Cells CD40-Mediated Development of Memory B Cell Latency Is ...

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Maintenance of Long Term γ-Herpesvirus B Cell Latency Is Dependent on CD40-Mediated Development of Memory B Cells In-Jeong Kim, Emilio Flaño, David L. Woodland, Frances E. Lund, Troy D. Randall and Marcia A. Blackman J Immunol 2003; 171:886-892; ; doi: 10.4049/jimmunol.171.2.886 http://www.jimmunol.org/content/171/2/886

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The Journal of Immunology

Maintenance of Long Term ␥-Herpesvirus B Cell Latency Is Dependent on CD40-Mediated Development of Memory B Cells1 In-Jeong Kim, Emilio Flan˜o, David L. Woodland, Frances E. Lund, Troy D. Randall, and Marcia A. Blackman2

he human ␥-herpesviruses, EBV and Kaposi’s sarcomaassociated herpesvirus (KSHV),3 establish lifelong latency and have been associated with the onset of malignancies such as Burkitt’s lymphoma, Hodgkin’s disease, Kaposi’s sarcoma, and primary effusion lymphoma. Thus, it is important to understand mechanisms by which the viruses maintain latency reservoirs in immunocompetent hosts. EBV latency is maintained predominantly in B cells, whereas KSHV latency is more promiscuous, in that the virus is harbored long term in B cells, macrophages, and dendritic cells (1–3). Although little is known about KSHV latency, EBV latency in B cells has been well described. EBV avoids immunosurveillance by a progressive shutdown of viral genes nonessential for the maintenance of the episomal viral DNA during cellular division (4). EBV latency is harbored predominantly in resting circulating memory B cells (5, 6). Based on this observation, it has been suggested that the virus gains entry into the memory B cell pool and maintains latency by exploiting host mechanisms for maintaining memory B cells, avoiding a requirement for viral reactivation and reinfection (6 –9). We have directly tested this hypothesis, using an experimental mouse model. The murine ␥-herpesvirus-68 (MHV-68) is structurally and biologically related to the human ␥-herpesviruses and provides a valuable in vivo experimental model for a ␥-herpesvirus in its natural host (10 –13). Intranasal infection with MHV-68 causes an acute respiratory infection that is controlled predominantly by virus-specific CD8⫹ T cells. Although the lytic virus is cleared, the virus establishes latency and persists in a quiescent state. Whereas

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B cells, macrophages, dendritic cells, and possibly lung epithelial cells have been shown to harbor latent virus in immunocompetent mice (14 –17), B cells are central to the establishment of the latent state (18, 19). Analogous to EBV, the major reservoirs of long term B cell latency are in the germinal center and memory B cells (20). To experimentally test the hypothesis that maintenance of ␥-herpesvirus latency is dependent on viral access to the long-lived memory B cell pool, we took advantage of the well-established requirement for CD40 in the differentiation and survival of memory B cells. CD40 is expressed on a variety of APC, including B cells. CD40-CD40 ligand (CD40L) interactions between T and B cells play important roles at several stages of T cell-dependent B cell activation, differentiation, and survival (21–25). Importantly, CD40 delivers essential survival signals to germinal center B cells, which allows them to become long-lived memory B cells (24, 26, 27). To test the requirement for the development of memory B cells in the maintenance of ␥-herpesvirus latency, we generated mixed bone marrow chimeras with distinct pools of CD40⫹ and CD40⫺ B cells using wild-type C57BL/6 (CD40⫹/⫹) and CD40 knockout (CD40⫺/⫺) mice. The data showed that after infection, CD40⫹ B cells selectively entered germinal centers. Longterm latency was preferentially maintained in isotype-switched CD40⫹ germinal center/memory B cells and was progressively lost in the short-lived CD40⫺ B cells that failed to become memory B cells. These data experimentally confirm the hypothesis that maintenance of ␥-herpesvirus latency is dependent on the virus gaining access into the long-lived memory B cell pool.

Trudeau Institute, Saranac Lake, NY 12983

Materials and Methods

Received for publication March 4, 2003. Accepted for publication May 1, 2003.

Mice and viral infection

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 is supported by Grant AI42927 from the National Institutes of Health (to M.A.B.) and the Trudeau Institute. 2 Address correspondence and reprint requests to Dr. Marcia A. Blackman, Trudeau Institute, 100 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: [email protected] 3 Abbreviations used in this paper: KSHV, Kaposi’s sarcoma-associated herpesvirus; MHV-68, murine ␥-herpesvirus 68; ORF, open reading frame; PNA, peanut agglutinin; CD40L, CD40 ligand.

