The organs in which lymphocytes function are low in oxygen (<5% oxygen) and even lower oxygen levels may be more prevalent in inflammatory tissues. In this thesis the effects of environmental oxygen on human CD4+ memory T lymphocyte function in vitro have been investigated. The level of oxygen in normal air (21%) which historically has been used for most in vitro experiments with immune cells was found result in suboptimal responses of this cell type, especially with regards to proliferation. At physiologically more appropriate oxygen levels of 8.5%, optimal proliferation was observed which coincided with an increase in Th2-associated markers. At 3% oxygen, the average level found in the inflamed joint in rheumatoid arthritis, a more sustained pro-inflammatory response was observed. In 1% oxygen, cytokine production was not maintained over time paralleling observations of CD4+ T lymphocyte behaviour in both the tumour and chronic inflammatory environment. This comparison was further supported by the increased expression of the activation marker CD69 and the depression of CD4+ T lymphocyte proliferation. A model of reperfusion injury also highlighted the effect that varying oxygen levels can have on CD4+ memory T lymphocytes. Proximal T cell receptor signalling was found to be altered after equilibration at different oxygen levels, and preliminary experiments investigating the potential role that redox plays in regulating CD4+ memory T lymphocyte functions were performed. It is concluded that environmental oxygen levels significantly influence CD4+ memory T lymphocyte responses, have implications for their function in inflammatory sites in vivo, and need to be considered when designing or interpreting in vitro experiments.

Infection and cardiovascular disease represent the two most important sources of mortality in ANCA associated vasculitis (AAV). Expansions of CD4+CD28null T-cells that are only present in cytomegalovirus (CMV) positive individuals have previously been associated with increased infection and mortality in AAV, and cardiovascular disease in other inflammatory diseases. The work described in this thesis examines the hypothesis that subclinical CMV reactivation in AAV drives the expansion of CD4+CD28null T-cells thereby leading to the observed adverse outcomes. To investigate this, a proof of concept clinical trial of 6 months valaciclovir treatment or no additional therapy was designed and implemented in CMV seropositive AAV patients in remission. Valaciclovir treatment successfully blocked CMV reactivation and in turn this led to a reduction in the proportion of CD4+CD28null T-cells in the treated patients together with favourable changes in other associated CMV induced changes on the immune system. CD4+CD28null T-cells in AAV were identified as Th1, proinflammatory cytotoxic T-cells, able to target endothelial cells and were independently associated with increased arterial stiffness, an established marker of cardiovascular risk. These findings implicate subclinical CMV reactivation as a potentially reversible cause of vascular pathology in inflammatory disease and open novel therapeutic opportunities.

The passage of leukocytes across blood vessel walls plays a key role in the immune response to infection in inflammatory conditions. ADAM10 is a ubiquitously expressed molecular scissor that proteolytically cleaves key cell surface proteins including vascular endothelial (VE)-cadherin and transmembrane chemokines. Their shedding by ADAM10 promotes leukocyte transmigration in cell line models, however the precise mechanism behind ADAM I O's involvement is unknown. ADAM10 associates with six different membrane organising tetraspanins (Tspan5/10/14/15/17/33) termed the TspanC8s. These tetraspanins regulate ADAM10 enzymatic maturation and trafficking to the cell surface and emerging evidence indicates that different TspanC8s can promote ADAM10 cleavage of specific substrates. It was hypothesised that ADAM10 promotes leukocyte transmigration by cleaving one of its endothelial substrates and one or more of the TspanC8s could facilitate this process. The aim of this thesis was to test this hypothesis using in vitro leukocyte adhesion assays with primary human leukocytes and human umbilical vein endothelial cells (HUVECs). siRNA knockdown or pharmacological inhibition of ADAM10 on HUVECs impaired the transmigration of lymphocytes, but not neutrophils or monocytes. ADAM10 knockdown/inhibition caused a reduction in VE-cadherin shedding and an increase in VE-cadherin surface expression. Partial knockdown of VE-cadherin, in the presence of ADAM10 knockdown/inhibition, reduced VE-cadherin levels to normal and restored basal lymphocyte transmigration. Systematic knockdown of TspanC8s in HUVECs revealed that the presence of either Tspan5 or Tspan17 was sufficient to maintain basal lymphocyte transmigration and reduced VE-cadherin surface levels. Tspan5 and Tspan17 are functionally uncharacterised, but they are the most highly related TspanC8s by sequence (78% amino acid identity) and may share a common role in lymphocyte transmigration by regulation of ADAM10 and VE-cadherin.

Atherosclerosis is an asymptomatic disease which is regarded as one of the most fatal diseases. However, the mechanism of the immune response is not well understood. There is accumulated evidence supporting the idea that inflammatory response initiates the disease. A new novel peptide has been discovered in our lab which down-regulates T cell recruitment during inflammation called PEPITEM (Peptide Inhibitor of Trans Endothelial Migration). We are interested in testing the action of PEPITEM on PBL isolated from atherosclerosis patients. We first demonstrated that PEPITEM did not affect the levels of adhesion of PBL from either diseased or healthy donors. Interestingly however, we did observe that PBL isolated from atherosclerosis patients adhere more readily than those isolated from healthy control subjects. Therefore, we studied the surface expression of certain adhesion molecules and chemokine receptors on the PBL of atherosclerosis patients. We found significantly higher surface expression of Beta-receptor family (Beta-1 and Beta-2) and PSGL-1 receptors in some PBL subsets in atherosclerosis patients. In addition, we looked at the effect of PEPITEM and adiponectin (AQ) treatment on the migration of PBL and we revealed for the first time based on our knowledge that there was no effect of treatment on PBL isolated from atherosclerosis patients. These observations will contribute to understanding the potential therapeutic applications of PEPITEM on atherosclerosis.

Invariant natural killer T (iNKT) cells are endowed with features of both innate and adaptive immunity. They are activated by the recognition of glycolipid agonists presented by CD1d which makes them excellent candidates for cellular therapies. In order to investigate the ability of iNKT cells to suppress experimental acute Graft versus host disease (GVHD), iNKT cells were first expanded in vitro, which is likely to be required prior to their use as a cellular therapeutic. Interestingly, the expanded cells showed increased frequencies of IL-10, IL-13 and IL-17 producing cells and were found to robustly suppress alloreactive T cell proliferation in vitro compared to freshly isolated cells. However, in a model of acute GVHD induced by alloantigen-reactive TCR-transgenic T cells neither freshly isolated nor expanded iNKT cells suppressed GVHD, although some survival benefit was seen following the activation of host iNKT cells. These data indicate that iNKT cells can be expanded ex vivo, that they can acquire different functional properties and that such cells robustly suppress alloreactive T cell proliferation in vitro. Therefore, further investigation into the suppressive behaviour of these cells is warranted despite a failure to suppress acute GVHD in the current study.

Much work has been done to elucidate the role of costimulatory molecules in CD4⁺ T cell responses. However the advent of major histocompatibility class II tetramers now allows endogenous polyclonal populations to be tracked from the naive pool through the primary response to the memory phase. I have utilised this method to dissect the role of OX40, ICOS and CD28 in the 2W1S:I-Aᵇ⁺ CD4⁺ T cell response. Additionally I have developed a model of local immunisation in photoconvertible Kaede transgenic mice, allowing the migration of antigen-specific memory cells to be tracked from a given site. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 ligation specifically expanded T effector (Teff) cells which resulted in a memory cell pool skewed towards T effector memory (Tem) cells, while T follicular helper (TFH) cell and germinal centre formation was abrogated. ICOS was required only for the formation of TFH cells in the primary response and the subsequent generation of both T central memory and Tem cells. However signals through OX40, ICOS and CD28 were dispensable for the persistence of memory cells once formed. Upon secondary challenge again OX40 and ICOS were specifically required for the proliferation of Teff and TFH cells respectively while CD28 had a more general role in the optimal expansion of all T cell subsets. Thus a complex set of temporally separated costimulatory molecule interactions are required for optimal CD4⁺ memory T cells responses in vivo. My results indicate that amongst this CD4⁺ memory T cell pool, a secondary lymphoid tissue resident population reside.

