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中华临床医师杂志(电子版)

中华临床医师杂志(电子版) ›› 2023, Vol. 17 ›› Issue (09) : 932 -938. doi: 10.3877/cma.j.issn.1674-0785.2023.09.002

临床研究

NK和NKT细胞对急性髓系白血病患者的临床影响
杨琬芳, 许晶, 张耀方, 王青, 杨智超, 任方刚, 王宏伟†()   
  1. 030001　太原，山西医科大学第二医院血液科实验室;030619　太原，山西中医药大学基础医学院（现工作单位）
    030001　太原，山西医科大学
    030001　太原，山西医科大学第二医院血液科实验室
    030619　太原，山西中医药大学基础医学院（现工作单位）
  • 收稿日期:2023-02-10 出版日期:2023-09-15
  • 通信作者: 王宏伟
  • 基金资助:
    山西省自然科学基金项目(202103021224299)

Clinical significance of NK and NKT cells on patients with acute myeloid leukemia

Wanfang Yang, Jing Xu, Yaofang Zhang, Qing Wang, Zhichao Yang, Fanggang Ren, Hongwei Wang†()   

  1. The Second Hospital of Shanxi Medical University, Taiyuan 030001, China;Shanxi University of Chinese Medicine, Taiyuan 030619, China
    Shanxi Medical University, Taiyuan 030001, China
    The Second Hospital of Shanxi Medical University, Taiyuan 030001, China
    Shanxi University of Chinese Medicine, Taiyuan 030619, China
  • Received:2023-02-10 Published:2023-09-15
  • Corresponding author: Hongwei Wang
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引用本文：

杨琬芳, 许晶, 张耀方, 王青, 杨智超, 任方刚, 王宏伟. NK和NKT细胞对急性髓系白血病患者的临床影响[J]. 中华临床医师杂志(电子版), 2023, 17(09): 932-938.

Wanfang Yang, Jing Xu, Yaofang Zhang, Qing Wang, Zhichao Yang, Fanggang Ren, Hongwei Wang. Clinical significance of NK and NKT cells on patients with acute myeloid leukemia[J]. Chinese Journal of Clinicians(Electronic Edition), 2023, 17(09): 932-938.

目的

探讨急性髓系白血病（AML）患者中NK和NKT细胞的数量和功能及其与临床生物学特征和预后的关系。

方法

回顾性收集2018年1月至2020年1月山西医科大学第二医院血液科血液病研究所初诊的172例成年AML患者，同期体检中心健康人105例作为做对照，采用流式细胞术检测外周血NK和NKT细胞的百分比，并分析其与AML患者临床生物学特征、预后的关系，荧光定量PCR和Western Blot检测NK和NKT细胞激活受体DNAM-1和抑制受体TIGIT、TACTILE的mRNA和相应分子的表达，分析其与AML患者病理特征的关系。

结果

和健康者相比，AML患者的NK和NKT细胞中位百分比较低8.77% vs 10.93%（P=0.006），0.55% vs 1.15%（P=0.003）；NK细胞百分比与骨髓中原始细胞比例成负相关性（r=-0.313，P=0.044）；NK和NKT细胞的中位百分比越大其危险度分层越低（P<0.05）；NKT细胞的百分比较高的AML患者有较好的OS和DFS（P<0.05）；NK细胞的百分比与AML患者的OS和DFS的关系无显著统计学意义（P>0.05）；NK和NKT细胞的激活受体DNAM-1mRNA和蛋白表达较低（P<0.05），而抑制受体TIGIT和TACTILE的mRNA和蛋白表达较高（P<0.05）；诱导治疗缓解、部分缓解和难治患者NK和NKT细胞中位百分比分别为10.32%、11.04%与10.56%，0.37%、0.41%和0.34%，差别无统计学意义（P>0.05）。

结论

AML患者的NK和NKT细胞百分比降低具有特异性临床特点和预后相关，同时其激活受体DNAM-1表达较低而抑制受体TIGIT、TACTILE的表达较高，这可能限制其细胞毒性并促进了AML的免疫逃逸；NK和NKT细胞的百分比可以作为预后评价，甚至有望成为治疗AML的潜在靶标。

关键词: NK细胞, NKT细胞, AML, DNAM-1, TIGIT
Objective

To investigate the number and function of natural killer (NK) and natural killer T (NKT) cells in AML patients and to analyze their relationship with clinical biological characteristics and prognosis.

Methods

One hundred and seventy-two adult AML patients initially diagnosed at the Hematology Department and Hematology Research Institute of the Second Hospital of Shanxi Medical University from January 2018 to January 2020 were collected retrospectively, and 105 healthy individuals from the physical examination center were included as controls. The percentage of NK and NKT cells in peripheral blood was detected by flow cytometry, and the relationship between NK and NKT cells and clinical biological characteristics and prognosis of AML patients was analyzed. Fluorescence quantitative PCR and Western Blot were used to detect the mRNA and protein expression of the activating NK and NKT cell receptor DNAM-1 and their inhibitory receptors TIGIT and TACTILE. The relationship between the expression of these molecules and the pathological characteristics of AML patients was analyzed.

