Characterization of Molecular Partners of the Programmed Death (PD)-1/Programmed Death Ligand (PD-L)1 Immune Checkpoint
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Authors
Loh, Harrison
Date
Type
thesis
Language
eng
Keyword
PD-1 , PD-L1 , CD274 , BioID , BirA* , interacting partners , cytoplasmic interacting partners , cytoplasmic interacting partners of PD-L1 , intracellular , PD-L1 interacting partners , PD-L1 binding partners , Immune checkpoint , PD-1/PD-L1 , PD-1/PD-L1 immune checkpoint , reverse signalling , reverse signaling , PD-L1 reverse signalling , PD-L1 reverse signaling , cancer , tumour , tumor , bi-directional
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Abstract
Since its discovery, the Programmed Death (PD)-1/Programmed Death-Ligand (PD-L)1 immune checkpoint has been an area of interest in cancer research due to its role in conferring immune escape to tumour cells. Most research on the PD-1/PD-L1 receptor signalling axis in tumour progression is centered around inactivation of immune effector phenotypes. However, we have recently demonstrated bi-directionality of PD-1/PD-L1 signalling resulting in acquisition of drug resistance and metastasis-promoting phenotypes in tumour cells. Thus, we hypothesized that PD-1-induced PD-L1 signalling in tumour cells results in cytoplasmic PD-L1-protein interactions which are linked to the acquisition of malignant properties. To uncover interacting partners of the cytoplasmic tail of PD-L1, an in vivo biotinylation technique called BioID wherein BirA* (R118G), a promiscuous form of the prokaryotic biotin ligase (BirA), ligated to the cytoplasmic region of PD-L1 was used. Cells were treated with recombinant PD-1 to activate BirA*-PD-L1 and incubated with biotin. Controls consisted of cells incubated in the absence of PD-1, as well as of cells transfected with the BirA* construct without PD-L1. Biotinylated proteins were isolated via streptavidin binding affinity. Nanostring® Analysis was also performed to provide exploratory information on differential mRNA expression. We provide evidence that our BirA*-PD-L1 transgene product is functional and found that PD-L1-cytoplasmic protein interactions occurred more often upon PD-L1 stimulation with recombinant PD-1. This supports our hypothesis that cytoplasmic interacting proteins facilitate PD-L1 intratumoural signalling. Results of this research may help lead to the identification of druggable targets as alternatives or adjuvants to immune checkpoint blockers as novel anti-cancer therapeutics.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
CC0 1.0 Universal