Brett Charlton

The Th1/Th2 balance in autoimmunity

The study of autoimmune disease in the context of T-helper type 1 (Th1) and T-helper type 2 (Th2) CD4+ T-cell responses demonstrates that the relative contribution of either T-cell type to the development of a particular autoimmune response can influence whether or not this response leads to clinical disease. Moreover, this influence can be quite different depending on whether the particular disease process is cell mediated or antibody mediated. Recent studies have demonstrated that the development of Th1 and Th2 responses may be significantly influenced by the costimulatory molecules recognized by responding CD4 T cells, and by other undefined factors in the genetic background. It has also been demonstrated that autoreactive Th2 CD4+ cells can regulate the activity of disease-causing Th1 CD4+ T cells in vivo. Control of autoimmune disease may thus be achieved by procedures that regulate the relative contribution of Th1/Th2 CD4 T cells to an autoimmune response.

Cyclophosphamide-Induced Diabetes in NOD/WEHI Mice: Evidence for Suppression in Spontaneous Autoimmune Diabetes Mellitus

Nonobese diabetic (NOD) mice spontaneously develop a lymphocytic infiltration of pancreatic islets (insulitis) that may progress to overt diabetes. Virtually all NOD/WEHI mice develop insulitis, but very few progress to diabetes. However, cyclophosphamide (CY) can promote the onset of diabetes in NOD mice, including the NOD/WEHI strain. The means by which CY produces diabetes was investigated in NOD/WEHI mice, in which it was hypothesized that active suppression mechanisms prevented the progression from insulitis to diabetes. A study of the time course of insulitis in the islets after CY was given showed that insulitis was initially reduced but rapidly increased over 16 days, and T-lymphocytes were predominant in the lesion. This suggested a compression of the normal time course of the disease seen in NOD mice. CY did not produce diabetes in any of 11 non-NOD strains studied. Fetal isografts in NOD mice given CY several days before were subjected to lymphocytic infiltration and β-cell destruction. These findings suggested that CY was not directly (β-cell toxic and that altered β-cells were not essential for β-cell destruction. This was further demonstrated with subdiabetogenic doses of streptozocin, which significantly damaged β-cells but did not increase the severity of insulitis or induce diabetes as did CY. Most important, the transfer of mononuclear cells from nondiabetic NOD mice to mice given CY prevented diabetes, which indicated that the likely effect of CY was via immunomodulation, possibly by allowing poised effector cells to act on (β-cells. The NOD/WEHI mice appear to have suppressor mechanisms acting to halt the progression of the early insulitis lesion and so preventing diabetes occurring in most mice. We propose that CY removes these suppressors and thereby induces a rapid progression to diabetes by compressing the normal immune destruction process into a 2-wk period. This model affords the opportunity to study the process in ways not practicable in the usual time course of events.

Beta cell MHC class I is a late requirement for diabetes

Type 1 diabetes occurs as a result of an autoimmune attack on the insulin-producing beta cells. Although CD8 T cells have been implicated both early and late in this process, the requirement for direct interaction between these cells and MHC class I on the beta cells has not been demonstrated. By using nonobese diabetic mice lacking beta cell class I expression, we show that both initiation and progression of insulitis proceeds unperturbed. However, without beta cell class I expression, the vast majority of these mice do not develop hyperglycemia. These findings demonstrate that a direct interaction between CD8 T cells and beta cells is not required for initiation or early disease progression. The requirement for class I on beta cells is a relatively late checkpoint in the development of diabetes.

Monoclonal T Cells Identified in Early NOD Islet Infiltrates

To examine the hypothesis that a single initiating antigen was recognized by a monoclonal T cell population leading to subsequent inflammatory insulitis in non-obese (NOD) mouse islets, we examined the T cell receptor TCR V beta repertoire of islet-infiltrating T cells in very young (2-week-old) NOD mice. In independent experiments, we repeatedly identified one monoclonal TCR V-beta 8.2 gene product expressed by T lymphocytes infiltrating the islets of NOD mice at 2 weeks of age. The resultant inflammatory response quickly obscures the monoclonal nature of the initiating event. These data suggest that autoimmune diabetes in NOD mice may be initiated by recognition of a single autoantigen.

