NOD Model Research Articles

Blockade of the Programmed Death-1 (PD1) Pathway Undermines Potent Genetic Protection from Type 1 Diabetes

Aims/HypothesisInhibition of PD1-PDL1 signaling in NOD mice accelerates onset of type 1 diabetes implicating this pathway in suppressing the emergence of pancreatic beta cell reactive T-cells. However, the molecular mechanism by which PD1 signaling protects from type 1 diabetes is not clear. We hypothesized that differential susceptibility of Idd mouse strains to type 1 diabetes when challenged with anti PDL1 will identify genomic loci that collaborate with PD1 signaling in suppressing type 1 diabetes.MethodsAnti PDL1 was administered to NOD and various Idd mouse strains at 10 weeks of age and onset of disease was monitored by measuring blood glucose levels. Additionally, histological evaluation of the pancreas was performed to determine degree of insulitis. Statistical analysis of the data was performed using Log-Rank and Student's t-test.ResultsBlockade of PDL1 rapidly precipitated type 1 diabetes in nearly all NOD Idd congenic strains tested, despite the fact that all are moderately (Idd5, Idd3 and Idd10/18) or highly (Idd3/10/18 and Idd9) protected from spontaneous type 1 diabetes by virtue of their protective Idd genes. Only the Idd3/5 strain, which is nearly 100% protected from spontaneous disease, remained normoglycemic following PDL1 blockade.ConclusionsThese results indicate that multiple Idd loci collaborate with PD1 signaling. Anti PDL1 treatment undermines a large portion of the genetic protection mediated by Idd genes in the NOD model of type 1 diabetes. Basal insulitis correlated with higher susceptibility to type 1 diabetes. These findings have important implications since the PD1 pathway is a target for immunotherapy.

Protective effects of polyamine depletion in mouse models of type 1 diabetes: implications for therapy

The underlying pathophysiology of type 1 diabetes involves autoimmune-mediated islet inflammation, leading to dysfunction and death of insulin-secreting islet β cells. Recent studies have shown that polyamines, which are essential for mRNA translation, cellular replication, and the formation of the hypusine modification of eIF5A may play an important role in the progression of cellular inflammation. To test a role for polyamines in type 1 diabetes pathogenesis, we administered the ornithine decarboxylase inhibitor difluoromethylornithine to two mouse models--the low-dose streptozotocin model and the NOD model--to deplete intracellular polyamines, and administered streptozotocin to a third model, which was haploinsufficient for the gene encoding the hypusination enzyme deoxyhypusine synthase. Subsequent development of diabetes and/or glucose intolerance was monitored. In the low-dose streptozotocin mouse model, continuous difluoromethylornithine administration dose-dependently reduced the incidence of hyperglycemia and led to the preservation of β cell area, whereas in the NOD mouse model of autoimmune diabetes difluoromethylornithine reduced diabetes incidence by 50%, preserved β cell area and insulin secretion, led to reductions in both islet inflammation and potentially diabetogenic Th17 cells in pancreatic lymph nodes. Difluoromethylornithine treatment reduced hypusinated eIF5A levels in both immune cells and islets. Animals haploinsufficient for the gene encoding deoxyhypusine synthase were partially protected from hyperglycemia induced by streptozotocin. Collectively, these studies suggest that interventions that interfere with polyamine biosynthesis and/or eIF5A hypusination may represent viable approaches in the treatment of diabetes.

IL-21 Promotes CD8+ CTL Activity via the Transcription Factor T-bet

CD8(+) T cells are fundamental for immune-mediated clearance of viral infections and contribute to immune pathology in autoimmune diseases such as type 1 diabetes. To execute these functions, CD8(+) T cells must differentiate into CTLs, a process that is precisely regulated by a variety of cytokines, costimulatory molecules, and transcription factors. IL-21 is an IL-2 family cytokine and a growth factor for multiple lymphocyte effector lineages, including cytotoxic CD8(+) T cells. Recent studies demonstrate that loss of IL-21 signaling results in reduced viral clearance in models of lymphocytic choriomeningitis virus infection, and also protection from type 1 diabetes in the NOD model. This is most likely the result of impaired CD8(+) CTL function in the absence of IL-21 signaling. Currently, the mechanisms by which IL-21 promotes CTL differentiation in CD8(+) T cells remain unclear, particularly the identity of the relevant transcription factor(s). We show that IL-21 promotes CTL function in vitro and killing of pancreatic islets in vivo via the use of transgenic mice expressing IL-21 in pancreatic β cells. We demonstrate that IL-21 induces the expression of the transcription factor T-bet in CD8(+) T cells, predominantly via STAT1, and that T-bet is required for the induction of cytolytic molecules, including perforin and granzyme B in response to IL-21. Finally, we show that IL-21-induced CTL function is T-bet dependent, as T-bet deficiency results in defective IL-21-dependent cytotoxicity in CD8(+) T cells in vitro and in vivo. Thus, IL-21 drives CD8(+) CTL differentiation via the actions of the transcription factor T-bet.

