A polyclonal increase in free light chains (FLCs) has been observed in a number of chronic inflammatory and autoimmune conditions and may be considered a biomarker of adaptive immune activation. The aim of this study was to examine whether FLCs could be a useful clinical biomarker in alpha-1 antitrypsin deficiency (A1ATD) and chronic obstructive pulmonary disease (COPD). Combined (\(\kappa\) & \(\lambda\)) FLCs (cFLCs) were measured in patients with AlATD and usual COPD in the stable state and assessed for association with clinical phenotype, disease severity, bacterial colonisation and mortality. The relationship of FLCs to total immunoglobulin levels was also examined in the COPD cohort. In addition, FLCs were measured in a small cohort of patients with bronchiectasis to further examine the relationship to bacterial colonisation. Circulating cFLCs were static in the stable state in both A1ATD and usual COPD. Levels were significantly higher in patients with chronic bronchitis and airway bacterial colonisation in AlATD. After adjusting for renal function and age the relationship between cFLCs and lung function was weak, however cFLC levels greater than normal significantly associated with mortality in both COPD cohorts. In conclusion, cFLCs may be a promising biomarker for risk stratification in AlATD and COPD.

T-cells engineered to target tumour antigens through surface-expressed chimeric antigen receptors (CARs) are highly effective in treating some leukaemias. The challenge is to extend this success to solid tumours. Tumour endothelial marker 8 (TEM8) is a conserved transmembrane protein overexpressed on the vasculature of many solid tumours but low or undetectable on healthy tissues, making it a potential CAR T -cell target. This thesis explores the safety and therapeutic efficacy of this approach by generating five human TEM8-specific CARs, expressing them in T-lymphocytes, and characterising their functional responses to TEM8 in vitro. Four of the five CARs showed unexpected reactivity to control cells, and in mouse studies some of these proved toxic while most were selectively lost from the circulation, an effect that was TEM8-dependent. Only one CAR selectively responded to target cells overexpressing human TEM8 in vitro but was unable to recognise mouse TEM8, so further in vivo studies were not possible. These results highlight the sensitivity and potency of CAR -engineered T -cells and demonstrate the need for additional safety measures if targeting TEM8. The thesis also demonstrates that another TEM, CLEC14A, is overexpressed in some inflammatory liver diseases, and identifies a suitable mouse model for exploring the therapeutic potential ofCLEC14A-specific CAR-expressing regulatory T-cells.

This project sought to identify paediatric tumour-specific phosphopeptide antigens on Post-transplantation lymphoproliferative disease samples (PTLD) and hepatic tumour samples. Tumour-specific T-cells are difficult to maintain in long-term culture, therefore the secondary aim of this project was exploring modalities for capturing T-cell receptor (TCR), important in recognising tumour-specific antigens. This may result in a tumour-specific product. Potential modalities included T-cell hybridomas and Human Induced Pluripotent Stem Cell (hIPSc) technology to immortalise tumour specific T-cells. As a result, we have developed a technology for expanding these and differentiating them into a T-cell of interest with potential for future clinical application in paediatric tumours, using OP9 DL1 mouse feeder cell system to support differentiation of hIPSc towards haemopoietic lineage, demonstrating functionality of the end stage ‘T-cell product’. In summary we have identified a number of novel phosphopeptide antigens in vitro as well as on patient tissues. This information has been used to identify potential T-cell targets and by formation of hIPSc we have established a method for expanding specific T-cell’s in vitro. Using OP9DL1 cells we have also identified a method for differentiating these cells into lymphocyte-like cells with T-cell functionality therefore representing a possible methodology for expanding tumour-specific T-cells for clinical application.

Systemic Salmonella Typhimurium infection induces an atypical antibody response in which the extrafollicular and germinal centre reactions develop at discordant rates. Dysregulated or atypical antibody responses represent a potential source of autoantibodies. In this thesis, we show that systemic infection induces pathogen-specific and self-reactive IgM and lgG antibodies early after infection, but with differing kinetics, suggesting that they are distinct responses. To characterize these two responses the antibody response was assessed in a range of genetically altered mice. We identified a role for TLR4 in this process, but this is dependent upon the genetic background of the murine host. There is strong reactivity of self-reactive antibodies from wild-type mice with stomach, muscle and liver. These antibodies can persist long after infection is cleared from the liver and spleen. Our investigation into the induction of these antibodies suggest that IL-6 from stromal cells may play a role in this response, but ICOSL, TNF, IFN-γ and Th1 responses are dispensable. Critically, CD80/CD86 are essential for the induction of the anti-self, but not the pathogen-specific response. This data suggests that pathogenspecific and self-reactive antibody responses are independently regulated and thus it may be possible to modulate anti-self-responses without compromising anti-pathogen immunity.

