1、T细胞中过表达Akt抵抗肿瘤免疫抑制和增强抗肿瘤活性 2、小鼠CT26肿瘤模型中多周期氟尿嘧啶化疗损伤细胞毒性T细胞

发布时间：2018-05-15 22:24

  本文选题：肿瘤 + 免疫抑制　； 参考：《北京协和医学院》2016年博士论文

【摘要】：背景肿瘤在其形成中产生了独特的微环境,具有异质性,结构复杂,能分泌刺激性生长因子和细胞因子,募集不同类型的细胞,通过多种机制抑制免疫应答,逃避免疫监视和免疫系统的攻击。复杂的炎症和免疫抑制环境会大大影响肿瘤反应性T细胞活性, 回输的T细胞也会被肿瘤微环境所抑制,降低治疗的有效性。针对某一种抑制策略的疗法通常不能广泛和长期有效,我们通过增强回输的T细胞活性,使之能够抵抗肿瘤中已知和未知的免疫抑制因子,可能会是一种有效的方法。对T细胞的信号通路进行分析,发现丝氨酸/苏氨酸蛋白酶Akt(蛋白激酶B, PKB)在T细胞发挥效应功能中处于信号中枢节点,通过调节Akt来提高T细胞抗肿瘤活性有显著的研究意义。方法我们在表达卵清蛋白(OVA)抗原的黑色素瘤(B16-OVA)模型中,检测肿瘤微环境里T细胞的Akt磷酸化水平,然后通过逆转录病毒转导Akt至特异性靶向OVA的OT-1小鼠的T细胞中,检测转导Akt后OT-1细胞的体内外抗肿瘤活性。在人前列腺癌PC3M模型中,我们构建了联合表达靶向上皮细胞粘附分子(EpCAM)的嵌合抗原受体(CAR)和Akt的逆转录病毒载体,转导至人外周血细胞,检测过表达Akt的肿瘤特异性T细胞的体内外抗肿瘤活性。结果肿瘤微环境中T细胞的Akt磷酸化水平是受抑制的,OT-1过表达Akt后在B16-OVA细胞的刺激下分泌的IFN-γ增多且增殖能力增强。回输过表达Akt的OT-1细胞治疗B16-OVA肿瘤,其抑制肿瘤的生长能力更强且延长了小鼠的生存期。PC3M细胞也表现多种免疫抑制表型,相比靶向P3CM表面抗原EpCAM的CAR-T细胞,过表达Akt的CAR-T细胞在PC3M微环境中增殖受到的抑制最少,凋亡水平降低,体外杀伤能力提高；回输过表达Akt的CAR-T细胞治疗免疫缺陷的NOD/SCID荷瘤小鼠时,对PC3M肿瘤生长的抑制能力更强。结论肿瘤特异性T细胞中过表达Akt能帮助其抵抗肿瘤微环境对T细胞活性的抑制,增强肿瘤微环境中T细胞的增殖和杀伤活性,提高回输T细胞的体内抗肿瘤效果。我们的研究为改善过继T细胞回输治疗的疗效提供了一种研究策略,是未来临床转化的一种思路。背景肿瘤细胞异于正常细胞的快速增殖特性使其可以被某些化疗药物相对特异性杀伤；化疗常分多个周期进行以降低毒性,但在化疗的休息阶段,机体正常组织恢复的同时,某些肿瘤组织也在恢复,伴随耐药、复发和转移的隐患。有些化疗药物有骨髓抑制的副作用,对分裂增殖的骨髓和免疫细胞同样有毒性。有临床研究发现对晚期肿瘤病人进行化疗不能提高生活质量和延长生存期。另一方面近期研究揭示有些化疗药物可以诱导肿瘤细胞免疫原性死亡,减少免疫抑制性细胞,激发抗肿瘤免疫。我们对不同化疗周期后荷瘤小鼠的免疫状态进行研究,探究化疗过程中抗肿瘤免疫功能的变化,以期更为精准的联合免疫治疗,增强疗效。方法我们建立了多周期5-FU治疗小鼠CT26肿瘤的模型,观察不同周期化疗对肿瘤生长的抑制和小鼠生存期的影响。我们检测了荷瘤小鼠一到三周期化疗后,体外抗肿瘤活性,重点比较一周期和三周期化疗后,脾脏中各免疫细胞亚群的细胞数和肿瘤中免疫细胞的比例,及外周血中细胞因子的变化；在肿瘤抗原刺激下各亚群细胞的增殖和分泌的细胞因子变化,及脾细胞对CT26的杀伤；并探究早期化疗联合细胞因子诱导的杀伤细胞(CIK)和PD-L1抗体治疗的抗肿瘤效果。结果5-FU多周期化疗相比一周期抑瘤生长效果更明显,但各治疗组在总生存率上没有显著差异。一周期和三周期化疗后,相比同期荷瘤对照组,脾脏中CD8+T细胞和NK细胞绝对数没有显著变化；但三周期化疗后,混合淋巴细胞肿瘤细胞培养(MLTC)中增殖的CD8+T细胞减少,分泌的IFN-γ也减少,脾细胞对CT26细胞的杀伤水平降低。而一周期化疗后,肿瘤组织中浸润的CD8+T细胞比例升高；脾细胞体外杀伤CT26增强,MLTC中增殖的CD8+T细胞数增多,分泌的IFN-γ也增多。化疗后免疫抑制因子如TGF-β、IL-10和肿瘤组织表达的PD-L1上升；一周期化疗联合CIK细胞和PD-L1抗体治疗,相比四周期的化疗和单纯免疫治疗,肿瘤抑制效果和总生存率都提高。结论一周期5-FU化疗促进了CT26肿瘤特异性免疫应答,而多周期化疗损伤了性抗肿瘤的细胞免疫功能。化疗停止后虽然免疫细胞亚群数量可以恢复,但有效的抗肿瘤免疫应答受损,化疗早期即联合免疫治疗可以提高抗肿瘤疗效。伴随肿瘤免疫治疗的发展,我们的研究以期为更合理有效的进行肿瘤化疗和联合免疫治疗提供思路。
[Abstract]:Background tumor produces a unique microenvironment in its formation, which is heterogeneous and complex. It can secrete stimulating growth factors and cytokines, raise different types of cells, inhibit immune response through a variety of mechanisms and escape the attack of immune surveillance and immune system. Complex inflammation and immunosuppression environment will greatly affect tumor reaction. In response to the activity of the T cells, the retransfused T cells can also be suppressed by the tumor microenvironment, reducing the effectiveness of the treatment. A therapy against a certain inhibitory strategy is usually not widely and long-term. We can be effective by enhancing the retransmission of T cell activity to enable it to resist known and unknown immunosuppressive factors in the tumor. Methods. The signal pathway of T