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.

CD137 Signaling Regulates Acute Colitis via RALDH2-Expressing CD11b-CD103+ DCs.

CD137, a potent costimulatory receptor for CD8+ T cells, is expressed in various non-T cells, but little is known about its regulatory functions in these cells. In this study, we show that CD137 signaling, specifically in intestinal CD11b-CD103+ dendritic cells (DCs), restricts acute colitis progression. Mechanistically, CD137 engagement activates TAK1 and subsequently stimulates the AMPK-PGC-1α axis to enhance expression of the Aldh1a2 gene encoding the retinoic acid (RA) metabolizing enzyme RALDH2. RA can act on CD11b+CD103- DCs and induce SOCS3 expression, which, in turn, suppresses p38MAPK activation and interleukin-23 (IL-23) production. Administration of RA in DC-specific CD137-/- mice represses IL-23-producing CD11b+CD103- DCs and TH17 cells, indicating that RA is a major inhibitory effector molecule against intestinal CD11b+CD103- DCs. Additionally, the therapeutic effect of the anti-CD137 antibody is abrogated in DC-specific CD137-/- mice. Taken together, our results define a mechanism of paracrine immunoregulation operating between adjacent DC subsets in the intestine.

Concomitant PIK3CD and TNFRSF9 deficiencies cause chronic active Epstein-Barr virus infection of T cells.

Infection of T cells by Epstein-Barr virus (EBV) causes chronic active EBV infection (CAEBV) characterized by T cell lymphoproliferative disorders (T-LPD) of unclear etiology. Here, we identified two homozygous biallelic loss-of-function mutations in PIK3CD and TNFRSF9 in a patient who developed a fatal CAEBV. The mutation in TNFRSF9 gene coding CD137/4-1BB, a costimulatory molecule expressed by antigen-specific activated T cells, resulted in a complete loss of CD137 expression and impaired T cell expansion toward CD137 ligand-expressing cells. Isolated as observed in one sibling, CD137 deficiency resulted in persistent EBV-infected T cells but without clinical manifestations. The mutation in PIK3CD gene that encodes the catalytic subunit p110δ of the PI3K significantly reduced its kinase activity. Deficient T cells for PIK3CD exhibited reduced AKT signaling, while calcium flux, RAS-MAPK activation, and proliferation were increased, suggestive of an imbalance between the PLCγ1 and PI3K pathways. These skewed signals in T cells may sustain accumulation of EBV-infected T cells, a process controlled by the CD137-CD137L pathway, highlighting its critical role in immunity to EBV.

CD137 deficiency causes immune dysregulation with predisposition to lymphomagenesis.

Dysregulated immune responses are essential underlying causes of a plethora of pathologies including cancer, autoimmunity, and immunodeficiency. We here investigated 4 patients from unrelated families presenting with immunodeficiency, autoimmunity, and malignancy. We identified 4 distinct homozygous mutations in TNFRSF9 encoding the tumor necrosis factor receptor superfamily member CD137/4-1BB, leading to reduced, or loss of, protein expression. Lymphocytic responses crucial for immune surveillance, including activation, proliferation, and differentiation, were impaired. Genetic reconstitution of CD137 reversed these defects. CD137 deficiency is a novel inborn error of human immunity characterized by lymphocytic defects with early-onset Epstein-Barr virus (EBV)-associated lymphoma. Our findings elucidate a functional role and relevance of CD137 in human immune homeostasis and antitumor responses.

Direct CD137 costimulation of CD8 T cells promotes retention and innate-like function within nascent atherogenic foci.

