Activation of Natural Killer (NK) T Cells during Murine Cytomegalovirus Infection Enhances the Antiviral Response Mediated by NK Cells
ABSTRACT
NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and α-galactosylceramide (α-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jα281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by α-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-γ), have revealed that the antiviral effects of α-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of α-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-γ.
Innate immune responses are critical in the process of immune surveillance, with natural killer (NK) and NK1.1+ T (NKT) cells being among the main mediators of early host responses. The role of NK cells in these processes has been the subject of intensive investigation, and it is clear that they play a crucial role as effectors in tumor control (38, 41, 46) as well as in limiting the spread of viral infections (4, 5, 42). In particular, infection of mice by the herpesvirus murine cytomegalovirus (MCMV) has proven to be one of the best systems for evaluating the role of NK cells following a viral challenge (29, 43). In contrast, NKT cells have been shown to play a central role in early innate responses to tumors (35, 37), but their relevance in response to challenges with viral pathogens is still largely unclear (5). Here, we have examined the relevance of classical, T-cell receptor (TCR)-restricted NKT cells in controlling MCMV infection. Because of the similarity in structure and biology between MCMV and human cytomegalovirus, the former provides a unique model of human disease and, importantly, it permits the study of in vivo infection in the natural host (1).
Classical murine NKT cells are a specialized subset of T lymphocytes that express a number of NK cell markers, including NK1.1 (3, 14), together with a restricted TCR repertoire, consisting of a single invariant TCR α chain encoded by Vα14Jα281 in association with a Vβ8.2, Vβ7, or Vβ2 TCR β chain. The resultant semi-invariant TCRαβ is specific for CD1d, a nonclassical class I molecule (3) and recognizes glycolipid antigens, the nature of which is currently unclear. A marine sponge-derived glycosphingolipid, α-galactosylceramide (α-GalCer), has been identified as a ligand that specifically binds the semi-invariant TCR of NKT cells in association with CD1d (8, 21). Following the association of the α-GalCer/CD1d complex with the Vα14 TCR, NKT cells become activated (8, 21), rapidly release immunoregulatory cytokines, such as gamma interferon (IFN-γ) or interleukin 4 (IL-4), and increase their cytotoxic capacity (3, 21, 22).
Rapid release of cytokines by NKT cells suggests their role in the regulation of immune responses and highlights their potential as targets for immunotherapy. Indeed, α-GalCer-stimulated NKT cells have been shown to drive immune responses with both Th1 and Th2 biases. For example, repeated α-GalCer administration can enhance Th2-mediated immune responses (9, 24, 33), which have been shown to protect against experimentally induced Th1-type colitis (31) and type I diabetes (17, 32). Conversely, α-GalCer has also been shown to protect against malaria (15) and to suppress the ability of the B16 melanoma to form liver and lung metastases by a Th1- and IFN-γ-dependent mechanism (22, 27, 36, 45). Importantly, it is now appreciated that the antitumor effects of α-GalCer are mediated in a collaborative manner by both NK and NKT cells (26, 34, 36).
The induction of antitumor immune responses following α-GalCer therapy has been proposed to occur in a defined sequence of events (35). Briefly, production of IFN-γ by α-GalCer-stimulated NKT cells induces IL-12 production by CD11c+ dendritic cells (DC) in a CD40/CD40L-dependent manner, thus establishing a positive feedback loop for IFN-γ production (23, 44). IFN-γ and IL-12, resulting from activation of NKT cells, then selectively induce NK cell proliferation, IFN-γ secretion, and cytotoxicity (12, 35). NKT cells activated by α-GalCer can subsequently promote bystander activation of NK cells, B cells, CD4+ T cells, CD8+ cytotoxic T lymphocytes, and DC (9, 10, 12, 23, 24, 28).
The relevance of α-GalCer-activated classical NKT cells in the control of herpesvirus infections is unknown. Here, we have utilized the MCMV model of viral infection to determine the relevance of NKT cells in the early immune responses that limit the spread of this herpesvirus infection in visceral organs. The cytotoxic function of NK cells is known to control MCMV replication in visceral organs during acute-phase infection (7, 43) and limit viral spread and replication prior to the development of adaptive CD8+-cytotoxic-T-lymphocyte responses which resolve productive virus infection (25, 30). In this report, we show that although NKT cells do not play a role in the natural control of MCMV infection, their activation, following α-GalCer therapy, results in improved viral clearance through the activation of bystander NK cells.
MATERIALS AND METHODS
Mice.
Wild-type (WT) inbred C57BL/6J (B6) and BALB/c mice were purchased from the Animal Resources Centre (Perth, Australia) or from the Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia). B6 TCRJα281-deficient (B6.Jα281−/−) mice (originally provided by M. Taniguchi, Chiba University Graduate School of Medicine, Chiba, Japan), BALB/c perforin-deficient (BALB/c.pfp−/−) mice, BALB/c IFN-γ-deficient (BALB/c.IFN-γ−/−) mice (Genentech, San Francisco, Calif.), and BALB/c mice deficient for both perforin and IFN-γ (BALB/c.pfp−/−.IFN-γ−/−) were bred at the Peter MacCallum Cancer Institute (Melbourne, Australia). Adult female mice (8 to 12 weeks old) were used in all experiments. All animal experimentation was performed with the approval of the Animal Ethics and Experimentation Committee of the University of Western Australia and according to the guidelines of the National Health and Medical Research Council of Australia.
