While the inflammatory immune response mounted against viral infections is usually believed to enhance autoimmune processes, we discovered that it triggers two key regulatory phenomena that synergize to promote tolerance in type 1 diabetes (T1D). Acute infection of prediabetic non‐obese diabetic (NOD) mice with lymphocytic choriomeningitis virus (LCMV) significantly delayed autoimmune diabetes and reduced disease incidence. The delay of T1D was due to transient up‐regulation of Programmed death‐ligand 1 (PD‐L1) which was induced, at least in part, by interferon, and caused the elimination of PD‐1‐expressing, diabetogenic CD8+ T cells. The reduction of T1D incidence was due to increased number and function of CD4+CD25+Foxp3+ regulatory T cells (Tregs) which produced TGF‐β and maintained long‐term tolerance. PD‐L1 and virally enhanced Tregs conferred marked protection from T1D by acting together in synergy. LCMV induced the regulatory mechanisms that prevented T1D by utilizing Toll‐like receptor 2 and dendritic cells. These results reconcile current discordance about the role of viruses in T1D and provide novel insight into disease etiology and potential treatment.

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells in culture, but are also present in serum. They contain a wide array of protein ligands on their surface, which has led to the hypothesis that they might mediate intercellular communication. Indeed, data support that exosomes can transfer Ags to dendritic cells (DC), and, interestingly, that these DC can subsequently induce T cell priming or tolerance. We have investigated whether this concept can be expanded to antiviral immunity. We isolated exosomes from supernatant of cultured bone marrow-derived DC (BMDC) that were infected with lymphocytic choriomeningitis virus (LCMV) or loaded with an immunodominant LCMV peptide, and characterized them by flow cytometry upon binding to beads. We then incubated the exosome preparations with BMDC and looked at their potential to activate LCMV gp33-specific naive and memory CD8 T cells. We found that exosomes do not significantly contribute to CD8 T cell cross-priming in vitro. Additionally, exosomes derived from in vitro-infected BMDC did not exhibit significant in vivo priming activity, as evidenced by the lack of protection following exosome vaccination. Thus, DC-derived exosomes do not appear to contribute significantly to CTL priming during acute LCMV infection.

Type 1 Diabetes (T1D) is an autoimmune disease that results from the selective destruction of insulin‐producing beta cells in the pancreas. While it is generally believed that viral infections are responsible for inducing autoimmunity in individuals at risk, experimental evidence indicates that viruses can efficiently prevent T1D. We have identified two mechanisms by which acute lymphocytic choriomeningitis virus (LCMV) infection hampers autoimmune diabetes development in the mouse.The first mechanism is the direct consequence of the early release of inflammatory mediators following LCMV challenge which rapidly confer leukocytes from infected mice the capacity to diminish T1D. The second mechanism involves longer‐term modification of CD4+CD25+ regulatory T cells which become capable of preventing both spontaneous and virally induced T1D as a consequence of TGF‐beta production. Importantly, hampering of autoimmunity by viral infection does not appear to impair antiviral immunity and viral clearance.These findings provide novel mechanistic insight into the ability of viral infection to abort autoimmune diabetes. Induction of such mechanisms during infection may account for the capacity of viruses to prevent T1D as well as other autoimmune disorders.

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking 14-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F, hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the inummodominance hierarchy of CD8(+) T cell responses.

The primary CD8+ T cell response against lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice is directed against 28 H‐2b‐restricted epitopes derived from the glycoprotein, nucleoprotein and RNA polymerase L protein. Within these 28 epitopes a clear immunodominance hierarchy is observed. Some major epitopes dominate the response, while some epitopes are intermediate and others are relatively minor contributors to the overall response. To probe the mechanisms regulating CD8+ T cell immunodominance, we manipulated the number of epitopes seen during acute LCMV infection. Deletion of epitope‐specific responses, either by using a LCMV viral variant lacking 4 major epitopes, or C57BL/6J mice altogether lacking expression of H‐2Kb, resulted in an overall decrease of the response, minor compensatory effects, and no detectable responses against new epitopes. Increasing the number of epitopes available for recognition by use of H‐2b F1 hybrid mice, or delivery of LCMV antigens by recombinant vaccinia virus also did not dramatically alter the immunodominance pattern. These data suggest that immunodominance hierarchy might be intrinsic to the epitope and its antigen of origin. We are currently investigating these issues by directly measuring the hierarchies in MHC binding affinities, the yield of epitope production as a result of cellular processing, and by quantifying the existing naïve T cell repertoire by the use of a sensitive tetramer‐based enrichment method.

