Murine Gammaherpesvirus 68 Infection Is Associated with Lymphoproliferative Disease and Lymphoma in BALB {beta}2 Microglobulin-Deficient Mice

Human gammaherpesvirus infections are associated with developmentof lymphoproliferative disease. Understanding of the mechanismsof gammaherpesvirus lymphomagenesis during chronic infectionin a natural host has been limited by the exquisite speciesspecificity of human gammaherpesviruses and the expense of primates.Murine gammaherpesvirus ${gamma}$ HV68 is genetically and biologicallyrelated to human gammaherpesviruses and herpesvirus saimiriand has been reported to be associated with lymphoproliferativedisease in mice (N. P. Sunil-Chandra, J. Arno, J. Fazakerley,and A. A. Nash, Am. J. Pathol. 145:818-826, 1994). We reportthe development of an animal model of ${gamma}$ HV68 lymphomagenesis inBALB/c ß2 microglobulin-deficient mice (BALB ß2m^–/–). ${gamma}$ HV68 infection induced two lymphoproliferative lesions: B-celllymphoma and atypical lymphoid hyperplasia (ALH). ALH lesionhistology resembled lesions of Epstein-Barr virus-associatedposttransplant lymphoproliferative disease and was characterizedby the abnormal infiltration of the white pulp with cells expressingthe plasma cell marker CD138. Lymphomas observed in ${gamma}$ HV68-infectedanimals were B220⁺/CD3^– large-cell lymphomas. ${gamma}$ HV68-infectedcells were common in ALH lesions as measured by in situ hybridizationwith a probe specific for viral tRNAs (vtRNAs), but they werescarce in ${gamma}$ HV68-infected spleens with normal histology. UnlikeALH lesions, ${gamma}$ HV68 vtRNA-positive cells were rare in lymphomas. ${gamma}$ HV68 infection of BALB ß2m^–/– mice resultsin lymphoproliferation and lymphoma, providing a valuable toolfor identifying viral and host genes involved in gammaherpesvirus-associatedmalignancies. Our findings suggest that ${gamma}$ HV68 induces lymphomasvia hit-and-run oncogenesis, paracrine effects, or stimulationof chronic inflammation.

INTRODUCTION

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Introduction

Gammaherpesviruses establish lifelong latency in lymphoid andmyeloid cells of the host. Importantly, many gammaherpesviruses,including human Epstein-Barr virus (EBV), Kaposi's sarcoma-associatedherpesvirus (KSHV), and simian herpesvirus saimiri (HVS) areassociated with a variety of lymphoproliferative diseases (LPDs)(13, 31, 71). In addition to Kaposi's sarcoma, a tumor of endothelialcell origin, KSHV has also been associated with at least twotypes of lymphoproliferative B-cell diseases, multicentric Castleman'sdisease and primary effusion lymphoma (PEL) (6). EBV induceshyperproliferation of infected B cells that can evolve intolymphoma in an immunocompromised host (7, 14). Other lymphomaswith a strong link to EBV infection include Burkitt's lymphoma,Hodgkin's lymphoma, and AIDS-associated lymphomas (40, 71).HVS is associated with T-cell lymphomas in New World primatesand in certain rabbits (31). Immunocompromised hosts have anincreased incidence of gammaherpesvirus-associated lymphoproliferativedisease, suggesting a critical role of the immune system incontrolling lymphoproliferation and lymphomagenesis (71).

The mechanism of gammaherpesvirus-induced lymphomagenesis inanimal models is not clear. The four major models of virus-associatedoncogenesis include (i) direct transformation, (ii) "hit andrun," (iii) paracrine, and (iv) chronic inflammation. Directtransformation is the only model that requires viral presencein a majority of malignant cells (38). Such is the case in earlyonset posttransplant lymphomas that are uniformly associatedwith EBV (71). EBV genes expressed in infected B cells of theselymphomas represent the latency III program of EBV gene expressionthat provides the necessary signals for uncontrolled B-cellproliferation. In other cases, such as EBV-associated Hodgkin'sdisease, virus is present in a minority of cells found in thetumor (71). HVS-induced T-cell lymphomas uniformly retain viralgenome (10). According to the "hit-and-run" model, the presenceof the virus is only required to initiate events leading tothe transformation of infected cells (1). Once a fully transformedstate is achieved, virus is lost (44). In support of the "hit-and-run"mechanism, cell lines derived from the primary Kaposi's sarcomalesions lose viral episome upon passage in vitro (24, 47). Invivo, EBV-associated Hodgkin's lymphoma may no longer harborvirus upon relapse (15). The existence of the paracrine mechanismof lymphomagenesis is supported by studies showing that cell-type-specificexpression of the KSHV G protein-coupled receptor (vGPCR) inlymphocytes of transgenic mice induces angioproliferative lesionscharacteristic of Kaposi's sarcoma (69). KSHV vGPCR expressionupregulates expression of vascular endothelial growth factorand thus contributes to the transformation of human endothelialcells in vitro (2). Although the role of chronic inflammationin the development of gammaherpesvirus-associated malignancieshas not been examined, other chronic inflammatory stimuli, suchas Helicobacter pylori infection, are major contributors tothe development of cancer (12). EBV-specific CD8⁺ T cells isolatedfrom latently infected individuals have immediate effector functions(direct ex vivo cytotoxic activity and epitope-specific cytokineproduction) (30, 59), suggesting continuous stimulation by viralantigens. Therefore, chronic inflammation associated with persistentEBV and KSHV infections might contribute to gammaherpesviruslymphomagenesis. While the direct transformation model requiresthe continuous presence of virus in most malignant cells, theother three potential mechanisms of gammaherpesvirus-associatedlymphomagenesis require short-term or no viral infection oflymphoma cells.

