MECHANISMS OF BONE MARROW GRAFT REJECTION

描述：（改编自申请人的摘要）：本申请的目标 应用是为了阐明骨髓移植的机制， 在小鼠中被拒绝。 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方法设计 克服排斥反应以获得治疗益处。 本申请基于 在上一个项目期间取得的成果， 骨髓移植急性排斥反应的机制被证明是 可能是由于共表达NK 1和CD 3的细胞。 的目的 应用程序是测试几个假设，其中第一个是， NK 1 CD 3细胞表达特异性细胞毒性。 其他的是NK 1 CD 3 细胞在排斥反应中利用T细胞受体或NK细胞受体， 细胞溶解功能和MHC I类抗原被识别。 NK1+ 将通过荧光光度法从正常或裸鼠的脾脏中纯化CD 3+。 细胞分选 补充细胞因子以刺激细胞生长的培养物 将建立增殖和细胞毒性。细胞溶解活性 对肿瘤和淋巴母细胞靶点的影响， 特殊性 体外靶细胞裂解的特异性为 与骨髓排斥反应的特异性相比。 为了探索参与 TCR的F（ab '）2片段可阻断其功能。如果 当观察到抑制时，来自TCR转基因小鼠的NK 1 + CD 3+细胞将被 检查分离和细胞毒性特异性，并与 NK 1 + CD 3-细胞。 NK 1 + CD 3+细胞上NK受体的功能将 通过使用NK受体特异性的F）ab ′）2片段进行探测，即， NK1.1、Ly 49、5E 6来阻断靶识别和裂解。 NK1+ CD3+ 将测定NK 1 + CD 3-细胞对MHC靶点的细胞毒性 重组或转基因小鼠以定位特异性或转染子 表达多种MHC I类特异性。 MHC I类特异性 F（ab '）2片段将用于体外阻断以定位表位 特殊性 来自缺乏TCR转基因小鼠的NK 1 + CD 3+细胞 不同的TCR特异性将用于检查MHC-I类是否 抗原被识别以及它们的特异性是什么。 尝试将 干扰体内MHC I类分子的识别， NK 1 + CD 3+细胞的发育，并将研究这如何影响 骨髓移植排斥反应的特异性和 效应器 NK 1 + CD 3-细胞在骨髓排斥反应中的作用将被进一步研究。 通过探索SCID小鼠骨髓排斥反应的特异性来检查 如果它是MHC特异性的，它将被映射到特定的MHC， 地区 将分离来自SCID小鼠的NK 1 + CD 3-细胞，并进行特异性分析。 与骨髓排斥反应相比，靶细胞溶解的程度更低。 还将确定效应物的精确MHC表位特异性。 最后，正常人NK 1 + CD 3-和NK 1 + CD 3+细胞的特异性 将小鼠与来自TCR转基因小鼠的细胞进行比较， 研究正常小鼠的NK 1受体是否在 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.