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This project investigated the effect of β2-adrenergic receptor (β2-AR) stimulation on the function of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells in vitro. Catecholamines have been previously shown to increase efflux of these cells into the blood, but the effects on cell function are unclear. In this thesis three aspects of function have been addressed. The results of the studies presented in this thesis showed that: (1) β2-adrenergic stimulation by salmeterol reduced the percentage of IFN-γ producing CD4+ and CD8+ T cells activated by Staphylococcus Aureus enterotoxin type B (SEB) superantigen, cytomegalovirus lysate (CMV) or CMV pp65 (pp65) recombinant protein. (2) salmeterol, at high concentrations, increased rolling behaviour and decreased stationary behaviour of peripheral blood lymphocytes (PBLs) on human microvascular cell line (HMEC-1) and on vascular cell adhesion molecule-1 (VCAM-1), in both flow and static assays. (3) adrenergic stimulation impaired the activation and cytotoxic function of CD8+ T and NK cells, as indicated by lower expression of CD107a (a marker of CD8+ T and NK cell activation and function) following incubation of peripheral blood mononuclear cells (PBMCs) with human erythromyeloblastoid leukemia (K562) cell line or MHC class I chain-related gene A (MICA*009) transfected Chinese hamster ovary cells (T-CHO) was analysed. The results presented in this thesis showed that adrenergic stimulation influences a number of cellular functions, such as those related to migration, cytokine production and cytotoxic function. Together the above studies may contribute to our understanding about how stress affects the ability of the cytotoxic cells.

The interaction between the nervous and immune system is well documented, although is still not fully understood - particularly the impact of neurotransmitter receptors on immune cell function. 5-HT\(_3\)A receptor expression was identified on activated regulatory T cells (Treg) but not on effector T cells. Incubation of human peripheral blood mononuclear cells (PBMC) with the 5-HT\(_3\) receptor agonist DDP733 and the positive allosteric modulator 5-chloroindole increased the percentage of CD25+FoxP3+ lymphoyctes (Treg phenotype). Proliferation of PBMC was inhibited by DDP733 plus 5-chloroindole indicating functional impact by the 5-HT\(_3\) receptor. The GPR55 receptor was also expressed by human T cells. The GPR55 agonist lysophosphatidylinositol increased cell viability by preventing apoptosis. However, the induced response was not blocked by the GPR55 receptor antagonist cannabidiol casting doubt over the GPR55 receptor mediating the response. Cannabidiol was demonstrated to have a pro-apoptotic effect in its own right, although whether this effect is mediated by GPR55 or the CB2 cannabinoid receptor is unknown. Further experiments are required to elucidate the role of the 5-HT\(_3\)A receptor in lymphocyte function and the mechanism responsible for the immunoprotective role of lysophosphatidylinositol.

The recruitment of peripheral blood lymphocytes (PBL) to sites of inflammation and their subsequent traffic into the lymphatic circulation is important in host defense. However, surprisingly little is known about their recruitment from the blood vasculature into inflamed tissue, and almost nothing about their egress from inflamed tissue via the lymphatic circulation. We showed that both human macrovascular and microvascular endothelial cells stimulated by TNF\(\alpha\) and IFN\(\gamma\), preferentially recruited memory T-lymphocytes (CD45RO positive cells) from a mixed pool of PBL. T-cells that had migrated across vascular endothelial cells subsequently utilised a combination of \(\beta\)1 and \(\beta\)2 integrins to traverse cytokine activated lymphatic endothelium. In addition we provide evidence that PGD2 was critical for the transmigration of lymphocytes through vascular endothelium. The process of trans-lymphatic migration was also significantly retarded in the presence of a function neutralising antibody against CCR7. Most importantly, we observed that memory T-cells showed a markedly enhanced capacity to migrate across lymphatic endothelium if they had first traversed a vascular endothelial cell barrier. We have shown that addition of exogenous PGD2 to isolated lymphocytes is able to restore the enhanced migration capacity of lymphocytes that have previously migrated through a vascular monolayer. The nature of the priming signal delivered by the process of migration across blood vessel endothelium remains to be fully identified, but is likely to be important in regulating the dynamics of an inflammatory response.

TNF receptor/ligand superfamily members signal through pathways giving rise to proteins that regulate lymphocyte proliferation, activation, differentiation and survival. Absence of TNF ligands OX40L and CD30L impairs survival of GC T cells and affinity maturation of antibody responses. Direct effects of these molecules on B cells in antibody responses are not characterised. I dissected roles of OX40 and CD30 for B cells using T-independent type II (TI-II) antigen NP-Ficoll. Humoral immunity is impaired in OX40 deficiency. Defects in class switched and non-class switched antibody production are due to reduced development of antigen-specific switched and non-switched plasma cells. CD30 has an opposing role, deficiency results in similar or higher switched and non-switched antibody titres and higher numbers of antigen-specific plasma cells that develop rapidly. This may explain why in OX40/CD30 double deficiency, there is a less pronounced defect than in OX40 single deficiency. B cell intrinsic roles are revealed for OX40 and CD30 that suggest OX40 on B cells is critical for TI-II plasmablast differentiation or survival and B cell CD30 inhibits onset of plasmablast differentiation.

Secondary lymphoid organs have a key role in the initiation of adaptive immune responses to infection. Organogenesis occurs in foetal development, and the use of genetic tools, imaging technologies, and ex vivo culture systems has provided significant insights into the cellular components and associated signalling pathways that are involved. However such approaches tend to be reductionist and descriptive, focusing on the contribution of individual components, and cannot fully explain how lymphoid organs develop through interaction between biological components. In this study, a set of simulation and statistical tools have been developed that provide further insights into the molecular and biophysical mechanisms of lymphoid tissue organogenesis. Specifically, the formation of Peyer’s Patches, gut-associated secondary lymphoid organs, is examined. In collaboration with experimental immunologists, a structured process in the design and calibration of a computer simulation of the biological process has been conducted, leading to the development of a publicly accessible scientific tool where cell behaviour emerges that is statistically similar to that observed in ex vivo culture. Robust biological hypotheses can be generated through use of the tool to perform in silico experimentation that simulates different physiological conditions. A lack of available statistical tools to analyse in silico simulation results has been addressed through the development and release of the spartan toolkit, a set of techniques that can suggest the influence that pathways and components have on simulation behaviour, offering valuable biological insight into the system being explored. An analysis of simulation results using spartan suggests the influence of biological pathways on tissue formation changes during development, in contrast to hypotheses in the literature that suggest the process is chemokine driven. Data presented suggests the development period is biphasic, with cell adhesion the key factor early in development, and chemokine expression influential at later point. Through novel application of the statistical tools in spartan to perform a time-lapse analysis of cell behaviour, it is suggested this change in phase occurs between hours 24 and 36. Novel in silico experimentation performed has suggested the key biological factors in causing cell aggregation, and suggested a role for LTin cells in limiting size and number of Peyer’s Patches. A range of potential laboratory investigations have been suggested that could validate whether these simulation derived hypotheses are valid.

Neutrophils are phagocytic white blood cells which act as the first line of defence against entry of foreign microorganisms. Neutrophils are recruited to their target site through the process of spreading, extravasation and phagocytosis involving complex signal transduction within the cells, which might include the activation of the cytosolic Ca2+ activated protease, calpain-1. The work described here investigates the role of calpain-1 in regulating neutrophil functions such as spreading, trans-endothelial migration, chemotaxis, phagocytosis and Ca2+ signalling. Through the work done at European Mutant Mouse Archive (EMMA), Oxford, and by using intracellular sperm injection (ICSI) of calpain-1 deleted gene from mice generated in the USA, and with a selective genotype breeding programme, a colony of homozygous calpain-1 KO mouse has been generated in Cardiff. Homozygous calpain-1 KO neutrophils appeared to have a smaller surface spreading area and their recruitment into the peritoneal cavity of the mouse in vivo was disrupted. In vitro experiments showed significant defects in their ability to cross the ICAM-1 expressing endothelial cells in trans-endothelial migration assay. Disruption in this transmigration was only evident with ICAM-1 upregulated (TNF-treated) endothelial cells, suggesting a specific defect in the β2 integrin-ICAM-1 signalling process. Calpain-1 absence did not affect signal transduction as neutrophils were able to signal cytosolic Ca2+ in response to β2 integrin engagement (C3bi-opsonised zymosan) and also to release intracellular Ca2+ store upon IP3 uncaging. This showed that the IP3 pathway in the cells was not affected by knocking-out calpain-1 and continued to be functional. The key signalling mechanisms from β2 integrin also remained intact and this is consistent with calpain-1 activation by Ca2+ being an important event in trans-endothelial migration. In conclusion, calpain-1 absence has significantly affected the ability of neutrophils to undergo trans-endothelial migration and this effect is directed towards the event which happens downstream to the increase in cytosolic free Ca2+ concentration.