Cells.
Primary mouse embryo fibroblasts were cultured in minimal essential medium (Gibco BRL, Grand Island, N.Y.) supplemented with 10% neonatal calf serum (Gibco BRL) and antibiotics (100 μg of penicillin [CSL]/ml and 40 μg of gentamicin [Pharmacia and Upjohn, Sydney, Australia]/ml).
Treatment of mice.
For the pathogenesis studies, mice were infected intraperitoneally (i.p.) with 104 PFU of salivary gland-propagated stocks of the virulent MCMV strain K181-Perth. Control animals were mock infected with mouse osmolarity-buffered saline (MOBS)-5% fetal calf serum (FCS). Mice were sacrificed at various times postinfection, and tissues were collected (see below). For α-GalCer therapy, mice received 2 μg of α-GalCer diluted in MOBS-5% FCS by i.p. injection on days 0 and 4 relative to virus inoculation. On day 0, mice were inoculated with α-GalCer 1 h after virus inoculation. Control mice were injected with an identical volume of vehicle solution (0.025% polysorbate) diluted in MOBS-5% FCS. α-GalCer was kindly provided by Kirin Brewery Pharmaceutical Research Laboratory, Gunma, Japan. To deplete NK cells, mice were inoculated i.p. with 100 μl of MOBS-0.5% FCS containing 200 μg of anti-asialoGM1 antibody (Wako Pure Chemical Industries Ltd., Osaka, Japan), as described previously (34), on days −1, 0, and 6 relative to virus inoculations.
In vivo growth of MCMV.
Mice were inoculated with the virulent MCMV strain K181-Perth, as described above. At designated times postinoculation (p.i.), mice were sacrificed and spleens, livers, lungs, and salivary glands were removed. All organs were individually weighed, homogenized in cold minimal essential medium-2% neonatal calf serum, and centrifuged at 3,000 × g for 15 min at 4°C. The supernatants were stored at −80°C, and virus titers were subsequently quantified on mouse embryo fibroblasts by standard plaque assay (2). Virus titers are expressed as log means ± standard errors of the mean (SEM). Statistical analyses were performed by using the nonparametric Mann-Whitney test.
RESULTS
α-GalCer therapy improves clearance during acute-phase MCMV infection.
To determine the relevance of activated NKT cells in anti-MCMV responses, we studied the effect of α-GalCer therapy on the ability of MCMV to replicate in visceral organs and salivary glands. MCMV replicates to high titers in the visceral organs of susceptible mice during the acute stage of infection (days 2 to 6). Treatment with α-GalCer significantly (P < 0.001) reduced virus titers in the spleens of susceptible BALB/c WT mice, at days 2, 4, and 6 postinfection (Fig. 1). At these times, MCMV titers were reduced by >1.0 log (day 2), >2.0 log (day 4), and >0.5 log (day 6) when treated and untreated mice were compared. In the liver, the therapeutic effect of α-GalCer was also significant at days 2 and 4 p.i. (P < 0.001) and day 6 p.i. (P ≤ 0.05) but less pronounced with an ∼1.0 log reduction in virus titers observed at day 2 p.i. and a ≤0.5 log reduction at days 4 and 6 p.i. in α-GalCer-treated mice compared to controls (Fig. 1). In the lungs and salivary glands, sites of viral persistence for MCMV, viral replication is not detectable until at least days 4 and 6 p.i., respectively. At these early times, when virus infection is first established in these organs, α-GalCer significantly reduced viral replication (Fig. 1). However, no therapeutic effects of α-GalCer were observed during the persistent phase of virus infection in either the lungs or the salivary glands (Fig. 1).
A therapeutic effect of α-GalCer was also observed in B6 WT mice infected with 105 PFU of MCMV. At this virus dose, MCMV replicates effectively in MCMV-resistant B6 WT mice. As observed in BALB/c WT mice, α-GalCer treatment resulted in reduced viral replication in visceral organs (Fig. 2). In the spleens of α-GalCer-treated B6 mice, a significant >1.0 log reduction (P < 0.005) in virus titer was observed at day 2 p.i. (Fig. 2). In the liver, virus titers were significantly (P < 0.005) reduced at days 2 and 4 (>1.0 log reduction) (Fig. 2). A transient increase in virus titer was reproducibly observed at day 6 p.i. in the livers of α-GalCer-treated B6 mice. However, by day 10 p.i., virus titers in visceral organs were equivalent in α-GalCer-treated B6 and untreated control B6 mice. Titers from salivary glands were comparable at day 10 p.i. in α-GalCer-treated mice and vehicle-treated control mice (data not shown).
