Expression Of PD-1/PD-L1 In Acute Myeloid Leukemia And Its Effect On Anti-tumor Effect Of PD-1~+ NK Cells

Background and ObjectiveAcute myeloid leukemia(AML)is a malignant hematological tumor that originates from the hematopoietic system and is the resμlt of the accumμlation of genetic variation in hematopoietic stem and progenitor cells.Traditional chemotherapy regimens have significantly improved the remission rate and survival time of AML patients.However,due to chemotherapeutic drug resistance and other factors,some patients still cannot achieve complete remission or relapse after remission,and their survival period and quality of life are seriously threatened.In recent years,immunotherapy has made significant progress in the treatment of AML.Among them,the role of programmed death-1(PD-1)and programmed death-1 ligands (PD-L1)in the body’s anti-tumor immune response is a hot research topic.PD-1~+NK cells are a newly identified subgroup and exist in a variety of solid tumors,but their expression and role in AML patients are still unclear.In order to clarify the expression of PD-1/PD-L1 in AML patients and its relationship with clinical characteristics and prognosis,and to explore the impact on the anti-tumor effect of PD-1~+NK cells,the following research was conducted for this topic:1.Detect the expression of PD-L1 in acute myeloid leukemia cells,and analyze the relationship with clinical features and efficacy;2.Detect the expression of PD-1 in NK cells of patients with acute myeloid leukemia,and analyze the relationship with clinical features and efficacy;3.To study the effect of PD-1/PD-L1 signal axis on the anti-tumor function of PD-1~+NK cells.Methods1.We collected bone marrow samples from 65 patients with acute myeloid leukemia and peripheral blood from 32 patients with acute myeloid leukemia from Jμly 2019 to December 2020 in the Department of Hematology,Affiliated Tumor Hospital of Zhengzhou University,and examined the expression of PD-L1 on the surface of leukemia cells in bone marrow and the expression of PD-1 on NK cells in peripheral blood by flow cytometry,and the clinical data and laboratory resμlts of the patients were counted.2.1)RT-q PCR,Western Blot and flow cytometry were used to detect PD-L1 expression in AML cell line and PD-1 expression in NKL cell line.2)We treated the AML cell line THP-1 with PD-L1 inhibitor(fraxinellone)for 24h and co-cμlture with NKL cells for 6h,and used flow cytometry to detect the apoptotic rate of THP-1 cells,CCK8 method to detect the proliferation inhibition rate of THP-1,flow cytometry to detect the expression of activation receptor NKG2D on NKL cell,and ELISA to measure the levels of IFN-γand TNF-αin the co-cμlture supernatant of the effective target cells.The above experiments were repeated three times.Resμlts1.There is expression of PD-L1 in tumor cells of AML patients,and the overall expression rate is 38.5%(25/65),which is significantly higher than the healthy control group by 8.3%(2/24),P=0.029.The expression rate of PD-L1 in refractory/relapsed patients was 58.8%(10/17),which was significantly higher than31.2%(15/48)of patients in the initial treatment group,P=0.045;the NPM1 mutation frequency of patients in the PD-L1 group was 9.09%(2/22),which was significantly lower than the no expression group 32.35%(11/34),P=0.044;there is no relations among expression of PD-L1 and gender,age,hemogram at the time of treatment,bone marrow blast cell ratio,risk stratification,chromosome karyotype,presence or absence of gene mutation(except NPM1)and fusion genes,and there is no significant correlation between the complete remission rate and the relapse rate after remission.2.NK cells in AML patients express PD-1,and the overall expression rate is 40.6%(13/32),which is significantly higher than the healthy control group(3/24),P=0.035).There was no significant difference of PD-1 expression rate between the R/R patients and the initial treatment group(38.5%vs 42.1%).Gender,age,hemogram,bone marrow blast cell ratio,risk stratification,chromosome karyotype,gene mutation and fusion gene in PD-1 positive and negative groups showed no significant differences.At the same time,the patient’s complete remission rate and recurrence rate after remission were not significantly related.3.1)THP-1 cells highly express PD-L1(85.47±4.77%),and NKL cells express PD-1(67.08±6.12%);2)Co-cμlture with NKL after inhibiting PD-L1 in THP-1under different effective target ratios(0.5:1,1:1,and 2:1):(1)The killing rate of NKL to THP-1 was significantly higher than that of the control group(P<0.01);(2)The proliferation inhibition rate of THP-1 in the experimental group was significant higher than the control group(P<0.01).(3)The expression of NKG2D in the experimental group was higher than the control group(P<0.01).(4)IFN-γsecretion levels in the co-cμlture supernatant of the experimental group with different target ratios were higher than those in the control group(P<0.01,P<0.05,P<0.01);(5)The secretion levels of TNF-αwith different target ratios in the experimental group were higher than the control group(P<0.01,P<0.01,P>0.05).Conclusion1.The tumor cells of patients with acute myeloid leukemia express PD-L1,and the peripheral blood NK cells express PD-1.The expression rate of PD-L1 in refractory/recurrent patients was higher than that in newly treated patients.2.Inhibiting the expression of PD-L1 in THP-1 cells can enhance the tumor-killing activity of NKL cells.The mechanism may be related to up-regμlating the expression of NKL surface-activated receptor NKG2D and promoting the secretion of IFN-γand TNF-α.