Damage control, rather than unresponsiveness, effected by protective DX5+ T cells in autoimmune diabetes.

The progression of autoimmune diabetes is regulated. We examined here the cellular controls exerted on disease that developed in the BDC2.5 T cell receptor-transgenic model. We found that all BDC2.5 mice with a monoclonal, beta cell-reactive, T cell repertoire developed diabetes before 4 weeks of age; transfer of splenocytes from young standard NOD (nonobese diabetic) mice into perinatal monoclonal BDC2.5 animals protected them from diabetes. The protective activity was generated by CD4+ alphabeta T cells, which operated for a short time at disease initiation, could be partitioned according to DX5 cell surface marker expression and split into two components. Protection did not involve clonal deletion or anergy of the autoreactive BDC2.5 cells, permitting their full activation and attack of pancreatic islets; rather, it tempered the aggressiveness of the insulitic lesion and the extent of beta cell destruction.

NK T cell-induced protection against diabetes in V alpha 14-J alpha 281 transgenic nonobese diabetic mice is associated with a Th2 shift circumscribed regionally to the islets and functionally to islet autoantigen.

The onset of autoimmune diabetes is related to defective immune regulation. Recent studies have shown that NK T cells are deficient in number and function in both diabetic patients and nonobese diabetic (NOD) mice. NK T cells, which are CD1d restricted, express a TCR with an invariant V alpha 14-J alpha 281 chain and rapidly produce large amounts of cytokines. V alpha 14-J alpha 281 transgenic NOD mice have increased numbers of NK T cells and are protected against diabetes onset. In this study we analyzed where and how NK T cells interfere with the development of the anti-islet autoimmune response. NK T cells, which are usually rare in lymph nodes, are abundant in pancreatic lymph nodes and are also present in islets. IL-4 mRNA levels are increased and IFN-gamma mRNA levels decreased in islets from diabetes-free V alpha 14-J alpha 281 transgenic NOD mice; the IgG1/IgG2c ratio of autoantibodies against glutamic acid decarboxylase is also increased in these mice. Treatment with IL-12 (a pro-Th1 cytokine) or anti-IL-4 Ab abolishes the diabetes protection in V alpha 14-J alpha 281 NOD mice. The protection from diabetes conferred by NK T cells is thus associated with a Th2 shift within islets directed against autoantigen such as glutamic acid decarboxylase. Our findings also demonstrate the key role of IL-4.

Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex.

The NK1.1 cell surface receptor, which belongs to the NKR-P1 gene cluster, has been bred onto nonobese diabetic (NOD) mice for two purposes. The first was to tag NK and NKT cells for easier experimental identification of those subsets and better analysis of their implication in type 1 diabetes. The second was to produce a congenic strain carrying Idd6, a susceptibility locus that has been repeatedly mapped in the vicinity of the NKR-P1 gene cluster and the NK complex, to explore the impact of this locus upon autoimmune diabetes. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice manifest reduced disease incidence and improved NK and NKT cell performance, as compared with wild-type NOD mice. The association of those two features in the same congenic strain constitutes a strong argument in favor of Idd6 being associated to the NK complex. This could explain at the same time the multiple alterations of innate immunity reported in NOD mice and the fact that disease onset can be readily modified by boosting the innate immune system of the mouse.

Prion diseases and the immune system.

Transmissible spongiform encephalopathies are caused by unusual infectious agents that are purported to contain a single type of macromolecule, a modified host glycoprotein. The term prion has been applied to this group of agents. Surprisingly, the immune system appears to behave as a Trojan's horse rather than a protective fortification during prion infections. Because prions seem to be essentially composed of a protein, PrP(Sc), identical in sequence to a host encoded protein, PrP(C), the specific immune system displays a natural tolerance. However, lymphoid organs are strongly implicated in the preclinical stages of the disease. Certain immunodeficient animals are resistant to prions after peripheral inoculation. In normal subjects, cells of the immune system support the replication of prions and/or allow neuroinvasion. A better understanding of these aspects of prion diseases could lead to immunomanipulation strategies aimed at preventing the spread of infectious agents to the central nervous system.

Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells.

alpha-Galactosylceramide (alpha-GalCer) is a glycolipid with potent antitumor properties that binds to CD1d molecules and activates mouse Valpha14 and human Valpha24 NKT cells. Surprisingly, we found that, as early as 90 min after alpha-GalCer injection in vivo, NK cells also displayed considerable signs of activation, including IFN-gamma production and CD69 induction. NK activation was not observed in RAG- or CD1-deficient mice, and it was decreased by pretreatment with anti-IFN-gamma Abs, suggesting that, despite its rapid induction, it was a secondary event that depended on IFN-gamma release by NKT cells. At later time points, B cells and CD8 T cells also began to express CD69. These findings identify a high-speed communication network between the innate and adaptive immune systems in vivo that is initiated upon NKT cell activation. They also suggest that the antitumor effects of alpha-GalCer result from the sequential recruitment of distinct innate and adaptive effector lymphocytes.

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.

Male gonadal environment paradoxically promotes dacryoadenitis in nonobese diabetic mice.

Similar to pancreatic islets, submandibular glands are more rapidly infiltrated in female NOD mice than in males. The present comparative analysis of cellular infiltrations in lacrimal glands, however, revealed the opposite finding. At 12 wk of age, approximately 25% of male lacrimal tissue area is infiltrated, whereas age-matched female NOD mice still lack major signs of inflammation. T cells predominate in early stages of invasion, but B cells accumulate promptly in more advanced stages, and ultimately dominate over T cells. Dacryoadenitis is promoted by sex hormones, as suggested by the reduced infiltrations seen in orchidectomized NOD males (P < 0.01). It is also controlled by the local environment provided by the lacrimal tissue. Splenocytes from 4- and 20-wk-old female NOD mice cause massive lesions upon adoptive transfer into NOD male recipients while, conversely, female recipients develop barely any histological sign of infiltration, even after transfer of splenocytes from 20-wk-old donor males. These observations provide strong evidence for a dacryoadenitis-promoting role of male gonadal hormones in NOD mice, a finding that contrasts the known androgen-mediated protective effects on insulitis and submandibulitis in the same strain and on dacryoadenitis in other animal models of Sjögren's syndrome.

Non-obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP).

Rodents develop inflammatory, non-infectious, prostatitis upon autoimmuniz-ation with male accessory gland (MAG) extracts in complete Freund's adjuvant (CFA). Although there appears to be differences among strains, with respect to susceptibility to induction, specific details are not known about the genetic bases of such differences. Because NOD mice have inherited a genetic predisposition to autoimmune lesions affecting, apart from the islets of Langerhans, a large array of secretory glands such as salivary glands, thyroid, parathyroids and adrenal cortex, we selected this strain to assess the influence of inherited genes upon experimentally-induced autoimmune prostatitis (EAP). Indeed, MAG extracts injected into young NOD males in association with CFA cause a severe inflammatory reaction in the prostate, accompanied by a humoral and T cell-mediated response. NOD mice develop a more aggressive form of EAP than Wistar rats, the strain of reference used to establish the model. In NOD mice, disease begins earlier, affects 100% of the animals, does not require boosting and leads to florid infiltrates circumscribed to lateral and dorsal prostatic lobes. Immune mice develop a T cell-mediated response to MAG assessed by in vitro proliferation and accompanied by the release of IFN-gamma, whereas IL-4 is not detectable in the same culture super-natants. To assess the influence of the NOD background genes upon EAP susceptibility, we tested C57BL/6.H2(g7) mice in parallel. NOD mice are considerably more susceptible to EAP induction than congenic C57BL/6.H2(g7) mice. Both strains demonstrate a detectable humoral and cell-mediated response against MAG, but the histopathological manifestations are considerably more dramatic in NOD than in the C57BL/6.H2(g7) strain. Our results thus support the notion that NOD mice have background genes which favour severe autoimmune manifestations, irrespective of the target tissue.

Analysis of susceptibility of NOD mice to spontaneous and experimentally induced thyroiditis.