Analysis of the mechanisms responsible for gammaherpesvirus-associatedlymphomagenesis in vivo has been hampered by the exquisite speciesspecificity of human gammaherpesviruses and the expense andavailability of primates. Murine ${gamma}$ HV68 readily infects rodents,including laboratory mice (3). Genetically, ${gamma}$ HV68 is relatedto the human viruses EBV and KSHV and the primate virus HVS(21, 63) and shares important features of its biology with itshuman and primate relatives. During acute infection, ${gamma}$ HV68 lyticallyreplicates in lymphoid and nonlymphoid compartments, includinglung epithelial cells (52). Upon resolution of the acute infection, ${gamma}$ HV68 latency is primarily maintained in B cells, macrophages,and dendritic cells (25, 53, 67, 68). CD8⁺ and CD4⁺ T cellsare important for controlling acute ${gamma}$ HV68 replication and ${gamma}$ HV68reactivation during latency (5, 9, 22, 46, 48, 58, 60, 61, 64).

Similar to human gammaherpesviruses, ${gamma}$ HV68 infection is reportedto be associated with lymphoproliferative disease in wild-typemice; however, ${gamma}$ HV68-associated lymphomas develop at a low incidence(9%) and after a prolonged incubation period (up to 3 years)(51). Only a few cells in tumors were reported to harbor virusas determined by in situ hybridization (ISH) with probes specificfor ${gamma}$ HV68 sequences (51). ${gamma}$ HV68 can infect and establish persistentinfection in cultured B-cell lines (54), raising the possibilitythat ${gamma}$ HV68-positive lymphoma cells isolated from a tumor in previousstudies could have arisen from infection of virus negative tumorcells during the isolation process. Cyclosporine treatment wasreported to increase the incidence of lymphoproliferative diseasein ${gamma}$ HV68-infected mice, suggesting the critical role for immunecontrol of gammaherpesvirus-associated lymphomagenesis (51).However, these initial studies have not been extended and arobust model for ${gamma}$ HV68-induced lymphomas is still needed. Interestingly,while infection with ${gamma}$ HV68 activates and stimulates proliferationof primary murine B cells both in vitro and in vivo, attemptsto use ${gamma}$ HV68 to transform primary B cells in vitro have to datebeen unsuccessful (20, 37a, 49).

We considered the hypothesis that development of lymphomas after ${gamma}$ HV68 infection may be dependent on both immune status and mousegenetic background. Therefore, we asked whether the absenceof ß2 microglobulin (ß2m) expression inmice could potentiate ${gamma}$ HV68-associated lymphoproliferative disease.ß2 microglobulin is required for control of both latentand persistent ${gamma}$ HV68 infection (11, 64; unpublished observation).In this report, we demonstrate that ${gamma}$ HV68 infection of BALB ß2microglobulin-deficient mice (BALB ß2m^–/–),but not 129 ß2m^–/– mice, led to a highincidence and accelerated development of B-cell lymphoma anda second lymphoproliferative lesion we termed atypical lymphoidhyperplasia (ALH). ALH lesions were characterized by the abnormalexpansion of the white pulp infiltrated with cells bearing theplasma cell CD138 marker and significant numbers of ${gamma}$ HV68-positivecells. Lymphomas observed in ${gamma}$ HV68-infected animals were of B-cellorigin with a propensity to disseminate. Unlike cells in ALHlesions, few lymphoma cells expressed viral tRNAs (vtRNAs),as measured by in situ hybridization. These findings argue thatlymphomagenesis in ${gamma}$ HV68-infected immunocompromised hosts doesnot require the continuous presence of virus in malignant cells.

MATERIALS AND METHODS

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Materials and Methods

Animals. BALB/cJ (BALB) and ß2m knockout mice (B2m^tm1Unc) onthe BALB/cJ (BALB ß2m^–/–) genetic backgroundwere obtained from Jackson Laboratories (Bar Harbor, Maine)and bred at Washington University, St. Louis, MO. 129Pas mice(129-ß2m^–/–) were derived from 129/BL6-ß2m^–/–mice (Jackson Laboratories, Bar Harbor, Maine) and were subsequentlybred to 129/Pas (19) for 10 generations. All mice were housedin a specific-pathogen-free barrier facility at Washington Universityin accordance with federal and institutional guidelines. Micewere infected between 1.9 and 2.4 months of age.

Virus infections. ${gamma}$ HV68 WUMS (ATCC VR1465) was passaged and the titer was determinedon NIH 3T12 cells (64). All injections were done intraperitoneallywith 10⁷ PFU ${gamma}$ HV68 diluted in 500 µl of Dulbecco's modifiedEagle's medium supplemented with 10% fetal calf serum. Mockinfections were performed with 500 µl of Dulbecco's modifiedEagle's medium containing 10% fetal calf serum.

Organ harvest and tissue processing. At various times postinfection, mice were sacrificed, and thespleen, liver, lungs, and thymus as well as any enlarged lymphnodes or macroscopically abnormal organs were removed. Portionsof the organ samples were fixed in 10% buffered formalin (FisherScientific). Additionally, small fragments of splenic tissueand lymph nodes, when available, were snap-frozen in liquidnitrogen and stored at –70°C.

