Secondary lymphedema is a debilitating condition driven by impaired regeneration of lymphatic vasculature following lymphatic injury, surgical removal of lymph nodes in cancer patients, or infection. However, the extent to which collecting lymphatic vessels regenerate following injury remains unclear. Here, we employed a novel mouse model of lymphatic injury in combination with state-of-the-art lymphatic imaging to demonstrate that the implantation of an optimized fibrin gel following lymphatic vessel injury leads to the reconnection of the injured lymphatic vessel network through sprouting lymphangiogenesis of initial-like lymphatic vessels from the ends of the collecting lymphatic vessels, resulting in the restoration of lymph flow to the draining lymph node. Mechanistically, we found that fibrin implantation elevates the tissue levels of CCL5, a potent immune cell-recruiting chemokine. Notably, injured vessels in CCL5-KO mice made fewer connections following fibrin gel implantation. These novel findings shed light on the mechanisms underlying lymphatic regeneration and suggest that enhancing CCL5 signaling may be a promising therapeutic strategy for enhancing lymphatic regeneration.

Background Intrinsic lymphatic contractility is essential for tissue fluid balance, immunity and organ function, yet no FDA-approved pharmacologic treatments specifically restore lymphatic contractility. Lymph is returned to the circulation by ion channel-driven cyclic contractions of collecting lymphatic vessels. Although voltage-gated sodium (NaV) channels drive cardiomyocyte excitability, their role in lymphatic muscle cell (LMC) physiology is not well defined. We identified NaV1.3, a NaV channel historically viewed as developmentally restricted and limited in adult tissues, as unexpectedly and selectively expressed in adult lymphatic muscle but absent from heart, vascular smooth muscle, and mature brain. We tested whether selective NaV1.3 activation restores impaired lymphatic pumping in aging and radiation injury. Methods NaV1.3 expression in LMCs was confirmed through single-cell RNA sequencing analysis and immunostaining of mouse and human lymphatic vessels. Lymphatic contractility was quantified by in vivo fluorescence lymphangiography and interstitial fluid clearance was measured with a new bioluminescence assay. NaV1.3 function was assessed in young, aged, and radiation-injured mice. NaV1.3 knockout ( Scn3a-/- ) mice established the requirement of NaV1.3 for basal lymphatic excitability and responsiveness to the NaV1.3-specific activator, Tf2. Results In mouse and human lymphatic vessels, NaV1.3 is expressed in adult LMCs. Although dispensable for basal lymphatic contractions, NaV1.3 acted as a pharmacologically recruitable reserve that amplified contractile output. Acute NaV1.3 activation with Tf2 increased lymphangion ejection fraction and accelerated interstitial fluid clearance. Tf2 fully restored lymphatic pumping in aged mice and partially rescued radiation-induced contractile deficits. All Tf2 responses were abolished in Scn3a-/- mice, confirming NaV1.3 dependence. Conclusions NaV1.3 is a selectively druggable ion channel in adult lymphatic muscle that can be recruited to restore lymphatic pump function across aging and injury. Targeted NaV1.3 activation provides a molecular entry point for treating diseases characterized by lymphatic pump failure, a domain with no existing pharmacologic therapies. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, R21AG072205, R01CA284372, R01CA284603, K08GM155886, T32-GM007592 United States Department of Defense, HT94252410100 MGH Research Institute, Rullo Family MGH Research Scholar Award International Anesthesia Research Society, https://ror.org/0252rqe04, IMRA Harvard Medical School, https://ror.org/03wevmz92, Eleanor and Miles Shore Award

The lymphatic system plays a crucial role in maintaining tissue fluid balance, immune surveillance, and the transport of lipids and macromolecules. Lymph is absorbed by initial lymphatics and then driven through lymph nodes and to the blood circulation by the contraction of collecting lymphatic vessels. Intraluminal valves in collecting lymphatic vessels ensure the unidirectional flow of lymph centrally. The lymphatic muscle cells that invest in collecting lymphatic vessels impart energy to propel lymph against hydrostatic pressure gradients and gravity. A variety of mechanical and biochemical stimuli modulate the contractile activity of lymphatic vessels. This review focuses on the recent advances in our understanding of the mechanisms involved in regulating and collecting lymphatic vessel pumping in normal tissues and the association between lymphatic pumping, infection, inflammatory disease states, and lymphedema.

