Identification of T cell epitopes from ovarian cancer associated anti-idiotype antibody

目的 鉴定卵巢上皮性癌(卵巢癌)抗独特型抗体6B11的T细胞表位,探讨其诱导抗卵巢癌细胞免疫的分子基础.方法 利用表位预测和12结合力分析实验筛选6B11的互补决定区(CDR)人白细胞抗原(HLA)A0201结合肽.以负载肽的抗原递呈细胞刺激HLA-A2阳性的自体淋巴细胞,获得特异性细胞毒淋巴细胞(CTL),经51Cr释放实验确定6B11的CTL表位.以6B11特异性CTL为反应细胞、负载肽的树突状细胞为刺激细胞,经细胞增殖实验确定6B11的辅助性T细胞(Th)表位.经细胞因子含量测定和干扰素(IFN)γ分泌细胞频数分析,进一步验证获得的表位.结果 6B11轻链可变区CDR3肽(VL CDR3)诱导的CTL能杀伤靶抗原阳性的卵巢癌细胞,该杀伤作用能被抗人主要组织相容性复合物(MHC)Ⅰ类分子抗体阻断,具有MHC-Ⅰ类分子依赖性,为6B11的CTL表位.6B11重链可变区CDR3肽(VH CDR3)能诱导6B11特异性CTL的增殖,该增殖作用大部分能被抗人MHC-Ⅱ类分子抗体阻断,具有MHC-Ⅱ类分子依赖性,为6B11 Th表位.6B11及其表位联合诱导的CTL与卵巢癌细胞共育后均分泌高水平白细胞介素(IL)2(分别为1630、1503 ng/L)、IFN-γ(分别为5620、5421 ng/L)和低水平IL-4(分别为253、274 ng/L),且存在特异性IFN-γ分泌细胞,细胞频数分别为196个/1 ×106个T细胞和184个/1×106个T细胞.结论 6B11具有模拟卵巢癌抗原的CTL和Th表位,对于抗独特型抗体作为抗肿瘤疫苗应用具有重要的理论和实践价值.Objective To identify the T cell epitopes from ovarian cancer associated anti-idiotypic antibody 6B11in order to explore the molecular basis of 6B11 induced cellular immune responses against ovarian cancer.Methods Potential human leukocyte antigen(HLA)A0201 ligands were predicted by using SYFPEITHI algorithm and tested by the T2 binding assay for screening of HLA-A2 binding peptides from 6B11 complimentary determining region(CDR).Cytotoxic T lymphocytes(CTL)to 6B11 or peptides were generated by 3 rounds of in vitro stimulation with 6B11 or peptide-pulsod dendritic cells(DC),and then tested by 51Cr-release assay to ascertain the CTL epitope of 6B11.Cell proliferation assay was performed by using 6B11 specific CTL as responder cells and peptide-pulsed DC as stimulators to ascertain the helper T lymphocyte(Th)epitope of 6B11.Cytokine assay and interferon-γ ELISPOT assay were performed to verify the CTL and Th epitope of 6B11 further.Results Light chain CDR3 peptide(VL CDR3)of 6B11 induced specific CTL to kill HLA-A2 and target antigen positive ovarian cancer cells,which could be blocked by anti human major histocompatibility complex(MHC)Ⅰ antibody.Heavy chain CDR3 peptide(VH CDR3)of 6B11 stimulated the proliferation of 6B11-primed CTL,which could be blocked mainly by anti human MHC-Ⅱ antibody,and further experiments showed that part of the VH CDR3 peptide-primed CTL killed K562 cells.Peptides in VL CDR3 and VH CDR3 were the CTL and Th epitope mimicking the original antigen of 6B11 respectively.Collaboration of 6B11 CTL and Th epitope,or 6B11 CTL epitope and keyhole limpet hemocyanin(KLH),the former was more powerful in inducing specific cellular immune responses against ovarian cancer.6B11 or corresponding CTL and Th epitope specific CIL secreted high levels of interleukin-2 (1630,1503 ng/L)and interferon-γ(5620,5421 ng/L),while basal level of interleukin-4 was detected (253,274 ng/L).ELISPOT assay confirmed the existence of specific interferon-γ secreting cells in 6811 or epitopes specific CTL(196/1×106 T cell,184/1×106 T cell).Conclusions VL CDR3 and VH CDR3 peptides of 6B11 are the CTL and Th epitopes mimicking original antigen which could duplicate the activity of intact 6B11 to induce the cellular immune responses against ovarian cancer.The results have significant theoretical and practical value in application of anti-idiotypic antibody ag anti tumor vaccine.The acquired CTL and Th epitopes as constituents of future multiple peptides against ovarian cancer could be used in peptide vaccine based ovarian cancer therapy.国家自然科学基金PubMed中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)010764-7694