Slingluff C L, Cox A L, Henderson R A, Hunt D F, Engelhard V H
Department of Surgery, University of Virginia, Charlottesville 22908.
J Immunol. 1993 Apr 1;150(7):2955-63.
HLA-A2.1-associated peptides were extracted from human melanoma cell lines and used to study epitopes for melanoma-specific HLA-A2.1-restricted CTL. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic A2.1+ melanoma cells. These CTL lysed autologous melanoma plus four allogeneic HLA-A2.1+ melanomas, including an HLA-A2.1-transfected melanoma. K562, HLA-A2- melanomas and HLA-A2+ nonmelanomas were not lysed. HLA-A2.1 molecules were purified from human melanoma cell lines by immunoaffinity column chromatography of detergent-solubilized cell pellets. Peptides bound to the MHC molecules were acid eluted and fractionated by reversed phase HPLC. Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by addition to the HLA-A2.1+ Ag-processing mutant, 721.174XCEM.T2 (T2). Five peaks of reconstitution were observed. Second dimension HPLC separations of reconstituting fractions revealed evidence for two distinct reconstituting peptides within one of these peaks. Based on these data, a minimum of six distinct peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL are present on these different melanoma lines. These data document the presence of multiple peptide-defined CTL epitopes that are shared by at least three unrelated human melanoma cell lines.
从人黑色素瘤细胞系中提取与HLA - A2.1相关的肽段，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A2.1 +黑色素瘤细胞。这些CTL可裂解自体黑色素瘤以及四种同种异体HLA - A2.1 +黑色素瘤，包括一种HLA - A2.1转染的黑色素瘤。K562、HLA - A2 -黑色素瘤和HLA - A2 +非黑色素瘤细胞未被裂解。通过对去污剂溶解的细胞沉淀进行免疫亲和柱层析，从人黑色素瘤细胞系中纯化HLA - A2.1分子。与MHC分子结合的肽段经酸洗脱，并用反相高效液相色谱法进行分离。通过添加到HLA - A2.1 +抗原加工突变体721.174XCEM.T2（T2）中，评估各个级分重构黑色素瘤特异性表位的能力。观察到五个重构峰。对重构级分进行二维高效液相色谱分离，结果显示其中一个峰内存在两种不同的重构肽段。基于这些数据，在这些不同的黑色素瘤细胞系上存在至少六种与HLA - A2.1相关且被黑色素瘤特异性CTL识别的不同肽段。这些数据证明了存在多个由肽段定义的CTL表位，这些表位至少被三种不相关的人黑色素瘤细胞系所共有。
