Regulatory T cells (Tregs) are essential to suppress unwanted immunity or inflammation. After islet allo-transplant Tregs must migrate from blood to allograft, then via afferent lymphatics to draining LN to protect allografts. Here we show that Tregs but not non-Treg T cells use lymphotoxin (LT) during migration from allograft to draining LN, and that LT deficiency or blockade prevents normal migration and allograft protection. Treg LTαβ rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTβR. These results demonstrate a form of T-cell migration used only by Treg in tissues that serves an important role in their suppressive function and is a unique therapeutic focus for modulating suppression.

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies. To obtain insight into the molecular pathogenesis of PDC, we constructed a retroviral cDNA expression library with total RNA isolated from the PDC cell line MiaPaCa-2. Screening of this library with the use of a focus formation assay with NIH 3T3 mouse fibroblasts resulted in the identification of 13 independent genes with transforming activity. One of the cDNAs thus identified encodes an NH(2)-terminally truncated form of the lymphotoxin-beta receptor (LTBR). The transforming activity of this short-type LTBR in 3T3 cells was confirmed by both an in vitro assay of cell growth in soft agar and an in vivo assay of tumorigenicity in nude mice. The full-length (wild-type) LTBR protein was also found to manifest similar transforming activity. These observations suggest that LTBR, which belongs to the tumor necrosis factor receptor superfamily of proteins, may contribute to human carcinogenesis.

Previously, we reported that adoptively transferred perforin k/o (PKO), and IFN-gamma k/o (GKO), or perforin/IFN-gamma double k/o (PKO/GKO) effector T cells mediated regression of B16BL6-D5 (D5) pulmonary metastases and showed that TNF receptor signaling played a critical role in mediating tumor regression. In this report we investigated the role of lymphotoxin-alpha (LT-alpha) as a potential effector molecules of tumor-specific effector T cells.

We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTbeta-R and TNF-R signaling. Lymph nodes developed in LTalpha-/- mice treated in utero with agonist anti-LTbeta-R monoclonal antibody. Thus, LTbeta-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTalphabeta/LTbeta-R interaction were generated. Normal mice treated in utero with LTbeta-R-Ig and TNF-R55-Ig or anti-TNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTalpha-/- mice had disrupted cellular organization. Therefore, LTbeta-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes.

The formation of germinal centers (GCs) represents a crucial step in the humoral immune response. Recent studies using gene-targeted mice have revealed that the cytokines tumor necrosis factor (TNF), lymphotoxin (LT) alpha, and LTbeta, as well as their receptors TNF receptor p55 (TNFRp55) and LTbetaR play essential roles in the development of GCs. To establish in which cell types expression of LTbetaR, LTbeta, and TNF is required for GC formation, LTbetaR-/-, LTbeta-/-, TNF-/-, B cell-deficient (BCR-/-), and wild-type mice were used to generate reciprocal or mixed bone marrow (BM) chimeric mice. GCs, herein defined as peanut agglutinin-binding (PNA+) clusters of centroblasts/centrocytes in association with follicular dendritic cell (FDC) networks, were not detectable in LTbetaR-/- hosts after transfer of wild-type BM. In contrast, the GC reaction was restored in LTbeta-/- hosts reconstituted with either wild-type or LTbetaR-/- BM. In BCR-/- recipients reconstituted with compound LTbeta-/-/BCR-/- or TNF-/-/BCR-/- BM grafts, PNA+ cell clusters formed in splenic follicles, but associated FDC networks were strongly reduced or absent. Thus, development of splenic FDC networks depends on expression of LTbeta and TNF by B lymphocytes and LTbetaR by radioresistant stromal cells.

Regulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αβ and LEC LTβ receptor (LTβR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTβR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.

The lymphotoxin axis is important for the maintenance of several specialized lymphoid microenvironments in secondary lymphoid tissue. Lymphoid-tissue architecture is highly plastic and requires continual homeostatic signaling to maintain its basal functional state. The cellularity of lymph nodes in adult mice was reduced by systemic blockade of lymphotoxin-beta receptor (LTbeta R) signaling with a soluble decoy receptor both in resting and reactive settings. This reduction in cellularity resulted from greatly impaired lymphocyte entry into lymph nodes due to decreased levels of peripheral lymph node addressing (PNAd) and MAdCAM on high endothelial venules (HEV). LTbeta R signaling was required to maintain normal levels of RNA expression of MAdCAM, and also of PNAd by regulating the expression of key enzymes and scaffold proteins required for its assembly. Thus, the homeostatic maintenance of functional HEV status in adult mice relies largely on LTbeta R signaling.

The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial interleukin-23 (IL-23) production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4(-)CCR6(+)T-bet(-) RAR-related orphan receptor gamma t (RORγt)(+) lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage.

