Discrete monoclonal antibodies define functionally important epitopes in the CD200 molecule responsible for immunosuppression function.
BACKGROUND
Both murine and human CD200 fusion proteins (CD200Fc) act as immunosuppressants after engagement of cell-bound receptors (CD200R). Anti-CD200 monoclonal antibodies (mAbs) augment activity in mixed leukocyte cultures (MLCs) (increased cytotoxic T lymphocyte/cytokine production) after neutralization of endogenous CD200 activity. Previous studies documented critical regions in the N-terminal domains of both CD200 and CD200R1 for ligand:receptor binding and defined a number of synthetic CD200 and CD200R peptides that antagonize that interaction.


METHODS
We used a panel of mAbs to mouse and human CD200Fc to compare the rank activities of antibodies for binding (flow cytometric analysis [FACS] or enzyme-linked immunoadsorbent assay [ELISA]) to CD200 with their abilities to augment immune reactivity in MLCs.


RESULTS
Only mAbs defining epitopes in the N-terminal domain could augment MLC reactivity (or block immunosuppression by soluble CD200Fc), whereas mAbs targeting C-domain epitopes, although reactive in ELISA or FACS (targeting cell surface CD200), were inactive in MLCs.


CONCLUSIONS
In addition to defining the importance of N-terminal epitopes for CD200 function, rank comparison of mAbs for FACS staining of CD200 expressed on various cell types indicates heterogeneity in expressed CD200.
Chen
Dang-Xiao
DX
The Toronto Hospital, University Health Network, Toronto, Canada.
Gorczynski
Reginald M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD; D000954:Antigens, Surface / Q000276:immunology*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D016207:Cytokines / Q000032:analysis / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007165:Immunosuppression Therapy / Q000379:methods; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D013154:Spleen / Q000276:immunology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000954:Antigens, Surface; D016207:Cytokines; D000939:Epitopes; D008070:Lipopolysaccharides; D010446:Peptide Fragments; C042739:antigens, CD200