Anti-CD40诱导的炎性E-cadherin阳性树突状细胞在lewis肺腺癌模型免疫微环境中的作用及机制

发布时间：2018-05-29 15:35

本文选题：E-cadherin + 树突状细胞　；参考：《华中科技大学》2015年博士论文

【摘要】：研究背景及目的：肺癌是我国最常见的恶性肿瘤之一,据肿瘤登记中心报道,2010年,中国新发肺癌病例60.59万,居恶性肿瘤首位,同期我国肺癌死亡人数为48.66万,占恶性肿瘤死因的24.87%。目前针对肺癌的治疗方式主要有手术,放疗,化疗及靶向治疗等治疗方式,但是目前还没有一种传统的治疗方式可以根治肺癌。随着治疗方式的不断发展和更新,免疫治疗及对肺癌免疫微环境的发病机制的研究已进入新的时代,这为肺癌的个体化治疗提供了新的治疗思路和彻底治愈肺癌带来了希望。由于肺癌这类实体肿瘤,与恶性黑色素瘤不同,不具备免疫原性,在肿瘤微环境中不易被T细胞发现及结合,同时自身能促进调节T细胞(Treg)分化及其相应细胞因子的分泌,从而抑制自身抗肿瘤T细胞反应。目前肺癌免疫治疗针对增强抗肿瘤T细胞反应的主要方法是用单克隆抗体(如anti-PD-1和anti-CTLA-4。)阻滞抑制T细胞激活通路的检查点。但最近共刺激单克隆抗体anti-CD40为肺癌免疫治疗提供了又一新的免疫治疗思路,而且目前已经在胰腺癌,弥漫大B细胞淋巴瘤一期临床研究中展现出良好的抗肿瘤效果。但是anti-CD40刺激机体后又具备细胞因子释放综合征,血栓等不良反应,从而限制了其广泛的应用。我们知道,Anti-CD40具备激活抗原提呈细胞(APC)诱导炎症反应及肿瘤微环境中具备良好抗肿瘤的双重作用。为了弥补anti-CD40的不足,研究anti-CD40介导的炎症反应中炎性树突状细胞抗肿瘤的机制研究将为anti-CD40单克隆抗体更好的应用于肺癌树突状细胞免疫治疗领域提供良好的理论依据。同时,以树突状细胞(DCs)为基础的肺癌免疫治疗手段已经成为新的研究热点。但不同DCs亚群生物学功能及其调控机制尚未明确,这将成为导致其临床应用疗效不佳的重要原因。而最新研究发现Anti-CD40诱导的炎症反应中,一类新型的炎症树突状细胞亚型E-cadherin阳性树突状细胞被Fiona Powrie教授发现。但是此类树突状细胞在肺癌免疫微环境中的作用及机制未知,本实验主要是探索该新型炎性E-cadherin阳性树突状细胞在lewis市癌模型抗肿瘤T细胞免疫中的作用及机制。材料与方法：1.利用FGK45细胞上清制备鼠anti-CD40抗体。然后利用rag1KO小鼠及anti-CD40抗体制备anti-CD40炎症动物模型。2.分别取Lewis肺癌模型,C57/BL6小鼠,Rag1KO小鼠及其anti-CD40炎症动物模型小鼠的肺组织,提取此4种免疫状态的肺组织及其单个细胞,分别利用共聚焦技术和流式检测技术检测炎性E-cadherin阳性树突状细胞(DC)的分布及表型。3.提取anti-CD40炎症动物模型脾脏来源的单个细胞,流式分选出E-cadherin+DC,并携带CEA421-435抗原肽与初始(naive) CD4+T细胞共培养,利用胞内,胞外细胞因子检测法分析anti-CD40诱导的炎性E-cadherin+DC在肿瘤微环境下激活初始CD4+T细胞向效应(effector) T细胞(Thl, Th2及Th17)及调节T细胞(Treg)分化情况。4.将E-cadherin+DC携带CEA526-533抗原肽与初始(naive) CD8+T细胞共培养,利用ELISA技术分析其刺激效应CD8+T细胞产生IFN-γ的水平。5.将anti-CD40诱导的E-cadherin+DCs负载CEA526-533抗原肽通过尾静脉注入lewis肺癌原位模型,检测其体内CEA tetramer+(四聚体)CD8+T细胞动态变化及CD103+CD8+T细胞变化。同时在第28天取肺癌及肺组织检测其微环境CD4+T细胞亚群变化。结果：1.成功制备高纯度anti-CD40抗体和anti-CD40炎症动物模型。2.E-cadherin+DC主要大量聚集在anti-CD40炎症动物模型的肺部,而在lewis市癌模型,正常C57/BL6小鼠及Rag1KO小鼠肺部未见明显聚集。肺部anti-CD40诱导的炎性E-cadherin+DC类似脾脏炎性E-cadherin+DC表型,都是E-cadherin+CD11chighCD11b+CD103-CD4-.3. E-cadherin+DC携带CEA抗原肽在体外促进了初始CD4+T细胞向Thl, Th17细胞分化,抑制了Th2和Treg细胞的分化。