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* Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239;
Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006;
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and
Max von Pettenkofer Institute, Department for Virology, Ludwig-Maximilians-Universität, Munich, Germany
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Abstract |
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m4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both m4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MCMV is an excellent model in which to dissect the real evolutionary role of immune evasion. It is fully sequenced (7), and the expression of its genome as a bacterial artificial chromosome (BAC) has revolutionized functional genetic analysis (8). Since its isolation in 1954 (9), its immunobiology has been extensively studied, revealing complex and redundant immunological control mechanisms (4, 10, 11). Any immunological investigation of MCMV needs to take into account the marked strain difference in susceptibility to MCMV, which is mediated by NK cells. The most important resistance locus (Cmvr-1) has been recently mapped to the Ly49H gene (12, 13, 14), which C57BL/6 mice express and BALB/c mice do not. Ly49H is an activating NK cell receptor that recognizes the product of the MCMV m157 gene (15, 16). In consequence, MCMV replicates to much higher titers in BALB/c than B6 mice, especially in the spleen. Most of the previous work on MCMV T cell immunology has been conducted in the BALB/c strain, and much less is known about the immunobiology of this virus in B6 mice.
MCMV encodes three known VIPRS, all of which are glycoproteins expressed in the early (E) phase of viral gene expression. m152/gp40 causes class I MHC to be retained in the endoplasmic reticulum/Golgi intermediate compartment (ERGIC) (17). m6/gp48 binds to class I and directs it to the lysosome for destruction (18). m4/gp34 is primarily endoplasmic reticulum resident and associates with class I there; a small amount of m4/gp34 complexed with class I travels to the cell surface where they remain stably associated (19). Each of these three VIPRs has been shown to inhibit the ability of MCMV-specific CD8 CTLs to lyse infected cells expressing H-2b MHC (20, 21); gp40 has also been shown to inhibit H-2d-restricted Ag presentation (22). The relative potency of the individual VIPRs varies for different allelic variants of MHC class I (6, 20). Mutagenesis of an MCMV BAC to delete these genes alone and in combination indicated that there are no other MCMV genes that have a significant impact on cell surface MHC class I (6).
The impact of VIPR function on MCMV infection remains largely unexplored, and is not readily predicted from the literature on the role of CD8 T cells in controlling MCMV infection. For instance, even when VIPRs are functional, CD8 T cells can impair wild-type MCMV replication in vivo. Adoptively transferred CD8 T cells reduce virus titers in the lungs, spleen, liver, and adrenal glands of acutely infected, irradiated BALB/c mice (23, 24) Also, prior immunization with CD8 T cell epitopes protects BALB/c mice against a lethal dose of virus (25, 26). In contrast, animals can be completely depleted of CD8 T cells in acute (27) or chronic (28) infection without impacting virus control, because of redundant mechanisms contributed by CD4 T cells and NK cells. We predicted that removing VIPRs would enable CD8 T cells to play a much more dominant and effective role in controlling virus.
There have been three reports of the effect of m152 on acute infection in vivo with MCMV. First, Jonjic and colleagues (22) showed that m152 affects the ability of CD8 T cells to control acute infection, and that this led to 
1 log lower virus titers from days 8 to 10 postinfection. This effect was seen in both BALB/c and B6 mice. To increase the dependence of host defense on CD8 T cell-mediated control, all of the experiments reported in that paper used immunocompromised mice that were either neonatal, B cell deficient, or irradiated. Even so, the effect of m152 removal was modest, with at best a 1.5 log reduction in titer. In view of the recent discovery that m152 can also affect NK function, it should be noted that this paper clearly showed that m152 affects the function of CD8 T cells: removing m152 from the virus reduced virus titers at day 10 postinfection in normal B6 mice but had no effect on virus titers on day 10 postinfection in mice lacking 
2-microglobulin or CD8 (22).
Second, it has recently been discovered that, in addition to inhibiting MHC class I-restricted Ag presentation, m152 also inhibits the expression of the RAE-1 family of ligands for the NK cell-activating receptor NKG2D (29, 30). Jonjic and colleagues (29) found that, in BALB/c mice, this inhibition of NK function resulted in higher virus titers on day 3 postinfection. Thus, in addition to being a VIPR, m152 can mediate anti-NK cell immune evasion.
