头颈部鳞癌是世界上最常见的恶性肿瘤之一，其转移率高，预后差，常给患者带来严重的面部损伤，以及进食、发音等功能障碍。自然杀伤细胞是先天免疫系统的效应细胞，具有直接识别并杀死肿瘤细胞的功能。自然杀伤细胞活化水平与头颈部鳞癌的预后密切相关，头颈部鳞癌的发生发展常伴随自然杀伤细胞的数量减少或功能被抑制。重新激活自然杀伤细胞可提高头颈部鳞癌的疗效。本文就自然杀伤细胞在头颈部鳞癌发展及治疗中的应用进行综述。

头颈部鳞癌是世界上最常见的恶性肿瘤之一，其转移率较高，生存率较低，常伴有发音、进食等功能的障碍和面部外形的破坏[1]。研究发现头颈部鳞癌表现出免疫抑制的特点[2]。自然杀伤（nature killer，NK）细胞作为肿瘤免疫的第一道防线，在早期清除肿瘤细胞及激活下游免疫细胞上有重要作用，其功能的抑制往往导致肿瘤的免疫逃逸。本文就NK细胞的生物学特性、抗肿瘤机制、在头颈部鳞癌中的活化水平、基于NK细胞的免疫逃逸及目前与NK细胞相关免疫疗法的研究进展进行综述。

1 NK细胞的生物学特性及抗肿瘤机制

NK细胞是先天免疫系统的重要效应细胞，约占所有外周淋巴细胞的10% ~ 15%。静止的NK细胞多存在于外周血中，肝脏、脾脏及腹膜腔内也有NK细胞存在。被细胞因子激活后，NK细胞能够迁移并渗入大多数含有病原体感染或恶性细胞的组织中[3-4]。

NK细胞可根据表面标记物的差异性表达划分为具有不同功能特性的亚群。一般来说，NK细胞被定义为CD3-CD56+细胞，并可通过抗原CD56的丰度分为CD56dim和CD56bright两个亚群。其中，CD56dim的NK细胞约占总数的90%，多存在于外周血循环中；而约占总数10%的CD56brightNK细胞则主要位于次级淋巴组织中 [5-7]。CD16是另一种常用的NK细胞表面标记物，其表达规律与CD56的丰度有关：CD56dim NK细胞常表达高水平的CD16（CD16high），而CD56bright NK细胞伴随CD16的表达缺失或低水平表达（CD16-/low）。从功能上讲，CD16使NK细胞能够检测抗体包被的靶细胞并发挥抗体依赖性细胞毒性（antibody-dependent cellular cytotoxicity，ADCC）[8]。此外，CD56bright NK细胞表面稳定表达抑制性主要组织相容性复合体（major histocompatibility complex，MHC） I类结合受体CD94/NKG2A，但缺乏杀伤细胞免疫球蛋白样受体（killer cell immunoglobulin-like receptors，KIRs）和激活性MHC I类结合受体CD94/NKG2C的表达。而CD56dim NK细胞则显示出CD94/NKG2A、CD94/NKG2C和KIRs的多样化表达[9]。这种不同亚群间表面标记物的差异性表达与他们不同的功能直接相关：CD56dim CD16highNK细胞识别靶细胞并分泌高水平的穿孔素和颗粒酶，有较强的细胞毒性，可发挥免疫监控的作用[10-11]；CD56bright CD16-/lowNK细胞则具有较弱的细胞毒性和介导ADCC的功能，更多地通过响应单核细胞分泌的炎症因子产生大量的细胞因子和趋化因子，如干扰素γ（interferon-γ，IFN-γ）、肿瘤坏死因子β（tumor necrosis factor-β，TNF-β）及白细胞介素10（interleukin-10，IL-10）等，发挥免疫调节的作用[12-13]。

NK细胞的主要功能为裂解靶细胞和提供免疫调节。NK细胞与T细胞同为直接杀伤细胞，但应答过程无需识别抗原预先致敏，具有应答快速的特点。NK细胞的活性受表面抑制性受体如CD94/NKG2A、KIRs、T 细胞免疫球蛋白和粘蛋白结构域3（T cell immunoglobulin and mucin domain-3，TIM-3），以及活化性受体如CD16（FcγRIIIa）、NKG2D、NKp30、NKp44、NKp46等的共同调节。生理状态下，正常细胞表面的配体结合NK细胞的抑制性受体，抑制性受体信号转导，NK细胞处于静息状态，避免杀伤正常细胞；病理状态下，肿瘤细胞表面的抑制性配体减少或活化性配体增加，NK细胞活化，通过胞吐穿孔素和颗粒酶、人凋亡相关因子配体（factor associated suicide ligand，FasL）和TNF相关的细胞凋亡诱导配体（TNF-related apoptosis-inducing ligand，TRAIL）的激活，或通过ADCC诱导靶细胞的凋亡[5]。NK细胞还可以通过分泌细胞因子和趋化因子，激活下游适应性免疫反应，发挥免疫调节的作用[8]。

