The latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus induces B cell hyperplasia and lymphoma

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA's contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+ IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.

Figure 1

Development of LANA transgenic mice. (A) The LANA transcription locus (14) and transgene construct carrying LacZneo (27) as previously described and the LANA transgene construct used here. SV40 pA, simian virus 40 poly A; β-geo, β-galactosidase-neomycin; TK, thymidine kinase. (B) Southern blot analysis of tail DNA, digested with BamHI and hybridized with a LANA-specific probe to reveal the founder-specific integration sites. Lane 1 shows the transgene plasmid as a positive control (Pos.), lane 5 contains the DNA from a C57BL/6 mouse as negative control (Neg.), and remaining lanes represent DNA from LANA transgenic animals belonging to lines i, ii, and iii (animals 4 and 6 are siblings). Arrowhead indicates a fragment specific for all transgenic animals; other bands are integration-site specific. (C and D) Real-time quantitative RT-PCR analysis of spleen samples for LANA transgene mRNA or apoB housekeeping mRNA resolved on a 2% tris-borate-EDTA–agarose gel and (C) subsequent Southern blot analysis with a probe specific for LANA (LANA-P). Lanes i–v refer to spleen RNA from 5 different transgene-positive animals. NTC, nontemplate control; MW, 100-bp molecular weight marker. (D) Lanes 1–3 show transgene-negative, and lanes 4–6 transgene-positive, littermates after backcross into the C57BL/6 background.

Figure 2

LANA expression in spleens. (A) Immunohistochemistry using a rat mAb against LANA (right panels) or rabbit polyclonal antibody against PCNA (left panels) and visualization with NovaRed (brown) and hematoxylin (blue) counterstain in a transgene-negative and a LANA-positive animal. Arrowhead indicates the localization of LANA-positive cells. Magnification, ×400. (B) Diameter of LANA-positive foci for wild-type and transgenic animals (P ≤ 0.01, n = 11). The box represents the interquartile range, the line within the box indicates the median and whiskers indicate the highest and lowest values.

Figure 3

Analysis of GC development in a representative LANA and littermate wild-type mouse. Markers are indicated on the left (No 1°, no primary antibody). All sections are counterstained blue with hematoxylin. Magnification, ×400 (bottom 2 panels); ×100 (remaining panels).

Figure 4

GC response to NP(30)-KLH. Spleen sections of wild-type and LANA transgenic mice with or without NP(30)-KLH immunization (-I) were stained with PNA. Arrowheads indicate GCs. Magnification, ×40 (top panels); ×100 (bottom panels).

Figure 5

Anaplastic plasmacytoma in a LANA transgenic animal. (A–D) IgG-κ expression (surface and intracellular) in a lymphoma from LANA transgenic (A and C) and nontransgenic (B and D) littermates. (E and F) CXC-chemokine receptor 4 (CXCR4) for expression in LANA transgenic (E) or control (F) mice. Magnification, ×100 (A, B, E, and F); ×400 (C and D). All sections are counterstained blue with hematoxylin. (G and H) Flow cytometry analysis of the anaplastic plasmacytoma. (I and J) Flow cytometry analysis of spleen of a nontransgenic littermate. (G and I) Gates represent CD138⁺B220⁺CD27^– lymphoma cells at 16% frequency and normal spleen cells at 3% frequency. (H and J) Gates represent IgG-κ⁺B220^–CD27^– lymphoma cells at 20% frequency and normal spleen cells at 2% frequency.

Figure 6

H&E stain of wild-type mice and phenotypes in LANA transgenic mice — LPD, MZ lymphoma (MZ), and anaplastic plasmacytoma (PC). Arrowheads indicate individual cells that exemplify the phenotype in panels E, G, and H. Magnification, ×100 (A–D); ×400 (E–H).

Figure 7

PCR analysis of VDJ_H and DJ_H rearrangements in tumors of LANA transgenic mice. Total spleen genomic DNAs were used in the PCR reactions analyzing tumors. (A–C) PCR analysis of total spleen genomic DNAs from 4 LANA transgenic mice with tumors, 2 C57BL/6 mice older than 300 days (WT), and 2 different murine B cell lines resolved on a 1.5% Tris-acetate-EDTA–agarose gel, used as controls for the clonality. WT lanes demonstrate VDJ_H1, VDJ_H2, VDJ_H3, and VDJ_H4 rearrangements (A and B) using V_H7183 (A) or V_HJ558 primers (B) and DJ_H1, DJ_H2, DJ_H3, and DJ_H4 rearrangements (C) using Dq52 primers. The primers were designed to amplify all rearrangement products between J_H4 and V_H7183, V_HJ558, or Dq52; 4 different bands would be expected by the combination of each primer set. M, 100-bp DNA Ladder (New England Biolabs Inc.) (D) DNA samples used for the Ig rearrangement PCRs were quantified using real-time quantitative PCR to demonstrate equal total DNA concentrations.