We document here that infection of prediabetic mice with a virus expressing an H-2Kb-restricted mimic ligand to a self epitope present on beta cells accelerates the development of autoimmune diabetes. Immunization with the mimic ligand expanded autoreactive T cell populations, which was followed by their trafficking to the islets, as visualized in situ by tetramer staining. In contrast, the mimic ligand did not generate sufficient autoreactive T cells in naive mice to initiate disease. Diabetes acceleration did not occur in H-2Kb-deficient mice or in mice tolerized to the mimic ligand. Thus, arenavirus-expressed mimics of self antigens accelerate a previously established autoimmune process. Sequential heterologous viral infections might therefore act in concert to precipitate clinical autoimmune disease, even if single exposure to a viral mimic does not always cause sufficient tissue destruction.

ABSTRACT CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP −/− ) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1β, IFN-γ-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2 b or H-2 d molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest K b tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-γ) or tumor necrosis factor alpha (TNF-α) cytokines and were unable to lyse PrP −/− embryo fibroblasts or macrophages coated with 51 Cr-labeled mPrP peptide. These results suggest that the expression of PrP sc in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-γ and TNF-α expression or release or lytic activity.

Systemic treatment with antibody to CD40 ligand (aCD40L) can prevent autoimmunity and transplant rejection in several animal models and is currently under evaluation in clinical trials. While it is known that aCD40L administration inhibits expansion and effector functions of aggressive T cells, it is still unclear whether additional regulatory mechanisms are operative. Here we demonstrate that a single episode of CD40L blockade during development of the autoaggressive immune response completely prevented autoimmune disease in the RIP-LCMV mouse model for virally induced type 1 diabetes. Interestingly, protection could be transferred by a highly potent, bitypic cell population sharing phenotypic and functional properties of both natural killer (NK) and dendritic cells (DC). Furthermore, protection of prediabetic recipients was autoantigen specific and did not result in generalized immunosuppression. The origin, function, and therapeutic potential of these bitypic NK/DC regulatory cells is discussed.

Treatment with nonmitogenic CD3 Ab reverses established autoimmune diabetes in nonobese diabetic mice by restoring self-tolerance, and is currently under clinical evaluation in patients presenting recent onset type I diabetes. Due to the immunosuppressive potential of this strategy, it was relevant to explore how this treatment would influence the outcome of concomitant viral infections. In this study, we used a transgenic model of virally induced autoimmune diabetes (rat insulin promoter-lymphocytic choriomeningitis virus) that allows for more precise tracking of the autoaggressive response and choice of the time point for initiation of autoimmunity. CD3 was most effective during a clearly defined prediabetic phase and prevented up to 100% of diabetes by drastically lowering activation of autoaggressive CD8 lymphocytes and their production of inflammatory cytokines. Interestingly, reversion of established disease could be achieved as well, when nonmitogenic CD3 was administered late during pathogenesis to overtly diabetic recipients. Most importantly, competence to clear viral infections was maintained. Thus, administration of nonmitogenic CD3 prevents diabetes by sufficient systemic reduction of (auto)aggressive lymphocytes, but without compromising antiviral immune competence.

Immunization with agonist peptides recognized by autoaggressive lymphocytes has been used successfully in several animal models for type 1 diabetes (T1D) or multiple sclerosis (MS) to prevent disease. Depending on the timing of immunization, use of adjuvant and route of administration either elimination of autoaggressive T cells or induction of regulation reflected by cytokine shifts were described. Since it was also reported that such agonist peptides could enhance autoimmunity by activating aggressive lymphocytes, our goal was to re-evaluate their efficacy in an antigen-specific model of virally-induced T1D that allowed us to precisely track the autoaggressive response. We find that rather than the route of administration (oral versus sc) the precise timing is important for inducing tolerance to self-antigens. Tolerance is transient and only immunization during a susceptible phase 10 to 20 days prior to the induction of disease but not in prediabetic mice resulted in protection. Further, use of a stronger adjuvant (CFA) compared to IFA enhanced the protective effect. Mechanistically, a transient loss of autoaggressive T cells was responsible for preventing disease, the effect was quantitative and no regulatory lymphocytes or cytokine shifts were induced by any of our treatments. Thus, MHC class I-restricted agonist peptides might only find a limited use in treating autoimmune disorders, because tolerance induction is transient and treatment has to be given very early, ideally prior to activation of the aggressive response.

