To clarify the mechanism for development of Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms, we focused on the costimulatory receptor CD137. We detected high expression of CD137 gene and its protein on EBV-positive T- or NK-cell lines as compared with EBV-negative cell lines. EBV-positive cells from EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs) patients also had significantly higher CD137 gene expression than control cells from healthy donors. In the presence of IL-2, whose concentration in the serum of EBV-T/NK-LPDs was higher than that of healthy donors, CD137 protein expression was upregulated in the patients' cells whereas not in control cells from healthy donors. In vitro EBV infection of MOLT4 cells resulted in induction of endogenous CD137 expression. Transient expression of LMP1, which was enhanced by IL-2 in EBV-T/NK-LPDs cells, induced endogenous CD137 gene expression in T and NK-cell lines. In order to examine in vivo CD137 expression, we used EBV-T/NK-LPDs xenograft models generated by intravenous injection of patients' cells. We identified EBV-positive and CD8-positive T cells, as well as CD137 ligand-positive cells, in their tissue lesions. In addition, we detected CD137 expression on the EBV infected cells from the lesions of the models by immune-fluorescent staining. Finally, CD137 stimulation suppressed etoposide-induced cell death not only in the EBV-positive T- or NK-cell lines, but also in the patients' cells. These results indicate that upregulation of CD137 expression through LMP1 by EBV promotes cell survival in T or NK cells leading to development of EBV-positive T/NK-cell neoplasms.

A 22-year-old male was admitted for a sustained fever of 2 months, lymphadenopathy, and liver dysfunction. Anti-VCA-IgM antibody was positive, with elevated Epstein-Barr virus (EBV)-DNA load in the peripheral blood. Liver biopsy revealed infiltration of CD8-positive and EBV-positive cells. Most peripheral blood mononuclear cells (PBMCs) were also positive for CD8, and showed detectable levels of EBV-DNA. Monoclonal proliferation of EBV-infected cells was detected in the PBMCs by Southern blotting for EBV-terminal repeat (EBV-TR). Although EBV-positive T-cell lymphoproliferative disease (EBV-T-LPD) was suspected, the symptoms spontaneously resolved within 12 months. Anti-VCA-IgM antibody and the clonal band of EBV-TR were negative 1 year after the onset, while anti-EBNA antibody was positive. The final diagnosis was thus confirmed as infectious mononucleosis (IM). Our results indicate that EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells may be temporally detected in IM. EBV-T-LPDs should be carefully excluded in such cases.

Introduction Epstein–Barr virus (EBV) infects B cells and rarely T or NK cells, causing EBV-positive T/NK-cell lymphoproliferative neoplasms (EBV-T/NK-neoplasm), such as extranodal NK/T-cell lymphoma, aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are fatal disorders presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV infects B cells making them immortal and resulting in B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies has not been elucidated yet. Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin genes. Deregulated AID expression acts as a genomic mutator leading to the development of B-cell lymphoma. In addition, EBV infection induces AID expression in B cells. These findings indicate that AID has a role in EBV-induced lymphomagenesis in B cells. However, of interest, AID transgenic mice developed T-cell lymphoma (J. Exp. Med., 197, p.1173, 2003). Recently, Nakamura et al. reported that AID expression was upregulated in the peripheral blood mononuclear cells (PBMCs) of EBV-T/NK-LPDs patients (Eur. J. Dermatol., 21, p.780, 2011). Therefore, we hypothesized that EBV infection induces AID expression in T or NK cells contributing to the tumor development. Objectives We designed this study to investigate AID expression and its contribution to EBV-T/NK-neoplasms development. Materials and Methods EBV-positive T- and NK-cell lines, SNT8, SNK6, SNT13, SNT15, and SNT16, were examined. The EBV-negative T-cell line HPB-ALL, Jurkat and human primary T cells derived from cord blood were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. AID expression in tissue lesions were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid, IL-2R γKO mice. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to HPB-ALL or Jurkat cells (Proc. Natl. Acad. Sci., 100, pp.7836-40, 2003). To determine AID-induced mutation of the gene, DNA was extracted from SNT13, SNK6, or EBV-infected Jurkat cells, and c-myc gene was cloned by TA cloning, which was then sequenced. We compared the number of the mutation with that of Jurkat cells. Results We detected AID expression in EBV-positive T- or NK-cell lines by RT-PCR, western blotting, and immunological staining using confocal microscopy, whereas it was not detected in the control. Furthermore, we validated the results in EBV-infected T or NK cells derived from 12 EBV-T/NK-LPDs patients (infected cell types: CD4, 5; CD8, 3; and CD56, 4). Quantitative RT-PCR demonstrated that AID expression was upregulated in EBV-infected T or NK cells compared with the control. Expression was confirmed by immunological staining using confocal microscopy in 5 patients. We also detected AID expression by histopathological staining in EBV-infected cells in lesions of 4 patients and the EBV-T/NK-LPDs xenograft models. Subsequently, we examined the role of EBV for AID expression in T cells. It was demonstrated that AID expression was induced by in vitro EBV infection in the T-cell line HPB-ALL and primary T cells. Moreover, it was demonstrated by the luciferase assay that the viral protein LMP1 upregulated AID promoter activity in HPB-ALL cells. Finally, we found that SNT13, SNK6, and EBV-infected Jurkat cells had increased number of the mutation of c-myc gene compared with Jurkat cells. Conclusions We previously reported that EBV infection of T or NK cells enhances survival of the infected cells by the activation of NF-κB. The results of the present study suggest that EBV contributes to the development of EBV-T/NK-neoplasms through NF-κB-induced cell survival and AID-induced mutagenesis leading to clonal evolution and expansion. Disclosures: No relevant conflicts of interest to declare.

