Perforin binding to cells and lipid membranes determined by a simple competition assay

Perforin-mediated lysis consists of at least three steps: perforin binding to the target cell, insertion into the plasma membrane, and polymerization to form pores. Perforin binding, the first step, is critical for pore formation. Accordingly, a competition assay was here established for detecting the perforin-binding activities of nucleated cells and lipid membrane vesicles such as cytoplasts or liposomes. The competition assay has certain advantages over the 51Cr release assay, since no isotope and less perforin are needed for the competition assay, and the perforin-binding activity of liposomes and proteolytic enzyme-treated and fixed nucleated cells can also be detected. The competition assay was used to study the mechanism of resistance of cytolytic T lymphocytes (CTL) to perforin-mediated lysis. The results from this assay indicate that perforin-binding activity is not a function of membrane rigidity, and that there is a direct correlation between the ability of cells to bind perforin and their susceptibility to lysis by perforin, i.e., resistant CTL and their corresponding cytoplasts bind perforin much less effectively than susceptible tumor cells and their cytoplasts. A model is proposed whereby a surface molecule or complex of molecules on CTL interferes with perforin-binding activity, thus protecting CTL from perforin-mediated lysis.