Tenatoprazole-induced Mouse T Cell Killing Tumor Cells By Regulating IDO2

Background:The immune system can effectively identify and kill tumor in vivo,but some tumor cells can use the immune suppression mechanism to help themselves escape the surveillance of it.The tryptophan metabolism enzyme that include tryptophan2,3-dioxygenase?TDO?,indoleamine 2,3-dioxygenase?IDO?1 and IDO2 is an immunosuppressive protease,and its high expression leads to excessive degradation of tryptophan and accumulation of its metabolites,which cause T cell dysfunction and lead to tumor immune escape.IDO2 is a newly discovered tryptophan metabolism enzyme mainly expressed in antigen-presenting cells?APC?,but some researches have shown that it is abnormally expressed in a variety of tumor cells.In addition,a report have showed that Tenatoprazole can specifically inhibit the enzyme activity of IDO2.Currently,no studies have been found on the use of Tenatoprazole for tumor immunotherapy.Therefore,our study was to investigate the regulation of IDO2 by Tenatoprazole by constructing the 293T cell line expressing IDO2,and to investigate the effect of Tenatoprazole on the regulation of mouse T cells by IDO2.This may provide a new therapeutic drug for tumor immunotherapy.Methods:?1?we recombined the IDO2 gene into the eukaryotic expression vector pIRES-EGFP plasmid by genetic engineering technology to structure SBP-IDO2 plasmid.Then,we transfected the SBP-IDO2 plasmid into 293T cells by cell transfection,and detected the transfection efficiency by qRT-PCR and Western Blot.?2?The 293T-IDO2 cells were treated with different concentrations of tenatoprazole.The half maximal inhibitory concentration(IC₅₀)of the drug on 293T-IDO2 cells was detected by MTT assay and the appropriate treatment time and concentration were selected by this result.The effect of drugs on the expression of IDO2 was detected by Western blot assay,and the regulation of IDO2 enzyme activity was detected by Ehrlich's assay.?3?293T-IDO2 cells were co-cultured with mouse T lymphocytes and treated with different concentrations of tenatoprazole and then detected the killing ability of T cells by CCK-8 assay.Result:?1?Through DNA sequencing tests we successfully constructed the SBP-IDO2plasmid.The results of qRT-PCR and Western Blot showed that the relative expression of IDO2 in 239T cells transfected with SBP-IDO2 plasmid was significantly increased at RNA level and protein level?P<0.01?.?2?The IC₅₀ value of the drug was 50?M by MTT assay.The gray value analysis of the results of Western Blot showed that the expression of IDO2was inhibited by Tenatoprazole and the inhibitory effect increased with the increase of Tenatoprazole concentration?P<0.05?.The result of Ehrlich's assay show that Tenatoprazole could inhibit the activity of IDO2 in a dose-dependent manner.?3?The results of cell co-culture and Flow Cytometry showed that Tenatoprazole reduced the ability of293T-IDO2 cell induced T cell transform to Treg cell and decreased the number of Treg cell?P<0.05?.The results of cell co-culture and CCK-8 tumor killing assay indicated that the lethality of T cells which co-cultured with 293T-IDO2 were evidently increased?P<0.01?.Conclusion:Tenaporazole could effectively inhibit the expression and enzyme activity of IDO2 protein in a dose-dependent manner.Tenatoprazole could decrease the number of Treg cells and improve the killing ability of mouse T lymphocytes to tumor cells by inhibiting IDO2.So,We infered that Tenatoprazole has the potential to participate in tumor immunotherapy.