cells was analyzed. It was found that serine / threonine protease Akt (protein kinase B, PKB) was in the signal center node of T cells, and it was significant to improve the anti-tumor activity of T cells by regulating Akt. Methods we were expressing the ovalbumin (OVA) antigen of melanoma (B16-OVA). In the model, the Akt phosphorylation level of T cells in the tumor microenvironment was detected and then the antitumor activity of the OT-1 cells after the transduction of Akt was detected by retroviral transduction Akt to the T cells of the specifically targeted OVA OT-1 mice. In the PC3M model of human prostate cancer, we constructed a joint expression target epithelial cell adhesion molecule (EpCAM). The retroviral vector of the chimeric antigen receptor (CAR) and Akt, transduced to the human peripheral blood cells, detected the antitumor activity of the tumor specific T cells expressing Akt. Results the level of Akt phosphorylation of T cells in the tumor microenvironment was inhibited, and the IFN- gamma secreted by B16-OVA cells increased and increased after the OT-1 overexpressed Akt. Increased colonization ability. OT-1 cells transfused with Akt expressed B16-OVA tumor, which inhibited tumor growth and extended the survival time of.PC3M cells to a variety of immunosuppressive phenotypes. Compared to CAR-T cells targeting the P3CM surface antigen EpCAM, the CAR-T cells that overexpressed Akt were inhibited at least in PC3M microenvironment. The level of apoptosis and the ability to kill in vitro increase in vitro, and when the CAR-T cells transfused with Akt expressed in the NOD/SCID bearing mice with immune deficiency, the inhibition of PC3M tumor growth is stronger. Conclusion the overexpression of Akt in the tumor specific T cells can help it resist the inhibition of the activity of T cells in the tumor microenvironment and enhance the T cells in the tumor microenvironment. Our study provides a research strategy to improve the efficacy of adoptive T cell retransmission therapy, and it is a way of thinking in the future clinical transformation. Background tumor cells are different from normal cells in the rapid proliferation of T cells, which can be relatively specific to some chemotherapeutic drugs. Kill; chemotherapy is often divided into several cycles to reduce toxicity, but in the rest stage of the chemotherapy, while the body's normal tissue is restored, some tumor tissues are also recovering, accompanied by resistance, recurrence and metastasis. Some chemotherapeutic drugs have side effects of bone marrow suppression and are also toxic to the split and proliferating bone marrow and immune cells. It is found that chemotherapy on advanced cancer patients can not improve the quality of life and prolong life. On the other hand, recent studies have revealed that some chemotherapeutic drugs can induce