Background: Antigen presenting cells (APCs) such as macrophages play a crucial role in orchestrating immune responses against foreign materials. The activation status of macrophages can determine the outcome of an immune response following implantation of synthetic materials, towards either healing or inflammation. A large range of biomaterials are used in the fabrication of implantable devices and drug delivery systems. These materials will be in close contact with APCs and characteristics such as surface chemistry may have a critical role in polarising macrophages towards pro- or anti-inflammatory immune phenotype. Each phenotype can be characterised by their cytokine profile, transcription factors, surface markers or even morphology. Objectives: The overall objective of this study was identifying novel chemistries that are able to induce differentiation of human monocytes towards macrophages with distinct pro or anti-inflammatory phenotypes. To achieve this, a combination of different surface chemistries has been generated using oxygen plasma etching as well as acrylate and acrylamide polymer libraries. Methods: Fluorescent microscopy, real time-PCR, multiplex assay, ELISA, macrophage phagocytic activity were used for macrophage phenotype identification. Libraries of acrylates and acrylamide polymer microarrays (first generation microarray of 141 polymers and second generation of 442 polymers), and oxygen plasma etching of polystyrene used as two different techniques for making different surface chemistries. CellProfiller software was used for analysing images and was used for machine learning for phenotype identification. Results: polystyrene with highly hydrophobic surfaces are shown to suppress expression of M1-associated surface markers and cytokines while promoting M2-associated markers. However, highly hydrophilic surfaces seem to have the opposite effect as evidenced by promoting M1-associated marker expression and pro-inflammatory cytokine production while suppressing M2-associated marker expression and anti-inflammatory cytokine production. Also, the protein thickness was proportional with the hydrophilicity of the surface, which had impact on cell polarisation. Furthermore, co-polymers 157 from the second generation array was the most M2 biased polymer among the first and second generation of microarray polymers by induction of MR (M2 marker) cell expression, while co-polymers 217 and 123 from the second generation had impact to increase calprotectin (M1 marker). Also, cell adherence and morphology were affected by polymers surface chemistry. Conclusion: Surface chemistry without using polarising cytokine can polarise macrophage towards pro-inflammatory and anti-inflammatory phenotypes.

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.

PRH is a transcriptional repressor that regulates development of the haematopoietic and vascular systems. VEGF is a mitogen that stimulates cell survival via cell surface receptors including VEGFR1 and VEGFR2. This thesis identifies Vegf, Vegfr1, and Vegfr2 as bona fide PRH target genes and shows PRH can control cell survival through the modulation of VEGF and VEGF receptor signalling. CK2 is a stress-activated protein kinase with pleiotropic activity and CK2 phosphorylation of PRH inhibits its DNA binding activity. Here I show that CK2 can antagonise PRH induced cell death and transcriptional regulation. Furthermore I show that CK2 reduces PRH stability and decreases nuclear retention of PRH. The oncogenic BCR-ABL fusion protein increases CK2 activity. I show that Inhibition of BCR-ABL in CML cells results in decreased PRH phosphorylation and the down-regulation of PRH target genes. Reestablishment of gene control by PRH is partly responsible for the therapeutic effects of BCR-ABL inhibition in CML and that the reestablishment of PRH function could be valuable for the treatment of leukaemias with elevated CK2 activity. These data show that PRH is a key regulator of the VEGF family and loss of PRH transcriptional activity, through elevated CK2, has a role in leukaemogenesis.

RhoJ is an endothelial expressed Rho GTPase that localises to focal adhesions and regulates cell migration and tube formation. Previous work in our laboratory determined that RhoJ activation negatively regulates focal adhesion (FA) numbers, and interacts with the FA proteins GIT1 and β-PIX. The studies presented in this thesis aimed to characterise the role of RhoJ in modulating FA dynamics, further investigate its interactions with the GIT/PIX complex, and generate a RhoJ knockout mouse to determine its function in vivo. Silencing RhoJ led to prolonged FA disassembly in migrating human umbilical vein endothelial cells (HUVEC), while adhesions in cells expressing a dominant active (da) mutant of RhoJ disassembled more rapidly. Interactions between daRhoJ and GIT1, GIT2 and β-PIX were identified, and the localisation of any member of the RhoJ/GIT/PIX complex to FAs was found to be dependent on each of the other components. daRhoJ expression increased levels of GIT2 protein, while silencing RhoJ reduced GIT2 Tyr 392 phosphorylation. In vivo, the growth of subcutaneous tumours was reduced in RhoJ knockout mice compared to wild type controls. In conclusion, the data presented in this thesis show that RhoJ regulates FA disassembly, most likely in concert with the GIT/PIX proteins. This in turn influences endothelial cell migration and ultimately angiogenesis.

Thymic microenvironments act to control T-cell development and selection. While thymic epithelial cells are key regulators of these processes, the thymus also contains multiple accessory cells that influence its function. Considering this, the major aim of this thesis is to examine the roles of dendritic cells and eosinophils in thymus biology. As regulation of thymic DC has previously been correlated to the thymic medulla we focused on the medullary regulator LTβR and generated cell type specific LTβR deficient mice. We found LTβR signalling regulates mTEC in a cell intrinsic manner, independent of thymic DC maintenance. Thymic DC alternatively require LTβR signalling on thymic mesenchyme for their regulation. Additionally, disruption of the medulla in Ltbr-/- mice was distinct from tolerance breakdown, which we instead found to correlate to reduced DC and impaired negative selection. Further we found the CCR7 ligand CCL21 was also required to control thymic DC, suggesting a link between these two pathways. Finally, dblGATA mice were used as a model of eosinophil deficiency and we identify a role for eosinophils in thymus recovery following sublethal irradiation damage. Collectively these findings shed new light on accessory cells during key aspects of thymus tolerance and regeneration.

