Investigating gene regulatory networks in immune cells of children who differ in susceptibility to malaria infection using single-cell approaches

项目总结/摘要 疟疾每年造成40多万人死亡，这突出表明需要一种高效的疟疾疫苗。更好的 了解免疫恶性疟原虫，最致命的人类疟疾寄生虫，在自然 暴露人群可以为更有效疫苗的合理设计提供信息。可靠的免疫 预防疟疾的相关因素仍然难以捉摸，关于预防疟疾的免疫力也知之甚少。 P.鉴于通过自然疟疾暴露持续保护免受寄生虫血症， 很少被观察到。在我们的K 08资助的项目中，我们将系统生物学方法应用于一个独特的子集， 在一个疟疾季节里，尽管进行了密集的治疗， 监测，包括每2周采集一次血液，并在患病访视期间进行基于PCR的筛查 作为7个月监测期间寄生虫暴露的抗体证据。这很少被观察到， “寄生虫血症”表型代表了在自然暴露的寄生虫中最接近不育免疫的表型。 人口和一个独特的机会，研究宿主控制寄生虫血症。本R21提案的目标是 扩展我们先前对这些寄生虫血症患者全血进行批量转录组学分析的结果。 使用更高分辨率技术的儿童，包括单细胞（sc）测序和多参数流 细胞仪我们建议对获得的基线外周血单核细胞（PBMC）使用scRNA-seq， 从这些孩子身上得到的信息当与转座酶的单细胞测定整合时， 可及染色质测序（scATAC-seq），评估全基因组染色质可及性， scRNA-seq可以识别“开放”的染色质区域，也可以提供对基因表达调控的深入了解。 将整合的单细胞方法与功能测定相结合可以帮助识别基因调控网络 在特定的细胞类型，可以作为免疫相关的保护免受疟疾感染。我们 假设相对于寄生虫血症儿童，寄生虫血症儿童将表现出抗原增加 记忆B和T细胞亚群内的受体信号传导和增强的恶性疟原虫特异性T细胞记忆 尽管T细胞的比例降低，但这可能反映了T细胞的运输， 将外周血输送至靶器官，如肝或脾。我们将通过以下内容来解决这一假设 具体目的：1）确定宿主免疫基因的差异调节和表达，使用整合的 接触疟疾儿童PBMC的单细胞基因表达和染色质可及性分析 在对恶性疟原虫寄生虫血症的易感性方面存在差异，以及2）鉴定和表征免疫表型， 对恶性疟原虫寄生虫血症易感性不同的儿童的PBMC功能差异。成功 这些目标的完成将有助于确定与疟疾有关的基因特征和基因调控网络 免疫力，这将成为未来研究的基础，旨在阐明疟疾保护机制， 人类的细胞反应。

Project Summary/Abstract Malaria kills over 400,000 people annually, underscoring the need for a highly effective malaria vaccine. A better understanding of immunity to Plasmodium falciparum, the most lethal of the human malaria parasites, in naturally exposed populations can inform the rational design of more effective vaccines. However, reliable immune correlates of protection against malaria remain elusive, and relatively little is known about immunity that prevents P. falciparum infection given that sustained protection from parasitemia through natural malaria exposure is rarely observed. In our K08-funded project, we have applied systems biology approaches to a unique subset of children who remained free of P. falciparum parasitemia during a single malaria season despite intensive surveillance consisting of PCR-based screening of blood collected every 2 weeks and during sick visits as well as antibody evidence for parasite exposure during a 7-month surveillance period. This rarely observed “aparasitemic” phenotype represents the closest approximation to sterile immunity in a naturally exposed population and a unique opportunity to study host control of parasitemia. The goal of this R21 proposal is to expand on findings from our prior bulk transcriptomic analysis of whole blood obtained from these aparasitemic children using higher resolution technologies that include single-cell (sc) sequencing and multiparameter flow cytometry. We propose using scRNA-seq on baseline peripheral blood mononuclear cells (PBMCs) obtained from these children just prior to the surveillance period. When integrated with single-cell assay for transposase- accessible chromatin sequencing (scATAC-seq), which assesses genome-wide chromatin accessibility to identify “open” chromatin regions, scRNA-seq can also provide insight into the regulation of gene expression. Combining integrated single-cell approaches with functional assays can help identify gene regulatory networks within specific cell types that could serve as immune correlates of protection against malaria infection. We hypothesize that, relative to parasitemic children, aparasitemic children will demonstrate increased antigen receptor signaling within memory B and T cell subsets and enhanced P. falciparum-specific T-cell memory responses despite having decreased proportions of T cells, which may reflect trafficking of T cells out of the peripheral blood to target organs such as the liver or spleen. We will address this hypothesis with the following specific aims: 1) to determine host immune genes that are differentially regulated and expressed using integrated single-cell gene expression and chromatin accessibility profiling of PBMCs from malaria-exposed children who differ in susceptibility to P. falciparum parasitemia and 2) to identify and characterize the immunophenotypic and functional differences in PBMCs from children who differ in susceptibility to P. falciparum parasitemia. Successful completion of these aims will help identify gene signatures and gene regulatory networks relevant to malaria immunity that would form the basis of future studies aimed at elucidating the mechanisms of malaria-protective cellular responses in humans.