Heterogeneity in VEGFR3 levels drives lymphatic vessel hyperplasia through cell-autonomous and non-cell-autonomous mechanisms

Incomplete delivery to the target cells is an obstacle for successful gene therapy approaches. Here we show unexpected effects of incomplete targeting, by demonstrating how heterogeneous inhibition of a growth promoting signaling pathway promotes tissue hyperplasia. We studied the function of the lymphangiogenic VEGFR3 receptor during embryonic and postnatal development. Inducible genetic deletion of Vegfr3 in lymphatic endothelial cells (LECs) leads to selection of non-targeted VEGFR3(+) cells at vessel tips, indicating an indispensable cell-autonomous function in migrating tip cells. Although Vegfr3 deletion results in lymphatic hypoplasia in mouse embryos, incomplete deletion during post-natal development instead causes excessive lymphangiogenesis. Analysis of mosaically targeted endothelium shows that VEGFR3(-) LECs non-cell-autonomously drive abnormal vessel anastomosis and hyperplasia by inducing proliferation of non-targeted VEGFR3(+) LECs through cell-contactdependent reduction of Notch signaling. Heterogeneity in VEGFR3 levels thus drives vessel hyperplasia, which has implications for the understanding of mechanisms of developmental and pathological tissue growth.Peer reviewe

Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels.

The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow-responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial-specific Tie2-Cre or the tamoxifen-inducible LEC-specific Prox1-CreER <sup>T2</sup> line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow-induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel-type-specific endothelial cell identities

Intralymphatic CCL21 promotes tissue egress of dendritic cells through afferent lymphatic vessels

To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient

A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

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

cKit Lineage Hemogenic Endothelium-Derived Cells Contribute to Mesenteric Lymphatic Vessels

Peer reviewe

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology

pMHC affinity controls duration of CD8+ T cell&-DCinteractions and imprints timing of effector differentiationversus expansion

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinityclones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue andhow low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymphnodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, theduration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC)affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulatedfactor 4) induction and timing of effector differentiation, as low affinity&-primed T cells acquired cytotoxic activity earlierthan high affinity&-primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vesselsfor egress, whereas high affinity&-stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner forsustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cellelimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cellactivation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during earlymicrobial containment.This work was supported by a Novartis Research grant (to A.J. Ozga) and Swiss National Science Foundation grants (31003A_135649 and CR23I3_156234 to J.V. Stein and CRS II3_141918 to J.V. Stein and J. Sharpe). J. Ripoll acknowledges support from European Commission FP7 Career Integration Grants (HIGH-THROUGHPUT TOMO), European Commission FP7 Marie Curie Actions (grant 2PM), and the Ministerio de Economía y Competitividad (grant FIS2013-41802-R). J. Sharpe acknowledges support from the Ministerio de Economía y Competitividad, Centro de Excelencia Severo Ochoa 2013–2017 (grant SEV-2012-0208)

Endogenous TNF alpha orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation

This work was supported by funds from Arthritis Research UK (19913 to M.-B.V.) and the Wellcome Trust (098291/Z/12/Z to S.N.). S. A. was supported by a QMUL Principal’s Award PhD Studentship

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fya/Fyb blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40–47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core

Cardiac lymphatics in health and disease

The lymphatic vasculature, which accompanies the blood vasculature in most organs, is indispensable in the maintenance of tissue fluid homeostasis, immune cell trafficking, and nutritional lipid uptake and transport, as well as in reverse cholesterol transport. In this Review, we discuss the physiological role of the lymphatic system in the heart in the maintenance of cardiac health and describe alterations in lymphatic structure and function that occur in cardiovascular pathology, including atherosclerosis and myocardial infarction. We also briefly discuss the role that immune cells might have in the regulation of lymphatic growth (lymphangiogenesis) and function. Finally, we provide examples of how the cardiac lymphatics can be targeted therapeutically to restore lymphatic drainage in the heart to limit myocardial oedema and chronic inflammation.Peer reviewe