Histology and immunohistochemistry. Organs were embedded in paraffin, and 5-µm sections werecut and stained with hematoxylin and eosin (H&E). For immunohistochemistry,sections were deparaffinized in Citri-Solv buffer (Fisher Scientific)and antigen retrieval was performed by boiling in 10 mM citratebuffer (pH 6.0) or 1 mM EDTA (pH 8.0) in a microwave oven. Endogenousperoxidase was quenched with 3% H₂O₂ in methanol for 30 min,and the sections were blocked 30 min in a blocking buffer (1%bovine serum albumin, 0.2% powdered skim milk, 0.3% Triton-Xin phosphate-buffered saline [PBS]). Primary antibodies wereapplied to the sections overnight at 4°C in a humid chamber.Horseradish peroxidase (HRP)-conjugated secondary antibody dilutedin the blocking buffer (goat anti-rabbit; BD Pharmingen, SanJose, CA) or donkey anti-rat (Jackson ImmunoResearch, West Grove,PA) was applied for 1 h at room temperature. Signal was enhancedwith one round of tyramide signal amplification using the Trypticasesoy agar-biotin system (Perkin-Elmer Life Sciences, Inc., Boston,MA), and color development was performed with diamidinobenzidine(DAB) substrate kit (Vector laboratories, Burlingame, CA). Immunostainedslides were counterstained with hematoxylin (Sigma, St. Louis,MO). The following antibodies were used: B-cell marker (purifiedrat anti-B220, BD Pharmingen), plasma cell marker (purifiedrat anti-CD138/Syndecan-1, BD Pharmingen), T-cell marker (purifiedrat anti-CD3; Serotec, Oxford, United Kingdom), and rabbit polyclonalanti- ${gamma}$ HV68 (65).

In situ hybridization. Sections were deparaffinized and rehydrated using xylene washesfollowed by ethanol gradients. Postfixation was performed for20 min in fresh 4% paraformaldehyde (pH 7.4), followed by 0.3%H₂O₂ in PBS for 30 min, acetylation in two 10-min incubationsin 0.1 M triethanolamine and 0.25% acetic anhydride, and permeabilizationwith proteinase K (40 µg/ml; Sigma, St. Louis, MO) inPBS at room temperature for 20 min. Hybridization was conductedfor 40 h at 37°C using a hybridization mix containing 4xSSC (1x SSC is 0.15 M NaCl plus 0.015M sodium citrate); 20%dextran sulfate; 50% formamide; 0.25 mg/ml each of poly(A),salmon sperm DNA, and tRNA; 5x Denhardt's solution (all fromSigma); and 100 mM dithiothreitol (Fisher). GreenStar digoxigenin-labeledoligonucleotide probes (Genedetect, Auckland, New Zealand) specificfor vtRNAs were used at a final concentration of 200 ng/ml inhybridization mix. A cocktail of probes antisense to vtRNAs1 to 4 was utilized (vtRNA1, nucleotides [nt] 12 to 41; vtRNA2,nt 13 to 42; vtRNA3, nt 3 to 32; and vtRNA4, nt 33 to 62). Stringentposthybridization washes were performed with 1x and 0.5x SSCat 55°C, and the hybridized probes were detected using agoat HRP-conjugated anti-digoxigenin Fab fragment (Dako) followedby a round of tyramide signal amplification and DAB substratecolor development.

Molecular analysis of clonality. DNA was extracted from frozen spleen fragments using standardprocedures. Analysis of the rearranged status of the immunoglobulinheavy chain (IgH) locus was performed by PCR amplification usingthe family-specific degenerate forward primers V_h558, V_h7183,V_hQ52, and D_hL with the reverse primer J_h3 (35, 43). Amplifiedproducts were separated by agarose gel electrophoresis. Whena predominant fragment was observed, it was cut, purifed, andreamplified in a second round using the appropriate primers.The resulting product was cloned in pGEM-T and sequenced. Sequenceanalysis was performed by aligning the sequenced product usingV-QUEST search on the Immunogenetics database (http://imgt.cines.fr:8104/textes/vquest/).

Statistical analysis. The data were analyzed using GraphPad Instat software (San Diego,CA). All results were compared using unpaired Student's t testor chi-square tests.

RESULTS

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Results

${gamma}$ HV68 infection is associated with an increased incidence of lymphoproliferative diseases in BALB-ß2m-deficient mice. Gammaherpesvirus-associated lymphoproliferative disease is increasedin immunocompromised hosts. Given the importance of CD8 T cellsand ß2 microglobulin in the control of ${gamma}$ HV68 replicationand latency (11, 22, 58, 61, 64), we assessed the developmentof lymphoproliferative disease in mock-infected and ${gamma}$ HV68-infectedsex- and age-matched BALB/c mice lacking ß2 microglobulinexpression. Several defects are attributed to the ß2microglobulin deficiency, including lack of classical CD8 Tcells, hepatic iron overload, and abnormal IgG homeostasis dueto the inefficient expression of neonatal Fc receptor (28, 42,70, 73). Mice were infected between 1.9 and 2.4 months of ageand sacrificed at the indicated times (Table 1). We then assessedlymphoid histology in spleens, lymph nodes, livers, lungs, andkidneys.

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TABLE 1. Demographics of animals used in this study

Two lymphoproliferative lesions were observed disproportionatelyin infected compared to uninfected BALB ß2m^–/–mice: atypical lymphoid hyperplasia with plasmacytosis (ALH),and large-cell lymphomas. The pathology of these lesions isdescribed below. Follicular hyperplasia, as defined by the presenceof a prominent germinal center reaction with otherwise normalhistology, was occasionally observed and was not scored as lymphoproliferativedisease. Overall 37/55 (67%) of the infected BALB ß2m^–/–mice developed LPD (ALH or lymphoma) compared to 11/50 (22%)of the mock-infected controls (P < 0.0001) (Fig. 1A). Therewere significantly more lymphomas (29% versus 6%, P = 0.0022)and atypical lymphoid hyperplasias (38% versus 16%, P = 0.0157)in the ${gamma}$ HV68-infected group as compared to the mock-infectedanimals (Fig. 1A).

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FIG. 1. ${gamma}$ HV68 infection increases incidence of lymphoproliferative disease in BALB ß2 microglobulin-deficient mice. Age- and sex-matched BALB ß2m animals were mock infected or infected with ${gamma}$ HV68 and monitored for the development of LPD. Total incidence of LPD (A) was subdivided into incidence of each of the two primary lesions (ALH and lymphoma) in male and female animals. (B and C) Incidence of LPD at indicated times postinfection in ${gamma}$ HV68 (B)- and mock (C)-infected groups. n, number of mice.

