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. 2013 Apr 11;121(15):2952-63.
doi: 10.1182/blood-2012-03-415620. Epub 2013 Jan 30.

Viral latency locus augments B-cell response in vivo to induce chronic marginal zone enlargement, plasma cell hyperplasia, and lymphoma

Sang-Hoon Sin  1 , Dirk P Dittmer
Affiliations

Viral latency locus augments B-cell response in vivo to induce chronic marginal zone enlargement, plasma cell hyperplasia, and lymphoma

Sang-Hoon Sin et al. Blood. .

Abstract

Kaposi sarcoma (KS) is associated with KS-associated herpesvirus (KSHV). This virus also causes B-cell lymphoma and B-cell hyperplasia. There exists no in vivo model for KSHV-associated B-cell malignancies or premalignant persistence in B cells. We generated a transgenic mouse that expresses multiple viral latent genes, including LANA, vFLIP, vCYC, all viral micro RNAs, and kaposin under the transcriptional control of their natural regulatory region. This promoter is B-cell specific, though it is a weak promoter. Mature B cells were chronically activated, leading to hyperglobulinemia triggered by increased plasma cell frequency and marginal zone (MZ) B-cell hyperplasia. The mice had an augmented response to T-dependent antigen as well as the TLR4 ligand LPS, leading to exacerbated MZ and germinal center responses and increased CD138(+) plasma cells. It is the first model to assess the viral micro RNA function in vivo. These data support a potentially novel mechanism of viral persistence in which virally infected B cells become hyper-responsive to coincident, but unrelated, pathogen exposure, leading to preferential expansion and ultimately lymphoma in a small subset of cases.