The above results indicate that α-GalCer-activated NKT cells play a significant role in limiting MCMV replication during early acute-phase infection of visceral organs. The phenotype observed in MCMV-infected mice after α-GalCer treatment is consistent with the timing of an NK cell response, which is induced shortly after MCMV infection (2, 7, 43).
Role of NKT cells in the antiviral effects of α-GalCer.
Having shown that α-GalCer has an antiviral effect in vivo during the acute stages of MCMV infection, we wanted to determine whether the observed therapeutic effects are mediated directly by activated, cytolytic NKT cells. To address the specific involvement of NKT cells in antiviral responses, we analyzed MCMV pathogenesis in B6.Jα281−/− mice that are deficient in classical NKT cells (11). It must be noted that CD1d−/− mice, also deficient in NKT cells, were not used in these experiments as it would not be possible to discriminate between a role of (i) NKT cells themselves, (ii) the CD1d molecule, and/or (iii) polyclonal CD1d-reactive T cells. Since the therapeutic effects of α-GalCer were observed in both BALB/c and B6 mice, the specific role of classical NKT cells was analyzed by using Jα281−/− mice on the B6 genetic background. In B6 mice, the loss of innate effectors required for effective MCMV clearance would result in increased viral pathogenesis, with viral replication expected to be equivalent to that observed in susceptible mice, which have inefficient innate immune responses and are thus unable to clear MCMV infection effectively (7). For these experiments, B6.Jα281−/− and B6 WT control mice were infected with 104 PFU of MCMV. This virus dose was chosen so that, in the event that NKT cells were required for MCMV clearance, viral replication and disease would at most be equivalent to those observed in susceptible mice but would not lead to death. If NKT cells were found to be necessary for effective MCMV clearance, a higher dose of virus (105 PFU) would result in mortality. Following MCMV infection, NKT-cell-deficient B6.Jα281−/− mice showed viral replication which was overall equivalent to that observed in B6 WT mice (Fig. 3). The loss of NKT cells did not result in significant differences in MCMV titers in the spleen or liver at either day 2, day 4, or day 6 p.i. (Fig. 3), indicating that NKT cells per se do not play a critical role in the natural clearance of virus from visceral organs during acute-phase MCMV infection. A consistent increase in virus titer was observed in the livers of B6.Jα281−/− mice compared to those of B6 WT controls at day 10 p.i. This difference, although significant (P < 0.005), was transient and disappeared by day 12 p.i. (Fig. 3). No differences in virus titers were observed in the salivary glands and lungs of B6.Jα281−/− mice compared to those of B6 WT mice at days 6, 10, and 12 p.i. (data not shown).
The antiviral effects of α-GalCer therapy were then tested in NKT-cell-deficient B6.Jα281−/− mice. As expected, in B6.Jα281−/− mice, viral replication was not altered by α-GalCer therapy and virus titers were equivalent in treated and untreated mice (Fig. 4). The effects of α-GalCer therapy were tested in B6.Jα281−/− mice infected with both 104 and 105 PFU of MCMV (Fig. 4A and B, respectively). In B6 WT mice infected with 105 PFU of MCMV, the therapeutic effects of α-GalCer in both the spleen and the liver are maximal at day 2 p.i. (Fig. 2). At the same virus dose and same time point, virus titers in the spleen and liver were equivalent in untreated B6.Jα281−/− mice and B6.Jα281−/− mice that received α-GalCer (Fig. 4B).
Based on these results, we conclude that NKT cells are involved in inducing the antiviral effects of α-GalCer, despite the fact that they do not appear to possess direct antiviral activity during a natural infection with MCMV.
Role of NK cells in the antiviral effects of α-GalCer.
Since it appears that NKT cells play an indirect role in restricting viral replication after α-GalCer treatment, possibly by activating NK cells, we specifically assessed the role of NK cells in the observed antiviral effect of α-GalCer therapy. Viral pathogenesis was assessed in α-GalCer-treated WT mice that were selectively depleted of NK cells, but not NKT cells, by anti-asialoGM1 administration, as described previously (34). MCMV-susceptible BALB/c WT mice were used in this experiment, resulting in naturally high virus titers in visceral organs at early times postinfection. Depletion of NK cells by anti-asialoGM1 treatment resulted in a 0.5 log elevation of MCMV titers in the liver at day 6 p.i. (P < 0.05) (Fig. 5), whereas virus titers in the spleen (Fig. 5) and salivary gland (data not shown) remained equivalent to those of untreated control animals.
