis of clinical diabetes, high ICA levels were detected in 18 (58%), high IAA levels in 7 (23%), both in 5 (15%), and either in 19 (61%); GADAb were detected in 26 (84%), IA2Ab in 13 (42%), both in 11 (35%), and either in 28 (90%). First phase insulin release (FPIR) less than 50mU/L was very strongly associated with progression to diabetes. In relatives with FPIR initially greater than 50mU/L who eventually developed diabetes, there was a gradual and continuous reduction in FPIR over time before diagnosis. Conclusions: Type 1 diabetes can be diagnosed in the preclinical stage. The recently described antibodies to glutamic acid decarboxylase and tyrosine phosphatase IA2 appear superior to ICA as screening tools for the preclinical diagnosis of type 1 diabetes.

To determine whether the predictive value of islet cell antibodies (ICA) and insulin autoantibodies (IAA) is increased by measurement of glutamic acid decarboxylase antibodies (GADAb) in first-degree relatives of patients with insulin-dependent diabetes mellitus (IDDM), we measured GADAb in those developing IDDM and in relatives found to be ICA- or IAA-positive in our family screening study. First-degree relatives (n=2904) were followed for 2.4 (median, range 0.04-5.8) years. Of the subjects developing IDDM, 11/14 (78%) had ICA≥20JDF units, 1/14 (7%) had IAA≥100 nU/ml and 6/14 (43%) had GADAb (≥460nU/ml, measured by precipitation of enzymatic activity). Of the four subjects with ICA<20 and IAA<100 nU/ml who developed IDDM, one had elevated GADAb. Significant inhibition of GAD enzymatic activity by serum immunoglobulins, a potential cause of false-negative results in our immunoprecipitation assay, was not detected in seven subjects who developed IDDM in the absence of GADAb. Sixty-nine of the 2904 subjects with ICA≥20 or IAA>100 were followed for 3.1 (median range 0.1-5.4) years. Survival analysis showed that diabetes-free survival this group was not influenced significant by GADAb positivity. In conclusion, GADAb in the absence of ICA and IAA are uncommon in first-degree relatives who progress to IDDM and the presence of GADAb does not increase the risk for IDDM in ICA- or IAA-positive relatives. Copyright 1994, 1999 Academic Press

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in β-cell destruction and immune regulation. A major target of β-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of β-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1β (1 ng/ml, 24 h), tumour necrosis factor α (TNF-α; 200 units/ml, 24 h) or interferon γ (IFN-γ; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-α (1000 units/ml), TNF-β (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor β2 (TGF-β2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1β, TNF-α or IFN-γ was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1β and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1β, TNF-α or IFN-γ by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of β-cell destruction in IDDM.

Pancreatic islet β-cell destruction leading to insulin-dependent diabetes mellitus (IDDM) is an autoimmune T cell-mediated process. Peripheral blood T cells, which proliferate to islet antigens such as glutamic acid decarboxylase (GAD), (pro)insulin or tyrosine phosphatase IA-2, can be detected in at-risk, first degree relatives of people with IDDM. However, cross-sectional studies cannot define the relationship between T cell responses and progression to IDDM. Longitudinal studies were therefore undertaken on 50 at-risk, first degree relatives tested at least yearly for up to 4 years, during which time five developed IDDM. Peripheral blood T cell responses to a GAD67(aa208–404)-glutathione-S-transferase (GST) fusion protein, GST, insulin and tetanus toxoid were measured, together with antibodies to islet cells, GAD, insulin and IA-2. High levels of antibodies to GAD or insulin were generally associated with low T cell responses to these antigens. Relatives who developed IDDM were characterized by high levels of antibodies to insulin and/or islet cells, and high T cell responses to GAD67-GST and tetanus, but not insulin, in the 24 months before clinical diagnosis. Cross-sectionally, T cell responses to GAD67(aa208–404)-GST and to full-length GAD65-GST were highly correlated (r=0.75, P<0.002). In conclusion, increased cellular immunity to the mid region of GAD67 was a marker of late pre-clinical IDDM, but appears to reflect a more general, transient state of cellular immune hyperresponsiveness.

Background: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24–36) that bears marked similarity to a peptide of GAD65 (amino acids 506–518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM. Materials and Methods: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay. Results: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p&lt; 0.008; GAD: p&lt; 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects. Conclusions: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.