During an autoimmune process, the autoaggressive response spreads from the initiating autoantigen to other antigens expressed in the target organ. Based on evidence from experimental models for multiple sclerosis, such “antigenic spreading” can play an important role in the exacerbation of clinical disease. We evaluated whether pathogenesis of spontaneous diabetes in NOD mice could be accelerated in a similar way when a novel autoantigen was expressed in pancreatic β-cells. Unexpectedly, we found that the expression of the lymphocytic choriomeningitis virus nucleoprotein only led to marginal enhancement of diabetes, although such NOD-nucleoprotein mice were not tolerant to nucleoprotein. Although the frequency of nucleoprotein-specific CD8 T-cells in the pancreatic draining lymph node was comparable with the frequency of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific T-cells, more IGRP-specific CD8 T-cells were found both systemically and in the islets where there was a fourfold increase. Interestingly, and in contrast to nucleoprotein-specific CD8 T-cells, IGRP-specific T-cells showed increased CXCR3 expression. Thus, autoreactivity toward de novo–expressed β-cell autoantigens will not accelerate autoimmunity unless large numbers of antigen-experienced autoreactive T-cells expressing the appropriate chemokine receptors are present.

Abstract While viral infections may be capable of triggering autoimmune diabetes (T1D) in genetically susceptible individuals, accumulating evidence indicates that viruses can also prevent T1D. However, the underlying mechnisms are only partially understood. Here, we show that activation of CD4+CD25+ Tregs by acute lymphocytic choriomeningitis virus (LCMV) infection provides these cells with the capacity to prevent both spontaneous and virally induced T1D in the mouse. We found that CD4+CD25+ Tregs activated by LCMV in vivo or in vitro produce the regulatory cytokine TGF-b and are capable of protecting NOD and RIP-LCMV mice from spontaneous and LCMV-induced diabetes, respectively. Our results suggest that LCMV-modulated CD4+CD25+ Tregs act in a bystander fashion independent of beta-cell antigens and suppress TNF-a production by autoreactive CD8+ T cells, leading to prevention of T1D. These findings provide a novel mechanistic explanation for the previously reported ability of viral infections to prevent autoimmune diabetes. Furthermore, our results support a model explaining the differential ability of viral infections to modulate diabetes. Activation of Tregs during viral infections may be a general feature accounting for the ability of viruses to prevent T1D as well as other autoimmune disorders.

We examine here the outcome of viral encephalomyelitis; [mouse hepatitis virus (MHV) A59, Theiler's encephalomyelitis virus, and Coxsackievirus B3] in mice with autoantibodies to a central nervous system (CNS)-specific antigen, myelin oligodendrocyte glycoprotein, that usually develop no clinical disease. Morbidity and mortality of the acute viral CNS disease was augmented by the presence of the autoantibodies in all three viral infections. Transfer of serum containing the autoantibodies at the time of infection with MHV was sufficient to reproduce the exacerbated disease. The presence of the autoantibodies was found to result in increased infiltration of mononuclear cells into the brain. Early demyelination was severely augmented in brains and spinal cords of MHV-infected mice with CNS-specific autoantibodies. The antibody-mediated exacerbation was shown to be independent of the complement system but to require expression of Fc receptors, because it was observed in C'-3-deficient but not in Fc receptor-deficient mice. Our study illustrates die possibility that infections can lead to much more profound inummopathology in the presence of an otherwise latent autoinmume condition.

