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Journal of Virology, December 2008, p. 11970-11975, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01053-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Primary Clearance of Murine Gammaherpesvirus 68 by PKC^–/– CD8 T Cells Is Compromised in the Absence of Help from CD4 T Cells

Peter Dias, Ashley L. Shea, Chandra Inglis, Francesca Giannoni, Lian Ni Lee, and Sally R. Sarawar^*

Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121

Received 20 May 2008/ Accepted 16 September 2008

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CD4 T cells are dispensable for acute control of murine gammaherpesvirus 68 (MHV-68) but are necessary for effective long-term control of the virus by CD8 T cells. In contrast, protein kinase C (PKC) is not essential for either acute or long-term viral control. However, we found that while either CD4 or CD8 T cells could mediate the clearance of MHV-68 from the lungs of PKC^+/+ mice, PKC^–/– mice depleted of either subset failed to clear the virus. These data suggest that there are two alternative pathways for MHV-68 clearance, one dependent on CD4 T cells and the other on PKC. Protection mediated by the latter appears to be short-lived. These observations may help to explain the differential requirement for PKC in various models of CD8 T-cell activation and differences in the costimulatory requirements for acute and long-term viral control.

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Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen with significant homology to the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (6, 10, 11, 35). The intranasal administration of MHV-68 results in acute productive infection of lung alveolar epithelial cells and a latent infection in B lymphocytes, dendritic cells, epithelial cells, and macrophages (12, 13, 28, 30-32, 34, 37). Infectious virus is cleared from the lungs 10 to 15 days after infection by a cytotoxic T-cell-mediated process (33). In the absence of CD8 T cells, CD4 T cells can mediate viral clearance via a gamma interferon (IFN-)-dependent mechanism (8). While CD4 T cells are not essential for primary control of lytic MHV-68, they are required for long-term control and the virus reactivates in the lungs of CD4 T-cell-deficient mice (7). Long-term control of MHV-68 can be mediated by CD4 T cells acting as effectors or by providing help to either CD8 T cells or B cells (25, 28).

Protein kinase C (PKC) is an isoenzyme of the PKC family that localizes to the immune synapse and mediates the activation of several transcription factors, including NF-B, NFAT, and AP-1, resulting in T-cell activation (1-3, 5, 20-22, 29). Early reports suggested that PKC was essential for T-cell activation. However, our previous studies showed PKC is not essential for the T-cell activation events that are required for either short- or long-term control of MHV-68 (15). Several other studies in different infectious disease models have also suggested that PKC is not always required for T-cell activation (4, 18, 19). In the current study, we dissected the individual roles of CD4 and CD8 T cells in the immune response to MHV-68 in PKC^–/– mice. Surprisingly, we found that while either CD4 or CD8 T cells could clear replicating MHV-68 in wild-type mice, in mice that lacked PKC, both subsets were required, suggesting that CD8 cytotoxic T lymphocyte (CTL) activity requires the help of CD4 T cells in the absence of PKC.

129/B6 mice that were heterozygous (PKC^+/–) for the disruption of the PKC gene were originally obtained from Dan Littman (Skirball Institute, New York, NY). The mice were bred and housed under specific pathogen-free conditions. 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- and sex-matched 6- to 15-week-old PKC^+/+ and PKC^–/– mice were used in all experiments.

Both CD4 and CD8 T cells are required for primary clearance of MHV-68 in PKC^–/– mice. To examine the role of PKC in CD4 or CD8 T-cell subsets during the response to MHV-68, we depleted either CD4 or CD8 T-cell subsets and measured lung viral titers at day 16 postinfection. As shown previously (7, 27), wild-type mice depleted of either subset showed effective short-term control of the virus. Major histocompatibility complex (MHC) class II^–/– mice were included as an additional control, as they are PKC^+/+ but lack functional CD4 T cells. Like the CD4-depleted wild-type mice, they showed effective viral clearance. However, PKC^–/– mice depleted of either subset failed to control the virus (Fig. 1). As expected, either wild-type or PKC^–/– mice depleted of both subsets failed to clear the virus and eventually died. To determine whether viral clearance was delayed, rather than blocked completely in PKC^–/– mice depleted of either the CD4 or CD8 T-cell subsets, lung viral titers were also determined at day 28 postinfection (Fig. 1). Similar results were obtained at this time point. Thus, CD8-depleted wild-type mice showed effective viral control, while PKC^–/– mice depleted of this subset had not cleared the virus. While, as expected, CD4 T-cell-depleted wild-type mice showed low levels of viral reactivation at day 28, CD4 T-cell-depleted PKC^–/– mice showed much higher viral titers. Some of the PKC^–/– mice that were depleted of either the CD4 or CD8 T-cell subsets had died by day 28 postinfection. In contrast, undepleted PKC^–/– mice had cleared the virus and did not show any evidence of viral reactivation or ill heath, in accordance with our previously published data (15).

