Cell circuits between leukemic cells and mesenchymal stem cells block lymphopoiesis by activating lymphotoxin beta receptor signaling.

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 α1ß2 and activate lymphotoxin beta receptor (LTßR) 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 α1ß2 expression in leukemic cells. Genetic or pharmacological disruption of LTßR 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.

Extracellular vesicles shed by follicular lymphoma B cells promote polarization of the bone marrow stromal cell niche.

Follicular lymphoma (FL) originates in the lymph nodes (LNs) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSCs) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid stromal cell signature, and an increased capacity to sustain FL B-cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been identified. In the current work, we demonstrate that FL B cells produce extracellular vesicles (EVs) that can be internalized by BM-MSCs, making them more efficient to support FL B-cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activate transforming growth factor ß-dependent and independent pathways in BM-MSCs and modify their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 and angiopoietin-1. Moreover, we provide the first characterization of BM FL B-cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV-primed BM-MSCs. This work identifies FL-derived EVs as putative mediators of BM stroma polarization and supports further investigation of their clinical interest for targeting the crosstalk between BM-MSCs and malignant B cells.

Differential Roles of LTßR in Endothelial Cell Subsets for Lymph Node Organogenesis and Maturation.

Cellular cross-talk mediated by lymphotoxin αß-lymphotoxin ß receptor (LTßR) signaling plays a critical role in lymph node (LN) development. Although the major role of LTßR signaling has long been considered to occur in mesenchymal lymphoid tissue organizer cells, a recent study using a VE-cadherincreLtbrfl/fl mouse model suggested that endothelial LTßR signaling contributes to the formation of LNs. However, the detailed roles of LTßR in different endothelial cells (ECs) in LN development remain unknown. Using various cre transgenic mouse models (Tekcre , a strain targeting ECs, and Lyve1cre , mainly targeting lymphatic ECs), we observed that specific LTßR ablation in Tekcre+ or Lyve1cre+ cells is not required for LN formation. Moreover, double-cre-mediated LTßR depletion does not interrupt LN formation. Nevertheless, TekcreLtbrfl/fl mice exhibit reduced lymphoid tissue inducer cell accumulation at the LN anlagen and impaired LN maturation. Interestingly, a subset of ECs (VE-cadherin+Tekcre-low/neg ECs) was found to be enriched in transcripts related to hematopoietic cell recruitment and transendothelial migration, resembling LN high ECs in adult animals. Furthermore, endothelial Tek was observed to negatively regulate hematopoietic cell transmigration. Taken together, our data suggest that although Tekcre+ endothelial LTßR is required for the accumulation of hematopoietic cells and full LN maturation, LTßR in VE-cadherin+Tekcre-low/neg ECs in embryos might represent a critical portal-determining factor for LN formation.

Lymphotoxin ß receptor-mediated NFκB signaling promotes glial lineage differentiation and inhibits neuronal lineage differentiation in mouse brain neural stem/progenitor cells.

