Defining Epigenetic States of Senescent Cells and Associated Tissue Environments in the Human Lymphoid Tissues

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PROJECT SUMMARY Cell senescence in human tissues is an irreversible cell cycle arrest state in otherwise proliferative cells, which is a hallmark of aging that alters the tissue environments via senescence-associated secretory phenotype (SASP) but may also play a beneficial role in tissue remodeling, regeneration, and wound healing. Lymphoid organs play a vital role in hematopoiesis and immune function. How cellular senescence in these tissues is associated with stress-induced or age-related functional impairment, what types or subtypes of senescent cells are present and their spatial heterogeneity and how these cells impact the tissue environments remain poorly understood, precluding the development of strategies to target senescent cells to improve healthspan/lifespan or harnessing these cells or secreted factors to promote tissue remodeling and repair. A recent commentary published in Cell by NIH identified five broad areas (atlases, imaging &visualization, biomarkers, model systems, perturbation and validation) that would help propel the field forward. Our application will assemble a multidisciplinary team to tackle all these areas and specifically, as a Tissue Mapping Center (TMC), will focus on generating the molecular and cellular maps of cellular senescence and associated tissue environments in 4 primary and secondary lymphoid organs. Specifically, it will (a) collect, analyze, annotate, and share high quality non-diseased human primary (bone marrow and thymus) and secondary (tonsil and lymph node) lymphoid tissues, (2) develop and deploy a suite of high-resolution, high-content and high-throughput single- cell & spatial omics technologies to characterize these specimens and paired biofluids, and (3) perform integrated informatics to identify biomarkers of senescent cell heterogeneity and to construct comprehensive molecular and cellular maps of cellular senescence and associated environments in these organs. Four major biological analysis pipelines are: (1) single-cell high-plex (>40) protein secretome profiling, (2) single-cell proteo-transcriptomic sequencing (scCITE-seq), (3) spatial proteo-transcriptomic sequencing at cellular level (DBiT-seq for co-mapping whole transcriptome and a panel of ~300 proteins at cellular level with 10µm pixel size), and (4) spatial molecular imaging (SMI) of ~1,000 molecular targets in FFPE tissues at single-molecule subcellular resolution. With these unique tools, we will (a) characterize functional SASP heterogeneity and identify biomarkers of SAPS in different cell types, (b) construct molecular and cellular maps in 4 human lymphoid tissues, and (c) identify biomarkers of cellular senescence in tissue and the associated environments, contributing to the resource building of SenNet. Since the immune function is central to the physiology of all major organs, our study will provide insights to the role of senescent immune cells in development, aging, or disease in other organs like gut, lung, liver, and kidney, representing a strong synergy within SenNet and the wider scientific community.