四种不同尤文肉瘤树突状细胞免疫疫苗的体内外抗肿瘤免疫应答研究

Comparison and analysis of different dendritic cell-based immunotherapeutic strategies for Ewing sarcoma: in vitro and in vivo induction on SCID mouse models

目的:制备4种尤文肉瘤树突状细胞(dendritic cell,DC)疫苗,并比较和分析其在体内外抗肿瘤免疫反应中的效果。方法:从健康供者体中分离和培养外周血单核细胞,利用粒-巨噬细胞集落刺激因子(granulocyte-macro-phage colony-stimulating factor,GM-CSF)和白细胞介素(interleukin-4,IL-4)将单核细胞诱生为DC,并对其表型进行流式细胞仪分析,分别制备4种DC疫苗:(1)融合瘤,将DC与A673细胞进行电融合,并通过流式细胞仪检测荧光双染色融合瘤,以确定其融合率;(2)负载肿瘤裂解物DC,将DC与A673细胞反复冻融物共培养;(3)EWS/FLI1修饰DC,用编码EWS/FLI1的腺病毒转染DC;(4)未作处理的成熟的DC。将各种DC疫苗与组织相容性白细胞抗原(histocom patibility leukocyte antigen,HLA)相配的CD8+T细胞共培养,用ELISA试剂盒检测刺激增殖细胞毒性T淋巴细胞(cytotoxicity T lymphocyte,CTL)分泌干扰素γ(interferon γ,IFN-γ)的量,用51Cr细胞杀伤试剂盒检测CTL在不同比例下对A673细胞的杀伤作用。通过腹腔注射人外周血淋巴细胞(peripheral blood lymphocyte,PBL),皮下接种A673细胞,构建重建人免疫系统的SCID鼠尤文肉瘤模型,应用ELISA试剂盒检测小鼠外周血中IgG的量,以确定免疫重建效果,并接种不同的DC疫苗,检测其对小鼠成瘤的影响。结果:流式细胞术检测出从外周血单核细胞诱生出的CD83、CD80、CD86及HLA-DR高表达的成熟DC。DCs/A673融合细胞的电融合效率达到16.32%。各种DC疫苗都可以诱导出有效的抗肿瘤免疫反应,IFN-γ分泌检测显示,融合瘤组较其他DC疫苗组产生CTL水平高,其后依次为肿瘤裂解物负载组、EWS/FLI1修饰组和未作处理组。而在体外杀伤A673实验中,融合瘤组杀伤效率最高,肿瘤裂解物负载组次之,EWS/FLI1修饰组与未作处理组最低,且后两组间差异无统计学意义。在抑制肿瘤生长的体内实验中,融合瘤组小鼠肿瘤最小,EWS/FLI1修饰组、肿瘤裂解物负载组和未作处理组之间差别不大。结论:尤文肉瘤DC疫苗可以诱导有效的抗肿瘤免疫反应,将各种尤文肉瘤DC疫苗进行体内外综合比较时发现,融合瘤是最佳的尤文肉瘤免疫疫苗。

Objective：To compare the efficacy of different immunotherapeutic strategies of loading dendritic cells （DCs） with the antigen of the Ewing sarcoma in vitro and in vivo. Methods： DCs were either electrofused with the whole Ewing sarcoma cells A673, pulsed with lysates of the tumor cell or modified with the characteristic EWS/FLI1 gene. Then we assessed the capacity of the stimulated cytoxicity T lymphocyte （CTLs） by the parameter of the interferon-γ （IFN-γ） secreted and the cytotoxicity to the A673. In vivo experiment, the human IgG serum concentrations of the SCID mice were measured to determine the mouse human immune system reconstitution, and the growths of the inoculated tumor were measured to assess the anti-tumor effect. Results： The data revealed that various DC-based strategies could induce specific immune responses to the tumor, with the hybrids showing superiority to the other strategies while there were no significant differences between the sets of the gene modified DCs and non-manipulated DCs in the cytotoxicity assay to A673 cells. Moreover, there were no significant differences among the sets of the gene modified DCs, lysate pulsed DCs and non-manipulated in vivo anti-tumor effect about the tumor volume on the SCID mice. Conclusion： The Ewing sarcoma had good responses to the DC-based immunotherapy and based on this experiment, we could also conclude that the product of electrofusion may be an optimal strategy for immunotherapy of Ewing sarcoma.