Insulin as an autoantigen in NOD/human diabetes

Although multiple islet autoantigens are recognized by T lymphocytes and autoantibodies before the development of type 1A (immune-mediated diabetes), there is increasing evidence that autoimmunity to insulin may be central to disease pathogenesis. Evidence is strongest for the NOD mouse model where blocking immune responses to insulin prevents diabetes, and insulin peptides can be utilized to induce diabetes. In man insulin gene polymorphisms are associated with disease risk, and autoantibodies and T cells reacting with multiple insulin/proinsulin epitopes are present. It is not currently clear why insulin autoimmunity is so prominent and frequent, and though insulin can be used to immunologically prevent diabetes of NOD mice, insulin-based preventive immunoregulation of diabetes in man is not yet possible.     

Cell-mediated immunity to pancreatic islet cells in the non-obese diabetic (NOD) mouse: in vitro characterization and time course study

The non-obese diabetic (NOD) mouse is an animal model of insulin-dependent diabetes mellitus (IDDM), in which 80% of the females become diabetic after the age of 12 weeks. Using an in vitro assay we investigated the capacity of spleen lymphocytes from NOD mice to inhibit the insulin secretion of normal islet cells after stimulation by theophylline plus arginine. Spleen cells from diabetic NOD mice inhibited the insulin release of DBA/2 islet cells. Depletion experiments using monoclonal antibodies demonstrated that inhibitory cells belonged to the Lyt2 positive T lymphocyte subset. The phenomenon was not restricted by the MHC class I K region, shared by NOD and DBA/2 mice, since lymphocytes from diabetic NOD mice also inhibited the insulin secretion of normal Wistar rat islet cells. Inhibitory T cells were detected in overtly diabetic mice but also in non-diabetic females aged 5-11 weeks indicating that they are not secondary to metabolic disturbances and might contribute to their onset. Conversely they were not found in male NOD mice although some of these mice show insulitis. The presence of these inhibitory T cells might thus represent an early and sensitive marker of anti-islet cell-mediated autoimmunity.     

Anti-serum with anti-autoantibody activity decreases autoantibody-positive B lymphocytes and type 1 diabetes of female NOD mice

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by an autoimmune-mediated loss of insulin secreting β-cells. Each B lymphocyte clone that escapes immune tolerance produces a specific antibody. No specific treatment against autoantibodies is available for autoimmune diseases. We have developed a strategy to produce an antiserum against autoantibodies for the treatment of T1DM. Non-obese diabetic (NOD) but not Balb/c mouse serum contains autoantibodies. Antisera were produced by immunizing Balb/c mice with affinity-purified IgG from NOD or BALB/c mice along with the immune adjuvant (hereafter, NIgG or BIgG, respectively). A bolus administration of NIgG significantly reduced serum autoantibodies, autoantibody-positive B lymphocytes in the spleens of NOD mice, mortality and morbidity of diabetes, blood glucose and islet immune infiltration, whereas it increased islet mass in NOD mice for at least 26 weeks. NIgG antiserum treatment has no significant effect on CD3⁺, CD4⁺ or CD8⁺ T cells and B220⁺ or CD19⁺ B cells. BIgG also imparted a moderate therapeutic effect, although it was considerably lower than that of NIgG. NIgG did not cross-react with allogeneic serum. NIgG showed no effect on Balb/c mice. The results show the feasibility of producing antiserum against autoantibodies to prevent and treat autoimmune-induced T1DM with a single bolus administration.     

Syngeneic T cell transfer of diabetes into NOD newborn mice: in situ studies of the autoimmune steps leading to insulin-producing cell destruction

To overcome the limitations of in situ studies during the chronic spontaneous autoimmune process leading to insulin cell destruction and diabetes in non-obese diabetic (NOD) mice, we have developed a model of acute transfer of diabetes into healthy syngeneic newborns. The injection of 20 x 10(6) T cells from adult diabetic mice produced synchronous insulitis within 3 weeks and diabetes within 4-5 weeks in young recipients, at a time when non-injected control mice do not even exhibit histological changes in their pancreases. Sequential studies of pancreases from T cell-transferred mice showed that lymphoid infiltration was preceded by a strong tissue expression of Ia antigen which was restricted to the vessel-associated cells limiting the islet of Langerhans, and which might play a role in the recruitment of circulating T cells inside the islets. Acute destruction of most of the insulin-producing cells, leading to diabetes, could take place within a few days after insulitis had begun. A majority of the inflammatory cells were T lymphocytes, approximately 30% of which expressed interleukin 2 receptors. L3T4+ T cells largely predominated at the early phase of islet invasion whereas the proportion of Ly-2+ T cells substantially increased later when beta cell destruction occurred. In contrast, only a minority of B cells and macrophages participated to the inflammatory process. These data are in keeping with previous demonstrations that both T cell subsets contribute to the autoimmune disease. Furthermore, they suggest that beta cell injury is mediated through a cytotoxic process, which requires the sequential involvement of L3T4+ (helper) and Ly-2+ (cytotoxic) T cells.     

