OX40-OX40L Interaction Research Articles

Effects of OX40–OX40L Interaction on the Nuclear Factor of Activated T Cells c1 in ApoE-Deficient Mice

We previously reported the emerging role of OX40-OX40L interaction in inflammation and atherosclerosis. However, the mechanism by which OX40-OX40L interaction contributes to pathogenesis is poorly understood. This study investigated the effects of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE(-/-) mice. Atherosclerotic plaque was induced via rapid perivascular carotid collar placement in ApoE(-/-) mice. The expression levels of OX40, OX40L, and NFATc1 in the lymphocytes were measured via real-time polymerase chain reaction and flow cytometry. The presence of NFATc1 in the atherosclerotic plaque was detected via immunohistochemistry, and the level of IL-4 was measured via enzyme-linked immunosorbent assay. The expression level of NFATc1 significantly increased in atherosclerotic lesion and in the leukocytes from the ApoE(-/-) mice. After stimulating OX40-OX40L interaction, the mRNA and protein expression levels of NFATc1 in the lymphocytes significantly increased. Meanwhile, anti-OX40LmAb significantly suppressed the expression of NFATc1 in the leukocytes and substantially elevated the level of IL-4. NFATc1 inhibitor markedly suppressed IL-4 production. This study suggests that OX40-OX40L interaction regulates the expression of NFATc1, which may play a critical role in atherosclerotic plaque formation, and may therefore have implications with pathophysiology of atherosclerosis.

A diametric role for OX40 in the response of effector/memory CD4+ T cells and regulatory T cells to alloantigen.

OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40 ligand (OX40L) pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined. In this article, we show that the alloantigen-mediated activation of naive and memory CD4(+) T cells results in the induction of OX40 expression and that blockade of OX40-OX40L interactions prevents skin allograft rejection mediated by either subset of T cells. Moreover, a blocking anti-OX40 had no effect on the activation and proliferation of T cells; rather, effector T cells failed to accumulate in peripheral lymph nodes and subsequently migrate to skin allografts. This was found to be the result of an enhanced degree of cell death among proliferating effector cells. In clear contrast, blockade of OX40-OX40L interactions at the time of exposure to alloantigen enhanced the ability of regulatory T cells to suppress T cell responses to alloantigen by supporting, rather than diminishing, regulatory T cell survival. These data show that OX40-OX40L signaling contributes to the evolution of the adaptive immune response to an allograft via the differential control of alloreactive effector and regulatory T cell survival. Moreover, these data serve to further highlight OX40 and OX40L as therapeutic targets to assist the induction of tolerance to allografts and self-Ags.

Blockade of OX40/OX40 Ligand to Decrease Cytokine messenger RNA Expression in Acute Renal Allograft Rejection In Vitro

AimThe aim of this study was to investigate cytokine messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) from renal recipients experiencing acute rejection by blocking OX40-OX40L interactions with recombinant human OX40-Fc fusion protein (rhOX40Fc) in vitro. MethodsPBMCs were isolated from 20 recipients experiencing acute rejection episodes (rejection group) and 20 recipients with stable graft function (stable group). Levels of Th1 (interferon [IFN]-γ) and Th2 (interleukin [IL]-4) mRNA expressions by PBMCs were measured using real-time reverse transcriptase-polymerase chain reactions. ResultsIFN-γ mRNA expression levels were significantly higher in the rejection than the stable group (P < .05). Levels of IL-4 mRNA expression were not significantly different. Among the rejection group, rhOX40Fc reduced significantly the expression of IFN-γ and IL-4 mRNA by anti-CD3-monoclonal antibody stimulated PBMCs (P < .05, and P < .01, respectively). ConclusionsBlocking of the interaction between OX40 and OX40L in vitro inhibited production of Thl and Th2 type cytokines.