The role of the alternative NF-\(\kappa\)B pathway in the development of secondary lymphoid organs (SLOs) has been well described. Tertiary lymphoid organs (TLOs) include intestinal cryptopatches (CPs) and isolated lymphoid follicles (ILFs). This thesis investigates the role of the alternative NF-\(\kappa\)B pathway in the development of colonic ILFs, using the p100\(\Delta\) mouse model, where the alternative pathway is constitutively active. We present compelling data that p100\(\Delta\) mice develop significantly more lymphoid aggregates in the colon, compared with WT littermates, and provide several lines of evidence showing that these aggregates are analogous to ILFs. Additionally, we demonstrate that in the p100\(\Delta\) a significant increase in the numbers of B and T cells, and DCs in the colon and show alterations in the subsets of colonic T cells and DCs. We also present evidence that constitutively active NF-\(\kappa\)B2 p100 in LT\(\alpha\) deficient mice induces recovery of B and T cell segregation in the spleen. Most strikingly, we show recovery of iLNs and mLN development in one of five \(p100\)\(\Delta\)\(LT\)\(\alpha^{-/-}\) mice generated. These findings demonstrate that the alternative NF-\(\kappa\)B pathway plays an important role in not only SLO development and splenic organisation, but also in the development of TLOs, specifically colonic ILFs.

CD4\(^+\) and CD8\(^+\) T cell populations can be divided into subpopulations based on expression of surface markers CCR7 and CD45RA. The resulting populations are referred to as naive, central memory, effector memory and effector memory RA\(^+\) (EMRA). The aim of this study was to identify potential lineage relationships between these subpopulations for both CD4\(^+\) and CD8\(^+\) T cells through microarray analysis. The genes found to distinguish between these subpopulations include many molecules with known functions in T cell differentiation, including CCR7, CD45RA, granzymes, L-selectin and TNF receptors. Several genes from the tetraspanin family of proteins were found to be differentially expressed at mRNA and protein level; suggesting a possible role for these genes in CD4\(^+\) and CD8\(^+\) T cell activation, migration and lysosomal function. Other genes identified, such as LRRN3 and CXCR5 which were expressed highest on naive and CM T cells respectively, provide interesting gene targets to follow up on their function in these T cell populations. Microarray data was validated through Real Time PCR and suggests that both CD4\(^+\) and CD8\(^+\) T cells differentiate along a linear pathway of naive to central memory to effector memory. The transcriptional programmes responsible for these differentiation steps were distinct between CD4\(^+\) and CD8\(^+\) T cells, although additional elements were common to both subsets.

Macrophages are present in virtually all tissues and account for approximately 10% of all body mass. Although classically credited as the scavenger cells of innate immune system, ridding a host of pathogenic material and cellular debris though their phagocytic function, macrophages also play a crucial role in embryogenesis, homeostasis, and inflammation. De-regulation of macrophage function is therefore implicated in the progression of many disease states including cancer, arthritis, and atherosclerosis to name just a few. The diverse range of activities of this cell can be attributed to its exceptional phenotypic plasticity i.e. it is capable of adapting its physiology depending on its environment; for instance in response to different types of pathogens, or specific cocktail of cytokines detected. This plasticity is exemplified by the macrophages capacity to adjust rapidly its transcriptional profile in response to a given stimulus. This includes interferons which are a group of cytokines capable of activating the macrophage by interacting with their cognate receptors on the cell. The different classes of interferons activate downstream signalling cascades, eventually leading to the expression (as well as repression) of hundreds of genes. To begin to fully understand the properties of a dynamic cell such as the macrophage arguably requires a holistic appreciation of its constituents and their interactions. Systems biology investigations aim to escape from a gene-centric view of biological systems. As such this necessitates the development of better ways to order, display, mine and analyse biological information, from our knowledge of protein interactions and the systems they form, to the output of high throughput technologies. The primary objectives of this research were to further characterise the signalling mechanisms driving macrophages activation, especially in response to type-I and type- II interferons, as well as lipopolysaccharide (LPS), using a ‘systems-level’ approach to data analysis and modelling. In order to achieve this end I have explored and developed methods for the executing a ‘systems-level’ analysis. Specifically the questions addressed included: (a) How does one begin to formalise and model the existing knowledge of signalling pathways in the macrophage? (b) What are the similarities and differences between the macrophage response to different types of interferon (namely interferon-β (IFN-β) and interferon-γ (IFN-γ))? (c) How is the macrophage transcriptome affected by siRNA targeting of key regulators of the interferon pathway? (d) To what extent does a model of macrophage signalling aid interpretation of the data generated from functional genomics screens? There is general agreement amongst biologists about the need for high-quality pathway diagrams and a method to formalize the way biological pathways are depicted. In an effort to better understand the molecular networks that underpin macrophage activation an in-silico model or ‘map’ of relevant pathways was constructed by extracting information from published literature describing the interactions of individual constituents of this cell and the processes they modulate (Chapter-2). During its construction process many challenges of converting pathway knowledge into computationally-tractable yet ‘understandable’ diagrams, were to be addressed. The final model comprised 2,170 components connected by 2,553 edges, and is to date the most comprehensive formalised model of macrophage signalling. Nevertheless this still represents just a modest body of knowledge on the cell. Related to the pathway modelling efforts was the need for standardising the graphical depiction of biology in order to achieve these ends. The methods for implementing this and agreeing a ‘standard’ has been the subject of some debate. Described herein (in Chapter-3) is the development of one graphical notation system for biology the modified Edinburgh Pathway Notation (mEPN). By constructing the model of macrophage signalling it has been possible to test and extensively refine the original notation into an intuitive, yet flexible scheme capable of describing a range of biological concepts. The hope is that the mEPN development work will contribute to the on-going community effort to develop and agree a standard for depicting pathways and the published version will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest. With a desire to better understand the transcriptional response of primary mouse macrophages to interferon stimulation, genome wide expression profiling was performed and an explorative-network based method applied for analysing the data generated (Chapter-4). Although transcriptomics data pertaining to interferon stimulation of macrophages is not entirely novel, the network based analysis of it provided an alternative approach to visualise, mine and interpret the output. The analysis revealed overlap in the transcriptional targets of the two classes of interferon, as well as processes preferentially induced by either cytokine; for example MHC-Class II antigen processing and presentation by IFN-γ, and an anti-proliferative signature by IFN-β. To further investigate the contribution of individual proteins towards generating the type-I (IFN-β) response, short interfering RNA (siRNA) were employed to repress the expression of selected target genes. However in macrophages and other cells equipped with pathogen detection systems the act of siRNA trasfection can itself induce a type-I interferon response. It was therefore necessary to contend with this autocrine production of IFN-β and optimise an in vitro assay for studying the contribution of siRNA induced gene-knock downs to the interferon response (described in Chapter-5). The final assay design incorporated LPS stimulation of the macrophages, as a means of inducing IFN-β autonomously of the transfection induced type-I response. However genome-wide expression analysis indicated the targeted gene knock-downs did not perturb the LPS response in macrophages on this occasion. The optimisation process underscored the complexities of performing siRNA gene knockdown studies in primary macrophages. Furthermore a more thorough understanding of the transcriptional response of macrophages to stimulation by interferon or by LPS was required. Therefore the final investigations of this thesis (Chapter-6) explore the transcriptional changes over a 24 hour time-course of macrophage activation by IFN-β, IFN-γ, or LPS and the contribution of the macrophage pathway model in interpreting the response to the three stimuli. Taken together the work described in this thesis highlight the advances to be made from a systems-based approach to visualisation, modelling and analysis of macrophage signalling.

