Upregulation Of Tumor-specific CD8~+CD39~+ T Cells By Artesunate Conjugated Plakoglobin

Objective:Artesunate(ART)is an antimalarial drug that has been shown to have potent anticancer properties;however,this effect is mostly focused on the antitumor cells themselves and remains unknown for CD8~+T cells in the immune microenvironment.CD39 is an ectonuclease localized in the cell membrane that exerts immunosuppressive effects by catalyzing the conversion of ATP to adenosine.Recent studies have shown that CD39 can serve as a biomarker for tumor-specific CD8~+T cells and that increasing CD8~+CD39~+T cells can promote antitumor immune responses.Therefore,this paper focuses on the effect of ART on specific anti-tumor CD8~+T cells and the regulatory mechanism to seek strategies to improve the therapeutic effect of lung cancer.Methods.(1)PBMC from peripheral blood of lung cancer patients were extracted and cultured by adding anti-CD3/CD28 or PMA activation,and the toxicity of ART on human PBMC and the effect on different CD8~+T cell subsets were detected by flow cytometry.(2)To further investigate the regulatory mechanism of ART on CD8~+T cells,the nucleation of NF-κB in CD8~+T cells after ART treatment was observed by immunofluorescence,and the activation of NF-κB by ART was verified by flow cytometry.The expression levels of p-PKA,p-AKT and p-ERK were detected by protein blotting(Western blot)after ART treatment of PBMC.PBMC were co-cultured with A549 cells and the apoptosis of A549 cells was detected by flow cytometry.(3)CD8~+T cells were sorted using immunomagnetic beads,and whether ART binds directly to the target protein was verified by SM pull down technique.(4)The sh JUP lentiviral vector was constructed to knock down the plakoglobin in CD8~+T cells,and the knock down efficiency was verified by Western blot and Real time quantitative PCR.The activation of the signaling pathway by ART and the expression level of CD39~+T cells after knockdown were detected by Western blot and flow cytometry.Results:(1)The IC50 of ART on PBMC was 229.9u M as determined by flow cytometry,and ART upregulated tumor-specific CD8~+CD39~+T cell subpopulation and enhanced secretion of CD8~+T cell granzyme B.Co-culture showed that ART enhanced the killing effect of effector T cells on A549 cells in lung cancer patients.(2)Immunofluorescence and flow cytometry assays showed that ART promoted p65entry of CD8~+T cells into the nucleus,and Western blot confirmed that ART could activate the p-ERK signaling pathway,indicating that ART enhanced the antitumor immune response of CD8~+CD39~+T cell subpopulation by activating the ERK/NF-κB pathway.(3)The SM pull down assay verified the direct interaction between ART and plakoglobin in.The ability of ART to upregulate the proportion of CD39~+T cells and promote the secretion of granzyme B by CD8~+T cells was decreased after lentiviral knockdown of plakoglobin pearl protein,indicating that ART binds to plakoglobin pearl protein to activate CD39~+CD8~+T cells and enhance the killing effect of effector T cells on tumor cells,further demonstrating that plakoglobin pearl protein is a direct target of ART.Conclusion:Artesunate enhances the antitumor immune response of effector T cells against lung cancer by activating the ERK/NF-κB pathway through binding to plakoglobin and upregulating the ratio of CD8~+CD39~+T cells.Figure 18 Table 0 Reference 54...