Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine

BACKGROUND: Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice. METHODS: Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation. RESULTS: Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice. CONCLUSIONS: KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.     

Interleukin-4 or interleukin-10 expressed from adenovirus-transduced syngeneic islet grafts fails to prevent beta cell destruction in diabetic NOD mice

BACKGROUND: We performed ex vivo adenoviral gene transfer in a mouse pancreatic islet transplant model to test the efficacy of this expression system. We then determined whether adenoviral-mediated expression of mouse interleukin (IL) 4 or IL-10 from transduced syngeneic islet grafts could prevent disease recurrence in diabetic nonobese diabetic (NOD) mice. METHODS: An adenoviral vector expressing beta-galactosidase (AdCMV betaGal) was used to transduce BALB/c islets (2.5 x 10(3) plaque-forming units/islet), which were analyzed for glucose responsiveness, islet cell recovery, and efficiency of gene transfer. In vivo function and reporter gene expression were examined with AdCMV betaGal-transduced islet grafts in alloxan-induced diabetic syngeneic recipients. Adenoviruses expressing either IL-4 or IL-10 were used in a similar fashion to infect NOD islets, which were characterized in vitro, as well as transplanted into diabetic syngeneic recipients. RESULTS: In vitro functional studies showed no significant difference between control or transduced islets, with 50+/-4% of AdCMV betaGal-infected islet cells staining positive for beta-galactosidase. Transplant recipients became nomoglycemic within 48 hr after transplant, and, although beta-galactosidase expression decreased over time, it was detectable in the graft for up to 8 weeks. Despite the nanogram quantities of IL-4 or IL-10 produced/day from each graft equivalent in vitro, transduced and transplanted NOD islets failed to prevent disease recurrence. CONCLUSIONS: These results suggest that adenoviruses are efficient for at least medium term gene expression from islets in vivo, but neither IL-4 nor IL-10 alone can prevent autoimmune disease recurrence in NOD mice.     

TNF-alpha down-regulates type 1 cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.     

Immunosuppression preventing concordant xenogeneic islet graft rejection is not sufficient to prevent recurrence of autoimmune diabetes in nonobese diabetic mice

BACKGROUND: We and others have reported previously that the immunosuppressant, leflunomide (Lef), can prevent allogeneic and xenogeneic islet graft rejection in streptozocin (STZ)-induced diabetic animals. However, whether Lef required to prevent islet graft rejection is sufficient to prevent the recurrence of autoimmune diabetes has not been addressed. METHODS: The effect of Lef on concordant xenogeneic islet graft in STZ-induced diabetic mice and autoimmune nonobese diabetic (NOD) mice were studied. Then, whether Lef prevents the onset of spontaneous diabetes in young NOD mice and the recurrence of diabetes after major histocompatibility complex (MHC)-matched islet transplantation in diabetic NOD mice were investigated. RESULTS: In STZ-induced diabetic BALB/c mice, Lef treatment significantly prolonged rat islet graft survival. However, Lef could not significantly prolong rat islet graft survival in autoimmune diabetic NOD mice. For prevention studies, treatment with Lef at 30 mg/ kg/day from 4 weeks to 20 weeks of age significantly reduced the incidence of spontaneous diabetes in NOD mice. However, when the NOD mice were treated from 8 to 24 weeks of age, the incidence of spontaneous diabetes was not significantly reduced as compared to the incidence of diabetes in the untreated female NOD mice at 28 weeks of age. Finally, in the MHC-matched islet transplant model, Lef could not significantly prolong MHC-matched nonobese diabetes-resistant mice islet graft survival in NOD mice. CONCLUSIONS: Lef preventing concordant xenogeneic islet graft rejection is not sufficient to prevent the recurrence of autoimmune diabetes in NOD mice. We believe that controlling autoimmunity after islet transplantation will lead the way to promote successful clinical islet transplantation in the future.     

