ELECTROPORATION OF EXOGENOUS ANTIGEN INTO THE CYTOSOL FOR ANTIGEN PROCESSING AND CLASS-I MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) PRESENTATION - WEAK BASE AMINES AND HYPOTHERMIA (18-DEGREES-C) INHIBIT THE CLASS-I MHC PROCESSING PATHWAY

While endogenous antigens are presented by class I major histocompatibility complex (MHC) molecules, exogenous antigens generally require a means for penetration into the cytosol for processing prior to class I MHC presentation.We have optimized conditions for electroporation as a means to experimentally introduce exogenous antigens into the cytosol, providing a system with a number of advantages for dissecting the class I MHC processing pathway. Presentation was assessed by the response of class I or class II MHC-restricted T hybridoma cells. Essentially instantaneous antigen delivery by electroporation facilitated kinetic analysis of the class I pathway and investigation of the effects of various inhibitors or hypothermic conditions on class I MHC antigen processing. This pathway was inhibited by weak base amines (e.g. chloroquine and NH4Cl), cycloheximide, and hypothermia (18-degrees-C, which inhibits certain intracellular vesicular processing pathways). The electroporation technique provides a simple, consistent approach for rapid cytosolic antigen delivery for analysis of class I MHC processing.