| Background: According to the "Global Cancer Report 2014" issued by the World Health Organization,the incidence and mortality rate of cancer increased world widely in 2012.Nearly 50% of newly diagnosed cancer cases were occurred in Asia and China was ranked first.Malignant tumors have become common and frequently occurring disease in China.Recent years,with the deepening understanding on the mechanism of tumor escaping immune surveillance,adoptive gamma delta(γδ)T cell therapy has become an important part of comprehensive tumor treatment.Adoptive γδT cell therapy has played an increasingly important role in preventing recurrence and improving disease-free survival,especially in the removal of minimal residual disease.Therefore,a comprehensive evaluation of whether γδT cell therapy combined with other therapeutic approaches can exert a better clinical therapeutic effect and further study of its mechanism are important for guiding the better use of γδT cells for tumor treatment.These are also urgent issues that need to be resolved in clinical practice.Decitabine(DAC)is a cytosine analog that was first used as an anti-cancer drug.With the development of epigenetic studies,the role of DNA methyltransferase inhibitors like DAC has gradually gained attention.DAC is metabolized to deoxyribonucleoside triphosphates,which replace cytosine during DNA replication and strongly bind to DNA methyltransferases to competitively inhibit their activity and block DNA methylation.Thereby,DAC activates tumor suppressor genes and induces cell differentiation.Clinical trials have shown that DAC can not only be used for the treatment of myelodysplastic syndrome(MDS)and acute myeloid leukemia(AML),but also exerted anti-tumor effects in clinical trials of liver cancer,lung cancer,and ovarian cancer.In addition,DAC enhances the sensitivity of tumor cells to chemotherapeutic drugs,increases the immunogenicity of tumor cells,and promotes the killing of tumor cells by immune cells.Regarding the effect of DAC on immune cells,the role of DAC on NK cells has been widely studied.Different concentrations of DAC were added to an in vitro NK cell culture system.NK cell viability,proliferative capacity,and NKG2 D expression significantly decreased as the concentration of DAC increased.However,the expression of KIR and NKp44 also increased and the activity of NK cells decreased initially and then recovered.After treatment with DAC in vitro,the cytotoxicity and cytokine secretion functions of NK cells were enhanced;however,the specific mechanism was not clear.In addition,DAC also caused an increase in the number of Treg cells owing to a decrease in the degree of methylation of Cp G islands in the Foxp3 gene.Regarding the effect of DAC on γδT cells,only one study reported that DAC induced γδTreg cells in vitro.However,the effects of DAC on the function of γδT cells have not been reported.In the clinical use of DAC to treat tumors,whether the function of γδT cells will be affected and the mechanism remains to be determined.Objective: In this study,a large number of γδT cells were obtained by in vitro expansion and the effects of DAC on γδT cell activity,proliferation,cell cycle,cytokine secretion,and cytotoxicity were evaluated.We aimed to determine the effect of DAC on γδT cells and elucidate its mechanism.These observations will broaden our knowledge of the effect of DAC on γδT cells and will be beneficial for determining the clinical use of DAC.Methods: 1.γδT cell expansion The γδT cells in this study were obtained using in vitro amplification.Peripheral blood mononuclear cells were isolated from the blood of patients with tumors,then γδT cells were expanded using IL-2 and zoledronic acid and cultured in vitro for 9 days.Thereafter,γδT cells were harvested and used for subsequent experiments.2.Effect of DAC on the biological function of γδT cells(1)Effect of DAC on γδT cell activity The expanded γδT cells were treated with different concentrations(0 μM,0.25 μM,0.5 μM,1 μM,2 μM,3 μM,4 μM,and 5 μM)of DAC for 48 h.Subsequently,the apoptosis of γδT cells was detected using an apoptosis detection kit according to the manufacturer’s protocol.After staining,the cells were analyzed using a BD FACS Calibur.