肝细胞癌（hepatocellular carcinoma，HCC）是全球最常见的恶性肿瘤之一。近年来发现程序性死亡因子１及其受体（programmed cell death-1 and its ligand，PD-1/PD-L1）与HCC的产生和发展密切相关，为免疫治疗提供了一个新方向。然而，抗PD-1/PD-L1免疫治疗缺乏有效的生物标志物。最新研究发现，三级淋巴结构（tertiary lymphoid structures，TLS）对HCC的抗PD-1/PD-L1免疫治疗效果有一定的预测价值。本文对TLS和PD-1/PD-L1信号通路的发生过程，以及二者在肝细胞癌中的表达和临床中的具体研究进展作一综述，以期为TLS和PD-1/PD-L1信号通路在肝细胞癌中的应用前景提供新的参考。

肝细胞癌（hepatocellular carcinoma，HCC）是全球最常见的恶性肿瘤之一，亚洲尤其严重，据世界卫生组织调查显示，2020年亚洲HCC新增人数占全球72.5%，相关死亡人数占全球73.3%[1]。现代医学手术和介入相结合的疗法对部分HCC患者产生了不错的疗效 [2-5]。但大多数患者发现HCC时已经出现转移，无法进行手术[6]。近年来，免疫疗法尤其是抗程序性死亡因子１及其受体（programmed cell death-1 and its ligand，PD-1/PD-L1）通路治疗对部分晚期HCC的预后有所改善[7-9]，但该疗法的预后缺乏可靠的预测指标[9]。如何筛选出适合PD-1/PD-L1的优势人群，从而节约医疗资源，降低患者医疗费用，已成为目前免疫治疗的热点。最新研究表明，三级淋巴结构（tertiary lymphoid structures，TLS）对HCC免疫治疗的预后有一定的指导作用。本文阐述了TLS和PD-1/PD-L1信号通路在HCC治疗中的最新进展及TLS对抗PD-1/PD-L1预后的预测价值。

1 TLS与HCC及其免疫治疗

1.1 TLS的基本结构

TLS是在非造血器官中形成的淋巴聚集物，该结构最初在黑色素瘤和非小细胞肺癌中发现，之后在多种肿瘤组织中被证实[10-11]。基本结构由生发中心、B细胞区和T细胞区构成，其中生发中心由成熟B淋巴细胞、树突状细胞（dendritic cell，DC）构成，B细胞区由B淋巴细胞构成，T细胞区由T淋巴细胞构成。成熟TLS的生发中心较大，T细胞区较小[12]。

1.2 TLS的形成机制

C-X-C趋化因子配体13 （C-X-C chemokine ligand 13，CXCL13）及其受体C-X-C趋化因子受体5（C-X-C chemokine receptor 5，CXCR5）与肺癌、结直肠癌、皮肤黑色素瘤及口腔癌等多种肿瘤的TLS形成有关[13-15]（具体机制详见图1）。此外，凋亡外泌体样囊泡可通过促进淋巴毒素和炎症因子的释放从而促进TLS形成[16]（具体机制详见图2）；有研究发现CXCL13会促进淋巴毒素分泌[16-17]，这可能是其产生和发展TLS的途径；Suematsu等人发现趋化因子与DC可促进TLS中淋巴细胞簇的形成，且趋化因子起主导作用，DC起增强作用[18]；Finkin等人发现与同月龄Alb-cre小鼠相比，IKK-β（EE）Hep小鼠（该小鼠能持续表达IKKB及NF-κB转录活性）肝脏中TLS较多[19]，说明IKK-NF-κB信号通路是肝脏TLS产生的重要途径。

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  图1 CXCL13促进TLS的形成

  Figure1.CXCL13 promotes the initiation of tertiary lymphoid structure development

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  图2 细胞凋亡促进TLS形成

  Figure2.Apoptosis promotes the initiation of tertiary lymphoid structure development

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1.3 TLS的测量和分级方法

目前临床上采用HE染色或HE染色联合免疫组化判定HCC组织中TLS的存在。HE染色切片中TLS的特征是成熟DC位于T细胞区并邻近生发中心[20]。TLS常见免疫组化标志物为CD21、CD20 、CD3、CD8 和CD208[21-22]。

在HCC的TLS分级方面，目前缺乏统一的分级标准。近年来比较有影响力的分级方法是 Julien Calderaro等人的方法：①淋巴细胞聚集体型TLS：模糊、定义不清的淋巴细胞簇；②初级淋巴滤泡型TLS：无生发中心形成的圆形淋巴细胞簇；③次级淋巴滤泡型TLS：含有生发中心的滤泡[19, 23-24]。此外，Nie等人根据成熟度将TLS分为聚集体（肿瘤样本仅显示聚集体而无淋巴滤泡）和淋巴滤泡型TLS（肿瘤样本显示至少一个淋巴滤泡或生发中心，有无聚集体均可）[22]。而在非小细胞肺癌的TLS相关研究中，Devi-Marulkar等人提出可以根据TLS内部免疫细胞比例的不同，如调节性T细胞（Treg）/B细胞和γδT细胞等对TLS进一步划分，优化分级方法[25]。

