Dendritic cells engineered to secrete anti-DcR3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro.
Dendritic cells engineered to secrete anti-DcR3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro.
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DOI：
10.3748/wjg.v23.i5.817
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发表时间：
2017-02-07
影响因子：
4.3
通讯作者：
Xu WD
中科院分区：
文献类型：
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作者：
Chen J;Guo XZ;Li HY;Zhao JJ;Xu WD
目的研究分泌抗DcR3单抗的树突状细胞(DC)体外诱导的抗胰腺癌(PC)细胞毒性T淋巴细胞反应。采集PC患者外周血中DC、T淋巴细胞和原代PC细胞。用电穿孔技术将人源化抗DcR3单抗重链和轻链mRNA和/或肿瘤总RNA(DC-Tumor-anti-DcR3 RNAor DC-Tull Tumor RNA)导入DC。用Western blotting和酶联免疫吸附试验检测DC-Tumor-anti-DcR3RNA分泌的抗DcR3mAb的种类、浓度和功能。用四甲基偶氮唑盐比色法和流式细胞仪检测分泌抗DcR3单抗对体外培养的自体PC细胞的活性和细胞凋亡率的影响。用~(51)Cr释放试验分析了分泌DC的抗DcR3单抗对PC抗原特异性免疫应答的增强作用。检测干扰素-γ、IL-10、IL-4、肿瘤坏死因子-α和IL-12等细胞因子水平，分析树突状细胞诱导的T细胞应答。DC分泌的抗DcR3单抗与重组人DcR3蛋白反应，产生一条相对分子质量为35 kDa的条带。DC-Tumor-anti-DcR3RNA与DC-Tumor-anti-DcR3 RNA共孵育一定时间后，自体PC细胞培养上清液中DcR3水平明显下调(P<；0.05)。与DC-总肿瘤RNA相比，分泌抗DcR3单抗的DC可提高细胞存活率，延缓RNA负载DC的凋亡(P<；0.01)。DC-Tumor-anti-DcR3RNA分泌的抗DcR3mAb对转导RNA的DC、原代肿瘤细胞和PC细胞株的细胞毒性T淋巴细胞(CTL)的诱导活性明显高于DC-总肿瘤RNA或对照组(P<；0.05)。同时，抗原特异性CTL反应是MHC-I类限制性的。CD_4~+T细胞和CD_8~+T细胞与分泌DC的抗DcR_3单抗孵育后产生的干扰素-γ水平显著高于DC-肿瘤总RNA组和对照组(P<0.01)，而IL-4水平显著低于对照组(P<0.01)。DcR3基因工程树突状细胞在体外可增强抗PC的CTL反应，其免疫增强作用可能与其下调DC细胞凋亡和调节Th1/Th2细胞因子网络有关。
To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs’ viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a 51Cr releasing test. T cell responses induced by RNA-loaded DCs were analyzed by measuring cytokine levels, including IFN-γ, IL-10, IL4, TNF-α and IL-12. The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DC-tumor-anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-γ and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.