生命科学   2017, Vol. 29 Issue (9): 845-854.  DOI: 10.13376/j.cbls/2017114.
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谭思雨, 梁晓红, 高立芬, 马春红. 居留NK细胞研究新进展及其在肿瘤细胞治疗中的意义. 生命科学, 2017, 29(9): 845-854. DOI: 10.13376/j.cbls/2017114.
[复制中文]
TAN Si-Yu, LIANG Xiao-Hong, GAO Li-Fen, MA Chun-Hong. Research progress on tissue resident NK cells and NK cell-based tumor immunology. Chinese Bulletin of Life Sciences, 2017, 29(9): 845-854. DOI: 10.13376/j.cbls/2017114.
[复制英文]

基金项目

国家自然科学基金杰出青年基金项目(81425012);山东省重点研发计划(2016ZDJS07A17)

作者简介

马春红,博士,教授,博士生导师。国家自然科学基金杰出青年基金、中组部“万人计划”领军人才、教育部“新世纪优秀人才支持计划”、霍英东基金会高等院校青年教师基金获得者,全国三八红旗手。现任山东大学基础医学院副院长。中华医学会微生物与免疫学分会青年委员会委员、山东免疫学会常务理事。。

长期从事肝病与免疫研究,致力于研究HBV、代谢等环境因素诱发肝脏炎症及其恶性转化机制。近期研究团队利用动物模型及临床标本,围绕免疫耗竭及肝癌微环境开展工作,揭示了免疫调节分子Tim-3介导CD8+T细胞、NK细胞耗竭及肿瘤相关巨噬细胞极化的新机制(Gut 2015,J Hepatol 2010,Cell Mol Immunol 2009);发现并鉴定了肝癌中的新型抑癌基因ZHX2及其调控的多个靶基因(Gastroenterology 2012,Int J Biochem Cell Biol 2015,Oncotarget 2015);发现HBV及其编码蛋白通过调节肝细胞中生长死亡相关基因和免疫调节基因(Oncogene 2014,Liver Int 2015,Int J Biochem Cell Biol 2013,Cell Death Dis 2009,Gut 2009,JImmunol 2007)促进肝细胞恶性转化和促癌免疫微环境的形成 。

通信作者

马春红, E-mail: machunhong@sdu.edu.cn

文章历史

收稿日期:2017-07-21
居留NK细胞研究新进展及其在肿瘤细胞治疗中的意义
谭思雨 , 梁晓红 , 高立芬 , 马春红     
(山东大学基础医学院免疫学系,济南 250100)
摘要:NK细胞亚群不同表型间存在效应功能的差异,在组织器官的分布也有显著差别。研究证明,肝脏、胸腺、肺脏、子宫、皮肤等组织内存在居留NK细胞,其发育、表型及功能与骨髓中发育、成熟后迁入外周器官的传统NK细胞不同,在维持组织稳态、调控多种重要病理生理过程中发挥作用,与I类固有样淋巴细胞具有部分重叠又不完全相同的表型及功能,并且与诱导NK细胞记忆性相关。针对NK细胞尤其是居留NK细胞的研究将为肿瘤免疫治疗提供新的机遇。
关键词居留NK细胞    发育    免疫记忆    Ⅰ类固有淋巴样细胞    肿瘤免疫治疗    
Research progress on tissue resident NK cells and NK cell-based tumor immunology
TAN Si-Yu , LIANG Xiao-Hong , GAO Li-Fen , MA Chun-Hong     
(Department of Immunology, Shandong University School of Basic Medical Science, Jinan 250100, China)
Abstract: NK cells are the first line of defense of the body. Accumulated data has been demonstrated that besides conventional NK cells in the peripheral, there are tissue resident NK cells in liver, thymus, lungs, skin and other tissues. The development, phenotypes and functions of the tissue resident NK cells are different from conventional NK cells, which matured in the bone marrow before moving to peripheral organs. Tissue resident NK cells shares overlapping but different phenotypes and functions with type 1 innate lymphoid cells and play roles in maintenance of tissue homeostasis and induction of NK cells memory. The research on tissue resident NK cells will provide new ideas for NK cells related tumor immunology.
Key words: tissue resident NK cells    development    immunological memory    type 1 innate lymphoid cells    tumor immunology    