Effector CD8 T cells infiltrate atherosclerotic lesions and are correlated with cardiovascular events, but the mechanisms regulating their recruitment and retention are not well understood. CD137 (4-1BB) is a costimulatory receptor induced on immune cells and expressed at sites of human atherosclerotic plaque. Genetic variants associated with decreased CD137 expression correlate with carotid-intimal thickness and its deficiency in animal models attenuates atherosclerosis. These effects have been attributed in part to endothelial responses to low and disturbed flow (LDF), but CD137 also generates robust effector CD8 T cells as a costimulatory signal. Thus, we asked whether CD8 T cell-specific CD137 stimulation contributes to their infiltration, retention, and IFNγ production in early atherogenesis. We tested this through adoptive transfer of CD8 T cells into recipient C57BL/6J mice that were then antigen primed and CD137 costimulated. We analyzed atherogenic LDF vessels in normolipidemic and PCSK9-mediated hyperlipidemic models and utilized a digestion protocol that allowed for lesional T-cell characterization via flow cytometry and in vitro stimulation. We found that CD137 activation, specifically of effector CD8 T cells, triggers their intimal infiltration into LDF vessels and promotes a persistent innate-like proinflammatory program. Residence of CD137+ effector CD8 T cells further promoted infiltration of endogenous CD8 T cells with IFNγ-producing potential, whereas CD137-deficient CD8 T cells exhibited impaired vessel infiltration, minimal IFNγ production, and reduced infiltration of endogenous CD8 T cells. Our studies thus provide novel insight into how CD137 costimulation of effector T cells, independent of plaque-antigen recognition, instigates their retention and promotes innate-like responses from immune infiltrates within atherogenic foci. NEW & NOTEWORTHY Our studies identify CD137 costimulation as a stimulus for effector CD8 T-cell infiltration and persistence within atherogenic foci, regardless of atherosclerotic-antigen recognition. These costimulated effector cells, which are generated in pathological states such as viral infection and autoimmunity, have innate-like proinflammatory programs in circulation and within the atherosclerotic microenvironment, providing mechanistic context for clinical correlations of cardiovascular morbidity with increased CD8 T-cell infiltration and markers of activation in the absence of established antigen specificity.

CD137 Plays Both Pathogenic and Protective Roles in Type 1 Diabetes Development in NOD Mice.

We previously reported that CD137 (encoded by Tnfrsf9) deficiency suppressed type 1 diabetes (T1D) progression in NOD mice. We also demonstrated that soluble CD137 produced by regulatory T cells contributed to their autoimmune-suppressive function in this model. These results suggest that CD137 can either promote or suppress T1D development in NOD mice depending on where it is expressed. In this study, we show that NOD.Tnfrsf9-/- CD8 T cells had significantly reduced diabetogenic capacity, whereas absence of CD137 in non-T and non-B cells had a limited impact on T1D progression. In contrast, NOD.Tnfrsf9-/- CD4 T cells highly promoted T1D development. We further demonstrated that CD137 was important for the accumulation of ß cell-autoreactive CD8 T cells but was dispensable for their activation in pancreatic lymph nodes. The frequency of islet-infiltrating CD8 T cells was reduced in NOD.Tnfrsf9-/- mice in part because of their decreased proliferation. Furthermore, CD137 deficiency did not suppress T1D development in NOD mice expressing the transgenic NY8.3 CD8 TCR. This suggests that increased precursor frequency of ß cell-autoreactive CD8 T cells in NY8.3 mice obviated a role for CD137 in diabetogenesis. Finally, blocking CD137-CD137 ligand interaction significantly delayed T1D onset in NOD mice. Collectively, our results indicate that one important diabetogenic function of CD137 is to promote the expansion and accumulation of ß cell-autoreactive CD8 T cells, and in the absence of CD137 or its interaction with CD137 ligand, T1D progression is suppressed.

Amelioration of Japanese encephalitis by blockage of 4-1BB signaling is coupled to divergent enhancement of type I/II IFN responses and Ly-6C(hi) monocyte differentiation.

BACKGROUND: Japanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model. METHODS: Infected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed. RESULTS: Blocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4(+) Th1 cells as well as increased infiltration of mature Ly-6C(hi) monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host. CONCLUSIONS: Blocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4(+) Th1 cells and mature Ly-6C(hi) monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.

Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb.

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.

CD137 facilitates the resolution of acute DSS-induced colonic inflammation in mice.

