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The Journal of Immunology, 2007, 178: 1692-1701.
Copyright © 2007 by The American Association of Immunologists, Inc.

Perturbation of B Cell Activation in SLAM-Associated Protein-Deficient Mice Is Associated with Changes in Gammaherpesvirus Latency Reservoirs1

In-Jeong Kim*, Claire E. Burkum*, Tres Cookenham*, Pamela L. Schwartzberg{dagger}, David L. Woodland* and Marcia A. Blackman2,*

* Trudeau Institute, Saranac Lake, NY 12983; and {dagger} Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of {gamma}HV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD+"naive" B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The oncogenic human gammaherpesviruses, EBV, and Kaposi’s sarcoma-associated herpesvirus (KSHV)3 are widely disseminated in the population. The initial infection is usually asymptomatic and is followed by the establishment of lifelong latency. Malignancies are largely associated with latent infection, so it is important to understand mechanisms involved in the establishment and long-term maintenance of viral latency (1). The murine gammaherpesvirus, gammaherpesvirus 68 ({gamma}HV68) or MHV68, is emerging as an important in vivo experimental model for elucidating virus/host interactions involved in the establishment and immune control of latency during a natural infection (2, 3).

A major reservoir of both EBV and {gamma}HV68 latency is the B cell (4, 5, 6, 7, 8, 9). Our recent studies have shown that {gamma}HV68 latency is established in lung B cells as early as 24 h after intranasal inoculation, concurrent with the lytic phase of infection (10). Whereas lytic virus is effectively cleared from the lung by virus-specific CD8 T cells by 12 days postinfection (dpi), latently infected cells escape immune elimination and are disseminated to the spleen and elsewhere (11). Latency peaks in the spleen at 14–15 dpi, corresponding with the peak of polyclonal B cell activation and germinal center formation. There is a subsequent rapid decline in numbers of latently infected spleen cells corresponding with the presence of CD8 T cells specific for a latency epitope, followed by stabilization at low, but persistent lifelong levels (12, 13).

Accumulating data support the hypothesis that EBV and {gamma}HV68 exploit the life cycle of the B cell to establish and maintain latency (4, 5, 6, 7, 8, 9, 14). We have shown that {gamma}HV68 preferentially establishes latency in activated germinal center B cells (15). This strategy provides access to the long-lived memory B cell compartment, resulting in a stable reservoir of lifelong latency. As has been described for EBV, memory B cells are a preferential reservoir of long-term {gamma}HV68 latency (6, 8, 16). Genetically manipulated mouse strains with defects in B cell activation and memory B cell development thus provide valuable models for studying {gamma}HV68 latency (7, 17).

In the current studies, we have taken advantage of SAP-deficient mice to further characterize the relationship between the life cycle of the B cell and the establishment and maintenance of {gamma}HV68 latency. SAP is an adaptor protein that binds to SLAM family proteins. The gene encoding SAP is altered in patients with X-linked lymphoproliferative (XLP) disease. Key manifestations of X-linked lymphoproliferative are hypogammaglobulinemia and an inability to control EBV, as well as other infections (18, 19). Thus, there has been much emphasis on the molecular characterization of the SAP defect. SAP-deficient mice have been generated independently by three groups (20, 21, 22). It has been shown that SAP-mediated signaling is involved in several stages of immune cell activation and effector function (reviewed in Ref. 23). SAP-deficient mice have defective Th2 cell development and abnormal Ig class-switching (20, 21, 24, 25, 26, 27). In addition, they generate reduced numbers of germinal centers and memory B cells (20, 21, 27, 28, 29), which may be due both to impaired CD4 T cell function as well as intrinsic B cell defects (27, 28, 29, 30). SAP-deficient mice have also been reported to develop enhanced Th1 responses characterized by excessive IFN-{gamma} production, and exaggerated CTL activity (20, 21, 29, 31, 32). As a consequence of the complex effects on signaling, SAP-deficient mice have been shown to have perturbations in both cellular and humoral immunity in response to a variety of infections (20, 21, 27, 29, 33).

The current studies focused on the impact of SAP-associated defects in T and B cell function and development on the establishment and maintenance of gammaherpesvirus latency. The data show both quantitative and qualitative perturbation in gammaherpesvirus latency reservoirs in {gamma}HV68-infected SAP-deficient mice that are associated with effects of the SAP mutation on B cell rather than T cell function. First, there is no defect in the ability of the virus to establish latency in memory B cells, although the reduced numbers of memory B cells in SAP-deficient mice translate into a reduced latent load. Second, two additional reservoirs of latency in wild type mice, germinal center and IgD+"naive" B cells, are absent in SAP-deficient mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal procedures and virus infection

{gamma}HV68, clone WUMS (Washington University School of Medicine) (34), was propagated and titered on NIH-3T3 fibroblasts (ATCC CRL1568). C57BL/6J mice were purchased from The Jackson Laboratory. SAP-deficient mice were generated as previously described (21), and mice backcrossed six generations to C57BL/6 mice were bred at Trudeau Institute. Mice were anesthetized with 2,2,2-tribromoethanol and intranasally infected with 30 µl of HBSS containing 400 PFU of {gamma}HV68. All infected mice were housed under specific pathogen-free conditions in ABSL3 annual biosafety level 3 containment. The Institutional Animal Care and Use Committee at the Trudeau Institute approved all studies described.

Viral assays

Plaque assay. Preformed, infectious virus was tittered by a standard plaque assay on NIH-3T3 fibroblasts, as described previously (35, 36).