1.4 诱导TLS形成的方法

目前大多数研究表明，TLS对HCC的预后起到积极作用，可考虑人工诱导TLS的形成以改善HCC预后。Suematsu等人研究将表达LTα或趋化因子的细胞系和DC嵌入到海绵状胶原支架并移植到小鼠的肾包膜下进行培养，成功构建出了人工TLS，并且证实了其结构和功能与一般TLS存在相似性[18, 26]。此外，Aoyama等人根据TLS的形成条件，总结了目前可能作为人工TLS支架的构建材料，如胶原基质、水凝胶、介孔二氧化硅棒和聚酰胺纤维[27]。

1.5 TLS对HCC免疫的影响

肿瘤相关TLS有助于肿瘤浸润淋巴细胞产生抗肿瘤免疫应答，从而改善肿瘤预后[28]。作为淋巴细胞产生局部免疫的“前沿堡垒”和“传导体”，淋巴细胞产生的多种抗肿瘤免疫作用需通过TLS启动和维持，包括T细胞免疫、B细胞体液免疫、DC抗原呈递或其他结构的作用。Lin等人发现TLS通过产生中枢记忆T淋巴细胞和浆细胞，在抗肿瘤特异性免疫反应中发挥关键作用[29]。Sautès-Fridman等人通过泛癌分析制作了T-分布随机邻域嵌入图，发现HCC组织中，肿瘤内TLS成熟度的增加则提示机体产生了长期的抗肿瘤免疫[30]。Wen等人通过单因素比例风险回归分析发现TLS内中性粒细胞/淋巴细胞比值与HCC总生存率呈正相关[21]。El-Rebey等人的研究表明HCC肿瘤TLS内的CD8+T、CD4+T和NK等免疫细胞发挥抗肿瘤作用[31]。Li等人通过对HCC患者的肿瘤组织进行免疫荧光和免疫组化染色，也发现TLS内CD20+B细胞和CD8+T细胞的数量与HCC患者总生存期呈明显正相关关系，CD3+T细胞和LAMP3+DCs的数量与患者无进展生存期显著正相关[32]。

然而，在HCC中，局部免疫不仅产生抗肿瘤作用，也具有加速肿瘤恶性程度进展的作用。这是因为在长期抗HBV的免疫过程中，免疫抑制介质例如肝脏驻留的Kupffer细胞分泌的白细胞介素-10，作用于HBV特异性CD8+T细胞，使其数量减少或活性丧失，导致免疫系统无法根除HBV感染[33-34]。但其他免疫细胞如CD4+T细胞仍然继续分泌细胞因子和生长因子，刺激肝脏持续产生炎症，导致肝损伤和肝细胞再生的重复循环，进而加速肿瘤进展。Finkin等人通过分析82例进行过肝切除的HCC患者的临床资料、组织学切片和肝TLS相关基因，发现TLS与HCC预后呈负相关[19]。研究认为IKK-β（EE）Hep小鼠肝组织中TLS表达增多，HCC发生率升高，而注射抗原Thy-1.2抗体（该种抗体通过消融T淋巴细胞和自然杀伤细胞减少了TLS的产生和发展）的小鼠HCC产生几率较低[19]。此外，TLS周围的肝细胞最先恶化为肿瘤细胞并表达多种HCC祖细胞标志物。组织学检测证实这是因为TLS中的T淋巴细胞和B淋巴细胞通过分泌LTα等细胞因子，促使其周围的肝细胞向恶性肿瘤细胞转化并获得自分泌相同细胞因子的能力。Zhao等人的研究发现TLS中Tregs产生促肿瘤作用，并会加速HCC的恶化[35]。

2 PD-1/PD-L1通路与HCC及其免疫治疗

2.1 PD-1/PD-L1通路与肿瘤免疫

PD-1是一种在免疫细胞表面表达的抑制性跨膜蛋白，PD-L1在多种组织类型的表面，而PD-L2大部分在造血细胞表面[36]。 该信号可调节T淋巴细胞，在免疫稳态、消炎、耐受等方面具有重要作用。PD-1在其胞质结构域中有两个酪氨酸基序，当与配体接合时，酪氨酸残基处被磷酸化，募集多种蛋白酪氨酸磷酸酶（protein tyrosine phosphatase，PTPs），如蛋白酪氨酸磷酸酶Src和同源蛋白2等。这些PTPs通过去磷酸化作用拮抗T细胞抗原受体（TCR）和CD28产生的阳性信号，从而影响T淋巴细胞的下游信号通路分子，例如磷酸肌醇3激酶（PI3K）-Akt、Ras、胞外信号调节激酶（ERK）、VAV和磷脂酶Cγ（PLCγ）、p27、p15[37-39]。

正常细胞表面表达PD-L1，通过结合PD-1而抑制淋巴细胞功能。当组织损伤时，细胞表面的PD-L1表达量减少，出现免疫反应[40]。较大程度的免疫反应会产生炎症，促进PD-L1表达量增加，减少免疫系统对正常组织的损伤，形成负反馈循环，既保证了免疫系统能发挥作用，又减少了对正常组织的损伤。肿瘤细胞高度表达PD-L1，通过PD-1/PD-L1通路减少淋巴细胞的杀伤作用和长期免疫作用， 产生免疫逃逸[41]。阻断PD-1/PD-L1通路可以增强肿瘤免疫治疗效果[42]（图3）。

