OX40-OX40 Ligand Research Articles

Rheumatoid Arthritis Treatment: Harnessing Novel Molecular Targets and Therapeutics for Improved Patient Outcomes

Rheumatoid arthritis (RA) is a chronic autoimmune disease that has significant unmet needs for improved treatment outcomes. This review provides an overview of emerging molecular targets and potential new therapies for managing RA. The discussed targets, such as CD40-CD40 ligand, programmed death protein 1 (PD-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor-associated kinases (IRAKs), tyrosine kinase 2 (TYK2), bradykinin receptor 1 (B1R), and OX40-OX40 ligand, have shown promising results in preclinical studies and clinical trials. These findings stimulate continued efforts to develop more effective and safer therapeutic options, encouraging stakeholders to contribute to improving RA management.

Noninvasive Imaging OX40+ Activated T Cells Provides Early Warning of Rheumatoid Arthritis

PurposeThe goal of this study was to develop an imaging probe—IRDye-680RD-OX40 mAb—that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA.MethodsOX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups.ResultsNIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring.ConclusionThe results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.Graphical

IL-10-/- Enhances DCs Immunity Against Chlamydia psittaci Infection via OX40L/NLRP3 and IDO/Treg Pathways.

Chlamydia psittaci (C. psittaci) is a common zoonotic agent that affects both poultry and humans. Interleukin 10 (IL-10) is an anti-inflammatory factor produced during chlamydial infection, while dendritic cells (DCs) are powerful antigen-presenting cells that induce a primary immune response in the host. However, IL-10 and DCs regulatory mechanisms in C. psittaci infection remain elusive. In vivo and in vitro investigations of the regulatory mechanisms were performed. IL-10−/− mice, conditional DCs depletion mice (zinc finger dendritic cell-diphtheria toxin receptor [zDC-DTR]), and double-deficient mice (DD, IL-10−/−/zDCDTR/DTR) were intranasally infected with C. psittaci. The results showed that more than 90% of IL-10−/− mice, 70% of wild-type mice, and 60% of double-deficient mice survived, whereas all zDC-DTR mice died. A higher lymphocyte proliferation index was found in the IL-10 inhibitor mice and IL-10−/− mice. Moreover, severe lesions and high bacterial loads were detected in the zDC-DTR mice compared with double-deficient mice. In vitro studies revealed increased OX40-OX40 ligand (OX40-OX40L) activation and CD4+T cell proliferation. Besides, the expression of indoleamine 2, 3-dioxygenase (IDO), and regulatory T cells were significantly reduced in the co-culture system of CD4+ T cells and IL-10−/− DCs in C. psittaci infection. Additionally, the activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome increased to facilitate the apoptosis of DCs, leading to rapid clearance of C. psittaci. Our study showed that IL-10−/− upregulated the function of deficient DCs by activating OX40-OX40L, T cells, and the NLPR3 inflammasome, and inhibiting IDO, and regulatory T cells. These effects enhanced the survival rate of mice and C. psittaci clearance. Our research highlights the mechanism of IL-10 interaction with DCs, OX40-OX40L, and the NLPR3 inflammasome, as potential targets against C. psittaci infection.