4.较E-cadherin-DCs,炎性E-cadherin+DC能促使初始(naive) CD8+T细胞产生更高浓度IFN-γ.5. E-cadherin+DC体内对CD4+T细胞亚群分化的作用类似体外,同时刺激了大量CEA特异性效应CD8+T细胞(第14天,E-ca+组VS E-cad-组2.01%VS1.31%,第21天,E-cad+组VS E-cad-组3.47%VS1.94%,第28天,E-cad+组VS E-cad-组4.85%VS2.57%)及CD103+CD8+T细胞(E-cad+组VS E-cad-组15.20%VS3.70%)的产生,发挥了良好的抗肿瘤效应。结论：anti-CD40诱导的E-cadherin+DCs能增强lewis肺癌模型抗肿瘤T细胞免疫反应并表现出良好的抗肿瘤能力。这为未来anti-CD40单克隆抗体应用于肺癌免疫治疗及肺癌树突状细胞疫苗治疗开辟了新的思路。
[Abstract]:Research background and objective: lung cancer is one of the most common malignant tumors in China. According to the cancer registration center, in 2010, 605 thousand and 900 new cases of lung cancer in China were the first malignant tumor, and the number of lung cancer deaths in China was 486 thousand and 600 in the same period, accounting for the cause of the death of malignant tumors, the main treatment methods for lung cancer were surgery, radiotherapy, chemotherapy and 24.87%.. However, there is no traditional therapy for the treatment of lung cancer. With the continuous development and renewal of the treatment, the immunotherapy and the pathogenesis of the immune microenvironment of lung cancer have entered a new era. This provides a new way of treatment for the individualized treatment of lung cancer and a thorough cure for the lung. Cancer has brought hope. Because of lung cancer, it is different from malignant melanoma and has no immunogenicity. It is not easy to be found and bound by T cells in the tumor microenvironment. At the same time, it can promote the regulation of the differentiation of T cells (Treg) and the secretion of corresponding cytokines, so as to inhibit the reaction of their own anti-tumor T cells. The main way to enhance the anti-tumor T cell response is to block the checkpoint of inhibition of T cell activation pathway with monoclonal antibodies (such as anti-PD-1 and anti-CTLA-4.).