NK细胞(natural killer cells)是固有免疫系统中的一类大颗粒淋巴细胞,无需致敏即可对肿瘤和病毒感染的靶细胞产生快速的应答。NK细胞不仅借助颗粒酶和穿孔素的胞吐作用以及FasL-Fas和TNFα-TNFαR介导的细胞凋亡杀伤靶细胞,而且还可分泌大量的促炎因子调节免疫应答,发挥其免疫监视及免疫清除功能。

长期以来认为NK由骨髓发育,成熟后随循环迁出骨髓,进入外周组织中发挥作用,此类NK细胞被称为传统NK细胞(conventional NK, cNK)。越来越多的研究证明,肝脏、胸腺、肺、子宫、皮肤等组织内存在一群组织居留NK (tissue resident NK, trNK)细胞,其发育、表型及功能与传统NK细胞不同,可能参与维持组织局部稳态,与多种重要病理生理过程有关,并可能与NK的记忆性有关[1]。深入研究传统NK细胞及居留NK细胞发育过程,阐明其来源分类特征及功能调控机制,不仅可以进一步深化对NK细胞的认识,还可以更好地利用其在免疫调节中的作用,为肿瘤治疗提供新思路。

1 传统NK细胞的发育及转录调控 1.1 NK细胞在骨髓内的发育过程

NK细胞由造血干细胞发育分化而来,经历NK细胞前体、不成熟NK细胞和成熟NK细胞等阶段。胚胎期,NK细胞的发育主要在肝脏内进行,出生后发育场所转移到骨髓[2]。骨髓内造血干细胞(hematopoietic stem cells, HSCs)经历共同淋巴系前体(common lymphoid precursors, CLPs)(Lin-c-kit+Flt3+ CD27+IL-7Rα+)、共同天然淋巴样细胞前体(common innate lymphoid cells precursors, CILPs)(α4β7+IL-7Rα+CXCR6+Nfil3+),发育为NK前体细胞(NK cell precursors, NKPs),此时尚不表达NK标记但表达IL-15R (CD122)(Lin-NK1.1-DX5-CD122+),且共表达CD27及CD244。NKPs在IL-15刺激下丢失IL-7Rα (CD127),获得NK1.1表达,发育为不成熟NK细胞(immature NK cells, iNK),表现为TRAIL+CD51+ NK1.1+DX5-。iNKs接受MHC-Ⅰ分子所提供的驯化信号发育为成熟NK细胞(mature NK cells, mNKs),表达CD11b、CD43、Ly49、DX5,并获得细胞毒性及分泌细胞因子的能力,迁出骨髓进入外周[3-5]

随着研究的进展,有关NK在骨髓内发育的研究逐渐深入。通过多色流式分析,Fathman等[6]在CLP向NKP发育过程中发现了一群pre-NKP细胞(NK cell-committed precursors),其表型为Lin-CD27+CD122-IL-7Ra+Flt3-CD244+。同时,Carotta等[7]通过转录因子分析,将表达Id2和IL-7R的细胞定义为NK细胞最早前体,称为pre-pro NK (the earliest committed NK cell precursors),其表型与pre-NKP极为相似,并与某些ILC细胞表达相似的细胞受体。pre-pro NK启动CD122表达,发育为NKP,但pre-NK与pre-pro NK及CILP之间的关系尚有待研究。

伴随NK细胞的发育成熟,其表面表达系列活化性受体和抑制性受体,经历复杂的驯化(education)过程,NK细胞获得自身耐受性和功能反应性[8]。与T、B淋巴细胞不同,NK细胞受体表达不经历由RAG介导的基因重排。但最新研究显示,部分NK细胞发育过程中表达RAG (高表达于CLP,成熟时降低),并可能影响NK细胞功能亚群的分化[9]