The development of αβ T-cells is a step-wise process guided by unique stromal microenvironments within the thymus, which results in the formation of T-cells with a highly diverse repertoire of T-cell receptors (TCRs). In the latter stages of development, T-cell tolerance is established through the selective deletion of cells expressing auto-reactive TCRs, in addition to the generation of immunosuppressive regulatory T-cells (T-Reg). Central tolerance induction is mediated by interaction with functionality heterogeneous medullary thymic epithelial cells (mTEC). The aim of this study was to search for a novel mTEC-expressed functional molecules involved in medullary homeostasis and central tolerance. Here we identify two novel mTEC populations; cells expressing osteoprotegerin (OPG), a negative regulator of Rank-mediated mTEC maturation, and inducible nitric oxide synthase (iNOS), an immune-active enzyme catalysing the production of nitric oxide. Importantly, we found OPG to restrict the size of the mTEC compartment, which in contrast to previous findings had no impact on T-Reg production, and instead limited the influx of peripheral lymphocytes to the thymus. In contrast, iNOS appears to play only a minor role in the maturation of single positive thymocytes in the medulla. These findings highlight the importance of functionally distinct mTEC compartments in maintaining medullary homeostasis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that although primarily affecting the joints is recognised as a systemic disease with a variety of co-morbidities. One such co-morbidity is cardiovascular disease (CVD) and patients with RA are at a two-fold increased risk of developing CVD compared to the general population. Indeed, CVD, principally as a result of atherosclerosis, is recognised as the leading cause of death in RA patients. In this study the vascular adventitia and serum samples from patients with co-existing RA and CVD were investigated to identify factors that could explain the increased CVD morbidity observed in RA patients. This involved culture-independent identification of bacterial signatures and histological evaluation for pro-inflammatory molecules and potential RA-associated autoantigens in the aortic adventitia. Additionally, serum samples from healthy controls, patients with RA or CVD alone, and patients with co-existing RA and CVD, were screened for a broad range of cytokines, chemokines and growth factors. These data provided the basis for further studies into the effect of cytokines on foam cell formation. Histological evaluation of the aortic adventitia revealed that pronounced inflammatory infiltrates were detectable in all patients with CVD, regardless of rheumatic disease. To assess whether or not the inflammatory composition of the aortic adventitia differed between patients with or without RA, sections were examined using immunohistochemistry for a markers including TNFα, CD21 and heat shock proteins (HSP) 47 and 60, both of which have been implicated in the pathogenesis of RA and CVD. TNFα and HSP47 were confirmed in the aortic adventitia; however, neither CD21 nor HSP60 were detected. The expression of total TNFα was significantly elevated in RA+CVD compared to non-RA CVD patients. These data demonstrate that the aortic adventitia is a site of considerable immunological function and suggest a possible role for TNFα overexpression in the pathogenesis of CVD in RA. Bacterial infection has been implicated as a causative agent for both RA and CVD. The present study used culture-independent methods to identify bacteria in the aortic adventitia of RA+CVD and non-RA CVD patients. The presence of bacterial DNA was confirmed in three of 11 RA+CVD and four of 11 non-RA CVD patients. The bacterial flora of RA+CVD patients was significantly less heterogeneous compared to non-RA CVD patients. Methylobacterium oryzae was detected in every RA+CVD patient positive for the presence of bacterial DNA. Subsequent in vitro characterisation demonstrated that M.oryzae stimulates mild Toll-like receptor 2 (TLR2), but not TLR4, signalling and, upon challenge of primary human macrophages, produces a robust pro-inflammatory response. Importantly, these findings implicate M. oryzae infection as playing a potential role in the pathogenesis of CVD in RA patients. Previous studies have argued that the persistently high levels of systemic inflammation exhibited by RA patients are critical to elevated CVD risk. In the current study it was demonstrated that RA patients exhibit an altered systemic immune profile compared to non-RA CVD patients and healthy controls. The pro-inflammatory phenotype in co-morbind CVD and RA is suggestive of an environment that may promote atherosclerosis. Furthermore, RA+CVD patients were shown to possess an altered systemic immune phenotype compared to both RA and CVD patients. Notably, IL-1β, IL-2, IL-5, IL-8, IL-13, IL-17, Monokine induced by gamma interferon (MIG), Granulocyte colony-stimulating factor (G-CSF) and Granulocyte macrophage colony stimulating factor (GM-CSF) expression was greater in the serum of RA+CVD patients compared to the additive values of the RA and CVD patients. The formation of lipid-rich foam cells is a major feature of atherosclerosis pathogenesis and is partially dependent on the inflammatory environment. The present study demonstrated that GM-CSF stimulation significantly decreased the rate at which human primary macrophages could endocytose oxLDL and form foam cells. The effect of dextran sulphate, a known competitive inhibitor of scavenger receptors, on the ability of GM-CSF stimulated macrophages to differentiate into foam cells revealed that GM-CSF decreases the concentration of dextran sulphate required to successfully inhibit scavenger receptor-mediated ox-LDL uptake; suggesting that GM-CSF-stimulated macrophages express less scavenger receptors on the cell surface. However, subsequent investigation demonstrated that GM-CSF stimulation did not decrease expression of the currently recognised scavenger receptors that are capable of oxLDL recognition (SR-A1, SR-B1, CD36 and MARCO). These data imply that the overexpression of GM-CSF observed in RA and RA+CVD patients may provide some atheroprotective benefit. This has clinical implications for future anti-RA drugs which target GM-CSF function. In summary, the data presented in this thesis demonstrate that RA+CVD patients exhibit immunological and pathological alterations both in the aortic adventitia and systemically. Further research will, over time, provide insight into the mechanisms underlying increased CVD burden in RA.

