NK Cell Exosomes Alleviate PD-L1 Expression and Facilitate Tumor Immunity by Repressing PI3K-AKT-mTOR Signaling

Background: Liver cancer (LC) is a deadly malignancy with limited therapeutic options in recent years. Natural killer cell-derived exosomes (NK-exo), as an important bridge of information transmission between cells, also have a certain killing effect on tumor cells. On this basis, this study investigated the specific regulatory mechanism of NK-exo on LC cells.

Methods: NK-exo was collected by differential centrifugation. The diameter and size distribution were characterized by dynamic light scattering (DLS), respectively. Western Blot (WB) assay detected the expression levels of exosome marker protein, PD-L1, and PI3K-AKT-mTOR signal-related proteins. The effect of NK-exo treatment on LC cell viability was measured by the CCK-8. With the use of CFDA·SE, we assessed the proliferation ability of CD8⁺T cells in direct co-culture with LC cells. The content of cytokines secreted by CD8⁺T cells in each treatment group was determined by enzyme-linked immunosorbent assay (ELISA) kits. We employed flow cytometry to analyze the expression of PD-L1 protein on the surface of LC cells and CD8 level in mice tumor tissues.

Results: CCK-8 assay demonstrated that NK-exo repressed the cell viability of LC cells. WB uncovered that the protein expressions of PD-L1, p-AKT, and p-mTOR in NK-exo treated LC cells were decreased, which was returned to the control level after the addition of PI3K agonist. When NK-exo-treated LC cells were directly co-cultivated with CD8⁺T cells, the proliferation ability and cytokine secretion content of T cells were considerably elevated, and the expression of PD-L1 on LC cell surface was considerably reduced. However, these effects were restored to control levels by PI3K agonists.The in vivo experiments also confirmed that NK-exo could effectively inhibit the progression of LC, and the PI3K agonist could restore this effect to the level of the control group.

Conclusion: This study provided the first evidence that exosomes derived from NK cells inhibited the PI3K-AKT-mTOR signaling pathway in LC cells, and reduced PD-L1 expression, thereby promoting tumor immunity. In comparison to traditional immune checkpoint inhibitors, NK-exo possessed unique mechanisms of action and potential advantages. NK-exo holds the promise of becoming an innovative immunotherapy for the treatment of LC.