CD8+ T-cells are essential for the immune control of pathogens and the natural eradication of cancer. CD8+ T-cells also play a major role in the pathogenesis of autoimmunity and alloreactivity. CD8+ T-cells recognize short peptide fragments (8-13 amino acids) presented at the target cell surface bound to Major Histocompatability Class I (MHCI) molecules. Tcell antigen recognition is unique in nature because it involves the binding of a single ligand (peptide–MHC [pMHC]) by two receptors (TCR and CD8). The CD8 glycoprotein, which serves as the coreceptor on MHCI-restricted T-cells, acts to enhance the antigen sensitivity of T-cells by binding to a largely invariant region of MHCI at a site distinct from the TCR docking platform. CD8 has been shown to have multiple roles including enhancing effects on early T-cell activation events and also in controlling the level of T-cell cross-reactivity. The pMHCI/CD8 interaction is classified as having a very weak binding affinity and very fast kinetics. I discovered that this low solution binding affinity is essential in maintaining homeostasis as dramatically increasing the strength of this interaction resulted in total loss of T-cell specificity and activation independent of TCR engagement. This led me to examine the possibility that anti-CD8 antibodies could also bypass the normal requirements for T-cell activation. I identified one specific clonotype of antibody capable of this phenomenon but simultaneously discovered multiple effector phenotypes of other anti-CD8 antibodies. These included both enhancing and inhibitory effects on pMHCI tetramer binding and CD8+ T-cell activation. Subsequently, I explored the possibility of using these inhibitory anti-CD8 antibodies to block T-cell function in systems which are highly dependent on CD8 such as autoreactive CD8+ T-cells. I demonstrated that targeting CD8 can be used as a strategy to block autoreactive CD8+ T-cell activation in the absence of any effect on pathogen specific immunity. This highlights a novel therapeutic strategy that warrants further investigation. Finally, I demonstrated that CD8 can alter the functional avidity of a CD8+ T-cell for its agonists and act to re-arrange the relative potencies of each of its potential agonists, a novel “focussing mechanism” for CD8 in T cell activation. These results provide new insight to the biological role of CD8 in T-cells and even predict a novel mechanism for CD8 in controlling T-cell function. My results also highlight the potential of targeting CD8 for immunotherapeutic design in autoimmune disorders.

Using an IBV reverse genetics system a series of recombinant viruses were generated to investigate the potential for utilizing IBV as a vaccine vector. Through the replacement of non-essential regions of the IBV genome, with eGFP or hRluc, factors influencing the stability of recombinant viruses expressing heterologous genes were determined. Expression of heterologous proteins was possible from a variety of virus constructs. The stability of recombinant viruses varied depending on the genome location of the heterologous gene, with replacement of Gene 5 proving to be most stable following passage in cell culture. Stability was strongly influenced by the MOI at which viruses were passaged, with low MOIs resulting in increased stability. The replacement of Gene 5 with a heterologous virus gene may be a suitable target for development of a bivalent vaccine capable of protecting against IBV and a second avian viral disease. Analysis of recombinant IBV mRNA expression profiles led to an investigation into an uncharacterized RNA species, and its link to the IBV intergenic region. A novel subgenomic mRNA was identified associated with the intergenic region that was shown to be transcribed via a non-canonical transcription regulatory sequence. In contradiction to the current model of coronavirus transcription this mRNA has a transcription regulatory sequence derived mainly from the leader, and not the body, transcription regulatory sequence. The non-canonical sequence was shown to be responsible for reduced transcription levels of the intergenic region mRNA. This project proposes the presence of an additional IBV subgenomic mRNA, transcribed via a non-canonical mechanism, and encoding a novel 5th accessory protein of IBV and closely related gammacoronaviruses.

This Thesis presents an analysis of the role of interleukin (IL)-1α (IL-1α) as a potential future surrogate biomarker for AAA. It is the only research work to date to have looked into the role of IL-1α as a biomarker in AAA disease, correlating titres with different anatomical, morphological and patient-related factors. It is the first piece, in over 20 years of published literature, to have performed a robust methodology study on the measurement of IL-1a in serum samples using different techniques. A comparison study of commercially available immunoassays in the context of IL-1α has never been undertaken before, and we are the first to undertake one. Additionally the work on the natural history of AAA is one of the largest single-centre cohort studies to analyse AAA growth in surveillance. The work covers three main areas: identifying why current strategies for monitoring AAA are ineffective, analysis of different serum processing methodologies and commercially available immunoassays used to measure IL-1α, and linking IL-1α to different anatomical, morphological and patient-related AAA factors.

