Nancy H. Ruddle - US grants

The goal of these studies is an in-depth investigation of the thymus-derived lymphocyte (T cell). Murine T-cell clones and hybrids will be used as homogeneous sources of antigen-specific activities and lymphokines. Lymphokines are mediators of T-cell function and participate in delayed hypersensitivity and immunity to tumors, viral, and fungal diseases. Lymphokines to be studied include lymphotoxin (LT), migration inhibitory factor (MIF), interferon (IFN), and T-cell growth factor (IL2). Antigen-specific activities will include induction of replication and lymphokine production in T-cell clones. Conditions that regulate induction of lymphokines will be studied by determining the nature of the H2 restricting element in antigen-specific induction of LT production. Lymphotoxin messenger RNA will be isolated from T-cell clones and hybrids by immunological precipitation of polyribosomes with a monoclonal anti-LT antibody. Alternatively, polyadenylated RNA will be isolated and enriched for LT mRNA by size fractionation. In vitro translates (frog oocytes, rabbit reticulocytes, or wheat germ extract) will be assayed for LT, MIF, IFN, and IL2. LT cDNA probes will be prepared by taking advantage of the fact that a T-cell clone is induced to make 2000-fold more LT after induction with a T-cell mitogen. Cloning will be accomplished after purification of LT mRNA, cDNA preparation and insertion into pBR322 or pcDV1, and replication in E. coli. cDNA clones will be identified by hybridization selection with LT mRNA, by differential hybridization of cDNA probes prepared from mRNA of induced and uninduced T-cell clones, or by subtraction of common cDNA sequences by cascade hybridization. Genomic LT DNA probes will also be used to screen cDNA libraries. The LT gene will be transfected with a mammalian expression vector into fibroblasts or T cells by means of protoplast fusion, and its expression studied. Organization of T-cell genes will be studied with the LT cDNA probe and an additional T-cell probe for an antigen-binding protein from a T-cell hybrid. Genes for LT and other T-cell products will be mapped by somatic cell hybridization. These studies will provide basic information on the nature of genetic regulation of lymphokines and will provide information on the interrelationships of lymphokines with each other and with T-cell antigen receptors. (IS)

Lymphotoxin (LT), a product of activated T cells, kills many cell types including several tumors and activated T cells. This effect may contribute significantly to tumor surveillance and may also regulate the immune response. LT, and the functionally and structurally related molecule, tumor necrosis factor (TNF), may also be involved in the pathogenesis of disease, paticularly those of autoimmune etiology. LT and TNF have non cytotoxic effects on various other target cells. These include activation of polymorphonuclear leukocytes and induction of monocyte differentiation. The goal of the project outlined here is to elucidate the mechanism of LT's pleiotropic effects and to understand the basis of its cytotoxic effects on some cells and its induction of differentiation in others. First the mechanism of LT's cytotoxic effects will be probed. Recombinant derived murine LT, biosynthetically labeled naturally derived LT and TNF, and LT peptides will be analyzed for receptor binding, effects on lysosomes, changes in intracellular calcium and arachidonic acid metabolism, and induction of nuclear changes, especially DNA fragmentation. The effect of murine LT on monocyte differentiation will be studied with murine monocyte tumors. Once such a system has been established, the mechanism of LT induction of differentiation will be studied and compared with its mechanism of killing. These studies on the systematic analysis of the mechanisms of LT's effects will provide information which will allow its optimal use as an immunotherapeutic agent. They will also provide insight into methods to inhibit these effects when they contribute to disease pathogenesis.

