STAT3 Is Activated By EBV in T or NK Cells Leading to Development of EBV-T/NK-Lymphoproliferative Disorders

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-2Rγ^null 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.