封闭CD25和中和TGF-β下调胰腺癌微环境中Treg及增强新型MUC1 DNA疫苗治疗效果的研究

The prognosis is extremely poor, and is hopeful to be improved by tumor vaccine. Regulatory T cells (Treg) in tumor microenvironment might limit therapeutic efficacy of tumor vaccine through suppressing killing ability of effector T cells. In our previous research, it is proved that CD25 and TGF-β is respectively the key phenotypic marker and the critical cytokine in the differentiation and function of Treg. Therefore, it is suggested that CD25 and TGF-β would be potential combined intervention points to downregulate Tregs amount and function of suppressing CTL killing ability in pancreatic cancer microenvironment, and then to enhance therapeutic vaccine efficacy. At present, we have successfully established C57BL/6 murine model of pancreatic tumor expressing MUC1 and constructed new MUC1 DNA vaccine. In this study, anti-CD25 monoclonal antibody and anti-TGF-βmonoclonal antibody will be injected intraperitoneally to block CD25 and neutralize TGF-β respectively. Firstly, the pole on reduction and functionally depletion of Tregs in pancreatic cancer microenvironment will be explored. Secondly, we will investigate the effect of blocking CD25 and neutralizing TGF-β on murine pancreatic cancer treatment with new MUC1 vaccine. Finally, we analyze the influencing factor of CTL killing ability through cell experiment in Vitro. By means of above studies, the influence and mechanism will be elucidated and then lay a foundation of further clinical use of tumor vaccine, where blocking CD25 and neutralizing TGF-β could result in downregulating regulatory T cells in pancreatic cancer microenvironment and enhancing therapeutic efficacy of new MUC1 DNA vaccine.

胰腺癌预后极差，肿瘤疫苗有望改善预后。肿瘤微环境中调节性T细胞（Treg）抑制效应T细胞的杀伤功能，从而限制疫苗的治疗效果。我们前期研究证实，CD25和TGF-β分别是胰腺癌微环境中Treg分化、功能维持的关键表型和关键细胞因子。提示以此为联合干预靶点，有望从数量上和功能上阻断胰腺癌微环境中Treg抑制CTL杀伤功能的途径，从而增强疫苗的治疗效果。目前我们已成功构建了表达MUC1的小鼠胰腺癌模型和新型MUC1 DNA疫苗，本项目拟完成：①研究腹腔注射抗CD25单抗和抗TGF-β单抗对胰腺癌微环境中Treg的下调作用；②探讨封闭CD25和中和TGF-β对新型疫苗治疗胰腺癌效果的影响；③通过体外杀伤实验，分析CTL杀伤功能的影响因素。通过上述研究，力图阐明封闭CD25和中和TGF-β下调胰腺癌微环境中Treg对新型MUC1 DNA疫苗治疗效果的影响和作用机制，最终为肿瘤疫苗的临床应用奠定基础。

胰腺癌早期诊断困难、预后极差，免疫治疗有望改善其预后。本项目分别通过阻断TGF-beta及封闭CD25来研究对胰腺癌微环境内调节性T细胞（Tregs）数量及功能的影响，从而拟增强免疫治疗效果。通过构建胰腺癌小鼠模型，腹腔注射CD25抗体后，发现外周及微环境内Foxp3+Treg细胞显著降低（p<0.05），肿瘤组织内Treg功能性细胞因子IL-10呈显著下降趋势（p<0.05），但大剂量CD25抗体的应用，反而引起肿瘤组织内IFN-γ下调，提示肿瘤免疫功能同时也受到了一定地抑制；腹腔注射TGF-beta抗体后，发现微环境及脾脏内Foxp3+Treg细胞下调（p<0.05），肿瘤组织内TGF-beta水平显著降低（p<0.05）；而联合腹腔注射两种抗体后，发现除了外周及微环境内Foxp3+Treg细胞显著降低（p<0.05）外，TGF-beta、IL-10水平均下降，而IFN-γ水平增高（p<0.05）。综合以上数据可以推断出，以CD25和TGF-β 作为干预靶点，能够互补各自的不足，抑制肿瘤组织内Treg功能及数量效果更加显著，对调控免疫抑制有着重要作用，同时减少了CD25抗体的应用量，减少了对细胞毒性T细胞功能的抑制，充分提高了免疫效能，同时辅以现有免疫治疗，能够达到有效对抗胰腺癌的目标。通过上述研究，阐明联合封闭CD25和中和TGF-beta下调胰腺癌微环境中Treg的影响和作用机制，最终为新免疫治疗的临床应用奠定基础。

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