Prediabetic NOD Mice Research Articles

Costimulatory Blockade Enhances Engraftment and Prevents Anti-Donor Antibody Generation in Nonmyeloablative Conditioned Prediabetic NOD Mice

Chimerism induces tolerance to organ and tissue grafts. Although the toxicity of conditioning has been significantly reduced with nonmyeloablative approaches, it would be beneficial to further reduce the intensity of conditioning in organ transplant recipients. Studies in normal mice and autoimmune NOD mice have revealed that the primary role for conditioning is to control host-versus-graft alloreactivity, allowing for immune-based conditioning to establish chimerism. Recent insights into the mechanism of interaction and crossregulation between innate and adaptive immune responses have led to the development of new approaches for immune-based conditioning. We previously demonstrated that preconditioning of diabetes-prone NOD mice with anti-CD8 monoclonal antibody (mAb) combined with anti-CD154 mAb allowed chimerism to be established with 550 cGy total body irradiation (TBI). Here we investigated whether combined costimulatory blockade can improve the nonmyeloablative regimen and enhance bone marrow chimerism in autoimmune recipients. Prediabetic NOD (H-2d) mice were treated with anti-CD8 antibody (day -3). After 500 cGy TBI (day 0), recipient NOD mice were transplanted with 30 × 106 B10.BR (H-2k) bone marrow cells (day 0) and received anti-CD154, anti-OX40L, and anti- inducible co-stimulatory molecule (ICOS) mAbs intraperitoneally (IP) (day 0, 1, 2, 3). Chimerism and multilineage analysis were quantitated by flow cytometry. Chimeric animals produced multilineage donor chimerism. While 18 prediabetic NOD mice demonstrated an average PB chimerism of ~37% at 1 month and 61% engraftment, 15 of 18 mice lost PB chimerism by 6 months. NOD mice were then transplanted with B10.BR donor skin grafts 1 month post BM transplant. Flow cytometry cross-match assays were performed to detect anti-donor antibody (Ab) in the sera collected 1 month post skin grafting. 44% of chimeric NOD mice (n=9) accepted their skin grafts for over 90 days, while 100% of non-chimeric animals (n=4) rejected their grafts within 60 days. NOD mice transplanted with B10.BR skin grafts alone rejected their grafts within 2 weeks and generated significantly higher levels of donor-specific Abs (mean fluorescence intensity: 251.2 ± 61.5, P<0.0001, n=6) compared to chimeric NOD (4.5 ± 0.6, P<0.0005, n=17) or non-chimeric NOD (12.6 ± 2.4, P<0.0005, n=10). Both chimeric NOD and non-chimeric NOD mice transplanted with 3rd party B6 skin grafts rejected their grafts within 30 days. These results suggest that rejection of skin grafts in chimeric and nonchimeric animals is not associated with anti-donor antibody generation in preconditioned animals. Studies are currently underway to determine if impaired generation of effector/memory T cells (CD44high/CD62Llow/−) is responsible for enhanced engraftment and to determine the potential mechanisms that promote tolerance in prediabetic NOD mice after loss of PB chimerism.

Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes

Although regulatory T cells (Tregs) are well described, identifying autoaggressive effector T cells has proven more difficult. However, we identified CD4loCD40+ (Th40) cells as being necessary and sufficient for diabetes in the NOD mouse model. Importantly, these cells are present in pancreata of prediabetic and diabetic NOD mice, and Th40 cells but not CD4+CD40(-) T cells transfer progressive insulitis and diabetes to NOD.scid recipients. Nonobese-resistant (NOR) mice have the identical T cell developmental background as NOD mice, yet they are diabetes-resistant. The seminal issue is how NOR mice remain tolerant to diabetogenic self-antigens. We show here that autoaggressive T cells develop in NOR mice and are confined to the Th40 subset. However, NOR mice maintain Treg numbers equivalent to their Th40 numbers. NOD mice have statistically equal numbers of CD4+CD25+forkhead box P3+intrinsic Tregs compared with NOR or nonautoimmune BALB/c mice, and NOD Tregs are equally as suppressive as NOR Tregs. A critical difference is that NOD mice develop expanded numbers of Th40 cells. We suggest that a determinant factor for autoimmunity includes the Th40:Treg ratio. Mechanistically, NOD Th40 cells have low susceptibility to Fas-induced cell death and unlike cells from NOR and BALB/c mice, have predominantly low Fas expression. CD40 engagement of Th40 cells induces Fas expression but further confers resistance to Fas-mediated cell death in NOD mice. A second fundamental difference is that NOD Th40 cells undergo much more rapid homeostatic expansion than Th40 cells from NOR mice.

