Cytomegalovirus (CMV) infection is the most frequent and significant opportunistic infection in kidney transplant recipients. It is associated with direct (CMV disease) and indirect (rejection, poor graft survival) effects with resultant increases in morbidity and mortality. The mechanisms responsible for the indirect effects of CMV infection remain unclear. In this thesis, the indirect effects of cytomegalovirus infection in kidney transplantation are studied. Firstly, the mechanism of CMV infection is investigated. Secondly, the mechanism of CMV associated kidney transplant damage is explored. Thirdly, an assessment for the role of CMV in causing immunosenescence within the kidney transplantation cohort is undertaken. This thesis provides previously undescribed and direct evidence of immune hypo- responsiveness to latent CMV. I have shown CD4⁺CD27⁻CD28^null cells are pathognomonic of prior CMV exposure and have a role in glomerular endothelial cell damage, an effect which may be mediated by NKG2D. Higher CD4⁺CD27⁻CD28^null cell counts at 12 months post-transplantation predict a steeper decline in kidney allograft function thereafter. I provide novel insight into the ‘indirect’ effect of CMV in the pathogenesis of CD8⁺CD28^null cells. My study is the first to demonstrate a temporal association between elevated CD8⁺CD28^null cell frequencies and subsequent development of clinically relevant episodes of infection. The findings from this thesis set the scene for future interventional research and therapeutic strategies.

The innate immune system is a generic response to infection or injury. Evidence shows the innate response has immunological memory capable of altering subsequent responses to stimuli. Fibroblasts are ubiquitous stromal cells capable of responding to inflammatory triggers, and of orchestrating endothelial cell and leukocyte behaviour during inflammation. Repeated challenge with cytokines (such as tumour necrosis factor (TNF) a) induced an augmented second response to stimulation. Fibroblasts from multiple anatomical locales significantly increased cytokine secretion upon second challenge with TNFa. The precise mediators augmented depended on fibroblast site of origin. Depending on site, memory was inherent, or only present in fibroblasts from chronically-inflamed tissue. This suggests a phenomenon intrinsic to some sites but pathological in others. The secreted mediators from the fibroblast initial or memory responses exerted differing effects on leukocytes, dependent upon fibroblast site of origin. Finally, examination of intracellular signalling showed the augmented response was at least partly due to prolonged activity of nuclear factor (NF) KB during the memory response. Innate immune memory exists in fibroblasts from multiple tissues, but may be pathologically acquired in some. The altered response to second challenge may represent a fibroblast mechanism for altering the recruitment and behaviour of the inflammatory infiltrate.

The liver contains a number of tissue-associated lymphocyte populations, of which many have been implicated in the pathogenesis of chronic liver diseases. γδ T cells, particularly the Vδ2^neg subset, are known to comprise a substantial proportion of tissue-associated lymphocytes, although their immunobiology remains poorly understood. Here, the localisation, TCR diversity, immunophenotype and function of human intrahepatic γδ T cells was explored with an emphasis on highlighting any potential role in chronic liver disease and also to further understanding of tissue-associated γδ T cells, using the liver as a model tissue. Intrahepatic γδ T cells were predominantly localised in the sinusoids and did not increase in frequency with chronic inflammation. Vδ2^neg cells exhibited private TCR clonal focussing, with complex CDR3 regions suggestive of antigen-driven expansions, concordant with a loss of naive-like CD27^hi cells present in the periphery. Expanded clonotypes were phenotypically T_EM- or T_EMRA-like, with T_EMRA-like clonotypes shared between liver and blood and resembling vasculature-associated virus-specific CD8⁺ T cells while T_EM clonotypes were identified only in the liver and resembled tissue-resident CD8⁺ T cells. These findings suggest that disease has minimal impact on intrahepatic γδ T cells, while supporting an adaptive paradigm for these cells in the formation of tissue-associated subsets.

Known for over a hundred years, the human fungal pathogen, Cryptococcus neoformans causes cryptococcosis, a life threatening disease. Infection is acquired through inhalation of spores or dried yeast cells into the lungs from which the fungus can potentially transmit to all body parts of which the brain is the most affected organ. Once in the brain, the yeast C. neoformans causes meningoencephalitis, a fatal condition even with optimal treatment. The mechanism by which C. neoformans penetrates the normally impermeable blood brain barrier (BBB) to cause brain infection is not understood. This thesis presents two aspects of investigation: 1) the extent to which binding and uptake of cryptococci by brain microvascular endothelial cells (BMEC) explains transcytosis as a mechanism for cryptococcal traversal of the BBB and 2) the relationship between Cryptococcus – macrophage interaction and Cryptococcal meningoencaphalitis (CM) disease. We show that adherence and internalization of cryptococci by brain microvascular endothelial cells is a rare event characterized by a small number of cryptococci, an indication that C. neoformans most likely uses multiple routes to traverse the BBB. Secondly, by studying clinical isolates from cerebral spinal fluid (CSF) of HIV- associated CM patients, we demonstrate that high rate of cryptococci uptake by macrophages is associated with patient fungal burden whilst the intracellular proliferation rate is inversely associated with TNF- \(\alpha\) levels in the patient CSF. Interestingly, the high uptake – high fungal burden isolates were less encapsulated but more rapid melanin formers, traits known to modulate phagocytosis and protection from host-induced oxidative stress respectively. We therefore hypothesize that highly phagocytosed C. neoformans strains use phagocytes to disseminate faster to the brain resulting in high fungal burden.

