OX40-OX40L Interaction Research Articles

An OX-Tra'Ordinary Tale: The Role of OX40 and OX40L in Atopic Dermatitis.

The transmembrane glycoprotein OX40 receptor (OX40) and its ligand, OX40L, are instrumental modulators of the adaptive immune response in humans. OX40 functions as a costimulatory molecule that promotes T cell activation, differentiation, and survival through ligation with OX40L. T cells play an integral role in the pathogenesis of several inflammatory skin conditions, including atopic dermatitis (AD). In particular, T helper 2 (TH2) cells strongly contribute to AD pathogenesis via the production of cytokines associated with type 2 inflammation (e.g., IL-4, IL-5, IL-13, and IL-31) that lead to skin barrier dysfunction and pruritus. The OX40-OX40L interaction also promotes the activation and proliferation of other T helper cell populations (e.g., TH1, TH22, and TH17), and AD patients have demonstrated higher levels of OX40 expression on peripheral blood mononuclear cells than healthy controls. As such, the OX40-OX40L pathway is a potential target for AD treatment. Novel therapies targeting the OX40 pathway are currently in development, several of which have demonstrated promising safety and efficacy results in patients with moderate-to-severe AD. Herein, we review the function of OX40 and the OX40-OX40L signaling pathway, their role in AD pathogenesis, and emerging therapies targeting OX40-OX40L that may offer insights into the future of AD management.

Abstract 1027: Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs

Abstract Background. Tumor necrosis factor receptor superfamily member 4, or OX40, is an immunostimulatory receptor usually expressed in activated T-cells. The OX40-OX40L (ligand of OX40) interaction plays a crucial role in the enhancement of T-cell proliferation, survival, and cytokine production, including those in tumor microenvironment (TME) leading to increased anti-tumor immunity1. OX40 agonist antibodies have been widely tested for immunotherapy for solid tumors2,3. Interestingly, in the treatment of lymphoma, the situation may become more complicate, where OX40 could also be found to be expressed on lymphoma cells, in addition to TME. This phenomenon might or might not impact OX40 agonist therapy. This report aims at developing an immunohistochemistry (IHC) assay to assess tumor-associated OX40 by utilizing morphology approach to understand OX40 expression in lymphoma cells, as opposed on tumor-infiltrate T-lymphocytes (TILs) present in TME. FFPE samples from patient-derived xenografts (PDXs), recapitulating the main pathological features of the original tumors, but exclusive of human TILs, were used as the first stage of the assay development, avoiding TIL interference prior to human sample testing. Methods. Ninety leukemia and lymphoma PDXs are comprehensively annotated, including RNAseq and histopathology (HuBaseTM: https://hubase.crownbio.com). IHC staining of PDX FFPE slides or TMA (tissue microarray) of various cancers was performed using a commercial OX40 antibody (CST, #61637) and Bond RX Automated IHC/ISH Staining System (Leica Biosystems), followed by whole slide imaging by NanoZoomer NDP2.0-HT Digital Slide System (Hamamatsu) and quantitative analysis by HALOTM image analysis software (Indica labs). OX40 IHC scores were also compared to the RNA expression determined by RNAseq. Results. A total of 90 leukemia and lymphoma PDXs were comprehensively evaluated. RNASeq data (human mRNA) indeed revealed OX40 gene expression (cutoff as Log2 (FPKM)>3) in 8.9% of models (8/90 models); on the other hand, IHC data also confirmed these OX40 protein expression; and the IHC scores largely correlated with the RNA data. Together, our data firmly confirmed the expression of OX40 in certain hematological cancers, beyond just in TME. The ongoing studies is investigating the characteristics of the OX40 positive lymphoma/leukemia, and the preliminary data will be presented in the annual conference. Furthermore, we are also using the same assay to explore other cancer types beyond hematological cancers. Conclusion. A tumor associated OX40 expression IHC workflow was preliminarily established and validated to facility the OX40 immunotherapy development. Citation Format: Jie Lin, Xiaolong Tu, Likun Zhang, Hongjiang Yu, Cheng JIang, Tao Yang, Henry Qixiang Li. Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1027.

The Future of Atopic Dermatitis Treatment.

