泛素连接酶Cbl-b基因沉默联合PD-1通路阻断对小鼠恶性黑素瘤TRP-2肽疫苗治疗效应的影响及机制研究

Malignant melanoma, the most aggressive and deadly form of skin cancer, remains a management challenge. Immunotherapy seems to be a promising and relatively safer strategy for the treatment of cancer, and therapeutic vaccination is the most important form of active specific immunotherapy. To date, the clinical trials of melanoma vaccines have shown limited degrees of success. It is believed that the failure to mount an efficient immune response is due to a hostile tumor microenvironment dominated by immunosuppressive cells and cytokines. The expression of PD-L1 on melanoma cells suggests that the inhibitory PD-1/PD-L1 pathway is hijacked by melanoma cells to evade the host immune system. Our previous study showed that blocking PD-1 pathway by PD-1Ig, to some extent, can potentiate the therapeutic effects of TRP-2 peptide vaccination against the B16F10 melanoma in mice. Therefore, additional immune intervention may be required to stimulate the immune system's ability to attack melanoma cells. The E3 Ubiquitin Ligase Cbl-b functions as a negative regulator of antigen-specific T cell activation and is a critical mediator of T cell anergy. Recent study showed that therapeutic transfer of naive cbl-b?/? CD8+ T cells is sufficient to mediate rejection of established tumors. The objectives of the present study are to investigate the possibility of potentiating the therapeutic immunity of TPR-2 vaccination against melanoma by cbl-b siRNA plus soluble PD-1Ig, and elucidate its immunopharmacological mechanisms by enumurating and phenotyping TRP-2 specific CD8+ T cells. First, the dynamic of the expression of Cbl-b gene in tumor infiltrating lymphocytes will be analyzed. Then, the effects of Cbl-b siRNA and recombinant PD-1Ig, alone or in combination, will be investigated. Finally, TRP-2 specific CD8+ T cells, myeloid-derived suppressor cells, and Treg cells in tumor tissues will be enumurated, and both phenotype and function of the former cells will be analyzed to explore the mechanisms. The results may provide the basis for the development of new immunostimulants.

恶性黑素瘤是恶性程度最高的皮肤肿瘤，对放化疗不敏感，迫切需要发展新型治疗策略。免疫治疗可诱生高频率肿瘤特异性CTL，其疗效不理想与肿瘤微环境密切相关。研究提示泛素连接酶Cbl-b通过调节TGF-β敏感性、诱生Treg细胞等机制，在外周T细胞免疫无能方面起关键作用。PD-1通路则通过下调Th1和CTL应答参与T细胞功能耗竭和免疫逃逸。二者联合阻断能否增强小鼠黑素瘤肽疫苗的治疗效应尚未确定。本研究拟以小鼠黑素瘤为模型，首先分析TIL中Cbl-b的表达动力学；继而制备Cbl-b siRNA和PD-1Ig融合蛋白，联合应用以沉默Cbl-b基因并阻断PD-1通路，探讨其对TRP-2肽疫苗治疗效应的影响；最后检测TIL中TRP-2抗原特异性CTL、MDSC和Treg细胞，通过分析这些细胞的数量、表型及功能变化探讨Cbl-b siRNA和PD-1Ig的作用机制，为将其发展成为新型免疫增强剂提供实验依据。

皮肤恶性黑素瘤（Cutaneous Malignant Melanoma, CMM）是一种具有高度侵袭和转移性皮肤肿瘤，起源于皮肤黑素细胞异常增殖。该肿瘤对放射治疗和化学治疗均不敏感，缺乏有效的术后治疗，是皮肤肿瘤的主要死亡原因。同时近年来CMM发病率在世界范围内逐年上升。因此进一步研究黑素瘤发生发展机制仍为迫切。Cbl-b是Cbl家族成员之一。现有研究主要集中在Cbl-b在机体免疫中的作用，研究表明肿瘤微环境中浸润的淋巴细胞出现Cbl-b过表达，从而引起肿瘤免疫耐受。Cbl-b缺失则可打破免疫耐受，活化T细胞反应，从而可增强机体的抗肿瘤免疫反应。沉默Cbl-b可否增强机体抗黑素瘤的免疫能力尚未明确。另外在在前期研究结果中本课题组还发现相对于皮肤痣，Cbl-b在皮肤黑素瘤组织中 明显的高表达，且Cbl-b表达水平与影响预后因素（Clark分级、Breslow厚度）正相关，并随着肿瘤的侵袭和转移，其表达水平呈升高趋势，提示其在CMM中可能还发挥着癌基因的作用。在此基础上，本课题进行了以下研究：(1)cbl-b沉默可否增强小鼠对黑素瘤的免疫反应及机制如何。（2）从样本，细胞，动物以及分子水平阐明cbl-b人皮肤黑素瘤发生发展的关系以及探讨可能得作用机制。为黑素瘤的靶向治疗及诊断提供实验依据。