In this issue of JEM, Cleary et al. (https://doi.org/10.1084/jem.20241359) present a new intravital imaging technique using a 3D-printed window device that enables lung lymphatics and their participation in immune cell trafficking events to be visualized in action for the first time in mechanically ventilated mice.

Immune surveillance involves the continual migration of antigen-scavenging immune cells from the tissues to downstream lymph nodes via lymphatic vessels. To enable such passage, cells first dock with the lymphatic entry receptor LYVE-1 on the outer surface of endothelium, using their endogenous hyaluronan glycocalyx, anchored by a second hyaluronan receptor, CD44. Why the process should require two different hyaluronan receptors and by which specific mechanism the LYVE-1•hyaluronan interaction enables lymphatic entry is however unknown. Here we describe the crystal structures and binding mechanics of murine and human LYVE-1•hyaluronan complexes. These reveal a highly unusual, sliding mode of ligand interaction, quite unlike the conventional sticking mode of CD44, in which the receptor grabs free hyaluronan chain-ends and winds them in through conformational re-arrangements in a deep binding cleft, lubricated by a layer of structured waters. Our findings explain the mode of action of a dedicated lymphatic entry receptor and define a distinct, low tack adhesive interaction that enables migrating immune cells to slide through endothelial junctions with minimal resistance, while clinging onto their hyaluronan glycocalyx for essential downstream functions.

In adult mice, surgically-induced myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis, draining interstitial fluid and trafficking macrophages to mediastinal lymph nodes (MLNs), improving cardiac function. Given the importance of the adult cardiac lymphatics post-MI, we investigated their role across the neonatal “regenerative window”. At post-natal day 1 (P1) neonatal mice fully regenerate their heart following MI, in a macrophage-dependent manner, whereas equivalent injury at P7 leads to macrophage-driven scarring. We hypothesised that lymphatics respond differently during this window to clear macrophage subtypes depending upon their requirement for regeneration versus fibrotic repair. The response to injury revealed limited lymphangiogenesis and minimal macrophage clearance from P1 versus P7 infarcted hearts, coincident with maturation of lymphatic endothelial cell (LEC) junctions. Unbiased scRNA-Seq datasets from neonatal hearts post-MI demonstrated altered signalling between LECs and macrophages across the regenerative window, most notably of the lymphangiocrine factor Reelin Finally, in mice lacking the lymphatic endothelial receptor-1 (LYVE1), that exhibit impaired transmigration of macrophages to lymphatic vessels, MRI revealed surprising functional impairment in P1 mice post-MI. Given macrophages at P1 are not trafficked, this suggested a distinct role for LYVE1 in tissue-resident (TR) macrophages, where it is also expressed. Macrophage-specific deletion of Lyve1 revealed impaired heart regeneration post-MI, characterised by reduced neovascular response and function. Collectively, we reveal that cardiac lymphatics are developmentally compromised for clearance in early neonates, enabling retention of pro-regenerative macrophages, and that LYVE1 plays an essential role in macrophages to facilitate heart regeneration via the induction of coronary angiogenesis.

Systemic bacterial infection is typically attributed to direct blood vessel invasion or intracellular transit, while the role of the lymphatic system remains largely unexplored. We previously showed that Streptococcus pyogenes disseminates extracellularly via the lymphatics. Here, we extend this observation to a diverse group of important extracellular bacterial pathogens, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus , and Streptococcus agalactiae . In a murine soft tissue infection model, nearly all clinical isolates reached both local and distant draining lymph nodes and systemic organs, with minimal bacterial presence in non-draining nodes. This pattern demonstrates that diverse bacteria can traverse filtering lymph nodes and supports lymphatic rather than haematogenous spread as the primary driver of systemic invasion. Bacterial dissemination varied between species and among isolates of the same species, with capsular polysaccharides emerging as one key determinant. These findings establish lymphatic metastasis as a common but under-recognised route of bacterial invasion, with implications for pathogenesis, immunity, and treatment. ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, ,