Potential biological roles for two novel human glycan-binding proteins have been investigated. In the first part of the work, analysis of the scavenger receptor C-type lectin (SRCL) leads to the proposal that the primary function of this receptor is to clear glycoproteins released from neutrophils. The expression of SRCL was characterized by screening of cDNA libraries and by immunostaining of tissues. These results confirm and extend earlier reports that SRCL is found in a range of tissues and is expressed by cultured umbilical vein endothelial cells, and demonstrated that SRCL is localized primarily in endothelial cells. SRCL is thus positioned to interact with circulating glycoproteins and cells. Potential ligands were isolated from neutrophils by affinity chromatography and shown by mass spectrometry to comprise lactoferrin and other soluble granule proteins. The interaction between SRCL and lactoferrin was found to be dependent on fucose residues and analysis of lactoferrin N-glycans by mass spectrometry revealed multiple Lewisx epitopes borne on unsialylated triantennary glycans, consistent with selective binding of SRCL to the Lewisx trisaccharide. Transfected cells expressing SRCL were shown to internalize fluorescein-labelled lactoferrin. Taken together, these results provide support for the hypothesis that soluble glycoproteins in neutrophil secondary granules are tagged with the Lewisx epitope to direct their clearance by SRCL after release at sites of inflammation. Prolectin, a novel C-type lectin, was identified by screening of the human genome. Screening of cDNA libraries and immunostaining were used to ascertain that prolectin expression is restricted to dividing B cells in and around germinal centres of lymphoid tissues. This expression pattern, together with the presence of motifs in the cytoplasmic tail of prolectin that may interact with intracellular signalling molecules, leads to the suggestion that prolectin may have a role in the migration of B cells during the adaptive immune response.

Syndecan-4 (SDC-4), a transmembrane heparan sulphate proteoglycan and a co-receptor of integrins for fibronectin, has been reported to modulate the adhesion of cells to a range of extracellular matrix ligands. In addition to the modulation of integrins, SDC-4 has been recently reported to modify the interaction of some growth factors with their receptors. T cells are essential effectors of the adaptive immune response. When conjugating with antigen-presenting cells (APCs), T cells transform their shape to enable formation of a specialised morphological structure, called the immunological synapse (IS). The IS formation is associated with the polarisation of signalling and adhesive molecules towards the APC. The IS is formed and stabilized by similar adhesive forces and structures as in the motile fibroblasts - an extensively studied model of syndecan function. In this thesis I have investigated the role of SDC-4 in T cell - APC recognition and the IS. First, I confirmed the tight regulation of SDC-4 expression in T cells during the process of activation. Flow cytometry and PCR data demonstrate increased presence of SDC-4 in stimulated human T cells compared to their resting counterparts, indicating involvement of SDC-4 in the processes of T cell activation. Transient transfection of exogenous SDC-4 into Jurkat T cells with low endogenous SDC-4 expression was used as a model to study the effect of increased SDC-4 expression on T cell function. Using live cell imaging and advanced data image analysis I have quantitatively demonstrated that SDC-4 transfected Jurkat T cells are less likely to modify their shape during IS formation when compared to mock transfected Jurkat T cells. I also observed a delay in T cell antigenic response caused by SDC-4 over-expression. Moreover, I was able to visualise the exogenous SDC-4 in the IS using confocal microscopy and demonstrate the independence of this phenomenon on T cell receptor. In summary, my observations indicate a regulatory role for SDC-4 in T cell adhesion causing delays in the IS formation and reduced transformation of T cell shape during conjugate formation.

The αVβ5 integrin is a member of integrin family that binds to different ligands such as vitronectin, fibronectin and soluble CD23 in order to mediate different biological responses such as cell growth, adhesion and metastasis. It is expressed by B cell precursors and by different acute lymphocytic leukaemia cell lines such as SMS-SB cells. This thesis is an attempt to explain how the sCD23- αVβ5 integrin interaction stimulates SMS-SB cell growth and to study the role of the αVβ5 integrin and other receptors such as PDGF receptor and CXCR4 in B cell development in the bone marrow. The maturation and differentiation of B-cells occur due to several factors that impact on gene expression in its development program. This program is divided into two main phases, the antigen-independent B-cell development phase and antigen-dependent B-cell development phase, respectively. The antigen - independent phase of B cell development starts from the pluripotent haemopoietic stem cell (PHSC) and progresses through several successive stages which are identified by somatic recombination and rearrangement of both heavy and light chain genes. Soluble CD23 and LP (a synthetic peptide derived from soluble CD23) significantly stimulate SMS-SB growth while a smaller growth stimulation is caused by either SDF1-α or PDGFAB. There are different signalling targets involved in the αVβ5 integrin-mediated proliferation due to its binding to either sCD23 or LP. These ligands enhance the association between the αVβ5 integrin and the PDGF receptor which promote the phosphorylation of both Jak2 and STAT5. Moreover, cell growth was reduced and the phosphorylation of Jak2 and STAT5 was also knocked down with using either PDGF receptor inhibitor (AG1295) or Jak2 inhibitor (AG490). Both soluble CD23 and LP activate the STAT5-DNA binding and strongly increase its transcriptional activity. In addition, both ligands induce the phosphorylation of other different substrates such as STAT2, c-Src, c-yes and AMPKα2 which might be related to cell growth stimulation. The αVβ5 integrin ligands also promote the phosphorylation of ERK1/2, p90RSK and activate a SRF transfected reporter gene. However, ERK1/2 and p90RSK phosphorylation was completely blocked by the specific MEK inhibitor (U0126). In similar context, SDF1-α stimulates the transcriptional activity of SRF but not STAT5 while PDGFAB does the opposite. Finally, soluble CD23 induces the proliferation of 697 and BAF03 which are other pre-B cell line models. These data suggest that the αVβ5 integrin-ligated ligands stimulate SMS-SB cell growth by promoting different signalling pathways, mainly Jak2/STAT5 and MEK/ERK1/2 pathway. Further work is required to determine the role of STAT5, p90RSK, c-Src and SRF in stimulating either the proliferation or apoptosis that promoted by the αVβ5 integrin-sCD23 interaction and to investigate the relationship between the activation of these targets.