Preferential Costimulation by CD80 Results in IL-10-Dependent TGF-β1+-Adaptive Regulatory T Cell Generation

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.

Weak proinsulin peptide-major histocompatibility complexes are targeted in autoimmune diabetes in mice.

OBJECTIVE—Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a β-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes.RESEARCH DESIGN AND METHODS—The binding of a proinsulin epitope, proinsulin-1(47–64) (PI-1[47–64]), to the MHC class II molecules I-Ag7 and I-Ak was measured using purified class II molecules. T-cell reactivity to the proinsulin epitope was examined in I-Ag7+ and I-Ak+ mice.RESULTS—C-peptide epitopes bound very weakly to I-Ag7 molecules. However, C-peptide–reactive T-cells were induced after immunization in I-Ag7–bearing mice (NOD and B6.g7) but not in I-Ak–bearing mice (B10.BR and NOD.h4). T-cells reactive with the PI-1(47–64) peptide were found spontaneously in the peripancreatic lymph nodes of pre-diabetic NOD mice. These T-cells were activated by freshly isolated β-cells in the presence of antigen-presenting cells and caused diabetes when transferred into NOD.scid mice.CONCLUSIONS—These data demonstrate an inverse relationship between self-peptide–MHC binding and T-cell autoreactivity for the PI-1(47–64) epitope in autoimmune diabetes.

Preferential costimulation by CD80 results in IL‐10 dependent TGF‐beta1+ adaptive regulatory T cell generation

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.

First Signature of Islet β-Cell-Derived Naturally Processed Peptides Selected by Diabetogenic Class II MHC Molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet beta-cells selected by diabetogenic I-A(g7) molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-A(g7) were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in beta cells from normal islets. Peptides bound to I-A(g7) molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet beta-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.

HuFlt3‐L treatment does not protect RIP‐GP and RIP‐NP C57Bl/6 mice against LCMV‐induced diabetes.

Human Fms‐like Tyrosine Kinase3‐L (huFlt3‐L) treatment of prediabetic NOD mice has been shown to increase the intrinsically low numbers of myeloid dendritic cells in these mice, while decreasing insulitis and delaying the progression of diabetes. In this study, we investigated the influence of huFlt3‐L treatment on insulitis and diabetes onset in the LCMV‐induced RIP‐GP (fast onset) or RIP‐NP (slow onset) C57Bl/6 mouse models of T1D. We showed that these mice have no intrinsic abnormality in DC subset development during LCMV infection. As expected, huFlt3‐L treatment dramatically increased the total DC fraction and numbers in both spleen and pancreatic draining LN, with a preferential outgrowth of myeloid DCs (CD11c+B220−CD11b+) over plasmacytoid DCs (CD11c+B220+CD11b−). Moreover, NK cells and NK‐DC cells were also increased, but CD4 and CD8 T cell fractions were reduced. Strikingly, repeated huFlt3‐L administrations, initiated in the prediabetic phase, did not protect against diabetes onset in this model. It is possible that this is correlated with the observed reduced fraction of CD4+CD25+ T cells in the huFlt3‐L‐treated mice. Translation of these data suggests that huFlt3‐L might only be a useful therapeutic agent for T1D patiens with a demonstrated DC (subset) defect.

Purified MHC-Matched Hematopoietic Stem Cell Transplantation Following Total Lymphoid Irradiation and Anti-Thymocyte Globulin (TLI+ATG) Blocks EAE Pathogenesis.

