(381e) Direct Epitope Mapping of T Cells From Influenza A Virus Genome Using Yeasts Displaying MHC-Peptide Complexes as Artificial Antigen Presenting Cells

Cellular immunity is mediated by T cells, which respond to processed antigenic peptides in complex with major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). Identification of these peptides, namely T cell epitopes, is a critical, but often slow and difficult step in studying immune responses to cancers, infectious agents, and autoantigens, as well as in developing pertinent vaccines and therapeutic strategies. When the antigenic protein sequence is unknown, the conventional T cell epitope mapping method, which involves screening synthetic overlapping peptides, is not applicable. Instead, expression cloning and combinatorial peptide library-based approaches have been introduced. However, the expression cloning strategy has met limited success with MHC class II restricted antigens, since its processing pathway favors exogenous proteins; and few studies were able to identify native T cell epitopes from a combinatorial peptide library, due to its ?random? nature. In addition, most of these approaches rely on the use of autologous APCs that are often difficult to isolate, expand, and maintain.

To overcome the above-mentioned issues, we have developed a new approach that combines the advantages of expression cloning and random peptide library strategies by generating a pathogen-derived peptide library. More importantly, we demonstrated that yeast cells displaying the MHC-peptide complexes could be used as artificial APCs (AAPCs) to activate hybridoma T cells in a peptide-specific manner, avoiding the use of autologous APCs. Using human class II molecule HLA-DR1 and influenza A virus (X31/A/Aichi/68) as a model system, we generated a library of all possible peptides present in the virus in complex with wild-type HLA-DR1 and established a high throughput screening method for rapid and accurate epitope mapping. After as few as two rounds of fluorescence-activated cell sorting (FACS), the epitope-containing clones were enriched to ~1%. Following a second screening step using HA306-318-specific T hybridoma cells, the antigenic epitope PKYVKQNTLKLATGMRN (HA306-322) was pinpointed. Although the method was demonstrated by identifying a viral epitope, it should be generally applicable to the identification of T cell epitopes from other systems such as cancer and autoimmune diseases.