Autoimmune Diabetes In NOD Mice Research Articles

The Role of Toll‐Like Receptors 3 and 9 in the Development of Autoimmune Diabetes in NOD Mice

Innate immunity is mediated, at least in part, through a number of receptors known as Toll-like receptors (TLRs), which are activated by different microbial stimuli. Adaptive immunity, including autoimmunity, follows the innate response in a more specific manner. To investigate the roles of TLR3 and TLR9 in the development of type 1 diabetes, we generated NOD mice that are deficient in TLR3 and 9, respectively. There was no obvious difference in the incidence of spontaneous diabetes between TLR3-deficient mice and TLR3 heterozygous mice. However, TLR9-deficient mice were markedly protected from the disease compared to TLR9 heterozygous mice. Our results suggest that different TLRs play a varying role in autoimmune diseases.

In Vivo BLyS/BAFF Neutralization Ameliorates Islet-Directed Autoimmunity in Nonobese Diabetic Mice

B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR-->FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR-->FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.

Effects of the immunomodulator, VGX‐1027, in endotoxin‐induced uveitis in Lewis rats

VGX-1027 is a novel, low molecular weight, immunomodulatory compound that has shown efficacy against a variety of immuno-inflammatory disease models in animals including autoimmune diabetes in NOD mice, collagen-induced arthritis and chemically induced inflammatory colitis. Here, we have studied the effects of VGX-1027 on the development of endotoxin-induced uveitis (EIU) in male Lewis rats, as a model of inflammatory ocular diseases in humans. EIU was induced by a single footpad injection of 200 microg lipopolysaccharide (LPS). Groups of rats were treated with either VGX-1027 (25 mg kg(-1)) or its vehicle at different time points (30 min, 6 h or 12 h) after the challenge with LPS or, as positive control, with dexamethasone. The rats were killed within 16 h after LPS challenge, and the eyes and aqueous humor were collected to study serological, immunological and histological signs of EIU. The rats treated with VGX-1027 within 6 h after LPS challenge exhibited milder clinical, histological and laboratory signs of EIU than those treated with vehicle. This study provides the first evidence that systemic treatment with VGX-1027 counteracts the uveitis-inducing effect of LPS in rats and suggests that this drug may have potential in the treatment of immuno-inflammatory conditions of the eye in humans.

Inhibition of autoimmune diabetes in NOD mice with serum from streptococcal preparation (OK-432)-injected mice

We have recently reported that systemic and chronic administration of recombinant tumour necrosis factor alpha (TNF-alpha), as well as streptococcal preparation (OK-432), inhibits development of insulin-dependent diabetes mellitus (IDDM) in NOD mice and BB rats, models of IDDM. In this study we examined whether serum containing endogenous TNF induced by OK-432 injection could inhibit IDDM in NOD mice. Treatment twice a week from 4 weeks of age with OK-432-injected mouse serum, which contained endogenous TNF (75U), but not IL-1, IL-2 and interferon-gamma (IFN-gamma) activity, reduced the intensity of insulitis and significantly inhibited the cumulative incidence of diabetes by 28 weeks of age in NOD mice, as compared with the incidence in non-treated mice (P less than 0.01) and in mice treated with control serum (P less than 0.02). This inhibitory effect of the serum was diminished, although not significantly, by neutralization of serum TNF activity with anti-mouse TNF antibody. In the mice treated with the serum from OK-432-injected mice, Thy-1.2+ or CD8+ spleen cells decreased (P less than 0.01) and surface-Ig+ (S-Ig+) cells increased (P less than 0.05), whereas the proliferative response of spleen cells to concanavalin A (P less than 0.01) and lipopolysaccharide (P less than 0.05) increased. The results indicate that the inhibition by OK-432 treatment of IDDM in NOD mice was partially mediated by serum factors including endogenous TNF.