In prior studies, we demonstrated that transplantation of major histocompatibility complex (MHC) matched and mismatched purified hematopoietic cells (HSCs) can block diabetes pathogenesis in pre-diabetic NOD mice. Here we applied this treatment to experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis (MS). C57BL/6 (B6) strains congenic for the CD45 locus were immunized with MOG 35–55 to induce disease. Similar to our studies in NOD mice we observed that disease response after transplantation of MHC-mismatched hematopoietic cells in EAE affected mice was superior to those that received congenic cells. Importantly, mice transplanted with MHC-matched HSC or BM showed no significant improvement in disease symptoms when compared with congenic recipients or disease controls. This result was markedly different from the positive response observed in NOD mice transplanted with MHC-matched HSC. We then sought to optimize translation of this approach by giving total lymphoid irradiation (TLI) plus anti-thymocyte globulin (ATG), a non-myeloablative regimen and permits allogeneic hematopoietic cell engraftment with significant protective effect against GVHD. We found that TLI/ATG treatment in wild type C57BL/6 mice permit engraftment of purified HSC isolated from MHC-matched AKR/B donors and improve disease severity significantly. The level of stable mixed chimerism correlated with the clinical score. While the prior studies demonstrated that a class of regulatory cells that express both nature killer cell and T cell markers (NK-T) are present at high levels in TLI/ATG treated mice, we determined that regulatory CD4+CD25+Foxp3+ cells are also present at a disproportionately high levels as compared to wild type animals. To study the role of regulatory T cell in disease protection, a flexible lentiviral vector platform supporting constitutive expression of a Tet-transactivating component with a selection marker and coordinated conditional expression of miR-shRNA targeting Foxp3 gene products in stem cells is being developed. The detailed mechanism will be discussed. We hope a MHC-matched hematopoietic graft in conjunction with TLI/ATG conditioning shown here to successfully ameliorate EAE can be translated to the treatment of human autoimmune disease.

Role of ICOS pathway in autoimmune and alloimmune responses in NOD mice

Islet allografts are subject to alloimmune and autoimmune destruction when transplanted into autoimmune prone animals or humans. The ICOS-B7h pathway plays a role in alloimmune responses, but its function in autoimmunity against islet cells is controversial. We investigated the role of ICOS signaling in autoimmune and alloimmune responses in NOD mice. ICOS blockade prevents development of spontaneous disease in pre-diabetic NOD mice. Furthermore, while ICOS blockade prolongs graft survival in a fully mismatched non-autoimmune islet allograft model in C57BL/6 recipients, it has no beneficial effect in reversing diabetes in models of islet transplantation in NOD mice involving autoimmunity alone or both allo- and autoimmunity. Interestingly, ICOS blockade is effective in prolonging heart allograft (not subject to tissue-specific autoimmunity) survival in NOD mice. We conclude that in islet transplantation and autoimmune diabetes, ICOS blockade can be effective in inhibiting alloimmunity and preventing autoimmunity but is ineffective in inhibiting recurrence of autoimmunity.

Aberrant endometrial features of pregnancy in diabetic NOD mice.

Pregnant diabetic women are at a 4-12 times higher risk for preeclampsia, an urgent acute-onset complication of mid- to late gestation, than normal pregnant women. Hallmarks of preeclampsia are hypertension, proteinuria, and incomplete modification of endometrial spiral arteries. Transient proangiogenic lymphocytes called uterine natural killer (uNK) cells are implicated in human and rodent spiral artery modification. We studied mid- to late gestations in spontaneously type 1 diabetic NOD mice to investigate whether diabetes alters uNK cell homing and/or function. Normoglycemic, pre-diabetic, and diabetic NOD mice and controls were mated. Lymphocytes and endometrial endothelium and decidua were studied histologically and in functional assays. Conception accelerated progression to overt diabetes in NOD females who had limited spiral artery development, heavier placentas, and lighter fetuses displaying numerous birth defects compared with controls. UNK cell numbers were reduced in the decidua basalis of diabetic females, whereas interferon-gamma production was elevated. In diabetic NOD mice, decidual expression of the mucosal vascular addressin cell adhesion molecule (MAdCAM)-1 was aberrant in position, whereas vascular cell adhesion molecule (VCAM)-1 expression was reduced. Assays of lymphocyte adhesion to tissue sections under shear forces indicated that diabetes compromises the potential homing functions of both endometrial endothelium and peripheral NK cells. In diabetes, gestational endometrium has immune and vascular defects that likely contribute to murine fetal loss and birth defects. Analogous problems and preeclampsia in diabetic women may involve similar mechanisms.