Copyright © 2003 by The American Association of Immunologists, Inc.

CD40⫺/⫺ (24) and wild-type C57BL/6 mice were bred at Trudeau Institute (Saranac Lake, NY), and Ly5.1 congenic C57BL/6 (B6.SJL-PtprcA Pep3b/ BoyJ) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MHV-68 (WUMS strain; Ref. 28) was obtained from H. W. Virgin (Washington University School of Medicine, St. Louis, MO) and propagated and titered on NIH 3T3 fibroblasts (ATCC CRL1568), as described (29). Mice were anesthetized with 2,2,2-tribromoethanol and intranasally infected with 400 PFU MHV-68. After infection, mice were housed under specific pathogen-free conditions in BL3 containment. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Trudeau Institute. 0022-1767/03/$02.00


It has been proposed that the ␥-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40ⴙ and CD40ⴚ B cells from CD40ⴙCD40ⴚ mixed bone marrow chimera mice after infection with a murine ␥-herpesvirus, MHV-68. CD40ⴙ B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40ⴚ B cells and preferentially maintained in the long-lived, isotype-switched CD40ⴙ B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency. The Journal of Immunology, 2003, 171: 886 – 892.

The Journal of Immunology Generation of CD40-mixed bone marrow chimeric mice To distinguish residual CD40⫹ host B cells from CD40⫹ donor B cells, we took advantage of congenic mice for the allotypic marker, Ly5. Thus, wildtype C57BL/6 mice (Ly5.2) were lethally irradiated (950 rad) in two doses of 475 rad in a 6-h interval and reconstituted with bone marrow cells from both CD40⫹/⫹ and CD40⫺/⫺ mice. Bone marrow cells were flushed from the femurs of Ly5.2 CD40⫺/⫺ and Ly5.1 CD40⫹/⫹ mice, depleted of RBC, and mixed in a ratio of 2:8. A total of 1–2 ⫻ 107 cells were injected i.v. into the lateral tail veins of irradiated recipient mice (Ly5.2). Mice were rested for 2–3 mo to allow reconstitution, which was confirmed by flow cytometric analysis of peripheral blood lymphocytes, before MHV-68 infection. Chimeric mice received sulfamethoxazole and trimethoprim (GensiaSicor Pharmaceuticals, Irvine, CA)-treated water throughout the study to prevent opportunistic infections.

Flow cytometry analysis and cell purification

In vitro viral reactivation assay In vitro viral reactivation assays to detect lytic and latent virus were performed as described (30). Briefly, serial dilutions of splenocytes or lung homogenates were plated onto monolayers of NIH 3T3 cells and overlaid with carboxymethylcellulose. After 6 days of culture, cells were fixed and stained with Giemsa. Because this assay measures lytic virus as well as reactivating latent virus, samples were simultaneously assayed after a single freeze/thaw cycle to assess the contribution of lytic virus to the titers. Previous controls have shown that a single freeze/thaw cycle results in ⬍20% loss of lytic virus (30, 31). Latent virus levels in individual mice were calculated by subtracting virus levels of the freeze/thaw sample from virus levels of the duplicate, untreated sample.

Results

CD40⫹CD40⫺ mixed bone marrow chimeric mice control MHV-68 infection comparably with wild-type mice To directly compare latency reservoirs in CD40⫹ and CD40⫺ B cells in an immunocompetent host, we generated CD40⫹CD40⫺mixed bone marrow chimeric mice. Lethally irradiated C57BL/6 (Ly5.2) mice were reconstituted with an 80:20 ratio of Ly5.1 wildtype (CD40⫹/⫹) and Ly5.2 knockout (CD40⫺/⫺) bone marrow cells. The skewed ratio was chosen to bias the mouse toward the wild-type while providing a readily detectable population of CD40⫺ B cells for analysis. In addition, the Ly5.1 and Ly5.2 differences provided a second marker with which to distinguish CD40⫹ and CD40⫺ B cells. From 2 to 3 mo after generating the chimeras and before MHV-68 infection, flow cytometric analysis of PBL (Fig. 1A) and splenic B cells (Fig. 1B) showed that the bone marrow chimeric mice were reconstituted with the expected 80:20 ratio of Ly5.1⫹CD40⫹ and Ly5.2⫹CD40⫺ B cells. The observation that CD40 was exclusively expressed on Ly5.1⫹CD19⫹ B cells confirmed that there were no residual host CD40⫹ B cells (Ly5.2). Analysis of reconstituted mice at several time points after MHV-68 infection showed that the 80:20 ratio of CD40⫹ to CD40⫺ B cells was consistently maintained throughout the analysis (Fig. 1B). Thus, MHV-68 infection did not skew the ratio of CD40⫹ and CD40⫺ B cells in the chimeric mice. To study maintenance of MHV-68 latency in the chimeras, it was essential to show that immune control of virus and the establishment of latency are comparable in chimeric and wild-type mice. First, the kinetics of viral clearance from the lungs of mixed bone marrow chimeric and wild-type mice were compared. Maximal viral load in the lungs and the kinetics of viral clearance in wild-type and mixed bone marrow chimeric mice were virtually

