Anti-CD9 monoclonal antibodies induce homotypic adhesion of pre-B cell lines by a novel mechanism.

Anti-CD9 mAb are known agonists of platelet aggregation, but have not been implicated in cell-cell adhesion. We show here in an experimental system that the anti-CD9 mAb 50H.19, ALB6, and BA-2 can induce rapid, and irreversible, homotypic aggregation of the CD9-positive pre-B lymphoblastoid cell lines NALM-6 and HOON, but not of the CD9-negative B cell line Raji. The specificity of the response is indicated by the failure to effect aggregation with mAb directed to CD24, or to HLA class I Ag. The initiation of strong homotypic aggregates of lymphoid cells is a property ascribed to lymphocyte function-associated Ag-1 (LFA-1), a member of the beta 2 subfamily of leukocyte integrins. We show that CD9-induced aggregation is an active process which proceeds at 37 degrees C, but not at 4 degrees C, requires the expenditure of metabolic energy, and a functioning cytoskeleton, and is not inhibited by Arg-Gly-Asp-Ser peptide. These are properties described for LFA-1-mediated aggregation. However, because beta 2-integrins are not expressed on NALM-6 or HOON cells, they are not the mediators of CD9-induced aggregation. In contrast to LFA-1-mediated adhesion which is Mg2+ dependent, CD9-induced adhesion has an absolute requirement for Ca2+, but not Mg2+, indicating that a Ca2(+)-dependent event is sufficient for adhesion. However, Mg2+ enhances adhesion even at optimal concentrations of Ca2+, implicating an additional Mg2(+)-dependent event which requires Ca2+ to be effective. These findings suggest that CD9 Ag regulates a novel mechanism for promoting tight cell-cell adhesion which requires both Ca2+ and Mg2+ for optimal expression.