Identification of regulatory functions for 4-1BB and 4-1BBL in myelopoiesis and the development of dendritic cells.

The costimulatory molecule 4-1BB and its ligand 4-1BBL can control adaptive immunity, but here we show that their interaction also suppressed myelopoiesis. We found that 4-1BBL was expressed on hematopoietic stem cells, differentiating common myeloid progenitors and granulocyte-macrophage progenitors, and 4-1BB was inducible on activated myeloid progenitors. Steady-state numbers of granulocyte-macrophage progenitors, myeloid-lineage cells and mature dendritic cells were higher in 4-1BB- and 4-1BBL-deficient mice, indicative of a negative function, and we confirmed that result with bone marrow chimeras and in vitro, where the absence of interactions between 4-1BB and 4-1BBL led to enhanced differentiation into dendritic cell lineages. The regulatory activity was mediated by 4-1BBL, with binding by 4-1BB inhibiting differentiation of myeloid progenitors. Thus, 4-1BB and 4-1BBL have a previously unknown function in limiting myelopoiesis and the development of dendritic cells.

A disintegrin and metalloprotease-17 and galectin-9 are important regulators of local 4-1BB activity and disease outcome in rheumatoid arthritis.

OBJECTIVE: Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS: Stimulated mononuclear cells from patients with chronic RA (n = 12) were co-incubated with tissue inhibitor of metalloproteinase, 4-1BB ligand and Gal-9. Plasma samples were examined for soluble 4-1BB (s4-1BB) in newly diagnosed, treatment-naïve patients with RA (n = 97). The 28-joint DAS with CRP (28DAS-CRP), total Sharp score, erosion score and joint space narrowing were used to evaluate treatment outcome serially over a 2-year period. RESULTS: RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference-mediated knockdown of ADAM17 or the addition of an ADAM17 inhibitor reduced the 4-1BB shedding. Shedding of 4-1BB was not influenced by Gal-9. Plasma levels of s4-1BB were increased in early RA and correlated with the number of swollen joints at baseline. After 3 months of treatment, the plasma levels of s4-1BB were equal to those of the controls. Baseline plasma levels of s4-1BB were inversely correlated with DAS28-CRP after 2 years of treatment, but not with total Sharp score, erosion score or joint space narrowing. CONCLUSION: ADAM17 induces 4-1BB shedding in RA. Gal-9 is pivotal for the function of 4-1BB and induction of TNF-α. Furthermore, high plasma levels of s4-1BB were associated with the number of swollen joints, but also with a low DAS28-CRP after 2 years treatment in early RA.

Blockade of 4-1BB and 4-1BBL interaction reduces obesity-induced skeletal muscle inflammation.

Obesity-induced skeletal muscle inflammation is characterized by increased macrophage infiltration and inflammatory cytokine production. In this study, we investigated whether 4-1BB, a member of the TNF receptor superfamily (TNFRSF9) that provides inflammatory signals, participates in obesity-induced skeletal muscle inflammation. Expression of the 4-1BB gene, accompanied by increased levels of inflammatory cytokines, was markedly upregulated in the skeletal muscle of obese mice fed a high-fat diet, in muscle cells treated with obesity factors, and in cocultured muscle cells/macrophages. In vitro stimulation of 4-1BB with agonistic antibody increased inflammatory cytokine levels in TNFα-pretreated muscle cells, and this effect was absent in cells derived from 4-1BB-deficient mice. Conversely, disruption of the interaction between 4-1BB and its ligand (4-1BBL) with blocking antibody decreased the release of inflammatory cytokines from cocultured muscle cells/macrophages. Moreover, deficiency of 4-1BB markedly reduced macrophage infiltration and inflammatory cytokine production in the skeletal muscle of mice fed a high-fat diet. These findings indicate that 4-1BB mediates the inflammatory responses in obese skeletal muscle by interacting with its ligand 4-1BBL on macrophages. Therefore, 4-1BB and 4-1BBL may be useful targets for prevention of obesity-induced inflammation in skeletal muscle.