The development of ${gamma}$ HV68-associated lymphoproliferative lesionswas not detected in BALB (0% LPD, n = 23) or 129/Pas ß2m^–/–animals (0% LPD, n = 20) (Table 1), demonstrating that boththe ß2m deficiency and the genetic background of themice are key factors in determining susceptibility to ${gamma}$ HV68-associatedLPD.

Kinetics of lymphoproliferative disease development in ${gamma}$ HV68-infected and control BALB-ß2m^–/– mice. We analyzed the kinetics of lymphoproliferative lesions in ${gamma}$ HV68-infectedor mock-infected BALB ß2m^–/– mice (Fig.1B and C). Overall, atypical lymphoid hyperplasia and lymphomaswere detected at a higher incidence and at an earlier time inthe ${gamma}$ HV68-infected group as compared to the mock-infected group.The mean age of mice with a diagnosis of lymphoma was 11.6 monthsin the ${gamma}$ HV68-infected group compared to 15.8 months in the mock-infectedcontrols (P = 0.011). Atypical lymphoid hyperplasia was observedearlier and at an increased incidence in the ${gamma}$ HV68-infected group.At 6 to 7 months postinfection, over 50% of the animals hadatypical lymphoid hyperplasia lesions in the spleen, whereasnone of the mock-infected animals had detectable lymphoproliferativedisease (compare Fig. 1B and C). In both mock- and ${gamma}$ HV68-infectedgroups, atypical lymphoid hyperplasia was detected at an earliertime postinfection than lymphomas. Thus ${gamma}$ HV68 infection was associatedwith both an increased penetrance and an earlier onset of lymphoproliferativelesions.

Role of gender in development of lymphoproliferative disease. An increased incidence of spontaneous lymphomas has been previouslyobserved in BALB female but not male animals (23). Interestingly,we also observed a gender-dependent difference in the incidenceof lymphoproliferative disease in mock-infected BALB ß2m^–/–animals (Fig. 1A). Mock-infected male BALB ß2m^–/–animals had a low level of atypical lymphoid hyperplasia andno spontaneous lymphomas (Fig. 1A, middle panel). The incidenceof lymphoproliferative lesions was significantly increased in ${gamma}$ HV68-infected males. In contrast, while there was a trend towardsan increase in lymphoproliferative disease in ${gamma}$ HV68-infectedversus mock-infected females, the difference did not reach statisticalsignificance. Mock-infected females had a higher incidence andseverity of lymphoproliferative lesions (6 of 27 females withatypical lymphoid hyperplasia, 3 of 27 with lymphomas). Themean time of development of LPD in ${gamma}$ HV68-infected females withlymphomas was shorter (13.2 months) than that observed in mock-infectedfemales (15.8 months), suggesting that ${gamma}$ HV68 accelerates thedevelopment of lymphomas in female BALB ß2m^–/–animals. A larger study will be required to determine whether ${gamma}$ HV68 induces lymphoproliferative disease in female BALB ß2m^–/–mice.

Histopathology of atypical lymphoid hyperplasia. Four conditions were represented in spleens of the ${gamma}$ HV68-infectedmice in this study. Normal spleen sections displayed well-delineatedareas of white pulp containing tightly packed lymphocytes withdark-staining nuclei (Fig. 2A and B). Follicular hyperplasiawas scored based on the presence of reactive B-cell follicles(Fig. 2C and D, arrows) with otherwise normal splenic histology.Two examples of the atypical lymphoid hyperplasia lesions describedbelow are shown in Fig. 2E and F. Of note, atypical lymphoidhyperplasia lesions displayed a spectrum of severity rangingfrom only a mild effacement of a few white pulp areas on a section(Fig. 2E, outlined) to a dramatic expansion of most white pulpareas (Fig. 2F). Spleens with lymphomas displayed a loss ofnormal splenic architecture and expansion of large cells withmoderate amounts of cytoplasm and vesicular nuclei (Fig. 2G and H).

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FIG. 2. Splenic histology of ${gamma}$ HV68-infected BALB ß2m^–/– mice. H&E staining of formalin-fixed paraffin-embedded tissues. (A and B) Normal (x20). (C and D) Follicular hyperplasia (x20). Arrows point to germinal centers abundant throughout the section. (E and F) Atypical lymphoid hyperplasia (x10). (E) A hyperplastic lesion is outlined. (G and H) Lymphoma (x4).

Atypical lymphoid hyperplasia lesions presented with a partialdisruption of the normal splenic architecture with an irregularexpansion of the white pulp. In contrast to the lymphomas, thedistinction between red and white pulp remained apparent. Whiledisorganized in more severe lesions, the expansion involvedprimarily the periarteriolar lymphoid sheath and sometimes themarginal zone (Fig. 3A and B). Atypical lymphoid hyperplasialesions were also present in nonlymphoid organs, mainly thelungs, where they appeared as nodular infiltrates in parenchyma(Fig. 3C). The expansion reflected the presence of large lymphoidcells with moderate amounts of amphophilic cytoplasm and vesicularnuclei with clumped chromatin, features characteristic of plasmacyticdifferentiation (Fig. 3A to C, insets). Supporting the morphologicalfindings, immunohistochemistry demonstrated an increased numberof cells bearing the plasma cell CD138 marker in the areas ofALH lesions (Fig. 3D). B220 staining was confined to the otherwisenormal B-cell areas of the white pulp (Fig. 3E).

