Ai Li-Mei, Ren Han-Yun, Shi Yong-Jin
Department of Hematology, Peking University First Hospital, Beijing 100034, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2007 Dec;15(6):1247-52.
In order to investigate the cellular immunoresponses mediated by chimeric anti-CD20 monoclonal antibody (anti-CD20 McAb) through dendritic cells (DCs), mononuclear cells were isolated from human peripheral blood (PBMNC) and DCs from PBMNCs in vitro were generated with normal methods. Human CD20 positive lymphoma cell line-Raji cells were treated with different methods including treatment with anti-CD20 McAb (group R), treatment with heat-induced apoptosis (group A) and treatment with anti-CD20 McAb+heat-induced apoptosis (group R+A), then Raji cells treated with above-mentioned methods as tumor antigen were loaded on DCs. The phagocytosis of DCs to tumor antigen was observed by transmission electron microscope (TEM), the auto-mixed lymphocyte reaction was performed with antigen-primed DCs, the release of INF-gammafrom effector cells was detected by ELISPOT, the killing of effector cells on Raji cells was assayed by MTT. The results showed that under TEM, no pronounced phagocytosis of DCs was seen in group R, while the phagocytosis of apoptotic bodies could be easily seen in group A and the more cell fraqments were observed in group R+A. The FCM indicated that the expressions of CD80, CD86 and HLA-DR on DCs in 3 experimental groups were higher than those in group control (p<0.05), while expression positive rate in group R+A was higher than those in group R and A (p<0.05). The detection of lymphocyte surface antigen revealed that proportions of CD8+ cells in all experimental groups were higher than those in group control (p<0.05), count of CD56+ cells in group R and R+A increased, as compared with group A and control, difference was significant (p<0.05). ELISPOT assay indicated that amount of cells releasing IFN-gamma in all experimental groups was higher than that in group control, and also number of spots in group R+A significantly higher than that in other groups at effector-targetor ratio=1:10 (p<0.05). The results of killing assay demonstrated that killing rate on Raji cells in all experimental groups increased as compared with group control (p<0.05), while killing rate in group R+A was higher than that in group R and A. It is concluded that anti-CD20 McAb can mediate DC to induce cellular immunoresponse against lymphoma, that is, to stimulate and amplify specific CTLs and NK cells. Anti-CD20 McAb combined with DCs primed by heat-stressed tumor cells as antigen can further enhance cellular immunoresponse against lymphoma.
为了研究嵌合抗CD20单克隆抗体（抗CD20 McAb）通过树突状细胞（DCs）介导的细胞免疫反应，从人外周血中分离单个核细胞（PBMNC），并采用常规方法在体外从PBMNC中生成DCs。人CD20阳性淋巴瘤细胞系-Raji细胞用不同方法处理，包括抗CD20 McAb处理（R组）、热诱导凋亡处理（A组）和抗CD20 McAb+热诱导凋亡处理（R+A组），然后将经上述方法处理的Raji细胞作为肿瘤抗原负载于DCs上。通过透射电子显微镜（TEM）观察DCs对肿瘤抗原的吞噬作用，用抗原致敏的DCs进行自体混合淋巴细胞反应，通过ELISPOT检测效应细胞中INF-γ的释放，用MTT法检测效应细胞对Raji细胞的杀伤作用。结果显示，在TEM下，R组未见DCs有明显的吞噬作用，而A组可见凋亡小体的吞噬作用，R+A组观察到更多的细胞碎片。流式细胞术（FCM）表明，3个实验组DCs上CD80、CD86和HLA-DR的表达均高于对照组（p<0.05），而R+A组的表达阳性率高于R组和A组（p<0.05）。淋巴细胞表面抗原检测显示，所有实验组CD8+细胞比例均高于对照组（p<0.05），R组和R+A组CD56+细胞计数与A组和对照组相比增加，差异有统计学意义（p<0.05）。ELISPOT检测表明，所有实验组释放IFN-γ的细胞数量均高于对照组，在效应细胞与靶细胞比例为1:10时，R+A组的斑点数也显著高于其他组（p<0.05）。杀伤实验结果表明，所有实验组对Raji细胞的杀伤率均高于对照组（p<0.05），而R+A组的杀伤率高于R组和A组。结论是，抗CD20 McAb可介导DC诱导针对淋巴瘤的细胞免疫反应，即刺激和扩增特异性CTL和NK细胞。抗CD20 McAb与热应激肿瘤细胞致敏的DCs作为抗原联合应用可进一步增强针对淋巴瘤的细胞免疫反应。