Crucial role of OX40/OX40L signaling in a murine model of asthma

The aim of the present study was to explore the roles of OX40/OX40 ligand (OX40L) signaling and OX40+ T cells in ovalbumin (OVA)-induced mouse asthma model. Asthma was induced by OVA exposure and subsequent co-treatment with OX40L protein, neutralizing anti-OX40L blocking antibody, OX40+ T cells or PBS. The protein expression levels of interleukin (IL)-4, IL-6, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in bronchoalveolar lavage fluid (BALF) were examined using murine cytokine-specific ELISA. Eosinophil accumulation as well as proliferation and apoptosis of T cells in BALF were detected by Cell Counting kit-8 and flow cytometric assays. Expression of the apoptosis-related protein cleaved caspase-3 was examined in OX40+ T cells using western blot assay. Flow cytometric analysis revealed that OVA-treated mice that were co-treated with OX40L or OX40+ T cells exhibited higher eosinophil infiltration compared with control mice treated only with OVA, whereas neutralizing anti-OX40L blocking antibody inhibited eosinophil infiltration. ELISA assays demonstrated that the expression of IL-4, IL-6, IL-13, IL-17, TNF-α and IFN-γ in BALF in OX40L-treated and OX40+ T cell-treated mice was increased compared with expression levels in control mice. Treatment with OX40L protein effectively reduced apoptosis of T cells and the expression of cleaved caspase-3 in T cells. OX40L-treated and OX40+ T cell-treated mice exhibited increased asthma through OX40/OX40L signaling, which probably promoted inflammatory factor expression, eosinophil infiltration and T cell proliferation.

OX40L blockade protects against inflammation-driven fibrosis

Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40-OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation.

Small-molecule modulators of the OX40-OX40 ligand co-stimulatory protein-protein interaction.

The OX40-OX40L protein-protein interaction (PPI) is an important cell-surface signalling co-stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small-molecules. Here, we describe the identification of a small-molecule OX40 modulator and confirm its partial agonist character. Cell-free screening assays were developed and used to identify OX40-OX40L inhibitors. Modified versions of this assay were used to elucidate the binding partner and the binding nature of active compounds. OX40-transfected sensor cells with NF-κB reporters were constructed and used to confirm and characterize activity and specificity. Immunomodulatory activity and partial agonist nature were further confirmed by ex vivo T-cell polarization assays. Several compounds that concentration-dependently affected OX40-OX40L were identified. Cell assays indicated that they were partial agonists with low micromolar potency and adequate selectivity. Under polarizing conditions based on TGF-β, the most promising compound mimicked the effect of an agonistic anti-OX40 antibody in suppressing regulatory T-cell generation and diverting CD4(+) CD62L(+) Foxp3(-) cells to TH 9 phenotype in vitro. We identified, to our knowledge, the first small-molecule compounds able to interfere with OX40-OX40L binding and, more importantly, to act as partial agonists of OX40. This is particularly interesting, as small-molecule agonism or activation of PPIs is considered unusually challenging and there are only few known examples. These results provide proof-of-principle evidence for the feasibility of small-molecule modulation of the OX40-OX40L interaction and for the existence of partial agonists for TNFRSF-PPIs.

GW25-e4349 Effects of OX40-OX40 ligand interaction on the levels of ROS and Cyclophilin A in C57BL/6J mice' atherogenesis

GW25-e4349 Effects of OX40-OX40 ligand interaction on the levels of ROS and Cyclophilin A in C57BL/6J mice' atherogenesis

The upregulated expression of OX40/OX40L and their promotion of T cells proliferation in the murine model of asthma.

To investigate whether the expression of OX40/OX40 ligand (OX40L) was upregulated in a murine model of asthma and their significance in the pathogenesis of asthma. After an ovalbumin-sensitized/challenged murine model of asthma was established, the expressions of OX40, OX40L in peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage fluid (BALF) cell pellets were measured. Then T cell proliferation was analyzed by cell counting kit-8 (CCK8), and the protein levels of OX40 and OX40L in the lungs were determined by immunohistochemistry. The concentrations of IL-4 and IFN-γ in BALF and T cell culture supernatant were evaluated by ELISA. The percentages of CD4(+)OX40(+), CD19(+)OX40L(+), F4/80(+)OX40L(+) in PBMCs and BALF cell pellets were higher in asthma group than in control group (all P<0.01). The proliferation capacity of T cells in asthma group was higher than that in control group (P<0.05). In asthma group, stimulation of OX40 by anti-OX40 mAb obviously promoted T cell proliferation and secretion of IL-4 and IFN-γ. Immunohistochemistry assay showed that OX40 and OX40L protein levels were higher in asthma group than those in control group (all P<0.05). The expressions of OX40 and OX40L were upregulated in the murine asthmatic model. The upregulation of OX40/OX40L signals could induce the proliferation and cytokines secretion of T cells in asthmatic mice, indicating that OX40/OX40L signal was involved in the pathogenesis of asthma.