Tissue microenvironments within chronic inflammatory disease sites, such as the synovial compartment within rheumatoid arthritis (RA) patients contains a plethora of factors to drive immune responses. However, one specific characteristic that is commonly found at these sites is the presence of tissue hypoxia. Therefore, both resident and infiltrating immune cells need to adapt to these microenvironments in order to survive and function to promote chronic inflammation. Adaptation to hypoxia requires a level of metabolic reprogramming for this purpose. Therefore, this thesis aimed to examine what the metabolic consequences were for human monocytes that were adapting into hypoxic sites and to interrogate what role these metabolic pathways had in driving specific functions in hypoxic conditions. Metabolomic analyses reveal that hypoxia induces metabolic alterations in human monocytes, including the decrease in abundance of carnitine metabolites, important for subsequent fatty acid oxidation (FAO), and increases in glycolytic metabolites. Furthermore, hypoxia exacerbated the release of pro- inflammatory mediators in LPS activated monocytes, such as CCL20 and IL-1β. Manipulation of carnitine metabolites identify a role for FAO in the production of CCL20, and in the regulation of IL-1β release. To mimic the RA synovial environment more thoroughly in vitro, human monocytes were cultured in cell culture medium containing RA synovial fluid (RA-SF) under hypoxic conditions. Further metabolomics analysis revealed that monocytes accumulate a number of metabolites in comparison to untreated and LPS activated cells, suggesting that monocytes may enter a stasis-like phase when challenged with RA-SF. This was reflected by low level release of pro-inflammatory mediators under these conditions. Nevertheless, media supplementation with carnitine increased CCL20 production under RA-SF treatment, highlighting FAO may have a role in CCL20 release in several inflammatory contexts. This body of work shows that distinct metabolic pathways regulated by the extracellular environment may act in conjunction for the production of pro- inflammatory mediators in chronic inflammatory disease. This thesis highlights the influencing nature of tissue microenvironments on the functional capacity of myeloid cells by harnessing its metabolic machinery.

No description available.

The adaptive immune system must be able to respond to virtually any pathogen that the body encounters. T cell immunity is able to do so by developing a diverse repertoire of T cell receptors and maintaining large numbers of T cells. These two quantitative properties are fundamental for the ability of T cell-mediated immunity to clear infections and generate memory cells for future protection. The aims of this thesis are to quantify the sizes of T cell populations, to develop tools to measure the diversity of T cell repertoires, and to describe how T cell populations develop in neonatal mice. We studied the development of T cell populations in neonatal mice by measuring cell counts and Ki67 expression in thymocyte and peripheral T cell subsets from mice soon after birth to late adulthood. The presumed lymphopenic environment of the neonatal mouse is thought to cause T cells to undergo lymphopenia-induced proliferation, and we wanted to quantify the balance between thymic output and peripheral expansion in the naive T cell compartment during development with mathematical modeling. We also used modeling to find the most parsimonious description of differentiation within the thymus that explains the dynamically growing thymus. We then sought to quantify the sizes of the peripheral T cell compartments in the adult mouse. Understanding the characteristics of healthy T cell immunity requires knowing the precise numbers of the different T cell subsets found in the body. We performed thoracic duct cannulations in adult mice to collect recirculating T cells and reduce cell numbers in the lymph nodes and spleens; by counting the number of collected T cells and its effect on cell numbers on the secondary lymphoid organs, we sought to back-calculate the total number of T cells in the mouse. Finally, we developed tools that provide high-throughput and cost-effective methods for identifying paired TCR sequences. By using computational techniques, we were able to adapt standard sequencing protocols to identify many paired TCR sequences without resorting to large and expensive single-cell sequencing techniques. By leveraging experimental design with mathematical methods, we were able to quantify and characterize many properties of effective T cell immunity.

