Sato T, Sato N, Takahashi S, Okubo M, Yagihashi A, Torigoe T, Takahashi N, Okazaki M, Asaishi K, Kukuchi K
Department of Pathology, Sapporo Medical College, Japan.
Cancer Res. 1988 Jul 15;48(14):3892-7.
We demonstrated previously the establishment of a human cytotoxic T-cell clone, TcHMC-1, under culturing with recombinant interleukin that showed the specific cytotoxicity against an autologous breast tumor cell line, HMC-1-8. In the present study, the autologous tumor specific antigens that could be involved in this cytotoxicity were extracted by using n-butyl alcohol and were analyzed for their biochemical profiles. The cytotoxicity of TcHMC-1 against HMC-1-8 was inhibited by adding OKT3 and OKT8 monoclonal antibodies into the cultures, or by pre-sensitizing HMC-1-8 target cells by anti-major histocompatibility complex class I monoclonal antibodies. This suggests that T-cell antigen receptor molecule complexes Ti/T3 on TcHMC-1 and corresponding specific tumor antigens on HMC-1-8 are involved in the cytotoxicity under the restriction of major histocompatibility complex class I products. Precultures of TcHMC-1 with crude n-butyl alcohol extracts from HMC-1-8 cells enhanced the cytotoxic potentials of this clone as seen as mixed lymphocyte tumor cell cultures. This enhancement was dependent on dosage of crude n-butyl alcohol extracts and these TcHMC-1 cells were still cytotoxic specifically for HMC-1-8 targets, but not for other allogenic tumor lines including K562. However, HMC-1-8 crude n-butyl alcohol extracts could not enhance DNA synthesis of TcHMC-1 as assessed by incorporation of [3H]thymidine in the cells. Biochemical purification studies demonstrated that the HMC-1-8 tumor specific antigens were eluted into fractions containing molecules with molecular weights of approximately 200,000 on Sephadex G-200 column chromatography. The antigens were further separated into the fraction that was eluted with 0.4-0.5 M NaCl in an ionic strength on Mono Q fast protein liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this fraction demonstrated three molecules with molecular weights of 26,000, 30,000, and 32,000 under reduced molecular conditions. The data suggest that these molecules could be tumor specific antigens that are involved in the cytotoxicity of cytotoxic T-cells against a human autologous tumor.
我们先前证明了在用重组白细胞介素培养的条件下建立了一个人细胞毒性T细胞克隆TcHMC-1，该克隆对自体乳腺肿瘤细胞系HMC-1-8表现出特异性细胞毒性。在本研究中，使用正丁醇提取了可能参与这种细胞毒性的自体肿瘤特异性抗原，并分析了它们的生化特征。通过在培养物中加入OKT3和OKT8单克隆抗体，或通过用抗主要组织相容性复合体I类单克隆抗体预致敏HMC-1-8靶细胞，可抑制TcHMC-1对HMC-1-8的细胞毒性。这表明，在主要组织相容性复合体I类产物的限制下，TcHMC-1上的T细胞抗原受体分子复合物Ti/T3与HMC-1-8上相应的特异性肿瘤抗原参与了细胞毒性作用。如混合淋巴细胞肿瘤细胞培养所示，用HMC-1-8细胞的正丁醇粗提物对TcHMC-1进行预培养可增强该克隆的细胞毒性潜能。这种增强依赖于正丁醇粗提物的剂量，并且这些TcHMC-1细胞对HMC-1-8靶细胞仍具有特异性细胞毒性，但对包括K562在内的其他同种异体肿瘤细胞系则无细胞毒性。然而，通过细胞中[3H]胸腺嘧啶核苷掺入评估，HMC-1-8正丁醇粗提物不能增强TcHMC-1的DNA合成。生化纯化研究表明，在Sephadex G-200柱色谱上，HMC-1-8肿瘤特异性抗原被洗脱到含有分子量约为200,000的分子的级分中。在Mono Q快速蛋白质液相色谱上，通过离子强度用0.4-0.5M NaCl洗脱，可将抗原进一步分离到该级分中。在还原分子条件下，对该级分进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，显示出分子量分别为26,000、30,000和32,000的三个分子。数据表明，这些分子可能是参与细胞毒性T细胞对人自体肿瘤细胞毒性作用的肿瘤特异性抗原。
