Comprehensive characterization of immune signaling networks in single-cells by joint quantification of proteins, protein complexes and mRNA

蛋白质的准确和多重特征对于免疫学的基础和临床研究至关重要， 感染、发育和癌症。免疫发展、信号激活和药物的许多过程 抗性是由一小部分细胞和信号通路的可变激活驱动的，需要 单细胞测量。目前，单细胞DNA/rna检测具有较高的精度和较高的产量。 然而，在蛋白质，特别是它们的复合体的测量方面存在着重大的技术差距 在单独的细胞中。高通量方法结合同时测量蛋白质、复合体和 需要信使核糖核酸更好地理解和模拟个体的细胞反应，并发现新的细胞状态。 和功能。我们的建议有两个同样重要的协同目标：a)广泛地优化/适应 一种同时测定蛋白质、蛋白质复合体和mRNA的实用技术 数以千计的单个细胞(目标1)，和b)研究了关于细胞的功能和进化的几个关键假说 免疫发育过程中的信号网络(目标2和3)。我们的技术，称为细胞内接近- 测序(iProx-seq)，使用DNA条码邻近探针和单细胞测序进行多路传输 蛋白质及其复合体的测量。用iProx-seq标尺测量蛋白质复合体的数量 二次：以100个蛋白质为目标将能够测量每个蛋白质中5500个潜在的蛋白质复合体 手机。通过测序来定量蛋白质还有一个额外的好处，那就是整个转录组的mrna测量。 在同一个细胞中，所有都使用了坚固和广泛使用的测序流水线。我们提供了大量的初步数据 论证了我们整个技术方法和机制研究的可行性。 我们将结合iProx-seq、活细胞成像和数学建模，研究 生发中心的造血干细胞和B细胞的分化(见目标2和3)。我们会 测量信号受体、接头蛋白、转录因子、细胞因子、激活酶和蛋白质 并对免疫信号网络中的NF-κB、MAPK、PI3K和IRF3进行了全面的表征 单个人和小鼠HSC，单核细胞来源的巨噬细胞，粒细胞-单核细胞前体细胞，以及 生发中心B细胞。我们将回答的具体问题包括：受体水平如何变化，受体- 共受体复合体和细胞内复合体的形成解释单个巨噬细胞对炎症的敏感性 TLR信号？NF-κB、MAPK、PI3K和IRF3蛋白网络的发育性重构 调节整个造血系的信号特异性？什么是不同的蛋白质组和信号状态 在生发中心，蛋白质网络如何调节B细胞的分化？我们的建议将 带来强大而实用的单细胞分析技术，并改进了对功能和 蛋白质网络在免疫中的进化。我们的结果将对理解 免疫发展、免疫激活、出现抗药性。

Accurate and multiplexed characterization of proteins is essential to basic and clinical studies in immunity, infection, development, and cancer. Many processes in immune development, signal activation, and drug resistance are driven by a small subset of cells and variable activation of signaling pathways, necessitating single-cell measurements. Currently, there is high precision and throughput in measuring DNA/RNA in single cells, however a major technological gap exists in the measurement of proteins and especially their complexes in individual cells. High-throughput methods combining simultaneous measurement of proteins, complexes and mRNA are needed to better understand and model individual cellular responses, and to discover new cell states and functions. Our proposal has two, equally important, and synergistic goals: a) optimize/adapt a broadly applicable and practical technology that simultaneously measures proteins, protein-complexes and mRNA in thousands of individual cells (Aim 1), and b) study several key hypotheses on the function and evolution of signaling networks during immune development (Aims 2 and 3). Our technology, called Intracellular Proximity- Sequencing (iProx-seq), uses DNA barcoded proximity probes and single-cell sequencing for multiplexed measurement of proteins and their complexes. The number of protein complexes measured by iProx-seq scales quadratically: Targeting 100 proteins will enable the measurement of 5500 potential protein complexes in each cell. Protein quantification by sequencing has the additional benefit of transcriptome-wide mRNA measurements in the same cell, all using a robust and widely used sequencing pipeline. Extensive preliminary data we present demonstrated the feasibility of our entire technical approach and mechanistic studies. We will combine iProx-seq, live cell imaging and mathematical modeling and study key hypotheses in the differentiation of hematopoietic stem cells (HSCs) and B cells in the germinal center (see Aims 2 and 3). We will measure signaling receptors, adaptor proteins, transcription factors, cytokines, kinases, and protein modifications, and comprehensively characterize immune signaling networks NF-κB, MAPK, PI3K and IRF3 in single human and mouse HSCs, monocyte derived macrophages, granulocyte-monocyte progenitors, and germinal center B cells. Specific questions we will answer include: How do changes in receptor levels, receptor- coreceptor complexes, and intracellular complex formation explain single macrophage sensitivity to inflammatory TLR signals? How does the developmental remodeling of protein networks NF-κB, MAPK, PI3K and IRF3 regulate signal specificity across the hematopoietic lineage? What are the distinct proteomic and signaling states in the germinal center, and how do protein networks regulate the differentiation of B cells? Our proposal will result in a powerful and practical single-cell analysis technology and improved insight on the function and evolution of protein networks in immunity. Our results will make significant impact into the understanding of immune development, immune activation, and emergence of drug resistance.