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An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets
Published on: April 18, 2016
Discrete monoclonal antibodies define functionally important epitopes in the CD200 molecule responsible for
Dang-Xiao Chen1, Reginald M Gorczynski
1The Toronto Hospital, University Health Network, Toronto, Canada.
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.
Conclusion:
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.
