原发性肝癌外周血NK细胞受体NKG2D表达和NK细胞亚群分布的变化及意义
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摘要
研究背景
自然杀伤(natural killer ,NK)细胞是机体天然免疫系统的主要细胞成分之一,在抗病毒、抗肿瘤和移植排斥反应中起着十分重要的作用。自然杀伤细胞受体G2D(natural killer cells receptor group 2D,NKG2D)是NK细胞表面的一种活化性受体,可激活NK细胞对靶细胞的杀伤效应。NK细胞对肿瘤细胞和病毒感染细胞的杀伤机制主要是通过Fas(CD95)和FasL(CD95L)相互作用、抗体依赖性细胞介导的细胞毒作用( antibody-dependent cell-mediated cytotoxicity,ADCC)以及通过直接释放细胞毒效应分子穿孔素和颗粒酶B而破坏靶细胞。人类NK细胞的分化抗原很多,其中具有代表性的细胞表型为CD3-、CD56+、CD16+[7,8]。人外周血约80-90%的NK细胞低密度表达CD56(CD56dim),同时高水平表达CD16(CD16brigh),可有效地发挥细胞毒作用;约10-20%的NK细胞表型为CD56brighCD16dim或CD56brighCD16-,其功能主要是分泌细胞因子,参与机体的免疫调节。但在肝癌患者中,外周血NK细胞受体NKG2D和NK细胞亚群分布的变化如何,其对NK细胞杀伤活性的影响又如何,文献报道较少涉及,本研究就此进行探讨。
研究目的
1.探讨正常人外周血NK细胞亚群的生物学特性。
2.探讨原发性肝癌患者外周血NK细胞受体NKG2D表达及NK细胞亚群分布的变化,并分析其对NK细胞杀伤活性的影响。
研究方法
1.应用流式细胞仪在单个细胞水平上分析正常人外周血NK细胞亚群的多样性和生物学特征。
2.应用流式细胞仪检测分析20例原发性肝癌、23例乙型肝炎后肝硬化、20例乙型肝炎及20例健康者的外周血NK细胞数量、其受体NKG2D的表达及NK细胞亚群的分布情况,并用酶标仪检测分析各组样本NK细胞的细胞毒活性。
研究结果
1.根据CD56和CD16分子表达与否将NK细胞分为CD56+、CD56+CD16+和CD16+三个亚群。在健康人中,CD56+NK细胞表面CD95、NKG2D及细胞内穿孔素+颗粒酶B的表达分别为22.2(18.0,29.0)%、53.4(47.1,62.0)%、53.0(44.7,63.0)%,明显低于CD56+CD16+NK细胞[39.9(35.8,44.9)%、69.1(63.8、76.7)%、95.9(89.5,99.3)%]和CD16+NK细胞[39.1(32.9,46.0)%、67.9(63.1,72.1)%、93.6(85.7,96.3)%],差异有统计学意义,P均<0.01,而CD56+CD16+和CD16+NK细胞亚群之间则无明显差异,P均>0.05。因为NK细胞对肿瘤细胞和病毒感染细胞的杀伤机制主要是通过Fas(CD95)和FasL(CD95L)相互作用以及直接释放细胞毒效应分子穿孔素和颗粒酶B而破坏靶细胞,因此该结果说明NK细胞杀伤作用主要通过CD56+CD16+和CD16+NK细胞进行,其杀伤作用较单纯CD56+细胞明显强。(除平均值外,所有结果都是用中位数表示法即M(P25,P75),下同。)
2.肝癌和肝硬化组CD56+NK细胞比例分别为16(12,29)%、18(13,25)%,均高于正常组10(8,18)%和乙肝组13(10,15)%(均P<0.05),肝癌和肝硬化组CD56+CD16+NK细胞比例分别为75(62,78)%、70(57,81)%,均低于正常组80(72,85)%和乙肝组79(71,82)%(均P<0.05),而CD16+NK细胞四组间无明显差异[正常组8(3,12)%,乙肝组8(4,11)%,肝硬化组7(5,15)%,肝癌组9(6,12)%],均P>0.05。由于CD56+CD16+和CD16+NK细胞的杀伤作用较单纯CD56+ NK细胞明显强,因此本组结果提示肝癌和肝硬化患者NK细胞的杀伤作用较对照组及乙肝组明显减弱。
3.原发性肝癌患者的NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平及NK细胞数[(25±7)%、6(3,13)%、0.7(0.3,0.9)×107、15(11,30)、(1.1±0.6)×108]均明显低于正常组[(63±7)%、36(28,44)%、8.3(7.0,10.6)×107、116(99,153)、(2.7±1.1)×108]和乙肝组[(41±8)%、16(12,24)%、2.8(2.0,3.7)×107、49(31,72)、(1.9±1.1)×108],差异均有统计学意义(均P<0.05);与肝硬化组[(29±10)%、7(4,15)%、0.6(0.3,1.7)×107、29(16,42)、(1.5±1.2)×108]比较也略低,但只有NK细胞NKG2D的表达水平两组差异有统计学意义(P<0.05),其余指标包括NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数及NK细胞数两组间差异均无统计学意义(均P>0.05)。
4.进一步分级比较,肝癌组Child B级和C级NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平分别为Child B: (27±5) % , 6.1(3.2,11.9)%, 0.7(0.5,0.9)×107, 14(12,23);Child C: (17±2) %, 3.0(1.1,5.6)%, 0.2(0.1,0.3)×107, 6(4,18),均分别较肝硬化组[Child B: (39±9)%, 12.4(8.7,25.6)%, 2.0(1.3,3.0)×107, 39(30,83); Child C: (25±6) %, 4.5(2.2,11.0)%, 0.4(0.3,0.7)×107, 20(14,37)]低,其中NK细胞杀伤率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平在肝癌Child B与肝硬化Child B、肝癌Child C与肝硬化Child C两组间差异显著,P<0.05,但NK细胞NKG2D的阳性表达率两组间差异无显著性,P>0.05;肝癌组和肝硬化组Child B级NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平均较Child C级高,其中NK细胞杀伤率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平在肝癌Child B与肝癌Child C、肝硬化Child B与肝硬化Child C两组间差异显著,但NK细胞NKG2D的阳性表达率两组间差异无显著性,P>0.05。
5.肝癌组Child B级NK细胞数和NK细胞亚群CD56+NK、CD56+CD16+NK、CD16+NK构成比分别为:(1.2±0.6)×108、15.4(11.2,35.5)%、74.3(55.5,77.0)%、9.8(6.4,11.8)%,肝癌组Child C级分别为:(0.8±0.4)×108、16.4(15.3,17.7)%、76.4(70.3,77.9)%、8.5(4.4,13.6)%,肝硬化组Child B级分别为:(1.8±1.5)×108、19.7(12.9,22.5)%、72.1(56.0,80.4)%、13.2(4.2,21.5)%,肝硬化组Child C级分别为:(1.2±0.9)×108、16.7(13.0,30.0)%、70.2(57.6,82.9)%、6.7(4.0,14.1)%,其在肝癌Child B与肝硬化Child B、肝癌Child C与肝硬化Child C、肝癌Child B与肝癌Child C、肝硬化Child B与肝硬化Child C四组间差异无显著性,P均>0.05。
6. NK细胞的活性与NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞NKG2D的表达水平、NK细胞数及NK细胞中CD56+CD16+NK比例均呈正相关关系(n=83例)(r=0.770、0.927、0.734、0.657,0.287,均P <0.01),与NK细胞中CD56+细胞比例呈负相关关系(r=-0.301,P<0.01)。肝癌组和肝硬化组Child-pugh积分与NK细胞活性、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞NKG2D的表达水平及NK细胞数均呈负相关性(肝癌组:r=-0.647,-0.356,-0.652,-0.414,-0.380;P=0.002,0.124,0.002,0.069,0.098;肝硬化组:r=-0.517,-0.368,-0.566,-0.412,-0.311;P=0.012,0.084,0.005,0.051,1.490)。
结论
1. NK细胞乃异质性群体,随着CD56表达减少和CD16表达增加,其细胞杀伤功能逐渐增强。
2.肝癌患者外周血NK细胞活性降低与NKG2D的表达下调和NK细胞亚群分布变化有关。
3.肝功能可影响NK细胞NKG2D的表达和NK细胞的杀伤活性,但对NK细胞亚群分布无明显影响。
Background
Natural killer cell (NK)is one of the major component of native immune system in human organism,which plays fully important role in antivirus,antitumor and graft-rejection. NKG2D is one of NK cell’s actived receptors,which can active the killing effect of NK cells on target cells. The mechanism of NK cells’killing effect on tumor and vivus infection cells are based on Fas(CD95) and FasL(CD95L) , antibody-dependent cell-mediated cytotoxicity(ADCC)and the action of cytotoxic effect molecule such as perforin and Granzyme B through direct release to target cells. There are many different antigen in human NK cell , the representative cell phenotytes are CD3-,CD56+,CD16+. About 80-90% NK cells in human peripheral blood express low-density CD56 ( CD56dim ) and high-density CD16(CD16brigh).Therefor, NK cells can play the role of cytotoxicity.About 10-20% NK cells phenotyte are CD56brighCD16dim or CD56brighCD16-,which can secrete many cell fators and participate the immunoregulation.