1.2 NK细胞发育的转录调控

在NK细胞发育过程中,除IL-15等细胞因子外,多种转录因子协同作用,共同决定NK细胞的命运和发育过程(表 1)。其中Notch (Jagged1/Jagged2)、Ets-1及E4-binding protein 4 (E4BP4 or Nfil3)参与NK早期发育,决定NK谱系分化;ID2、T-bos等参与NK成熟;MITF-1、Blimp-1及CEBP-r调控NK细胞的终末成熟及穿孔素、颗粒酶、IFNγ的表达[10]。E4BP4是锌指蛋白转录因子家族成员,对NK细胞谱系决定和早期发育极为重要,是NK细胞谱系形成的决定因子。E4BP4表达水平随着NK细胞成熟逐渐升高,E4BP4缺陷导致pre-NKP和NKP大幅减少,mNK缺失[11]。Eomes属于转录因子T-box家族,可诱导CD122高表达,调控骨髓NK细胞发育,其突变会导致IL-15依赖的细胞发育受阻[12]

表 1 参与NK细胞发育、功能的转录调控因子
2 组织居留NK细胞

成年个体内,除骨髓外的多种组织可以产生NKP细胞,进而发育为组织居留NK细胞。NKP及组织微环境共同决定了trNK的发育及表型与功能。与cNK不同,多种trNK表达CD49a,组成性表达CD69,除IFNγ外,产生更多的TNFα和GM-CSF。

2.1 胸腺NK细胞

Sanchez等最早报道胸腺中存在一群与骨髓来源不同的NK细胞前体[10],随后更多研究证实了这一观点[28-29]。与传统NK细胞不同,胸腺发育来的NK细胞低水平表达Ly49、Mac-1和CD43,但表达高水平的IL-7R (CD127)[30]。CD127+胸腺NK细胞的发育同样依赖于IL-15,但其发育必需转录因子GATA-3。胸腺NK细胞表现为低细胞毒性、高细胞因子表达。成熟后这群细胞会迁移至腋窝和腹股沟等淋巴结,并维持其表型和功能,但不存在于脾脏和骨髓中。关于这群细胞是由胸腺前体细胞直接发育而来,还是骨髓NKP细胞迁移分化而来,尚不清楚。

2.2 子宫NK细胞

子宫中存在大量的NK细胞[31],其表面NK受体与传统NK细胞显著不同[32],并在胚胎植入及妊娠进程的子宫内膜及蜕膜内呈现动态变化[33],在维持子宫内的免疫稳态、调节胎盘血管再生及维持母胎耐受中发挥重要作用[34-36]。研究者发现,妊娠期子宫蜕膜中存在大量的特殊居留NK细胞。人子宫内CD56highCD16-NK细胞同时表达CD9、CD103,并高表达多种KIR分子和转录因子Eomes,但其发育不依赖转录因子T-bet [37],尽管子宫内居留NK细胞表达NKp46、NKp44、NKG2D等分子,但其细胞毒性相对较弱,而其产生的IFNγ对于启动胚胎植入后的蜕膜血管重塑至关重要[38]

子宫内NK细胞的表型与功能区别于其他组织亚群,推测与其局部微环境相关,如TGF-β可调控CD9、CD103、KIR等分子表达。目前,关于这群子宫居留NK细胞的来源并不清楚,推测可能是由外周血CD56highCD16-NK细胞通过CXCR4等趋化因子招募至子宫局部,亦可能由子宫脱膜层中CD34+细胞发育分化而来。

2.3 肝脏NK细胞

2013年,田志刚教授与Wayne M Yokoyama教授合作发现肝脏内存在组织居留NK细胞[39]。与传统的CD49a-DX5+NK不同,肝内居留NK细胞表现为CD49a+DX5-;联体小鼠实验证实,该群细胞仅存在于肝脏,并不迁出肝脏。CD49a+DX5-的trNK细胞在表型上与骨髓来源的未成熟NK (iNK)有相似之处,表现为NK1.1+NKp46+CD11blowDX5-,并高表达TRAIL[40];但与iNK显著不同的是,肝脏trNK细胞高表达CD49a,具有相应的细胞毒性,不需活化即表达CD69[39]。转录组及RNA-seq分析进一步证实肝脏trNK与cNK表型不同,属于两个不同亚群[41]

肝脏在胚胎时期是重要的造血器官,因此成熟肝内仍会有一些造血干细胞。有关肝脏trNK细胞的发育过程尚不十分清楚。GATA-3缺陷小鼠的NK细胞向肝脏迁移的能力显著降低,提示肝脏trNK与胸腺NK可能有共同前体[42]。然而胸腺切除裸鼠及GATA-3缺失小鼠的肝脏内CD49a+trNK细胞数目均不受影响,否定了上述假说[24]。进一步研究发现,肝脏trNK发育与cNK同样需要IL-15。然而与cNK不同,肝脏trNK的发育依赖于T-bet,但不依赖于Eomes和Nfil3[43-44]。除此之外,由PLZF标记的一类前体细胞可以生成trNk而不能生成cNK[45]。这些表明两种NK细胞亚群有着不同的发育分支。

