Abstracts (complete list) - Wissenschaft Online

Björn Kolbe, Ulrike Kolrep, Roland Wagner, Jürgen Schmitz, Ian C.D. Johnston
Characterization of the natural ligand of the plasmacytoid
dendritic cell-specific marker CD303
CD303, the human Blood Dendritic Cell Antigen (BDCA)-2, belongs to the Type II C-
Type Lectin family and is specifically expressed on Plasmacytoid Dendritic Cells (PDC).
The Type I IFN secretion of PDC plays a major role in inflammatory autoimmune
diseases such as Systemic Lupus Erythematosus. The natural ligand of CD303 may
prove to be a key molecule in understanding these diseases, as it has been shown that
cross linking of CD303 with the monoclonal antibody AC144 inhibits PDC IFN production.
In order to identify the CD303 ligand (CD303L), we generated a CD303-Fc fusion
protein containing the extracellular domain of CD303, an HA-tag and a human IgG1-Fc
domain for purification and detection of the protein. CD303 protein purified by ProteinA
chromatography can be detected by a panel of CD303 antibodies in ELISA studies and
inhibits the staining of PDC with AC144 antibody. However, as high titers of protein
were required in these assays, a second affinity purification step using AC144-coupled
Sepharose was introduced. CD303 purified via this 2-step protocol showed an at least
10x better binding to AC144 in ELISA than the ProteinA purified protein, indicating that
only a fraction of the original protein was in fact correctly processed and fully functional.
Many human and non-human cell lines express CD303L (determined by FACS analysis)
and the highly purified CD303 also stained these cells 200x more effectively than only
ProteinA purified protein. Therefore, high affinity binding of CD303 to AC144 correlates
with binding to the natural CD303L.
This protein will now be used to isolate the CD303L by immunoprecipitation from L+
cells, and as lectins are known to recognize pathogen carbohydrate structures (eg. DC-
SIGN), glycan binding studies will also be performed.