Allogeneic neonatal blood transfusion without recipient preconditioning is a novel strategy to treat autoimmune diabetes in NOD mice

The efficacy of neonatal blood derived from diabetes‐resistant mice to reverse autoimmune diabetes was evaluated in NOD mice under irradiation‐free and immunosuppression‐free conditions. Transfusion of allogeneic neonatal blood containing Sca‐1+ pluripotential stem/progenitor cells significantly delayed the onset and incidence of diabetes. Neonatal blood transfusion also reduced the mononuclear cell infiltration of the islets of Langerhans. Flow cytometry and RT‐PCR analysis revealed the induction of chimerism in neonatal blood transfused NOD mice. Splenocytes from most cured NOD mice failed to transfer diabetes into immunodeficient NODscid mice, indicating deletion/suppression of diabetogenic T cells in transfused mice. Protection afforded by neonatal blood transfusion was not simply due to alterations in T regulatory subsets. Splenic T cells from both cured and unprotected mice that received neonatal blood transfusion proliferated poorly and produced lower amounts of IL‐2 and IL‐17A when challenged with allogeneic antigens in vitro. These data indicate that neonatal blood transfusion can serve as a novel strategy to induce lymphoid chimerism, T cell tolerance and deletion/suppression of diabetogenic T cells in the absence of cytoreduction or costimulatory blockade.

A Th1-Recognized Peptide P277, When Tandemly Repeated, Enhances a Th2 Immune Response toward Effective Vaccines against Autoimmune Diabetes in Nonobese Diabetic Mice

Subunit immunogens containing tandemly repeated copies of T and B cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. To investigate whether the increased copies of the Th2-activated peptide sequence will enhance the Th2-like immune response, we compared the cytokine secreted in mice that inoculated with two immunogens containing one or six tandemly repeated copies of a Th2-activated peptide sequence P277. Immunization of mice with a 6xP277 fusion protein elicited much higher levels of Th2-type cytokines and lower Th1-type cytokines than with a fusion protein with one P277 peptide. The data of tandemly repeated peptide P277 potentiate the anti-inflammatory in NOD mice, most likely associated with a Th1 to Th2 cytokine shift specific for the autoimmune T cells, which suggested that application of multiple tandem repeats of a Th2-activated epitope is an efficient method to enhance the anti-inflammatory immune response by shifting the immune response from Th1-like to Th2-like. The subunit immunogens containing tandemly repeated copies of peptide P277 might be effective vaccines against autoimmune diabetes in NOD mice.

Gene delivery GAD 500 autoantigen by AAV serotype 1 prevented diabetes in NOD mice: Transduction efficiency do not play important roles

We previously found that adeno-associated viral vector serotype 2 (AAV-2) muscle gene delivery of GAD 500–585 autoantigen efficiently prevented autoimmune diabetes in NOD mice. Recent reports suggest that AAV vectors based on serotype 1 (AAV-1) transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2 to 1000-fold. To determine whether this increased efficacy of AAV-1 could result in increased therapeutic effects in mice, we constructed rAAV 1/GAD 500–585 vectors and compared their effects in preventing autoimmune diabetes in NOD mice with those of rAAV2/GAD 500–585 after muscle injection. rAAV 1/GAD 500–585 gene therapy prevented diabetes in NOD mice. However, although much higher level of GAD 500–585 expression was found in mice using AAV-1 as gene delivery vector than those using AAV-2, no increased efficiency of AAV-1 vectors were found in their capability to prevent autoimmune diabetes, as higher titers of rAAV 1/GAD 500–585 virus (3 × 10 11 v.g./mouse) were needed to obtain therapeutic effects in NOD mice, a titer not different from that of AAV-2. Protection resulted from rAAV 1/GAD 500–585 gene therapy were marked by enhanced Th2 immune response and up-regulated CD4 +Foxp3 +T regulatory cells, which might actively suppress effector T cells in NOD mice. As here we found that the therapeutic effects of AAV1 were not positively correlated to it's transduction efficiency, our data suggested that the safety and other factors besides efficiency should be considered when use different AAV serotype to treat autoimmune disease.

Human chorionic gonadotropin is an immune modulator and can prevent autoimmune diabetes in NOD mice

