Core D: Antigen Receptor Identification and Tracking Core

项目摘要 U19的总体目标是通过研究PD-1途径在人类免疫中的作用 治疗性PD-1阻断对慢性病毒免疫应答的影响及预防 疫苗(项目2)。为了监测患者的获得性免疫反应，我们需要跟踪复合体 T和B细胞的谱系，因为克隆类型可能随着时间的推移而表现不同，并且对感染和 心理治疗。虽然有几种追踪抗原特异性淋巴细胞的方法，但大量的血液是 由于需要监测多种抗原，活组织检查用于标准管道和克隆类型的材料有限。 与不同的抗原受体没有区别。TCR和BCR的标准批量测序可以提供 数量克隆型频率；然而，它的用途是有限的，因为靶抗原不是已知的 无法确定每个TCR/BCR以及每个克隆型的激活状态。核心D提供了 用于跟踪患者血液和组织中的抗原特异性T和B细胞的创新服务，允许项目1 2对淋巴细胞的频率、抗原特异性和活化状态进行定量。AIM 1提供了一个 通过对患者T、B细胞的抗原受体测序进行TCR和BCR与抗原配对的方法 用病毒或疫苗抗原刺激或标记的。AIM 2使用更具成本效益的批量TCR-和BCR-seq 为了量化接受抗PD-1治疗的患者系列样本中克隆类型的频率，但 目标1中的结果将来自谱系的抗原受体与特定病毒或疫苗连接的优势 抗原。Aim 3使用前沿液滴为基础的rna-seq对成对抗原受体链(tcrα)进行测序 &β；IGH和IgK/L)以及单个淋巴细胞中数以千计的mRNAs，将功能通路与 抗原受体。来自三个AIMS的结果数据集将被集成以提供频率， T和B细胞的抗原特异性和激活状态，使我们能够检验抗PD-1的假设 治疗对每个淋巴细胞亚群的增殖和激活有不同的影响，取决于其 抗原特异性和治疗前分化状态。简而言之，核心D使得能够监测抗原- 特定的淋巴细胞以前所未有的分辨率，并将有助于剖析免疫保护的变化 慢性感染(包括乙肝病毒、丙型肝炎病毒、巨细胞病毒和EB病毒)，以及抗PD-1抗体导致的流感疫苗接种 心理治疗。这些方法的建立和完善以及核心D基础设施的创建将 能够询问免疫机制，并提供对免疫的发展和持久性的见解 在检查站封锁或任何其他免疫疗法的背景下的免疫记忆。

Project Summary The overall objective of the U19 is to discover the role of the PD-1 pathway in human immunity by studying the effects of therapeutic PD-1 blockade on immune responses to chronic viruses (Project 1) and preventive vaccines (Project 2). To monitor adaptive immune responses in patients, we need to track the complex repertoire of T and B cells since clonotypes may behave differently over time and in response to infection and therapy. While there are several methods for tracking antigen-specific lymphocytes, large volumes of blood are needed for monitoring multiple antigens, biopsies have limited material for standard pipelines, and clonotypes with distinct antigen receptors are not distinguished. Standard bulk sequencing of TCRs and BCRs can provide quantitative clonotype frequencies; however, its utility is limited because the target antigens are not known for each TCR/BCR and the activation states of each clonotype cannot be determined. Core D provides an innovative service for tracking antigen-specific T and B cells in patient blood and tissues, allowing Projects 1 and 2 to quantify the frequency, antigen specificity and activation states of lymphocytes. Aim 1 provides an approach to match TCRs and BCRs to antigens by sequencing antigen receptors in patient T and B cells stimulated or tagged with viral or vaccine antigens. Aim 2 uses the more cost-effective bulk TCR- and BCR-seq to quantify the frequencies of clonotypes in serial samples of patients treated with anti-PD-1 therapy, but takes advantage of the results in Aim 1 to link antigen receptors from the repertoire with specific viral or vaccine antigens. Aim 3 uses leading-edge droplet-based RNA-seq to sequence paired antigen receptor chains (TCRα & β; IgH & IgK/L) along with thousands of mRNAs in single lymphocytes, linking functional pathways with antigen receptors. The resulting dataset from the three Aims will be integrated to provide the frequencies, antigen specificities and activation states of T and B cells, allowing us to test the hypothesis that anti-PD-1 therapy differentially impacts the proliferation and activation of each lymphocyte subset depending on its antigen specificity and pre-therapy differentiation state. In short, Core D enables the monitoring of antigen- specific lymphocytes at unprecedented resolution, and will help dissect changes in immune protection against chronic infections (including HBV, HCV, CMV, and EBV), and influenza vaccination as result of anti-PD-1 therapy. The establishment and refinement of these approaches and creation of a Core D infrastructure will enable interrogation of immune mechanisms and provide insights into development and durability of immunological memory in the context of checkpoint blockade or any other immunotherapies.