HCA587蛋白疫苗诱导的CD4+T细胞杀伤肝癌细胞的效应及其机制研究

Immunotherapy has been assessed an attractive alternative therapeutic treatment option for hepatocellular carcinoma, based on the sensitivity, specificity and fewer side-effects. Our published data showed that HCA587 protein vaccine was able to induce potent and significant antitumor effect. After removal of CD4+ T cells, but not CD8+ T cells, loss of antitumor activity was absolutely complete. Moreover, depletion of IFN-γ led to decrease of secretion of granzyme B from CD4+ T cells and no antitumor effect.So our hypothesis is the CD4+ T cells induced by HCA587 protein vaccine can secrete granzyme B to kill the hepatoma cells. On the basis of constructed hepatoma model in mice, we will confirm the killing activity of the CD4+ T cells by cytotoxic assay in vivo and in vitro. We plan to explore the importance of granzyme B mediating cytotoxic activity of CD4+ T cells by reverse transcription PCR, Western, flow cytometry, confocal microscopy and antibody blocking assay. Besides, enzymatic activity of granzyme B and the following apoptosis of hepatoma cells will be detected to clarify the effect of granzyme B from CD4+ T cells. The aim of the project is to confirm the killing activity and explore its mechanism of CD4+ T cells induced by HCA587 protein vaccine. In addition, this research could provide evidences for the future clinical trial of the vaccine and hold promise for cytotherapy of hepatocellular carcinoma.

免疫疗法以高度敏感性、特异靶向性及极少副作用等优势，正成为肝癌治疗的一个新的模式和热点。我们前期结果显示小鼠体内CD4+T细胞（而非CD8+T细胞）清除后，肝癌相关抗原HCA587蛋白疫苗诱导的抗肿瘤效应完全消失；IFN-γ缺失致CD4+T细胞分泌颗粒酶B的能力显著降低，且抗肿瘤效应也丧失。据此，我们推测该疫苗诱导的CD4+T细胞主要通过分泌颗粒酶B来杀伤肝癌。在已构建好肝癌模型的基础上，本课题拟通过体内和体外杀伤实验验证CD4+T细胞具有特异的杀伤功能；利用RT-PCR、Western Blot、流式细胞术、共聚焦显微镜和抗体阻断等方法，证明CD4+T细胞通过分泌颗粒酶B执行杀伤；通过酶活性和细胞凋亡的检测，证明分泌的颗粒酶B具有酶活性并致肝癌细胞凋亡。本课题旨在研究HCA587蛋白疫苗诱导的CD4+T细胞杀伤效应及机制，为该疫苗进入临床试验提供实验基础，并为免疫细胞治疗肝癌提供新思路。

癌-睾丸抗原（CT）是一种在多种肿瘤组织表达、而正常组织不表达（除免疫豁免的睾丸）的肿瘤抗原。该类抗原具良好的组织表达限制性，故被认为是肿瘤免疫治疗的理想靶点。HCA587是课题组前期从肝癌组织中克隆出来的一种新型CT抗原。.本课题探讨了HCA587蛋白疫苗对荷瘤小鼠的抗肿瘤效应，并揭示了抗肿瘤效应的免疫细胞亚群是CD4+T细胞。于C57BL/6小鼠左侧胁腹部皮下接种肿瘤细胞建立肿瘤模型，予HCA587 蛋白疫苗等免疫策略治疗2次，观察抗肿瘤效果。用苏木精-伊红（HE）染色法分析疫苗治疗后肿瘤组织中淋巴细胞的浸润。用SDS-PAGE电泳分析自行制备的抗CD4和CD8分子单抗的相对分子质量和纯度，并用流式细胞术对其体内阻断效果进行鉴定。将上述单抗腹腔内注射入小鼠体内，以实现免疫细胞在体清除。用野生型（WT）和IFN-γ敲除（IFN-γ KO）C57BL/6小鼠对比，研究HCA587蛋白疫苗抗肿瘤作用的改变及对疫苗诱导的总的肿瘤浸润淋巴细胞（TIL）和其亚群CD4+T细胞分泌颗粒酶（granzyme B）的影响。结果发现，与单一蛋白治疗组、佐剂对照组相比，HCA587蛋白疫苗具有显著的抗肿瘤作用（P<0.05）。在HE染色中，HCA587 蛋白疫苗治疗后肿瘤局部可见大量的淋巴细胞浸润。经流式细胞术鉴定，自行制备、纯化的抗CD4和CD8分子单抗在体能完全清除相应的CD4+T和CD8+T细胞亚群。当在体清除CD4+T细胞后，疫苗的抗肿瘤效应完全消失，而CD8+T细胞的清除不影响其抗肿瘤效应。在野生型C57BL/6小鼠中，相比未治疗组，疫苗治疗组不仅总TIL分泌颗粒酶B和肿瘤组织局部颗粒酶B的含量显著升高，而且TIL中CD4+T细胞分泌颗粒酶B的水平也大幅度提高。与野生型小鼠相比，在IFN-γ KO小鼠中，疫苗治疗组CD4+T细胞分泌颗粒酶B的水平显著下降。可见HCA587蛋白疫苗具有显著的抗肿瘤效应，其诱导的CD4+T细胞在抗肿瘤效应中起着至关重要的作用，且效应分子可能为颗粒酶B。本课题为该疫苗进入临床试验提供了实验基础，并为免疫细胞治疗肿瘤提供了新思路。