In comparing the incidence of virally induced type 1 diabetes in F(1) crosses of RIP-LCMV mice to three different mouse strains identical at the major histocompatibility complex H-2D(b) locus, we surprisingly found that disease development was reduced by 80% in F(1) crosses to the SV129 genetic background and by 60% after eight backcrosses to the original C57BL/6 RIP-LCMV mice. In this model, diabetes is strongly dependent on a virally induced H-2D(b)-restricted cytotoxic T-cell (CTL) response. Importantly, numbers and effector functions of autoaggressive CD4 and CD8 lymphocytes were not decreased in the protected mice, and CTLs were still able to kill syngeneic islet cells in vitro with equal efficacy compared with CTLs from the original RIP-LCMV strain. Furthermore, CTLs were able to extravasate into islets in vivo, and no evidence for induction of regulatory cells was observed. However, regeneration of beta-cells in islets under "attack" occurred only in the protected SV129-crossed animals, whereas it was not evident at any time in any mice that developed diabetes. Thus, genetic factors can "override" the diabetogenic potential of high numbers of autoaggressive lymphocytes through, for example, increased islet regeneration. This finding has important implications for interpreting numbers and pathogenicity of autoreactive lymphocytes in prediabetic patients of genetically diverse backgrounds.

Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic beta-cells. The main factors directly implicated in beta-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse beta-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the beta-cells. Unexpectedly, we found that the resulting perforin-mediated killing of beta-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of beta-cells also requires a direct effect of gamma-interferon (IFN-gamma), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-gamma receptor on their beta-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most beta-cells occurs as cytokine-mediated death and requires IFN-gama in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes.

Mice expressing lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a transgene in their beta cells develop insulin-dependent diabetes mellitus (IDDM) only after LCMV infection. Inoculation of plasmid DNA encoding the insulin B chain reduced the incidence of IDDM by 50% in this model. The insulin B-chain DNA vaccination was effective through induction of regulatory CD4 lymphocytes that react with the insulin B chain, secrete IL-4, and locally reduce activity of LCMV-NP-autoreactive cytotoxic T lymphocytes in the pancreatic draining lymph node. In contrast, similar vaccination with plasmids expressing the LCMV viral ("self") protein did not prevent IDDM, because no such regulatory cells were induced. Thus, DNA immunization with plasmids expressing self-antigens might constitute a novel and attractive therapeutic approach to prevent autoimmune diseases, if the antigens are carefully preelected for an ability to induce regulatory lymphocytes in vivo.

The role of the STAT4 signaling pathway in autoimmune diabetes was investigated using the rat insulin promoter lymphocytic choriomeningitis virus model of virally induced autoimmune diabetes. Abrogation of STAT4 signaling significantly reduced the development of CD4(+)-T cell-dependent but not CD4(+)-T cell-independent diabetes, illustrating the fine-tuned kinetics involved in the pathogenesis of autoimmunity, Hen-ever, the absence of STAT4 did not prevent the generation of autoreactive Th1/Tc1 T cell responses, as well as protective antiviral immunity, Protection from insulin-dependent diabetes mellitus was associated with decreased numbers of autoreactive CTL precursors in the pancreas and the spleen and a general as well as Ag-specific reduction of IFN-gamma secretion by T lymphocytes. A shift from Th1 to Th2 T cell immunity was not observed. Hence, our results implicate both CTL and cytokines in beta cell, destruction. Selective inhibition of the STAT4 signal transduction pathway might constitute a novel and attractive approach to prevent clinical insulin-dependent diabetes mellitus in prediabetic individuals at risk.

