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

最近新冠肺炎大流行的意外出现激起了人们对改善 对由体液(中和抗体)组成的SARS-CoV-2免疫记忆的理解 和细胞(T和B细胞)记忆。对SARS-CoV-2病毒关键免疫记忆的产生 取决于T细胞的反应。在病毒感染期间，CD4辅助T细胞在很大程度上分化为Th1细胞 协调I型抗病毒免疫反应或增强抗体的滤泡辅助细胞(TFH) 制作。CD8T细胞克隆扩增并获得效应器功能，直接杀死感染病毒的细胞。尽管 病毒特异性T细胞的异质性和克隆多样性，大多数效应性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，为脑出血提供长期保护 主持人。类似地，B细胞发育成记忆B细胞和能产生中和作用的长寿浆细胞 抗体。多参数流式细胞术在慢性粒细胞白血病发病过程中的快照观察 感染已经产生了丰富的T细胞表型和功能多样性的知识。然而，其方法如下 这未能解决病毒特异性T细胞的发展轨迹。这在人类身上变得更加明显 给出了通过基因改变或过继转移实验进行血统追踪的研究，这些实验很容易在老鼠身上完成， 在人类身上天生是困难或不可能的。在这项建议中，我们将首先结合新开发的单项- 细胞RNA测序(scRNA-seq)和T细胞受体测序(tcr-seq)技术 使用TCR序列作为自然条形码，直接“血统追踪”每个患者的SARS-CoV-2特异性CD4 而CD8T细胞效应器的反应和记忆的形成贯穿于单细胞水平的整个自然过程 感染。此外，我们将进行基因调控网络(GRN)分析，以确定哪些转录 各因子协同调节病毒特异性的CD4和CD8T细胞分化轨迹。接下来，我们将 检测新冠肺炎患者和健康人T细胞克隆多样性和T细胞记忆质量 人类控制。后者将被用来衡量可能存在的预先存在的免疫(PEI)的形式 由于对常见冠状病毒的交叉反应而产生的记忆T细胞。这些测量将使用高- 从Recall培养中获得的TCR扩增产物的RNA-seq和记忆T细胞的scRNA-seq作为输入 计算型TCR基序分析。最后，成功的疫苗研发有赖于对 在自然感染期间产生的TFH细胞的类型，以及它们如何与B细胞以及 人类产生抗SARS-CoV-2抗体的T调节细胞。为此，我们建议监测 循环Tfh细胞，包括三个主要群体(Th1、Th2和Th17样亚群)和T滤泡 SARS-CoV-2感染和健康对照受试者中的调节性(TFR)细胞。此外，我们还将 进行T细胞-B细胞共培养试验，分析TFH细胞各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。

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.