In adult mice, myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis (lymphangiogenesis), drain interstitial fluid and traffic macrophages to mediastinal lymph nodes (MLNs). This prevents edema and reduces inflammatory/fibrotic immune cell content to improve cardiac function. Here we investigated the role of cardiac lymphatics and macrophage clearance across the neonatal mouse regenerative window. The response to injury revealed limited lymphangiogenesis and clearance of macrophages from postnatal day 1 compared to postnatal day 7 infarcted hearts. This coincides with the maturation of lymphatic endothelial cell junctions from impermeable to permeable and with altered signaling between lymphatic endothelial cells and macrophages. Mice lacking the lymphatic endothelial receptor-1 (LYVE-1), where macrophage lymphatic trafficking is impaired in adults, experienced worse long-term outcomes after MI induced at postnatal day 1, suggesting an alternative role for LYVE-1 in macrophages. Macrophage-specific deletion of Lyve1 during neonatal heart injury impaired heart regeneration. This study demonstrates that immature cardiac lymphatics are impermeable to clearance in early neonates, ensuring retention of pro-regenerative LYVE-1-dependent macrophages.

Winning a scientific prize is always a welcome honor and receiving the Aselli Award in 2023 was a particularly pleasant surprise. The following text provides a brief summary of the research carried out by my group in Oxford that led to the discovery of the lymphatic endo-thelial marker LYVE-1 and its emerging role as a pivotal receptor controlling the entry of immune cells and metastatic tumor cells to lymphatic capillaries in peripheral tissues. As a basic scientist and relatively late comer to the field of lymphology, my hope is that a closer partnership of basic and clinical research will help explain the intricate workings of the lymphatics and improve the picture for patients suffering from much neglected lymphatic disorders.

Intercellular Cell Adhesion Molecule 1 (ICAM-1, CD54) is a type I transmembrane protein and a member of the immunoglobulin superfamily. ICAM-1 is involved in many key processes such as cell-cell interactions, signal transduction and leukocytes trans-endothelial migration. ICAM-1 is constitutively present at low levels on healthy tissues but highly expressed in some lymphoma, myeloma and some types of solid tumors including melanoma, non-small cell lung cancers and liver cancers. Previous attempt to target ICAM-1 with a monoclonal antibody bersanlimab (BI505, BioInvent) showed that it was well-tolerated but with limited clinical efficacy. To improve the effectiveness of targeting ICAM-1, we developed a novel ICAM-1 antibody drug conjugate (ADC). The ICAM-1 ADC is comprised of a humanized anti-ICAM-1 hIgG1 antibody conjugated to a novel topoisomerase I inhibitor through a cleavable linker at a drug-to-antibody ratio (DAR) of about 8. The antibody moiety of the ADC targets a unique epitope on ICAM-1 which is far from its ligand binding domain. Potent in vitro cytotoxicity of ICAM-1 ADC, with nanomolar to sub-nanomolar EC50, was observed on a panel of hematologic and solid tumor cell lines. In preclinical studies, one or two doses of ICAM-1 ADC at 5-6 mg/kg were generally sufficient to induce tumor regression in multiple cell line-derived xenograft models, including those of lung and liver cancers. In all models tested so far, the novel topoisomerase I inhibitor conjugated ICAM-1 ADC is at least as efficacious as the same ICAM-1 antibody conjugated to the benchmark topoisomerase I inhibitor linker payload deruxtecan (ICAM-1-DXd). Furthermore, in a repeat dose exploratory non-human primate safety study, the benchmark conjugate ICAM-1-DXd (DAR 8) was well-tolerated at 41 mg/kg, the highest dose tested. In conclusion, ICAM-1 is an attractive target for topoisomerase I inhibitor based ADC and warrants further investigation. Citation Format: Oi Kwan Wong, David Jackson, Lei Shi, Qi Fei, Xiaocheng Chen, Leonard Post. A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6298.

Most cancers and in particular carcinomas metastasise via the lymphatics to draining lymph nodes from where they can potentially achieve systemic dissemination by invasion of high endothelial blood venules (HEVs) in the paracortex [ 1 , 2 ]. Currently however, the mechanisms by which tumours invade and migrate within the lymphatics are incompletely understood, although it seems likely they exploit at least some of the normal physiological mechanisms used by immune cells to access lymphatic capillaries and traffic to draining lymph nodes in the course of immune surveillance, immune modulation and the resolution of inflammation [ 3 , 4 ]. Typically these include directional guidance via chemotaxis, haptotaxis and durotaxis, adhesion to the vessel surface via receptors including integrins, and junctional re-modelling by MMPs (Matrix MetalloProteinases) and ADAMs (A Disintegrin And Metalloproteinases) [ 5 – 7 ]. This short review focusses on a newly emerging mechanism for lymphatic entry that involves the large polysaccharide hyaluronan (HA) and its key lymphatic and immune cell receptors respectively LYVE-1 (Lymphatic Vessel Endothelial receptor) and CD44, and outlines recent work which indicates this axis may also be used by some tumours to aid nodal metastasis.