Objectives
1. To investigate the phenotypic and biological properties of NK cell subpopulations in peripheral blood of health control.
2. To investigate the expression of NK cells receptor NKG2D and the change of NK cell subpopulations in peripheral blood in patients with primary hepatic carcinoma and the relationship among NKG2D expression, NK cell subpopulations and cytotoxicity of NK cells.
Methods
1. Surface markers and intracellular cytotoxic molecules of peripheral blood mononuclear cells in healthy controls were stained by color-labeled monoclonal antibodies. The phenotypic and biological characteristics of different NK subsets were analyzed by flow cytometer.
2. Flow cytometry was used to determine the number of NK cells,the expression of NK cells receptor NKG2D and the change of NK cells subpopulations in peripheral blood in patients with primary hepatic carcinoma (20 cases),hepatitis B cirrhosis(23 cases),hepatitis B(20 cases) and healthy control(20 cases). The microplate reader was used to detect cytotoxicity of NK cells in all cases.
Results
1. Three distinct subpopulations (CD56+, CD56+CD16+and CD16+)of human NK cells were identified based upon the expression of CD56 and CD16 molecules.The expression of NKG2D,CD95,perforin and Granzyme B in CD56+NK cells [22.2(18.0,29.0)%,53.4(47.1,62.0)%,53.0(44.7,63.0)%] were obviously lower than those in CD56+CD16+NK cells[39.9(35.8,44.9)%,69.1(63.8,76.7)%,95.9(89.5,99.3)%]and CD16+NK cells[39.1(32.9,46.0)%,67.9(63.1,72.1)%,93.6(85.7,96.3)%](all P<0.01),but these values were similar between CD56+CD16+ cells group and CD16+NK cells group(P>0.05).
2. The constituent ratio of CD56+NK cells in NK cells in both the liver cancer group[16(12,29)%] and the hepatitis B cirrhosis group[18(13,25)%] were obviously higher than those in the healthy group[10 (8,18)%] and the hepatitis B group[13(10,15)%](P<0.05). On the contrary, the constituent ratio of CD56+CD16+NK cells in the liver cancer group[75(62,78)%] and the hepatitis B cirrhosis group[70(57,81)%] were much lower than those in the healthy group[80(72,85)%] and hepatitis B group[79(71,82)%](P<0.05). While the constituent ratio of CD16+NK cells were similar among four groups[healthy group :8(3,12)%,hepatitis B group :8(4,11)%,hepatitis B cirrhosis group :7(5,15)%,liver cancer group :9(6,12)%)](all P>0.05).
3. All the values of NK cell killing rate, NKG2D expression rate, the number of NKG2D+NK cells, NKG2D expression level and the number of NK cells in the liver cancer group[(25±7)%,6(3,13)%,0.7(0.3,0.9)×107,15(11,30),(1.1±0.6)×108] decreased significantly as compared with the healthy group[(63±7)%,36(28,44)%,8.3(7.0,10.6)×107,116(99,153),(2.7±1.1)×108] and the hepatitis B group [(41±8)%,16(12,24)%,2.8(2.0,3.7)×107,49(31,72),(1.9±1.1)×108](P<0.05). When compared with the hepatitis B cirrhosis group [(29±10)%,7(4,15)%,0.6(0.3,1.7)×107,29(16,42),(1.5±1.2)×108], the NKG2D expression level of NK cells in the liver cancer group decreased significantly(P<0.05). The other values decreased slightly(P>0.05).
4. All the values of NK cell killing rate,NKG2D expression rate, the number of NKG2D+NK cells and the NKG2D expression level of NK cells in the subgroups of liver cancer patients [Child B: (27±5) % , 6.1(3.2,11.9)%, 0.7(0.5,0.9)×107, 14(12,23);Child C: (17±2) %, 3.0(1.1,5.6)%, 0.2(0.1,0.3)×107, 6(4,18)] decreased significantly as compared with those in the hepatitis B cirrhosis group[Child B:(39±9)%, 12.4(8.7,25.6)%, 2.0(1.3,3.0)×107, 39(30,83); Child C: (25±6) %, 4.5(2.2,11.0)%, 0.4(0.3,0.7)×107, 20(14,37)],(P<0.05), except the expression rate of NKG2D in NK cells(P>0.05). These comparessions were Child B of the liver cancer group vs.Child B of the hepatitis B cirrhosis group, Child C of the liver cancer group vs. Child C of the hepatitis B cirrhosis group. while in the liver cancer group, all these values except the expression rate of NKG2D in NK cells in the Child B subgroup were much higher than those in the Child C subgroup, P<0.05. The same results were found in the hepatitis B cirrhosis group.
5. The number of NK cells and the constituent ratio of CD56+NK ,CD56+CD16+NK and CD16+NK cells in NK cells in Child B of the liver cancer subgroup were (1.2±0.6)×108,15.4(11.2,35.5)%,74.3(55.5,77.0)%,9.8(6.4,11.8)% and in Child C of the liver cancer subgroup were(0.8±0.4)×108,16.4(15.3,17.7)%,76.4(70.3,77.9)%,8.5(4.4,13.6)%;in Child B of the hepatitis B cirrhosis subgroup were(1.8±1.5)×108,19.7(12.9,22.5)%,72.1(56.0,80.4)%,13.2(4.2,21.5)%;in Child C of the hepatitis B cirrhosis subgroup were(1.2±0.9)×108,16.7(13.0,30.0)%,70.2(57.6,82.9)%,6.7(4.0,14.1)%.All of these values were similar between Child B of the liver cancer subgroup and Child B of the hepatitis B cirrhosis subgroup,Child C of the liver cancer subgroup and Child C of the hepatitis B cirrhosis subgroup,Child B of the liver cancer subgroup and Child C of the liver cancer subgroup, Child B of the hepatitis B cirrhosis subgroup and Child C of the hepatitis B cirrhosis subgroup, P>0.05.
6. The NK cell activity showed an obvious positive correlation to the NKG2D expression rate in NK cells, the number of NKG2D+NK cells,the NKG2D expression level in NK cells,the number of NK cell and the constituent ratio of CD56+CD16+NK cells in NK cells(n=83cases)(r=0.770,0.927,0.734,0.657,0.287,all P<0.01),while negative correlation with the constituent ratio of CD56+NK cells in NK cells(r=-0.301,P<0.01).Child-pugh score in the liver cancer group and the hepatitis B cirrhosis group were obviously negative related to the NK cells activity,the NKG2D expression rate in NK cells, the number of NKG2D+NK cells and NKG2D expression level in NK cells and the number of NK cell(the liver cancer group:r=-0.647,-0.356,-0.652,-0.414,-0.380;P=0.002,0.124,0.002,0.069,0.098;the hepatitis B cirrhosis group:r=-0.517,-0.368,-0.566,-0.412,-0.311;P=0.012,0.084,0.005,0.051,1.490)。
Conclusions
1. NK cells in healthy control’s peripheral blood mononuclear cells are phenotypically and biologically heterogenous. With the reducing of CD56 expression and the increasing of CD16 expression, the killing activity of NK cells strengthened gradually.