Third, we have studied the effect of m152 on the acute CD8 T cell response to MCMV in B6 mice, looking at its impact on the Ag specificity of the immune response. We found that m152 did not affect the immunodominance of the Db-restricted M45 HGIRNASFI epitope in acute infection (31). M45 is an early gene, expressed at the same time in the infectious cycle as the immune modulators. Because m152 profoundly inhibits the ability of M45-specific CD8 T cells to detect infected cells, including macrophages and DCs, we interpreted this result to suggest that CD8 T cells in acute MCMV infection are primed by cross-priming rather than by infected APCs.
Each of these three studies on the effect of m152 in vivo was limited to acute infection. Because all herpesviruses appear to encode VIPRs, and the hallmark of the herpesvirus lifestyle is lifelong survival in the infected host, it is generally assumed that VIPRs will be necessary for this lifelong survival. In other words, the main phenotype of a VIPR-deficient virus would be predicted to be in the chronic phase of infection. Indeed, VIPR-deficient mutants of the gamma-2-herpesvirus murine gammaherpesvirus-68 showed little defect in the acute phase of infection in the lungs, but were markedly impaired in their ability to establish splenic latency (32). After acute infection, MCMV establishes a true latent infection that is characterized by periodic reactivation, sometimes aborted at the immediate-early (IE) phase of gene expression, and at other times presumably progressing to replication (33, 34). However, unless mice are immunosuppressed, replicating virus is not detectable, and even the latent virus DNA load is near or below the threshold of detection, especially in B6 mice. Virus activity is believed to be kept to this very low level by effective immune surveillance.
The concept of active immune surveillance in the chronic phase of MCMV infection is supported by studies of the T cell response in BALB/c mice. Memory CD8 T cells numbers remain high, their antigenic specificity narrows with the duration of infection, and they express an effector memory phenotype, remaining CD62L– (26, 35, 36). Interestingly, a remarkably similar picture is emerging in studies of human CMV infection. In healthy CMV-seropositive adults, 2% of CD4 T cells (37) and 6% of CD8 T cells (11% of memory CD8 T cells) (L. Picker, unpublished observations) are specific for CMV for the life of the infected individual. In some healthy individuals, >30% of CD8 T cells are CMV specific (38, 39). Many human CMV-specific CD8 T cells have an effector memory phenotype (CD57+CD45RAbrightCD28–CD27–) (40, 41, 42). Thus, in the chronic phase of CMV infection in immunocompetent humans and mice, the size and phenotype of the CD8 T cell response may be the best indicator of virus activity.
In this study, we report a series of experiments designed to assess the effect of VIPRs on the course of MCMV infection in B6 mice, and on the CD8 T cell response to it. We compared wild-type infection with a virus lacking all three known VIPRs: m4, m6, and m152. Our expectation was that a virus lacking VIPRs would establish acute infection, and would probably be able to establish some latent pool during the 5-day window before effective CD8 T cell control develops. However, after that, we expected that the VIPR-deficient virus would be rapidly controlled, and as latent virus reactivated, it would also be recognized and eradicated. Over time, we expected that this would lead to a reduced or eliminated latent virus pool, and in consequence, we predicted that the MCMV-specific CD8s would come to display the familiar phenotype seen in cleared virus infections such as lymphocytic choriomeningitis virus (LCMV). We were surprised to find that removing VIPRs had little detectable effect on the course of virus infection or on the antiviral CD8 T cell response.
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Materials and Methods |
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Female C57BL/6 (B6) mice were purchased from Simonson (Gilroy, CA) or The Jackson Laboratory (Bar Harbor, ME); mice from a single vendor were used for each experiment. B cell-deficient (µmt) mice were purchased from The Jackson Laboratory. OT-1 transgenic mice were kindly provided by D. Hinrichs (Veterans Affairs Hospital, Portland, OR). All mice were housed in an isolation suite and used at age 6 wk or greater. Sentinel mice were routinely tested for mouse pathogens, and none were found in the course of this study.
Cells
Mouse embryo fibroblasts (MEFs) were grown from trypsin-digested day 12–14 mouse embryos, and used between passages 2 and 6. NIH 3T3 cells (CRL-1658), BALB 3T3 cells (CCL-163), and JAWSII cells (CRL-11904) were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM/10% FBS. JAWSII cells were maintained in 
-MEM supplemented with 20% FBS, sodium pyruvate, nonessential amino acids, and 5 ng/ml GM-CSF.