OX40-OX40L Interaction Promotes Proliferation and Activation of Lymphocytes via NFATc1 in ApoE-Deficient Mice

BackgroundOur previous studies have shown that OX40-OX40L interaction regulates the expression of nuclear factor of activated T cells c1(NFATc1) in ApoE−/− mice during atherogenesis. The aim of this study was to investigate whether OX40-OX40L interaction promotes Th cell activation via NFATc1 in ApoE−/− mice.Methods and ResultsThe lymphocytes isolated from spleen of ApoE−/− mice were cultured with anti-CD3 mAb in the presence or absence of anti-OX40 or anti-OX40L antibodies. The expression of NFATc1 mRNA and protein in isolated lymphocytes were measured by real time PCR (RT-PCR) and flow cytometry (FCM), respectively. The proliferation of lymphocytes was analyzed by MTT method,and the expression of IL-2, IL-4 and IFN-γ in the cultured cells and supernatant were measured by RT-PCR and enzyme-linked immunosorbent assary (ELISA), respectively. After stimulating OX40-OX40L signal pathway, the expression of NFATc1 and the proliferation of leukocytes were significantly increased. Anti-OX40L suppressed the expression of NFATc1 in lymphocytes of ApoE−/− mice. Anti-OX40L or the NFATc1 inhibitor (CsA) markedly suppressed the cell proliferation induced by anti-OX40. Moreover, the expression of IL-2 and IFN-γ was increased in lymphocytes induced by OX40-OX40L interaction. Blocking OX40-OX40L interaction or NFATc1 down-regulated the expression of IL-2 and IFN-γ, but didn’t alter the expression of IL-4 in supernatants.ConclusionThese results suggest that OX40-OX40L interaction promotes the proliferation and activation of lymphocytes through NFATc1.

The Role of OX40 and Its Ligand in Acute Myeloid Leukemia: Expression, Function and Modulation of NK Cell Anti-Leukemia Reactivity

AbstractAbstract 3548The TNF/TNF receptor (TNFR) family comprises various molecules that substantially influence cellular functions of both tumor and immune effector cells. The TNFR family member OX40 has been shown to influence proliferation and differentiation of T cells in autoimmune diseases. Here we studied the yet unknown role of OX40 in acute myeloid leukemia (AML). Substantial surface expression of OX40 was detected on malignant cells of AML patients in 24 of 60 (40%) investigated cases. Expression of OX40 mRNA and protein by leukemic cells was confirmed by analysis of AML cells lines, which displayed substantial surface levels in 6 of 7 investigated cases. Induction of OX40 signaling into AML cells by recombinant OX40L or agonistic antibodies lead to the release of cytokines like IL-10 and TNF which contribute to AML pathophysiology and stimulated metabolic activity (WST test) of the leukemia cells. Moreover, we found that NK cells, which play an important role in anti-tumor immunity and largely contribute to the clinical success of allogeneic stem cell transplantation (SCT) in AML, express OX40 ligand (OX40L) following activation, and OX40L triggering stimulated NK cell reactivity. Functional analyses with OX40 transfectants and OX40-negative controls confirmed that OX40L signaling promotes NK cell cytotoxicity and IFN-γ production. Furthermore, disruption of OX40-OX40L interaction by blocking OX40 F(ab)2 fragments resulted in reduced cytotoxicity and cytokine production of allogenic NK cells, thereby further confirming the stimulatory effect of OX40L on NK cell anti-leukemia reactivity when interacting with its AML-expressed counterpart. Our data suggest that OX40 is involved in disease pathophysiology of AML and identify OX40-OX40L interaction as previously unknown modulator of NK cell immunosurveillance in AML. Disclosures:No relevant conflicts of interest to declare.

OX40-OX40L INTERACTION TARGETS NFATC1 IN APOE−/− MICE DURING ATHEROGENESIS

ObjectivesWe investigated the effect of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE−/− mice.MethodsAtherosclerotic plaque was induced by rapid perivascular carotid collar placement in...

Direct demonstration of CD4 T cell cooperation in the primary in vivo generation of CD4 effector T cells

Many observations bear upon the cellular and molecular requirements for CD4 T cell activation. The interaction of CD4 T cells with dendritic cells (DC), central to the induction of most immune responses, is the most studied. However, leukocytes other than DC can dramatically affect the induction and differentiation of CD4 T cells into effector cells. We recently provided indirect evidence that in vivo CD4 T cooperation facilitates the activation of CD4 T cells. Here, we demonstrate that the activation of CD4 T cells, specific for the hen egg lysozyme (HEL)(105) (-120) peptide, is optimally achieved when BALB/c mice are immunized with additional MHC class II-binding HEL peptides in incomplete Freund's adjuvant. This cooperation cannot be mimicked by the coadministration of LPS or of an agonistic antibody to CD40, at the time of immunization. In contrast, OX40-OX40L interactions are necessary for CD4 T cell cooperation in that an OX40 agonistic antibody can replace, and an OX40L-blocking antibody can abrogate, CD4 T cell cooperation in situations where such cooperation would otherwise enhance the activation of CD4 T cells.