The therapeutic effect of α-GalCer was markedly diminished after depletion of NK cells by anti-asialoGM1 treatment. A marginal (<1 log) reduction in virus titer was observed in the spleens of anti-asialoGM1-treated animals that received α-GalCer at day 4 p.i. (P < 0.005) in comparison to the >2 log reduction (P < 0.0005) observed at the same time point in animals that received α-GalCer without NK cell depletion (Fig. 5). As expected, significant reductions in virus titers in the spleen were observed after α-GalCer treatment on days 2 (P < 0.0005) and 6 (P < 0.005) post-MCMV infection. In contrast, at day 2 and 6 p.i., α-GalCer did not lead to any reduction in virus titers in the spleens of mice that were also depleted of NK cells (Fig. 5). Similar to the observations made for the spleen, administration of α-GalCer in NK-cell-depleted BALB/c WT mice resulted in a marginal reduction of virus titers in the liver at day 4 p.i. only (P < 0.005). This reduction was only to levels equivalent to those observed in control, vehicle-treated mice (Fig. 5). In contrast, and as expected, significant reductions in viral replication in the liver were observed on days 2 (P < 0.005), 4 (P < 0.005), and 6 (P < 0.05) p.i., when mice received α-GalCer without anti-asialoGM1 to deplete NK cells.
The replication of MCMV in salivary glands was not altered by α-GalCer therapy in NK-cell-depleted mice compared with that in non-NK-cell-depleted mice (data not shown). These results indicated that in MCMV-susceptible BALB/c mice, NK cells are the primary mediators of the antiviral activity of α-GalCer in visceral organs. The minimal residual antiviral effect of α-GalCer in NK-cell-depleted mice may be mediated by other bystander cells or may represent an effect of the few NK cells (<10% as assessed by fluorescence-activated cell sorter analysis) (data not shown) not completely depleted by anti-asialoGM1 treatment.
Role of perforin and IFN-γ in the antiviral effects of α-GalCer.
Having shown that NK cells are the primary effector population mediating the antiviral effects of α-GalCer, we analyzed the specific roles of perforin and IFN-γ, these being the two major effector molecules used by NK cells to clear MCMV infection (29, 43). In addition, IFN-γ released by activated NKT cells activates NK cells to release more IFN-γ and up-regulates their perforin-mediated cytotoxic activity (12, 16, 36). To test the specific importance of perforin and IFN-γ in the enhanced antiviral host responses induced by α-GalCer, we investigated the efficacy of α-GalCer therapy in mice deficient in perforin (BALB/c.pfp−/−), IFN-γ (BALB/c.IFN-γ−/−), or perforin and IFN-γ (BALB/c.pfp−/−.IFN-γ−/−) (Fig. 6).
In BALB/c.pfp−/− mice, α-GalCer did not significantly reduce virus titers (Fig. 6). In spleens of α-GalCer-treated BALB/c.pfp−/− mice, virus titers were only slightly, and not significantly, reduced at day 4 p.i. (Fig. 6). In the liver and lung, virus titers in BALB/c.pfp−/− mice were not altered by α-GalCer therapy (Fig. 6). In BALB/c.IFN-γ−/− mice, a significant (P < 0.05) 1 log reduction in virus titer was observed at day 2 p.i. in the livers of α-GalCer-treated mice (Fig. 6). In other organs of BALB/c.IFN-γ−/− mice, virus titers were not reduced by α-GalCer therapy (Fig. 6) and, indeed, administration of α-GalCer increased virus titers at day 6 p.i. in the liver and lung (P < 0.05) (Fig. 6).
In BALB/c.pfp−/−.IFN-γ−/− mice, α-GalCer did not alter virus titers in the spleen at any time postinfection (Fig. 6). Similarly, α-GalCer therapy did not reduce virus titers in either the liver or the lung of BALB/c.pfp−/−.IFN-γ−/− mice (Fig. 6). In fact, in BALB/c.pfp−/−.IFN-γ−/− mice, α-GalCer therapy resulted in a transient, but significant (P < 0.05), increase in virus titer in the lung at day 4 p.i. (Fig. 6).
Although overall virus titers (except for those of the lung) were only marginally increased in the untreated gene-targeted mice examined compared to those in BALB/c WT mice, MCMV already replicates in the spleens and livers of BALB/c WT mice at the maximal load sustainable in these visceral organs. In fact, all of the gene-targeted mice tested were more susceptible to MCMV infection, and unlike BALB/c WT, none of which died as a result of MCMV infection, all of the gene-targeted mice succumbed to viral infection by day 8 or 9 p.i. (data not shown).
DISCUSSION
The contribution of NK cells in innate immune responses to viral infections is well established. In contrast, the involvement of NKT cells in viral immune surveillance remains poorly understood (5). In this study, we investigated the relevance of NKT cells during infection with the herpesvirus MCMV. The role of NKT cells was defined both in the context of a natural infection and after activation with α-GalCer. Treatment of mice with α-GalCer induced a significant antiviral response resulting in decreased virus titers in the visceral organs of MCMV-susceptible BALB/c WT mice. Similar results were obtained in B6 WT mice. Although normally resistant to MCMV infection, B6 WT mice show susceptibility to escalating doses of virus (2). At a dose of 105 PFU, which caused pathogenesis in B6 WT vehicle-treated mice, B6 WT mice receiving α-GalCer demonstrated reduced viral replication, with lower titers in both the spleen and liver, similar to the effects observed with BALB/c WT mice.