Infectious center assay. To determine the number of latently infected cells capable of spontaneous in vitro reactivation, a modification of the infective center assay was used, as described previously (35, 36). Duplicates of spleen cell samples at 15 dpi were plated in triplicate onto monolayers of NIH-3T3 cells in serial 10-fold dilutions in 12-well plates. One replica was left intact to determine total infected cells, and one was disrupted by freeze-thawing to determine lytically infected cells (virus can only reactivate from live cells). The monolayers were overlaid and the plaques quantitated as for the plaque assay (35, 36). The number of latently infected cells was calculated as the difference between total and lytically infected cells.

Limiting dilution-nested PCR (LDA/PCR). The frequency of lung or spleen cells containing the {gamma}HV68 genome was determined by a combination of LDA and PCR, using primers specific for {gamma}HV68 ORF50, as described previously (17, 37). Twelve replicates were assessed for viral genome on each cell dilution. Linear regression analysis was performed to determine the frequency (95% degree of confidence) of cells positive for the {gamma}HV68 genome. As controls of nested PCR, multiple wells of 104 NIH-3T3 cells with and without plasmid DNA containing the {gamma}HV68 ORF50 gene were included in each 96-well plate.

B cell staining and purification

Cells from lung and spleens were depleted of erythrocytes with buffered ammonium chloride. For mice >12 mo of age, whole lungs and spleens were incubated with collagenase D (5 mg/ml) for 45 min to generate single-cell suspensions. To isolate B cells, cells were incubated with a mixture of biotin-conjugated Abs against CD4, CD8, CD11b, CD11c, Gr1, DX5, and TER119 and subsequently incubated with streptavidin-conjugated microbeads (Miltenyi Biotec). The cells were added to magnetic columns and flow-through was collected as negatively enriched B cells. Cells were incubated with peanut lectin (agglutinin) (PNA)-FITC, CD95-PE, CD19-PE/Cy5, and CD38-biotin, followed by streptavidin-allophycocyanin. Rat-anti-mouse IgG was used as control. Activated B cells, including germinal center B cells, were separated by FACS as PNAhigh/CD19+ cells on day 15 after infection. For later time points, B cells were further stained with IgD (11-26c.2a)-FITC, CD38-PE, biotin-conjugated sIgG, including IgG1 (A85-1), IgG2ab (R2-40), and IgG3 (R40-82), followed by streptavidin-allophycocyanin. Memory B cells were sorted as IgD–/sIgG+/CD38high using a FACS Vantage SE/Diva sorter (BD Biosciences). Staining was done in the presence of Fc block, and the sort was conducted using a doublet discriminator to reduce the possibility that nonspecific cells were being carried along in the sort. In addition to PNA-FITC and CD19-PE/Cy5, PE-conjugated Abs against CD25, CD40, CD69, CD80, CD86, or CD95 (Fas) were used to further characterize B cell activation status. Abs were obtained from BD Pharmingen and eBioscience. Flow cytometry data were analyzed using FlowJo software (Tree Star).

Assays for virus-specific T cells

In vivo CTL assay. Targets were prepared by loading spleen cells from naive C57BL/6J mice with peptides specific for influenza virus nucleoprotein (NP)366–374, {gamma}HV68 ORF61524–531, and {gamma}HV68 ORF6487–495 and labeling with 2, 1, or 0.5 nM CFSE (Molecular Probes), respectively. Targets were mixed and 2 x 107 cells were injected i.v. into individual wild-type (SAP+/+) and SAP-deficient (SAP–/–) mice 10 or 14 dpi. Four hours later, spleen cells were analyzed for intensity of CFSE staining. Percent-specific killing was calculated as previously described (31), according to the following formula: percent-specific killing = (number of Flu NP-pulsed targets x A – number of ORF6- or ORF61-pulsed targets/number of Flu NP-pulsed targets x A) x 100, where A = (number of ORF6- or ORF61-pulsed targets/number of Flu NP-pulsed targets) in uninfected recipient mice.

T cell staining. Virus-specific CD8 T cells were analyzed at 14 dpi using allophycocyanin-conjugated tetrameric reagents obtained from the Trudeau Institute Molecular Core Facility (ORF6487–495/Db or ORF61524–531/Kb) in association with CD8-PerCp, as described previously (17).

ELISPOT assay for IFN-{gamma}. IFN-{gamma} secretion by CD4 and CD8 T cells was assessed in response to APCs pulsed with purified virus or virus-specific peptides (ORF6, ORF61, or gp15057–83), as described previously (17, 38, 39).

Statistical analyses

Student’s t test was used to determine significant differences in the percentage of immune cell populations, CTL activity, and IFN-{gamma} production, and the Mann-Whitney U test was used for statistical analysis of frequencies of virus genome-positive cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Peak levels of {gamma}HV68 latency in B cells are reduced in SAP-deficient compared with wild-type mice