Introduction Epstein–Barr virus (EBV) genome is positive not only in B-, but also T- or NK-lymphoid neoplasms: extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are disorders formerly called chronic active EBV infection presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV makes the infected B-cells immortal leading to B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of EBV-T/NK-neoplasms has not been elucidated yet. STAT3 is a transactivation factor which mediates proliferation and anti-apoptotic signaling. It was reported that a large variety of primary tumor cells as well as tumor-derived cell lines from patients harbored constitutively activated STAT3. In addition, tyrosine 705 (Y705) of STAT3 was constitutively phosphorylated in ENKL cells (Leukemia, 23, p1667, 2009). Objectives We designed this study to investigate STAT3 activation and its contribution to EBV-T/NK-LPDs development. Materials and Methods EBV-positive T-cell lines, SNT8, SNT15, SNT16, and NK-cell lines, SNK1, SNK6, SNK10, were examined. The EBV-negative T-cell lines HPB-ALL, Jurkat, MOLT4 and peripheral blood mononuclear cells (PBMCs) from healthy donors were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. STAT3 phosphorylation in EBV-T/NK-LPDs cells were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid/IL-2Rγnull mice. Mutation of STAT3 was examined by direct sequencing. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to MOLT4 cells (Proc Natl Acad Sci, 100, p7836, 2003). Results First, we investigated the activation of STAT3 in EBV-positive T- or NK-cell lines. Phosphorylation of STAT3 on Y705 and serine 727 (S727) was more clearly detected in comparison with that in EBV-negative cell lines by western blotting under their maintenance condition. STAT3 was localized in the nucleus in the EBV-T/NK-cells. These results indicated STAT3 was constitutively activated in EBV-T/NK-cells. We validated the results in EBV-infected T or NK cells derived from 5 EBV-T/NK-LPDs patients (infected cell types: CD4, 1; CD8, 2; and CD56, 2). Phosphorylation of Y705 and S727 of STAT3 was detected in EBV-infected T or NK cells in them. Immunohistological staining also detected the phosphorylation of EBV-positive cells in the tissue of the xenograft models. In these EBV-T/NK-cells, gene mutation was not identified in SH2 domain of STAT3. Next, we examined the direct effect of EBV on STAT3 activation by in virto EBV infection on MOLT4 cells. Immunofluorescence staining detected that STAT3 moved to the nucleus after the infection. Finally, STAT3 specific inhibitor STA-21 suppressed the proliferation of EBV-T/NK-cells. Conclusions STAT3 is activated by EBV leading to growth promoting effects on EBV-T/NK-LPDs. STAT3 can be an attractive molecular target of the treatment for the disorders. Disclosures No relevant conflicts of interest to declare.