PTPN22 Silencing in the NOD Model Indicates the Type 1 Diabetes–Associated Allele Is Not a Loss-of-Function Variant

PTPN22 encodes the lymphoid tyrosine phosphatase (LYP) and is the second strongest non-HLA genetic risk factor for type 1 diabetes. The PTPN22 susceptibility allele generates an LYP variant with an arginine-to-tryptophan substitution at position 620 (R620W) that has been reported by several studies to impart a gain of function. However, a recent report investigating both human cells and a knockin mouse model containing the R620W homolog suggested that this variation causes faster protein degradation. Whether LYP R620W is a gain- or loss-of-function variant, therefore, remains controversial. To address this issue, we generated transgenic NOD mice (nonobese diabetic) in which Ptpn22 can be inducibly silenced by RNA interference. We found that Ptpn22 silencing in the NOD model replicated many of the phenotypes observed in C57BL/6 Ptpn22 knockout mice, including an increase in regulatory T cells. Notably, loss of Ptpn22 led to phenotypic changes in B cells opposite to those reported for the human susceptibility allele. Furthermore, Ptpn22 knockdown did not increase the risk of autoimmune diabetes but, rather, conferred protection from disease. Overall, to our knowledge, this is the first functional study of Ptpn22 within a model of type 1 diabetes, and the data do not support a loss of function for the PTPN22 disease variant.

Enhanced Anti-Serpin Antibody Activity Inhibits Autoimmune Inflammation in Type 1 Diabetes

Intracellular (clade B) OVA-serpin protease inhibitors play an important role in tissue homeostasis by protecting cells from death in response to hypo-osmotic stress, heat shock, and other stimuli. It is not known whether these serpins influence immunological tolerance and the risk for autoimmune diseases. We found that a fraction of young autoimmune diabetes-prone NOD mice had elevated levels of autoantibodies against a member of clade B family known as serpinB13. High levels of anti-serpinB13 Abs were accompanied by low levels of anti-insulin autoantibodies, reduced numbers of islet-associated T cells, and delayed onset of diabetes. Exposure to anti-serpinB13 mAb alone also decreased islet inflammation, and coadministration of this reagent and a suboptimal dose of anti-CD3 mAb accelerated recovery from diabetes. In a fashion similar to that discovered in the NOD model, a deficiency in humoral activity against serpinB13 was associated with early onset of human type 1 diabetes. These findings suggest that, in addition to limiting exposure to proteases within the cell, clade B serpins help to maintain homeostasis by inducing protective humoral immunity.

The Role of Th17 cells in Type 1 Diabetes. (123.30)

Abstract Type 1 diabetes (T1D) has been considered to be elicited primarily by Th1 cells. However, the role of Th17 cells, which have been implicated in other autoimmune diseases, is controversial. Using the NOD model, we found that CD4 T cells infiltrate the pancreas by 2 weeks of age, a significant proportion of which expressed either IFNγ or IL-17, suggesting that islet-specific Th1 and Th17 both develop very early. Although serum IL-17 levels increased at diabetes onset, Th17 cells from spontaneously diabetic mice failed to transfer disease to NOD.Scid recipients whereas Th17-depleted CD4 cells elicited diabetes as efficiently as non-depleted cells. To further address the pathogenic potential of Th17 cells, we differentiated polyclonal or BDC2.5 Th17 cells in vitro and transferred them into NOD or NOD.Scid recipients. Polyclonal Th17 cells failed to induce diabetes, and the recovered donor cells maintained their phenotype, suggesting that Th17 cells can be stable. In contrast, BDC2.5 Th17 cells readily induced diabetes in both recipients, although a majority of the donor cells became IFNγ-producers. However, neither the acquisition of IFNγ production nor IFNγ itself were required for diabetes induction. Instead, TNFα was essential for their pathogenicity. Our results raise the possibility that Th17 cells do not make a critical contribution to spontaneous T1D unless re-differentiation that is marked by IFNγ production occurs as a consequence of local inflammation in the pancreas.

