Activation of mTOR Signal Bolster Natural Killer Cell Immunity Against EBV-Infected B Lymphocytes

Background: Epstein-Barr virus (EBV) is one of the most common human pathogens that infects around 95% of adults throughout their lifetime and is involved in the carcinogenesis process of several malignant diseases, including various types of lymphomas. EBV-associated lymphoproliferative disease is a devastating complication for patients with immunosuppressive conditions. Although current therapeutic approaches for this disorder, including tapering of immunosuppression, donor lymphocyte infusion, and rituximab, may be effective for this complication after hematopoietic cell transplantation (HST), severe adverse events such as fatal GVHD and infections could develop thereafter. Whereas EBV-specific T cells play a crucial role in immunity against EBV infection, various studies indicate that natural killer cells (NK cells) are also implicated, particularly in the early stages of EBV infection and during the malignant transformation of EBV-infected B cells.

Methods: To identify bioactive agents capable of enhancing the NK cell-mediated immunity against EBV-infected B cells, we carried out two step experiments in which peripheral blood mononuclear cells (PBMC-EBV) from healthy individuals are first infected with EBV (first step), and seven days later (second step), freshly isolated autologous NK cells (au-NK cells) are cocultured with PBMC-EBV in the presence or absence of various bioactive compounds. NK cell immunity against PBMC-EBV is assessed by the release of IFN-gamma and granzyme B in the co-culture supernatants and by their ability to prevent the transformation of B cells within PBMC-EBV into lymphoblastoid cells (LCLs).

Results: Among the 48 bioactive agents tested, we identified the compound Clausine K capable of increasing IFN-gamma secretion from au-NK cells cocultured with PBMC-EBV, although it had no effects on B cell transformation. Similarly, ascophyllan, a marine polysaccharide purified from Ascophyllum nodosum and currently available for human use as a health supplement, enhanced IFN-gamma and granzyme B secretion from au-NK cells, and dramatically reduced the ability of PBMC-EBV to generate LCLs. To identify the mechanisms underlying the activator effects of ascophyllan on NK cells, we next screened various signaling pathways, including JAK-STAT, MAPK/ERK, mTOR signaling, Wnt, and PI3K/AKT/mTOR signaling pathways. Our analysis showed that ascophyllan activates the mammalian target of rapamycin (mTOR) signal in NK cells, as demonstrated by the hyperphosphorylation of the mTOR protein itself (at Ser2448 phosphorylation site) and its downstream proteins p70S6K and 4E-BP1 in response to NK cells exposure to ascophyllan. In line with these observations, granzyme B secretion induced by ascophyllan in NK cells was severely impaired when NK cells were exposed to the mTOR inhibitor rapamycin. As expected, rapamycin attenuated, in a dose-dependent manner, the hyperphosphorylation of mTOR, p70S6K, and 4E-BP1 in NK cells exposed to ascophyllan. Finally, flow cytometry studies showed that NK cells treated with ascophyllan for 48 hours showed higher immune activation features, as illustrated by their higher expression levels of CD69, CD107, NKG2D, and CD137 on their cell surface, compared with their untreated counterparts.

Conclusions: These results suggest that mTOR signal activation plays important roles in granzyme B secretion by NK cells, and in their ability to eliminate EBV-infected B cells, and thus ascophyllan may have potential roles in preventing EBV-associated lymphoproliferative diseases in immune compromised patients. Since ascophyllan is a nontoxic, inexpensive, and clinically available agent, it may be useful in preventing EBV infection and reactivation after HCT.

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No relevant conflicts of interest to declare.