Establishment and Maintenance of Long-Term Murine Gammaherpesvirus 68 Latency in B Cells in the Absence of CD40

Murine gammaherpesvirus 68 ( ${gamma}$ HV68), like Epstein-Barr virus (EBV),establishes a chronic infection in its host by gaining accessto the memory B-cell reservoir, where it persists undetectedby the host's immune system. EBV encodes a membrane protein,LMP1, that appears to function as a constitutively active CD40receptor, and is hypothesized to play a central role in EBV-drivendifferentiation of infected naive B cells to a memory B-cellphenotype. However, it has recently been shown that there isa critical role for CD40-CD40L interaction in B-cell immortalizationby EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama,and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840,2003), indicating that LMP1 does not adequately recapitulateall of the necessary functions of CD40. The role of CD40 receptorexpression on B cells for the establishment and maintenanceof ${gamma}$ HV68 latency is unclear. Data previously obtained with acompetition model, demonstrated that in the face of CD40-sufficientB cells, ${gamma}$ HV68 latency in CD40-deficient B cells waned over timein chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E.Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892,2003). To further investigate the role of CD40 in ${gamma}$ HV68 latencyin vivo, we have characterized the infection of CD40 knockout(CD40^–/–) mice. Here we report that, consistentwith previous observations, ${gamma}$ HV68 efficiently established a latentinfection in B cells of CD40^–/– mice. Notably, unlikethe infection of normal C57BL/6 mice, significant ex vivo reactivationfrom splenocytes harvested from infected CD40^–/–mice 42 days postinfection was observed. In addition, in contrastto ${gamma}$ HV68 infection of C57BL/6 mice, the frequency of infectednaive B cells remained fairly stable over a 3-month period postinfection.Furthermore, a slightly higher frequency of ${gamma}$ HV68 infection wasobserved in immunoglobulin D (IgD)-negative B cells, which wasstably maintained over a period of 3 months postinfection. Thepresence of virus in IgD-negative B cells indicates that ${gamma}$ HV68may either directly infect memory B cells present in CD40^–/–mice or be capable of driving differentiation of naive CD40^–/–B cells. A possible explanation for the apparent discrepancybetween the failure of ${gamma}$ HV68 latency to be maintained in CD40-deficientB cells in the presence of CD40-sufficient B cells and the stablemaintenance of ${gamma}$ HV68 B-cell latency in CD40^–/– micecame from examining virus replication in the lungs of infectedCD40^–/– mice, where we observed significantly higherlevels of virus replication at late times postinfection comparedto those in infected C57BL/6 mice. Taken together, these findingsare consistent with a model in which chronic virus infectionof CD40^–/– mice is maintained through virus reactivationin the lungs and reseeding of latency reservoirs.

INTRODUCTION

Top

Introduction

Gammaherpesviruses establish a lifelong latent infection withinlymphocytes of their respective hosts. Significantly, thereis a close association between gammaherpesvirus infection andthe development of a diverse set of lymphomas and other tumors.The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi'ssarcoma-associated herpesvirus (KSHV; also called human herpesvirus8) have been implicated in the development of a wide range ofcancers. EBV is often associated with the development of Burkitt'slymphoma, Hodgkin's disease, gastric carcinoma, and nasopharyngealcarcinoma (for reviews, see references 31, 33, 48, 51, and 64),and there are close associations of KSHV with Kaposi's sarcoma,primary effusion lymphomas, and a variant of multicentric Castleman'sdisease (11-13, 21, 42).

For both EBV and KSHV, B lymphocytes represent the major reservoirof latent virus; however, KSHV is also known to establish latencyin macrophages and dendritic cells (3, 15, 16, 38, 49, 50).Although little is known about KSHV latency in vivo, EBV latencyhas been relatively well characterized. In sites of active viralreplication, such as the tonsils, EBV is found in a varietyof B-cell subsets; however, long-term EBV latency in the peripheryresides almost exclusively in resting memory B cells (2, 3,24, 28, 34). Recent data support a model by which EBV gainsaccess to this subset of long-lived B cells by appropriatingcontrol of normal B-cell differentiation (reviewed in references39, 49, and 51). This model suggests that EBV is able to driveB-cell activation through the expression of nine viral geneproducts (six nuclear antigens [EBNAs] and three membrane proteins[LMPs]) in what is referred to as the "growth program" or latencyIII program of viral gene expression (4, 27). Some of theseEBV growth-transformed lymphoblasts are hypothesized to formgerminal centers, with a concomitant downregulation of viralgene expression with expression of EBNA1, LMP1, and LMP2a (4,5, 9). LMP1 and LMP2A are able to provide surrogate CD40 andBCR engagement signals, respectively, which play important rolesin B-cell differentiation and survival during the germinal centerreaction and thus facilitating differentiation into memory Bcells.

