ACCESSORY MOLECULES--ANTIGEN-SPECIFIC T-CELL ACTIVATION

辅助分子——抗原特异性 T 细胞激活

• 批准号: 3307494
• 负责人: ADRIENNE A BRIAN
• 金额: $ 20.51万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307494
• 关键词: B lymphocyte; T cell receptor; T lymphocyte; antigen presentation; antigen presenting cell; athymic mouse; cell adhesion; cell adhesion molecules; clone cells; genetic library; genetically modified animals; hamsters; hybridomas; immunologic memory; integrins; laboratory mouse; laboratory rat; leukocyte activation /transformation; leukocyte adhesion molecules; monoclonal antibody; radioimmunoassay; receptor binding; receptor expression; transfection

The leukocyte integrin LFA-1 is critically involved in antigen-specific activation of T cells and B cells and in lymphocyte recirculation. In addition to its function as an adhesion receptor, LFA-1 appears to have a role in enhancing T cell activation, and T cell activation modulates the adhesion function of LFA-1. The chief aims of this project are (I) to characterize the equilibrium and kinetic properties of the LFA-1-ICAM-1 interaction, (II) to examine the function of ICAM-1 expression on T cells with respect to recognition of antigen and T cell activation, and (III) to develop reagents, especially monoclonal antibodies, for the study in murine antigen-specific systems, of other LFA-1 ligands. (I) The molecular basis for the modulation of LFA-1 adhesion during activation is unknown, but a possible mechanism is a transition to a conformation with an increased affinity for the LFA-1 counter-receptor, ICAM-1. The equilibrium and kinetic properties of the LFA-1-ICAM-1 interaction on resting and activated lymphocytes will be determined by using a soluble recombinant form of the ICAM-1 molecule in competitive binding assays in which ICAM-1 competes with radiolabeled Fab fragments of anti-LFA-1 monoclonal antibodies, or with radiolabeled ICAM-1, for binding to LFA-1 on cells. This approach has been adopted because of results from preliminary measurements that indicate that the affinity is low. The equilibrium and kinetic measurements will be used to determine (1) if LFA-1 on resting and activated lymphocytes differs in binding to ICAM-1 and (2) if parameters that affect adhesion, such as temperature, divalent metal ion concentration and presence of metabolic inhibitors, have direct effects on the LFA-1-ICAM-1 interaction or affect post-receptor events. (II) To examine the significance of ICAM-1 expression on T cells, cDNAs for the alpha and beta chains on LFA-1 and Mac-1 will be transfected into fibroblast cell lines that have been previously transfected with class II MHC genes. These fibroblast antigen- processing cell lines that express LFA-1, Mac-1, or both integrins will then be analyzed in functional assays for their ability to activate antigen-specific T cell lines. To determine their efficacy in activation of unprimed naive T cells the ability of the integrin-expressing fibroblast lines to activate T cells from T cell receptor transgenic mice will be analyzed. The binding of soluble ICAM-1 to these cell lines will be measured and these data, along with the functional data, will be examined for evidence that LFA-1 and Mac-1 act in a concerted fashion. (III) To determine the role of other LFA-1 ligands in antigen recognition, monoclonal antibodies against murine homologs of recently identified human FLA-1 counter receptors will be derived by immunizing hamsters with purified human proteins and selecting clones that cross-react on mouse. These reagents will be characterized and used in expression and functional studies using antigen-specific T cell clones and T cells from TCR transgenic mice.

白细胞整合素LFA-1在抗原特异性 T细胞和B细胞的活化以及淋巴细胞再循环。在 除了作为粘附受体的功能外，LFA-1似乎还具有 在增强T细胞活化中的作用，而T细胞活化调节 LFA-1的粘附功能。 该项目的主要目标是（一） 表征LFA-1-ICAM-1的平衡和动力学性质 相互作用，（II）检测T细胞上ICAM-1表达的功能 关于抗原识别和T细胞活化，和（III） 开发试剂，特别是单克隆抗体，用于小鼠的研究 其他LFA-1配体的抗原特异性系统。 (I)的分子基础 在活化过程中LFA-1粘附的调节是未知的，但 可能的机制是向构象的转变， 对LFA-1反受体ICAM-1的亲和力。 平衡位置以及 LFA-1-ICAM-1相互作用对静息和活化的 将通过使用可溶性重组形式的 ICAM-1分子在竞争性结合测定中的作用，其中ICAM-1与 放射性标记的抗LFA-1单克隆抗体的Fab片段，或与 放射性标记的ICAM-1，用于结合细胞上的LFA-1。 这种方法已经 由于初步测量结果表明， 亲和力低。 将使用平衡和动力学测量 为了确定（1）静息和活化淋巴细胞上的LFA-1是否在以下方面不同： 与ICAM-1的结合和（2）如果影响粘附的参数，例如 温度、二价金属离子浓度和代谢产物的存在 抑制剂，对LFA-1-ICAM-1相互作用有直接作用或影响 受体后事件 (II)为了检测ICAM-1的意义， T细胞上的表达、LFA-1上α和β链的cDNA以及 Mac-1将被转染到已被转染的成纤维细胞系中。 先前用II类MHC基因转染。 这些成纤维细胞抗原- 处理表达LFA-1、Mac-1或这两种整联蛋白的细胞系将 然后在功能测定中分析它们激活 抗原特异性T细胞系。 为了确定它们在激活 未引发的幼稚T细胞的整合素表达成纤维细胞的能力， 将使用来自T细胞受体转基因小鼠的活化T细胞的细胞系， 分析了 可溶性ICAM-1与这些细胞系的结合将是 这些数据将与功能数据一起进行沿着检查 寻找LFA-1和Mac-1协同作用的证据。 (III)到 确定其他LFA-1配体在抗原识别中的作用， 抗最近鉴定的人的鼠同源物的单克隆抗体 弗拉-1反受体将通过用以下物质免疫仓鼠来衍生： 纯化的人蛋白质和选择在小鼠上交叉反应的克隆。 这些试剂将被表征并用于表达和功能分析。 使用抗原特异性T细胞克隆和来自TCR的T细胞的研究 转基因小鼠。