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High endothelial venules (HEVs) are specialized postcapillary venules composed of cuboidal blood endothelial cells that express high levels of sulfated sialomucins to bind L-Selectin/CD62L on lymphocytes, thereby facilitating their transmigration from the blood into the lymph nodes (LN) and other secondary lymphoid organs (SLO). HEVs have also been identified in human and murine tumors in predominantly CD3 + T cell-enriched areas with fewer CD20 + B-cell aggregates that are reminiscent of tertiary lymphoid-like structures (TLS). While HEV/TLS areas in human tumors are predominantly associated with increased survival, tumoral HEVs (TU-HEV) in mice have shown to foster lymphocyte-enriched immune centers and boost an immune response combined with different immunotherapies. Here, we discuss the current insight into TU-HEV formation, function, and regulation in tumors and elaborate on the functional implication, opportunities, and challenges of TU-HEV formation for cancer immunotherapy.

Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venules are the point-of-least resistance at the BBB, and compared to capillaries, provide a more feasible route for nanoparticle drug carriers into the brain. Limited understanding of the interactions between nanoparticle drug carriers and the blood-brain barrier underlies many translational failures in treatments of brain disorders. Here the authors use two-photon microscopy in mice to characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery from the blood to the brain in vivo.

Weninger and colleagues show that perivascular macrophages are critical for neutrophil migration into skin infected with Staphylococcus aureus and that the pathogen uses hemolysin-dependent killing of these cells as an immune evasion strategy. Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus . Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Key Points The shuttling of leukocytes between the blood stream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. There are key concepts in the regulation of leukocyte migration through venular walls and motility in the extravascular tissue, with common and distinct mechanisms mediating these responses. Integrin-mediated adhesion of leukocytes to endothelial cells lining venular walls is a prerequisite to leukocyte crawling over and migration through endothelial cells. These responses are associated with great morphological changes in both leukocytes and endothelial cells and can support leukocyte transendothelial cell migration through both paracellular and transcellular routes. Leukocyte migration through endothelial cells is dependent on signalling events in both leukocytes and endothelial cells, events that can regulate leukocyte–endothelial cell interactions, as well as contacts between adjacent endothelial cells and/or endothelial cell vesicular trafficking. After endothelial cell migration, leukocytes need to penetrate the pericyte sheath and the venular basement membrane in which pericytes are embedded. Breaching the pericyte layer may occur through gaps between adjacent cells or in a transcellular manner. Migration through the venular basement membrane occurs through regions that may be biochemically or biophysically permissive. Once detached from the perivascular basement membrane, leukocytes approach their final destination by crawling within the three-dimensional interstitial space, which can either be a fibrillar network or a cell-packed environment like many organ parenchymas or lymphatic tissues. Leukocyte migration in the interstitium is driven by actin protrusion at the leading edge and is occasionally supported by actomyosin contraction at the trailing edge. The cytoskeletal forces can be transduced onto the environment either by integrins or by direct physical interaction of the cell body with the extracellular environment. This flexible mode of migration renders leukocytes largely independent of the molecular composition of the interstitium. Leukocytes use different strategies to migrate through the endothelium of venular walls and in interstitial tissues. These strategies are regulated by soluble and cell-bound signals. Studies have identified many of the cellular and subcellular events that govern transendothelial migration and are beginning to elucidate the nature of leukocyte interstitial motility. The shuttling of leukocytes between the bloodstream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. Recent studies have furthered our understanding of the rapidly advancing field of leukocyte migration, particularly regarding cellular and subcellular events at the level of the venular wall. Furthermore, emerging cellular models are now addressing the transition from an adherent mode to a non-adherent state, incorporating mechanisms that support an efficient migratory profile of leukocytes in the interstitial tissue beyond the venular wall.

Breast cancers present intricate microenvironments comprising heterotypic cellular interactions, yet a comprehensive spatial map remained to be established. Here, we employed the DNA nanoball-based genome-wide in situ sequencing (Stereo-seq) to visualize the geospatial architecture of 30 primary breast tumors and metastatic lymph nodes across different molecular subtypes. This unprecedented high-resolution atlas unveils the fine structure of the tumor vasculature, highlighting heterogeneity in phenotype, spatial distribution, and intercellular communication within both endothelial and perivascular cells. In particular, venular smooth muscle cells are identified as the primary source of CCL21/CCL19 within the microenvironment. In collaboration with ACKR1-positive endothelial cells, they create a chemokine-rich venular niche to synergistically promote lymphocyte extravasation into tumors. High venule density predicts increased immune infiltration and improved clinical outcomes. This study provides a detailed spatial landscape of human breast cancer, offering key insights into the venular regulation of tumor immune infiltration. This study utilizes Stereo-seq to comprehensively map the geospatial architecture of breast cancer, revealing venular niches that promote immune infiltration and better clinical outcomes, offering new insights into tumor microenvironment regulation.

