TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Lymphatics in Tumor Progression and Immunomodulation.

The lymphatic system consists of a unidirectional hierarchy of vessels responsible for fluid homeostasis, lipid absorption, and the transport of immune cells and antigens to secondary lymphoid organs. In cancer, lymphatics play complex and heterogenous roles that can promote or inhibit tumor growth. While lymphatic proliferation and remodeling promote tumor dissemination, functional lymphatics are necessary for generating an effective immune response. Recent reports have noted lymphatic-dependent effects on the efficacy of immunotherapy. These findings suggest that the impact of lymphatic vessels on tumor progression is organ- and context-specific and that a greater understanding of the interaction of tumor cells, lymphatics, and the tumor microenvironment can unveil novel therapies.

Tumor-Associated Lymphatics Upregulate MHC-II to Suppress Tumor-Infiltrating Lymphocytes.

Steady-state lymphatic endothelial cells (LECs) can induce peripheral tolerance by presenting endogenous antigens on MHC class I (MHC-I) molecules. Recent evidence suggests that lymph node LECs can cross-present tumor antigens on MHC-I to suppress tumor-specific CD8+ T cells. Whether LECs can act as immunosuppressive cells in an MHC-II dependent manner in the local tumor microenvironment (TME) is not well characterized. Using murine heterotopic and spontaneous tumor models, we show that LECs in the TME increase MHC-II expression in the context of increased co-inhibitory signals. We provide evidence that tumor lymphatics in human melanoma and breast cancer also upregulate MHC-II compared to normal tissue lymphatics. In transgenic mice that lack LEC-specific MHC-II expression, heterotopic tumor growth is attenuated, which is associated with increased numbers of tumor-specific CD8+ and effector CD4+ T cells, as well as decreased numbers of T regulatory CD4+ cells in the TME. Mechanistically, we show that murine and human dermal LECs can take up tumor antigens in vitro. Antigen-loaded LECs in vitro can induce antigen-specific proliferation of CD8+ T cells but not CD4+ T cells; however, these proliferated CD8+ T cells have reduced effector function in the presence of antigen-loaded LECs. Taken together, our study suggests LECs can act as immunosuppressive cells in the TME in an MHC-II dependent manner. Whether this is a result of direct tumor antigen presentation on MHC-II requires additional investigation.

Lymphatic blood filling in CLEC-2-deficient mouse models.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

Lymphedema: Pathogenesis and Novel Therapies.

Lymphedema affects up to 1 in 6 patients who undergo treatment for a solid tumor in the United States. Its prevalence has increased as more effective oncologic therapies have improved patient survival, but there remains no definitive cure. Recent research has elucidated new details in the pathogenesis of the disease and has demonstrated that it is fundamentally an immunologic process that ultimately results in inflammation, fibroadipose deposition, impaired lymphangiogenesis, and dysfunctional lymphatic pumping. These findings have allowed for the development of novel medical and surgical therapies that may potentially alter the standard of care for a disease that has largely been treated by compression. This review seeks to provide an overview of the emerging therapies and how they can be utilized for effective management of lymphedema.

T lymphocytes negatively regulate lymph node lymphatic vessel formation.

Lymph node lymphatic vessels (LNLVs) serve as a conduit to drain antigens from peripheral tissues to within the lymph nodes. LNLV density is known to be positively regulated by vascular endothelial growth factors secreted by B cells, macrophages, and dendritic cells (DCs). Here, we show that LNLV formation was negatively regulated by T cells. In both steady and inflammatory states, the density of LNLVs was increased in the absence of T cells but decreased when T cells were restored. Interferon-γ secretion by T cells suppressed lymphatic-specific genes in lymphatic endothelial cells and consequently caused marked reduction in LNLV formation. When T cells were depleted, recruitment of antigen-carrying DCs to LNs was augmented, reflecting a compensatory mechanism for antigen presentation to T cells through increased LNLVs. Thus, T cells maintain the homeostatic balance of LNLV density through a negative paracrine action of interferon-γ.

Histopathologic Features of Lymphedema: A Molecular Review.

