Qiu Jian, Li Guo-Wei, Sui Yan-Fang, Sun Yu-Jing, Huang Ya-Yu, Si Shao-Yan, Ge Wei
Department of General Surgery, Shaanxi Provincial People's Hospital, The Third Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, China.
Acta Oncol. 2007;46(7):961-8. doi: 10.1080/02841860601166941.
To select the MHC-I-binding epitope-rich sequence of mice telomerase reverse transcriptase (mTERT) and study the antitumor immune response induced by truncated TERT through mRNA-transfected dendritic cells (DCs) immunization in mice. The MHC-I-binding epitopes of TERT were predicted using bioinformatics software. The selected sequence of TERT (Truncated mTERT, TERT(t), mTERT cDNA 1776 bp-2942 bp encoding 584 aa-969 aa) was cloned from B16 mouse melanoma cells and inserted into pBluescriptIIKS(+) plasmid downstream of the T7 promoter. TERT(t) RNA was prepared through in vitro transcription. Bone marrow-derived DCs were electroporated with TERT(t) RNA and used to immunize syngeneic naïve mice. The quantity and cytotoxic activity of TERT-specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated using IFN-gamma enzyme-linked immunospot (ELISPOT) and Lactate dehydrogenase release assay. The immunoprophylactic effects against TERT positive tumor induced by TERT(t) RNA transfected DC in vivo were evaluated through an immunized-challenged mouse model. TERT(t) was cloned and in vitro transcribed into TERT(t) mRNA. As shown in FCM analysis, the efficiency of DC electroporation is 35.1% (29.7-41.2%). After electroporation, a subtle increase of costimulator and MHC-II molecules were expressed on the cell surface. Immunization of TERT(t) mRNA transfected DCs induced IFN-gamma-secreting CTLs which manifested specific cytotoxic activity against TERT-positive target cells. In a cancer mouse model, vaccination of TERT(t) mRNA-transfected DCs suppressed the growth of TERT positive tumors (p=0.001) and prolong the survival time of tumor-bearing animals (p=0.029). TERT(t) evokes an antitumor immune response in vivo which is targeted to TERT. TERT(t) can be used as an antigeneic sequence to produce anti-TERT tumor vaccine.
筛选小鼠端粒酶逆转录酶(mTERT)的MHC-I结合表位富集序列,并通过mRNA转染树突状细胞(DCs)免疫小鼠,研究截短的TERT诱导的抗肿瘤免疫反应。使用生物信息学软件预测TERT的MHC-I结合表位。从B16小鼠黑色素瘤细胞中克隆TERT的选定序列(截短的mTERT,TERT(t),mTERT cDNA 1776 bp - 2942 bp,编码584个氨基酸 - 969个氨基酸),并插入到T7启动子下游的pBluescriptIIKS(+)质粒中。通过体外转录制备TERT(t) RNA。用TERT(t) RNA电穿孔骨髓来源的DCs,并用于免疫同基因的未免疫小鼠。使用γ干扰素酶联免疫斑点法(ELISPOT)和乳酸脱氢酶释放试验评估小鼠脾脏中TERT特异性细胞毒性T淋巴细胞(CTLs)的数量和细胞毒性活性。通过免疫 - 攻击小鼠模型评估TERT(t) RNA转染的DCs在体内对TERT阳性肿瘤的免疫预防作用。克隆TERT(t)并体外转录为TERT(t) mRNA。如流式细胞术分析所示,DC电穿孔效率为35.1%(29.7 - 41.2%)。电穿孔后,细胞表面共刺激分子和MHC-II分子表达略有增加。TERT(t) mRNA转染的DCs免疫诱导分泌γ干扰素的CTLs,其对TERT阳性靶细胞表现出特异性细胞毒性活性。在癌症小鼠模型中,TERT(t) mRNA转染的DCs疫苗接种抑制了TERT阳性肿瘤的生长(p = 0.001),并延长了荷瘤动物的生存时间(p = 0.029)。TERT(t)在体内引发针对TERT的抗肿瘤免疫反应。TERT(t)可作为抗原序列用于生产抗TERT肿瘤疫苗。