The mammalian target of rapamycin (mTOR) complex is a key regulator of protein synthesis, with resistance exercise and protein ingestion both shown to increase mTOR activity in human skeletal muscle. It has recently been proposed that mTOR activity is regulated via its intracellular localization and protein complex interaction. However, no research to date has examined this process in human skeletal muscle. Accordingly, the aims of this thesis were to (1) develop immunofluorescent-based methodologies to study mTOR in human skeletal muscle, and (2) apply this approach to the study of mTOR in acute and chronic resistance exercise scenarios. This thesis describes a novel approach to study mTOR regulation in human skeletal muscle in vivo. Taking advantage of this approach, novel data was presented on mTOR distribution, translocation and association with regulators in response to resistance exercise in human skeletal muscle in vivo. It is hoped that this approach will provide insight into the cellular regulation of skeletal muscle protein synthesis and by extension the control of skeletal muscle mass in humans during scenarios of health and chronic disease.

Innate lymphoid cells (ILCs) are an emerging family of cells which have been well-characterised within the gut and peripheral tissues. Despite being implicated in shaping adaptive immune responses, relatively little is known about their function within lymph nodes (LNs), important sites for the generation of this type of response. The aim of this investigation was to characterise ILC populations within a range of different LNs, both at steady state and using a draining LN model to understand their role in an immune response. Mice in which a subset of ILC, or key functional molecules, are deficient will be used to better understand their function within LNs, with a focus on adaptive immune responses. My results reveal that ILCs are present in all LNs analysed, however, differences in the ILC composition of mucosal tissue and peripheral tissue-draining LNs indicate site-specific requirements for these cells. Differing dependencies on CCR7 for ILC entry into LNs were observed, consistent with migration of these cells into these secondary lymphoid tissues. Within LNs, group 3 ILCs were found to express major-histocompatibility complex class-II and specifically locate to sites where adaptive immune responses are initiated and maintained. Notably, ILCs accumulated in draining LNs following immunisation and whilst their roles here remain unclear they are unlikely to be involved in the priming of naïve CD4+ T cells. In summary, I show that subsets of ILC3 are enriched in LNs which drain mucosal sites and can be found to locate to crucial sites within LNs following their entry by a CCR7-dependent mechanism. These data support a role for ILC3 in adaptive immune responses.

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic beta cells. Preservation of beta cell function reduces the risk of the complications of T1D. Regular exercise preserves beta cell function rat models of T1D and patients with other forms of diabetes. We wished to examine whether exercise could preserve beta cell function in T1D and whether this was influenced by adipokine receptor expression and function. We undertook a qualitative study to explore barriers to exercise in patients newly diagnosed with T1D. This showed that patients lacked confidence managing diabetes for exercise, and were poorly supported by healthcare professionals. Using these results, we then undertook a pilot clinical trial aiming to determine recruitment and retention, adherence to exercise, and exploring whether exercise preserves beta cell function in patients newly diagnosed with T1D. We show successful recruitment to an unsupervised exercise intervention study. We did not detect a beneficial effect of exercise on beta cell function in this pilot trial, but identified several areas that will need to be addressed in designing a larger scale study. Finally, we demonstrate improved adiponectin receptor expression and adiponectin mediated suppression of T cell endothelial migration in the months after diagnosis with T1D.