Limiting dilution PCR assay The frequency of cells carrying the MHV-68 genome was determined by a limiting dilution-nested PCR assay for the open reading frame (ORF50) gene, as described (19, 32, 33). Briefly, sort-purified cells were serially diluted in NIH 3T3 cells in 12-well replicas in 96-well plates, and lysed; then, without a DNA isolation step, the ORF50 gene was directly amplified by PCR using specific primers as published (19). A 2-␮l aliquot of the product was then reamplified using nested primers. The final PCR product was then electrophoresed on 3% agarose gels and stained with ethidium bromide. This assay can detect a single copy of viral genome in 105 cells (19). As controls, 1 ⫻ 104 NIH 3T3 cells per well, with and without plasmid DNA containing the MHV-68 ORF50 gene, were included in each 96-well PCR assay. The reciprocal frequency of cells carrying viral genome was determined by linear regression analysis with a 95% degree of confidence.

Immunofluorescent staining of spleen sections Spleen tissue was embedded in OCT compound (Sakura Finetek, Torrance, CA), snap-frozen in liquid nitrogen, and stored at ⫺70°C until used. Frozen tissues were cut in 6-␮m-thick sections with a microcryostat. Spleen sections were fixed in acetone, air-dried, and washed with PBS. After blocking with Fc block, followed by an avidin/biotin blocking agent (Vector Laboratories, Burlingame, CA), serial sections were incubated with biotinylated Abs against Ly5.1 or Ly5.2, and stained with streptavidin conjugated with Alexa fluor 594 (red). Next, the sections were incubated with biotinylated anti-B220/CD45RB (RA3-6B2) Ab followed by streptavidinconjugated Alexa fluor 488 (green). Finally, the sections were incubated with biotinylated PNA (Sigma-Aldrich) followed by streptavidin conjugated with Alexa fluor 350 (blue). Slides were thoroughly washed four times with PBS between incubation steps, and sections were mounted using ProLong AntiFade kit (Molecular Probes, Eugene, OR). Each Ab was carefully titered and tested for specificity in multistep staining protocols. All streptavidin-conjugated Alexa fluors were purchased from Molecular Probes. Digital images were captured by AxioCam digital camera from Zeiss (Thornewood, NJ) and analyzed using the AxioVision software from Zeiss.

FIGURE 1. CD40⫹CD40⫺ chimeric mice are successfully reconstituted and exclusively express CD40 on Ly5.1⫹ B cells derived from wild-type mice. CD19⫹ PBL from CD40⫹CD40⫺ chimeric mice were examined 60 –90 days after reconstitution, before MHV-68 infection. A, The CD40⫹CD40⫺ chimeric mice expressed the input ratio (80:20) of Ly5.1⫹ and Ly5.2⫹ CD19⫹ B cells, and all CD40⫹ B cells expressed the Ly5.1 allotype of the donor CD40⫹ B cells. B, After intranasal infection with MHV-68, the 80:20 ratio of Ly5.1⫹CD19⫹ cells to Ly5.2⫹CD19⫹ cells in the spleens was consistently maintained through 90 days postinfection. Ten individual mice were examined at each time point (mean ⫾ SD).


