Deviation of pancreas-infiltrating cells to Th2 by interleukin-12 antagonist administration inhibits autoimmune diabetes

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)2 can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4+ cells to the Th2 pathway in vitro. Although (p40)2 does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4- cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)2 in NOD mice. (p40)2 administration to NOD mice inhibits interferon-gamma but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)2 treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)2 from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4+ but not CD8+ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)2 from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4+ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.     

Endogenous immune response to glutamic acid decarboxylase (GAD67) in NOD mice is modulated by adjuvant immunotherapy

We have shown that immunization of non-obese diabetic (NOD) mice with adjuvants (CFA or BCG) prevents the onset of diabetes by induction of regulatory cells. Since autoimmune responses to glutamic acid decarboxylase (GAD) are up-regulated in insulin-dependent diabetes mellitus (IDDM), in this study GAD67-specific antibody, T cell proliferation and lymphokine production patterns were analysed in the adjuvant-treated mice to characterize the regulatory mechanisms underlying the protection. We used both spontaneous diabetes and syngeneic islet transplantation models in NOD mice. Protection against spontaneous diabetes and prevention of syngeneic islet graft rejection by CFA or BCG treatment was found to be accompanied by the production of long lasting and high titre anti-GAD67 antibody of IgG1 isotype in the sera. Upon in vitro stimulation with GAD67, draining lymph node and spleen cells from CFA-immunized NOD mice or syngeneic islet-grafted and BCG-protected NOD mice produced much more IL-4, whereas there was no significant change in IFN-gamma production. The strong early T cell proliferative response to GAD67 in CFA or BCG-immunized NOD mice was followed by a low or unresponsiveness state. Taken together, these results suggest a shift in Th1/Th2 balance in the GAD67-specific endogenous immune response to a change in Th2 levels after adjuvant treatment. We postulate that the protective effect of CFA or BCG is due to the diversion of GAD-specific endogenous cellular immune response to a non-pathogenic humoral response.     

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.     

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.     

Beta-cell destruction may be a late consequence of the autoimmune process in nonobese diabetic mice

The NOD mouse is an animal model of IDDM that shows many of the characteristics of human IDDM. It has been proposed that beta-cell destruction in IDDM progresses over time in a linear manner. Recently, we and others have demonstrated that T helper type 1 (Th1) cells have pathogenic roles in the NOD model and proposed that cytokine balances change as the disease progresses. However, it has not been demonstrated how or when the cytokine balances change or how the beta-cell destruction progresses. We have recently demonstrated that the cytokine profiles of CD45RB(low) CD4+ cells correlate either with their pathogenic or with their protective roles in the NOD mouse. To further analyze this apparent correlation between the shift in cytokine level and IDDM, we examined the anti-CD3-induced cytokine profiles of this subset from NOD mice of various ages compared with that from age-matched I-Ak transgenic NOD and BALB/c mice as controls. A significantly higher ratio of anti-CD3-induced interferon-gamma/interleukin-4 was found in diabetic NOD mice (P < 0.0001) but not in age-matched nondiabetic NOD mice. This cytokine ratio did not change significantly until the onset of diabetes in NOD mice. Based upon these results, we propose that IDDM in the NOD mouse progresses as a predominant inflammatory beta-cell dysfunction without actual beta-cell destruction until late in the disease process. This supports the possibility that late-stage immunotherapy may preserve islet beta-cell mass.     