The effects of α-GalCer therapy are normally mediated by a potent and selective activation of NKT cells via association of the α-GalCer/CD1d complex with the Vα14 TCR (6, 8, 21). In MCMV infection, we have shown that NKT cells are necessary to induce the α-GalCer-mediated antiviral therapy, since the effects of α-GalCer were abolished in NKT-cell-deficient B6.Jα281−/− mice. Importantly, although activation of NKT cells results in improved viral clearance, these cells do not play a major role in controlling the early acute phase of natural infection with MCMV. Indeed, at early times postinfection, NKT-cell-deficient B6.Jα281−/− mice did not show increased susceptibility to MCMV, since viral replication in the visceral organs of these mice was equivalent to that observed in B6 WT mice. Similarly, NKT cells have been shown to be dispensable for the control of lymphocytic choriomeningitis virus infection (39). Although NKT cells do not appear to play a major role in restricting MCMV replication during the acute stage of infection, virus titers were transiently and slightly increased (<1.0 log) in the livers of B6.Jα281−/− mice 10 days after infection, suggesting that NKT cells may play a role in helping to activate adaptive immune responses that control viral replication during the later stages of acute-phase infection. This conclusion was corroborated by the finding that the effects observed in B6.Jα281−/− mice at day 10 p.i. were transient, with equivalent virus titers in B6.Jα281−/− and B6 WT mice by day 12 p.i.
Previous studies have reported on the antiviral effects of α-GalCer-activated NKT cells, with improved viral clearance or prevention of virus-induced disease described for hepatitis B virus (20), diabetogenic encephalomyocarditis virus (13), and respiratory syncytial virus (18). In the case of diabetogenic encephalomyocarditis virus (13) and respiratory syncytial virus (18) infections, the therapeutic effects of α-GalCer were accompanied by the demonstration that NKT cells also play a critical role in viral clearance without α-GalCer activation. Although it is not possible to conclusively ascribe the reported effects directly to classical NKT cells, as the studies were undertaken in CD1d−/− mice, it appears that there may be differences in the role played by NKT cells and α-GalCer-activated NKT cells in different viral infections. The studies reported here may be more analogous to those of Kakimi and colleagues, which demonstrated that the α-GalCer-induced inhibition of hepatitis B virus replication was principally mediated by the rapid induction of IFN-α/β and IFN-γ in the liver (20).
Thus, having shown that NKT cells are dispensable for the control of MCMV replication during a natural infection but essential for the antiviral efficacy of α-GalCer therapy, we wanted to establish whether the observed antiviral response was mediated directly through NKT cells or indirectly through activation of other immune effectors. It is known that activated NKT cells exhibit increased perforin-mediated cytotoxicity against tumor cell lines in vitro (10, 12) while also rapidly releasing immunoregulatory cytokines such as IL-4 and IFN-γ (3). Cytokines produced by activated NKT cells can in turn trigger bystander activation of NK cells, which involves proliferation, further IFN-γ production, and cytotoxicity (10, 12).
Depletion of NK cells prior to α-GalCer therapy confirmed the involvement of these effectors in the antiviral response induced by α-GalCer, with the efficacy of the therapy almost completely abolished in mice depleted of NK cells but possessing an intact NKT cell population. Having shown that the therapeutic effects of α-GalCer in MCMV infection were mediated by the activation of NK cells, we further examined the involvement of specific effector mechanisms, namely perforin and IFN-γ, in the antiviral response by utilizing gene-targeted mice. These studies demonstrated that the α-GalCer-induced antiviral response was dependent upon both perforin-mediated cytotoxicity and IFN-γ release, as mice deficient for one or both of these molecular effector systems were unable to mount a complete α-GalCer-induced antiviral response.
Recent studies in a B16 melanoma metastasis model demonstrated that the antitumor activity of α-GalCer requires the sequential production of IFN-γ by both NKT and NK cells, but it occurs independently of perforin release (36). This contrasts with our present findings, which clearly demonstrate that perforin is the major contributor to the α-GalCer-induced antiviral response. Although treatment of BALB/c.IFN-γ−/− mice with α-GalCer demonstrated that the production of IFN-γ was integral to the α-GalCer-mediated antiviral effect in the spleen, the available data cannot distinguish between the relative importance of IFN-γ produced by NKT cells and NK cells. The present studies have revealed that activated NKT cells are key mediators of the antiviral effects of α-GalCer, with NK cells, IFN-γ, and perforin all playing crucial roles. Interestingly, although it has been postulated that IFN-γ is the most important anti-MCMV mechanism in the liver (43), α-GalCer still showed an antiviral effect in these organs in IFN-γ-deficient mice. In contrast, α-GalCer therapy did not reduce viral replication in the livers of perforin-deficient mice, suggesting that perforin plays a crucial antiviral role at this site in α-GalCer-treated mice.