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.

Use of Genetically Modified Allograft to Deliver Local Immunomodulatory Molecule with Minimal Systemic Toxicity in a Rat Model of Allogeneic Skin Flap Transplantation

The effects of OX40-OX40 ligand (OX40L) costimulatory pathway blockade to prevent T-cell-mediated acute rejection were investigated in a rat model of allogeneic superficial inferior epigastric artery flap transplantation. An ex vivo gene transfer technique was used to modify allografts to locally deliver an immunomodulatory molecule. The flaps were separated from donors, perfused with an adenoviral vector expressing the OX40 immunoglobulin (AdOX40Ig) for 1 hour, and incubated at 37°C for 2 hours. Before transplantation, the flaps were flushed with phosphate-buffered saline solution to remove unincorporated viral particles. Recipients were randomly divided into 5 groups, and treated with topical OX40Ig gene transfer, a single low dose of rapamycin alone, or a combination of agents. Graft survival was assessed using histopathologic classification of skin rejection. All animals in the untreated group (n = 9) or the group treated with adenovirus expressing green fluorescence protein (n = 9) developed grade 3 clinical rejection by postoperative day 7. No significant difference was observed in graft survival between the locally treated AdOX40Ig groups (mean [SD], 8.1 [0.7] days) and the untreated groups (7.7 [1.2] days) could be observed ( P > .05, t test). Graft survival in the locally treated AdOX40Ig groups was extended to 18.7 (1.2) days when transduction was combined with a low dose of rapamycin, a significant improvement over survival with rapamycin treatment alone (13.2 [0.6] days) ( P < .01). These results demonstrated that local immunomodulation by the allograft itself and low-dose rapamycin treatment promote graft acceptance. This protocol may enable reduction of the dosage of immunosuppressive drugs needed for successful inhibition of acute rejection in the early postoperative period.

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.

Upregulation of OX40–OX40 Ligand System on T Lymphocytes in Patients With Acute Coronary Syndromes

To investigate whether upregulation of OX40-OX40 ligand (OX40L) system is related to stability of coronary atherosclerotic plaque in patients with coronary heart diseases. Thirty normal controls and 250 patients, including 80 with stable angina (SA), 110 with unstable angina (UA), and 60 with acute myocardial infarction (AMI), were enrolled in our study. The expression of OX40 and OX40L in peripheral CD4 T lymphocytes were analyzed by flow cytometry. Serum soluble OX40L (sOX40L) and C-reactive protein levels were measured by commercially available enzyme-linked immunosorbent assay kit. The expression of OX40 and OX40L in peripheral CD4 T lymphocytes in patients with UA [26.7 +/- 3.4 and 45.5 +/- 8.1 mean fluorescence intensity (MFI)] and AMI (27.4 +/- 4.6 and 55.7 +/- 9.4 MFI) were significantly higher than those in patients with SA (6.5 +/- 1.4 and 12.4 +/- 3.2 MFI) and controls (7.3 +/- 1.5 and 11.9 +/- 6.1 MFI). sOX40L in patients with UA (38.7 +/- 6.9 ng/mL) and AMI (39.5 +/- 7.3 ng/mL) were significantly higher than those in patients with SA (8.4 +/- 1.4 ng/mL) (P < 0.01) and controls (8.9 +/- 2.3 ng/mL) (P < 0.01). C-reactive protein level in serum in patients with UA (14.6 +/- 3.3 ng/mL) and AMI (15.0 +/- 4.3 ng/mL) were also higher than those in patients with SA (1.4 +/- 0.4 ng/mL) and controls (1.3 +/- 0.3 ng/mL). It was interesting that there was a peak level of sOX40L at 12 hours after AMI in patients with AMI. A positive correlation was found between sOX40L and serum C-reactive protein levels (r = 0.71; P < 0.0001). Patients with acute coronary syndromes showed increased coexpression of OX40 system, which may create a proinflammatory and prothrombotic milieu for aggravating the development of atherosclerosis and instability of atherosclerotic plaques, and sOX40L is a potential marker for predicting the severity of coronary heart diseases.