A Transgenic Model of Central Nervous System Autoimmunity Mediated by CD4+ and CD8+ T and B Cells

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.

Optimization of the Hot Forming Processes for the Connecting Rod

A three-dimensional thermo-mechanically coupled FEM-simulation of the production of a connecting rod has been performed between dies (pre- and final-forging). According to the part’s characters and its dimensions, a hammer die forging process was determined. According to the hammer die process, three-dimensional connecting nod model was built in UG software and two different cases for pre-forging was designed. Different forming case was simulated by Deform-3D FEM program, the effective stress field and effective strain field were analyzed by comparison; it proved that optimized performing process was reasonable and can be used as reference in production.

ICOS-Dependent Homeostasis and Function of Foxp3+ Regulatory T Cells in Islets of Nonobese Diabetic Mice

A progressive waning in Foxp3(+) regulatory T cell (Treg) functions is thought to provoke autoimmunity in the NOD model of type 1 diabetes (T1D). A deficiency in IL-2 is one of the main triggers for the defective function of Tregs in islets. Notably, abrogation of the ICOS pathway in NOD neonates or BDC2.5-NOD (BDC2.5) mice exacerbates T1D, suggesting an important role for this costimulatory pathway in tolerance to islet Ags. Thus, we hypothesize that ICOS selectively promotes Foxp3(+) Treg functions in BDC2.5 mice. We show that ICOS expression discriminates effector Foxp3(-) T cells from Foxp3(+) Tregs and specifically designates a dominant subset of intra-islet Tregs, endowed with an increased potential to expand, secrete IL-10, and mediate suppressive activity in vitro and in vivo. Consistently, Ab-mediated blockade or genetic deficiency of ICOS selectively abrogates Treg-mediated functions and T1D protection and exacerbates disease in BDC2.5 mice. Moreover, T1D progression in BDC2.5 mice is associated with a decline in ICOS expression in and expansion and suppression by intra-islet Foxp3(+) Tregs. We further show that the ICOS(+) Tregs, in contrast to their ICOS(-) counterparts, are more sensitive to IL-2, a critical signal for their survival and functional stability. Lastly, the temporal loss in ICOS(+) Tregs is readily corrected by IL-2 therapy or protective Il2 gene variation. Overall, ICOS is critical for the homeostasis and functional stability of Foxp3(+) Tregs in prediabetic islets and maintenance of T1D protection.

Involvement of SIRT1 in Zn2+, Streptozotocin, Non-Obese Diabetic, and Cytokine-Mediated Toxicities of β-cells.

To determine if the sirtuin pathway is involved in Zn2+-, streptozotocin-, or cytokine-mediated β-cell death in vitro, and streptozotocin-, or NOD induced T1DM in vivo. Sensitivity of MIN6 cells expressing empty vector, sirtuin protein-1 (SIRT1) or its siRNA, to Zn2+, streptozotocin, or cytokines, and effects on NAD+ levels were determined. Covariance of manipulating SIRT1 levels with diabetic incidence was tested in vivo. 1) sirtuin pathway inhibition or SIRT1 knockdown attenuated Zn2+-, STZ-, and cytokine-mediated toxicity and NAD+ loss in β-cells, 2) SIRT1 overexpression potentiated these toxicities, 3) young SIRT1 β-cell transgenic mice have improved glucose tolerance under basal conditions, but upon aging showed increased sensitivity to streptozotocin compared to SIRT1 +/- mice, and 4) SIRT1 +/- mice in an NOD background or exposed to streptozotocin trended toward reduced diabetic incidence and mortality compared to wildtype. These results have implicated SIRT1-mediated NAD+ loss in Zn2+, STZ, or cytokine toxicities of MIN6, and in NOD or streptozotocin T1DM animal models. Modulation of β-cell Zn2+ and NAD+ levels, and the sirtuin pathway could be novel therapeutic targets for T1DM.