Viruses
MCMV Smith was purchased from American Type Culture Collection (no. 1399-VR). MS94.5 (lacking ORFs m151–165) (43), wild-type BAC MCMV MW97.01 (44), and 04+m06+m152 (6) have been described. Viruses were grown on B6 MEFs. Stocks were prepared by sonication of infected cells. Virus was titered without centrifugal enhancement on BALB-3T3 cells to determine PFU for all stocks. Aliquots of the same virus stock were used for each experiment. Mice were infected i.p. with either 5 x 104 or 5 x 106 PFU of MCMV, as described in the figures. The lower dose was used in earlier experiments in which we assessed CTL responses only (see Figs. 1, 5, and 6). The higher dose was necessary to detect robust virus replication in B6 mice, and hence was used in the later experiments when virus load was assessed in addition to monitoring CD8 T cell responses (see Figs. 2–4). A recombinant vaccinia virus expressing chicken OVA (vv-ova) (a gift from J. Yewdell, National Institutes of Health, Bethesda, MD) was grown on L929 cells and titered on Vero cells.
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Organs were dissected and immediately flash frozen in liquid nitrogen (see Fig. 2b), or placed on ice and homogenized by passing through a cell strainer and frozen. DNA was extracted using DNA Blood mini-kit (no. 51106; Qiagen, Valencia, CA) or High Pure Viral Nucleic Acid kit (no. 1 858 874; Roche, Basel, Switzerland). Real-time PCR was performed to detect the MCMV IE1 gene exon 1 using the following primers: primer 1, TCG CCC ATC GTT TCG AGA; primer 2, TCT CGT AGG TCC ACT GAC GGA. PCR product was detected with the probe ACT CGA GTC GGA CGC TGC ATC AGA AT labeled with 6-FAM and black hole quencher-1, manufactured by Biosearch Technologies (Novato, CA). Copy numbers were determined by reference to a standard curve obtained using plasmid DNA expressing IE1, a gift from J. Nelson (Oregon Health and Science University).
Immunosuppression to reactivate latent MCMV
For the experiment in Fig. 5, mice were infected with 5 x 106 PFU of MCMV. Thirty-seven (for µmt mice) or 42 (for B6 mice) days later, mice were injected with cyclophosphamide (150 mg/kg) and 0.3 ml of anti-lymphocyte serum (M4529; Sigma-Aldrich, St. Louis, MO). They were then injected with 0.3 ml of anti-lymphocyte serum and hydrocortisone succinate (125 mg/kg) every 2 days until sacrifice 14 days later. To detect viable virus, salivary gland homogenates were plated onto MEFs until most of the monolayer showed cytopathic effect. Cells were then scraped and pelleted, and DNA was extracted using a Qiagen DNA Blood mini-kit. Viral genes were detected by PCR using the following primers: IE1, primer 1, CAC CAT GGA GCC CGC CGC A, and primer 2, CTT CTT GCT CTT CTT CTT GGG C; m4, primer 1, CAC CAT GTC TCT CGT ATG TCG GCT GGT GTT GGT G, and primer 2, GTT ACT CTT AAG CGG TTT GAA GTT C; m6, primer 1, CAC CAT GCC CAG TTG GAG CGA T, and primer 2, TTT GGT AAG CAA GGG GGA AGT; and m152, primer 1, CAC CAT GCT GGG CGC TAT CA, and primer 2, CCA CAC GCG GCA GTT GAT GTA
CTL clones and assay
MCMV-specific CTL clones have been previously described (20). The 51Cr release assay was conducted as previously described (14), using IFN-
-pretreated MEF targets infected for 16 h with virus at a multiplicity of infection of 50 in the presence of phosphonoacetic acid to prevent late gene expression.
OT-1 T cell transfer
A total of 2x107 whole splenocytes from naive OT-1 TCR transgenic mice was transferred i.v. into unmanipulated naive B6 mice that were either left uninfected or immediately injected i.p. with 5 x 104 PFU of MS94.5 or 2 x 105 PFU of vv-ova. Splenocytes were analyzed by FACS 7 days later.
