Adoptive Immunotherapy against Posttransplant Lymphoprolifrative Disorders

针对移植后淋巴细胞增殖性疾病的过继免疫疗法

• 批准号: 10557032
• 负责人: TAKADA Kenzo
• 金额: $ 7.42万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557032/
• 关键词: Epstein-Barr virus; Adoptive Immunotherapy; CTL; Transplantation; PTLD; 細胞傷害性Tリンパ球; リンパ増殖症

There is increasing interest in the use of immunotherapy with antigen-specific cytotoxic T cells (CTL) for the treatment of Epstein-Barr virus (EBV)-associated postransplant lymphoproliferative disorders (PTLD). The present study is aimed to develop a quantitative RT-PCR test for monitoring EBV reactivation in the peripheral blood and to test the safety, persistence and anti-tumor activity of in vitro generated CTL. EBV infection was quantified by realtime PCR assay using Lightcycler. The cellular DNA was purified by EZNA blood kit. The primer pair was set at the latent membrane protein 2A region. This method could detect 1 copy of EBV genome per one million peripheral mononuclear cells. The total time required was one hour. The EBV-specific CTL was induced by cocultivation of peripheral blood mononuclear cells with autologous EBV-immortalized lymphoblastoid cells (LCL) that were killed by γ ray irradiation. They were incubated in the presence of IL2 for 2 to 4 weeks and subjected to cytotoxicity test. The result indicated that the CTL was specific for autologous LCL.

使用抗原特异性细胞毒性T细胞（CTL）的免疫疗法治疗EB病毒（EBV）相关的移植后淋巴组织增生性疾病（PTLD）的兴趣越来越大。本研究旨在建立外周血EB病毒再激活的定量RT-PCR检测方法，并检测体外产生的CTL的安全性、持久性和抗肿瘤活性。使用Lightcycler通过实时PCR测定来定量EBV感染。用EZNA血液试剂盒纯化细胞DNA。引物对设置在潜伏膜蛋白2A区域。该方法每100万个外周血单个核细胞可检测到1个拷贝的EBV基因组。总共需要一个小时。将外周血单个核细胞与经γ射线灭活的EB病毒永生化淋巴母细胞（LCL）共培养，诱导出EB病毒特异性CTL。将它们在IL 2存在下孵育2至4周，并进行细胞毒性试验。结果表明，该CTL对自体LCL具有特异性。

项目成果

Sugawara Y.: "Detection of EBV in hepatocellular carcinoma tissue : a novel EBV latency characteized by absence of EBER expression"Virology. 256. 196-202 (1999)

Sukawara Y.：“肝细胞癌组织中 EBV 的检测：一种以 EBER 表达缺失为特征的新型 EBV 潜伏期”病毒学。

Imai, S.: "Cell-to-cell contact as an efficient mode of Epstein-Barr virus infection of diverse human epithelial cells." J.Virol.72. 4371-4378 (1998)

Imai, S.：“细胞间接触是 Epstein-Barr 病毒感染多种人类上皮细胞的有效模式。”

Sugawara, Y.: "Enhancement of hepatitis C virus replication by Epstein-Barr virus-encoded nuclear antigen"ENBO J.. 18. 5755-5760 (1999)

Sukawara, Y.：“Epstein-Barr 病毒编码核抗原增强丙型肝炎病毒复制”ENBO J.. 18. 5755-5760 (1999)

Takada, K.: "Epstein-Barr virus and gastric carcinoma (Review)"Epstein=Barr Virus Report. 6. 95-100 (1999)

Takada, K.：“Epstein-Barr 病毒和胃癌（评论）”Epstein=Barr 病毒报告。

Sugawara. Y.: "Detection of EBV in hepatocellular carcinoma tissue : a novel EBV latency characterized by absence of EBER expression."Virology. 256. 196-202 (1999)

菅原.