In rheumatoid arthritis (RA), chronic inflammation and destruction of the joint is driven by local production of cytokines. My aims were to characterise cytokine mRNA expression in multiple synovial fluid cell populations in early and established RA and to study these, in addition to whole synovial tissue, in early synovitis patients in relation to disease outcome. I established a novel method to determine cytokine mRNA expression in synovial fluid CD4 T cells, CD8 T cells, B cells, macrophages and neutrophils directly ex vivo. I made several novel observations, for example RA synovial fluid B cells expressed high levels of RANKL. As RANKL drives bone resorption, this suggests a potential role for B cells in bone erosion in RA. RANKL protein was expressed by B cells in synovial tissue, and mainly by memory B cells in synovial fluid. Furthermore, synovial RANKL levels were reduced after treatment with the B cell depleting therapy, rituximab. Overall, both in whole synovial tissue and in sorted cells, the cytokine mRNA expression profile was very similar in early synovitis patients who subsequently developed RA or had resolving synovitis, and in patients with early or established RA. In comparison, cytokines and chemokines were upregulated in early and established arthritis patients compared to uninflamed controls. The finding that cytokine mRNA expression is largely similar in early synovitis patients who develop RA or have resolving disease, and in the early and established phases of RA, suggests that cytokine expression is reflective of general synovial inflammation, rather than being specific for early synovitis outcome or stage of RA.

Parallel increases in many inflammatory diseases including atopy over the last 40 years suggest that common environmental changes may be promoting inflammatory immune responses. Modern diets have become increasingly rich in n-6 polyunsaturated fatty acids (PUFAs) and relatively deficient in n-3 PUFAs. These dietary changes are believed to promote a pro-sensitisation, pro-allergic and pro-inflammatory environment. Exposure to such an environment during pregnancy and in the very early life period is considered to influence subsequent patterns of the immature and developing neonatal immune system, and this may contribute to the increase in allergic disease in early life. As allergic diseases often first manifest in infancy, prevention strategies need to be targeted early, even in utero. Epidemiologic and experimental data provide a plausible link between dietary changes and increased incidence of childhood atopic disease. Although there have been studies examining the potential benefits of giving n-3 PUFA-rich fish oil supplements during pregnancy, there are no studies examining the effects of increased consumption of oily fish in pregnancy on neonatal immune responses and subsequent clinical outcomes. The Salmon in Pregnancy Study (SIPS) is the first randomised controlled trial of oily fish intervention during pregnancy. The hypotheses being investigated in SIPS is that increased intake of salmon, a source of long chain (LC) n-3 PUFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), in pregnancy will a) increase maternal LC n-3 PUFA intake, b) increase maternal and infant blood LC n-3 PUFA status, c) modulate fetal/neonatal immune responses and d) lower the risk of infant atopy determined at 6 months of age. The primary outcome measures of SIPS were the clinical signs of atopy in the offspring. Pregnant women (n=123) at high risk of having atopic offspring, and with low habitual intake of oily fish (≤ 2/month) were randomised at 20 weeks of pregnancy to either consuming 2 portions/week of farmed salmon (n=62) or continuing their habitual diet (n=61) until the end of pregnancy. The woman attended a clinic at 20 (n=123), 34 (n=110) and 38 (n=91) weeks of gestation at which fasting blood was collected and a food frequency questionnaire (FFQ) was administered (at 20 and 34 weeks). At delivery umbilical cord blood was collected (n=101) for fatty acid and immunological analysis. Infants attended a clinic at 6 months of age (n=86) for assessment of allergic sensitisation by skin prick testing (SPT) using various allergen extracts and of atopic dermatitis (SCORAD index). Maternal and cord plasma and cord blood mononuclear cell (CBMC) fatty acid compositions were determined by gas chromatography. Neonatal (cord) immune cell subsets were identified by flow cytometry. Ex-vivo cytokine production by CBMC in response to stimulants (allergen, mitogen, and toll-like receptor (TLR) ligands) was determined by cytometric bead array and flow cytometry. Ex-vivo prostaglandin E2 production by CBMC was determined by enzyme-linked immunosorbent assay. Immunoglobin E concentration was measured in cord blood plasma and in 6 month infant blood plasma. Eating oily fish twice a week during pregnancy resulted in a higher maternal intake of LC n-3 PUFAs (both EPA and DHA) and in higher maternal and cord blood plasma status of LC n-3 PUFAs (both EPA and DHA). LC n-3 PUFA content of CBMC was not significantly affected. CBMC production of interleukins-2, -4, -5, and -10 and tumour necrosis factor-α was lower in the salmon group. There was no effect of salmon on the atopic outcomes assessed at 6 months.