CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.

Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic approach to identify naturally-processed HPV16-derived HLA class I epitopes for immunotherapy development. Methods: K562 cells, which lack HLA expression, were transduced with each HPV16 antigen using lentivirus and supertransfected with HLA-A2 by nucleofection. Stable cell lines expressing each antigen were selected for and maintained throughout the investigation. In order to establish a Gateway-compatible vector for robust transient gene expression, a Gateway recombination expression cloning cassette was inserted into the commercial Lonza pMAX GFP backbone, which has been experimentally shown to display high transfection expression efficiency. GFP was cloned into the vector and plain K562 cells were transfected with the plasmid by nucleofection. Results: Expression of K562-A2 was tested at various time points by flow cytometry and A2 expression was confirmed. Protein expression was shown for the transduced K562 E7 by Western blot analysis. High transfection efficiency of the pMAX_GFP_Dest vector (up to 97% GFP+ cells) was obtained 48 hours post transfection, comparable to the commercial GFP-plasmid. Conclusion: We have established a rapid system for target viral antigen co-expression with single HLA molecules for analysis of antigen presentation. Using HPV as a model system, our goal is to identify specific antigenic peptide sequences to develop immunotherapeutic treatments for HPV-associated cancers.