The outcome of viral infections is dependent on the amount of tissue destruction caused either by direct lysis of infected cells and/or by immunopathology resulting from the immune response to the virus. We investigated whether induction of tolerance to only one viral protein could reduce immunopathology caused by nonlytic lymphocytic choriomeningitis virus (LCMV) in perforin-deficient hosts. Earlier studies had shown that LCMV infection results in aplastic anemia and death in most of these mice and that this is associated with bone marrow infiltration by antiviral cytotoxic T lymphocytes (CTL) that secrete inflammatory cytokines. We report here that perforin-deficient mice exhibit severe immunopathology in multiple organs that is characterized by infiltration of anti-LCMV CTL that secrete large amounts of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Importantly, this immunopathology is significantly reduced and long-term survival of LCMV infection is increased in perforin-deficient mice expressing LCMV nucleoprotein (NP) in the thymus (and therefore deleting most of their LCMV-NP CTL) compared to the situation in thymus nonexpressors. This is due to the selective reduction of NP-specific CTL responses and their inflammatory-cytokine (IFN-gamma and TNF-alpha) secretion and to a lack of pathogenetically relevant compensatory responses to other viral proteins. Thus, "selective reduction" of the antiviral immune response to only one viral protein can significantly reduce inflammatory immunopathology and might be a therapeutic possibility for certain nonlytic infections.

Targeted immune regulation can be achieved by use of tissue-specific T cells and offers the potential for organ-specific suppression of destructive autoimmune processes. Here, we report the generation and characterization of insulin B chain–specific “autoreactive” CD4+ regulatory T cells that locally suppress diabetogenic T cell responses against an unrelated self-antigen (viral transgene) in a virus-induced model for type 1 diabetes. Interleukin 4 (IL-4) is essential for prevention of diabetes since regulatory T cells cannot be induced in the absence of IL-4 or stat6 (IL-4 signaling pathway). Our observations demonstrate that autoreactive regulatory T cells can suppress autoreactive destructive T cell activity of differential antigenic specificity locally in the pancreatic draining lymph node, probably via cytokine-mediated modulation of antigen-presenting cells.

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2D(b) for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the D-b allele, which correlated directly with the affinity of the respective epitopes, Expression of the blocking peptide in the target cell lowered recognition of all D-b-restricted LCMV epitopes, In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-gamma secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV D-b-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on D-b-restricted anti-self(viral) CTL, Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-gamma and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.

The majority of the response of cytotoxic T-lymphocytes (CTL) to lymphocytic choriomeningitis virus (LCMV) in H-2dmice is directed toward one epitope located on the nucleoprotein (NP, aa 118–126), and usually no primary responses to other epitopes are detectable. Previous studies have shown that thymic expression of lymphocytic choriomeningitis virus-nucleoprotein (LCMV-NP) in H-2dtransgenic mice (Thy-NP mice) leads to deletion of high-affinity anti-LCMV-NP CTL by negative selection. Selection is incomplete, so that low-affinity NP-specific CTL pass through the thymus and are detectable in the periphery. To analyze the importance of interferon-γ (IFN-γ) in the ability of low-affinity antiviral CTL to clear an acute viral infection, double transgenic mice were generated that are IFN-γ deficient and express the NP of LCMV in the thymus (Thy-NP × IFN-γ −/− mice). When infected with LCMV, these bigenic mice were unable to clear the infection despite generating low-affinity primary antiviral CTL, and they became persistently infected. In contrast, IFN-γ competent Thy-NP mice cleared LCMV within 7–8 days and IFN-γ deficient mice that did not express NP in their thymus generated high-affinity CTL that terminated an acute LCMV infection within 10–12 days post-viral challenge. Persistently infected IFN-γ deficient mice selectively depleted LCMV-specific CTL and displayed reduced levels of antigen-presenting cells in the spleen, and 60% of these mice died at 2–3 months postinfection. Thus, IFN-γ is required for clearing an acute viral infection in the absence of a high-affinity CTL response. In the absence of IFN-γ persistent viral infection results despite the presence of low-affinity CTL.