Spleen cells were stained with peanut agglutinin (PNA; Sigma-Aldrich, St. Louis, MO), Abs against CD19, CD38, CD40, Fas (CD95), Ly5.1 (clone A20), Ly5.2 (clone 104), IgM, IgD, and a mixture of Abs against IgG1, IgG2a, IgG2b, IgG3, and IgA. All Abs were purchased from BD PharMingen (San Diego, CA). Analytical data were acquired on a FACScan or FACSCalibur and analyzed using CellQuest software (BD Biosciences, San Jose, CA) or Flo-Jo 3.6.1 software (Tree Star, San Carlos, CA). Cells were sorted on a FACSVantage DIVA (BD Biosciences). Purities after sorting were ⬎99%, unless otherwise indicated.

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CD40-DEPENDENT MAINTENANCE OF ␥-HERPESVIRUS LATENCY

FIGURE 2. Levels of lytic virus in the lung and latent virus in the spleen are comparable after MHV-68 infection of CD40⫹CD40⫺ chimeric and wildtype mice. A, Titers of lytic virus in the lungs from C57BL/6 mice (E) and CD40⫹CD40⫺ chimeric mice (F) after intranasal infection with MHV-68 were determined by plaque assay. B, Latent virus in the spleen was measured by an in vitro reactivation assay. Low levels of preformed lytic virus were detected in spleens from C57BL/6 (27 PFU/106 cells; 䡺) and CD40⫹CD40⫺ chimeric (15 PFU/106 cells; f) mice only at day 14 after MHV-68 infection. Data represent the mean values obtained from analysis of three mice at each time point (mean ⫾ SD). The differences in viral latency between the wildtype and chimeric mice are not significant (p ⬎ 0.05).

FIGURE 3. MHV-68 latency in CD40⫹CD40⫺ mixed chimeric mice is preferentially maintained in CD40⫹ B cells. At the indicated times after intranasal MHV-68 infection of CD40⫹CD40⫺ chimeric mice, three to five spleens were pooled and sorted for Ly5.1⫹CD40⫹ B cells (E), Ly5.2⫹CD40⫺ B cells (F), or total CD19⫹ B cells (f). The presence of the MHV-68 genome was detected in each B cell population using a limiting dilution PCR assay. The percent of wells positive for viral DNA in the CD40⫹ and CD40⫺ B cell subsets is shown at day 14 (A) and at day 90 postinfection (PI; B). The reciprocal frequency of virus genome-positive cells in each B cell subset was determined at 14, 30, 60, and 90 days after infection by linear regression analysis with a 95% confidence level (C). The absolute numbers of virus-infected cells per spleen were also determined (D). Data represent the mean of three to four experiments ⫾ SD. Asterisks indicate statistical significance; ⴱ, p ⬍ 0.0001; ⴱⴱ, p ⬍ 0.00003.

MHV-68 latency is preferentially maintained in CD40⫹ B cells


To characterize long term MHV-68 latency within CD40⫹ and CD40⫺ B cells, Ly5.1 (CD40⫹) and Ly5.2 (CD40⫺) B cells from the spleens of CD40⫹CD40⫺ mixed bone marrow chimeras obtained at intervals after MHV-68 infection were purified by FACS, and the frequency of virus-infected cells was determined. Long term latency is not reliably detectable with the in vitro virus reactivation assay used in Fig. 2 (19, 29). Therefore, to analyze long term latency in splenic B cells, a limiting dilution-PCR assay was used to determine the frequency of cells that harbor viral genome. In the absence of lytic virus, this assay has been used extensively to assess the frequency of latently infected cells (15, 19, 20, 33). Consistent with the analysis using the in vitro reactivation assay, analysis of cells harboring viral genome showed that viral latency

is established comparably 14 days postinfection in both the CD40⫹ and CD40⫺ B cell pools (Fig. 3A). Calculation of the frequencies of latently infected cells from such assays by linear regression showed that 1 in 329 and 1 in 367 CD40⫹ and CD40⫺ B cells, respectively, were latently infected, indicating that CD40 is not required for the establishment of latency. The comparable establishment of latency in the CD40⫹ and CD40⫺ B cells validates the use of this experimental model for examining cellular requirements for the maintenance of long term latency. To determine the requirements for the maintenance of latency reservoirs, latency was kinetically monitored within CD40⫹, CD40⫺, and total B cells at 30, 60, and 90 days postinfection. The data show a striking decrease in latency in the CD40⫺ B cells 90 days postinfection (Fig. 3B). A plot of the reciprocal frequencies of latency in CD40⫹ B cells determined by linear regression at various times after infection (Fig. 3C; frequencies of 1 in 329, 1 in 1,163, and 1 in 2,696 at 14, 30, and 90 days, respectively) reflects the normal kinetics of latency, characterized by an early decline followed by stabilization. The latency profile of CD40⫹ B cells reflected that of total CD19⫹ B cells. In contrast, there was a more substantial and continual decline in latency of CD40⫺ B cells, from a frequency of 1 in 367 cells at day 14 and 1 in 3,701 cells at day 30, to 1 in 65,151 cells at day 90 postinfection, accounting for a ⬎170-fold overall decrease in frequency. The decline in frequency of latency was mirrored by the decline in absolute numbers of latently infected cells per spleen (Fig. 3D). These data clearly show that although comparable frequencies of viral latency are established in CD40⫹ and CD40⫺ B cells, latency is preferentially maintained in CD40⫹ B cells.