This chapter thoroughly examines recent breakthroughs in atopic dermatitis (AD) treatment, with a primary focus on the medications in the development pipeline. Biologics agents targeting new interleukin receptors like interleukin-31, interleukin-22, and interleukin-2 are discussed along with the novel pathway looking at the OX40-OX40L interaction. Oral agents and small molecule therapies like Janus kinase inhibitors, sphingosine-1-phosphate modulators, and Bruton's tyrosine kinase inhibitors are also discussed along with the various new topical medications. Newly approved topicals like phosphodiesterase-4 and JAK inhibitors are highlighted while also discussing the potential of tapinarof and emerging microbiome-targeted therapies. Beyond conventional approaches, the chapter touches upon unconventional therapies currently being studied. The goal of this chapter is to discuss new advances in AD treatment from medications in the initial stages of development to those nearing FDA approval.

BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models.

OX40 is a costimulatory receptor that is expressed primarily on activated CD4+, CD8+, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.

392 Safety of amlitelimab in a Phase 2a clinical trial of patients with moderate-to-severe atopic dermatitis

Abstract Amlitelimab (SAR445229), a fully human, nondepleting, noncytotoxic, monoclonal antibody, binds to OX40Ligand (OX40L) on antigen-presenting cells to block OX40-OX40L interactions. Outcomes from a 36-week, double-blind, Phase 2a trial (NCT03754309) in patients with moderate-to-severe atopic dermatitis are described. This study aims to assess the safety of OX40-OX40L signaling blockade with amlitelimab in patients with moderate-to-severe atopic dermatitis. Eighty-nine patients were randomized to amlitelimab low-dose (LD; 200-mg loading/100-mg maintenance Q4W), high-dose (HD; 500 mg/250 mg Q4W) or placebo. The coprimary safety endpoint was treatment-emergent adverse event (TEAE) incidence to W16. Safety was assessed to W36. Amlitelimab was well tolerated. Related TEAE incidence was similar between amlitelimab [LD 35% (10/29); HD 20% (6/30)] and placebo [31% (9/29)] to W16 and W36 [LD 10% (2/20); HD 0 (0/22); placebo 0 (0/17)]. The TEAEs more common with amlitelimab vs. placebo (≥5% difference) to W16 included headache [LD 0; HD 3 (10%); placebo 1 (3.4%)], upper respiratory tract infection [3 (10.3%); 0; 1 (3.4%)], hyperhidrosis [0; 2 (6.7%); 0], pyrexia [2 (6.9%); 0; 0], aspartate aminotransferase increased [0; 2 (6.7%); 0] and iron-deficiency anemia [2 (6.9%); 0; 0]; and to W36, Herpes simplex [0; 2 (9.1%); 0]. Before W16, one severe adverse event (SAE) of infected dermal cyst was reported as possibly related; it resolved, and the patient completed the study. Post-W16, one unrelated SAE of sudden death occurred 87 days following the final amlitelimab dose. No hypersensitivity or tolerability events, or clinically meaningful changes in laboratory values, vital signs or electrocardiogram recordings were reported. Amlitelimab was well tolerated in a Phase 2a trial of patients with moderate-to-severe atopic dermatitis, confirming the unremarkable safety profile observed in Phase 1.

353 Amlitelimab reduces serum IL-13 in a phase 2a clinical trial in atopic dermatitis without impacting T-cell expansion in a T-cell recall assay

