B cell receptor endocytosis induced by divalent or multivalent ligands differs in human Naïve and Memory B cells

Antigen binding to the B cell antigen receptor (BCR) initiates endocytosis, which represents the first step of antigen processing and presentation to helper T cells. The receptor crosslinking model (CLM), in which BCRs freely diffuse on the cell surface and are clustered after multivalent antigen binding, was considered for a long time the only mechanism responsible for lymphocyte activation and BCR- antigen complex endocytosis. Recently, a different model of B cell activation, called dissociation- activation model (DAM), was described. In the DAM model, BCRs are organized in auto-inhibiting oligomers that, subsequently to antigen binding, dissociate and become activated. Here we investigated BCR endocytosis in human B cells by a novel flow cytometric assay making use of monoclonal antibodies (mAbs) for ligating surface Igs. We found that the binding of mAbs, a situation that might perhaps reproduce the DAM model of BCR activation, induced significant BCR endocytosis in naïve, IgM+ memory and switched B cells. Crosslinking of mAb-coated BCRs with F(ab’)2 goat-anti-mouse IgG (GAMIgG), which putatively reproduces the CLM model of activation by multivalent antigens, increased BCR endocytosis in all B cell subsets. Interestingly, when endocytosis was sequentially induced by DAM (addition of Ig-specific mAb and first step at 37°C) and then by CLM (addition of GAMIgG and second step at 37°C), a model defined as “double triggering”, nearly 100% endocytosis was obtained in switched memory but not in the other B cell subsets, suggesting distinctive features of BCR activation and endocytosis in these subsets. Thus, exploiting mAbs rather than the commonly used polyclonal antibodies for ligating surface Igs provides a simple and accurate way for the analysis of BCR endocytosis potentially useful for untangling the interplay of CLM and DAM.