Christina Bartholdy

Lambda Interferon (IFN-λ), a Type III IFN, Is Induced by Viruses and IFNs and Displays Potent Antiviral Activity against Select Virus Infections In Vivo

ABSTRACT Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-λ]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-λ1 and -λ2/3 in similar patterns. The IFN-λs were—unlike alpha/beta interferon (IFN-α/β)—induced directly by stimulation with IFN-α or -λ, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-λs have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-α potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-α reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-λ in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-λ completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-α, which had a more modest antiviral activity. Finally, pretreatment with IFN-λ enhanced the levels of IFN-γ in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-λ exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.

An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity.

Type III IFNs (IFN-λ/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-λ was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Rα-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR−/−) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA−/− and IFNAR−/− mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-αβ expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-λ expression, whereas IL-28Rα signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Rα only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-λ and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.

TLR2 and TLR9 Synergistically Control Herpes Simplex Virus Infection in the Brain

Viruses are recognized by the innate immune system through pattern recognition receptors (PRRs). For instance, HSV virions and genomic DNA are recognized by TLR2 and TLR9, respectively. Although several viruses and viral components have been shown to stimulate cells through TLRs, only very few studies have defined essential roles for single TLRs in innate immune defense in vivo. This could suggest that PRRs act in concert to mount the first line of defense against virus infections. To test this hypothesis we have examined the host response of C57BL/6, TLR2−/−, TLR9−/−, and TLR2/9−/− mice toward HSV-2 infection. After a systemic infection, the cytokine serum response was markedly reduced in the double knockout mice, but only partly affected in either strain of the single knockout mice. This was supported by in vitro data showing that HSV-induced cytokine expression relayed on TLR2 and TLR9 in a cytokine- and cell type-dependent manner. With respect to the cellular response to infection, we found that recruitment but not activation of NK cells was impaired in TLR2/9−/− mice. Importantly, the viral load in the brain, but not liver, was significantly higher in the brain of TLR2/9−/− mice whereas the viral loads in organs of single knockout mice were statistically indistinguishable from C57BL/6 mice. In the brain we found that TNF-α and the IFN-stimulated gene CXCL9 were expressed during infection and were dependent on either TLR2 or TLR9. Thus, TLR2 and TLR9 synergistically stimulate innate antiviral activities, thereby protecting against HSV infection in the brain.

Persistent Virus Infection despite Chronic Cytotoxic T-Lymphocyte Activation in Gamma Interferon-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus

ABSTRACT The role of gamma interferon (IFN-γ) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-γ-deficient (IFN-γ −/−) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-γ −/− mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-γ −/− mice. The prolonged effector phase in infected IFN-γ −/− mice was associated with elevated numbers of CD8+ T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8+ T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-γ −/− mice than in wild-type mice. These findings indicate that in the absence of IFN-γ, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-γ-mediated antiviral activity on the dynamics between virus replication and CTL activity.

CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell‐independent encephalitis, and infection resulted in a dramatic early up‐regulation of chemokine gene expression. Similar marked up‐regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV‐infected mice, only LCMV‐induced T cell‐dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus‐activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+‐dependent CNS inflammation.

Regulation of T cell migration during viral infection: role of adhesion molecules and chemokines

T cell mediated immunity and in particular CD8+ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8+ T cells to sites of viral infection.

Agonistic Anti-CD40 Antibody Profoundly Suppresses the Immune Response to Infection with Lymphocytic Choriomeningitis Virus

Previous work has shown that agonistic Abs to CD40 (anti-CD40) can boost weak CD8 T cell responses as well as substitute for CD4 T cell function during chronic gammaherpes virus infection. Agonistic anti-CD40 treatment has, therefore, been suggested as a potential therapeutic strategy in immunocompromised patients. In this study, we investigated whether agonistic anti-CD40 could substitute for CD4 T cell help in generating a sustained CD8 T cell response and prevent viral recrudescence following infection with lymphocytic choriomeningitis virus (LCMV). Contrary to expectations, we found that anti-CD40 treatment of MHC class II-deficient mice infected with a moderate dose of LCMV resulted in severe suppression of the antiviral CD8 T cell response and uncontrolled virus spread, rather than improved CD8 T cell immune surveillance. In Ab-treated wild-type mice, the antiviral CD8 T cell response also collapsed prematurely, and virus clearance was delayed. Additional analysis revealed that, following anti-CD40 treatment, the virus-specific CD8 T cells initially proliferated normally, but an increased cell loss compared with that in untreated mice was observed. The anti-CD40-induced abortion of virus-specific CD8 T cells during LCMV infection was IL-12 independent, but depended partly on Fas expression. Notably, similar anti-CD40 treatment of vesicular stomatitis virus-infected mice resulted in an improved antiviral CD8 T cell response, demonstrating that the effect of anti-CD40 treatment varies with the virus infection studied. For this reason, we recommend further evaluation of the safety of this regimen before being applied to human patients.

The Virus-Encoded Chemokine vMIP-II Inhibits Virus-Induced Tc1-Driven Inflammation

ABSTRACT The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8+ T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8+ T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.

Depletion of CD4+ T Cells Precipitates Immunopathology in Immunodeficient Mice Infected with a Noncytocidal Virus

IFN-γ-deficient (IFN-γ−/−) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4+ Th cells are required to establish and maintain this new equilibrium. The absence of IFN-γ does not impair the generation of IL-2-producing CD4+ cells, and depletion of these cells precipitates severe CD8+ T cell-mediated immunopathology in IFN-γ−/− mice, indicating an important role of CD4+ T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-γ−/− mice following CD4+ cell depletion. Initially the antiviral CTL response did not require CD4+ cells, but with time an impaired reactivity toward especially the glycoprotein 33–41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33–41 and nucleoprotein 396–404) CD8+ T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4+-depleted mice, especially in mice also deficient in IFN-γ. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-γ−/− mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-γ−/− hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8+ effector T cells is one important factor influencing the requirement for T cell help during viral infection.

The Role of CD80/CD86 in Generation and Maintenance of Functional Virus-Specific CD8+ T Cells in Mice Infected with Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis virus (LCMV)–specific CD8+ T cell responses are considered to be independent of CD28–B7 costimulation. However, the LCMV-specific response has never been evaluated in B7.1/B7.2−/− mice. For this reason, we decided to study the T cell response in B7.1/B7.2−/− mice infected with two different strains of LCMV, one (Traub strain) typically causing low-grade chronic infection, and another (Armstrong clone 53b) displaying very limited capacity for establishing chronic infection. Using Traub virus we found that most B7.1/B7.2−/− mice were unable to rid themselves of the infection. Chronic infection was associated with a perturbed CD8+ T cell epitope hierarchy, as well as with the accumulation of cells expressing markers of terminal differentiation and being unable to respond optimally to Ag restimulation. Examination of matched CD28−/− mice revealed a similar albeit less pronounced pattern of CD8+ T cell dysfunction despite lack of virus persistence. Finally, analysis of B7.1/B7.2−/− mice infected with Armstrong virus revealed a scenario quite similar to that in Traub infected CD28−/− mice; that is, the mice displayed evidence of T cell dysfunction, but no chronic infection. Taken together, these results indicate that B7 costimulation is required for induction and maintenance of LCMV-specific CD8+ T cell memory, irrespective of the LCMV strain used for priming. However, the erosion of CD8+ T cell memory in B7.1/B7.2−/− mice was more pronounced in association with chronic infection. Finally, virus-specific T cell memory was more impaired in the absence of B7 molecules than in the absence of the CD28 receptor, supporting earlier data suggesting the existence of additional stimulatory receptors for B7.