The key focus of these studies was to determine the impact of the SAP mutation on viral latency in B cells following infection with {gamma}HV68. Viral latency is first established in lung B cells as early as 24 h after the initial intranasal infection. Latently infected cells can subsequently be detected in the spleen several days after infection, attain peak levels at 14–15 dpi, and then rapidly decline over the next several weeks. Therefore, latency was assessed following {gamma}HV68 infection of SAP-deficient and wild-type (C57BL/6) mice in lung B cells at 5 dpi, and in splenic B cells at 10 and 15 dpi, using LDA/PCR to identify latently infected cells carrying viral genome. Samples from 15 dpi were also assayed for latency by an in vitro reactivation assay. The LDA/PCR showed that the frequency of latently infected cells in the lung and spleen was entirely comparable between wild-type and SAP-deficient mice at 5 and 10 dpi (Fig. 1, A and B). As the total numbers of B cells in the lung and spleen of SAP-deficient and wild-type mice was similar at these time points (data not shown), there were comparable numbers of latently infected B cells in SAP-deficient and wild-type mice. However, there was a striking decrease in the frequency of latently infected splenic B cells in SAP-deficient compared with wild-type mice at 15 dpi, the normal peak of splenic latency (Fig. 1C). Analysis of several independent experiments showed the reduced frequency of latency among B cells at this time point to be consistent, averaging 24-fold (Table I). As the total numbers of B cells in wild-type and SAP-deficient mice were also comparable at this time point, the reduction in latency frequency translated into a reduced latent load (Table I). This reduced latency in SAP-deficient compared with wild-type mice at 15 dpi as assessed by frequency of cells harboring viral genome was also reflected in latency as assessed by the in vitro reactivation assay (Fig. 1D).


Figure 1
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  		FIGURE 1. Impact of the SAP deficiency on early establishment of viral latency. CD19+ lung or splenic B cells were enriched to a purity of >97% and analyzed for viral latency at 5 dpi (lung, A), 10 dpi (spleen, B), and 15 dpi (spleen, C and D). The data in A–C show the mean ± SD of LDA/PCR analysis (percentage of wells positive for viral DNA by PCR plotted against numbers of cells analyzed) of four individual wild-type mice (SAP+/+, open symbols) and SAP-deficient mice (SAP–/–, closed symbols). From these data, the reciprocal frequency of CD19+ B cells harboring viral genome in wild-type and SAP-deficient mice, respectively, was determined: lung at 5 dpi was 379 ± 264 and 323 ± 124; spleen at 10 dpi was 1016 ± 634 and 471 ± 155; and spleen at 15 dpi was 288 ± 163 and 6882 ± 4051. The data in D show analysis of latently infected cells using the in vitro reactivation assay. Latent virus can only reactivate from live cells, and cells harboring latent virus were determined by subtracting the cells harboring preformed lytic virus (freeze-thaw samples) from the cells harboring total virus (duplicate untreated samples). Statistical analysis revealed a significant reduction in numbers of latently infected cells in SAP-deficient mice, determined both by LDA/PCR analysis shown in C (Mann-Whitney U test) and the viral reactivation assay shown in D (Student’s t test).

 

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  		Table I. Effect of the SAP mutation on the establishment of {gamma}HV68 latency in B cellsa

 
Peak levels of latency at 15 dpi declined rapidly in wild-type mice, as expected, with little decline in SAP-deficient mice, such that by 27–42 dpi the mean frequencies of latency among total B cells in wild-type and SAP-deficient mice varied only 3-fold (Table I). Thus, there was a dramatic effect of the SAP mutation on B cell latency at 14–15 dpi, the normal peak of latency in the spleen, but the differences compared with wild-type mice were minimal at earlier and later time points.

Lack of an effect of the SAP mutation on numbers and function of virus-specific CD8 T cells

The SAP mutation is associated with both T and B cell defects (40). The normal peak of splenic {gamma}HV68 latency in C57BL6 mice at days 14–15 dpi correlates both with the maximal B cell germinal center response and also the maximal virus-specific CD8 T cell response in the spleen (15, 41, 42). Thus, it is possible that the reduced latent load at 14–15 dpi in SAP-deficient mice is a consequence of hyperactive effector T cell activity resulting in enhanced elimination of virally infected target cells, or is a consequence of reductions in the optimal germinal center reservoir for the establishment of latency. It was recently suggested that reduced latency in SAP-deficient mice correlated with increased numbers of virus-specific CD8 T cells and hyperactive CTL activity (31). To independently assess {gamma}HV68 T cell function in SAP-deficient mice, we examined virus-specific CTL activity and IFN-{gamma} secretion and also assessed numbers of total and virus-specific T cells.

First, we measured CTL activity against two well-characterized immunodominant lytic epitopes, ORF6 and ORF61, in an in vivo CTL assay at both 10 and 14 dpi. Fig. 2A shows representative data from individual wild-type and SAP-deficient mice, and Fig. 2B shows mean values obtained from the analysis of multiple individual mice in two separate assays. The data show comparable CTL activity in wild-type and SAP-deficient mice at both time points, arguing against a role for enhanced CTL activity at 14–15 dpi in mediating reduced viral latency.


Figure 2
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  		FIGURE 2. Cytotoxicity against viral epitopes was comparable in wild-type and SAP-deficient mice at 10 and 14 dpi. Cytotoxicity was assessed by an in vivo CTL assay. Splenic B cells from naive C57BL/6 mice were loaded with peptides specific for Flu NP, {gamma}HV68 ORF61, and {gamma}HV68 ORF 6 and respectively labeled with 2, 1, or 0.5 nM CFSE before being injected into uninfected C57BL/6 (naive control), wild-type (SAP+/+), or SAP-deficient (SAP–/–) mice 10 or 14 dpi with {gamma}HV68. Killing was calculated 4 h later, as described in Materials and Methods. A, Representative data from analysis of individual mice. B, Compiled data (mean ± SD) from analysis of a minimum of three individual mice in each of two independent experiments. Statistical analysis (Student’s t test) revealed no differences in killing in wild-type and SAP-deficient mice.

 
Second, we assessed numbers of virus-specific cells, using tetrameric reagents for two lytic epitopes, ORF6 and ORF61. The data in Fig. 3 show no statistically significant differences in numbers of tetramer-positive CD8 T cells in SAP-deficient compared with wild-type mice at 14 dpi.