In immunotherapy, dendritic cells (DCs) can be used as powerful antigen-presenting cells to enhance or suppress antigen-specific immunity upon in vivo transfer in mice or humans. However, to generate sufficient numbers of DCs, most protocols include an ex vivo culture step, wherein the cells are exposed to heterologous serum and/or antigenic stimuli. In mouse models of virus infection and virus-induced autoimmunity, we tested how heterologous serum affects the immunomodulatory capacity of immature DCs generated in the presence of IL-10 by comparing fetal bovine serum (FBS)- or normal mouse serum (NMS)-supplemented DC cultures. We show that FBS-exposed DCs induce a systemic immune deviation characterized by reduction of virus-specific T cells, delayed viral clearance, and enhanced systemic production of interleukin 4 (IL-4), IL-5, and IL-10 to FBS-derived antigens, including bovine serum albumin (BSA). By contrast, DCs generated in NMS-supplemented cultures modulated immunity and autoimmunity in an antigen-specific fashion. These cells did not induce systemic IL-4, IL-5, or IL-10 production and inhibited generation of virus-specific T cells or autoimmunity only if pulsed with a viral antigen. These data underscore the importance of using autologous serum-derived immature DCs in preclinical animal studies to accurately assess their immunomodulatory potential in future human therapeutic settings, where application of FBS is not feasible.

ABSTRACTCTLA-4 is considered one of the most potent negative regulators of T-cell activation. To circumvent experimental limitations due to fatal lymphoproliferative disease associated with genetic ablation of CTLA-4, we have used radiation chimeras reconstituted with a mixture of CTLA-4+/+and CTLA-4−/−bone marrow that retain a normal phenotype and allow the evaluation of long-term T-cell immunity under conditions of intrinsic CTLA-4 deficiency. Following virus infection, we profiled primary, memory, and secondary CD8+and CD4+T-cell responses directed against eight different viral epitopes. Our data demonstrate unaltered antigen-driven proliferation, acquisition of effector functions, distribution of epitope hierarchies, T-cell receptor repertoire selection, functional avidities, and long-term memory maintenance in the absence of CTLA-4. Moreover, regulation of memory T-cell survival and homeostatic proliferation, as well as secondary responses, was equivalent in virus-specific CTLA4+/+and CTL-A-4−/−T-cell populations. Thus, lack of CTLA-4 expression by antigen-specific T cells can be compensated for by extrinsic factors in the presence of CTLA-4 expression by other cells. These findings have implications for the physiologic, pathological, and therapeutic regulation of costimulation.

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.

Viruses can cause but can also prevent autoimmune disease. This dualism has certainly hampered attempts to establish a causal relationship between viral infections and type 1 diabetes (T1D). To develop a better mechanistic understanding of how viruses can influence the development of autoimmune disease, we exposed prediabetic mice to various viral infections. We used the well-established NOD and transgenic RIP-LCMV models of autoimmune diabetes. In both cases, infection with the lymphocytic choriomeningitis virus (LCMV) completely abrogated the diabetic process. Interestingly, such therapeutic viral infections resulted in a rapid recruitment of T lymphocytes from the islet infiltrate to the pancreatic draining lymph node, where increased apoptosis was occurring. In both models this was associated with a selective and extensive expression of the chemokine IP-10 (CXCL10), which predominantly attracts activated T lymphocytes, in the pancreatic draining lymph node, and in RIP-LCMV mice it depended on the viral antigenic load. In RIP-LCMV mice, blockade of TNF-alpha or IFN-gamma in vivo abolished the prevention of T1D. Thus, virally induced proinflammatory cytokines and chemokines can influence the ongoing autoaggressive process beneficially at the preclinical stage, if produced at the correct location, time, and levels.

