SERINE ESTERASES IN HUMAN CYTOLYTIC LYMPHOCYTES

这项提议的目标是分离和鉴定基因 编码人类自然杀伤细胞丝氨酸酯酶(SE)和 为了探讨细胞毒性T淋巴细胞(CTL)的作用 这些分子在细胞内具有细胞毒性。它们的作用机制 NK细胞和CTL破坏其细胞靶点具有根本意义 对于我们理解细胞免疫的重要性。细胞 杀戮涉及杀手和目标之间的接触和捆绑 在大多数情况下，细胞分泌的内容物 成熟的细胞毒性淋巴细胞可见大的胞浆颗粒。 这些颗粒含有几种有潜力的多肽。 参与细胞溶解，包括一种形成孔洞的蛋白， 另一种抗原性与肿瘤相关的细胞毒素--穿孔素 坏死因子、淋巴毒素、溶核因子(S)以及 几种不同的丝氨酸蛋白酶(SE)。企业系统工程的功能 细胞毒细胞是未知的，但有一种可能性是它们可能 构成其他细胞毒素的细胞内激活途径 类似于细胞外看到的分子(如穿孔素) 与补体途径的自体反应有关。到目前为止，多达8个 不同但结构上相关的SE已从 然而，小鼠细胞毒性淋巴细胞，几乎没有任何信息 关于人类中相应的分子是可用的。因此， 我们建议：#1)克隆和鉴定cdna和基因组克隆 编码人类NK细胞和CTL的SE；#2)SE的特征 利用原核表达载体纯化杂交瘤蛋白 蛋白质并制备兔抗血清；#3)研究表达 人类淋巴细胞亚群中SE基因的差异 细胞毒性和非细胞毒性激活过程中的表达 淋巴细胞，以及参与这些基因调控的因素； #4)以具有生物活性的形式表达个体SE 详细的功能研究。

The objective of this proposal is to isolate and characterize genes encoding serine esterases (SEs) from human natural killer (NK) and cytotoxic T-lymphocytes (CTL) in order to investigate the role of these molecules in cellular cytotoxicity. The mechanisms by which NK cells and CTLs destroy their cellular targets is of fundamental importance for our understanding of cell mediated immunity. Cell killing involves contact and binding between killer and target cells followed in most instances by secretion of the contents of large cytoplasmic granules from mature cytotoxic lymphocytes. These granules contain several polypeptides with potential involvement in cytolysis including a pore-forming protein, perforin, another cytotoxin which is antigenically related to tumor necrosis factor and lymphotoxin, nucleolytic factor(s) as well as several different serine proteases (SEs). The functions of SEs in cytotoxic cells are unknown, but one possibility is that they may constitute an intracellular activation pathway for other cytotoxic molecules (e.g., perforin) analogous to that seen extracellularly with the SEs of the complement pathway. So far, as many as eight different but structurally related SEs have been isolated from murine cytotoxic lymphocytes, however, little if any information is available regarding the corresponding molecules in man. Thus we propose to: #1) Clone and characterize cDNA and genomic clones encoding SEs from human NK cells and CTLs; #2) characterize SE proteins using prokaryotic expression vectors, purify hybrid proteins and produce rabbit antisera; #3) Investigate expression of SE genes in human lymphocyte subsets, their differential expression during activation of cytotoxic and non-cytotoxic lymphocytes, and factors involved in the regulation of these genes; #4) Express individual SE in a biologically active form for detailed functional studies.