School of Basic Medical Science, Guangxi Medical University, Nanning, 530021, China.
Laboratory of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Guilin Medical University, Guilin, 541001, China.
Curr Med Sci. 2022 Jun;42(3):491-497. doi: 10.1007/s11596-022-2556-x. Epub 2022 Mar 15.
Cell division cyclin 25 homolog C (Cdc25C) is a tumor-associated antigen candidate gene, and this may be used as an effective target in cancer treatment. The present study aims to evaluate the lysis effect of cytotoxic T lymphocytes (CTLs) induced by dendritic cell line DC2.4 overexpressing Cdc25C, and the feasibility of Cdc25C as a component in hepatoma immunotherapy.
The mouse Cdc25C gene was ligated into a lentiviral vector, and transfected into DC2.4 cells. The DC2.4 cell phenotype and cytokine secretion were determined by flow cytometry and ELISA, respectively. CD8 T cells were sorted from the spleens of C57BL/6 mice using a magnetic bead sorting kit obtained from Miltenyi Biotech, Germany, and co-cultured with DC2.4 cells for one week as effector cells. Then, IL-2, granzyme B and perforin were detected in the CTL culture medium by ELISA. Next, time-resolved fluorescence immunoassay was used to detect the immune killing effect of Cdc25C-specific CTLs on target cells. Meanwhile, the effect of blocking MHC-I sites on target cells with a monoclonal anti-MHC-I antibody was evaluated.
The results revealed that Cdc25C could be stably overexpressed in DC2.4 cells by LV-Cdc25C infection. DC2.4 cells transfected with LV-Cdc25C secreted more IL-6, IL-12, TNF-α and IFN-γ, and had higher expression levels of CD40, CD86, CCR7 and MHC-II than unaltered DC2.4 cells. The elevated Cdc25C in dendritic cells also further increased the secretion of IL-2, granzyme B and perforin to elicit Cdc25C-specific CTLs, and induced the higher cytotoxicity in Hepa1-6 cell lines (P<0.05), but this had no effect on the target cells when MHC-I monoclonal antibodies were blocked.
DC2.4 cells transfected with LV-Cdc25C can induce specific CTLs, and result in a strong cellular immune response. The dendritic cells that overexpress Cdc25C may be useful for hepatoma immunotherapy.
细胞分裂周期蛋白 25 同源物 C（Cdc25C）是一种与肿瘤相关的候选抗原基因，可作为癌症治疗的有效靶点。本研究旨在评估过表达 Cdc25C 的树突状细胞系 DC2.4 诱导的细胞毒性 T 淋巴细胞（CTL）的裂解效应，以及 Cdc25C 作为肝癌免疫治疗组成部分的可行性。
将小鼠 Cdc25C 基因连接到慢病毒载体中，并转染到 DC2.4 细胞中。通过流式细胞术和 ELISA 分别测定 DC2.4 细胞表型和细胞因子分泌情况。使用德国 Miltenyi Biotech 公司的磁珠分选试剂盒从 C57BL/6 小鼠脾脏中分选 CD8 T 细胞，并与 DC2.4 细胞共培养一周作为效应细胞。然后通过 ELISA 检测 CTL 培养上清液中 IL-2、颗粒酶 B 和穿孔素的水平。接下来，使用时间分辨荧光免疫分析法检测 Cdc25C 特异性 CTL 对靶细胞的免疫杀伤作用。同时，评估用单克隆抗 MHC-I 抗体阻断靶细胞 MHC-I 位点的效果。
结果表明，LV-Cdc25C 感染可稳定过表达 Cdc25C 于 DC2.4 细胞中。转染 LV-Cdc25C 的 DC2.4 细胞分泌更多的 IL-6、IL-12、TNF-α 和 IFN-γ，并且 CD40、CD86、CCR7 和 MHC-II 的表达水平高于未改变的 DC2.4 细胞。树突状细胞中升高的 Cdc25C 进一步增加了 IL-2、颗粒酶 B 和穿孔素的分泌，从而引发 Cdc25C 特异性 CTL，并诱导 Hepa1-6 细胞系更高的细胞毒性（P<0.05），但当 MHC-I 单克隆抗体被阻断时，这对靶细胞没有影响。
转染 LV-Cdc25C 的 DC2.4 细胞可诱导特异性 CTL，并引起强烈的细胞免疫反应。过表达 Cdc25C 的树突状细胞可能对肝癌免疫治疗有用。