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Figures

Figure 1
Figure 1
Transcription of KSHV miRNAs. (A) Transcription of pre-miRNAs and other transgenes was represented as RT-qPCR Ct values from 2 transgenic mice from 2 independent lines or a wild-type (WT) mouse. BC3, a PEL cell line, was used as a positive control. (B) Transcription of pre-miRNAs was validated using Bioanalyzer. RT+ and RT− represent RT-PCR reactions and reactions without reverse transcriptase, respectively. (C) Expression of mature miRNAs was confirmed using the TaqMan assay. Amplified products were visualized via the LabChip system. BC3, a PEL cell line was used as a positive control and U6 was used as a positive control for TaqMan assay system.
Figure 2
Figure 2
Phenotype of KSHV latency locus transgenic mice. Splenocytes were subject to flow cytometric analyses. (A) Representative mature B-cell profiles from wild-type (WT), KSHV LANA transgenic (LANA), and KSHV latency locus transgenic mouse (Latency). Percentages of IgD+IgM+ lymphocytes among whole lymphocytes are indicated above each gate. (B) Profiles of activated (FSChi) mature B cells are shown. Gates represent CD19+IgD+IgM+FSChi-activated B cells. (C) MZ B cells are gated as IgD-IgM+. (D) Activated MZ B cell (CD19+IgD-IgM+FSChi) profiles are shown. (E) Comparison of activated mature B cells defined as CD19+IgD+IgM+FSChi from WT (n = 9), ko (CD19 knockout, n = 4), LANA (KSHV LANA transgenic, n = 13), and latency (KSHV latency locus transgenic, n = 23) mice. tg125 (n = 8) and tg151 (n = 5) mean 2 independent lines of the KSHV LANA transgenic mice. tg442 (n = 9), tg455 (n = 6), tg456 (n = 5), and tg633 (n = 3) represent 4 independent lines of the KSHV latency locus transgenic mice.
Figure 3
Figure 3
MZ expansion in KSHV latency locus transgenic mice. (A) Splenocytes from wild-type (WT) or latency transgenic mouse (TG) were analyzed by fluorescence-activated cell sorter (FACS). CD19+CD24+ cells were further gated with CD21 and CD23. Percentages of CD19+CD24+CD21hiCD23- cells among whole lymphocytes are indicated in FACS profiles. (B) Alternatively, CD19+IgD- cells were gated with CD21 and CD23 in an independent analysis. (C) Bar plot of flow cytometry data from panels A-B (n = 5 for WT and n = 9 for the transgenic mice). (D) MZ precursor cells were gated as CD19+IgD+CD21hiCD23+. Average percentages of 6 WT and 9 latency transgenic (TG) mice are shown with the standard deviation. (E) Frozen spleen sections were stained with B220 (green) for B cells and MOMA-1 for marginal zone macrophages (red). Magnification ×200. FL, follicle.
Figure 4
Figure 4
LPS-induced B-cell expansion in KSHV latency locus transgenic mice. (A-B) Frozen spleen sections were stained with B220 (green) and PNA (orange). Magnification ×200. KSHV latency transgenic mice were injected with LPS. On d 1 and 7 after injection, splenic cells were analyzed by fluorescence-activated cell sorter. (C-D) Percentages of activated GC B cells (CD19+CD71+PNAhi) and activated MZ B cells (CD19+CD21hiCD23-CD69+) are shown on d 1 after LPS injection. Averages of 6 wild-type (WT) and 6 latency transgenic (TG/Tg) mice are indicated with the standard deviation. (E) Expression of CD69, an activation marker on the CD19+CD21hiCD23- gated MZ B cells. Veh, vehicle.
Figure 5
Figure 5
TD antigen-driven GC response in KSHV latency locus transgenic mice. KSHV latency locus transgenic mice and littermate controls were immunized with NP-KLH or PBS (n = 6). After 10 d, splenocytes were subject to fluorescence-activated cell sorter (FACS) analysis. Blood was collected on d 0, 5, 10, and 14. (A) Activated GC (CD19+CD71+PNAhi) are plotted. (B) Levels of resting non-NP–specific Igs of 9 wild-type (WT; red triangle) and 6 transgenic mice (black circle) were plotted on the first or second column, respectively. * and *** represent significant difference with P < .05 and P < .005 by Student t test, respectively. (C) The frequency of plasma cells was analyzed by FACS. Cells from spleens (SP; n = 5) or BM (n = 6) in WT or latency transgenic mice (TG) were subject to FACS. CD19- cells were further gated with B220 and CD138. Percentages of CD19-B220-CD138+ cells among CD19- lymphocytes are plotted. (D) Representative flow cytometry profiles of plasmablasts (CD19-B220+CD138+) and plasma cells (CD19-B220-CD138+) from FACS analysis in (C) were shown.
Figure 6
Figure 6
Monoclonal IgH rearrangement in tumors from KSHV latency locus transgenic mice was evaluated using PCR. (A) Splenomegaly found in a KSHV latency locus transgenic mouse with the spleen of a wild-type (WT) mouse. Bar represents 0.5 cm. (B-D) PCR products of splenic genomic DNAs from latency transgenic mice, 2 mouse lymphoma cell lines (i and ii), and 1 C57BL/6 mouse (WT) were resolved on a 1% Tris-acetate-EDTA-agarose gel. The WT lane shows 4 rearrangements of VDJH1, VDJH2, VDJH3, and VDJH4 (B-D) using DH5 (B), Dq52 (C), or VH7183 primers (D). ApoB was amplified to demonstrate an equal amount of genomic DNA was used for PCR (F). Cell lines i and ii mean mouse lymphoma cell lines; K46 and M12. M; 1-kb or 100-bp DNA ladder. Spleen architectures of the same mouse as in Figure 6A (E, TG) and littermate control mouse (E, WT) revealed by hematoxylin and eosin staining. Magnification ×400. (G) Spleen sizes of the KSHV latency transgenic (TG) and littermate control mice (WT) were plotted.
Figure 7
Figure 7
Model for KSHV latency locus-mediated augmentation of B-cell response. The expression of KSHV latent genes in mouse B cells results in mature B-cell activation, expansion of MZ and plasma cells, and augmented B-cell response to antigen. The status of constant B-cell activation and subsequent hyper-responsiveness to antigen in vivo suggests that KSHV latency locus serves as a chronic antigenic stimulator to the viral lymphomagenesis. Ag, antigen; CB, centroblast; CC, centrocyte; FO, follicular B cell; PC, plasma cell; T, T cell.
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