BACKGROUND: Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LTα binds one or two membrane-associated LTß to form LTα2ß1 or LTα1ß2 heterotrimers. The predominant LTα1ß2 binds to LTß receptor (LTßR) primarily expressed in epithelial and stromal cells. Most studies on LTßR signaling have focused on the organization, development, and maintenance of lymphoid tissues. However, the roles of LTßR signaling in the nervous system, particularly in neurogenesis, remain unknown. Here, we investigated the role of LTßR-mediated NFκB signaling in regulating neural lineage differentiation. METHODS: The C57BL/6J wild-type and GFAP-dnIκBα transgenic mice were used. Serum-free embryoid bodies were cultured from mouse embryonic stem cells and further induced into neural stem/progenitor cells (NSCs/NPCs). Primary neurospheres were cultured from embryonic and adult mouse brains followed by monolayer culture for amplification/passage. NFκB activation was determined by adenovirus-mediated NFκB-firefly-luciferase reporter assay and p65/RelB/p52 nuclear translocation assay. LTßR mRNA expression was evaluated by quantitative RT-PCR and LTßR protein expression was determined by immunohistochemistry and Western blot analysis. Multilabeled immunocytochemistry or immunohistochemistry followed by fluorescent confocal microscopy and quantitative analysis of neural lineage differentiation were performed. Graphing and statistical analysis were performed with GraphPad Prism software. RESULTS: In cultured NSCs/NPCs, LTα1ß2 stimulation induced an activation of classical and non-classical NFκB signaling. The expression of LTßR-like immunoreactivity in GFAP+/Sox2+ NSCs was identified in well-established neurogenic zones of adult mouse brain. Quantitative RT-PCR and Western blot analysis validated the expression of LTßR in cultured NSCs/NPCs and brain neurogenic regions. LTßR expression was significantly increased during neural induction. LTα1ß2 stimulation in cultured NSCs/NPCs promoted astroglial and oligodendrocytic lineage differentiation, but inhibited neuronal lineage differentiation. Astroglial NFκB inactivation in GFAP-dnIκBα transgenic mice rescued LTßR-mediated abnormal phenotypes of cultured NSCs/NPCs. CONCLUSION: This study provides the first evidence for the expression and function of LTßR signaling in NSCs/NPCs. Activation of LTßR signaling promotes glial lineage differentiation. Our results suggest that neurogenesis is regulated by the adaptive immunity and inflammatory responses.

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.

Deficiency in lymphotoxin ß receptor protects from atherosclerosis in apoE-deficient mice.

RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.

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.

Tumor necrosis factor superfamily in innate immunity and inflammation.

The tumor necrosis factor superfamily (TNFSF) and its corresponding receptor superfamily (TNFRSF) form communication pathways required for developmental, homeostatic, and stimulus-responsive processes in vivo. Although this receptor-ligand system operates between many different cell types and organ systems, many of these proteins play specific roles in immune system function. The TNFSF and TNFRSF proteins lymphotoxins, LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes), lymphotoxin-ß receptor (LT-ßR), and HVEM are used by embryonic and adult innate lymphocytes to promote the development and homeostasis of lymphoid organs. Lymphotoxin-expressing innate-acting B cells construct microenvironments in lymphoid organs that restrict pathogen spread and initiate interferon defenses. Recent results illustrate how the communication networks formed among these cytokines and the coreceptors B and T lymphocyte attenuator (BTLA) and CD160 both inhibit and activate innate lymphoid cells (ILCs), innate γδ T cells, and natural killer (NK) cells. Understanding the role of TNFSF/TNFRSF and interacting proteins in innate cells will likely reveal avenues for future therapeutics for human disease.

The thymic microenvironment differentially regulates development and trafficking of invariant NKT cell sublineages.

The regulatory role of the thymic microenvironment during trafficking and differentiation of the invariant NKT (iNKT) cell lineage remains poorly understood. In this study, we show that fractalkine receptor expression marks emigrating subpopulations of the NKT1, NKT2, and NKT17 sublineages in the thymus and peripheral organs of naive mice. Moreover, NKT1 sublineage cells can be subdivided into two subsets, namely NKT1(a) and NKT1(b), which exhibit distinct developmental and tissue-specific distribution profiles. More specifically, development and trafficking of the NKT1(a) subset are selectively dependent upon lymphotoxin (LT)α1ß2-LTß receptor-dependent differentiation of thymic stroma, whereas the NKT1(b), NKT2, and NKT17 sublineages are not. Furthermore, we identify a potential cellular source for LTα1ß2 during thymic organogenesis, marked by expression of IL-7Rα, which promotes differentiation of the NKT1(a) subset in a noncell-autonomous manner. Collectively, we propose a mechanism by which thymic differentiation and retention of the NKT1 sublineage are developmentally coupled to LTα1ß2-LTß receptor-dependent thymic organogenesis.

Lymphotoxin-LIGHT pathway regulates the interferon signature in rheumatoid arthritis.