Activation of NKT cells by α-GalCer can lead to both IFN-γ and IL-4 secretion (9, 24, 33). IL-4 is involved in the induction of a Th2-mediated humoral immune response, which does not play a role in the clearance of MCMV infection (19). In BALB/c.IFN-γ−/− mice, α-GalCer therapy had detrimental effects in the liver and lung at day 6 postinfection. Due to the targeted deletion of the IFN-γ gene in these mice, α-GalCer-stimulated NKT cells only secrete IL-4. Consequently, in these animals the balance of the immune response induced by activated NKT cells will be biased towards the Th2 pathway, resulting in less-efficient clearance of MCMV, as observed.
It is important to note that our previous studies on the effects of α-GalCer in mediating tumor suppression, together with our present analysis of α-GalCer antiviral activity, have revealed that activated NKT cells can regulate different effector immune responses. Thus, the antitumor effects of α-GalCer are dependent on NK cells and IFN-γ, whereas the α-GalCer antiviral activity requires NK cells, IFN-γ, and perforin. These differences are most likely due to differences in the key factors associated with tumor suppression versus viral clearance. Thus, a requirement for perforin-mediated cytotoxicity may be unnecessary for tumor suppression when the antiangiogenic effects of IFN-γ alone may be sufficient to prevent tumor growth and metastasis (40). In contrast, perforin is likely to be essential in mediating the α-GalCer antiviral effects due to a requirement for direct cytolysis of virus-infected targets. Consistent with this hypothesis is the finding that the antiviral response induced by α-GalCer appears to be most potent during acute-phase MCMV infection, controlling virus replication most effectively in the spleen and liver. In both these organs, the early control of MCMV infection is highly reliant on innate effector mechanisms (7, 43). Thus, in α-GalCer therapy, the timing of the antiviral response elicited and the organs in which it occurs concur with our conclusion that the therapy specifically and potently enhances innate immune responses mediated principally by activated NK cells.
In this report we have provided the first evidence that, although TCR Vα14Jα281 NKT cells do not play a critical role in restricting early acute-phase natural infection with MCMV, activating them with α-GalCer induces a potent and specific antiviral response. We have shown that the antiviral response elicited by α-GalCer is dependent on Vα14Jα281 NKT cells, that the effectors are activated NK cells, and that both perforin and IFN-γ are critical molecular effector mechanisms. These studies have not only elucidated the role of different innate immune effectors in viral immune surveillance, but they provide the basis for the design of targeted antiviral therapies.
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Acknowledgments
We are grateful to Masaru Taniguchi for providing the B6.TCRJα281-deficient mice. We also thank the Kirin Brewery for kindly providing α-GalCer. Christine Hall and Donna Pead are thanked for assistance with animal husbandry. Anthony Scalzo, Dale Godfrey, and Geoffrey Shellam are thanked for helpful discussions and critical reading of the manuscript.
The project was supported by grants from the National Health and Medical Research Council of Australia (NH&MRC) and the Wellcome Trust. S.L.H.V.D. and H.A.T. are supported by grants from the NH&MRC and the Wellcome Trust, M.J.S. is supported by an NH&MRC Principal Research Fellowship, and M.A.D.-E. is supported by a Wellcome Trust Overseas Senior Research Fellowship in Biomedical Science in Australia.
REFERENCES
1.
Alford, C. A., and W. J. Britt. 1996. Cytomegalovirus, p. 629-660. In B. N. Fields (ed.), Virology. Raven Press, New York, N.Y.
2.
Allan, J. E., and G. R. Shellam. 1984. Genetic control of murine cytomegalovirus infection: virus titres in resistant and susceptible strains of mice. Arch. Virol. 81 : 139-150.
3.
Bendelac, A., M. N. Rivera, S. H. Park, and J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity and function. Annu. Rev. Immunol. 15 : 535-562.
4.
Biron, C. A. 1997. Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9 : 24-34.
5.
Biron, C. A., and L. Brossay. 2001. NK cells and NKT cells in innate defense against viral infections. Curr. Opin. Immunol. 13 : 458-464.
6.
Brossay, L., M. Chioda, N. Burdin, Y. Koezuka, G. Casorati, P. Dellabona, and M. Kronenberg. 1998. CD1d-mediated recognition of an α-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J. Exp. Med. 188 : 1521-1528.
7.
Brown, M. G., A. O. Dokun, J. W. Heusel, H. R. C. Smith, D. L. Beckman, E. A. Blattenberger, C. E. Dubbelde, L. R. Stone, A. A. Scalzo, and W. M. Yokoyama. 2001. Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 292 : 934-937.
8.
Burdin, N., L. Brossay, Y. Koezuka, S. T. Smiley, M. J. Grusby, M. Gui, M. Taniguchi, K. Hayakawa, and M. Kronenberg. 1998. Selective ability of mouse CD1 to present glycolipids: α-galactosylceramide specifically stimulates Vα14+ NK T lymphocytes. J. Immunol. 161 : 3271-3281.
9.
Burdin, N., L. Brossay, and M. Kronenberg. 1999. Immunization with α-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29 : 2014-2025.
10.
Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, and A. Bendelac. 1999. Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J. Immunol. 163 : 4647-4650.
