Theresa T. Lu

Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone

The factors directing marginal zone B cells to the splenic marginal zone are not well understood. Here we report that FTY720, a drug that targets sphingosine 1-phosphate (S1P) receptors, induced marginal zone B cell migration into follicles. Marginal zone B cells expressed S1P receptors 1 and 3 (S1P1 and S1P3, respectively). Using gene-targeted mice, we show that S1P1 but not S1P3 was required for localization in the marginal zone. In mice lacking the chemokine CXCL13, S1P1-deficient marginal zone B cells reacquired a marginal zone distribution. Exposure to lipopolysaccharide or antigen caused marginal zone B cells to downregulate S1P1 and S1P3 and to migrate into the splenic white pulp. These data suggest that marginal zone B cell localization to the marginal zone depends on responsiveness to the blood lysophospholipid S1P, with S1P1 signaling overcoming the recruiting activity of CXCL13.

Integrin-dependence of Lymphocyte Entry into the Splenic White Pulp

The steps involved in lymphocyte homing to the white pulp cords of the spleen are poorly understood. We demonstrate here that the integrins lymphocyte function associated (LFA)-1 and α4β1 make essential and mostly overlapping contributions necessary for B cell migration into white pulp cords. T cell entry to the white pulp is also reduced by blockade of LFA-1 and α4β1. The LFA-1 ligand, intercellular adhesion molecule 1 is critical for lymphocyte entry and both hematopoietic cells and radiation-resistant cells contribute to this requirement. Vascular cell adhesion molecule 1 contributes to the α4β1 ligand requirement and a second ligand, possibly fibronectin, also plays a role. By contrast with the entry requirements, antigen-induced movement of B cells from follicles to the outer T zone is not prevented by integrin blocking antibodies. Comparison of the distribution of integrin-blocked B cells and B cells treated with the Gαi inhibitor, pertussis toxin, early after transfer reveals in both cases reduced accumulation in the inner marginal zone. These observations suggest that chemokine receptor signaling and the integrins LFA-1 and α4β1 function together to promote lymphocyte transit from the marginal zone into white pulp cords.

Regulation of lymph node vascular growth by dendritic cells

Lymph nodes grow rapidly and robustly at the initiation of an immune response, and this growth is accompanied by growth of the blood vessels. Although the vessels are critical for supplying nutrients and for controlling cell trafficking, the regulation of lymph node vascular growth is not well understood. We show that lymph node endothelial cells begin to proliferate within 2 d of immunization and undergo a corresponding expansion in cell numbers. Endothelial cell proliferation is dependent on CD11c+ dendritic cells (DCs), and the subcutaneous injection of DCs is sufficient to trigger endothelial cell proliferation and growth. Lymph node endothelial cell proliferation is dependent on vascular endothelial growth factor (VEGF), and DCs are associated with increased lymph node VEGF levels. DC-induced endothelial cell proliferation and increased VEGF levels are mediated by DC-induced recruitment of blood-borne cells. Vascular growth in the draining lymph node includes the growth of high endothelial venule endothelial cells and is functionally associated with increased cell entry into the lymph node. Collectively, our results suggest a scenario whereby endothelial cell expansion in the draining lymph node is induced by DCs as part of a program that optimizes the microenvironment for the ensuing immune response.

Fibroblast-Type Reticular Stromal Cells Regulate the Lymph Node Vasculature

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)+ endothelial cells are less sensitive than PNAd− endothelial cells to VEGF blockade. Lymphotoxin β receptor (LTβR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTβR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTβR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd+ and PNAd− endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.

Platelet Endothelial Cell Adhesion Molecule-1 Is Phosphorylatable by c-Src, Binds Src-Src homology 2 Domain, and Exhibits Immunoreceptor Tyrosine-based Activation Motif-like Properties

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and inin vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 → Phe and Tyr686 → Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.

Coordinated Regulation of Lymph Node Vascular–Stromal Growth First by CD11c+ Cells and Then by T and B Cells

