B Cell-Activating Factor Promotes B Cell Survival in Ectopic Lymphoid Tissues in Nasal Polyps.

Ectopic lymphoid tissues (eLTs) characterized by B cell aggregation contribute to the local immunoglobulin production in nasal polyps (NPs). B cell-activating factor (BAFF) is vital for B cell survival, proliferation, and maturation. The purpose of this study is to investigate whether BAFF is involved in the B cell survival and eLT formation in NPs. The mRNA and protein levels of BAFF in NP tissues with and without eLTs were detected by PCR and ELISA assay, respectively. The cellular sources of BAFF and active caspase-3-positive B cells in NPs were studied by immunofluorescence staining. B cells purified from NP tissues were stimulated with BAFF and were analyzed by flow cytometry. Stromal cells purified from NP tissues were stimulated with lymphotoxin (LT) α1ß2, and BAFF levels in culture supernatants were analyzed by ELISA. Compared with those in control tissues and NPs without eLTs, the BAFF levels were elevated in NPs with eLTs. Abundant BAFF-positive cells and few active caspase-3-positive apoptotic B cells were found in NPs with eLTs, in contrast to those in NPs without eLTs. There was a negative correlation between the numbers of BAFF-positive cells and frequencies of apoptotic B cells in total B cells in NP tissues. BAFF protected nasal polyp B cells from apoptosis in vitro. Stromal cells were an important cellular source of BAFF in NPs with eLTs. LTα1ß2 induced BAFF production from nasal stromal cells in vitro. We propose that BAFF contribute to eLT formation in NPs by promoting B cell survival.

Type 3 innate lymphoid cell-derived lymphotoxin prevents microbiota-dependent inflammation.

Splenomegaly is a well-known phenomenon typically associated with inflammation. However, the underlying cause of this phenotype has not been well characterized. Furthermore, the splenomegaly phenotype seen in lymphotoxin (LT) signaling-deficient mice is characterized by increased numbers of splenocytes and splenic neutrophils. Splenomegaly, as well as the related phenotype of increased lymphocyte counts in non-lymphoid tissues, is thought to result from the absence of secondary lymphoid tissues in LT-deficient mice. We now present evidence that mice deficient in LTα1ß2 or LTßR develop splenomegaly and increased numbers of lymphocytes in non-lymphoid tissues in a microbiota-dependent manner. Antibiotic administration to LTα1ß2- or LTßR-deficient mice reduces splenomegaly. Furthermore, re-derived germ-free Ltbr-/- mice do not exhibit splenomegaly or increased inflammation in non-lymphoid tissues compared to specific pathogen-free Ltbr-/- mice. By using various LTß- and LTßR-conditional knockout mice, we demonstrate that retinoic acid-related orphan receptor γT-positive type 3 innate lymphoid cells provide the required active LT signaling to prevent the development of splenomegaly. Thus, this study demonstrates the importance of LT-mediated immune responses for the prevention of splenomegaly and systemic inflammation induced by microbiota.

Bimodal Expansion of the Lymphatic Vessels Is Regulated by the Sequential Expression of IL-7 and Lymphotoxin α1ß2 in Newly Formed Tertiary Lymphoid Structures.

Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)ßR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1ß2/LTßR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.

Treg engage lymphotoxin beta receptor for afferent lymphatic transendothelial migration.

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 LTαß rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTßR. 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.

CXCL13 responsiveness but not CXCR5 expression by late transitional B cells initiates splenic white pulp formation.

Secondary lymphoid organs (SLO) provide the structural framework for coconcentration of Ag and Ag-specific lymphocytes required for an efficient adaptive immune system. The spleen is the primordial SLO, and evolved concurrently with Ig/TCR:pMHC-based adaptive immunity. The earliest cellular/histological event in the ontogeny of the spleen's lymphoid architecture, the white pulp (WP), is the accumulation of B cells around splenic vasculature, an evolutionarily conserved feature since the spleen's emergence in early jawed vertebrates such as sharks. In mammals, B cells are indispensable for both formation and maintenance of SLO microarchitecture; their expression of lymphotoxin α1ß2 (LTα1ß2) is required for the LTα1ß2:CXCL13 positive feedback loop without which SLO cannot properly form. Despite the spleen's central role in the evolution of adaptive immunity, neither the initiating event nor the B cell subset necessary for WP formation has been identified. We therefore sought to identify both in mouse. We detected CXCL13 protein in late embryonic splenic vasculature, and its expression was TNF-α and RAG-2 independent. A substantial influx of CXCR5(+) transitional B cells into the spleen occurred 18 h before birth. However, these late embryonic B cells were unresponsive to CXCL13 (although responsive to CXCL12) and phenotypically indistinguishable from blood-derived B cells. Only after birth did B cells acquire CXCL13 responsiveness, accumulate around splenic vasculature, and establish the uniquely splenic B cell compartment, enriched for CXCL13-responsive late transitional cells. Thus, CXCL13 is the initiating component of the CXCL13:LTα1ß2 positive feedback loop required for WP ontogeny, and CXCL13-responsive late transitional B cells are the initiating subset.

