NKLAM--A NOVEL GENE REQUIRED FOR NK FUNCTION

自然杀伤(NK)细胞是一种能溶解肿瘤的大颗粒淋巴细胞 以及没有事先致敏的病毒感染细胞。他们的歌词 体外培养可使活性提高20-30倍 干扰素(IFN)α和β或白介素2(IL-2)。一个 了解这一激活过程将通过以下方式来促进 检测干扰素或白介素2对基因表达的影响 增强NK功能。我们已经分离出56个cdna克隆 干扰素-β激活的NK细胞表达上调 干扰素-B诱导的NK细胞的差异性筛选 C DNA文库。其中46个克隆编码以前未描述过的基因 基因。其中一个被命名为NKLAM的新基因是由IL-2诱导的 在外周血NK和T细胞中，但也不表达 未受刺激或干扰素-β-，与已知的没有明显的同源性 基因。非常令人兴奋的是，对NK细胞的治疗 用NKLAM反义寡核苷酸完全废除这两种 未刺激和IL-2激活的细胞溶解活性。这是直接的 说明NKLAM参与了溶血过程。我们现在建议：1)评估 反义NKLAM在NK和T细胞介导的裂解中的作用 寡核苷酸和基因转移。反义寡核苷酸治疗 表达Bote正义和反义基因的细胞及NK和CTL细胞株 将评估构建体在靶细胞结合和裂解方面的变化， 颗粒胞吐和胞嘧啶的产生。NKLAM抗血清 蛋白质的产生将决定蛋白质的位置和结构 NKLAM的。我们将研究NKLAM RNA在NK和T细胞亚群中的表达 来自一组正常的捐赠者，使用各种细胞溶解增强剂和 抑制剂。NKLAM的基因组同源物将被分离，以便 定义它的遗传结构，并最终研究它的调节。 2)检测文库中其他独特基因的表达 RNA印迹法检测细胞毒作用。表达基因的cdna将被 已排序。将使用反义寡核苷酸来识别这些 潜在参与细胞溶解过程的基因。这些基因将 如NKLAM所述，可进一步确定其特征。 这项工作将为深入了解其分子机制提供帮助。 增强NK细胞溶解能力，这最终将有助于临床医生 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.