Granulocyte-Macrophage Colony-Stimulating Factor Prevents Diabetes Development in NOD Mice by Inducing Tolerogenic Dendritic Cells that Sustain the Suppressive Function of CD4+CD25+ Regulatory T Cells

Autoimmune diabetes results from a breakdown of self-tolerance that leads to T cell-mediated beta-cell destruction. Abnormal maturation and other defects of dendritic cells (DCs) have been associated with the development of diabetes. Evidence is accumulating that self-tolerance can be restored and maintained by semimature DCs induced by GM-CSF. We have investigated whether GM-CSF is a valuable strategy to induce semimature DCs, thereby restoring and sustaining tolerance in NOD mice. We found that treatment of prediabetic NOD mice with GM-CSF provided protection against diabetes. The protection was associated with a marked increase in the number of tolerogenic immature splenic DCs and in the number of Foxp3+CD4+CD25+ regulatory T cells (Tregs). Activated DCs from GM-CSF-protected mice expressed lower levels of MHC class II and CD80/CD86 molecules, produced more IL-10 and were less effective in stimulating diabetogenic CD8+ T cells than DCs of PBS-treated NOD mice. Adoptive transfer experiments showed that splenocytes of GM-CSF-protected mice did not transfer diabetes into NOD.SCID recipients. Depletion of CD11c+ DCs before transfer released diabetogenic T cells from the suppressive effect of CD4+CD25+ Tregs, thereby promoting the development of diabetes. These results indicated that semimature DCs were required for the sustained suppressive function of CD4+CD25+ Tregs that were responsible for maintaining tolerance of diabetogenic T cells in NOD mice.

Continuous stimulation of human glucagon-like peptide-1 (7–36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes

We examined the effect of glucagon-like peptide-1 (GLP-1) on the development of diabetes and islet morphology in NOD mice by administering GLP-1 to prediabetic mice. Eight-week-old female NOD mice were infused subcutaneously with human GLP-1 via a mini-osmotic pump for 4 or 8 weeks. In mice treated with GLP-1 for 4 weeks, blood glucose levels and body weight were measured. An intraperitoneal glucose tolerance test (IPGTT) and evaluation of insulitis score were also performed. Beta cell area, proliferation, apoptosis, neogenesis from ducts and subcellular localisation of forkhead box O1 (FOXO1) were examined by histomorphometrical, BrdU-labelling, TUNEL, insulin/cytokeratin and FOXO1/insulin double-immunostaining methods, respectively. Mice treated with human GLP-1 for 4 weeks had lower blood glucose levels until 2 weeks after completion of treatment, showing improved IPGTT data and insulitis score. This effect continued even after cessation of the treatment. In addition to the increase of beta cell neogenesis, BrdU labelling index was elevated (0.24 vs 0.13%, p < 0.001), while apoptosis was suppressed by 54.2% (p < 0.001) in beta cells. Beta cell area was increased in parallel with the translocation of FOXO1 from the nucleus to the cytoplasm. The onset of diabetes was delayed in mice treated with GLP-1 for 4 weeks, while mice treated with GLP-1 for 8 weeks did not develop diabetes by age 21 weeks compared with a 60% diabetes incidence in control mice at this age. Continuous infusion of human GLP-1 to prediabetic NOD mice not only induces beta cell proliferation and neogenesis, but also suppresses beta cell apoptosis and delays the onset of type 1 diabetes.

Resistance to the Induction of Mixed Chimerism in Spontaneously Diabetic NOD Mice Depends on the CD40/CD154 Pathway and Donor MHC Disparity

Blockade of CD40/CD154 pathway has proven effective in promoting the induction of allogeneic mixed chimerism. Using NOD mouse model of human type 1 diabetes, we investigated whether allogeneic mixed chimerism can be induced in prediabetic NOD mice and in spontaneously diabetic NOD mice under nonmyeloablative and irradiation-free conditioning therapy and anti-CD154 mAb as a short-term posttransplant treatment. We found that spontaneously diabetic NOD mice are more resistant to the induction of allogeneic mixed chimerism than prediabetic NOD mice under our nonmyeloablative and irradiation-free conditioning therapy. This alloresistance in spontaneously diabetic NOD mice is dependent on the CD40/CD154 pathway and donor MHC disparity.

