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

Abstract

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

“…Our previous findings also indicate that LMP2A increases the levels of IL-10 RNA transcripts (Incrocci, McCormack, and Swanson-Mungerson, 2013) and therefore, we hypothesized that PI3K activation may result in the activation of a transcription factor that regulates IL-10 RNA levels. Since Signal Transducer and Activator of Transcription 3 (STAT3) increases IL-10 transcription (Gabrysova et al, 2014) and is regulated by PI3K (Hart et al, 2011), we hypothesized that the LMP2A-dependent activation of PI3K would result in STAT3 phosphorylation, which is a pre-requisite for STAT3 induction as a transcription factor (Harrison, 2012).…”

“…LMP2A is a BCR mimic that directly promotes B cell survival through a RAS/PI3K dependent pathway (Portis and Longnecker, 2004) and indirectly by inducing IL-10 production in both LMP2A-expressing primary murine transgenic B cells and in B cell lymphomas (Incrocci, McCormack, and Swanson-Mungerson, 2013). An additional study at the time indicated that the Syk inhibitor, R406, blocked the enhancement of IL-10 production in EBV-positive tumor cells from post-transplant lymphoproliferative disease (PTLD) patients (Hatton et al, 2011).…”

“…Our previous work using the PI3K inhibitor, Ly294002, suggested that LMP2A utilizes PI3K to increase IL-10 production (Incrocci, McCormack, and Swanson-Mungerson, 2013). However, due to the identification that Ly294002 can also affect NF-kB activation (Avni, Glucksam, and Zor, 2012), we wanted to confirm our previous findings through the use of the more PI3K-specific inhibitor Wortmannin (Avni, Glucksam, and Zor, 2012).…”

“…Briefly, the BJAB B cell lymphoma line was transduced with either the vector backbone alone or the vector backbone with LMP2A. Transduced cells were selected using hygromycin and gentamycin and LMP2A expression was identified in all selected cells by immunofluorescence and found to be similar in levels when compared to lymphoblastoid cell lines (Incrocci, McCormack, and Swanson-Mungerson, 2013). Independent clones were isolated and maintained in cRPMI media supplemented with hygromycin (0.4 ug/ml) (EMD Millipore) and gentamycin (2 ug/ml) (Sigma Aldrich) at 37°C/5% CO 2 .…”

“…Additional studies demonstrate that LMP2A signaling in B cells directly results in an increase in anti-apoptotic factors, such as BCL-2 and BCL-xL (Bultema, Longnecker, and Swanson-Mungerson, 2009; Portis and Longnecker, 2004; Swanson-Mungerson, Bultema, and Longnecker, 2010). More recently, it has become appreciated that LMP2A indirectly promotes B cell survival by increasing the production of pro-survival cytokines, such as IL-10 (Incrocci, McCormack, and Swanson-Mungerson, 2013). Due to the redundant expression of LMP2A throughout many phases of the EBV life cycle, targeting its pro-survival abilities in EBV-associated tumors may be of therapeutic benefit.…”