【作者】 夏瑞；

【导师】 张学光； 卢斌峰；

【作者基本信息】 苏州大学 ， 免疫学， 2013， 硕士

【摘要】 肿瘤是一个包含有肿瘤细胞、浸润的免疫细胞与间质细胞，以及微环境中一系列分子组成的生态系统。在肿瘤微环境中，浸润的免疫细胞是肿瘤发生发展的重要因素之一。效应T细胞、调节性T细胞、无能T细胞、耗竭性T细胞、巨噬细胞、粒细胞和髓系来源抑制细胞（MDSC）等免疫细胞的浸润在肿瘤微环境中发挥不同的作用，影响着肿瘤进展的全过程。近年对肿瘤微环境和肿瘤免疫逃逸机制的深入研究发现，一组介导免疫调节的重要负性共刺激分子——免疫卡控点（immune checkpoints），如负性B7家族分子、CTLA-4和TIM-3等，异常表达在一系列肿瘤组织和/或浸润的免疫细胞，参与肿瘤免疫逃逸，并与肿瘤临床病理和预后密切相关，是构成肿瘤微环境的重要成分。TIM-3， T细胞免疫球蛋白粘蛋白分子（T cell immunoglobulin-andmucin-domain-containing molecules，TIM）家族的一员，主要表达在分化成熟的Th1细胞，细胞毒性CD8+T细胞、单核细胞、树突状细胞等，也被认为是应答性CD8+T细胞耗竭/功能障碍的重要标志性分子之一，而其在肿瘤免疫中的作用则正越来越受到关注。肿瘤发展的不同阶段，肿瘤微环境浸润的T淋巴细胞亚群和抑制性免疫细胞（如MDSC）借助其表达的负性共刺激分子/免疫卡控点介导抗肿瘤的免疫应答和肿瘤免疫逃逸。深入探讨负性共刺激分子TIM-3在肿瘤浸润T淋巴细胞（TIL）和髓系来源抑制细胞（MDSC）的表达特征、与临床的相关性及其参与肿瘤免疫逃逸的机制可为以干预TIM-3信号为基础的肿瘤免疫治疗提供新的理论基础和靶点。【目的】探讨负性共刺激分子TIM-3在人非小细胞肺癌T细胞和髓系来源抑制细胞（MDSC）上的表达及其临床意义。【方法】收集51例手术切除的诊断明确的非小细胞肺癌（NSCLC）病人癌组织及癌旁组织，采用消化液消化和机械研磨的方法获取单细胞悬液，经淋巴细胞分层液分离，经免疫荧光标记和流式细胞仪分析检测TIM-3在肿瘤浸润T淋巴细胞和MDSC的表达。同时采集同一患者和健康志愿者外周血，进行免疫荧光标记并溶血，经流式细胞仪分析检测TIM-3在外周血T淋巴细胞和MDSC的表达。分析TIM-3在非小细胞肺癌患者T细胞的表达与患者临床病理因素之间的相关性。【结果】（1）与癌旁正常组织浸润淋巴细胞和外周血T细胞相比，非小细胞肺癌患者肿瘤组织浸润淋巴细胞（TILs）表面TIM-3的表达明显上调；（2）统计学分析显示，TIM-3分子在NSCLC患者肿瘤组织浸润CD4+T淋巴细胞的表达与淋巴结转移及患者分期有关；（3）与健康对照组相比，非小细胞肺癌患者外周血MDSC高表达负性共刺激分子TIM-3；与癌旁组织相比，TIM-3在同一患者肿瘤组织的表达也明显上调。【结论】TIM-3在非小细胞肺癌患者肿瘤浸润CD4+和CD8+T淋巴细胞上有较高的表达，并且其在非小细胞肺癌肿瘤浸润CD4+T细胞的表达与肿瘤的淋巴结转移及患者分期有关。非小细胞肺癌患者外周血和肿瘤组织MDSC也表达负性共刺激分子TIM-3。所获结果为进一步探讨TIM-3分子在肿瘤微环境中的负性调控作用提供了新的思路和机制。更多还原

