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Lipopolysaccharide (LPS), also known as endotoxin, is a constituent of the outer membrane of gram-negative bacteria which is toxic for humans and other animals. LPS probably plays a key part in the pathogenesis of Gram-negative bacteraemia and sepsis syndrome in humans. Cross-reactive antibodies to LPS may play a part in natural host defences, and may also be useful in the treatment of Gram-negative bacteraemia and sepsis syndrome. The structure of LPS, its toxicity, its role in Gram-negative bacteraemia and sepsis syndrome in humans, and the potential value of cross-reactive antibodies to LPS are reviewed. The antibody response in recipients of typhoid vaccine was studied, with particular reference to the possibility that typhoid vaccine might induce the production of antibodies to the core region of LPS (LPS-core). In most recipients however the response observed was directed against specific antigens. Urine samples from patients with suspected UTI were tested for IgG antibodies to LPS-core. Such antibodies were found to be associated with the presence of bacteriuria, although the association was not strong enough for antibody assay to be useful as a diagnostic test. Total urinary IgG was equally strongly associated with bacteriuria. This suggested that the antibodies were probably present because of non-specific leakage of serum components into the urine as a result of inflammation. A large number of murine monoclonal antibodies (MAbs) to LPS-core had been produced by a collaborative group in Edinburgh and Basel, in the hope of producing a cross-reactive MAb which would be useful therapeutically. The binding of some of these MAbs to a collection of blood-culture isolates of Gram-negative bacteria was studied.

Regulatory T cells (Tregs) play an important role in immune regulation. Their development in the thymus requires interaction of their TCR with self-peptide-MHC and the induction of Foxp3. The downstream signals from the TCR that lead to commitment to the regulatory lineage and subsequent up-regulation of Foxp3 are unclear. The development of regulatory T cells has been shown to occur at the DP stage of thymocyte development where positive and negative selection occurs and it is thought that Tregs differentiate from positively selected thymocytes. The three classical MAPK cascades and their targets have been implicated in both positive and negative selection. Thus they represent an opportunity to gain further insights into the development of regulatory T cells. Here I have compared the requirements of positive selection and regulatory T cell development through the use of knockout and transgenic animals defective in Raf signalling, and components of the SRF regulatory network including the ternary complex factors (TCFs) and SRF itself. Whilst the TCF deficient mice display severe defects in positive selection, Treg development was unimpaired. However depletion of SRF resulted in a complete block in positive selection and Treg development suggesting that positive selection consists of both TCF-dependent and TCF-independent events. Inhibition of Raf signalling by the dominant interfering DN Raf derivative reduced both Foxp3+ and Foxp3" CD4+ populations. TCR crosslinking efficiently induced ERK activation in regulatory T cells but induction of the TCF target gene was impaired. Nevertheless, both TCF-deficient and DN Raf CD4+CD25+ Tregs effectively suppressed CD4+CD25" T cell proliferation in vitro. Finally SAP-l"7" CD4XD25" Tregs are functional in an in vivo model of colitis. Thus the signalling requirements for development of Tregs in the thymus are distinct from those required for conventional T cell positive selection.

Proteolysis of NF-kB1 p105 is vital for its function as a precursor to p50 and as an IkB. This occurs in two ways, both mediated by the proteasome. A constitutive proteolytic removal of the p105 C-terminus, termed processing, generates the mature transcription factor p50. In contrast, a signal-induced p105 proteolysis is triggered by phosphorylation of serines 927 and 932 in the p105 PEST region by the IKK complex. This promotes p105 poly-ubiquitination and subsequent complete degradation. IKK-induced p105 proteolysis has been demonstrated to regulate the kinase activity of the MAP3K TPL-2, since all detectable TPL-2 is found in a complex with p105. Furthermore, NF-kB1 p105 retains Rel subunits in the cytoplasm via interaction with the p105 C-terminal ankyrin repeat region. However, it is unclear whether IKK-induced p105 proteolysis contributes to NF-kB activation, though this process would be expected to release Rel subunits to translocate into the nucleus. A large body of evidence exists to suggest a major role for NF-kB in T cell development and function. To investigate the significance of IKK-induced p105 degradation in T cells, a knock-in mouse strain, Nfkb1S927A'S93ZA, in which serines 927 and 932 of NF-kB1 p105 were mutated to alanine residues was analysed. Previous work has shown that constitutive processing of pio5S927A S932A to p50 occurs normally, but this mutated p105 is refractory to IKK-induced proteolysis. Work presented here demonstrates that whilst p105 mutation did not affect thymic differentiation of CD4+ and CD8+ T cells, numbers of CD4+CD25+ regulatory T cells, memory-phenotype CD4+ T cells and thymic NKT cells were significantly reduced. Analysis of BM chimeras revealed cell autonomous and non-haematopoietic defects required for generation of these sub-populations. In vitro experiments indicated that TCR-induced proliferation was significantly impaired in /Vflcb7S927A S932A CD4+ T cells, due partly to reduced interleukin-2 production. In contrast, p105 mutation had no effect on CD4+ T cell survival. These defects were not due to a lack of TPL-2 activity, based on analysis of TPL-2-deficient mice. This study presents evidence to suggest a critical role for IKK-induced p105 proteolysis in regulating NF-kB activation in T lymphocytes.