identical, with virus being cleared from the lungs by day 14 postinfection (Fig. 2A). Second, the establishment of splenic latency was assessed by an in vitro virus reactivation assay. In the absence of preformed infectious virus, this assay detects latently infected cells for ⬃1 mo after infection by their ability to spontaneously reactivate when plated on a susceptible monolayer in vitro. The data show that viral reactivation in the spleens of chimeric and wildtype mice were not significantly different (Fig. 2B). Thus, the mixed bone marrow chimeric mice were able to control the acute viral infection and establish splenic latency comparably with wildtype mice, validating the use of the CD40⫹CD40⫺ mixed bone marrow chimera model to assess mechanisms involved in the long term maintenance of splenic B cell latency.

The Journal of Immunology CD40⫹ B cells are selectively activated to become germinal center cells

FIGURE 4. CD40⫹ B cells are selectively localized in the germinal centers of CD40⫹CD40⫺ chimeric mice. Spleens were removed from CD40⫹CD40⫺ chimeric mice at day 14 postMHV-68 infection. Frozen serial sections from three individual spleens were prepared, and three different regions of each spleen were examined. A and C, Overlay of staining of a representative germinal center with anti-B220 (green), antiLy5.1 or anti-Ly5.2, respectively (red), and PNA (blue), as described in Materials and Methods. B and D, Analysis of the corresponding boxed areas in A and C. Arrows indicate yellow Ly5.2⫹ cells (derived from CD40⫺/⫺ donors) lacking PNA expression (red ⫹ green) in a germinal center area, contrasted with white Ly5.1⫹ cells expressing PNA (red ⫹ green ⫹ blue) in B. Magnification is indicated by the bar.

fection was also analyzed by flow cytometry (Fig. 5). CD40⫹ and CD40⫺ B cells were stained for B cell markers associated with differentiation to germinal center B cells, including isotypeswitched surface Ig (IgG1, IgG2, IgG3, and IgA), PNA, and Fas (CD95). Isotype switching is associated with the development of germinal center or memory B cells that have transitioned through the germinal center, and we find that only CD40⫹ cells stain positive for a mixture of Abs specific for isotype-switched surface Igs (Fig. 5A). In addition, consistent with the observations that expression of PNA and Fas is up-regulated on germinal center B cells (34, 35), the staining shows that CD40⫹ cells are exclusively represented among the PNAhigh and Fashigh cells (Fig. 5, B and C). Taken together, flow cytometric analysis showing that B cells with a germinal center phenotype are exclusively CD40⫹ and immunofluorescent staining of spleen sections showing the selective CD40⫹ B cell localization in the germinal center areas, support the conclusion that CD40⫺ B cells do not become germinal center B cells following MHV-68 infection of mixed CD40⫹CD40⫺ chimeric mice. Thus, viral infection does not overcome the requirement for CD40 in the participation of B cells in germinal center reactions. Isotype-switched, postgerminal center CD40⫹ B cells are the major reservoirs of long term MHV-68 latency The data are consistent with the idea that latency is preferentially maintained in the CD40⫹ population of B cells that is capable of developing into memory B cells via germinal center reactions. To directly confirm this, latency frequencies were determined in purified populations of cells from mixed chimeric mice 128 days post-MHV-68 infection. Five-color staining was used to sort CD19⫹ B cells into five phenotypically distinct populations based on their expression of CD40, surface Ig, and CD38. Latency was assessed in each population using limiting dilution/PCR, and the frequency of latently infected cells was determined by linear regression (Table I). CD40⫺ B cells and the majority of the CD40⫹ B cells had the characteristics of naive B cells, in that they were


