PD1+肿瘤浸润性CD8+T淋巴细胞的分离与鉴定.docVIP

PD1+肿瘤浸润性CD8+T淋巴细胞的分离与鉴定 李小红，陈陵，朱波 （400037 重庆，第三军医大学新桥医院全军肿瘤研究所 ） 【摘要】目的 从肿瘤局部分离衰竭性CD8+T淋巴细胞（CD8+PD1+T cell），并对其功能进行研究。初步探讨机体免疫系统无法有效杀伤和清除肿瘤的原因。方法 C57BL/6小鼠皮下注射Lewis肺癌细胞株建立小鼠肿瘤模型。将肿瘤制成单细胞悬液后，染细胞表面标志。采用流式细胞术从肿瘤局部分选CD8+PD1+T淋巴细胞及CD8+PD1-T淋巴细胞，体外经非特异性刺激后行细胞内因子染色，检测IFN-γ分泌能力。CFSE细胞染色检测其增殖活性。结果 肿瘤局部成功分离得浸润性CD8+T淋巴细胞，其中将近80%的细胞高表达衰竭表型PD1+。CFSE结果显示CD8+PD1+T cell较CD8+PD1-T cell增殖活性明显下降；细胞内因子染色后流式细胞术检测结果显示肿瘤局部CD8+PD1+T cell较CD8+PD1-T cell IFN-γ分泌能力降低约50%。结论 肿瘤局部存在一群免疫衰竭性T细胞，可能参与了肿瘤的免疫逃逸。 【关键词】 肿瘤；T 淋巴细胞；PD1 【中图法分类号】 R392.12；R73-35+4；R730.3 Identification of tumor-infiltrating CD8+PD1+T-lymphocytes in Lewis lung cancer Li Xiaohong；Chen Ling；Zhu Bo （Cancer Center of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China） [Abstract] Objective To identify the biological function of tumor-infiltrating CD8+ PD1+T-lymphocytes in Lewis lung cancer. Methods The implanted tumor model was established by injection of Lewis lung carcinoma cells in C57BL/6 mice subcutaneously. CD8+PD1+T-lymphocytes and CD8+PD1-T-lymphocytes were isolated from transplanted tumors by flow cytometry and labeled with CFSE to follow their proliferation. The IFN-γ level of isolated cells was analyzed by intracellular cytokine staining. Results CD8+T-lymphocytes existed in the transplanted tumors. Some of those CD8+T-lymphocytes also expressed PD-1 which has been identified as a marker of exhausted T cells. In comparison with CD8+PD1-T-lymphocytes, the proliferation rate of CD8+PD1+T-lymphocytes was significantly decreased and IFN-γ secretion was suppressed. Conclusion The tumor-infiltrating CD8+T-lymphocytes that express PD-1 do not have a normal function, which may be the reason that TIL can not kill tumor cells effectively. [Key words] Tumor；T lymphocyte；PD1 Corresponding author: Zhu Bo, E-mail: oncology_bozhu@ 肿瘤现已成为发达国家的首要致死因素，发展中国家的次要致死因素。机体T细胞免疫在控制肿瘤的发生发展过程中起到了重要作用[1-4]。其中肿瘤局部浸润性CD8+ T[5]，妨碍机体对肿瘤的清除，导致肿瘤长期定植，表现为T淋巴细胞功能障碍与增殖能力减弱[6]。 PD1已被公认为是衰竭性T淋巴细胞的表面标志[7]。阻断PD1-PD1L通路可以部分恢复衰竭