Abstract Amlitelimab (SAR445229), a fully human non-depleting, non-cytotoxic, monoclonal antibody, binds to OX40Ligand (OX40L) on antigen-presenting cells (APC) to block OX40-OX40L interactions. In a 16-week, double-blind, Phase 2a trial (NCT03754309), amlitelimab induced clinically meaningful disease activity improvement in patients with moderate-to-severe atopic dermatitis (AD). To assess the effects of amlitelimab on interleukin (IL)-13 and T-cell recall responses. In the trial, 89 patients were randomized to amlitelimab low dose (LD; 200 mg loading/100 mg maintenance every 4 weeks [Q4W]), high dose (HD; 500 mg loading/250 mg maintenance Q4W), or placebo. Serum IL-13 levels were assessed by single molecule immunoassay (259 samples). Using human peripheral blood mononuclear cells from five healthy donors, a T-cell recall assay was performed. At baseline in the trial, IL-13 levels significantly correlated with disease severity (Eczema Area and Severity Index; r = 0.4784, P < 0.0001). Week 16 IL-13 levels were significantly reduced with amlitelimab vs. baseline but not placebo (median fold change [95% confidence interval, CI] from baseline to week 16 with P-values based on two-way Analysis of Variance [ANOVA] of log10-transformed fold change for patients with a complete dataset at week 16: LD 0.345 [0.23–0.44], P < 0.0001; HD 0.390 [0.30–0.63], P = 0.0002; placebo 0.835 [0.34–1.12], P = 0.1544). In a T-cell recall assay, amlitelimab significantly reduced IL-13 protein levels at days 3 and 6 without negatively impacting T-cell expansion, based on the percentage of proliferating CD4+ T-cells vs. isotype control. Amlitelimab decreased IL-13 levels in patients with AD and in a T-cell recall assay without negative effects on T-cell expansion, thus reducing inflammation without blocking T-cell proliferation during recall responses.

Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome.

Mycosis fungoides (MF) and Sézary syndrome (SS), the most common types of cutaneous T-cell lymphoma (CTCL), are characterized by proliferation of mature CD4+ T-helper cells. Patients with advanced-stage MF and SS have poor prognosis, with 5-year survival rates of 52%. Although a variety of systemic therapies are currently available, there are no curative options for such patients except for stem cell transplantation, and thus the treatment of advanced MF and SS still remains challenging. Therefore, elucidation of the pathophysiology of MF/SS and development of medical treatments are desired. In this study, we focused on a molecule called OX40. We examined OX40 and OX40L expression and function using clinical samples of MF and SS and CTCL cell lines. OX40 and OX40L were co-expressed on tumor cells of MF and SS. OX40 and OX40L expression was increased and correlated with disease severity markers in MF/SS patients. Anti-OX40 antibody and anti-OX40L antibody suppressed the proliferation of CTCL cell lines both in vitro and in vivo. These results suggest that OX40–OX40L interactions could contribute to the proliferation of MF/SS tumor cells and that the disruption of OX40–OX40L interactions could become a new therapeutic strategy for the treatment of MF/SS.

OX40L blockade cellular nanovesicles for autoimmune diseases therapy

Current clinical agents for autoimmunity disorders treatment often cause substantial adverse effects and safety concerns, owing to non-specific immune modulation. Due to the prominent contribution of effector T cells in pathogenesis of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and preferential location of co-stimulatory receptor-ligand pair OX40-OX40L at the inflamed sites, selectively targeting autoaggressive T cells by blockade OX40-OX40L, might represent an alternative strategy. Herein, we developed a new strategy to antagonize OX40-OX40L interaction by engineering a cell membrane derived nanovesicles (NVs) expressing OX40 receptors (OX40 NVs), and explored their potential for autoimmune disorders therapy. OX40 NVs showed specific binding capability to inflamed HUVECs in vitro, it also possessed distinct arthritic-targeting capacity in RA inflamed joints, and preferential accumulation in IBD inflamed colon. OX40 NVs efficiently suppressed the progression of both RA and IBD diseases through reducing CD4+OX40+ T cells population, and proinflammatory cytokines (i.e., TNF-α and IL-1β), while reinforcing Tregs immune-suppressive effect, with superior therapeutic efficacy than anti-OX40L. Additionally, dexamethasone (DEX) loading can further enhance the potential of OX40 NVs for RA treatment. Owing to their preferential localization to inflamed sites, and potent immune-suppression ability, targeting OX40-OX40L blockade by OX40 NVs for autoimmune therapy is highly promising.

OX40 and OX40L Interaction in Cancer.