immunogenic death of tumor cells, reduce immunosuppressive cells and stimulate anti-tumor immunity. We study the immune status of tumor bearing mice after different chemotherapy weeks. We studied the changes in anti tumor immune function during the chemotherapy, in order to make a more accurate combined immunotherapy and enhance the effect. Methods we established a multi cycle 5-FU model of CT26 tumor in mice, and observed the inhibition of tumor growth and the effect of mice survival on different cycles of chemotherapy. External antitumor activity, the number of cells in the spleen of the spleen and the proportion of immune cells in the tumor, and the changes in the cytokines in the peripheral blood after the one cycle and three cycles of chemotherapy, and the changes in the proliferation and secretion of the subgroups of the subgroups under the stimulation of the tumor antigen, and the killing of the spleen cells to CT26. The effect of chemotherapy combined with cytokine induced cytokine induced killer cells (CIK) and PD-L1 antibody was found. Results the effect of 5-FU multi cycle chemotherapy was more obvious than one cycle of tumor growth, but there was no significant difference in the total survival rate of each treatment group. One cycle and three cycles of chemotherapy were compared with the same period of the tumor bearing control group, the CD8+T cells in the spleen and the NK fine. After three cycles of chemotherapy, the proliferation of CD8+T cells in the mixed lymphocyte tumor cell culture (MLTC) decreased, the secretion of IFN- gamma decreased and the killing level of the spleen cells decreased to CT26 cells. After a period of chemotherapy, the proportion of CD8+T cells infiltrated in the tumor tissues increased and the spleen cells killed CT26 in vitro. The number of CD8+T cells proliferated in MLTC increased and the secretion of IFN- gamma increased. After chemotherapy, the immunosuppressive factors such as TGF- beta, IL-10 and the expression of PD-L1 in tumor tissues increased; one cycle chemotherapy combined with CIK cell and PD-L1 antibody, compared with four cycles of chemotherapy and simple immunotherapy, the effect of swelling and total survival increased. Conclusion a cycle of 5 -FU chemotherapy promotes the specific immune response of CT26 tumor, while multi cycle chemotherapy injure the cellular immune function of sexual antitumor. Although the number of immune cell subgroups can be recovered after chemotherapy, the effective anti tumor immune response is impaired, and early chemotherapy combined with immunotherapy can improve the antitumor effect. Development, our research will provide ideas for more rational and effective chemotherapy and combined immunotherapy.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.3
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本文编号：1894211