2.4 其他组织内居留NK细胞

除胸腺、子宫、肝脏外,皮肤、淋巴结、小肠、肺等多种组织中存在NK细胞。但目前的联体小鼠实验仅对子宫、皮肤内的NK细胞进行了研究,并证实在子宫及皮肤内同样存在CD49a+Dx5- trNK,提示CD49a可能是多种组织居留NK细胞的共同标记[43]。与肝脏trNK类似,皮肤NK细胞发育依赖于T-bet,提示皮肤NK与肝脏trNK更为接近。

2.5 人类组织居留NK细胞

根据细胞表面标志CD56及CD16的表达水平,人类NK细胞被分为CD56brightCD16+/-和CD56dimCD16+两个亚群。传统观点认为,CD56dimCD16+NK细胞主要存在于外周血,具有更强的细胞毒性,被认为是成熟NK细胞,而CD56brightCD16-NK细胞主要存在于组织,具有更强的细胞因子分泌能力。然而,越来越多的研究发现,与小鼠类似,人的子宫、肝脏等多种组织内也存在居留NK细胞[46-49]。与循环cNK不同,CD56bright居留NK细胞通过特定趋化因子定居于组织[50],并组成性表达CD69。人类trNK中研究较为清楚的是肝脏trNK[46]。研究显示,人类肝脏trNK表达CD49a及CXCR6[51],并高表达NKp46和CCR5,呈现较低水平的细胞毒性[52-53]

3 trNK与NK细胞的记忆性

2006年,在半抗原诱发的接触性皮炎(contact hypersensitivity, CHS)模型中,首次发现NK细胞对预先接触的特异性抗原具有记忆性。研究表明,过继转输已致敏的供体鼠肝脏NK细胞可以使初始受体鼠获得不依赖于T、B细胞的接触性过敏反应能力,并且这一记忆应答能力可以长期持续[54-55]。转输实验证明只有肝脏CXCR6+NK细胞具有记忆性[56]。后续的研究进一步发现,肝脏内CD49a+Dx5-trNK是介导CHS记忆性的NK细胞,提示trNK在NK细胞的记忆性中发挥重要作用[57-58]

多种病原体(小鼠巨细胞病毒mCMV、小鼠单纯庖疹病毒mHSV、流感病毒、人巨细胞病毒hCMV、灵长类免疫缺陷病毒SLV)均可诱导记忆性NK细胞产生。其中研究比较透彻的是mCMV感染模型。该模型中NK细胞活化性受体Ly49H识别CMV的m157病毒蛋白,介导NK免疫记忆,并呈现抗原特异性NK细胞扩增、增殖减降、记忆维持及记忆应答四个阶段[59]。与naive NK细胞相比,记忆性NK细胞具有产生更多IFNγ的能力,并且能够迅速发挥杀伤功能[60]

NK细胞活化性受体DNAM-1(CD226)及其下游Fyn信号和PKCη信号对于记忆性NK细胞的形成非常重要[61],提示与T细胞类似,记忆性NK细胞的活化和扩增需要共刺激活化信号。同样,记忆性NK细胞活化需要细胞因子。Ly49H+NK细胞活化扩增依赖于IL-12及其下游STAT4信号通路[62]。体外实验表明,抗原递呈细胞分泌的多种细胞因子(IL-1、IL-12、IL-15、IL-18)均会活化NK细胞形成记忆样NK细胞[60]。此外,记忆性NK细胞的扩增依赖于转录因子zbtb32。通过上调zbtb32表达,抑制抗增殖因子Blimp-1表达,从而促进NK细胞增殖及增强抗病毒能力[63]

