Project description:Tracing early mammalian lineage decisions by single cell genomics. Lewis Wolpert famously called gastrulation the most important time in your life. During this fascinating process, a pluripotent stem cell population in the early embryo gives rise to the three germ layers from which all organ systems develop. Cell signalling and transcriptional networks are known to regulate aspects of gastrulation, but the precise mechanisms have not been investigated at the single cell level. Thus, new principles remain to be discovered that govern the exit from naïve pluripotency, epigenetic priming, stochasticity in transcriptional programmes, symmetry breaking, and acquisition of heritable transcriptome patterns. We have brought together a consortium of experts in single cell genomics, mammalian postimplantation development, and computational biology to comprehensively tackle this challenge. We will apply recently established single cell genomics techniques for DNA, RNA, and DNA methylation to profile the majority of the cells in mouse postimplantation embryos. This will result in epigenetic and gene expression maps of most cells together with experimentally determined lineage relationships, hence populating a Waddingtonian landscape. Based on such maps, combinations of transcription factors and epigenetic modifiers will be used to experimentally direct differentiation in human iPS cells.
Project description:Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune evasion. While lymphatic vessels facilitate T cell priming, they also exert immune suppressive effects in lymph nodes at steady-state. Therefore, we hypothesized that peripheral lymphatic vessels acquire suppressive mechanisms to limit local effector CD8+ T cell accumulation in murine skin. We demonstrate that nonhematopoietic PD-L1 is largely expressed by lymphatic and blood endothelial cells and limits CD8+ T cell accumulation in tumor microenvironments. IFNγ produced by tissue-infiltrating, antigen-specific CD8+ T cells, which are in close proximity to tumor-associated lymphatic vessels, is sufficient to induce lymphatic vessel PD-L1 expression. Disruption of IFNγ-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in infected and malignant skin leading to increased viral pathology and tumor control, respectively. Consequently, we identify IFNγR as an immunological switch in lymphatic vessels that balances protective immunity and immunopathology leading to adaptive immune resistance in melanoma.
Project description:To address the requirement for lymphatic capillaries in dendritic cell (DC) mobilization from skin to lymph nodes (LNs), we used mice bearing one inactivated allele of vascular endothelial growth factor receptor 3 (VEGFR3) where skin lymphatic capillaries are reported absent. Unexpectedly, DC mobilization from the back skin to draining LNs was similar in magnitude, and kinetics to control mice and humoral immunity appeared intact. By contrast, DC migration from body extremities, including ear and forepaws, was ablated. An evaluation in different regions of skin revealed rare patches of lymphatic capillaries only in body trunk areas where migration was intact. That is, whereas the ear skin was totally devoid of lymphatic capillaries, residual capillaries in the back skin were present though retained only at ?10% normal density. This reduction in density markedly reduced the clearance of soluble tracers, indicating that normal cell migration was spared under conditions when lymphatic transport function was poor. Residual lymphatic capillaries expressed slightly higher levels of CCL21 and migration of skin DCs to LNs remained dependent on CCR7 in Chy mice. DC migration from the ear could be rescued by the introduction of a limited number of lymphatic capillaries through skin transplantation. Thus, the development of lymphatic capillaries in the skin of body extremities was more severely impacted by a mutant copy of VEGFR3 than trunk skin, but lymphatic transport function was markedly reduced throughout the skin, demonstrating that even under conditions when a marked loss in lymphatic capillary density reduces lymph transport, DC migration from skin to LNs remains normal.
Project description:Angiogenesis and lymphangiogenesis participate in many inflammatory diseases, and their reversal is thought to be beneficial. However, the extent of reversibility of vessel remodeling is poorly understood. We exploited the potent anti-inflammatory effects of the corticosteroid dexamethasone to test the preventability and reversibility of vessel remodeling in Mycoplasma pulmonis-infected mice using immunohistochemistry and quantitative RT-PCR. In this model robust immune responses drive rapid and sustained changes in blood vessels and lymphatics. In infected mice not treated with dexamethasone, capillaries enlarged into venules expressing leukocyte adhesion molecules, sprouting angiogenesis and lymphangiogenesis occurred, and the inflammatory cytokines tumor necrosis factor and interleukin-1 increased. Concurrent dexamethasone treatment largely prevented the remodeling of blood vessels and lymphatics. Dexamethasone also significantly reduced cytokine expression, bacterial burden, and leukocyte influx into airways and lungs over 4 weeks of infection. In contrast, when infection was allowed to proceed untreated for 2 weeks and then was treated with dexamethasone for 4 weeks, most blood vessel changes reversed but lymphangiogenesis did not, suggesting that different survival mechanisms apply. Furthermore, dexamethasone significantly reduced the bacterial burden and influx of lymphocytes but not of neutrophils or macrophages or cytokine expression. These findings show that lymphatic remodeling is more resistant than blood vessel remodeling to corticosteroid-induced reversal. We suggest that lymphatic remodeling that persists after the initial inflammatory response has resolved may influence subsequent inflammatory episodes in clinical situations.
Project description:Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system.Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8-10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up.High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages.Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.
Project description:The lymphatic system is essential for the generation of immune responses by facilitating immune cell trafficking to lymph nodes. Dendritic cells (DCs), the most potent APCs, exit tissues via lymphatic vessels, but the mechanisms of interaction between DCs and the lymphatic endothelium and the potential implications of these interactions for immune responses are poorly understood. In this study, we demonstrate that lymphatic endothelial cells (LECs) modulate the maturation and function of DCs. Direct contact of human monocyte-derived DCs with an inflamed, TNF-alpha-stimulated lymphatic endothelium reduced expression of the costimulatory molecule CD86 by DCs and suppressed the ability of DCs to induce T cell proliferation. These effects were dependent on adhesive interactions between DCs and LECs that were mediated by the binding of Mac-1 on DCs to ICAM-1 on LECs. Importantly, the suppressive effects of the lymphatic endothelium on DCs were observed only in the absence of pathogen-derived signals. In vivo, DCs that migrated to the draining lymph nodes upon inflammatory stimuli, but in the absence of a pathogen, showed increased levels of CD86 expression in ICAM-1-deficient mice. Together, these data demonstrate a direct role of LECs in the modulation of immune response and suggest a function of the lymphatic endothelium in preventing undesired immune reactions in inflammatory conditions.
Project description:Systemic delivery of an anti-cancer agent often leads to only a small fraction of the administered dose accumulating in target sites. Delivering anti-cancer agents through the lymphatic network can achieve more efficient drug delivery for the treatment of lymph node metastasis. We show for the first time that polymeric gold nanorods (PAuNRs) can be delivered efficiently from an accessory axillary lymph node to a tumor-containing proper axillary lymph node, enabling effective treatment of lymph node metastasis. In a mouse model of metastasis, lymphatic spread of tumor was inhibited by lymphatic-delivered PAuNRs and near-infrared laser irradiation, with the skin temperature controlled by cooling. Unlike intravenous injection, lymphatic injection delivered PAuNRs at a high concentration within a short period. The results show that lymphatic administration has the potential to deliver anti-cancer agents to metastatic lymph nodes for inhibition of tumor growth and could be developed into a new therapeutic method.
Project description:Lymphatic endothelial cells are important for efficient flow of antigen-bearing fluid and antigen-presenting cells (APCs) from peripheral sites to lymph nodes (LNs). APC movement to LNs is dependent on the constitutive chemokine receptor CCR7, although how conflicting inflammatory and constitutive chemokine cues are integrated at lymphatic surfaces during this process is not understood. Here we reveal a previously unrecognized aspect of the regulation of this process. The D6 chemokine-scavenging receptor, which is expressed on lymphatic endothelial cells (LECs), maintains lymphatic surfaces free of inflammatory CC-chemokines and minimizes interaction of inflammatory leukocytes with these surfaces. D6 does not alter the level of CCR7 ligands on LECs, thus ensuring selective presentation of homeostatic chemokines for interaction with CCR7(+) APCs. Accordingly, in D6-deficient mice, inflammatory CC-chemokine adherence to LECs results in inappropriate perilymphatic accumulation of inflammatory leukocytes at peripheral inflamed sites and draining LNs. This results in lymphatic congestion and impaired movement of APCs, and fluid, from inflamed sites to LNs. We propose that D6, by suppressing inflammatory chemokine binding to lymphatic surfaces, and thereby preventing inappropriate inflammatory leukocyte adherence, is a key regulator of lymphatic function and a novel, and indispensable, contributor to the integration of innate and adaptive immune responses.