Because of the restricted host ranges of EBV and KSHV and thelack of suitable nonhuman primate models, it is difficult todirectly address questions pertaining to the mechanisms involvedin the establishment of long-term latency in vivo. Murine gammaherpesvirus68 ( ${gamma}$ HV68; also known as MHV-68) infection of mice provides atractable small-animal model in which central questions pertainingto gammaherpesvirus biology, pathogenesis, and lymphomagenesiscan be addressed in the context of a natural host. ${gamma}$ HV68 wasoriginally isolated from bank voles in Slovakia (7) and wasrecently shown to be endemic in European wood mice (6). On thebasis of the genomic sequence of ${gamma}$ HV68, the virus is closelyrelated to both EBV and KSHV (57). Infection of mice with ${gamma}$ HV68results in an acute, productive infection within the lungs,followed by rapid clearance (46). Concomitant with this process,the virus establishes latency in a variety of tissues, includingthe spleen, lymph nodes, and bone marrow. B lymphocytes, macrophages,dendritic cells, and possibly lung epithelial cells are allknown latency reservoirs (19, 44, 47, 60). As observed withEBV infection, long-term ${gamma}$ HV68 latency is narrowly confined tocells expressing a memory B-cell phenotype (20, 62).

It is unclear if ${gamma}$ HV68 gains access to the memory B-cell compartmentin a manner similar to that proposed for EBV. In a recent reportanalyzing ${gamma}$ HV68 latency in CD40⁺/CD40^– mixed bone marrowchimera mice, the authors demonstrated that viral latency wasprogressively lost in cells lacking CD40 (30). Furthermore,as expected, they only detected CD40⁺ B cells in the germinalcenters of these infected chimeric mice. These results suggestthat access of ${gamma}$ HV68 to the memory B-cell compartment may bemore reliant upon conventional B-cell activation and differentiationpathways than is the case for EBV. However, these authors didnot assess whether ${gamma}$ HV68, at least transiently, accesses thememory B-cell reservoir in CD40^– B cells and thus theycould not rule out the possibility that there is a role forCD40 in the maintenance of ${gamma}$ HV68 latency in memory B cells ratherthan an essential role for CD40 in accessing the memory B-cellreservoir.

The studies described here were aimed at addressing the requirementfor CD40 in ${gamma}$ HV68 gaining access to memory B cells, as well asto evaluate the ability of ${gamma}$ HV68 to establish and maintain long-termlatency in mice lacking CD40. CD40 is a surface glycoproteinbelonging to the tumor necrosis factor receptor family (43,53) and is expressed on a wide variety of cell types such asB lymphocytes and specialized antigen-presenting cells, includingmacrophages and dendritic cells (56). CD40 interactions withits cognate ligand (CD40L [CD154/gp39]) on T cells underpinmany of the significant T-cell-dependent B-cell differentiation,survival, and antibody production pathways. As a consequence,CD40-deficient mice are unable to form germinal centers, haveimpaired memory B-cell formation, and do not generate isotype-switchedantibodies in a T-cell-dependent manner (29). We have examined ${gamma}$ HV68 latency and reactivation from latency in bulk splenocytesand various sorted splenocyte populations in CD40-deficientmice.

MATERIALS AND METHODS

Top

Materials and Methods

Virus and tissue culture. ${gamma}$ HV68 clone WUMS (ATCC VR1465) was passaged in and its titerswere determined on NIH 3T12 cells as described previously (14).NIH 3T12 and mouse embryonic fibroblast (MEF) cells were maintainedin Dulbecco's modified Eagle's medium supplemented with 10%fetal calf serum, 100 U of penicillin per ml, 100 mg of streptomycinper ml, and 2 mM L-glutamine (cMEM). MEF cells were obtainedas described previously (36), by homogenizing C57BL/6J day 12embryos, plating the resulting homogenate in tissue cultureflasks for 7 to 14 days, and harvesting adherent cells by trypsinization.

Mice, infections, and organ harvests. CD40^–/– (29) and wild-type C57BL/6J mice (catalogno. 002928/000664; The Jackson Laboratory, Bar Harbor, Maine)were housed and bred at the Yerkes Vivarium in accordance withall federal, university, and facility regulations. Mice wereanesthetized with isoflurane and inoculated intranasally with1,000 PFU of ${gamma}$ HV68 in 20 µl of cMEM. Mice were anesthetizedprior to sacrifice by cervical dislocation, and spleens andlungs were harvested and treated as described below. Sentinelmice were assayed every 3 months and were negative for adventitiousmouse pathogens by parasitology and serology.

Plaque assays. Plaque assays were performed as described previously, with modifications(55). Briefly, six-well plates were seeded with 2 x 10⁵ NIH3T12 cells 1 day prior to infection. Lung samples were thawedand mechanically disrupted in 1 ml of medium containing 100µl of 1.0-mm-diameter zirconia-silica beads (Biospec Products,Inc., Bartlesville, Okla.) with a Mini-Beadbeater-8 (BiospecProducts, Inc.) for four 1-min pulses. Serial 10-fold dilutionsof lung homogenates were plated onto NIH 3T12 monolayers ina 200-µl volume and allowed to adsorb for 1 h at 37°Cwith periodic rocking. Samples were overlaid with 5 ml of mediumcontaining 2% methylcellulose and maintained at 37°C. Plaqueswere scored microscopically 5 to 7 days postplating. All titerswere determined in parallel with a laboratory standard witha known titer. The limit of detection of this assay is 50 PFUper organ.

Cell preparation and flow cytometry cell purification. Single-cell suspensions were generated by disrupting spleenswith a TenBroek homogenizer and filtering the homogenate througha 100-um-pore-size nylon cell strainer (Becton Dickinson, FranklinLakes, N.J.). Following red blood cell lysis, cells were collectedby centrifugation, washed, refiltered, enumerated, and assayedfor viability with trypan blue (Sigma, St. Louis, Mo.). Ratanti-mouse CD16/CD32 (Fc Block) was used to block Fc receptorsprior to staining. Cells were stained for fluorescence-activatedcell sorter (FACS) analysis with a phycoerythrin-conjugatedantibody directed against CD19 and a fluorescein isothiocyanate-conjugatedantibody to immunoglobulin D (IgD; BD Pharmingen). Stained cellpopulations were sorted expeditiously with a FACSVantage SE(Becton Dickinson, Mountain View, Calif.) or MoFlo (Cytomation,Fort Collins, Colo.) flow cytometer. Data were analyzed withFloJo software (TreeStar, Inc., San Carlos, Calif.). The resultantpurified cell populations were resuspended in cMEM supplementedwith 10% dimethyl sulfoxide and stored at –80°C forlimiting-dilution PCR assays or at 4°C in cMEM for limited-dilutionex vivo reactivation assays as described below. Pooled splenocytesfrom 5 to 15 mice were used in all experiments.

Frequency of reactivation of virus from latency in cells upon explant culture. The frequency of reactivation of virus from latency in cellswas determined by a limiting-dilution ex vivo reactivation assay(58, 60). Briefly, unsorted splenocytes or purified cell populationswere obtained from mice at 16, 42, and 84 days postinfection(dpi). Cells were resuspended in cMEM and plated in serial twofolddilutions (starting with 10⁵ cells) onto MEF monolayers in 96-wellplates. Twenty-four replicate wells were plated per dilution,and 12 dilutions were plated per sample. The appearance of acytopathic effect (CPE) was monitored microscopically at days14 and 21 postplating. The contribution to reactivation frompreformed infectious virus was determined by parallel platingof mechanically disrupted cells (latent virus cannot reactivatefrom killed cells). This procedure kills >99% of the cellsbut has only negligible effects on virus titers (58-60).

Frequency of ${gamma}$ HV68 viral genome-positive cells. The frequency of cells harboring the ${gamma}$ HV68 genome was determinedby a single-copy sensitivity nested PCR assay directed againstthe ${gamma}$ HV68 ORF50 gene sequence as described previously (59, 60).Briefly, the bulk of FACS-sorted splenocytes generated at 16,42, and 84 dpi were thawed, counted, and resuspended in isotonicbuffer. A series of six threefold serial dilutions, startingwith 10⁴ cells per well, were plated in a background of 10⁴uninfected NIH 3T12 cells in 96-well PCR plates. Cells werelysed prior to nested PCR by overnight treatment at 65°Cin the presence of detergent and proteinase K. Each experimentalplate included both negative and positive control wells to ensureoptimal assay sensitivity. All cell lysis and PCR protocolswere performed as described previously (59, 60) on a PrimusHTthermal cycler (MWG Biotech). All of the assays reported hereindemonstrated near-single-copy sensitivity in the absence ofany false-positive PCRs. Twelve replicate PCRs were performedfor each cell dilution per sample per experiment.

Measurement of chronic viral persistence in the lungs. Persistent viral replication in the lungs was measured by amodified form of the limiting-dilution ex vivo reactivationassay described above. Briefly, the lower left lobe of the lungfrom each animal was pooled into 500 µl of cMEM and mechanicallydisrupted as described previously (55). The homogenate was broughtup to 5 ml with cMEM and plated in twofold serial dilutionsonto MEFs in 96-well tissue culture plates. Six dilutions of24 replicate wells were plated. The appearance of a CPE wasmonitored microscopically and read 10 to 12 days postplating.Pooled lung lobes from five mice were used in all experiments.

Statistical analysis. GraphPad Prism software (GraphPad Software, San Diego, Calif.)was used for all data analysis. Frequencies of viral genome-positivecells or reactivation from latency were calculated on the basisof a Poisson statistical distribution in which the intersectof 63.2% represents a single-cell event. Nonlinear regressionanalysis with a best-fit sigmoidal dose-response algorithm wasapplied to the experimental data to obtain single-cell frequencies.