CCX-CKR is an atypical chemokine receptor for the homeostatic chemokines CCL19, CCL21 and CCL25. CCL19 and CCL21 are also ligands for CCR7 and are crucial for the induction of antigen specific immunity and tolerance, whereas CCL25 is the sole ligand for CCR9 and is involved in the recruitment of immune effector cells to the small intestine. CCX-CKR does not signal after binding its ligands, as determined by a failure to induce the rapid increase in intracellular calcium that is typical of G-protein mediated signalling. CCX-CKR also does not become desensitised to chemokine binding and therefore is proposed to act as a scavenger receptor that can regulate the activity of CCR7 and CCR9 by affecting the availability of their ligands in vivo. At the time of starting my project, there were no published reports describing the biological function of CCX-CKR in vivo and the principal aim of my thesis was to characterise the immune system of the recently generated CCX-CKR KO mouse with particular focus on the intestinal immune compartment where all three of the chemokine ligands are expressed. Firstly, as described in Chapter 3, I analysed the cellular composition of the secondary lymphoid organs of CCX-CKR KO mice. These studies revealed normal proportions and absolute numbers of lymphocytes, CD11c+ dendritic cells (DC), macrophages and natural killer (NK) cells in the absence of CCX-CKR. The proliferative responses of lymphocytes to mitogenic or TCR stimulation in vitro were also normal, although there was a decreased production of IFNγ by CD4+ T cells from CCX-CKR KO mice. Although most of the phenotypic subsets of conventional DC were present in comparable numbers in the mesenteric lymph nodes (MLN) of CCX-CKR KO and WT mice, there was a consistent and dramatic reduction in the numbers of CD11cloPDCA-1+ plasmacytoid DC (pDC) in CCX-CKR KO MLN. In parallel, fewer CD11cloB220+ cells from CCX-CKR KO MLN than WT expressed CCR9, despite this marker being expressed normally by lymphocytes in these mice. The proportions of pDC in CCX-CKR KO inguinal lymph nodes (ILN) were also significantly reduced compared to WT and pDC from CCX-CKR KO MLN, ILN and spleen all appeared to express higher levels of class II MHC than WT pDC. These data suggest that CCX-CKR may play an important role in the recruitment and/or survival of pDC in the LN and that in its absence, pDC in secondary lymphoid organs may have a more mature phenotype. In Chapter 4, I examined the cellularity of the intestinal immune compartment in resting CCX-CKR KO mice, as well as the effects of Flt3L administration in vivo. Although CCX-CKR KO mice displayed no histological abnormalities in their small intestinal architecture and had normal numbers of T cells in the lamina propria, they did have significantly reduced numbers of intra-epithelial lymphocytes (IEL) as well as decreased proportions of CD19+ B cells and increased proportions of CD11c+ cells in the lamina propria compared with WT mice. The proportions and absolute numbers of CD103+ DC were normal in the lamina propria and MLN of CCX-CKR KO mice, suggesting that CCX-CKR has little to no role in regulating DC migration from the lamina propria to the MLN, a process that is critically dependent on CCR7. Although the proportions and absolute numbers of B cells and CD11c+ cells were normal in CCX-CKR KO Peyer’s patches (PP), there were significantly decreased proportions of pDC compared with WT PP. In vivo treatment of CCX-CKR KO mice with the DC differentiation factor Flt3L, expanded CD11c+ DC numbers dramatically in both CCX-CKR KO and WT small intestinal lamina propria, ILN, spleen, MLN and PP. Although Flt3L abolished the apparent defects in pDC populations in the ILN and PP, this was not the case for CCX-CKR KO MLN, which remained significantly deficient in pDC compared to WT MLN. Work in Chapter 6 examined parallel effects in the blood and bone marrow. As the CCX-CKR ligands CCL19 and CCL21 are involved in the development of all adaptive immune responses, and together with the other CCX-CKR ligand, CCL25, orchestrate immune responses to antigen encountered in the gut, I next investigated the development of antigen specific immunity and tolerance in CCX-CKR KO mice. There were no significant differences in systemic immune responses to subcutaneous immunisation with ovalbumin (OVA) emulsified in complete Freunds adjuvant (CFA) between CCX-CKR KO and WT mice when assessed in vivo or in vitro. However, the development of oral tolerance in CCX-CKR KO mice was impaired, with no suppression of OVA specific delayed type hypersensitivity (DTH) responses or of serum OVA specific IgG2a as was seen in WT mice. In parallel with defective systemic tolerance after feeding OVA, CCX-CKR KO mice appeared to be more susceptible to priming of systemic and local antibody responses after feeding OVA with cholera toxin (CT) as a mucosal adjuvant. In addition, there was some evidence of priming of OVA specific antibody responses in CCX-CKR KO mice fed OVA alone, which was not seen in WT mice. Despite this evidence of abnormal mucosal immunity, CCX-CKR KO mice developed DSS colitis normally, with all indices of disease being identical in CCX-CKR KO and WT animals. Together, these data suggest that there are selective defects in the regulation of antigen specific mucosal immune responses in the small intestine of CCX-CKR KO mice that may predispose these animals towards exaggerated active immune responses. Finally, I performed some preliminary experiments to try and relate DC function to the immune dysregulation I observed in CCX-CKR KO mice and to explore the basis of the pDC defect in the MLN. Bone marrow derived and splenic DC from CCX-CKR KO mice showed a reduced ability to process and present intact protein antigens although endocytic activity was normal. MLN DC from normal and Flt3L treated CCX-CKR KO mice responded similarly to in vitro stimulation with the synthetic TLR7 agonist R848, showing an expansion in the numbers of CD11chiPDCA-1+ cells that nearly all expressed CD40 and CD86. In addition, in vivo administration of R848 triggered identical migration of CD11chiclassIIMHChiCD103+ DC into the MLN of CCX-CKR KO and WT mice, suggesting that lamina propria pDC can effectively mobilise local DC to the MLN in the absence of CCX-CKR. There were no differences in the proportions or absolute numbers of pDC in the liver of CCX-CKR KO and WT mice despite the fact that liver pDC have been implicated in other studies of oral tolerance. Although the proportions of pDC were normal in the bone marrow of resting and Flt3L treated CCX-CKR KO mice, there did appear to be a defective recruitment of Flt3L expanded pDC into the blood of these animals. I used an adoptive transfer approach to study the in vivo localisation of pDC into lymphoid organs and although these experiments were not entirely conclusive, they indicated that WT pDC could enter the MLN and other lymphoid tissues of CCX-CKR KO mice normally and that pDC from CCX-CKR KO mice may have a defect in their ability to enter WT MLN. Taken together, my data suggest that CCX-CKR is involved in the entry and/or survival of pDC in secondary lymphoid organs, a process that normally involves migration across high endothelial venules (HEV). This was associated with impaired oral tolerance induction and heightened immune responses to antigen delivered orally, indicating that CCX-CKR contributes to the regulation of mucosal immunity and tolerance by an as yet unclear mechanism. Further study of these animals will hopefully better define the relationship between pDC and the regulation of mucosal immune responses.

As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.

Lymph nodes contain complex vascular networks composed of lymphatic and blood vessels. The blood vasculature contains specialised high endothelial venules functioning to permit the efficient entry of blood-borne lymphocytes into the node, while the lymphatics contain antigen-presenting cells draining from tissues. In contrast to the well understood cellular interactions and signalling mechanisms driving development of the stromal networks upon which immune cell interactions occur, the processes by which the complex vascular networks develop are poorly characterised. This study aimed to determine the mechanisms by which vascularisation of lymph node anlagen occurs during development. The structure of developing lymph nodes was studied using confocal microscopy to observe the organisation of basement membranes and the different cell types involved in the process. Expression of angiogenesis-related genes was studied using quantitative real-time PCR and microarrays. No evidence for vascularisation of the anlagen was found, though the markers used may not have stained nascent vessels. The lymph sac surrounding the anlagen was shown to exist as a two-layered structure composed of anastomosing blood and lymphatic endothelium. In vitro models of vasculogenesis and angiogenesis were developed utilising human umbilical vein endothelial cells in three dimensional collagen gels, with and without smooth muscle coverage. A spheroid-based model of angiogenesis was used to study the net angiogenic environment in developing E14.5–E17.5 anlagen, which determined that a net anti-angiogenic environment existed at all timepoints in the lymph nodes and thymus, but not skin. Additionally, tensional forces were observed to affect angiogenic sprouting in addition to soluble growth factors. As a consequence of the double-layered lymph sac observed in vitro, and the influence of anti-angiogenic factors and tensional forces observed ex vivo, a model of lymph node development involving anlagen patterning and vascularisation as a result of condensation-induced tensional forces was proposed, complementary to soluble growth factor-driven angiogenesis.