Beside diabetes, non-obese diabetic (NOD) mice develop sporadic lymphoid infiltration of the thyroid gland, mimicking Hashimoto's thyroiditis. We have examined the prevalence of those manifestations in NOD mice, the influence of the major histocompatibility complex (MHC) and the association with autoantibodies. The incidence at 1 year is of 14.3% in wild-type NOD mice versus 19.6% in congenic NOD.H2k mice. The moderate, but statistically significant difference, based on the analysis of 161 NOD and 169 NOD.H2k mice, suggests that MHC genes partially control spontaneous NOD thyroiditis. Autoantibodies against thyroglobulin (Tg) are mouse specific and their presence correlates closely with thyroiditis. The strong correlation between cellular and humoral anomalies therefore resembles Hashimoto's thyroiditis. NOD and NOD.H2k mice actively immunized against Tg develop severe chronic lesions with epithelium necrosis and interstitial tissue fibrosis. Most interestingly, those lesions do not regress spontaneously as in CBA/J mice. Paradoxically, the response to Tg of lymph node cells from NOD mice is weaker both in proliferation and cytokine production. The defect is most evident for interferon-gamma-producing T cells and is reflected in the marked deficit in IgG2a antibodies. Thus a moderate anti-Tg response seems to favor chronicity of thyroiditis. In conclusion, NOD and NOD.H2k mice offer a unique opportunity of analyzing the factors leading to immune chronicity in a genetic context which promotes autoimmune endocrinopathies.

Immunological tolerance to a pancreatic antigen as a result of local expression of TNFalpha by islet beta cells.

Recent experiments have suggested that tumor necrosis factor alpha (TNFalpha) can down-regulate islet-specific T cells and prevent the development of autoimmune diabetes. Here we demonstrate that transgenic mice expressing both TNFalpha and the Leishmania major LACK antigen in the pancreas (RIP-TNFalpha/RIP-LACK) exhibit an impaired ability to mount a CD4+ T cell response against LACK. In addition, peripheral CD4+ T cells from TCR transgenic mice (TCR-LACK/RIP-TNFalpha/RIP-LACK) produced reduced interleukin-2 but elevated levels of T helper 2 cytokines in response to LACK peptide in vitro. Taken together, our data suggest that TNFalpha may act in vivo to modulate a potentially damaging self-reactive T cell response by inducing tolerance to pancreatic antigens.

Treatment with neutralizing antibodies specific for IL-1beta prevents cyclophosphamide-induced diabetes in nonobese diabetic mice.

Interleukin-1 (IL-1) has been shown to be involved in the pathogenesis of IDDM, but it is not clear which form, IL-1alpha or IL-1beta, is predominantly implicated. In this study, we have evaluated the contribution of IL-1beta by treating diabetes-prone nonobese diabetic (NOD) mice with specific neutralizing antibodies. First, we assessed the neutralizing potential of these antibodies in C57BL/6 mice under acute septic shock by measuring IL-1beta in sera 4 h after lipopolysaccharide injection. One milligram and 0.1 mg of anti-IL-1beta antibodies (Abs) were capable of neutralizing the IL-1beta produced, and the effect persisted for at least 5 days. Second, we evaluated the role of IL-1beta in the cyclophosphamide (CY)-accelerated model of diabetes. Nondiabetic male NOD mice were injected with 200 mg/kg CY and treated twice weekly with anti-IL-1beta Ab. The incidence of diabetes reached 76 and 100% in the control groups treated with 0.25 and 0.1 mg rabbit IgG, respectively. In contrast, only 34% of mice treated with 0.25 mg of anti-IL-1beta Ab became diabetic. In the group treated with 0.1 mg of anti-IL-1beta Ab, 89% of the mice became diabetic in the same period of time, demonstrating that the protective effect was dose dependent. Our results show that IL-1beta is a critical effector molecule in this model of IDDM and that its specific inhibition could be an attractive target for therapeutic intervention.

Prevention of autoimmune diabetes mellitus in NOD mice by transgenic expression of soluble tumor necrosis factor receptor p55.

The non-obese diabetic (NOD) mouse represents a relevant animal model of autoimmunity for insulin-dependent diabetes mellitus. The pathogenic role of tumor necrosis factor (TNF) in insulitis and beta cell destruction observed in these mice remains controversial, since injections of TNF or of anti-TNF antibodies have been reported to exert protection or acceleration of diabetes, depending on the timing of administration. In this study, we demonstrate that, in contrast to the non-transgenic littermates, NOD mice with permanent neutralization of TNF by high blood levels of soluble TNF receptor p55-human FcIgG3-fusion molecules resulting from the expression of a transgene are protected from spontaneous diabetes. They are also protected from accelerated forms of disease caused by transfer of NOD spleen cells or cyclophosphamide injections. This protection is associated with a marked decrease in the severity and incidence of insulitis and in the expression of the adhesion molecules MAdCAM-1 and ICAM-1 on the venules of pancreatic islets. These data suggest a central role for TNF-alpha in the mediation of insulitis and of the subsequent destruction of insulin-secreting beta-cells observed in NOD mice. They may be relevant to cell-mediated autoimmune diseases in general, in which treatment with soluble TNF receptors might be beneficial.

Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse.

An immunoregulatory role has recently been attributed to the discrete subset of major histocompatibility complex class I-restricted NK1+ mature heat-stable antigen- (HSA-) thymocytes expressing an unusual Vbeta8-biased T cell receptor repertoire. NK1+ T cells are the main interleukin (IL)-4 producers upon priming. We have studied the size and the function of this subset in the nonobese diabetic (NOD) mouse, a model of spontaneous T cell-mediated autoimmune insulin-dependent diabetes. This study was complicated by the absence in this strain of the NK1.1 allele, the only one for which an antibody is available. To circumvent this difficulty, the cells, hereafter designated the NK1+-like T subset, were characterized by the use of monoclonal antibodies which showed the Vbeta8 bias in the CD44+ Ly-49+ MEL-14- 3G11- thymocyte subset of non-autoimmune strains and of its absence in class I-deficient (beta2-microglobulin-/-) mice. A clear deficit in the number of NK1+-like cells was evidenced at 3 weeks of age in NOD mice. It was still present at 8 weeks of age in the double-negative CD4-CD8- population. The functional anomaly was even more striking: NOD mouse NK1+-like thymocytes virtually lacked the ability to produce IL-4 at 3 weeks and still showed a very reduced capacity at 8 weeks. NK1+ T cell deficiency was also suggested in the periphery by the reduction of Ly-49A+ cells in the spleen of 3- and 8-week-old NOD mice and the absence of short-term production of IL-4 in vitro by NOD mouse spleen cells 90 min after the administration of anti-CD3 antibody, a response attributed to NK1+ T cells. Taken together, these data demonstrate a very early defect in NK1+-like T cells which could be involved in the genesis of autoimmunity in NOD mice through a deficiency in Th2 cell function.

Genetic control of giant perivascular space formation in the thymus of NOD mice.

The immune system of NOD mice exhibits several anomalies, one being the intrathymic formation of giant perivascular spaces (PVSs) filled with mature thymocytes and some B-cells, intermingled within a network of extracellular matrix. The abnormal retention of thymocytes on their way to the periphery could have a profound impact on the nature of the exported cells and the regulation of autoimmune events. In the present study, we evaluated the appearance of this defect into F1 hybrids, the association with some of the known diabetes susceptibility loci (Idd genes) in a panel of NOD and reciprocal C57BL congenic strains, and the relative contribution of epithelial versus hematopoietic stroma. The analysis of F1 hybrid thymuses reveals a dominant expression of thymic giant PVS that is only marginally influenced by the outcross strain. Moreover, giant PVS expression in major histocompatibility complex (MHC) and Idd congenic mice is determined by the genetic background. All of the NOD congenics express the anomaly, irrespective of the Idd resistance alleles that have been introgressed, whereas none of the C57BL congenic mice present abnormal PVS. Finally, the expression of giant PVS in parental --> F1 bone marrow chimeras is predominantly controlled by the thymic NOD-derived hematopoietic microenvironment. In conclusion, the giant PVS formation in the NOD mouse thymus is a dominantly inherited anomaly associated with hematopoietic-derived tissue and with non-MHC genes. The exact contribution of PVS to the autoimmune process remains to be definitively established.

Inhibition of submandibular and lacrimal gland infiltration in nonobese diabetic mice by transgenic expression of soluble TNF-receptor p55.

Besides a prominent mononuclear cell infiltration of the islets of Langerhans, nonobese diabetic (NOD) mice also show massive cellular infiltrates of the submandibular and lacrimal glands concomitant with histological signs of tissue damage. To obtain insights into the mechanisms operative during the initiation and progression of tissue damage, we followed by in situ hybridization the appearance of cells containing mRNA of the gene encoding the proinflammatory cytokine TNF-alpha in the cellular infiltrates. Cells expressing TNF-alpha are mainly located in infiltrates, are absent in nonaffected glands, and are preferentially found among CD4 T cells. Secretion of TNF-alpha by gland-infiltrating cells was confirmed by an ELISPOT procedure. Direct evidence for an instrumental role of TNF-alpha in initiation and progression of submandibular and lacrimal gland infiltration is provided by the observed significant reduction in the extent of infiltration in nonobese diabetic mice transgenic for a soluble TNF receptor p55 fused to the Fc part of human IgG3. This protection from infiltration is paralleled by decreased expression of the adhesion molecules ICAM-1 and VCAM-1 in submandibular and lacrimal glands. These data suggest a central role of TNF-alpha in the initiation and progression of autoimmune tissue destruction of salivary glands and indicate beneficial effects of soluble TNF receptors in the treatment of organ-specific autoimmune diseases.