2. The decreasing of NK cells activity in patients with primary hepatic carcinoma is closely related to the lower expression of NKG2D and the change of NK cells subpopulations.
3. Liver function can affect the expression of NKG2D and the activity of NK cells,but can not change the constituent ratio of NK cells subpopulations.
自然杀伤(natural killer ,NK)细胞是机体天然免疫系统的主要细胞成分之一,在抗病毒、抗肿瘤和移植排斥反应中起着十分重要的作用。自然杀伤细胞受体G2D(natural killer cells receptor group 2D,NKG2D)是NK细胞表面的一种活化性受体,可激活NK细胞对靶细胞的杀伤效应。NK细胞对肿瘤细胞和病毒感染细胞的杀伤机制主要是通过Fas(CD95)和FasL(CD95L)相互作用、抗体依赖性细胞介导的细胞毒作用( antibody-dependent cell-mediated cytotoxicity,ADCC)以及通过直接释放细胞毒效应分子穿孔素和颗粒酶B而破坏靶细胞。人类NK细胞的分化抗原很多,其中具有代表性的细胞表型为CD3-、CD56+、CD16+[7,8]。人外周血约80-90%的NK细胞低密度表达CD56(CD56dim),同时高水平表达CD16(CD16brigh),可有效地发挥细胞毒作用;约10-20%的NK细胞表型为CD56brighCD16dim或CD56brighCD16-,其功能主要是分泌细胞因子,参与机体的免疫调节。但在肝癌患者中,外周血NK细胞受体NKG2D和NK细胞亚群分布的变化如何,其对NK细胞杀伤活性的影响又如何,文献报道较少涉及,本研究就此进行探讨。
研究目的
1.探讨正常人外周血NK细胞亚群的生物学特性。
2.探讨原发性肝癌患者外周血NK细胞受体NKG2D表达及NK细胞亚群分布的变化,并分析其对NK细胞杀伤活性的影响。
研究方法
1.应用流式细胞仪在单个细胞水平上分析正常人外周血NK细胞亚群的多样性和生物学特征。
2.应用流式细胞仪检测分析20例原发性肝癌、23例乙型肝炎后肝硬化、20例乙型肝炎及20例健康者的外周血NK细胞数量、其受体NKG2D的表达及NK细胞亚群的分布情况,并用酶标仪检测分析各组样本NK细胞的细胞毒活性。
研究结果
1.根据CD56和CD16分子表达与否将NK细胞分为CD56+、CD56+CD16+和CD16+三个亚群。在健康人中,CD56+NK细胞表面CD95、NKG2D及细胞内穿孔素+颗粒酶B的表达分别为22.2(18.0,29.0)%、53.4(47.1,62.0)%、53.0(44.7,63.0)%,明显低于CD56+CD16+NK细胞[39.9(35.8,44.9)%、69.1(63.8、76.7)%、95.9(89.5,99.3)%]和CD16+NK细胞[39.1(32.9,46.0)%、67.9(63.1,72.1)%、93.6(85.7,96.3)%],差异有统计学意义,P均<0.01,而CD56+CD16+和CD16+NK细胞亚群之间则无明显差异,P均>0.05。因为NK细胞对肿瘤细胞和病毒感染细胞的杀伤机制主要是通过Fas(CD95)和FasL(CD95L)相互作用以及直接释放细胞毒效应分子穿孔素和颗粒酶B而破坏靶细胞,因此该结果说明NK细胞杀伤作用主要通过CD56+CD16+和CD16+NK细胞进行,其杀伤作用较单纯CD56+细胞明显强。(除平均值外,所有结果都是用中位数表示法即M(P25,P75),下同。)
2.肝癌和肝硬化组CD56+NK细胞比例分别为16(12,29)%、18(13,25)%,均高于正常组10(8,18)%和乙肝组13(10,15)%(均P<0.05),肝癌和肝硬化组CD56+CD16+NK细胞比例分别为75(62,78)%、70(57,81)%,均低于正常组80(72,85)%和乙肝组79(71,82)%(均P<0.05),而CD16+NK细胞四组间无明显差异[正常组8(3,12)%,乙肝组8(4,11)%,肝硬化组7(5,15)%,肝癌组9(6,12)%],均P>0.05。由于CD56+CD16+和CD16+NK细胞的杀伤作用较单纯CD56+ NK细胞明显强,因此本组结果提示肝癌和肝硬化患者NK细胞的杀伤作用较对照组及乙肝组明显减弱。
3.原发性肝癌患者的NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平及NK细胞数[(25±7)%、6(3,13)%、0.7(0.3,0.9)×107、15(11,30)、(1.1±0.6)×108]均明显低于正常组[(63±7)%、36(28,44)%、8.3(7.0,10.6)×107、116(99,153)、(2.7±1.1)×108]和乙肝组[(41±8)%、16(12,24)%、2.8(2.0,3.7)×107、49(31,72)、(1.9±1.1)×108],差异均有统计学意义(均P<0.05);与肝硬化组[(29±10)%、7(4,15)%、0.6(0.3,1.7)×107、29(16,42)、(1.5±1.2)×108]比较也略低,但只有NK细胞NKG2D的表达水平两组差异有统计学意义(P<0.05),其余指标包括NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数及NK细胞数两组间差异均无统计学意义(均P>0.05)。
4.进一步分级比较,肝癌组Child B级和C级NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平分别为Child B: (27±5) % , 6.1(3.2,11.9)%, 0.7(0.5,0.9)×107, 14(12,23);Child C: (17±2) %, 3.0(1.1,5.6)%, 0.2(0.1,0.3)×107, 6(4,18),均分别较肝硬化组[Child B: (39±9)%, 12.4(8.7,25.6)%, 2.0(1.3,3.0)×107, 39(30,83); Child C: (25±6) %, 4.5(2.2,11.0)%, 0.4(0.3,0.7)×107, 20(14,37)]低,其中NK细胞杀伤率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平在肝癌Child B与肝硬化Child B、肝癌Child C与肝硬化Child C两组间差异显著,P<0.05,但NK细胞NKG2D的阳性表达率两组间差异无显著性,P>0.05;肝癌组和肝硬化组Child B级NK细胞杀伤率、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平均较Child C级高,其中NK细胞杀伤率、NKG2D+NK细胞数、NK细胞中NKG2D的表达水平在肝癌Child B与肝癌Child C、肝硬化Child B与肝硬化Child C两组间差异显著,但NK细胞NKG2D的阳性表达率两组间差异无显著性,P>0.05。
5.肝癌组Child B级NK细胞数和NK细胞亚群CD56+NK、CD56+CD16+NK、CD16+NK构成比分别为:(1.2±0.6)×108、15.4(11.2,35.5)%、74.3(55.5,77.0)%、9.8(6.4,11.8)%,肝癌组Child C级分别为:(0.8±0.4)×108、16.4(15.3,17.7)%、76.4(70.3,77.9)%、8.5(4.4,13.6)%,肝硬化组Child B级分别为:(1.8±1.5)×108、19.7(12.9,22.5)%、72.1(56.0,80.4)%、13.2(4.2,21.5)%,肝硬化组Child C级分别为:(1.2±0.9)×108、16.7(13.0,30.0)%、70.2(57.6,82.9)%、6.7(4.0,14.1)%,其在肝癌Child B与肝硬化Child B、肝癌Child C与肝硬化Child C、肝癌Child B与肝癌Child C、肝硬化Child B与肝硬化Child C四组间差异无显著性,P均>0.05。
6. NK细胞的活性与NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞NKG2D的表达水平、NK细胞数及NK细胞中CD56+CD16+NK比例均呈正相关关系(n=83例)(r=0.770、0.927、0.734、0.657,0.287,均P <0.01),与NK细胞中CD56+细胞比例呈负相关关系(r=-0.301,P<0.01)。肝癌组和肝硬化组Child-pugh积分与NK细胞活性、NK细胞NKG2D的阳性表达率、NKG2D+NK细胞数、NK细胞NKG2D的表达水平及NK细胞数均呈负相关性(肝癌组:r=-0.647,-0.356,-0.652,-0.414,-0.380;P=0.002,0.124,0.002,0.069,0.098;肝硬化组:r=-0.517,-0.368,-0.566,-0.412,-0.311;P=0.012,0.084,0.005,0.051,1.490)。
结论
1. NK细胞乃异质性群体,随着CD56表达减少和CD16表达增加,其细胞杀伤功能逐渐增强。
2.肝癌患者外周血NK细胞活性降低与NKG2D的表达下调和NK细胞亚群分布变化有关。
3.肝功能可影响NK细胞NKG2D的表达和NK细胞的杀伤活性,但对NK细胞亚群分布无明显影响。
Background
Natural killer cell (NK)is one of the major component of native immune system in human organism,which plays fully important role in antivirus,antitumor and graft-rejection. NKG2D is one of NK cell’s actived receptors,which can active the killing effect of NK cells on target cells. The mechanism of NK cells’killing effect on tumor and vivus infection cells are based on Fas(CD95) and FasL(CD95L) , antibody-dependent cell-mediated cytotoxicity(ADCC)and the action of cytotoxic effect molecule such as perforin and Granzyme B through direct release to target cells. There are many different antigen in human NK cell , the representative cell phenotytes are CD3-,CD56+,CD16+. About 80-90% NK cells in human peripheral blood express low-density CD56 ( CD56dim ) and high-density CD16(CD16brigh).Therefor, NK cells can play the role of cytotoxicity.About 10-20% NK cells phenotyte are CD56brighCD16dim or CD56brighCD16-,which can secrete many cell fators and participate the immunoregulation.