The peripheral T cell compartment is maintained at a constant size, resulting from a balance of cell development, survival, proliferation and death. Transmission of signals through the TCR and IL-7R on the T cell surface is involved in regulating all these processes, however the precise manner in which these signals together maintain T cell homeostasis is unclear. To investigate the contribution of TCR and IL-7 signals to naive T cell homeostatic responses we established two model systems. To specifically address the role of homeostatic TCR signalling in the development and maintenance of the T cell compartment, we generated transgenic mice that conditionally express the Syk family tyrosine kinase Zap70. The transduction of TCR signals by Zap70 is essential for thymic development and T cell activation. Given the importance of Zap70 expression in T cell antigen receptor signalling, we investigated whether Zap70 was also essential for the transmission of TCR signals, required for the steady state survival of the peripheral naive T cell compartments. Zap70 deficient mice exhibit a complete block in thymopoiesis at the DP stage in the thymus and as a consequence lack mature peripheral T cells. For this reason we generated mice that express Zap70 in a conditional manner, using the tetracycline responsive gene regulatory system. Thymic selection proceeded normally in these mice, however ablation of Zap70 expression resulted in the disappearance in the peripheral naive CD4+ and CD8+ T cells, with the naive CD8+ T cell compartment appearing most affected by the loss of Zap70 expression. This data suggests an important role for Zap70 signalling in the transmission of homeostatic TCR survival signals. Unexpectedly we also found that TCR signals transmitted by Zap70 had the capacity to influence IL-7R expression and importantly revealed a novel role for positive selection signals in the regulation of peripheral IL-7R expression and therefore the competitive fitness of peripheral T cell clones. The second model system examined the homeostatic responses of class I MHC restricted F5+/+ TCR transgenic Rag1A CD8+ T cells following transfer into MHC class I or IL-7 deficient hosts, to specifically quantify the contribution of TCR and IL-7 signals in naive T cell homeostasis. Our data reveals that IL-7 signals are more essential than homeostatic TCR signals for the survival of the naive T cell compartment in conditions of lymphopenia. Interestingly we also demonstrate that IL-7 may exert part of its homeostatic effects by modulating TCR engagement with MHC ligands by regulating T cell - APC interactions in vivo. In conclusion the data presented in this thesis confirms that TCR and IL-7 signals are essential for naive T cell homeostasis, but also reveals extensive cross-talk between homeostatic TCR and IL7 signals in the control of peripheral T cell homeostasis.

The default pathway for developing thymocytes is apoptosis they must be actively instructed to survive and proliferate. Approximately 95% of developing thymocytes undergo apoptosis only 5% survive to enter the periphery. Thymocytes commit to apoptosis either because of a lack of survival signalling (death by neglect), or by encountering high avidity T cell receptor signalling that might predicate potential autoimmunity (negative selection). We have performed two Affymetrix DNA microarray screens to attempt to identify candidate regulatory genes for thymocytes undergoing apoptosis in response to the synthetic glucocorticoid dexamethasone and in the F5 TCR Rag-1"7" Tap-1"" transgenic model for antigen-mediated negative selection. The up-regulation and expression of genes identified in these screens has been confirmed by qPCR. The action of these genes was assayed in both foetal thymic organ culture (FTOC) and OP9-Delta-Like 1 (OP9-DL1) thymocyte co-culture systems. We have used modified pMSCV (murine stem cell virus) over-expression and knockdown retroviral vectors to infect haematopoietic progenitor cells (HPCs). These cells were then used to reconstitute depleted FTOCs or placed into culture on OP9-DL1 stromal cell layers. Transduced HPCs then differentiate into thymocytes, allowing us to assay the effect of target genes upon apoptosis and the T cell development program. We assayed target genes from both screens for their effect on thymocyte apoptosis in the presence and absence of apoptosis inducing agents, and have identified several that may be critical in the context of thymocyte apoptosis. One gene, Tnfaip8, has been shown to markedly enhance apoptosis in response to dexamethasone, and to inhibit apoptosis when knocked down using RNA interference. This gene is both up-regulated at the double negative (DN) to double positive (DP) transition and highly up-regulated by glucocorticoids. We have also demonstrated that Krox24, a highly induced gene in our F5 TCR7" Rag- lv" Tap-1A antigen screen, appears to have a role in enhancing apoptosis induced by negative selection in over-expression studies.

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.

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.

Human CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidised low density lipoprotein (oxLDL) and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation and ten putative N-linked glycosylation sites situated in the large extracellular loop of the protein have been identified, however their utilisation and role in the folding and function of the protein have not been characterised. Using mass spectrometry on purified and PNGaseF-deglycosylated CD36, and also by comparing the electrophoretic mobility of different glycosylation-site mutants, this study determined that nine of the ten sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells, established that glycosylation is necessary for trafficking to the plasma membrane. Minimally-glycosylated mutants that supported trafficking were identified and indicated the importance of carboxy-terminal sites N247, N321 and N417 and amino-terminal sites N102 and N205. However, unlike the related mouse scavenger receptor SR-BI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally-glycosylated mutants appear to be predominantly core glycosylated indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified LDL.

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