2α-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D3 protects the insulin 2 knockout non-obese diabetic mouse from developing type 1 diabetes without hypercalcaemia

Type 1 diabetes (T1D) is an autoimmune disease that destroys the insulin-producing beta-islet cells of the pancreas. Currently, there are no treatment modalities for prevention of T1D, and the mechanisms influencing disease inception and early progression are not well understood. We have used the insulin 2(-/-) non-obese diabetic (Ins2(-/-) NOD) model to study stages of T1D and to examine the protective effects of a potent analogue of 1α,25-dihydroxyvitamin D(3), 2α-methyl-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2AMD). Pancreatic tissues from control and 2AMD-treated Ins2(-/-) NOD mice were obtained weekly from 5 to 16 weeks of age. Using immunohistochemical (IHC) analysis, samples were analysed for changes in beta cell survival, islet structure and T cell invasion. Weekly intraperitoneal glucose tolerance tests (IPGTT) were performed to assess comparative beta cell function in control and treated animals. IHC demonstrated progressive beta cell destruction in control mice. In contrast, 2AMD treatment preserved islet cell architecture, arrested intra-islet T cell invasion and prevented the transition from insulitis to diabetes. IPGTT results revealed progressive impairment of beta cell function with increasing age in control mice, while 2AMD treatment resulted in normal beta function throughout the study. These results demonstrate that the Ins2(-/-) NOD model provides a rapid and effective method for studying T1D and for assessing efficacy of anti-diabetic agents.

The Soluble CTLA-4 Splice Variant Protects From Type 1 Diabetes and Potentiates Regulatory T-Cell Function

OBJECTIVECTLA4 gene variation associates with multiple autoimmune disorders, including type 1 diabetes. The CTLA4 susceptibility allele was found to generate decreased levels of mRNA encoding soluble CTLA-4 (sCTLA-4) relative to the full-length isoform, the functional consequence of which is as yet unknown. In this study, we investigated the contribution of sCTLA-4 to immune regulation with the aim to elucidate the functional basis of the disease association of CTLA4.RESEARCH DESIGN AND METHODSTo model the disease-associated splicing variation of CTLA4, we generated NOD mice in which sCTLA-4 mRNA is silenced by RNA interference.RESULTSWe found that loss of sCTLA-4 impairs the function of regulatory T (Treg) cells. This functional defect could be attributed, at least in part, to the failure of sCTLA-4 knockdown (KD) Treg cells to downregulate dendritic cell costimulation. sCTLA-4 KD Treg cells, in contrast with wild-type Treg cells, failed to inhibit colitis induced by transfer of CD4+CD45RBhi cells into NOD.SCID animals. Furthermore, diminished sCTLA-4 expression accelerated the onset of autoimmune diabetes in transgenic mice.CONCLUSIONSOur results demonstrate that sCTLA-4 participates in immune regulation by potentiating the function of Treg cells. The functional outcome of silencing this splice variant in the NOD model provides an explanation for the association of CTLA4 variation with autoimmunity. Lower sCTLA-4 expression from the susceptibility allele may directly affect the suppressive capacity of Treg cells and thereby modulate disease risk. Our unprecedented approach establishes the feasibility of modeling splicing variations relevant to autoimmunity.

High Levels of Adenosine Deaminase on Dendritic Cells Promote Autoreactive T Cell Activation and Diabetes in Nonobese Diabetic Mice

Adenosine has been established as an important regulator of immune activation. It signals through P1 adenosine receptors to suppress activation of T cells and professional APCs. Adenosine deaminase (ADA) counters this effect by catabolizing adenosine. This regulatory mechanism has not been tested in a disease model in vivo. Questions also remain as to which cell types are most sensitive to this regulation and whether its dysregulation contributes to any autoimmune conditions. We approached this issue using the NOD model. We report that ADA is upregulated in NOD dendritic cells, which results in their exuberant and spontaneous activation. This, in turn, triggers autoimmune T cell activation. NOD DCs deficient in ADA expression have a greatly reduced capacity to trigger type I diabetes. We also provide evidence that although many cell types, particularly T cells, have been implicated as the suppression targets by adenosine in an in vitro setting, DCs also seem to be affected by this regulatory mechanism. Therefore, this report illustrates a role of ADA in autoimmunity and suggests a potential target for therapeutic intervention.

