Title: Development and Characterization of Mouse Monoclonal Antibodies Reactive with Chicken CD83 Authors
|Lee, Kyung Woo|
|Hong, Yeong Ho|
Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 12, 2012
Publication Date: December 11, 2012
Citation: Lee, S.H., Lillehoj, H.S., Jang, S.I., Lee, K., Baldwin, C., Tomkpins, D., Wagener, B., Lillehoj, E., Hong, Y. 2012. Development and characterization of mouse monoclonal antibodies reactive with chicken CD83. Veterinary Immunology and Immunopathology. 145(1-2):527-533. Interpretive Summary: Significant gaps in our ability to carry out comprehensive immune assessment in poultry hinder our progress in poultry disease research. To address this problem, ARS scientists in collaboration with the scientists of the United States Veterinary Immune Reagent Consortium, identified a cell surface marker of chicken dendritic cells which is designated as CD83. CD83 is a cell surface glycoprotein predominantly expressed on mature human and mouse dendritic cells (DCs) and its chicken counterpart was not identified so far. This paper describes many immunological properties of chicken CD83 antigen that will enable scientists from academia and industry to identify chicken dendritic cells. This information will advance poultry immunology field.
Technical Abstract: This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese hamster ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83+ cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83+ cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII+ cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.