CD137 Expression Is Enhanced in EBV-Infected T or NK Cells by Viral Protein LMP1 and Mediates Anti-Apoptotic Intracellular Signaling through NF-ĸB Activation.

Abstract 1929

Poster Board I-952

Epstein-Barr virus (EBV) can infect not only B cells but also T or NK cells uncommonly and causes lymphoid malignancies, such as extranodal NK/T-cell lymphoma nasal type (ENKL), aggressive NK-cell leukemia, and EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), which is also known as chronic active EBV infection. However, why and how EBV infects T or NK cells and the mechanism of action responsible for these EBV-induced malignancies have not been elucidated to date. To clarify the molecular mechanism underlying development of EBV-T/NK-LPD, we focused on costimulatory receptor CD137, which is expressed on the surface of activated T cells and plays a pivotal role in their proliferation, survival, and differentiation. We investigated CD137 expression on the surface of EBV-infected T/NK cells (EB-T/NK cells) by flow cytometry. First, three EBV-positive T and NK cell lines, SNT8, SNK6, and SNT16, were obtained for examination. These cell lines had been established from primary lesions of ENKL patients (SNT8 and SNK6) and peripheral blood of an EBV-T/NK-LPD patient (SNT16). CD137 expression was confirmed on the cell surface of these cells, whereas the EBV-negative T and NK cell line, Jurkat and KHYG1 cells, respectively, were negative for CD137. Next, we investigated expression on the surface of EB-T/NK cells derived from EBV-T/NK-LPD patients. EBV-T/NK-LPD was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in the peripheral blood (PB), and detection of EBV-infected T or NK cells. To detect the infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. Next, we measured the EBV-DNA titer of each fraction by quantitative RT-PCR. Nine patients (aged 8–41 years; 4 male, 5 female; 4 T and 5 NK cell types) were diagnosed with EBV-T/NK-LPD. Then, we examined surface CD137 expression of the infected cells of each patient. Expression was detected in 7 of 9 patients. Control cells (PB mononuclear cells of a healthy donor, who was negative for EBV-DNA titer in the PB) did not express the molecule. We also examined transcription of CD137 mRNA by RT-PCR assay and detected it in all the 12 EB-T/NK-cell samples described above. From these results we concluded that CD137 expression was induced at the level of both mRNA and protein in EB-T/NK cells. To investigate the molecular mechanism of CD137 overexpression in EBV-T/NK cells, we examined the influence of viral proteins on CD137 expression. EB-T/NK cells express EBV-encoded proteins, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We cotransfected expression plasmids for these proteins with a luciferase reporter plasmid containing the CD137 gene promoter in Jurkat cells and performed a luciferase assay. LMP1 significantly upregulated the CD137 promoter activity, although the other molecules did not. Furthermore, in a transient expression assay of these viral proteins using Jurkat cells, transcription of endogenous mRNA of CD137 was upregulated only in the LMP1 transfectant. These results indicate that LMP1 may transactivate CD137 transcription and expression in EBV-T/NK cells. Next, we investigated the role of CD137 in developing EBV-T/NK-LPD. We cultured the above-mentioned CD137-expressing EBV-T/NK cells on CHO cells that stably express human CD137L on the cell surface. NF-ĸB activation was detected in CD137-positive EBV-T/NK cells that were cocultured with CD137L-expressing CHO cells. We confirmed that both p50 and p52 translocated to the nucleus, indicating that both canonical and non-canonical pathways for NF-ĸB activation were activated downstream of CD137. Finally, we investigated the role of CD137-mediated NF-ĸB activation in the development of EBV-T/NK-LPD. We cocultured EB-T/NK cells on CHO-wt or CHO-CD137L with VP-16 for 48 h and determined apoptosis by measuring DiCO6 uptake. We noted that stimulation of CD137 significantly suppressed VP-16-induced apoptosis of these cells. Together, these results indicate that EBV-infected T/NK-cells express CD137 on the cell surface, which may be induced by LMP1 and activate the anti-apoptotic intracellular signaling pathway through NF-ĸB activation. This pathway may contribute to immortalization of the infected cells and development of EBV-T/NK-LPD.