Selective training of the vastus medialis muscle using electrical stimulator for chondromalacia patella

Islet allografts transplanted into Type I diabetic recipients may be destroyed by allorejection or recurrent autoimmune diabetes. We studied islet transplantation in three murine models in order to determine the relative sensitivity of autoimmunity and alloimmunity to two immunosuppressive agents that may be useful in clinical islet transplantation: 15-deoxyspergualin (DSG) and anti-CD4 antibody (GK 1.5). In the model in which only allorejection occurs (BALB/c islets transplanted into streptozotocin-induced diabetic CBA or streptozotocin-induced diabetic NOD recipients), both DSG and anti-CD4 antibody treatment led to indefinite survival of allogeneic islets (>100 days in both treatments). In the second model in which only recurrent autoimmunity can destroy islet grafts (islets from NOD donors transplanted into spontaneously diabetic NOD recipients), only anti-CD4 treatment caused prolonged graft survival [MST 36.7 +/- 6.8 days vs 9.8 +/- 1.8 days (controls), P < 0.0002]. Treatment with DSG did not cause any increase in graft survival (MST 12.6 +/- 5.4 days, NS). Finally, using a model in which both autoimmunity and allorejection may occur (BALB/c to spontaneously diabetic NOD mice), treatment with anti-CD4 caused marked graft prolongation [42.0 +/- 14.5 days vs 7.2 +/- 0.8 days (control), P < 0.002] while DSG again did not prolong graft survival with respect to untreated recipients (9.8 +/- 3.0, NS). We conclude that recurrent autoimmunity in the NOD mouse involves a CD4+ T cell that is not sensitive to DSG. Anti-CD4 antibody may be useful in human clinical islet transplantation trials because it seems to prevent both allorejection and recurrent autoimmunity.     

Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism

Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.     

TGF-beta1 alters APC preference, polarizing islet antigen responses toward a Th2 phenotype

TGF-beta1, expressed in the pancreatic islets, protects the nonobese diabetic (NOD) mouse from insulin-dependent diabetes mellitus (IDDM). The islet antigen-specific T cell response of ins-TGF-beta1 mice relied on different antigen-presenting cells (APC) from those used by NOD T cells. T cells from NOD mice utilized B cells to present islet antigen, whereas T cells from ins-TGF-beta1 mice utilized macrophages. In addition, the islet antigen-specific T cell repertoire of ins-TGF-beta1 mice was distinct and deviated toward an IL-4-producing Th2 phenotype. When ins-TGF-beta1 mice were treated with anti-iL-4 antibody, islet antigen-specific IFNGamma-producing Th1 cells were unleashed, and the incidence of diabetes increased to the level of NOD mice. This suggests active suppression of a diabetogenic T cell response. This study describes a novel mechanism in which expression of TGF-beta1 in the context of self-antigen shifts APC preference, deviating T cell responses to a Th2 phenotype, preventing IDDM.     

IL-1 receptor antagonist inhibits recurrence of disease after syngeneic pancreatic islet transplantation to spontaneously diabetic non-obese diabetic (NOD) mice

The effect of an IL-1 receptor antagonist on recurrence of hyperglycaemia after syngeneic pancreatic islet transplantation to spontaneously diabetic female NOD mice was investigated. The transplanted animals were treated with either the receptor antagonist (8.0 mg/kg body weight per day for 12-14 days) or PBS, delivered by subcutaneously implanted osmotic pumps. In the control animals, a transient normoglycaemia was achieved, but hyperglycaemia was generally observed 6 days after islet transplantation. Administration of IL-1 receptor antagonist had a clear protective effect against recurrence of hyperglycaemia until day 14, but after cessation of drug delivery hyperglycaemia re-appeared. The results indicate that continuous administration of the IL-1 receptor antagonist can prevent recurrence of the diabetogenic process in NOD mice. IL-1 receptor antagonist may therefore become a useful adjuvant immunomodulating therapy after human islet transplantation in insulin-dependent diabetes mellitus.     

The role of lymphocyte subsets in accelerated diabetes in nonobese diabetic-rat insulin promoter-B7-1 (NOD-RIP-B7-1) mice

B7-1 transgene expression on the pancreatic islets in nonobese diabetic (NOD) mice leads to accelerated diabetes, with >50% of animals developing diabetes before 12 wk of age. The expression of B7-1 directly on the pancreatic beta cells, which do not normally express costimulator molecules, converts the cells into effective antigen-presenting cells leading to an intensified autoimmune attack. The pancreatic islet infiltrate in diabetic mice consists of CD8 T cells, CD4 T cells, and B cells, similar to diabetic nontransgenic NOD mice. To elucidate the relative importance of each of the subsets of cells, the NOD-rat insulin promoter (RIP)-B7-1 animals were crossed with NOD.beta2microglobulin -/- mice which lack major histocompatibility complex class I molecules and are deficient in peripheral CD8 T cells, NOD.CD4 -/- mice which lack T cells expressing CD4, and NOD.muMT -/- mice which lack B220-positive B cells. These experiments showed that both CD4 and CD8 T cells were necessary for the accelerated onset of diabetes, but that B cells, which are needed for diabetes to occur in normal NOD mice, are not required. It is possible that B lymphocytes play an important role in the provision of costimulation in NOD mice which is unnecessary in the NOD-RIP-B7-1 transgenic mice.     

