Peptide Binding to Class I MHC

描述（由申请人提供）：物理相互作用定义了TCR如何识别抗原，但不是机制。这种相互作用如何导致特定的T细胞反应尚不清楚。决定细胞溶解性T细胞（CTL）是否杀死抗原呈递细胞的主要关键事件是T细胞受体（TCR）和肽/MHC复合物（pMHC）之间的相互作用。我们已经观察到p1049/A2和AHIII 12.2 TCR的异反应性对以正交而不是对角线的方式停靠在其他TCK:pMHC对中。我们假设这种非正统的结合是由于AHIII12.2 T细胞对HLA-A2.1缺乏T细胞选择的结果。已经研究了一组与同基因MHC H-2Db结合并被AHIII12.2识别的肽。第二组被P14 T细胞识别的多肽也已经进行了测试。这两组肽和两个TCR将用于探测控制T细胞活性的生化参数。pMHC和TCR之间的亲和度（KD）或微观速率常数（koff或kon）是否是控制T细胞活性的关键因素，目前还存在重大分歧。我们假设，这些分歧源于迄今为止对pMHC与单个TCR结合常数的少量观察。本应用程序使用定义良好的T细胞模型系统和大量pMHC配体探索pMHC和TCR之间的物理相互作用。这种方法是强大的，因为要分析的pMHC样本量大，而且将使用一套跨学科的物理工具和免疫学分析方法。这些工具包括：蛋白质晶体学，表面等离子体共振（SPR）和定点诱变。生物物理测量将与T细胞反应相关，如：钙通量、增殖、细胞因子分泌和细胞溶解活性。我们将测试从TCR和pMHC的物理相互作用中得出的T细胞反应定量模型的假设。在Aim 1中，我们询问非对角线定向是否为未在限制性MHC上经历胸腺选择的T细胞所共有。在目标2中，pMHC和TCR之间的结合特性被发现是预测T细胞反应的研究。在Aim 3中，我们将观察扩展到自身免疫性糖尿病，并确定自身免疫性双反应性TCR是否以对角线方式与pMHC相互作用，就像A2与AHIII12.2结合时看到的那样。这些研究将被证明对自身免疫、慢性病毒感染、癌症和移植的免疫治疗有用。

DESCRIPTION (provided by applicant): Physical interactions define how a TCR recognizes an antigen, but not the mechanism. How that interaction results in specific T cell responses is unknown. The primary critical event that determines whether a cytolytic T cell (CTL) kills an antigen-presenting cell is the interaction between the T cell receptor (TCR) and the peptide/MHC complex (pMHC). We have observed that the xenoreactive pair of p1049/A2 and AHIII 12.2 TCR dock in an orthogonal rather than diagonal orientation seen in other TCK:pMHC pairs. We hypothesize that this unorthodox binding is a result of the absence of T cell selection on HLA-A2.1 by the AHIII12.2 T cell. A large panel of peptides that bind to the syngeneic MHC H-2Db and are recognized by AHIII12.2 has been examined. A second set of peptides, recognized by the P14 T cell, has also been tested. These two sets of peptides and two TCR will be used to probe the biochemical parameters that control T cell activity. There are significant disagreements as to whether affinity (KD) or microscopic rate constants (koff or kon) between pMHC and TCR are the key factors that control T cell activity. We hypothesize that the disagreements stem from the small number of observations made to date of pMHC binding constants to individual TCR. This application explores the physical interactions between pMHC and TCR using a well-defined T cell model systems and a large set of pMHC ligands. This approach is powerful because of the large sample size of pMHC to be assayed and because an interdisciplinary set of physical tools and immunological assays will be used. These tools include: protein crystallography, surface plasmon resonance (SPR) and site-directed mutagenesis. The biophysical measurements will be correlated with T cell responses such as: Ca flux, proliferation, cytokine secretion and cytolytic activity. A hypothesis for a quantitative model of T cell responses derived from the physical interactions of TCR and pMHC will be tested. In Aim 1, we ask whether non-diagonal orientations are common to T cells that have not gone through thymic selection on the restricting MHC. In aim 2, which binding properties between pMHC and TCR are found to be predictive of T cell responses is studied. In Aim 3, we extend our observations to autoimmune diabetes and determine if autoimmune dual-reactive TCR interact with pMHC in a diagonal fashion like that seen for A2 binding to AHIII12.2. These studies will prove to be useful in immunological therapy of autoimmunity, chronic viral infection, cancer and transplantation.