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）和CD 8辅助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。 这种相互作用对积极和消极都很重要 T细胞分化和活化过程中胸腺中的选择 成熟的T细胞。 CD 8由两个不同的 但紧密相连的基因α和β， α/α同二聚体或α/β异二聚体 或分泌的分子。 而TCR结合多态性α 1 和α 2结构域的MHC I类，CD 8 α/α结合到 非多态性α 3结构域。 最近，晶体结构的 解析CD 8 α/α同源二聚体，表明CD 8具有以下结构： 类似于免疫球蛋白。 然而，目前尚不清楚CD 8是否结合 I类MHC以类似于免疫球蛋白的方式;即， 对应于互补决定区（CDR）的环。 我们已发表的关于CD 8 α突变的研究表明，CDR 1和CDR 2的表达与CD 8 α的表达有关。 CDR 2环对于结合是重要的。 在本提案中，我们计划 基于晶体对这些回路进行更详细的分析 结构和测试分子的其他区域，如CDR 3和D-E 循环 另一个目的是确定CD 8 α/β异二聚体是否 能够与MHC I类结合，如果是，检查 在每种形式和MHC I类之间的相互作用的亲和力。 分析CD 8 α与MHC相互作用的重要区域 I类，我们计划进行氨基酸的突变分析， 溶剂可接近的，并且位于环中或环中。 框架. 此外，我们将检验氨基酸 在CDR 1和CDR 2环中， 用人的环取代鼠的环。 的影响 CD 8 α/α结合任一HLA类别的能力的突变 I或非经典MHC分子HLA-G将使用 瞬时细胞-细胞粘附测定。 该试验还将用于 确定是否CD 8 α/β可以结合MHC I类，通过连接 α链和嵌合β-α链的cDNA（细胞外 部分是β）进入COS 7细胞，并询问抗CD 8 β 抗体可以阻断。 研究CD 8的一个问题是， 当α和β基因在同一细胞中表达时， α/α和α/β二聚体将在细胞表面表达 (beta不能被运送到细胞表面而不与 α）。 为了避免解释上的困难，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分析以确定与任一MHC类别的结合亲和力 I与β 2-微球蛋白或与β 2-微球蛋白对应的肽结合。 在α 3结构域中的带负电荷的环。 T细胞在检测和破坏病毒感染中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以下问题的理解： 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.