Date of Award

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Paul G. Thomas, Ph.D.

Committee

Mari H. Dallas, Pradyot Dash, Terrence L. Geiger, Tony N. Marion, Maureen A. McGargill

Abstract

"Influenza virus is a significant pathogen in humans and animals with the ability to cause extensive morbidity and mortality. Exuberant immune responses associated with immune cell migration/activation and cytokine/chemokine release, can be induced after infections. Recent studies have painted a complex picture of viral clearance and tissue repair in adults. The immune responses in neonatal influenza infections and the potential methods to engineer antigen-specific T cells for immunotherapy against cancers and infections will be the focus of this dissertation." "Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were positively correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis." "In addition, transgenic expression of antigen-specific T cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific TCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these α and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCR-null) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics." "Overall, this dissertation addresses the protective roles of γδ T cells in murine and human neonatal influenza infections, and establishes the rapid system for TCR characterization, cloning and expression, which may shed light on T cell adoptive transfer immunotherapy for viral infections."

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0002-5564-3287

DOI

10.21007/etd.cghs.2019.0483

2019-008-Guo-DOA.pdf (435 kB)
Declaration of Authorship

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