PROJECT SUMMARY/ABSTRACT Studies of the thymic microenvironment have focused largely on the central role of thymic epithelial cells (TECs) during development, disease and aging. However recent evidence suggests that the thymic vascular compartment also plays an active and complex role within the thymus beyond that of a simple conduit for hematopoietic cells. This role is particularly evident during the neonatal period when robust thymopoiesis is accompanied by a profound though transient phase of angiogenesis, followed by rapid maturation of TECs. This research proposal builds on the recent and novel finding that the vasculature of the neonatal thymus is in fact dominated by lymphatic endothelial cells (LECs) rather than blood endothelial cells (BECs), the latter becoming dominant by early adulthood. The application of classical LEC-associated cell surface markers is problematic in the thymus in which these markers are either undetectable or expressed on other cell types. The few reports describing the existence of lymphatics in the thymus have studied the adult organ in which LECs are very rare. As a result, the existence of the lymphatic compartment of the thymus has remained largely ignored and the function of LECs and the vasculature they form is unknown. The recent development of key technical advances now allows us to explore this poorly understood cellular compartment of the thymus. Reliable mouse models developed to study LECs in other organs, will now be applied for the first time to identify and manipulate LECs in the thymus. Single cell transcriptomics, including spatial gene expression and proteomics, will allow the detailed simultaneous analysis of LECs, BECs and the other compartments of the thymus during development and after in vivo perturbation of LECs. By combining these powerful technologies, we will test the hypothesis that the lymphatic vasculature of the neonatal period plays a critical role in both the remodeling and maturation of the murine thymic architecture and the differentiation and egress of T cells during the same period. The transcriptional signature of thymic LECs so developed in the mouse will allow direct comparative transcriptional studies of the LECs in the human thymus that would be impossible by standard assays. Specifically, we aim to: 1. Define the lymphatic compartment of the thymus during development at the cellular and transcriptional level 2. Determine if inhibition of lymphangiogenesis affects development of the thymic microenvironment 3. Define the role of lymphangiogenesis in T cell differentiation and egress from the developing thymus In summary we will use the unique window of the murine neonatal period to define the factors that control the growth and regression of the lymphatic endothelium in the thymus, as well as the role of LECs in the dramatic concurrent changes seen in the other thymic compartments. The ultimate goal of these studies is to uncover novel pathways that may be explored for enh...