Supplementary Figures 1-8 from A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

DCs play a vital role in immunity by conveying antigens from peripheral tissues to draining lymph nodes, through afferent lymphatic vessels. Critical to the process is initial docking to the lymphatic endothelial receptor LYVE-1 via its ligand hyaluronan on the DC surface. How this relatively weak binding polymer is configured for specific adhesion to LYVE-1, however, is unknown. Here, we show that hyaluronan is anchored and spatially organized into a 400-500 nm dense glycocalyx by the leukocyte receptor CD44. Using gene knockout and by modulating CD44-hyaluronan interactions with monoclonal antibodies in vitro and in a mouse model of oxazolone-induced skin inflammation, we demonstrate that CD44 is required for DC adhesion and transmigration across lymphatic endothelium. In addition, we present evidence that CD44 can dynamically control the density of the hyaluronan glycocalyx, regulating the efficiency of DC trafficking to lymph nodes. Our findings define a previously unrecognized role for CD44 in lymphatic trafficking and highlight the importance of the CD44:HA:LYVE-1 axis in its regulation.

Macrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1(+) myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.

Entry to the afferent lymphatics marks the first committed step for immune cell migration from tissues to draining lymph nodes both for the generation of immune responses and for timely resolution of tissue inflammation. This critical process occurs primarily at specialised discontinuous junctions in initial lymphatic capillaries, directed by chemokines released from lymphatic endothelium and orchestrated by adhesion between lymphatic receptors and their immune cell ligands. Prominent amongst the latter is the large glycosaminoglycan hyaluronan (HA) that can form a bulky glycocalyx on the surface of certain tissue-migrating leucocytes and whose engagement with its key lymphatic receptor LYVE-1 mediates docking and entry of dendritic cells to afferent lymphatics. Here we outline the latest insights into the molecular mechanisms by which the HA glycocalyx together with LYVE-1 and the related leucocyte receptor CD44 co-operate in immune cell entry, and how the process is facilitated by the unusual character of LYVE-1 • HA-binding interactions. In addition, we describe how pro-inflammatory breakdown products of HA may also contribute to lymphatic entry by transducing signals through LYVE-1 for lymphangiogenesis and increased junctional permeability. Lastly, we outline some future perspectives and highlight the LYVE-1 • HA axis as a potential target for immunotherapy.

Recent advances in our understanding of the lymphatic system, its function, development, and role in pathophysiology have changed our views on its importance. Historically thought to be solely involved in the transport of tissue fluid, lipids, and immune cells, the lymphatic system displays great heterogeneity and plasticity and is actively involved in immune cell regulation. Interference in any of these processes can be deleterious, both at the developmental and adult level. Preclinical studies into the cardiac lymphatic system have shown that invoking lymphangiogenesis and enhancing immune cell trafficking in ischaemic hearts can reduce myocardial oedema, reduce inflammation, and improve cardiac outcome. Understanding how immune cells and the lymphatic endothelium interact is also vital to understanding how the lymphatic vascular network can be manipulated to improve immune cell clearance. In this Review, we examine the different types of immune cells involved in fibrotic repair following myocardial infarction. We also discuss the development and function of the cardiac lymphatic vasculature and how some immune cells interact with the lymphatic endothelium in the heart. Finally, we establish how promoting lymphangiogenesis is now a prime therapeutic target for reducing immune cell persistence, inflammation, and oedema to restore heart function in ischaemic heart disease.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity.

PURPOSE.We recently reported that the glycosaminoglycan hyaluronan (HA), which promotes inflammatory angiogenesis in other vascular beds, is an abundant component of the limbal extracellular matrix.Consequently, we have explored the possibility that HA contributes to lymphangiogenesis in the inflamed cornea. METHODS.To study the role of HA on lymphangiogenesis, we used mice lacking the hyaluronan synthases and injury models that induce lymphangiogenesis. RESULTS.Here we report that HA regulates corneal lymphangiogenesis, both during post-natal development and in response to adult corneal injury.Furthermore, we show that injury to the cornea by alkali burn upregulates both HA production and lymphangiogenesis and that these processes are ablated in HA synthase 2 deficient mice.CONCLUSION.These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis might prevent the corneal scarring and rejection that frequently results from corneal transplantation.