B lymphocytes are critical antigen-presenting cells for the initiation of T cell-mediated autoimmune diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice genetically deficient in B lymphocytes (NODJg mu(null)) are resistant to T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM). Ig infusions from diabetic NOD donors did not abrogate IDDM resistance in NODJg mu(null) mice. However, T cell responses to the candidate pancreatic beta cell autoantigen glutamic acid decarboxylase (GAD), but not the control Ag keyhole limpet hemocyanin, were eliminated in NODJg mu(null) mice. To initially test whether they contribute to IDDM as APC, NOD B lymphocytes were transferred into NODJg mu(null) recipients. B lymphocytes transferred into unmanipulated NODJg mu(null) recipients were rejected by MHC class I-restricted T cells. Stable T and B lymphocyte repopulation was achieved in irradiated NODJg mu(null) mice reconstituted with syngeneic bone marrow admixed with NOD B lymphocytes. IDDM susceptibility was restored in NODJg mu(null) mice reconstituted with syngeneic marrow plus B lymphocytes, but not with syngeneic marrow only. T cell responses to GAD were restored only in NODJg mu(null) mice reconstituted with syngeneic marrow plus B lymphocytes. Hence, B lymphocytes appear to contribute to IDDM in NOD mice as APC with a preferential ability to present certain beta cell Ags such as GAD to autoreactive T cells.     

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.     

Differential expression of ICAM-1 and LFA-1 versus L-selectin and VCAM-1 in autoimmune insulitis of NOD mice and association with both Th1- and Th2-type infiltrates

The infiltration of pancreatic islets by mononuclear cells is the hallmark of the development of insulin dependent diabetes mellitus (IDDM) in the NOD mouse, an animal model for human IDDM. The aim, of this study was to correlate adhesion molecule expression with the degree of islet infiltration and to compare Th1- and Th2-driven islet inflammation. Cryostat sections of NOD mouse pancreata before and after diabetes development were analysed by semiquantitative immunohistochemistry. NOD mouse islets did not show the expression of ICAM-1, LFA-1, L-selectin and VCAM-1 prior to infiltration by mononuclear cells. Furthermore, islets with early stage insulitis (grade 1, periinsular location of small infiltrates) still were devoid of adhesion molecule expression. ICAM-1 and LFA-1 were first demonstrable in islets with strong periinsular infiltrates (insulitis grade 2) while L-selectin and VCAM-1 were only seen in islets with mild or strong intraislet infiltration (grade 3-4). Adhesion molecules were demonstrable in areas of macrophage and T-lymphocyte infiltrates but not in adjacent endocrine islet tissue. Islets of all infiltration stages contained Th2 lymphocytes (positive for IL-4). Substantial numbers of Th1 cells (positive for IFN-gamma, TNF-alpha, IL-2 and/or IL-2 receptor) were observed only after acceleration of diabetes development by a single injection of cyclophosphamide (250 mg/kg i.p.). Interestingly, the adhesion molecule expression pattern in islets with "Th1' versus "Th2 insulitis' was not different. In conclusion, the expression of adhesion molecules in islets during the development of autoimmune diabetes does not precede mononuclear infiltration but probably occurs in response to the activation of initial small infiltrates. ICAM-1 and LFA-1 expression is seen prior to L-selectin and VCAM-1. However, adhesion molecule expression during Th1 versus Th2 cell infiltration is very similar, suggesting similar adhesion molecule requirements of the two Th subsets.     

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.     

Analysis of the role of variation of major histocompatibility complex class II expression on nonobese diabetic (NOD) peripheral T cell response

The current paradigm of major histocompatibility complex (MHC) and disease association suggests that efficient binding of autoantigens by disease-associated MHC molecules leads to a T cell-mediated immune response and resultant autoimmune sequelae. The data presented below offer a different model for this association of MHC with autoimmune diabetes. We used several mouse lines expressing different levels of I-Ag7 and I-Ak on the nonobese diabetic (NOD) background to evaluate the role of MHC class II in the previously described NOD T cell autoproliferation. The ratio of I-Ag7 to I-Ak expression correlated with the peripheral T cell autoproliferative phenotype in the mice studied. T cells from the NOD, [NOD x NOD. I-Anull]F1, and NOD I-Ak transgenic mice demonstrated autoproliferative responses (after priming with self-peptides), whereas the NOD.H2(h4) (containing I-Ak) congenic and [NOD x NOD. H2(h4) congenic]F1 mice did not. Analysis of CD4(+) NOD I-Ak transgenic primed lymph node cells showed that autoreactive CD4(+) T cells in the NOD I-Ak transgenic mice were restricted exclusively by I-Ag7. Considered in the context of the avidity theory of T cell activation and selection, the reported poor peptide binding capacity of NOD I-Ag7 suggested a new hypothesis to explain the effects of MHC class II expression on the peripheral autoimmune repertoire in NOD mice. This new explanation suggests that the association of MHC with diabetes results from "altered" thymic selection in which high affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in humans) in susceptibility to insulin-dependent diabetes mellitus.     