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FIG. 1. Both CD4 and CD8 T cells are required for the primary clearance of MHV-68 in PKC–/– mice. Groups of PKC+/+ and PKC–/– mice were infected intranasally with MHV-68 (105 PFU/mouse). CD4 and CD8 T cells were depleted by the intraperitoneal injection of 0.5 mg monoclonal antibody GK1.5 or 2.43, respectively, every 2 to 3 days, commencing 3 days before infection. Lung virus titers were determined at days 16 or 28 postinfection by plaque assay on NIH 3T3 fibroblasts as previously described (7). 1d, one dead mouse; 2d, two dead mice. Data represent lung titers from individual mice and are combined from two independent experiments that gave similar results.

CD8 T-cell expansion and trafficking to the lungs is not affected by the absence of CD4 T cells. Leukocyte numbers in bronchoalveolar lavage (BAL) or spleen cells were determined at day 16 after infection with MHV-68 (Fig. 2A). In addition, BAL or spleen cell phenotypes were examined by flow cytometry (Fig. 2B). The data showed that the antibody treatment was effective in depleting CD4 and CD8 T cells. There was no significant difference between the wild-type and PKC^–/– mice in leukocyte subset distributions or cell numbers in BAL or spleen cells within a treatment group (control, CD4-depleted, and CD8-depleted). The frequency of virus-specific CD8 T cells was also examined by staining with MHC peptide tetramers (obtained from the NIH tetramer facility). There was no significant difference between the wild-type and PKC^–/– mice in the undepleted or CD4-depleted groups (Fig. 2C). Taken together, these data show that the compromised viral clearance in PKC^–/– mice depleted of CD4 T cells results from a failure in CD8 T-cell effector function (33), rather than from defective expansion or trafficking of CD8 T cells to the lung.

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FIG. 2. Cell numbers and leukocyte subsets in the BAL and spleen cells of PKC+/+ and PKC–/– mice. Effect of CD4 or CD8 T-cell depletion. Mice were infected with MHV-68 and depleted of CD4 or CD8 T cells as described in the legend to Fig. 1. Single cell suspensions were prepared from BAL specimens and spleens. (A) Viable cell counts in BAL specimens or spleens were determined at day 16 after infection by trypan blue exclusion. (B) BAL or spleen cells were stained with phycoerythrin (PE) or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies. The resulting populations were examined by flow cytometry using a lymphocyte gate. TCR, T-cell receptor. (C) Frequency of virus-specific CD8 T cells. BAL or spleen cells collected on day 16 postinfection were stained with PE-conjugated H-2Db p56 or H-2Kb p79 MHC peptide tetramers (p56 and p79 are prominent MHV-68 epitopes for CD8 T cells [17, 26]) and an FITC-conjugated monoclonal antibody to CD8. Data are the means and standard deviations for groups of three to four mice at each time point.

CD8 CTL activity is compromised in the absence of PKC and CD4 T-cell help. To demonstrate directly that CD8 T-cell effector function was reduced in CD4 T-cell-depleted PKC^–/– mice, we performed CTL assays using two different approaches. For the first approach, we used a redirected chromium release assay in which anti-CD3 was used to facilitate the interaction of CD8⁺ T-cell effectors from the BAL cells of MHV-68-infected mice with FcR^{+ 51}Cr-loaded P815 target cells as previously described (7). Previous data suggest that CD8 T cells mediate the majority of the CTL activity in this type of assay. The second approach employed a modification of the in vivo CTL assay described by Fuse et al. (14), in which syngeneic MHV-68 peptide-pulsed or control splenocyte target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) or Orange Tracker fluorophores (Invitrogen) and incubated for 8 h with BAL cells from MHV-68-infected mice. The death of target cells (resulting in a relative loss of CFSE-labeled cells) was determined by flow cytometry. Both assays showed a significant reduction in CD8 CTL activity in CD4-depleted PKC^–/– mice compared with that in CD4-depleted wild-type mice (Fig. 3A and B), consistent with their relative abilities to clear MHV-68 (P < 0.001 for each assay by the paired t test). Additional groups included in the redirected assay showed that CTL activity was significantly decreased in CD4 T-cell-depleted PKC^–/– mice compared with that in intact PKC^–/– mice (Fig. 3A; P < 0.0001 by the paired t test), which again is consistent with their relative abilities to clear virus. In addition, as previously noted (7), CD8 T-cell depletion of either the PKC^+/+ or PKC^–/– mice ablated CTL activity.