In order to prevent autoimmune diseases the immune system must be tightly regulated. One mechanism that the immune system uses to prevent autoimmunity is through the use of regulatory receptors on the surface of T lymphocytes. In this thesis I have concentrated on the role of one such receptor, CD5, in T lymphocyte activation. CD5 is a member of the scavenger receptor cysteine rich superfamily and is expressed, along with the closely related CD6, on T cells and a subset of B cells. CD5 is known for its inhibition of thymocyte selection but its function in peripheral T cell activation remains unclear. In this thesis I demonstrate that OX19, a rat CD5 monoclonal antibody (mAb), will block extracellular engagement of CD5 in vitro resulting in an increase in proliferation following T cell activation. In demonstrating the mode of action of OX19 I show that extracellular engagement of CD5 will inhibit T cell activation in vitro. I also demonstrate that OX19 does not modulate CD5 from the surface in vitro whereas it does modulate CD5 in vivo. Another key finding in this thesis is that differential phosphorylation of the three tyrosines in the CD5 cytoplasmic tail can account for positive and negative signalling by CD5 during T cell activation. Evidence here demonstrates that CD5 Y1 is required for positive signalling which becomes more apparent in the absence of costimulation by the coreceptor, CD2. CD5 Y2 and Y3 are required for the CD5 mediated inhibition of T cell activation. Phosphorylation of these tyrosines is also necessary for CD5 regulation of CD3 down modulation following TCR triggering. These data point to a potential molecular mechanism involving CD5 regulation of CD3 down modulation creating control of T cell activation as well as a potential for cross talk between another coreceptor CD2. Direct and indirect associations of the phosphorylated tyrosines in CD5 was analysed here using a combination of a screen of binding affinities of SH2 domains with CD5 phospho-peptides and mass spectrometry of CD5 associated proteins. SH2 domains measured in this thesis did not show a high binding capacity to tyrosine phosphorylated CD5 peptides indicating that CD5 mediates transient interactions with signalling proteins. The region in the cytoplasmic tail in CD6, CD6Y3Y4, which lies at the same distance from the membrane as the C-terminus of CD5, will bind to Itk and Lck. Given CD5 and CD6 are often coexpressed this would allow phosphorylation of CD5 by these two kinases providing a molecular mechanism underlying cooperation between CD5 and CD6.

Natural Killer (NK) cells are lymphocytes of the innate immune system. However, there is increasing evidence that they can also play important roles in the adaptive immune system; as initiators, through antigen presentation; as effectors, via early release of IFN-y; and as immunoregulators, by eliminating over-activated macrophages. The functions of NK cells in these roles are intimately linked to their interactions with other cells during an immune response, for example recognition of target cells via activating receptors. The activating receptor NKG2D recognises proteins that are not normally expressed at the surface of most cells but are up-regulated during a cellular ‘stress’ response. However, NKG2D ligands can also be induced on human macrophages by TLR stimulation, leading to NK cell-mediated lysis. Here, I clarify that ligation of TLR4 preferentially up-regulates MICA but not MICB, TLR7/8 ligation up-regulated both MICA and MICB, while ligating TLR3, signalling through a MyD88-independent pathway, up-regulated neither. TLR4 stimulation decreased expression of microRNAs, miR-17-5, miR- 20a and miR-93, which target MICA, implicating a novel role for microRNAs in post-transcriptional regulation of NKG2D ligand expression. Moreover, the pathway involved IL-12/TNF- a-mediated autocrine signalling, thus incorporating an intrinsic mechanism for NK cell-mediated elimination of particularly activated macrophages. In addition to this immunoregulatory role, NK cell activity can shape a subsequent adaptive immune response. Subsets of NK cells can have distinct functions. Here, I demonstrate that following culture with IL-2, >25% of human peripheral blood NK cells express HLA-DR, due to an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLADR is upregulated on previously negative cells. HLA-DR-expressing NK cells exhibited enhanced degranulation to susceptible target cells and expression of the chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggestive of an immunological role, stimulation of PBMCs with Mycobacterium bovis BCG triggered dramatic expansion of HLADR- expressing NK cells. In addition, the magnitude of the NK cell IFN-y secretion response in PBMC triggered by BCG was associated with the proportion of HLA-DR-expressing NK cells ex vivo. A direct contribution to the immune response was determined by specifically enriching the HLA-DR-expressing NK cell compartment, which substantially augmented the total NK cell IFN-y secretion response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in peripheral blood but readily expanded by IL-2, that can play a significant role during immune responses.

Cathelicidins are cationic host defence peptides (CHDP) with essential roles in the innate defence system. These peptides have antimicrobial potential and the capacity to modulate innate immunity and inflammatory processes. Neutrophils (PMN) are the main reservoir of cathelicidins and play key roles in first line defence against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and the efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. In this thesis I demonstrate that the human cathelicidin LL-37 rapidly induced secondary necrosis of apoptotic human PMN and identify the essential C-terminal region of LL-37 required for this activity. In addition to the induction of secondary necrosis, higher concentrations of LL-37 also promoted PMN granule contents release. LL-37-induced secondary necrosis did not affect PMN ingestion by human monocyte-derived macrophages and, in contrast to expectation, was not proinflammatory. Interestingly, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated where LL-37-mediated secondary necrosis induced anti-inflammatory granule content release. Consistent with the results of in vitro studies, in vivo murine sterile peritonitis model revealed the same phenomenon: LL-37-induced secondary necrosis diminished inflammatory responses with decreased PMN influx. I also present data on LL-37- mediated modulation of innate immune effector cell cytokines responses to inflammatory signals. I propose that during acute inflammation LL-37 can modulate innate immune responses through its activity on cytokine production, and that LL-37-mediated secondary necrosis of apoptotic PMN has anti-inflammatory effects, but may also mediate host damage by promoting the release of potentially harmful intracellular contents under chronic or dysregulated conditions.

Fibroblasts are the most abundant cell type of the stroma, producing extracellular matrix components which provide mechanical strength to tissues. Recent work has shown that fibroblasts are not simply passive structural cells but active participants in the immune response. In this study fibroblast heterogeneity within the murine lymphoid system was investigated by analysing gene and protein expression. Fibroblasts were isolated from a range of lymphoid and peripheral sites. Our findings show that fibroblasts grown from different sites show heterogeneous gene and protein expression and are functionally different in their response to lymphotoxin α treatment in vitro and in their capacity to recruit host leucocytes in vivo. A novel in vivo functional assay has been developed using a collagen sponge as a three-dimensional structure in the kidney capsule transfer model, to assess the ability of fibroblasts to form and maintain lymphoid structures. We conclude that murine fibroblasts isolated from lymphoid tissues, display site specific features. They are phenotypically distinct with site specific gene and protein expression profiles. This suggests that murine lymphoid fibroblasts have positional memory and help impart anatomical identity. An important implication of these findings is the effect this diversity may have in conveying site specificity to immune responses.

Germinal centres (GCs) are the sites where V-gene hypermutation and B cell selection are taking place. Testing specificity and affinity of GC B cell receptor by interaction with antigen on follicular dendritic cells (FDCs) may be an important selection process to select high affinity B cell clone. As antigen on FDC is present in the form of antigen-antibody immune complex, GC B cells are expected to have to compete with antibody to get access antigen. Initially this antibody will be of low affinity. However, during the course of an immune response, this affinity may increase. We have tested this competitive selection model by following the replacement of antibodies in the GC over the course of an immune response. The speed of this replacement is dependent on affinity. Antibody added during an ongoing GC reaction can replace antibody in the GC, but only, if it is of high enough affinity. Presence of high or low affinity antibodies on FDC influences centrocyte selection, leading to variations in apoptosis within the GC, serum affinity, and plasma cell output. Parallel in silico experiments support the idea that a dynamic GC selection threshold, dependent on the affinity of GC output cells increases affinity maturation, because it enhances selection efficiency over a longer period during the course of a GC reaction. A dynamic selection threshold may explain the termination of the GC reaction, when affinity of new B cell variants is not sufficient to overcome the affinity of antibodies produced outside the GC. IRF4 is essential for the plasma cell differentiation and Ig class switch. IRF4 mRNA and protein rapidly upregulate within one hour after naive B cells get stimulation with NP-Ficoll in QM×C57BL/6 mice, and then activated B blasts expressing intermediate level of IRF4 either go into the red pulp to form the early extrafollicular response by upregulating high level of IRF4, or travel into the follicle to differentiate into GC founding cells. IRF4 completely shuts down when cells becomes proliferating centrocytes. But IRF4 expresses again in centorcytes, which have been committed to differentiate into the plasmablasts. Its high level expression shows the GC emigrants. Here IRF4 is selected as the marker for the early plasmablats appearance on the GC-T zone interface at the beginning of the GC reaction. And further experiments by using cytokines such as IL-6, IL-10, IL-21, costimulatory signals OX40, CD30 deficient mice show that these signals can affect the development of these early IRF4\(^+\) plasmablasts on the GC-T zone interface in the TD antigen response.