BACKGROUND: CD137 and its ligand (CD137L) are potent immunoregulatory molecules that influence activation, proliferation, differentiation and cell death of leukocytes. Expression of CD137 is upregulated in the lamina propria cells of Crohn's disease patients. Here, the role of CD137 in acute Dextran-Sodium-Sulfate (DSS)-induced colitis in mice was examined. METHODS: We induced acute large bowel inflammation (colitis) via DSS administration in CD137(-/-) and wild-type (WT) mice. Colitis severity was evaluated by clinical parameters (weight loss), cytokine secretion in colon segment cultures, and scoring of histological inflammatory parameters. Additionally, populations of lamina propria mononuclear cells (LPMNC) and intraepithelial lymphocytes (IEL) were characterized by flow cytometry. In a subset of mice, resolution of intestinal inflammation was evaluated 3 and 7 days after withdrawal of DSS. RESULTS: We found that both CD137(-/-) and WT mice demonstrated a similar degree of inflammation after 5 days of DSS exposure. However, the resolution of colonic inflammation was impaired in the absence of CD137. This was accompanied by a higher histological score of inflammation, and increased release of the pro-inflammatory mediators granulocyte macrophage colony-stimulating factor (GM-CSF), CXCL1, IL-17 and IFN-γ. Further, there were significantly more neutrophils among the LPMNC of CD137(-/-) mice, and reduced numbers of macrophages among the IEL. CONCLUSION: We conclude that CD137 plays an essential role in the resolution of acute DSS-induced intestinal inflammation in mice.

CD137 ligand reverse signaling skews hematopoiesis towards myelopoiesis during aging.

CD137 is a costimulatory molecule expressed on activated T cells. Its ligand, CD137L, is expressed on the surface of hematopoietic progenitor cells, and upon binding to CD137 induces reverse signaling into hematopoietic progenitor cells promoting their activation, proliferation and myeloid differentiation. Since aging is associated with an increasing number of myeloid cells we investigated the role of CD137 and CD137L on myelopoiesis during aging. Comparing 3 and 12 months old WT, CD137­/­ and CD137L­/­ mice we found significantly more granulocytes and monocytes in the bone marrow of older WT mice, while this age­dependent increase was absent in CD137­/­ and CD137L­/­ mice. Instead, the bone marrow of 12 months old CD137­/­ and CD137L­/­ mice was characterized by an accumulation of hematopoietic progenitor cells, suggesting that the differentiation of hematopoietic progenitor cells became arrested in the absence of CD137L signaling. CD137L signaling is initiated by activated CD137­expressing, CD4+ T cells. These data identify a novel molecular mechanisms underlying immune aging by demonstrating that CD137­expressing CD4+ T cells in the bone marrow engage CD137L on hematopoietic progenitor cells, and that this CD137L signaling biases hematopoiesis towards myelopoiesis during aging.

Cutting edge: 4-1BB controls regulatory activity in dendritic cells through promoting optimal expression of retinal dehydrogenase.

Dendritic cells (DC) in the gut promote immune tolerance by expressing retinal dehydrogenase (RALDH), an enzyme that promotes retinoic acid, which aids differentiation of Foxp3+ inducible regulatory T cells (iTreg) in the intestinal mucosa. How RALDH expression is regulated is unclear. We found that 4-1BB (CD137), a member of the TNFR family, together with CD103, marked mesenteric lymph node DC with the highest level of RALDH activity, and ligation of 4-1BB maintained RALDH expression in these gut DC. Moreover, 4-1BB signals synergized with those through TLR2 or GM-CSFR to promote RALDH activity in undifferentiated DC. Correspondingly, 4-1BB-deficient mice were impaired in their ability to generate iTreg in the GALT when exposed to oral Ag, and 4-1BB-deficient mesenteric lymph node DC displayed weak RALDH activity and were poor at promoting iTreg development. Thus, our data demonstrate a novel activity of 4-1BB in controlling RALDH expression and the regulatory activity of DC.

Deficiency for costimulatory receptor 4-1BB protects against obesity-induced inflammation and metabolic disorders.

OBJECTIVE: Inflammation is an important factor in the development of insulin resistance, type 2 diabetes, and fatty liver disease. As a member of the tumor necrosis factor receptor superfamily (TNFRSF9) expressed on immune cells, 4-1BB/CD137 provides a bidirectional inflammatory signal through binding to its ligand 4-1BBL. Both 4-1BB and 4-1BBL have been shown to play an important role in the pathogenesis of various inflammatory diseases. RESEARCH DESIGN AND METHODS: Eight-week-old male 4-1BB-deficient and wild-type (WT) mice were fed a high-fat diet (HFD) or a regular diet for 9 weeks. RESULTS: We demonstrate that 4-1BB deficiency protects against HFD-induced obesity, glucose intolerance, and fatty liver disease. The 4-1BB-deficient mice fed an HFD showed less body weight gain, adiposity, adipose infiltration of macrophages/T cells, and tissue levels of inflammatory cytokines (e.g., TNF-α, interleukin-6, and monocyte chemoattractant protein-1 [MCP-1]) compared with HFD-fed control mice. HFD-induced glucose intolerance/insulin resistance and fatty liver were also markedly attenuated in the 4-1BB-deficient mice. CONCLUSIONS: These findings suggest that 4-1BB and 4-1BBL may be useful therapeutic targets for combating obesity-induced inflammation and metabolic disorders.