Cyclophosphamide treatment of the NOD mouse reveals quasi-public dominant T cell clones (130.34)

Abstract To define the major components of the autoreactive T cell repertoire that arise spontaneously in the NOD mouse, we used CDR3 length spectroscopy to study the T cell repertoire in PLN from pre-diabetic NOD mice to identify public dominant clones; andto identify potentially pathogenic clones in islet-infiltrating cells that persist after Cyclophosphamide (Cyp) treatment. We found that any public T cell expansions were identified in PLN of female NOD mice at 4 and 10 weeks of age. Two expansions in the rearrangements Vb5.2/Jb2.5 and Vb10/Jb1.5 were found in 60% and 50% of 4 week old NOD mice. These expansions can be useful in the study of how the T cell repertoire can be modified after islet transplantation. After Cyp-treatment, the Vb4, Vb5.2, Vb10 and Vb12 families showed dominant expansions. Interestingly, we found a unique Vb4-"195 nucleotide" peak that was found in 10 out of 11 (91%) Cyp-treated mice. In contrast, this peak was not detected in untreated NOD mouse islets. In the Vb12 family, a "200" peak was prominent in 6 out of 10 (60%) Cyp-treated mice. These expansions may represent the more pathogenic clones. Currently, we are sequencing these expansions to establish whether the Cyp-induced expansions represent truly unique clones. We believe that these Cyp experiments will provide the possibility of distinguishing between regulatory and aggressive clones. This work was supported by JDRF 3-2005-336.

Systemic Expression of Heme Oxygenase-1 Ameliorates Type 1 Diabetes in NOD Mice

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.

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.

TRPV1 + Sensory Neurons Control β Cell Stress and Islet Inflammation in Autoimmune Diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.

Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: Role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W ( N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, l-NAME ( N G-nitro- l-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78 ± 4 vs. 88 ± 2%, respectively, P < 0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic ( P < 0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals ( P < 0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased ( P < 0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.

Control of Autoimmune Myocarditis and Multiorgan Inflammation by Glucocorticoid-Induced TNF Receptor Family-Related Proteinhigh, Foxp3-Expressing CD25+ and CD25− Regulatory T Cells

Anomalies of naturally occurring CD4+ regulatory T cells (Treg) cause severe autoimmune/inflammatory diseases in humans and rodents. The transcription factor Foxp3 is currently the most specific marker for natural CD4+ Treg, but it would be useful if other Treg markers, particularly cell surface molecules, could be elucidated. We demonstrate in this study that the vast majority of Foxp3-expressing CD4+ T cells (whether CD25+ or CD25-) show constitutive high-level expression of glucocorticoid-induced TNFR family-related gene/protein (GITR). Transfer of T cell or thymocyte suspensions depleted of GITR(high) cells produces in BALB/c nude mice a wider spectrum and more severe forms of autoimmune diseases than does transfer of similar cell suspensions depleted of CD25+ CD4+ T cells only. Notably, mice that receive cells depleted of GITR(high) populations develop severe multiorgan inflammation that includes fatal autoimmune myocarditis resembling giant cell myocarditis in humans, accompanying high-titer anti-myosin autoantibodies. Similar transfer of GITR(high)-depleted cells from prediabetic NOD mice to NOD-SCID mice accelerates the development of diabetes and induces skeletal muscle myositis and other autoimmune/inflammatory diseases. We conclude that GITR(high), Foxp3-expressing natural Treg, containing both CD25+ and CD25- cell populations, contribute to preventing a variety of autoimmune/inflammatory diseases, and depletion of these cells allows the activation of even weak or rare autoreactive T cells yielding widespread severe autoimmune disease. Diseases induced in this way include many which have been suspected of an autoimmune etiology in humans without much evidence. GITR(high), Foxp3-expressing natural Treg represent a potential target for the treatment and prevention of these diseases.

Survival of Microencapsulated Islets at 400 Days Posttransplantation in the Omental Pouch of NOD Mice

The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, beta-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.