Prevention of cyclophosphamide-induced diabetes by anti-Vβ8 T-lymphocyte-receptor monoclonal antibody therapy in NOD/Wehi mice

Walter & Eliza Hall Institute nonobese diabetic (NOD/Wehi) mice exhibit a low incidence of spontaneous diabetes mellitus, but one large dose of cyclophosphamide (CY) can lead to a rapid progression to overt diabetes. Macrophages and Lyt-2+ and L3T4+ cells have been demonstrated to be involved in β-cell destruction in this model. The role of a specific subset of T-lymphocytes expressing a particular T-lymphocyte–receptor segment was examined in CY-induced diabetic NOD mice with a mouse anti-Vβ8 T-lymphocyte–receptor monoclonal antibody (F23.1). After administration of CY, only 4 of 51 treated mice became hyperglycemie compared to 23 of 47 untreated mice, 13 of 26 mice treated with an isotype-matched control ascites, and 4 of 6 mice given antibody-negative ascites. Insulitis was significantly reduced in the F23.1-treated group, and immunocytochemistry revealed the absence of Vβ8 expression on cells in the lymphoid organs and insulitis of these mice. This investigation revealed that Vβ8+ cells were implicated in CY-induced diabetes in NOD/ Wehi mice.

The role of Fas ligand in beta cell destruction in autoimmune diabetes of NOD mice

Abstract: Fas ligand (FasL), a type 2 membrane protein belonging to the TNF family, plays an important role in the induction of cell death. Ligation of Fas receptors by FasL results in apoptosis of the Fas‐expressing cell. Autoimmune diabetes results from β cell destruction by islet‐reactive T cells, a process that involves β cell apoptosis. This raises the question of whether the FasL‐Fas pathway plays a major role in β cell death. To address this issue it is important to know whether β cells express Fas and/or FasL and, if so, whether induction of these molecules leads to β cell death. In fact, both Fas and FasL have been demonstrated to be expressed by β cells in response to cytokine stimulation, although there remains an argument in the literature as to whether β cells truly express FasL. This is largely because FasL expression has only been demonstrable by immunohistochemistry and not by flow cytometry. Transgenic NOD mice with β cells expressing a FasL transgene develop an accelerated form of diabetes. We show here that β cells from FasL transgenic NOD mice are more susceptible to cytokine‐induced apoptosis than wild‐type β cells, consistent with the hypothesis that if β cells express FasL then Fas‐FasL interaction on the β cell surface is able to mediate β cell self‐death in the absence of T cells. Interventions that block the Fas‐FasL pathway may be useful, therefore, in the prevention or treatment of type 1 diabetes.

Inhibition of Nitric Oxide Synthase Initiates Relapsing Remitting Experimental Autoimmune Encephalomyelitis in Rats, Yet Nitric Oxide Appears to be Essential for Clinical Expression of Disease

Myelin basic protein-CFA-induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats is an acute monophasic disease from which animals recover. In this model, spontaneous relapses do not occur and rats develop a resistance to further active reinduction of disease. Previously, we reported that oral administration of the NO synthase inhibitor N-methyl-l-arginine acetate (l-NMA) to recovered rats precipitated a second episode of disease in 100% of animals. Further studies now show that this second clinical episode is actually a chronic relapsing disease that persists for months. This occurs only in rats that have recovered from actively induced EAE and not in rats recovered from passively induced EAE, suggesting the need for a peripheral Ag depot to induce secondary disease. We have also determined that clinical signs of EAE in l-NMA-treated recovered rats do not appear until l-NMA treatment has stopped. This is despite the fact that, at the same time point, CNS inflammatory lesions in symptomless animals receiving l-NMA are qualitatively and quantitatively similar to those with severe disease symptoms from whom l-NMA treatment has been withdrawn. The latter animals have significantly higher levels of reactive nitrogen intermediates in the cerebrospinal fluid than the former group. This study examines the mechanism of reinduction of disease by l-NMA treatment, and the findings suggest a dual role for NO in regulation of pathology in EAE that is dependent on site and timing of NO production.