2 NK细胞在头颈部鳞癌中的表达、活化水平及与预后的关系

NK细胞参与了肿瘤防御的第一道防线，而头颈部鳞癌中常出现NK细胞数量减少、低水平浸润或功能丧失。研究显示，头颈部鳞癌患者的癌组织中仅能检测到低水平的NK细胞浸润，且多为调节性的CD56brightNK细胞而不是具有细胞毒性的CD56dimNK细胞[14-15]。此外，肿瘤浸润性NK细胞活性升高的患者预后更加良好，且区域淋巴结转移和远处转移的发生率也更低[16-17]。相对的，低NK细胞毒性的患者区域和远处转移的发生率更高，且死亡风险更高。分析发现这类患者的外周血中存在一种免疫复合物，可抑制NK细胞的功能[18]。人乳头瘤病毒（human papilloma virus，HPV）阳性患者的CD56dimNK细胞肿瘤浸润比率要高于HPV阴性患者，这可能是HPV阳性患者临床预后较好的原因[19]。与CD56类似，表面抗原CD57与良好预后、低淋巴结转移率成正相关[20-21]。对外周血循环中的NK细胞进行检测发现，早期肿瘤患者外周血循环中的NK细胞数量高于晚期肿瘤患者[22]。同肿瘤组织浸润的NK细胞类似，循环NK细胞的数量[23-24]及细胞毒性水平降低[25]，CD56brightNK细胞的数量多于CD56dimNK细胞；进一步研究发现CD56dimNK细胞较CD56brightNK细胞优先发生自发凋亡[26]。虽然也有研究报道循环NK细胞中CD56dimNK细胞的比例上升，但仍检测到了总体循环NK细胞数量的减少和细胞毒性的抑制[27]。提示随着肿瘤发展进程，NK细胞的数量和功能可能受到抑制。

放疗联合化疗是局部晚期和转移的头颈部鳞癌的治疗方案。放化疗的全身性副作用包括对免疫功能的抑制。一项对23例仅接受放疗的患者的调查发现，放疗前后循环NK细胞的水平无明显变化[28]，另一研究发现化疗（顺铂/紫杉醇与卡铂/多西他赛联合方案）后NK细胞数量减少，而同期放化疗后NK细胞数量增多[29]。由于目前对NK细胞的活化水平不明确，故NK细胞对放化疗疗效的影响仍待进一步研究。

NK细胞的部分配体也可作为头颈部鳞癌预后及预测临床病理特征的指标。抑制性受体癌胚抗原相关细胞黏附分子1（carcinoembryonic antigen cell adhesion molecule 1， CEACAM1）的高表达与生存率降低和预后不良有关[30-31]，CEACAM1还抑制活化受体NKG2D的信号传导，并因此抑制NK细胞的抗肿瘤功能[32]。而CEACAM1的配体RCAS1可诱导NK细胞凋亡，进而在肿瘤细胞的免疫逃逸中起作用。研究显示其在肿瘤细胞中的高表达预示着较高的肿瘤分级及淋巴结转移[33]。

3 基于NK细胞的头颈部鳞癌免疫逃逸

头颈部鳞癌的免疫系统多处于抑制或失活状态[34]。NK细胞通过细胞毒性和免疫调节发挥对肿瘤细胞的监管作用，而肿瘤细胞则通过一系列方式逃逸NK细胞的监管作用。头颈部鳞癌细胞产生各种细胞因子，或使抑制性受体信号转导增强，或使活化性受体信号阻断，或通过肿瘤微环境（tumor microenvironment，TME）抑制NK细胞募集，最终导致NK细胞抗肿瘤功能的下降。

抑制NK细胞表面的活化性受体是头颈部鳞癌细胞免疫逃逸的常见原因。程序性死亡1（programmed death 1，PD-1）在NK细胞上高表达时是一种NK细胞的激活表型，高水平的循环PD-1阳性NK细胞与更好的总体生存率相关。PD-1阳性NK细胞富集于肿瘤组织中，但其对NK细胞的活化作用在与TME中的程序性死亡配体1（programmed death ligand 1，PD-L1）结合后丧失[22]。大多数头颈部鳞癌过表达表皮生长因子受体（epidermal growth factor receptor，EGFR），而PD-L1的表达受EGFR及其下游JAK2/STAT1依赖性诱导，最终过表达PD-L1的头颈部鳞癌逃避NK细胞的监视[35]。NKG2D是NK细胞表面的活化性受体，在免疫监视中起着重要的作用，因此头颈部鳞癌细胞通过分泌各种NKG2D配体（NKG2DLs）来逃避NK细胞的监视。高水平NKG2DLs存在下NK细胞不能浸润肿瘤组织且细胞毒性降低，而从头颈部鳞癌患者血浆中去除脱落的NKG2DLs可以恢复NK细胞功能[14]。复发的头颈部鳞癌中可溶性主要组织相容性复合物I类链相关肽A和转化生长因子-β的表达升高，并抑制依赖NKG2D的NK细胞活化[36]。同为活化性受体的NKp30和NKp46也有表达下降的报道[37]。