Correction of diabetic nod mice with insulinomas implanted within Baxter immunoisolation devices

Insulin replacement by injection is clearly not a cure for Insulin Dependent Diabetes Mellitus (IDDM). Replacement of the destroyed islets by pancreas or islet allograft transplantation can achieve the good metabolic control required to prevent diabetic complications, but tissue supply is limited. The problem of islet supply to treat the 1 million IDDM patients in the USA could be overcome by using immortalized islet beta-cells as a donor source. However, before either allogeneic or xenogeneic immortalized beta-cells are used, some major problems have to be overcome: control of immortalized cell growth, allograft or xenograft rejection and recurrence of autoimmunity. To tackle these problems we have used a cell impermeable immunoisolation device containing mouse insulinoma cells. Transplantation of devices with insulinomas from NOD mice carrying the Rat-insulin promoter regulated SV40 T-Antigen transgene (RIP-TAg), normalized the blood glucose levels of diabetic NOD mice. Insulinomas from allogeneic CBA/NOD-RIP-TAg mice were also capable of normalizing diabetic NOD mice. Not only were non-fasting blood glucoses normalized but when given an intraperitoneal injection of glucose, the corrected mice had a near normal clearance of glucose from the blood. When the devices were removed from normalized mice they became diabetic again, demonstrating that the immunoisolation device was capable of protecting against both alloimmune and autoimmune destruction. The results with allogeneic mouse beta-cells suggest the possibility that immortalized human beta-cells could be an effective source of tissue to correct diabetes in IDDM patients without the use of immunosuppression.     

IL-12 gene therapy protects mice in lethal Klebsiella pneumonia

IL-12 is a proinflammatory cytokine that has recently been shown to have beneficial effects in the setting of acquired host immunity. To determine the role of IL-12 in innate immunity against Gram-negative bacterial organisms, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally (i.t.), resulting in the time-dependent expression of IL-12 mRNA (p35 and p40) and protein within the lung. Passive immunization of animals with anti-IL-12 serum i.p. at the time of K. pneumoniae inoculation resulted in a 12-fold increase in K. pneumoniae CFU in lung homogenates at 48 h, as compared with animals receiving control serum. In addition, treatment of Klebsiella-infected mice with anti-IL-12 Abs significantly decreased both short and long term survival. To assess the effect of compartmentalized IL-12 overexpression on outcome in Klebsiella pneumonia, animals were treated i.t. with 5 x 10(8) PFU of a nonreplicating adenoviral vector containing a human cytomegalovirus promoter and cDNAs coding for the p35 and p40 subunits of IL-12 inserted into the E1 and E3 domains (Ad5mIL-12), respectively. In vivo transfection with Ad5mIL-12 resulted in 45% long term survival in Klebsiella pneumonia, whereas no animals with Klebsiella pneumonia receiving control adenovirus survived. Moreover, treatment with anti-IFN-gamma Abs or soluble TNF receptor:Ig construct partially and completely attenuated survival benefits observed in animals receiving Ad5mIL-12, respectively. In conclusion, endogenous IL-12 is a critical component of antibacterial host defense, and the compartmentalized overexpression of IL-12 using recombinant adenoviral gene therapy represents a safe and effective approach to deliver IL-12 to the lung in the setting of murine Klebsiella pneumonia.     

Protection of nonobese diabetic mice from autoimmune diabetes by reduction of islet mass before insulitis

Nonobese diabetic mice spontaneously develop diabetes that is caused by autoimmune cell-mediated destruction of pancreatic beta cells. Here we report that surgical removal of 90% of pancreatic tissue before onset of insulitis induced a long-term diabetes-free condition in nonobese diabetic mice. Pancreatectomy after development of moderate insulitis had no effect on the course of diabetes. The effect of pancreatectomy was abrogated with subsequent development of diabetes by infusion of islet-cell-specific T lymphocytes and by transplantation of pancreatic islets. Lymphocytes from pancreatectomized diabetes-free mice exhibited low response to islet cells but responded normally to alloantigens. These results suggest that the islet cell mass plays a critical role in development of autoimmune diabetes.     