High endothelial vessels (HEVs) provide the conduit for blood-borne leukocytes to enter lymph nodes. Butcher and colleagues report transcriptional profiles of various endothelial cell populations that can explain functional differences of homing-molecule modifications. Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV–selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.

During inflammation neutrophils roll along the vascular endothelium; here, previously unknown structures called ‘slings’, which appear and persist at the front of rolling cells in vivo and in vitro , are described. What keeps neutrophils on a roll? During inflammation, neutrophils — the white blood cells that fight microbial infection — roll along the blood-vessel endothelium, come to a stop and then migrate into tissues. Neutrophil rolling under forces of high shear stress is not fully understood. In this study, Klaus Ley and colleagues describe novel structures called 'slings' (an analogy to the ancient weapon) that provide a 'carpet' of adhesive substrate in front of rolling neutrophils. The slings are derived from membrane tethers, and express the adhesion molecule PSGL-1 in a spatial pattern that results in stepwise peeling of the slings. Most leukocytes can roll along the walls of venules at low shear stress (1 dyn cm −2 ), but neutrophils have the ability to roll at tenfold higher shear stress in microvessels in vivo 1 , 2 . The mechanisms involved in this shear-resistant rolling are known to involve cell flattening 3 and pulling of long membrane tethers at the rear 4 , 5 , 6 . Here we show that these long tethers do not retract as postulated 6 , 7 , but instead persist and appear as ‘slings’ at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro , where P-selectin-glycoprotein-ligand-1 (PSGL-1) is found in discrete sticky patches whereas LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The ‘step-wise peeling of slings’ is distinct from the ‘pulling of tethers’ reported previously 4 , 5 , 6 , 8 . Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.

Erythrodermic psoriasis (EP) is a life-threatening variant of psoriasis. In this study, we contrasted the vascular endothelial cells (ECs) in EP lesions against those in psoriasis vulgaris and healthy controls. Utilizing single-cell RNA sequencing, immunofluorescence, and flow cytometry on human and mouse samples, we observed a marked increase and activation of EP ECs, which upregulated genes relative to angiogenesis, leukocyte adhesion and antigen presentation. This was particularly evident in the subpopulation post-capillary venules (PCV), especially the cluster from EP. Cell–cell communication studies revealed intensified interactions between PCV and leukocytes, mediated by SELE and ICAM1, predominantly in EP. Trajectory analysis suggested differentiation direction of venules-PCV-CAP. 1 with a concomitant reduction in NF2R2 expression. Elevated and activated PCVs were found in EP patient biopsies and mouse models. These findings underscore the significance of PCV in EP pathogenesis, presenting new therapeutic avenues for this debilitating disease.

Background Microsatellite-unstable (MSI) tumours show a high load of mutational neo antigens, as a consequence of DNA mismatch repair deficiency. Consequently, MSI tumours commonly present with dense immune infiltration and develop immune evasion mechanisms. Whether improved lymphocyte recruitment contributes to the pronounced immune infiltration in MSI tumours is unknown. We analysed the density of high endothelial venules (HEV) and postcapillary blood vessels specialised for lymphocyte trafficking, in MSI colorectal cancers (CRC). Methods HEV density was determined by immunohistochemical staining of FFPE tissue sections from MSI ( n  = 48) and microsatellite-stable (MSS, n  = 35) CRCs. Associations with clinical and pathological variables were analysed. Results We found elevated HEV densities in MSI compared with MSS CRCs (median 0.049 vs 0.000 counts/mm 2 , respectively, p  = 0.0002), with the highest densities in Lynch syndrome MSI CRCs. Dramatically elevated HEV densities were observed in B2M- mutant Lynch syndrome CRCs, pointing towards a link between lymphocyte recruitment and immune evasion (median 0.485 vs 0.0885 counts/mm 2 in B2M -wild-type tumours, p  = 0.0237). Conclusions Our findings for the first time indicate a significant contribution of lymphocyte trafficking in immune responses against MSI CRC, particularly in the context of Lynch syndrome. High HEV densities in B2M -mutant tumours underline the significance of immunoediting during tumour evolution.