Intracellular cytokine staining (ICS) and tetramer staining
For ICS, JAWSII cells were infected with MCMV (MS94.5 at a multiplicity of infection of 30–50) for 16 h in the presence of phosphonoacetic acid. Effector splenocytes were isolated from MCMV-infected mice and incubated at a ratio of 1:1 with infected or uninfected APCs for 6 h in the presence of brefeldin A (GolgiPlug; BD PharMingen, San Diego, CA). Cells were washed, incubated with FcBlock (BD Biosciences, Mountain View, CA), and surface stained with Abs to the CD8 chain conjugated to PE-Cy5 (no. 15-0081-83; eBioscience, San Diego, CA) and to NKG2A/C/E using the mAb 20d5 (no. 550520; BD Biosciences), or to CD43 (no. 558761; BD Biosciences), CD11c (no. 557400; BD Biosciences) or CD62L (no. 553150; BD Biosciences), all FITC conjugated. Cells were then fixed and permeabilized using BD PharMingen’s Cytofix/Cytoperm kit before staining with an Ab to IFN- (no. 12-7311-82; BD Biosciences). For BrdU staining, the BD PharMingen BrdU Flow kit (2345KK) was used according to the manufacturer’s protocol. CD8 T cells were analyzed by flow cytometry using CellQuest software (BD Biosciences). All further analyses were performed using FlowJo software (Treestar, San Carlos, CA). Kb-SIINFEKL tetramers were purchased from the Trudeau Institute Molecular Biology Core Facility (Saranac Lake, NY). Db-HGIRNASFI tetramers were used to stain T cells specific for the immunodominant M45 epitope (31). Tetramers were generated using HGIRNASFI peptide, following the previously described protocols (45, 46), and coupled to streptavidin-PE.
BrdU labeling
Mice infected for 1 day or 30 wk were injected i.p. with 1.8 mg of 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich) on day 1 and subsequently fed 0.8 mg/ml BrdU in their drinking water for 7 days before sacrifice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we examined the course of infection and immune responses of B6 mice following infection with wild-type MCMV and compared this to the response mounted against viruses lacking effective VIPR function. The virus MS94.5 lacks ORFs 151–165 (43) and is readily detected by MCMV-specific CTL (20, 43). Recently, BAC technology was used to construct a virus lacking all three VIPRs, m4+m6+m152 (6). m4+m6+m152 has identical growth kinetics in vitro to its parental BAC-derived wild-type virus, and is unimpaired in growth in vivo in immune-depleted animals (6). Whereas wild-type MCMV-infected cells were not lysed by MCMV-specific CTL clones, cells infected with m4+m6+m152 were readily lysed, indicating a substantial loss of VIPR function (Fig. 1a).
We adapted the ICS assay to quantitatively assess the total CD8 T cell response to MCMV using virus-infected cells (Fig. 1b). For APCs, JAWSII dendritic cells were infected with the Ag presentation-competent mutant MCMV MS94.5. The region deleted in MS94.5 does not contain immunodominant epitopes for MCMV in B6 mice (M. W. Munks, manuscript in preparation). In some experiments, we also assessed the response to the immunodominant Db-restricted M45985–93 epitope (31). A Db-M45985–93 tetramer was constructed, and Fig. 1c demonstrates the specificity of this reagent: only CD8 T cells from MCMV-infected mice are stained by this tetramer.
Because ICS using infected APCs may underestimate the true number of MCMV-specific CD8 T cells, we also assessed expression of the inhibitory receptor NKG2A as an indication of Ag experience. NKG2A is expressed after activation on a high percentage of CD8 T cells and remains expressed long after clearance of Ag on a high percentage of Ag-specific CD8 T cells (47, 48, 49, 50), although the level of expression decreases with time after Ag exposure (50). To determine the usefulness of NKG2A expression as a surrogate marker for MCMV specificity, we assessed expression of NKG2A on bystander CD8 T cells during MCMV infection. OVA-specific CD8 T cells from OT-1 TCR transgenic mice were adoptively transferred into naive B6 recipients. In the experiment shown in Fig. 1d, MCMV induced expression of NKG2A on only 4% of OT1 T cells, and in a second experiment (not shown), there was no increase in the percentage of OT1 T cells expressing NKG2A after MCMV infection. We conclude that the increase in percentage of CD8 cells expressing NKG2A after MCMV infection is a useful surrogate marker for MCMV-specific CD8 T cells.