11.
Cui, J. Q., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors. Science 278 : 1623-1626.
12.
Eberl, G., and H. R. MacDonald. 2000. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur. J. Immunol. 30 : 985-992.
13.
Exley, M. A., N. J. Bigley, O. Cheng, S. M. A. Tahir, S. T. Smiley, Q. L. Carter, H. F. Stills, M. J. Grusby, Y. Koezuka, M. Taniguchi, and S. P. Balk. 2001. CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus. J. Leukoc. Biol. 69 : 713-718.
14.
Godfrey, D. I., K. J. L. Hammond, L. D. Poulton, M. J. Smyth, and A. G. Baxter. 2000. NKT cells: facts, functions and fallacies. Immunol. Today 21 : 573-583.
15.
Gonzalez-Aseguinolaza, G., C. de Oliveira, M. Tomaska, S. Hong, O. Bruna-Romero, T. Nakayama, M. Taniguchi, A. Bendelac, L. Van Kaer, Y. Koezuka, and M. Tsuji. 2000. Alpha-galactosylceramide-activated Vα14 natural killer T cells mediate protection against murine malaria. Proc. Natl. Acad. Sci. USA 97 : 8461-8466.
16.
Hayakawa, Y., K. Takeda, H. Yagita, S. Kakuta, Y. Iwakura, L. Van Kaer, I. Saiki, and K. Okumura. 2001. Critical contribution of IFN-γ and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of α-galactosylceramide. Eur. J. Immunol. 31 : 1720-1727.
17.
Hong, S., M. T. Wilson, I. Serizawa, L. Wu, N. Singh, O. V. Naidenko, T. Miura, T. Haba, D. C. Scherer, J. Wei, M. Kronenberg, Y. Koezuka, and L. Van Kaer. 2001. The natural killer T-cell ligand α-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice. Nat. Med. 7 : 1052-1056.
18.
Johnson, T. R., S. M. Hong, L. Van Kaer, Y. Koezuka, and B. S. Graham. 2002. NK T cells contribute to expansion of CD8+ T cells and amplification of antiviral immune responses to respiratory syncytial virus. J. Virol. 76 : 4294-4303.
19.
Jonjic, S., I. Pavic, B. Polic, I. Crnkovic, P. Lucin, and U. H. Koszinowski. 1994. Antibodies are not essential for the resolution of primary cytomegalovirus infection but limit dissemination of recurrent virus. J. Exp. Med. 179 : 1713-1717.
20.
Kakimi, K., L. G. Guidotti, Y. Koezuka, and F. V. Chisari. 2000. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J. Exp. Med. 192 : 921-930.
21.
Kawano, T., J. Q. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, H. Koseki, and M. Taniguchi. 1997. CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides. Science 278 : 1626-1629.
22.
Kawano, T., J. Q. Cui, Y. Koezuka, I. Toura, Y. Kaneko, H. Sato, E. Kondo, M. Harada, H. Koseki, T. Nakayama, Y. Tanaka, and M. Taniguchi. 1998. Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Vα14 NKT cells. Proc. Natl. Acad. Sci. USA 95 : 5690-5693.
23.
Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, T. Kawano, M. Taniguchi, and T. Nishimura. 1999. The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189 : 1121-1128.
24.
Kitamura, H., A. Ohta, M. Sekimoto, M. Sato, K. Iwakabe, M. Nakui, T. Yahata, H. X. Meng, T. Koda, S. Nishimura, T. Kawano, M. Taniguchi, and T. Nishimura. 2000. α-Galactosylceramide induces early B-cell activation through IL-4 production by NKT cells. Cell. Immunol. 199 : 37-42.
25.
Lathbury, L. J., J. E. Allan, G. R. Shellam, and A. A. Scalzo. 1996. Effect of host genotype in determining the relative roles of natural killer cells and T cells in mediating protection against murine cytomegalovirus infection. J. Gen. Virol. 77 : 2605-2613.
26.
Metelitsa, L. S., O. V. Naidenko, A. Kant, H. W. Wu, M. J. Loza, B. Perussia, M. Kronenberg, and R. C. Seeger. 2001. Human NKT cells mediate antitumor cytotoxicity directly by recognizing target cell CD1d with bound ligand or indirectly by producing IL-2 to activate NK cells. J. Immunol. 167 : 3114-3122.
27.
Nakagawa, R., K. Motoki, H. Nakamura, H. Ueno, R. Iijima, A. Yamauchi, S. Tsuyuki, T. Inamoto, and Y. Koezuka. 1998. Antitumor activity of α-galactosylceramide, KRN7000, in mice with EL-4 hepatic metastasis and its cytokine production. Oncol. Res. 10 : 561-568.
28.
Nishimura, T., H. Kitamura, K. Iwakabe, T. Yahata, A. Ohta, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, T. Kawano, M. Taniguchi, M. Nakui, M. Sekimoto, and T. Koda. 2000. The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes. Int. Immunol. 12 : 987-994.
29.