Objectives
1. To investigate the phenotypic and biological properties of NK cell subpopulations in peripheral blood of health control.
2. To investigate the expression of NK cells receptor NKG2D and the change of NK cell subpopulations in peripheral blood in patients with primary hepatic carcinoma and the relationship among NKG2D expression, NK cell subpopulations and cytotoxicity of NK cells.
Methods
1. Surface markers and intracellular cytotoxic molecules of peripheral blood mononuclear cells in healthy controls were stained by color-labeled monoclonal antibodies. The phenotypic and biological characteristics of different NK subsets were analyzed by flow cytometer.
2. Flow cytometry was used to determine the number of NK cells,the expression of NK cells receptor NKG2D and the change of NK cells subpopulations in peripheral blood in patients with primary hepatic carcinoma (20 cases),hepatitis B cirrhosis(23 cases),hepatitis B(20 cases) and healthy control(20 cases). The microplate reader was used to detect cytotoxicity of NK cells in all cases.
Results
1. Three distinct subpopulations (CD56+, CD56+CD16+and CD16+)of human NK cells were identified based upon the expression of CD56 and CD16 molecules.The expression of NKG2D,CD95,perforin and Granzyme B in CD56+NK cells [22.2(18.0,29.0)%,53.4(47.1,62.0)%,53.0(44.7,63.0)%] were obviously lower than those in CD56+CD16+NK cells[39.9(35.8,44.9)%,69.1(63.8,76.7)%,95.9(89.5,99.3)%]and CD16+NK cells[39.1(32.9,46.0)%,67.9(63.1,72.1)%,93.6(85.7,96.3)%](all P<0.01),but these values were similar between CD56+CD16+ cells group and CD16+NK cells group(P>0.05).
2. The constituent ratio of CD56+NK cells in NK cells in both the liver cancer group[16(12,29)%] and the hepatitis B cirrhosis group[18(13,25)%] were obviously higher than those in the healthy group[10 (8,18)%] and the hepatitis B group[13(10,15)%](P<0.05). On the contrary, the constituent ratio of CD56+CD16+NK cells in the liver cancer group[75(62,78)%] and the hepatitis B cirrhosis group[70(57,81)%] were much lower than those in the healthy group[80(72,85)%] and hepatitis B group[79(71,82)%](P<0.05). While the constituent ratio of CD16+NK cells were similar among four groups[healthy group :8(3,12)%,hepatitis B group :8(4,11)%,hepatitis B cirrhosis group :7(5,15)%,liver cancer group :9(6,12)%)](all P>0.05).
3. All the values of NK cell killing rate, NKG2D expression rate, the number of NKG2D+NK cells, NKG2D expression level and the number of NK cells in the liver cancer group[(25±7)%,6(3,13)%,0.7(0.3,0.9)×107,15(11,30),(1.1±0.6)×108] decreased significantly as compared with the healthy group[(63±7)%,36(28,44)%,8.3(7.0,10.6)×107,116(99,153),(2.7±1.1)×108] and the hepatitis B group [(41±8)%,16(12,24)%,2.8(2.0,3.7)×107,49(31,72),(1.9±1.1)×108](P<0.05). When compared with the hepatitis B cirrhosis group [(29±10)%,7(4,15)%,0.6(0.3,1.7)×107,29(16,42),(1.5±1.2)×108], the NKG2D expression level of NK cells in the liver cancer group decreased significantly(P<0.05). The other values decreased slightly(P>0.05).
4. All the values of NK cell killing rate,NKG2D expression rate, the number of NKG2D+NK cells and the NKG2D expression level of NK cells in the subgroups of liver cancer patients [Child B: (27±5) % , 6.1(3.2,11.9)%, 0.7(0.5,0.9)×107, 14(12,23);Child C: (17±2) %, 3.0(1.1,5.6)%, 0.2(0.1,0.3)×107, 6(4,18)] decreased significantly as compared with those in the hepatitis B cirrhosis group[Child B:(39±9)%, 12.4(8.7,25.6)%, 2.0(1.3,3.0)×107, 39(30,83); Child C: (25±6) %, 4.5(2.2,11.0)%, 0.4(0.3,0.7)×107, 20(14,37)],(P<0.05), except the expression rate of NKG2D in NK cells(P>0.05). These comparessions were Child B of the liver cancer group vs.Child B of the hepatitis B cirrhosis group, Child C of the liver cancer group vs. Child C of the hepatitis B cirrhosis group. while in the liver cancer group, all these values except the expression rate of NKG2D in NK cells in the Child B subgroup were much higher than those in the Child C subgroup, P<0.05. The same results were found in the hepatitis B cirrhosis group.
5. The number of NK cells and the constituent ratio of CD56+NK ,CD56+CD16+NK and CD16+NK cells in NK cells in Child B of the liver cancer subgroup were (1.2±0.6)×108,15.4(11.2,35.5)%,74.3(55.5,77.0)%,9.8(6.4,11.8)% and in Child C of the liver cancer subgroup were(0.8±0.4)×108,16.4(15.3,17.7)%,76.4(70.3,77.9)%,8.5(4.4,13.6)%;in Child B of the hepatitis B cirrhosis subgroup were(1.8±1.5)×108,19.7(12.9,22.5)%,72.1(56.0,80.4)%,13.2(4.2,21.5)%;in Child C of the hepatitis B cirrhosis subgroup were(1.2±0.9)×108,16.7(13.0,30.0)%,70.2(57.6,82.9)%,6.7(4.0,14.1)%.All of these values were similar between Child B of the liver cancer subgroup and Child B of the hepatitis B cirrhosis subgroup,Child C of the liver cancer subgroup and Child C of the hepatitis B cirrhosis subgroup,Child B of the liver cancer subgroup and Child C of the liver cancer subgroup, Child B of the hepatitis B cirrhosis subgroup and Child C of the hepatitis B cirrhosis subgroup, P>0.05.
