Electroporation of CTL clones: a useful method to investigate signalling pathways leading to the expression of effector functions

Abstract

Signal transduction mechanisms leading to effector functions in mouse cytolytic T lymphocyte (CTL) clones were studied following the introduction of exogenous molecules by electroporation. Conditions were defined in which the application of an electric pulse permeabilized the CTL without affecting functions such as antigen-dependent or antibody-mediated cytotoxicity. When a non-permeant Ca2+ chelator such as EGTA was added in the external medium during the electric pulse, it inhibited subsequent target cell cytolysis carried out in the presence of external Ca2+, thereby indicating the efficiency of EGTA uptake. Results obtained in this system, using a 13 amino-acid protein kinase C (PKC) pseudo-substrate peptide, indicated that it selectively inhibited cytolysis, whereas a substrate peptide with one amino-acid substitution was not inhibitory. This suggests that the technique could be used to study the signal transduction mechanisms of CTL clones which lead to the expression of effector functions.