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Journal of Virology, June 2005, p. 6808-6813, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6808-6813.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Protein Kinase C Is Not Essential for T-Cell-Mediated Clearance of Murine Gammaherpesvirus 68

Francesca Giannoni, Ashley B. Lyon, Mark D. Wareing, Peter B. Dias, and Sally R. Sarawar^*

Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, California 92121

Received 2 November 2004/ Accepted 18 January 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen with significant homology to human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. T cells are essential for primary clearance of MHV-68 and survival of mice following intranasal infection. Previous reports have suggested that protein kinase C (PKC) is essential for T-cell activation and cytokine production in vitro. To determine the role of this molecule in vivo during the immune response to a viral infection, PKC^–/– mice were infected with MHV-68. Despite the essential role of T cells in viral clearance, PKC^–/– mice survived infection, cleared lytic virus, and maintained effective long-term control of latency. CD8 T-cell expansion, trafficking to the lung, and cytotoxic activity were similar in PKC^+/+ and PKC^–/– mice, whereas antiviral antibody and T-helper cell cytokine production were significantly lower in PKC^–/– mice than in PKC^+/+ mice. These studies demonstrate a differential requirement for PKC in the immune response to MHV-68 and show that PKC is not essential for the T-cell activation events leading to viral clearance.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein kinase C (PKC) is an isoenzyme of the PKC family that is selectively expressed in T lymphocytes (2, 19, 20). In mature T cells, stimulation with antigen and CD28 induces PKC translocation from the cytosol into plasma membrane lipid rafts, where it colocalizes with T-cell receptor at the central core of the immune synapse (4, 21). PKC subsequently mediates activation of several transcription factors, including NF-B, NFAT, and AP-1, resulting in T-cell activation and increased interleukin 2 (IL-2) gene expression (reviewed in references 1 and 11). Studies of cell lines have led to the conclusion that PKC is essential for T-cell activation and that this isoenzyme of PKC appears to function by integrating signals from CD28 and the T-cell receptor (reviewed in references 1 and 11). Studies in mice deficient in PKC have tended to support this view and have shown that PKC is critical for NF-B activation in mature T lymphocytes (29), NFAT activation (22), pulmonary allergic hypersensitivity responses (17), TH2 responses to Nippostrongylus brasiliensis (17), and the induction of T-cell activation versus tolerance in vivo (3). However, recent studies have demonstrated T-cell responses to lymphocytic choriomeningitis virus (3) and Leishmania major (17) in PKC^–/– mice, suggesting that some infectious agents may be able to overcome the requirement for PKC in T-cell activation.

To determine the role of PKC in T-cell activation during the immune response to MHV-68, we infected wild-type or PKC^–/– mice with the virus, a rodent pathogen (5), which is closely related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (8, 36). Intranasal administration of MHV-68 results in acute productive infection of lung alveolar epithelial cells and a latent infection in various cell types, including B lymphocytes (9, 10, 28, 30, 35, 37). The virus induces an inflammatory infiltrate in the lungs, splenomegaly, and an increase in the number of activated CD8 T cells in the blood (30, 33). Virus-specific CD8 T cells traffic to the lungs and clear infectious virus by a cytolytic mechanism 10 to 15 days after infection (30, 31), while both B and T cells appear to function in the long-term control of latent virus (12, 28).