Expression of T helper (Th)1 cytokine mRNA in pregnant women is known to be inversely correlated with serum human chorionic gonadotropin (hCG). Type 1 diabetes is a Th1-mediated autoimmune disease, in which intervention at an early stage of the autoimmune process can prevent disease progression. We hypothesised that immune modulation by treating young NOD mice with hCG may prevent diabetes. Female NOD mice were treated with hCG or recombinant hCG from 3 to 15 weeks of age and the incidence of diabetes and development of insulitis was determined. CD4(+) and CD8(+) T cell populations, T cell proliferation, cytokine production and CD4(+)CD25(+) regulatory T cells were examined and adoptive transfer experiments were performed. Both purified and recombinant hCG prevented development of diabetes in NOD mice. hCG decreased the proportion and number of CD4(+) and CD8(+) T cells and inhibited T cell proliferative responses against beta cell antigens. hCG treatment suppressed IFN-gamma production, but increased IL-10 and TGF-beta production in splenocytes stimulated with anti-CD3 antibody. hCG treatment also suppressed TNF-alpha production in splenocytes stimulated with lipopolysaccharide. Furthermore, hCG treatment increased the CD4(+)CD25(+)/CD4(+) T cell ratio in spleen and pancreatic lymph nodes. Depletion of CD4(+)CD25(+) T cells from splenocytes of hCG-treated NOD mice abolished their preventive effect on diabetes transfer. We conclude that hCG has an immunomodulatory effect by downregulating effector cells, including Th1 cells, CD8(+) T cells and macrophages, and increasing the CD4(+)CD25(+)/CD4(+) T cell ratio, thus preventing autoimmune diabetes in NOD mice.

The Role of Vitamin D in the Pathogenesis of NOD Mouse Diabetes

The existence of β-cell dysfunction and insulin resistance in vitamin D-deficient individuals on top of the demonstrated presence of receptors for 1,25-dihydroxyvitamin D3, the active metabolite of vitamin D, in pancreatic islet β- cells and immune cells have lead to scientific and clinical interest in vitamin D with respect to its potential role in the pathogenesis of type 1 diabetes. Also, its therapeutic potential in the prevention of type 1 diabetes has been studied, especially since the availability of synthetic analogues of the molecule that lack its calcemic effects. Solid evidence is available on the detrimental effects of vitamin D deficiency on insulin synthesis and secretion in animal models as well as in humans. Interventions with pharmacological doses of 1,25-dihydroxyvitamin D3 are able to delay onset of autoimmune diabetes in NOD mice mainly through immune modulation. Keywords: Vitamin D, 1,25(OH)2D3, type 1 diabetes, β-cell, NOD mouse, prevention, vitamin D deficiency, analogue

Role of Fas in Autoimmune Diabetes (128.30)

Abstract We have studied the role of Fas in the induction of autoimmune diabetes in NOD mice and found that although Fas is required for the development of diabetes, early overexpression of this molecule on pancreatic β cells delays diabetes onset and reduces diabetes incidence in NOD mice. Moreover, Fas overexpression on β cells is not sufficient to restore the diabetic phenotype in Fas-deficient NOD mice. On the other hand we show that induction of β cell death by CD4 T cells is dependent on the expression of Fas on β cells. IL-1β and IFNγ are key cytokines involved in Fas up-regulation on mouse β cells. However, here we demonstrate that IL-1β is not required for either spontaneous t or adoptively transferred diabetes. Interestingly we have also observed that FasL overexpression on β cells seems to confer immune-privilege to islet cells against diabetogenic CD4 T cells. We hypothesize that early, immune-independent, Fas expression on β cells protects from diabetes by promoting immune tolerance to islet antigens, while late, cytokine-mediated induction of Fas has the opposite effect by promoting massive β cell death and diabetes. There is redundancy regarding the cytokines involved in Fas up-regulation on β cells in vivo.

Expansion of GAD65–Specific Immunoregulatory Effector Cells by Biolistic-Mediated Gene Delivery Prevents Autoimmune Diabetes in NOD Mice (131.14)

Abstract Intramuscular (i.m.) vaccination with plasmid DNA (pDNA) encoding beta cell autoantigens such as glutamic acid decarboxylase 65 (GAD65) is an approach to suppress ongoing Type 1 diabetes (T1D) in NOD mice. However, protection is dependent on co-treatment with pDNAs encoding IL-4 and/or IL-10, and the subsequent induction of type 2 CD4+ T effectors. To enhance the therapeutic efficacy of pDNA vaccination, we explored epidermal delivery of pDNA via “gene gun”. Notably, reports have shown induction of a preferential type 2-like CD4+ T cell response when pDNA encoding antigen-only is delivered to the epidermis via gene gun. We hypothesized that biolistic delivery of pDNA would preferentially induce type 2 CD4+ T effectors and suppress established beta cell autoimmunity in NOD mice. Groups of 10 wk-old NOD female mice were treated four times with pDNA encoding GAD65-Ig (pGAD65) administered i.m. or delivered by gene gun on 1.6 uM gold particles. Whereas i.m. injection of pGAD65 resulted in preferential induction of GAD65-specific CD4+ Th1 cells and no protection against T1D, the majority of NOD mice treated with gene gun delivered pGAD65 remained diabetes-free and protection correlated with increased GAD65-specific IL-4 secreting CD4+ T cells. These results demonstrate that gene-gun delivered pDNA encoding beta cell autoantigen-only is an effective strategy to induce immunoregulatory CD4+ T cells and suppress T1D.