OX40-OX40L interaction targets NFATc1 in ApoE-/- mice during atherogenesis

BackgroundWe have previously reported the emerging role of OX40-OX40L interaction in inflammation and atherosclerosis. However, the mechanism of OX40-OX40L interaction contribution towards pathogenesis has been poorly understood.AimTo investigate the effect...

OX40/OX40L in systemic lupus erythematosus: Association with disease activity and lupus nephritis

BACKGROUND AND OBJECTIVES:OX40-OX40L interaction is implicated in the pathogenesis of systemic lupus erythematosus (SLE). We evaluated the role of OX40/OX40L as markers of disease activity and nephritis in SLE patients.DESIGN AND SETTING:Case-control study conducted in 2009 on SLE patients attending the outpatient clinics of Ain Shams University Hospital, Egypt.PATIENTS AND METHODS:We assessed the percentage of CD4+ T-lymphocytes expressing OX40 by flowcytometry, and serum OX40 ligand (OX40L) levels in 40 patients with SLE (20 with lupus nephritis and 20 without) and in 20 healthy controls. Disease activity was assessed by the University of Toronto SLE disease activity index (SLEDAI).RESULTS:The percentage of CD4+ T-lymphocytes expressing OX40 was significantly higher in SLE patients than in controls, and in patients with lupus nephritis than in those without. OX40 expression correlated positively with both serum creatinine levels and SLEDAI. OX40 expression was the highest in patients with class V lupus nephritis and lowest in class II. Serum OX40L levels were significantly higher in SLE patients than in controls, and in patients with nephritis than in those without. Serum OX40L levels correlated with serum creatinine levels but not with SLEDAI. OX40 expression on CD4+ T-cells had a higher sensitivity and specificity in diagnosing lupus nephritis than both OX40L and anti–double-stranded DNA levels.CONCLUSION:OX40-OX40L interaction plays a role in the pathogenesis of SLE. The expression of OX40 on CD4+ T-lymphocytes and the serum level of OX40L may act as markers of lupus nephritis. Measurements of percentages of CD4+ T-lymphocytes expressing OX40 may serve as an indicator of disease activity in SLE.

OX40L mediates susceptibility to chronic Leishmania donovani infection (40.26)

Abstract OX40 is an antigen-specific T cell cosimulatory molecule belonging to the TNF receptor superfamily that is involved in both inflammatory and autoimmune responses. Its ligand, OX40L, is primarily expressed on antigen-presenting cells. The role of OX40-OX40L interactions during visceral leishmaniasis (VL) is currently unclear. Previous studies have reported that OX40-OX40L interactions contribute to increased parasite burdens and pathology of cutaneous leishmanias caused by L. major by increasing Th2 cytokine responses. However, numerous studies have found that during VL, mixed Th1/Th2 cytokine responses are key to disease resolution. In fact, one study has shown that OX40 agonist, in conjunction with anti-CTLA-4 treatment, enhances granuloma formation and parasite clearance during VL. In this study, we examined disease outcome in OX40L-deficient C57BL/6 mice infected with VL caused by L. donovani. We found that while these mice had similar parasite burdens in their livers and spleens early post-infection, they exhibited accelerated parasite clearance during late, chronic stages of disease. Ex vivo antigen stimulation of splenocytes showed that this rapid drop in parasite levels was associated with increased IFNγ production. We therefore propose that OX40-OX40L interactions play a role in the development and pathogenesis of chronic disease caused by visceral leishmaniasis, and their blockade may represent a viable therapeutic target during clinical disease.

Control of Immunity by the TNFR-Related Molecule OX40 (CD134)

TNFR/TNF superfamily members can control diverse aspects of immune function. Research over the past 10 years has shown that one of the most important and prominent interactions in this family is that between OX40 (CD134) and its partner OX40L (CD252). These molecules strongly regulate conventional CD4 and CD8 T cells, and more recent data are highlighting their ability to modulate NKT cell and NK cell function as well as to mediate cross-talk with professional antigen-presenting cells and diverse cell types such as mast cells, smooth muscle cells, and endothelial cells. Additionally, OX40-OX40L interactions alter the differentiation and activity of regulatory T cells. Blocking OX40L has produced strong therapeutic effects in multiple animal models of autoimmune and inflammatory disease, and, in line with a prospective clinical future, reagents that stimulate OX40 signaling are showing promise as adjuvants for vaccination as well as for treatment of cancer.

