title: Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.
abstract: The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-β2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1β, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-β2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.
authors.affiliation: *Laboratory of Immunology, MIGAL-Galilee Research Institute, Kiryat Shmona †Sharett Institute of Oncology, Hadassah Hebrew University Hospital, Jerusalem ‡Department of Immunology, Weizmann Institute of Science, Rehovot §Tel-Hai College, Upper Galilee, Israel.
authors.forename: Noam
authors.initials: N
authors.lastname: Levin
authors.forename: Aviad
authors.initials: A
authors.lastname: Pato
authors.forename: Gal
authors.initials: G
authors.lastname: Cafri
authors.forename: Galit
authors.initials: G
authors.lastname: Eisenberg
authors.forename: Tamar
authors.initials: T
authors.lastname: Peretz
authors.forename: Alon
authors.initials: A
authors.lastname: Margalit
authors.forename: Michal
authors.initials: M
authors.lastname: Lotem
authors.forename: Gideon
authors.initials: G
authors.lastname: Gross
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D015703:Antigens, CD / Q000378:metabolism; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism*; D019496:Cancer Vaccines / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000502:physiology*; D005818:Genetic Engineering; D006649:Histocompatibility Antigens / Q000378:metabolism; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D016350:Leucine Zippers / Q000235:genetics; D008213:Lymphocyte Activation; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D055503:Protein Multimerization / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D015703:Antigens, CD; D050976:Basic-Leucine Zipper Transcription Factors; D019013:CD40 Antigens; D019496:Cancer Vaccines; D016207:Cytokines; C501391:GCN4 protein, S cerevisiae; D006649:Histocompatibility Antigens; D018836:Inflammation Mediators; D016328:NF-kappa B; D029701:Saccharomyces cerevisiae Proteins