Comparison of VIPR-deficient and wild-type virus during acute infection
We have previously reported that the peak size and immunodominance of the CD8 T cell response to M45 does not differ between wild-type infection and infection with a virus lacking the single VIPR m152. In this study, we compared wild-type infection with the m4+m6+m152 virus, which lacks all three VIPRs, and performed a kinetic analysis of virus levels and the CD8 T cell response during the first 2 wk of infection. Fig. 2a shows an experiment in which mice were infected with 5 x 106 PFU of wild-type or m4+m6+m152 MCMV. Over a 14-day period, the CD8 T cell response to M45 was measured by tetramer staining, and total MCMV-specific CD8s assessed by ICS for IFN- after incubation with infected APCs. The percentage of CD8 T cells expressing NKG2A is also shown. Virus levels were assessed in the lungs, an organ in which virus is particularly sensitive to CD8 T cell control. Fig. 2b shows a second experiment, in which the CD8 response to M45 was assessed by ICS after incubation with the M45 peptide. Virus levels in kidneys, liver, and lungs are shown.
As has been previously described, virus levels peaked at day 3 and declined thereafter. The CD8 T cell response peaked later, at day 7, and then declined. The peak of NKG2A positivity was a little slower, because CD8 T cells only express NKG2A after several days of activation. In multiple assays, we observed that size of the peak CD8 T cell response to m4+m6+m152 was frequently (e.g., Fig. 2b) but not always (e.g., a) greater than the response to wild-type viruses. Also, small differences in viral titers were sometimes seen between wild-type and m4+m6+m152 at some time points. However, overall, the remarkable feature of these experiments was the similarity between wild-type and m4+m6+m152 infections.
Comparison of VIPR-deficient and wild-type viruses during the chronic/latent phase of infection
We were surprised that VIPR-mediated impairment of infected cells had little effect on the course of acute MCMV infection and on the antiviral CD8 T cell response. However, as with many other virus infections, the initial control of MCMV infection may be primarily a function of NK cells: an effective CD8 response does not develop until day 5 postinfection, a time at which virus titers are already falling (Fig. 2). Furthermore, we have previously suggested that, in MCMV infection, naive CD8 T cells may be initially primed in vivo primarily through cross-presented Ag (31), which would explain why VIPR function had little effect on the size of the initial CD8 T cell response. However, because cells infected with m4+m6+m152 are readily detected and lysed by CTL (Fig. 1a), we expected that the virus would be eventually cleared and that the numbers of MCMV-specific CD8 T cells would then decline and show the characteristic features of a CD8 T cell response to a cleared virus infection. Specifically, we expected that MCMV-specific CD8 T cell numbers in the memory phase would be 5–10% of those seen at the peak, and the phenotype would revert in time to a resting, central memory phenotype (CD62L+). In contrast, we expected that mice infected with wild-type virus in which the VIPRs are functional would maintain a higher level of virus activity, resulting in higher numbers of memory CD8 T cells, and that these cells would display the characteristic effector memory phenotype (CD62L–) that has been described in chronic CMV infection in mice and humans.
Fig. 3 shows an experiment in which the number and activation status of MCMV-specific CD8 T cells was assessed at 8 and 24 wk postinfection. The M45985–93 response becomes very subdominant during chronic infection (M. W. Munks and M. C. Gold, unpublished data), and so Ag-specific responses could only be measured by the ICS assay with infected APCs. During chronic infection, we have not observed CD69 expression on MCMV-specific CD8s (data not shown). Staining for CD43, CD11c, NKG2A, and CD62L is shown. We also quantified the level of CD8 expression. The CD8 coreceptor is down-regulated after T cell activation, but returns to near naive levels within 30–60 days after the acute response to cleared pathogens such as LCMV and Listeria monocytogenes (45, 51). We assigned a CD8low gate to determine the percentage of cells whose intensity of CD8 staining was clearly below that seen on naive cells.
The top panel of Fig. 3 shows the CD8 and CD62L staining of cells incubated with uninfected APCs, where alteration in staining levels cannot be attributed to in vitro TCR triggering. These plots demonstrate the dramatic, permanent alteration in the CD8 T cell compartment engendered by MCMV infection: mice infected with MCMV had a much expanded CD62L–CD8low effector memory CD8 T cell population at both 8 and 22 wk. Strikingly, the pattern was very similar for wild-type and m4+m6+m152 viruses. The rest of the panels show staining of cells exposed to infected APCs. Around 13% of CD8 T cells made IFN- in response to infected APCs at both 8 and 22 wk. Most of these cells were NKG2A+, indicating Ag experience. About one-half expressed CD11c, which is up-regulated on effector CD8 T cells (52, 53, 54). About one-half were CD8low, suggesting that they had encountered Ag within 60 days. Only a small percentage (6–17%) displayed the acute effector marker CD43. Interestingly, consistent with previous reports, the IFN-+ CD8 T cells were almost exclusively CD62L negative, which is considered the hallmark of the effector memory phenotype. Although some CD62L loss may have occurred during the 6-h incubation with brefeldin A, most of this loss is likely to have occurred in vivo, as a high proportion of CD8 T cells were CD62L– without exposure to Ag (Fig. 3, top panel). Thus, a large proportion of the MCMV-specific CD8 T cells in chronic infection were CD62L–, NKG2Ahigh, and CD8low, characteristic of an effector memory phenotype and suggestive of repeated exposure to viral Ag. Remarkably, the number and phenotype of the CD8 T cells in m4+m6+m152-infected mice were strikingly similar to wild-type infection.