Lymphatic transport facilitates the presentation of cancer antigens in tumor-draining lymph nodes (tdLNs), leading to T cell activation and the generation of systemic anti-cancer immune surveillance. Surgical removal of tdLNs to control cancer progression is routine in clinical practice. However, whether removing tdLNs impairs immune checkpoint blockade (ICB) is still controversial. Our analysis demonstrates that melanoma patients remain responsive to PD-1 checkpoint blockade after regional LN dissection. We were able to recapitulate the persistent response to ICB after regional LN resection in murine melanoma and mammary carcinoma models. Mechanistically, soluble antigen is diverted to distant LNs after tdLN dissection. Consistently, robust ICB responses in patients with head and neck cancer after primary tumor and tdLN resection correlated with the presence of reactive LNs in distant sites. These findings indicate that distant LNs sufficiently compensate for the removal of direct tdLNs and sustain the response to ICB.

Background:Intrinsic lymphatic contractility is essential for tissue fluid balance, immunity and organ function, yet no FDA-approved pharmacologic treatments specifically restore lymphatic contractility. Lymph is returned to the circulation by ion channel-driven cyclic contractions of collecting lymphatic vessels. Although voltage-gated sodium (Na V ) channels drive cardiomyocyte excitability, their role in lymphatic muscle cell (LMC) physiology is not well defined. We identified Na V 1.3, a Na V channel historically viewed as developmentally restricted and limited in adult tissues, as unexpectedly and selectively expressed in adult lymphatic muscle but absent from heart, vascular smooth muscle, and mature brain. We tested whether selective Na V 1.3 activation restores impaired lymphatic pumping in aging and radiation injury. Methods:Na V 1.3 expression in LMCs was confirmed through single-cell RNA sequencing analysis and immunostaining of mouse and human lymphatic vessels. Lymphatic contractility was quantified by in vivo fluorescence lymphangiography and interstitial fluid clearance was measured with a new bioluminescence assay. Na V 1.3 function was assessed in young, aged, and radiation-injured mice. Na V 1.3 knockout ( Scn3a -/- ) mice established the requirement of Na V 1.3 for basal lymphatic excitability and responsiveness to the Na V 1.3-specific activator, Tf2. Results:In mouse and human lymphatic vessels, Na V 1.3 is expressed in adult LMCs. Although dispensable for basal lymphatic contractions, Na V 1.3 acted as a pharmacologically recruitable reserve that amplified contractile output. Acute Na V 1.3 activation with Tf2 increased lymphangion ejection fraction and accelerated interstitial fluid clearance. Tf2 fully restored lymphatic pumping in aged mice and partially rescued radiation-induced contractile deficits. All Tf2 responses were abolished in Scn3a -/- mice, confirming Na V 1.3 dependence. Conclusions:Na V 1.3 is a selectively druggable ion channel in adult lymphatic muscle that can be recruited to restore lymphatic pump function across aging and injury. Targeted Na V 1.3 activation provides a molecular entry point for treating diseases characterized by lymphatic pump failure, a domain with no existing pharmacologic therapies.

Chronic lymphedema is a progressive, disfiguring disease that results from dysfunction of the lymphatic vasculature, causing distal accumulation of interstitial fluid, localized development of tissue edema, and expansion of subcutaneous adipose tissue (SAT). As the molecular mechanisms governing SAT remodeling in this disease are unclear, we performed single-nucleus RNA sequencing on paired control and affected SAT biopsies from patients with unilateral lymphedema. Lymphedema samples were characterized by expansion of SAA + adipocytes, pro-adipogenic stem cells, and proliferation of lymphatic capillaries. A GRIA1 + lymphedema-enriched stromal cell population expressing VEGFC , ADAMTS3 , and CCBE1 was identified, suggesting an enhanced axis of communication between adipose stem and progenitor cells (ASPCs) and lymphatic endothelial cells. Furthermore, lymphedema ASPC-conditioned media promoted lymphatic endothelial tube elongation in vitro . These findings indicate a critical role for ASPCs in regulating adipocyte differentiation and lymphatic vascular remodeling in lymphedema, and provide a valuable resource for better understanding this disease.

A diverse naive CD8 T cell repertoire is essential to provide broad protection against infection and cancer. Here, we uncover a sex-biased mechanism of immune aging in which male mice exhibit early depletion of naive CD8 T cells through accelerated, antigen-agnostic differentiation into virtual memory cells. This depletion, compounded by androgen-driven thymic atrophy, leads to contraction of lymph nodes and a reduced local naive T cell repertoire, limiting antigen recognition capacity. Therapeutic thymus regeneration via androgen ablation repopulates naive CD8 T cells in lymph nodes and reinvigorates tumor recognition in middle-aged male mice. These findings reveal the crucial impact of sex and age on locoregional naive T cell repertoires in lymph nodes and suggest strategies to restore immune competence in aging males.