Paget's disease is a chronic skeletal disease characterized by abnormally rapid bone turnover. Little is known about the causative agents, etiology or pathogenesis of the disease except for a possible link with slow virus infection. Bone pathology identified in mice that are transgenic for the human T lymphotropic virus I (HTLV-1 trans acting gene tax includes a very high and coupled bone turnover, reminiscent of Paget's disease. HTLV-I is associated in humans with activation of lymphotoxin (LT, TNF-beta) and GM-CSF, two cytokines associated with bone cell activation. Preliminary evidence tax mice suggests that cytokine gene activation occurs. The hypothesis to be tested is that the presence of the tax gene activates bone cells or induces increased production of a specific set of cytokines which result in accelerated bone turnover with increased osteoblast and osteoclast activity. The pathophysiology of high bone turnover of tax mice will be characterized by biochemical and hormone analysis, histomorphometry, and ultrastructure. Calcitonin will be administered. The mechanisms by which high bone turnover is induced in HTLV-I tax mice will be analyzed by evaluating osteoblast and osteoclast functional characteristic and differentiation. Tax mice will be analyzed for increased cytokine production (LT, TNF-a IL-1, IL-2, IL-4, GM-CSF, IL-6, PTHrP) by standard biological assays and by their ability to activate osteoblasts and osteoclasts. The cellular origin of increased cytokine production will be determined and correlated with expression of the Tax protein by Western and Northern blot analysis. The molecular mechanism of activation of cytokines contributing to high bone turnover in tax mice will be evaluated using LT as a model, by transfection of constructs consisting of LT 5' sequences driving expression of a bacterial reporter gene, chloramphenicol acetyl transferase into Tax expressing cell lines. LT 5' sequence activation will be evaluated by crossing LTCAT transgenic mice with tax mice. Additional transgenics will be prepared to allow further analysis of the LT gene, and other cytokine genes induced in tax mice. The accomplishments of these goals could help in understanding the pathogenesis of Paget's disease and the mechanism by which viral genes can induce high bone turnover, and will also contribute to basic understanding concerning the mechanism of trans activation of cytokine genes.

Lymphotoxin (LT;TNF-beta) contributes to defense against tumors and virus-infected cells on the one hand and to disease pathogenesis on the other. It may also play a role in T and B cell development in its ability to induce apoptosis and proliferation. Our long range goals are to define the molecular basis of LT regulation and to understand how this impacts on the protein's biologic significance. The focus is on LT, but its special relationship with the functionally similar and genetically linked TNF-alpha will also be considered. Defined genetic elements that regulate LT expression in T and B cell lines are transcriptional start sites, minimal promoters, and upstream negative and positive regulatory elements. These include a NF-kappa-B site important for constitutive LT production in T cells and a poly (dA-dT) rich HMG-1 binding sequence that contributes to LT promoter activity in pre B cells. LT's tight linkage to the TNF-alpha gene, and the fact that in many cells the two cytokines are activated by the same signals, gives rise to the hypothesis that the region between the genes (IGR) contains shared regulatory elements that may influence expression of both within the context of their promoters. The Specific Aims are: 1. To evaluate the role in LT regulation of sequences shared between the LT and TNF-alpha genes. 2. To evaluate the role of HMG-1 in LT regulation. 3. To identify additional regulatory elements within and outside-the LT gene. 4. To evaluate the biologic role of LT and LT and TNF-alpha particularly in the development of T and B cell competence. DNA protein binding studies, functional analyses by transient transfections and in transgenic mice, and homologous recombination and gene knock-out techniques will be used. These experiments will contribute to our understanding of LT's biological significance and enhance our ability to harness the expression of this potent cytokine in clinical situations. Experiments that test the consequences of elimination of LT expression will provide information concerning its biological role and its redundancy with TNF-alpha.

Paget's disease is a chronic skeletal disease characterized by abnormally rapid bone turnover. Little is known about the causative agents, etiology or pathogenesis of the disease except for a possible link with slow virus infection. Bone pathology identified in mice that are transgenic for the human T lymphotropic virus I (HTLV-1 trans acting gene tax includes a very high and coupled bone turnover, reminiscent of Paget's disease. HTLV-I is associated in humans with activation of lymphotoxin (LT, TNF-beta) and GM-CSF, two cytokines associated with bone cell activation. Preliminary evidence tax mice suggests that cytokine gene activation occurs. The hypothesis to be tested is that the presence of the tax gene activates bone cells or induces increased production of a specific set of cytokines which result in accelerated bone turnover with increased osteoblast and osteoclast activity. The pathophysiology of high bone turnover of tax mice will be characterized by biochemical and hormone analysis, histomorphometry, and ultrastructure. Calcitonin will be administered. The mechanisms by which high bone turnover is induced in HTLV-I tax mice will be analyzed by evaluating osteoblast and osteoclast functional characteristic and differentiation. Tax mice will be analyzed for increased cytokine production (LT, TNF-a IL-1, IL-2, IL-4, GM-CSF, IL-6, PTHrP) by standard biological assays and by their ability to activate osteoblasts and osteoclasts. The cellular origin of increased cytokine production will be determined and correlated with expression of the Tax protein by Western and Northern blot analysis. The molecular mechanism of activation of cytokines contributing to high bone turnover in tax mice will be evaluated using LT as a model, by transfection of constructs consisting of LT 5' sequences driving expression of a bacterial reporter gene, chloramphenicol acetyl transferase into Tax expressing cell lines. LT 5' sequence activation will be evaluated by crossing LTCAT transgenic mice with tax mice. Additional transgenics will be prepared to allow further analysis of the LT gene, and other cytokine genes induced in tax mice. The accomplishments of these goals could help in understanding the pathogenesis of Paget's disease and the mechanism by which viral genes can induce high bone turnover, and will also contribute to basic understanding concerning the mechanism of trans activation of cytokine genes.