Reactivation of wild-type and m4+m6+m152 viruses from mice infected for 6 wk
During chronic infection, virus cannot be cultured from mice (55). We have occasionally detected viral DNA from homogenized organs by real-time PCR in both wild-type and VIPR-deficient infections, but frequency of positive samples for both infections is too low in our hands for statistical comparison.
To assess whether the virus persisted but was undetectable due to the ongoing immune response, we immunosuppressed chronically infected mice to enable reactivating virus to replicate. In addition to B6 mice, we used B cell-deficient (µmt) mice, in which reactivating virus is easier to detect because it cannot be eliminated by neutralizing Ab (28). Mice were infected with either wild-type or m4+m6+m152 MCMV for 6 wk. They were then immunosuppressed for 2 wk with a combination of cyclophosphamide, hydrocortisone, and anti-lymphocyte globulin. After sacrifice, viral load in liver, lungs, kidneys, and salivary glands was measured by real-time PCR. For µmt mice, virus was detected in four of four wild-type and four of four m4+m6+m152-infected mice. For B6 mice, virus was detected in three of three wild-type and three of four m4+m6+m152-infected mice. Salivary gland extracts from B6 mice were plated onto monolayers until viral CPE was detected; DNA was extracted and analyzed for presence of viral genes. In each case, the reactivated virus was of the expected genotype: Fig. 4 shows a representative PCR for two animals. These experiments do not allow us to quantify the level of latent or persistent virus in the animals before immunosuppression, but they provide formal proof that m4+m6+m152 virus was not eradicated by 6 wk postinfection in six of seven animals.
Comparison of wild-type infection with a virus lacking 15 ORFs (m151–m165)
We also analyzed infections with the virus MS94.5, which lacks the VIPR m152 and also lacks m157 and 13 other ORFs, many of which are likely to be immunomodulatory (four have MHC class I homology (16)). Unlike wild-type virus, MS94.5 is readily detected in vitro by MCMV-specific CTL (20, 43). Fig. 5 shows two experiments. At the peak of the response to each virus, 5 million CD8+ splenocytes were virus specific, based on IFN- production following in vitro stimulation with MCMV-infected cells. This resolved to a chronic response that was relatively stable over the time assessed (over 1 year). Many of these cells displayed a CD8low phenotype. The small number of animals in these experiments does not allow statistically significant comparison between the two infections. However, we can conclude both from the size of the chronic response relative to the acute response, and from the phenotype of the cells, that the response to both viruses suggested ongoing virus infection. Furthermore, over the multiple time points analyzed, the kinetics of the response appears similar for both viruses, suggesting that a similar dynamic equilibrium between virus and host immune response was reached in both infections. These experiments are of interest because 1) they provide the first report of the CD8 T cell response in B6 mice to MCMV lacking m157, 2) they will also be of interest as the function of the other genes missing from MS94.5 is determined, and 3) we performed more detailed and protracted kinetic analyses of the CD8 T cell response to this virus than to the more recent m4+m6+m152 virus.