Defensiveness and perception of external inspiratory resistive loads in asthma

This paper introduces a model which incorporates fetal thymus organ culture (FTOC) from NOD mice to replicate thymic development of diabetogenic T cells. NOD fetal pancreas organ culture (FPOC) co-cultured with 13-16 day NOD FTOC for an additional 14-21 days produced less insulin than FPOC cultured alone. Insulin production from the FTOC of non-diabetic strains C57BL/6 and BALB/c was not inhibited by co-culture with FTOC from their syngeneic counterparts. Sections of the NOD co-cultures showed peri-islet infiltration with lymphocytes. Insulin reduction by FTOC/FP co-culture was prevented by co-culture of the NOD FT with FT from immunologically incompetent C.B-17 SCID/SCID mice. Co-culture of NOD FP with NOD FT prior to the development of T cells prevented generation of diabetogenic FTOC. Thus, early exposure of NOD T cell precursors to the thymic stromal elements of C.B-17 SCID/SCID FT or to islet antigens can negatively select for diabetogenic T cells or activate immuno-regulatory cells that can suppress diabetogenic T cell activity. The addition of blocking F(ab')2 fragments of anti-CD3epsilon monoclonal antibody to NOD FTOC/FP co-cultures prevented insulin reduction, implicating a role for TcR-mediated recognition in this "in vitro IDDM" model. The addition of activating whole anti-CD3epsilon caused the complete ablation of insulin production in FTOC/FP co-cultures from all strains tested. Transfer of unprimed syngeneic FTOC cells to prediabetic NOD mice prevented the onset of IDDM while transfer of islet-cell primed FTOC/FP cells slightly increased disease incidence. These data suggest that while diabetogenic T cells are present in the FT, they are normally suppressed, even after organ culture. However, these cells can induce the destruction of islet cells, in vitro and in vivo, if they are appropriately activated with pancreatic tissue.     

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.     

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.     

An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that beta-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNF alpha, and IFN alpha) and type 1 cytokines (IFN gamma, TNF beta, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGF beta). Cytokines (IL-1, TNF alpha, TNF beta and IFN gamma) may be directly cytotoxic to beta-cells by inducing nitric oxide and oxygen free radicals in the beta-cells. In addition, cytokines may sensitize beta-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the beta-cells (an action of IFN gamma), and inducing Fas (CD95) expression on beta-cells (actions of IL-1, and possibly TNF alpha and IFN gamma). Transgenic expression of cytokines in beta-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFN alpha, IFN gamma, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNF alpha. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4+ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFN gamma-producing), but other islet-specific Th1 clones that produce TGF beta can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFN gamma genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFN gamma, also IL-1, TNF alpha, IL-2, and IL-6--by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs--significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNF alpha, and IL-6) and type 1 cytokines (IFN gamma, IL-2 and IL-12) in IDDM development.     

Meiotic crossing over between nonhomologous chromosomes affects chromosome segregation in yeast

