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-¿B, 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-¿B, 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.