NOD小鼠自然状态下CD4~+CD25~+T细胞的动态变化及意义

研究自发的1型糖尿病雌鼠模型(NOD)在自然状态下发生1型糖尿病过程中CD4+CD25+T细胞的动态变化,旨在初步探讨调节性T细胞参与1型糖尿病发病的可能机制。采用雌性NOD小鼠作动物模型,每2周尾静脉采血1次,采用三色流式细胞术测定NOD小鼠外周血中CD4+CD25+T细胞(CD3+CD4+CD25+)的百分率。在32周时,对比发生糖尿病和未发生糖尿病NOD小鼠不同脏器中的CD4+CD25+T细胞阳性率。HE法检测胰岛炎。结果显示:(1)自第6周起NOD小鼠CD4+CD25+T细胞百分率逐渐降低。发生糖尿病NOD小鼠CD4+CD25+T细胞比率低于未发病NOD小鼠对照组(外周血分别为0.94%±0.21%、1.62%±0.23%,P=0.01;脾脏2.09%±0.14%、2.77%±0.36%,P=0.019),提示糖尿病NOD小鼠外周血中存在异常比例的CD4+CD25+T细胞;(2)32周龄糖尿病NOD小鼠与未发病NOD小鼠的CD4+CD25+T细胞抑制功能减低,与阳性对照组有显著性差异;(3)HE染色结果示糖尿病NOD小鼠胰岛结构完全破坏,胰岛炎程度较未发病NOD小鼠严重。该结果提示NOD小鼠发生糖尿病时免疫功能紊乱与CD4+CD25+T细胞参与调节及T细胞亚群变化相关,糖尿病的发生受致病性T细胞和调节性T细胞的调节。     

Autoantibodies against pancreatic beta-cells: characterization by western blot analysis in the non-obese diabetic (NOD) mouse.

The NOD mouse is a relevant model for studying autoimmune diabetes. As in human insulin-dependent diabetes mellitus, the nature of the autoantigen towards which the immune system is directed remains to be clarified. It has been shown that T cells are central to the disease process. However, autoantibodies may be used as a probe to identify islet autoantigens to which self tolerance is defective. Using Western blot analysis, we characterized autoantibodies which are specific for a 58 kDa islet antigen and a 29 kDa antigen. The 58 kDa autoantigen was present in cellular extracts prepared from rat tumoral insulin-secreting cells (Rin5F) and NOD islets but not from most other non-insulin-secreting cell lines. By contrast the 29 kDa antigen was a ubiquitous antigen expressed in all cell lines tested and was not further characterized since it is very likely to be responsible for secondary immunization rather than play any role in the NOD disease process. Anti-58 kDa autoantibodies were detected in all diabetic male and female NOD animals as well as in sera from old non-diabetic NOD animals. Anti-58 kDa antibodies were not detected in sera from young NOD mice (less than 6 weeks of age) or in sera from other conventional laboratory strains of mice including autoimmune prone animals such as MRL/lpr and (NZB x NZW)F1 mice. A monoclonal antibody (72.2) specific for the 58 kDa structure was obtained, which allowed further characterization of the corresponding islet cell antigen. The expression of the 58 kDa antigen was evidenced by Western blot analysis in normal islets and in a mouse neuroblastoma cell line.     