Thymic stromal lymphopoietin, OX40‐ligand, and interleukin‐25 in allergic responses

Allergic diseases are often triggered by environmental allergens that induce dominant type 2 immune responses, characterized by the infiltrated T-helper type 2 (TH2) lymphocytes, eosinophils, and elevated TH2 cytokines. In addition to TH2 type immune responses, epithelial stress and injury linked to tissue remodelling are often observed, suggesting that epithelial cells may play important role in regulating allergic responses. Dendritic cells (DCs), the professional antigen-presenting cells with the capabilities of sampling allergens, are considered as the key player on instructing TH2 immune responses. Whether inflamed epithelium can regulate innate immunity, such as macrophages and DCs, which in turn instructs adaptive immunity has long been hypothesized. Studies of thymic stromal lymphopoietin (TSLP), an epithelial cells-derived cytokine, that can strongly activate DCs, provide important evidences that the epithelial barrier can trigger allergic diseases by regulating immune responses. The finding that OX40/OX40Ligand (OX40L) interactions are the molecular trigger responsible for the induction and maintenance of TH2 responses by TSLP-activated DCs provides a plausible molecular explanation for TSLP-mediated allergy. Recent progresses in characterizing the pro-inflammatory IL-17 cytokine family have added an additional layer of complexity on the regulation of allergic inflammation. TSLP-DCs can induce a robust expansion of TH2 memory cells and strengthen functional attributes by up-regulating their surface expression of IL-17RB (IL-25R), the receptor for cytokine IL-17E (IL-25), a distinct member of IL-17 cytokine family. IL-17E (also known as IL-25) produced by epithelial cells, and other innate cells, such as eosinphils, basophils, and mast cells, are shown to regulate adaptive immunity by enhancing TH2 cytokine productions. These exciting findings expand our knowledge of the complex immunological cascades that result in allergic inflammation and may provide novel therapeutic approaches for the treatment of allergic diseases.

NKT Cell-Plasmacytoid Dendritic Cell Cooperation via OX40 Controls Viral Infection in a Tissue-Specific Manner

Invariant natural killer T (iNKT) cells promote immune responses to various pathogens, but exactly how iNKT cells control antiviral responses is unclear. Here, we showed that iNKT cells induced tissue-specific antiviral effects in mice infected by lymphocytic choriomeningitis virus (LCMV). Indeed, iNKT cells inhibited viral replication in the pancreas and liver but not in the spleen. In the pancreas, iNKT cells expressed the OX40 molecule and promoted type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) through OX40-OX40 ligand interaction. Subsequently, this iNKT cell-pDC cooperation attenuated the antiviral adaptive immune response in the pancreas but not in the spleen. The dampening of pancreatic anti-LCMV CD8(+) T cell response prevented tissue damage in transgenic mice expressing LCMV protein in islet beta cells. Thus, this study identifies pDCs as an essential partner of iNKT cells for mounting an efficient, nondeleterious antiviral response in peripheral tissue.

OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity

Signals through the OX40 costimulatory receptor on naive CD4 T cells are essential for full-fledged CD4 T cell activation and the generation of CD4 memory T cells. Because the ligand for OX40 is mainly expressed by APCs, including activated B cells, dendritic cells, and Langerhans cells, the OX40-OX40 ligand (OX40L) interaction has been thought to participate in T cell-APC interactions. Although several reports have revealed the expression of OX40L on T cells, the functional significance of its expression on them is still unclear. In this study, we demonstrate that Ag stimulation induced an increase in the surface expression and transcript levels of OX40L in CD4 T cells. Upon contact with OX40-expressing T cells, the cell surface expression of OX40L on CD4 T cells was markedly down-regulated, suggesting that OX40-OX40L binding occurs through a novel T cell-T cell interaction. To investigate the function of this phenomenon, we examined the proliferative response and survival of OX40L-deficient CD4 T cells when challenged with Ag. In vitro studies demonstrated markedly less CD3-induced proliferation of OX40L-deficient CD4 T cells compared with wild-type CD4 T cells. When using TCR transgenic CD4 T cells upon Ag stimulation, survival of OX40L-deficient T cells was impaired. Furthermore, we show that upon antigenic stimulation, fewer OX40L-deficient CD4 T cells than wild-type cells survived following transfer into wild-type and sublethally irradiated recipient mice. Taken together, our findings indicate that OX40L-expressing T cells have an autonomous machinery that provides OX40 signals through a T cell-T cell circuit, creating an additional mechanism for sustaining CD4 T cell longevity.