Figure 3
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  		FIGURE 3. Comparable numbers of virus-specific CD8 T cells are generated following {gamma}HV68 infection of wild-type and SAP-deficient mice. Spleen cells were analyzed by tetramer staining at 14 dpi for either for allophycocyanin-conjugated ORF6487–495/Db or ORF61524–531/Kb and CD8-PerCP. Representative flow cytometry analysis is shown in A. The numbers in the gated area indicate the percent of virus-specific T cells among the CD8 T cells. The mean percentage of virus-specific T cells ± SD of five mice per group is shown in B. The mean absolute numbers of virus-specific T cells ± SD of five mice per group is shown in C. No statistical difference was detected between wild-type and SAP-deficient mice (Student’s t test).

 
Finally, we assessed IFN-{gamma} secretion of virus-specific CD4 and CD8 T cells at three time points after infection. Analysis of numbers of IFN-{gamma}-secreting CD4 cells following in vitro restimulation with whole virus (Fig. 4A) or an I-Ab epitope, gp15057–83 (Fig. 4B) (38, 43), and CD8 cells following in vitro restimulation with ORF6 (Fig. 4C) and ORF61 (Fig. 4D) epitopes, showed no statistically significant differences in the response of SAP-deficient compared with wild-type mice at any time point measured.


Figure 4
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  		FIGURE 4. Numbers of virus-specific IFN-{gamma}-secreting T cells in the spleen are comparable in wild-type and SAP-deficient mice. Virus-specific IFN-{gamma}-secreting effector T cells in the spleen were quantitated by ELISPOT assay at the indicated dpi. CD4 or CD8 T cells were enriched and cocultured with APC pulsed with virus or peptides for 48 h. The number of CD4 T cells per spleen producing IFN-{gamma} in response to whole virus (A) or gp15057–88/I-Ab (B) and CD8 T cells per spleen producing IFN-{gamma} in response to ORF6487–495/Db (C) or ORF61524–531/Kb (D) are shown. The percentage of CD4 or CD8 splenic T cells is shown in E and F, respectively. Each bar graph indicates the mean of three individual mice ± SD. Asterisks indicate significant differences between groups assessed by the Student t test.

 
Despite the lack of an effect on the virus-specific T cell response, there were statistically significant differences in numbers of total CD4 and CD8 T cells following {gamma}HV68 infection of SAP-deficient and wild-type mice. The analysis revealed a generalized reduction in percentage of total CD4 T cells (Fig. 4E) and an increase in percent of total CD8 T cells (Fig. 4F).

{gamma}HV68-induced polyclonal activation fails to bypass SAP-associated defects in germinal center formation and class-switched Ab production

Next, we determined whether reduced latency in SAP-deficient mice correlated with SAP-associated B cell defects. B cell activation and function have been shown to be impaired in SAP-deficient mice, manifest by reduced numbers of germinal centers and failure to generate class-switched Abs and memory B cells following infection with a variety of pathogens (20, 21, 22, 28, 29, 33). There is evidence to suggest that both inherent B cell defects and defects in CD4 T cells contribute to the impaired B cell function (27, 28, 29, 30). Infection with {gamma}HV68 induces a striking splenomegaly, due to concordant increases in splenic B and T cells (44), and a strong CD4-dependent polyclonal B cell response (45). Thus, it was important to determine whether viral infection was able to bypass the characteristic SAP-associated B cell defects. To assess the impact of the SAP mutation on B cell activation and function following {gamma}HV68 infection, we examined B cell numbers, activation phenotype, and development of germinal center cells in SAP-deficient compared with wild-type mice following {gamma}HV68 infection.

First, we examined the effect of the SAP mutation on the splenomegaly and polyclonal B cell activation associated with {gamma}HV68 infection. Comparative analysis of numbers of splenocytes in naive and infected SAP-deficient and wild-type mice (Fig. 5A) and the distribution of CD4 T cells, CD8 T cells, and B cells within the spleen at 15 dpi (Fig. 5B) showed no effect of the SAP mutation on the characteristic splenomegaly associated with the establishment of splenic latency. There was, however, a decrease in generalized activation of CD4 cells and an increase in generalized activation of CD8 T cells assessed by CD25 expression (Fig. 5C). In addition, analysis of B cell phenotype at 15 dpi (Fig. 6) showed activation among PNAhigh (activated) B cells to be entirely comparable in {gamma}HV68-infected SAP-deficient and wild-type mice, in that expression of cell surface molecules associated with B cell activation, including CD40, CD80, CD86, and the MHC class II molecule, I-Ab, was up-regulated to the same extent in SAP+/+ and SAP–/– B cells. Up-regulation of early activation markers CD25 and CD69 in SAP+/+ and SAP–/– B cells was also comparable.


Figure 5
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  		FIGURE 5. Splenomegaly associated with {gamma}HV68 infection is unaffected by the SAP mutation. The data show numbers of spleen cells from naive and infected wild-type (SAP+/+) and SAP-deficient (SAP–/–) mice at 15 dpi (A), percentage of CD4, CD8, and B cells isolated from the spleens of wild-type (SAP+/+) and SAP deficient (SAP–/–) at 15 dpi (B), and percentage of CD4 and CD8 T cells from wild-type (SAP+/+) and SAP-deficient (SAP–/–) expressing CD25 at 15 dpi (C). Data are the mean (±SD) of four to five infected mice analyzed at each time point. Statistical significance was determined by the Student t test.

