Novel approaches to generating cytotoxic T lymphocyte target structures

grantor: University of TorontoCytotoxic T lymphocytes (CTLs) are important effectors in the immune response against viral infections and tumors. CTL function is critically dependent upon the recognition of a specific molecular target: a class I major histocompatibility complex (MHC) molecule with bound peptide antigen. Considerable effort has been focused on elucidating the structure, biochemistry, and cell biology of class I molecules. This information has provided a solid foundation for understanding antigen presentation, a basic process central to cellular immune recognition. In addition, an intimate knowledge of CTL target structure formation is proving useful for the development of new vaccine and immunotherapy strategies. This thesis describes two novel approaches for generating class I MHC/peptide complexes, which share the common feature of coupling MHC and peptide antigen. They were undertaken within the framework of developing new tools for probing class I antigen presentation and devising unique strategies for consideration in disease prevention and treatment. In the first approach, the biosynthesis and endoplasmic reticulum (ER) translocation of MHC and peptide were transiently coupled by inserting a CTL epitope into the signal sequence of a class I MHC heavy chain. It was speculated that such an integrated peptide would be liberated by ER enzymes and preferentially form CTL target structures. Although the signal sequence-incorporated epitope could be presented by its restricting class I molecule, this presentation was unexpectedly observed to require TAP (transporter-associated with antigen processing) transport. This result indicated a cytosolic origin for the signal sequence peptide, and suggested a lack of ER processing. The second strategy achieved a more permanent peptide-MHC linkage by covalently coupling CTL epitopes to the class I light chain, beta 2-microglobuhn (ß2m). Such peptide-ß2m proteins could efficiently form CTL target structures when expressed endogenously or added to cells exogenously. Importantly, covalent linkage to ß2m enhanced the MHC stability and antigenicity of suboptimal class I epitopes. This strategy, therefore, offers the potential to generate high levels of stable, defined class I MHC/peptide complexes, which could be valuable in vaccine and immunotherapy design.Ph.D