Date of Completion
high-throughput screening, cytotoxic T lymphocyte, immune modulation, phenotypic assay, flow cytometry
Field of Study
Molecular and Cell Biology
Doctor of Philosophy
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.
Zhao, Ziyan, "Multiplexed High-throughput Screenings for Immune Modulators of Cytotoxic T Lymphocyte" (2018). Doctoral Dissertations. 1797.