Prevention of autoimmune but not allogeneic destruction of grafted islets by different therapeutic strategies

Grafting autoimmune-diabetic recipients with allogeneic islets, graft rejection and disease recurrence as major problems of reaching indefinite survival and tolerance induction have to be solved. Anti-CD25 and anti-CD4 monoclonal antibodies were successfully used after allogeneic islet transplantation in experimentally diabetic rats. A temporary anti-CD25 therapy also prevented disease recurrence in autoimmune-diabetic BB rats, while this was not yet reported for an anti-CD4 treatment. In autoimmune-diabetic NOD mice disease recurrence can be successfully treated using an anti-CD4 monoclonal antibody. We, therefore, compared the efficacy of a short-term anti-CD25 and anti-CD4 treatment regarding the prevention of allograft rejection and disease recurrence in autoimmune-diabetic BB/OK rats. Both monoclonal antibodies were combined with low doses of Cyclosporin A. Untreated BB/OK rats relapsed into hyperglycaemia within 3 weeks independent of the islet donor, LEW.1A, LEW.1BB/OK or BB/OK rats. However, after grafting MHC-identical allogeneic (LEW.1BB/OK) or syngeneic (BB/OK) islets we observed about 30% spontaneous acceptance. Both the anti-CD25 and anti-CD4 therapy significantly prolonged the survival of allogeneic grafted islets. After MHC-identical allogeneic and syngeneic islet transplantation the temporary immunotherapy increased the proportion of permanent acceptors to 63% and 75%, respectively. The efficacy of both treatment strategies in prolonging allograft survival and prevention of disease recurrence was identical. In summary, anti-CD25 as well as anti-CD4 therapy prevented autoimmune but not allogeneic islet destruction in autoimmune-diabetic BB/OK rats. In conclusion, targeting different immune cells by monoclonal antibodies with different specificities can lead to very similar results with respect to an interruption of allograft rejection and autoimmune reaction.     

IL-10 impacts autoimmune diabetes via a CD8+ T cell pathway circumventing the requirement for CD4+ T and B lymphocytes

IL-10 is essential for an early phase of diabetes in nonobese diabetic (NOD) mice, but later becomes protective against its development. The mechanism by which IL-10 mediates the pathway to diabetes in these mice is unknown. Herein, we dissected the cellular and costimulation requirements for diabetes in transgenic (tg) NOD mice that expressed IL-10 in their pancreatic islets (IL-10-NOD mice). We found that IL-10 alone did not cause diabetes because the offspring (IL-10-NOD-scid mice) from back-crosses of IL-10-NOD mice with NOD-scid mice had no diabetes. Moreover, these IL-10-NOD-scid mice were free of lymphocytic infiltration. Treatment of IL-10-NOD mice with depleting anti-CD4 mAb or control mAb had no effect on diabetes. Surprisingly, depletion of CD8+ T cells by treatment with the corresponding mAb inhibited diabetes without attenuating insulitis, demonstrating a critical role for CD8+ T cells in the disease process. Interestingly, B cell-deficient IL-10-NOD mice readily developed diabetes with kinetics and incidence similar to those observed in wild-type mice, demonstrating that B lymphocytes as APCs were not required in the disease process. Administration of anti-CD40 ligand (CD40L) mAb did not prevent disease, indicating that CD40/CD40L costimulation is not required for diabetes in IL-10-NOD mice. Immunization of IL-10-NOD mice with CFA or heat-shock protein 65, known to block diabetes in NOD mice, had no effect on their diabetes. We demonstrate that IL-10 contributes early to the pathology of diabetes via a CD8+ T cell pathway, eliminating the requirement for B lymphocytes and CD40-CD40L costimulation. Our findings provide a mechanism for the participation of IL-10 in the early development of diabetes.     