The lymphatic vasculature plays essential roles in fluid balance, immunity, and lipid transport. Chronic, low-grade inflammation in peripheral tissues develops when lymphatic structure or function is impaired, as observed during aging. While aging has been associated with a broad range of heart pathophysiology, its effect on cardiac lymphatic vasculature has not been characterized. Here, we analyzed cardiac lymphatics in aged 20-month-old mice versus young 2-month-old mice. Aged hearts showed reduced lymphatic vascular density, more dilated vessels, and increased inflammation and fibrosis in peri-lymphatic zones. As exercise has shown benefits in several different models of age-related heart disease, we further investigated the effects of aerobic training on cardiac lymphatics. Eight weeks of voluntary wheel running attenuated age-associated adverse remodeling of the cardiac lymphatics, including reversing their dilation, increasing lymph vessel density and branching, and reducing perilymphatic inflammation and fibrosis. Intravital lymphangiography demonstrated improved cardiac lymphatic flow after exercise training. Our findings illustrate that aging leads to cardiac lymphatic dysfunction, and that exercise can improve lymphatic health in aged animals.

Kahn and colleagues reveal that lymph nodes (LN) provide an intrinsically immunosuppressive niche that prevents effector function of activated CD8+ T cells in LNs and allows immunogenic tumor cells to survive and drive cancer progression, independent of tumor-derived preconditioning. By locally suppressing IL2 availability, regulatory T cells in LNs impair CD8+ T-cell cytotoxicity-a mechanism with important implications for immune checkpoint therapy and LN-targeted immunomodulation. See related article by Kahn et al., p. 1949.

Antigen-based tumor vaccines rely on adjuvants to stimulate local inflammation, recruit antigen-presenting cells (APCs), and enhance immune activation. However, the complex interplay between antigen transport, lymphatic drainage, and immune cell dynamics across organs remains poorly understood, limiting the rational design of vaccination strategies. Here, we present a multiscale compartmental Physiologically Based Pharmacokinetic (PBPK) model of antigen vaccination that integrates systemic circulation, lymphatic connectivity, and immune cell activation at the whole-body level. The model incorporates arterial, venous, and lymphatic flows, organ-specific interstitium and lymph node (LN) networks, and a superficial skin network. The model reproduces spatiotemporal distributions of antigen and suppressive factors, APC activation, and nT priming across activation sites, including LNs and spleen. Our results show that the sensitivity of vaccination-induced immunity is highly related to antigen and suppressive factor production by the tumor, and that early-stage vaccination, enhances immunity. Since the model is able to identify optimal vaccination administration over the course of tumor growth for each patient with certain levels of antigen and immune suppressive factors, it can serve as the foundation for digital twins of patients to help inform anti-cancer vaccination strategies. Teaser:A PBPK model links body-wide immune transport to optimize cancer vaccine design and delivery.

Lymphatic muscle cells orchestrate the contraction of collecting lymphatic vessels in mice.

To identify vulnerable upper extremity regions in native lymphatic anatomy that predispose women to the development of breast cancer-related lymphedema. In addition, to identify currently available imaging technologies that could be repurposed for in-vivo lymphatic imaging of these anatomic regions and pathways. Breast cancer-related lymphedema remains an incurable complication of breast cancer treatment, but improvements to knowledge of upper extremity lymphatic anatomy and imaging can unlock new techniques for prevention and treatment. “Bringing to Light the Invisible Lymphatic Anatomy of the Human Body” was a 2-day accelerator workshop held in May 2024 at the Harvard Radcliffe Institute attended by sixteen experts in lymphatic anatomy and imaging, including 4 lymphatic anatomists, 5 imaging clinicians, 3 lymphatic scientists, and 3 program officers from the National Heart, Lung, and Blood Institute (NHLBI) and Advanced Research Projects Agency for Health (ARPA-H). Collateral pathways of the superficial lymphatic system, perforating lymphatic vessels, and the deep lymphatic system were implicated in preventing or reducing the severity of BCRL. Several strategies were proposed for repurposing existing imaging technology and developing new imaging technology that can improve understanding of the anatomy, function, and connectivity of lymphatic vessels in these 3 regions of the arm. Advancements in lymphatic imaging are central to refining our knowledge of lymphatic anatomy. Key challenges to lymphatic imaging are visualization of the deep lymphatic system and perforating lymphatic vessels.