Our previous studies (14) showed that CD28 is not essential for cytotoxic T-lymphocyte (CTL) responses to MHV-68 or for primary viral clearance. However, lack of CD28 resulted in a significant reduction in antiviral antibody titers. Furthermore, Kim et al. (12) demonstrated that the compromised antiviral antibody response in CD28^–/– mice was ineffective in the long-term control of latent MHV-68, whereas T-cell responses remained effective. To further delineate signaling pathways in T-cell activation during MHV-68 infection, in the current study we utilized PKC^–/– mice to determine whether T-cell activation and viral clearance were mediated via PKC in a CD28-independent pathway or whether an alternative PKC-independent pathway was utilized.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. 129/B6 mice that were heterozygous (PKC^+/–) for the disruption of the PKC gene (29) were kindly provided by Amnon Altman, La Jolla Institute for Allergy and Immunology, San Diego, Calif., with the prior permission of Dan Littman, Skirball Institute, New York. Mice were bred and housed under specific-pathogen-free conditions in the vivarium at the La Jolla Institute for Allergy and Immunology or Torrey Pines Institute for Molecular Studies. PKC^–/– homozygous knockout mice and PKC^+/+ littermates were obtained from pairings of heterozygous mice. The genotypes of the progeny were determined by PCR on tail snips. Age-matched 6- to 15-week-old female PKC^+/+ and PKC^–/– mice were used in all experiments.

Viral infection and sampling. MHV-68 (clone G2.4) (5, 30) was propagated in BHK-21 cells (ATCC CCL-10). Mice were anesthetized with Avertin (2,2,2-tribromoethanol) and infected intranasally with 10⁵ PFU of the virus in phosphate-buffered saline. At various intervals after infection, the mice were terminally anesthetized with Avertin. The lungs were removed and homogenized in medium on ice prior to virus titration. Single-cell suspensions were prepared from the bronchoalveolar lavage (BAL) or spleen, and titers of replicating virus were determined by plaque assay on NIH 3T3 cells (ATCC CRL1658) as described previously (7). The detection limit of this assay is 10 PFU/ml of a 10% tissue homogenate based on data for plaques recovered from homogenates of uninfected tissues spiked with known amounts of virus.

Cytotoxicity assays. Cytotoxic T-cell activity was determined using a redirected chromium release assay. Suspensions of spleen or BAL cells from MHV-68-infected animals were incubated with ⁵¹Cr-labeled FcR⁺ P815 (ATCC TIB-64) target cells in the presence of 2 µg/ml 2C11 anti-CD3 antibody for 6 to 8 h at 37°C as previously described (7). The level of specific ⁵¹Cr release is a measure of total (virus-specific and nonspecific) cytotoxicity.

Measurement of cytokine responses. The frequency of MHV-68-specific CD8 T cells in spleen or BAL was determined by intracellular staining for gamma interferon (IFN-). Restimulation of spleen or BAL cells with MHV-68 peptides p56 (AGPHNDMEI from ORF6) and p79 (TSINFVKI from ORF61) and dual staining for IFN- and CD8 were performed as described by Stevenson et al. (26). These peptides have been identified as major epitopes (16, 26). Cytokine levels in culture supernatants from cells that had been restimulated in vitro with irradiated virus-infected splenic antigen-presenting cells were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) as described previously (24). All reagents were obtained from BD PharMingen (San Diego, CA).

Flow cytometric analysis. Cells were stained with phycoerythrin- or fluorescein isothiocyanate-conjugated monoclonal antibodies, as described previously (14). All antibodies were purchased from BD PharMingen (San Diego, CA). Isotype controls were included in each assay.

ELISA for virus-specific antibody. Serum antibody titers were determined by ELISA, as described previously (15), using peroxidase-labeled secondary reagents from Southern Biotechnology (Birmingham, Ala.). Sera from uninfected mice and positive-control sera were included in each assay.

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC^–/– mice can clear replicating MHV-68 from their lungs and maintain effective long-term control of the virus. Despite the essential role of T cells in viral clearance, mice homozygous for a targeted disruption of the PKC gene were able to clear lytic MHV-68 with normal kinetics (Fig. 1). Both PKC^–/– and PKC^+/+ mice had cleared infectious virus from their lungs by day 10 after intranasal infection with 10⁵ PFU MHV-68. Thus, in this in vivo model, PKC did not appear to be essential for the T-cell activation events leading to viral clearance. The lungs of both PKC^–/– and PKC^+/+ mice remained clear of replicating virus at days 35 and 50 after infection (Fig. 1), demonstrating effective long-term control of the virus.