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FIG. 3. Histopathology of atypical lymphoid hyperplasia. (A and B) ALH lesions in spleens (x4). The splenic architecture is partially disrupted with expanded white pulp areas. (C) Lung lymphocytic infiltrates seen in ${gamma}$ HV68-infected animals with ALH lesions (x4). (Insets in panels A to C) Prominent plasmacytic features of cells in ALH lesions (x100). (D) The majority of the ALH-associated cells stain with the plasma cell marker CD138 (x10). (E) B220 is detected in residual B-cell follicles (x10). Adjacent spleen sections obtained from ${gamma}$ HV68-infected BALB ß2m^–/– mice were stained with hematoxylin and eosin (F and H) or hybridized with antisense digoxigenin-labeled vtRNA probe (G, I, and J) and counterstained with hematoxylin. Sections originated from spleens exhibiting follicular hyperplasia (F and G) or atypical lymphoid hyperplasia (H, I, and J). Magnification: F to I, x4; J, x40.

The presence of ${gamma}$ HV68 in lymphoproliferative lesions. To determine whether ${gamma}$ HV68 was present in lymphoproliferativelesions, we performed ISH for expression of viral tRNAs on spleensections harvested from ${gamma}$ HV68-infected BALB ß2m^–/–animals. Sections were hybridized to a digoxigenin-labeled probeantisense to the viral tRNA transcripts. ${gamma}$ HV68 tRNA genes areexpressed during viral latency and are a robust target for detectionby ISH (4, 45, 50). Spleen sections from ${gamma}$ HV68-infected BALBmice (16 days postinfection) and naïve animals were utilizedas positive and negative controls. No staining was observedwith vtRNA-specific probes on mock-infected spleen sectionsor with the control sense probe on ${gamma}$ HV68-infected spleen sections(data not shown).

In order to test the sensitivity of detection of ${gamma}$ HV68-infectedcells by in situ hybridization, we calculated the frequencyof vtRNA-positive cells on spleen sections obtained from ${gamma}$ HV68-infectedBL6 mice at 16 days postinfection. The nature of ${gamma}$ HV68 latencyin these mice is well established in the literature. At 16 dayspostinfection, approximately 1 in 1,000 splenocytes are ${gamma}$ HV68genome positive as determined by limited dilution nested PCR(56). Out of over 12,500 cells counted in random fields of spleensections stained with vtRNA-specific probe, on average, 1 in407 nucleated cells was vtRNA positive (range 1 in 186 cellsto 1 in 1,256 cells). Therefore, the sensitivity of detectionof the ${gamma}$ HV68-infected cell by in situ hybridization using a vtRNAprobe is comparable to the sensitivity of established methods,such as limited dilution PCR. These data show that in situ hybridizationis not likely to significantly underestimate the number of vtRNA-expressingcells in tumors.

When spleens from BALB ß2m^–/– mice wereexamined by in situ hybridization, no or a few small vtRNA-positivefoci were consistently detected in spleens from ${gamma}$ HV68-infectedanimals in the absence of pathological lesions (defined as normalor follicular hyperplasia). Consistent with a previous report(50), the positive cells, when present, colocalized with B-cellfollicles on the adjacent section stained with H&E (Fig.3F and G, arrow).

Interestingly, spleen sections derived from ${gamma}$ HV68-infected animalsdiagnosed with atypical lymphoid hyperplasia showed a greaterabundance of vtRNA-positive foci. Based on the H&E-stainedadjacent spleen section, the positive cells were localized tothe B-cell follicles with some positive cells present in theabnormally expanded T-cell zone (Fig. 3H and I, arrow). Theintensity of vtRNA staining of cells in a positive focus wasvariable, ranging from intense to low (Fig. 3J). Based on thenuclear morphology, the positive staining was detected in lymphocytesas well as cells with monocytoid-like nuclei.

Histopathology of splenic lymphomas found in ${gamma}$ HV68-infected BALB ß2m^–/– mice. Lymphomas were characterized by nodular and/or diffuse proliferationof pleiomorphic large cells, showing a mixture of immunoblasticand centroblastic features (Fig. 4A and B). The involved spleenshad a largely disrupted architecture with a massively expandedwhite pulp and a general loss of the distinction between B-and T-cell areas. The malignant proliferation involved mainlythe white pulp. The malignant cells had large nuclei with openchromatin and single prominent nucleoli (immunoblasts) or severalsmall but conspicuous nucleoli (centroblasts). Aside from thelarge cells, some of the lymphomas showed various degrees ofplasmacytosis. These lesions were histologically aggressiveas judged by the high mitotic index and the number of apoptoticbodies present.

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FIG. 4. Histopathology of splenic lymphomas. (A and B) Lymphoma-bearing spleens (x4). The general architecture of the spleen is disrupted by the massive expansion of the white pulp. (Insets in panels A and B) Large lymphoma cells with irregular nuclei with conspicuous nucleoli (x40). Frequent mitotic figures are present. (C) Metastasis to the liver (x10). Lymphoma cells are preferentially localized in the portal areas of the liver surrounding the bile ducts. (Inset) Tumor cells adjacent to a bile duct (x40). (D) Metastasis to the lung (x10). (E to G) HRP immunostaining with hematoxylin counterstain. Magnification at x10 (insets at x40). (E) Expression of B-cell marker B220. (F) Expression of the plasma cell marker CD138. (G) Expression of T-cell marker CD3. Peritumoral and tumor-infiltrating small lymphocytes but not lymphoma cells stain positive. (H) Lymphoma-bearing spleen section was hybridized with antisense digoxigenin-labeled vtRNA probe and counterstained with hematoxylin (x40).

Although lymphomas were primarily detected in the spleens, metastaticdissemination was also observed. Two of the nine lymphomas foundin ${gamma}$ HV68-infected males displayed metastases to the liver (periportaldistribution, Fig. 4C); six out of nine lymphomas metastasizedto the lung (peribronchiovascular distribution, Fig. 4D).