Oncolytic viruses have strong potential as immunotherapies. By causing cancer cells to die and relieve antigens, these viruses can stimulate robust, systemic immune responses that may eliminate disseminated disease and prevent recurrence. Unfortunately, clinical trials using oncolytic viruses have not induced clearance of metastasis or protection from recurrence. Likewise, the combination of the only FDA-approved oncolytic virus-Talimogene laherparapvec-with immune checkpoint blockade did not improve progression-free or overall survival. Because of these disappointing clinical trials, we sought to measure the ability of oncolytic viruses to induce cancer antigen presentation and the elicitation of cancer antigen-specific immune responses. Our data revealed that despite improved antigen presentation by dendritic cells, priming of cancer antigen-specific T cells was limited. However, viral antigen-specific T cells did develop and were in a phenotypic state to induce an effective response against virally infected cells. These preclinical results were mirrored in human peripheral blood samples. Overall, these data show that oncolytic virus treatment induces a response against the virus itself, but not cancer antigen, explaining the lack of response in metastatic disease. These interesting findings identify a critical mechanism that needs to be overcome to increase the efficacy of oncolytic virus therapy.

Background Immunotherapies have revolutionized cancer care in recent decades, but approved therapies often fail and currently only target specific steps in the generation of anti-cancer immune responses. Notably, the majority of approved immunotherapies do not target antigen processing and presentation, which are key steps in the development of immune responses and harbor potential as targets to improve immunotherapy. Here, we hypothesize that tumor-mediated alterations in cytokine concentrations alter antigen presentation, which can be normalized by locoregional cytokine delivery or targeted immunological adjuvant delivery.Methods We used mouse models of breast cancer, with analysis by flow cytometry, immunofluorescence, confocal imaging, and single-cell RNA sequencing to address the impacts of tumors on locoregional antigen presentation, along with mechanisms to remedy these impacts.Results Here, we demonstrate that breast tumors induce locoregional impairments in dendritic cell antigen presentation that limits anti-cancer antigen-specific T cell responses. Antigen processing was not impaired in dendritic cells within the tumor-draining lymph node. A reduction of the cytokine IL-1β in tumor-draining lymph nodes was responsible for impairments in antigen presentation by dendritic cells. As such, we tested the ability of dendritic cells in lymph nodes at various distances from the primary tumor to be activated utilizing an antigen-agnostic immunological adjuvant delivery strategy. We observed improved antitumor T cell responses when immunological adjuvant was delivered to cancer antigen-positive lymph nodes distant from the tumor, suggesting that these lymph nodes can be targeted to improve anti-cancer immune responses. When combined with immune checkpoint blockade, delivery of immunological adjuvant to distant lymph nodes led to long-term survival and protection from recurrence. Antigen presentation by dendritic cells and T cell responses could also be recovered by exogenous delivery of IL-1β via intratumoral injection, with improved survival when combined with immune checkpoint blockade.Conclusions This study demonstrates that tumor-induced impairments in antigen presentation in tumor-draining lymph nodes can be overcome by the appropriate introduction of immunological adjuvant to tumor-distant lymph nodes or by restoring IL-1β to the tumor-draining lymph node. These strategies can induce high-quality, durable immune responses and have clinical implications for expanding the efficacy of immunotherapies.

Despite significant strides in lymphatic system imaging, the timely diagnosis of lymphatic disorders remains elusive. This is driven by the absence of standardized, non-invasive, reliable, quantitative methods for real-time functional analysis of lymphatic contractility with adequate spatial and temporal resolution. Here, we address this unmet need by integrating near-infrared fluorescence lymphangiography imaging with an innovative analytical workflow that combines data acquisition, signal processing, and statistical analysis to integrate traditional peak-and-valley analysis with advanced wavelet time-frequency analyses. Variance component analysis was used to evaluate the drivers of variance attributable to each experimental variable for each lymphangiography measurement type. Generalizability studies were used to assess the reliability of measured parameters and how reliability improves as the number of repeat measurements per subject increases. This allowed us to determine the minimum number of repeat measurements needed per subject for acceptable measurement reliability. This approach not only offers detailed insights into lymphatic pumping behaviors across species, sex and age, but also significantly boosts the reliability of these measurements by incorporating multiple regions of interest and evaluating the lymphatic system under various gravitational loads. For example, the reliability of the peak-and-valley analysis of human lymphatic vessels was increased 3-fold using the described approach. By addressing the critical need for improved imaging and quantification methods, our study offers a new standard approach for the imaging and analysis of lymphatic function that can improve our understanding, diagnosis, and treatment of lymphatic diseases. The results highlight the importance of comprehensive data acquisition strategies to fully capture the dynamic behavior of the lymphatic system.