6. The NK cell activity showed an obvious positive correlation to the NKG2D expression rate in NK cells, the number of NKG2D+NK cells,the NKG2D expression level in NK cells,the number of NK cell and the constituent ratio of CD56+CD16+NK cells in NK cells(n=83cases)(r=0.770,0.927,0.734,0.657,0.287,all P<0.01),while negative correlation with the constituent ratio of CD56+NK cells in NK cells(r=-0.301,P<0.01).Child-pugh score in the liver cancer group and the hepatitis B cirrhosis group were obviously negative related to the NK cells activity,the NKG2D expression rate in NK cells, the number of NKG2D+NK cells and NKG2D expression level in NK cells and the number of NK cell(the liver cancer group:r=-0.647,-0.356,-0.652,-0.414,-0.380;P=0.002,0.124,0.002,0.069,0.098;the hepatitis B cirrhosis group:r=-0.517,-0.368,-0.566,-0.412,-0.311;P=0.012,0.084,0.005,0.051,1.490)。
Conclusions
1. NK cells in healthy control’s peripheral blood mononuclear cells are phenotypically and biologically heterogenous. With the reducing of CD56 expression and the increasing of CD16 expression, the killing activity of NK cells strengthened gradually.
2. The decreasing of NK cells activity in patients with primary hepatic carcinoma is closely related to the lower expression of NKG2D and the change of NK cells subpopulations.
3. Liver function can affect the expression of NKG2D and the activity of NK cells,but can not change the constituent ratio of NK cells subpopulations.
引文
1 Bancroft GJ.The role of natural killer cells in innate resistance to infection.CurrOp in Immunol,1993,5:503-510.
2 Smyth MJ,Hayakawa Y,Takeda K et al. New aspects of natural killer cell surveillance and therapy of cancer [J].Nat Rev Cancer,2002,2:850-861.
3田志刚.NK细胞新理论对重大疾病的新解释[J].中国免疫学杂志,2002,18:75-78.
4 French AR,Yokoyama WM.Natural killer cells and viral infections [J].Curr Opin Immunol,2003,15:45-51.
5 Natarajan K,Dimasi N,Wang J,et al.Structure and function of natural killer cell receptors : Multiple molecular solutions to self , nonself discrimination [J].Annu Rev Immunol,2002,20:853 - 885.
6 Vivier E,Tomasello E,Paul P.Lymphocyte activation via NKG2D:towards a new paradigm in immune recognition [J].Curr Opin Immunol,2002, 14:306-311.
7 Cooper MA,Fehniger TA,Caligiuri MA.The biology of human natural killer cell subsets [J].Trends Immunol,2001,22:633-640.
8金伯泉,主编.细胞和分子免疫学[M].第2版.北京:科学出版社,2001:9
9陆再英,钟南山,主编.内科学,第七版.北京,人民卫生出版社,2008,4: 457-462
10中华医学会肝病学分会、中华医学会感染病学分会.慢性乙型肝炎防治指南.中华传染病杂志,2005,6:421-431.
11 Trapani JA,Smyth MJ.Functional significance of the perforin/granzyme cell death pathway [J].Nat Rev Immunol,2002,2:735-747.
12 Diefenbach A,Tomasello E,Lucas M,et al.Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D[J]. Nat Immunol,2002,3:1142-1149.
13 Coudert JD,Held W.The role of the NKG2D receptor for tumor immunity.Semin Cancer Biol 2006,16:333–343.
14 Conejo-Garcia JR,Benencia F,Courreges MC et al.Letal,a tumor assoc- iated NKG2D immunoreceptor ligand,induces activation and expansion of effector immune cells. Cancer Biol Ther 2003:2:446–451.
15 Pende D,Cantoni C,Rivera P et al.Role of NKG2D in tumor cell lysis mediated by human NK cells:cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin. Eur J Immunol,2001:31:1076–1086.
16 Bauer S,Groh V,Wu J,et al.Activation of natural killer cells and T cells by NKG2D,a receptor for stress-inducible MICA.Science,1999:285: 727–730.
17 Groh V,Wu J,Yee C,et al.Tumor-derived soluble MIC ligands impair eexpression of NKG2D and T cell activation[J].Nature,2002,419:734- 738.
18梅家转,牛新清,郭坤元等.K562和K562/AO2细胞HLA I类分子与MICA/B的表达及其对NK细胞杀伤活性的影响.中国实验血液学杂志.2007,15(2):288-291
19 Diefenbach A,Jensen ER,Jamieson AM,et al.Rae1 and H60 ligands of the NKG2D recep tor stimulate tumour immunity.Nature,2001,413:165-171.
20 Cosman D,Mullberg J,Sutherland CL,et al.ULBPs,novel MHC class I related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor.Immunity 2001:14:123–133.
21 Pende D,Rivera P,Marcenaro S,et al.Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent nat- ural killer cell cytotoxicity.Cancer Res 2002:62:6178–6186.
22 Raffaghello L,Prigione I,Airoldi I et al.Downregulation and/or release of NKG2D ligands as immune evasion strategy of human neuroblastoma. Neoplasia 2004:6:558–568.
23 Wu JD, Higgins LM,Steinle A,et al.Prevalent expression of the immuno- stimulatory MHC class I chain-related molecule is counteracted byshedding in prostate cancer.J Clin Invest,2004,114:560-568.
24张彩,冯进波,王郡甫等.膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响.中华微生物学和免疫学杂志,2004,24:107-111.
25 Doubrovina ES,Doubrovin MM,Vider E,et al.evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. J Immunol,2003,171:6891-6899.
26邬鹏,魏海明,田志刚.IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D.中华微生物学和免疫学杂志,2006,26:684-687
27李晓红,马健,汪菲菲等.体外扩增高纯度的人外周血来源的NK细胞的研究.中国实验血液学杂志,2007,15:373-377
1田志刚.NK细胞新理论对重大疾病的新解释[J].中国免疫学杂志,2002,18:75-78.
2 Natarajan K,Dimasi N,Wang J,et al.Structure and function of natural killer cell receptors : Multiple molecular solutions to self , nonself discrimination [J].Annu Rev Immunol,2002,20:853-885.
3 Vivier E,Tomasello E,Paul P.Lymphocyte activation via NKG2D:towards a new paradigm in immune recognition [J].Curr Opin Immunol,2002, 14:306-311.
4 Houchins JP ,Yabe T,McSherry C,et al.DNA sequence analysis of NKG2D,a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells[J].J Exp Med,1991,173: 1017-1020.
5 Bauer S,Groh V,Wu J,et al.Activation of natural killer cells and T cells by NKG2D,a receptor for stress-inducible MICA.Science 1999:285:727–730.
6 Wu J,Song Y,Bakker AB,et al.An activating immunoreceptor complex formed by NKG2D and DAP10.Science 1999,285:730-732.
7 Coudert JD,Held W.The role of the NKG2D receptor for tumor immunity. Semin Cancer Biol 2006,16:333–343.
8 Glienke J,Sobanov Y,Brostjan C,et al.The genomic organization of NKG2C,E,F and D receptor genes in the human natural killer gene complex[J].Immunogenetics,1998,48:163-173.
9 Diefenbach A,Tomasello E,Lucas M,et al.Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D[J].Nat Immunol,2002,3:1142-1149.
10 ShumBP,Flodin LR,Muir DG,et al.Conservation and variation in human and common chimpanzee CD94 and NKG2 genes[J].J Immunol,2002,168:240- 252.
11 Ding Y,Sumitran S,Holgersson J.Direct binding of purified HLA class I antigens by soluble NKG2/CD94 C-type lectins from natural killer cells.[J].Scand J Immunol,1999,49:459-465.
12 Jamieson AM,Diefenbach A,McMahon CW,et al.The role of the NKG2D immunoreceptor in immune cell activation and natural killing [J]. Immunity,2002,17:19-29.
13 Diefenbach A,Jamieson AM,Liu SD,et al.Ligands for the murine NKG2D receptor:expression by tumor cells and activation of NK cells and macrophages[J].Nat Immunol,2000,1:119-126.
14邬鹏,魏海明,田志刚.IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D.中华微生物学和免疫学杂志,2006,26:684-687.
