Rationale:: The formation of the blood vasculature is achieved via 2 fundamentally different mechanisms, de novo formation of vessels from endothelial progenitors (vasculogenesis) and sprouting of vessels from pre-existing ones (angiogenesis). In contrast, mammalian lymphatic vasculature is thought to form exclusively by sprouting from embryonic veins (lymphangiogenesis). Alternative nonvenous sources of lymphatic endothelial cells have been suggested in chicken and Xenopus, but it is unclear whether they exist in mammals. Objective:: We aimed to clarify the origin of the murine dermal lymphatic vasculature. Methods and Results:: We performed lineage tracing experiments and analyzed mutants lacking the Prox1 transcription factor, a master regulator of lymphatic endothelial cell identity, in Tie2 lineage venous–derived lymphatic endothelial cells. We show that, contrary to current dogma, a significant part of the dermal lymphatic vasculature forms independently of sprouting from veins. Although lymphatic vessels of cervical and thoracic skin develop via sprouting from venous-derived lymph sacs, vessels of lumbar and dorsal midline skin form via assembly of non– Tie2 -lineage cells into clusters and vessels through a process defined as lymphvasculogenesis. Conclusions:: Our results demonstrate a significant contribution of nonvenous-derived cells to the dermal lymphatic vasculature. Demonstration of a previously unknown lymphatic endothelial cell progenitor population will now allow further characterization of their origin, identity, and functions during normal lymphatic development and in pathology, as well as their potential therapeutic use for lymphatic regeneration.

Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular endothelial growth factor receptor 3 (VEGFR-3)/p110α PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma.

Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not previously recognized as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates the known features and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells; (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation; and (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization, and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN.

Gliomas are brain tumors characterized by an immunosuppressive microenvironment. Immunostimulatory agonistic CD40 antibodies (αCD40) are in clinical development for solid tumors, but are yet to be evaluated for glioma. Here, we demonstrate that systemic delivery of αCD40 in preclinical glioma models induces the formation of tertiary lymphoid structures (TLS) in proximity of meningeal tissue. In treatment-naïve glioma patients, the presence of TLS correlates with increased T cell infiltration. However, systemic delivery of αCD40 induces hypofunctional T cells and impairs the response to immune checkpoint inhibitors in pre-clinical glioma models. This is associated with a systemic induction of suppressive CD11b ⁺ B cells post-αCD40 treatment, which accumulate in the tumor microenvironment. Our work unveils the pleiotropic effects of αCD40 therapy in glioma and reveals that immunotherapies can modulate TLS formation in the brain, opening up for future opportunities to regulate the immune response.