OBJECTIVE:To identify vulnerable upper extremity regions in native lymphatic anatomy that predispose women to the development of breast cancer-related lymphedema. In addition, to identify currently available imaging technologies that could be repurposed for in-vivo lymphatic imaging of these anatomic regions and pathways. BACKGROUND:Breast cancer-related lymphedema remains an incurable complication of breast cancer treatment, but improvements to knowledge of upper extremity lymphatic anatomy and imaging can unlock new techniques for prevention and treatment. METHODS:"Bringing to Light the Invisible Lymphatic Anatomy of the Human Body" was a 2-day accelerator workshop held in May 2024 at the Harvard Radcliffe Institute attended by sixteen experts in lymphatic anatomy and imaging, including 4 lymphatic anatomists, 5 imaging clinicians, 3 lymphatic scientists, and 3 program officers from the National Heart, Lung, and Blood Institute (NHLBI) and Advanced Research Projects Agency for Health (ARPA-H). RESULTS:Collateral pathways of the superficial lymphatic system, perforating lymphatic vessels, and the deep lymphatic system were implicated in preventing or reducing the severity of BCRL. Several strategies were proposed for repurposing existing imaging technology and developing new imaging technology that can improve understanding of the anatomy, function, and connectivity of lymphatic vessels in these 3 regions of the arm. CONCLUSION:Advancements in lymphatic imaging are central to refining our knowledge of lymphatic anatomy. Key challenges to lymphatic imaging are visualization of the deep lymphatic system and perforating lymphatic vessels.

Lymphatic muscle cells (LMCs) within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to LMCs disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the gene transcriptional signatures in LMCs and how they relate to lymphatic function in normal and disease contexts is largely missing. We have generated a comprehensive transcriptional single-cell atlas—including LMCs—of peripheral collecting lymphatic vessels from mice across the lifespan. We identified genes that distinguish LMCs from other types of muscle cells, characterized the phenotypical and transcriptomic changes in LMCs in aged vessels, and identified a proinflammatory microenvironment that suppresses the contractile apparatus in LMCs from advanced-aged mice. Our findings provide a valuable resource to accelerate future research for the identification of potential drug targets on LMCs to improve lymphatic vessel function.

Overly dense microvascular networks are treated by selective reduction of vascular elements. Inappropriate manipulation of microvessels could result in loss of host tissue function or a worsening of the clinical problem. Here, experimental, and computational models were developed to induce blood flow changes via selective artery and vein laser ablation and study the compensatory collateral flow redistribution and vessel diameter remodeling. The microvasculature was imaged non-invasively by bright-field and multi-photon laser microscopy, and Optical Coherence Tomography pre-ablation and up to 30 days post-ablation. A theoretical model of network remodeling was developed to compute blood flow and intravascular pressure and identify vessels most susceptible to changes in flow direction. The skin microvascular remodeling patterns were consistent among the five specimens studied. Significant remodeling occurred at various time points, beginning as early as days 1-3 and continuing beyond day 20. The remodeling patterns included collateral development, venous and arterial reopening, and both outward and inward remodeling, with variations in the time frames for each mouse. In a representative specimen, immediately post-ablation, the average artery and vein diameters increased by 14% and 23%, respectively. At day 20 post-ablation, the maximum increases in arterial and venous diameters were 2.5x and 3.3x, respectively. By day 30, the average artery diameter remained 11% increased whereas the vein diameters returned to near pre-ablation values. Some arteries regenerated across the ablation sites via endothelial cell migration, while veins either reconnected or rerouted flow around the ablation site, likely depending on local pressure driving forces. In the intact network, the theoretical model predicts that the vessels that act as collaterals after flow disruption are those most sensitive to distant changes in pressure. The model results match the post-ablation microvascular remodeling patterns.

The lymphatic system plays a vital role in maintaining fluid balance in living tissue and serves as a pathway for the transport of antigen, immune cells, and metastatic cancer cells. In this study, we investigate how the movement of cells through a contracting lymphatic vessel differs from steady flow, using a lattice Boltzmann-based computational model. Our model consists of cells carried by flow in a 2D vessel with regularly spaced, bi-leaflet valves that ensure net downstream flow as the vessel walls contract autonomously in response to calcium and nitric oxide levels regulated by stretch and shear stress levels. The orientation of the vessel with respect to gravity, which may oppose or assist fluid flow, significantly modulates cellular motion due to its effect on the contraction dynamics of the vessel, even when the cells themselves are neutrally buoyant. Additionally, our model shows that cells are carried along with the flow, but when the vessel is actively contracting, they move faster than the average fluid velocity. We also find that the fluid forces cause significant deformation of the compliant cells, especially in the vicinity of the valves. Our study highlights the importance of considering the complex, transient flows near the valves in understanding cellular motion in lymphatic vessels.