Ge Hai-Liang, Wang Ying, Wang Shu-Jun, Zhang Yong
Department of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Immunology, Shanghai 200025, China.
Acta Biochim Biophys Sin (Shanghai). 2006 Feb;38(2):110-8. doi: 10.1111/j.1745-7270.2006.00134.x.
HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and allogeneic HLA-A2.1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.
从人黑色素瘤细胞中提取的HLA - A2.1相关肽，通过表位重构、活性肽序列表征和合成肽验证，用于研究黑色素瘤特异性HLA - A2.1限制性细胞毒性T淋巴细胞（CTL）的表位。通过体外刺激从肿瘤累及的淋巴结中产生CTL，最初用自体黑色素瘤细胞，随后用同种异体HLA - A2.1阳性黑色素瘤细胞。这些CTL能够裂解自体和同种异体HLA - A2.1阳性黑色素瘤，但不能裂解HLA - A2.1阴性黑色素瘤或HLA - A2.1阳性非黑色素瘤。黑色素瘤的裂解可被抗CD3、抗HLA I类和抗HLA - A2.1单克隆抗体抑制。通过免疫亲和柱色谱从去污剂溶解的人黑色素瘤细胞中纯化HLA - A2.1分子，并通过反相高效液相色谱进一步分级分离。评估这些级分与HLA - A2.1阳性抗原加工突变体T2细胞重构黑色素瘤特异性表位的能力。在乳酸脱氢酶释放试验中观察到三个重构峰。使用质谱和离子交换高效液相色谱分析来鉴定肽表位。质荷比为948的肽通常由九个氨基酸残基组成。重构实验的数据证实，合成肽包含表位，并且与HLA - A2.1相关且被黑色素瘤特异性CTL识别的肽存在于这些不同的黑色素瘤细胞中。这些肽可能在基于肽的新型抗肿瘤疫苗中用于CTL免疫治疗。
