Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.
Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.
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DOI：
10.4049/jimmunol.146.9.3074
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发表时间：
1991-05
影响因子：
4.4
通讯作者：
S. Wolf;P. Temple;Michiko Kobayashi;D. Young;M. Dicig;L. Lowe;R. Dzialo;L. Fitz;C. Ferenz;R. Hewick
中科院分区：
文献类型：
--
作者：
S. Wolf;P. Temple;Michiko Kobayashi;D. Young;M. Dicig;L. Lowe;R. Dzialo;L. Fitz;C. Ferenz;R. Hewick
此前，我们报道了从 EBV 转化的 B 细胞系 RPMI 8866 中纯化和表征一种新型细胞因子。该因子称为自然杀伤细胞刺激因子 (NKSF)，具有多效活性，包括从 PBL 诱导 IFN-γ、增强 NK 细胞的细胞毒性以及刺激 PBL 的增殖。纯化的 NKSF 被发现是一种二硫键连接的异二聚体蛋白，由 35 kDa 和 40 kDa 亚基（p35 和 p40）组成。我们现在报告从 RPMI 8866 细胞 RNA 中分子克隆 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 均源自人类基因组。 [35S]蛋氨酸标记表明用p35或p40 cDNA转染的cos-1细胞产生了适当大小的独特蛋白质种类。用 p35 加 p40 cDNA 共转染的 cos-1 细胞的蛋氨酸标记产生了在非还原凝胶上迁移在 70 至 90 kDa 之间的宽带。这种高分子量物质的还原产生了与 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 生物活性。通过观察培养物上清液中的生物活性在非还原性凝胶中以 70 至 90 kDa 迁移，表明高分子量材料造成了这种活性。此外，抗p40血清能够阻断重组和天然NKSF的生物活性，这表明它是活性蛋白的组成部分。相反，在仅用p40或p35 cDNA转染的cos-1细胞的上清液中检测不到活性。共转染的 cos-1 细胞产生的生物活性谱与从 RPMI 8866 细胞系纯化至同质的 NKSF 相同。通过添加 IL-2 或共刺激剂 PHA 或佛波二酯，发现其中一些反应具有协同增强作用。 NKSF 与 IL-2 对 T 和 NK 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。
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.