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异性T细胞反应尚不清楚。决定溶细胞性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的异种反应性对以正交方向而不是在其他TCK：pMHC对中看到的对角方向对接。我们假设这种非常规结合是由于AHIII12.2 T细胞对HLA-A2.1缺乏T细胞选择性的结果。已经检查了与同基因MHC H-2Db结合并被AHIII 12.2识别的大量肽。还测试了由P14 T细胞识别的第二组肽。这两组肽和两种TCR将用于探测控制T细胞活性的生化参数。对于pMHC和TCR之间的亲和力（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存在显著分歧。我们推测，分歧源于少数的观察，迄今为止的pMHC结合常数个别TCR。本申请使用定义明确的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，因为大样本量的pMHC进行测定，因为一个跨学科的物理工具和免疫学测定集将被使用。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细胞应答相关，例如：Ca通量、增殖、细胞因子分泌和细胞溶解活性。将检验从TCR和pMHC的物理相互作用导出的T细胞应答的定量模型的假设。在目标1中，我们问非对角线方向是否是共同的T细胞，没有经过胸腺选择的限制性MHC。在目的2中，研究了pMHC和TCR之间的结合特性被发现是T细胞应答的预测。在目标3中，我们将我们的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是否与pMHC以对角方式相互作用，就像A2与AHIII 12.2结合一样。这些研究将为自身免疫、慢性病毒感染、肿瘤和移植等疾病的免疫治疗提供参考。

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.