Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvementof the cytosolic class I antigen processing pathway

A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing an d presentation pathway. To test the ability of pertussis toxin (PT) to deli ver peptides to the class I pathway for CTL recognition, we constructed fus ions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the in sertion of peptides, allowing efficient assembly and secretion of the holot oxin fusion by Bordetella pertussis, Target cells incubated with these fusi on proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldi n A, suggesting a dependence on intracellular trafficking events, but was n ot inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl -L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mu tant antigen-presenting cells lacking the transporter associated with antig en processing, which transports peptides from the cytosol to the endoplasmi c reticulum for association with MWC class I molecules. PT may therefore by pass the proteasome-dependent cytosolic pathway for antigen presentation an d deliver epitopes to class I molecules via an alternative route.