与T细胞一样,活化后的细胞凋亡是记忆性NK细胞形成的关键时期。研究发现,凋亡相关分子Bim[64-65]、Noxa和SOCS1[66]调控的凋亡对记忆性NK细胞的形成非常重要。此外,Atg3信号启动线粒体自噬途径,诱导BNIP3/BNIP3L信号分子表达,并可通过ROS促进NK细胞的存活,从而产生记忆性NK细胞[67]。存活下来的NK细胞分为KLRG1-Ly49H+和KLRG+Ly49H+两群,其中,KLRG1+Ly49H+NK细胞表达RAG基因,在mCMV感染时表现出较强的DNA损伤修复功能,适应能力提高,具有更强的增殖能力,从而在感染后期可以长期存活并形成记忆性NK细胞[68]

近期研究表明,人的CD57+CD56dimNK细胞在应对病原体如hCMV的过程中表现出记忆性的特征,其活化性受体CD94/NKG2C上调,抑制性受体Siglec-7表达下调[69]。该群细胞表型为CD56dimCD16brightLIR-1+KIR+NKG2A-,可在机体内稳定存活并表现出适应性免疫细胞的特征,如在IFNγ基因座位点经历与记忆性T淋巴细胞相同的表观遗传学修饰,失去部分信号;如FcεRγ分子表达,使其能够比传统NK细胞更加有效地应对hCMV感染[70]。此外,共刺激信号(CD2)参与调控记忆性NK细胞的抗hCMV作用[71]

越来越多的研究表明,NK细胞除作为固有免疫系统重要组成之外,表现出的功能性特征与适应性免疫细胞具有共通性,尤其是具有记忆性的特征。记忆性NK细胞最重要的功能特点在于在某些慢性病毒感染过程中可以特异性地通过各种信号分子及转录因子的相互作用,提高ADCC杀伤作用及产生大量IFNγ作用于病原[60]。另一方面,相比于传统NK细胞,由于某些信号因子(如NKp46和NKp30)表达缺失及细胞因子靶位点的激活,这些记忆性NK细胞对其他特异类型的肿瘤的杀伤作用下降,表现出特异性的特征[69]。对记忆性NK细胞的研究将为连接固有免疫及适应性免疫提供思路,并为NK细胞在感染性疾病及肿瘤治疗方面提供新机遇。

4 NK细胞与ILC1s

固有淋巴样细胞(innate lymphoid cells, ILCs)是新近发现的一群固有免疫细胞,与T、B淋巴细胞具有共同的前体细胞(CLPs),但其抗原受体表达不依赖于基因重排。根据表型和功能,可以将ILCs分为三群:Ⅰ类ILCs分泌IFNγ,依赖于转录因子T-bet调控发育、发挥功能;Ⅱ类ILCs分泌IL-4、IL-5、IL-9和IL-13,其功能发挥及发育依赖于GATA-3和RORα;Ⅲ类ILCs是分泌IL-22及IL-17的淋巴样细胞,其必需转录因子为RORγt[72]。由于NK细胞高水平分泌IFNγ,也被归为Ⅰ类ILCs。因此,Ⅰ类ILC1包括细胞毒性的NK细胞和ILC1s。但在发育过程中NK细胞并非严格依赖T-bet,与ILC1s有明显的区别,但又具有相似特征,特别在感染及炎症发生过程中,区分这两类细胞的表面分子会发生改变,使得它们难以被区分。

一般认为ILC1s区分于NK细胞的特征在于其缺乏杀伤活性[55]。研究发现,NK细胞表面的很多受体如NKp46、NK1.1、NKp44、NKG2D和Ly9等,均可以在ILC1s及ILC3s亚群表达[73]。小鼠NK细胞与ILC1s均表达CD49b,而CD127只表达于人类及小鼠ILC1s。胸腺NK细胞表达CD127,并且依赖于转录因子GATA-3,因此,胸腺NK细胞被归类为ILC1s[74]

NK细胞与ILC1s区别的关键在于转录因子T-bet和Eomes。一般而言,ILC1s发育过程中依赖于T-bet而不需要Eomes,与之相反,NK细胞发育依赖于Eomes[75]。人类绝大多数成熟NK细胞被归类为CD3-CD127-CD7+CD56+(或NKp46+)T-bet+Eomes+淋巴细胞。小鼠成熟NK细胞为CD3-CD127-NK1.1+(或NKp46+)T-bet+Eomes+淋巴细胞。人类外周血CD56highCD16dim亚群NK细胞表达Eomes,但是具有高细胞因子分泌活性的CD56dimCD16highNK细胞则低水平表达,甚至不表达Eomes。因此,在人类及小鼠中均没有单一存在的marker可以区分开NK细胞及ILC1s细胞。

