Molecular Analysis of CD8, MHC Class I Interaction

描述(申请人提供)：CD8辅受体是T细胞活化和发育的关键分子，与T细胞受体和多肽-MHC-I类(PMHC1)形成三分子复合体。由于CD8ab异源二聚体作为辅助受体的效率是CD8aa的100倍，我们正在研究CD8b在辅助受体功能中的作用。我们确定了免疫球蛋白样域CD8b蛋白与pMHC1结合所需的CDR样环上的关键残基，以及导致结合增强的突变体。基于我们的研究，我们提出了CD8ab与MHC I类分子相互作用的两种结合模式，由于缺乏结构信息，对CD8ab-pMHCI相互作用的分子基础的理解受到阻碍。在目标1中，我们将通过核磁共振光谱从结构上确定CD8ab和pMHCI是如何相互作用的，并验证我们的假设，即存在两种结合模式。在目标2中，我们将通过表面等离子体共振研究进行亲和力测量来进一步表征小鼠CD8b增强突变体的特征，并将使用CD8b-TCR相互作用的FRET分析来比较协同受体活性与激动剂、弱激动剂和拮抗剂多肽的增强作用。此外，我们将使用一种方法来鉴定人CD8b增强突变体，该方法包括对CDR-环中单个氨基酸残基进行随机突变，将质粒转入COS7细胞进行瞬时表达，以及FACS分选表达CD8b突变体的细胞，增强其与HLAI类四聚体的结合。与鼠CD8b不同，人CD8b蛋白有多种异构体，具有不同的细胞质尾巴，来自选择性剪接。我们发现四种人类CD8b剪接变异体(M1-4)在发育期间、激活后和记忆亚群的mRNA水平上存在差异表达。其中一种亚型(M-2)的表面表达受泛素化的调节。我们推测这些异构体对人类CD8 T细胞反应的不同调节有贡献。在目标3中，我们将研究异构体之间的功能差异，并确定这些差异的分子基础。不同的小鼠和人类细胞株将被使用，包括来自抗黑色素瘤克隆的人CD8T细胞和抗病毒巨细胞病毒特异性株。细胞质尾巴中暗示分子机制的基序，如磷酸化、二亮氨酸、Grb2结合，将通过在基序中产生突变并进行功能分析、与接头蛋白的结合、辅受体内化和信号转导来研究。拟议的研究结果将为CD8细胞毒性T细胞杀死肿瘤细胞和感染细胞内病原体的细胞提供关键的蛋白质-蛋白质相互作用CD8-pMHC1的见解。我们获得的信息将作为设计一种“优化的”CD8b的基础，该CD8b具有增强的与pMHC1的结合和/或可能用于肿瘤过继细胞免疫治疗的最佳信号。 公共卫生相关性：长期目标是分离一个增强的人类CD8b突变体，并确定不同细胞质尾巴的人CD8b剪接变异体的功能意义，以便设计一种优化的CD8b蛋白，可能用于过继细胞免疫治疗。

DESCRIPTION (provided by applicant): The CD8 coreceptor is a critical molecule for T cell activation and development forming a trimolecular complex with the T cell receptor, and peptide-MHC class I (pMHC1). As the CD8ab heterodimer is 100 fold more efficient than CD8aa as a coreceptor, we are studying the role of CD8b in coreceptor function. We identified critical residues on the CDR-like loops of the immunoglobulin-like domain CD8b protein required for binding to pMHC1 as well as mutants that led to enhanced binding. Based on our studies we proposed that CD8ab has two binding modes for interaction with MHC class I. Understanding the molecular basis for CD8ab-pMHCI interaction is hampered due to a lack of structural information. In aim 1, we will determine structurally how CD8ab and pMHCI interact by NMR spectroscopy and test our hypothesis that there are two binding modes. In aim 2, we will further characterize the murine CD8b enhancing mutants by making affinity measurements with surface plasmon resonance studies and will compare the enhancement of coreceptor activity with agonist, weak agonist, and antagonist peptides using FRET analysis of CD8b-TCR interaction. In addition, we will identify human CD8b enhancing mutants using an approach that involves random mutagenesis at individual amino acid residues in CDR- loops, transfection of plasmids into COS7 cells for transient expression, and FACS sorting for cells expressing CD8b mutants with enhanced binding to an HLA class I tetramer. The human CD8b protein has multiple isoforms with different cytoplasmic tails arising from alternative splicing, unlike murine CD8b. We found differential expression at the level of mRNA of the four human CD8b splice variants (M1-4) during development, after activation, and in memory subsets. and surface expression of one of the isoforms (M-2) was regulated by ubiquitination. We hypothesize that these isoforms contribute to differential regulation of CD8 T cell responses in humans. In aim 3, we will study functional differences between the isoforms and determine the molecular basis for those differences. Different murine and human cell lines will be used including human CD8 T cells from an anti-melanoma clone and an anti-viral cytomegalovirus specific line. Motifs within the cytoplasmic tails suggesting molecular mechanisms, e.g. phosphorylation, dileucine, Grb2 binding, will be studied by creating mutations in the motifs and performing functional assays, association with adaptor proteins, coreceptor internalization, and signal transduction. The outcome of the proposed studies will provide insights into a critical protein-protein interaction, CD8-pMHC1, for the CD8 cytotoxic T cells that kill tumor cells and cells infected with intracellular pathogens. The information we obtain will serve as a basis for designing an "optimized"CD8b with enhanced binding to pMHC1 and/or optimal signaling that potentially could be used for adoptive cellular immunotherapy against tumors. PUBLIC HEALTH RELEVANCE: The long-term objective is to isolate an enhanced human CD8b mutant, and to determine the functional significance of human CD8b splice variants with different cytoplasmic tails in order to design an optimized CD8b protein that potentially can be used for adoptive cellular immunotherapy.