Killer immunoglobulin-like receptors (KIRs) are a family of proteins expressed on human natural killer cells and a subset of T cells. Several inhibitory KIRs have been shown to recognise MHC class I molecules (predominantly HLA-C), with their engagement preventing target cell lysis. The ligand(s) and function(s) of activating KIRs, however, are less well characterised. Genetic studies of the association of KIRs with disease have identified an association with viral infections and autoimmune disease and this implicates that these proteins are important in human health. This thesis was concerned with an investigation of the factors that determine KIR expression on lymphocytes, and how this might influence the cellular functional response. In my initial work I produced soluble recombinant forms of activating and inhibitory KIRs and studied the biophysical interaction of these proteins with HLA-C molecules. I saw some evidence that KIR2DS2 binds to the HLA-C group 1 allele HLA-Cw*0702, supporting the idea that HLA-C alleles are a true ligand for stimulatory KIRs. I then went on to make a detailed 11 colour flow cytometric analysis of the expression of KIR proteins in healthy individuals. I was able to show that total, and individual, KIR protein expression was correlated and defined a pattern of dominance on lymphoid subsets. I then went on to study the distribution of KIR expression on discrete memory T cell subsets and showed that they were found predominantly on late differentiating CD45RA+ T cells. Interestingly there was also considerable expression on central memory CD8+ T cells although the biological basis for this is unclear. I demonstrated that age and CMV infection have a marked effect on KIR expression and I speculate on the reason for this. Finally I studied KIR expression on CMV-specific T cell clones in order to undertake a functional analysis of the consequence of KIR expression. I observed that KIR expression increased when cells were cultured in vitro but I could not detect any difference in cytokine production or cytotoxicity between KIR+ and KIR- cells. My work has contributed to the literature on KIR biology in relation to lymphoid cells and will have direct relevance to a number of clinical studies.

Glioblastoma is a malignant form of brain cancer. This type of tumour is resistant to medical treatment and often yields a poor prognosis. It is important to understand the molecular basis of glioblastoma for developing new therapies. This study used U87 human glioblastoma cells for the investigation. The glioma cell line displayed the hallmarks of a tumour cell and corresponded on various cellular functions that are regulated by Rho family GTPases. This study intended to analyse the role of one member from this family - RhoE in U87 cells. RNA interference (RNAi) method was applied to knock down RhoE. The result showed that the loss of RhoE expression is associated with the decrease in cell cycle progression and increase apoptosis in U87 cells. Endogenous RhoE was found localised to the nucleus in U87 cells. RhoE is known can interact with serine/threonine kinases called ROCK1 and PKC. The application of RNAi that knocks down ROCK1 has led to the stabilisation of RhoE expression in U87 cells. Y27632 was used to inhibit ROCK1 whereas Gӧ6976 blocked the function of PKC. Both pharmacological inhibitors prevent individual interaction with RhoE. The activation of novel PKC by PMA exhibit an increased the upper band of RhoE (29kD) and it remains more in the nucleus consistent with the active conventional PKC during ROCK1 inhibition. A co-immunoprecipitation (Co-IP) technique was used to pull down FLAG-RhoE from the transfected U87 cells with wild-type construct. As a result, the translocation of RhoE to the nucleus was facilitated by the interaction with importin  (Imp). A bioinformatics study on protein-protein interactions using DAVID on mass spectrometry data elucidated that RhoE could be indirectly bound to nuclear proteins. In conclusion, RhoE plays a pivotal role in regulating glioblastoma at the post-translational level.

Alterations to metabolic pathways, in particular post-translational modification, are a recognised hallmark of diseases such as autoimmunity, inflammation and cancer, and potentially provide a source of altered self antigens that can stimulate immune responses. Most notably, phosphorylated peptides have emerged as a group of tumour associated antigens which can be presented by MHC molecules and recognised by T-cells, and therefore represent promising candidates for future cancer immunotherapy strategies. However, how antigen phosphorylation impacts upon antigen presentation and recognition remains unclear. During this study I demonstrated that the phosphate moiety of phosphopeptides bearing the canonical P4 phosphorylation is more structurally diverse in its binding mode than previously thought. Strikingly, two epitopes exhibited a major conformational change upon addition of the phosphate moiety, effectively creating “conformational neoantigens”. This occurred through a similar mechanism for each epitope, whereby the presence of the phosphate moiety raised the position of the P4 Serine, allowing phosphate-mediated contacts with MHC residues and distorting the conformation of the central epitope region most critical for T- cell receptor recognition. Finally, I found that recognition of phosphopeptides can be both phosphate-dependent and epitope-specific at the level of the T-cell receptor. Therefore, this study shows that phosphorylation can have a profound and diverse effect on antigen binding, epitope identity and T-cell receptor recognition. In summary, my studies suggest that phosphopeptides are not only tumour associated but also highly antigenically distinct, establishing them as attractive candidates for cancer immunotherapy strategies.