This study addresses the nature of the pathogenic effector T cell in experimental autoimmune uveoretinitis and the effect of different cytokines on these cells in vitro. Lymph node cells of B10.RIII mice immunized with the uveitogenic peptide 161-180 of interphotoreceptor retinoid binding protein were cultured with the peptide with or without IL-12, IL-4, or anti-IL-4. An antigen-specific T cell line was subsequently derived from these cells. Primary cultures of immune lymph node cells stimulated with the peptide proliferated and produced IL-2 and some IL-4, but no IFN-gamma. The addition of recombinant IL-12 resulted in abundant production of IFN-gamma, which was blocked by the addition of IL-4 and was enhanced by anti-IL-4. Only those cultures that produced IFN-gamma in vitro were uveitogenic in vivo. A long-term uveitogenic T cell line, initially derived in the presence of IL-12, produced IFN-gamma and IL-2, but not IL-4, and was CD4+ (Th1-like). Antigen-specific proliferation and IFN-gamma production of the line were enhanced by exogenous IL-4, TGF-beta, IL-2, IL-6, IL-7, and IL-9 and were inhibited by IL-10 and TNF-alpha. Our results provide support for the hypothesis that the uveitogenic effector T cell has a Th1-like phenotype. Furthermore, the data suggest that the effects of the cytokine milieu on fully differentiated Th1 effectors may differ considerably from their effects on less mature stages of antigen-specific T cells.     

Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.     

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.     

Subcongenic analysis of the Idd13 locus in NOD/Lt mice: evidence for several susceptibility genes including a possible diabetogenic role for beta 2-microglobulin

Although they share approximately 88% of their genome with NOD mice including the H2g7 haplotype, NOR mice remain free of T cell-mediated autoimmune diabetes (IDDM), due to non-MHC genes of C57BLKS/J (BKS) origin. NOR IDDM resistance was previously found to be largely controlled by the Idd13 locus within an approximately 24 cM segment on Chromosome 2 encompassing BKS-derived alleles for H3a, B2m, Il1, and Pcna. NOD stocks carrying subcongenic intervals of NOR Chromosome 2 were utilized to more finely map and determine possible functions of Idd13. NOR- derived H3a-Il1 (approximately 6.0 cM) and Il1-Pcna (approximately 1.2 cM) intervals both contribute components of IDDM resistance. Hence, the Idd13 locus is more complex than originally thought, since it consists of at least two genes. B2m variants within the H3a-Il1 interval may represent one of these. Monoclonal Ab binding demonstrated that dimerizing with the beta 2m(a) (NOD type) vs beta 2m(b) isoform (NOR type) alters the structural conformation, but not total expression levels of H2g7 class I molecules (e.g. Kd, Db). Beta 2m-induced alterations in H2g7 class I conformation may partially explain findings from bone marrow chimera analyses that Idd13 modulates IDDM development at the level of non-hematopoietically derived cell types controlling selection of diabetogenic T cells and/or pancreatic beta cells targeted by these effectors. Since trans-interactions between relatively common and functionally normal allelic variants may contribute to IDDM in NOD mice, the search for Idd genes in humans should not be limited to functionally defective variants.     

Cytokines and their roles in pancreatic islet beta-cell destruction and insulin-dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is a disease that results from autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The autoimmune response against islet beta-cells is believed to result from a disorder of immunoregulation. According to this concept, a T helper 1 (Th1) subset of T cells and their cytokine products, i.e. Type 1 cytokines--interleukin 2 (IL-2), interferon gamma (IFNgamma), and tumor necrosis factor beta (TNFbeta), dominate over an immunoregulatory (suppressor) Th2 subset of T cells and their cytokine products, i.e. Type 2 cytokines--IL-4 and IL-10. This allows Type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in beta-cell destruction. Type 1 cytokines activate (1) cytotoxic T cells that interact specifically with beta-cells and destroy them, and (2) macrophages to produce proinflammatory cytokines (IL-1 and TNFalpha), and oxygen and nitrogen free radicals that are highly toxic to islet beta-cells. Furthermore, the cytokines IL-1, TNFalpha, and IFNgamma are cytotoxic to beta-cells, in large part by inducing the formation of oxygen free radicals, nitric oxide, and peroxynitrite in the beta-cells themselves. Therefore, it would appear that prevention of islet beta-cell destruction and IDDM should be aimed at stimulating the production and/or action of Type 2 cytokines, inhibiting the production and/or action of Type 1 cytokines, and inhibiting the production and/or action of oxygen and nitrogen free radicals in the pancreatic islets.