Because CD40-CD40L interactions are required for B cell activation to germinal center cells and the development of memory B cells, a likely explanation for the preferential maintenance of latency in CD40⫹ B cells is that they selectively differentiate into long-lived memory B cells via germinal center reactions. However, it has been shown that EBV encodes a functional homolog of CD40, LMP1 (4). Therefore, it is possible that MHV-68 infection bypasses the CD40 requirement for the development of memory cells through germinal centers. Thus, we analyzed the ability of CD40⫹ and CD40⫺ B cells to become germinal center B cells after viral infection by two independent techniques, immunofluorescent staining of frozen spleen sections and flow cytometry. Consistent with polyclonal B and T cell activation after infection (10, 11), analysis of spleen sections from mice 14 days after infection showed that the spleens of chimeric mice contained many germinal centers and PNAhigh B cells. To visualize the participation of CD40⫹ and CD40⫺ B cells in individual germinal centers, serial spleen sections were examined. Germinal center B cells with high PNA binding are indicated by the overexposed appearance (white) of the B220⫹PNAhighLy5 triple-stained (green ⫹ blue ⫹ red) cells. Analysis of serial sections stained with B220 and PNA and either Ly5.1 (CD40⫹; Fig. 4, A and B) or Ly5.2 (CD40⫺; Fig. 4, C and D) show that the majority of B220⫹PNAhigh B cells in the germinal centers are Ly5.1⫹ (CD40⫹) (Fig. 4B), whereas the CD40⫺ B cells (Ly5.2) predominantly reside in B cell follicles that are not PNA-positive (Fig. 4C). Similar analysis of serial sections from three independent mice failed to reveal any germinal centers in which CD40⫺ B cells predominated (data not shown). Although some Ly5.2⫹CD40⫺ B cells, indicated by the arrows in Fig. 4D, were occasionally detected in germinal center areas, it was clear that these B220⫹ Ly5.2⫹ cells did not express PNA, because they were yellow (red ⫹ green) rather than white (red ⫹ green ⫹ blue). As an independent assessment, MHV-68-induced activation of B cells from the spleens of mixed chimeric mice 14 days postin-

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IgD⫹ and did not express class-switched Ig. The latency frequencies were comparable among the naive CD40⫹ and CD40⫺ B cells (1 in 61,461 and 1 in 44,260, respectively). Approximately 4% of the CD40⫹ B cells expressed isotype-switched surface Ig, characteristic of Ag-experienced B cells, and this population had a latency frequency of 1 in 469. The isotype-switched CD40⫹ B cells could be divided into CD38high memory B cells and CD38low germinal center B cells (36, 37) and, consistent with our previous findings in C57BL/6 mice (20), were the preferential reservoirs of latency, expressing frequencies of 1 in 544 and 1 in 331, respectively. Together, these data verify that latency in CD40⫹CD40⫺ mixed chimeric mice is preferentially maintained in CD40⫹ B cells that have matured through germinal centers and express germinal center and memory phenotypes.

Discussion These studies directly demonstrate that efficient maintenance of MHV-68 latency in splenic B cells requires the CD40-dependent development of memory B cells via germinal center reactions. Analysis of CD40⫹ and CD40⫺ B cells from immunocompetent CD40⫹CD40⫺ mixed bone marrow chimeras showed that latency declined rapidly in CD40⫺ B cells incapable of entering germinal centers and was preferentially maintained in CD40⫹ post-germinal center B cells. The observation that long term ␥-herpesvirus la-

Table I. Long term MHV-68 latency in CD40⫹ and CD40⫺ B cells from mixed bone marrow chimeric micea

CD40 Phenotype

B Cells

CD40⫹ CD40⫹ CD40⫹ CD40⫹ CD40⫺

Isotype-switchedc Germinal centerc Memorye Non-isotype-switchedc Non-isotype-switchedc

