Biochemical and functional analysis of COS3A, a novel CD63-specific monoclonal antibody.

BACKGROUND: Monoclonal antibodies (mAbs) have become essential tools in life science research and in medicine, because of their extreme specificity. Several mAbs against leukocyte surface molecules have been generated in our laboratory. From these, mAb COS3A was selected for biochemical and functional analysis. OBJECTIVE: To analyze the properties and function of the mAb COS3A. METHOD: Cellular distribution was analyzed by immunofluorescence staining and flow cytometry. Biochemical characterization of the molecular target of COS3A was approached by immunoprecipitation, Western blotting and amino acid sequencing using LC-MS. N-glycosidase F treatment of COS3A-precipitated protein and culture of U937 cells in the presence of tunicamycin before cell lysate preparation were used to study the glycosylation state of proteins. Phagocytosis was examined by flow cytometry. RESULTS: MAb COS3A bound specifically to a molecule expressed on the surface of various human hematopoietic cells and cell lines but not on erythrocytes. The antigen had a molecular weight of 30-70 kDa, which was reduced to 25 kDa by elimination of N-linked glycan. LC-MS data and immunoprecipitation indicated that mAb COS3A bound specifically to the CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis. CONCLUSIONS: The mAb COS3A recognized the CD63 molecule and strongly diminished granulocyte phagocytosis of E. coli, suggesting that CD63 may play a crucial role in the initial step of phagocytosis. MAb COS3A is, therefore, suitable for both biochemical and functional studies of CD63, and may be used for further study of the mechanism of phagocytosis and also in therapeutic approaches.

Anti-human CD63 monoclonal antibody COS3A upregulates monocyte-induced IL-10 excretion leading to diminution of CD3-mediated T cell response.

Human CD63 has been reported to play a role either as an inhibitor or as a co-stimulator of T- cell responses, although the mechanism of this is unclear. In this study, an anti-human CD63 monoclonal antibody (mAb) COS3A was used to monitor the role of CD63 in T-cell activation. MAb COS3A could inhibit CD3-mediated T-cell proliferation and CD25 expression in peripheral blood mononuclear cells (PBMCs), used as a study model, but the suppressive effect was not observed when purified T-cells were used instead of PBMCs. The inhibitory phenomenon was associated with downregulation of IL-2 and IFN-γ by T-cells, but upregulation of IL-10 by monocytes. Neutralizing IL-10 with anti-IL-10 mAb improved the T-cell response, indicating the role of IL-10 in T-cell suppression. In this study, monocytes were demonstrated to play a role in impeding T-cell activation by the anti-CD63 mAb COS3A. This is the first evidence that anti-CD63 mAb induces IL-10 secretion by monocytes, which later play a role in T-cell hypo-responsiveness.