A subset of patients with autoimmune diseases including rheumatoid arthritis (RA) and lupus appear to be exposed continually to interferon (IFN) as evidenced by elevated expression of IFN induced genes in blood cells. In lupus, detection of endogenous chromatin complexes by the innate sensing machinery is the suspected driver for the IFN, but the actual mechanisms remain unknown in all of these diseases. We investigated in two randomized clinical trials the effects on RA patients of baminercept, a lymphotoxin-beta receptor-immunoglobulin fusion protein that blocks the lymphotoxin-αß/LIGHT axis. Administration of baminercept led to a reduced RNA IFN signature in the blood of patients with elevated baseline signatures. Both RA and SLE patients with a high IFN signature were lymphopenic and lymphocyte counts increased following baminercept treatment of RA patients. These data demonstrate a coupling between the lymphotoxin-LIGHT system and IFN production in rheumatoid arthritis. IFN induced retention of lymphocytes within lymphoid tissues is a likely component of the lymphopenia observed in many autoimmune diseases. ClinicalTrials.gov NCT00664716.

Dimerization of LTßR by LTα1ß2 is necessary and sufficient for signal transduction.

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1ß2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1ß2 trigger signaling via LTß Receptor (LTßR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα1ß2 possesses two binding sites for LTßR with distinct affinities and that dimerization of LTßR by LTα1ß2 is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα1ß2, LTßR, and the fab fragment of an antibody that blocks LTßR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα1ß2 reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTßR.

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.

B cell maintenance of subcapsular sinus macrophages protects against a fatal viral infection independent of adaptive immunity.

Neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (VSV). Utilizing mice that possess B cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) VSV challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. However, B cells were absolutely required to provide lymphotoxin (LT) α1ß2, which maintained a protective subcapsular sinus (SCS) macrophage phenotype within virus draining lymph nodes (LNs). Macrophages within the SCS of B cell-deficient LNs, or of mice that lack LTα1ß2 selectively in B cells, displayed an aberrant phenotype, failed to replicate VSV, and therefore did not produce type I interferons, which were required to prevent fatal VSV invasion of intranodal nerves. Thus, although B cells are essential for survival during VSV infection, their contribution involves the provision of innate differentiation and maintenance signals to macrophages, rather than adaptive immune mechanisms.

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.

Induction of the alternative NF-κB pathway by lymphotoxin αß (LTαß) relies on internalization of LTß receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin ß receptor (LTßR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTßR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTßR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTßR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTßR cellular trafficking as a process required for specific biological functions of NF-κB.

Sources and signals regulating type I interferon production: lessons learned from cytomegalovirus.

Type I interferons (IFN-αß) are pleiotropic cytokines critical for antiviral host defense, and the timing and magnitude of their production involve a complex interplay between host and pathogen factors. Mouse cytomegalovirus (a ß-herpesvirus) is a persistent virus that induces a biphasic IFN-αß response during the first days of infection. The cell types and molecular mechanisms governing these 2 phases are unique, with splenic stromal cells being a major source of initial IFN-αß, requiring communication with B cells expressing lymphotoxin, a tumor necrosis factor family cytokine. Here we review the factors that regulate this lymphotoxin-IFN-αß "axis" during cytomegalovirus infection, highlight how stroma-derived IFN-αß contributes in other models, and discuss how deregulation of this axis can lead to pathology in some settings.

Adiponectin inhibits lymphotoxin-ß receptor-mediated NF-κB signaling in human umbilical vein endothelial cells.

Adiponectin exerts anti-diabetic and anti-atherogenesis properties through its 2 receptors (AdipoR1 and AdipoR2). However, the signaling pathways responsible for the anti-inflammatory effects of adiponectin are largely unknown. In this study, we identified the lymphotoxin (LT)-ß receptor (LTBR) as an interacting partner of human AdipoR1 by using a yeast two-hybrid screening. The interaction between LTBR and AdipoR1 was confirmed by co-immunoprecipitation and co-localization analysis. Furthermore, adiponectin incubation inhibited lymphotoxin-induced NF-κB activation and the expression of adhesion molecules in human umbilical vein endothelial cells. These results indicated that AdipoR1 interacted with LTBR and mediated the inhibition of LTBR-activated NF-κB pathway.

