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.

大多数临床相关血液样本的特征在于可用于分析的细胞数量有限 结合低频率的感兴趣的T细胞群体。目前的分析方法 这些样品通常需要大量的体外操作和/或关于抗原特异性的假设 和功能。该项目的总体目标是开发新技术 这将改善T细胞特异性和功能的离体分析， 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， 应用质谱分析的最新进展来鉴定天然加工的MHC结合肽， 测量MHC-肽动力学寿命而不是平衡结合亲和力。目标2：开发新的 MHC寡聚体用于扩展四聚体染色技术以表征异源免疫 中等亲和力和低亲和力T细胞的应答和表征。目的3：研制MHC多肽 阵列和ARC阵列，这些阵列将用于T细胞的功能表征， 抗原特异性捕获和/或活化。目的4是使用非特异性优化T细胞的离体扩增 扩增和抗原特异性刺激方案，并开发抗原特异性 T细胞群的富集、扩增、永生化。目标5是制定方法， 高通量T细胞克隆和分析，包括微规模培养方法的开发， 高通量筛选方法在T细胞表征中的应用。一旦开发出来， 经过验证，这些技术将应用于T细胞鉴定和分析实验中， 相关研究项目。