Characterization of Molecular Partners of the Programmed Death (PD)-1/Programmed Death Ligand (PD-L)1 Immune Checkpoint

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.