Novel approaches to generating cytotoxic T lymphocyte target structures

by Uger, Robert A.

Novel Approaches to Generating Cytotoxic T lymphocyte Target Structures O 1999by Robert A. Uger. Thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy, Graduate Department of Immunology, University of Toronto Cytotoxic T lymphocytes (CTLs) are important effectors in the immune response against viral infections and tumors. CIZ function is critically dependent upon the recognition of a specific molecular target: a class I major histocompatibility complex (MHC) rnolecule with bound peptide antigen. Considerable effort has been focused on elucidating the structure, biochemistry, and ce11 biology of class 1 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 1MHCfpeptide 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 1antigen 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 1 MHC heavy chah. It was speculated that such an integrated peptide would be liberated by ER enzymes and preferentially form Cn target structures. Although the signal sequence-incorporated epitope could be presented by its restricting class 1 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-microglobulin (B,m). Such peptide-P,m proteins could efficiently fonn CIZ target structures when expressed endogenously or added to cells exogenously. Importantly, covalent linkage to p,m enhanced the MHC stability and antigenicity of suboptimd class 1 epitopes. This strategy. therefore, offers the potential to generate high levels of stable, defined class 1 MHC/peptide complexes, which could be valuable in vaccine and immunotherapy design. iii