Activated T cells express the OX40 ligand: requirements for induction and costimulatory function

OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T-cell activation. OX40 expression is transiently up-regulated on T cells following T-cell receptor engagement, while OX40L is expressed on antigen-presenting cells following activation. Although expression of the OX40L by T cells has been reported, the requirements for induction of OX40L on T cells have not been studied in detail. Here, we demonstrate that the OX40L can be induced on murine CD4(+) and CD8(+) T cells after 6 days of culture under T helper type 1 (Th1) conditions, but not under Th2 conditions. Induction of OX40L expression required a high concentration of interleukin-12 (IL-12), was not seen in the presence of interferon-gamma, and was dependent on signal transducer and activator of transcription type 4 (STAT4). Notably, induction of OX40L on T cells was only seen at very low concentrations of antigen or anti-CD3. T-cell-expressed OX40L was fully capable of delivering a potent costimulatory signal that enhanced the proliferation of CD4(+) T cells as well as promoted their differentiation to Th2 cells. OX40L expression could also be induced on CD4(+) T cells in vivo following immunization with low-dose antigen and an IL-12 inducer. OX40/OX40L interactions between antigen-specific T cells may occur in T-cell zones in lymph node and spleen when OX40L expression has diminished on APC. Costimulation by T-cell-expressed OX40L may result in deviation of a Th1 response to a Th2 response under conditions where T cells are exposed to low concentrations of foreign or autoantigens in the presence of high concentrations of IL-12.

OX40 interactions in gastrointestinal nematode infection

The immune expulsion of gastrointestinal nematode parasites is usually associated with T helper type 2 (Th2) responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. The intestinal inflammatory response accompanying infection with gastrointestinal helminths is thought to be a contributory factor leading to the expulsion of the parasite. However, we have shown that the intestinal inflammation, which is controlled by interleukin (IL)-4, is not required for parasite expulsion. OX40-OX40 ligand (L) signals have been shown to be important for the development of Th2 immune responses but are also involved in a number of inflammatory diseases including those of the intestine. Here, we have investigated the effect of OX40 and OX40L fusion protein treatment on the induction of protective Th2 responses and enteropathy following infection with the gastrointestinal nematode Trichinella spiralis. Treatment with an OX40-immunoglobulin (Ig) blocking fusion protein resulted in enhanced expulsion of the parasite and an increase in the accompanying mastocytosis, despite unaltered levels of Th2 cytokines. Furthermore, there was a delay in the villus atrophy and crypt hyperplasia usually associated with this infection. In contrast, levels of Th2 cytokines were greatly up-regulated in mice treated with an OX40L-Ig activating fusion protein, yet the expulsion of the parasite and the enteropathy were unaffected. Therefore, OX40 ligation potentiates the Th2 response without enhancing host protective immune responses, whereas blocking the OX40-OX40L interaction enhances host protection without promoting Th2 cytokine responses during Trichinella spiralis infection.

