Gao Qian, Liu Hui-Ting, Xu Yu-Qin, Zhang Lin, Liu Yuan-Ru, Ren Qianqian, Sheng Ju-Ping, Zhang Zhen-Xin
Medical College of Nantong University, Nantong, China.
Traditional Chinese Medicine Hospital of Kunshan, Kunshan, China.
Cancer Cell Int. 2021 Oct 30;21(1):584. doi: 10.1186/s12935-021-02294-z.
Hypopharyngeal cancer (HPC) is associated with a poor prognosis and a high recurrence rate. Immune escape is one of the reasons for the poor prognosis of malignant tumors. Programmed cell death ligand 1 (PD-L1) and programmed cell death-1 (PD-1) have been shown to play important roles in immune escape. However, the role of PD-1/PD-L1 in HPC remains unclear. In this experiment, we investigated the effect of exosomes from HPC patient serum on CD8+ T cell function and PD-1/PD-L1 expression and, thus, on prognosis. We hope to provide guidance for the identification of new targets for HPC immunotherapy.
PD-1 and CD8 expression in 71 HPC tissues and 16 paracarcinoma tissues was detected by immunohistochemistry. Concurrently, the clinicopathological data of the patients were obtained to conduct correlation analysis. Exosomes were isolated from serum and then identified by Western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Flow cytometry was used to assess the activity of CD8+ T cells after exosome stimulation. The effects of exosomes on the ability of CD8+ T cells to kill FaDu cells were assessed by CCK-8 assay. The expression of IL-10 and TGF-β1 was measured by enzyme-linked immunosorbent assay (ELISA). PD-L1 expression in HPC tissue samples was evaluated by immunohistochemistry, and the relationship between PD-1/PD-L1 expression and prognosis was investigated with patient specimens.
PD-1 expression was significantly upregulated on CD8+ T cells in tumor tissues compared with those in normal tissues. The overall survival (OS) and disease-free survival (DFS) of PD-1-overexpressing patients were decreased. Serum exosomes from patients can elevate PD-1 expression on CD8+ T cells and suppress their killing capacity and secretory function. The rate of positive PD-L1 expression was increased in HPC tissues compared with paracancerous tissues. The DFS and OS of the PD-1(+)-PD-L1(+) group were significantly lower than those of the PD-1(-)-PD-L1(-) group.
Our findings indicate that serum exosomes from HPC patients can inhibit CD8+ T cell function and that the PD-1-PD-L1 pathway plays an important role in the immune escape of HPC. Exosomes combined with immunotherapy may guide the treatment of patients with advanced disease in the future.
下咽癌（HPC）预后较差且复发率高。免疫逃逸是恶性肿瘤预后不良的原因之一。程序性细胞死亡配体1（PD-L1）和程序性细胞死亡蛋白1（PD-1）已被证明在免疫逃逸中起重要作用。然而，PD-1/PD-L1在下咽癌中的作用仍不清楚。在本实验中，我们研究了下咽癌患者血清来源的外泌体对CD8+ T细胞功能及PD-1/PD-L1表达的影响，进而对预后的影响。我们希望为下咽癌免疫治疗新靶点的识别提供指导。
采用免疫组织化学法检测71例下咽癌组织和16例癌旁组织中PD-1和CD8的表达。同时，获取患者的临床病理资料进行相关性分析。从血清中分离外泌体，然后通过蛋白质免疫印迹法（WB）、透射电子显微镜（TEM）和纳米颗粒跟踪分析（NTA）进行鉴定。采用流式细胞术评估外泌体刺激后CD8+ T细胞的活性。通过CCK-8法评估外泌体对CD8+ T细胞杀伤FaDu细胞能力的影响。采用酶联免疫吸附测定（ELISA）检测白细胞介素-10（IL-10）和转化生长因子-β1（TGF-β1）的表达。通过免疫组织化学评估下咽癌组织样本中PD-L1的表达，并利用患者标本研究PD-1/PD-L1表达与预后的关系。
与正常组织相比，肿瘤组织中CD8+ T细胞上的PD-1表达显著上调。PD-1过表达患者的总生存期（OS）和无病生存期（DFS）降低。患者的血清外泌体可提高CD8+ T细胞上的PD-1表达，并抑制其杀伤能力和分泌功能。与癌旁组织相比，下咽癌组织中PD-L1阳性表达率升高。PD-1(+)-PD-L1(+)组的DFS和OS显著低于PD-1(-)-PD-L1(-)组。
我们的研究结果表明，下咽癌患者的血清外泌体可抑制CD8+ T细胞功能，且PD-1-PD-L1通路在下咽癌免疫逃逸中起重要作用。外泌体联合免疫治疗可能为未来晚期下咽癌患者的治疗提供指导。