The nematode Caenorhabditis elegans has been a powerful experimental organism for almost half a century. Over the past ten years, researchers have begun to exploit the power of C. elegans to investigate the biology of a number of human pathogens. This work continues to uncover mechanisms of host immunity and pathogen virulence that are either analogous to those involved during pathogenesis in alternative animal hosts or mechanisms which are, thus far, unique to the worm. In this thesis, we present data that describes an immunological balance in C. elegans, whereby heightened tolerance to one pathogen, the enteric bacteria Salmonella Typhimurium, comes at the cost of increased susceptibility to another, the fatal fungal human pathogen Cryptococcus neoformans. We find that this susceptibility trade-off is mediated by the reciprocal activity of two immune genes: the lysozyme lys-7 and the tyrosine kinase abl-1. We suggest that ABL-1 controls two different DAF-16-dependent pathways to regulate this balance. Both pathways are necessary for wild type resistance to C. neoformans, whilst the activity of only one pathway is a requirement for the tolerance phenotype to S. Typhimurium. We infer from sequence data that LYS-7 has an atypical mode of action in C. elegans, which we hypothesise to be detrimental to the worm during S. Typhimurium pathogenesis and thus a contributing factor to the tolerance phenotype. Furthermore, we find that this tolerance has a Salmonella-dependency which we propose to be under the control of the alternative sigma factor, RpoS. Taken together, we describe an immunological balance in C. elegans for the first time, one that is mediated by both host and pathogen factors. We therefore suggest that the innate immune response of C. elegans has a higher level of immune complexity than previously believed, and that such trade-offs are evolutionarily ancient mechanisms.

Fibrocytes are monocyte-derived cells that morphologically look like fibroblasts, express both stromal and haematopoietic markers, and have been reported as being involved in wound healing and fibrosis. In-vitro derived fibrocytes can be differentiated in both serum-containing and serum-free environments and we wanted to study the relationship between these two fibrocytes; which potentially could be involved at different time points at a wound healing site. To investigate the relationship between serum-free and serum-containing derived fibrocytes monocytes were differentiated without serum. When these cells were placed in a serum-containing environment they became round, losing their fibroblast-like morphology. However when the reverse experiment was done on fibrocytes derived in a serum-containing environment there was no apparent effect on their morphology. The relationships between these two fibrocytes, as well as macrophages and fibroblasts was also examined using transcriptome analysis of 37000 genes, clustering the samples based on all the genes, and identifying those that were significantly different between the populations. This demonstrated that both fibrocyte populations are distinct from each other, as well as from both fibroblasts and macrophages. These data demonstrate that these two fibrocytes have different characteristics, suggesting that they may have different roles in the modulation of fibrosis in inflammation.

The autoimmune regulator (AIRE), a key player in negative selection of developing thymocytes, acts as a transcriptional regulator, inducing the expression of tissue restricted antigens (TRA) within medullary thymic epithelial cells in a process known as promiscuous gene expression (PGE). Here we demonstrate how AIRE influences PGE through a direct impact on post-translational modifications of core histones which are associated with the regulation of transcription. Through native chromatin immunoprecipitation on thymic epithelial cells transfected with AIRE, we show how in vitro, TRA are enriched in active acetylation and methylation of core histones, yet retain silencing modifications. Furthermore, across a cluster of AIRE-regulated genes, histone modifications were deposited across the entire domain, dependent upon the expression profile of each gene, suggesting a role for domain-wide epigenetic regulation by AIRE. Extension of these studies in vivo, utilising the recently developed carrier ChIP technique, allowed examination of the epigenetic status of TRA throughout the thymic developmental pathway. We report how poised TRA are marked with combinations of active and silent modifications early in thymic development and that the chromatin signatures re-organise as the cells differentiate. The epigenetic patterning differs on a gene by-gene basis, however their significance is implied upon disruption to normal development as the predictive pattern of modifications is lost.

Over-expression studies in cultured B cells showed that both TIS11b and TIS11d were able to block plasma cell differentiation in vitro. This action was specific as class switch recombination was not affected. TIS11b conditional knockout mice showed normal lymphocyte development and maturation and a normal humoral immune response. TIS11b conditional knockout mice lacked marginal zone B cells and had reduced numbers of peritoneal B1 cells. However they mounted a normal humoral response to immunisation with NP-KLH, with normal affinity maturation and a normal memory response. Single knockouts were inter-crossed to create double knockout (dKO) mice. Unexpectedly, close to 100% of dKO mice developed T-lymphoblastic leukaemia from three months of age. Pre-leukaemic mice showed arrested B cell development at the pro-B stage and significantly perturbed thymic development. This included the aberrant passage of thymocytes through the β-selection checkpoint in the absence of expression of a functional T cell receptor β chain. A genome-wide approach to identify deregulated TIS11b/d targets in pre-leukaemic mice showed elevated expression of the oncogenic transcription factor Notch1. Bioinformatic analysis revealed potentials binding sites for TIS11b and TIS11d within a highly conserved region of the Notch1 3’UTR. The ability of TIS11b and TIS11d to interact and suppress expression of Notch1 was demonstrated by electromobility shift assay (EMSA) and luciferase reporter assays. Furthermore, development and proliferation of T-ALL was Notch1 dependent, both in vivo and in vitro.

Injury, infection and tissue malfunction are triggers of inflammation which if not regulated may acquire new characteristics that result in pathological outcomes. Since innate immunity plays a key role in the resolution of acute inflammation knowledge of the regulation of this component of the host response is relevant to understanding processes in disease progression and therefore has potential clinical benefits. In this thesis I have applied zebrafish as a model organism to investigate the response of innate immune cells to qualitatively distinct inflammatory signals in the absence of adaptive immunity. Using a zebrafish embryo wound injury model I have investigated leukocyte migration profiles by in vivo imaging. In response to wound alone leukocytes migrated to the site of injury with predominantly random walk behaviour. However, the addition of lipopolysaccharide (LPS) enhanced recruitment and influenced the directionality of leukocyte migration to the wound. I demonstrate that leukocyte dynamic behaviour is also dependent on the location of the cells. The LPS enhanced directionality and reduced the random walk behaviour of the leukocytes, and these effects were ablated in the presence of the p38 mitogenactivated protein kinase (MAPK) specific inhibitor SB203580. Cytokine gene profiling in adult zebrafish leukocytes reveals that LPS can stimulate a pro-inflammatory response via the activation of p38 MAPK characteristic of mammalian innate immune responses. It is documented in mammalian innate immune cells that LPS can modulate Notch mediated signalling and thereby cell function. Using zebrafish with null mutations in Notch, which provide an unbiased in vivo model, I have investigated the influence of Notch signalling on leukocyte recruitment and demonstrate that migration to a wound injury is reduced. However, this effect is due to decreased cell numbers and not altered function as the Notch signalling inhibitor DAPT had no effect of recruitment to wound injury. The defect in myelomonocyte numbers was also present in adult zebrafish and this was partially compensated for by an increase in lymphocytes. The experimental results that I report here highlight zebrafish as a model 2 organism for studying the function and regulation of innate immunity. The unique optical translucency, which permits in vivo imaging of host responses in real-time, facilitates the analysis of the innate immune response to different inflammatory signals and immune modulators.