Peripheral 4-1BB signaling negatively regulates NK cell development through IFN-gamma.

Stimulation of 4-1BB (CD137) was shown to produce strong anticancer effects in vivo. In contrast, 4-1BB-deficient (4-1BB(-/-)) B6 mice are remarkably resistant to tumor growth. We set out to determine the mechanisms involved in these seemingly contradictory observations. We found that the therapeutic effects of 4-1BB triggering were mainly dependent on CD8(+) T cells and partially on NK cells, whereas CD8(+) T and NK cells were equally needed to suppress tumor growth in 4-1BB(-/-) mice. Cellular analysis showed that the frequency and number of NK cells in the spleen and bone marrow were decreased by 4-1BB triggering but were increased in the absence of 4-1BB signaling in tumor-challenged mice. The 4-1BB-mediated downregulation of NK cell development was primarily dependent on IFN-gamma, which was produced by peripheral CD8(+) T and NK cells. The suppression of NK cell development by 4-1BB-mediated IFN-gamma production occurred in the bone marrow. As 4-1BB signaling increased in the periphery, more CD8(+) T cells but fewer NK cells contributed to the antitumor immunity. As 4-1BB signaling decreased, more NK cells participated in the antitumor immunity. We conclude that 4-1BB signaling results in a shift of the dominant type of immune cell in antitumor immunity from the innate NK cell to the adaptive CD8(+) T cell and that the level of IFN-gamma is critical for this 4-1BB-mediated shift.

Dendritic cells and Stat3 are essential for CD137-induced CD8 T cell activation-induced cell death.

Agonistic anti-CD137 mAbs either positively or negatively regulate T cell function. When administered at the beginning of lymphocytic choriomeningitis virus Armstrong infection anti-CD137 induced immunosuppression and T cell deletion, and in the case of influenza infection led to increased mortality. In contrast, 72 h delay in anti-CD137 treatment led to an enhanced virus-specific CD8 T cell response and rapid viral clearance. Virus-specific CD8 T cells in anti-CD137-injected mice rapidly upregulate Fas expression, and although necessary, was insufficient to induce CD8 T cell deletion. Strikingly, CD137 signaling in T cells was found to be insufficient to induce suppression or deletion. Rather, immunosuppression and T cell deletion was only observed if CD137 signals were provided to T cells and dendritic cells (DCs). In vitro CD137 crosslinking in DCs led to phosphorylation of Stat3, and importantly, anti-CD137 treatment of lymphocytic choriomeningitis virus Armstrong infected Stat3 conditional knock-out mice induced neither immune suppression or T cell deletion. Taken together, these data suggest that CD137 signaling in DCs can regulate CD8 T cell survival through a Stat3 and Fas-mediated pathway.

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.

ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo.

During an acute immune response, CD8 T cells undergo rapid expansion followed by a contraction phase during which the majority of activated T cells die, leaving a few survivors to persist as memory cells. The regulation of T cell survival is critical at each stage of this response. 4-1BB, a TNFR family member, has been implicated in prolonging the survival of activated and memory CD8 T cells; however, the precise mechanisms by which 4-1BB sustains T cell survival are incompletely understood. Upon aggregation on T cells, 4-1BB associates with two TNFR-associated factors (TRAF), TRAF1 and TRAF2. TRAF2 is essential for downstream signaling from 4-1BB; however, the role of TRAF1 in 4-1BB signaling has not been elucidated and there have been conflicting data as to whether TRAF1 provides a positive or a negative signal in T cells. In this study, we report that TRAF1 plays a critical role in survival signaling downstream of 4-1BB during CD8 T cell expansion in response to viral infection in vivo. Further analysis reveals that TRAF1-deficient cells are impaired in their ability to up-regulate the prosurvival Bcl-2 family member Bcl-x(L) and show increased levels of the proapoptotic Bcl-2 family member Bim following 4-1BB signaling. TRAF1-deficient CD8 T cells fail to activate ERK in response to 4-1BB ligation and inhibition of ERK signaling downstream of 4-1BB in wild-type cells leads to increased Bim levels. Thus, TRAF1 has a prosurvival effect in CD8 T cells via the 4-1BB-mediated up-regulation of Bcl-x(L) and ERK-dependent Bim down-modulation.