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FIG. 3. T-cell responses in PKC+/+ and PKC–/– mice. Effect of CD4 or CD8 T-cell depletion. Mice were infected with MHV-68 and depleted of CD4 or CD8 T cells as described in the legend to Fig. 1. (A) Redirected cytotoxicity assay. BAL cells were harvested on day 16 after infection with MHV-68. BAL cells 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 h as previously described (7). The level of 51Cr release is a measure of total (virus-specific and nonspecific) cytotoxicity. The mean percent specific lysis is shown for four separate experiments. Groups of two to four mice were used in each experiment. There was a highly statistically significant difference in CTL activity for PKC–/– versus PKC–/– CD4–, PKC–/– CD4– versus wild-type (WT) CD4–, WT versus WT CD8–, and PKC–/– versus PKC–/– CD8– (P < 0.001, in each case, by paired t test). (B) MHV-68 peptide-specific cytotoxicity assay. BAL cells were harvested from groups of mice at day 13 postinfection. Splenocytes from naive mice were stained with either Orange Tracker dye (control) or with CFSE and pulsed with both p56 and p79 MHV-68 CTL epitope peptides for use as targets. BAL cells were mixed with the splenocyte targets and incubated for 8 h. Controls with no effectors were included. The ratio of control:peptide pulsed targets was determined by flow cytometric analysis. The mean percentages of specific lysis for groups of three mice are shown (% specific lysis = 100 – [1 – (Rcontrol/Reffectors)], where R is the ratio of Orange Tracker [unpulsed]/CFSE [peptide-pulsed] targets). There was a highly statistically significant difference in the CTL activity for the two groups (P < 0.001 by paired t test). (C) Virus-specific CD8 T-cell cytokine responses. BAL cells were harvested 13 days after infection and stimulated for 6 h with both p56 and p79 MHV-68 peptides in the presence of brefeldin A prior to staining for CD8 and either IFN- or TNF. Results are the means and standard deviations for percentages of the CD8 population producing cytokine for groups of three mice. There was a significant reduction in the percentage of CD8 T cells producing IFN- or TNF in the CD4-depleted PKC–/– group versus the undepleted PKC–/– group (P < 0.05 for each cytokine by Student's t test). (D) Recall IFN- response (CD4 T cells). Spleens were harvested from wild-type, control, CD4 T-cell-depleted, or CD8 T-cell-depleted PKC–/– mice 16 days after infection with MHV-68. Splenocytes were restimulated with MHV-68-infected antigen-presenting cells, and IFN- production was determined by enzyme-linked immunosorbent assay (24). Data are expressed as mean cytokine concentrations (pg/ml) plus standard deviations for three to four PKC+/+ or PKC–/– mice per group and are combined from two independent experiments that gave similar results.

As a further test of CD8 T-cell function, CD8 T cells from BAL specimens were restimulated in vitro with a mixture of the MHV-68 p56 and p79 peptides, which are known CD8 T-cell epitopes (17, 26), and the production of the cytokines tumor necrosis factor (TNF) and IFN- by CD8 T cells was analyzed using intracellular staining as previously described (26). A significantly lower percentage of CD8 T cells produced TNF and IFN- in the CD4 T-cell-depleted PKC^–/– groups than in the undepleted PKC^–/–, CD4-depleted, or undepleted wild-type mice (Fig. 3C; P < 0.05 for each cytokine by Student's t test). Again, this shows that CD8 T-cell function is compromised when both CD4 T-cell help and PKC are absent. Although there was a trend toward reduced mean fluorescence intensities for IFN- and TNF production by CD8 T cells, which can be used as a measure of the level of cytokine production by these cells, the difference was not statistically significant (data not shown). The assay described above examines cytokine production by CD8 T cells, which produce rather low levels of the cytokines, although the latter may have important functions at short range. However, the major producers of cytokines, such as IFN-, are CD4 T cells, whose function is examined in the following section.

Defective IFN- production by CD4 T cells in the absence of PKC. We also examined IFN- production after the in vitro restimulation of lymphocytes from MHV-68-infected PKC^+/+ and PKC^–/– mice with virus-infected antigen-presenting cells. Previous studies of wild-type mice have shown that CD4 T cells produce the majority of the IFN- in this system (16). Furthermore IFN- has been shown to be essential for viral clearance by CD4 T cells in wild-type (PKC^+/+) mice in the absence of CD8 T cells. The control, CD4-depleted, and CD8-depleted groups of PKC^–/– mice all showed very low or undetectable levels of IFN- (Fig. 3D), confirming our previous data (15) showing that this process is PKC dependent.

