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T细胞(CTL)是否杀死抗原提呈细胞的主要关键事件是T细胞受体(TCR)和多肽/MHC复合体(PMHC)之间的相互作用。我们观察到p1049/A2和AhiII 12.2TCR的异种反应对在其他TCK：pMHC对中看到的是垂直的而不是对角的对接。我们推测，这种非常规结合是由于AHIII12.2T细胞缺乏对HLA-A2.1的T细胞选择所致。研究了一大组与同基因MHC H-2DB结合并被AHIII12.2识别的多肽。另一组由P14 T细胞识别的多肽也已经进行了测试。这两组多肽和两个TCR将被用来探索控制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)是控制T细胞活性的关键因素，存在着显著的分歧。我们假设，这些分歧源于到目前为止观察到的少量pMHC与单个TCR的结合常数。这个应用程序使用定义明确的T细胞模型系统和大量的pMHC配体来探索pMHC和TCR之间的物理相互作用。这种方法是有效的，因为要检测的pMHC样本量很大，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：蛋白质结晶学、表面等离子共振(SPR)和定点突变。生物物理测量将与T细胞反应相关，如：CA流量、增殖、细胞因子分泌和细胞溶解活性。从TCR和pMHC的物理相互作用中推导出T细胞反应的量化模型的假设将被检验。在目标1中，我们询问未通过限制性MHC上胸腺选择的T细胞是否是常见的非对角取向。在目的2中，研究了pMHC和TCR之间的结合特性对T细胞应答的预测作用。在目标3中，我们将观察扩展到自身免疫性糖尿病，并确定自身免疫双反应TCR是否以对角方式与pMHC相互作用，就像A2与AHIII12.2结合一样。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.