Insulin dependent diabetes mellitus (IDDM) also known as Type l diabetes is an autoimmune disease of unknown etiology is manifested as destruction of beta cells in the pancreatic islets of Langerhans. T cells are implicated in the pathogenesis of this multigenic inflammatory disease. From an immunological point of view, two hypotheses (at least) can be suggested for the development of human IDDM. The first states that beta cell destruction is an autoantigen targeted process; the second that beta cell destruction is the result of an inflammatory process that is not specifically directed to those cells but results in their destruction because of their susceptibility to cytokines produced by infiltrating cells. Transgenic mice will be used to evaluate these hypotheses and to study mechanisms of inflammation. The long term goal of this project is to evaluate the role of tumor necrosis factor-alpha (cachectin) and beta (lymphotoxin) in the pathogenesis of IDDM. Mice transgenic for the rat insulin promoter II (RIP) driving TNF-alpha or TNF-beta express the transgene in their islets. Both types of RIP-TNF mice exhibit periinsulitis and insulitis, but no beta cell destruction, insulin reduction, or diabetes. These animals appear to be locked in the first stages of IDDM. The specific aims and methods to evaluate the cells and mechanisms of inflammation and pathogenesis in a transgenic model of IDDM are: A) To determine the requirements for progression from periinsulitis and insulitis to diabetes in TNF transgenic mice by testing whether TNF under the control of the glucagon promoter also induces inflammation and then analyze the response of RIP-TNF and GLU-TNF mice to activation signs (anti-CD3, anti-CD 28, superantigens) delivered in vivo or in vitro. TNF transgenic mice will be compared to NOD mice, a well established model of human IDDM and crossed to mice that possess the NOD but lack the background genes to determine if TNF expression in the islets can substitute for these genes; B)To determine the role of T cells specific or not for beta antigens in inflammation and beta cell destruction in TNF transgenic mice by analyzing T cells specific for GAD, BSA, and additional islet antigens or highly purified homogeneous populations of cells activated to cytochrome c or ovalbumin; C)To determine the mechanism of inflammation and beta cell destruction in TNF transgenic mice by analyzing the role of T and B cells, adhesion molecules, MHC antigens, cytokines, and chemokines. Transgenic mice that express TNF at inappropriate levels in islet tissue represent an interesting model system to test the mechanism of inflammation and beta cell destruction in IDDM and the role of particular immunologic cells in this process. They provide a unique opportunity to investigate the effect of targeted expression of a single cytokine gene on autoimmune disease pathogenesis.

DESCRIPTION (Adapted from the Investigator's abstract): The goals of this project are the delineation of the biological roles and molecular regulation of lymphotoxin (LT; LT alpha; TNF beta) as an entity in itself and with regard to its relationship to the other members of the LT/TNF family, tumor necrosis factor (TNF alpha) and LT beta. LT alpha can assume two molecular forms: a secreted homotrimer, or a cell-associated heterotrimeric LT alpha/beta complex. LT alpha has been implicated in the pathogenesis of autoimmune diseases and participates in defense against cancer through its ability to kill cells by apoptosis and promote inflammation. Mice genetically deficient in LT alpha have no lymph nodes or Peyer's patches and exhibit splenic disorganization indicating a crucial role in lymphoid organ development. Mice transgenic for LT alpha under the control of the insulin promoter (RIPLT) exhibit inflammation at the sites of transgene expression. This inflammation has most of the characteristics or organized lymphoid tissue, suggesting that LT-induced chronic inflammation is lymphoid neogenesis. The RIPLT transgene restores LN but not splenic architecture to LT alpha -/- mice. The specific aims are to: 1) Determine whether LT beta contributes to lymphoid organ development; 2) Identify the temporal limits and physical forms by which ectopic LT restores lymphoid organs; 3) Identify the mechanisms by which LT induces lymphoid organogenesis; 4) Determine which cells produce LT alpha in development and define the molecular basis of its regulation. These aims will be accomplished by analyzing LT beta -/- mice; by studying the reconstitution of LN in RIPLT.LT alpha -/- mice and in a new LTtet inducible system; by analyzing LT's effects in reconstituted RIPLT.LT alpha -/- and doxycycline induced tetLT.LT alpha -/- mice on adhesion molecules, chemokines, and dendritic cells; and analyzing developmentally regulated LT expression in mice transgenic for LT alpha regulatory regions driving expression of human CD8 alpha or beta-galactosidase reporter genes.

The long term objective of this work is to understand the pathogenesis of Type I diabetes mellitus with an emphasis on elucidating mechanisms of inflammation and determinant spreading. The hypothesis to be tested is that tertiary lymphoid organs at the local site, which are characteristic of many autoimmune diseases, are crucial for IDDM. Mice transgenic for lymphotoxin (LTalpha; TNFbeta) under the control of the insulin promoter (RIPLT mice) express the transgene in the pancreas and kidney and develop inflammation at those sites. The insulitis and nephritis have lymph node (LN) characteristics, including T, B cell compartmentalization, antigen presenting cells (FDC and DC), vessels with the morphologic and antigenic (MAdCAM-1 and PNAd) characteristics of high endothelial venules (HEV), and plasma cells secreting specific IgG after immunization. This process, termed lymphoid neo-organogenesis, requires TNFR1 and may be a model for LT's role in development since mice deficient in members of the LT/TNF family have profound defects in lymphoid organs. LTbetaR contributes to PNAd expression and influences the ratio of naive cells in the tertiary lymphoid organ. Chemokines (RANTES, MCP-1, and IP-10) are induced in RIPLT islets. When RIPLT mice are crossed to RIPB7 mice, diabetes occurs. The specific aims are to: 1. Identify the role of chemokines in islet tertiary lymphoid organs by crossing RIPLT mice with mice deficient in chemokines induced in RIPLT islets (MCP-1) or identified to be involved in lymphoid organ development and mononuclear trafficking (BRL-1) and by in situ hybridization; 2. Characterize the kinetics of tertiary lymphoid organs with a beta cell specific LT tetracycline (Dox) inducible system; 3. Determine whether the islet infiltrate is a functional lymphoid organ capable of presenting endogenous (Y-Ae) and exogenous antigen; 4. Determine whether the islet infiltrate is a functional lymphoid organ capable of responding to antigen; 5. Discover whether determinant spreading occurs in LT-induced diabetes and if maintenance of a tertiary lymphoid organ is required for this process. These studies which will provide insight into how and where antigens are presented in inflammatory autoimmunity concentrate on identifying cellular, molecular, and anatomic requirements for this process. They also provide insight into the mechanism of cytokine induction of lymphoid organs in development. Understanding how islet tertiary lymphoid organs are established and function will allow identification of therapeutic targets for determinant spreading and tissue damage in IDDM.