The thymic stroma primarily comprises epithelial, mesenchymal and endothelial cells, interspersed with those of haematopoietic origin. Thymic epithelial cells (TECs) are highly heterogeneous, but can be divided into two broad lineages, cortical and medullary, based on phenotype, functionality and location. A population of Plet1+ TEC progenitors have been identified which, when isolated from mouse E12.5 or E15.5 fetal thymus, reaggregated, and grafted, can produce a functional thymus. However, the potential of individual progenitors to form cortex and/or medulla is undefined. The main aim of this thesis was to use retrospective clonal analysis to ascertain the point during thymus ontogeny at which the cortical and medullary lineages diverge. To this end, I used transgenic mice carrying a ubiquitous ROSA26laacZ reporter gene (where a duplication within lacZ encodes non-functional b-galactosidase). Here, rare, random laacZ-lacZ genetic recombinations result in heritable expression of functional b-gal, producing labelled clones. As this occurs at a known frequency, determination of TEC numbers would enable calculation of the expected number of TEC clones present throughout ontogeny. Due to the lack of quantitative data on all thymic cell populations, I determined the size not only of TEC (lin-EpCAM+), but also haematopoietic (CD45+), mesenchymal (lin-PDGFRa+ and/or lin-PDGFRb+) and endothelial (lin-CD31+) populations from E12.5 until E17.5. I then showed that the absolute number of Plet1+ TECs remains constant during this time, although the proportion of Plet1+ cells in cycle decreases. From these collective data, I propose a model for the role of the Plet1+ population in thymus development, in which Plet1+ cells continually give rise to Plet1- TECs in a self-renewing manner. Finally, I present a ‘dual origin coefficient’ strategy for analysis of a library of prospective TEC clones. I calculated the number of TEC lacZ+ clones expected to be present throughout thymus ontogeny, selecting an appropriate developmental stage for analysis. Although I observed several clones of apparent mesenchymal origin, supporting a single origin for intrathymic and capsular mesenchyme at E15.5, I observed no TEC clones in this extensive analysis. The CpG content of the ROSA26 promoter suggests a possibility of methylation-induced silencing brought about by de novo methylation of the lacZ reporter gene.

The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.

The published articles [p. 268-278] are not available in the electronic version of this thesis due to third party copyright restrictions. The full version can be consulted at the University of Leicester Library.

The dramatic rise in the incidence of allergic/asthmatic disorders in the past three decades has placed a major socio-economic burden on global health care. Exocytosis of mediators causing allergic responses follows the binding, and subsequent receptor cross-linking by cognate allergen of immunoglobulin (lg) E antibodies to Fe-receptors (FcERI), expressed predominantly on mast cells and basophils. Fe-receptors have an invariant predominantly hydrophobic ammo acid motif (LFAVDTGL) in their transmembrane (TM) domain but contain a hydrophilic aspartic acid residue (D194). The function of this potentially energetically unstable residue in the TM of the human (hu) Fci,Rla subunit was targeted by transfecting the Rat Basophilic Cell line (RBL-2H3.1) with cDNA constructs encoding the gene for native and mutant huFcERia subunits and assessing receptor expression and signalling events. RBL-2H3 transfected with cDNA constructs encoding a medium-sized polar residue hufci,Rla (D194T) demonstrated the formation of a functional rat/human chimeric receptor complex which, when activated via hulgE and antigen, supports mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of ychain and Syk kinase. Transfection with mutant huFci,Rla subunit cDNA constructs encoding non-polar Ile (D1941) and V (D194V), larger sized polar Arg (D194R), smaller sized polar Ser (D194S) and non-polar Ala (D194A) abrogated surface expression of huFcERia and degranulation. An established RBL y-chain deficient cell line served as a model for assessing a- and y-chains interactions through the effect of mutations introduced into the y-chain. Compared to parental RBL-2H3 cells, the RBL y-chain deficient cells supported reduced levels of FcERI expression and mediator release. Transfection of RBL y-chain deficient cells with wild-type and mutant (T22A and T22S) y-chain cDNA constructs restored FctRI expression to levels observed in the parental cell line, but only partially restored mediator release, indicating a defective secretory response. Collectively this study identifies D194 in FctRia as a potential target for developing future anti-allergic drugs that act by inhibiting the FctRI signalling cascade.

The pre-T cell receptor (preTCR) is required for αβ T cell development and hence efficient adaptive immunity. Composed of a rearranged TCRβ chain paired with the invariant preTCR α chain (pTα), it drives immature thymocytes through the “β-selection” checkpoint, promoting differentiation, proliferation and survival. Although two functional splice isoforms of pTα exist, non-redundant roles for pTαa and pTαb have yet to be ascribed. This thesis demonstrates that pTαa and pTαb display non-identical expression patterns in immature thymocyte subsets. Moreover, it demonstrates that preTCRa and preTCRb promote divergent T cell development; preTCRa drives prolonged expansion of post-β-selection thymocytes, while preTCRb drives rapid maturation of TCRαβ(+) cells. Importantly, by mutating charged residues in the extracellular domain of pTα, it is shown that pTα oligomerization is not required for preTCR signalling per se, as had previously been proposed. Instead, the capacity for oligomerization regulates surface preTCR levels, which in turn dictates subsequent developmental potential. This thesis also presents preliminary evidence for a novel role for the preTCR; that preTCR signalling sensitises the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway that in turn regulates the signalling thresholds for positive and negative selection in CD4(+)CD8(+) double positive (DP) cells. Indeed, it is suggested that this process may be critical for central tolerance in the thymus. Finally, this thesis describes the generation and initial characterisation of pTαa and pTαb BAC transgenic mice that will facilitate the separate investigation of pTαa and pTαb in a pTα-deficient background in vivo. Collectively, the results presented in this thesis ascribe non-redundant roles for the two isoforms of pTα and necessitate a fresh examination of the mechanism by which the preTCR initiates signalling. The implications of these results for αβ T cell development and the immune system as a whole are discussed.

Recent research has revealed a role of NF-B in the resolution of inflammation. Using Cre-lox mediated gene targeting, IKK was selectively deleted in macrophages (IKKβ∆Mye). From in vitro studies, LPS stimulated IKKMye macrophages increased STAT1 phosphorylation, iNOS, MHC II and IL-12 production, suggesting negative cross talk between NF-B and STAT1 signalling pathways. Since IKK is required for TNF gene expression and TNF signalling, I investigated the hypothesis that TNF inhibits ‘classical’ macrophage activation through IKK activation. Macrophages from p55-/- and mice treated with anti-TNF antibody show increased STAT1 activation and IL-12 expression after LPS and IFN stimulation. BMDM infected with adenovirus expressing IKKβ dominant negative rescued the inhibitory effect of TNFα on IL-12p40 production, indicating TNFα inhibits IL-12p40 via IKKβ activation. Macrophages are antigen presenting cells while IL-12 and MHC II are critical factors for TH1 cell development. I thus investigate the inhibitory effects of IKKβ∆Mye macrophages in TH1 responses. FACS analysis showed higher MHC II, costimulatory molecules expression on IKKβ∆Mye macrophages after LPS stimulation. In a DTH model, recall assay has shown increased antigen-specific IFN production from IKKMye splenocytes compared to IKKβF/F splenocytes. Furthermore, IFN production was greatly enhanced by CD4+ OTII T cells co-cultured with IKKMye macrophages. Further analysis of CD4+ OTII T cells with qRT-PCR showed increased TH1 genes including IRF1, IFN, IL-12R1 and IL-12R2 and reduced TH2 marker IL-4. In addition to the enhanced antigen-specific T cell responses, IKKMye macrophages also increased anti-tumour immunity. Injection of H-Y positive MB49 tumour cells into IKKF/F and IKKMye female mice has shown tumour rejection, but no tumours were rejected after CD8+ T cells depletion, suggesting tumour rejection is associated with enhanced CTL activity. Taken together, these studies demonstrated the negative regulatory roles of IKK in macrophage activation and their impact to the innate and adaptive immunity.