Mechanisms of Accelerated Immune-Mediated Diabetes Resulting from Islet β Cell Expression of a Fas Ligand Transgene

Nonobese diabetic (NOD) mice transgenic for Fas ligand (FasL) on islet β cells (HIPFasL mice) exhibit an accelerated diabetes distinct from the normal autoimmune diabetes of NOD mice. This study was undertaken to define the mechanism underlying accelerated diabetes development in HIPFasL mice. It was found that diabetes in HIPFasL mice is dependent on the NOD genetic background, as HIPFasL does not cause diabetes when crossed into other mice strains and is lymphocyte dependent, as it does not develop in HIPFasLSCID mice. Diabetes development in NODSCID recipients of diabetic HIPFasL splenocytes is slower than when using splenocytes from diabetic NOD mice. β cells from HIPFasL mice are more susceptible to cytokine-induced apoptosis than wild-type NOD β cells, and this can be blocked with anti-FasL Ab. HIPFasL islets are more rapidly destroyed than wild-type islets when transplanted into nondiabetic NOD mice. This confirms that FasL+ islets do not obtain immune privilege, and instead NOD β cells constitutively expressing FasL are more susceptible to apoptosis induced by Fas-FasL interaction. These findings are consistent with the accelerated diabetes of young HIPFasL mice being a different disease process from the autoimmune diabetes of wild-type NOD mice. The data support a mechanism by which cytokines produced by the insulitis lesion mediate up-regulation of β cell Fas expression, resulting in suicide or fratricide of HIPFasL β cells that overexpress FasL.

An increase in the survival of murine H-2-mismatched cultured fetal pancreas allografts using depleting or nondepleting anti-CD4 monoclonal antibodies, and a further increase with the addition of cyclosporine.

Depletion of CD4+ T lymphocytes with monoclonal antibodies (mAbs) has been shown to prolong allograft survival in mice. In this study, two rat anti-CD4 mAbs, H129.19 and GK1.5, were administered either alone or in combination with cyclosporine (CsA) to recipients of MHC-mismatched (H-2k to H-2d) cultured fetal pancreas allografts to determine their effect on graft survival. When compared with control mice, splenic CD4+ cells of GK1.5-treated mice were depleted by greater than 95%, but in H129.19-treated mice no depletion of CD4+ cells occurred. Instead, rat Ig was present on the surface of CD4+ cells in H129.19-treated mice. Anti-CD4 therapy with either H129.19 or GK1.5 prolonged fetal pancreas allograft survival to a similar extent, but did not lead to indefinite survival. Blockade of the CD4 antigen by the mAb H129.19 was as effective as the depletion of CD4+ cells by GK1.5 in prolonging allograft survival. Rejection of grafts by day 28 posttransplantation occurred in the absence of CD4+ cells, as determined by both flow cytometric examination of spleen cells and immunoperoxidase staining of the graft site. CsA alone did not prolong graft survival, but its addition to either H129.19 or GK1.5 mAb treatment significantly increased the survival rate of grafts at 28 days compared with mAb treatment alone. These results suggest that CD4+ cell depletion is not essential for effective anti-CD4 mAb therapy--and, further, that CsA may have a direct inhibitory effect on CD8+ cells during allograft rejection.

Prevention of Autoimmune Diabetes through Immunostimulation with Q Fever Complement‐Fixing Antigen

Abstract: The most promising strategies for prevention of type 1 diabetes seem to be in the categories of immunomodulation (e.g., nondepleting anti‐CD3, Diapep, linomide) and/or immunostimulation (e.g., QFA, BCG). We are currently undertaking a research program directed toward better understanding of immunostimulants to help maximize the likelihood of success of future human clinical trials for diabetes prevention. This program is focused on the key areas of optimization of vaccine dose and route of administration, development of surrogate immune markers, and elucidation of the mechanism of protection. The mechanism whereby QFA protects against diabetes currently is not known. The elucidation of the mechanism should help identify the optimal way in which to administer QFA to provide diabetes protection. It may also assist the development of even more potent immunostimulatory vaccines.