Intracellular Delivery of a Cytolytic T-Lymphocyte Epitope Peptide by Pertussis Toxin to Major Histocompatibility Complex Class I without Involvement of the Cytosolic Class I Antigen Processing Pathway

Abstract

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 and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions 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 insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin andN-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.

Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells

“…However, we (and others) hypothesize that a fraction of the PT (or at least the S1 subunit) that enters cells undergoes retrograde transport to the ER, from where S1 gains access to the cytosol. The hypothesis of transport of PT to the ER is based partly on our recent finding that PT can deliver major histocompatibility complex (MHC) class I-binding epitopes to MHC class I molecules without utilizing the endogenous cytosolic antigen-processing machinery for delivery of peptides to class I molecules, and therefore may deliver the epitopes directly to nascent class I molecules in the ER (Carbonetti et al, 1999). In addition, there is evidence that other toxins (Shiga toxin and ricin) are transported to the ER (Sandvig et al, 1992;Rapak et al, 1997).…”

Traffic of Proteins and Peptides across Membranes for Immunosurveillance by CD8+ T Lymphocytes: A Topological Challenge

“…Again, this has been reported for a variety of antigens that use different types of endocytosis to enter the cell. However, these cells probably lack the capacity to translocate antigens to the cytosol, as only TAP‐independent presentation of both soluble and particulate exogenous antigens has been documented (71–80) (Table 1). Interestingly, in two cases the processing proteases involved were ER‐resident signal peptidase (72) or trans Golgi network furin (73,74), as in TAP‐independent endogenous pathways (Table 1).…”

Role of Furin in Delivery of a CTL Epitope of an Anthrax Toxin‐Fusion Protein

“…To deliver a cytotoxic T lymphocyte (CTL) epitope to CTL, bacterial toxins, such as anthrax toxin (2,9) and pertussis toxin (7) have been employed as delivery vehicles in the form of fusion proteins. Anthrax toxin (20,23) is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF).…”