Abstract: Objective To investigate the transcriptional activation potential of Mestranol (Mes) on acylglycerol Kinase (AGK) and its role in enhancing CD8+ T cell effector function. Methods Public databases were utilized to analyze the correlation between AGK expression and CD8+ T cell effector function genes, and its association with CD8+ T cell infiltration levels across the tumor microenvironments. The effects of Mestranol on AGK mRNA and protein expression levels, as well as on the activation of the AGK downstream signaling pathway were assessed using quantitative PCR (qPCR) and Western blot. The half maximal inhibitory concentration (IC50) of Mestranol was determined using the CCK⁃8 assay. An AGK overexpression plasmid was transfected into human T lymphocyte leukemia Jurkat cells and CD8⁺ T cells by electroporation, and a luciferase reporter gene assay was employed to examine the impact of Mestranol on AGK promoter transcriptional activity. Enzyme⁃linked immunosorbent assay (ELISA) was used to quantify the levels of  interferon⁃γ (IFN⁃γ) and tumor necrosis factor⁃α (TNF⁃α) secreted by CD8+ T cells under following AGK overexpression, along with Mestranol's effects on these parameters. The effects of Mestranol on the cytotoxic activity of CD8+ T cells was assessed in vitro using flow cytometry.  Results Public databases analysis revealed that AGK expression was significantly positively correlated with CD8+T cell effector function genes, negatively correlated with inhibitory function genes, and positively associated with CD8+ T cell infiltration levels in various tumor microenvironments （all P<0.05）. The IC50 of Mestranol was 36 μmol/L. Mestranol significantly upregulated AGK expression at both mRNA and protein levels in Jurkat cells in a dose⁃dependent manner across various concentrations （all P<0.01）. Furthermore, luciferase reporter assays demonstrated that Mestranol significantly enhanced the transcriptional activity of the AGK promoter（P<0.001）. Mestranol elevated the phosphorylation levels of AKT protein at Ser473, enhanced the secretion of IFN⁃γ and TNF⁃α in treated CD8⁺T cells (all P<0.001), and increased their tumor⁃killing capacity in vitro (P<0.01).  Conclusions Mestranol up⁃regulates AGK expression by enhancing the transcriptional activity of the AGK promoter, thereby augmenting CD8+ T cell effector function, which provides a new insight for tumor immunotherapy targeting AGK.

Key words: Mestranol, Acylglycerol kinase, CD8+ T cell, Tumor immunity