RESULTS

Top

Results

Acute ${gamma}$ HV68 replication in the lungs is unaffected by the absence of CD40 following intranasal inoculation. To assess the potential effects of CD40 on acute viral replication,we infected CD40^–/– and wild-type C57BL/6J miceintranasally with 1,000 PFU of wild-type ${gamma}$ HV68. Lungs were harvestedfrom these mice at both 4 and 9 dpi, and viral titers were determinedby plaque assay on murine NIH 3T12 fibroblast monolayers asdescribed in Materials and Methods. Figure 1 demonstrates thatat early times postinfection, virus replication in the lungsof CD40^–/– mice was indistinguishable from thatobserved in C57BL/6J mice (ca. 447 versus 385 PFU/ml at 4 dpiand 13,680 versus 11,803 PFU/ml at 9 dpi, respectively). Thesefindings indicate that acute viral replication in the lungsis unaffected by the absence of CD40.

View larger version (14K):
[in this window]
[in a new window]
FIG. 1. Acute-phase ${gamma}$ HV68 titers are unaffected by the host mouse strain. C57BL/6J and CD40^–/– mice were inoculated with 1,000 PFU of wild-type ${gamma}$ HV68, and their lungs were harvested at 4 and 9 dpi. The data shown were compiled from single experiments with 9 or 10 individual mice. Virus titers from lung homogenates were determined by plaque assay on NIH 3T12 monolayers as described in Materials and Methods. Each point represents the virus titer from an individual mouse. The solid line represents the mean virus titer for each group of mice, and the dashed line represents the limit of detection of this assay (50 PFU).

Reactivation from latency in ${gamma}$ HV68-infected CD40^–/– mice. Spleens were harvested from ${gamma}$ HV68-infected CD40^–/–mice at various time points, and single-cell suspensions ofsplenocytes were generated as described in Materials and Methods.The frequency of reactivation of virus from latency in cellswas determined by a limiting-dilution ex vivo reactivation assay(58, 59). For this analysis, splenocytes were enumerated andassessed for viability by trypan blue exclusion and live spleencells were plated in serial twofold dilutions onto permissiveindicator monolayers of MEFs. Spontaneous virus reactivationresulted in a pronounced CPE, which was scored microscopically.To evaluate the contribution to reactivation from preformedinfectious virus in the samples, a parallel series of cellswere plated following mechanical disruption to kill cells butleave preformed virus intact, as previously described (58, 59).

Examination of infected splenocytes at 16 dpi demonstrated readilydetectable levels of reactivation (ca. 1 in 39,000) (Fig. 2and Table 1). This level of reactivation is approximately 5-to 20-fold lower than we have routinely observed in ${gamma}$ HV68-infectedC57BL/6J mice at 16 dpi (23, 35, 52, 62). It is possible thatthis decrease in the reactivation frequency can be attributedto an inherent reactivation defect in latently infected CD40^–/–cells, a defect in the ability of ${gamma}$ HV68 to establish latency,or both. However, on the basis of the acute virus replicationdata (Fig. 1), it is unlikely that this defect reflects alteredseeding of latency reservoirs.

View larger version (18K):
[in this window]
[in a new window]
FIG. 2. ${gamma}$ HV68 reactivation from latency in CD40^–/– mice. Unsorted splenocytes or purified splenic cell populations were obtained from ${gamma}$ HV68-infected CD40^–/– mice at the times indicated and subjected to analysis for reactivation upon explant culture. Serial dilutions of intact (live) cells were plated onto MEF indicator monolayers in parallel with samples that had been mechanically disrupted. The level of reactivation that could be attributed to preformed infectious virus was either undetectable or very low (data not shown). Best-fit curves were derived from nonlinear regression analysis, and each symbol represents the mean percentage of wells positive for a virus-induced CPE ± the standard error of the mean. The dashed line represents 63.2%, from which the frequency of genome-positive cells or the frequency of cell reactivation of virus was calculated with a Poisson distribution. The data shown represent at least two independent experiments with pooled cells from 10 to 15 mice per experimental group.

View this table:
[in this window]
[in a new window]
TABLE 1. Frequency of cells reactivating ${gamma}$ HV68 in explant cultures of unsorted splenocytes

Although the frequency of splenocyte reactivation of virus at16 dpi in latently infected CD40^–/– mice was decreasedcompared to that in wild-type C57BL/6 mice, virus reactivationfrom CD40^–/– mouse splenocytes was readily detectable(ca. 1 in 80,000) at 42 dpi (Fig. 2 and Table 1). This is incontrast to numerous reports indicating an inability to detectreactivation of wild-type ${gamma}$ HV68 from latently infected splenocytesat later time points (e.g., 42 dpi) (26, 58, 59, 62). Furthermore,low levels of reactivation were also seen as late as 84 dpifrom infected CD40^–/– mice (Fig. 2 and Table 1).In all of our assays, the levels of preformed infectious virusin the splenocyte samples were either undetectable or too lowto quantitate (data not shown). The latter is consistent withthe reported efficient clearance of acutely replicating virusfrom the spleen by 12 to 14 dpi (46). Thus, the observed virusgrowth in ex vivo limiting-dilution analyses could be exclusivelyattributed to reactivation from latency.

${gamma}$ HV68 infection of the spleen is efficiently maintained over a 3-month period in CD40^–/– mice. Although reactivation frequencies in CD40^–/– miceat 16 dpi were slightly lower than those seen for infectionof wild-type mice (62), it was unclear if this was a consequenceof a defect in the ability of ${gamma}$ HV68 to establish latency in thisparticular knockout strain. To assess this, we determined thefrequency of splenocytes harboring the viral genome with a limiting-dilutionPCR assay, as described in Materials and Methods. Spleens frominfected mice were harvested at 16, 42, and 84 dpi. Bulk splenocyteswere serially diluted and proteinase K treated, and the resultinglysate was subjected to a nested PCR assay to detect the viralgenome. By calculating the percentage of replicate wells scoringpositive for a PCR product at each dilution and applying a Poissondistribution, we were able to determine the frequency of cellsharboring the viral genome at various time points postinfection.The results of this analysis indicated the presence of latentvirus at all of the time points tested. The frequency of viralgenome-positive cells at 16 dpi was approximately 1 in 3,750splenocytes (Fig. 3 and Table 2), and this frequency remainedrelatively constant over the time course of the study. Frequenciesof viral genome-positive cells at 42 and 84 dpi were determinedto be approximately 1 in 2,450 and 1 in 5,400, respectively(Fig. 3 and Table 2).

View larger version (32K):
[in this window]
[in a new window]
FIG. 3. ${gamma}$ HV68 splenic latency in CD40^–/– mice. Unsorted and FACS-sorted splenocytes were obtained from CD40^–/– mice at 16, 42, and 84 dpi. The frequency of viral genome-positive cells within these cell populations was determined by a limiting-dilution PCR assay as described in Materials and Methods. The data shown represent at least two independent experiments with pooled cells from 10 to 15 mice per experimental group. Curve fit lines were derived from nonlinear regression analysis, and symbols represent mean percentages of wells positive for viral DNA. The error bars represent the standard error of the mean. The dotted line represents 63.2%, from which the frequency of viral genome-positive cells was calculated with a Poisson distribution.

View this table:
[in this window]
[in a new window]
TABLE 2. Analysis of frequency of cells harboring ${gamma}$ HV68 in splenocyte populations

By combining results obtained from the reactivation assay withdata obtained from this limiting-dilution PCR assay, we couldcalculate the efficiency of reactivation at each time point.The calculated efficiency of reactivation at 16 dpi was 9.6%of latently infected cells that underwent spontaneous reactivationupon explant culture. This number decreased to 3.1% by 42 dpi.The ability of ${gamma}$ HV68 to reactivate from latency in CD40^–/–mice is not markedly different from that of 10.8% reported forwild-type mice at 16 dpi (62). Thus, the decreased reactivationfrequency observed in CD40^–/– mice compared to historicaldata on C57BL/6J mice at 16 dpi can be largely accounted forby a decrease in the frequency of cells harboring virus at 16dpi in CD40^–/– mice. However, by 42 dpi the observedfrequency of latently infected splenocytes in CD40^–/–mice was very similar to that historically observed in C57BL/6Jmice (1 in 2,450 versus 1 in ca. 3,000, respectively).

B cells are the predominant splenic latency reservoir in CD40^–/– mice. From the analysis presented above, ${gamma}$ HV68 is able to establishand maintain long-term latency in the spleen. Although ${gamma}$ HV68can establish latency in a variety of cell types and maintainlong-term splenic latency in memory B cells in wild-type mice,it was unclear what subset(s) of splenocytes harbored latent ${gamma}$ HV68 in CD40^–/– mice. Initially we purified B-celland non-B-cell populations by FACS, following the staining ofbulk splenocytes with an antibody directed against the pan-B-cellmarker CD19 (data not shown). CD19⁺ (B) and CD19^– (non-B)cells were purified to mean purities of 97.4 and 99.4%, respectively,and subjected to limiting-dilution PCR analyses to determinethe frequency of cells harboring the viral genome in each population(Fig. 3). This analysis revealed a marked propensity for ${gamma}$ HV68to establish latency within the B-cell population. Notably,B cells harbored high levels of latent virus at early timespostinfection, and this frequency of viral genome-positive cellsremained constant over the time course of the study (ca. 1 in2,200 to 2,500) (Fig. 3 and Table 2). In contrast, the frequencyof latent virus in the non-B-cell subset was either very lowor undetectable, which precluded any valid frequency analysis(Fig. 3 and Table 2). On the basis of calculations of the totalnumbers of latently infected B cells compared to the total numberof latently infected cells within bulk unsorted splenocytes,the virus present within B cells accounts for most of the viruspresent in bulk splenocytes (Table 2).