Molecular transfer of CD40 and OX40 ligands to leukemic human B cells induces expansion of autologous tumor–reactive cytotoxic T lymphocytes

AbstractClinical benefits from monoclonal antibody therapy for B-chronic lymphocytic leukemia (B-CLL) have increased interest in developing additional immunotherapies for the disease. CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory antigen-specific T cells. We expressed both CD40L and OX40L on B-CLL cells by exploiting the phenomenon of molecular transfer from fibroblasts overexpressing these ligands. We analyzed the effects of the modified B-CLL cells on the number, phenotype, and cytotoxic function of autologous T cells in 7 B-CLL patients. Transfer of CD40L and OX40L was observed in all and was followed by the up-regulation of B7-1 and B7-2. The culture of CD40L/OX40L-expressing B-CLL cells with autologous T cells generated CD4+/CD8+ cytotoxic T-cell lines, which secreted interferon-γ (IFN-γ) and granzyme-B/perforin in response to autologous, but not to allogeneic, B-CLL cells or to autologous T-cell blasts. CD40L or OX40L alone was insufficient to expand tumor-reactive T cells. The combination of CD40L and OX40L on B-CLL cells may allow the generation of therapeutic immune responses to B-CLL, either by active immunization with modified tumor cells or by adoptive immunotherapy with tumor-reactive autologous T cells.

Molecular Transfer of Human CD40 and OX40 Ligands to Leukemic Human B-CLL Cells Extensively Expands Tumor-Reactive Cytotoxic T-Lymphocytes.

CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory T cells. We expressed both human CD40L and OX40L on B-Chronic Lymphocytic Leukemia (B-CLL) cells, by exploiting the phenomenon of molecular transfer from fibroblasts engineered to over-express both of these TNF-receptor superfamily members. We analyzed the effects of the modified B-CLL cells on the number, phenotype and cytotoxic function of autologous T cells in seven B-CLL patients. Transfer of CD40L and OX40L to B-CLL cells was observed in all patients (mean value from 1% pre to 76% post for CD40L; from 0.7% pre to 88% post for OX40L). Subsequent up-regulation of the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) was obtained after engagement of the endogenous CD40 receptor on B-CLL by the transferred CD40L molecules (mean value from 8% pre to 64% post for CD80; from 36% pre to 95% post for CD86). Co-culture of modified and unmodified B-CLL cells with autologous T cells revealed profound differences in the immune responses they induced. With unmodified B-CLL cells, or cells expressing either CD40L or OX40L individually, less than a 10-fold expansion of autologous T cells was observed, with a <100-fold expansion in tumor reactive T cells (measured by IFN-gamma Elispot with autologous B-CLL cells as stimulators, and allogeneic B-CLL cells as controls). By contrast, co-culture with B-CLL cells expressing both CD40L and OX40L induced a >40 fold expansion of autologous T cells - including both CD8+ T cells and CD4+ T cells with a Th1 pattern of cytokine release - and a >2500-fold increase in leukemia-reactive T cells. These expanded T cells were also directly cytotoxic to B-CLL targets, producing a mean 48% B-CLL killing at an E:T ratio of 10:1. A proportion of these tumor-reactive CD8+ T cells were specific for survivin, a B-CLL associated tumor antigen. Hence the combination of CD40L and OX40L expression by B-CLL cells allows generation of potent immune responses to B-CLL, which may be exploitable either by using active immunization with CD40L/OX40L-modified tumor cells or by adoptive immunotherapy with CD40L/OX40L generated tumor-reactive T cells.

Cross-talk between activated human NK cells and CD4+ T cells via OX40-OX40 ligand interactions.

It is important to understand which molecules are relevant for linking innate and adaptive immune cells. In this study, we show that OX40 ligand is selectively induced on IL-2, IL-12, or IL-15-activated human NK cells following stimulation through NKG2D, the low affinity receptor for IgG (CD16) or killer cell Ig-like receptor 2DS2. CD16-activated NK cells costimulate TCR-induced proliferation, and IFN-gamma produced by autologous CD4+ T cells and this process is dependent upon expression of OX40 ligand and B7 by the activated NK cells. These findings suggest a novel and unexpected link between the natural and specific immune responses, providing direct evidence for cross-talk between human CD4+ T cells and NK receptor-activated NK cells.