 

Figure 6
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  		FIGURE 6. B cell activation after {gamma}HV68 infection of wild-type and SAP-deficient mice. B cells were analyzed from wild-type and SAP-deficient mice at 15 dpi. Representative FACS profiles for a series of phenotypic markers within gated PNAhigh and PNAlow B cells from wild-type (SAP+/+, dotted line) and SAP-deficient (SAP–/–, solid line) mice are shown.

 
Next, we examined the ability of SAP-deficient mice to generate germinal center B cells following {gamma}HV68 infection. Germinal center B cells characteristically bind high levels of PNA, down-regulate CD38 expression, and up-regulate CD95 (Fas) expression (46, 47). Phenotypic analysis at 15 dpi show that the percentage of PNAhigh B cells was generally reduced, but not statistically significantly different in SAP-deficient and wild-type mice (Fig. 7). However, there was a 10-fold reduction in the subset of PNAhigh B cells that expressed a germinal center phenotype (CD95 (Fas)+ and CD38low) in SAP-deficient mice (Fig. 7B).


Figure 7
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  		FIGURE 7. SAP-deficient mice fail to generate germinal center B cells following {gamma}HV68 infection. Spleen cells from wild-type (SAP+/+) and SAP-deficient (SAP–/–) mice were assessed for their ability to bind PNA and stained with Abs against CD19, CD38, and CD95 on 15 dpi. Left panels show representative FACS data, and the right panels show bar graphs depicting the mean of four individual mice (±SD) in each group. A, CD19+ B cells were analyzed for their activation status assessed by up-regulation of PNA binding. The percentage of PNAhigh B cells from wild-type and SAP-deficient mice is directly compared in the right panel. B, CD38 and CD95 expression by cells in the PNAhigh B cell gate was quantitated by FACS analysis. The percentage of CD38lowCD95high B cells within the PNAhigh gate from wild-type and SAP-deficient mice is directly compared in the right panel. The difference in the percentage of CD38lowCD95high cells among PNAhigh B cells in SAP-deficient and wild-type mice was statistically significant, as determined by Student’s t test.

 
Thus, polyclonal B cell activation associated with {gamma}HV68 infection was intact in SAP-deficient mice, as indicated by generalized expansion of B cell numbers and by B cell activation phenotype within PNAhigh B cells. However, viral infection failed to bypass the well-characterized defect in the ability of SAP-deficient mice to develop germinal center cells. In addition, we confirmed the previous observation of others (22, 28) that Ig class switching fails to occur following {gamma}HV68 infection (data not shown).

The frequency of viral latency in activated PNAhigh B cells is comparable in SAP-deficient and wild-type mice despite reduced numbers of germinal center B cells

The dramatic reduction in peak levels of latently infected B cells in SAP-deficient mice (Table I and Fig. 1, C and D), which failed to correlate with significant enhancement of virus-specific T cell numbers or effector function (Figs. 2–4) raised the possibility that SAP-associated effects on B cell activation caused a deficiency in the normal reservoir for the establishment of latency. We have previously shown that B cell latency is established preferentially in activated germinal center B cells (15). However, full maturation to germinal center cells is apparently not required, as we have also previously reported that latency can be efficiently established in PNAhigh activated B cells from CD28-deficient mice, which, like SAP-deficient mice, have a deficiency in germinal center development (17). To determine whether the activated B cells in SAP-deficient mice provided an adequate reservoir for the establishment of latency, we analyzed latency frequencies in highly purified populations of PNAhigh B cells from SAP-deficient and wild-type mice at 14–15 dpi. In contrast to the ~24-fold reduction in latency frequency among total CD19+ B cells from SAP-deficient compared with wild-type mice, the latency frequencies were comparable (within 2-fold) among the subset of highly activated, PNAhigh B cells analyzed at the same time point (Table I). Thus, latency was established efficiently within the subset of activated cells, with little effect of the SAP mutation on frequencies of peak levels of latency and only a modest (~6-fold) reduction in numbers of latently infected cells in SAP-deficient compared with wild-type mice (Table I), which is consistent with the general reduction in PNAhigh cells in SAP-deficient mice (Fig. 7A).

Quantitative impact of the SAP mutation on long-term latency in memory B cells

We next addressed the impact of the SAP mutation on cellular reservoirs of long-term latency. Because of the paucity of germinal center and memory B cells in SAP-deficient mice (29, 32, 48), both of which are preferential reservoirs of long-term latency (6), we anticipated that maintenance of long-term latency would be defective, as we had previously shown for CD40–/– B cells within CD40+/CD40– chimeric mice (7). However, unexpectedly, the frequencies of latency in CD19+ B cells from SAP-deficient and wild-type mice between 90 and 210 dpi were remarkably comparable, with the SAP-deficient mice showing only modest reductions ranging between 1.4- and 4-fold (circles in Fig. 8). To identify SAP-associated effects on preferential long term reservoirs of latency, we measured latency frequencies in class-switched splenic B cells (containing both memory and germinal center subsets) from mice between 90 and 210 dpi (squares in Fig. 8). The results showed comparable frequencies of latency in class-switched B cells from both SAP-deficient and wild type mice that varied less than 2-fold. In addition, in both strains of mice, the latency frequency in class-switched B cells was ~100-fold higher than in total (CD19+) B cells, confirming the class-switched B cells as a preferential latency reservoir.