Activation of T cells requires recognition by T-cell receptors of specific peptides bound to major histocompatibility complex (MHC) molecules on the surface of either antigen-presenting or target cells. These peptides, T-cell epitopes, have potential therapeutic applications, such as for use as vaccines. Their identification, however, usually requires that multiple overlapping synthetic peptides encompassing a protein antigen be assayed, which in humans, is limited by volume of donor blood. T-cell epitopes are a subset of peptides that bind to MHC molecules. We use an artificial neural network (ANN) model trained to predict peptides that bind to the MHC class II molecule HLA-DR4(*0401). Binding prediction facilitates identification of T-cell epitopes in tyrosine phosphatase IA-2, an autoantigen in DR4-associated type1 diabetes. Synthetic peptides encompassing IA-2 were tested experimentally for DR4 binding and T-cell proliferation in humans at risk for diabetes. ANN-based binding prediction was sensitive and specific, and reduced the number of peptides required for T-cell assay by more than half, with only a minor loss of epitopes. This strategy could expedite identification of candidate T-cell epitopes in diverse diseases.

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 βcells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to βcells in spontaneously diabetic NOD mice. We created a model of accelerated β-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in βcells. Materials and Methods: NOD.scid(severe combined immunodeficiency) mice were crossed with bml mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their βcells, to generate B7-1 NOD.scidmice. 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.scidmice caused diabetes in recipients within 12–16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scidmice than in non-transgenic NOD.scidmice. Dual immunolabeling for TUNEL and either B7-1 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in βcells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic βcells was estimated to be less than 6 min. Conclusions: B7-1 transgenic βcells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that βcells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.

Tumour necrosis factor (TNF) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). To investigate a possible role for TNF in IDDM we compared endogenous TNF production in two lines of non-obese diabetic (NOD) mice, NOD/Lt and NOD/WEHI, that have a high and low incidence of diabetes, respectively. Preliminary experiments had shown that the lower syngeneic mixed lymphocyte reaction (SMLR) in NOD/Lt mice could be corrected by TNF-α. Plasma TNF-α was measured in 8 week-old female non-diabetic mice primed with 1000 units IV of murine interferon gamma (IFN-γ) followed after 3 hours by 5 μmlg IV of lipopolysaccharide (LPS). Two hours later plasma was collected and TNF measured by ELISA. Plasma TNF in NOD/Lt mice was 9.2>2.4ng/ml (meanseM, n 16) compared to 2.5>0.5 ng/ml in NOD/WEHI mice (n 15) and 7.6>1.0ng/ml in BALB/c mice (n 14). Time course studies demonstrated higher levels of both immunoreactive and bioactive TNF in NOD/Lt compared to NOD/WEHI mice up to 4 hours post-stimulation. A separate group of female NOD/Lt mice had IFN-γ/LPS-stimulated plasma TNF-α measured at 10 weeks and were followed to age 30 weeks. The mean stimulated plasma TNF-α level wsa consistently higher in those mice that developed diabetes compared to those that remained non-diabetic, the difference being significant when mice were 21 weeks of age. These results suggest that endogenous TNF-α production may be a trait marker of IDDM susceptibility.

Autoantibodies to glutamic acid decarboxylase (GAD) are present in humans before and after the onset of clinical insulin-dependent diabetes (IDD). The non-obese diabetic (NOD) mouse, a model of human IDD, develops mononuclear cell infiltration of the pancreatic islets ('insulitis') associated particularly in females with T cell-mediated destruction of the islet β cells. In NOD mice of both sexes we detected serum antibodies to GAD (GAD Ab) that precipitate mouse brain GAD enzymatic activity. Antibodies in NOD sera also precipitate a M_r 65,000 protein from Triton X-100 extracts of ³⁵S-methionine-labelled NOD islets, identical in size to that precipitated by a monoclonal antibody to GAD. GAD Ab were not detected in other mouse strains. There were significant differences in the frequency, level and age at initial detection of GAD Ab between females of the NOD/Lt and NOD/WEHI lines, previously shown to have a higher and lower incidence of diabetes, respectively. Comparing NOD/Lt (n=26) and NOD/WEHI (n=20) females, in which diabetes occurred in 38% and 20% by 150 days, the frequency of elevated GAD Ab was 50 vs. 80%, the mean maximum GAD Ab levels 21.1 vs. 30.6% and the mean age at which GAD Ab were first detected 94 vs. days. No significant differences in these parameters were observed between male mice of either line. There was a significant negative correlation between the level of GAD Ab and the degree of insulitis in female mice from both lines. GAD Ab were not a prerequisite for the development of diabetes. In 7 of 10 female mice the onset of diabetes was preceded by a decrease of GAD Ab levels into the normal range. These findings indicate that, while GAD is a target of autoimmunity in the NOD mouse, GAD Ab do not necessarily correlate with the development of diabetes. Indeed, the difference between the two NOD lines and the inverse relationship with insulitis suggests that a strong humoral response to GAD may be associated with a less destructive pathology, as proposed in humans 'at-risk' for IDD. Copyright 1994, 1999 Academic Press

Host macrophages are abundant within fetal pig pancreas xenografts undergoing rejection, but their role is unknown. Therefore, we examined the effect of host macrophage depletion on xenograft rejection.

The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes.