Date of Completion

5-3-2018

Embargo Period

5-3-2018

Keywords

high-throughput screening, cytotoxic T lymphocyte, immune modulation, phenotypic assay, flow cytometry

Major Advisor

Adam Zweifach

Associate Advisor

Michael Lynes

Associate Advisor

Marcy Balunas

Associate Advisor

James Cole

Associate Advisor

Charles Giardina

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Immune modulation is an important therapeutic approach. Down regulation using immune inhibitors can be used to treat autoimmune diseases and prevent transplant rejection, whereas up regulation using immune augmentors can increase cancer cell killing and better clear viral and bacterial infections. Currently available immune modulators are limited by number and mechanisms, so there is an unmet medical need to look for more immune modulators. A cell-based assay was devised using cytotoxic T lymphocytes (CTL) as a model cell system in high-throughput screens to look for active compounds. Activation of cells results in the externalization of lysosome-associated membrane protein to the cell surface, which can be detected by fluorescent antibody present in extracellular solution in flow cytometry. By measuring the final output of a cell response, active hits can target any molecular pathways including the novel ones. Testing molecular mechanism of action led to the identification of eight novel immunosuppressant compounds that can be developed into chemical probes for target identification. By using different methods to activate cells in an increasingly multiplexed design, the assay gains more power to look for interesting compounds as immune modulators. The enhanced screen of a natural product library led to the identification of teleocidin as an enhancer of CTL exocytosis. Further screen of a small molecule library at multiple treatment times revealed seven immune enhancers. Multiplexed assays of CTL can be used in high-throughput screens to look for immune modulators.

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