Figure 8
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  		FIGURE 8. Frequency of latency among splenic B cells at various times postinfection. CD19+ B cells (circles) and class-switched sIgG+ B cells (squares) sorted from wild-type (SAP+/+, open symbols) and SAP-deficient (SAP–/–, closed symbols) mice were analyzed for the frequency of virus genome-positive cells at various dpi using LDA/PCR. Each symbol represents analysis of a pool of four to eight mice. The data for 10 and 15 dpi are the same as shown in Fig. 1. The difference in latency between the wild-type and SAP-deficient mice at day 15 was statistically significant, as determined by the Mann-Whitney U test (p = 0.0286).

 
Absolute numbers of CD19+ splenic B cells in SAP-deficient mice were variable, largely due to variation in spleen size and cellularity in SAP-deficient mice (Table II). Initially, it appeared that numbers in SAP deficient were progressively declining with the age of the mouse and/or time after infection. However, when more care was taken to dissociate spleen cells with collagenase D to maximize recovery, the large differences in cell numbers between SAP-deficient and wild-type mice noted at 150 and 210 dpi were reduced, and at 420 and 540 dpi, absolute numbers of B cells between SAP-deficient and wild-type mice were within 2-fold. Despite relatively comparable numbers of CD19+ B cells and consistent with the previously characterized deficit in memory B cells in SAP-deficient mice, the reduction in memory B cell numbers in SAP-deficient compared with wild-type mice at various times following {gamma}HV68 infection ranged between ~10- and 35-fold. In summary, the frequencies of latency from 90 to 210 dpi were remarkably comparable among both total B cells and class-switched B cells (Fig. 8), but the long-term latent load was reduced in SAP-deficient mice because of the reduction in numbers of memory B cells (Table II), a preferential reservoir of long-term latency.


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  		Table II. Number of cells in the spleen of {gamma}HV68-infected wild-type and SAP-deficient micea

 
The data in Fig. 8 show analysis of long-term latency in total CD19+ B cells and IgG class-switched B cells. Class-switched B cells consist of memory and germinal center cells, both of which are preferential long-term reservoirs of {gamma}HV68 latency (6). To more precisely define the reservoir of long-term latency in SAP-deficient mice, we further sorted splenic B cells from mice at 150 and 210 dpi into class-switched CD38high (memory phenotype), class-switched CD38low (germinal center phenotype), and IgD+ (mostly naive phenotype) subsets, as reported previously (6, 46, 47, 49) (Fig. 9). It can be seen that, although the frequency of class-switched B cells was 10-fold lower in SAP-deficient mice compared with wild-type mice (Fig. 9A), we were able to sort a highly enriched population of class-switched CD38 high (memory) B cells from SAP-deficient mice (Fig. 9B). The frequencies of latency among class-switched, CD38high (memory phenotype) B cells from SAP-deficient and wild-type mice were entirely comparable (1/355 compared with 1/399, and 1/1481 compared with 1/1002 for wild-type and SAP-deficient mice, respectively), at 5 and 7 mo postinfection (Table III). However, the absolute numbers of memory phenotype B cells were reduced 10- to 30-fold in SAP-deficient compared with wild-type mice (Table II). Thus, despite comparable frequencies, absolute numbers of latently infected memory B cells were severely impacted by the SAP deficiency (Table III).


Figure 9
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  		FIGURE 9. Flow cytometry analysis and sorting of spleen cells from wild-type and SAP-deficient mice 7 mo postinfection. Spleen cells from wild-type (SAP+/+) and SAP-deficient (SAP–/–) mice obtained 7 mo postinfection were enriched for B cells, and gated CD19+ B cells were sorted on the basis of CD38, IgD, and a mixture of class-switched Igs (IgG1+IgG2a/b+IgG3). A, Presort analysis of CD19+ B cells, IgD+sIgG–, and IgD–sIgG+ B cells. B, Postsort analysis of isotype switched (IgD–sIgG+) germinal center (CD38low) and memory (CD38high) B cells from wild-type mice and memory (CD38high) B cells from SAP-deficient mice.

 

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  		Table III. Maintenance of {gamma}HV68 latency in B cell subsetsa

 
Qualititative impact of the SAP mutation on long-term latency reservoirs

Another important reservoir of long-term latency is germinal center B cells (6). Unfortunately, we were unable to accurately assess latency in the germinal center cells at late times after infection because there were insufficient germinal center B cells (class-switched surface Ig+, CD38low) from SAP-deficient mice to sort (Fig. 7). Thus, due to paucity of germinal center cells, this reservoir of latency is deficient in SAP-deficient mice.

A third reservoir of long-term latency is IgD+ naive B cells (6). Although IgD+ B cells express a low frequency of latency, because they are the most abundant B cell subset in the spleen, they constitute an important reservoir of latently infected cells (6). Unexpectedly, although we were able to sort sufficient numbers of IgD+ B cells from both strains of mice, the frequency of latency in naive B cells was below the level of detection in SAP-deficient mice, whereas this subset made a substantial contribution to the latent reservoir in wild type mice (Table III). Thus, unexpectedly, this reservoir of long-term latency is also deficient in SAP-deficient mice.

