Ortaldo J R, Winkler-Pickett R, Morgan A C, Woodhouse C, Kantor R, Reynolds C W
Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research Facility, MD 21701-1013.
J Immunol. 1987 Nov 1;139(9):3159-65.
Natural killer cytotoxic factor (NKCF) is produced as a result of the interaction of murine, rat, or human natural killer (NK) cells with NK-susceptible targets. This factor has been linked to the target cell lysis mediated by the NK effector cell. In the present results, culture supernatants from rat large granular lymphocyte (LGL) tumors exhibited NKCF activity which lysed the susceptible targets, MBL-2 and YAC-1. NKCF production from these rat tumor lines was spontaneous and was not significantly increased by co-incubation of the LGL tumors with target cells, target cell membranes, or by preincubation of the LGL tumor cells with interferon or interleukin 2. In addition to NKCF activity, the supernatants lysed L929, indicating the presence of tumor necrosis factor (TNF) in these preparations. The presence of this latter cytokine was verified using specific antibodies to recombinant murine TNF which neutralized the L929 activity while not affecting the NKCF activity against MBL-2 or YAC-1. Mouse monoclonal antibodies (mAb) A0287, A0462, and A0316) which significantly inhibit the NKCF cytolytic activity of these LGL-derived supernatants were also produced. These antibodies were shown to cross-react with human NKCF in a manner similar to that seen in the rat. Interestingly these same mAb demonstrated no inhibition of L929 cytotoxicity from either LGL-derived supernatants or by recombinant murine or human TNF. To examine further the specificity of these antibodies, they were chemically linked to Sepharose 4B and found to remove a significant proportion of the NKCF cytolytic activity from LGL supernatants, while not affecting the TNF reactivities in these preparations. In addition, these antibodies demonstrated significant inhibition of cell-mediated cytotoxicity by rat LGL against YAC-1 target cells. Biochemical analysis of labeled NKCF-containing supernatants indicated the major protein recognized by these anti-NKCF mAb to be approximately 12,000 m.w. The use of these mAb against NKCF should be very useful in further purification and biochemical characterization of NKCF and in studying its role in a variety of cell-mediated cytotoxicity assays.
自然杀伤细胞细胞毒素因子（NKCF）是小鼠、大鼠或人类自然杀伤（NK）细胞与NK敏感靶细胞相互作用的产物。该因子与NK效应细胞介导的靶细胞裂解有关。在本研究结果中，大鼠大颗粒淋巴细胞（LGL）肿瘤的培养上清液表现出NKCF活性，可裂解敏感靶细胞MBL-2和YAC-1。这些大鼠肿瘤细胞系自发产生NKCF，将LGL肿瘤与靶细胞、靶细胞膜共同孵育，或用干扰素或白细胞介素2预孵育LGL肿瘤细胞，均不会显著增加NKCF的产生。除了NKCF活性外，上清液还能裂解L929，表明这些制剂中存在肿瘤坏死因子（TNF）。使用针对重组小鼠TNF的特异性抗体验证了后一种细胞因子的存在，该抗体可中和L929活性，同时不影响针对MBL-2或YAC-1的NKCF活性。还制备了能显著抑制这些LGL来源上清液的NKCF细胞溶解活性的小鼠单克隆抗体（mAb）A0287、A0462和A0316）。这些抗体与人类NKCF发生交叉反应，其方式与在大鼠中观察到的相似。有趣的是，这些相同的mAb对LGL来源上清液或重组小鼠或人类TNF的L929细胞毒性均无抑制作用。为了进一步研究这些抗体的特异性，将它们化学连接到琼脂糖4B上，发现可从LGL上清液中去除相当一部分NKCF细胞溶解活性，同时不影响这些制剂中的TNF反应性。此外，这些抗体显著抑制大鼠LGL对YAC-1靶细胞的细胞介导细胞毒性。对标记的含NKCF上清液的生化分析表明，这些抗NKCF mAb识别的主要蛋白质分子量约为12,000。使用这些针对NKCF的mAb在NKCF的进一步纯化和生化特性研究以及研究其在各种细胞介导细胞毒性试验中的作用方面应该非常有用。