Having established that B cells represent the predominant splenicreservoir of ${gamma}$ HV68 latency in CD40^–/– mice, we extendedthe above analysis to assess virus infection of specific B-cellsubsets. In wild-type mice, both germinal-center and memoryB cells harbor very high frequencies of latent virus at earlytimes postinfection, and memory B cells are known to be a criticalreservoir for long-term latency (20, 62). However, because CD40^–/–mice exhibit a profound defect in the development of memoryB cells, it was unclear if ${gamma}$ HV68 would preferentially partitioninto any specific B-cell subset in CD40^–/– mice.Thus, B cells were sorted on the basis of cell surface expressionof IgD (Fig. 4). Mature, naive B cells express high levels ofIgD on their surface, and this expression is down-regulatedduring B-cell differentiation within the germinal center. Spleencells were isolated at various times postinfection and stainedwith antibodies directed against CD19 and IgD and sorted intonaive (CD19⁺ IgD⁺) and CD19⁺ IgD^– B cells (Fig. 4). Notably,CD40^–/– mice have a significant population of surfaceIgD^– B cells (ca. 5%), which is only slightly reducedcompared to that of C57BL/6 mice. Analysis of virus infectionin these B-cell populations revealed that both naive and IgD^–B cells harbor high levels of latent ${gamma}$ HV68 (Fig. 3). Interestingly,IgD^– B cells harbored a ca. 15- to 30-fold higher frequencyof latent virus than that in naive B cells (Fig. 3 and Table2). Importantly, the purity of the CD19⁺ IgD^– B-cell populationwas 95% and CD19^– non-B cells, which harbor a very lowfrequency of latent virus, were the major contaminating population(Fig. 4). Thus, latency observed in the IgD^– B-cell populationcould not be accounted for by contaminating naive B cells. Furthermore,the frequency of viral genome-positive cells within each cellpopulation did not deviate significantly over a 3-month timecourse (Fig. 3; Table 2), in contrast to infection of normalC57BL/6J mice, in which naive B-cell infection rapidly declinesby 3 months (20, 23) and is not detectable by 6 months postinfection(62). The total number of latently infected cells observed withinthe sorted IgD^– and IgD⁺ B-cell populations did not completelyaccount for that observed in the bulk B-cell fraction; however,this is most likely due to the loss of virus-infected cellsbecause conservative FACS gates were set.

View larger version (36K):
[in this window]
[in a new window]
FIG. 4. FACS analysis of splenocyte populations in CD40^–/– mice. Splenocytes collected from CD40^–/– mice were isolated and prepared as described in Materials and Methods. Purification of B-cell and non-B-cell fractions was based on surface expression of the pan-B-cell marker CD19 (data not shown). Purified B cells (CD19⁺) were further fractionated into naive (IgD⁺) and IgD^– B cells with a fluorescein isothiocyanate-conjugated antibody to IgD. Representative FACS plots from one replicate experiment are shown. (A) Presorting analysis of total splenocytes stained with antibodies directed against CD19 and IgD. (B) Postsorting FACS analysis indicating the purity of sorted lymphocyte subsets. Mean postsorting purities were as follows: CD19⁺, 97.4% ± 2.0%; CD19^–, 99.4% ± 0.9%; CD19⁺ IgD⁺, 95.0% ± 5.2%; CD19⁺ IgD^–, 94.8% ± 0.6.%. The contaminating fractions for B-cell and non-B-cell fractions were 2.5 and 0.6%, respectively. Contamination within the naive B-cell fraction was 0.45% IgD^– B cells, and contamination within the IgD^– B-cell population with naive B cells was 2.5%.

CD40 knockout mice are unable to clear persistent ${gamma}$ HV68 replication from the lungs. As noted above, one intriguing observation was that latent viruswas maintained in all B-cell populations at significant levelsand did not appear to decrease markedly over the duration ofthe study. It is noteworthy that ${gamma}$ HV68 was able to maintain moderatelevels of latent virus in the naive B-cell population to atleast 3 months postinfection. This is in sharp contrast to ${gamma}$ HV68infection of wild-type mice, in which latent ${gamma}$ HV68 infectionof naive B cells decreases rapidly from 16 to 42 dpi and isundetectable by 6 months postinfection (62). Although ${gamma}$ HV68 infectionin wild-type mice is usually cleared from the lungs by 10 to12 dpi, CD40^–/– mice have impaired T-cell-dependentantibody responses and clearance of virus from sites of acutereplication may be compromised in these animals. An inabilityto clear lytic virus from the lungs could provide a source ofvirus, which then serves to seed sites of latency, such as thespleen, and thus maintain latency reservoirs that have a relativelyshort half-life (e.g., naive B cells).

To examine ongoing virus replication in the lungs of infectedmice, we harvested infected lung tissue from both CD40^–/–and wild-type C57BL/6J control mice, prepared lysates as describedin Materials and Methods, and limiting dilution plated theselysates onto MEFs. This analysis revealed that, as previouslyshown (55), by 42 dpi C57BL/6J mice have very low levels ofreplicating virus in the lungs (Fig. 5). However, significantlymore virus was detected in the lungs of CD40^–/–mice at both 42 and 84 dpi (Fig. 5).