Salmonella typhimurium is one of the most common causes of bacteraemia in children in sub-Saharan Africa and is prevalent in HIV-infected individuals. However, symptoms of this systemic infection are unclear, and while fatalities are frequent, how infection kills is unknown. Here we use a mouse model of systemic (but resolving) infection to investigate physiological and immunological aspects of the host response to infection. The liver is colonised during systemic infection, and in the model used, bacterial numbers peak at day 7 and are largely resolved within a month. Inflammatory lesions, consisting of multiple leukocyte populations, develop within the liver. These persist and are more severe once bacterial clearance is established. Whilst lesions can develop in the absence of T and B cells, these cells contribute to the regulation of inflammatory foci. In the absence of interferon-γ, lesions do not develop and inflammation in the liver is largely absent. In parallel, extensive platelet thrombosis occurs in the liver venous system and the shared kinetics with lesion formation suggest these phenotypes may be co-regulated. Here we describe how parenchymal and vascular inflammation are anchored by inflammatory up-regulation of podoplanin expression in the liver. Thrombosis is substantially abrogated in the absence of C-like lectin-type receptor-2 (CLEC-2) expression on platelets and we show that podoplanin (the physiological ligand for CLEC-2) expression on clodronate-sensitive myeloid populations is necessary for thrombus development. Therefore, the parallel association between inflammation and platelet activation could be the basis for developing novel treatments for systemic bacterial infections in humans.

The bacterial pathogen Chlamydia trachomatis causes Trachoma, the worlds leading cause of preventable blindness and is also responsible for the most common curable sexually transmitted disease in the UK and United States. C. trachomatis is an obligate intracellular organism characterised by a unique and complex growth cycle. Its study presents many challenges since it has historically been recalcitrant to genetic manipulation and growth in the absence of a host cell. Nevertheless, the sequencing of the C. trachomatis genome and its relatively small size by comparison to genomes from other bacterial pathogens, has paved the way for studies at the proteomic level. This thesis describes the development and application of proteomic approaches to study C. trachomatis L2. To survey the expressed chlamydial proteome, a combination of the qualitative approaches, 2-DGE, MudPIT and GeLC-MS/MS; and the quantitative approaches AQUA, iTRAQ and LC-MSE were used. Collectively, the approaches efficiently identified 648 expressed proteins, representing ~72% of the predicted proteome of C. trachomatis L2, from both the infectious (elementary body, EB) and replicating (reticulate body, RB) form of the pathogen. In the infectious EB, the entire set of predicted glycolytic enzymes were detected, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. Further, proteomic analysis of the RB form also uncovered biosynthetic enzymes for chlamydial cell wall synthesis, indicating that peptidoglycan is produced in some form during growth in host cells. Comparison of the quantitative approaches iTRAQ and LC-MSE demonstrated that LC-MSE quantitative data was significantly more robust and extensive relative to iTRAQ data. In addition to information on relative amounts of these proteins between the two forms, LC-MSE data also yielded the cellular concentration (molecules per cell) for 489 proteins. This extensive set of absolute quantitation data permits estimates of the energy invested in the synthesis of various classes of proteins. The results indicate that C. trachomatis devotes most of its energy into maintenance of the translational machinery. However, it also expends significant amounts of energy into making cell envelope components and a set of hitherto hypothetical proteins. These proteins, which account for the bulk of the energy invested by the intracellular RB form of the pathogen as it converts to the extracellular EB form, highlight the importance of absolute quantitation data for understanding the biological processing status of the cell. The datasets also revealed a large number of proteins that were differentially expressed between replicating RBs and infectious EBs, ranging from 8.4-fold down-regulation to 3.5-fold up-regulation. Consistent with transcriptomic studies (Belland et al., 2003), proteins involved in protein synthesis, ATP generation, central metabolism, secretion and nutrient uptake were predominant in the metabolically active RB at 15 h PI. Although many of the proteins in these functional categories were down-regulated in EBs, proteins required for glycolysis, central metabolism, protein synthesis, and type III secretion were present in significant amounts in EBs suggesting that the infectious EB is primed ‘ready-to-go’ upon contact with the host cell.