By immunohistochemistry, lymphoma cells expressed the B220 isoformof CD45, although the staining was often weaker than that onthe remaining normal B cells (Fig. 4E). Staining with a monoclonalantibody to CD138 specific for plasma cells indicated that someof the atypical cells had detectable levels of CD138 expression(Fig. 4F). CD3 immunostaining showed only interspersed T cellsbut was consistently negative on the tumor cells (Fig. 4G).

In contrast to atypical lymphoid hyperplasia lesions, spleensderived from lymphoma-bearing ${gamma}$ HV68-infected animals displayeda very limited vtRNA staining. Overt vtRNA-positive foci werenot detected in the tumors analyzed. Instead, a small numberof individual cells in the tumors stained vtRNA positive (Fig.4H). The ${gamma}$ HV68-harboring cells were scattered throughout thelesion, with most of the positive cells showing low levels ofvtRNA expression. Occasionally, small vtRNA-positive lymphocyteswere found outside the lesions in the tumor-bearing spleens(data not shown). It was not possible to determine with certaintywhether these virus-positive tumor cells were malignant.

Clonality of splenic lymphomas. Because of the apparent B-cell nature of the ${gamma}$ HV68-associatedlymphomas in infected animals, clonality of lymphomas was assessedby PCR analysis of the IgH locus (43). Although this clonalityassay is quite robust in detection of specific rearrangements,it is not comprehensive in detection of all possible heavy chainrearrangements. In this assay, rearranged, but not germ line,heavy chain sequences are amplified using forward primers homologousto conserved framework region 3 of three murine V_h gene families(Fig. 5, lanes 1, 2, and 3 throughout) or primers homologousto a majority of known murine D minigenes (lanes 4). A reverseprimer specific for the J_h3 sequence is used in all cases. Basedon the utilization of the specific J region during heavy chainrearrangement (J_h1, J_h2, or J_h3), up to three major PCR productsare obtained in each reaction from a heterogeneous populationof splenic B cells (Fig. 5, normal spleen). A variety of sequencesare expected to be present in any of the major PCR productsobtained from a nonclonal population of B cells (Fig. 5, normalspleen and summary in the table below). The primers selectedfor the PCR analysis of the IgH locus are capable of amplifyingonly a select number of V_h families and are unable to detectheavy chain rearrangements utilizing J_h4.

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FIG. 5. Clonality assessment of ${gamma}$ HV68-associated lymphomas. VH-to-DJH gene rearrangement was analyzed by PCR using forward primers specific for members of the V_h558 (lane 1), V_h7183 (lane 2), and V_hQ52 (lane 3) families. DH-to-JH gene rearrangements were analyzed with the D_hL primer (lane 4). A J_h3 reverse primer was used in all cases. PCR products were analyzed on a 1% agarose gel. M, 100-bp molecular size marker. Bands indicated by boxes were excised from the gel, reamplified using the same primers, cloned, and sequenced. The table shows the sequencing results.

Importantly, out of seven lymphomas analyzed, three tumors werefound to be clonal based on the limited number of sequencespresent in the amplified major PCR product (Fig. 5). Specifically,two lymphomas were monoclonal; three sequences were detectedupon analysis of the third lymphoma with majority of the sequences(11/13) belonging to one clone. Detection of more than one sequencecould be due to amplification of the heavy chain of normal Bcells present in the lymphoma-bearing spleen or due to the presenceof several clonal transformed populations. The clonality ofthe remaining four lymphomas was indeterminate.

DISCUSSION

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Discussion

Gammaherpesvirus infection is associated with development ofmalignancies in immunocompromised hosts. To facilitate analysisof mechanisms and pathogenesis of this process, we have soughta murine model for gammaherpesvirus induction of lymphoproliferativedisease. In this report, we demonstrate that ${gamma}$ HV68 infectioninduces two lymphoproliferative diseases, each with histologicsimilarities to human diseases associated with gammaherpesvirusinfection. We found that lack of the immunologically importantmolecule ß2 microglobulin, genetic background, andgender all contributed to the risk of lymphoproliferative disease.The role of genetic background is of particular interest since,while immunocompromise is a clear risk factor for gammaherpesvirus-associatedtumors, the reason that these tumors arise in only a small proportionof infected hosts is obscure. Interestingly, ${gamma}$ HV68 infectionwas undetectable in most tumor cells, suggesting that directtransformation is not responsible for induction of malignanciesin this model.

ß2 microglobulin deficiency of ${gamma}$ HV68-infected BALB mice contributes to development of lymphoproliferative disease. BALB ß2m^–/– animals developed lymphoproliferativelesions in response to ${gamma}$ HV68 infection, while their wild-typecounterparts did not. One explanation for this is the paucityof CD8⁺ T cells capable of recognizing classical major histocompatibilitycomplex class I-antigen complex in ß2m^–/–mice (73). CD8⁺ T cells play an important role in the controlof ${gamma}$ HV68 replication, latency, and persistent replication andcan contribute to vaccination against latency in addition tocontrol of lymphoproliferative disease associated with humangammaherpesviruses (5, 9, 22, 46, 48, 58, 60, 61, 64). CD4⁺T cells also play a role in control of latent and persistent ${gamma}$ HV68 infection (5, 9, 22, 46, 48, 58, 60, 61, 64). CD4⁺ T cellsinfiltrated and were critical for control of the ${gamma}$ HV68-infectedS11 tumor cell line in nude mice (41). A profound deficiencyof CD8⁺ T cells and disruptions of immunoglobulin homeostasisdue to the absence of neonatal Fc receptor are also observedin ß2 microglobulin-deficient mice (28, 70, 73). AsB cells and antibody play a role in regulating ${gamma}$ HV68 latency(27, 32, 57, 65, 66), we cannot rule out a contribution of abnormalimmunoglobulin homeostasis to tumorigenesis in BALB ß2m^–/–mice. Interestingly, ß2 microglobulin deficiency ona C57BL/6 background resulted in a dramatic increase of polyomavirus-induced tumors in spite of equivalent levels of persistentviral gene expression and serum virus neutralizing antibodiesin ß2m^–/– and wild-type mice (18).