Lymph node blood vessels play important roles in the support and trafficking of immune cells. The blood vasculature is a component of the vascular–stromal compartment that also includes the lymphatic vasculature and fibroblastic reticular cells (FRCs). During immune responses as lymph nodes swell, the blood vasculature undergoes a rapid proliferative growth that is initially dependent on CD11c+ cells and vascular endothelial growth factor (VEGF) but is independent of lymphocytes. The lymphatic vasculature grows with similar kinetics and VEGF dependence, suggesting coregulation of blood and lymphatic vascular growth, but lymphatic growth has been shown to be B cell dependent. In this article, we show that blood vascular, lymphatic, and FRC growth are coordinately regulated and identify two distinct phases of vascular–stromal growth—an initiation phase, characterized by upregulated vascular–stromal proliferation, and a subsequent expansion phase. The initiation phase is CD11c+ cell dependent and T/B cell independent, whereas the expansion phase is dependent on B and T cells together. Using CCR7−/− mice and selective depletion of migratory skin dendritic cells, we show that endogenous skin-derived dendritic cells are not important during the initiation phase and uncover a modest regulatory role for CCR7. Finally, we show that FRC VEGF expression is upregulated during initiation and that dendritic cells can stimulate increased fibroblastic VEGF, suggesting the scenario that lymph node-resident CD11c+ cells orchestrate the initiation of blood and lymphatic vascular growth in part by stimulating FRCs to upregulate VEGF. These results illustrate how the lymph node microenvironment is shaped by the cells it supports.

Dual regulation of IRF4 function in T and B cells is required for the coordination of T–B cell interactions and the prevention of autoimmunity

Female mice lacking DEF6 and SWAP70 develop a lupus-like syndrome through dysregulation of IRF4 in activated B cells and plasma cells.

Inhibition of Neovascularization to Simultaneously Ameliorate Graft-vs-Host Disease and Decrease Tumor Growth

BACKGROUND Blood vessels are formed either by sprouting of resident tissue endothelial cells (angiogenesis) or by recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs, vasculogenesis). Neovascularization has been implicated in tumor growth and inflammation, but its roles in graft-vs-host disease (GVHD) and in tumors after allogeneic BM transplantation (allo-BMT) were not known. METHODS We analyzed neovascularization, the contribution of endothelial cells and EPCs, and the ability of anti-vascular endothelial-cadherin antibody, E4G10, to inhibit neovascularization in mice with GVHD after allo-BMT using immunofluorescence microscopy and flow cytometry. We examined survival and clinical and histopathologic GVHD in mice (n = 10-25 per group) in which GVHD was treated with the E4G10 antibody using immunohistochemistry, flow cytometry, and cytokine immunoassay. We also assessed survival, the contribution of green fluorescent protein-marked EPCs to the tumor vasculature, and the ability of E4G10 to inhibit tumor growth in tumor-bearing mice (n = 20-33 per group) after allo-BMT using histopathology and bioluminescence imaging. All statistical tests were two-sided. RESULTS We found increased neovascularization mediated by vasculogenesis, as opposed to angiogenesis, in GVHD target tissues, such as liver and intestines. Administration of E4G10 inhibited neovascularization by donor BM-derived cells without affecting host vascularization, inhibited both GVHD and tumor growth, and increased survival (at 60 days post-BMT and tumor challenge with A20 lymphoma, the probability of survival was 0.29 for control antibody-treated allo-BMT recipients vs 0.7 for E4G10-treated allo-BMT recipients, 95% confidence interval = 0.180 to 0.640, P < .001). CONCLUSIONS Therapeutic targeting of neovascularization in allo-BMT recipients is a novel strategy to simultaneously ameliorate GVHD and inhibit posttransplant tumor growth, providing a new approach to improve the overall outcome of allogeneic hematopoietic stem cell transplantation.

Role of the Lymphotoxin/LIGHT System in the Development and Maintenance of Reticular Networks and Vasculature in Lymphoid Tissues

Lymphoid organs are meeting zones where lymphocytes come together and encounter antigens present in the blood and lymph or as delivered by cells migrating from the draining tissue bed. The exquisite efficiency of this process relies heavily on highly specialized anatomy to direct and position the various players. Gated entry and exit control access to these theaters and reticular networks and associated chemokines guide cells into the proper sections. Lymphoid tissues are remarkably plastic, being able to expand dramatically and then involute upon resolution of the danger. All of the reticular scaffolds and vascular and lymphatic components adapt accordingly. As such, the lymph node (LN) is a wonderful example of a physiologic remodeling process and is potentially a guide to study such elements in pathological settings such as fibrosis, chronic infection, and tumor metastasis. The lymphotoxin/LIGHT axis delivers critical differentiation signals that direct and hone differentiation of both reticular networks and the vasculature. Considerable progress has been made recently in understanding the mesenchymal differentiation pathways leading to these specialized networks and in the remodeling that occurs in reactive LNs. In this article, we will review some new advances in the area in terms of developmental, differentiation, and maintenance events mediated by this axis.

A Dendritic-Cell-Stromal Axis Maintains Immune Responses in Lymph Nodes

Within secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTβR) ligands were critical mediators, and LTβR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTβR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases.