Generation and use of an antigen-specific hybrid to study B-cell function.

Abstract

Several important events are known to occur following the crosslinking of membrane-associated immunoglobulin (mIg) on B-lymphocytes. Among these include the internalization and re-expression of mIg. We have addressed two questions regarding the re-expression of mIg following an antigen-induced clearance of this B-cell receptor. 1) How long does it take for a cell to re-express its receptors after the first or second exposure to antigen, and 2) is this re-expression dependent upon the synthesis of new mIg? These questions, which in the past have been addressed primarily with heterogeneous populations of cells and using anti-immunoglobulin instead of antigen, are vital to understanding B-cell activation as well as antigen processing events. In order to address these questions, we have produced and characterized an antigen-specific B-cell hybrid 2C3E1 that expresses a membrane-associated form of immunoglobulin. A hybrid specific for a charged hapten (phthalate) was selected to avoid the possibility of nonspecific hydrophobic interactions of the binding ligand with the plasma membrane. After establishing the presence of mIg biochemically and demonstrating the ability of phthalate-keyhole limpet hemocyanin to induce the clearance of the phthalate-specific receptor from the plasma membrane, it was determined that re-expression of antigen-specific receptors was detected within one hour and completed by six hours after the first or second pulse with antigen and that this re-expression did not require the synthesis of new receptors. This later finding is novel and suggests that B-cell receptors are re-utilized or that a presynthesized pool of mIg exists within the cytoplasm of these cells. During the characterization of the 2C3E1 cell line, it was determined that this hybrid also produced a secreted form of the phthalate-specific immunoglobulin (sIg). An unexpected subsequent observation was that sIg was found associated with the cell surface in addition to membrane Ig. A likely source of this unexpected surface-associated sIg was observed as a vesicle on the surface of many cells that is associated with a high concentration of sIg. This finding and its possible relevance to Ig secretion per se are considered further in the paper which follows this one.