BrdU staining of CD8 T cells in mice infected with wild-type and MS94.5 virus
To assess whether MCMV-specific CD8 T cells were actively dividing, mice that had been infected for 1 or 30 wk were fed BrdU for 1 wk before being sacrificed. Splenocytes from these mice were restimulated as above, and costained for CD8, IFN-, and BrdU. Fig. 6 shows that at wk 1 after infection with either wild-type or MS94.5, 90% of Ag-specific cells had incorporated BrdU, indicating they had recently replicated their DNA. However, at 30 wk, BrdU incorporation in IFN-+ CD8 T cells was not distinguishable from naive animals. Thus, these cells were not cycling rapidly enough to be detected by this assay, suggesting that the CD8 T cell pool was not turning over rapidly.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m4+m6+m152 virus, the size and phenotype of the chronic CD8 response was characteristic of chronic virus infection, or at least repeated Ag presentation. At 6 wk postinfection, we could readily detect both viruses after immunosuppression, formally demonstrating that the m4+m6+m152 virus was not eradicated by an effective CD8 T cell response. Some sort of VIPR function has been described for all herpesviruses looked at closely, implying that this function is crucial for the herpesvirus lifestyle (2, 3, 5). It has generally been assumed that VIPRs would be necessary for herpesviruses to persist in an immunocompetent host. However, our results show that, even though MCMV VIPRs effectively inhibit lysis of infected cells by MCMV-specific CTL in vitro, a virus without these genes was still able to establish latent or persistent infection. Furthermore, there was no discernible effect of VIPR removal on the CD8 T cell response to infection in vivo.
These results are obviously very different from those we had expected, which was that the m4+m6+m152 virus would be cleared and that the CD8 T cell response would in turn become typical in size and phenotype of a cleared virus infection. This has caused us to look more closely at the assumptions that formed the basis of this expectation. In the following, we list some of the issues that we believe need to be clarified or explored further.
Demonstration of the ability of VIPRs to impact CD8 T cell recognition relevant cell types in vivo
The MCMV genes m4, m6, and m152 have a profound effect on the ability of CTL to lyse infected cells in Cr release assays in several cell types (Fig. 1; Refs. 20, 21, 22 and 43), and an m152-deletant virus was better controlled by CD8 T cells in vivo (22, 56), indicating that VIPRs do indeed function as predicted in important tissues in vivo. However, the strength of VIPR-mediated inhibition of Ag presentation in vivo to CD8 T cells of different specificities, and in APCs as well as somatic cells, should be determined.
Correlation of the effector memory phenotype of CD8 T cells in MCMV infection with virus activity
The size of the CD8 T cell response to CMV and the characteristic effector memory phenotype of the cells in both humans and mice has been accepted to mean that the T cells have been repeatedly restimulated, either by replicating virus or at the very least by proteins produced by abortively reactivating virus. Because CMV is known to persist, even though it is often undetectable without immunosuppression, this explanation of the CD8 T cell phenotype seems logical. In fact, it has been suggested that the CD8 T cell response may be the best indicator of virus activity in chronic CMV infection (39). We have formally demonstrated persistent viable m4+m6+m152 at 6 wk postinfection. When the same immunosuppression protocol was used on B6 mice that had been infected for 6 mo, we did not detect virus. Detection of reactivating virus from long-term infected B6 mice is difficult, perhaps due to the presence of neutralizing Ab, and we are attempting to improve our protocols. However, in none of these experiments did we find any significant difference in our ability to detect virus in animals infected in parallel with wild-type or m4+m6+m152 virus. Thus, beyond 6 wk postinfection, the phenotype of the CD8 T cell response is our main evidence suggesting that virus activity in chronic infection is not affected by VIPR removal. The relationship between the CD8 T cell phenotype and virus activity in MCMV infection needs to be formally demonstrated. The phenotype of CD8 T cells in peripheral organs that support CMV reactivation, such as salivary gland and lungs, and the extent to which T cells recirculate between peripheral tissues and central lymphoid organs will also be of interest. It has been well documented in other models, particularly acute LCMV infection, that in the absence of continued antigenic stimulation, memory CD8 T cells come in time to re-express CD62L and to up-regulate CD8 (51, 54, 57). Thus, it seems likely that the CD62L–CD8low phenotype in MCMV infection—both m4+m6+m152 and wild-type—does indeed result from continued Ag exposure. If this proves not to be the case, a different problem will need to be addressed, i.e., how does CD8 T cell priming in MCMV infection differ from other models to cause CD8 T cells to retain this unique phenotype?