The goal of these studies is to understand the mechanisms and relationships of lymphoid organ development and inflammation as mediated by LTalpha3 and LTalphaB. The hypothesis to be tested is that the processes are similar by which LT/TNF cytokines induce lymphoid organs in ontogeny (secondary lymphoid organs) (lymphoid organogenesis) and inflammation (tertiary lymphoid organs) (lymphoid neogenesis) and that these lymphoid organs have common functions, i.e. presentation and response to antigen. Mice transgenic for LTalpha under the control of the rat insulin promoter (RIPLT mouse) that expresses LTalpha constitutively in the islets of Langerhans, the kidney, and skin, exhibit inflammation at the sites of transgene expression that has many characteristics of lymphoid organs with regard to expression of adhesion molecules, chemokines, and cellular composition. Some (MAdCAM-1, SLC, and BLC) are mediated by LTalpha3, whereas others (PNAd; L-selectin hi cells) require LTalphabeta as well. These data and others from mice selectively deficient in cytokines or their receptors suggest that LTalpha induces mesenteric and peripheral LN and LTalphabeta induces peripheral LN. The RIPLT mouse is a unique reagent that provides an accessible system to study mechanisms, kinetics, and molecular requirements for lymphoid organogenesis which will be used to answer the following questions: 1. Does the simultaneous expression of LTalpha and LTalphabeta result in neogenesis with the characteristics of peripheral lymph nodes? The infiltrates of mice transgenic for RIPLTalpha and RIPLTbeta will be analyzed for expression of PNAd and L-selectin, chemokines, DC, and FDC. 2. How precisely does the LT-induced infiltrate resemble a functional lymphoid organ? The ability of the infiltrates to present and respond to endogenous (Y-Ae) and exogenous antigen will be evaluated. 3. What are the individual roles of LTalpha and LTbeta in the kinetics of lymphoid neogenesis? Does LT production need to be sustained to maintain a lymphoid organ? RIP specific and inducible systems that express LTalpha and/or LTbeta will be developed, turned on and off at will and evaluated for lymphoid neogenesis. 4. What is the role of LT-induced NFkappaB activation in lymphoid organogenesis? RIPLT mice will be crossed with mice expressing a superinhibitor Ikappabalpha and the effect on lymphoid neogenesis evaluated. These studies will provide information regarding the mechanisms of cytokine induced inflammation and identify targets for therapeutics in disease and provide insight into the process and importance of determinant spreading by elucidating the establishment of a new lymphoid organ at the local site in inflammatory autoimmune diseases.

DESCRIPTION (provided by the applicant): The goal of this project is to gain an understanding of the regulation and biological roles of the members of the immediate lymphotoxin (LT)/tumor necrosis factor (TNF) family. These molecules contribute to inflammation through induction of chemokines and adhesion molecules. Individual ligands (LTalphax3, LTalpha1Beta2; TNFalpha) and receptors (TNFR1 and LTBetaR) play crucial and non-overlapping roles in development of lymph nodes, Peyer's patches, splenic organization, and nasal associated lymphoid tissue (NALT). Multiple different ligand receptor pairs interact to accomplish development of a full complement of lymphoid organs. A goal is to determine whether and how LT regulates development of high endothelial venules (HEV) and the switch in expression in peripheral lymph (PLN) HEV from mucosal addressin (MAdCAM-1) to peripheral node addressin (PNAd) in the perinatal period. Differences between LTalpha and LTBeta with regard to the NALT development and function will be studied as will the temporal and kinetic expression of LT and putative upstream and downstream genes and their interactions. The specific aims are: I. Determination of LT' s role in mucosal immunity; II. Determination of the temporal and spatial regulation of LTalpha and LTBeta in development and determination of the cells of origin; III. Identification of upstream regulators of LT; IV. Identification of the downstream target genes of LT in development; V. Determination of the mechanism by which LT regulates downstream target genes. These aims will be approached by evaluating NALT structure and function in mice deficient in the various LT/TNF family members; producing and evaluating expression of LT expression in embryogenesis and the perinatal and postnatal periods; monitoring expression of a reporter gene (Beta galactosidase or yellow fluorescent protein) knocked into the LTa locus; analyzing the role of Ikaros, a chromatin remodeling protein, in regulation of LTa and LTBeta with Ikaros knock-out and Ikaros. GFP reporter genes; evaluating LT family members in the regulation of lymphoid chemokines in pit mice and in the generation of the L-selectin ligand by the HEV sulfotransferase, HEC-GlcNAc6ST: and molecular characterization of HEV sulfotransferase regulation. Understanding the mechanisms of LT regulation of lymphoid organs in development will provide insight into common themes in inflammation and autoimmune disease and provide information for development of vaccines and identification of therapeutic targets.

