ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal, Humanized , CD40 Antigens , Humans , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/immunology , Binding Sites , CD40 Antigens/chemistry , CD40 Antigens/immunology , CD40 Antigens/metabolism , CD40 Ligand/chemistry , CD40 Ligand/metabolism , CD40 Ligand/immunology , Crystallography, X-Ray , Models, Molecular , Protein Binding , Protein ConformationABSTRACT
The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.
Subject(s)
AIDS Vaccines , HIV Infections , Interferon-gamma , Protein Subunit Vaccines , nef Gene Products, Human Immunodeficiency Virus , Animals , Humans , Mice , Adjuvants, Immunologic/pharmacology , AIDS Vaccines/immunology , AIDS Vaccines/chemistry , CD40 Ligand/immunology , CD40 Ligand/chemistry , Computer Simulation , Epitopes/immunology , Epitopes/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/chemistry , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1 , Interferon-gamma/metabolism , Interferon-gamma/immunology , Molecular Docking Simulation , nef Gene Products, Human Immunodeficiency Virus/immunology , nef Gene Products, Human Immunodeficiency Virus/chemistry , Protein Subunit Vaccines/chemistry , Protein Subunit Vaccines/immunologyABSTRACT
The X-linked hyper-IgM syndrome (X-HIGM1) is a rare primary immunodeficiency disorder (PID) caused by mutations in the gene encoding the CD154 protein, also known as CD40 ligand (CD40LG). X-HIGM1 is characterized by normal or elevated serum levels of IgM in association with decreased levels of IgG, IgA, and IgE. The CD40LG protein expressed on activated T cells interacts with its receptor protein, CD40, on B lymphocytes and dendritic cells. Mutations in the CD40LG gene lead to the production of an abnormal CD40L protein that fails to attach to its receptor, CD40 on B cells resulting in failure to produce IgG, IgA, and IgE antibodies. In the present study, we investigated the molecular defects underlying such a PID in a patient presenting with clinical history of pneumonia and acute respiratory distress syndrome (ARDS) at 7 months of age and diagnosed as transient hypogammaglobulinemia with decreased levels of IgG and increased levels of IgM. We have identified a novel and yet to be reported frame shift deletion of a single base pair (c.229delA) in exon 2 (p.Arg77AspfsTer6) of the CD40L gene ensuing the premature truncation of the protein by 6 amino acids by targeted gene sequencing. This frame shift mutation identified as a CD40L variant was found to be pathogenic which was also validated by Sanger sequencing. The in-silico analysis of c.229 del A mutation also predicted the change to be pathological affecting the structure and function of the CD40L (CD40L, CD154) protein and its protein-protein interaction properties.
Subject(s)
Hyper-IgM Immunodeficiency Syndrome, Type 1 , Humans , Hyper-IgM Immunodeficiency Syndrome, Type 1/genetics , Hyper-IgM Immunodeficiency Syndrome, Type 1/diagnosis , CD40 Ligand/genetics , CD40 Ligand/chemistry , Ligands , Mutation , Immunoglobulin M/genetics , Immunoglobulin A/genetics , Immunoglobulin E , Immunoglobulin G/geneticsABSTRACT
CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.
Subject(s)
CD40 Ligand/genetics , CD40 Ligand/immunology , Cichlids/genetics , Cichlids/immunology , Fish Diseases/immunology , Gene Expression Regulation/immunology , Immunity, Humoral/genetics , Amino Acid Sequence , Animals , Base Sequence , CD40 Ligand/chemistry , Female , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Profiling/veterinary , Phylogeny , Sequence Alignment/veterinary , TranscriptomeABSTRACT
Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.
Subject(s)
Amino Acids/chemistry , Binding Sites, Antibody , CD40 Ligand/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Immunoglobulin Fab Fragments/chemistry , Amino Acids/metabolism , CD40 Ligand/metabolism , Crystallography, X-Ray , Humans , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Protein ConformationABSTRACT
Cell-cell interactions involving specific membrane proteins are critical triggers in cellular development. Ex vivo strategies to mimic these effects currently use soluble proteins or (recombinant) presenter cells, albeit with mixed results. A promising alternative are bacterial magnetosomes, which can be selectively transformed into cell-free membrane-protein presenters by genetic engineering. In this study, the human CD40 Ligand (CD40L), a key ligand for B cell activation, is expressed on the particle surface. Functionality is demonstrated on sensor cells expressing the human CD40 receptor. Binding of CD40L magnetosomes to these cells triggers a signaling cascade leading to the secretion of embryonic alkaline phosphatase. Concomitantly, the CD40-CD40L interaction is strong enough to allow cell recovery by magnetic sorting. Overall, this study demonstrates the potential of magnetosomes as promising cell-free tools for cellular biotechnology, based on the display of membrane-bound target molecules, thereby creating a biomimetic interaction.
Subject(s)
Biomimetic Materials , Magnetosomes , Membrane Proteins , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Biotechnology , CD40 Antigens/chemistry , CD40 Antigens/metabolism , CD40 Ligand/chemistry , CD40 Ligand/metabolism , Cell Line , Humans , Magnetosomes/chemistry , Magnetosomes/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nanoparticles/chemistry , Nanoparticles/metabolism , Signal TransductionABSTRACT
The antagonistic anti-CD40 antibody, 2C10, and its recombinant primate derivative, 2C10R4, are potent immunosuppressive antibodies whose utility in allo- and xenotransplantation have been demonstrated in nonhuman primate studies. In this study, we defined the 2C10 binding epitope and found only slight differences in affinity of 2C10 for CD40 derived from four primate species. Staining of truncation mutants mapped the 2C10 binding epitope to the N-terminal portion of CD40. Alanine scanning mutagenesis of the first 60 residues in the CD40 ectodomain highlighted key amino acids important for binding of 2C10 and for binding of the noncross-blocking anti-CD40 antibodies 3A8 and 5D12. All four 2C10-binding residues defined by mutagenesis clustered near the membrane-distal tip of CD40 and partially overlap the CD154 binding surface. In contrast, the overlapping 3A8 and 5D12 epitopes map to an opposing surface away from the CD154 binding domain. This biochemical characterization of 2C10 confirms the validity of nonhuman primate studies in the translation of this therapeutic antibody and provides insight its mechanism of action.
Subject(s)
Antibodies, Monoclonal/metabolism , CD40 Antigens/metabolism , CD40 Ligand/metabolism , Epitopes/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , CD40 Antigens/chemistry , CD40 Antigens/genetics , CD40 Antigens/immunology , CD40 Ligand/chemistry , CD40 Ligand/genetics , CD40 Ligand/immunology , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Humans , Macaca mulatta , Mutation , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
CD40/CD154-interaction is critical in the development of Experimental Autoimmune Encephalomyelitis (EAE; mouse model of Multiple Sclerosis). Culprit CD4+CD40+ T cells drive a more severe form of EAE than conventional CD4 T cells. Blocking CD40/CD154-interaction with CD154-antibody prevents or ameliorates disease but had thrombotic complications in clinical trials. We targeted CD40 using a CD154-sequence based peptide. Peptides in human therapeutics demonstrate good safety. A small peptide, KGYY6, ameliorates EAE when given as pretreatment or at first symptoms. KGYY6 binds Th40 and memory T cells, affecting expression of CD69 and IL-10 in the CD4 T cell compartment, ultimately hampering disease development.
Subject(s)
CD40 Antigens/antagonists & inhibitors , CD40 Ligand/chemistry , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Oligopeptides/therapeutic use , T-Lymphocyte Subsets/drug effects , Animals , Drug Administration Schedule , Drug Evaluation, Preclinical , Female , Immunologic Memory , Mice , Mice, Inbred C57BL , Oligopeptides/administration & dosage , Oligopeptides/chemistry , T-Lymphocyte Subsets/immunologyABSTRACT
The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.
Subject(s)
Caspases/metabolism , Peptides/pharmacology , Proto-Oncogene Proteins c-bcl-6/metabolism , YY1 Transcription Factor/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , CD40 Antigens/metabolism , CD40 Ligand/chemistry , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Molecular Mimicry , Peptides/chemistry , Porins/chemistryABSTRACT
Simultaneous blockade of immune checkpoint molecules and co-stimulation of the TNF receptor superfamily (TNFRSF) is predicted to improve overall survival in human cancer. TNFRSF co-stimulation depends upon coordinated antigen recognition through the T cell receptor followed by homotrimerization of the TNFRSF, and is most effective when these functions occur simultaneously. To address this mechanism, we developed a two-sided human fusion protein incorporating the extracellular domains (ECD) of PD-1 and OX40L, adjoined by a central Fc domain, termed PD1-Fc-OX40L. The PD-1 end of the fusion protein binds PD-L1 and PD-L2 with affinities of 2.08 and 1.76 nM, respectively, and the OX40L end binds OX40 with an affinity of 246 pM. High binding affinity on both sides of the construct translated to potent stimulation of OX40 signaling and PD1:PD-L1/L2 blockade, in multiple in vitro assays, including improved potency as compared to pembrolizumab, nivolumab, tavolixizumab and combinations of those antibodies. Furthermore, when activated human T cells were co-cultured with PD-L1 positive human tumor cells, PD1-Fc-OX40L was observed to concentrate to the immune synapse, which enhanced proliferation of T cells and production of IL-2, IFNγ and TNFα, and led to efficient killing of tumor cells. The therapeutic activity of PD1-Fc-OX40L in established murine tumors was significantly superior to either PD1 blocking, OX40 agonist, or combination antibody therapy; and required CD4+ T cells for maximum response. Importantly, all agonist functions of PD1-Fc-OX40L are independent of Fc receptor cross-linking. Collectively, these data demonstrate a highly potent fusion protein that is part of a platform, capable of providing checkpoint blockade and TNFRSF costimulation in a single molecule, which uniquely localizes TNFRSF costimulation to checkpoint ligand positive tumor cells.
Subject(s)
CD40 Ligand/metabolism , Immunoglobulin Fc Fragments , Immunomodulation , Neoplasms/metabolism , Neoplasms/therapy , Programmed Cell Death 1 Receptor/metabolism , Recombinant Fusion Proteins/metabolism , Animals , B7-H1 Antigen/metabolism , Biomarkers, Tumor , CD40 Ligand/chemistry , Cell Line , Cytotoxicity, Immunologic , Humans , Immunoglobulin Fc Fragments/immunology , Lymphocyte Activation/immunology , Mice , Neoplasms/immunology , Neoplasms/mortality , Programmed Cell Death 1 Receptor/chemistry , Protein Binding , Receptors, OX40/metabolism , Recombinant Fusion Proteins/chemistry , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses.
Subject(s)
CD40 Ligand/chemistry , CD40 Ligand/pharmacology , Dendritic Cells/drug effects , Sigmodontinae/immunology , Amino Acid Sequence , Animals , Antigens, CD/genetics , Antigens, CD/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Base Sequence , Blood Platelets/cytology , Blood Platelets/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , CD40 Ligand/genetics , CD40 Ligand/immunology , Cloning, Molecular , Dendritic Cells/cytology , Dendritic Cells/immunology , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Granulocytes/cytology , Granulocytes/immunology , HeLa Cells , Humans , Interleukin-6/genetics , Interleukin-6/immunology , Macrophages/cytology , Macrophages/immunology , Mesocricetus , Mice , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
We report the design, synthesis, and testing of novel small-molecule compounds targeting the CD40â»CD154 (CD40L) costimulatory interaction for immunomodulatory purposes. This protein-protein interaction (PPI) is a TNF-superfamily (TNFSF) costimulatory interaction that is an important therapeutic target since it plays crucial roles in the activation of T cell responses, and there is resurgent interest in its modulation with several biologics in development. However, this interaction, just as all other PPIs, is difficult to target by small molecules. Following up on our previous work, we have now identified novel compounds such as DRI-C21091 or DRI-C21095 that show activity (IC50) in the high nanomolar to low micromolar range in the binding inhibition assay and more than thirty-fold selectivity versus other TNFSF PPIs including OX40â»OX40L, BAFFR-BAFF, and TNF-R1-TNFα. Protein thermal shift (differential scanning fluorimetry) assays indicate CD154 and not CD40 as the binding partner. Activity has also been confirmed in cell assays and in a mouse model (alloantigen-induced T cell expansion in a draining lymph node). Our results expand the chemical space of identified small-molecule CD40â»CD154 costimulatory inhibitors and provide lead structures that have the potential to be developed as orally bioavailable immunomodulatory therapeutics that are safer and less immunogenic than corresponding biologics.