Lymphotoxin-ß receptor activation by lymphotoxin-α(1)ß(2) and LIGHT promotes tumor growth in an NFκB-dependent manner.

Lymphotoxin beta receptor (LTßR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTßR, namely LTα(1) ß(2) and LIGHT, are involved in the activation of LTßR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTßR in these fibrosarcoma tumors, we used conditional LTß-deficient mice that specifically lack LTß expression either on T cells (T-LTß(-/-)) or on B cells (B-LTß(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTßR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTßR in BFS-1 fibrosarcomas. LTßR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTßR activation. Our data support the idea that the ablation of LTßR signaling should be considered for cancer treatment.

Lymphotoxin-alphabeta heterotrimers are cleaved by metalloproteinases and contribute to synovitis in rheumatoid arthritis.

Tumor necrosis factor-superfamily (TNF-SF) members, lymphotoxin (LT)-alpha and LTbeta, are proinflammatory cytokines associated with pathology in rheumatoid arthritis. LTalpha3 homotrimers are secreted, whereas LTalpha(1)beta(2) heterotrimers are expressed on the surface of activated lymphocytes. As many TNF-SF members are actively cleaved from cell membranes, we determined whether LTalphabeta heterotrimers are also cleaved, and are biologically active in rheumatoid arthritis (RA) patients. LTalphabeta heterotrimers were detected in culture supernatants from activated human T-helper (Th) 0, Th1, and Th17 cells, together with LTalpha3 and TNFalpha. The heterotimers were actively cleaved from the cell surface by ADAM17 metalloproteinase (MMP) and MMP-8, and cleavage was inhibited by TAPI-1, a TNF-alpha converting enzyme (TACE) inhibitor. Soluble LTalphabeta was detected in serum from both normal donors and RA patients, and was elevated in synovial fluid from RA patients compared to osteoarthritis (OA) patients. Levels of LTalphabeta in RA patient synovial fluid correlated with increased TNFalpha, IL-8, IL-12, IL-1beta, IFN-gamma, and IL-6 cytokines. Moreover, recombinant LTalpha1beta2-induced CXCL1, CXCL2, IL-6, IL-8, VCAM-1, and ICAM-1 from primary synovial fibroblasts isolated from RA patients. Therefore, soluble LTalphabeta in synovial fluid is associated with a proinflammatory cytokine milieu that contributes to synovitis in RA.

CD70 expression by dendritic cells plays a critical role in the immunogenicity of CD40-independent, CD4+ T cell-dependent, licensed CD8+ T cell responses.

The stimulation of DC by CD4(+) T cells is known to condition DC to activate naïve CD8(+) T cells, predominantly via CD40-CD40L interactions. It has been proposed that a critical consequence of DC conditioning is the induction of CD70 expression. Whether and how CD70 induction contributes to CD8(+) T cell responses in the absence of CD40-CD40L interactions are unknown. CD8(+) T cell responses to adenoviral- or DC-based immunization of CD40-deficient mice revealed a CD40-independent, CD4(+) T cell-dependent pathway for CD70 induction on conventional DC. This pathway and subsequent CD8(+) T cell responses were enhanced by, but not dependent on, concomitant activation of TLR and in part, used TRANCE and LIGHT/LTalphabeta stimulation. Blocking TRANCE and LIGHT/LTalphabeta during stimulation reduced the immunogenicity of CD40-deficient DC. These data support the hypothesis that induction of CD70 expression on DC after an encounter with activated CD4(+) T cells is a major component of CD4(+) T cell-mediated licensing of DC. Further, multiple pathways exist for CD4(+) T cells to elicit CD70 expression on DC. These data in part explain the capacity of CD40-deficient mice to mount CD8(+) T cell responses and may provide additional targets for immunotherapy in situations when CD40-mediated licensing is compromised.