(2)Effect of DAC on the proliferation of γδT cells The γδT cells were pre-labeled with Carboxyfluorescein Succinimidyl Ester(CFSE)and then treated with different concentrations(0 μM,0.25 μM,0.5 μM,1 μM,2 μM,3 μM,4 μM,and 5 μM)of DAC for 5 days.Flow cytometry was used to detect the effect of DAC on the proliferation of γδT cells.(3)Effect of DAC on the γδT cell cycle After treatment with various concentrations of(0 μM,0.25 μM,0.5 μM,1 μM,2 μM,3 μM,4 μM,and 5 μM)DAC for 48 h,expanded γδT cells were fixed with cold 70% ethanol overnight at-20°C and then washed once with cold phosphate-buffered saline(PBS).The fixed cells were treated with RNase and stained with propidium iodide.The stained cells were analyzed by flow cytometry using Mod Fit LT software.(4)Effect of DAC on the killing function of γδT cells The γδT cells were treated with 0.5 μM and 1 μM DAC for 48 h,then γδT cells were collected.The cytotoxicity of DAC-treated γδT cells was determined using a Calcein-AM release assay.(5)Effect of DAC on the surface molecules of γδT cells Expanded γδT cells treated with 0.5 μM DAC for 48 h were stained with DNAM-1-PE,NKG2D-APC,Vγ9-FITC,KIR2DL2/3-PE,CD3-Per CP,KIR2DL1-PE,CD279-APC,KIR2DS4-APC,and KIR3DL1-APC.Appropriate isotype-matched antibodies(Abs)were used as controls.Data were analyzed by flow cytometry.(6)Effect of DAC on the secretion of γδT cytokines The γδT cells were treated with 0.5 μM DAC and γδT cells were collected for 48 h.After incubation with different target cells for 4 h,γδT cells were stained with intracellular antibodies against IFN-γ and TNF-α.Data were obtained by flow cytometry.3.The mechanism of the anti-tumor effect of DAC on γδT cells The γδT cells were treated with 0.5 μM DAC for 48 h,then incubated with KIR2DL2/3 antibody for 30 min to block the interaction between KIR2DL2/3 and its ligands.A Calcein-AM release assay was used to detect the cytotoxicity of DAC treated and untreated γδT cells.KIR2DL2/3+ and KIR2DL2/3-γδT cells were sorted from these cultured cells using a flexible BD Influx? cell sorter.The cytotoxicity of these two types of γδT cells on tumor target cells was measured using a Calcein-AM release assay to detect whether there was a difference in the anti-tumor effects between KIR2DL2/3+ and KIR2DL2/3-γδT cells.At the same time,KIR2DL2/3+ and KIR2DL2/3-γδT cells were treated with 0.5 μM DAC for 48 h and the expression of KIR2DL2/3 on the surface of γδT cells was detected by flow cytometry.Finally,IFN-γ was used to increase the expression of KIR2DL2/3 ligand on the surface of target tumor cells and the Calcein-AM release assay was used to detect the cytotoxicity of KIR2DL2/3+ and KIR2DL2/3-γδT cells against INF-γ-treated tumor targets.4.The mechanism of DAC on the expression of KIR2DL2/3 in γδT cells Genomic DNA and total RNA from γδT cells treated with and without DAC were isolated using a DNeasy? Blood and Tissue kit and an Easy Pure RNA kit,respectively.The effects of DAC on the expression of KIR2DL2 and KIR2DL3 genes in γδT cells were evaluated using q RT-PCR.Bisulfite-treated sequencing analysis was conducted to detect the methylation levels of KIR2DL2/3 and NKG2 D promoter regions in DAC-treated γδT cells.Chromatin immunoprecipitation assays were used to analyze the effects of DAC on transcription factors Sp-1 and H3K4me3 in the promoter regions of KIR2DL2/3 and NKG2 D of γδT cells.Finally,q RT-PCR,co-immunoprecipitation and flow cytometry were conducted to determine whether Sp-1 inhibitors could reverse the up-regulation of KIR2DL2/3 gene and protein expression in γδT cells caused by DAC.Results: We successfully induced γδT cells from the peripheral blood of 7 patients with hematological tumors.The purity of the γδT cells was 95.2%(90% to 98.7%).In the study of the effects of DAC on the biological function of γδT cells,we found that:(1)Different concentrations of DAC promoted γδT cell apoptosis,but this effect was weak and did not occur with the DAC concentration increasing;(2)DAC significantly inhibited the proliferation of γδT cells,but this effect was