Loudon W G, Abraham S R, Owen-Schaub L B, Hemingway L L, Hemstreet G P, DeBault L E
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Cancer Res. 1988 Apr 15;48(8):2184-92.
When peripheral blood lymphocytes (PBL) are incubated with interleukin 2 (IL 2), a novel cytotoxic lymphocyte subpopulation, termed lymphokine activated killers (LAK), arises. LAK are functionally defined as IL 2 responsive cells demonstrating major histocompatibility antigen-unrestricted cell-mediated cytotoxicity against fresh solid tumors and other natural killer cell-resistant and -sensitive tumor targets in the absence of prior antigen priming. Flow cytometric analysis of IL 2 activated PBL using forward and right angle light scatter and fluorescence intensity identified the emergence of a large, optically dense, autofluorescent cell population which paralleled the generation of LAK activity. These unique IL 2 induced lymphocytes have been named giant autofluorescent lymphocytes (GAL). These cells are readily distinguished from the small nonfluorescent lymphocytes (SNL) observed in fresh PBL, unstimulated cultured PBL, and those cells remaining after incubation with IL 2 which have not acquired GAL characteristics. In this investigation, LAK cultures were sorted on days 4, 5, and 6 into GAL and SNL populations and were tested for oncolytic activity against the natural killer-resistant Daudi and RC-1 tumor targets. Against these targets, lymphocytes from non-IL 2 activated PBL or the sorted SNL population expressed less than 2% of the oncolytic activity (measured in lytic units) exhibited by GAL effectors. The SNL and GAL populations were cultured in IL 2 for up to 48 h following the sorting procedure and then reassayed for tumor cytolytic activity. During this culture period, GAL but not SNL continued to express LAK killing against natural killer-resistant tumor targets. Using gamma-irradiation to prevent further cell cycling, it was shown that the functional half-life of the LAK effector was approximately 8.5 h. Therefore, the cytotoxicity expressed by the sorted GAL population after 48 h in culture (equivalent to five functional half-lives) must be expressed by progeny of the originally plated lymphocytes. These results indicate that in addition to the LAK effector, the GAL population contains a self-sustaining, recycling intermediate responsible for generating new LAK. Our data indicate that analysis of IL 2 activated PBL using GAL light-scattering properties has application in phenotyping LAK, monitoring of cellular kinetics, cell sorting, and enrichment of the LAK effector population, and in the clinical monitoring of IL 2 therapy.
当外周血淋巴细胞（PBL）与白细胞介素2（IL-2）一起孵育时，会产生一种新的细胞毒性淋巴细胞亚群，称为淋巴因子激活的杀伤细胞（LAK）。LAK在功能上被定义为对IL-2有反应的细胞，在没有预先抗原致敏的情况下，表现出对新鲜实体瘤以及其他对自然杀伤细胞有抗性和敏感性的肿瘤靶标的主要组织相容性抗原非限制性细胞介导的细胞毒性。使用前向和直角光散射以及荧光强度对IL-2激活的PBL进行流式细胞术分析，确定出现了一个大的、光学密度高的、自发荧光的细胞群体，这与LAK活性的产生平行。这些独特的IL-2诱导的淋巴细胞被命名为巨大自发荧光淋巴细胞（GAL）。这些细胞很容易与新鲜PBL、未刺激培养的PBL中观察到的小的非荧光淋巴细胞（SNL）以及与IL-2孵育后未获得GAL特征的那些细胞区分开来。在这项研究中，在第4、5和6天将LAK培养物分选成GAL和SNL群体，并测试其对自然杀伤细胞抗性的Daudi和RC-1肿瘤靶标的溶瘤活性。针对这些靶标，来自未用IL-2激活的PBL或分选的SNL群体的淋巴细胞表现出的溶瘤活性（以裂解单位衡量）不到GAL效应细胞的2%。分选后，将SNL和GAL群体在IL-2中培养长达48小时，然后重新测定肿瘤细胞溶解活性。在这个培养期间，GAL而非SNL继续表现出对自然杀伤细胞抗性肿瘤靶标的LAK杀伤作用。使用γ射线照射以防止进一步的细胞周期循环，结果表明LAK效应细胞的功能半衰期约为8.5小时。因此，分选的GAL群体在培养48小时后（相当于五个功能半衰期）所表现出的细胞毒性必定是由最初接种的淋巴细胞的后代所表现出来的。这些结果表明，除了LAK效应细胞外，GAL群体还包含一个负责产生新的LAK的自我维持、循环利用的中间体。我们的数据表明利用GAL光散射特性对IL-2激活的PBL进行分析，在LAK表型分析、细胞动力学监测、细胞分选、LAK效应细胞群体富集以及IL-2治疗的临床监测中都有应用价值。