The role of t/b lymphocyte collaboration in the regulation of autoimmune and alloimmune responses

The present review highlights the two areas of research pursuit in our laboratory: (1) the regulation of the autoimmune T cell response to pancreatic islet β cells using the nonobese diabetic (NOD) mouse model of type 1 diabetes and (2) the regulation the T cell response to alloantigens. Our work has established a central role for B lymphocytes in driving both autoimmune and allo-immune T cell responses. Our studies indicate that: (1) B cell-deficient NOD mice are protected from autoimmune diabetes; (2) targeted disruption of cognate T/B cell collaboration via major histocompatibility complex (MHC) class II prevents both T cell-mediated islet destruction and allograft rejection; and (3) maternal transmission of islet-reactive autoantibodies potentiates the activation of diabetogenic T cells, highlighting the important role of B cells in the early targeting of islet β cells.     

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 β-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 β-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-γ)-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.     

The postnatal maternal environment influences diabetes development in nonobese diabetic mice

When nonobese-diabetic (NOD) mouse embryos were implanted into pseudopregnant mothers of a nonautoimmune mouse strain, the progeny had a reduced type 1 diabetes (T1D) incidence, suggesting that transmission of maternal autoantibodies is important for T1D development. Whether eliminating islet autoantibody transmission in utero, or postnatally (through milk), prevented T1D is unknown. Herein, we show that fostering newborn NOD mice on B-cell deficient NOD.Igmu-/- dams does not prevent T1D, demonstrating that postnatally transmitted islet autoantibodies are not required for disease pathogenesis. Additionally, NOD.Igmu-/- mice reared on NOD dams did not develop T1D, indicating that autoantibody transmission to B-cell deficient NOD neonates is insufficient to trigger T1D. Interestingly, newborn NOD mice that were reared by ICR (but not NOD or C57BL/6) dams had reduced T1D incidence, although not as reduced as that reported after embryo transfer to ICR mice, suggesting that both prenatal and postnatal factors contribute to the observed reduction in T1D incidence. Thus, NOD mice have different risks for developing T1D depending on the strain of their foster mother, and both prenatal and postnatal maternal factors, other than islet autoantibodies, influence their T1D incidence. The results may be relevant for understanding the increasing incidence of T1D and designing interventions.     

Paradoxical lessening of autoimmune processes in non-obese diabetic mice after infection with the diabetogenic variant of encephalomyocarditis virus

In order to gain insight into the interaction between autoimmunity and viral infection in the onset of insulin-dependent diabetes, non-obese diabetic (NOD) mice which spontaneously develop autoimmune diabetes were inoculated with the diabetogenic variant of the encephalomyocarditis virus (EMCV-D) before the onset of the disease. The pre-diabetic period was divided into two phases: the early phase (days 88 to 116) during which development of spontaneous diabetes is rare and the late phase (day 123 to 200) during which the incidence of spontaneous diabetes is high. As controls ICR mice of common ancestry were also inoculated. During the early phase diabetes was observed in 4/10 inoculated, 0/13 control NOD and 7/13 inoculated ICR males vs. 6/12 inoculated, 1/11 control NOD and 0/15 inoculated ICR females. However, in NOD female, virus-induced diabetes prevalence was variable from one experiment to another. In parallel the flow cytometric analysis showed a high percentage of L3T4+ T lymphocytes in the pancreas of inoculated female NOD mice 10 days after the infection. At this time a large proportion of both L3T4+ and Ly-2+ cells expressed the interleukin 2 receptor. During the late phase no new case of diabetes occurred in inoculated NOD mice but one case was observed in control NOD males and five in control NOD females. This prevention of autoimmune diabetes was constantly found in other experiments. Insulitis was milder in inoculated NOD mice of both sexes than in control NOD. Adoptive transfer of diabetes into irradiated 8-week-old males by splenocytes from 28-week-old females was successful in five out seven attempts with control splenocytes and in zero out of six attempts with splenocytes from inoculated mice. This immunosuppression was specific as the ability of lymphocytes to respond to soluble or allogeneic antigens was preserved. In the early phase EMCV-D precipitated the onset of diabetes in females NOD mice by amplifying L3T4+ T lymphocyte-mediated immune mechanisms. During the late phase viral infection had lessened immune processes in animals which had resisted or recovered from virus-induced diabetes.     

Predominance of T lymphocytes in pancreatic islets and spleen of pre-diabetic non-obese diabetic (NOD) mice: a longitudinal study.