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  图3 肿瘤细胞通过PD-1/PD-L1通路抑制免疫细胞的杀伤作用

  Figure3.Tumor cells inhibit the killing effect of immune cells through PD-1/PD-L1 pathway

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2.2 PD-1/PD-L1免疫治疗的不足

目前分子靶向治疗和免疫治疗是晚期HCC唯一的治疗方案。免疫治疗，尤其是抗PD-L1联合抗血管内皮生长因子（vascular endothelial growth factor，VEGF）疗法显示出更高的缓解率（约30%），并显著延长HCC患者生存期[43]，例如卡瑞利珠单抗、纳武单抗或帕博利珠单抗联合索拉非尼疗法[44-47]。尽管如此，大多数晚期HCC患者对靶向治疗反应仍不佳[48-49]。实际临床治疗中，抗PD-1/PD-L1药物应答率普遍较低，如纳武单抗的应答率仅20%[45]。且有研究表明，抗PD-1/PD-L1治疗甚至会增加非酒精性脂肪肝所致HCC的恶性程度[50-51]。因此，在进行PD-1/PD-L1免疫治疗前需检测生物标志物预测治疗效果，以降低治疗成本并避免治疗相关不良事件。目前对抗PD-1/PD-L1免疫治疗HCC预后的评估指标——免疫组化、肿瘤突变负荷和微卫星不稳定状态的准确性均不足[9, 45, 47]。由于肝癌组织本身很少发生基因突变，因此后两项并不作为主要评判指标，尤其是肿瘤突变负荷，不仅尚未得到验证，甚至在HCC免疫治疗中的作用也未完全阐明[52-53]。虽然在多种肿瘤中，PD-L1表达程度的增加与PD-1阻断治疗反应率的增加和肿瘤预后的改善有关[54-56]，但在HCC免疫治疗中PD-L1的表达却不能准确预测疗效，例如在KEYNOTE-224以及CheckMate 040试验中，PD-L1就不具有预测价值[45, 47]。Yan等人在探索HCC免疫治疗生物标志物的研究中同样观察到PD-L1的表达程度不足以对HCC中抗PD-1/PD-L1免疫治疗效果进行准确预测[57]。因此，迫切需要开发更有效的生物标志物，以准确预测抗PD-1/PD-L1免疫治疗对晚期HCC患者的疗效。

3 TLS与HCC抗PD-1免疫治疗

TLS作为肿瘤免疫环境的首要特征[58]，反映抗肿瘤免疫应答的程度[20, 59] ，对抗PD-1/PD-L1免疫治疗效果有一定的预测价值[12, 59]。2020年以来均有研究发现在多种肿瘤中，成熟TLS与免疫治疗的更好反应性有关[60-61]。Helmink发现TLS中的B细胞可通过与其他免疫成分相互作用并分泌细胞因子（包括TNF、IL-2、IL-6和IFNγ）激活和招募其他免疫细胞如T淋巴细胞，以增强肿瘤对免疫治疗的反应[62]。Clubb等人也发现TLS形成与头颈癌抗PD-1/PD-L1免疫治疗有协同作用，这与TLS维持免疫反应微环境中的作用有关，并据此提出可通过诱导产生TLS或增加其成熟度以增强免疫治疗作用的观点[63]。此外，Li等在HCC与TLS相关性的研究中发现，淋巴细胞特异性蛋白酪氨酸激酶作为通过调控细胞因子信号通路、趋化因子信号通路和T细胞活化参与TLS形成和成熟的分子，其表达水平与HCC患者对HCC免疫治疗的敏感性呈正相关[32]。Zhong等以高密度TLS作为判断条件，对伴有门静脉瘤栓的HCC实行抗PD-1治疗联合索拉非尼，发现预测效果良好，表明TLS有作为预测免疫治疗反应指标的价值[64]。因此，在研究抗PD-1/PD-L1免疫治疗效果时，可考虑将TLS视为一个生物标志物。

4 结语

未来HCC免疫治疗的研究方向可能为：①从TLS入手，提出预测HCC术后复发的循证医学证据；②将导致HCC、亚临床HCC和有恶化为HCC风险的肝脏疾病的病因作为一个考虑因素，评判HCC患者联合治疗时是否应当加入抑制PD-1通路的免疫治疗以及将其作为主要治疗方式或辅助治疗，可通过以上两点，更新HCC患者免疫治疗的评判量表，依据病情提出提升患者生存时间和生存质量的方案；③目前预测抗PD-1/PD-L1疗效的指标单一，将TLS及其密度、与肿瘤位置的远近、组成成分和成熟程度作为抗PD-1/PD-L1疗效的评判指标可提高预测准确度；④HCC中TLS存在及成熟度与抗PD-1/PD-L1疗效呈负相关，因此，诱导TLS形成或成熟可能成为提高HCC抗 PD-1/PD-L1疗效的方法之一。

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