An estimated 5 million people in the United States are affected by secondary lymphedema, with most cases attributed to malignancies or malignancy-related treatments. The pathogenesis of secondary lymphedema has historically been attributed to lymphatic injury or dysfunction; however, recent studies illustrate the complexity of lymphedema as a disease process in which many of its clinical features such as inflammation, fibrosis, adipogenesis, and recurrent infections contribute to on-going lymphatic dysfunction in a vicious cycle. Investigations into the molecular underpinning of these features further our understanding of the pathophysiology of this disease and suggests new therapeutics.

Inflammatory Manifestations of Lymphedema.

Lymphedema results from lymphatic insufficiency leading to a progressive inflammatory process that ultimately manifests as discomfort, recurrent infections, and, at times, secondary malignancy. Collectively, these morbidities contribute to an overall poor quality of life. Although there have been recent advances in microsurgical interventions, a conservative palliative approach remains the mainstay of treatment for this disabling disease. The absence of a cure is due to an incomplete understanding of the pathophysiological changes that result in lymphedema. A histological hallmark of lymphedema is inflammatory cell infiltration and recent studies with animal models and clinical biopsy specimens have suggested that this response plays a key role in the pathology of the disease. The purpose of this report is to provide an overview of the ongoing research in and the current understanding of the inflammatory manifestations of lymphedema.

Exercise training improves obesity-related lymphatic dysfunction.

KEY POINTS: Obesity results in perilymphatic inflammation and lymphatic dysfunction. Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells. Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. ABSTRACT: Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti-inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR-3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity-induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications.

Obesity-induced lymphatic dysfunction is reversible with weight loss.

KEY POINTS: Obesity induces lymphatic leakiness, decreases initial lymphatic vessel density, impairs collecting vessel pumping and decreases transport of macromolecules. Obesity results in perilymphatic inducible nitric oxide synthase (iNOS) expression and accumulation of T cells and macrophages. Deleterious effects of obesity on the lymphatic system correlate with weight gain. Weight loss restores lymphatic function in obese animals and decreases perilymphatic iNOS and inflammatory cell accumulation. ABSTRACT: Although clinical and experimental studies have shown that obesity results in lymphatic dysfunction, it remains unknown whether these changes are permanent or reversible with weight loss. In the current study, we used a mouse model of diet-induced obesity to identify putative cellular mechanisms of obesity-induced lymphatic dysfunction, determine whether there is a correlation between these deleterious effects and increasing weight gain, and finally examine whether lymphatic dysfunction is reversible with diet-induced weight loss. We report that obesity is negatively correlated with cutaneous lymphatic collecting vessel pumping rate (r = -0.9812, P < 0.0005) and initial lymphatic vessel density (r = -0.9449, P < 0.005). In addition, we show a significant positive correlation between weight gain and accumulation of perilymphatic inflammatory cells (r = 0.9872, P < 0.0005) and expression of inducible nitric oxide synthase (iNOS; r = 0.9986, P < 0.0001). Weight loss resulting from conversion to a normal chow diet for 8 weeks resulted in more than a 25% decrease in body weight and normalized cutaneous lymphatic collecting vessel pumping rate, lymphatic vessel density, lymphatic leakiness, and lymphatic macromolecule clearance (all P < 0.05). In addition, weight loss markedly decreased perilymphatic inflammation and iNOS expression. Taken together, our findings show that obesity is linearly correlated with lymphatic dysfunction, perilymphatic inflammation and iNOS expression, and that weight loss via dietary modification effectively reverses these deleterious effects.

Regulation and implications of inflammatory lymphangiogenesis.

Lymphatic vessels (LVs) are highly dynamic structures that intimately interact with their surrounding microenvironment. They have a profound influence on the immune system and therefore can manipulate inflammatory processes. Inflammation is a major cause of adulthood lymphangiogenesis and LV remodeling. In turn, LVs can reciprocally manipulate inflammatory processes. For instance, LV growth and/or activation regulate antigen presentation and inflammatory cell recruitment to lymph nodes (LNs), and therefore critically affect adaptive immunity. The vascular endothelial growth factor (VEGF)-C-VEGF receptor-3 and VEGF-A-VEGF receptor-2 signaling pathways are particularly important in inflammatory lymphangiogenesis. LVs contribute to the pathophysiology of various inflammatory conditions. Knowledge of lymphatic biology can be applied to manipulate inflammatory disorders and divert immune responses. This review summarizes basic concepts of inflammation-relevant lymphatic biology, and describes recent progress and practical implications.