We examined sequential changes in the subsets of mononuclear cells infiltrating the pancreatic islets and splenic lymphocytes in pre-diabetic non-obese diabetic (NOD) mice, an animal model for type I diabetes, using immunofluorescent techniques. In the pancreas, a predominant infiltration by activated T lymphocytes, including helper inducer and cytotoxic suppressor T cells, was observed in the early stage of insulitis. Natural killer cells were also detected in the lesions. Immunoglobulin bearing cells tended to increase in number with the progression of insulitis. T lymphocytes were localized close to islet cells, while immunoglobulin bearing cells appeared adjacent to blood vessels and around T cell clusters. Immunoglobulin deposition or Ia expression on islet cells was not observed. The percentage of splenic T lymphocytes was markedly increased in the initial stage of insulitis as compared with control ICR mice and this elevated proportion of T cells continued throughout the observation period. As for splenic T cell subsets, cytotoxic suppressor T cells were increased in NOD mice. These results suggest that T lymphocytes play an important role in the initiation of insulitis long before the onset of overt diabetes. Moreover, NOD mice seem to have characteristic immunological features different from the BB rat or a reported case with human type I diabetes.     

Insulin-specific tolerance in diabetes.

At present it is possible to predict the development of type 1A diabetes (immune-mediated diabetes) in man and prevent the disorder in animals. Studies of immunity to insulin play a prominent role in both disease prediction and disease prevention. For both man and the NOD mouse, insulin autoantibodies usually precede the development of diabetes and can be utilized to assist in disease prediction. T cells clones recognizing insulin, both CD4 and CD8, can transfer disease to young mice or immunodeficient animals. Specific insulin peptides reacting with these clones have been identified, their crystal structure when bound to a human "diabetogenic" MHC allele has been determined, and specific peptides can be used either to induce or to prevent disease. Clinical trials of both insulin and an altered peptide ligand of insulin to prevent islet beta-cell destruction are underway. Insulin is one of a number of islet autoantigens, but it is likely that immune responses to insulin will be central to both pathogenesis and immunologic protection.     

A multivalent vaccine for type 1 diabetes skews T cell subsets to Th2 phenotype in NOD mice

Previous studies by our group, using an experimental autoimmune thyroiditis (EAT) model in Strain 13 inbred guinea pigs, resulted in T cell-mediated delayed hypersensitivity; however, autoantibodies proved not to be cytotoxic to thyroid epithelial cells in the presence or absence of complement proteins. Albeit, T cell-mediated lymphocyte cytotoxicity began to diminish sharply concomitantly with increasing titers of circulating autoantibodies, indicating a skewing of the self-reactive response and amelioration of the EAT. Furthermore, immunization of guinea pigs with thyroglobulin in incomplete Freund's adjuvant (IFA) generated a high titer of antithyroglobulin antibodies and proved to inhibit thyroiditis. These observations indicated that the shift in the immune response from Th1 to Th2 and the production of antibodies were likely responsible for ameliorating EAT. Based upon these results, we extrapolated our studies to design a multivalent vaccine, which shows promise in preventing/reversing T1D in NOD mice. A small pilot study was conducted in which a total of 34 mice, 20 non-immunized controls and 14 immunized with syngeneic islet lysate, were monitored for mean day to diabetes for a total of 28 weeks. Immunization of NOD animals with syngeneic islet lysates resulted in a significant delay in diabetes onset (P < 0.001) as compared to non-immunized controls. To further assess the vaccine's efficacy, robustness, and delay of disease, a large-scale experiment was conducted and monitored for 32 weeks using 106 mice, 64 non-immunized controls and 42 immunized with syngeneic islet lysate. At the end of the study, 90% of the non-immunized group developed diabetes, while less than 25% of the immunized group became diabetic (P < 0.0001). The protective effect, as a result of vaccination, correlated with an increase in the levels of IL-10 and IL-4 cytokines as well as a skewing to Th2-dependent isotype antibodies in serum. Strikingly, adoptive transfer of spleen cells from immunized animals into NOD.scid recipients provided protection against transfer of diabetes by diabetogenic spleen cells. The results of this study provide evidence that vaccination with islet lysate leads to a Th2-dependent skewing of the immune response to islet beta cells as a possible mechanism of protection. This strategy may be implemented as a possible vaccination protocol for arresting and/or preventing T1D in patients.     

