| Objective: Recently,with the application of FDA approved anti CTLA-4and PD-1 antibodies in malignant solid tumor.Immunotherapy,as an effective and less side effects therapy,has been widely used in clinic after surgery,chemotherapy and radiotherapy.By mobilizing the body’s own immune system against the tumor,rather than the tumor itself,people revolutionize the thinking of new drug research and development.The combination of anti-PD-1monoclonal antibody and current therapeutic drugs is a potential research and development direction.Almost all of the cancer drugs,which have more or less effect on the immune system,have an important impact on their efficacy and safety.Whether DAC has effect on the immune system,the research is less.Myelodysplatic sydrome(MDS)and Leukemia are malignant tumors in hematopoietic system.Inactivation of tumor genes caused by DNA methylation is one of the major mechanisms of tumor.DNA methylation is widespread in hematonosis.Various studies found that DNA methylation can be reversed by drugs,which makes the ehancement of remission rate of the drug resistance as well as relapse tumors to be possible.DAC,methyltransferas inhibitor,has been recommended by FAB for the treatment of MDS,elderly AML and CMML.However,there are still relapse and drug resistance.Recently,the role of PD-1/PD-L1 signaling system in hematological malignancies has been reported,the use of PD-1 antibodies in hematological malignancies have also been achieved encouraging results.Therefore,the study of the effect of DAC on PD-1/PD-L1,combined with the application of both,is expected to become a new treatment for hematological malignancies.This study was designed to investigate the changes of PD-1/PD-L1 in MDS and AML before and after treatment with DAC and the changes of PD-L1 in SKM-1 and HL-60 cell lines in order to lay a foundation for exploring new therapeutic methods.Methods:1 This study collected the clinical date form Hematology Department of the second hospital of Hebei Medical University from January 2016 to January2017.According to WHO 2008 typing,18 cases who first diagnosis is MDS with WPSS in the prognosis of stratified middle-risk group and high-risk(5cases were in the MDS middle-risk group and the other 13 cases in high-risk group)and received DAC treatment,20 cases who first diagnosed with AML and treated by DAC,and 5 cases of hematological diseases were compared.MDS patients received a 20mg/m2 of DAC 5 day program,AML patients received DAC 20mg/m2 3 days plus CAG(Acla 20 mg d4-6,Ara-c 10-20mg/m2d4-10,G-CSF 300 ug subcutaneous injection d4-1)chemotherapy.Peripheral blood and bone marrow cells were collected before and after DAC application for 5 and 3 days(bone marrow cells of AML patients were collected for tenth days of DAC+CAG).Flow cytometry by Per-CP CD45 and SSA was used to determine the distribution of lymphocytes and primitive cells.The changes before and after treatment with DAC were detected of PD-1 in peripheral blood lymphocytes of CD3+CD4+T and CD3+CD8+T cells and PD-L1 in bone marrow progenitor cells.The changes in relative expression of PD-1mRNA and PD-L1 mRNA in peripheral blood and bone marrow mononuclear cells before and after DAC treatment were detected by real-time fluorescence quantitative RT-PCR.2 SKM-1 and HL-60 cell line was cultured in vitro,and the suitable DAC concentration was explored by CCK-8 metho,Flow cytometry was used to detect the changes of PD-L1 before and after the intervention of DAC,Real time fluorescent quantitative RT-PCR was applied to explore the changes of PD-L1 mRNA,PD-L1 mRNA after DAC intervention.Results:1 FCM method was used to detect the changes of PD-1 and PD-L1 in peripheral blood T lymphocytes and bone marrow mononuclear cells of MDS middle-risk group,high-risk group and AML patients before DAC treatment:(Table 1,Fig 1)The average expressions of PD-1 on CD3+CD4+T lymphocytes,CD3+CD8+T lymphocytes of MDS middle-risk group,high-risk group and AML before DAC treatment are higher than that of normal control group,no difference in the expression of PD-L1 in bone marrow mononuclear cells and normal control group.2 FCM method was used to detect the changes of PD-1 and PD-L1 in peripheral blood T lymphocytes and bone marrow mononuclear cells of MDS middle-risk group,high-risk group and AML patients after DAC treatment:(Table 2,Fig 1)The average expressions of PD-1 on peripheral blood CD3+CD4+T lymphocytes,CD3+CD8+T lymphocytes after DAC treatment are higher than that of normal control group and before DAC treatment,The expression of PD-L1 on bone marrow mononuclear cells was higher than that of normal control group and before treatment.3 FCM method was used to detect the expression of PD-1/PD-L1 in MDS/AML remission group and non remission group:(Table 3,Fig 2)MDS remission group and non-remission group: The expressions of PD-1on peripheral blood CD3+CD4+T lymphocytes,CD3+CD8+T lymphocytes and PD-L1 on bone marrow mononuclear were significantly higher than those in the remission group,the difference was statistically significant(P=0.001).AML remission group and non-remission group: The expression of PD-1on peripheral blood CD3+CD4+T lymphocytes,CD3+CD8+T lymphocytes and PD-L1 on bone marrow