But recently co stimulation of monoclonal antibody anti-CD40 provides a new way of immunotherapy for lung cancer immunotherapy, and it has shown good antitumor effects in a phase study of pancreatic cancer and diffuse large B cell lymphoma. But anti-CD40 stimulates the body with cytokine release syndrome, thrombus and other adverse reactions. It is known that Anti-CD40 has the dual role of activating the antigen presenting cell (APC) to induce the inflammatory reaction and the good antitumor effect in the tumor microenvironment. In order to make up for the deficiency of anti-CD40, the study of the mechanism of the anti-tumor mechanism of inflammatory dendritic cells in the anti-CD40 mediated inflammatory reaction will be anti-CD 40 monoclonal antibodies are better used in the field of immunotherapy for lung cancer dendritic cells. At the same time, the immunotherapy of lung cancer based on dendritic cells (DCs) has become a new research hotspot. However, the biological functions and regulatory mechanism of different DCs subgroups are not clear, which will lead to the clinical application and treatment. The new study found that in the Anti-CD40 induced inflammatory response, a new type of inflammatory dendritic cell subtype E-cadherin positive dendritic cells were found by Professor Fiona Powrie. But the role and mechanism of this kind of dendritic cells in the immune microenvironment of lung cancer is unknown. This experiment is mainly to explore the new type of inflammation. The role and mechanism of E-cadherin positive dendritic cells in the anti-tumor T cell immunity of Lewis cancer models.
Materials and methods: 1. using FGK45 cell supernatant to prepare mouse anti-CD40 antibody, and then use rag1KO mice and anti-CD40 antibody to prepare anti-CD40 inflammation animal model.2. to take Lewis lung cancer model, C57/BL6 mouse, Rag1KO mouse and anti-CD40 inflammation animal model mice lung tissue, extract the 4 kinds of immune state of lung tissue and its single The distribution of E-cadherin positive dendritic cells (DC) and phenotypic.3. were used to detect the distribution of DC and the phenotypic.3. to extract the single cells of the spleen from the animal model of the inflammatory animal. The flow formula was used to isolate the E-cadherin+DC, and the CEA421-435 antigen peptides were co cultured with the initial (naive) CD4+T cells, and the intracellular and cellular cells were used. The analysis of anti-CD40 induced inflammatory E-cadherin+DC induced the initial CD4+T cell effect (effector) T cells (Thl, Th2 and Th17) and the differentiation of T cells (Treg) in the tumor microenvironment. The stimulation effect CD8+T cells produce IFN- gamma level.5., and anti-CD40 induced E-cadherin+DCs loaded CEA526-533 antigen peptide is injected into the situ model of Lewis lung cancer through the tail vein to detect the dynamic changes of CEA tetramer+ (four polymer) CD8+T cells in the body and the change of CD103+CD8+T cells in the body. At the same time, the microenvironment CD is detected by lung cancer and lung tissue on the twenty-eighth day. Changes in 4+T cell subsets.
Results: 1. the successful preparation of high purity anti-CD40 antibody and anti-CD40 inflammation animal model.2.E-cadherin+DC mainly concentrated in the lungs of the animal model of anti-CD40 inflammation, while in the Lewis City cancer model, no obvious aggregation was found in the lungs of normal C57/BL6 mice and Rag1KO mice. The inflammatory E-cadherin+DC induced by the lung anti-CD40 was similar to the spleen inflammatory E-ca. The dherin+DC phenotype, which is the E-cadherin+CD11chighCD11b+CD103-CD4-.3. E-cadherin+DC carrying CEA antigen peptide in vitro, promotes the differentiation of initial CD4+T cells to Thl, Th17 cells, and inhibits the differentiation of Th2 and Treg cells,.4. is higher than E-cadherin-DCs. The effect of +DC on the differentiation of CD4+T cell subsets was similar in vitro, and a large number of CEA specific effect CD8+T cells were stimulated at the same time (Fourteenth days, 2.01%VS1.31% of group VS E-cad- in group E-ca+, twenty-first days, VS E-cad- group 3.47%VS1.94% of E-cad+ group, twenty-eighth days. A good antitumor effect.
Conclusion: anti-CD40 induced E-cadherin+DCs can enhance the anti tumor T cell immune response of Lewis lung cancer model and exhibit good anti-tumor ability, which opens up a new idea for the application of anti-CD40 monoclonal antibody in the treatment of lung cancer immunotherapy and the treatment of lung cancer dendritic cell vaccine.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

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