In Vivo Blockade of OX40 Ligand Prevents Respiratory Syncytial Virus (RSV)-Induced Airway Hyperresponsiveness (AHR) and Inflammation

RATIONALE: OX40-OX40L interactions appear crucial for T cell activation and survival, and for the generation of memory T cells from activated effector T cells. Recent studies revealed the critical role for OX40L expressed on dendritic cells (DC) for Th2 priming and polarization. However, the role of OX40-OX40L interactions in the response to RSV infection is not defined.

Serial OX40 Engagement on CD4+T Cells and Natural Killer T Cells Causes Allergic Airway Inflammation

OX40-OX40 ligand (OX40L) interactions have been proposed to support induction of allergic airway inflammation, which may be attributable to OX40 signaling in CD4(+) helper T cells for adaptive immune responses. However, a possible involvement of natural killer T (NKT) cells in the pathogenesis suggests that the underlying mechanisms are not yet fully elucidated. We aimed to characterize the OX40-modulated cellular contribution to allergic airway inflammation in a mouse model of house dust mite (HDM) allergen exposure. Mice were sensitized to HDM and, 3 weeks later, challenged with HDM on three consecutive days through the airways. Two days after the last exposure, bronchoalveolar lavage fluids and blood samples and lung tissues were evaluated for the airway inflammation. The development of HDM-induced eosinophilic airway inflammation was dependent on OX40 of both CD4(+) T cells and NKT cells; OX40 engagement on CD4(+) T cells in the sensitization led to pulmonary OX40L augmentation after the allergen challenge, which stimulated pulmonary NKT cells through OX40 to provide the pathogenic cytokine milieu. This was ablated by OX40L blockade by inhalation of the neutralizing antibody during the challenge, suggesting the therapeutic potential of targeting pulmonary OX40-OX40L interactions. Moreover, OX40 expression in CD4(+) T cells, but not in NKT cells, was reciprocally regulated by the helper T cell type 1-skewing transcription factor Runx3. OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. Notably, pulmonary blockade of OX40 ligation on NKT cells has therapeutic implications.

OX40–OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol–protein kinase C (DAG–PKC) and the inositol trisphosphate (IP 3)–intracellular free calcium ([Ca 2+] i ) pathway. The aim of this work was to investigate whether OX40–OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP 3 level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [γ- 32P]ATP to lysine-rich histone. [Ca 2+] i concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40–50 s and 12–16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP 3 formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca 2+] i responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG–PKC and IP 3–[Ca 2+] i signal pathway activation in HUVEC. In conclusion, the data suggested that OX40–OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.

The significance of OX40 and OX40L to T‐cell biology and immune disease

OX40 (CD134) and its binding partner, OX40L (CD252), are members of the tumor necrosis factor receptor/tumor necrosis factor superfamily and are expressed on activated CD4(+) and CD8(+) T cells as well as on a number of other lymphoid and non-lymphoid cells. Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen. OX40 additionally suppresses the differentiation and activity of T-regulatory cells, further amplifying this process. OX40 and OX40L also regulate cytokine production from T cells, antigen-presenting cells, natural killer cells, and natural killer T cells, and modulate cytokine receptor signaling. In line with these important modulatory functions, OX40-OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for intervention in the clinic. Conversely, stimulating OX40 has shown it to be a candidate for therapeutic immunization strategies for cancer and infectious disease. This review provides a broad overview of the biology of OX40 including the intracellular signals from OX40 that impact many aspects of immune function and have promoted OX40 as one of the most prominent costimulatory molecules known to control T cells.

