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Wahid, Mussarat (2019) Generation of endogenous soluble PD-1 as a potential therapeutic strategy for cancer treatment. PhD thesis, University of Nottingham.

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Abstract

The Programmed cell death (PD-1) pathway is an immunoinhibitory T cell pathway that is involved in maintaining peripheral T cell tolerance and regulation of inflammation. PD-1 binds to its ligand, PD-L1 which is expressed normally on a variety of cells and aberrantly on cancer cells. Binding of PD-1 to PD-L1 is a dominant immune checkpoint pathway operative in the tumour microenvironment; its normal function is to control immune homeostasis but is exploited by the cancer cells to evade immune attack. Targeting this pathway by blocking antibodies (nivolumab and pembrolizumab) has given impressive anti-cancer responses in patients with a range of cancer types. Blocking of these pathways have successfully shown to restore the function of exhausted T cells.

PD-1 is alternatively spliced to form two types of protein, a transmembrane signalling receptor (fl-PD-1) that mediate T cell death by binding to the ligand, PD-L1 and an alternatively spliced variant, soluble PD-1 that lacks the transmembrane domain and is secreted by the T cells and could inhibit PD-L1 from acting on the receptor. The soluble form of PD-1 is generated by skipping exon 3 from the PD-1 mRNA by alternative splicing. These splicing decisions in the cells are made by RNA binding proteins called SR Splicing Factors (SRSFs). SRSF1, among the other SRSFs, is a key player of constitutive and alternative splicing. Nuclear localisation and activation of SRSF1 for performing its role in alternative splicing requires its phosphorylation by SR protein kinases (SRPKs). The aim of this study is to understand the factors and mechanisms that are involved in the regulation of PD-1 splicing.

RT-PCR analysis of Jurkat T cells showed the expression of both fl-PD-1 and sPD1. Our bioinformatics analysis indicated multiple potential binding site motifs of SRSF1 adjacent to PD-1 splicing sites. However, RNA immunoprecipitation of SRSF1 followed by RT-PCR showed pull down of only fl-PD-1 indicating the role of SRSF1 in promoting splicing of fl-PD-1. Jurkat cells transfected with the nuclear localised SRSF1 plasmid resulted in the increased expression of fl-PD-1. Site directed mutagenesis of the important SRSF1 splicing regulatory regions in the PD-1 gene resulted in reduced PD-1 expression. Furthermore, inhibition of SRPK1 by lentiviral knockdown and by small molecule inhibitor resulted in reduced mRNA expression of fl-PD-1 and increased production of IL-2 by Jurkat cells. In conclusion, switching of the splicing decision from fl-PD-1 to sPD1 by targeting SRPK1 could represents a potential novel mechanism of immune checkpoint inhibition for cancer therapy.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Bates, David Patel, Poulam
Keywords:	Programmed cell death pathway; Tumour microenvironment; Immune homeostasis; Antibody blocking; Anti-cancer responses; Splicing decision; Immune checkpoint inhibition
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QZ Pathology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	56199
Depositing User:	Wahid, Mussarat
Date Deposited:	25 Jul 2019 08:18
Last Modified:	07 May 2020 11:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/56199

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