Harnessing the Fas pathway for type 1 diabetes therapy (128.29)

Abstract The Fas pathway is not required for T cell activation yet loss-of-function mutation that inactivates Fas (lpr) or FasL (gld) prevents autoimmune diabetes in NOD mice without interfering with normal immune responses. However, the Fas pathway has not been considered a viable therapeutic option because these mutations also cause double negative (DN) T cell lymphoproliferation and lupus-like syndrome. Using bone marrow chimeras and adoptive transfer, we show that gld mutation on either hemopoietic or nonhemopoietic compartment is equally protective from autoimmune diabetes; however, gld mutation on hemopoietic cells is responsible for most of DN T cell lymphoproliferation. Therefore, limiting gld mutation to the nonhemopoietic tissue completely controls diabetogenicity of wild type T cells without causing lymphoproliferation. The protection is recapitulated therapeutically by treating NOD-wt mice with FasL neutralizing antibody. Anti-FasL treatment curtailed insulitis and did not lead to lupus-like disease or DN T cell lymphoproliferation. These results suggest the potential of generating a new class of immunotherapy that is based on targeting T cell apoptosis inducing ligand rather than costimulatory pathways.

Modulating Protective and Pathogenic CD4+ Subsets via CD137 in Type 1 Diabetes

CD137 (TNFRSF9) is an activation-inducible T-cell costimulatory molecule and a member of the tumor necrosis factor (TNF) receptor superfamily. Cd137 is also a candidate gene (in the Idd9.3 interval) for autoimmune diabetes in NOD mice. Here, we demonstrate that anti-CD137 treatment protects NOD mice from diabetes. Anti-CD137-treated mice are not protected from insulitis and still harbor pathogenic T-cells, as demonstrated by transfer studies. Transfer of CD4(+), but not CD8(+), cells from anti-CD137-treated pre-diabetic NOD mice into NOD-scid mice delayed diabetes onset. Anti-CD137 treatment significantly increased the number of CD4(+)CD25(+) cells, which demonstrated intracellular Foxp3 expression and in vitro suppressive activity. The CD4(+)CD25(+) cell subset from anti-CD137-treated mice transferred complete protection from diabetes, whereas the CD4(+)CD25(-) cell subset offered no significant protection. Anti-CD137 treatment of NOD-scid recipients of diabetic spleen cells, however, hastened the onset of disease, showing that the effect of anti-CD137 treatment depends on the balance of pathogenic and protective cells. These results support a critical role for CD137 acting in the early phase of autoimmune diabetes to enhance regulatory cell production. Disease-associated CD137 alleles are likely ineffectual at stimulating a regulatory T-cell population sufficient to prevent disease.

A Potent Immunomodulatory Compound, (S,R)-3-Phenyl-4,5-dihydro-5-isoxasole Acetic Acid, Prevents Spontaneous and Accelerated Forms of Autoimmune Diabetes in NOD Mice and Inhibits the Immunoinflammatory Diabetes Induced by Multiple Low Doses of Streptozotocin in CBA/H Mice

(S,R)-3-Phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027) is an isoxazole compound that exhibits various immunomodulatory properties. The capacity of VGX-1027 to prevent interleukin (IL)-1beta plus interferon-gamma-induced pancreatic islet death in vitro prompted us to evaluate its effects on the development of autoimmune diabetes in preclinical models of human type 1 diabetes mellitus (T1D). Administration of VGX-1027 to NOD mice with spontaneous or accelerated forms of diabetes induced either by injection of cyclophosphamide or by transfer of spleen cells from acutely diabetic syngeneic donors markedly reduced the cumulative incidence of diabetes and insulitis. In addition, VGX-1027 given either i.p. or p.o. to CBA/H mice made diabetic with multiple low doses of streptozotocin successfully counteracted the development of destructive insulitis and hyperglycemia. The animals receiving VGX-1027 exhibited reduced production of the proinflammatory mediators tumor necrosis factor-alpha, IL-1beta, macrophage migration inhibitory factor, and inducible nitric-oxide synthase-mediated nitric oxide generation in both pancreatic islets and peripheral compartments. These results indicate that VGX-1027 probably exerts its antidiabetogenic effects by limiting cytokine-mediated immunoinflammatory events, leading to inflammation and destruction of pancreatic islets. VGX-1027 seems worthy of being considered as a candidate drug in the development of new therapeutic strategies for the prevention and early treatment of T1D.

