B CHAIN OF I-A: STRUCTURE AND FUNCTION

这项拨款提案的总体目标是了解哪种氨基酸 I-A不同功能作用的胞质（Cy）结构域 在对抗原（Ag）的免疫应答中。 这一目标将在以下两个方面实现： 体外和体内。 体外方法将使用定点诱变来引入氨基 在β链的Cy结构域中进行酸突变。 转染型B淋巴瘤 将测试表达突变I-A分子的细胞的能力， 跨膜信号，蛋白激酶c（PKC）的转位， 原子核 体外研究的第二个方面是测量 突变体I-A的翻译扩散（Dlat）的变化 分子，与野生型I-A分子相比。 变异分子 分析Dlat的变化将包含Dlat基因中的点突变， β链或α链的截短。 转移的能力 PKC与突变型I-A分子的Dlat之间存在相关性。 决赛 体外研究将调查β和α基因突变的作用， 链对I-A分子提呈Ag的能力有影响。 T细胞杂种 特异于鸡蛋溶菌酶肽将用于探测 转染子呈递肽Ag。 PKC变化的结果 易位、Dlat和呈递Ag的能力将是相关的。 这项拨款提案的体内方面将利用转基因小鼠 表达突变的I-A分子，具有两种不同的表型， 具有野生型Dlat值和改变的PKC易位动力学， 具有比野生型高3倍的Dlat值和改变的PKC易位 反应 利用这些小鼠，T淋巴细胞的Ag受体的亲和力 (TCR)将被探测。 Ag体外研究的基本原理 在这种情况下，突变的I-A分子更善于将Ag呈递给 表达高亲和力TCR的细胞。 使用转基因小鼠作为 环境中成熟的T淋巴细胞，然后产生T细胞杂交， 将允许评估TCR亲和力，其反映了TCR的亲和力。 第二种方法来衡量的变化， TCR库是分析胸腺细胞的Vbeta分布， 通过转基因胸腺成熟的成熟T淋巴细胞。 的 确定突变体I-A的影响的最终体内功能测定 分子的耐受性，将利用Mls系统。 一般来说，老鼠 在适当的单倍型背景上表达I-A将删除 表达V β 6作为其TCR的一部分的胸腺细胞。 在这些研究中， 将分析表达野生型或突变体I-A（如上所述）的细胞 因为他们有能力删除Vbeta 6。 这些综合研究将提供详细的生化和功能 有证据证明了I-A的β链和α链上的氨基酸， 这是Ia在Ag驱动的免疫应答中的作用所需的。

The general aim of this grant proposal is to understand which amino acid structures of the cytoplasmic (Cy) domains of I-A's varied functional roles in an immune response to antigen (Ag). This aim will be approached both in vitro and in vivo. The in vitro approach will use site-directed mutagenesis to introduce amino acid mutations into the beta chain's Cy domain. Transfected B lymphoma cells, expressing mutant I-A molecules will be tested for their ability to transmembrane signal, translocation of the enzyme protein kinase c (PKC) to the nucleus. The second aspect of the in vitro studies, will be to measure the changes in the translational diffusion (Dlat) of the mutant I-A molecules, compare to wildtype I-A molecules. Mutant molecules that will be analyzed for changes in Dlat will contain either point mutations in the beta chain or truncations of the alpha chain. The ability to translocate PKC and the Dlat of mutant I-A molecules will be correlated. The final in vitro studies will investigate the role the mutations in the beta and alpha chains have on the ability of I-A molecules to present Ag. T-cell hybrids specific for hen egg lysozyme peptides will be used to probe the ability of transfectants to present peptide Ag. The results of changes, in PKC translocation, Dlat and the ability to present Ag will be correlated. The in vivo aspect of this grant proposal will make use of transgenic mice that express mutant I-A molecules that have two distinct phenotypes, one has wildtype Dlat values and altered PKC translocation kinetics, the other has a Dlat value 3-fold above wildtype and an altered PKC translocation response. Using these mice, the affinity of the T lymphocyte's Ag receptor (TCR) will be probed. The rationale for the in vitro studies of Ag presentation is that mutant I-A molecules are better at presenting Ag to cells expressing high affinity TCR. Using the transgenic mice as an environment to mature T lymphocytes in and then generating T-cell hybrids, will allow an assessment of the TCR affinity that is a reflection of the type of I-A it was selected on. The second way to measure changes in the TCR repertoire is to analyze the Vbeta distribution of thymocytes and mature T lymphocytes that have matured through the transgenic thymi. The final in vivo functional assay to determine the affect of mutant I-A molecules on tolerance, will take advantage of Mls system. Normally, mice expressing I-A on an appropriate haplotype background will delete thymocytes that express Vbeta6 as part of their TCR. In these studies mice expressing either wildtype or mutant I-A (described above) will be analyzed for their ability to delete Vbeta6. These combined studies will provide detailed biochemical and functional evidence to identify the amino acids on the beta and alpha chain of I-A, required for Ia's role in an Ag driven immune response.