The source of viral Ag and the nature of Ag presentation that drives the CD8 T cell response in chronic infection
The fact that we are forced to rely on the CD8 T cell response to compare m4+m6+m152 and wild-type virus activity in chronic infection raises some important issues. We are using a single measurement to assess the effect of VIPRs on two separate processes: T cell priming, and the impact of effector CD8 T cells on virus load. CD8 T cells can be primed either by directly infected APCs or by APCs that take up proteins synthesized in infected cells and cross-present them. VIPR function should affect priming by directly infected cells, but not by cross-presentation. It follows that the size and phenotype of the CD8 T cell response would not be affected by VIPR function 1) if cross-presentation were the only means by which CD8 T cells could be primed and 2) if the amount of viral protein being made were the same in wild-type and m4+m6+m152 infections. It has been shown that MCMV encodes immune evasion genes other than VIPRs that impair the ability of infected dendritic cells to prime T cells (58, 59), so cross-priming may indeed be the only mechanism by which MCMV-specific CD8 T cells can be primed. Even so, we would think that the m4+m6+m152 virus should be more readily controlled by CD8 T cells, limiting the amount of viral protein available for cross-presentation. In contrast, the amount of viral protein available for cross-presentation during chronic infection with both viruses could be the same if the major source of viral Ag is abortively reactivating virus, and the size of the latent virus pool, established early during acute infection, is similar for both viruses. Another explanation could be that, if directly infected cells do play a role in priming, a smaller amount of m4+m6+m152 virus in chronic infection would be able to activate more CD8 T cells. In other words, opposite effects on T cell priming and virus control could lead to a similar final picture. These permutations highlight the need to define experimentally 1) the source and amount of viral Ag in chronic infection, 2) the relative importance of direct and cross-priming, and 3) the ability of CD8 T cells to affect the amount of wild-type and m4+m6+m152 virus load in chronic infection.
The evolutionary advantage of VIPRs
VIPR functions are too widely expressed among herpesviruses (2, 3, 5) for us to readily dismiss the notion of interference with Ag presentation as laboratory artifact. At present, our favored explanation is that VIPRs function in concert with other types of immunoevasins, and that removing one layer of a complex strategy has not been enough to have a major impact on viral fitness. This may especially be the case because of the artificial context of inbred laboratory mice and experimental infection conditions (high virus dose and i.p. infection).
Nevertheless, the lack of impact of VIPR removal causes us to at least wonder whether we have looked in the wrong place to understand their evolutionary advantage for this virus. It is possible that the greatest impact of VIPRs may be on virus transmission. In other words, VIPR function may be necessary for latent virus to occasionally replicate sufficiently in the salivary gland to generate an infectious inoculum and spread to a new host. The observation that, in the salivary gland, wild-type (VIPR-containing) MCMV is particularly resistant to CD8 T cell control (60) might support this hypothesis. Another possibility is that VIPRs may lessen the risk of immunopathology engendered by the massive size of the CMV-specific memory CD8 T cell population. Persistent viruses depend on their hosts’ health for their own persistence and transmission. CD8 T cell immune responses can be hazardous. Although we saw no gross pathology in our chronically infected animals, it is possible that, in some circumstances, CD8 T cell effector functions, such as recurrent IFN- and TNF- release (which would presumably occur more often in VIPR-deficient infections), could damage host tissues such as great vessels and lungs over a lifetime. Yet another possibility is that VIPRs are necessary for superinfection. A strange feature of CMV epidemiology, demonstrated in mice (61, 62), humans (63), and monkeys (64), is the ability to reinfect an already-infected animal. Although the evolutionary advantage of superinfection remains to be determined, VIPR function may be necessary for it to occur.
Conclusions
CMVs have been coevolving with their mammalian hosts at least since the mammalian radiation 60 million years ago (65, 66). It is apparent that, in this time, they have fine-tuned a complex relationship that we are far from understanding. Given the ubiquity of CMV infection and the size of the immune response to it, understanding its immunobiology must be part of understanding the normal immune system. Although there are differences in genetic mechanisms, at a functional level, the mouse model seems to closely parallel human infection. This is fortunate because sorting out most of the issues raised above requires in vivo experiments. Understanding the normal immunobiology of CMV will be important as we attempt to manipulate or exploit it through such interventions as vaccination.
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2 Address correspondence and reprint requests to Dr. Ann B. Hill, Department of Molecular Microbiology and Immunology L220, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239. E-mail address: hillan{at}ohsu.edu
3 Abbreviations used in this paper: VIPR, viral gene that inhibits Ag presentation; MCMV, murine CMV; BAC, bacterial artificial chromosome; LCMV, lymphocytic choriomeningitis virus; IE, immediate early; MEF, mouse embryo fibroblast; vv-ova, recombinant vaccinia virus expressing chicken OVA; ICS, intracellular cytokine staining; BrdU, 5-bromo-2'-deoxyuridine.
Received for publication February 11, 2003. Accepted for publication March 26, 2004.
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-herpesvirus. Nat. Immunol. 3:733.[Medline]
