*Purpose: The immunosuppressive regimen currently used after organ transplantation efficiently reduces the adaptive immune components. During suppressed adaptive immunity, the innate immune cells may play pivotal roles in immune surveillance and defense against microbes and neoplastic cells after transplantation. In the present study, we aimed to determine the possible impact of everolimus (EVR), an mTOR inhibitor, on NK cells, which are innate cells providing a first line of defense in a mouse model.

*Methods: B6 mice were treated with EVR by intraperitoneal injection at one of two different doses (0.0125 and 0.25 mg/kg/day) for 1 and 4 weeks. Peripheral blood, spleen, and liver lymphocytes were obtained from EVR-treated and untreated mice. NK cells were separated by high-gradient magnetic sorting. In cytotoxicity assay with the isolated NK cells, mouse lymphoma cells (YAC-1) and mouse hepatoma cells (Hepa 1-6) were used as target cells.

*Results: In mice that received EVR for seven days the proportion of splenic NK cells was unchanged, whereas the number of liver NK cells and TNF-related apoptosis inducing ligand (TRAIL)⁺ NK cells increased for any doses of EVR. Consistently, liver resident NK cells from the EVR-treated mice displayed enhanced cytotoxicity against TRAIL-sensitive neoplastic cells. The sequential development of NK cell subsets can be classified based on the surface expression of CD11b and CD27: CD11blow CD27high (CD27 SP), CD11bhigh CD27 high (DP), and CD11b high CD27low (CD11b SP). We found that EVR-treatment impaired the transition of CD27 SP to DP NK cells and enhanced TRAIL expression on the immature CD27 NK cells. To address the difference of EVR-sensitivity in the sequential development of NK cell subsets, we analyzed FoxO1 and T-bet levels on each NK cell subset. The negative regulator of NK cells FoxO1 was activated as a consequence of impaired mTORC2-dependent AKT phosphorylation. Activated FoxO1 both reduced T-bet expression and induced TRAIL expression, thereby inhibiting NK cell maturation and promoting the antitumor activity of the immature subset of liver NK cells in response to EVR-treatment.

*Conclusions: These findings indicate that EVR-treatment enhances the antitumour activity of immature liver-resident NK cells through TRAIL upregulation. Immature and mature subsets of liver NK cells have different levels of sensitivity to EVR-treatment, mainly because of differential AKT activity and FoxO1 expression.

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