Adoptive cell therapy using antigen-specific CD4−CD8− T regulatory cells to prevent autoimmune diabetes and promote islet allograft survival in NOD mice

A new differentiation pathway for CD4(-)CD8(-) (DN) T cells has recently been identified that exhibits the potent function of peripheral converted DN T cells in suppressing immune responses and provides the potential to treat autoimmune diseases. The aim of this study was to determine if the DN T cells converted from CD4(+) T cells of NOD mice retain the antigen-specific regulatory capacity and prevent autoimmune diabetes in vivo. We also sought to determine if the combination of DN T cells with rapamycin promotes islet allograft survival in autoimmune diabetic NOD recipients. NOD CD4(+) T cells were converted to DN T cells in an in vitro mixed-lymphocyte reaction, with or without GAD65 peptide, as previously reported. The antigen-specific DN T cells were adoptively transferred to NOD/SCID mice, new-onset diabetic NOD mice or islet-allograft-recipient NOD mice as the part of cell-based therapy. The development of diabetes and allograft survival was assessed by monitoring blood glucose levels. NOD CD4(+) T cells were converted in vitro to DN T cells at a rate of 50% and expressed unique cell features. The DN T cells from NOD donors blocked autoimmunity and prevented diabetes in NOD models, and these effects were even greater for GAD65-peptide-primed DN T cells. DN T cells acted in conjunction with rapamycin to suppress alloantigen-triggered T cell proliferation, promoted apoptosis and prolonged islet allograft survival in NOD recipients. Administration of the islet beta cell antigen-specific DN T cells can prevent the development of autoimmune diabetes and promote islet allograft survival in NOD mice.

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome

IntroductionIn Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome.MethodsMale NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores.ResultsLTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01).ConclusionsBlockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.

Anti-CD3 therapy permits regulatory T cells to surmount T cell receptor–specified peripheral niche constraints

Treatment with anti-CD3 is a promising therapeutic approach for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (T reg) cells may be involved, but the evidence has been conflicting. We investigated this issue in mice derived from the NOD model, which were engineered so that T reg populations were perturbed, or could be manipulated by acute ablation or transfer. The data highlighted the involvement of Foxp3+ cells in anti-CD3 action. Rather than a generic influence on all T reg cells, the therapeutic effect seemed to involve an ∼50–60-fold expansion of previously constrained T reg cell populations; this expansion occurred not through conversion from Foxp3− conventional T (T conv) cells, but from a proliferative expansion. We found that T reg cells are normally constrained by TCR-specific niches in secondary lymphoid organs, and that intraclonal competition restrains their possibility for conversion and expansion in the spleen and lymph nodes, much as niche competition limits their selection in the thymus. The strong perturbations induced by anti-CD3 overcame these niche limitations, in a process dependent on receptors for interleukin-2 (IL-2) and IL-7.

Pre-existing autoimmunity determines type 1 diabetes outcome after Flt3-ligand treatment

Redirection of immune responses by manipulation of antigen-presenting cells is an emerging strategy for immunosuppressive treatment of autoimmune diseases. In vivo expansion of dendritic cells (DC) by Fms-like tyrosine kinase-3 (Flt3)-Ligand (FL) treatment was shown to delay diabetes onset in the NOD model of autoimmune diabetes. However, we show here that Flt3 stimulation actually accelerates autoimmunity when autoreactive CD8 T cells are detectable in blood prior to treatment. With autoreactive CD8 cells present, the capacity of FL to expand DCs and induce Treg remained intact, but both numbers and the functional response of islet-specific CD8s were boosted. Also, the inhibitory receptor PD-1 on (autoreactive) CD8 T cells and its ligand PD-L1 on Treg were no longer upregulated. These data highlight the need to pre-screen for T cell autoreactivity prior to generalized DC expansion and illustrate how accelerated disease can occur when the intended initiation of regulatory mechanisms is impaired later in diabetogenesis.

Blockade of OX40 Signals Enhance Survival of Xenoislet Grafts in Spontaneously Diabetic NOD Mice

