MECHANISMS OF BONE MARROW GRAFT REJECTION

描述：(改编自申请人的摘要)：这一目标 应用是为了阐明骨髓移植的机制 在小鼠体内被排斥。实现这一目标将有助于理解拒绝 人类同种异体骨髓移植的研究和治疗方法的设计 克服排斥反应，以获得治疗益处。此应用程序基于 关于在前一个项目期内取得的成果，在该项目期内 骨髓移植急性排斥反应的机制被证明是 可能是由于细胞同时表达NK1和CD3。这样做的目的是 应用程序是测试几个假设，第一个是 NK1 CD3细胞具有特异性的细胞毒作用。其他的是NK1 CD3 细胞利用T细胞受体或NK细胞受体进行排斥反应和 细胞溶解功能和MHC I类抗原被识别。NK1+ 用荧光法从正常或裸鼠的脾中提纯CD3+ 细胞分类。添加细胞因子以刺激细胞的培养 增殖和细胞毒性将被确定。细胞溶解活性 将对肿瘤和淋巴母细胞靶点进行检测以调查 具体细节。体外靶细胞裂解的特异性将是 与骨髓排斥反应的特异性相比。关于参与性的探讨 对于TCR，其功能将被抗TCR F(ab‘)2片段所阻断。如果 TCR转基因小鼠的NK1+CD3+细胞将受到抑制 检测分离的和细胞毒性的特异性，并与 NK1+CD3-细胞。NK受体在NK1+CD3+细胞上的作用 通过使用F)AB‘)2个针对NK受体的片段来探测，即， NK1.1、Ly49、5E6阻断目标识别和裂解。NK1+CD3+ 并将检测NK1+CD3-细胞对MHC靶点的细胞毒作用 重组或转基因小鼠以定位特异性或转染体 表达各种MHC I类特异性。特定于MHC I类 将F(ab‘)2片段用于体外封闭以映射表位 具体细节。缺乏TCR转基因小鼠的NK1+CD3+细胞 将使用不同的TCR特异性来检查MHC I类 识别抗原以及它们的特异性是什么。尝试次数将是 在体内干扰MHC I类分子的识别 NK1+CD3+细胞的发育及其影响 骨髓移植排斥反应的特异性和细胞毒性的特异性 效应器。NK1+CD3-细胞在骨髓排斥反应中的作用 探讨SCID小鼠骨髓排斥反应的特异性 如果它是特定于MHC的，它将被映射到特定的MHC 区域。SCID小鼠NK1+CD3-细胞的分离和特异性 靶细胞裂解与骨髓排斥反应的比较。 还将确定效应器的精确MHC表位特异性。 正常人NK1+CD3-和NK1+CD3+细胞的特异性 小鼠将与TCR转基因小鼠的细胞进行比较 检查正常小鼠的NK1受体是否可能在 急性骨髓移植排斥反应。

DESCRIPTION: (Adapted from the Applicant's abstract): The goal of this application is to elucidate mechanisms by which bone marrow grafts are rejected in mice. Achieving this goal will help understand the rejection of allogeneic marrow grafts in man and the design of approaches to overcome rejection for therapeutic benefit. This application is based on results obtained during the previous project period in which a mechanism responsible for acute rejection of marrow grafts was shown to be due to cells that co-expresses NK1 and CD3. The objective of this application is to test several hypotheses, the first of which is that NK1 CD3 cells express specific cytotoxicity. Others are that NK1 CD3 cells utilize T-cell receptors or NK cell receptors in rejection and cytolytic function and that MHC class I antigens are recognized. NK1+ CD3+ will be purified from spleen of normal or nude mice by fluorometric cell sorting. Cultures supplemented with cytokines to stimulate cell proliferation and cytotoxicity will be established. Cytolytic activity on tumor and lymphoblast targets will be assayed to investigate specificities. Specificity of in vitro target cell lysis will be compared to specificity of marrow rejection. To probe the participation of TCR, its function will be blocked by anti-TCR F(ab')2 fragments. If inhibition is seen, NK1+ CD3+ cells from TCR transgenic mice will be isolated and cytotoxic specificities examined and compared to those of NK1+ CD3- cells. The function of NK receptors on NK1+ CD3+ cells will be probed by using F)ab')2 fragments specific for NK receptors, i.e., NK1.1, Ly49, 5E6 to block target recognition and lysis. Both NK1+ CD3+ and NK1+ CD3- cells will be assayed for cytotoxicity on targets from MHC recombinant or transgenic mice to map specificity or transfectants expressing various MHC class I specificities. MHC class I specific F(ab')2 fragments will be used for in vitro blocking to map epitope specificities. NK1+ CD3+ cells from TCR transgenic mice lacking distinct TCR specificities will be used to examine whether MHC class I antigens are recognized and what their specificity is. Attempts will be made to interfere with recognition of MHC class I in vivo during development of NK1+ CD3+ cells and it will be examined how this affects specificity of marrow graft rejection and cytotoxic specificity of effectors. The role of NK1+ CD3- cells in marrow rejection will be examined by exploring the specificity of marrow rejection in SCID mice and in case it is MHC specific, it will be mapped to a specific MHC region. NK1+ CD3- cells from SCID mice will be isolated and specificity of target cell lysis compared with that of bone marrow rejection. Precise MHC epitope specificity of effectors will also be determined. Finally, the specificity of NK1+ CD3- and NK1+ CD3+ cells from normal mice will be compared with that of cells from TCR transgenic mice to examine whether in normal mice NK1 receptors may play a dominant role in acute marrow graft rejection.