Taken together, there are both quantitative and qualitative changes in long-term {gamma}HV68 latency reservoirs in SAP-deficient mice. Despite defects in germinal center and memory B cell formation in SAP-deficient mice, the virus is able to access the memory B cell reservoir and establish latency in memory B cells with comparable frequency to wild-type mice, although the reduced numbers of memory B cells in SAP-deficient mice translate into a significantly reduced long-term latent load. In addition, SAP-deficient mice lack two other important reservoirs of long-term latency, germinal center and IgD+ B cells, reflecting both a qualitative impact on latency reservoirs and also quantitative effects as a consequence of further reduction in the overall long-term latent load.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have kinetically analyzed the establishment and maintenance of {gamma}HV68 latency in B cell reservoirs of SAP-deficient and wild-type mice. We have defined both quantitative (reduced latent load) and qualitative (altered reservoirs of latency) effects of the SAP mutation on long-term latency. First, there are reduced numbers of latently infected memory B cells in SAP-deficient mice and, second, two substantial reservoirs of long-term latency in wild-type mice, germinal center and IgD+ naive B cells, are not detected in SAP-deficient mice. We have interpreted the data in light of our current understanding of the association between the virus and the B cell. It has been proposed that the virus exploits the life cycle of the B cell by establishing latency preferentially in highly activated germinal center B cells, allowing efficient entry into the pool of memory B cells (5, 14, 50, 51, 52). Because they are long-lived and are maintained homeostatically by cellular mechanisms (53), memory B cells potentially can provide a stable reservoir of latency without an absolute requirement for viral reactivation and re-infection.

Early latency frequencies in CD19+ B cells in the lung (5 dpi) and spleen (10 dpi) were remarkably unaffected by the SAP mutation. In striking contrast, at the peak of latency at 14–15 dpi, there was a greater than 20-fold decrease in the frequency of latently infected CD19+ B cells in SAP-deficient compared with wild-type mice (Fig. 1). Despite this, analysis of latency at the same time within the subset of activated PNAhigh B cells showed only a 2-fold decrease in latency frequency (Table I). This was not explained by the ability to bypass the SAP-associated defect in B cell activation as a consequence of virus-induced polyclonal B cell activation, as {gamma}HV68-infected SAP-deficient mice developed only 10% of the number of germinal center B cells induced in wild-type mice (Fig. 7), which is consistent with the previous histological (29) and phenotypic (32) analysis of lymphocytic choriomeningitis virus-infected SAP-deficient mice. However, despite the defect in germinal center cell development, B cells in SAP-deficient mice expanded (Fig. 5) and become activated (Fig. 6) sufficiently to provide an adequate reservoir for the establishment of latency. Although germinal center cells are the preferential reservoir for the establishment of latency in wild-type mice (15), they are apparently not an absolute requirement, and latency can be efficiently established in highly activated B cells. For example, we previously showed that latency was efficiently established in CD28-deficient mice, which are defective in the ability to form germinal centers (15, 17). In addition, in mixed bone marrow chimeric mice, latency was established comparably in CD40+ and CD40– B cells, despite the inability of CD40– B cells to localize into germinal centers (7). Thus, despite the reduced numbers of germinal center B cells in SAP-deficient mice, the establishment of latency in highly activated B cells was efficient and sufficient to allow viral access to and establishment of latency in the memory B cell pool (Table III). This reflects a quantitative, but not qualitative, impact of SAP on the establishment of latency in memory B cells.

Although hyperactive CTL activity has been proposed as an explanation for reduced latency in {gamma}HV68-infected SAP-deficient mice in an independent study (31), our data are more consistent with a SAP-mediated effect on B cells. First, we were unable to reproduce data from Chen et al. (31), showing elevated numbers, increased IFN-{gamma} secretion, and enhanced cytolytic activity of CD8 T cells specific for two lytic viral epitopes (Figs. 2–4). There was, however, an increased overall activation status of CD8+ T cells from SAP-deficient mice compared with wild-type mice (Fig. 5C). Although we cannot explain the discrepancy in the experimental results, it is difficult to assess precisely the biological significance of the statistical difference in quantitative CTL activity reported previously (31). It is not clear that the less than 2-fold differences in the levels of cytolysis of cells expressing a particular epitope have physiological relevance in terms of viral control and the establishment of latency. In addition, {gamma}HV68-specific CTL activity is complex in that we and others (41, 54, 55) have shown that peak levels of CD8 T cells specific for ORF6 and ORF61 vary differentially during the infection, corresponding to kinetic differences in levels of expression of the two lytic epitopes, and presentation of the epitopes by different cell types. Our observation that latency in total B cells at 5 and 10 dpi is unaffected by the SAP mutation, but is dramatically reduced in SAP-deficient mice at 15 dpi (Fig. 1), does not correlate with these previously described kinetic differences in epitope-specific CTL activity or viral epitope expression.

One puzzling aspect of the data is the selective deficiency in peak latency levels among total (CD19+) B cells in SAP-deficient mice, despite comparable levels among activated PNAhigh B cells at the same time point (Table I). This could be explained by either SAP-associated B cell or CD8 T cell defects and remains unresolved by our current studies. On the one hand, it is possible that a particular stage of B cell development strongly affected by the SAP mutation provides an important, as yet unidentified, early reservoir for latency. In contrast, it is possible that an early stage of latency is uniquely susceptible to enhanced CTL killing in SAP-deficient mice. As discussed above, our in vivo CTL data did not show evidence for generalized enhancement of CTL activity in the spleens of SAP-deficient mice (Fig. 2). However, it is possible that there is differential expression of virus-specific T cell epitopes on cells in early and peak stages of latency, leading to differential susceptibility to CTL control. This would be consistent with the proposal that there are distinct forms of latency in the spleen associated with differential viral gene expression at specific time points after infection (12, 13, 16, 56, 57). Unfortunately, this possibility cannot currently be experimentally addressed because lytic and latent T cell epitopes expressed by different subsets of B cells during the establishment of latency have not been defined.