Induction of tolerance in murine autoimmune diabetes by transient blockade of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 pathway

The present study demonstrated that a short-term administration of mAbs against leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) at critical periods resulted in complete protection of autoimmune diabetes in non-obese diabetic (NOD) mice. When these mAbs were administered for only 6 days at 2 wk of age, neither diabetes nor insulitis was observed at 30 wk of age. It appears that the tolerance against beta cell Ag(s) was induced by this transient blockade of the LFA-1/ICAM-1 pathway. Protective suppressor activity was not enough to prevent diabetes because co-transfer of splenocytes from female NOD mice, which had received these mAbs at 2 wk of age, resulted in only a short delay of the diabetic onset caused by adoptive transfer of splenocytes from acutely diabetic NOD mice. Transfer of these splenocytes to young NOD mice could not also abrogate the spontaneous diabetes and insulitis. Furthermore, cyclophosphamide treatment could not abrogate the protection. When splenocytes from the treated NOD mice were transferred to NOD-SCID mice, none of the recipient mice developed significant insulitis and subsequent overt diabetes, suggesting the absence or the inactivation of diabetogenic effector T cells. However, splenic T cells from the insulitis-free NOD mice that had received the mAb treatment preserved proliferative responses to both islet cells and 65-kDa glutamic acid decarboxylase (GAD65) in vitro. These results suggest that a unique peripheral tolerance was induced by the transient blockade of the LFA-1/ICAM-1 pathway in an early age of NOD mice.     

Love, courage, and honor

OBJECTIVES: Despite the increasing success of whole-organ pancreas transplantation, the success of clinical islet allografts has remained limited. One of the factors limiting the success is the difficulty in monitoring islet allografts after transplantation. The aim of these studies is to develop a method of "biopsying" human islet allografts using a forearm islet implantation site. METHODS: A subtherapeutic number of isolated human islets were placed in the forearm under the muscle fascia in three human recipients with Type I insulin dependent diabetes. All of the recipients had undergone successful cadaveric renal transplantation at least one year prior and were maintained on their baseline immunosuppression. Aliquots of the islet grafts were removed 7 and 14 days to assess engraftment and graft infiltrate. To verify that the islets were viable, 400 were handpicked and transplanted into B6-scid mice made diabetic with streptozotocin. RESULTS: The biopsy site was found in all three cases. In one patient, no islets were recovered. In two other patients, viable islet tissue was recovered 7 days after transplantation. Immunohistology at 7 days showed the presence of both insulin and glucagon-staining cells in the islets. At 14 days in these two patients, a mononuclear cell infiltrate was observed in the explanted islet biopsies. Immunohistology showed the relative absence of insulin-staining cells with intact glucagon-staining cells. This finding is consistent with recurrent autoimmunity in the islet grafts. DISCUSSION: This preliminary study shows that the forearm biopsy site is a useful method to retrieve human islet grafts after transplantation. The islets engraft and are easily found in the first weeks after transplantation. These data suggest that recurrent autoimmunity may affect islet allografts. Further studies will be needed to determine if the histology in the forearm will correlate with the fate of intraportal or intraperitoneal islet allografts. Although they were shown to reduce the incidence of early allograft failure, their influence on the long-term graft survival remains to be proven.     

The pathogenicity of islet-infiltrating lymphocytes in the non-obese diabetic (NOD) mouse

The aim of the present study was to investigate the pathogenic properties of islet-infiltrating lymphocytes related to the severity of the autoimmune destruction of islet beta-cells in the NOD mouse. We analysed the development of insulin-dependent diabetes mellitus (IDDM) produced by adoptive transfer of islet lymphocytes from NOD into NOD.scid mice. Here we show that the transfer was most effective when both CD4+ and CD8+ T cells were present in the infiltrate, but CD4+ T cells alone were sufficient to cause the disease. Islet lymphocytes from both females and males transferred diabetes effectively, but the severity of IDDM was higher when female islet lymphocytes were used. Unexpectedly, the sensitivity of male islets to beta-cell damage was greater than that of female islets. Treatment of NOD females with a peptide of heat shock protein (hsp)60, p277, known to protect NOD mice from IDDM, reduced the pathogenicity of the islet lymphocytes. In contrast, administration of cyclophosphamide to males, a treatment that accelerates the disease, rendered the islet lymphocytes more pathogenic. More severe disease in the recipient NOD.scid mice was associated with more interferon-gamma (IFN-gamma)-secreting islet T cells of the NOD donor. The disease induced by islet lymphocytes was strongly inhibited by co-transfer of spleen cells from prediabetic mice, emphasizing the regulatory role of peripheral lymphocytes. Thus, the cellular characteristics of the islet infiltrate and the pathogenicity of the cells are subject to complex regulation.     