ILC1s和NK细胞在不同组织中具有部分重叠又不完全相同的表型及功能。肠道固有层ILC1s表型为CD49a+CD49b-,与NK细胞相比,其表达高水平IFNγ但表现出较弱的杀伤作用,不表达Eomes[75]。固有层ILC1s的细胞毒性由凋亡配体TRAIL介导,而非依赖于穿孔素的作用[75]。唾液腺中发现的另外一类ILC1s亚群同样表达CD49a和TRAIL,但是仅有极低水平的Eomes和CD49b,不表达IFNγ[76]

肝脏trNK与肠道固有层ILC1s在表型上有很多共性,两者均表达CD49a、CXCR6及CD69,但低表达或不表达Dx5、CD62L和CD11b[77]。从发生上,肝脏trNK与ILC1s均来自于PLZFhigh前体细胞[45],发育过程依赖于IL-15及T-bet的存在[78]。同样,肝脏trNK与肠固有层ILC1s相似,表现为较低的细胞毒性和高水平分泌IFNγ和TNF-α的能力。因此,肝脏trNK与ILC1s更接近。然而,两者并不完全相同。固有层ILC1s普遍表达CD27,并表达高水平IL-7Rα[79];而肝脏trNK中仅中等水平表达IL-7Rα,约有一半细胞表达CD27[42]。多个实验证明,肝脏trNK介导半抗原及病毒诱导的NK记忆性反应,并可以通过高表达TRAIL介导靶细胞凋亡[57];然而,ILC1s是否同样表达TRAIL以及是否具有免疫记忆尚有待进一步的研究。

综上所述,在小鼠中,尽管NK细胞和ILC1s有不同的发育起源,这两类细胞表达模式仍具有高度相似性。目前而言,还不能通过单一的marker对不同组织或者不同分化、活化阶段,特别是在感染或者炎症性疾病中的NK细胞及ILC1s细胞进行区分,深入研究这两类细胞的表观遗传及转录特征有望为区分NK细胞及ILC1s确定更好的marker。

5 NK细胞在肿瘤细胞治疗中的新策略

由于NK细胞不需要特异性抗原预先活化,其杀伤作用不受MHC限制,因此相较于T细胞,NK细胞在抗肿瘤免疫治疗中有其独特优势。

5.1 NK识别肿瘤细胞的机制

NK细胞主要通过多种活化性及抑制性受体之间的相互调控识别肿瘤细胞(图 1)。目前认为主要有三种识别模式——“丢失自我(missing-self)”、“非我(non-self)”和“压力诱导(stress-induced-self)”模式[79]。NK细胞表面均表达识别MHC-Ⅰ分子的抑制性受体,比如KIRs、Ly49、CD94/NKG2A等,这类受体的胞浆区含有ITIMs,可以招募胞内酪氨酸磷酸化酶,介导免疫抑制作用[68]。病毒感染或癌变细胞表面MHC-Ⅰ分子表达下降或缺失,NK细胞抑制性受体无法发挥作用,从而激活NK细胞发挥杀伤功能(missing-self)。同时,细胞压力和DNA损伤会导致肿瘤细胞高表达NK活化受体的配体,介导NK细胞活化(stress-induced-self)。此外,NK细胞通过其表面非克隆性分布的模式识别受体直接识别非己成分,称为Non-self识别,如Ly49H识别mCMV感染引起的病毒蛋白m157,激活KARAP/DAP12信号通路,达到清除病原微生物的作用[69]

图 1 NK细胞表面受体
5.2 基于NK细胞的肿瘤治疗新策略

肿瘤细胞可以通过逃逸NK细胞的识别过程或诱导NK细胞失能逃逸NK细胞的识别和攻击。临床研究及动物实验结果显示,在病毒感染和肿瘤发生过程中均出现NK细胞失能和耗竭的现象:NK细胞高表达抑制性受体,增殖能力及细胞因子表达水平降低,细胞毒杀伤功能缺陷[81-83]。拯救失能NK细胞成为NK细胞杀伤肿瘤的关键。

