Wolf S F, Temple P A, Kobayashi M, Young D, Dicig M, Lowe L, Dzialo R, Fitz L, Ferenz C, Hewick R M
Genetics Institute, Inc., Cambridge, MA 02140.
J Immunol. 1991 May 1;146(9):3074-81.
Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.
此前我们报道了从EB病毒转化的B细胞系RPMI 8866中纯化和鉴定一种新型细胞因子。这种因子被称为自然杀伤细胞刺激因子（NKSF），具有多种活性，包括诱导外周血淋巴细胞（PBL）产生γ干扰素、增强自然杀伤细胞的细胞毒性以及刺激PBL增殖。纯化后的NKSF是一种由35 kDa和40 kDa亚基（p35和p40）通过二硫键连接的异源二聚体蛋白。我们现在报道从RPMI 8866细胞RNA中克隆NKSF两个亚基的cDNA。cDNA序列表明这两个基因都是新的，Southern印迹分析证实这两个cDNA均来源于人类基因组。[35S]甲硫氨酸标记表明，用p35或p40 cDNA转染的cos - 1细胞产生了大小合适的独特蛋白质。用p35加p40 cDNA共转染的cos - 1细胞进行甲硫氨酸标记，在非还原凝胶上产生了一条在70至90 kDa之间迁移的宽带。还原这种高分子量物质后产生了与p35和p40基因产物相关的条带。只有共转染的cos - 1细胞的培养上清液具有高水平的NKSF生物学活性。在非还原凝胶中培养上清液中的生物学活性在70至90 kDa处迁移，这一观察结果表明这种高分子量物质负责这种活性。此外，抗p40血清能够阻断重组NKSF和天然NKSF的生物学活性，这表明它是活性蛋白的一个组成部分。相比之下，单独用p40或p35 cDNA转染的cos - 1细胞的上清液中未检测到活性。共转染的cos - 1细胞产生的生物学活性谱与从RPMI 8866细胞系纯化至同质的NKSF相同。通过添加白细胞介素-2或共刺激剂PHA或佛波酯，发现其中一些反应有协同增强作用。NKSF加白细胞介素-2对T细胞和自然杀伤细胞功能的协同刺激支持了这些细胞因子可能在癌症治疗中有用的可能性。
