摘要目的:探讨程序性死亡受体1（PD-1）/程序性死亡受体配体1（PD-L1）通路在急性髓系白血病（AML）患者中的表达及其与临床特征、预后的关系，并体外验证其对PD-1阳性自然杀伤（NK）细胞抗AML细胞的影响。方法:前瞻性收集2019年7月至2020年12月于郑州大学附属肿瘤医院确诊的65例AML患者骨髓标本及32例AML患者外周血，同时留取24名健康人外周血作为健康对照。流式细胞术检测骨髓肿瘤细胞PD-L1、外周血NK细胞PD-1表达水平，分析骨髓肿瘤细胞PD-L1与NK细胞PD-1表达与患者临床病理特征及疗效、预后的关系。流式细胞术检测AML细胞株THP-1（靶细胞）的PD-L1、NK细胞株NKL（效应细胞）的PD-1表达水平。经及未经PD-L1抑制剂梣酮（25 μmol/L）处理的THP-1细胞分别为实验组和对照组，分别与NKL细胞按不同效靶比共培养。流式细胞术检测THP-1细胞凋亡情况和NKL细胞NKG2D的表达水平，CCK-8法检测细胞增殖情况并计算增殖抑制率，酶联免疫吸附试验（ELISA）检测共培养体系上清中干扰素γ（IFN-γ）、肿瘤坏死因子α（TNF-α）水平。结果:AML患者骨髓肿瘤细胞PD-L1阳性表达者比例高于健康对照组[38.5%（25/65）比8.3%（2/24）， P＝0.029]，AML患者外周血NK细胞PD-1阳性表达者比例高于健康对照组[40.6%（13/32）比12.5%（3/24）， P＝0.035]。骨髓肿瘤细胞PD-L1阳性和阴性患者间及外周血NK细胞PD-1阳性和阴性患者间性别、年龄、就诊时血象、原始细胞比例、危险度分层、染色体核型、有无基因突变（除外NPM1基因）、融合基因及法、美、英协作组（FAB）分型的构成差异均无统计学意义（均 P>0.05）。复发难治患者中骨髓肿瘤细胞PD-L1阳性表达者比例高于初治患者[58.8%（10/17）比31.2%（15/48）， P＝0.045]，复发难治患者与初治患者间外周血NK细胞PD-1阳性表达者比例的差异无统计学意义[38.5%（5/13）比42.1%（8/19）， P＝0.837]。PD-L1阳性与阴性患者完全缓解（CR）率差异无统计学意义[69.6%（16/23）比74.3%（26/35）， P>0.05]；PD-1阳性与阴性患者CR率差异无统计学意义[66.7%（8/12）比70.6%（12/17）， P>0.05]。PD-L1阳性与阴性患者CR后复发率差异无统计学意义[12.5%（2/16）比19.2%（5/26）， P>0.05]；PD-1阳性与阴性患者CR后复发率差异无统计学意义[25.0%（2/8）比16.7%（2/12）， P>0.05]。流式细胞术检测示，NKL细胞PD-1阳性率为（67±6）%，THP-1细胞PD-L1阳性率为（85±5）%。与NKL细胞共培养后，与对照组相比，实验组THP-1细胞凋亡率和增殖抑制率均明显增高，NKL细胞表面NKG2D表达升高，共培养上清中IFN-γ、TNF-α水平升高。 结论:AML患者骨髓肿瘤细胞PD-L1表达水平较高，外周血NK细胞PD-1表达水平亦较高，复发难治AML患者骨髓肿瘤细胞PD-L1表达水平高于初治患者。体外抑制THP-1细胞PD-L1表达可增强NKL细胞肿瘤杀伤活性，其机制可能是抑制THP-1细胞PD-L1表达后上调了NKL细胞活化型受体NKG2D的表达以及促进了IFN-γ和TNF-α的分泌。

abstractsObjective:To investigate the expression of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway in patients with acute myeloid leukemia (AML) and its relationship with clinical features and prognosis, and to examine its effect on PD-1-positive natural killer (NK) cells against AML cells in vitro.Methods:The bone marrow samples of 65 AML patients and the peripheral blood of 32 AML patients diagnosed in Affiliated Cancer Hospital of Zhengzhou University from July 2019 to December 2020 were prospectively collected, and the peripheral blood of 24 healthy people was taken as healthy control. The expression level of PD-L1 in bone marrow tumor cells and expression level of PD-1 in peripheral blood NK cells were detected by flow cytometry. The correlations of PD-1 expression in bone marrow tumor cells and PD-1 expression in NK cells with the clinicopathological features, curative effect and prognosis of patients were analyzed. Flow cytometry was used to detect the expression level of PD-L1 in AML cell line THP-1 (target cells) and the expression level of PD-L1 in NK cell line NKL (effector cells). THP-1 cells treated with and without 25 μmol/L of PD-L1 inhibitor fraxinellone were used as experimental group and control group, and co-cultured with NKL cells at different effector-to-target ratios. The apoptosis of THP-1 cells and the expression of NKG2D in NKL cells were detected by flow cytometry, the cell proliferation status was detected by CCK-8 and the cell proliferation inhibition rate was calculated; the levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant of co-culture system were detected by enzyme-linked immunosorbent assay (ELISA).Results:The proportion of AML patients with PD-L1-positive expression in bone marrow tumor cells was higher than that in the healthy control group [38.5% (25/65) vs. 8.3% (2/24), P = 0.029]. The proportion of AML patients with PD-1-positive expression in peripheral blood NK cells was higher than that in the healthy control group [40.6% (13/32) vs. 12.5% (3/24), P = 0.035]. There were no statistical differences in sex, age, hemogram, proportion of primordial cells, risk stratification, chromosomal karyotype, gene mutation (except NPM1 gene), fusion gene and French-American-British cooperative group (FAB) typing between patients with PD-L1 positive and negative in bone marrow tumor cells and between patients with PD-1 positive and negative in peripheral blood NK cells (all P > 0.05). In relapsed/refractory patients, the proportion of patients with PD-L1-positive expression in bone marrow tumor cells was higher than that in newly treated patients [58.8% (10/17) vs. 31.2% (15/48), P = 0.045]. There was no significant difference in the proportion of patients with PD-1-positive expression in peripheral blood NK cells between relapsed/refractory patients and newly treated patients [(38.5% (5/13) vs. 42.1% (8/19), P = 0.837]. There was no statistical difference in complete remission (CR) rate between PD-L1 positive and negative patients [69.6% (16/23) vs. 74.3% (26/35), P > 0.05]. There was no statistical difference in CR rate between PD-1 positive and negative patients [66.7% (8/12) vs. 70.6% (12/17), P > 0.05]. There was no statistical difference in recurrence rate after CR between PD-L1 positive and negative patients [12.5% (2/16) vs. 19.2% (5/26), P > 0.05]. There was no statistical difference in recurrence rate after CR between PD-1 positive and negative patients [25.0% (2/8) vs. 16.7% (2/12), P > 0.05]. Flow cytometry showed that the positive rate of PD-1 in NKL cells was (67±6)% and the positive rate of PD-L1 in THP-1 cells was (85±5)%. After co-culture with NKL cells, the apoptotic rate and proliferation inhibition rate of THP-1 cells were higher in the experimental group compared with the control group, the expression of NKG2D on the surface of NKL cells was elevated, and the levels of IFN-γ and TNF-α in the co-culture supernatant were increased. Conclusions:In AML patients, the expression of PD-L1 in bone marrow tumor cells is high, and the expression of PD-1 in peripheral blood NK cells is also high. The expression of PD-L1 in bone marrow tumor cells of relapsed/refractory AML patients is higher than that of newly treated patients. Inhibition of PD-L1 expression in THP-1 cells can enhance the tumor killing activity of NKL cells in vitro. The mechanism may be that inhibition of PD-L1 expression in THP-1 cells up-regulates the expression of NKL cell activated receptor NKG2D and promotes the secretion of IFN- γ and TNF- α.