Biochemistry and Pharmacology - Theses
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ItemInvestigating immune checkpoint expression in immune cells( 2021)Programmed cell death ligands (PD-L) 1 and PD-L2 are immune checkpoint molecules that play a critical role in regulating T cell immunity. If T cells recognise the ligands by programmed cell death-1 (PD-1) receptor, their activity is inhibited. This axis functions to limit immune responses in a healthy immune system and its dysregulation has implications in many disease settings. While PD-L1 and PD-L2 can be expressed by numerous cell types, expression by immune cells, particularly dendritic cells (DCs), is critical. Regulation of PD-L1 and PD-L2, however, is poorly understood. To gain insights into the expression and regulation of the immune checkpoint molecules PD-L1 and PD-L2 in immune cells, the molecules were studied under non-inflammatory and inflammatory conditions by different approaches. In Chapter 3, findings highlight vast differences in the regulation of PD-L1 and PD-L2 in DCs. While PD-L2 shows restricted expression, PD-L1 is constitutively expressed on DCs and further induced under inflammatory conditions. To study ligand expression under different inflammatory conditions, TLR agonists and cytokines, as major sources of DC activation, were applied in vitro. Results were also confirmed in vivo by injecting inflammatory stimuli to mice. In Chapter 4, a B cell lymphoma model was used to study PD-L1, PD-L2, and PD-1 expression in response to T cell immunotherapy. Expression on tumour cells and immune cells in the tumour microenvironment (TME) was assessed. Two cell populations were identified with an immune suppressive phenotype, including a population of myeloid derived suppressor cells that expressed PD-L2 and a subset of CD4+ T cells that showed increased PD-1 expression. Due to their suppressive phenotype, both have the potential to be critical in determining success of T cell immunotherapy. In Chapter 5, ubiquitin specific peptidase 9 X-linked (USP9x), interferon regulatory factor 2 (IRF2), and IRF2-binding protein 2 (IRF2bp2) were identified to regulate PD-L1 in DCs. While USP9x and IRF2bp2 repress PD-L1 expression, IRF2 can function to promote or repress PD-L1 expression depending on the stimuli encountered. To study PD-L1 regulation, single-gene knockout cell lines were generated using the CRISPR-Cas9 system. In summary, this thesis contributes to the understanding of immune checkpoint regulation in immune cells by providing new insights into how PD-L1 and PD-L2 are regulated in DCs. The findings will enhance knowledge of how T cell responses are regulated, a prerequisite critical for the improvement and development of novel therapies.