View larger version (14K):
[in this window]
[in a new window]
FIG. 5. Persistent ${gamma}$ HV68 viral replication in the lungs of CD40^–/– mice. Lung homogenates from ${gamma}$ HV68-infected CD40^–/– and C57BL/6J mice were generated at 42 and 84 dpi as described in Materials and Methods. Serial dilutions of lung homogenates were plated onto MEF indicator monolayers and scored microscopically for the presence of a CPE 10 to 12 days postplating. Twenty-four replicates were plated per dilution. The data represent two replicate experiments with pooled lungs from five mice per experiment.

DISCUSSION

Top

Discussion

The prototypic gammaherpesvirus EBV establishes and maintainslife-long latency within memory B lymphocytes in its human host.It is widely accepted that EBV gains access to this cell typevia manipulation of normal B-cell differentiation pathways.Activation of B cells in response to antigen relies upon specificinteractions with T-helper cells, costimulation, and the appropriatecytokine milieu. Activated B lymphocytes traffic to primarylymphoid follicles at anatomic locations such as the spleenor lymph nodes and initiate rapid rounds of proliferation, resultingin the de novo generation of a germinal center. It is withinthis germinal center that isotype switching, affinity maturation,commitment to plasma cell lineage, and generation of B-cellmemory take place. The ability of EBV to manipulate this processrelies in part upon two virus-encoded gene products, LMP1 andLMP2a. Together, these gene products are able to provide requisitesignals for B-cell activation, differentiation, and survivalduring this highly selective process.

${gamma}$ HV68 infection of mice, which is gaining popularity as a small-animalmodel for human gammaherpesvirus infections, exhibits some strikingsimilarities to EBV latency. Acute infection of mice with ${gamma}$ HV68manifests itself as a mononucleosis-like syndrome, followedby the establishment of life-long latency (46). The developmentof lymphomas has also been reported in mice infected with ${gamma}$ HV68(45). Importantly, like that of EBV, long-term latency of ${gamma}$ HV68is narrowly restricted to memory B cells (20, 62), which suggeststhat gammaherpesviruses may have evolved common mechanisms forgaining access to this particular cell type. Memory B cellsare known to be long-lived, can traffic throughout the hostvia the peripheral blood, and are able to take up residencein numerous anatomic locations, including the spleen, bone marrow,and thymus. However, in contrast to EBV, ${gamma}$ HV68 does not appearto encode homologues of LMP1 and LMP2a (57) and it is unclearwhether ${gamma}$ HV68 actively promotes B-cell activation and differentiationto gain access to the memory B-cell reservoir. Recent experimentsmonitoring ${gamma}$ HV68 infection and latency within CD40 mixed bonemarrow chimeric mice demonstrated a role for CD40 in the persistenceof virus infection (30). These results suggest that ${gamma}$ HV68 mayrely to a greater extent upon non-virus-encoded B-cell differentiationpathways to gain access to and/or persist within memory B cells.It is also possible that ${gamma}$ HV68 gains access to this compartmentvia direct infection of memory B cells. However, it is importantto note with respect to the role of CD40 in gammaherpesvirusinfection of B cells that it has recently been shown that CD40plays an important role in EBV infection even though EBV LMP1mimics many aspects of CD40 signaling (25). The latter studiesshowed that EBV infection of naive B cells results in upregulationof CD40L expression on EBV B lymphoblasts, which appears toplay a critical role by engaging CD40 on the surface of infectedB lymphoblasts. Thus, the requirement of CD40 to persist withinthe infected host may reflect a role for CD40 in maintaininglatency within memory B cells.

The experiments described here were aimed at further definingthe requirement for CD40 in ${gamma}$ HV68 infection of murine B cellsin vivo. Although we observed at early times postinfection adecreased frequency of latently infected B cells in CD40^–/–mice than has been routinely observed in normal C57BL/6J mice, ${gamma}$ HV68 was able to establish a stable chronic infection in thesemice. This decreased frequency of latently infected cells observedat early times postinfection was not due to an inherent replicationor packaging defect in CD40^–/– mice, as ${gamma}$ HV68 replicatedto comparable titers in the lungs of CD40^–/– andCD57Bl/6 mice during the acute phase of infection. Notably,infection of both naive IgD⁺ B cells and more mature IgD^–B cells was observed and, as has been previously reported forinfection of immunocompetent mice, infection was biased towardthe IgD^– population of B cells (62). Thus, in the absenceof competition from CD40-sufficient B cells, ${gamma}$ HV68 can persistin CD40^–/– B cells. However, it also seems likelythat chronic infection of B cells in CD40^–/– miceis driven by persistent virus replication in the lungs and reseedingof B-cell latency.

The data presented here are in good agreement with a recentreport by Kim et al. (30) demonstrating efficient establishmentof latency in cells lacking CD40 and suggest that involvementof CD40 is not required at least for the early stages of latency.In contrast to a prior report (32), we did not observe any deathsin CD40^–/– mice as a result of infection with ${gamma}$ HV68within the first 6 months postinfection. This discrepancy mostlikely represents the difference between the inoculating dosesused, with our studies using 10-fold less virus. Importantly,we have observed a 50% mortality rate from 8 to 10 months postinfection,while no deaths were observed in control infected C57BL/6 mice(data not shown).

