The cellular mechanisms of immunological memory development in COVID-19 patients

COVID-19患者免疫记忆发展的细胞机制

• 批准号: 10745497
• 负责人: WEIGUO CUI
• 金额: $ 32.14万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10745497
• 关键词: cellular; mechanisms; immunological; memory; development

The recent unexpected emergence of the COVID-19 pandemic has spurred significant interest in an improved understanding of immunological memory to SARS-CoV-2, which consists of humoral (neutralizing antibodies) and cellular (T and B cells) memory. The generation of immunological memory to the SARS-CoV-2 virus critically depends on T cell responses. During a viral infection, CD4 helper T cells differentiate largely into either Th1 cells that orchestrate a type I antiviral immune response or follicular helper (Tfh) cells that enhance antibody production. CD8 T cells clonally expand and acquire effector function to directly kill virus-infected cells. Despite the heterogeneity and clonal diversity of virus-specific T cells, the majority of effector T cells die after viral clearance and only a small portion of them develop into memory T cells that provide long-lasting protection for the host. Similarly, B cells develop into memory B cells and long-lived plasma cells that produce neutralizing antibodies. Snapshot observations with multi-parameter flow cytometry-based assays along the course of infection has yielded abundant knowledge of T cell phenotypic and functional diversity. However, approaches as such fail to address the developmental trajectory of virus-specific T cells. This becomes more obvious in human studies given that lineage tracing by genetic alterations or adoptive transfer experiments, done easily in mice, are inherently difficult or impossible in humans. In this proposal, we will first combine newly developed single- cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (TCR-seq) techniques on the same cells and use TCR sequences as natural barcodes to directly “lineage trace” each patient’s SARS-CoV-2-specific CD4 and CD8 T cell effector response and memory formation at the single-cell level throughout the course of natural infection. Furthermore, we will perform gene regulatory network (GRN) analysis to delineate which transcription factors collaboratively regulate virus-specific CD4 and CD8 T cell differentiation trajectories. Next, we will measure T cell clonal diversity and the quality of T cell memory from COVID-19 patients as well as healthy human controls. The latter will be used to gauge the possible presence of pre-existing immunity (PEI) in the form of memory T cells derived from cross-reactivity to common coronaviruses. These measurements will use high- throughput RNA-seq of TCR amplicons and scRNA-seq of memory T cells from recall cultures as inputs for computational TCR motif analysis. Lastly, successful vaccine development relies on an advanced understanding of the types of Tfh cells that are generated during natural infection and how they interact with B cells as well as T regulatory cells for anti-SARS-CoV-2 antibody production in humans. To this end, we propose to monitor circulating Tfh cells, including three major populations (Th1-, Th2- and Th17-like subsets), and T follicular regulatory (Tfr) cells in both SARS-Cov-2-infected and healthy human control subjects. In addition, we will perform T cell-B cell coculture assays to dissect the functional contributions of each subset of Tfh cells and how they interact with Tfr cells in regulating anti-SARS-CoV-2 neutralizing antibody responses.

最近出人意料的 COVID-19 大流行引发了人们对改善环境的浓厚兴趣。 了解 SARS-CoV-2 的免疫记忆，该记忆由体液（中和抗体）组成 SARS-CoV-2 病毒的免疫记忆的产生至关重要。 取决于 T 细胞反应 在病毒感染期间，CD4 辅助 T 细胞主要分化为 Th1 细胞。 协调 I 型抗病毒免疫反应或增强抗体的滤泡辅助 (Tfh) 细胞 尽管如此，CD8 T 细胞仍会克隆扩增并获得效应功能以直接杀死病毒感染的细胞。 由于病毒特异性T细胞的异质性和克隆多样性，大多数效应T细胞在病毒感染后死亡 清除，其中只有一小部分发育成记忆 T 细胞，为细胞提供持久的保护。 同样，B 细胞发育成记忆 B 细胞和产生中和作用的长寿命浆细胞。 抗体的快照观察基于多参数流式细胞仪的检测过程。 感染已经产生了关于 T 细胞表型和功能多样性的丰富知识，然而，方法如下。 这样无法解决病毒特异性 T 细胞的发育轨迹，这在人类中变得更加明显。 研究表明，通过基因改变或过继转移实验进行谱系追踪很容易在小鼠身上完成， 在这个提案中，我们将首先结合新开发的单 对相同细胞进行细胞 RNA 测序 (scRNA-seq) 和 T 细胞受体测序 (TCR-seq) 技术 使用 TCR 序列作为天然条形码来直接“谱系追踪”每个患者的 SARS-CoV-2 特异性 CD4 在整个自然过程中，CD8 T 细胞效应反应和单细胞水平的记忆形成 进一步感染，我们将进行基因调控网络（GRN）分析来描绘哪些转录 因素协同调节病毒特异性 CD4 和 CD8 T 细胞分化轨迹。 测量 COVID-19 患者以及健康人的 T 细胞克隆多样性和 T 细胞记忆质量 后者将用于衡量是否存在预先存在的免疫力（PEI）。 这些测量将使用高-来自与常见冠状病毒的交叉反应的记忆T细胞。 吞吐量 TCR 扩增子的 RNA-seq 和来自回忆培养物的记忆 T 细胞的 scRNA-seq 作为输入 最后，成功的疫苗开发依赖于先进的理解。 自然感染过程中产生的 Tfh 细胞类型以及它们如何与 B 细胞相互作用以及 为此，我们建议监测人类抗 SARS-CoV-2 抗体产生的 T 调节细胞。 循环 Tfh 细胞，包括三个主要群体（Th1、Th2 和 Th17 样亚群）和滤泡 T 此外，我们还将研究 SARS-Cov-2 感染者和健康人类对照受试者中的调节 (Tfr) 细胞。 进行 T 细胞-B 细胞共培养测定，以剖析每个 Tfh 细胞亚群的功能贡献以及如何 它们与 Tfr 细胞相互作用，调节抗 SARS-CoV-2 中和抗体反应。