Role of OX40 blockade was investigated in islet xenograft model in chemically diabetic C57B1/6 and spontaneously diabetic NOD mice. The effect of OX40/OX40L blockade on effector function of diabetogenic T cells was studied in an adoptive transfer model. 2000 IEQ of porcine islets were transplanted under the kidney capsule of diabetic C57B1/6 or NOD mice and were treated i.p. with control Ig, anti-OX40L, or CTLA4Ig. Graft survival was assessed using blood glucose (BG) measurement. Cells from diabetic NOD spleens and pancreatic lymphnodes were injected i.v. into NOD.scid mice. Recipients were treated i.p. with anti-OX40L, CTLA4Ig alone, or in combination; control mice received Ig. Diabetes incidence was assessed using BG measurement. Anti-OX40L treatment delayed xenoislet rejection significantly in chemically diabetic animals, although CTLA4Ig delayed rejection even further. Neither treatment completely prevented rejection. In spontaneously diabetic NOD mice, rejection of xenoislet graft was delayed by anti-OX40L treatment but not by CTLA4Ig. Anti-OX40L alone and in combination with CTLA4Ig significantly delayed diabetes transfer by activated diabetogenic T cells, compared with control and CTLA4Ig treatment. Preliminary mechanistic studies suggest that anti-OX40L treatment preserves Treg numbers, unlike CTLA4Ig that diminishes Tregs. Our studies show that OX40 blockade offers better xenoislet graft survival than CTLA4Ig in spontaneous autoimmune NOD model, suggesting that preactivated T cells use alternate costimulatory pathways, independent of CD28. Results from adoptive transfer studies further support the role of OX40 signaling in effector function of diabetogenic T cells. These data suggest that OX40/OX40L may offer novel therapeutic target for xenoislet graft protection in type 1 diabetic patients.

Cyclin-Dependent Kinase 4 Hyperactivity Promotes Autoreactivity in the Immune System but Protects Pancreatic β Cell Mass from Autoimmune Destruction in the Nonobese Diabetic Mouse Model

Cyclin-dependent kinase 4 (Cdk4) plays a central role in perinatal pancreatic beta cell replication, thus becoming a potential target for therapeutics in autoimmune diabetes. Its hyperactive form, Cdk4R24C, causes beta cell hyperplasia without promoting hypoglycemia in a nonautoimmune-prone mouse strain. In this study, we explore whether beta cell hyperproliferation induced by the Cdk4R24C mutation balances the autoimmune attack against beta cells inherent to the NOD genetic background. To this end, we backcrossed the Cdk4R24C knockin mice, which have the Cdk4 gene replaced by the Cdk4R24C mutated form, onto the NOD genetic background. In this study, we show that NOD/Cdk4R24C knockin mice exhibit exacerbated diabetes and insulitis, and that this exacerbated diabetic phenotype is solely due to the hyperactivity of the NOD/Cdk4R24C immune repertoire. Thus, NOD/Cdk4R24C splenocytes confer exacerbated diabetes when adoptively transferred into NOD/SCID recipients, compared with NOD/wild-type (WT) donor splenocytes. Accordingly, NOD/Cdk4R24C splenocytes show increased basal proliferation and higher activation markers expression compared with NOD/WT splenocytes. However, to eliminate the effect of the Cdk4R24C mutation specifically in the lymphocyte compartment, we introduced this mutation into NOD/SCID mice. NOD/SCID/Cdk4R24C knockin mice develop beta cell hyperplasia spontaneously. Furthermore, NOD/SCID/Cdk4R24C knockin females that have been adoptively transferred with NOD/WT splenocytes are more resistant to autoimmunity than NOD/SCID WT female. Thus, the Cdk4R24C mutation opens two avenues in the NOD model: when expressed specifically in beta cells, it provides a new potential strategy for beta cell regeneration in autoimmune diabetes, but its expression in the immune repertoire exacerbates autoimmunity.

Anti-CD20 Therapy in NOD Model of Type 1 Diabetes (131.26)

Abstract B cells play an important role in the pathogenesis of T1D although T1D is primarily a T cell mediated autoimmune disease. Apart from producing antibodies, B cells are important APCs and a rich source for production of various cytokines. Nonetheless, the precise roles of B cells in promoting diabetes onset and progression have not been defined. Rituximab targets human CD20 causing rapid and specific B cell depletion. A clinical trial is currently launched in treating T1D patients with Rituximab. However, there has not been a single preclinical study to test this particular therapy in T1D. To evaluate the efficacy of anti-CD20 in treating T1D, we have generated a transgenic NOD mouse expressing human CD20 on their B cells. This allows the use of anti-hCD20 antibody (2H7), which is closely related to Rituximab. Our preliminary data showed that a single cycle of anti-hCD20 treatment (4 injections within 10 days) to temporarily deplete B cells significantly delays and/or reduces the onset of diabetes in hCD20 transgenic NOD mice. This was seen at both the disease initiation and pre-diabetic stages. The same treatment also rendered 36% (5/14) of diabetic mice euglycemic, either transiently or for a long period. Anti-hCD20 treatment appears to induce and/or expand regulatory T cells and enhance TGF-β production. Our results provide extremely valuable in vivo information for the human studies using a similar approach.