New Tools for T Cell Identification and Analysis

大多数临床相关的血液样本的特征是可供分析的细胞数量有限。 结合低频率的感兴趣的T细胞群体。当前用于分析的方法 这些样本通常需要广泛的体外操作和/或关于抗原特异性的假设。 以及感兴趣的细胞群体的功能。这个项目的总体目标是开发新技术 这将改进对T细胞特异性和功能的体外分析，因为它利用了 生物信息学、蛋白质组学、蛋白质工程和阵列技术。有五个具体目标。目标1是 为了改进表位发现实践，通过开发基于生物信息学的表位预测算法，通过 应用质谱学的最新进展来鉴定自然加工的MHC结合肽，并通过 测量MHC-多肽的动力学寿命，而不是平衡结合亲和力。目标2是开发小说 MHC寡聚体将四聚体染色技术扩展到异种免疫的表征 中等亲和力和低亲和力T细胞的反应和特性。目标3是开发MHC多肽 阵列和ARC阵列，这些阵列将用于T细胞的功能表征后 抗原特异性捕获和/或激活。目标4是优化非特异性T细胞的体外扩增 扩增和抗原特异性刺激方案，并开发抗原特异性的新方法 丰富、扩增、永生化T细胞群。目标5是为以下方面开发方法学 高通量T细胞克隆和分析，包括微型培养方法和 高通量筛选方法在T细胞鉴定中的应用。一旦开发和使用 经过验证，这些技术将应用于T细胞鉴定和分析实验中 联合研究项目。

Most clinically relevant blood samples are characterized by a limited number of cells available for analysis combined with a low frequency of the T cell populations of interest. Current methodologies for analysis of these samples often require extensive in vitro manipulation and/or assumptions about the antigen specificity and function of the cell populations of interest. The overall goal of this project is to develop new technology that will improve ex vivo analysis of T cell specificity and function, by taking advantage of advances in bioinformatics, proteomics, protein engineering, and array technology. There are five specific aims. Aim 1 is to improve epitope discovery practice, by developing bioinformatics-based epitope prediction algorithms, by applying recent advances in mass spectrometry to identify naturally processed MHC-bound peptides, and by measuring MHC-peptide kinetic lifetimes rather than equilibrium binding affinities. Aim 2 is to develop novel MHC oligomers to extend tetramer staining technology to characterization of heterologous immune responses and characterization of moderate-affinity and low-avidity T cells. Aim 3 is to develop MHCpeptide arrays and ARC arrays, The arrays will be used for functional characterization of T cells after antigen-specific capture and/or activation. Aim 4 is to optimize ex vivo expansion of T celts using nonspecific expansion and antigen-specific stimulation protocols, and to develop new methods for antigenspecific enrichment, expansion, immortalization of T cell populations. Aim 5 is to develop methodology for high-throughput T cell cloning and analysis, including development of microscale culture methods and application of high-throughput screening methodology to T cell characterization. Once developed and validated, these technologies will be applied to T cell identification and analysis experiments in the associated Research Projects.