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

项目摘要 疫苗是人类医学史上最伟大的发明之一。天花的根除 在1979年宣布，这一成功激发了人们继续努力开发疫苗， 其他毁灭性疾病，如艾滋病毒、癌症和流感大流行（1-4）。可惜 依赖于抗体应答的常规疫苗设计已经显示在这些疾病中是不充分的 这表明激活人类适应性免疫系统的另一个分支也很关键 - T细胞应答（5-7）。与医生可以常规监测的抗体反应不同， 没有明确的标准来告诉他们T细胞反应有多好（5，8-10）。因此，定义 这样的标准可以大大提高我们预测疫苗有效性的能力， 免疫监测标准。 定义健康T细胞应答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， 其特异性结合一种类型的T细胞（11）。由于荧光染料的固有限制， 同时测量超过15种不同的T细胞是非常困难的（12，13）。另外这款 方法也显示出有限的灵敏度- T细胞的丰度小于0.01%（约几十个T细胞）。 细胞）不能被可靠地检测。因此，本提案的目标是制定一个高度 多重和敏感的方法来分析T细胞库。具体来说，我们将标记 一种蛋白质复合物，将特定的T细胞与独特的DNA分子结合。因此，每一个T细胞都将 用独特的DNA序列“条形码化”，然后可以使用DNA芯片检测。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 几个数量级，这要归功于DNA杂交的精确特异性和高 DNA芯片的吞吐量数据处理（可以分析数百万种不同的DNA分子， 使用单个芯片并行）。此外，检测灵敏度可以通过以下方式显著提高： 包括DNA扩增步骤（仅从一个拷贝就可以获得10亿个DNA分子 在芯片分析之前的几个小时内）。该方法的成功开发将使我们能够 可以同时检测多达数万种不同的T细胞，也可以只检测一种T细胞。

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.