High endothelial venules (HEV) are specialized post capillary portals for lymphocyte entry into lymphoid tissues and sites of chronic inflammation from the blood. They regulate immune cell trafficking in physiologic and pathologic settings including autoimmune diseases and cancer. HEV in lymph nodes draining sites of immune challenge proliferate extensively to support enhanced lymphocyte recruitment, but the endothelial precursors that give rise to high endothelium and the molecular pathways that control their proliferation and differentiation are unclear. Our transcriptomic and immunologic studies reveal that capillary endothelial cells (CapEC) express multiple markers classically associated with stem and progenitor cells, leading to the hypothesis that capillary phenotype EC comprise a population of stem cell-like blood endothelial progenitors that contribute to HEV expansion during the immune response. Under Aim 1 we will apply state-of-the-art single cell immunofluorescence and high dimensional mass label (CyTOF) flow cytometry to uncover the diversity of EC subsets in LN and in extra lymphoid sites of immune cell recruitment, define the kinetics and subset-specificity of their proliferative responses to immune challenge, and reveal the time course of emergence of transitional phenotypes leading to the amplification and maturation of functional HEV. Trajectory analyses will reveal developmental relationships of identified subsets including identified progenitors, and immunofluorescence histology and confocal tissue imaging will define their location within the vasculature. In Aim 2, innovative fate mapping approaches will elucidate precursor-product relationships among BEC subsets and will define clonal contributions of precursors to capillary and high endothelium. Aim 3 will apply transcriptional profiling of induced EC subsets in combination with pan-EC and novel capillary EC-specific inducible gene targeting systems to define molecular mechanisms of HEV homeostasis and pathways regulating progenitor cell activation, amplification and contribution to HEV expansion in the immune response. Comprehensive phenotypic analyses of blood endothelial cell subsets and their responses to immunization will open up new areas of investigation in vascular biology and immunology. Elucidation of the mechanisms of endothelial cell specialization and homeostasis, including mechanisms regulating endothelial cells that control lymphocyte traffic, will lead to novel targets and approaches for the control of autoimmune inflammation and for therapeutic regulation of immune cell traffic for vaccination and cancer immunity.