Harnessing spatial transcriptomics to study fate decisions and lineage specification in T cell development

Abstract T cells are critical components of the adaptive immune system; they develop from bone-marrow derived precursors that migrate to the thymus. There, precursors undergo a sequence of lineage specification and commitment steps that give rise to multiple T cell lineages serving essential functions in defenses against infection and immune homeostasis. The diversity of thymocytes, and their complex interactions with the thymic stroma, including epithelial cells and other hematopoietic compartments, have hampered our understanding of T cell development. For greater insight into intrathymic developmental trajectories and their transcriptional control, we and others have utilized single cell RNA- and ATAC-sequencing (scRNA-seq and scATAC-seq). However, single-cell approaches do not assess contextual cell-cell interactions. To address this limitation, we used in-situ transcriptomics, specifically the 10× Genomics “Visium” technology, which assays in-situ gene expression in 55-micron spots spanning whole mouse thymus lobes. We will present initial results of these experiments, including mapping of thymocyte differentiation stages, location of developmental bifurcations, and the spatial progression of lineage specific gene expression. Briefly, we use statistical models informed by scRNA-seq data to infer the location, relative to the thymic architecture, of lineage decisions. Additionally, we combine positional information of thymocytes and stromal cells to infer cell-cell interactions that direct lineage decisions. Collectively, this analysis sheds light on the interactions that guide development of the diverse T cell family from a common precursor and characterizes the journey of these cells to reach maturity.