Immune regulatory molecules are well recognized for their importance in the maintenance of immune homeostasis by their ability to exert local fine control of immunological responses. CD200 is a transmembrane immunoregulatory glycoprotein expressed by a variety of different cell types. The receptor for CD200 (CD200R) is expressed on cells of the myeloid lineage, NK cells and T cells and imparts an inhibitory signal in these cells upon binding of CD200. Research has shown that CD200-CD200R interaction is involved in immune regulation in transplant tolerance models, autoimmune disease and cancer. This research has been focused on a certain isoform of CD200, which was first identified in rats and mice. In humans the corresponding protein is translated from a mRNA splice variant lacking exon 2. Our database search show that CD200 mRNA containing this exon only exists in primates. The translation of this full-length mRNA could result in an alternative CD200 isoform with a rather hydrophobic N-terminus compared to the hitherto studied CD200 isoform.
In this study we aim to characterize this alternative isoform of CD200 by identifying the significance of exon 2 in N-terminal protein sequence, binding to the CD200R and function of CD200 in regulation of NK cells.
Understanding the role of different isoforms of CD200 in immunomodulation is important for the development of methods to manipulate this immunoregulatory pathway. This may prove valuable in clinical settings where CD200 is involved in the pathophysiology (transplantation, autoimmune diseases, cancer cell evasion of immune attack) or the target for therapy (multiple myeloma, chronic lymphocytic leukaemia).
PCR amplification, cloning and generation of expression vectors for transfection of HEK293T cells with CD200 isoforms and the CD200R; purification of His-tagged proteins from cell culture medium; detection and quantification of purified protein by western blotting and ELISA. Receptor binding studies by surface plasmon resonance in collaboration with MD Jonas Borch-Jensen (BMB). Transfection of K562 cells with CD200 expression vectors, IFN-γ ELISPOT and 51Cr-releasing assay for NK cell regulation study. Production of CD200 isoform-specific antibodies in collaboration with Associated Professor Søren Hansen (IMM). CD200 splice variant-specific real-time PCR and flow cytometry.
The studie is performed in collaboration with Department of Biochemistry and Molecular Biology (BMB) and Institute of Molecular Medicine (IMM).