mononuclear were significantly higher than those in the remission group,the difference was statistically significant(P=0.001).These results suggest that the high expression of PD-1/PD-L1 may be related to the drug resistance of DAC,which is an indicator of poor prognosis.4 PT-PCR method was used to detect the relative expression level of PD-1mRNA and PD-L1 mRNA in peripheral blood and bone marrow mononuclear cells of MDS middle-risk group,high-risk group and AML patients before DAC treatment:(Table 4,Fig 3-4)The expression of PD-1mRNA in peripheral blood mononuclear cells ofMDS middle-risk group,high-risk group and AML group was higher than that of normal control group before DAC treatment,the expression of PD-L1 mRNA on bone marrow mononuclear cells was not different from that in normal control group.5 PT-PCR method was used to detect the relative expression level of PD-1mRNA and PD-L1 mRNA in peripheral blood and bone marrow mononuclear cells of MDS middle-risk group,high-risk group and AML patients after DAC treatment:(Table 5,Fig 3-4)The relative expression level of PD-1mRNA in peripheral blood mononuclear cells and PD-L1 mRNA in bone marrow mononuclear cells after DAC treatment in patients with MDS middle-risk group,high-risk group and AML group were significantly higher than those in the control group and before treatment.6 RT-PCR method was used to detect the expression of PD-1mRNA/PD-L1 mRNA in MDS/AML remission group and non remission groups:(Table 5,Fig 5)MDS remission group and non-remission group: The expression of PD-1mRNA on peripheral mononuclear cells,bone marrow were significantly higher than those in the remission group,the difference was statistically significant(P=0.001).AML remission group and non-remission group: The expression of PD-1mRNA on peripheral mononuclear cells,bone marrow were significantly higher than those in the remission group,the difference was statistically significant(P=0.001).It is also indicated that the high expression of PD-1/PD-L1 may be related to the drug resistance of DAC,which is an indicator of poor prognosis.7 Inhibitory effect of DAC on proliferation of SKM-1 and HL-60 cells:The effect of DAC on proliferation and inhibition of SKM-1 and HL-60 cells was detected by CCK-8 assay,With 0,0.5,5,50,500umol/l concentration gradient of SKM-1,HL-60 cells,24 h,48h,72 h after DAC,The results showed that DAC could inhibit the expression of SKM-1 and HL-60 cells,and had the characteristics of time and dose dependence;SKM-1 and HL-60 48 h IC50 close to 50umol/l,72 h IC50 close to 0.5umol/l,FCM and RT-PCR were detected by 0,0.5,50,500umol/l concentration gradient of DAC,respectively.8 FCM method was used to detect the changes of PD-L1 at different time and different concentrations of DAC in SKM-1 and HL-60:(table 7/ fig 6-7)The expression of PD-L1 in MDS/leukemia SKM-1 and HL-60 cell line increased with the concentration of DAC(0,0.5,50,500umol/l)and the time(24h,48 h,72h),and the 50umol/l of DAC was the most obvious.9 RT-PCR method was used to detect the relative expression of PD-L1 mRNA with different concentrations of DAC(0,0.5,50,500umol/l)at different time in SKM-1,HL-60:(table 8-9/ fig 8-9)The relative expression of PD-L1 mRNA in MDS/leukemia SKM-1 and HL-60 cells increased with the concentration of DAC(0,0.5,50,500umol/l)and time(24h,48h),When the concentration of DAC was 50umol/l,the relative expression level of PD-L1 was the highest.Conclusions:1 There was expression of PD-1/PD-L1 in peripheral blood T lymphocytes and bone marrow mononuclear cells.2 There was no difference in the expression of PD-L1 in bone marrow mononuclear cells between the MDS middle-risk group and control group,the expression of PD-1 on peripheral blood T lymphocytes was higher than that in control group;The expression of PD-1/PD-L1 increased significantly after DAC treatment.3 There was no difference in the expression of PD-L1 in bone marrow mononuclear cells between the MDS high-risk group and control group,the expression of PD-1 on peripheral blood T lymphocytes was higher than that in control group;After treatment with DAC,the expression of PD-1/PD-L1 was significantly higher than that in control group and MDS middle-risk group.4 The expression of PD-L1 in bone marrow mononuclear cells before DAC treatment in patients with AML was not different from that in controlgroup,the expression of PD-1 on peripheral blood T lymphocytes was higher than that in control group;The expression of PD-1/PD-L1 was significantly increased after DAC treatment.5 PD-1/PD-L1 and prognosis: The expression of PD-1 on peripheral blood T lymphocytes and PD-L1 on bone marrow mononuclear cells,the expression of PD-1mRNA and PD-L1 mRNA in peripheral blood and bone marrow mononuclear cells of MDS and AML non remission group were significantly higher than those in remission group.Increased expression of PD-1/PD-L1 may mediate DAC resistance,which can be used as an index to evaluate the prognosis and therapeutic effect.6 There was PD-L1 in MDS/leukemia cell line SKM-1 and HL-60 cell lines,and the expression was increased after treatment with DAC,The highest expression was the concentration of DAC 50umol/l. |
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