NKLAM--A NOVEL GENE REQUIRED FOR NK FUNCTION

自然杀伤（NK）细胞是一种大颗粒淋巴细胞，具有杀伤肿瘤的作用 和病毒感染的细胞而没有预先致敏。 他们的裂解 在体外，通过与以下物质温育，活性可以提高20-30倍 干扰素（IFN）α和β或白细胞介素-2（IL-2）。 一个 理解这个激活过程将有助于 检测IFN或IL-2引起的基因表达变化， 增强NK功能。 我们分离了56个cDNA克隆， IFN-β激活的NK细胞表达升高， 未受刺激的NK细胞，通过IFN-B诱导的NK细胞的差异筛选， cDNA文库 这些cDNA克隆中有46个编码以前未描述的 基因. 这些新基因之一，命名为NKLAM，是由IL-2诱导的 在外周血NK和T细胞中，但也不表达 未受刺激的或IFN-β-，并显示与已知的 基因. 令人兴奋的是， 用NKLAM反义寡核苷酸完全消除了 未刺激和IL-2激活的细胞溶解活性。 这直接 表明NKLAM参与了裂解过程 我们现在建议：1）评估 NKLAM在使用反义核酸的NK和T细胞介导的溶解中的功能作用 寡核苷酸和基因转移。 反义寡核苷酸处理 表达bote正义基因和反义基因的细胞以及NK和CTL系 评估构建体的靶细胞结合和裂解的变化， 颗粒胞吐和胞嘧啶产生。 NKLAM抗血清 蛋白质将产生，以确定位置和结构 关于NKLAM 我们将研究NKLAM RNA在NK和T细胞亚群中的表达， 使用多种细胞溶解增强剂从一组正常供体中， 抑制剂的 将分离NKLAM的基因组同源物，以 确定其遗传结构，并最终研究其调节。 2)检查文库中其他独特基因的表达， 通过RNA印迹分析细胞毒性细胞。表达基因的cDNA将被 测序 反义寡核苷酸将用于鉴定那些 可能参与细胞溶解过程的基因。 这些基因将 如NKLAM所述进一步表征。 这项工作将提供深入了解的分子机制， 增强NK细胞溶解，这将最终帮助临床医生 以最有效的方式使用这些细胞进行治疗。

Natural killer (NK) cells are large granular lymphocytes which lyse tumor and virus-infected cells without prior sensitization. Their lytic activity can be enhanced 20-30 fold in vitro by incubation with interferons (IFNs) alpha and beta or interleukin-2 (IL-2). An understanding of this activation process would be facilitated by examining changes in gene expression that occur upon IFN or IL-2 augmentation of NK function. We have isolated 56 cDNA clones with elevated expression in IFN-beta-activated NK cells compared with unstimulated NK cells, by differential screening of an IFN-B induced NK cDNA library. 46 of these cDNA clones encode previously undescribed genes. One of these novel genes, designated NKLAM, is induced by IL-2 in peripheral blood NK and T cells, but is not expressed either unstimulated or IFN-beta-and shows no significant homology to known genes. Of great excitement is the finding that treatment of NK cells with NKLAM antisense oligonucleotides completely abolishes both unstimulated and IL-2 activated cytolytic activity. This directly implicates NKLAM in the lytic process. We now propose to: 1) Assess the functional role of NKLAM in NK and T cell-mediated lysis using antisense oligonucleotides and gene transfer. Antisense oligonucleotide-treated cells and NK and CTL lines expressing bote sense and antisense gen constructs will be assessed for changes in target cell binding and lysis, granule exocytosis and cytosine production. Antisera to the NKLAM protein will be produced in order to determine the location and structure of NKLAM. We will study NKLAM RNA expression in NK and T cell subsets from a panel of normal donors using a variety of cytolytic enhancers and inhibitors. The genomic homolog of NKLAM will be isolated in order to define its genetic structure and ultimately, to study its regulation. 2) Examine the expression of other unique genes in the library in cytotoxic cells by RNA blot analysis. cDNA of expressed genes will be sequenced. Antisense oligonucleotides will be used to identify those genes potentially involved in the cytolytic process. These genes will be further characterized as described for NKLAM. This work will provide insight into the molecule mechanism of augmentation of NK cytolysis, which will ultimately aid the clinician in using these cells therapeutically in the most effective way.