A long-standing paradox in cellular immunology concerns the conditional requirement for CD4 + T-helper (T H ) cells in the priming of cytotoxic CD8 + T lymphocyte (CTL) responses in vivo . Whereas CTL responses against certain viruses can be primed in the absence of CD4 + T cells, others, such as those mediated through ‘cross-priming’ by host antigen-presenting cells, are dependent on T H cells 1 , 2 , 3 , 4 . A clearer understanding of the contribution of T H cells to CTL development has been hampered by the fact that most T H -independent responses have been demonstrated ex vivo as primary cytotoxic effectors, whereas T H -dependent responses generally require secondary in vitro re-stimulation for their detection. Here, we have monitored the primary and secondary responses of T H -dependent and T H -independent CTLs and find in both cases that CD4 + T cells are dispensable for primary expansion of CD8 + T cells and their differentiation into cytotoxic effectors. However, secondary CTL expansion (that is, a secondary response upon re-encounter with antigen) is wholly dependent on the presence of T H cells during, but not after, priming. Our results demonstrate that T-cell help is ‘programmed’ into CD8 + T cells during priming, conferring on these cells a hallmark of immune response memory: the capacity for functional expansion on re-encounter with antigen.

Negative selection is designed to purge the immune system of high-avidity, self-reactive T cells and thereby protect the host from overt autoimmunity. In this in vivo viral infection model, we show that there is a previously unappreciated dichotomy involved in negative selection in which high-avidity CD8(+) T cells specific for a dominant epitope are eliminated, whereas T cells specific for a subdominant epitope on the same protein preferentially escape deletion. Although this resulted in significant skewing of immunodominance and a substantial depletion of the most promiscuous T cells, thymic and/or peripheral deletion of high-avidity CD8(+) T cells was not accompanied by any major change in the TCR Vbeta gene family usage or an absolute deletion of a single preferred complementarity-determining region 3 length polymorphism. This suggests that negative selection allows high-avidity CD8(+) T cells specific for subdominant or cryptic epitopes to persist while effectively deleting high-avidity T cells specific for dominant epitopes. By allowing the escape of subdominant T cells, this process still preserves a relatively broad peripheral TCR repertoire that can actively participate in antiviral and/or autoreactive immune responses.

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.

Administration of autoantigens through DNA immunizations or via the oral route can prevent progression of islet destruction and lower the incidence of type 1 diabetes in animal models. This beneficial effect is mediated by autoreactive regulatory CD4 lymphocytes, and it is known that their induction depends on the precise dose and route of antigen administration. However, it is not clear which endogenous factors determine when such immunizations lead to activation of regulatory versus aggressive autoreactive lymphocytes and how a deleterious outcome can be avoided. Here we describe novel observations made in an animal model for virally induced type 1 diabetes, showing that the endogenous expression levels of the islet antigens and glutamic acid decarboxylase determine whether immunization with these antigens is beneficial or detrimental. Lower expression levels in beta-cells support immune regulation resulting in induction of autoreactive, regulatory cells characterized by increased IL-4 production (Th2-like), whereas higher levels favor Th1-like autoaggressive responses characterized by augmented IFN-gamma generation. Co-immunization with an IL-4-expressing plasmid reduces the risk of augmenting autoaggression and in this way increases the safety margin of this immune-based therapy. Our findings will be of importance for designing safe antigen-specific interventions for human type 1 diabetes.

In vivo administration of APC expressing Fas ligand (Fas-L(+) dendritic cells (DCs)) has shown promise in dampening allergic reactions and transplant rejection. Since the effect in these studies was mainly on CD4 lymphocytes, our goal was to evaluate the ability of such killer DCs to eliminate antiviral CD8 lymphocytes and in this way ameliorate viral immunopathology or, conversely, impede viral clearance. Intravenous administration of Fas-L(+) DCs resulted in a 50% reduction of lytic CD8 precursors following intracerebral infection with lymphocytic choriomeningitis virus (LCMV), and accordingly, immunopathology and survival of LCMV meningitis were improved, whereas viral clearance remained unaffected. In transfer studies the effect of the Fas-L(+) DCs was only quantifiable on experienced, not naive, CD8 lymphocytes. Importantly, loading of Fas-L(+) DCs with viral Ag before therapy was not necessary to achieve this effect, indicating that non-LCMV-infected Fas-L(+) DCs acquired viral Ag during acute LCMV infection in vivo. Our studies delineate important aspects for the clinical use of Fas-L(+) DCs in vivo. One should expect that they acquire viral Ags and suppress antiviral CD8 responses to some degree when given while an acute infection is ongoing. In terms of safety it is encouraging that resolution of the infection, at least in the case of LCMV, is not inhibited.