Subject(s)
CD40 Antigens/metabolism , CD40 Ligand/metabolism , Chemistry Techniques, Synthetic , Drug Design , Immunologic Factors/chemical synthesis , Immunologic Factors/pharmacology , Protein Binding/drug effects , Animals , CD40 Antigens/chemistry , CD40 Ligand/chemistry , Cell Line , Humans , Immunologic Factors/chemistry , Immunomodulation/drug effects , Mice , Models, Molecular , Protein Conformation , Protein MultimerizationABSTRACT
OBJECTIVE: The aim of this study was to evaluate whether the concentration of sCD40L, a product of platelet activation, correlates with the presence of Pseudomonas aeruginosa in the airway of patients with cystic fibrosis (CF) and to determine its possible clinical association. METHODS: Sixty patients with CF, ranging in age from 2 months to 36 years, were studied. The demographics, cystic fibrosis transmembrane conductance regulator (CFTR) genotype, spirometry measurements, radiographic and tomographic scans, platelet count in peripheral blood, sCD40L, IL-6, TNF-α and ICAM1 data were collected. Infection-colonization of the airway was evaluated using sputum and throat swab cultures; the levels of anti-Pseudomonas aeruginosa antibodies (Anti-PaAb) were evaluated. RESULTS: Patients with CF and chronic colonization had anti-PaAb values of 11.6 ± 2.1 ELISA units (EU) and sCD40L in plasma of 1530.9 ±1162.3 pg/mL; those with intermittent infection had 5.7 ± 2.7 EU and 2243.6 ± 1475.9 pg/mL; and those who were never infected had 3.46 ± 1.8 EU (p≤0.001) and 1008.1 ± 746.8 pg/mL (p≤0.01), respectively. The cutoff value of sCD40L of 1255 pg/mL was associated with an area under the ROC (receiver operating characteristic curve) of 0.84 (95% CI, 0.71 to 0.97), reflecting P. aeruginosa infection with a sensitivity of 73% and a specificity of 89%. Lung damage was determined using the Brasfield Score, the Bhalla Score, and spirometry (FVC%, FEV1%) and found to be significantly different among the groups (p≤0.001). CONCLUSION: Circulating sCD40L levels are increased in patients with cystic fibrosis and P. aeruginosa infection. Soluble CD40L appears to reflect infection and provides a tool for monitoring the evolution of lung deterioration.
Subject(s)
CD40 Ligand/blood , CD40 Ligand/chemistry , Cystic Fibrosis/blood , Cystic Fibrosis/microbiology , Pseudomonas Infections/complications , Pseudomonas aeruginosa/physiology , Adolescent , Child , Child, Preschool , Cohort Studies , Cystic Fibrosis/complications , Female , Humans , Infant , Male , ROC Curve , Solubility , Young AdultABSTRACT
The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with â¼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model.
Subject(s)
CD40 Antigens/biosynthesis , Amino Acid Sequence , Animals , CD40 Antigens/chemistry , CD40 Antigens/genetics , CD40 Antigens/pharmacology , CD40 Ligand/chemistry , Cell Line, Tumor , Cells, Cultured , Cloning, Molecular , Gene Expression , Glycosylation , Humans , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/physiology , Macaca mulatta , Mice , Molecular Sequence Data , Pichia , Protein Binding , Protein Multimerization , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
BACKGROUND: Non-invasive blood-based biomarkers are eagerly awaited for the diagnosis of Alzheimer's disease (AD). The present study aimed to evaluate the individual and combined diagnostic value of soluble CD40 ligand (sCD40L) and vascular endothelial growth factor (VEGF) for AD. METHODS: Fifty patients with AD and forty gender and age-matched control participants with standardized clinical assessments and neuroimaging measures were enrolled. VEGF and sCD40L were qualified in 90 subjects using immunomagnetic beads assay. RESULTS: To evaluate the individual and combined diagnostic value of sCD40L and VEGF for AD, receiver operating characteristic curves were generated and logistic regression analysis was conducted. The AUCs (area under ROCs) of sCD40L and VEGF and their corresponding 95% confidence interval (CI) were 0.824 (95% CI: 0.737-0.910) and 0.731 (95% CI: 0.622-0.839), respectively. Combined ROC analysis based on these 2 biomarkers revealed an elevated AUC of 0.858 (95% CI: 0.775-0.941), which indicates an additive effect in the diagnostic value of these two biomarkers. CONCLUSIONS: We identified the feasibility of a blood-based biomarker approach in AD diagnostics though the results warrant validation in large-scale studies. A combination of sCD40L and VEGF could be a useful diagnostic biomarker for future clinical trials with AD and may act as a suitable add-on biomarker to the panel of markers already existing for AD.
Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/diagnosis , CD40 Ligand/blood , CD40 Ligand/chemistry , Vascular Endothelial Growth Factor A/blood , Aged , Biomarkers/blood , Case-Control Studies , Female , Humans , Male , Sensitivity and Specificity , SolubilityABSTRACT
Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4(+) T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4(+) T cell responses but also CD8(+) cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8(+) T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4(+) and CD8(+) T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens.
Subject(s)
Adjuvants, Immunologic/administration & dosage , CD40 Ligand/administration & dosage , Cattle Diseases/prevention & control , Vaccination/veterinary , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/genetics , Animals , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD40 Antigens/metabolism , CD40 Ligand/chemistry , CD40 Ligand/genetics , Cattle , Cattle Diseases/immunology , Cloning, Molecular , Endothelial Cells/immunology , Female , In Vitro Techniques , Lymphocyte Activation , Mice , Protein Structure, Quaternary , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Staphylococcus aureus/immunology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
CD40, as a single receptor that binds CD154 (CD40-ligand or CD40L), regulates counteractive effector functions such as production of pro- and anti-inflammatory cytokines. Therefore, we examined whether such dual messages are encrypted in CD40L. As such message encryption was never investigated, we hypothesized that mutation of certain amino acid residues should in principle enhance pro-inflammatory cytokine production whereas mutation of some others would enhance anti-inflammatory cytokine secretion. We mutated six such residues, which were previously showed to participate in CD40L function. Here, we report that the mutant CD154 129EâV was superior to the wild-type CD154 in killing of Leishmania donovani, induction of inducible nitric oxide synthase (iNOS) and production of IL-12 and relative phosphorylation of p38MAPK and ERK-1/2 in PBMC-derived macrophages. By contrast, 128SâV promoted L. donovani survival, reducing iNOS, but increasing IL-10 expression and predominant ERK-1/2 phosphorylation. The mutant 144GâV did not have significant effects. Other mutants (142EâV, 143KâA, 145YâF) mimicked the wild-type CD154. Molecular dynamics simulation suggested that these mutations induced differential conformational changes in the CD40-CD154 complex. Therefore, assortment of the contrasting messages encrypted in a given ligand performing counteractive functions presents a novel fundamental biological principle that can be used for devising various therapies.
Subject(s)
CD40 Ligand/metabolism , CD40 Antigens/chemistry , CD40 Antigens/genetics , CD40 Antigens/metabolism , CD40 Ligand/chemistry , CD40 Ligand/genetics , Cloning, Molecular , Cytokines/metabolism , Gene Expression , Humans , Leishmania donovani/immunology , Leishmania donovani/metabolism , Macrophages/immunology , Macrophages/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Models, Molecular , Mutation , Nitrites/metabolism , Phosphorylation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs/genetics , Surface Plasmon Resonance , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark (Scyliorhinus canicula). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies.
Subject(s)
B-Cell Activating Factor/genetics , CD40 Ligand/genetics , Fish Proteins/genetics , Sharks/genetics , Amino Acid Sequence , Animals , B-Cell Activating Factor/chemistry , B-Cell Activating Factor/metabolism , CD40 Ligand/chemistry , CD40 Ligand/metabolism , Fish Proteins/chemistry , Fish Proteins/metabolism , Leukocytes/immunology , Mitogens/pharmacology , Pathogen-Associated Molecular Pattern Molecules/pharmacology , Phylogeny , Sequence Alignment/veterinary , Sharks/immunology , Sharks/metabolismABSTRACT
Viruses use spatial control of constituent proteins as a means of manipulating and evading host immune systems. Similarly, precise spatial control of proteins encapsulated or presented on designed nanoparticles has the potential to biomimetically amplify or shield biological interactions. Previously, we have shown the ability to encapsulate a wide range of guest proteins within the virus-like particle (VLP) from Salmonella typhimurium bacteriophage P22, including antigenic proteins from human pathogens such as influenza. Expanding on this robust encapsulation strategy, we have used the trimeric decoration protein (Dec) from bacteriophage L as a means of controlled exterior presentation on the mature P22 VLP, to which it binds with high affinity. Through genetic fusion to the C-terminus of the Dec protein, either the 17 kDa soluble region of murine CD40L or a minimal peptide designed from the binding region of the "self-marker" CD47 was independently presented on the P22 VLP capsid exterior. Both candidates retained function when presented as a Dec-fusion. Binding of the Dec domain to the P22 capsid was minimally changed across designed constructs, as measured by surface plasmon resonance, demonstrating the broad utility of this presentation strategy. Dec-mediated presentation offers a robust, modular means of decorating the exposed exterior of the P22 capsid in order to further orchestrate responses to internally functionalized VLPs within biological systems.
Subject(s)
Bacteriophage P22/chemistry , Capsid Proteins/chemistry , Recombinant Fusion Proteins/genetics , Virion/chemistry , Animals , Bacteriophage P22/genetics , Bacteriophage lambda/chemistry , Bacteriophage lambda/genetics , CD40 Ligand/chemistry , CD40 Ligand/genetics , CD47 Antigen/chemistry , CD47 Antigen/genetics , Capsid Proteins/genetics , Humans , Mice , Peptides/chemistry , Peptides/genetics , Recombinant Fusion Proteins/chemistry , Salmonella typhimurium/virology , Virion/geneticsABSTRACT
BACKGROUND: Serum soluble CD40 ligand (sCD40L) concentrations are increased in patients with nasopharyngeal carcinoma (NPC). This study further evaluated the relationship between plasma sCD40L concentrations and long-term survival of NPC. METHODS: Plasma sCD40L concentrations of 312 patients and 312 healthy controls were determined using an ELISA. The associations of plasma sCD40L concentrations with 5-year overall survival, progression-free survival, distant metastasis-free survival, and locoregional relapse-free survival were investigated by univariate and multivariate analyses. RESULTS: Plasma sCD40L concentrations were substantially higher in patients than in healthy subjects and also correlated highly with tumor classification, lymph node classification and tumor node metastasis stage. sCD40L emerged as an independent predictor for 5-year overall survival, progression-free survival, distant metastasis-free survival, and locoregional relapse-free survival using univariate and multivariate Cox regression analysis. CONCLUSIONS: High plasma sCD40L concentration is correlated with stage progression of NPC as well as associated with poor survival of NPC. It is suggested that sCD40L should have the potential to be a prognostic biomarker for NPC.