Interactions of immune cells and lymphatic vessels.

In addition to fluid and lipid absorption, immune cell trafficking has now become recognized as one of the major functions of the lymphatic system. Recently, several critical roles of the lymphatic vessels (LVs) in modulating immune reactions during both physiological and pathological conditions have been emerging. As LVs serve as conduits for immune cells, they come to closely interact with macrophages/monocytes, dendritic cells, and T and B lymphocytes. Accumulating evidences indicate that reciprocal interactions between the LVs and immune cells exist which cause considerable influence over the process of immune cell migration, LV growth, and ultimately certain immune reactions. This chapter discusses on the interactions of macrophages/monocytes and dendritic cells with peripheral LVs and on those of sinusoidal macrophages and T and B lymphocytes with lymph node LVs.

Lymphatic-Dependent Modulation of the Sensitization and Elicitation Phases of Contact Hypersensitivity.

Allergic contact dermatitis is a common inflammatory skin disease comprising 2 phases. During sensitization, immune cells are activated by exposure to various allergens, whereas repeated antigen exposure induces local inflammation during elicitation. In this study, we utilized mouse models lacking lymphatics in different skin regions to characterize the role of lymphatics separately in the 2 phases, using contact hypersensitivity as a model of human allergic inflammatory skin diseases. Lymphatic-deficient mice exhibited no major difference to single antigen exposure compared to controls. However, mice lacking lymphatics in both phases displayed reduced inflammation after repeated antigen exposure. Similarly, diminished immune response was observed in mice lacking lymphatics only in sensitization, whereas the absence of lymphatics only in the elicitation phase resulted in a more pronounced inflammatory immune response. This exaggerated inflammation is driven by neutrophils impacting regulatory T cell number. Collectively, our results demonstrate that skin lymphatics play an important but distinct role in the 2 phases of contact hypersensitivity. During sensitization, lymphatics contribute to the development of the antigen-specific immunization, whereas in elicitation, they moderate the inflammatory response and leukocyte infiltration in a neutrophil-dependent manner. These findings underscore the need for novel therapeutic strategies targeting the lymphatics in the context of allergic skin diseases.

Breast Cancer-Related Lymphedema Results in Impaired Epidermal Differentiation and Tight Junction Dysfunction.

Breast cancer-related lymphedema (BCRL) is characterized by skin changes, swelling, fibrosis, and recurrent skin infections. Clinical studies have suggested that lymphedema results in skin barrier defects; however, the underlying cellular mechanisms and the effects of bacterial contamination on skin barrier function remain unknown. In matched biopsies from patients with unilateral BCRL, we observed decreased expression of FLG and the tight junction protein ZO-1 in skin affected by moderate lymphedema or by subclinical lymphedema in which dermal backflow of lymph was identified by indocyanine green lymphography, relative to those in the controls (areas without backflow and from the unaffected arm). In vitro stimulation of keratinocytes with lymph fluid obtained from patients undergoing lymphedema surgery led to the same changes as well as increased expression of keratin 14, a marker of immature keratinocytes. Finally, using mouse models of lymphedema, we showed that similar to the clinical scenario, the expression of skin barrier proteins was decreased relative to that in normal skin and that colonization with Staphylococcus epidermidis bacteria amplified this effect as well as lymphedema severity. Taken together, our findings suggest that lymphatic fluid stasis contributes to skin barrier dysfunction in lymphedema.

TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells.

Rationale: TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function. Objective: To characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics. Methods and Results: Pressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for ≥1 minute and in some instances remained for ≥4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a controlled and spatially defined single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of Trpv4 compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of Lyve1 , Cd163 , Folr2 , Mrc1 , Ccl8 , Apoe , Cd209f , Cd209d , and Cd209g ; and at least half of these macrophages also expressed Timd4. This subset of macrophages also highly expressed Txa2s , which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction. Conclusions: Clinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema.

Therapy-Induced Senescence Contributes to the Efficacy of Abemaciclib in Patients with Dedifferentiated Liposarcoma.