Phenotype

IgD⫺IgG/A⫹ IgD/M⫺IgG/A⫹CD38low IgD/M⫺IgG/A⫹CD38high IgD⫹IgG/A⫺ IgD⫹IgG/A⫺

Reciprocal Frequency of Virus-Infected Cellsb

469 (271– 801)d 331 (201– 489) 544 (288 –1,419) 61,461 (44,300 –127,811) 44,260 (26,033–1,212,685)

a Latent virus in FACS-sorted B cell subsets from a pool of 5–7 mice was analyzed at days 110 –128 after infection. Data are compiled from two independent experiments with similar results. b Latency in each B cell subset was detected using LDA/PCR. Frequency of virus-infected cells was determined using linear regression analysis. c Spleen cells were stained for CD19, CD40, IgD, and isotype-switched surface Igs, including IgG1, IgG2a, IgG2b, IgG3, and IgA (IgG/A). Postsorting purities for CD40⫹ isotype-switched, CD40⫹ non-isotype-switched, and CD40⫺ non-isotype-switched B cells were 97.3, 98.3, and 98.6%, respectively. d Numbers in parentheses, 95% degree of confidence levels. e Spleen cells were stained for CD19, CD40, IgD, and IgM (IgD/M), and isotype-switched surface Igs (IgG/A). Isotype-switched CD40⫹ B cells were further sorted into germinal center and memory B cell subsets on the basis of CD38 expression. Postsorting purities for CD40⫹ germinal center and memory B cells were 93.3 and 96.8%, respectively.


FIGURE 5. CD40⫹ B cells from CD40⫹CD40⫺ chimeric mice are selectively driven to differentiate. At day 14 after MHV-68 infection, spleen cells from CD40⫹CD40⫺ chimeric mice were stained with Abs against CD19, CD40, and several maturation markers associated with germinal center B cells. The distribution of CD40⫹ and CD40⫺ cells among populations positive and negative for isotype-switched surface Ig, including IgG1, IgG2a, IgG2b, IgG3, and IgA (A), PNA (B), and Fas (CD95, C), were determined. Data are representative of phenotypic analysis of three individual mice.

tency is maintained preferentially in memory B cells (5, 6, 20) is consistent with the hypothesis, originally proposed for EBV (6 –9), that the virus has developed strategies to exploit the host B cell biology to maintain latency. However, it has not been possible to experimentally test the hypothesis in humans. The current data provide the first direct test of the hypothesis in an experimental mouse model. The progressive and selective loss of latency in a subset of B cells that fail to develop into long-lived memory B cells demonstrates that maintenance of latency is dependent on whether the virus gains access to a long-lived pool of memory B cells. This clever strategy developed by the virus allows viral persistence independent of viral reactivation and reinfection, a process that exposes the virus to immunosurveillance. The current studies show that latency was established comparably 14 days postinfection in CD40⫹ and CD40⫺ B cells from bone marrow chimeras, despite the fact that CD40⫺ B cells did not enter germinal center reactions. These data agree with our previous studies showing that germinal centers are not absolutely required for establishing MHV-68 latency (16, 33). Thus, the observations by us and others that latency is preferentially established in germinal center B cells (16, 20, 38 – 40) may instead reflect a requirement for CD40-independent events in B cell activation in establishing latency. In this regard, MHV-68 is a potent polyclonal B cell activator (10, 11). Germinal center B cells may be a preferential reservoir for the establishment of latency because they represent a pool of highly activated B cells. The B cell activation signals required for the establishment of latency remain to be identified. The key point for the present study is that latency is established comparable in CD40⫹ and CD40⫺ B cells, yet CD40⫺ B cells cannot become germinal center/memory B cells, providing an experimental model in which to directly test the requirement of memory B cells for maintenance of B cell reservoirs of ␥-herpesvirus latency. The current studies show that, unlike EBV, MHV-68 does not express a viral protein to mimic CD40 signaling. CD40⫺ B cells become latently infected but fail to undergo isotype switching, enter germinal center reactions, or differentiate to memory B cells after MHV-68 infection. This points out several fundamental differences between the human ␥1-herpesvirus, EBV, and the murine ␥2-herpesvirus, MHV-68. Whereas they are both B-lymphotropic viruses and share the property of co-opting B cell memory to maintain latency, the viruses gain access to the memory B cell pool by two fundamentally different processes. EBV encodes several viral genes, such as LMP1, LMP2a, and EBNA2, which mimic normal cellular signaling. LMP1 is a homolog of CD40 and serves as a surrogate for normal T cell help by mimicking many events triggered by CD40-CD40L interactions between B and T cells (41),

The Journal of Immunology

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6. 7. 8. 9. 10.

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13. 14.

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16.

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19. 20.

21. 22. 23.

24.

25.

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27.

Acknowledgments We thank Simon Monard for cell sorting, Jean Brennen for digital image processing, and Alan Roberts, Patricia Lederman, and John Moore for technical assistance.

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