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.
Harding
C V
CV
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000941:Antigens / Q000276:immunology*; D002738:Chloroquine / Q000494:pharmacology; D003080:Cold Temperature; D003600:Cytosol / Q000276:immunology*; D006056:Golgi Apparatus / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL
D000941:Antigens; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D000643:Ammonium Chloride; D002738:Chloroquine