OX40 (CD134) and its binding partner, OX40L (CD252), are expressed on activated CD4, CD8 T-cells, and several other lymphoid and non-lymphoid cells. OX40L belongs to a TNF family member, a 34 kDa type II transmembrane protein. The crystallized complex of human OX40 and OX40L is a trimeric contableuration of one OX40L (trimer) and three OX40 monomers. OX40 and OX40L regulate cytokine production from T-cells, antigen-presenting cells, and natural killer (NK) cells, and modulate cytokine receptor signaling. In this review, an updated overview of the structural features of OX40/OX40L and their interactions with cancer are provided. Recent studies have shown that stimulation of OX40 is useful for therapeutic immunization strategies for cancer. OX40 serves as a secondary costimulatory immune checkpoint molecule; the binding of OX40 to its ligand enhances the augmentation, survival, memory formation, effector function, and recall responses of both CD4+ and CD8+ T-cells. This review highlights that OX40-OX40L interactions play crucial roles in both CD4+ and CD8+ T-cells. Signals through OX40 can abolish the suppressive activity of Tregs, prevent the induction of Tregs from effector T-cells, reduce Foxp3 expression, and induce the proliferation of memory and effector T lymphocytes. Additionally, when transferred into tumor-bearing recipients, they generate proliferation capability and successfully eliminate the established tumor.

Production and characterization of a novel site-specific-modifiable anti-OX40-receptor single-chain variable fragment for targeted drug delivery

OX40 receptor (tumor necrosis factor receptor superfamily, member 4; CD134) is a T-cell co-stimulatory molecule that plays an important role in T-cell activation and survival. OX40 receptor is activated by its ligand, OX40L; and modulation of the OX40–OX40L interaction is a promising target for the treatment of autoimmune diseases and cancers. Here, we generated a high-affinity anti-OX40 single-chain variable fragment carrying a C-terminal cysteine residue (scFvC). Physicochemical and functional analyses revealed that the scFvC bound to OX40-expressing cells and was internalized via OX40-mediated endocytosis without inducing phosphorylation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), an important complex in the classical NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway. In addition, mutation of the 36th cysteine residue in variable region of light chain enabled site-specific chemical modification to carboxy terminal cysteine and improved the thermal stability of the scFvC. These results suggest that this novel high-affinity anti-OX40 scFvC may be useful as a transporter for targeted delivery of small compounds, proteins, peptides, liposomes, and nanoparticles, into OX40-expressing cells for the treatment of autoimmune diseases and cancers.

Dual Blockade of OX40 and CD30 Signaling Reduces Memory Th2-driven Lung Inflammatory Responses to House Dust Mite Allergen

Abstract Allergic asthma is an inflammatory disease that is induced by immune responses to airborne allergens in the lungs. Several studies have identified T cells as being important players in asthma, with Type 2 helper T cells (Th2) being a critical subset. The selective removal of pre-existing pathogenic memory Th2 cells could be a key process leading to enhanced tolerance. We have attempted to identify signaling molecules in the TNF superfamily that regulate memory Th2 cells in a house dust mite (HDM) model of allergic lung inflammation. C57BL/6J mice up-regulated OX40L to higher levels than other TNF family members measured in the lungs in response to HDM exposure. We then tested whether OX40-OX40L interactions were required for memory Th2 cell responses driven by HDM. While OX40-deficient mice displayed strongly reduced overall lung inflammation and Th2 development when challenged with HDM, therapeutic blocking of OX40L at the time of memory T cell reactivation did not result in similar inhibition of allergic responses, implying OX40L may have been important for initiation but not the memory response. Similar negative results were gained by targeting another TNF superfamily protein, CD30L, that was also up-regulated by HDM exposure. However, we observed significant reduction in airway inflammation when OX40L was simultaneously neutralized with CD30L. Dual blockade was found to target memory Th2 cell expansion and effector function but did not prevent accumulation of pTreg cells. This suggests that persisting memory responses to complex allergens are controlled by several co-stimulatory interactions, and only abrogation of signaling through multiple TNF superfamily members might promote airway tolerance.

OX40 and 4-1BB downregulate Kaposi’s sarcoma-associated herpesvirus replication in lymphatic endothelial cells, but 4-1BB and not OX40 inhibits viral replication in B-cells.

Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the human gammaherpesvirus subfamily and is associated with malignancies of endothelial origin (Kaposi’s sarcoma, KS) and B-cell origin [primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD)]. Viral lytic replication is known to be required for KS and MCD. As KSHV-related tumours mostly develop in human subjects when the immune system is compromised by immunosuppressive regimen, human immunodeficiency virus infection or some genetic deficiencies, KSHV-specific immune responses are believed to be important in the control of KSHV replication. However, analysis of the roles of immune cells in viral pathogenesis has been difficult due to the lack of an adequate animal model. Recently, congenital OX40 deficiency, as determined by genome-wide exome sequencing, was shown to be associated with aggressive childhood KS in a patient, suggesting that disrupted OX40–OX40L interactions might be implicated in disease development. Here, we report that interaction of recombinant OX40 protein with OX40L expressed on endothelial cells severely impaired KSHV lytic replication. Furthermore, 4-1BB–4-1BBL interactions were also capable of efficiently inhibiting viral replication in B-cells and endothelial cells. To the best of our knowledge, this is the first direct evidence that ligation of tumour necrosis factor superfamily members and their cognate receptors is important for the control of viral lytic replication. These data are likely to pave the way for the development of KSHV-specific therapies for KS and MCD, in which viral lytic replication is a disease-determining factor.

Treatment with anti-OX40L or anti-TSLP does not alter the frequency of T regulatory cells in allergic asthmatics.

OX40-OX40L interactions and thymic stromal lymphopoietin (TSLP) are important in the induction and maintenance of Th2 responses in allergic disease, whereas T regulatory cells (Treg) have been shown to suppress pro-inflammatory Th2 responses. Both OX40L and TSLP have been implicated in the negative regulation of Treg. The effect of anti-asthma therapies on Treg is not well known. Our aim was to assess the effects of two monoclonal antibody therapies (anti-OX40L and anti-TSLP) on Treg frequency using a human model of allergic asthma. We hypothesized that the anti-inflammatory effects of these therapies would result in an increase in circulating Treg (CD4(+) CD25(+) CD127(low) Foxp3(+) cells) frequency. We measured Treg using flow cytometry, and our results showed that neither allergen challenge nor monoclonal antibody therapy altered circulating Treg frequency. These data highlight the need for assessment of airway Treg and for a more complete understanding of Treg biology so as to develop pharmacologics/biologics that modulate Treg for asthma therapy.

OX40, OX40L and Autoimmunity: a Comprehensive Review.

The tumour necrosis factor receptor OX40 (CD134) is activated by its cognate ligand OX40L (CD134L, CD252) and functions as a T cell co-stimulatory molecule. OX40-OX40L interactions have been proposed as a potential therapeutic target for treating autoimmunity. OX40 is expressed on activated T cells, and in the mouse at rest on regulatory T cells (Treg). OX40L is found on antigen-presenting cells, activated T cells and others including lymphoid tissue inducer cells, some endothelia and mast cells. Expression of both molecules is increased after antigen presentation occurs and also in response to multiple other pro-inflammatory factors including CD28 ligation, CD40L ligation and interferon-gamma signaling. Their interactions promote T cell survival, promote an effector T cell phenotype, promote T cell memory, tend to reduce regulatory function, increase effector cytokine production and enhance cell mobility. In some circumstances, OX40 agonism may be associated with increased tolerance, although timing with respect to antigenic stimulus is important. Further, recent work has suggested that OX40L blockade may be more effective than OX40 blockade in reducing autoimmunity. This article reviews the expression of OX40 and OX40L in health, the effects of their interactions and insights from their under- or over-expression. We then review OX40 and OX40L expression in human autoimmune disease, identified associations of variations in their genes (TNFRSF4 and TNFSF4, respectively) with autoimmunity, and data from animal models of human diseases. A rationale for blocking OX40-OX40L interaction in human autoimmunity is then presented along with commentary on the one trial of OX40L blockade in human disease conducted to date. Finally, we discuss potential problems with clinical use of OX40-OX40L directed pharmacotherapy.

Basophil-associated OX40 Ligand Participates in the Initiation of Th2 Responses during Airway Inflammation

Asthma is characterized by increased airway submucosal infiltration of T helper (Th) cells and myeloid cells that co-conspire to sustain a chronic inflammation. While recent studies have demonstrated that the myeloid basophils promote Th2 cells in response to various types of allergens, the underlying mechanisms are poorly understood. Here, we found for the first time that in a mouse model of allergic asthma basophils highly expressed OX40 ligand (OX40L) after activation. Interestingly, blockade of OX40-OX40L interaction suppressed basophils-primed Th2 cell differentiation in vitro and ameliorated ovalbumin (OVA)-induced allergic eosinophilic inflammation mediated by Th2 activation. In accordance, the adoptive transfer of basophils derived from mediastinal lymph nodes (MLN) of OVA-immunized mice triggered a robust Th2 response and eosinophilic inflammation in wild-type mice but largely muted in OX40(-/-) mice and mice receiving OX40L-blocked basophils. Taken together, our results reveal a critical role of OX40L presented by the activated basophils to initiate Th2 responses in an allergic asthma model, implicating OX40-OX40L signaling as a potential therapeutic target in the treatment of allergic airway inflammation.