15 Wu J,Cherwinski H,Spies T,et al.DAP10 and DAP12 form distinct,but functionally cooperative,receptor complexes in natural killer cells [J].J Exp Med,2000,192:1059-1068.
16 Upshaw JL,Leibson PJ.NKG2D mediated activation of cytotoxic lympho- cytes:unique signaling pathways and distinct functional outcomes. Semin Immunol 2006,18:167–175.
17 Ho EL,Carayannopoulos LN ,Poursine2Laurent J,et al.Costimulation of multiple NK cell activation receptors by NKG2D.[J].J Immunol,2002,169: 3667-3675.
18 Zompi S,Hamerman JA,Ogasawara K,et al.NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases.[J].Nat Immunol, 2003,4:565-572.
19 Gilfillan S,Ho EL,Cella M,et al.NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation.[J].Nat Immunol,2002, 3:1150-1155.
20 Andreas D,Jennifer KH,Ming-Yu BH,et al.A novel ligand for the NKG2D receptor activates NK cells and macrophages and induces tumor immunity [J].Eur J Immunol,2003,33:381-391.
21 Stephens HA.MICA and MICB genes:can the enigma of their polymorphismbe resolved? Trends Immunol 2001,22:378–385.
22 Bahram S,Inoko H,Shiina T,Radosavljevic M. MIC and other NKG2D ligands: from none to too many. Curr Opin Immunol 2005,17:505–509.
23 Elsner HA, Schroeder M, Blasczyk R. The nucleotide diversity of MICA and MICB suggests the effect of overdominant selection. Tissue Antigens 2001,58:419–21.
24 Steinle A,Li P,Morris DL,et al.Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. Immunogenetics 2001,53:279–287.
25 Bahram S,Bresnahan M,Geraghty DE,et al.A second lineage of mammalian major histocompatibility complex class I genes.Proc Natl Acad Sci USA 1994,96:6879–6884.
26 Groh V,Bahram S,Bauer S,et al.Cell stress-regulated human major his- tocompatibility complex class I gene expressed in gastrointestinal epithelium[J].Proc Natl Acad Sci USA,1996,93:12445-12450.
27 Cosman D.The human cytomegalovirus (HCMV) glycoproteinm UL16,bind to the MHC class I-related protein,MICB/ PERB11,and to two novel,MHC class I-related molecules,ULBP1and ULBP2[J].FASEB J,2000,14:A1018.
28 Cosman D,Mullberg J,Sutherland CL,et al.ULBPs,novel MHC class I related molecules,bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor.Immunity 2001,14:123–133.
29 Radosavljevic M,Cuillerier B,Wilson MJ et al.A cluster of ten novel MHC class I related genes on human chromosome 6q24.2-q25.3.Genomics 2002,79:114–123.
30 Jan Chalupny N,Sutherland CL,Lawrence WA,et al.ULBP4 is a novel ligand for human NKG2D[J].Biochem Biophys Res Commun,2003,305:129-135.
31 Bacon L,Eagle RA,Meyer M,et al.Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol 2004,173: 1078–1084.
32 Nomura M,Zou Z,Joh T,et al.Genomic structures and characterization ofRAE1 family members encoding GPI-anchored cell surface proteins and expressed predominantly in embryonic mouse brains. J Biochem (Tokyo) 1996,120:987–995.
33 Zou Z, Nomura M,Takihara Y,et al.Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells:a novel cDNA family encodes cell surface proteins sharing partial homology with MHC class I molecules. J Biochem (Tokyo) 1996,119:319–328.
34 Nowbakht P,Ionescu MC,Rohner A et al.Ligands for natural killer cell activating receptors are expressed upon the maturation of normal myelomonocytic cells but at low levels in acute myeloid leukemias. Blood 2005,105:3615–3622.
35 Molinero LL,Fuertes MB,Girart MV,et al.NF-kappa B regulates expression of the MHC class I-related chain A gene in activated T lymphocytes.J Immunol 2004,173:5583–5590.
36 Jinushi M,Takehara T,Kanto T et al.Critical role of MHC class I-related chain A and B expression on IFN-alpha-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection. J Immunol 2003,170:1249-1256.
37 Nedvetzki S,Sowinski S,Eagle RA,et al. Reciprocal regulation of human natural killer cells and macrophages associated with distinct immune synapses.Blood 2007,109:3776–3785.
38 Poggi A,Prevosto C,Massaro AM et al.Interaction between human NK cells and bone marrow stromal cells induces NK cell triggering: role of NKp30 and NKG2D receptors.J Immunol 2005,175:6352–6360.
39 Spaggiari GM,Capobianco A,Becchetti S,et al.Mesenchymal stem cell (MSC) /natural killer (NK) cell interactions:evidence that activated NK cells are capable of killing MSC while MSC can inhibit IL-2-induced NK cell proliferation.Blood 2005,107:1484–1490.
40 Carayannopoulos LN,Naidenko OV,Fremont DH,et al.Cutting edge: Murine UL16-binding protein-like transcript 1:a newly described transcript encodinga high-affinity ligand for murine NKG2D.J Immunol 2002,169: 4079–4083.
41 Jan Chalupny N,Sutherland CL,Lawrence WA,et al.ULBP4 is a novel ligand for human NKG2D.Biochem Biophys Res Commun 2003,305:129–135.
42 Pende D, Rivera P, Marcenaro S et al. Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent nat- ural killer cell cytotoxicity. Cancer Res 2002,62:6178–6186.
43 Eagle RA,Traherne JA,Ashiru O,Wills MR,et al.Regulation of NKG2D ligand gene expression. Hum Immunol 2006,67:159–169.
44 Vankayalapati R,Garg A,Porgador A et al.Role of NK cell activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium.J Immunol 2005,175:4611– 4617.
45 Tieng V,Le Bouguenec C,Du Merle L,et al.Binding of Escherichia coli adhesin AfaE to CD55 triggers cell- surface expression of the MHC class I-related molecule MICA.Proc Natl Acad Sci USA 2002,99:2977–2982.
46 Rolle A,Mousavi-Jazi M,Eriksson M,et al.Effects of human cytomega- lovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein(ULBP) 1 and ULBP2 is counteracted by the viral UL16 protein.J Immunol 2003,171:902–908.
47 Conejo-Garcia JR,Benencia F,Courreges MC et al.Letal, a tumor assoc- iated NKG2D immunoreceptor ligand, induces activation and expansion of effector immune cells.Cancer Biol Ther 2003,2:446–451.
48梅家转,牛新清,郭坤元等.K562和K562/AO2细胞HLA I类分子与MICA/B的表达及其对NK细胞杀伤活性的影响.中国实验血液学杂志.2007,15:288-291
49 Diefenbach A,Jensen ER,Jamieson AM,et al.Rae1 and H60 ligands of the NKG2D recep tor stimulate tumour immunity.Nature,2001,413:165-171.
50 Groh V,Wu J,Yee C,et al.Tumor-derived soluble MIC ligands impair eexpression of NKG2D and T cell activation[J].Nature,2002,419:734- 738.
51 Wu JD,Higgins LM,Steinle A,et al.Prevalent expression of the immuno stimulatory MHC class I chain-related molecule is counteracted by shedding in prostate cancer.J Clin Invest,2004,114:560-568.
52张彩,冯进波,王郡甫等.膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响.中华微生物学和免疫学杂志,2004,24(2):107-111.
53 Doubrovina ES,Doubrovin MM,Vider E,et al.evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. J Immunol,2003,171:6891-6899.
54 Dunn C,Chalupny NJ,Sutherland CL,et al.Human cytomegalovirus glyco- protein UL16 causes intracellular sequestration of NKG2D ligands, protecting against natural killer cell cytotoxicity. J Exp Med 2003, 197:1427–1439.
55 Wu J,Chalupny NJ,Manley TJ,et al.Intracellular retention of the MHC class I-related chain B ligand of NKG2D by the human cyto megalovirus UL16 glycoprotein.J Immunol 2003,170:4196–4200.