Innate lymphoid cells facilitate NK cell development through a lymphotoxin-mediated stromal microenvironment.

Natural killer (NK) cell development relies on signals provided from the bone marrow (BM) microenvironment. It is thought that lymphotoxin (LT) α1ß2 expressed by the NK cell lineage interacts with BM stromal cells to promote NK cell development. However, we now report that a small number of RORγt(+) innate lymphoid cells (ILCs), and not CD3(-)NK1.1(+) cells, express LT to drive NK development. Similar to LT(-/-) or RORγt(-/-) mice, the mice conditionally lacking LTα1ß2 on RORγt(+) ILCs experience a developmental arrest at the immature NK stages, between stages of NK development to the mature NK cell stage. This developmental block results in a functional deficiency in the clearance of NK-sensitive tumor cells. Reconstitution of Thy1(+) ILCs from BM or purified RORγt(+) ILCs from lamina propria lymphocytes into LT-deficient RORγt(+) BM cultures rescues NK cell development. These data highlight a previously undiscovered role of RORγt(+) ILCs for NK cell development and define LT from ILCs as an essential molecule for the stromal microenvironment supporting NK cell development.

Tumor targeting properties of antibody fusion proteins based on different members of the murine tumor necrosis superfamily.

The tumor necrosis factor superfamily (TNFSF) consists of more than 20 members that can modulate cellular and immunological functions, including cell survival and the stimulation of an inflammatory response. Many TNF superfamily members display potent anticancer activity when used as recombinant proteins in vitro and in vivo. While TNF, TRAIL and FasL have already been used as payloads in antibody-based pharmacodelivery strategies, most TNF superfamily members have not yet been investigated as antibody payloads. Here, we report the cloning, production and characterization of eight novel antibody fusion proteins based on CD40L, FasL, TRAIL, LiGHT, VEGI, lymphotoxin alpha, lymphotoxin beta and lymphotoxin alpha1/beta2. The monoclonal antibody F8 was chosen as fusion partner of proven tumor targeting performance, which recognizes the alternatively-spliced EDA domain of fibronectin, a marker of angiogenesis. A quantitative biodistribution analysis performed with radioiodinated protein preparations in tumor-bearing mice revealed that TRAIL and lymphotoxin alpha1/beta2 were able to selectively accumulate at the tumor site, while all other members of the TNF superfamily abrogated the selective tumor targeting performance of the parental antibody or accumulated also in healthy tissues. The study indicates that even cytokines, which are closely related in terms of structure and function, may have a substantially different impact on the biodistribution and functional properties of the corresponding fusions with disease-homing antibodies.

Human CD4+CD3- innate-like T cells provide a source of TNF and lymphotoxin-αß and are elevated in rheumatoid arthritis.

Innate lymphoid cells encompass a diverse array of lymphocyte subsets with unique phenotype that initiate inflammation and provide host defenses in specific microenvironments. In this study, we identify a rare human CD4(+)CD3(-) innate-like lymphoid population with high TNF expression that is enriched in blood from patients with rheumatoid arthritis. These CD4(+)CD3(-) cells belong to the T cell lineage, but the lack of AgR at the cell surface renders them nonresponsive to TCR-directed stimuli. By developing a culture system that sustains survival, we show that CD4(+)CD3(-) innate-like T cells display IL-7-dependent induction of surface lymphotoxin-αß, demonstrating their potential to modify tissue microenvironments. Furthermore, expression of CCR6 on the CD4(+)CD3(-) population defines a CD127(high) subset that is highly responsive to IL-7. This CD4(+)CD3(-) population is enriched in the peripheral blood from rheumatoid arthritis patients, suggesting a link to their involvement in chronic inflammatory disease.

Lymphotoxin α1ß2 expression on B cells is required for follicular dendritic cell activation during the germinal center response.

CD19-deficient mice were used as a model to study follicular dendritic cell (FDC) activation because these mice have normal numbers of FDC-containing primary follicles, but lack the ability to activate FDCs or form GCs. It was hypothesized that CD19 expression is necessary for B-cell activation and upregulation of membrane lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM-1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19(+) wild-type B cells into CD19-deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19(+) donor B cells lacking mLT were unable to induce VCAM-1 expression on FDCs, furthermore FcγRII/III upregulation was impaired in FDCs stimulated with mLT-deficient B cells. VCAM-1 expression on FDCs, but not FcγRII/III, was rescued when CD19-deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19(+) , mLT-deficient B cells, suggesting that FDC activation requires the CD19-dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.

