The viral latency-associated nuclear antigen augments the B-cell response to antigen in vivo

Abstract

Gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV), establish latency in B cells. We hypothesized that the KSHV latency-associated nuclear antigen (LANA/orf73) provides a selective advantage to infected B cells by driving proliferation in response to antigen. To test this, we used LANA B-cell transgenic mice. Eight days after immunization with antigen without adjuvant, LANA mice had significantly more activated germinal center (GC) B cells (CD19(+) PNA(+) CD71(+)) than controls. This was dependent upon B-cell receptor since LANA did not restore the GC defect of CD19 knockout mice. However, LANA was able to restore the marginal zone defect in CD19 knockout mice.

FIG. 1.

LANA augments the B-cell response to immunization. (A) Box plot of flow cytometry results. The frequency of triple-positive (CD19⁺ PNA⁺ CD71⁺) among CD19⁺ was determined and plotted. The transgene status is shown on the bottom, and the treatment is shown at the top of the graph. A dot indicates the median, the box indicates the interquartile range (IQR), bars denote the range, and open dots denote outliers (>1.5 × IQR). NP-KLH, 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin; LANA, LANA transgenic; WT, wild type. (B) Immunohistochemistry against GL7 antigen (brown) and hematoxylin (blue). Shown are representative images of spleen (magnification, ×40) for wild-type (WT), LANA transgenic (LANA), wild-type NP-KLH-immunized (WT-KLH), and LANA NP-KLH-immunized (LANA-KLH) animals.

FIG. 2.

LANA augments the proliferative capacity of B cells in culture. (A to D) Examples of flow cytometric analysis of purified cultured B cells labeled with CFSE after 4-day stimulation with LPS (gray density) or nonstimulation control (open diagram). Stimulation is measured by the fraction of cells that lose fluorescent intensity. The horizontal line (and number) indicates the gate that was used for quantification. The CFSE fluorescence intensity is shown on the horizontal axis, and the number of cells as a percentage of the maximum is shown on the vertical axis. Results with B cells from LANA transgenic mice are shown in panels C and D, and results from wild-type littermate control animals are shown in panels A and B. Animals in panels B and D were in vivo stimulated by NP-KLH immunization prior to harvest. (E) Box-and-whisker plot of the results from multiple animals. A dot indicates the median, a box indicates the interquartile range (IQR), bars indicate the range, and open dots denote outliers (>1.5 × IQR). The three asterisks indicate a significant difference between groups of six LANA and six wild-type mice at P ≤ 0.005 as determined by using the Wilcoxon rank sum test.

FIG. 3.

LANA does not complement the B-cell deficiency in CD19 knockout mice. (A) Shown is a representative picture of an ethidium bromide-stained 1% agarose-TAE gel of PCR products using mouse tail DNA. One hundred-base-pair molecular weight markers are shown on the left (MW); inferred genotypes are shown at the top. WT, wild type; NTC, nontemplate control. (B) Box plot of flow cytometry results (n = 30, animals are not incorporated in Table 1). The frequency of triple-positive (B220⁺ IgD⁺ PNA⁺) was determined and plotted. The LANA transgene status is shown at the bottom, and the CD19 status is shown at the top of the graph. Dot indicates the median, a box indicates the interquartile range (IQR), bars indicate the range, and open dots denote outliers (>1.5 × IQR). del, deletion for CD19 (CD19^−/−); wt (top), CD19^+/+; LANA, LANA transgenic; wt, wild-type (LANA^−/−). The morphology of the spleen in wild-type (C and D), LANA transgenic (E and F), and CD19^−/−/LANA^+/− (G and H) mice is shown. Representative H&E-stained images are presented. The upper row is at ×100 magnification, the lower row is at ×40 magnification. MZ, marginal zone, GC, germinal center. The asterisk in panel G denotes the absence of GC in CD19^−/−/LANA^+/− mice. Immunohistochemistry for PNA (brown) counterstained with hematoxylin (blue) was performed. Representative sections for wild-type (I), LANA transgenic (J), and CD19^−/−/LANA^+/− (K) mice are presented.

FIG. 4.

LANA complements the MZ defect in CD19 knockout mice. (A) Flow cytometry analysis of B220⁺ B cells in a 15-week-old LANA^−/−/CD19^−/− mouse spleen. (B) Flow cytometry analysis of B220⁺ B cells in an age-matched LANA^+/−/CD19^−/− mouse spleen. (A and B) Gates represent B220⁺ CD21^hi CD23⁻ MZ B cells. (C) Percentage of MZ B cells (B220⁺ CD21^hi CD23⁻) in LANA^−/−/CD19^−/− mice (n = 6) and LANA^+/−/CD19^−/− mice (n = 9). (D) Immunofluorescence for MOMA-1 (a marginal zone macrophage marker) in red and B220 (a B-cell marker) in green. The panels show results from wild-type C57BL/6, no-primary-antibody-control, CD19^−/−, and LANA^+/−/CD19^−/− mice. Arrowheads indicate that MZ architecture was rescued partially in a LANA^+/−/CD19^−/− mouse. Magnification, ×200.