Absence of 4-1BB increases cell influx into the peritoneal cavity in response to LPS stimulation by decreasing macrophage IL-10 levels.

Peritoneal injection of lipopolysaccharide (LPS) increased the influx of polymorphonuclear leukocytes and macrophages into the peritoneal cavity (PEC), with significantly higher cell numbers in the 4-1BB-deficient (KO) mice than in wild-type (WT) mice. The peritoneal macrophages of KO mice contained less IL-10 transcripts and protein than those of WT after LPS treatment, and immobilization of 4-1BB-Fc increased the level of IL-10. Injection of IL-10 resulted in lower cell numbers into the PEC of KO mice, suggesting that lower level of IL-10 is responsible for stimulated cell influx in KO mice due to lack of 4-1BB and 4-1BBL interaction.

Role of endogenous 4-1BB in the development of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies directed against nuclear antigens including nucleosomes and DNA. To determine the role of T-cell costimulatory molecule 4-1BB in the regulation of SLE, MRL-Fas(lpr) (lpr) mice deficient in 4-1BB (lpr/4-1BB(-/-)) were generated and their disease phenotype was compared to that of control lpr mice. The main finding of this study is that the lpr/4-1BB(-/-) mice had more pronounced skin lesions which appeared earlier, increased lymphadenopathy, increased renal damage, and higher mortality than 4-1BB-intact control lpr mice. The increased severity of lesions in lpr/4-1BB(-/-) mice was closely associated with increases in CD4(+) T, CD3(+) B220(+) double-negative T cells, serum immunoglobulin, anti-dsDNA autoantibodies, and tissue immunoglobulin deposits. These data suggest that the 4-1BB-4-1BB ligand signalling pathway plays an important role in SLE and that deletion of 4-1BB confers susceptibility to lpr mice, leading to accelerated induction of disease and early mortality.

Enhanced osteoclastogenesis in 4-1BB-deficient mice caused by reduced interleukin-10.

UNLABELLED: Enhanced osteoclastogenesis was observed in bone marrow-derived macrophage cells from 4-1BB-deficient mice than in those from wildtype mice. 4-1BB and 4-1BB ligand interaction may play a role at a certain stage of osteoclast formation through increased level of IL-10, a negative regulator of osteoclastogenesis. INTRODUCTION: 4-1BB is an inducible T-cell costimulatory molecule and a member of the TNF receptor family. The expression pattern of 4-1BB and 4-1BB ligand (4-1BBL) has suggested that 4-1BB plays a role not only in various responses related to innate immunity but also in bone metabolism. MATERIALS AND METHODS: Osteoclast formation was evaluated in bone marrow-derived macrophage cells (BMMs) from wildtype and 4-1BB-deficient (4-1BB-/-) mice. Expression of interleukin-10 (IL-10) during osteoclast formation was analyzed at the mRNA and protein levels. RESULTS: Expression of IL-10 was higher in RANKL-stimulated wildtype BMMs than 4-1BB-/- BMMs. When 4-1BBL was stimulated with 4-1BB-Fc fusion protein, the expression of IL-10 in BMMs increased. Neutralization of IL-10 was not as effective in preventing inhibition by IL-10 of osteoclast differentiation in 4-1BB-/- BMMs as in wildtype BMMs. When IL-10 was added to the culture medium, osteoclast formation was inhibited more efficiently in the 4-1BB-/- BMMs than in the wildtype BMMs. CONCLUSIONS: Interaction of 4-1BB and 4-1BBL stimulates IL-10 production through 4-1BBL signaling. 4-1BBL plays a role at a certain stage of osteoclast formation, and IL-10 may mediate this effect. The elevated level of osteoclastogenesis in 4-1BB-/- BMMs may thus be caused, in part, by a lower level of IL-10.