Discoveries of antibiotic resistant pathogens and failure in some conventional modern cancer treatments have led to the re-evaluation of ancient therapeutic remedies such as honey. In recent years, Malaysian Tualang honey (MTH) had been proven scientifically to possess many beneficial properties such as antimicrobial, antioxidant, anticancer, anti-inflammatory and wound healing potential. However, little scientific evidence about its immunomodulatory property has been published, therefore this study aimed to investigate the immunomodulatory effect of MTH. Initially, human monocytic cell lines (THP-1 and U-937) were first cultured and tested against MTH as part of methodology optimization before proceeding to peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. PBMCs were used in this study because these cells can mimic the in vivo system of immune responses more closely. The cytotoxic effect of MTH on THP-1, U-937 and PBMCs was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolim bromide (MTT) assay. It was found that the combination of 0.125 % to 2 % MTH and incubation durations 16, 24 and 48 hours yielded at least 90 % cell viability in PBMCs (except 2 % MTH for 48 hours of incubation). Hence, 0.125 % MTH was selected to treat the PBMCs in subsequent experiments. Using microarray approach, the gene expression profile in MTH-treated PBMCs were studied and it was found that 361 genes were significantly regulated by at least two fold changes (p < 0.05). Among these genes, the expression of immunorelated genes such as IFNG, IL10, IL20, IL24, CXCL1, CXCL3, CXCL9, IL2 and IL4 were validated using reverse transcription quantitative real time polymerase chain reaction (RT-qPCR). Functionally, these genes play crucial role in wound healing by facilitating the recruitment of leukocytes to sites of infection, supporting the wound healing activity in honey. Using flow cytometry, the immunomodulatory effect of MTH in activating PBMCs subpopulations was also determined using cell surface marker cluster of differentiation (CD) 69. It was found that MTH did not possessed any immunosuppressive effect in regulating cell activation in helper T cells (CD3+ CD4+), cytotoxic T cells (CD3+ CD4-) and B cells (CD3- CD19+). The production of interferon gamma (IFN-γ) and interleukin (IL)-10 from mitogen-stimulated and non-stimulated PBMCs after MTH treatment were also quantified using enzyme-linked immunosorbent assay (ELISA). IL-10 (immunoregulatory cytokine) and IFN-γ (pro-inflammatory cytokine) production were found elevated significantly (p < 0.05). These results suggested that MTH involved in early immunoregulation and late pro-inflammatory responses, supporting the antimicrobial activity of honey. This is the first study ever conducted to investigate the gene expression profile in PBMCs treated with MTH. Overall, present findings showed that MTH possessed immunomodulatory effect by regulating the expression of immune related genes, leading to significant increase in the production of type 1 cytokine (IFN-γ) and type 2 cytokine (IL-10) in PBMCs. This suggested that MTH possessed immunomodulatory effect that can potentially contribute to the antimicrobial and wound healing activities in honey. These findings can further justify the application of MTH as topical dressing especially in wound management potentially by eliminating wound infection as well as promoting wound healing process.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause infections in patients with compromised immunity including patients with neutropenia, HIV infection, burns, cancer, organ transplant surgery or in intensive care as well as Cystic Fibrosis (CF) patients. CF patients may have a T-helper (Th)-2 and Th-17-biased immune response which suggest that, either the absence of Th-1 and/or enhanced Th-17 responses may contribute to chronic PA infection with a deterioration of lung function in CF. A previous study in our laboratory showed that interferon (IFN)-γ production by peripheral blood mononuclear cells (PBMCs) from CF patients positively correlates with lung function, particularly in patients chronically infected with PA. In contrast, IL-17A levels correlated negatively with lung function especially in patients chronically infected with PA. These results agreed with the notion that IFN-γ and IL-17A play protective and detrimental roles, respectively, in CF disease. To explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control PA growth, resist the cytotoxicity induced by PA or promote inflammation was investigated previously in our laboratory. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF did not alter bacterial growth or macrophage survival upon PA infection, but made the macrophages more pro-inflammatory. As neutrophils have been reported to be the key effector cells for the clearance of PA, there was therefore the need to include neutrophils in the macrophage cultures so as to determine how inflammatory conditions affect PA clearance. Although neutrophils are essential for protection against extracellular bacteria, neutrophil-dominated inflammation can compromise organ function by causing tissue damage. Optimal anti-bacterial immunity should harness the microbicidal activity of neutrophils while minimising their potential for causing injury. However, neutrophils do not act in isolation, they respond to cues provided by other cells such as macrophages, which influence their activation and life span. In vivo models have dominated the study of anti-bacterial immunity and there is a need of human models for the dissection of macrophage/neutrophil communication during bacterial infection. This work describes the development of a human macrophage/neutrophil infection assay and its use to model Th-1 (IFN-γ) - and Th-17 (IL-17A)-driven microbicidal activity and inflammatory potential upon infection. Results show that IFN-γ and IL-17A have opposite effects on the killing ability of macrophages and neutrophils co-cultures; bacterial killing was reduced by IFN-γ and promoted by IL-17A. In addition, macrophages and neutrophils specifically collaborated for the production of IL-1β and IL-1α and IFN-γ-treated co-cultures generated significantly less IL-1β and IL-1α compared to those treated with IL-17A. This effect was not observed with other cytokines such as TNF-α, IL-6, MIP-1α and MCP-1. Also, there was increased production of IL-1β upon addition of neutrophils to infected macrophage cultures and this depended on caspase-1 activity. Thus phagocyte co-cultures provide a suitable model of human anti-microbial immunity and unveil an unappreciated collaboration between macrophages and neutrophils in the promotion of IL-1-mediated inflammation. The reduction of inflammation in the presence of IFN-γ highlights the potential of IFN-γ as a therapeutic strategy in extracellular bacterial infections such as infections with PA.

The efficient functioning of the immune system is dependent on the coordinated movement and positioning of immune cells. These cells patrol the body and facilitate the clearance of pathogens, whilst maintaining self-tolerance and inducing adaptive immunity. The coordinated migration of cells into and within tissues is mediated by chemokines, a family of small chemotactic cytokines that are potent inducers of cellular movement. Chemokines and their cognate receptors have been shown to play key roles in health, and in a broad spectrum of diseases. After their secretion, chemokines can be controlled by proteases and interactions with atypical chemokine receptors that structurally resemble conventional chemokine receptors but cannot couple to the signaling pathways that they use. Instead, they are thought to degrade, transport or buffer extracellular chemokines to regulate their access to cells bearing conventional chemokine receptors. However, their functions in vivo remain to be fully defined. CCRL1, a member of the atypical chemokine receptor family, binds to CCL19, CCL21 and CCL25 and is proposed to be a scavenger of these chemokines. Post-translational regulation of these chemokines is important because their interactions with cognate, conventional receptors CCR7 and CCR9, are critical for the development and functioning of the immune system. The work in this thesis primarily explores the importance of CCRL1 in regulating CCR7 ligands, but also considers the impact of protease-mediated ‘clipping’ on the function of CCL21 and other extended chemokines. Whilst in vitro evidence, and a growing body of in vivo evidence, supports the idea that CCRL1 serves an important role in the chemokine control, the true biological function of CCRL1 remains unclear. Therefore, the principal aim of this thesis was to further our understanding of the biology of CCRL1, mainly through the characterisation of CCRL1-deficient mice. Firstly, the effect of CCRL1 deletion on dendritic cell (DC) migration from the skin was investigated. Four distinct subsets of migratory DCs were examined in skin-draining lymph nodes at steady state and after induction of cutaneous inflammation. Under inflammatory conditions, skin-derived DCs were identified by FITC painiting or by photoconverting the skin of Kaede transgenic mice. CCRL1 deficiency was found to result in a specific reduction in the abundance of Langerin+ skin-derived DCs in inguinal lymph nodes at rest, and although the initial inflammation-driven arrival of DCs at skin-draining lymph nodes showed no requirement for CCRL1, DCs that took longer to reach these organs were substantially reduced in CCRL1-deficient mice. All DC subsets were affected, but overall it was epidermal Langerhans cells that showed the greatest requirement for CCRL1. Defective DC arrival at lymph nodes was accompanied by DC retention in resting and inflamed skin, and these cells struggled to leave CCRL1 deficient skin in ex vivo ‘crawl out’ assays. Further experiments demonstrated that, as expected, DC arrival at lymph nodes draining inflamed skin was heavily dependent on CCR7 in the models being used, and that increased levels of bioavailable CCL19 and CCL21 accompanied CCRL1 deficiency in inflamed skin. This suggested CCRL1 regulates chemokine to prevent DCs from becoming disorientated in the skin and failing to efficiently egress this tissue. I hypothesised that dysregulation of CCL19 rather than CCL21 may be particularly significant because of its greater diffusivity, and strikingly, inflammation-driven DC migration defects observed in CCRL1 deficient mice were completely reversed by genetic deletion of CCL19 in the same animals. To place these findings in an anatomical context, expression of CCRL1 in skin and lymph node was explored using CCRL1+/gfp ‘knock-in’ receptor mice and anti-CCRL1 antibodies. In skin, CCRL1 was abundantly expressed by keratinocytes, and found on lymphatic endothelial cells (LECs) that are traversed by migrating DC as they leave the skin. In lymph nodes draining the skin, LECs in the supcapsular sinus (SCS) were strongly CCRL1+. Although some leukocytes were found to express very low levels of eGFP in CCRL1+/gfp, the data suggested that CCRL1-mediated scavenging of CCL19 by keratinocytes and LECs facilitates CCR7-driven DC migration from resting and inflamed skin. CCRL1 expression