Subsets of macrophages and dendritic cells in nonobese diabetic mouse pancreatic inflammatory infiltrates: correlation with the development of diabetes

Islet-specific T cells are essential in the development of type I diabetes. The role of non-lymphoid cells is relatively unclear, although infiltration of dendritic cells and macrophages is the first sign of islet autoimmunity in diabetes-prone nonobese diabetic (NOD) mice. BDC2.5 is one of the autoreactive T cell clones isolated from NOD mice. Transfer of BDC2.5 T cells into young NOD mice accelerates diabetes development, whereas transgenic expression of the BDC2.5 T cell receptor on NOD T cells (BDC2.5 TCR-Tg NOD) markedly reduces diabetes development. We show that, although the same antigen-specificity is involved, both models differ significantly in insulitis. BDC2.5 TCR-Tg NOD mice develop an extensive, but non-aggressive, peri-insulitis by 3 weeks of age. In these large peri-islet infiltrates, resembling secondary lymphoid tissue, BM8+ macrophages (Mphi) are virtually absent. In contrast, BDC2.5 T cell clone transfer results in an aggressive insulitis with small infiltrates, but relatively large numbers of BM8 Mphi. Infiltration of BM8+ Mphi therefore correlates with islet destruction. This is, however, not observed for all Mphi; Monts-4+ Mphi follow a reverse pattern and are present in higher numbers in BDC2.5 TCR-Tg than in transferred mice. ER-MP23+ Mphi are reduced in both transferred and transgenic mice compared with wild-type NOD. Thus, this study underlines and extends previous data suggesting that Mphi are implicated in both early and late phases in diabetes development. Furthermore, our data imply that subsets of non-lymphoid cells have different roles in diabetes development. It is, therefore, important to recognize this heterogeneity when interpreting both in vivo and in vitro studies concerning non-lymphoid cells in diabetes.     

Increased CD5-positive B lymphocytes in type I diabetes.

CD5+ B lymphocytes have been implicated in the production of polyspecific and monospecific antibodies that bind self-antigens, and increased proportions of this B cell subset occur in patients with some autoimmune diseases. We investigated the proportion of peripheral blood CD5+ B lymphocytes in type I diabetic patients. Compared with 18 age-matched healthy subjects, 11 out of 28 (39.2%) type I diabetic patients had increased proportions of circulating CD5. B lymphocytes with no alterations in the numbers of circulating B and T lymphocytes. Although all patients with increased CD5 B lymphocytes also had serum islet cell antibodies and/or insulin autoantibodies, the occurrence of increased proportions of CD5+ B lymphocytes and serum autoantibodies was not significantly correlated. Increased proportions of CD5+ B lymphocytes was not related to the time elapsed since the clinical onset of diabetes. In addition, regardless of being increased or normal, the proportion of CD5+ B lymphocytes appeared as a relatively constant phenotype after 1 year of follow-up studies at 3-month intervals in eight patients. Although the significance of these findings remains to be established, the possibility exists that CD5+ cells play a role in the pathogenesis of type I diabetes.     

The role of interferon alpha in initiation of type I diabetes in the NOD mouse

Type 1 diabetes (T1D) is an autoimmune disease in both humans and the nonobese diabetic (NOD) mouse, in which the insulin-producing-cells of the pancreatic islets are destroyed by a beta islet cell-specific T cell immune response. We recently reported that interferon (IFN)-α is an early trigger of the T1D process in NOD mice. Here, we show that extensive blockade of IFN-α action by a monoclonal antibody specific to IFN-α receptor 1 results in nearly complete prevention of T1D in NOD mice. Whether professional IFN-α producing cells, plasmacytoid dendritic cells (pDCs), are responsible for the initiation of T1D has been unclear. Here we demonstrate that depletion of pDCs in NOD mice by a specific mAb given at 15-25 days of age significantly delays the onset and decreases the incidence of T1D. These findings indicate that pDC and pDC-derived IFN-α are the prime initiators of the pathogenesis of T1D in NOD mice.     

T-cell-mediated immune response in murine malaria: differential effects of antigen-specific Lyt T-cell subsets in recovery from Plasmodium yoelii infection in normal and T-cell-deficient mice.

