Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

James L. Riley

Abstract

Due to the success of antiretroviral therapy (ART), Human Immunodeficiency Virus (HIV) infection has transformed from a terminal disease into a manageable chronic illness. However, ART is not curative, and individuals must endure the side-effects of long-term treatment and the social stigma associated with HIV infection. As such, the development of therapeutic strategies capable of eradicating HIV would improve the lives of infected individuals. Herein, we investigated the in vivo therapeutic potential of HIV-specific Chimeric Antigen Receptor (CAR) T cell therapy as a means to reconstitute and optimize the antiviral T cell response. Through iterative in vivo development, we generated a novel T cell product that expressed two independent CD4-based CARs containing distinct costimulatory domains (4-1BB and CD28) on the T cell surface. These Dual-CAR T cells accentuated in vivo antigen-driven proliferation mediated by 4-1BB costimulation and preserved ex vivo effector functions mediated by CD28 costimulation. Notably, Dual-CAR T cells mitigated disease pathogenesis by reducing both plasma viremia and tissue viral burden, as well as preserving CD4+ T cells from virus-induced depletion. Moreover, through extensive in vitro and in vivo characterization, we uncovered the antiviral role of HIV-specific CAR-modified CD4+ T cells including their ability to both directly suppress virus replication and confer T cell-help to other virus-specific lymphocytes. Lastly, we developed an image-based cytometry method used to measure several quality control metrics of the CAR T cell manufacturing process, including ex vivo expansion criteria and cytotoxic function. We demonstrated that image-based cytometry overcomes several limitations associated with industry-standard equipment and assays, which could serve to improve the efficiency for identifying high-quality CAR T cell products with therapeutic utility. Collectively, our work focused on cell-based immunotherapy for the treatment of HIV; however, both the development of a next-generation CAR T cell product and the in-depth characterization of CAR-modified CD4+ T cells has broad applications for other disease indications.

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