4-1BB/4-1BBL interaction promotes obesity-induced adipose inflammation by triggering bidirectional inflammatory signaling in adipocytes/macrophages.

Obesity-induced adipose inflammation is characterized by recruitment of macrophages to adipose tissue and release of inflammatory cytokines. 4-1BB, a costimulatory receptor, modulates inflammatory processes through interaction with its ligand 4-1BBL on immune cell surfaces. In this study, we examined whether a 4-1BB/4-1BBL interaction between adipocytes and macrophages participates in obesity-induced adipose inflammation. We found that 4-1BB was expressed on adipocytes and was upregulated by obesity-related factors, which also enhanced 4-1BBL expression on macrophages. 4-1BB and/or 4-1BBL agonists, respectively, activated inflammatory signaling molecules (MAPK/IκBα and MAPK/Akt) in adipocytes and macrophages and enhanced the release of inflammatory cytokines (MCP-1, TNF-α, and IL-6). Moreover, disruption of the 4-1BB/4-1BBL interaction decreased the release of inflammatory cytokines from contact cocultured adipocytes/macrophages. These findings indicate that 4-1BB/4-1BBL-mediated bidirectional signaling in adipocytes/macrophages promotes adipose inflammation. 4-1BB and 4-1BBL may be useful targets for protection against obesity-induced adipose inflammation.

The structure of the trimer of human 4-1BB ligand is unique among members of the tumor necrosis factor superfamily.

Binding of the 4-1BB ligand (4-1BBL) to its receptor, 4-1BB, provides the T lymphocyte with co-stimulatory signals for survival, proliferation, and differentiation. Importantly, the 4-1BB-4-1BBL pathway is a well known target for anti-cancer immunotherapy. Here we present the 2.3-A crystal structure of the extracellular domain of human 4-1BBL. The ectodomain forms a homotrimer with an extended, three-bladed propeller structure that differs from trimers formed by other members of the tumor necrosis factor (TNF) superfamily. Based on the 4-1BBL structure, we modeled its complex with 4-1BB, which was consistent with images obtained by electron microscopy, and verified the binding site by site-directed mutagenesis. This structural information will facilitate the development of immunotherapeutics targeting 4-1BB.

CD137 (4-1BB) deficiency reduces atherosclerosis in hyperlipidemic mice.

BACKGROUND: The tumor necrosis factor receptor superfamily, which includes CD40, LIGHT, and OX40, plays important roles in atherosclerosis. CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in human atherosclerotic lesions. However, limited information is available on the precise role of CD137 in atherosclerosis and the effects of blocking CD137/CD137 ligand signaling on lesion formation. METHODS AND RESULTS: We generated CD137-deficient apolipoprotein E-knockout mice (ApoE(-/-) CD137(-/-)) and LDL-receptor-knockout mice (Ldlr(-/-)CD137(-/-)) to investigate the role of CD137 in atherogenesis. The deficiency of CD137 induced a reduction in atherosclerotic plaque lesions in both atherosclerosis mouse models, which was attributed to the downregulation of cytokines such as interferon-gamma, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha. CD137 signaling promoted the production of inflammatory molecules, including monocyte chemoattractant protein-1, interleukin-6, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, in endothelial cells. Stimulation of CD137 ligand signaling activated monocytes/macrophages and augmented the production of proinflammatory cytokines in atherosclerotic vessels. CONCLUSIONS: CD137/CD137 ligand signaling plays multiple roles in the progression of atherosclerosis, and thus, blockade of this pathway is a promising therapeutic target for the disease.

Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

Therapeutic and tumor-specific immunity induced by combination of dendritic cells and oncolytic adenovirus expressing IL-12 and 4-1BBL.

Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-gamma levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-gamma-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.

CD137 induces proliferation of murine hematopoietic progenitor cells and differentiation to macrophages.

