Role of meningeal lymphatic vasculature in neuroimmune communication development

The objectives of the proposal are to determine, for the first time, how developing meningeal lymphatic vessels (MLVs) impact neuroimmune communication in early postnatal life, and if MLV- dependent neuroimmune communication can be harnessed by VEGF-C signaling in young mice at steady state and in a model of pediatric brain tumor such as medulloblastoma, the most common tumor affecting children. Our previous work and preliminary data show that MLVs develop during early postnatal life, that their growth requires Vascular Endothelial Growth Factor-C (VEGF-C) and the VEGF-C co- receptor Neuropilin2 (Nrp2), and that enhancing MLV growth enables adaptive immune responses against glioblastoma in the adult. These findings prompt us to investigate mechanims how MLV-mediated neuroimmune connection establishes and to characterize the transport of antigen-presenting cells (APCs) to CNS-collecting lymph nodes (cLNs) and the MLV-dependent education of T cells in developing cLNs. Experimental approaches to successfully complete this project include new lymphatic-specific knockout lines, new procedures of three-dimensional whole-head imaging by light sheet fluorescence microscopy, and unbiased molecular analysis by mass spectrometry and single cell- RNA sequencing. This multidisciplinary study will bring together complementary expertise in the fields of meningeal lymphatics (Jean-Leon Thomas, Yale), developmental angiogenesis (Anne Eichmann, Yale), and immunology (Akiko Iwasaki, Yale), with additional advisors in pediatric oncology (Asher Marks, Director of Yale Pediatric Neuro-Oncology), liquid-chromatography mass spectometry (Tukiet Lam, Director of Keck MS & Proteomics Resource at Yale), and TCR repertoire analysis (Le Zhang, Yale Neurology). We expect to characterize the development of MLV-dependent neuroimmune communication, to identify Nrp2 as a new mandatory regulator of MLV sprouting, identify APC subpopulation(s) required for CNS antigen transport into cLNs, and to determine, as a proof of principle, the potential of VEGF-C-driven MLV growth to promote early T cell education against abnormal CNS antigens, with perspectives for the treatment of aggressive pediatric brain tumors.