【Abstract】 Tumor is a kind of ecosystem consisting of tumor cells, infiltration of immune cellsand stromal cells and the a series of molecules in microenvironment. Infiltratingimmune cells in the tumor microenvironment is one of the important factors for thedevelopment of tumors. infiltration of immune cells including effector T cells,regulatory T cells, anergic T cells, exhausted T cells, macrophages, granulocytes andmyeloid-derived suppressor cells (MDSC) in the tumor microenvironment play adifferent role and affect the whole process of tumor progression.In-depth study of the tumor microenvironment and tumor immune escapemechanisms in recent years, a group of negative costimulatory molecules mediatingimmune regulation were found and were called immune checkpoints, such as negativeB7family molecules, CTLA-4, TIM-3and so on. Those molecules abnormallyexpressed on a range of tumor tissue and/or infiltrating immune cells involving intumor immune escape, and closely related with clinical pathology and prognosis,constitute an important component of the tumor microenvironment.TIM-3, a number of the TIM family(T cell immunoglobulin andmucin-domain-containing molecules), mainly expressed on the differentiated Th1cellsand cytotoxic CD8+T cells, monocytes, cells, dendritic cells, is also considered to be animportant marker of the CD8+T cell exhaustion/anergy, and its role in tumor immunityis getting more and more attention.In different stages of tumor development, infiltrating T lymphocyte subsets andinhibitory immune cells (such as MDSC) in tumor microenvironment by expressingimmune checkpoints mediate anti-tumor immune response and tumor immune escape.The deeply research of expression characteristics of negative costimulatory moleculeTIM-3on infiltrating T lymphocyte subsets and myeloid-derived suppressor cells, therelevance to clinical factor and their participation in tumor immune escape mechanismcan provide a new theoretical basis and target for tumor immunotherapy based onintervention TIM-3signaling pathways. Objective: To explore the expression and clinical significance of negativecostimulatory molecules TIM-3on non-small cell lung cancer T cells andmyeloid-derived suppressor cells.Methods: Obtain cancer tissues and adjacent cancer tissues surgical removed from51non-small cell lung cancer (NSCLC) patients. By digesting and mechanicalpulverizing obtain a single cell suspension. Using lymphocyte separation medium getmononuclear cells.By immunofluorescence and flow cytometry detect TIM-3’sexpression on tumor-infiltrating T lymphocytes and MDSC. At the same time collectedthe same patients and healthy volunteers’ peripheral blood.Byimmunofluorescence,hemolysis and flow cytometry detect TIM-3’s expression onperipheral blood T lymphocytes and MDSC. Analysis the relevance between TIM-3’sexpression on non-small cell lung cancer patients’ T-cell and clinicopathological factors.Results:(1) Compared to adjacent normal tissue-infiltrating lymphocytes andperipheral blood T cells, expression of TIM-3were significantly increased on tumortissue TILs surface in patients with non-small cell lung cancer;(2) Statistical analysisshowed that the expression of TIM-3molecules on tumor-infiltrating CD4+Tlymphocytesin patients with NSCLC is correlated with lymph node metastasis and stage;(3) Compared to healthy controls,MDSC in peripheral blood of patients with non-smallcell lung cancer highly expressed negative costimulatory molecule TIM-3; Comparedto adjacent tissues, expression of TIM-3was also significantly up-regulated in thetumor tissue of the same patient.Conclusion: TIM-3on tumor infiltrating CD4+and CD8+T lymphocytesin inpatients with non-small cell lung cancer have high expression and TIM-3on tumorinfiltrating CD4+T cells in non-small cell lung cancer is correlated with tumor lymphnode metastasis and patient’s pathological stage.MDSC in peripheral blood and tumortissue of patients with non-small cell lung cancer highly expressed negativecostimulatory molecule TIM-3.The results obtained provide new ideas and mechanismsto further explore the negative regulatory role of TIM-3molecules in the tumormicroenvironment.更多还原