Node Remodeling Research Articles

Immune Function оf the Lymphatic System

Abstract—The lymphatic system plays a critical role in immunity, going far beyond the simple transport of immune cells and antigens. The endothelial cells in the various parts of this vasculature are highly specialized to perform various specific functions. Lymphatic capillaries express chemokines and adhesion molecules that in tissues promote the recruitment and transmigration of immune cells. Signaling molecules produced by endothelial cells of lymphatic capillaries during inflammation modulate the migration of lymphocytes through venules with high endothelium from the blood into the parenchyma of lymph nodes. Lymphatic vessels provide active regulated transport of immune cells and antigens to the lymph nodes. In the lymph nodes, with their complex structure organized by stromal cells, optimal conditions are created for the contacts of antigen-presenting cells with lymphocytes. Different subpopulations of lymph node endothelial cells perform specific functions according to lymph node location and contribute to both innate and adaptive immune responses through antigen presentation, lymph node remodeling, and regulation of leukocyte entry and exit.

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity.

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anticancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal antitumor T cell responses in both the tumor-draining lymph nodes (TDLNs) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into mesenteric lymph nodes (MLNs) and TDLNs, diminished DC and effector CD8+ T cell responses, and attenuated responses to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anticancer immunity.

Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular, physical, and structural properties and collectively determine tissue mechanics of lymph nodes, an immunologically relevant adult tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive expansion upon immune challenge. We find that in murine models of immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that C-type lectin-like receptor 2 (CLEC-2)–podoplanin signaling regulates the cell surface mechanics of fibroblasts, providing a mechanically sensitive pathway to regulate lymph node remodeling. Perturbation of fibroblast mechanics through genetic deletion of podoplanin attenuates T cell activation. We find that increased tissue tension through the fibroblastic stromal meshwork is required to trigger the initiation of fibroblast proliferation and restore homeostatic cellular ratios and tissue structure through lymph node expansion.

The Regulation of Lymph Node Pre-Metastatic Niche Formation in Head and Neck Squamous Cell Carcinoma.

In many distinct forms of malignancies, there is a close relationship between lymph node (LN) metastases and further dissemination to distant organs, and this is a critical prognostic factor. At the beginning of the process, the original tumor secretes soluble substances or releases extracellular vesicles (EVs) that are carried through lymphatic channels to draining (sentinel) LN. The tumor-derived factors then drive LN remodeling. These significant alterations occur prior to the emergence of the first metastatic cell, bringing about the development of a pre-metastatic niche that allows metastatic cells to survive and thrive. In this review, we discuss current information available about the regulation of lymph node pre-metastatic niche in head and neck squamous cell carcinoma (HNSCC), and the role of EVs in forming the pre-metastatic niche.

A Single‐Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

Single-cell transcriptomics promises to revolutionize our understanding of the vasculature. Here, we illustrate these methods in analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not recognized previously as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates known and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells, (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation, (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization and endothelial cell responses.

In Sickness and in Health: The Immunological Roles of the Lymphatic System.

The lymphatic system plays crucial roles in immunity far beyond those of simply providing conduits for leukocytes and antigens in lymph fluid. Endothelial cells within this vasculature are distinct and highly specialized to perform roles based upon their location. Afferent lymphatic capillaries have unique intercellular junctions for efficient uptake of fluid and macromolecules, while expressing chemotactic and adhesion molecules that permit selective trafficking of specific immune cell subsets. Moreover, in response to events within peripheral tissue such as inflammation or infection, soluble factors from lymphatic endothelial cells exert “remote control” to modulate leukocyte migration across high endothelial venules from the blood to lymph nodes draining the tissue. These immune hubs are highly organized and perfectly arrayed to survey antigens from peripheral tissue while optimizing encounters between antigen-presenting cells and cognate lymphocytes. Furthermore, subsets of lymphatic endothelial cells exhibit differences in gene expression relating to specific functions and locality within the lymph node, facilitating both innate and acquired immune responses through antigen presentation, lymph node remodeling and regulation of leukocyte entry and exit. This review details the immune cell subsets in afferent and efferent lymph, and explores the mechanisms by which endothelial cells of the lymphatic system regulate such trafficking, for immune surveillance and tolerance during steady-state conditions, and in response to infection, acute and chronic inflammation, and subsequent resolution.

Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats.

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3–4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4–5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy.

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature.

Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not previously recognized as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates the known features and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells; (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation; and (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization, and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN.

Dendritic cells in remodeling of lymph nodes during immune responses.

A critical hallmark of adaptive immune responses is the rapid and extensive expansion of lymph nodes. During this process, the complex internal structure of the organs is maintained revealing the existence of mechanisms able to balance lymph node integrity with structural flexibility. This article reviews the extensive architectural remodeling that occurs within lymph nodes during adaptive immune responses and how it is regulated by dendritic cells (DCs). In particular we focus on previously unappreciated functions of DCs in coordinating remodeling of lymph node vasculature, expansion of the fibroblastic reticular network and maintenance of lymphoid stromal phenotypes. Our increased understanding of these processes indicates that DCs need to be viewed not only as key antigen-presenting cells for lymphocytes but also as broad-acting immune sentinels that convey signals to lymphoid organ stroma and thereby facilitate immune response initiation at multiple levels.

Rapid activation of lymph node lymphangiogenesis (IRC4P.458)

Abstract The lymphatic system delivers fluid and molecules from tissues to the lymph node (LN) to mediate immune system surveillance of self and foreign antigens. LN lymphatic endothelial cells (LECs) function in orchestration of the immune response, and in reinforcement of peripheral tolerance. Extensive LN lymphatic sinus growth (lymphangiogenesis) is involved in multiple diseases including cancer, arthritis, and inflammation. Lymphocytes and leukocytes regulate this lymphangiogenesis; however, the mechanism is not well defined. We identified a monoclonal antibody (10.1.1) that recognizes the LEC protein mCLCA1. Our studies identified mCLCA1 as an interacting partner for LFA-1 and Mac-1 that mediates lymphocyte and leukocyte adhesion. The 10.1.1 Ab blocks adhesion of lymphocytes to LECs in vitro to a greater extent than ICAM-1 neutralizing Ab, suggesting that while ICAM-1 interactions predominate in vascular endothelium, mCLCA1 interaction is more important for adhesion to LECs. In addition to mediating adhesion, the 10.1.1 Ab (but not control Ab) induces rapid LN lymphangiogenesis within 23 hours after injection into mice, which involves proliferation of LN LECs. This lymphangiogenesis is accompanied by LN remodeling involving multiple stromal cell types, resembling the LN response to inflammation. These findings suggest that 10.1.1 Ab mimics lymphocyte or leukocyte B2-integrin binding to LEC mCLCA1, to drive LN remodeling and lymphangiogenesis during immune responses.

Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 (“SmyleDCpp65”). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4+ and CD8+ T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34+ cells obtained from PB and CB were transplanted into irradiated NOD.Rag1−/−.IL2γc−/− mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8+ T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4+/CD8+ T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

PI3Kα activates integrin α4β1 to establish a metastatic niche in lymph nodes

Lymph nodes are initial sites of tumor metastasis, yet whether the lymph node microenvironment actively promotes tumor metastasis remains unknown. We show here that VEGF-C/PI3Kα-driven remodeling of lymph nodes promotes tumor metastasis by activating integrin α4β1 on lymph node lymphatic endothelium. Activated integrin α4β1 promotes expansion of the lymphatic endothelium in lymph nodes and serves as an adhesive ligand that captures vascular cell adhesion molecule 1 (VCAM-1)(+) metastatic tumor cells, thereby promoting lymph node metastasis. Experimental induction of α4β1 expression in lymph nodes is sufficient to promote tumor cell adhesion to lymphatic endothelium and lymph node metastasis in vivo, whereas genetic or pharmacological blockade of integrin α4β1 or VCAM-1 inhibits it. As lymph node metastases accurately predict poor disease outcome, and integrin α4β1 is a biomarker of lymphatic endothelium in tumor-draining lymph nodes from animals and patients, these results indicate that targeting integrin α4β1 or VCAM to inhibit the interactions of tumor cells with the lymph node microenvironment may be an effective strategy to suppress tumor metastasis.

IL-7–producing stromal cells are critical for lymph node remodeling

AbstractNonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7–Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7–producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin–mediated depletion of IL-7–producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7–producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

The role of core TNF/LIGHT family members in lymph node homeostasis and remodeling

Lymph nodes (LNs) maintain active homeostasis at steady state. However, in response to changes in the local environment, such as local infection, cancer, vaccination, and autoimmune disease, dramatic remodeling of LN occurs. This remodeling includes changes in size, lymph and blood flow, immune cell trafficking and cellularity, lymphatic and blood vessel growth and activation, as well as microarchitecture. Therefore, inflammatory conditions often lead to enlarged nodes; after local inflammation resolves, LNs actively regress in size and return to steady state. Remodeling of lymphatic vessels (LVs) and blood vessels (BVs) during both the expansion and regression phases are key steps in controlling LN size as well as function. The cells, membrane-associated molecules, and soluble cytokines that are essential for LV and BV homeostasis as well as dynamic changes in the expansion and regression phases have not been well defined. Understanding the underlying cellular and molecular mechanisms behind LN remodeling would help us to better control undesired immune responses (e.g. inflammation and autoimmune diseases) or promote desired responses (e.g. antitumor immunity and vaccination). In this review, we focus on how the closely related tumor necrosis factor (TNF) members: LIGHT (TNFSF14), lymphotoxin-αβ, and TNF-α contribute to the remodeling of LNs at various stages of inflammation.

Compartmentalization of Immune Responses in Human Tuberculosis: Few CD8+ Effector T Cells but Elevated Levels of FoxP3+ Regulatory T Cells in the Granulomatous Lesions

Immune responses were assessed at the single-cell level in lymph nodes from children with tuberculous lymphadenitis. Tuberculosis infection was associated with tissue remodeling of lymph nodes as well as altered cellular composition. Granulomas were significantly enriched with CD68+ macrophages expressing the M. tuberculosis complex-specific protein antigen MPT64 and inducible nitric oxide synthase. There was a significant increase in CD8+ cytolytic T cells surrounding the granuloma; however, CD8+ T cells expressed low levels of the cytolytic and antimicrobial effector molecules perforin and granulysin in the granulomatous lesions. Quantitative real-time mRNA analysis revealed that interferon-gamma, tumor necrosis factor-alpha, and interleukin-17 were not up-regulated in infected lymph nodes, but there was a significant induction of both transforming growth factor-beta and interleukin-13. In addition, granulomas contained an increased number of CD4+FoxP3+ T cells co-expressing the immunoregulatory cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumor necrosis factor receptor molecules. Low numbers of CD8+ T cells in the lesions correlated with high levels of transforming growth factor-beta and FoxP3+ regulatory T cells, suggesting active immunosuppression at the local infection site. Compartmentalization and skewing of the immune response toward a regulatory phenotype may result in an uncoordinated effector T-cell response that reduces granule-mediated killing of M. tuberculosis-infected cells and subsequent disease control.

Gene Expression Model of Survival and Transformation in Follicular Lymphoma (FL): A Study by the LLMPP.

Background: FL is a common NHL that has a broad spectrum of clinical outcomes. Over time some pts will transform to an aggressive histology (Tly) associated with inferior survival. In 2004, the LLMPP constructed a model that was predictive of overall survival (OS) based on the gene expression profiles (GEP) of 191 specimens taken from pts with untreated FL. The genes associated with survival were derived from the non-neoplastic immune response (IR) cells. However the risk of developing Tly was not addressed in this study. Thus we re-analyzed the GEP with updated clinical data. Our goal was to validate our previous model with extended follow-up and to create a model that would predict the risk of developing TLy.Methods: 170 of 191 previously untreated FL pts had updated clinical information but only 142 had transformation outcome. Transformation was defined as biopsy proven DLBCL or clinically based on the presence of at least one of the following: hypercalcemia, a sudden rise in LDH >twice baseline, unusual extranodal growth or rapid discordant nodal growth. Raw CEL files from Affymetrix U133A arrays were pre-processed and normalized using Bioconductor's GCRMA package. Models were developed using SignS package (http://signs/bioinfo.cnio.es/), with 10 times cross-validation. All gene lists produced in these analyses were then re-tested for association with outcome using Bioconductor's Globaltest package. Over Representation Analysis of signature components was performed using Dchip.Results: The median OS of these patients was 8 yrs. A new 7-component survival model (85 genes) was developed that was significantly associated with survival (p= 2.9×10−13). In Globaltest, these gene lists were associated with survival at a level of (p=2.6×10−5). The previous model using IR-1 and IR-2 signatures was associated with survival at a level of p=2.6×10−4. Although there is little overlap between the 2 models, the new model confirms the importance of IR genes and extracellular matrix genes as being prognostically important. Interestingly, one component containing 10 genes on chromosome 6q was associated with a superior survival (p<1×107). 27% developed Tly over a median follow-up time of 11.2 yrs (69% biopsy proven). Our transformation model included 53 genes divided into 3 components (p=0.001). The Globaltest analysis for association of these genes with transformation was significant (p=0.018). 54 genes overlapped between the survival genes and transformation genes that were present in >1 cross validation run. These were significantly enriched in genes important in immune response like T cell and macrophage activation.Conclusion: Our survival model is stable and confirms the importance of key genes involved in the immune response and lymph node remodeling. It also introduces new genes that are potentially important for survival. Our transformation model may shed light on the mechanisms involved in the progression of FL to DLBCL but it is less stable and less reliable than our survival model at predicting outcome.

HIV-Infected Lymphocytes Regulate Fibronectin Synthesis by TGFβ1 Secretion

Alterations in lymph node architecture occur with HIV infection and contribute to immunological derangements. We previously showed that matrix fibronectin stabilized HIV and increased HIV infection of PBL. We showed increased fibronectin deposition in lymph nodes of HIV-infected patients. However, we did not detect a difference in fibronectin synthesis between uninfected and infected PBL. Therefore, we hypothesized that interactions of HIV-infected cells with fibroblasts resulted in increased fibronectin deposition. We detected increased fibronectin deposition by immunofluorescence on fibroblasts cocultured with HIV-infected PBL. We also found a 6-fold increase in fibronectin mRNA levels in fibroblasts cocultured with HIV-infected PBL by real-time PCR. Furthermore, when HIV-infected PBL were added to reporter fibroblasts stably transfected with a fibronectin promoter, we found a 1.5- to 2-fold increase in promoter activity. Since conditioned medium from HIV-infected PBL also increased fibronectin promoter activity, we hypothesized that a soluble factor such as TGFbeta was responsible for increased fibronectin secretion. Pretreatment of supernatant from HIV-infected PBL with a neutralizing Ab to TGFbeta1 abrogated the increased fibronectin promoter activity. We confirmed that HIV-infected PBL produced increased TGFbeta1 by ELISA. Using Mv1Lu reporter cells, we found a 2- to 3-fold increase in biologically active TGFbeta in supernatants of HIV-infected PBL. Finally, we determined that HIV infection did not change the percentage of active TGFbeta. Our data suggest that HIV-infected lymphocytes indirectly contribute to lymph node remodeling by secretion of TGFbeta1, which increases fibronectin synthesis by fibroblasts.