SERINE ESTERASES IN HUMAN CYTOLYTIC LYMPHOCYTES

这项建议的目的是分离和表征基因 编码来自人自然杀伤（NK）的丝氨酸酯酶（SE），和 细胞毒性T淋巴细胞（CTL），以研究的作用 这些分子的细胞毒性。 的机制 NK细胞和CTL破坏它们的细胞靶是最基本的 对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结合 在大多数情况下，细胞分泌的内容物 成熟细胞毒性淋巴细胞的大细胞质颗粒。 这些颗粒含有几种多肽， 参与细胞溶解，包括孔形成蛋白， 穿孔素是另一种与肿瘤抗原相关的细胞毒素 坏死因子和光敏素、溶核因子以及 几种不同的丝氨酸蛋白酶（SE）。 社会企业的职能 细胞毒性细胞是未知的，但一种可能性是，他们可能 构成其它细胞毒性的细胞内活化途径 分子（例如，穿孔素）类似于细胞外所见 与补体途径的SE结合。 到目前为止，多达8个 不同但结构相关的SE已从 然而，小鼠细胞毒性淋巴细胞，几乎没有任何信息， 在人体内的相应分子。因此， 我们建议：#1）克隆和表征cDNA和基因组克隆 编码来自人NK细胞和CTL的SE;#2）表征SE 蛋白质使用原核表达载体，纯化杂合 蛋白质并产生兔抗血清;#3）研究表达 人淋巴细胞亚群中SE基因的表达及其差异表达 在细胞毒性和非细胞毒性的活化期间表达 淋巴细胞和参与这些基因调节的因子; #4）以生物活性形式表达个体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.