CD137 is a member of the TNFR family, and reverse signaling through the CD137 ligand, which is expressed as a cell surface transmembrane protein, costimulates or activates APCs. CD137 and CD137 ligand are expressed on small subsets of bone marrow cells. Activation of bone marrow cells through CD137 ligand induces proliferation, colony formation and an increase in cell numbers. Compared with total bone marrow cells, the small subpopulation of progenitor cells that express no lineage markers but express CD117 cells (or Lin(-), CD117(+) cells) responds with the same activities to CD137 ligand signaling, but at a significantly enhanced rate. Concomitantly to proliferation, the cells differentiate to CFU granulocyte-macrophage and CFU macrophage, and then to monocytes and macrophages but not to granulocytes or dendritic cells. Hematopoietic progenitor cells differentiated in the presence of CD137 protein display enhanced phagocytic activity, secrete high levels of IL-10 but little IL-12 in response to LPS, and are incapable of stimulating T cell proliferation. These data demonstrate that reverse CD137 ligand signaling takes place in hematopoietic progenitor cells, in which it induces proliferation, an increase in cell numbers, colony formation, and differentiation toward monocytes and macrophages.

CD137 Ligand-CD137 Interaction is Required For Inflammasome-Associated Brain Injury Following Ischemic Stroke.

The CD137L-CD137 axis is a potent co-stimulatory immune checkpoint regulator that forms a bidirectional signaling pathway between the CD137 ligand (CD137L) and CD137 receptor to regulate immunological activities. This study investigated the potential involvement of the CD137L-CD137 axis on inflammasome-associated brain injury and neurological deficits in a mouse model of focal ischemic stroke. Cerebral ischemia was induced in male C57BL/6J wild-type (WT), CD137L-deficient (CD137L KO) and CD137-deficient (CD137 KO) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (6 h and 24 h). Brain infarct volume and neurological deficit scores were significantly lower in both CD137L KO and CD137 KO mice compared to WT controls. Moreover, CD137L-deficient brains had significantly lower levels of the pyroptotic protein, NT-Gasdermin D, while CD137-deficient brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3, pyroptotic protein, NT-Gasdermin D, and of the secondary pyroptotic protein NT-Gasdermin E, following ischemic stroke. This protection by CD137L and CD137 deletion was associated with a significant decrease in inflammasome signaling. In conclusion, our data provide evidence for the first time that the CD137L-CD137 axis contributes to brain injury and neurological deficits by activating the inflammasome signaling pathway following ischemic stroke.

[Immunological killing effect of recombicant adenovirus vector rAD-mTERT-m4-1BBL on mouse hepatoma cell line Hepa1-6 cells co-cultured with T lymphocytes].

OBJECTIVE: To study the immunological suppressing effect of recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL (rAD-mTERT) on mouse hepatoma cell line Hepa1-6 cells in co-culture with T lymphocytes. METHODS: Adding recombinant adenovirus rAD, rAD-CMV-m4-1BBL (rAD-CMV) and rAD-mTERT to Hepa1-6 and L929 cells, respectively, to observe the effect of these adenoviruses on growth and apoptosis of these cells in co-culture with T lymphocytes. RESULTS: Adding adenovirus significantly suppressed the growth and slightly increased apoptosis of the two types of cells (P < 0.05). rAD-mTERT promotor-m4-1BBL showed only pro-apoptotic effect on Hepa1-6 cells. When co-cultured with T lymphocytes, rAD-CMV-m4-1BBL showed promoting effect on apoptosis of the cells. Compared with that of T cells pre-co-culture, CD4(+) and CD8(+) T cells were proliferated, and the ratio of CD4/CD8 was significantly reduced (from 1.27 to 1.08). CONCLUSION: Adding the recombinant adenoviruses only suppresses the cell growth, but not promotes their apoptosis. In co-culture with T lymphocytes, recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL can targetingly suppress the growth and induce apoptosis of Hepa1-6 cells. The apoptosis is induced through the immunological killing effect of T lymphocytes.

Distinct approaches to investigate the importance of the murine 4-1BB 4-1BBL interaction in the antibody response to Streptococcus pneumoniae.