NK细胞抑制性受体及其配体的过度表达也是头颈部鳞癌细胞免疫逃逸的原因。CD56dimNK细胞受其表面抑制受体KIRs的控制。研究显示头颈部鳞癌较其他实体瘤表达更高水平的KIRs[2]，意味着NK细胞的活性可能因此受到抑制，头颈部鳞癌患者更有可能在抗KIRs抗体治疗中获益。顾小军等的研究发现口腔鳞癌患者外周血NK细胞表面抑制性受体TIM-3表达上升，并与肿瘤临床分期、分化程度及淋巴结转移显著相关，提示其与口腔鳞癌的发生发展有关[38]。

Ludwig等的研究显示，头颈部鳞癌患者的血浆中分离出的外泌体可下调NKG2D表达水平并抑制NK细胞的细胞毒性作用[39]，而前述NKG2DLs通过外泌体释放后较单体NKG2DLs引起更多的NKG2D下调[40]；Theodoraki等也报道了头颈部鳞癌患者血浆中可分离出携带PD-L1的外泌体，且外泌体内的PD-L1水平与肿瘤的活动性和淋巴结状态相关[41]，提示肿瘤免疫逃逸的新机制及外泌体作为癌症进展的非侵入性标志物和免疫功能低下指标的潜力。

除上述调节方式外，NK细胞与头颈部鳞癌肿瘤患者的其他免疫效应细胞如树突状细胞（dendritic cell，DC）、调节性T细胞及调节性B细胞等之间还存在复杂的调节网络，有待进一步的研究[42-46]。

4 基于NK细胞的免疫疗法在头颈部鳞癌治疗方案中的应用

目前，基于NK细胞的免疫疗法侧重于恢复或增强NK细胞的细胞毒性作用。主要方法包括以下几类：①基于西妥昔单抗（Cetuximab ）的联合用药方案；②基于白细胞介素家族细胞因子的联合用药方案；③经工程改造的NK细胞产物疗法等。

Cetuximab已被列入过表达EGFR的头颈部鳞癌（约占总数90%）的治疗方案。Cetuximab包被EGFR阳性HNSCC细胞后，通过与NK细胞表面的活化性受体FcγRIIIa结合激活NK细胞，进而NKG2D依赖性激发DC成熟、效应因子IFN-γ及趋化因子Th1分泌[47]。然而Cetuximab的临床应答率仅为10% ~ 15%。Baysal等报道，Cetuximab耐药性可能被基于NK细胞的免疫反应克服[48]。Faden等的研究发现，Cetuximab无应答的患者存在较高的人白细胞抗原-C（human leukocyte antigen-C，HLA-C）失活性突变，而HLA-C与NK细胞表面的KIRs结合后能够激活NK细胞，针对HLA-C/KIRs轴的联合用药方案（如抗KIRs单克隆抗体Lirilumab与Cetuximab）可以取得更好的临床治疗效果[49]。Jie等报道Cetuximab上调CTLA-4+ 调节性T细胞（regulatory T cells，Treg），使ADCC受到抑制；使用Ipilimumab消除肿瘤内的CTLA-4+ Treg，增强Cetuximab诱导的NK细胞ADCC[50]。体外实验中一种Toll样受体8的激动剂也可以通过激活NK细胞及下游ADCC作用增强Cetuximab对头颈部鳞癌细胞的杀伤作用[51]。Concha-Benavente等发现Cetuximab增加NK细胞上活化受体PD-1的表达，但被外源性PD-L1封闭；使用阻断PD-1-PD-L1轴的Nivolumab可显著增强Cetuximab对过表达PD-L1的头颈部鳞癌细胞的毒性[22]。Bochen等的研究显示，维生素D缺乏常见于头颈部鳞癌患者，进行维生素D的替代治疗后，肿瘤组织内及周边的NK细胞浸润增多，单独用药或与Cetuximab联用均可增强NK细胞毒性[52]；STING激动剂与Cetuximab联用也在体外实验中展示了较单药更好的抗肿瘤作用[53]。