The long-term goal of this project is to identify the biological roles of the LT/TNF family. These cytokines contribute importantly to inflammation and lymphoid organ development, including regulation of high endothelial venules (HEVs) in lymph nodes (LNs). In the next years, regulation of lymphatic vessels (LVs) will be studied. Though the ontogenic process of lymphangiogenesis has been described, the initiators of the process are unknown. Lymphangiogenesis in inflammation is even less well understood. Several cell types, including B cells and macrophages have been invoked, either as sources of VEGFs or as precursors of lymphatic endothelial cells. Lymphangiogenesis at different sites and under different conditions may be regulated differently. Aim 1. To identify the mechanism of inflammation-induced lymphangiogenesis in LNs. Remodeling of the LN vasculature that occurs during inflammation includes changes in expression of HEV genes, lymphangiogenesis, and the appearance of vessels positive for both HEV and LV markers. To test the hypothesis that LVs arise from HEVs in lymphangiogenesis mice that express green fluorescent protein under the regulation of a HEV unique gene, HEC-6ST, and mice to be developed that express red fluorescent protein under the regulation of a LV gene, Prox-1, will be evaluated by confocal and multiphoton intravital microscopy. Experiments will be carried out to determine if information is exchanged between the vessels. The roles of cells, signals, and cytokines in this process will be identified. Aim 2. To identify the mechanism of inflammation-induced lymphangiogenesis in the skin. Lymphangiogenesis occurs in the skin after immunization with ovalbumin and complete Freund[unreadable]s adjuvant. This is enhanced in LTβ-/- mice. A remarkable mononuclear infiltrate is present in the region of the immunization site. The hypothesis, that both LTαand TNFαderived from cells in that site, contribute to skin lymphangiogenesis, will be tested by identifying the cells, signals, and cytokines, and other lymphangiogenic factors required for the process. Testing the hypothesis that LT/TNF family members, key molecules in lymphoid organ development and inflammation, can induce lymphatic vessels will provide an understanding of the factors that can induce lymphangiogenesis and thus provide therapeutic insight into the pathology of lymphedema. The data will suggest modalities for treatment of lymphatic vessel insufficiency by controlled local administration of cytokines. The analysis of interactions between lymphatic vessels and HEVs will provide insight into how these two vascular systems are regulated, maintained, and cooperate during an immune response will provide insight into lymphangiogenesis of inflammation in transplantation, autoimmunity, infectious diseases, and cancer.

DESCRIPTION (provided by applicant): The long term goal of this R21 project is to develop tools for imaging lymphatic vessels (LVs) in living mice. Lymphatic vessels drain interstitial fluid by means of valve-like openings, they transport lipids and they participate in the immune system. They transport antigen and antigen presenting cells to lymph nodes and cells from the lymph node to the circulation. When lymphatic vessels are disrupted, lymphedema, the accumulation of lymph in the interstitial tissues, occurs. Immunological functions are thwarted. Lymphangiogenesis occurs in inflammation at the site of immunization and in lymph nodes (secondary lymphoid organs) where there is an interaction between LVs and high endothelial venules (HEVs). Lymphangiogenesis occurs in chronic inflammation in lymphoid accumulations in ectopic sites, called tertiary lymphoid tissues (TLOs) that have many characteristics of lymph nodes. Mice transgenic for the rat insulin promoter (RIP) driving lymphotoxin-1 (RIPLT1) exhibit such TLOs in the pancreas. Techniques that have been used to develop mice whose HEVs can be imaged via green fluorescent protein will be used here to develop mice whose LVs can be similarly imaged via a red fluorescent protein. To attain this goal, the following specific aims will be accomplished: 1. To develop transgenic mice whose lymphatic vessels express fluorescent proteins. The pCLASPER recombineering technology will be used to isolate LV genes (prox1, lyve-1, or podoplanin), insert red fluorescent reporter genes, such as tdTomato, and produce transgenic mice. 2. To evaluate lymphatic reporter transgenic mice for fidelity of expression. Mice transgenic for lymphatic vessel reporter constructs will be analyzed for co- expression of endogenous LV genes and transgenes in several tissues in the steady state, in lymph nodes after immunization, and in TLOs in RIPLT1 mice. 3. To evaluate LVs in living mice. Multiphoton in vivo microscopy will be used to evaluate fluorescent LVs. Interactions between (green) high endothelial venules and (red) lymphatic vessels in the lymph nodes of immunized mice will be analyzed in real time. A dynamic understanding of lymphangiogenesis and LV function will provide therapeutic insight into lymphatic function and dysfunction in lymphedema. The development of imaging tools for lymphatic vessels will provide insight into how LVs and HEVs are regulated, maintained, and cooperate during an immune response and provide insight into lymphangiogenesis in inflammation, transplantation, autoimmunity, infectious diseases, and cancer. PUBLIC HEALTH RELEVANCE: A thorough understanding of lymphangiogenesis and lymphatic vessel function will provide therapeutic insight into lymphatic function and dysfunction in conditions such as lymphedema. Imaging tools for lymphatic vessels will provide insight into how this vascular system is maintained and regulated during an immune response and provide insight into lymphangiogenesis in inflammation, transplantation, autoimmunity, infectious diseases, and cancer. )