For analysis of the role of immune T cells in protective immunity against murine malaria, Plasmodium yoelii-immune Lyt T-cell subsets were functionally characterized in vitro and in vivo. Selected Lyt2- and Lyt2+ T cells from P. yoelii-immune C57BL/10 mice differed in their capability to proliferate in response to P. yoelii antigen in vitro. Only the Lyt2- T-cell population produced T-cell growth factor upon restimulation, and none of the selected T-cell subsets produced detectable amounts of macrophage activating factor. Lyt2- but not Lyt2+ lymphocytes were capable of transferring protection to normal C57BL/10 mice. When transferred into T-cell-deficient C57BL/6-nu/nu mice, adoptive resistance to P. yoelii by Lyt2- lymphocytes was only demonstrable after prior reconstitution of recipients with normal T cells. These results suggest an interaction between P. yoelii-immune Lyt2- T cells and normal T lymphocytes via T-cell growth factor in the development of protective immunity to malaria.     

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

Islet-infiltrating lymphocytes of individual male and female non-obese diabetic (NOD) mice were examined with the purpose of determining the differences that lead to a predominance of diabetes in female versus males NOD mice. When normalized for the amount of islet lymphocytes recovered, the infiltrating lymphocytes of female NOD mice were indistinguishable from those of male NOD mice. The only observed difference was that islet inflammation progressed at an increased rate in female compared to male NOD mice. There was no difference in the composition of islet infiltrates in male and female NOD mice. Unexpectedly, the ratio of CD4⁺:CD8⁺ T cells was tightly controlled in the islets throughout diabetogenesis. The frequency of IL-4⁺ CD4⁺ T cells started high but quickly fell to 3% of the population that was maintained with increasing inflammation. A significant portion of the CD8⁺ T cells were islet-specific glucose-6-phosphatase catalytic subunit-related protein specific in both male and female NOD mice and this population was antigen experienced and increased at high levels of islet inflammation. Surprisingly, a large pool of antigen inexperienced naïve T cells was detected in the islets. We conclude the underlying immunological processes in both male and female NOD mice are similar while the rates differ and the presence of naïve T cell in the islets may contribute to epitope spreading.     

Spontaneous peripheral T-cell responses to the IA-2beta (phogrin) autoantigen in young nonobese diabetic mice

Phogrin (IA-2beta), a major autoantigen in type 1 diabetes in man is recognized by peripheral T cells in the nonobese diabetic (NOD) mouse. CD4(+) T-cell clones derived from immunized NOD animals elicit islet destruction in a disease transfer model. Spontaneous proliferative responses to the protein and derived peptide epitopes were detected in peripheral lymph node cells (LNC) of unprimed NOD mice but not BALB/c controls as early as 4 weeks of age at a time point when insulitis in NOD animals is minimal. Responses to irradiated NOD islet cells but not irradiated NOD spleen cells were observed for both male and female NOD animals. Insulin, phogrin and phogrin-peptide 7 (aa 755-777) but not phogrin-peptide 2 (aa 640-659) or tetanus toxin peptide were recognized as antigens. Islet cell-reactive and phogrin peptide 7-specific CD4(+) T-cell lines were generated from splenocytes of unprimed 4-week-old NOD females and shown to secrete Th1-type cytokines. The results show that the phogrin molecule is targeted early in the course of disease in NOD animals at a time when circulating autoantibodies are absent and insulitis is minimal.     

The Expression of Cytokine Genes in the Peritoneal Macrophages and Splenic CD4- and CD8-Positive Lymphocytes of the Nonobese Diabetic Mice

Type 1 diabetes is an autoimmune disease that results from the destruction of the insulin-producing pancreatic beta islet cells, probably via the influence of cytokines. However, direct correlation between the expression of selected cytokines by various immune cells at different time points during the progression of the disease has not yet been clearly demonstrated. In this study, we showed that the mRNA expression of the pro-inflammatory cytokines, TNF-alpha, IL-1 beta, IL-6, and GM-CSF, were increased while the anti-inflammatory cytokine, TGF-beta, decreased in the peritoneal macrophages of nonobese diabetic (NOD) mice. IL-6 expression however decreased when the mice became diabetic. Surprisingly the expression of IFN-gamma and IL-2 by splenic CD4+ cells were lower in 5-week-old NOD mice as compared to the nonobese diabetic resistant (NOR) control mice, but their expression was higher in older NOD mice. The expression of IL-4 and IL-10 decreased in splenic CD4-positive lymphocytes. Splenic CD8-positive lymphocytes expressed increased levels of IFN-gamma and IL-10 but the latter decreased sharply when diabetes occurred. The relevance of these findings to the pathogenesis of type 1 diabetes is discussed.