In contrast to ${gamma}$ HV68 infection of wild-type mice, we could readilydetect virus reactivation at 42 dpi, and low levels of reactivatingvirus could be seen as late as 3 months postinfection. The abilityto detect viral reactivation at later times postinfection likelystems from the inability of these mice to effectively clearlytic virus from the lungs. In wild-type mice, acutely replicatingvirus is efficiently cleared from the lungs by 10 to 12 dpi(46). The clearance is largely effected by an efficient antiviralantibody response and CD8 T-cell response (17). Although Usherwoodet al. (54) reported that antiviral antibodies are not requiredfor resolution of acute infection in the lungs, these studieswere conducted with mice that lacked B cells, the very celltype within which ${gamma}$ HV68 preferentially establishes latency. Itis difficult to directly address the role of antiviral antibodiesin B-cell knockout mice. We have shown previously that adoptivetransfer of immune serum from ${gamma}$ HV68-infected C57BL/6J mice canefficiently control lytic-virus titers in mice that lack B cells(µMT) and thus cannot generate appropriate antiviral antibodies(22). We confirmed that CD40^–/– mice have only verylimited antibody production compared to wild-type C57BL/6J miceby analyzing total antibody levels by enzyme-linked immunosorbentassay in both mock- and ${gamma}$ HV68-infected CD40^–/– andcontrol wild-type mice (data not shown). It is also formallypossible that the inability to control lytic virus in the lungsover the duration of this study may be due in part to a less-than-optimalCD4 T-cell response. Stimulation via CD40 is an important componentof this T-cell response, and this may also contribute to failureto control chronic virus replication in the lungs. Finally,engagement of CD40 has been shown for ${gamma}$ HV68, as well as EBV andKSHV, to suppress virus reactivation from B cells (1, 41). Thus,the observed chronic virus production in the lungs of CD40^–/–may, at least in part, be due to the absence of appropriatesuppression of virus reactivation mediated through CD40. Furthermore,the latter could also explain the attrition of latently infectedCD40^–/– B cells in the experiments performed withmixed bone marrow chimeric mice (30).

It is notable that both surface IgD⁺ naive B cells and surfaceIgD^– B cells are reservoirs of latent virus and that thesurface IgD^– subset harbors approximately 15- to 30-foldhigher frequencies of latent virus than naive B cells at allof the time points examined. Surface IgD is a faithful markerfor naive B cells, which is downregulated during the germinal-centerresponse in wild-type mice. However, mice lacking CD40 are largelyunable to generate germinal centers, and in this case the specificidentity of these B cells is difficult to ascertain. It is possiblethat these IgD^– B cells may represent marginal-zone Bcells or B1 cells (reviewed in reference 40), both of whichcan exhibit low levels of surface IgD. Moreover, it is formallypossible that isotype-switched B cells could be generated inthe absence of CD40-CD40L signaling. Indeed, with a cocktailof antibodies directed at identifying isotype-switched IgD^–/IgM^–B cells we were able to detect a small percentage of bona fidememory B cells (data not shown). This phenomenon has been reportedpreviously in both CD40- and CD40L-deficient mice, which havebeen shown to generate IgA and IgG isotypes in response to T-cell-independentantigens (10, 29, 37, 63). Furthermore, isotype-switched antibodiesare also generated in mice lacking CD40-CD40L signaling in responseto various pathogens, including vaccinia virus, lymphocyticchoriomeningitis virus, and Borrelia burgdorferi (8, 18, 61).It is formally possible that ${gamma}$ HV68 infection of CD40^–/–mice results in the generation of isotype-switched antibodiesin a T-cell-independent manner, perhaps through reprogrammingof B-cell differentiation. However, the small percentage ofthese cells within the spleen precluded examination of the frequencyof viral genome-positive cells in this population.

It remains to be determined whether ${gamma}$ HV68 is able to drive differentiationof latently infected naive CD40^–/– B cells. Furtheranalysis of ${gamma}$ HV68 infection of CD40^–/– mice, coupledwith an effective approach to stem the contribution to long-termlatency from acute lytic virus in the lungs, may offer a moredefinitive picture of the requirement for memory B cells inthe maintenance of long-term latency. Experiments are underway to generate replication-defective viruses to address thisissue.

ACKNOWLEDGMENTS

S. H. Speck was supported by NIH grants CA43143, CA52004, CA58524,and CA87650. David O. Willer is a Research Fellow of the NationalCancer Institute of Canada supported by funds provided by theTerry Fox Run.

We thank Robert E. Karaffa II and Mike Hulsey for cell sortingand analysis and members of the Speck laboratory for helpfulcomments and discussions.

FOOTNOTES

* Corresponding author. Mailing address: Center for Emerging Infectious Diseases, Yerkes National Primate Research Center, 954 Gatewood Rd. N.E., Atlanta, GA 30329. Phone: (404) 727-7665. Fax: (404) 727-7768. E-mail: sspeck{at}rmy.emory.edu.

REFERENCES

Top