56 Sp reu J,Stehle T,Steinle A.Human cytomegalovirus-encoded UL16 discriminates MIC molecules by their alpha 2 Domains.J Immunol,2006, 177:3143-3149.
57 Chalupny NJ, Rein-Weston A,Dosch S,et al.Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142. Biochem Biophys Res Commun,2006,346:175-181.
58 Lodoen MB,Abenes G,Umamoto S,et al.The cytomegalovirusm155 gene product subverts NK cell antiviral p rotection by disrup tion of H60-NKG2D interactions.J Exp Med,2004,200:1075-1081.
59 Malarkannan S.The balancing act:inhibitory Ly49 regulate NKG2D- mediated NK cell functions. Semin Immunol 2006,18:186–192.
2 Smyth MJ,Hayakawa Y,Takeda K et al. New aspects of natural killer cell surveillance and therapy of cancer [J].Nat Rev Cancer,2002,2:850-861.
3田志刚.NK细胞新理论对重大疾病的新解释[J].中国免疫学杂志,2002,18:75-78.
4 French AR,Yokoyama WM.Natural killer cells and viral infections [J].Curr Opin Immunol,2003,15:45-51.
5 Natarajan K,Dimasi N,Wang J,et al.Structure and function of natural killer cell receptors : Multiple molecular solutions to self , nonself discrimination [J].Annu Rev Immunol,2002,20:853 - 885.
6 Vivier E,Tomasello E,Paul P.Lymphocyte activation via NKG2D:towards a new paradigm in immune recognition [J].Curr Opin Immunol,2002, 14:306-311.
7 Cooper MA,Fehniger TA,Caligiuri MA.The biology of human natural killer cell subsets [J].Trends Immunol,2001,22:633-640.
8金伯泉,主编.细胞和分子免疫学[M].第2版.北京:科学出版社,2001:9
9陆再英,钟南山,主编.内科学,第七版.北京,人民卫生出版社,2008,4: 457-462
10中华医学会肝病学分会、中华医学会感染病学分会.慢性乙型肝炎防治指南.中华传染病杂志,2005,6:421-431.
11 Trapani JA,Smyth MJ.Functional significance of the perforin/granzyme cell death pathway [J].Nat Rev Immunol,2002,2:735-747.
12 Diefenbach A,Tomasello E,Lucas M,et al.Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D[J]. Nat Immunol,2002,3:1142-1149.
13 Coudert JD,Held W.The role of the NKG2D receptor for tumor immunity.Semin Cancer Biol 2006,16:333–343.
14 Conejo-Garcia JR,Benencia F,Courreges MC et al.Letal,a tumor assoc- iated NKG2D immunoreceptor ligand,induces activation and expansion of effector immune cells. Cancer Biol Ther 2003:2:446–451.
15 Pende D,Cantoni C,Rivera P et al.Role of NKG2D in tumor cell lysis mediated by human NK cells:cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin. Eur J Immunol,2001:31:1076–1086.
16 Bauer S,Groh V,Wu J,et al.Activation of natural killer cells and T cells by NKG2D,a receptor for stress-inducible MICA.Science,1999:285: 727–730.
17 Groh V,Wu J,Yee C,et al.Tumor-derived soluble MIC ligands impair eexpression of NKG2D and T cell activation[J].Nature,2002,419:734- 738.
18梅家转,牛新清,郭坤元等.K562和K562/AO2细胞HLA I类分子与MICA/B的表达及其对NK细胞杀伤活性的影响.中国实验血液学杂志.2007,15(2):288-291
19 Diefenbach A,Jensen ER,Jamieson AM,et al.Rae1 and H60 ligands of the NKG2D recep tor stimulate tumour immunity.Nature,2001,413:165-171.
20 Cosman D,Mullberg J,Sutherland CL,et al.ULBPs,novel MHC class I related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor.Immunity 2001:14:123–133.
21 Pende D,Rivera P,Marcenaro S,et al.Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent nat- ural killer cell cytotoxicity.Cancer Res 2002:62:6178–6186.
22 Raffaghello L,Prigione I,Airoldi I et al.Downregulation and/or release of NKG2D ligands as immune evasion strategy of human neuroblastoma. Neoplasia 2004:6:558–568.
23 Wu JD, Higgins LM,Steinle A,et al.Prevalent expression of the immuno- stimulatory MHC class I chain-related molecule is counteracted byshedding in prostate cancer.J Clin Invest,2004,114:560-568.
24张彩,冯进波,王郡甫等.膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响.中华微生物学和免疫学杂志,2004,24:107-111.
25 Doubrovina ES,Doubrovin MM,Vider E,et al.evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. J Immunol,2003,171:6891-6899.
26邬鹏,魏海明,田志刚.IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D.中华微生物学和免疫学杂志,2006,26:684-687
27李晓红,马健,汪菲菲等.体外扩增高纯度的人外周血来源的NK细胞的研究.中国实验血液学杂志,2007,15:373-377
1田志刚.NK细胞新理论对重大疾病的新解释[J].中国免疫学杂志,2002,18:75-78.
2 Natarajan K,Dimasi N,Wang J,et al.Structure and function of natural killer cell receptors : Multiple molecular solutions to self , nonself discrimination [J].Annu Rev Immunol,2002,20:853-885.
3 Vivier E,Tomasello E,Paul P.Lymphocyte activation via NKG2D:towards a new paradigm in immune recognition [J].Curr Opin Immunol,2002, 14:306-311.
4 Houchins JP ,Yabe T,McSherry C,et al.DNA sequence analysis of NKG2D,a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells[J].J Exp Med,1991,173: 1017-1020.
5 Bauer S,Groh V,Wu J,et al.Activation of natural killer cells and T cells by NKG2D,a receptor for stress-inducible MICA.Science 1999:285:727–730.
6 Wu J,Song Y,Bakker AB,et al.An activating immunoreceptor complex formed by NKG2D and DAP10.Science 1999,285:730-732.
7 Coudert JD,Held W.The role of the NKG2D receptor for tumor immunity. Semin Cancer Biol 2006,16:333–343.
8 Glienke J,Sobanov Y,Brostjan C,et al.The genomic organization of NKG2C,E,F and D receptor genes in the human natural killer gene complex[J].Immunogenetics,1998,48:163-173.
9 Diefenbach A,Tomasello E,Lucas M,et al.Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D[J].Nat Immunol,2002,3:1142-1149.
10 ShumBP,Flodin LR,Muir DG,et al.Conservation and variation in human and common chimpanzee CD94 and NKG2 genes[J].J Immunol,2002,168:240- 252.
11 Ding Y,Sumitran S,Holgersson J.Direct binding of purified HLA class I antigens by soluble NKG2/CD94 C-type lectins from natural killer cells.[J].Scand J Immunol,1999,49:459-465.
12 Jamieson AM,Diefenbach A,McMahon CW,et al.The role of the NKG2D immunoreceptor in immune cell activation and natural killing [J]. Immunity,2002,17:19-29.
13 Diefenbach A,Jamieson AM,Liu SD,et al.Ligands for the murine NKG2D receptor:expression by tumor cells and activation of NK cells and macrophages[J].Nat Immunol,2000,1:119-126.
14邬鹏,魏海明,田志刚.IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D.中华微生物学和免疫学杂志,2006,26:684-687.
15 Wu J,Cherwinski H,Spies T,et al.DAP10 and DAP12 form distinct,but functionally cooperative,receptor complexes in natural killer cells [J].J Exp Med,2000,192:1059-1068.
16 Upshaw JL,Leibson PJ.NKG2D mediated activation of cytotoxic lympho- cytes:unique signaling pathways and distinct functional outcomes. Semin Immunol 2006,18:167–175.
17 Ho EL,Carayannopoulos LN ,Poursine2Laurent J,et al.Costimulation of multiple NK cell activation receptors by NKG2D.[J].J Immunol,2002,169: 3667-3675.
18 Zompi S,Hamerman JA,Ogasawara K,et al.NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases.[J].Nat Immunol, 2003,4:565-572.