IgE and IgA produced by OX40–OX40L or CD40–CD40L interaction in B cells–mast cells re-activate FcεRI or FcαRI on mast cells in mouse allergic asthma

Mast cells are major effector cells of allergic diseases related to IgE. This study was undertaken to determine whether IgE or IgA, produced by CD40–CD40L or OX40–OX40L interactions between B cells and mast cells, re-activate FcεRI or FcαRI on mast cell surface. C57BL mice were sensitized and subjected to OVA challenge to induce asthma. Bone marrow-derived mast cells (BMMCs) and primary B cells were co-cultured. Mast cell recruitment into airways was stained by May-Grünwald Giemsa, the expression of markers or signaling molecules were determined by immunohistochemistry or Western blotting, and co-localization of B cells and mast cells by immunofluorescence. Anti-CD40 plus anti-OX40L Abs synergistically reduced IgE and IgA production, and mediators (histamine, LTs and cytokines) released in mast cells, and additively reduced other responses, such as, numbers of mast cells, the expression of markers (tryptase, mMCP5, B220 and CD19), surface molecules (CD40, CD40L, OX40 and OX40L), FcεRI or FcαRI and the co-localization of BMMCs and B cells, and IgE- or IgA-producing cells, as compared with individual blocking Ab treatment which reducedresponses in BAL cells or lung tissues of OVA-challenged mice or in co-culture of B and mast cells. The data suggest that IgE and IgA, produced by OX40–OX40L or CD40–CD40L interaction between B cells and mast cells, may re-activate receptors of FCεRI and FcαRI on mast cell surfaces, followed by more mediator release, and furthermore, that treatment with anti-CD40 plus anti-OX40L Abs offers a potential treatment for allergic asthma.

The role of OX40-OX40L interactions in atherosclerosis pathogenesis

The role of OX40-OX40L interactions in atherosclerosis pathogenesis

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.

Effects of OX40–OX40 ligand interaction on the levels of ROS and Cyclophilin A in C57BL/6J mice atherogenesis

BackgroundAn increasing amount of evidence shows that the OX40–OX40L interaction serves an important function in atherosclerosis. However, the mechanism of the OX40 signaling pathway remains unclear. This study investigates the effect of OX40–OX40L interaction on the levels of intracellular reactive oxygen species (ROS) and the secretion of Cyclophilin A (CyPA) in C57BL/6J mice atherogenesis. MethodsThe atherosclerotic plaque model was established by placing a rapid perivascular carotid collar on C57BL/6J mice fed with a western-type diet. In vivo, the expressions of CyPA in mouse plaque and lymphocytes were detected by immunohistochemical and Western blot analyses, respectively. In vitro, the expression of CyPA protein in cultured lymphocytes of C57BL/6J mice was assessed by using Western blot analysis. The level of ROS was detected through flow cytometry. ResultsCyPA expression was significantly increased in the atherosclerotic lesions and lymphocytes from C57BL/6J mice. The ROS levels in OX40+-lymphocytes were increased in vitro and in vivo. After stimulating the OX40–OX40L interaction, the ROS and CyPA levels in lymphocytes were obviously increased in vitro, whereas anti-OX40L mAb significantly down-regulated the anti-OX40 mAb-induced ROS generation and inhibited CyPA secretion in lymphocytes. ConclusionThe OX40–OX40L interaction up-regulates intracellular levels of ROS in C57BL/6J mice and increases CyPA secretion in lymphocytes. Increased CyPA secretion may serve an important function in atherosclerotic plaque formation.

Antigen presenting B cells facilitate CD4 T cell cooperation resulting in enhanced generation of effector and memory CD4 T cells.

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of “epitope-spreading” seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.