Autotransporters represent a diverse family of virulence effectors that are secreted from Gram-negative bacteria by the Type V Secretion System. Their initial description coined the term ‘Autotransporter’ to embody the notion that their three-part architecture governs their navigation through the bacterial cell envelope. The Pet cytotoxic autotransporter is secreted by the diarrhoeal pathogen, Enteroaggregative Escherichia coli (EAEC) and was used as a model to study autotransporter biogenesis. Following a global transposon mutagenesis of EAEC, novel accessory factors were identified that are required for Pet biogenesis, including the transcription factors CRP and Fis, periplasmic chaperones and components of the β-barrel assembly machinery (BAM) complex. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation and show that this co-activation mechanism extends to functionally similar autotransporters. Furthermore, we highlight novel components of the BAM complex required for AT secretion. This work builds on previous studies that, in recent years, have challenged the ‘auto’ nature of this secretion process causing a paradigm shift towards a much more complex mechanism of AT secretion than initially suggested.

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

Haemopoietic Stem Cell Transplant (HSCT) is used as a treatment for a number of conditions particularly leukaemias. Following conditioning and HSCT, there is a ‘resetting’ of the immune system, which reconstitutes over a number of months. Graft-versus-Host Disease (GvHD) is a life-threatening complication of HSCT that includes severe, sight-threatening dry eye disease. In GvHD transplanted immune cells mount an immune response against the host. This thesis investigated how the immune cells of the conjunctiva are affected by HSCT and how the ocular surface leukocytes reconstitute. A non-invasive technique, ocular surface impression cytology (OSIC), was used to demonstrate that whilst there was no apparent depletion of innate immune cells in the conjunctiva, there was a marked reduction in the lymphocytes, which gradually reconstituted, returning to normal levels at the 6 months timepoint. Secondly OSIC was used to profile the leukocyte population in a cohort of patients post-HSCT with and without eye disease. In patients with dry eye disease following HSCT, the conjunctiva contained increased CD8+ lymphocytes, macrophages and neutrophils; a pattern that was distinct to that found in patients with dry eye disease following HSCT.

The humoral immune response is characterised by the production of antibody secreting B cells. Some of these cells have cycled through the germinal centre, diversifying and optimising their antigen receptors to produce affinity-matured, class switched antibody. As this is a relatively slow process, the first class switched antibody is produced by non-mutated B blasts that have differentiated independently of the germinal centre reaction outside B-cell follicles. Extrafollicular foci of plasmablasts provide the first line of defence within the adaptive antibody response. IRF4 has been shown to be essential for Ig class switching and plasma-cell differentiation. Two expression levels of IRF4 were reported, with intermediate levels proposed to regulate CSR in the GC, while high levels regulate plasma-cell differentiation. We have correlated the two phases of IRF4 induction with specific stages of B-cell differentiation. Following immunisation of quasi-monoclonal mice with NP-Ficoll, intermediate levels of IRF4 protein are expressed by all B blasts as they move to the outer T zone and before the formation of germinal centres or plasma cells. This is followed by expression of AID and CSR. In contrast, plasma-cell differentiation occurs with high level expression of IRF4, expression of Blimp1 and complete suppression of AID and CSR. The NFκB family signalling molecules C-REL and NFκB1 have been shown to be required for the induction of IRF4 protein in B cells following stimulation. We show that these signalling molecules are not necessary to induce the early rapid intermediate level expression of IRF4, and extrafollicular expression of AID and CSR occur normally when they are absent. IRF4-high expression and plasma-cell differentiation, however, are blocked in the absence of these molecules.

The thymus is a heterogeneous mix of hematopoietic and stromal cells that function to generate a functional, self-tolerant T-cell pool. Although many of these populations are well studied, the role of non-epithelial stroma remains unclear. Thymic mesenchyme has been identified as an important regulator of T-cell egress. Studies of lymphotoxin beta-receptor LTβR have revealed its critical role in T-cell egress as well as the development and function of lymph node mesenchyme. We hypothesized that LTβR regulation of thymic mesenchyme is critical forT-cell egress. To test this we generated \(Wnt-1^{cre}Ltbr^{flox}\) mice to delete LTβR on thymic mesenchyme and revealed this to be non-essential forT-cell egress. Moreover, we generated \(Foxn-1^{cre}Ltbr^{flox}\) mice to delete LTβR on thymic epithelial cell (TEC). Despite the critical role of LTβR in medullary TEC development, T-cell egress was normal. However, deleting LTβR on thymic endothelium using \(Flk-1^{cre}Ltbr^{flox}\) mice revealed an essential role of LTβR regulation of endothelium to control T-cell egress. Our analysis also revealed that T-cell entry into the perivascular space during T-cell egress occurs stochastically. Collectively our findings highlight a novel role for LTβR regulation of thymic endothelium as a critical pathway of T-cell egress.