Orange, J. S., B. Wang, C. Terhorst, and C. A. Biron. 1995. Requirement for natural killer cell-produced interferon γ in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J. Exp. Med. 182 : 1045-1056.
30.
Reddehase, M. J., W. Mutter, K. Munch, H. J. Buhring, and U. H. Koszinowski. 1987. CD8+ T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity. J. Virol. 61 : 3102-3108.
31.
Saubermann, L. J., P. Beck, Y. P. De Jong, R. S. Pitman, M. S. Ryan, H. S. Kim, M. Exley, S. Snapper, S. P. Balk, S. J. Hagen, O. Kanauchi, K. Motoki, T. Sakai, C. Terhorst, Y. Koezuka, D. K. Podolsky, and R. S. Blumberg. 2000. Activation of natural killer T cells by α-galactosylceramide in the presence of CD1d provides protection against colitis in mice. Gastroenterology 119 : 119-128.
32.
Sharif, S., G. A. Arreaza, P. Zucker, Q. S. Mi, J. Sondhi, O. V. Naidenko, M. Kronenberg, Y. Koezuka, T. L. Delovitch, J. M. Gombert, M. Leite-De-Moraes, C. Gouarin, R. Zhu, A. Hameg, T. Nakayama, M. Taniguchi, F. Lepault, A. Lehuen, J. F. Bach, and A. Herbelin. 2001. Activation of natural killer T cells by α-galactosylceramide treatment prevents the onset and recurrence of autoimmune type 1 diabetes. Nat. Med. 7 : 1057-1062.
33.
Singh, N., S. Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg, Y. Koezuka, and L. Van Kaer. 1999. Activation of NK T cells by CD1d and α-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype. J. Immunol. 163 : 2373-2377.
34.
Smyth, M. J., N. Y. Crowe, and D. I. Godfrey. 2001. NK cells and NKT cells collaborate in host protection from methylcholanthrene-induced fibrosarcoma. Int. Immunol. 13 : 459-463.
35.
Smyth, M. J., N. Y. Crowe, Y. Hayakawa, K. Takeda, H. Yagita, and D. I. Godfrey. 2002. NKT cells—conductors of tumor immunity? Curr. Opin. Immunol. 14 : 165-171.
36.
Smyth, M. J., N. Y. Crowe, D. G. Pellicci, K. Kyparissoudis, J. M. Kelly, K. Takeda, H. Yagita, and D. I. Godfrey. 2002. Sequential production of interferon-γ by NK1.1+ T cells and natural killer cells is essential for the antimetastatic effect of α-galactosylceramide. Blood 99 : 1259-1266.
37.
Smyth, M. J., and D. I. Godfrey. 2000. NKT cells and tumor immunity—a double-edged sword. Nat. Immunol. 1 : 459-460.
38.
Smyth, M. J., K. Y. T. Thia, E. Cretney, J. M. Kelly, M. B. Snook, C. A. Forbes, and A. A. Scalzo. 1999. Perforin is a major contributor to NK cell control of tumor metastasis. J. Immunol. 162 : 6658-6662.
39.
Spence, P. M., V. Sriram, L. Van Kaer, J. A. Hobbs, and R. R. Brutkiewicz. 2001. Generation of cellular immunity to lymphocytic choriomeningitis virus is independent of CD1d1 expression. Immunology 104 : 168-174.
40.
Strasly, M., F. Cavallo, M. Geuna, S. Mitola, M. P. Colombo, G. Forni, and F. Bussolino. 2001. IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk. J. Immunol. 166 : 3890-3899.
41.
Talmadge, J. E., K. M. Meyers, D. J. Prieur, and J. R. Starkey. 1980. Role of NK cells in tumour growth and metastasis in beige mice. Nature 284 : 622-624.
42.
Tay, C. H., E. Szomolanyi-Tsuda, and R. M. Welsh. 1998. Control of infections by NK cells. Curr. Top. Microbiol. Immunol. 230 : 193-220.
43.
Tay, C. H., and R. M. Welsh. 1997. Distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells. J. Virol. 71 : 267-275.
44.
Tomura, M., W. G. Yu, H. J. Ahn, M. Yamashita, Y. F. Yang, S. Ono, T. Hamaoka, T. Kawano, M. Taniguchi, Y. Koezuka, and H. Fujiwara. 1999. A novel function of Vα14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J. Immunol. 163 : 93-101.
45.
Toura, I., T. Kawano, Y. Akutsu, T. Nakayama, T. Ochiai, and M. Taniguchi. 1999. Inhibition of experimental tumor metastasis by dendritic cells pulsed with α-galactosylceramide. J. Immunol. 163 : 2387-2391.
46.
Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47 : 187-376.
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© 2003 American Society for Microbiology.
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Received: 29 July 2002
Accepted: 28 October 2002
Published online: 1 February 2003
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2003. Activation of Natural Killer (NK) T Cells during Murine Cytomegalovirus Infection Enhances the Antiviral Response Mediated by NK Cells. J Virol 77:.
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