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

最近意外出现的COVID-19大流行激发了人们对改善 了解SARS-CoV-2的免疫记忆，它由体液（中和抗体）组成 和细胞（T和B细胞）记忆。SARS-CoV-2病毒免疫记忆的产生 取决于T细胞的反应。在病毒感染过程中，CD 4辅助性T细胞主要分化为Th 1细胞， 或滤泡辅助细胞（Tfh）， 生产CD 8 T细胞克隆扩增并获得效应子功能以直接杀死病毒感染的细胞。尽管 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。 它们中只有一小部分发育成记忆T细胞，为免疫系统提供持久的保护。 主持人类似地，B细胞发育成记忆B细胞和长寿的浆细胞，它们产生中和 抗体的沿着使用基于多参数流式细胞术的检测进行的快照观察 感染产生了关于T细胞表型和功能多样性的丰富知识。然而，作为 这不能解决病毒特异性T细胞的发育轨迹。这一点在人类身上表现得更为明显。 研究表明，通过遗传改变或过继转移实验进行谱系追踪，在小鼠中很容易完成， 在人类身上是很难或不可能的。在这个建议中，我们将首先联合收割机新开发的单- 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，直接“谱系追踪”每个患者的SARS-CoV-2特异性CD 4 和CD 8 T细胞效应反应和记忆形成在单细胞水平的整个过程中的自然 感染此外，我们将进行基因调控网络（GRN）分析，以描绘哪些转录 这些因子协同调节病毒特异性CD 4和CD 8 T细胞分化轨迹。接下来我们就 测量COVID-19患者和健康人的T细胞克隆多样性和T细胞记忆质量 人类控制。后者将用于衡量形式中可能存在的预先存在的免疫力（PEI） 来自对普通冠状病毒的交叉反应的记忆T细胞。这些测量将使用高- TCR扩增子的通量RNA-seq和来自回忆培养物的记忆T细胞的scRNA-seq作为输入， 计算TCR基序分析。最后，成功的疫苗开发依赖于先进的理解， 在自然感染过程中产生的Tfh细胞的类型，以及它们如何与B细胞相互作用， T调节细胞用于人类抗SARS-CoV-2抗体的产生。为此，我们建议监测 循环Tfh细胞，包括三个主要群体（Th 1-、Th 2-和Th 17-样亚群），和T滤泡细胞， 调节（Tfr）细胞在SARS-Cov-2感染和健康人对照受试者。此外，我们将 进行T细胞-B细胞共培养测定以剖析Tfh细胞的每个亚群的功能贡献以及如何 它们在调节抗SARS-CoV-2中和抗体应答中与Tfr细胞相互作用。

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.