Engineered Bacteriophage-Based In Situ Vaccine Remodels a Tumor Microenvironment and Elicits Potent Antitumor Immunity.

In situ vaccines (ISVs) utilize the localized delivery of chemotherapeutic agents or radiotherapy to stimulate the release of endogenous antigens from tumors, thereby eliciting systemic and persistent immune activation. Recently, a bioinspired ISV strategy has attracted tremendous attention due to its features such as an immune adjuvant effect and genetic plasticity. M13 bacteriophages are natural nanomaterials with intrinsic immunogenicity, genetic flexibility, and cost-effectiveness for large-scale production, demonstrating the potential for application in cancer vaccines. In this study, we propose an ISV based on the engineered M13 bacteriophage targeting CD40 (M13CD40) for dendritic cell (DC)-targeted immune stimulation, named H-GM-M13CD40. We induce immunogenic cell death and release tumor antigens through local delivery of (S)-10-hydroxycamptothecin (HCPT), followed by intratumoral injection of granulocyte-macrophage colony stimulating factor (GM-CSF) and M13CD40 to enhance DC recruitment and activation. We demonstrate that this ISV strategy can result in significant accumulation and activation of DCs at the tumor site, reversing the immunosuppressive tumor microenvironment. In addition, H-GM-M13CD40 can synergize with the PD-1 blockade and induce abscopal effects in cold tumor models. Overall, our study verifies the immunogenicity of the engineered M13CD40 bacteriophage and provides a proof of concept that the engineered M13CD40 phage can function as an adjuvant for ISVs.

Co-stimulators CD40-CD40L, a potential immune-therapy target for atherosclerosis: A review.

The interaction between CD40 and CD40 ligand (CD40L) a crucial co-stimulatory signal for activating adaptive immune cells, has a noteworthy role in atherosclerosis. It is well-known that atherosclerosis is linked to immune inflammation in blood vessels. In atherosclerotic lesions, there is a multitude of proinflammatory cytokines, adhesion molecules, and collagen, as well as smooth muscle cells, macrophages, and T lymphocytes, particularly the binding of CD40 and CD40L. Therefore, research on inhibiting the CD40-CD40L system to prevent atherosclerosis has been ongoing for more than 30 years. However, it's essential to note that long-term direct suppression of CD40 or CD40L could potentially result in immunosuppression, emphasizing the critical role of the CD40-CD40L system in atherosclerosis. Thus, specifically targeting the CD40-CD40L interaction on particular cell types or their downstream signaling pathways may be a robust strategy for mitigating atherosclerosis, reducing potential side effects. This review aims to summarize the potential utility of the CD40-CD40L system as a viable therapeutic target for atherosclerosis.

Crystal structures of human CD40 in complex with monoclonal antibodies dacetuzumab and bleselumab.

CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Utilizing murine dendritic cell line DC2.4 to evaluate the immunogenicity of subunit vaccines in vitro.

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies.

IFNγ insufficiency during mouse intra-vaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions.

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th1 response is abated while Treg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M2 macrophages with low CD40 expression promote Th2 and Treg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

Machine Learning Links T-cell Function and Spatial Localization to Neoadjuvant Immunotherapy and Clinical Outcome in Pancreatic Cancer.

Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC.

Electron transport chain and mTOR inhibition synergistically decrease CD40 signaling and counteract venetoclax resistance in chronic lymphocytic leukemia.

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.

CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment.

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-ß (TGF-ß) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-ß signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-ß-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-ß-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-ß-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.

Harnessing the potential of CD40 agonism in cancer therapy.

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily of receptors expressed on a variety of cell types. The CD40-CD40L interaction gives rise to many immune events, including the licensing of dendritic cells to activate CD8+ effector T cells, as well as the facilitation of B cell activation, proliferation, and differentiation. In malignant cells, the expression of CD40 varies among cancer types, mediating cellular proliferation, apoptosis, survival and the secretion of cytokines and chemokines. Agonistic human anti-CD40 antibodies are emerging as an option for cancer treatment, and early-phase clinical trials explored its monotherapy or combination with radiotherapy, chemotherapy, immune checkpoint blockade, and other immunomodulatory approaches. In this review, we present the current understanding of the mechanism of action for CD40, along with results from the clinical development of agonistic human CD40 antibodies in cancer treatment (selicrelumab, CDX-1140, APX005M, mitazalimab, 2141-V11, SEA-CD40, LVGN7409, and bispecific antibodies). This review also examines the safety profile of CD40 agonists in both preclinical and clinical settings, highlighting optimized dosage levels, potential adverse effects, and strategies to mitigate them.

CD40/CD40L expression and its prognostic value in cervical cancer.