Gangliosides, complex sialic acid-containing glycolipids, and other glycosphingolipids are active physiological membrane components with an array of functions in development and disease. Altered profiles are found in many disorders including those of neurological and cancerous aetiologies. Glycosphingolipids are also important for lateral membrane organization, cell communication and as binding sites for extra-cellular components. These lipids have long been implicated as targets in autoimmune diseases such as Guillain-Barré Syndrome (GBS) and Multifocal Motor Neuropathy. In GBS, auto-antibodies bind native membrane gangliosides signalling immune-mediated breakdown of nerves causing acute flaccid paralysis. While the fundamental pathology is understood, differences in clinical presentation, and preference for motor over sensory nerves, have yet to be explained. Understanding the precise nature of native gangliosides, including low abundance species and modifications, is an important first step. Meanwhile genetically engineered mouse models are under development that should increase our understanding of disease pathogenesis. To be truly functional it is essential these models contain a full range of complex and simple glycosphingolipids in the neurological tissue. Mass spectrometry has recently been applied with great effect to lipidomics; the comprehensive profiling of all lipids involved in a system. However, heavily glycosylated, low abundance and chemically unusual lipids such as the gangliosides tend to be neglected in otherwise thorough lipidomic studies. It was the aim here to optimise separation and mass spectrometry methodologies for ganglioside analysis. Workflows were developed for high performance thin layer chromatography (HPTLC) combined with direct imaging mass spectrometry (IMS) detection and identification, and for high performance liquid chromatography (HPLC) with online high resolution mass spectrometry detection and identification with dissociation to confirm structures (MSMS). A range of lipid standards were analysed using this second method to build a database of characteristic ionization behaviour, retention times, and product ion spectra to aid the analysis of unknowns in complex mixtures. Methods were then applied to molecular phenotyping in novel mouse models of GBS, and to glycosphingolipidomics in peripheral sensory and motor nerves. Finally the recently developed technique of imaging mass spectrometry, using matrix assisted laser desorption ionisation (MALDI) and secondary ion mass spectrometry (SIMS) ion sources, was investigated for its capability for direct ganglioside analysis in brain and spinal cord tissue sections. Results are presented below demonstrating the significant benefits of the mass spectrometry-based workflows over more conventional profiling methods as well as comparing and contrasting the two techniques developed here. Limitations and potential areas for future development are debated. Findings from profiling knockout and rescue mouse models and from single nerve glycosphingolipidomics are discussed along with further experiments and directions for these studies. The discovery of a full range of complex gangliosides in neurological tissue from rescue mice, albeit at low levels compared to the wild type, confirmed their molecular usefulness for modelling neurological autoimmune diseases. The sensitivity and reproducibility of the mass spectrometry technique enabled relative quantitation, revealing details into the abundance of different ganglioside species and inclusion of ceramide structures in each mouse type. The ability to detect very low abundance lipids with an additional dimension of structural description also suggested that O-acetylation of the second sialic acid on native disialylated lipids is more prevalent than previously thought. Finally imaging mass spectrometry results are presented. Although sensitivity was limited, both simple and complex gangliosides were detected in spinal cord sections; the first known IMS detection of these lipids outside of the brain. Results also demonstrate the abundance of parallel lipidomic information that can be obtained using these methods. Possible solutions to increasing the sensitivity limit are discussed that may increase IMS usefulness to glycosphingolipid studies in future.

Natural killer (NK) cells reconstitute rapidly following allogeneic stem cell transplantation (allo-SCT) at a time when alloreactive T cell immunity is being established. Important differences are seen in the patterns of reconstitution between T cell deplete, T cell replete and umbilical cord stem cell transplants. 82 patients who received T cell-deplete allo-SCT were studied to determine the functional and transcriptional profile of the reconstituting NK cells and to assess the relationship with clinical outcome. NK cells at day 14 (D14-NK) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. D14-NK cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of clinical outcome and suggest NK cells may suppress development of the T cell-mediated alloreactive immune response through production of IL-10.

The liver has a unique connection with the immune system; harbouring vast numbers of lymphocytes, able to instigate secondary lymphoid organ-independent naive T cell activation, and promoting potent immune tolerance. We set out to determine the effect of this unique microenvironment on the biology of CD4+ T cells at three key interaction points: following migration into the parenchyma, after short-term hepatocyte contact, and at long-term tissue-residency. Modelling transmigration through hepatocytes revealed intrinsic, disease-specific cytokine responses in blood-derived CD4+ T cells, not discernible through static co-culture. However, short-term co-culture did induce activation-independent CD69 upregulation, reliant upon cell-cell contact. This phenotype mimicked the similar hepatic CD4+ CD69INT cells that we discovered in liver tissue. Unlike CD69HI cells which represented the tissue-resident memory T cells (TRM) of the liver, CD69INT cells were the most activated population, likely able to migrate to many liver and gut niches, and singularly able to produce IL-4 and IL-10. By contrast, CD69HI TRM displayed a resting phenotype, marked for more restricted movement, and produced the best multifunctional TH1 responses following stimulation. These data demonstrate the importance of studying migration, and provide detailed characterisation of CD69HI TRM and novel CD69INT cells, along with their proposed roles and generation pathways.

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