not dose-dependent;(3)DAC had no significant effect on the cell cycle and the secretion of IFN-γ and TNF-α in γδT cells;(4)It was particularly important that DAC reduced the cytotoxicity of γδT cells to SKM-1(45.83 ± 8.01% vs.54.65 ± 6.9%,p < 0.05)and Raji(10.86 ± 3.65% vs.16.86 ± 5.05%,p < 0.05)cells.However,DAC did not affect the ability of γδT cells to kill K562 cells;(5)DAC increased the expression of KIR2DL2/3 and KIR2DL1,the surface inhibitory ligands of γδT cells,but had no effect on the expression of the activating receptors NKG2 D,DNAM-1,and KIR2DS4.The KIR2DL2/3 antibody can block the interaction between KIR2DL2/3 and its ligand,prevent the binding of KIR2DL2/3 and their ligands,and inhibit inhibitory signals to γδT cells.Through antibody blocking experiments,we found that KIR2DL2/3 blocking antibody rescued the inhibitory effect of DAC on the anti-tumor function of γδT cells.KIR2DL2/3+ γδT cells had weaker killing capacity against SKM-1 and Raji cells than KIR2DL2/3-cells.However,KIR2DL2/3-blocking antibody had no effect on the cytotoxicity of γδT cells against K562 cells.After incubation with 0.5 μM DAC for 48 h,some KIR2DL2/3-γδT cells expressed KIR2DL2/3.However,DAC did not affect KIR2DL2/3 expression on KIR2DL2/3+ γδT cells(Fig.3C).We further determined the expression of HLA-I molecules on target tumor cells and found that the HLA-I molecular expression level was low on SKM-1 cells,high on Raji cells,and very low on K562 cells.Interferon(IFN)-γ has been reported to enhance HLA-I molecular expression on tumor cells.We found that IFN-γ increased HLA-I molecular expression on SKM-1 cells,but not on Raji and K562 cells.Meanwhile,compared to untreated SKM-1 cells,KIR2DL2/3+ γδT cells were less cytotoxic to IFN-γ-treated SKM-1 cells.However,the cytotoxicity of KIR2DL2/3-γδT cells was not different against IFN-γ-treated and untreated SKM-1 cells.These findings indicated that the decreased cytotoxicity of DAC-treated γδT cells was dependent on the interaction between KIR2DL2/3 and their ligands.DAC can not only increase the transcription level of the KIR2DL2/3 gene in γδT cells,but also reduce the degree of methylation in the promoter region of the KIR2DL2/3 gene.However,DAC has no effect on the methylation status of the NKG2 D promoter region.DAC can demethylate the promoter region of the KIR2DL2/3 gene and expose the binding sites for transcription factor Sp-1,thereby enriching the transcription factor Sp-1 in the promoter region of the KIR2DL2/3 gene.To ascertain whether DAC increased KIR2DL2/3 expression through the ligation of Sp-1 and its binding sites in the Cp G islands of the KIR2DL2/3 promoter,the Sp-1 inhibitor mithramycin was added to γδT cells treated with DAC.Mithramycin prevented KIR2DL2 and KIR2DL3 upregulation mediated by DAC in γδT cells.Flow cytometry and western blot assays indicated that mithramycin substantially inhibited the upregulated KIR2DL2/3 expression induced by DAC.These findings indicated that DAC enhanced KIR2DL2/3 expression in γδT cells by increasing the binding of Sp-1 to its binding sites in the Cp G islands of the KIR2DL2/3 promoter.Conclusions: 1.The DNA methyltransferase inhibitor,DAC,induced apoptosis of γδT cells,although this effect was weak.2.DAC had no effect on the cell cycle of γδT cells,but inhibited the proliferation of γδT cells and their killing effect on tumor cells.3.DAC upregulated the expression of γδT cell surface inhibitory receptors,KIR2DL2/3 and KIR2DL1.However,DAC had no effect on the expression of the activating receptors,NKG2 D and DNAM-1.4.DAC reduced the killing effect of γδT cells on tumor cells by increasing the expression of KIR2DL2/3 on the surface of γδT cells.5.The methylation level in the KIR2DL2/3 promoter was relatively high and DAC caused demethylation.However,the methylation level in the promoter of NKG2 D was relatively low and DAC had no obvious demethylation effect on this region.6.DAC promoted the gene transcription and protein expression of KIR2DL2/3 by increasing the degree of transcription factor Sp-1 in the promoter of the KIR2DL2/3 gene. |
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