The Life Marker Chip (LMC) instrument is an antibody assay-based system which will attempt to detect molecular signatures of Life in the Martian subsurface as part of the payload on board the European Space Agency (ESA) ExoMars mission rover, currently scheduled for launch in 2018. The LMC will have the ability to detect up to 25 different molecular targets of different origins that are associated with meteoritic in-fall, extinct or extant Life, prebiotic chemistry and spacecraft contamination. Regolith / crushed rock samples will be collected for the LMC by the rover and subjected to solvent extraction to extract organic molecules for analysis by the immunoassays. One of the key stages in the development of the LMC is the selection of antibodies to be used in the flight instrument. The challenge lies in the nature of the molecules or classes of molecules that are LMC targets and the need for antibodies that remain functional in the extreme conditions during a planetary exploration mission, especially the radiation environments. The work described within focuses on two main aspects of the search for LMC-relevant antibodies; the effect of space radiation on antibody performance [in the form of both ground-based and Low Earth Orbit (LEO)-set studies] and the development of ―customised‖antibodies against some of the molecules that are being investigated as potential LMC targets. The need to study the effects of space radiation on antibodies arose due to lack of any heritage of their use in interplanetary missions. For all the antibodies in the LMC, the ability to resist inactivation due to space radiation seen during a Mars mission will be a prerequisite. The objective of the ground-based radiation studies was to expose a number of LMC-relevant antibodies to simulated Mars mission radiation in the form of proton and neutron radiation which are the components of the mission radiation environment that are expected to have the dominant effect on the operation of the LMC. Cont/d.

Macrophages play an important role in both innate and adaptive immunity by engulfing intruding pathogens and damaged or infected cells, antigen presentation, and by secretion of immune mediators. Interleukin 10 (IL-10) is a potent anti-inflammatory cytokine mainly produced by macrophages in response to cell activation, which serves to suppress immune reactions and inflammation. Hypoxia refers to oxygen deprivation. It is a common condition found in pathological tissues. The relationship between hypoxia and the production of IL-10 by macrophage is not yet thoroughly understood. In previous unpublished work by Staples and Burke et. al., it was found that both basal and LPS-induced IL-10 mRNA and protein are reduced in hypoxia. In the present study, it was shown that the transcription factor Hypoxia Inducible Factor 1 (HIF-1) appears to be involved in this reduction of IL-10 production in hypoxia. Although the regulatory elements on the IL-10 promoter responsible for this blockage effect were not definitively identified, our results indicated that the activity of a -4kb IL-10 luciferase reporter adenovirus was significantly reduced in cells treated with the HIF-1 inducing agents, cobalt chloride and desferrioxamine. The activity of a -195bp IL-10 luciferase reporter adenovirus was also decreased in the HIF-1inducing agent treated samples. These data imply that either by indirect interaction or physically binding to the IL-10 promoter or gene, HIF-1 does play a role in blocking IL-10 expression in hypoxia. Sequence and transcription factor analysis indicated the presence of a HIF-1 consensus sequence hypoxia responsive element (HRE) located in the -2,171bp to -2,187bp position on the IL-10 promoter. This result suggests that HIF-1 may affect IL-10 production, at least in part, by physically binding to its promoter. Lastly, we showed that the effect of hypoxia on IL-10 expression is observed with a range of different toll-like receptor ligands, and is not limited to induction by LPS.

The immune system has long been attributed cognitive capacities such as "recognition" of pathogenic agents; "memory" of previous infections; "regulation" of a cavalry of detector and effector cells; and "adaptation" to a changing environment and evolving threats. Ostensibly, in preventing disease the immune system must be capable of discriminating states of pathology in the organism; identifying causal agents or ``pathogens''; and correctly deploying lethal effector mechanisms. What is more, these behaviours must be learnt insomuch as the paternal genes cannot encode the pathogenic environment of the child. Insights into the mechanisms underlying these phenomena are of interest, not only to immunologists, but to computer scientists pushing the envelope of machine autonomy. This thesis approaches these phenomena from the perspective that immunological processes are inherently inferential processes. By considering the immune system as a statistical decision maker, we attempt to build a bridge between the traditionally distinct fields of biological modelling and statistical modelling. Through a mixture of novel theoretical and empirical analysis we assert the efficacy of competitive exclusion as a general principle that benefits both. For the immunologist, the statistical modelling perspective allows us to better determine that which is phenomenologically sufficient from the mass of observational data, providing quantitative insight that may offer relief from existing dichotomies. For the computer scientist, the biological modelling perspective results in a theoretically transparent and empirically effective numerical method that is able to finesse the trade-off between myopic greediness and intractability in domains such as sparse approximation, continuous learning and boosting weak heuristics. Together, we offer this as a modern reformulation of the interface between computer science and immunology, established in the seminal work of Perelson and collaborators, over 20 years ago.

Evidence suggesting that Hsp72 is actively participating in cellular signalling as well interacting with immune system dynamics has been increasing. This is true in healthy, stressed and diseased cells but to different degrees. Modulation of the plasma membrane association and secretion in the extracellular environment by different types of stressors is the key event that leads to different degrees of immune system activation. Hence a better understanding of the mechanisms of Hsp72 secretion and association with plasma membrane is crucial. This thesis investigated the tissue source and mechanism of Hsp72 surface presentation to plasma membrane structures and release in relation with different cellular and physiological stressors. In vivo models confirmed that different tissue types determine specific Hsp72 responses following the same stress and increase serum Hsp72 dependant on intensity and duration of the stress. Diseases models confirm that Hsp72 responses in specific cell populations is related to disease progression, while in vitro models clearly showed that there are multiple mechanisms of secretion and surface presentation, dependent on the nature of the stressor as well as the intensity and duration. This observations clearly change the view of extracellular Hsp72 as a danger signal and lead to a revision of the original danger model. It also suggests that manipulation of Hsp72 translocation through the different pathways involved may prove effective therapeutically.

The early B cell response to antigen is analyzed, probing the mechanisms of divergent differentiation into plasmablasts or germinal centre (GC) B cells. Heterozygous QMxB6 mice, in which 5% of B cells are specific for the hapten 4-hydroxy-3-nitrophenol (NP), were immunized with NP-Ficoll. Varying the antigen dose altered the proportion of B cells that entered the extra follicular or GC responses and the amount of class switch recombination (CSR). The expression of phenotypic markers and switched antibody protein in parallel with gene array analysis and single cell real time RT-PCR in responding B cells was used to identify GC and plasmablast precursors and when and where CSR occurred. The results suggest CSR occurs in B blasts before GC B cells or plasmablasts emerge in both thymus dependent and thymus independent type II responses. The protein IRF4, which is essential for CSR and plasmablast differentiation, is expressed in all responding cells immediately after immunization and selectively upregulated to high levels in plasmablasts. A hierarchy of gene expression was identified as B cells differentiate into plasmablasts whereby high IRF4 mRNA expression precedes CD138 protein, which in turn precedes Blimp1 expression

The thymus is organised into distinct microenvironments, and trafficking through these regions enables thymocytes to receive essential signals for the generation of a diverse and self-tolerant T-cell repertoire. Thymic epithelial cells (TEC) represent a key stromal cell type during defined stages in T cell development, yet the mechanisms regulating their development are only partly understood. An ontogenetic approach was employed to study stages of cortical thymic epithelial cell (cTEC) development. This study identifies a previously unreported population of cTEC progenitors expressing CD205 and 5T and has defined distinct checkpoints in the development of the cTEC lineage. Furthermore, the importance of thymic crosstalk during specific stages of cTEC development and also the requirement for RANK-RANKL signalling for the development of various medullary thymic epithelial cell (mTEC) subsets has also been defined. Additionally, the importance of chemokine-mediated signalling for the establishment and compartmentalisation of the thymus has been highlighted by employing laser capture microdissection and studying thymus microenvironments in mice deficient for particular chemokine related signalling pathways. Overall this study has provided novel insight into the development of the thymic cortex and will help to understand how these cells become specialised in their ability to support positive selection of developing T cells.

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.

The intention of the work described in this thesis was to identify whether the Notch signalling pathway is utilized by antigen presenting cells in order to influence CD4+ adaptive immune responses. The notion that Notch proteins may be involved in polarising CD4+ T cells is relatively recent and most of the work that had been done in this area so far has concentrated on the consequences of Notch signalling within T cells. In contrast, the work that I have done has focussed on Notch ligand expression by antigen presenting cells and addresses the question whether Notch signalling is a redundant, necessary or irrelevant tool in the arsenal of antigen presentation.