It is also possible that the inability of BALB ß2m^–/–animals to control ${gamma}$ HV68-induced lymphoproliferative diseaseis due instead to a lack of surface expression of nonclassicalß2m-dependent class I molecules. These molecules arerecognized by nonclassical CD8⁺ T cells, as well as NK cells,and have been shown to be important in tumor rejection (8, 34).Other abnormalities associated with the loss of ß2microglobulin expression, including iron overload, could havealso played a role in the development of lymphoproliferativelesions in this strain (28, 42, 70, 73).

Role of mouse genetic background in susceptibility to lymphoproliferative disease. The differences in oncogenic properties of gammaherpesviruseswithin species have been extensively documented for the HVSinfection of primates. While HVS is ubiquitously present inT cells of healthy squirrel monkeys (36), it rapidly inducesT-cell lymphomas in New World monkeys and marmosets (37). Wealso examined the role of background genes in development oflymphoproliferative disease. In contrast to the robust tumorigenesisobserved in ${gamma}$ HV68-infected BALB-ß2m^–/–mice, no tumors were detected in 129-ß2m^–/–mice, demonstrating that background genes contribute to susceptibilityto or control of ${gamma}$ HV68-associated lymphomagenesis.

The fact that mock-infected BALB ß2m^–/–mice develop similar lymphoproliferative disease as ${gamma}$ HV68-infectedmice (Fig. 1), albeit at a lower incidence and after a longerincubation time, suggests that the role of ${gamma}$ HV68 infection maybe to simply exacerbate an existing propensity to develop lymphoproliferativelesions which clearly seems to depend on the background andgender of the animal. It is interesting to speculate that theclear effect of background genes in our system may reflect amore general role for allelic variation in the susceptibilityto gammaherpesvirus-associated disease. In this scenario, thereason that some hosts develop lymphoproliferative disease whenimmunosuppressed while others do not is that polymorphic susceptibilitygenes play a role. For example, polymorphisms in the gamma interferon(IFN- ${gamma}$ ) gene have been associated with development of human posttransplantlymphoproliferative disease (PTLD), a lesion that is histologicallysimilar to the atypical lymphoid hyperplasia described here.In a small study, an IFN- ${gamma}$ gene polymorphism associated withlow production of IFN- ${gamma}$ positively segregated with developmentof PTLD in renal transplant patients (62). Importantly, IFN- ${gamma}$ is a key regulator of ${gamma}$ HV68 reactivation from latency (47a).Recently this same gene polymorphism was associated with thedevelopment of EBV⁺ lymphoproliferative disease in the humanperipheral blood lymphocyte-SCID mouse model (16).

The BALB/c background is associated with a functional polymorphismof the coding region of DNA dependent protein kinase catalyticsubunit involved in DNA repair following ionizing radiationexposure (72). The BALB variant of this gene produces a kinasewith decreased activity and, therefore, increases susceptibilityof BALB mice to ionizing radiation-induced genomic instability(72). Another functional coding polymorphism unique to BALB/cresults in decreased activity of p16INK4a, a cyclin-dependentkinase inhibitor (17). Crossing of BALB/c, but not other strains,to p53-null mice was important to establish the mouse modelreflecting the critical role of p53 heterozygosity in breastcancer development (33). In our study, the presence of atypicallymphoid hyperplasia in uninfected BALB ß2m^–/–mice revealed that ß2m deficiency combined with theBALB background predisposes to lymphoproliferative disease.Although ${gamma}$ HV68-dependent lymphoproliferative disease has beenreported in BALB/c mice (51), the shorter duration of this study,differences in mouse strains/suppliers, and animal housing conditionscould have contributed to the absence of tumors in BALB/c ${gamma}$ HV68-infectedanimals in our study.

Relationship between ${gamma}$ HV68-associated atypical lymphoid hyperplasia and lymphoma. ${gamma}$ HV68 infection accelerated development and increased incidenceof two lymphoproliferative lesions in BALB ß2m^–/–mice: lymphoma and atypical lymphoid hyperplasia. Compared tolymphomas, atypical lymphoid hyperplasia lesions appeared ata higher frequency and at an earlier time postinfection in animalsinfected with ${gamma}$ HV68. Expression of CD138, which we observed inatypical lymphoid hyperplasia lesions, has also been reportedto be present at high frequency in KSHV-associated primary effusionlymphoma (26).

Atypical lymphoid hyperplasia lesions have significant similaritiesto lesions of human PTLD. We have selected the term ALH ratherthan PTLD to emphasize that there is no current proof that themechanisms underlying ${gamma}$ HV68-associated ALH are shared with humanPTLD. Importantly, atypical lymphoid hyperplasia lacked featurestypical for malignant lesions. ALH lesions were spatially restrictedwithin the disrupted white pulp area and lacked mitotic figuresand apoptotic bodies. Because of the earlier appearance of ALHas compared to lymphomas in ${gamma}$ HV68-infected animals and becauseof the retention of some CD138⁺ cells in the ${gamma}$ HV68-associatedlymphomas, it is tempting to speculate that ALH is a precursorto malignancy. However, a causal relationship between the twolymphoproliferative diseases cannot be established by thesemorphological studies.