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FIG. 1. Lung virus titers in PKC+/+ and PKC–/– mice. PKC–/– or PKC+/+ mice were infected intranasally with 105 PFU MHV-68. At various times after infection, lungs were harvested and virus titers determined in lung homogenates by plaque assay. Data are expressed as PFU/0.1 g lung tissue for individual mice.

PKC is not required for CD8 T-cell expansion or trafficking to the lungs. Intranasal infection with MHV-68 induces an inflammatory infiltrate in the lungs comprising mainly T cells and monocytes/macrophages. There was no significant difference in the total number of cells recovered in the BAL in PKC^–/– and PKC^+/+ mice (Fig. 2A) or in the percentages of CD4 and CD8 T cells in this population determined by flow cytometric analysis (Fig. 2C). In both PKC^–/– and PKC^+/+ mice, CD8 T cells greatly outnumbered the CD4 T cells. MHV-68 also induces splenomegaly by a mechanism that is dependent on both T and B cells (7, 9, 34, 35). Lack of PKC did not diminish MHV-68-induced splenomegaly (Fig. 2B). In contrast, splenic cellularity was significantly increased in PKC^–/– mice on day 15 after infection. However, this appeared to be a temporary phenomenon, and the cell numbers were similar in PKC^–/– and PKC^+/+ mice by day 35 after infection (Fig. 2B). The percentages of CD4 and CD8 T cells and B cells were similar in the spleens of PKC^–/– and PKC^+/+ mice (Fig. 2D). The cell numbers and phenotypes were similar in the spleens of uninfected PKC^–/– and PKC^+/+ mice (data not shown).

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FIG. 2. Cell numbers and lymphocyte subsets in the BAL or spleens of PKC+/+ and PKC–/– mice. The numbers of cells in the BAL (A) or spleen (B) were determined at intervals after intranasal infection of PKC+/+ and PKC–/– mice with MHV-68. Data are means ± standard deviations (error bars) of cell counts for two separate experiments on cells 15 days after infection and for one experiment on cells 35 days after infection. Cell counts from three or four individual mice were performed at each time point. BAL (C) or spleen cells (D) were stained with phycoerythrin- or fluorescein isothiocyanate-conjugated monoclonal antibodies, and the resulting populations were analyzed by flow cytometry. The means ± standard deviations (error bars) of data from two separate experiments on cells 15 days after infection and one experiment each on cells 10 and 35 days postinfection are shown. Groups of two to four mice were used at each time point. BAL cells were pooled in each experiment, whereas individual spleen cell suspensions were analyzed. The asterisk denotes that there was a statistically significant difference in spleen cell numbers at day 15 after infection in PKC–/– and PKC+/+ mice (P < 0.05 by Student's t test).

The proportion of virus-specific CD8 T cells in the BAL and spleen was also determined by evaluating the number of CD8 cells producing IFN- in response to stimulation with two different viral peptide epitopes by intracellular staining. There was no significant difference in the percentages of CD8 T cells responding to the p56 and p79 viral epitopes in the BAL (Fig. 3A) or in the spleens of infected animals (Fig. 3B). Taken together, these data indicate that CD8 T cells from PKC^–/– mice are capable of expansion and trafficking to an inflammatory site in response to MHV-68 infection.

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FIG. 3. CD8 T-cell responses in PKC–/– mice. Frequency of virus-specific CD8 T cells in BAL (A) and spleen (B). BAL or spleen cells were harvested 15 days after infection and stimulated for 6 h with either p56 or p79 MHV-68 peptide in the presence of monensin prior to staining for CD8 and IFN-. Results are means ± standard deviations (error bars) for two separate experiments. Cell suspensions from three or four mice were analyzed individually in each experiment. CTL responses in BAL (C) and spleen (D). Single-cell suspensions were prepared from BAL or spleens at day 10 after intranasal infection with MHV-68. CTL activity was determined in a 6-h 51Cr release assay. Mean percentages of specific lysis ± standard deviations (error bars) are shown for two separate experiments. Groups of two or three mice were used in each experiment. BAL cells were pooled in each experiment, whereas spleen cell suspensions were analyzed individually.

