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- Creators: LaBaer, Joshua
Description
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.
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.
ContributorsKrishna, Sri (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Jacobs, Bertram L (Committee member) / Lake, Douglas F (Committee member) / Arizona State University (Publisher)
Created2017
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
MicroRNAs (miRNAs) are short non-coding RNAs that play key roles during metazoan development, and are frequently misregulated in human disease. MiRNAs regulate gene output by targeting degenerate elements primarily in the 3´ untranslated regions of mRNAs. MiRNAs are often deeply conserved, but have undergone drastic expansions in higher metazoans, leading to families of miRNAs with highly similar sequences. The evolutionary advantage of maintaining multiple copies of duplicated miRNAs is not well understood, nor has the distinct functions of miRNA family members been systematically studied. Furthermore, the unbiased and high-throughput discovery of targets remains a major challenge, yet is required to understand the biological function of a given miRNA.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
ContributorsWolter, Justin M (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Kusumi, Kenro (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2016