19 Gilfillan S,Ho EL,Cella M,et al.NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation.[J].Nat Immunol,2002, 3:1150-1155.
20 Andreas D,Jennifer KH,Ming-Yu BH,et al.A novel ligand for the NKG2D receptor activates NK cells and macrophages and induces tumor immunity [J].Eur J Immunol,2003,33:381-391.
21 Stephens HA.MICA and MICB genes:can the enigma of their polymorphismbe resolved? Trends Immunol 2001,22:378–385.
22 Bahram S,Inoko H,Shiina T,Radosavljevic M. MIC and other NKG2D ligands: from none to too many. Curr Opin Immunol 2005,17:505–509.
23 Elsner HA, Schroeder M, Blasczyk R. The nucleotide diversity of MICA and MICB suggests the effect of overdominant selection. Tissue Antigens 2001,58:419–21.
24 Steinle A,Li P,Morris DL,et al.Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. Immunogenetics 2001,53:279–287.
25 Bahram S,Bresnahan M,Geraghty DE,et al.A second lineage of mammalian major histocompatibility complex class I genes.Proc Natl Acad Sci USA 1994,96:6879–6884.
26 Groh V,Bahram S,Bauer S,et al.Cell stress-regulated human major his- tocompatibility complex class I gene expressed in gastrointestinal epithelium[J].Proc Natl Acad Sci USA,1996,93:12445-12450.
27 Cosman D.The human cytomegalovirus (HCMV) glycoproteinm UL16,bind to the MHC class I-related protein,MICB/ PERB11,and to two novel,MHC class I-related molecules,ULBP1and ULBP2[J].FASEB J,2000,14:A1018.
28 Cosman D,Mullberg J,Sutherland CL,et al.ULBPs,novel MHC class I related molecules,bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor.Immunity 2001,14:123–133.
29 Radosavljevic M,Cuillerier B,Wilson MJ et al.A cluster of ten novel MHC class I related genes on human chromosome 6q24.2-q25.3.Genomics 2002,79:114–123.
30 Jan Chalupny N,Sutherland CL,Lawrence WA,et al.ULBP4 is a novel ligand for human NKG2D[J].Biochem Biophys Res Commun,2003,305:129-135.
31 Bacon L,Eagle RA,Meyer M,et al.Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol 2004,173: 1078–1084.
32 Nomura M,Zou Z,Joh T,et al.Genomic structures and characterization ofRAE1 family members encoding GPI-anchored cell surface proteins and expressed predominantly in embryonic mouse brains. J Biochem (Tokyo) 1996,120:987–995.
33 Zou Z, Nomura M,Takihara Y,et al.Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells:a novel cDNA family encodes cell surface proteins sharing partial homology with MHC class I molecules. J Biochem (Tokyo) 1996,119:319–328.
34 Nowbakht P,Ionescu MC,Rohner A et al.Ligands for natural killer cell activating receptors are expressed upon the maturation of normal myelomonocytic cells but at low levels in acute myeloid leukemias. Blood 2005,105:3615–3622.
35 Molinero LL,Fuertes MB,Girart MV,et al.NF-kappa B regulates expression of the MHC class I-related chain A gene in activated T lymphocytes.J Immunol 2004,173:5583–5590.
36 Jinushi M,Takehara T,Kanto T et al.Critical role of MHC class I-related chain A and B expression on IFN-alpha-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection. J Immunol 2003,170:1249-1256.
37 Nedvetzki S,Sowinski S,Eagle RA,et al. Reciprocal regulation of human natural killer cells and macrophages associated with distinct immune synapses.Blood 2007,109:3776–3785.
38 Poggi A,Prevosto C,Massaro AM et al.Interaction between human NK cells and bone marrow stromal cells induces NK cell triggering: role of NKp30 and NKG2D receptors.J Immunol 2005,175:6352–6360.
39 Spaggiari GM,Capobianco A,Becchetti S,et al.Mesenchymal stem cell (MSC) /natural killer (NK) cell interactions:evidence that activated NK cells are capable of killing MSC while MSC can inhibit IL-2-induced NK cell proliferation.Blood 2005,107:1484–1490.
40 Carayannopoulos LN,Naidenko OV,Fremont DH,et al.Cutting edge: Murine UL16-binding protein-like transcript 1:a newly described transcript encodinga high-affinity ligand for murine NKG2D.J Immunol 2002,169: 4079–4083.
41 Jan Chalupny N,Sutherland CL,Lawrence WA,et al.ULBP4 is a novel ligand for human NKG2D.Biochem Biophys Res Commun 2003,305:129–135.
42 Pende D, Rivera P, Marcenaro S et al. Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent nat- ural killer cell cytotoxicity. Cancer Res 2002,62:6178–6186.
43 Eagle RA,Traherne JA,Ashiru O,Wills MR,et al.Regulation of NKG2D ligand gene expression. Hum Immunol 2006,67:159–169.
44 Vankayalapati R,Garg A,Porgador A et al.Role of NK cell activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium.J Immunol 2005,175:4611– 4617.
45 Tieng V,Le Bouguenec C,Du Merle L,et al.Binding of Escherichia coli adhesin AfaE to CD55 triggers cell- surface expression of the MHC class I-related molecule MICA.Proc Natl Acad Sci USA 2002,99:2977–2982.
46 Rolle A,Mousavi-Jazi M,Eriksson M,et al.Effects of human cytomega- lovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein(ULBP) 1 and ULBP2 is counteracted by the viral UL16 protein.J Immunol 2003,171:902–908.
47 Conejo-Garcia JR,Benencia F,Courreges MC et al.Letal, a tumor assoc- iated NKG2D immunoreceptor ligand, induces activation and expansion of effector immune cells.Cancer Biol Ther 2003,2:446–451.
48梅家转,牛新清,郭坤元等.K562和K562/AO2细胞HLA I类分子与MICA/B的表达及其对NK细胞杀伤活性的影响.中国实验血液学杂志.2007,15:288-291
49 Diefenbach A,Jensen ER,Jamieson AM,et al.Rae1 and H60 ligands of the NKG2D recep tor stimulate tumour immunity.Nature,2001,413:165-171.
50 Groh V,Wu J,Yee C,et al.Tumor-derived soluble MIC ligands impair eexpression of NKG2D and T cell activation[J].Nature,2002,419:734- 738.
51 Wu JD,Higgins LM,Steinle A,et al.Prevalent expression of the immuno stimulatory MHC class I chain-related molecule is counteracted by shedding in prostate cancer.J Clin Invest,2004,114:560-568.
52张彩,冯进波,王郡甫等.膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响.中华微生物学和免疫学杂志,2004,24(2):107-111.
53 Doubrovina ES,Doubrovin MM,Vider E,et al.evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. J Immunol,2003,171:6891-6899.
54 Dunn C,Chalupny NJ,Sutherland CL,et al.Human cytomegalovirus glyco- protein UL16 causes intracellular sequestration of NKG2D ligands, protecting against natural killer cell cytotoxicity. J Exp Med 2003, 197:1427–1439.
55 Wu J,Chalupny NJ,Manley TJ,et al.Intracellular retention of the MHC class I-related chain B ligand of NKG2D by the human cyto megalovirus UL16 glycoprotein.J Immunol 2003,170:4196–4200.
56 Sp reu J,Stehle T,Steinle A.Human cytomegalovirus-encoded UL16 discriminates MIC molecules by their alpha 2 Domains.J Immunol,2006, 177:3143-3149.
57 Chalupny NJ, Rein-Weston A,Dosch S,et al.Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142. Biochem Biophys Res Commun,2006,346:175-181.
58 Lodoen MB,Abenes G,Umamoto S,et al.The cytomegalovirusm155 gene product subverts NK cell antiviral p rotection by disrup tion of H60-NKG2D interactions.J Exp Med,2004,200:1075-1081.
59 Malarkannan S.The balancing act:inhibitory Ly49 regulate NKG2D- mediated NK cell functions. Semin Immunol 2006,18:186–192.