PKC is not required for the development of CTL responses during MHV-68 infection. MHV-68 is cleared from the lungs by cytotoxic T cells 10 to 13 days after infection (31). The normal kinetics of viral clearance in PKC^–/– mice suggested an intact CTL response. To verify this, CTL assays were performed on BAL and spleen cell populations from infected animals. Previous studies in this model have shown that the majority of the CTL activity is mediated by CD8 T cells (7, 31). There was no significant difference in CTL activity in either the spleen or BAL from PKC^–/– and PKC^+/+ mice (Fig. 3C and D). Therefore, PKC is not essential for the CTL response to MHV-68 infection.

Reduced T-helper cell cytokine responses in PKC^–/– mice. To determine the effect of absence of PKC on cytokine production during the immune response to viral infection, splenocytes from virus-infected mice were restimulated in vitro with virus-infected splenic antigen-presenting cells as described previously (24). Recall cytokine production was measured in the supernatant by ELISA. IFN- production was dramatically reduced in splenocytes from PKC^–/– mice (Fig. 4A). Subset depletion experiments have shown that CD4 T cells are the predominant cell type producing IFN- in these cultures (14). There was also a significant reduction in IL-10 production (Fig. 4B) while no IL-2, IL-4, or IL-5 was detected in the culture supernatants of cells from either PKC^+/+ or PKC^–/– mice (data not shown).

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FIG. 4. Cytokine responses to MHV-68 in PKC–/– mice. Spleens were harvested 15 or 35 days after infection with MHV-68. Splenocytes were restimulated with MHV-68-infected antigen-presenting cells, and IFN- (A) or IL-10 (B) concentrations were determined in culture supernatants by ELISA. Data are expressed as mean cytokine concentrations (pg/ml) ± standard deviations (error bars) for two separate experiments of cells, one 15 days after infection and one 35 days after infection. Splenocyte cultures from two to four individual PKC–/– or PKC+/+ mice were tested in each experiment. Asterisks denote that there was a statistically significant difference in cytokine responses in PKC–/– and PKC+/+ mice (P < 0.05 [*] and P < 0.01 [**] by the Mann-Whitney rank sum test).

Reduced serum antibody responses to MHV-68 in PKC^–/– mice. Substantial changes in the antibody response to MHV-68 were also noted in PKC^–/– mice. Virus-specific serum antibody titers were determined by ELISA 50 days after infection with MHV-68. There was a significant reduction in the level of virus-specific antibody in PKC^–/– mice compared to that in wild-type mice (Fig. 5). There was a more dramatic reduction in immunoglobulin G1 (IgG1) than in IgG2a or IgG2b. Virus-specific IgM and IgG3 were detected at low levels in both PKC^+/+ and PKC^–/– mice. Similar observations were made at days 15 and 35 after infection (data not shown). Thus, like CD28^–/– mice, PKC^–/– mice can mount humoral responses to MHV-68, and class switching still occurs. However, the response is significantly reduced compared to that in wild-type mice.