PURPOSE: We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans. PATIENTS AND METHODS: We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion. RESULTS: Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response. CONCLUSIONS: Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649.

The Vicious Circle of Stasis, Inflammation, and Fibrosis in Lymphedema.

SUMMARY: Lymphedema is a progressive disease of the lymphatic system arising from impaired lymphatic drainage, accumulation of interstitial fluid, and fibroadipose deposition. Secondary lymphedema resulting from cancer treatment is the most common form of the disease in developed countries, affecting 15% to 40% of patients with breast cancer after lymph node dissection. Despite recent advances in microsurgery, outcomes remain variable and, in some cases, inadequate. Thus, development of novel treatment strategies is an important goal. Research over the past decade suggests that lymphatic injury initiates a chronic inflammatory response that regulates the pathophysiology of lymphedema. T-cell inflammation plays a key role in this response. In this review, the authors highlight the cellular and molecular mechanisms of lymphedema and discuss promising preclinical therapies.

Keratinocytes coordinate inflammatory responses and regulate development of secondary lymphedema.

Epidermal changes are histological hallmarks of secondary lymphedema, but it is unknown if keratinocytes contribute to its pathophysiology. Using clinical lymphedema specimens and mouse models, we show that keratinocytes play a primary role in lymphedema development by producing T-helper 2 (Th2) -inducing cytokines. Specifically, we find that keratinocyte proliferation and expression of protease-activated receptor 2 (PAR2) are early responses following lymphatic injury and regulate the expression of Th2-inducing cytokines, migration of Langerhans cells, and skin infiltration of Th2-differentiated T cells. Furthermore, inhibition of PAR2 activation with a small molecule inhibitor or the proliferation inhibitor teriflunomide (TF) prevents activation of keratinocytes stimulated with lymphedema fluid. Finally, topical TF is highly effective for decreasing swelling, fibrosis, and inflammation in a preclinical mouse model. Our findings suggest that lymphedema is a chronic inflammatory skin disease, and topically targeting keratinocyte activation may be a clinically effective therapy for this condition.

Topical captopril: a promising treatment for secondary lymphedema.

Transforming growth factor-beta 1 (TGF-ß1)-mediated tissue fibrosis is an important regulator of lymphatic dysfunction in secondary lymphedema. However, TGF-ß1 targeting can cause toxicity and autoimmune complications, limiting clinical utility. Angiotensin II (Ang II) modulates intracellular TGF-ß1 signaling, and inhibition of Ang II production using angiotensin-converting enzyme (ACE) inhibitors, such as captopril, has antifibrotic efficacy in some pathological settings. Therefore, we analyzed the expression of ACE and Ang II in clinical lymphedema biopsy specimens from patients with unilateral breast cancer-related lymphedema (BCRL) and mouse models, and found that cutaneous ACE expression is increased in lymphedematous tissues. Furthermore, topical captopril decreases fibrosis, activation of intracellular TGF-ß1 signaling pathways, inflammation, and swelling in mouse models of lymphedema. Captopril treatment also improves lymphatic function and immune cell trafficking by increasing collecting lymphatic pumping. Our results show that the renin-angiotensin system in the skin plays an important role in the regulation of fibrosis in lymphedema, and inhibition of this signaling pathway may hold merit for treating lymphedema.

The Future of Lymphedema: Potential Therapeutic Targets for Treatment.

Purpose of Review: This review aims to summarize the current knowledge regarding the pharmacological interventions studied in both experimental and clinical trials for secondary lymphedema. Recent Findings: Lymphedema is a progressive disease that results in tissue swelling, pain, and functional disability. The most common cause of secondary lymphedema in developed countries is an iatrogenic injury to the lymphatic system during cancer treatment. Despite its high incidence and severe sequelae, lymphedema is usually treated with palliative options such as compression and physical therapy. However, recent studies on the pathophysiology of lymphedema have explored pharmacological treatments in preclinical and early phase clinical trials. Summary: Many potential treatment options for lymphedema have been explored throughout the past two decades including systemic agents and topical approaches to decrease the potential toxicity of systemic treatment. Treatment strategies including lymphangiogenic factors, anti-inflammatory agents, and anti-fibrotic therapies may be used independently or in conjunction with surgical approaches.