Tumor Cells from EBV-Positive T- or NK-Cell Neoplasms Induce Differentiation and Activation of Macrophages By Secreting Humoral Factors

Introduction: Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms (EBV-T/NK neoplasms), such as extranodal NK-/T-cell lymphoma, aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV) are often accompanied with severe systemic inflammation. During their progression, hemophagocytic lymphohistiocytosis (HLH) may develop, which is a lethal condition.To clarify the mechanisms of HLH development in EBV-T/NK neoplasms, we investigated the direct effects of EBV-T/NK cells on the differentiation and activation of macrophages.

Methods: The following EBV-T/NK-cell lines established from EBV-T/NK neoplasms were used: SNT8, SNT15, and SNT16 as T-cell lines (SNT cells), and SNK1, SNK6, and SNK10 as NK-cell lines (SNK cells). For investigating the effects of EBV-positive T or NK cells on the differentiation and activation of macrophages, the culture supernatant of these EBV-positive cells was used. U937 and THP1 cells, human monocytic cell lines, were cultured with the supernatant, and morphologic examination and flow cytometry (FCM) were used to examine their differentiation into the macrophage. Phorbol 12-myristate 13-acetate was used as a positive control for induction of the differentiation. FCM was used to determine the phagocytic activity based on the uptake of fluorescent beads. For detecting the cytokine production, the mRNA expression and cytokine concentrations were examined using TaqMan^® Gene Expression Assays and enzyme-linked immunosorbent assay, respectively. The EBV-derived microRNA expression was investigated using the next-generation sequencer (MiSeq^®)and TaqMan^® Gene Expression Assays.

Results: After culturing with the supernatant from the EBV-positive T or NK cells, the macrophage-like morphological change was observed in the THP1 and U937 cells. Interestingly, the effects were clear in the cells treated with the supernatant from the SNK cells, whereas the effects of the supernatant from the SNT cells were minimal. Consistent with the morphological changes, the co-culture with the SNK-cell supernatant significantly enhanced the CD11b expression, which is a marker for macrophages, on the THP1 and U937 cells. Next, the macrophage-like function in the differentiated cells was examined. We found that phagocytosis was significantly enhanced after the co-culture with the SNK-cell supernatant. In addition, the mRNA expression of the inflammatory cytokines IL-6, IL-8, and TNF-α was induced after the co-culture. These results indicated that the SNK-cell supernatant induced the differentiation of THP1 and U937 cells into the M1-macrophages and their activation. Finally, supernatant factors responsible for the differentiation and activation were also examined. The high concentration of TNF-α, which is known to induce macrophage activation, was determined in the SNK-cell culture supernatant, whereas it was not detected in the SNT-cell supernatant. In addition, the expression of several miR-BARTs that was detected in the SNK cells was reduced in the SNT cells. Interestingly, miR-BARTs were confirmed in the U937 cells cultured with the SNK-cell supernatant.

Conclusions: The findings of this study indicate that TNF-α and miR-BARTs secreted from EBV-positive SNK cells may induce the differentiation and activation of macrophages. They may be utilized as molecular targets in the treatment of HLH with EBV-positive T- or NK-cell neoplasms.