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FIG. 5. MHV-68-specific antibody responses in PKC–/– mice. Serum samples were collected from PKC–/– mice 50 days after infection with MHV-68. Virus-specific antibody responses were determined by ELISA. Data are expressed as mean absorbance values ± standard deviations (error bars) for two separate experiments. Sera from four or five individual mice were tested in each experiment. Asterisks denote that there was a statistically significant difference in antibody titers in PKC–/– and PKC+/+ mice (P < 0.01 [**] and P < 0.001 [***] by Student's t test). Ig (H&L), heavy and light chains of Ig.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Data from previous in vitro studies (reviewed in references 1 and 11) have led to the conclusion that PKC is essential for T-cell activation, and it is widely accepted that PKC functions as a pivotal convergence point for pathways of T-cell activation. As T-cell activation is a prerequisite for the clearance of MHV-68, in the current study we examined the role of PKC in the immune response to this virus. In contrast with the previous in vitro studies, we showed that PKC is not essential for the T-cell activation events required for clearance of MHV-68. Our data show that both PKC-dependent and -independent pathways of T-cell activation operate during the immune response to a viral infection. Thus, in our in vivo model, normal expansion of CD8 T cells occurred in response to MHV-68 infection, and similar frequencies of CD8 T cells responding to major viral epitopes were detected. In addition, virus-specific CD8 T cells were able to migrate successfully to the lungs in PKC^–/– mice, indicating that upregulation of the required chemokine receptors and adhesion molecules was not dependent on PKC. Clearance of lytic MHV-68 from the lungs of wild-type mice is mediated primarily by CD8 T cells via a cytolytic mechanism (7, 31). In accordance with the ability of PKC^–/– mice to clear infectious MHV-68 from the lungs with normal kinetics, CTL activity was not significantly affected in these mice. In this respect, PKC^–/– mice resemble CD28^–/– mice, which are able to clear lytic MHV-68 from the lungs (12, 14). Also, like CD28^–/– mice, PKC^–/– mice are able to maintain effective long-term control of latent MHV-68, whereas mice lacking CD4 T cells, CD40, or CD40L show viral reactivation in the lungs (6, 7, 14, 25). In the case of CD28^–/– mice, it has been shown that T cells are the effector cells maintaining long-term control of latent MHV-68 (12), whereas both antibody and T-cell responses are compromised in mice lacking CD4 or CD40 costimulation (6, 7, 25).

Our preliminary data (M. D. Wareing and S. R. Sarawar, unpublished) suggest that the nonessential role of PKC in viral clearance is not limited to MHV-68, as PKC^–/– mice also survive infection with influenza virus, whereas T-cell-deficient mice rapidly succumb to both viruses. Like MHV-68, influenza virus is cleared from the lungs by cytotoxic T cells (32). Furthermore, in a recent study, Berg-Brown et al. (3) were also able to demonstrate a CTL response to lymphocytic choriomeningitis virus in virally infected PKC^–/– mice. Our studies are in agreement with this finding and, in addition, show that the CTL response to MHV-68 is effective in mediating viral clearance. Thus, collectively, these studies demonstrate that viral infections can circumvent the requirement for PKC in CTL activation.

However, not all aspects of T-cell function in response to MHV-68 infection were independent of PKC. We showed that T-helper cell cytokine production following MHV-68 infection was significantly reduced in PKC^–/– mice compared to that in wild-type mice. Thus, we observed a dramatic reduction in IFN- production, which was sustained in PKC^–/– mice, whereas our previous studies (14) showed only a temporary reduction in IFN- production in CD28^–/– mice. This suggests a role for PKC beyond CD28 signaling in cytokine production. We did not observe a switch to a TH2 profile in PKC^–/– mice, and no IL-4 or IL-5 was detected in cultures from either wild-type or PKC^–/– mice. However, the effect was not limited to TH1 cytokines, as IL-10 production was also reduced. Recently, Marsland et al. (17) reported a decrease in TH2 responses to ovalbumin and Leishmania in PKC^–/– mice. However, IFN- responses were intact in these models, in contrast to our current findings, suggesting that the nature of the antigenic stimulus may dictate the dependence of TH1 responses on PKC.