Early defect of immunoregulatory T cells in autoimmune diabetes.

A potential immunoregulatory function has recently been attributed to the discrete subset of major histocompatibility complex (MHC) class I-restricted TCR-alpha beta mature thymocytes expressing an unusual V beta 8-biased T cell receptor repertoire. This T cell subset which also selectively express the CD44 marker is the main IL-4 producer in the thymus. Nonobese diabetic (NOD) mice were found to have a marked deficit in the number and functional capacity of CD44+ TCR-alpha beta+ thymocytes from as early as 3 weeks of age. The deficiency in IL-4 production was completely corrected after incubation with interleukin-7 (IL-7), a selective growth factor for CD44+ TCR-alpha beta+ mature thymocytes. This abnormality in T cell differentiation could explain the Th2 functional deficiency that may be a key element in the emergence of Th1-driven autoimmune disease in NOD mice.

The contribution of animal models to the understanding of the pathogenesis of type 1 diabetes.

The autoimmune nature of insulin-dependent diabetes mellitus (type 1 diabetes) has been definitively established during the past ten years only, owing essentially to the development of the NOD and the BB rat models. Three of the four criterias required for defining an autoimmune disease have been demonstrated in these animal models. IDDM is accompanied by immunological stigmatas including circulating autoantibodies and insulitis (lymphocytic infiltration of the islets of Langerhans), it is attenuated or prevented by immunosuppressors and it is transferable from diabetic to non-diabetic mice or rats, via T lymphocytes. Only the fourth criterium, namely the induction of the disease by immunization with an autoantigen, has so far not been met. As is the case for many, if not most, autoimmune diseases, the pathogenesis of IDDM is, however, far from being totally understood. Many aspects including the circumstances favoring escape from self-tolerance, the role of the genetic background, the nature of the pancreatic antigens involved in the initiation and perpetuation of the disease, the effector mechanisms responsible for the elimination of the insulin cells and, most importantly, the conditions for restoring tolerance are at the forefront of immunopathologists' concerns. We provide in this review an account of the present situation in these different areas of research. There is no doubt that a cure or a prevention of the disease will be available in the forseeable future. Experiments on animal models have already initiated several clinical trials and epidemiological studies, and this is probably only the beginning of a long list.

Accelerated beta-cell destruction in adoptively transferred autoimmune diabetes correlates with an increased expression of the genes coding for TNF-alpha and granzyme A in the intra-islet infiltrates.

Autoimmune destruction of beta-cells in nonobese diabetic (NOD) mice is greatly accelerated by adoptive cotransfer of syngeneic CD4+ and CD8+ T-cells from diabetic animals into newborn NOD mice. We followed, by in situ hybridization, the appearance of mRNA of the tumor necrosis factor (TNF)-alpha gene and, as a marker for activated cytotoxic T-cells, of the serine protease granzyme A gene in the cellular infiltrates generated by cell transfer at birth. Cells expressing the genes for granzyme A or TNF-alpha were seen in considerable numbers already on day 14, after adoptive transfer. These numbers gradually increased in the intra-islet infiltrates from day 14 through day 30 after adoptive transfer. Compared with our previous findings in NOD mice developing spontaneous insulin-dependent diabetes mellitus (IDDM) (Held W, MacDonald HR, Weissman IL, Hess MW, Mueller C: Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes. Proc Natl Acad Sci USA 87:2239-2243, 1990), frequencies of cells with TNF-alpha and granzyme A mRNA were 2- and 12-fold higher, respectively, in transferred IDDM (trIDDM). TNF-alpha mRNA positive cells were predominantly found in the CD4+ T-cell subset of the pancreas-infiltrating cells, whereas granzyme A mRNA positive cells were mainly observed in the CD4- T-cell subset. The effects of the observed enhanced TNF expression upon the pathogenesis of trIDDM are as yet unknown.(ABSTRACT TRUNCATED AT 250 WORDS)