MOLECULAR ANALYSIS OF CD8-MHC CLASS I INTERACTION

T细胞识别中最重要的相互作用之一涉及 T细胞受体(TCR)与CD8辅受体之间的相互作用 含肽的主要组织相容性复合体(MHC)I类 分子。这种互动对积极和消极都很重要 胸腺在T细胞分化和活化过程中的选择 外周血中的成熟T细胞。CD8由两个不同的 但紧密相连的基因阿尔法和贝塔会导致 细胞表面的α/α均二聚体或α/β异二聚体 或者是一种分泌分子。而TCR与多态的α1结合 和MHC I类的α2结构域，CD8α/α结合到 非多态的α3结构域。最近，该化合物的晶体结构 CD8α/α同源二聚体的溶解表明CD8具有结构 类似于免疫球蛋白。然而，目前尚不清楚CD8是否与 I类MHC以类似于免疫球蛋白的方式；即 对应于互补决定区域(CDR)的环。 我们发表的关于CD8α突变的研究表明，CDR1和CD8α CDR2循环对于绑定很重要。在这份提案中，我们计划 根据晶体对这些环路进行更详细的分析 结构和测试分子的其他区域，如CDR3和D-E 循环。另一个目的是确定CD8α/β异二聚体 能够绑定到MHC I类，如果是，请检查其中的差异 每种形式与MHC I类之间相互作用的亲和力。 分析CD8α与MHC相互作用的重要区域 第一类，我们计划对下列氨基酸进行突变分析 可访问的溶剂，位于循环中或位于 框架。此外，我们还将检验这样一个假设，即氨基酸 在CDR1和CDR2环中通过以下方式决定结合的物种特异性 用人类的循环取代了老鼠的循环。美国政府的影响 CD8α/α与任一类人类白细胞抗原结合能力的突变 I或非经典MHC分子人类白细胞抗原-G将使用 瞬时细胞-细胞黏附试验。这项检测还将被用于 通过共转染CD8α/β来确定CD8α/β能否与MHC I类分子结合 α链和嵌合的β-α链(细胞外)的cDNA 部分是β)进入COS7细胞，并询问抗CD8β抗体是否 抗体可以阻断。研究CD8的一个问题是， 当阿尔法和贝塔基因在同一细胞中表达时，两者 α/α和α/β二聚体将在细胞表面表达 (在没有配对的情况下，不能将Beta传输到细胞表面 Alpha)。为了避免口译的困难，我们将创建 这些分子的纯化的可溶形式。然后我们可以执行一个 Scatchard分析以确定结合到任一MHC类的亲和力 I与β2-微球蛋白或与 α3结构域中的负电荷环路。 T细胞在检测和摧毁病毒感染方面起着至关重要的作用 和恶性细胞。建议的工作将扩大我们对 免疫识别的基本机制，这是我们防御的关键部分 对抗癌症和感染。

One of the most important interactions in T cell recognition involves the interplay between the T cell receptor (TCR) and the CD8 coreceptor with a peptide-containing major histocompatibility complex (MHC) class I molecule. This interaction is important for both positive and negative selection in the thymus during T cell differentiation and for activation of mature T cells within the periphery. CD8 is encoded by two distinct but closely linked genes alpha and beta which give rise to either an alpha/alpha homodimer or an alpha/beta heterodimer on the cell surface or to a secreted molecule. While the TCR binds to the polymorphic alpha1 and alpha2 domains of MHC class I, CD8alpha/alpha binds to the nonpolymorphic alpha3 domain. Recently, the crystal structure of the CD8alpha/alpha homodimer was solved showing that CD8 has a structure similar to immunoglobulin. However, it is unclear whether CD8 binds class I MHC in a manner analogous to immunoglobulin; that is, with the loops corresponding to the complementarity determining regions (CDR). Our published work on mutations in CD8alpha indicate that the CDR1 and CDR2 loops are important for binding. In this proposal we plan to perform a more detailed analysis of these loops based on the crystal structure and test other regions of the molecule such as the CDR3 and D-E loops. Another aim is to determine whether the CD8alpha/beta heterodimer is capable of binding to MHC class I and if so, examine the difference in affinity of interaction between each form and MHC class I. To analyze the regions of CD8alpha important for interaction with MHC class I, we plan to perform a mutational analysis of amino acids that are solvent accessible and that are located either in loops or in the framework. In addition, we will test the hypothesis that the amino acids in the CDR1 and CDR2 loops determine species specificity of binding by replacing the murine loops with the human loops. The effect of the mutations on the ability of CD8alpha/alpha to bind to either HLA class I or the non-classical MHC molecule HLA-G will be tested using a transient cell-cell adhesion assay. This assay will also be used to determine if CD8alpha/beta can bind to MHC class I by cotransfecting the cDNAs for the alpha chain and a chimeric beta-alpha chain (extracellular portion is beta) into the COS7 cells and asking whether an anti-CD8beta antibody can block. One of the problems in studying CD8, is that whenever the alpha and beta genes are expressed in the same cell, both alpha/alpha and alpha/beta dimers will be expressed on the cell surface (beta cannot be transported to the cell surface without pairing with alpha). To avoid the difficulties in interpretation, we will create purified soluble forms of these molecules. We can then perform a Scatchard analysis to determine affinity of binding to either MHC class I with beta2-microglobulin or to a peptide corresponding to the negatively charged loop in the alpha3 domain. T cells play an essential role in detecting and destroying virus-infected and malignant cells. The work proposed will extend our understanding of the basic mechanisms of immune recognition, a crucial part of our defense against cancer and infection.