Protection against infection with Streptococcus pneumoniae is based mainly on the generation of antibodies to the pneumococcal capsular polysaccharides (caps-PS). Although caps-PS are considered thymus-independent antigens, there is a growing body of evidence that T lymphocytes and costimulatory molecules are involved in the regulation of the antibody response to caps-PS. We investigated whether the interaction between 4-1BB and 4-1BB ligand (4-1BBL) is involved in the modulation of the antibody response to caps-PS after immunization with Pneumovax or with intact heat-killed S. pneumoniae. Treatment with agonistic anti-4-1BB mAb, which mimics engagement of 4-1BB by 4-1BBL, had no effect on the IgG and IgM immune response to caps-PS (Serotype 3) after immunization with Pneumovax or with S. pneumoniae Serotype 3. However, anti-4-1BB treatment strongly inhibited the IgG response to pneumococcal surface protein A (PspA). By contrast, the IgG anti-caps-PS (Serotype 3) antibody response was reduced strongly in 4-1BBL(-/-) mice immunized with S. pneumoniae Serotype 3. The IgG anti-PspA antibody response in the 4-1BB(-/-) mice was comparable with the immune response in the wild-type mice. We conclude that distinct pathways are involved in the humoral antibody response to pneumococcal antigens, depending on the nature of the antigen and the context in which the different antigens are presented. The 4-1BB-4-1BBL interaction is not involved in the antibody response to soluble caps-PS. The influence of the 4-1BB-4-1BBL interaction in the immune reaction to S. pneumoniae Serotype 3 depends on the experimental system used.

TNFSF9 exerts an inhibitory effect on hepatocellular carcinoma.

OBJECTIVE: Tumor necrosis factor superfamily member 9 (TNFSF9), also known as 4-1BBL and CD137L, has been implicated in cancer immunotherapy due to its function as a T-cell co-stimulator. We aimed to investigate the role of TNFSF9 in the cancer pathogenesis in hepatocellular carcinoma (HCC). METHODS: TNFSF9 expression was examined by immunohistochemistry in 106 pairs of HCC and adjacent non-tumorous tissues, and by quantitative polymerase chain reaction and Western blot in HCC cell lines. The impact of TNFSF9 on the proliferation, migration and invasion of HCC cells was determined using the 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and transwell assays in vitro. We also assessed the influence of TNFSF9 on the growth and metastasis of HCC tumors in an orthotopic mouse model of human HCC. RESULTS: TNFSF9 expression was downregulated in approximately 70% of HCC tissues. A decreased expression of TNFSF9 was also consistently observed in all the four HCC cell lines. Either the overexpression of TNFSF9 or treatment with recombinant TNFSF9 protein could significantly inhibit the proliferation, migration and invasion of Huh7 and SMMC-7721 HCC cells in vitro. The inhibitory effect of TNFSF9 on HCC was further confirmed in vivo. Mice orthotopically transplanted with TNFSF9-overexpressing Huh7 cells developed significantly smaller tumors with less intrahepatic metastasis and distant metastasis compared with the control group. CONCLUSIONS: TNFSF9 may be a tumor suppressor in HCC. Based on its immune stimulatory aspect and the tumor inhibition property, TNFSF9 may be a promising therapeutic target for HCC.

4-1BB ligand-mediated imbalance of helper 17 T cells and regulatory T cells in patients with allergic asthma.

OBJECTIVES: To investigate the presence of 4-1BB ligand (4-1BBL) in the peripheral blood of patients with allergic asthma and evaluate its role in controlling the balance between helper 17 T (T(h)17) and regulatory T (T(reg)) cells. METHODS: Soluble 4-1BBL (s4-1BBL) was quantified by enzyme-linked immunosorbent assay in plasma from patients with asthma (n = 45) and from healthy control subjects (n = 35). The proportion of monocytes positive for membrane-bound 4-1BBL (m4-1BBL) was determined by flow cytometry. Peripheral blood mononuclear cells from patients with asthma were incubated with anti-4-1BB monoclonal antibody in vitro. Concentrations of interleukin (IL)-17 and transforming growth factor (TGF)-ß(1) in the culture supernatant were analysed. RESULTS: Plasma s4-1BBL concentrations and the proportion of m4-1BBL-positive monocytes were significantly lower in patients with asthma than in control subjects. The culture supernatant concentration of TGF-ß(1) was increased and that of IL-17 was decreased by incubation with anti-4-1BB monoclonal antibody. CONCLUSIONS: Both soluble and membrane-bound 4-1BBL were reduced in patients with allergic asthma compared with control subjects. 4-1BBL/4-1BB signalling may play an important role in allergic asthma by regulating the T(h)17/T(reg) balance.