Costimulation Blockade of Both Inducible Costimulator and CD40 Ligand Induces Dominant Tolerance to Islet Allografts and Prevents Spontaneous Autoimmune Diabetes in the NOD Mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4+CD25+ T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into naïve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti–CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.

OR.92. Ligand Independent Form of CTLA-4 Induced by Antisense Exon Skipping Alters T‐Cell Activity and Inhibits Autoimmune Diabetes in Nod Mice

OR.92. Ligand Independent Form of CTLA-4 Induced by Antisense Exon Skipping Alters T‐Cell Activity and Inhibits Autoimmune Diabetes in Nod Mice

Costimulation Blockade of Both Inducible Costimulator and CD40 Ligand Induces Dominant Tolerance to Islet Allografts and Prevents Spontaneous Autoimmune Diabetes in the NOD Mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into naïve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.

Insulin-dependent diabetes loci Idd5 and Idd9 increase sensitivity to experimental autoimmune encephalomyelitis

The spontaneous development of autoimmune diabetes in NOD mice suggests that they are unable to establish and maintain immunologic self-tolerance. Congenic NOD mice expressing B10-derived alleles are protected from pancreatic beta cell destruction and autoimmune diabetes. To determine if the B10 alleles in loci Idd5 and Idd9 could influence susceptibility to autoimmunity in other organs, we compared MOG35–55-induced EAE in NOD mice to that of diabetes-resistant NOD.B10.Idd5 and NOD.B10.Idd9 mice. Surprisingly, the severity and chronicity of EAE were enhanced in the diabetes-resistant congenic mice. Our findings indicate that some alleles may influence susceptibility to immune-mediated damage in an organ or tissue-specific fashion, and highlight the necessity of disease-specific investigations.

Strain differences in allele and expression levels of CD72 on B-lymphocytes from NOD, AKR, NON and C57BL/6 mice

The B-lymphocyte marker CD72 has multiple alleles and serves crucial and nonredundant roles in B-cell development and activation. B-lymphocytes play an important role in development of autoimmune diabetes in NOD mice, therefore we examined the patterns of expression of CD72 and its alloantigens on splenic lymphocytes from 4-week-old NOD/LtJ mice. Comparisons of CD72 expression were made between NOD mice and three non-diabetic strains, NON/LtJ mice, C57BL/6J and AKR/J. Use of allele-specific monoclonal antibodies revealed that the previously uncharacterized NON strain expresses either the a or d allele, whereas NOD and AKR mice were confirmed to express the rare c allele. Flow cytometric analysis revealed differential expression between the strains. Whereas NON, C57BL/6 and AKR mice expressed CD72 on 98, 94 and 92% of their B-lymphocytes (CD19 + cells), the NOD mice only expressed this regulatory marker on 78% of their B-lymphocytes. Furthermore, CD72 expression levels on CD72 positive cells were lower in NOD mice than in other three non-diabetic strains. The presence of the CD72c allele, as well as its low level of expression in NOD mice at 4 weeks of age, may be associated with B-lymphocyte hyper-responsiveness and resistance to activation-induced cell death.

Cathepsin L is essential for onset of autoimmune diabetes in NOD mice.

Lysosomal proteases generate peptides presented by class II MHC molecules to CD4+ T cells. To determine whether specific lysosomal proteases might influence the outcome of a CD4+ T cell-dependent autoimmune response, we generated mice that lack cathepsin L (Cat L) on the autoimmune diabetes-prone NOD inbred background. The absence of Cat L affords strong protection from disease at the stage of pancreatic infiltration. The numbers of I-A(g7)-restricted CD4+ T cells are diminished in Cat L-deficient mice, although a potentially diabetogenic T cell repertoire persists. Within the CD4+ T cell compartments of Cat L-deficient mice, there is an increased proportion of regulatory T cells compared with that in Cat L-sufficient littermates. We suggest that it is this displaced balance of regulatory versus aggressive CD4+ T cells that protects Cat L-deficient mice from autoimmune disease. Our results identify Cat L as an enzyme whose activity is essential for the development of type I diabetes in the NOD mouse.