Analysis of long-term latency in SAP-deficient mice gave unexpected results. Our initial hypothesis was that SAP-deficient mice would be compromised in their long-term maintenance of latency, because of the well-characterized SAP-associated defect in the development of memory B cells, a major reservoir of long-term latency (6, 8). Surprisingly, we found that long-term latency frequencies among class-switched B cells were remarkably comparable between wild-type and SAP-deficient mice as late as 210 dpi, although the numbers of latently infected cells was consistently 10- to 20-fold lower in SAP-deficient mice (Table III), due to reduced numbers of memory B cells (Table II). Thus, latency frequencies are maintained in memory B cells in SAP-deficient mice, indicating preservation of this important reservoir of latency, but there are quantitative effects due to reduced numbers of memory B cells.

Detailed analysis of long-term latency in other B cell subsets, however, revealed qualitative effects of the SAP mutation on latency reservoirs (Table III). First, the germinal center latency reservoir was deficient due to the paucity of germinal center cells in SAP-deficient mice. Second, despite adequate numbers of IgD+ B cells, latent virus was below the limits of detection in this population in SAP-deficient mice. Taken together with reduced numbers of latently infected memory B cells in SAP-deficient mice, these differences translate into a 25- to 50-fold reduction in total numbers of latently infected B cells in SAP-deficient mice between 5 and 7 mo postinfection. The significance of long-term latency in germinal center and IgD+ naive B cells is unknown, but the data are consistent with the possibility that viral reactivation and reinfection make an important contribution to maintenance of long-term latency.

The role of viral reactivation and reinfection in the maintenance of gammaherpesvirus latency is unknown. Whereas it is well-established that EBV reactivation occurs at low levels in the tonsil and is essential for viral transmission, the significance of this for maintenance of long-term viral latency is unclear (1). The role of reactivation and reinfection in the maintenance of {gamma}HV68 latency has only been studied in B cell-deficient mice and is poorly understood (58, 59). Recently, however, an essential role for lytic viral replication for the maintenance of KSHV latency was demonstrated (60). It is clear that this important issue merits further investigation.

The identification of germinal center and IgD+ naive B cells as additional long-term reservoirs of latency (6) support the possibility that activation of memory B cells, through cognate Ag or nonspecific mechanisms, leads to the generation of plasma cells via secondary germinal center reactions and consequent viral reactivation. In support of this, differentiation into plasma cells has been associated with reactivation of EBV (61, 62). We propose that viral reactivation results in de novo infection of a subset of IgD+ naive B cells, providing a mechanism for maintaining the long-term viral reservoir in wild-type mice. This possibility is supported by the observation that EBV infection of naive tonsillar B cells correlated with active viral replication in the tonsils (63). We hypothesize that memory B cell triggering fails to occur in SAP-deficient mice because of SAP-associated defects in B cell activation, resulting in the absence of virally infected naive B cells. The absence of viral genome in B cells with a naive phenotype in SAP-deficient mice was an unexpected result that may lead to important insight into mechanisms for the maintenance of long-term gammaherpesvirus latency. Further studies to test the significance of this reservoir for long-term latency maintenance in wild-type mice are in progress.

Although much can be learned from the experimental mouse model about gammaherpesvirus latency in general, it is important to bear in mind that each of the gammaherpesviruses has coevolved intimately and uniquely with its host. Sequence analysis of the EBV, KSHV, and {gamma}HV68 genomes reveals large colinear gene blocks, but the genes controlling latency and transformation lie outside of these regions of homology and are virus-specific (34). In many cases, however, the viruses exploit the same basic strategies via different mechanisms. For example, both {gamma}HV68 and EBV induce B cell activation, but they do it in different ways. EBV accomplishes this directly through the expression of several viral genes, including latent membrane protein 1, LMP2a, and EBV-encoded nuclear Ag 2 (64, 65, 66, 67, 68), whereas {gamma}HV68-driven B cell activation appears to rely on normal Ag and CD4 T cell signaling mechanisms (7, 45, 69, 70). However, despite using a different mechanism for B cell activation, {gamma}HV68 latency fits the original model proposed for EBV (14) that the virus exploits the life cycle of the B cell, in that latency is preferentially established in highly activated, germinal center B cells, allowing the virus to gain access to the long-lived memory B cell compartment.

In conclusion, despite quantitative effects of the SAP mutation on memory B cells, the virus was able to access the population of activated B cells and effectively establish and maintain latency in the memory B cell pool in SAP-deficient mice. Two additional key findings of the current study—that SAP-deficient mice are deficient in two understudied reservoirs of long-term virus latency, germinal center, and naive IgD+ B cells—support the possibility that latently infected memory B cells can be induced to reactivate virus, resulting in reinfection of naive B cells. The current analysis of {gamma}HV68 latency in SAP-deficient mice thus makes fundamental contributions to our understanding of mechanisms involved in maintaining long-term gammaherpesvirus latency.


   Acknowledgments
 
We thank Drs. Fran Lund, Troy Randall, and Larry Johnson for helpful discussions and Simon Monard and Branden Sells for FACS sorting.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI51602 and AI42927 (to M.A.B.), intramural funds from National Human Genome Research Institute, National Institutes of Health Grant (to P.L.S.), and by the Trudeau Institute. Back

2 Address correspondence and reprint requests to Dr. Marcia A. Blackman, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: mblackman{at}trudeauinstitute.org Back

3 Abbreviations used in this paper: KSHV, Kaposi’s sarcoma-associated herpesvirus; dpi, days postinfection; {gamma}HV68, gammaherpesvirus 68; LDA/PCR, limiting dilution-nested PCR assay; PNA, peanut lectin (agglutinin); SAP, SLAM-associated protein; SLAM, signaling lymphocyte activation molecule; NP, nucleoprotein. Back

Received for publication July 21, 2006. Accepted for publication November 15, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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