CAR-T技术中的嵌合抗原受体(chimeric antigen receptors, CARs)是由特异性识别某种肿瘤抗原的抗体可变区(或单链抗体)与激活T细胞必需的第一信号(CD3-ζ链)、第二信号胞内结构域偶联形成的嵌合蛋白。CAR-T技术通过基因转导的方法在T细胞表面表达嵌合抗原受体,形成具有靶向性并可以克服肿瘤免疫逃逸的效应细胞,以达到治疗癌症的目的。目前,CAR-T细胞治疗技术已经得到美国FDA批准,在多个国家和地区得到临床治疗研究的许可,并在靶向治疗CD19、CD20淋巴瘤和白血病[84-85]以及CD138多发性骨髓瘤中取得了良好效果[86]。除血液性肿瘤,靶向CEA的CAR-T也在胃癌、乳腺癌等实体瘤中具有良好前景[87]

相对于CD8+T细胞,NK细胞的抗肿瘤作用不需事先致敏,具有更短暂的生理周期和更广泛的杀瘤能力。理论上同样可以采用CAR修饰提高NK细胞的抗肿瘤效应。构建CAR-NK的目的是通过基因编辑手段建立新的活化NK细胞的激活通路,增强其抗肿瘤效应。CAR-NK的设计基本与CAR-T相同,胞外区利用单链可变区片段(single-chain variable fragment, scFv)引导NK细胞靶向肿瘤抗原,胞内选择CD3ζ及共刺激分子基序(如CD28或CD137)传递活化信号。与T细胞不同,进行CAR修饰的NK细胞可以来自于外周血细胞[88],也可以利用NK细胞系[89]。与自体NK细胞相比,异体NK细胞KIRs与患者HLA-Ⅰ类分子不匹配,因此并不会产生抑制信号引起干扰,对CAR靶向的肿瘤细胞具有足够的杀伤活性。NK-92作为一种成熟的NK细胞系,其表面的抑制性受体表达很低,使得其对多种肿瘤有很好的杀伤能力[90-91]。而且体外扩增NK-92细胞并不涉及分选纯化步骤,获得的细胞群体一致性更好,成为在CAR-NK中研究最为广泛的NK细胞系,但NK-92来自于霍奇金淋巴瘤,具有潜在的致瘤性,必须经过辐照后才能使用。虽然已经针对CD20、CD19、HER2等多种抗原开展了CAR-NK研究[90],但目前CAR-NK的临床试验仍然较少,疗效尚不可知,进一步优化CAR-NK实验设计,特别是针对胞内刺激分子的优化设计,对于激活更有效的NK细胞十分必要。

免疫检查点阻断(immune checkpoints blockage)利用针对免疫抑制受体PD-1/PD-L1[92]、CTLA-4[93]的阻断性抗体可以有效恢复耗竭的CD8 T细胞功能,被美国FDA批准进入临床后在实体肿瘤治疗中取得了良好的疗效,并在2013年被评为Science杂志十大科技进展之首,目前免疫检查点疗法已经在多种肿瘤中应用,成为肿瘤免疫治疗的热点。

越来越多的研究发现,多种T细胞表达的免疫抑制性受体,如2B4、PD-1、Tim-3、LAG-3等同样在NK细胞表达,并调控NK细胞功能[94-96]。多种肿瘤患者外周血NK细胞中检测到高水平Tim-3、PD-1的表达。本课题组的研究发现,Tim-3高表达于肝癌患者外周NK细胞、肿瘤浸润NK细胞(tumor infiltrated NK cells, TINK),尤其是CD49a+TINK同样表达高水平Tim-3,阻断Tim-3信号可以恢复TINK功能并在体内抑制HCC生长(未发表)。上述研究提示,与T细胞类似,高表达免疫抑制受体是耗竭NK细胞的重要表现。免疫检查点阻断疗法可以有效恢复NK功能,已经在动物实验中获得良好的抗肿瘤效果[97],然而相关临床研究尚未见报道。

6 结语

NK细胞的生物学特征、功能及调控机制已得到广泛的关注与研究。目前而言,基于NK细胞的肿瘤免疫疗法仍存在挑战,准确而全面地了解NK细胞的特征将对NK细胞的生物治疗发展起到积极的推动作用。

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