Deciphering T cell repertoire using DNA-barcoded MHC-peptide tetramers

使用 DNA 条形码 MHC 肽四聚体破译 T 细胞库

• 批准号: 8622232
• 负责人: Fei Wen
• 金额: $ 7.06万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622232
• 关键词: Alleles; Amines; Antibody Formation; Antigens; Binding; Biological Markers; Biotin; Caucasians; Caucasoid Race; Cells; Cellular Immunity; Communicable Diseases; Complex; Crosslinker; Cyclohexanes; Cysteine; Cytomegalovirus; DNA; DNA Microarray Chip; DNA Sequence; DNA amplification; Databases; Detection; Development; Disease; Dyes; Economic Burden; Elements; Endopeptidase K; Epitopes; Flow Cytometry; Fluorescent Dyes; Fluorescent Probes; Gene Frequency; Goals; HIV; History of Medicine; Hour; Housing; Human; Immune system; Immunity; Immunologic Monitoring; Incidence; Infection; Insecta; Invaded; Label; Lead; Major Histocompatibility Complex; Malignant Neoplasms; Measurement; Measures; Methods; Modern Medicine; Molecular Sieve Chromatography; Monitor; Nucleotides; Oligonucleotides; One-Step dentin bonding system; Peptide/MHC Complex; Peptides; Phycoerythrin; Production; Proteins; Reagent; Reporting; Research; Site; Smallpox; Specificity; Staining method; Stains; Streptavidin; System; T cell response; T-Cell Immunologic Specificity; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; Time; Vaccination; Vaccine Design; Vaccines; Validation; Work; arm; base; carboxylate; computerized data processing; cyanine dye 5; cytokine; flexibility; flu; gel mobility shift assay; improved; influenzavirus; interest; mutant; pandemic disease; pandemic influenza; pathogen; protein complex; protein expression; public health relevance; response; scale up; stem; success; vaccine effectiveness

Project Summary Vaccination is one of the greatest inventions in human medicine history. The eradication of smallpox was announced in 1979 and this success inspired continuing efforts in developing vaccines against other devastating diseases, such as HIV, cancer, and influenza pandemics (1-4). Unfortunately, the conventional vaccine design relying on antibody responses has been shown to be inadequate in these cases, suggesting that it is also critical to activate the other arm of the human adaptive immune system - T cell responses (5-7). Unlike antibody responses that could be routinely monitored by doctors, there is no defined standard for them to tell how good a T cell response is (5, 8-10). Therefore, the definition of such a standard could greatly improve our ability to predict vaccine effectiveness and help set new standards for immune monitoring. One major impediment to defining a healthy T cell response is that there is no high throughput method available to measure how many different T cells (each recognizes a specific pathogenic peptide) are activated, in other words, the breadth of the T cell response. The current best method for measuring different T cells simultaneously uses a mixture of fluorescent protein complexes (tetramers), each of which specifically binds one type of T cell (11). Due to the intrinsic limitations of the fluorescent dyes, it is very difficult to measure more than 15 different T cells simultaneously (12, 13). In addition, this method also showed limited sensitivity - T cells with less than 0.01% abundance (~ a few dozens of T cells) could not be reliably detected. Therefore, the goal of this proposal is to develop a highly multiplexed and sensitive method to profile the T cell repertoire. Specifically, we will tag the protein complex that binds a specific T cell with a unique DNA molecule. As a result, each T cell will be "barcoded" with a unique DNA sequence, which can then be detected using a DNA chip. The use of DNA as barcode offers us the potential to improve the multiplexed detection capacity of different T cells by several orders of magnitude, thanks to the exquisite specificity of DNA hybridization and high throughput data processing of DNA chips (millions of different DNA molecules can be analyzed in parallel using a single chip). In addition, the detection sensitivity could be significantly improved by including a DNA amplification step (one billion DNA molecules can be obtained from just a single copy within hours) before chip analysis. The successful development of the proposed method will enable us to detect as many as tens of thousands of different T cells simultaneously, and as few as just one T cell.

项目概要 疫苗是人类医学史上最伟大的发明之一。消灭天花 于 1979 年宣布，这一成功激发了人们继续努力开发针对该疾病的疫苗 其他毁灭性疾病，例如艾滋病毒、癌症和流感大流行 (1-4)。不幸的是， 依赖抗体反应的传统疫苗设计已被证明在这些方面是不够的 案例表明激活人类适应性免疫系统的另一臂也至关重要 - T 细胞反应 (5-7)。与医生可以常规监测的抗体反应不同， 对于他们来说，没有明确的标准来判断 T 细胞反应有多好 (5, 8-10)。因此，定义 这样一个标准可以极大地提高我们预测疫苗有效性的能力，并帮助制定新的疫苗有效性标准。 免疫监测标准。 定义健康 T 细胞反应的一个主要障碍是没有高通量方法 可用于测量有多少不同的 T 细胞（每个细胞识别特定的致病肽） 换句话说，激活了 T 细胞反应的广度。目前最好的测量方法 不同的 T 细胞同时使用荧光蛋白复合物（四聚体）的混合物，每个 它特异性结合一种类型的 T 细胞 (11)。由于荧光染料本身的局限性， 同时测量超过 15 个不同的 T 细胞是非常困难的 (12, 13)。此外，这 该方法还显示出有限的灵敏度 - T 细胞丰度低于 0.01%（~几十个 T 细胞）无法可靠地检测到。因此，本提案的目标是开发一个高度 用于分析 T 细胞库的多重且灵敏的方法。具体来说，我们将标记 将特定 T 细胞与独特 DNA 分子结合的蛋白质复合物。结果，每个 T 细胞将 用独特的 DNA 序列“条形码”，然后可以使用 DNA 芯片进行检测。使用 DNA 作为条形码为我们提供了提高不同 T 细胞多重检测能力的潜力 得益于 DNA 杂交的精确特异性和高 DNA 芯片的吞吐量数据处理（可以分析数百万个不同的 DNA 分子） 使用单芯片并行）。此外，通过以下方法可以显着提高检测灵敏度： 包括 DNA 扩增步骤（仅从单个拷贝中即可获得十亿个 DNA 分子） 数小时内）在芯片分析之前。所提出方法的成功开发将使我们能够 同时检测多达数万个不同的 T 细胞，少至仅一个 T 细胞。