New Tools for T Cell Identification and Analysis

T 细胞识别和分析的新工具

• 批准号: 7701546
• 负责人: Lawrence J. Stern
• 金额: $ 72.6万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7701546
• 关键词: Affinity; Algorithms; Animals; Antigens; Area; Avidity; Binding; Bioinformatics; Biological Assay; Biological Models; Blood specimen; Categories; Cell Count; Cell Culture Techniques; Cell physiology; Cellular Immunity; Cloning; Development; Elements; Epitopes; Equilibrium; Flavivirus; Frequencies; Goals; Hantavirus; Herpesviridae Infections; Human; Immune response; Immune system; Immunity; In Vitro; Kinetics; Leukocytes; Lymphocyte Function; MHC Class II Genes; MHC binding peptide; Mass Spectrum Analysis; Measures; Methodology; Methods; Peptide/MHC Complex; Peptides; Population; Poxviridae; Process; Protein Engineering; Proteins; Proteomics; Protocols documentation; Research Project Grants; Sampling; Screening procedure; Specificity; Staining method; Stains; Study models; System; T-Cell Immunologic Specificity; T-Lymphocyte; T-Lymphocyte Epitopes; Technology; Testing; Vaccination; Virus; Virus Diseases; antigen processing; base; biodefense; clinically relevant; high throughput screening; human disease; improved; influenzavirus; interest; new technology; novel; novel therapeutics; pathogen; peripheral blood; programs; research study; response; technology development; tool

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.

大多数临床相关的血液样本的特征是有限的细胞可用于分析 结合较低的感兴趣的T细胞群体。当前分析方法 这些样品通常需要广泛的体外操作和/或关于抗原特异性的假设 感兴趣的细胞群体的功能。该项目的总体目标是开发新技术 这将通过利用进步来改善T细胞特异性和功能的体外分析 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 通过开发基于生物信息学的表位预测算法来改善表位发现实践， 应用质谱法的最新进展来识别自然加工的MHC结合肽，并通过 测量MHC肽动力学寿命，而不是平衡结合亲和力。目标2是开发小说 MHC低聚物将四聚体染色技术扩展到异源免疫的表征 中等亲和力和低野合T细胞的反应和表征。目标3是开发MHCPEPTIDE 阵列和电弧数组，阵列将用于T细胞的功能表征 抗原特异性捕获和/或激活。 AIM 4是使用非特异性优化T CELTS的离体扩展 扩展和抗原特异性刺激方案，并开发新方法的抗原特异性方法 T细胞种群的富集，扩张，永生化。目标5是开发方法 高通量T细胞克隆和分析，包括显微镜培养方法的发展和 将高通量筛选方法应用于T细胞表征。一旦开发了 经过验证，这些技术将应用于T细胞识别和分析实验 相关研究项目。