白细胞介素家族中的数个因子被报道能够促进NK细胞的活化。Pinette等的研究表明，IL-15具有促进NK细胞增殖、分化、存活和活化的功能，提高NK细胞分泌IFN-γ和T细胞趋化因子RANTES和IL-8的水平。体内实验证实IL-15激动剂ALT-803和Cetuximab联合用药，可明显减少裸鼠肿瘤体积[54]；I期临床试验中也观察到了ALT-803对NK细胞数量和活性的良好的上调作用[55]。与IL-15有类似结构和作用的IL-12也在治疗不可切除的原发或复发头颈部鳞癌患者的I/II期临床试验中表现出了和Cetuximab的联合治疗作用[56]，一种携带IL-12的疫苗在裸鼠头颈部鳞癌移植瘤模型中完全阻止了肿瘤复发[57]。同为IL-12家族的IL-27也可激活NK细胞，并通过诱导ADCC来抑制NK细胞抗性头颈部鳞癌[58]。IL-2是已经证实的NK细胞活化性配体，在一项36例无法切除的头颈部鳞状细胞癌病例的IIb期临床试验中观察到IL-2的经皮和结节内注射能够提升NK细胞的肿瘤浸润率和细胞毒性[59]。主要活性成分是IL-2，IL-1β，IFNγ和TNFα的细胞因子生物制剂IRX-2通过恢复活化性受体NKp30和NKp46的表达水平来恢复NK细胞功能，IIa期临床试验中观察到了良好的耐受性和对总生存率的延长[37]。

PD-L1 t-haNK细胞是一种经过工程改造的高亲和力NK细胞（high affinity NK cells，haNK细胞），表达CD16高亲和力FcγRIIIa（158V）受体，可以内源性合成IL-2[60]及靶向PD-L1的特异性嵌合抗原受体，检测发现该细胞可分泌更高水平的穿孔素和颗粒酶。PD-L1 t-haNK细胞的这些特征使其可以通过三种不同的机制靶向肿瘤细胞：嵌合抗原受体介导的杀伤、ADCC介导的杀伤和天然NK细胞受体介导的杀伤。体外实验中，PD-L1 t-haNK细胞对来自乳腺、肺、结肠、泌尿生殖系统等20种组织的恶性肿瘤细胞都有较haNK细胞更良好的裂解作用，在与抗PD-1抗体和IL-15激动剂ALT-803联用后对裸鼠头颈部鳞癌移植瘤的生长有明显的抑制作用[61-62]；将晚期HPV阴性头颈部鳞癌患者的外周血白细胞与PD-L1 t-haNK一起体外孵育24小时，显著降低高表达PD-L1的巨噬细胞和CD14+ / CD15+骨髓细胞亚群[63]。这些结果提示了PD-L1 t-haNK细胞在治疗高表达PD-L1的头颈部鳞癌患者中的应用前景。

其他用药方案还包括：对NK细胞表面受体抑制剂的开发如NKG2A的单克隆抗体Monalizumab[64]或NKp44的抑制剂[65]；基于细胞周期检查点的开发如WEE1激酶抑制剂逆转G2/M期细胞周期检查点的激活，最终增加头颈部鳞癌细胞对NK细胞裂解作用的敏感性[66]；使用CXCR1 / 2小分子抑制剂SX-682抑制骨髓来源的抑制性细胞的转运，进而增强NK细胞的肿瘤浸润和细胞毒性[67]；使用同样具有激活NK细胞功能的Nimotuzumab替代Cetuximab，避免对TIM-3和PD-L1上调及Tregs细胞百分比上升造成的免疫逃逸[68-69]等。

5 结语

总体来看，NK细胞在头颈部鳞癌中的功能受到抑制，使免疫系统无法及时清除肿瘤细胞，可能是导致头颈部鳞癌发展及转移的机制。考虑到颌面部组织保留的重要性，减少肿瘤体积以及由转移或复发导致的多次手术具有重要意义。相比较其他手段，免疫疗法通过重新激活NK细胞的抗肿瘤能力，靶向清除头颈部鳞癌细胞，能够更好地保留颌面部的形态和功能。然而NK细胞受抑制的机制复杂，既包括肿瘤细胞本身分泌的各种细胞因子在肿瘤微环境及外周血循环中对NK细胞的抑制，也涉及免疫系统各组分间复杂的调节网络。目前的基于NK细胞的免疫治疗方案，虽在体外及体内实验上展现了良好的抗肿瘤能力，但由于NK细胞复杂的调控机制，与实际的临床应用还有较大距离，NK细胞在头颈部鳞癌治疗中的应用有待进一步的探索。

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