CD40, a member of the tumor necrosis factor receptor (TNFR) family, is known to be involved in immune system regulation, acting as a costimulatory molecule, and in antitumor responses against cancer cells. It is a protein that is expressed in different types of cells, including immune cells and cancer cells (e.g., cervical cancer, breast cancer, melanoma). In this study, we investigated CD40/CD40L transcriptional and protein levels in cervical cancer cell lines and tumors. Higher CD40 expression was observed in cervical cancer cell lines derived from squamous cell carcinomas than from adenocarcinomas. Search of CD40/CD40L expression in cervical cancer tissues in public data sets revealed that about 83% of squamous cell carcinomas express CD40 compared to other cervical tumor subtypes. Moreover, expression of CD40 and CD40L in squamous cervical carcinomas is associated with better overall survival. Therefore, these proteins could be explored as prognostic markers in cervical cancers.

TRAF6 and TRAF2/3 Binding Motifs in CD40 Differentially Regulate B Cell Function in T-Dependent Antibody Responses and Dendritic Cell Function in Experimental Autoimmune Encephalomyelitis.

Expression of the costimulatory molecule CD40 on both B cells and dendritic cells (DCs) is required for induction of experimental autoimmune encephalomyelitis (EAE), and cell-autonomous CD40 expression on B cells is required for primary T-dependent (TD) Ab responses. We now ask whether the function of CD40 expressed by different cell types in these responses is mediated by the same or different cytoplasmic domains. CD40 has been reported to possess multiple cytoplasmic domains, including distinct TRAF6 and TRAF2/3 binding motifs. To elucidate the in vivo function of these motifs in B cells and DCs involved in EAE and TD germinal center responses, we have generated knock-in mice containing distinct CD40 cytoplasmic domain TRAF-binding site mutations and have used these animals, together with bone marrow chimeric mice, to assess the roles that these motifs play in CD40 function. We found that both TRAF2/3 and TRAF6 motifs of CD40 are critically involved in EAE induction and demonstrated that this is mediated by a role of both motifs for priming of pathogenic T cells by DCs. In contrast, the TRAF2/3 binding motif, but not the TRAF6 binding motif, is required for B cell CD40 function in TD high-affinity Ab responses. These data demonstrate that the requirements for expression of specific TRAF-binding CD40 motifs differ for B cells or DCs that function in specific immune responses and thus identify targets for intervention to modulate these responses.

Potential role of soluble CD40 receptor in chronic inflammatory diseases.

The CD40 receptor and its ligand CD154 are widely expressed in various immune-competent cells. Interaction of CD154 with CD40 is essential for B-cell growth, differentiation, and immunoglobulin class switching. Many other immune-competent cells involved in innate and adaptive immunity communicate through this co-stimulatory ligand-receptor dyad. CD40-CD154 interaction is involved in the pathogenesis of numerous inflammatory and autoimmune diseases. While CD40 and CD154 are membrane-bound proteins, their soluble counterparts are generated by proteolytic cleavage or alternative splicing. This review summarises current knowledge about the impact of single nucleotide polymorphisms in the human CD40 gene and compensatory changes in the plasma level of the soluble CD40 receptor (sCD40) isoform in related pro-inflammatory diseases. It discusses regulation patterns of the disintegrin metalloprotease ADAM17 function leading to ectodomain shedding of transmembrane proteins, such as pro-inflammatory adhesion molecules or CD40. The role of sCD40 as a potential biomarker for chronic inflammatory diseases will also be discussed.

CD40-CD40 ligand interaction between periodontal ligament cells and cementoblasts enhances periodontal tissue remodeling in response to mechanical stress.

OBJECTIVE: We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L-expressing cells under mechanical stress. Furthermore, we investigated whether CD40-CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. BACKGROUND: PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40-CD40L interaction induces by converting the force to biological functions between the cement-PDL complex, are not fully understood. METHODS: The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel-titanium closed-coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP-expressing CD40L plasmid vector was transfected into HCEM (CD40L-HCEM). CD40L-HCEM was co-cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co-culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation- and extracellular matrix (ECM)-related genes was analyzed by real-time PCR. RESULTS: Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro-stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co-culturing CD40L-HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co-culturing CD40L-HCEM with hPDS upregulated cytodifferentiation- and ECM-related genes but did not affect cell viability and proliferation. CONCLUSION: We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis.

Inhibition of PI3K p110δ activity reduces IgE production in IL-4 and anti-CD40 stimulated human B cell cultures.

Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.

Decoding the contextual duality of CD40 functions.

Previously, we established that as a function of its mode of interaction with its ligand or cellular conditions such as membrane lipids, preexisting signaling intermediates activation status, a transmembrane receptor, as represented here with CD40, can induce counteractive cellular responses. Using CD40-binding peptides, recombinant mutated CD40-ligands, and an agonistic antibody, we have established the functional duality of CD40. CD40 builds up two constitutionally different signalosomes on lipid raft and non-raft membrane domains initiating two different signaling pathways. Although this initial signaling may be modified by the pre-existing signaling conditions downstream and may be subjected to feed-forward or negative signaling effects, the initial CD40-CD40L interaction plays a crucial role in the functional outcome of CD40. Herein, we have reviewed the influence of interaction between the CD40-CD40L evoking the functional duality of CD40 contingent upon different physiological states of the cells.

Ectodomain Shedding by ADAM17 Increases the Release of Soluble CD40 from Human Endothelial Cells under Pro-Inflammatory Conditions.

BACKGROUND: Homozygosity for the C allele of the -1T>C single nucleotide polymorphism (SNP) of the CD40 gene (rs1883832) is associated with susceptibility to coronary heart disease (CHD), enhanced CD40 expression, and shedding. The disintegrin metalloprotease ADAM17 can cleave various cell surface proteins. This study investigates an association between ADAM17-mediated CD40 shedding and inflammation in CC genotype human endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) carrying the CC genotype were stimulated with soluble CD40 ligand (sCD40L) or tumor necrosis factor-α (TNFα). Messenger RNA and protein expression were determined with standard methods. Levels of high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), and sCD40 in plasma samples from patients with CHD were assessed using ELISA. RESULTS: ADAM17 surface abundance was elevated following stimulation with CD40L and TNFα just as its regulator iRhom2. Inhibition of ADAM17 prevented TNFα-induced sCD40 and soluble vascular cell adhesion molecule-1 release into the conditioned medium and reinforced CD40 surface abundance. Secondary to inhibition of ADAM17, stimulation with CD40L or TNFα upregulated monocyte chemoattractant protein-1 mRNA and protein. Levels of sCD40 and the inflammatory biomarkers hs-CRP and IL-6 were positively correlated in the plasma of patients with CHD. CONCLUSIONS: We provide a mechanism by which membrane-bound CD40 is shed from the endothelial cell surface by ADAM17, boosting sCD40 formation and limiting downstream CD40 signaling. Soluble CD40 may represent a robust biomarker for CHD, especially in conjunction with homozygosity for the C allele of the -1T>C SNP of the CD40 gene.

RNA aptamers with specific binding affinity to CD40 (CD40Apt) represents a promising antagonist of the CD40-CD40L signaling for thyroid-associated ophthalmopathy (TAO) treatment in mouse.

Thyroid-associated ophthalmopathy (TAO) is the most common autoimmune inflammatory diseases of the orbit. The CD40-CD40L pathway has been regarded as a potential molecular mechanism contributing to the development and progression of TAO, and RNA aptamers with specific binding affinity to CD40 (CD40Apt) represents a promising inhibitor of the CD40-CD40L signaling in TAO treatment. In this study, CD40Apt was confirmed to specifically recognize mouse CD40-positive ortibtal fibroblast. Mouse orbital fibroblasts were isolated from TAO mice model orbital tissues and validated. In TGF-ß-induced orbital fibroblast activation model in vitro, CD40Apt administration inhibited TGF-ß-induced cell viability, decreased TGF-ß-induced α-SMA, Collagen I, Timp-1, and vimentin levels, and suppressed TGF-ß-induced phosphorylation of Erk, p38, JNK, and NF-κB. In TAO mice model in vivo, CD40Apt caused no significant differences to the body weight of mice; furthermore, CD40Apt improved the eyelid broadening, ameliorated inflammatory infiltration and the hyperplasia in orbital muscle and adipose tissues in model mice. Concerning orbital fibroblast activation, CD40Apt reduced the levels of CD40, collagen I, TGF-ß, and α-SMA in orbital muscle and adipose tissues of model mice. Finally, CD40Apt administration significantly suppressed Erk, p38, JNK, and NF-κB phosphorylation. In conclusion, CD40Apt, specifically binds to CD40 proteins in their natural state on the cell surface with high affinity, could suppress mouse orbital fibroblast activation, therefore improving TAO in mice model through the CD40 and downstream signaling pathways. CD40Apt represents a promising antagonist of the CD40-CD40L signaling for TAO treatment.

CD40 Signaling in Mice Elicits a Broad Antiviral Response Early during Acute Infection with RNA Viruses.

Macrophages are critical in the pathogenesis of a diverse group of viral pathogens, both as targets of infection and for eliciting primary defense mechanisms. Our prior in vitro work identified that CD40 signaling in murine peritoneal macrophages protects against several RNA viruses by eliciting IL-12, which stimulates the production of interferon gamma (IFN-γ). Here, we examine the role of CD40 signaling in vivo. We show that CD40 signaling is a critical, but currently poorly appreciated, component of the innate immune response using two distinct infectious agents: mouse-adapted influenza A virus (IAV, PR8) and recombinant VSV encoding the Ebola virus glycoprotein (rVSV-EBOV GP). We find that stimulation of CD40 signaling decreases early IAV titers, whereas loss of CD40 elevated early titers and compromised lung function by day 3 of infection. Protection conferred by CD40 signaling against IAV is dependent on IFN-γ production, consistent with our in vitro studies. Using rVSV-EBOV GP that serves as a low-biocontainment model of filovirus infection, we demonstrate that macrophages are a CD40-expressing population critical for protection within the peritoneum and T-cells are the key source of CD40L (CD154). These experiments reveal the in vivo mechanisms by which CD40 signaling in macrophages regulates the early host responses to RNA virus infection and highlight how CD40 agonists currently under investigation for clinical use may function as a novel class of broad antiviral treatments.