Results

Compared with healthy people, the median percentage of NK and NKT cells in AML patients was lower (8.77% vs 10.93%, P=0.006; 0.55% vs 1.15%, P=0.003). The percentage of NK cells was negatively correlated with the proportion of blasts in bone marrow (r=-0.313, P=0.044). The higher the median percentage of NK and NKT cells, the lower the risk grade (P<0.05). AML patients with a higher percentage of NKT cells had better overall survival (OS) and disease-free survival (DFS) (P<0.05). The relationship between the percentage of NK cells and OS and DFS in AML patients was not statistically significant (P>0.05). The mRNA and protein expression of the activating receptor DNAM-1 in NK and NKT cells was higher (P<0.05). However, the mRNA and protein expression of the inhibitory receptors TIGIT and TACTILE receptor was low (P<0.05). The median percentages of NK and NKT cells in patients with induced remission, partial remission, and refractoriness to treatment were 10.32%, 11.04%, and 10.56%, and 0.37%, 0.41%, and 0.34%, respectively, with no statistical significance.

Conclusion

The decrease in the percentage of NK and NKT cells in AML patients is related to specific clinical characteristics and prognosis. The expression of DNAM-1 in NK and NKT cells is lower, while that of TIGIT and TACTILE is higher, which might limit their cytotoxicity and promote the immune escape of AML. The percentage of NK and NKT cells can be used as a prognostic index, and may be a potential target for the treatment of AML.

Key words: NK cells, NKT cells, DNAM-1, TIGIT, AML
图1 NK和NKT细胞亚群的选择。图a为白细胞的选择；图b为T细胞的选择；图c为NK细胞和NKT细胞的选择 注：NK为自然杀伤细胞；NKT为自然杀伤T细胞
表1 荧光定量PCR正向和反向引物序列
基因 Forwards sequence 5'-3' Reverse sequence 5'-3'
DNAM-1 GATGTTGGCTACTATTCCTGCTC CTGAACCACCTGTATCACCTTC
TIGIT TCTGCATCTATCACACCTACCC CCACCACGATGACTGCTGT
TACTILE GTCTATCATCCCCAATACGGCT CTTCCACTGACTGAACAAGACAT
表2 AML患者的临床特征
一般资料 AML患者（172例） 健康人（105例） P值
年龄（岁） 42.5（19~82） 45.6（24~79） 0.563
性别[男性，例（%）] 94（54.65） 54（51.43） 0.293
NK（%） 8.77（0.10~33.92） 10.93（2.6~27） 0.006a
NKT（%） 0.55（0~6.20） 1.15（0.23~5.74） 0.003a
NEU（×109/L） 11.52（0~127.42） 7.43（4.16~10.02） 0.012a
BM blasts（%） 54.13（1.6~96.5） - -
CR[例（%）] 84（48.84） - -
PR[例（%）] 48（27.91） - -
RF[例（%）] 19（11.05） - -
图2 AML患者和健康供者外周血的NK和NKT细胞比例。图a为AML患者NK和NKT细胞的选择；图b为健康者NK和NKT细胞的选择；图c为AML患者和健康者的NK细胞比例；图d为AML患者和健康者的NTK细胞比例 注：**P<0.01；AML为急性髓系白血病；NK为自然杀伤细胞；NKT为自然杀伤T细胞
表3 NK和NKT细胞对诱导治疗的反应
CR PR RF aP值 bP值 cP值
NK（%） 10.32（0~27.44） 11.04（0.14~33.92） 10.56（0~26.81） 0.632 0.575 0.331
NKT（%） 0.37（0.1~4.33） 0.41（0~3.76） 0.34（0~6.20） 0.769 0.596 0.297
图3 AML患者的NK和NKT细胞与风险分层关系 注：*P<0.05；ns：P>0.05；AML为急性髓系白血病；NK为自然杀伤细胞；NKT为自然杀伤T细胞
图4 NK和NKT细胞的ROC曲线及AML患者预后的关系。图a、b为AML患者NK细胞和NKT细胞的ROC曲线；图c、d为NK high组和NK low组的OS和DFS；图e、f为NKT high组和NKT low组的OS和DFS 注：NK为自然杀伤细胞；NKT为自然杀伤细胞；AML为急性髓系白血病；OS为总生存期；DFS为无病生存期
图5 NK和NKT细胞中活化受体和抑制受体mRNA及蛋白表达。图a、b、c为AML患者和健康对照者DNAM-1，TIGIT和TACTILE mRNA表达相对量；图d、e、f为DNAM-1，TIGIT和TACTILE蛋白表达及相对量 注：*P<0.05；**P<0.01；ns：P>0.05；NK:为自然杀伤细胞；NKT为自然杀伤T细胞
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