Primary clearance of MHV-68 by PKC^–/– CD8 T cells is compromised in the absence of CD4 T-cell help. A key question was whether CD4 T cells induced a change in the function of the CD8 T cells, enabling them to effectively clear virus, or whether alternatively CD4 and CD8 T cells cooperated in acute viral clearance, with both cell types acting as effectors. IFN- has been shown to be essential for the CD4 T-cell-mediated clearance of MHV-68 in the absence of CD8 T cells (8) but does not appear to be essential for CD8-mediated clearance (9, 23, 36). As IFN- responses to MHV-68 are very low in PKC^–/– mice, regardless of whether the CD4 or CD8 subsets have been depleted (Fig. 3D), the inability of CD4 T cells alone to mediate viral clearance in these mice was not unexpected. Another mechanism by which CD4 T cells might function cooperatively with CD8 T cells is by providing help for anti-viral antibody production. In this case, we would expect to see significant antiviral antibody titers in undepleted PKC^–/– mice at the time of viral clearance (day 10 postinfection [15]). However, when we examined the antibody response to MHV-68 in both intact and CD4 T-cell-depleted PKC^–/– mice at day 10 postinfection, we found that antiviral antibody titers were very low in both groups at this time point (data not shown). Furthermore, our data show that CD8 CTL and cytokine responses are significantly reduced when both PKC and CD4 T cells are absent. Thus, taken together, our data support the view that CD4 T cells provide help for CD8 T cells rather than contributing toward CD8 T-cell-independent mechanisms of viral clearance. To test this hypothesis more directly, we performed an adoptive transfer experiment. In this experiment, CD8 T cells that had developed in either the presence or absence of CD4 T cells in PKC^–/– donors were transferred to PKC^–/– CD4 T-cell-depleted recipients (Fig. 4). The ability of the recipient mice to clear virus was determined by a plaque assay on lung homogenates. There was a significant reduction in viral titers in the recipients of CD8 T cells that had been primed in the presence of CD4 T cells compared with those that were primed in CD4 T-cell-depleted PKC^–/– donors (P < 0.01 by Student's t test). These data show that, as expected, based on our other data, CD4 T cells induce a functional change in CD8 T cells in PKC^–/– mice, enabling them to clear virus more effectively.

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FIG. 4. CD4 T-cell help induces a functional change in CD8 T cells in MHV-68-infected PKC–/– mice. Groups of CD4 T-cell-depleted or untreated donor PKC–/– mice or CD4 T-cell-depleted recipient PKC–/– mice were infected intranasally with 105 PFU MHV-68. Untreated PKC–/– mice were also infected as controls. Donor mice were infected 2 days earlier than recipient and control mice. Mice in the CD4-depleted groups were treated with GK1.5 anti-CD4 antibody (0.5 mg/mouse intraperitoneally) every 2 to 3 days, commencing 3 days before infection, to maintain CD4 T-cell depletion. On day 10 after infection, donor mice were killed and CD8 T cells were purified by negative selection from pooled BAL, lymph node, and spleen cells from each group, using CD8 T-cell purification columns (R&D Systems, Minneapolis, MN). Erythrocytes were lysed in splenocyte preparations prior to purification. A total of 106 purified CD8 T cells were administered intravenously to the recipient mice on day 8 postinfection. Lung virus titers were determined by plaque assay at day 18 after infection. Data represent the titers for individual mice. There was a highly statistically significant difference between the lung virus titers in mice that had received CD8 cells from control and CD4 T-cell-depleted PKC–/– donors (P = 0.0089 by Student's t test).

The inability of CD8 T cells from CD4 T-cell-depleted PKC^–/– mice to mediate primary viral clearance, in contrast to those from wild-type mice, was unexpected and suggests that PKC and CD4 T-cell costimulation define alternative pathways of CD8 T-cell activation. However, activation via the PKC pathway appears to be short-lived. Thus, CD4 T-cell help is required for long-term control of the virus, regardless of whether PKC is present. Our previous studies suggest that CD4 T-cell help for CD8 T cells in the long-term control of MHV-68 in vivo is mediated via dendritic-cell activation (15a). The results of the present study may help to explain some of the differences in the requirement for PKC for CD8 T-cell activation in vitro and in vivo and why the acute control of MHV-68 is CD4 T cell independent, whereas long-term control is CD4 T cell dependent. Further elucidating these pathways has implications for vaccine design, as it might be preferable to target the CD4 T-cell-dependent pathway, rather than the short-lived PKC dependent pathway.

 
This work was supported by grant AI50810 from the National Institutes of Health and by a grant from the Infectious Disease Science Center.

We thank Erin Lum and Peter Yu for excellent technical assistance.

 
* Corresponding author. Mailing 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

Published ahead of print on 25 September 2008.

Present address: Novartis, 500 Technology Square, Cambridge, MA 02139.

Present address: Department of Neuroscience, UCSD, 9500 Gilman Drive # 0624, La Jolla, CA 92093-0624.

Present address: Division of Research Immunology and Bone Marrow Transplantation, CHLA, 4650 Sunset Blvd, Los Angeles, CA 90027.

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Journal of Virology, December 2008, p. 11970-11975, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01053-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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