目的：分析鼻咽癌（NPC）肿瘤微环境（TME）中的肿瘤相关中性粒细胞（TAN）浸润与患者的预后和临床病理特征之间的相关性，初步探讨EBV阳性NPC的TAN对CD8+ T细胞活化的作用。方法：收集2008年至2012年间中山大学肿瘤防治中心收治的118例初治且无转移的EBV阳性NPC患者的肿瘤组织标本，通过免疫组织化学染色法观察并分析NPC组织中CD8+T细胞、TAN浸润和EBV感染之间的关系，检测NPC组织标本中的TAN浸润程度并分析其与患者预后和临床病理特征之间的关系；流式细胞术检测HK1-EBV细胞培养上清液对TAN极化的N2型标志物（CD182和CD206）表达水平的影响，并进一步检测极化后的TAN对CD8+ T细胞活化标志物（CD69和PD-1）表达水平的影响。结果：EBV阳性NPC组织中CD8+T细胞和TAN浸润增多且两者在数量上呈负相关关系（P=0.005 2）；EBV阳性NPC组织中高水平浸润的TAN与患者的不良预后密切相关（OS：P=0.025，PFS：P=0.027），TAN浸润水平是EBV阳性NPC患者总生存时间（P=0.035）的独立预后因子；与对照组相比，HK1-EBV细胞培养上清液可诱导TAN极化并高表达N2型标志物（CD182：P<0.001；CD206：P<0.01）；极化后的TAN可抑制CD8+T细胞CD69的表达并促进PD-1的表达（P<0.001）。结 论：EBV阳性NPC能够促进TME中的TAN浸润增多并使其极化成N2型，从而抑制CD8+ T细胞的活化，发挥免疫抑制作用，与NPC患者的不良预后密切相关。

Objective: To analyze tumor-associated neutrophils (TANs) infiltration in EBV-positive nasopharyngeal carcinoma (NPC) tumor microenvironment (TME) and its correlation with the prognosis and clinicopathological features of patients with NPC, and to preliminarily investigate the role of TANs in activating CD8+ T cells in EBV-positive NPC. Methods: Tumor tissue specimens from 118 patients with EBV-positive NPC without metastases admitted to Sun Yat-sen University Cancer Center from 2008 to 2012 were collected. Immunohistochemistry was performed to analyze CD8+ T cells infiltration and TANs infiltration in NPC and their correlation with EBV infection, and to evaluate the infiltration of TANs in 118 human EBV-positive NPC tissue samples and analyze its correlation with the prognosis and clinicopathological features of NPC patients; flow cytometry was used to detect the expression of N2 marker (CD182 and CD206) of neutrophils stimulated by HK1-EBV-conditioned medium and the expression of CD69 and PD-1 of CD8+ T cells co-cultured with TANs. Results: There were more CD8+ T cells and neutrophils infiltrating in EBV-positive NPC where they were negatively correlated（P=0.0052）; densely infiltrated TANs in EBV-positive NPC tissues were positively correlated with poor prognosis of patients (P=0.025 in OS; P=0.027 in PFS); the infiltration of TANs was proved to be an independent risk factor for overall survival (P=0.035) in patients with NPC; compared with Control group, freshly isolated neutrophils cultured in HK1-EBV-conditioned medium showed an increased expression of N2 markers (P<0.001 in CD182; P<0.01 in CD206) which could suppress the expression of CD69 and promote the expression of PD-1 of CD8+ T cells. Conclusion: More TANs infiltrate in EBV-positive NPC where they polarize into N2 neutrophils, exerting an immunosuppressive effect by suppressing the activation of CD8+ T cells and having a positive correlation with the poor prognosis of NPC patients.

R739.6；R730.3

国家自然科学基金资助项目（No.81874129）