The lymphatics fulfill a vital physiological function as the conduits through which leucocytes traffic between the tissues and draining lymph nodes for the initiation and modulation of immune responses. However, until recently many of the molecular mechanisms controlling such migration have been unclear. As a result of careful research, it is now apparent that the process is regulated at multiple stages from initial leucocyte entry and intraluminal crawling in peripheral tissue lymphatics, through to leucocyte exit in draining lymph nodes where the migrating cells either participate in immune responses or return to the circulation via efferent lymph. Furthermore, it is increasingly evident that most if not all leucocyte populations migrate in lymph and that such migration is not only important for immune modulation, but also for the timely repair and resolution of tissue inflammation. In this article, I review the latest research findings in these areas, arising from new insights into the distinctive ultrastructure of lymphatic capillaries and lymph node sinuses. Accordingly, I highlight the emerging importance of the leucocyte glycocalyx and its novel interactions with the endothelial receptor LYVE-1, the intricacies of endothelial chemokine secretion and sequestration that direct leucocyte trafficking and the significance of the process for normal immune function and pathology.

Tumour necrosis factor (TNF) is a multi-functional cytokine with profound and diverse effects on physiology and pathology. Identifying the molecular determinants underlying the functions and pathogenic effects of TNF is key to understanding its mechanisms of action and identifying new therapeutic opportunities based on this important molecule. Previously, we showed that some evolutionarily conserved peptides derived from TNF could induce cell death (e.g. apoptosis and/or necrosis), a feature of immune defence mechanisms shared by many vertebrates. In this study, we demonstrated that necrosis-inducing peptide P16 kills human glioblastoma cancer cells and primary human hepatoma or renal cancer cells isolated from patients who had not responded to standard treatments. Importantly, we show that the necrosis-inducing peptide P1516 significantly improves survival by inhibiting tumour metastasis in a 4T1 breast cancer syngeneic graft mouse model. Because the lymphatic system is an important metastatic route in many cancers, we also tested the effect of TNF-derived peptides on monolayers of primary human lymphatic endothelial cells (hDLEC) and found that they increased junctional permeability by inducing cytoskeletal reorganization, gap junction formation and cell death. Transmission electron microscopy imaging evidence, structural analysis and in-vitro liposome leakage experiments strongly suggest that this killing is due to the cytolytic nature of these peptides. P1516 provides another example of a pro-cytotoxic TNF peptide that probably functions as a cryptic necrotic factor released by TNF degradation. Its ability to inhibit tumour metastasis and improve survival may form the basis of a novel approach to cancer therapy.

LYVE-1, a close relative of the leucocyte receptor, CD44, is the main receptor for hyaluronan (HA) in lymphatic vessel endothelium and a widely used marker for distinguishing between blood and lymphatic vessels. Enigmatic for many years because of its anomalous HA-binding characteristics, the function of LYVE-1 has just recently been identified as that of a lymphatic docking receptor for dendritic cells, selectively engaging with their surface HA glycocalyx to regulate entry to peripheral lymphatics and migration to downstream lymph nodes for immune activation. Furthermore, LYVE-1 mediates the trafficking of macrophages, and is also exploited by HA-encapsulated Group A streptococci for lymphatic invasion and host dissemination. Consistent with a role in lymphatic trafficking, the interaction of LYVE-1 with HA and its degradation products can also activate intracellular signalling pathways for endothelial junctional retraction and lymphatic endothelial proliferation. Here we outline the latest findings on the receptor in the context of its peculiar biochemical properties and speculate on how the interaction of LYVE-1 with different HA sizes and conformations might variably influence cell function as a consequence of avidity and receptor crosslinking. Finally, we evaluate evidence that LYVE-1 can also bind growth factors and associate with kinase-linked growth factor receptors and conclude on how the LYVE-1·HA axis may be exploited as a target to either block inflammation or tissue allograft rejection, or potentiate vaccine and drug delivery.