Cancer is an important cause of mortality among the recipients of organ transplantation. Cancer transmitted from the donors has poor outcome and the fear of such transmission results in non-acceptance of certain organs. Study of the recipients in the UK over 10 years identified 15 cases of transmitted cancers. The rate of cancer transmission was 0.05%.The risk of cancer transmission was 9 times higher from donors older than 45 years. A comparison of the organ donor data with the guidelines classifying the donor’s risk showed that a selected cohort of donors, who are classed as high risk of cancer transmission, could safely donate their organs resulting in valuable additional survival for the recipients, with low risk of cancer transmission. These results provide evidence, for modification of donor classification guidelines resulting in increased availability of safe organs for transplantation. The risk of recurrence after transplantation of cancers treated before transplantation was low in selected recipients undergoing transplantation after a 2 year-wait following the diagnosis of cancer. No association was found between the donor-recipient CMV status and the risk of post-transplant cancer. This research estimated the risk of cancer transmission to the organ transplant recipients enabling improved risk assessment in transplantation.

Multiple sclerosis (MS) is a debilitating disease in which the immune system aberrantly targets the central nervous system (CNS). There is compelling evidence that Epstein-Barr virus (EBV) is associated with MS development but the pathogenic mechanisms are unknown. The molecular mimicry hypothesis suggests the immune response to EBV, which normally would restrain the virus infection, mistakenly targets CNS components. This thesis characterised humoural and cellular responses to the virus in healthy controls (HC) and MS patients, increasing the range of EBV and CNS proteins investigated and seeking evidence of crossreactivity as predicted by the hypothesis. Compared to HC, patients had elevated EBNA1 and virus capsid antigen-specific antibody responses. EBNA3-specific antibody responses were also more frequently detected in patients, a previously undescribed observation. Both groups had similar frequencies of circulating Tcells specific for autologous lymphoblastoid cell lines (LCL) or EBNA1, although minor differences in cytokine profile were detected. LCL-specific T-cell cultures established from both patients and HC exhibited cross-reactivity to CNS antigens. This result supports a role for molecular mimicry but also suggests that other unknown or more complex factors must influence MS development. While such T-cells are a necessary prerequisite for the molecular mimicry hypothesis, their presence in HC suggests other factors must influence MS development. Identification of these factors must be a priority for future studies.

Nuclear receptors (NR) are ligand dependent transcription factors. In the current study, expression of VDR and Farnesoid-X-receptor (FXR) protein is demonstrated along with relative mRNA expression of a range NRs and co-repressors in four bladder cancer cell lines. Nuclear co-repressor 1 (NCoR1) is over-expressed in RT-112 (1.6 fold) and EJ-28 cells (2.6 fold). This correlates with reduced sensitivity to NR ligands in EJ-28 cells. Stable over-expression of NCoR1 in sensitive RT-4 cells (lowest relative NCoR1 expression) led to reduced sensitivity to NR ligands; treatment with lithocholic acid (LCA - FXR and VDR ligand) led to expression of a cohort of genes consistent with a xenobiotic protective response (ABC transporter proteins, metabolizing enzymes and cell cycle arrest proteins) as assessed by microfluidic quantitative real-time reverse transcription polymerase chain reaction. NCoR1 over-expression was targeted with co-treatment with NR ligand and the histone deacetylase inhibitor Suberoylanilide hydroxamic acid (SAHA), resulting in strongly additive anti-proliferative responses to FXR, VDR and PPAR-γ ligands in NCoR1 over-expressing cells; confirmed as a G1/S phase cell cycle arrest in EJ-28. Microarray profiling revealed unique regulation of genes involved in cell proliferation. This study suggests NCoR1 acts as a selective regulator of NR function.