The present study also showed that there was a significant reduction in antiviral serum antibody titers in PKC^–/– mice. Since PKC is expressed in both CD4 and CD8 T cells, whereas little or none is expressed in B cells (2, 19, 20), this likely reflects a defect in CD4 T-cell help. CD28^–/– mice also show a significant reduction in anti-MHV-68 serum titers (12, 14). Interestingly, Kim et al. (12) showed that the compromised antibody response in CD28^–/– mice was ineffective in preventing reactivation of latent MHV-68 in the lungs, whereas the antibody response in wild-type mice controlled the virus effectively. They showed that it was the T-cell response that maintained effective control of latency in CD28^–/– mice (12). It is likely that this is also the case in PKC^–/– mice, although it is possible that the reduced antibody response is still capable of exerting some control of latency. Our data showed that lack of PKC had a more dramatic effect on IgG1 than IgG2b or IgG2a. A similar differential effect on antiviral IgG1 versus IgG2 was noted following MHV-68 infection of CD28^–/– mice in our earlier study (14) and by McAdam et al. (18) in vesicular stomatitis virus (VSV)-infected CD28^–/– mice. However, Berg-Brown et al. (3) reported that PKC was not required for an in vitro neutralizing antibody response to VSV, although they did not examine the subclass distribution of the antiviral antibody response. The reason for this difference in the PKC dependence of the antibody response during MHV-68 and VSV infection is not readily apparent. However, the antibody responses are somewhat different in the two viral infections. The helper-dependent IgG response develops much more rapidly following infection with VSV (3) than with MHV-68 (23, 27). It is possible that VSV is able to trigger costimulatory molecules that utilize PKC-independent signaling pathways for B-cell help, while MHV-68 may be unable to trigger such signals.

The reason that PKC is dispensable for certain T-cell functions during a viral infection in vivo but is required for T-cell activation in vitro is currently unclear. However, similar disparities between the in vitro and in vivo requirements for T-cell activation have been observed in other systems. For example, studies in IL-2^–/– mice showed that, although IL-2 is essential for T-cell proliferation in vitro, in vivo it does not appear to be required for T-cell expansion and CTL activity in response to viral infection (13). This likely reflects substitution of IL-2 function in vivo by other cytokines that also signal through the common gamma chain and by the ability of viruses to trigger costimulatory events. Viral infection activates the innate immune system, leading to the production of proinflammatory cytokines and other mediators, while viral components can act as ligands for cell surface receptors. These events can induce potent NF-B activation, and it is possible that PKC-independent NF-B activation, triggered by innate immune responses, can partially compensate for the lack of PKC in adaptive immunity to viruses.

In summary, our data provide novel insights into the role of PKC in T-cell activation during the immune response to a viral infection. No previous studies have examined T-cell trafficking, cytokine production, or antiviral IgG subclass distribution during a viral infection in CD28^–/– mice. Furthermore, no previous studies have examined viral clearance or the long-term control of persistent virus in CD28^–/– mice. Our data show that PKC is not universally required for T-cell activation in vivo—rather it is differentially required for different arms of the immune response. The idea that distinct signaling pathways, not all of which converge at the level of PKC, regulate T-cell activity, suggests that it may be possible to selectively block certain T-cell functions while preserving others. In this respect, it of interest that Marsland et al. (17) have recently shown that PKC is required for pulmonary allergic inflammation. Taken together with our findings, these data suggest further evaluation of PKC as a possible target for novel immunosuppressive drugs that could block T-cell functions leading to lung damage, without compromising viral clearance.

 
This work was supported in part by a grant from the Infectious Disease Science Center.

We thank Amnon Altman for helpful advice and discussion and Svetlana Lebedeva and Erin Campbell for assistance with the maintenance and genotyping of the PKC mice.

 
* Corresponding author. Present address: Viral Immunology, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. Phone: (858) 909-5139. Fax: (858) 909-5141. E-mail: ssarawar{at}tpims.org.

Present address: Viral Immunology, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121.

Present address: Department of Medicine, Karolinska Institute, Huddinge University Hospital, SE-141 86 Stockholm, Sweden.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, June 2005, p. 6808-6813, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6808-6813.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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