in other secondary lymphoid tissues was examined and its role in regulating leukocyte populations residing in or around the SCS was explored. As in the inguinal lymph node, CCRL1 was restricted to the SCS LECs in other lymph nodes, such as the mesenteric lymph nodes that drains the intestine. In the spleen, endothelial cells lining venules adjacent to the white pulp marginal zone specifically expressed CCRL1. The overall cellularity and microanatomy of lymph nodes and the spleen appeared normal in CCRL1-deficient mice, as was the recruitment of CCR7+ cells into the spleen two hours after adoptive transfer. However, NK cells, iNKT cells and γδ T cells, which are thought to reside in intrafollicular regions, were less abundant in CCRL1-deficient inguinal and mesenteric lymph nodes, although they were present at normal frequency in the spleen. CD169+ macrophages are intimately associated with CCRL1+ endothelial cells in the spleen. CCRL1 deficiency had no clear impact on these cells in the inguinal lymph nodes, and only resulted in a small increase in the abundance of these cells in the spleen, without obviously affecting their position. Strikingly, however, in mesenteric lymph nodes, CD169+ macrophages, and LECs with which they associate, were far more abundant when CCRL1 had been deleted and were aberrantly distributed throughout the lymph node parenchyma. The reason this phenotype is restricted to the mesenteric lymph node is unclear, but it might be related to the fact the intestine, unlike other non-lymphoid tissues, is an abundant source of CCL25. This, along with the immunological implications of these observations, requires further investigation. There is a lot of evidence that macrophages regulate lymphangiogenesis. The close association between LECs and CD169+ macrophages in lymph nodes, and the phenotype in CCRL1-deficient mesenteric lymph nodes, stimulated experiments to explore functional interactions between these cells. Inflammation in the footpad induced lymphangiogenesis in draining popliteal and inguinal lymph nodes. When clodronated liposomes were used to specifically deplete macrophages from the popliteal lymph node of WT mice, lymphangiogenesis appeared suppressed while it continued unabated in the inguinal lymph node where the CD169+ macrophage population was intact. WT and CCRL1-deficient mice responded similarly, and although preliminary, these data suggest that CD169+ macrophages play an important role in stimulating lymph node lymphangiogenesis. In addition to the CCRL1 studies above, other mechanisms that might regulate chemokines after their secretion were explored. Work published at the beginning of my PhD showed that CCL21, which carries an extended C-terminus that anchors it to the extracellular matrix, can be cleaved by bone marrow-derived DCs (BMDCs) to release a more freely diffusible version. These findings were reproduced here using in vitro-derived human or mouse DCs. DCs were far better at cleaving CCL21 than other leukocytes, and they could also cleave CCL2, a pro-inflammatory chemokine with an extended C-terminus. Interestingly, a truncated version of CCL21 was detected in mouse secondary lymphoid tissues. It was larger than the version generated by BMDCs in vitro, and the nature of its truncation is not clear, but this shows that CCL21 processing occurs in vivo. If this form of CCL21 is more diffusible than the full-length protein, then it might be more available for regulation by CCRL1. / However, neither forms of CCL21 were more abundant in CCRL1-deficient secondary lymphoid tissues than equivalent tissues from WT mice. CCR7 plays a critical role in directing DC egress from tissues, their entry into lymph nodes, and their movement within these tissues. The work presented in this thesis provides evidence of two mechanisms that regulate CCR7 ligands: CCRL1-mediated CCL19 scavenging and DC-mediated CCL21 cleavage. It reveals that, under certain circumstances, CCRL1 is critical for facilitating DC egress from peripheral tissues to the lymph nodes, and plays an indispensible role in regulating LECs and CD169+ macrophages in lymph nodes. These studies extend our understanding of CCRL1 and the chemokine networks at work in lymph nodes and other tissues, and form the foundation on which to explore the immunological importance of the regulation of extracellular chemokines.