Functional consequences of a decreased potassium affinity in a potassium channel pore. Ion interactions and C-type inactivation

We have shown previously that the inactivation of macrophages in nonobese diabetic (NOD) mice results in the prevention of diabetes; however, the mechanisms involved remain unknown. In this study, we found that T cells in a macrophage-depleted environment lost their ability to differentiate into beta cell-cytotoxic T cells, resulting in the prevention of autoimmune diabetes, but these T cells regained their beta cell-cytotoxic potential when returned to a macrophage-containing environment. To learn why T cells in a macrophage-depleted environment lose their ability to kill beta cells, we examined the islet antigen-specific immune response and T cell activation in macrophage-depleted NOD mice. There was a shift in the immune balance, a decrease in the T helper cell type 1 (Th1) immune response, and an increase in the Th2 immune response, due to the reduced expression of the macrophage-derived cytokine IL-12. As well, there was a deficit in T cell activation, evidenced by significant decreases in the expression of Fas ligand and perforin. The administration of IL-12 substantially reversed the prevention of diabetes in NOD mice conferred by macrophage depletion. We conclude that macrophages play an essential role in the development and activation of beta cell-cytotoxic T cells that cause beta cell destruction, resulting in autoimmune diabetes in NOD mice.     

Grafted immunoisolated human benign insulinoma reduces the incidence of diabetes in young NOD mice without abolishing the auto-immunity

Exogenous insulin may prevent the auto-immunity of diabetes in rodents. We studied the preventive effect of a safe endogenous insulin delivery in the diabetes-prone NOD mouse by immuno-protected human insulinoma grafts. Perm-selective macrocapsules seeded with human insulinoma were implanted in 34 young NOD mice, 4 and 8 weeks old. The animals were observed 18 months and compared to 34 NOD mice grafted with empty fibers and 25 simply observed. Before grafting, the capacity of the macrocapsules to release insulin was assessed in vitro by perifusion studies and by implantation to 12 diabetic NOD mice. At perifusion, the insulin release of the macrocapsules responded to step changes in glucose. During the in vivo study, the capsules reduced the glycemia of diabetic mice from 18+/-3.5 to 7.3+/-2.1 mmol/L. In the study groups, the survival rate without diabetes (50-70%) was statistically different from controls (10-20%). Recipient's splenocytes transplanted to irradiated male NOD mice transferred the autoimmunity in 75-83% of grafted mice and 86-100% of controls. Insulitis was persistent in all, although milder in the grafted mice. Encapsulated insulinoma prevents diabetes in the NOD mouse without abolishing the auto-immunity. The quantity and quality of the tissues needed and the best moment to graft them have to be determined. The prevention of diabetes by encapsulated pancreatic tissue is appealing because of its simplicity and safety.     

Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells

Lately, TNF alpha has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however, still unclear. To analyze the effects of TNF alpha in insulin-dependent diabetes mellitus (IDDM), we have generated nonobese diabetic (NOD) transgenic mice expressing TNF alpha under the control of the rat insulin II promoter (RIP). In transgenic mice, TNF alpha expression on the islets resulted in massive insulitis, composed of CD4+ T cells, CD8+ T cells, and B cells. Despite infiltration of considerable number of lymphoid cells in islets, expression of TNF alpha protected NOD mice from IDDM. To determine the mechanism of TNF alpha action, splenic cells from control NOD and RIP-TNF alpha mice were adoptively transferred to NOD-SCID recipients. In contrast to the induction of diabetes by splenic cells from control NOD mice, splenic cells from RIP-TNF alpha transgenic mice did not induce diabetes in NOD-SCID recipients. Diabetes was induced however, in the RIP-TNF alpha transgenic mice when CD8+ diabetogenic cloned T cells or splenic cells from diabetic NOD mice were adoptively transferred to these mice. Furthermore, expression of TNF alpha in islets also downregulated splenic cell responses to autoantigens. These data establish a mechanism of TNF alpha action and provide evidence that local expression of TNF alpha protects NOD mice from autoimmune diabetes by preventing the development of autoreactive islet-specific T cells.