Werkmeister J A, Triglia T, Burns G F
Cell Immunol. 1985 Apr 15;92(1):123-33. doi: 10.1016/0008-8749(85)90070-x.
Mechanisms involved in the lysis of tumor cells by natural killer (NK) cells were investigated by using mutagenized K562 targets resistant to the effects of NK cells. K562 cells were treated with the mutagen methyl methanesulfonate (MMS) and, to select for resistant mutants, rabbit anti-idiotypic (anti-id) antibodies were used. This anti-id was raised to a monoclonal antibody 9.1C3 which itself blocked lysis by NK cells by binding to the effector cells; the anti-id inhibited killing by binding to the K562 targets, presumably to a cell surface protein relevant to a secondary event in the NK lytic pathway. MMS-derived mutants showed a heterogeneity of staining with the anti-id, allowing the antibody to be used with flow cytometry to select a population of K562 cells relatively negative in antigen expression. The degree of reactivity of K562 cultures with the anti-id antiserum and the resistance to lysis by NK cells were inversely related. Cultures of NK-resistant K562 cells with low expression of the anti-id structure were cloned by limiting dilution: 96 clones were analyzed and one subclone, C9/2, which was six-to sevenfold less sensitive to lysis than the parental K562 cell line, was used in further studies by cold target inhibition and single cell binding assays. The increased resistance to lysis of C9/2 was not due to a reduced expression of target recognition structures, and resistance could not be overcome by prolonging the time allowed for lysis to 18 hr nor by adding exogenous recombinant leukocyte interferon. Killing of the NK-resistant variant was inhibited by mannose-6-phosphate but not by the monoclonal antibody against which the anti-id antibody was raised. It is therefore suggested that the structure on the K562 cells recognized by the anti-id antibodies is a novel secondary receptor which is important in the later stages of the NK cell cytolytic cascade.
通过使用对自然杀伤（NK）细胞作用具有抗性的诱变K562靶细胞，研究了NK细胞裂解肿瘤细胞的机制。用诱变剂甲磺酸甲酯（MMS）处理K562细胞，并使用兔抗独特型（抗独特型）抗体来选择抗性突变体。这种抗独特型抗体是针对单克隆抗体9.1C3产生的，9.1C3本身通过与效应细胞结合来阻断NK细胞的裂解作用；抗独特型抗体通过与K562靶细胞结合来抑制杀伤作用，推测是与NK裂解途径中二级事件相关的细胞表面蛋白。MMS诱导的突变体在用抗独特型抗体染色时表现出异质性，这使得该抗体可用于流式细胞术，以选择抗原表达相对阴性的K562细胞群体。K562培养物与抗独特型抗血清的反应程度与对NK细胞裂解的抗性呈负相关。通过有限稀释法克隆了抗NK的K562细胞培养物，这些细胞抗独特型结构的表达较低：分析了96个克隆，其中一个亚克隆C9/2对裂解的敏感性比亲代K562细胞系低6至7倍，用于进一步的冷靶抑制和单细胞结合试验研究。C9/2对裂解抗性的增加不是由于靶标识别结构表达的降低，延长裂解时间至18小时或添加外源性重组白细胞干扰素均不能克服其抗性。抗NK变体的杀伤作用受到6-磷酸甘露糖的抑制，但不受抗独特型抗体所针对的单克隆抗体的抑制。因此，提示抗独特型抗体识别的K562细胞上的结构是一种新型的二级受体，在NK细胞溶细胞级联反应的后期起重要作用。
