Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro

Abstract

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.

FIG. 1.

Increased number of total cells and B cells in E/HEL-Tg Mice. (a and b) Spleens were asceptically removed and dissociated as described in Materials and Methods. Total cell number was determined by trypan blue exclusion. (c) Total spleen cell suspensions were stained for CD19 and the IgM^a transgene and analyzed by flow cytometry. The number of B cells was calculated by multiplying the percentage of CD19⁺ IgM⁺ B cells by the total number of spleen cells for each mouse. For panels b and c, each symbol indicates the result from an independent mouse. WT, wild type.

FIG. 2.

Architecture of spleens from HEL-Tg and E/HEL-Tg mice. (a to d) Spleens were isolated either before or 10 days after immunization with 100 μg/ml hen egg lysozyme. (a to d) Spleens were snap-frozen before sectioning and stained with anti-B220 before visualization with horseradish peroxidase-dependent oxidation of diaminobenzidine to produce a brown substrate. (c and d) Polarized follicles in germinal centers from immunized E/HEL-Tg mice contain many more B cells than germinal centers from immunized HEL-Tg mice (see red arrows in panel d versus c) (e) Spleens from immunized mice were made into single-cell suspensions before being stained with phycoerythrin-labeled anti-CD19 antibody and fluorescein isothiocyanate-labeled anti-GL7 antibody and analyzed by flow cytometric analysis. The graph shows the average number of CD19⁺ GL7⁺ B cells from three to four independent mice.

FIG. 3.

Antibody response in E/HEL-Tg mice in vivo. (a) Sera were collected before immunization and 10 days after immunization and analyzed for IgM levels as described in Materials and Methods. PI refers to preimmune sera, and D10 refers to sera collected 10 days after immunization. (b) Spleens from immunized mice were dissociated into single-cell suspensions, and equal numbers of cells were plated for ELISPOT analysis. The spots were developed and counted using an ELISPOT counter as described in Materials and Methods. The data represent the average number of antibody-secreting cells (ASC) from three HEL-Tg mice and three E/HEL-Tg mice after immunization. (c) Spleens from immunized mice were made into single-cell suspensions before staining with phycoerythrin-labeled anti-CD19 antibody and fluorescein isothiocyanate-labeled anti-CD138 antibody and analyzed by flow cytometric analysis. The graph is the average number of CD19⁺ CD138⁺ B cells from three to four independent mice.

FIG. 4.

Antibody response of E/HEL-Tg B cells in vitro. Equal numbers of HEL-Tg and E/HEL-Tg B cells were activated with 10 μg/ml hen egg lysozyme and 1 μg/ml anti-CD40 antibody for 3, 5, or 7 days at 37°C with 5% CO₂. (a) Supernatants from individual wells for each time point were harvested and analyzed for the production of cumulative HEL-specific IgM by ELISA as described in Materials and Methods. The data are an average of the optical density (OD) readings of multiple wells for HEL-specific IgM ± standard error. (b and c) The total cell number and percent viability were determined by trypan blue exclusion at the indicated time points. (d) HEL-Tg and E/HEL-Tg B cells were activated for either 3 or 5 days, and viable cell numbers were determined. Equal numbers of viable cells were plated for ELISPOT analysis for 4 h as described in Materials and Methods. The percentage of plasma cells was determined by the following equation: (no. of spots counted/no. of cells plated) × 100. The data are an average of multiple wells ± standard error. The percentage of plasma cells was not determined on day 7 by ELISPOT analysis due to the lack of an adequate number of viable cells for the assay. For panels a to d, the data are representative of two to three experiments with similar results.