Michelle Swanson-Mungerson

Epstein-Barr Virus LMP2A Alters In Vivo and In Vitro Models of B-Cell Anergy, but Not Deletion, in Response to Autoantigen

ABSTRACT A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-κB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-κB nuclear translocation independent of BCR cross-linking. Since NF-κB is required to bypass tolerance induction, this LMP2A-dependent NF-κB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.

Epstein–Barr virus LMP2A accelerates MYC-induced lymphomagenesis

Despite the identification of Epstein–Barr virus (EBV) in tumors of Burkitts lymphoma (BL) over 40 years ago, the exact contribution of EBV to BL is undefined. EBV encodes for multiple proteins in latent B cells that affect B cell survival and activation. One such protein, latent membrane protein 2A (LMP2A), protects B cells from numerous pro-apoptotic stimuli. Therefore, we tested whether LMP2A protects B cells from apoptosis induced by aberrant c-MYC expression that precedes and dominates BL. We crossed LMP2A-transgenic mice (LMP2A-Tg), in which all B cells express LMP2A, to a transgenic mouse that expresses a BL translocation of myc (λ-MYC-Tg mice). LMP2A promotes proliferation and protects B cells from MYC-induced apoptosis in λ-MYC-Tg mice. LMP2A also accelerates the development of lymphoma in LMP2A/λ-MYC-Tg mice. Finally, LMP2A increases the expression of Bcl-XL in both pre-tumor B cells and tumor cells, suggesting a mechanism for LMP2A-mediated B cell survival in the presence of MYC. These results support a hypothesis that EBV LMP2A promotes tumor development by protecting pre-tumor B cells that would normally apoptose after the c-myc translocation.

Epstein-Barr virus in Burkitt's lymphoma: A role for latent membrane protein 2A

Burkitt’s lymphoma (BL) is characterized by translocation of the MYC gene to an immunoglobulin locus. Transgenic mouse models have been used to study the molecular changes that are necessary to bypass tumor suppression in the presence of translocated MYC. Inactivation of the p53 pathway is a major step to tumor formation in mouse models that is also seen in human disease. Human BL is often highly associated with Epstein-Barr virus (EBV). The EBV latency protein latent membrane protein 2A (LMP2A) is known to promote B cell survival by affecting levels of pro-survival factors. Using LMP2A transgenic mouse models, we have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work uncovers a contribution of EBV to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.

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.

Epstein-Barr virus LMP2A imposes sensitivity to apoptosis.

In cell lines, the Epstein–Barr virus (EBV)-encoded protein latent membrane protein 2A (LMP2A) protects B-cells from apoptosis by blocking B-cell receptor (BCR) signalling. However, EBV-infected B-cells in vivo are extremely different from cell lines. This study used a murine transgenic model in which B-cells express LMP2A and a BCR specific for hen egg lysozyme to determine whether LMP2A protects resting and antigen-activated B-cells from apoptosis. LMP2A allows BCR signal transduction and induces constitutive activation of NF-κB to increase Bcl-2 levels that afford LMP2A-mediated protection from apoptosis in the absence or presence of antigen. In contrast, low levels of NF-κB inhibitor only affected Bcl-2 and Bcl-xL levels and increased apoptosis in LMP2A-negative B-cells after BCR cross-linking. These data suggest that LMP2A uniquely makes resting B-cells sensitive to NF-κB inhibition and apoptosis and suggest that NF-κB may be a novel target to eradicate latently EBV-infected B-cells.

Epstein–Barr virus LMP2A increases IL-10 production in mitogen-stimulated primary B-cells and B-cell lymphomas

Epstein-Barr virus (EBV) latently infected B-cells are the precursors of EBV-associated malignancies. EBV-infection induces the production of pro-survival and anti-inflammatory cytokines that may be important in the transition between latency and malignancy. One EBV protein, LMP2A, can be detected in both latently infected resting B-cells and in EBV-associated malignancies. Therefore, we tested the ability of LMP2A to influence cytokine production using both LMP2A-Tg primary B-cells and LMP2A-expressing B-cell lines. Our data demonstrate that LMP2A does not globally alter B-cell-produced cytokine levels, but specifically targets IL-10. Additional studies using ELISA and real-time-RT-PCR confirm that LMP2A utilizes PI3-kinase to increase IL-10 levels. Finally, the data demonstrate that LMP2A-expressing B-cell lines are more dependent on IL-10 for survival in comparison to LMP2A-negative B-cell lines. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumour survival and anti-tumour responses.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 production through the activation of Bruton's tyrosine kinase and STAT3

Previous data demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 to promote the survival of LMP2A-expressing B cell lymphomas. Since STAT3 is an important regulator of IL-10 production, we hypothesized that LMP2A activates a signal transduction cascade that increases STAT3 phosphorylation to enhance IL-10. Using LMP2A-negative and -positive B cell lines, the data indicate that LMP2A requires the early signaling molecules of the Syk/RAS/PI3K pathway to increase IL-10. Additional studies indicate that the PI3K-regulated kinase, BTK, is responsible for phosphorylating STAT3, which ultimately mediates the LMP2A-dependent increase in IL-10. These data are the first to show that LMP2A signaling results in STAT3 phosphorylation in B cells through a PI3K/BTK-dependent pathway. With the use of BTK and STAT3 inhibitors to treat B cell lymphomas in clinical trials, these findings highlight the possibility of using new pharmaceutical approaches to treat EBV-associated lymphomas that express LMP2A.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.