CD4+CD25+ Regulatory T Cells Suppress Mast Cell Degranulation and Allergic Responses through OX40-OX40L Interaction

T regulatory (Treg) cells play a role in the suppression of immune responses, thus serving to induce tolerance and control autoimmunity. Here, we explored whether Treg cells influence the immediate hypersensitivity response of mast cells (MCs). Treg cells directly inhibited the FcvarepsilonRI-dependent MC degranulation through cell-cell contact involving OX40-OX40L interactions between Treg cells and MCs, respectively. When activated in the presence of Treg cells, MCs showed increased cyclic adenosine monophosphate (cAMP) concentrations and reduced Ca(2+) influx, independently of phospholipase C (PLC)-gamma2 or Ca(2+) release from intracellular stores. Antagonism of cAMP in MCs reversed the inhibitory effects of Treg cells, restoring normal Ca(2+) responses and degranulation. Importantly, the in vivo depletion or inactivation of Treg cells caused enhancement of the anaphylactic response. The demonstrated crosstalk between Treg cells and MCs defines a previously unrecognized mechanism controlling MC degranulation. Loss of this interaction may contribute to the severity of allergic responses.

OX40-OX40L interactions: a promising therapeutic target for allergic diseases?

Recent advances in understanding the cellular and molecular mechanisms of atopy have shed light on potential targets for the development of new therapies for allergic diseases. In this issue of the JCI, Seshasayee et al. provide direct in vivo evidence that OX40 has critical roles in allergic inflammation mediated by thymic stromal lymphopoietin (TSLP) (see the related article beginning on page 3868). Blockade of interactions between OX40 on Th2 cells and OX40 ligand (OX40L) on TSLP-activated DCs using an OX40L-specific monoclonal antibody, inhibited Th2 cell-mediated immune responses in both mouse and nonhuman primate models of allergic inflammation. The results point to potential therapeutic approaches to targeting the cellular and molecular mechanism underlying TSLP-mediated allergic inflammation.

Spontaneous pulmonary hypertension in mice associated with OX40 ligand overexpression (B82)

Abstract [Background] Ligation of OX40 receptor on T cells provides potent co-stimulatory signals associated with memory T cell generation, Ag-specific T cell migration, and enhanced inflammatory responses. OX40L is expressed on antigen presenting cells and vascular endothelial cells. Previous studies have shown that OX40L transgenic mice (OX40L-Tg) spontaneously developed ulcerative colitis-like disease and undetermined lung disease, which were evident in C57BL/6 (B6) but not BALB/c (BALB) genetic background. [Objective] The aim of this study is histopathological and genetic characterizations of the lung disease in OX40L-Tg. [Methods] We generated 351 OX40L-Tg from (BALB x B6) x B6 OX40L-Tg backcrosses. Severity of pulmonary arterial lesion was evaluated with microscopic examinations (0-1-2 grades). Genome-wide scan and other genetic analyses were carried out based on microsatellite polymorphisms. [Results] Pulmonary arterial lesion characterized by intimal and medial thickening that was observed in B6 but not BALB OX40L-Tg. Pulmonary hypertension (PH) in OX40L-Tg was shown by the elevation of right ventricular systolic pressure. We identified a novel susceptibility locus to the PH with suggestive linkage (P=0.0012, LOD=1.8) on chromosome 5. [Conclusion] Augmented OX40-OX40L interaction predisposes mice to spontaneous onset of PH under the influence of particular genetic background.

Targeting OX40 and OX40L for the Treatment of Autoimmunity and Cancer

The optimal activation of naïve T cells requires TCR-mediated recognition of cognate peptide-MHC complexes on antigen presenting cells in the presence of costimulatory signals. Although signals provided via CD28-B7 interactions are important for enhancing the initial T-cell response, other costimulatory signals are required for sustaining the response and promoting both T-cell differentiation and survival. In particular, engagement of OX40 (CD134) by its natural ligand OX40L (CD134L) or OX40 agonists has been shown to provide key signals that can augment CD4 and CD8 T-cell responses. Importantly, numerous studies have highlighted the ability of OX40-specific agonists or antagonists to enhance antitumor immunity or ameliorate autoimmune disease, respectively. On the basis of these studies, the development of OX40- and OX40L-specific reagents has been pursued for clinical use. Given the emerging role of OX40 and OX40L as novel therapeutic targets, this review will focus on the cellular and molecular mechanisms of OX40-mediated T-cell costimulation with a special emphasis on the role of OX40-OX40L interactions in the etiology and treatment of autoimmunity and cancer.

The Crystal Structure of the Costimulatory OX40-OX40L Complex

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.