去甲基化诱导肿瘤特异性CD8+T细胞识别杀伤骨肉瘤

摘要：目的探索肿瘤特异性免疫细胞杀伤骨肉瘤的实现方法,并分析使用过继性细胞输注免疫治疗对骨肉瘤产生的治疗效果。方法使用地西他滨(Decitabine,DAC)作为去甲基化药物对HOS及U20S骨肉瘤细胞系进行治疗，采用PCR及Western Blot检测治疗后肿瘤-睾丸抗原(cancer-testis antigen,CTA)的表达情况。在动物实验中,使用转染了萤火虫荧光素酶和HLA-A0201的人源肿瘤细胞系HOS在免疫缺陷NOD-SCID小鼠上成瘤,并使用DiR染色标记体外培养扩增的CTA特异性对应HLA人源CD8+T细胞输注,通过小动物活体成像系统检测T细胞在小鼠体内的分布，同时监测治疗过程中小鼠瘤块的生长情况。使用鼠源骨肉瘤细胞系K7M2在免疫健全鼠BALB/c成瘤，进行去甲基化，使用流式细胞仪分析治疗前后肿瘤块内淋巴细胞的比例、数量以及CD8+T细胞的活化情况，判断去甲基化对自体淋巴细胞浸润、活化的作用。结果使用DAC对骨肉瘤细胞系HOS及U20S进行去甲基化后,所有被检测的CTA表达均不同程度的提高。在体外培养的骨肉瘤细胞经过至少5d的DAC治疗后能被CTA特异性CD8+T细胞显著杀伤，DAC本身对骨肉瘤细胞系生长可产生抑制作用。在免疫缺陷动物模型中，经过DAC去甲基化预治疗的荷瘤小鼠能观测到明显的CTA特异性T细胞向肿瘤组织归巢现象，同时，去甲基化预治疗联合CTA特异性T细胞输注治疗显著抑制了骨肉瘤移植瘤的生长。在免疫健全鼠肿瘤模型中,去甲基化能促进自体T细胞向肿瘤组织的归巢，并提高肿瘤内CD8+T细胞的活性及其分泌的干扰素γ、颗粒酶B、穿孔素水平。结论去甲基化能在抑制骨肉瘤细胞生长的同时，提高骨肉瘤细胞的CTA表达，为以CTA为靶点的特异性细胞免疫输注治疗提供条件。

Abstract：

Objective To investigate feasible immunotherapy strategy using tumor specific cell against osteosarcoma,and to analyze the therapeutic effect of adoptive cellular infusion therapy on osteosarcoma.Methods Decitabine (DAC) was employed as a hypomethylating agent for the treatment in osteosarcoma cell lines HOS and U2OS.After treatment,the expression of cancer-testis antigen (CTA) was evaluated by PCR and Western Blot.In animal studies,human osteosarcoma cell line HOS,which was transfected by luciferase and HLA-A0201 in previous,was inoculated into immune deficient NOD-SCID mice to establish osteosarcoma xenografts.Ex-vivo expanded CTA specific homo CD8+T-ells were labeled with DiR and injected into the mice via the tail vein.In vivo imaging system was utilized to detect the distribution of administrated CD8+ T-cells.In addition,the progression of tumor xenografts was monitored.Moreover,mouse K7M2 osteosarcoma cell line was used to establish animal models in immune competent BALB/c mice.Immune competent models were utilized to evaluate the effectiveness of hypomethylating treatment in regarding to spontaneous immune attack against tumors.Flow cytometry was used to analyze the proportion of intratumoral lymphocytes and the status of these effector antitumor immune cells,and to reveal the effect of hypomethylating treatment in facilitating lymphocyte infiltration and activation.Results The expression of all the evaluated cancer/testis antigens were elevated in HOS and U2OS osteosarcoma cell lines after hypomethylating treatment with DAC.The proliferation of in vitro cultured osteosarcoma cells can be significantly suppressed after at least 5 d treatment with DAC.Besides,DAC alone controlled osteosarcoma cell proliferation.In immune deficient mouse models,hypomethylating pre-treatment resulted in successful T-cell homing to tumor sites.Moreover,the combination treatment with DAC and CTA specific T-cell adoptive transfer significantly suppressed tumor proliferation.In immune competent mouse models,hypomethylating treatment with DAC improved autologous T-cell infiltration into the tumor,and strengthened the activity of intratumoral CD8+ T-cells,elevated the secretion of IFN-gamma,granzyme B and perforin by CD8+ T-cells.Conclusion Hypomethylating treatment is able to suppress osteosarcoma cell proliferation,improve the expression of CTA in osteosarcoma cells,and consequently provide optimal environment for CTA specific T-cell adoptive therapy.