T-cells are key orchestrators of airways inflammation, but the relative roles of different human T-cell subsets remain unclear. The aim of my PhD was to carry out a detailed investigation of T cell phenotypes in asthma in relation to severity and virus-induced exacerbations, with particular focus on interleukin-17 and TH 17 cells, and the recently described mucosal associated invariant T (MAlT) cells, to improve characterisation of severe asthma versus milder forms of asthma. A role for interleukin-17 secreting TH17 cells in asthma has been suggested by several groups. I used clinical and physiologic phenotyping to compare T-cell subsets in health and a spectrum of different asthma severities. Samples obtained via sputum induction, phlebotomy, and bronchoscopy were phenotyped using 9-colour flow-cytometry/sorting, RT-qPCR and multiplex ELlSA. The results of my thesis confirm the pre-eminence of TH2 cells in asthma and provide further evidence of a deficiency of bronchoalveolar Treg in severe asthma, as well as new evidence of a role for CD8+ Tc2 cells in eosinophilic disease. Conversely, the data do not indicate a significant role for TH17 or yo-17 cells in asthma. Mucosal immunity is intrinsically linked to the associated commensal or pathogenic microbes. In an exploratory study of these interactions I employed deep-sequencing to characterise the whole microbial and viral metagenome of the airways in asthma and health. MAlT cells are novel innate-like T-cells which express an invariant TCRa chain and recognise the highly-conserved restriction molecule MR1. I observed a selective deficiency of MAlT cells in asthma, which was not related to age, but exacerbated by systemic corticosteroids and subject to seasonal variation, indicating their possible regulation by vitamin D. I established MAlT cell-lines and observed heterogeneity of cytokine expression profiles. These findings open exciting new avenues for research in this emerging area of T cell biology.

Human schistosomiasis is one of the helminthic neglected tropical diseases. It leads to serious health problems and imposes a huge burden on communities. All interactions between the parasite and the human body occur at a molecular and cellular level. Therefore, the study of the molecular aspects of infection and the immune response is a very active area of research. It has been known for decades that there is a direct relationship between protection against infection, or reinfection after treatment, and parasite-specific Immunoglobulin E (IgE) antibody in infected individuals. This study aims to investigate the allergenicity of S. mansoni antigens using RS-ATL8 reporter cell line assay. Towards this goal, I first identified the best reporter cell line for allergenicity assessment. This was done by the characterisation of the transgenic human FcԑRIα chain’s gene copy number and by comparing the levels of human FcԑRI receptor surface expression. The second goal was the optimisation of RS-ATL-8 reporter cell line. This was achieved by the optimisation of fundamental conditions such as the cell density, sensitising agent’s optimum dilution and the stimulant optimum concentration. Once a robust standard operating procedure (SOP) had been established, I investigated the allergenicity of the four expressed S. mansoni antigens using the optimised reporter cell line RS-ATL8.