Reverse signaling through the co-stimulatory ligand, CD137L, as a critical mediator of sterile inflammation.

CD137 (also called 4-1BB and TNFRSF9) has recently received attention as a therapeutic target for cancer and a variety of autoimmune and inflammatory diseases. Stimulating CD137 in vivo enhances CD8(+) T cell-activity and results in strong immunosuppression in some contexts. This paradoxical phenomenon may be partially explained by the ability of CD137-stimulating reagents (usually agonistic monoclonal antibodies against CD137) to overactivate T cells and other CD137-expressing cells. This over-activity is associated with deleting pathogenic T cells and B cells or generating a tolerogenic microenvironment. Recent studies, however, suggest that the biology of CD137 and its ligand (CD137L) are more complex, mainly due to bidirectional signaling between CD137 and CD137L. For example, signaling through CD137L in non-hematopoietic cells such as epithelial cells and endothelial cells has been shown to play an essential role in sterile inflammation by regulating immune cell recruitment. One outstanding, and clinically important, issue is understanding how bidirectional signaling through CD137 and CD137L controls the vicious cycle of sterile inflammation (e.g., ischemia-reperfusion tissue injury and meta-inflammatory diseases).

Triple expression of B7-1, B7-2 and 4-1BBL enhanced antitumor immune response against mouse H22 hepatocellular carcinoma.

OBJECTIVES: Costimulatory signals are essential for T-cell activation and hence play a very important role in antitumor immunity. B7 and 4-1BBL which belongs to tumor necrosis factor (TNF) family provide costimulatory interaction for T-cell activation and function. This study investigated the role of B7 and 4-1BBL in the amplification of tumor immunity by transduction of the B7-1, B7-2 and 4-1BBL into mouse hepatocellular carcinoma cell line H22. METHODS: The tumorigenicity of H22 variants expressing either B7-1, B7-2 (H22/B7-1/B7-2) or 4-1BBL was compared with an H22 variant expressing B7-1, B7-2 and 4-1BBL (H22/B7-1/B7-2/4-1BBL). The study next investigated whether the combination of B7-1/B7-2 and 4-1BBL cell injection induced cytotoxic T lymphocyte (CTL) response and IL-2/IFN-γ secretion. The immune mechanisms underlying this combination treatment were then analyzed. RESULTS: Syngeneic BALB/c mice injected with H22/B7-1/B7-2/4-1BBL cells that expressed elevated levels of B7-1, B7-2 and 4-1BBL showed a tumor development frequency of 50% compared with 100% in mice injected with the H22 parental line, H22/neo, H22/B7-1/B7-2 and H22/4-1BBL. Mice inoculated with H22 tumor cells expressing B7-1, B7-2 and 4-1BBL developed a strong cytotoxic T lymphocyte response and long-term immunity against wild-type tumor, suggesting a synergistic effect between the B7 and 4-1BBL costimulatory pathways. Results showed that H22/B7-1/B7-2/4-1BBL tumor vaccines probably protect the infiltrating lymphocytes from apoptosis and induce NF-κB activation to improve T-cell-mediated antitumor response. CONCLUSIONS: In this study, the antitumor consequences of using B7-1, B7-2 and 4-1BBL gene transfer have demonstrated the therapeutic potential of gene therapy approach for hepatocellular carcinoma.

The change of immunoactivity of dendritic cells induced by mouse 4-1BBL recombinant adenovirus.

PURPOSE: The purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells. MATERIALS AND METHODS: Mouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4- 1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E.coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary molecules (CD80 and CD86) on DCs were analyzed by flow cytometry. RESULTS: The levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs. CONCLUSION: The results indicated the recombinant mouse 4-1BBL can effectively activate DCs.