Biology and signal transduction pathways of the Lymphotoxin-αß/LTßR system.

This review focuses on the biological functions and signalling pathways activated by Lymphotoxin α (LTα)/Lymphotoxin ß (LTß) and their receptor LTßR. Genetic mouse models shed light on crucial roles for LT/LTßR to build and to maintain the architecture of lymphoid organs and to ensure an adapted immune response against invading pathogens. However, chronic inflammation, autoimmunity, cell death or cancer development are disorders that occur when the LT/LTßR system is twisted. Biological inhibitors, such as antagonist antibodies or decoy receptors, have been developed and used in clinical trials for diseases associated to the LT/LTßR system. Recent progress in the understanding of cellular trafficking and NF-κB signalling pathways downstream of LTα/LTß may bring new opportunities to develop therapeutics that target the pathological functions of these cytokines.

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-αß, and TNF-α contribute to the remodeling of LNs at various stages of inflammation.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.

Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) beta-receptor-dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTalpha(1)beta(2)-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A-independent, LT- and B cell-dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

The lymphotoxin LTalpha(1)beta(2) controls postnatal and adult spleen marginal sinus vascular structure and function.

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.

Targeting lymphocyte activation through the lymphotoxin and LIGHT pathways.

SUMMARY: Cytokines mediate key communication pathways essential for regulation of immune responses. Full activation of antigen-responding lymphocytes requires cooperating signals from the tumor necrosis factor (TNF)-related cytokines and their specific receptors. LIGHT, a lymphotoxin-beta (LTbeta)-related TNF family member, modulates T-cell activation through two receptors, the herpesvirus entry mediator (HVEM) and indirectly through the LT-beta receptor. An unexpected finding revealed a non-canonical binding site on HVEM for the immunoglobulin superfamily member, B and T lymphocyte attenuator (BTLA), and an inhibitory signaling protein suppressing T-cell activation. Thus, HVEM can act as a molecular switch between proinflammatory and inhibitory signaling. The non-canonical HVEM-BTLA pathway also acts to counter LTbetaR signaling that promotes the proliferation of antigen-presenting dendritic cells (DCs) within lymphoid tissue microenvironments. These results indicate LTbeta receptor and HVEM-BTLA pathways form an integrated signaling circuit. Targeting these cytokine pathways with specific antagonists (antibody or decoy receptor) can alter lymphocyte differentiation and activation. Alternately, agonists directed at their cell surface receptors can restore homeostasis and potentially reset immune and inflammatory processes, which may be useful in treating autoimmune and infectious diseases and cancer.

Inhibition of the lymphotoxin pathway as a therapy for autoimmune disease.

SUMMARY: The lymphotoxin (LT) system is part of the tumor necrosis factor family and is required for lymph node development. It has provided a wonderful tool for the dissection of processes critical not only for lymphoid organ development but also the maintenance of the adult immune architecture and the formation of ectopic organized lymphoid tissues in chronically inflamed sites. A soluble lymphotoxin-beta receptor-immunoglobulin (LTbetaR-Ig) fusion protein can block this pathway and is currently being tested in the treatment of autoimmune disease. This review focuses on the immunological consequences of combined LT and LIGHT inhibition with LTbetaR-Ig administration as distinct from the developmental biology.

TNF receptor-associated factor 2-dependent canonical pathway is crucial for the development of Peyer's patches.

Activation of the noncanonical pathway through the interaction of lymphotoxin (LT)-alpha(1)beta(2) and LT-betaR is essential for the development of secondary lymphoid organs including lymph nodes (LN) and Peyer's patches (PP). Although TNFR-associated factor (TRAF) 2 and TRAF5 were identified as signal transducers for the LT-betaR, roles for TRAF2 and TRAF5 in the development of secondary lymphoid organs remain obscure. In this study, we show that PP but not mesenteric LN development is severely impaired in traf2(-/-) and traf2(-/-)traf5(-/-) mice. Development of VCAM-1(+) and ICAM-1(+) mesenchymal cells and expression of CXCL13, a crucial chemokine for the development of PP, are severely impaired in PP anlagen in the intestines of traf2(-/-) mice. Surprisingly, TNF-alpha stimulation potently up-regulates cxcl13 mRNA expression in wild-type murine embryonic fibroblasts, which is impaired in traf2(-/-) and relA(-/-) murine embryonic fibroblasts. Moreover, RelA is recruited to the promoter of cxcl13 gene upon TNF-alpha stimulation and PP development is impaired in TNFR type 1 (tnfr1)(-/-) mice. These results underscore a crucial role for the TNFR1-TRAF2-RelA-dependent canonical pathway in the development of PP through up-regulation of cxcl13 mRNA.