Role of ${gamma}$ HV68 infection in the development of atypical lymphoid hyperplasia. Clusters of vtRNA-positive cells were observed in atypical lymphoidhyperplasia lesions, but not in spleens with normal histologytaken from ${gamma}$ HV68-infected animals. It is not clear at this pointwhether these cells represent expansion of latent or lyticallyinfected cells. Interestingly, EBV-triggered PTLD in humansis characterized by increased numbers of EBV-positive cellsand increased viral replication and leads to malignancies ifnot controlled (55). Perhaps lymphoma cells arise from the vtRNA-positivecells associated with atypical lymphoid hyperplasia. In thefuture, it will be important to determine the type of cell expressingvtRNAs in ALH lesions.

Role of ${gamma}$ HV68 infection in the development of lymphoma. The increased incidence and accelerated development of lymphoproliferativelesions in ${gamma}$ HV68-infected BALB ß2m^–/– mice(Fig. 1) strongly argues for the role of ${gamma}$ HV68 infection in thegenesis of lymphomas. However, unlike certain EBV-associatedtumors, only a few tumor cells in ${gamma}$ HV68-infected animals werepositive for vtRNA expression. Our results are in agreementwith previously published studies of ${gamma}$ HV68-associated lymphomagenesisin wild-type mice, where only a small proportion of malignantcells were reported to harbor ${gamma}$ HV68 in infected animals (39,51). Thus, the direct transformation model that operates incertain EBV-associated tumors is clearly not applicable to ${gamma}$ HV68-associatedlymphomagenesis in BALB ß2m^–/– mice.

The role of ${gamma}$ HV68 vtRNA-positive cells in lymphomas is not clear.They may be incidental to the malignancy process. However, itis also possible that these cells are driving the malignancyprocess. For example, although only a small portion of cellsin Hodgkin's lymphoma are EBV positive, it is thought that thesecells are the malignant cells surrounded by nonmalignant inflammatorycell infiltrate. Interestingly, it was reported that EBV waslost from a malignant Hodgkin's tumor upon relapse of the samemalignancy, raising an intriguing possibility that EBV presencemay not be required to maintain a malignant phenotype of tumorin vivo (15). Unfortunately, a true case of hit-and-run oncogenesismay be difficult to prove for human gammaherpesviruses, especially,in the case of a "clean exit" where the viral genome is completelylost (1).

Although the direct transformation model with continuous presenceof viral genome in malignant cells is not applicable to ourtumor system, several other mechanisms of virus-associated lymphomagenesismay contribute to the ${gamma}$ HV68-induced lymphoproliferative disease. ${gamma}$ HV68-infected cells might stimulate proliferation and transformationof uninfected malignant B cells through release of growth factors(paracrine model). A similar situation may occur in Kaposi'ssarcomas where release of vascular endothelial growth factorby KSHV-infected cells expressing viral G protein-coupled receptorcan stimulate angiogenesis and proliferation of uninfected cellsin the lesion (6).

Alternatively, chronic inflammation associated with ${gamma}$ HV68 infectionmay drive proliferation of antigen-specific and bystander Bcells that could subsequently acquire deleterious mutationsleading to transformation. The monoclonal nature of some ${gamma}$ HV68-associatedtumors suggests that a single transformed B cell gave rise tothe observed malignancies. Interestingly, in humans, B-cellreceptors of chronic lymphocytic lymphoma cells are strikinglysimilar among several independent cases and resemble monoclonalantibodies reactive with carbohydrates found on pathogens (29),suggesting that chronic B-cell stimulation may play a role inhuman lymphomagenesis. If chronic inflammation is indeed theprimary driving force behind ${gamma}$ HV68-associated lymphomagenesisin our tumor model, then other mouse pathogens that cause chronicinfections (i.e., mouse cytomegalovirus) may also trigger lymphoproliferativedisease in BALB ß2m^–/– mice. Therefore,monitoring of lymphoproliferative disease in BALB ß2m^–/–mice chronically infected with other viruses is an importantpriority for future studies.

In summary, ß2m deficiency in BALB/c mice greatlyincreases susceptibility to ${gamma}$ HV68-associated lymphomagenesis. ${gamma}$ HV68 infection of these animals leads to increased incidenceand accelerated development of atypical lymphoid hyperplasiaand B-cell lymphoma and represents a valuable tool to studyhost and viral factors involved in gammaherpesvirus-associatedpathogenesis in a natural host. Because ${gamma}$ HV68 is geneticallyand biologically related to the human gammaherpesviruses, EBVand KSHV, identification of mechanisms of lymphomagenesis associatedwith ${gamma}$ HV68 infection in vivo might contribute to the understandingof lymphomagenesis associated with human gammaherpesviruses.The availability of a model in which one can study potentialmechanisms of tumorigenesis such as hit and run, paracrine,and chronic inflammation should enhance chances of understandingfundamental mechanisms responsible for virus-associated tumors.

ACKNOWLEDGMENTS

H.W.V. and S.H.S. were supported by National Institutes of Health(NIH) grant CA74730. V.L.T. was supported by an NIH traininggrant T32 CA 09547 and NIH/NRSA fellowship F32 AI065079-01.F.S. was supported by and American Cancer Society postdoctoralfellowship. S.A.T. is a Leukemia and Lymphoma Society specialfellow. We acknowledge the assistance of core histopathologyfacilities funded by DRTC and NIH grant P30 DK52514.

We thank Darren Kreamalmayer for his outstanding expertise inthe breeding of knockout animals and all members of the Virginlab for helpful discussions. A special acknowledgment goes toStacy Efstathiou, who selflessly provided reagents and helpfulsuggestions for analysis of tumors by in situ hybridization.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology and Immunology, Washington University, 660 S. Euclid Ave, Box 8118, St. Louis, MO, 63110. Phone: (314) 362-9223. Fax: (314) 362-0369. E-mail: virgin{at}wustl.edu.

V.L.T. and F.S. contributed equally to this study.

REFERENCES

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