The immune signaling molecule 4-1BB stimulation reduces adiposity, insulin resistance, and hepatosteatosis in obese mice.

Immune cells (e.g. macrophages and T cells) in adipose tissue play a crucial role in the development of obesity-induced inflammation and metabolic disorders. Here we report findings suggesting that the immune signaling molecule 4-1BB/CD137 is a novel target for treatment of obesity and metabolic disorders. 4-1BB stimulation with agonistic antibody reduced body weight and adiposity and markedly improved glucose intolerance and hepatosteatosis in diet-induced obese mice and genetically obese/diabetic mice. Increases in lymphoid T cell expansion/activation and adipose/hepatic CD8+ T cell recruitment were evident in the anti-4-1BB antibody-treated obese mice. Glycolysis, ß-oxidation, and oxygen consumption rates also increased in the treated mice. These findings suggest that 4-1BB-stimulation accompanied by CD8+ T cell expansion/activation enhances glucose/lipid metabolism, leading to increased energy expenditure. Manipulation of 4-1BB may provide a unique immunological strategy against obesity and metabolic disorders.

The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling.

The 4-1BB is a costimulatory molecule similar to the receptor activator of NF-kappaB ligand (RANKL), both of which are key factors for the differentiation of osteoclasts and are expressed mainly by activated T cells. The 4-1BB shares common signaling pathways with RANK, suggesting a potential role in osteoclastogenesis. In this study, the role of 4-1BB and 4-1BB ligand (4-1BBL) in osteoclastogenesis was investigated using 4-1BB(-/-) and 4-1BB(+/+) mice. Osteoclast precursors normally express 4-1BB and 4-1BBL after exposure to RANKL, which was confirmed by semi-quantitative RT-PCR and flow cytometry. The 4-1BB(-/- )mice had a slightly increased bone mass accompanied by a reduced osteoclastogenic ability of 4-1BB(-/-) bone marrow-derived macrophages (BMM) ex vivo. In addition, 4-1BB(-/-) BMM demonstrated hypophosphorylation of JNK and p38 and decreased induction of c-Fos in response to RANKL stimulation. Retroviral transduction of wild-type as well as partial-length 4-1BB, which lacks TNF receptor-associated factor 2-binding sites for signaling, restored the osteoclastogenic ability of 4-1BB(-/-) BMM. Furthermore, both recombinant 4-1BB and 4-1BBL enhanced RANKL-induced osteoclastogenesis by 4-1BB(+/+) BMM and the induction of c-Fos and NFATc1.Together, these results indicate that 4-1BBL and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling, findings that may delineate the complex nature of the 4-1BBL and 4-1BB interaction.

CD8+ T cell dysfunction and increase in murine gammaherpesvirus latent viral burden in the absence of 4-1BB ligand.

Studies of costimulatory receptors belonging to the TNFR family have revealed their diverse roles in affecting different stages of the T cell response. The 4-1BB ligand (4-1BBL)/4-1BB pathway has emerged as a receptor-ligand pair that impacts not the initial priming, but later phases of the T cell response, such as sustaining clonal expansion and survival, maintaining memory CD8(+) T cells, and supporting secondary expansion upon Ag challenge. Although the role of this costimulatory pathway in CD8(+) T cell responses to acute viral infections has been well-studied, its role in controlling chronic viral infections in vivo is not known to date. Using the murine gammaherpesvirus-68 (MHV-68) model, we show that 4-1BBL-deficient mice lack control of MHV-68 during latency and show significantly increased latent viral loads. In contrast to acute influenza infection, the numbers of MHV-68-specific memory CD8(+) T cells were maintained during latency. However, the virus-specific CD8(+) T cells showed defects in function, including decreased cytolytic function and impaired secondary expansion. Thus, 4-1BBL deficiency significantly affects the function, but not the number, of virus-specific CD8(+) T cells during gammaherpesvirus latency, and its absence results in an increased viral burden. Our study suggests that the 4-1BB costimulatory pathway plays an important role in controlling chronic viral infections.