To assess the role of lymphotoxin-beta receptor (LTbetaR) in diabetes pathogenesis, we expressed an LTbetaR-Fc fusion protein in nonobese diabetic (NOD) mice. The fusion protein was expressed in the embryo, reached high levels for the first 2 wk after birth, and then declined progressively with age. High expression of LTbetaR-Fc blocked diabetes development but not insulitis. After the decline in chimeric protein concentration, mice became diabetic with kinetics similar to the controls. Early expression of fusion protein resulted in disrupted splenic architecture. However, primary follicles and follicular dendritic cells, but not marginal zones, developed in aged mice. Hence, LTbetaR signaling is required for diabetes development and regulates follicular and marginal zone structures via qualitatively or quantitatively distinct mechanisms.

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1β2 and activate lymphotoxin beta receptor (LTβR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1β2 expression in leukemic cells. Genetic or pharmacological disruption of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage.

The development of spontaneous insulin-dependent diabetes mellitus is preceded by the organization of tertiary lymphoid organ (TLO) in situ, but its role in the development of tissue destruction and the cytokines that control such structures have not been fully defined. We have now observed that TNF superfamily 14 (TNFSF14) is upregulated in aged nonobese diabetic (NOD) pancreas with the appearance of TLO. Blockade of TNFSF14 signaling caused a substantial reduction in the expression of lymphotoxin beta receptor (LTbetaR)-controlled migration factors within the islets and disrupts organization of tertiary structures, leading to prevention of diabetes. Consistently, enhancing LTbetaR signaling by transgenic expression of TNFSF14 in the islets of NOD mice rapidly promoted de novo formation of local TLO, resulting in diabetes, even in the absence of draining lymph nodes (LN). Thus, the TNFSF14-LTbetaR pathway appears to be critical in the development and maintenance of TLO for the onset of diabetes.

We have recently shown that de novo formation of lymphoid structures resembling B-cell follicles occurs in the inflamed central nervous system (CNS) meninges in a subset of patients with secondary progressive multiple sclerosis and in SJL mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Because lymphotoxin (LT) alpha(1)beta(2) is essential for lymphoid tissue organization, we used real-time PCR to examine LTbeta and LTbeta receptor (LTbetaR) gene expression in the CNS of SJL mice immunized with PLP 139-151 peptide. Moreover, we used the decoy receptor LTbetaR-immunoglobulin fusion protein to block the interaction of lymphotoxin (LT) alpha(1)beta(2) with the LTbeta receptor (LTbetaR) in mice with established EAE and evaluate the effect of systemic and local treatments with the fusion protein on disease progression, CNS lymphocytic infiltration and formation of meningeal B-cell follicles. The present findings indicate that both LTbeta and LTbetaR are upregulated at EAE onset and during subsequent relapses and that systemic and local blockade of the LT pathway with LTbetaR-Ig results in protracted and transient inhibition of EAE clinical signs, respectively. LTbetaR-Ig treatment also reduces T- and B-cell infiltration and prevents the induction of the chemokines CXCL10 and CXCL13 and the formation of organized ectopic follicles in the EAE-affected CNS. Targeting of molecules involved in lymphoid organogenesis could represent a valid strategy to inhibit CNS inflammation and formation of ectopic follicles, which may play a role in maintaining an abnormal, intrathecal humoral immune response in CNS autoimmune disease.

Lymphotoxin beta receptor (LTβR) is a promising therapeutic target in autoimmune and infectious diseases as well as cancer. Mice with genetic inactivation of LTβR display multiple defects in development and organization of lymphoid organs, mucosal immune responses, IgA production and an autoimmune phenotype. As these defects are imprinted in embryogenesis and neonate stages, the impact of LTβR signaling in adulthood remains unclear. Here, to overcome developmental defects, we generated mice with inducible ubiquitous genetic inactivation of LTβR in adult mice (iLTβRΔ/Δ mice) and redefined the role of LTβR signaling in organization of lymphoid organs, immune response to mucosal bacterial pathogen, IgA production and autoimmunity. In spleen, postnatal LTβR signaling is required for development of B cell follicles, follicular dendritic cells (FDCs), recruitment of neutrophils and maintenance of the marginal zone. Lymph nodes of iLTβRΔ/Δ mice were reduced in size, lacked FDCs, and had disorganized subcapsular sinus macrophages. Peyer`s patches were smaller in size and numbers, and displayed reduced FDCs. The number of isolated lymphoid follicles in small intestine and colon were also reduced. In contrast to LTβR-/- mice, iLTβRΔ/Δ mice displayed normal thymus structure and did not develop signs of systemic inflammation and autoimmunity. Further, our results suggest that LTβR signaling in adulthood is required for homeostasis of neutrophils, NK, and iNKT cells, but is dispensable for the maintenance of polyclonal IgA production. However, iLTβRΔ/Δ mice exhibited an increased sensitivity to C. rodentium infection and failed to develop pathogen-specific IgA responses. Collectively, our study uncovers new insights of LTβR signaling in adulthood for the maintenance of lymphoid organs, neutrophils, NK and iNKT cells, and IgA production in response to mucosal bacterial pathogen.

