title: Molecular cloning and characterization of a novel anti-TLR9 intrabody.
abstract: Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.
authors.affiliation: Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Reimer
authors.forename: Stefan
authors.initials: S
authors.lastname: Somplatzki
authors.forename: Diana
authors.initials: D
authors.lastname: Zegenhagen
authors.forename: Svenja
authors.initials: S
authors.lastname: Hänel
authors.forename: Alina
authors.initials: A
authors.lastname: Fels
authors.forename: Thorsten
authors.initials: T
authors.lastname: Bollhorst
authors.forename: Ludger Grosse
authors.initials: LG
authors.lastname: Hovest
authors.forename: Stefan
authors.initials: S
authors.lastname: Bauer
authors.forename: Carsten J
authors.initials: CJ
authors.lastname: Kirschning
authors.forename: Thomas
authors.initials: T
authors.lastname: Böldicke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D001483:Base Sequence; D037281:Calnexin / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D016328:NF-kappa B; D057127:Single-Chain Antibodies; D051217:Toll-Like Receptor 9; D014409:Tumor Necrosis Factor-alpha; D037281:Calnexin; D008156:Luciferases
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