Epstein-Barr virus (EBV) is associated with B- and epithelial cell malignancies. It is also associated with lymphoproliferations and malignancies of T- and natural killer (NK) cells. The global impact of these conditions is significant, and although rare, they are aggressive and are often resistant to treatment. Diagnosis is often delayed, and evidence-based treatment strategies are limited due to their rarity. Viral gene expression in extranodal T- and NK-cell lymphoma (ENKTL), chronic active Epstein-Barr virus (CAEBV) and haemophagocytic lymphohistiocytosis (HLH) is limited. The viral latent membrane proteins LMP1, LMP2A and LMP2B-TR-TR have growth-transforming properties in B- and epithelial cells. Their effects on cellular gene expression in primary NK cells included pathways involved in cell cycle and stress responses. LMP1 and LMP2B-TR expression by ENKTL and CAEBV cell lines is associated with increased survival in the absence of relevant growth factors, but also with increased susceptibility to apoptosis. This cannot be fully explained by variation in the expression of proteins involved in the intrinsic apoptotic pathway. Finally, we describe PrimeFlow RNA, a new protocol for identification of the EBV-infected lymphocyte subset. Importantly, this technique means that we can begin to identify druggable targets on the EBV-infected cells directly from patient blood samples.

Wnt signalling is one the most important pathways involved in embryonic development. It controls a number of processes including cellular proliferation, stem cells maintenance, cell fate decisions and establishment of tissue polarity. It is also frequently deregulated in human cancers. Frizzled-7 is a member of the Frizzled family responsible for the signal transduction in Wnt signalling. Frizzled-7 has been reported to be upregulated in several types: of cancer. Furthermore, recent reports suggest that endocytosis of Frizzled may play a critical enhancing role in Wnt signal transduction, thus facilitating cancer development. We demonstrate here that the C-terminal PDZ binding motif (PDZ-BM) of Frizzled-7 contributes to signalling triggered by the receptor. We also explore the interaction between Frizzled-7 and syntenin-1, a PDZ domain containing protein that controls endocytic trafficking of various transmembrane proteins. We demonstrate that syntenin-1 regulates Frizzled-7 cell distribution and also modulates canonical Wnt signalling in epithelial breast cancer cells. Further, we report that the C-terminal PDZ-BM of Fz7 is indispensable for the receptor interaction with a number of PDZ proteins that control protein trafficking and cell polarity. Among these PDZ proteins are LNXl and LNX2, E3 ubiquitin ligases which are known to control trafficking of transmembrane proteins. In this study we characterize the interaction between Frizzled-7 and LNX2. We demonstrate that LNX2 influences ubiquitylation of Frizzled-7 and has the ability to moderate signal transduction within the canonical Wnt pathway in breast cancer cells.

In this thesis we validate the efficacy of a new adenoviral construct in prostate cancer cell lines in preparation for a gene and immunotherapy clinical trial in prostate cancer. By demonstrating the constructs ability to infect prostate cancer cells and cause them to die with the introduction of the prodrug, CB1954 as well as releasing a biologically active cytokine, GMCSF we secured GTAC approval to proceed to a phase I/II clinical trial in patients with local relapse after treatment with curative intent for prostate cancer. A tertiary endpoint in the trial is evidence of immune responses relating to treatment. To measure this we have modified an interferon gamma ELISpot assay to measure T cell mediated immune responses. We have then used this assay on 38 patients with suspected or diagnosed prostate cancer. In this study we have found the assay to be acceptable to patients and deliverable within the setting of the clinical trial. We found evidence of strong immune responses in patients with low and intermediate risk prostate cancer based on D’Amico’s classification with these responses declining in more advanced patients. We found that some interventions lead to an increase in immune responses and these observations warrant further exploration.

Nuclear receptors (NRs) are essential transcription factors that participate in a diverse number of cellular functions. Many have attractive chemotherapeutic potential due to their ability to govern pathways of cellular differentiation, growth arrest and programmed cell death. There are numerous examples of NR signaling becoming disrupted in human malignancies including the prostate. Mechanisms that give rise to impaired receptor signalling are investigated herein, including pre-receptor regulation of NR ligand and epigenetically mediated hypoacetylation. Furthermore, attempts either to overcome or circumvent their disruption are investigated. In parallel to these one member of the NR subfamily was chosen for further analysis. The vitamin D receptor and its target gene CDKN1A were examined for recruitment of VDR, nuclear corepressor (NCOR1) and ppolymerase II to response elements. Using the non-malignant prostate epithelial cell line RWPE-1, and PC-3 prostate cancer cell line to represent stages of prostate disease progression the spatio-temporal binding characteristics in response to ligand were measured. These findings identified aberrant nuclear corepressor recruitment to the transcription start site of CDKN1A in malignant disease. In addition examination of coregulation of a microRNA known to target CDKN1A revealed a mechanism of NR sensitivity that is also perturbed in prostate cancer. MiR106b also showed elevated tumour and serum expression in prostate cancer, suggesting a new biomarker of VDR responsiveness.