Lymphotoxin alpha (LT alpha)-deficient mice revealed critical roles for LT alpha in lymphoid organogenesis, but it is not clear whether LT alpha functions through an LT alpha homotrimer (LT alpha3) or LT alpha/beta heterotrimers. We generated LTbeta-deficient mice and found them to lack Peyer's patches, peripheral lymph nodes, splenic germinal centers, and follicular dendritic cells. Unlike LT alpha-deficient mice, LT beta-deficient mice had cervical and mesenteric lymph nodes. Furthermore, the mesenteric lymph nodes had germinal center-like regions, although these structures appeared to lack follicular dendritic cells. The absence of cervical and mesenteric lymph nodes in LT alpha-deficient mice, and yet their presence in LT beta-deficient mice and in mice deficient in tumor necrosis factor receptor types I and II, suggest that LT alpha3 may signal via an as yet unidentified receptor.

In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome.

Lymphotoxin alpha (LTalpha) signals via tumor necrosis factor receptors (TNFRs) as a homotrimer and via lymphotoxin beta receptor (LTbetaR) as a heterotrimeric LTalpha1beta2 complex. LTalpha-deficient mice lack all lymph nodes (LNs) and Peyer's patches (PPs), and yet LTbeta-deficient mice and TNFR-deficient mice have cervical and mesenteric LN. We now show that mice made deficient in both LTbeta and TNFR type 1 (TNFR1) lack all LNs, revealing redundancy or synergism between TNFR1 and LTbeta, acting presumably via LTbetaR. A complete lack of only PPs in mice heterozygous for both ltalpha and ltbeta, but not ltalpha or ltbeta alone, suggests a similar two-ligand phenomenon in PP development and may explain the incomplete lack of PPs seen in tnfr1-/- mice.

Clusterin is a multifunctional protein that participates in tissue remodeling, apoptosis, lipid transport, complement-mediated cell lysis and serves as an extracellular chaperone. The role of clusterin in cancer and neurodegeneration has been extensively studied, however little is known about its functions in the immune system. Using expression profiling we found that clusterin mRNA is considerably down-regulated in mouse spleen stroma upon knock-out of lymphotoxin β receptor which plays pivotal role in secondary lymphoid organ development, maintenance and function. Using immunohistochemistry and western blot we studied clusterin protein level and distribution in mouse spleen and mesenteric lymph nodes in steady state and upon immunization with sheep red blood cells. We showed that clusterin protein, represented mainly by the secreted heterodimeric form, is present in all stromal compartments of secondary lymphoid organs except for marginal reticular cells. Clusterin protein level rose after immunization and accumulated in light zones of germinal centers in spleen--the effect that was not observed in lymph nodes. Regulation of clusterin expression by the lymphotoxin beta signaling pathway and its protein dynamics during immune response suggest a specific role of this enigmatic protein in the immune system that needs further study.

Over 1.5 million individuals in the United States are afflicted with inflammatory bowel disease (IBD). While the progression of IBD is multifactorial, chronic, unresolved inflammation certainly plays a key role. Additionally, while multiple immune mediators have been shown to affect pathogenesis, a comprehensive understanding of disease progression is lacking. Previous work has demonstrated that a member of the TNF superfamily, TNFSF14 (LIGHT), which is pro-inflammatory in several contexts, surprisingly plays an important role in protection from inflammation in mouse models of colitis, with LIGHT deficient mice having more severe disease pathogenesis. However, LIGHT is a single member of a complex signaling network. It signals through multiple receptors, including herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTβR); these two receptors in turn can bind to other ligands. It remains unknown which receptors and competing ligands can mediate or counteract the outcome of LIGHT-signaling during colitis. Here we demonstrate that LIGHT signaling through LTβR, rather than HVEM, plays a critical role in the progression of DSS-induced colitis, as LTβR deficient mice exhibit a more severe disease phenotype. Further, mice deficient in LTαβ do not exhibit differential colitis progression compared to WT mice. However, deletion of both LIGHT and LTαβ, but not deletion of both LTαβ and LTβR, resulted in a reversal of the adverse effects associated with the loss of LIGHT. In sum, the LIGHT/LTαβ/LTβR signaling network contributes to DSS colitis, but there may be additional receptors or indirect effects, and therefore, the relationships between these receptors and ligands remains enigmatic.

The lymphotoxin LTalpha(1)beta(2) supports the development and maintenance of several aspects of spleen structure, but its significance for marginal sinus (MS) vascular organization is unclear. We showed here that, in early postnatal lymphotoxin-deficient mice, the developing Flk-1+ white pulp vessels failed to organize or upregulate MAdCAM-1, leading to altered spatial rearrangement of both the white pulp endothelial cells and the smooth muscle actin-expressing cells. In vitro, MAdCAM-1 directed the reorganization of LTbeta receptor+ endothelial cells grown on Matrigel. LTalpha(1)beta(2) also regulated the maintenance of both MAdCAM-1 expression and mature MS structure in adult mice, contributing importantly to normal trafficking of CD11b+ cells in response to bacterial antigens. Together, our studies demonstrate that LTalpha(1)beta(2) and LTbeta receptor signals control proper development and maintenance of the mature MS structure and implicate MAdCAM-1 in the structuring of the MS endothelial cells that is important for the movement of immune cells within the spleen.