Expression of the vitamin D receptor on mononuclear phagocytes renders them susceptible to modulation by 1,25(OH)2D3, which is metabolised from its precursor 25(OH)D3 by the enzyme CYP27B1. Modulation of dendritic cells (DC) with 1,25(OH)2D3 has been shown in vitro to suppress their ability to prime proinflammatory T cells responses, suggesting extra renal synthesis of 1,25(OH)2D3 from the precursor 25(OH)D3 may promote tolerance in vivo. I sought to gain a greater mechanistic understanding of these phenomena at molecular, cellular and intercellular levels in the human system. I found that 1,25(OH)2D3 stimulation of DC results in a selective preservation of NFκB-mediated transcriptional responses to LPS, although these gene expression changes were diminished in magnitude. I also found enhanced p38 signalling in 1,25(OH)2D3 treated ex vivo monocytes, as well as those from 25(OH)D3 supplemented vitamin D deficient patients. 1,25(OH)2D3 suppresses expression of genes relating to antigen presentation which can be rescued by IFNγ stimulation. This treatment also results in a rescue of phenotypic maturation in DC in response to LPS, but without rescue of T cell activation. Preliminary experiments suggest there may be a defect in MDDC-T cell contact formation and maintenance. I find that DC are incapable of synthesising 1,25(OH)2D3 sufficient to inhibit their maturation, unlike macrophages which are able to bring about gene expression changes in DC by paracrine activation. This is due to reduced expression of CYP27B1 protein in DC due to significant expression of truncated transcript, which can be rescued by LPS stimulation. DC also readily induce CYP24A1 to degrade bioactive 1,25(OH)2D3 to limit VDR-driven transcription. Overall macrophages are a possible physiological source of 1,25(OH)2D3, and as such sufficient 1,25(OH)2D3 to inhibit DC activity will only arise after initiation of adaptive immunity and monocyte recruitment. Overall, my thesis provides deeper mechanistic insight into the role of vitamin D in immunomodulation of DC with regard to their interactions with T cells in the human system.

Retroviruses possess an RNA genome that is reverse transcribed into DNA and becomes integrated into the host genome during infection. Human immunodeficiency virus 1 (HIV-1) belongs to the genus lentivirus of the retroviridae family and is a major human pathogen with more than 50 million infected people worldwide to date. Lentiviruses have very narrow host ranges. Cross-species lentiviral transmissions are rare, due to the ability of mammals to counteract viral infections by the innate immune system, which includes intracellular factors that block virus replication. Of these, the murine protein Fv1 and the tripartite (RBCC) motif protein family member TRIM5α potently inhibit retroviral infections early after virus cell entry. This study presents evidence that antiviral activity exerted by TRIM5α is conserved across mammals. Rabbit cells are strongly non-permissive to infections by certain retroviruses including HIV-1. The study identifies a TRIM5α orthologue in rabbits, which possesses antiretroviral activity. Phylogenetic analysis demonstrates that rabbit TRIM5 is a true orthologue and clusters with other mammalian TRIM5 genes. Remarkably, TRIM5 fusion proteins with the prolyl-peptidyl isomerase cyclophilin A (CypA) have independently evolved twice by retrotransposition of a CypA cDNA into the TRIM5 genomic locus, generating a viral restriction factor with a CypA binding domain. This study shows that CypA fusion proteins with RBCC domains of other TRIM proteins also function as restriction factors against HIV-1. Furthermore, fusion of CypA to Fv1 enables the restriction of HIV-1 and FIV. Insights into the mechanism of TRIM5α restriction are also provided in this study. Functional analyses of TRIM5α and TRIMCyp deletion mutants, as well as fusion proteins of Fv1Cyp with the TRIM5α RING and B-box domains, indicate that the RING and B-box domains mediate the proteasome dependent block to reverse transcription during TRIM5α/TRIMCyp restriction. The results suggest that the major antiviral activity exerted by TRIM5α is a RING dependent block to reverse transcription, whereas the major activity of TRIMCyp is independent of RING function.

CD4+CD8+ double positive (DP) precursors undergo positive selection in the thymus, and subsequently commit to either the CD4 or CD8 single positive (SP) lineage. Mice lacking Zap70, a crucial kinase involved in T-cell receptor (TCR) signal transduction are developmentally arrested at the DP stage, showing that this process depends on TCR signalling. Furthermore, the resultant SP populations are major histocompatibility (MHC) matched, as CD4 lineage cells recognise MHC-II whereas CD8 lineage T cells recognise MHC-I. There are currently two favoured models describing how the MHC restriction of the TCR correlates with lineage choice. These suggest that differences in either the TCR signalling strength or signalling length underlie the CD4/CD8 lineage decision during the DP stage. Both models posit that differences in signalling strength/length are properties that are conferred by the different signalling capacities of the CD4/CD8 coreceptors. However, questions remain over the mechanisms behind this process, including how the selecting cell interprets the proximal differences in TCR activation and whether the quality of this signal impacts on its future homeostatic survival potential. A major obstacle in addressing these questions is the lack of tools to facilitate understanding of the kinetic regulation of positive selection. We therefore sought to examine the kinetics of T-cell development using a tetracycline inducible Zap70 mouse model (TetZap70 hereon). In the absence of the tetracycline derivative doxycycline (dox), T-cells were arrested at the DP stage, prior to positive selection. However, the administration of dox induced positive selection of a synchronized wave of positively selecting thymocytes, enabling the resolution of intermediate populations of positively selecting DPs. We found that CD4 and CD8 lineage development occurred with temporal distinction, from phenotypically disparate populations of DP thymocytes. Furthermore, we found that endogenous Zap70 was developmentally regulated in different DP populations of WT mice, and loss of this regulation in TetZap70 mice corresponded to an impairment in CD8 lineage generation. Thus we suggest that temporal regulation of T-cell signalling sensitivity during thymic development facilitates the resolution of strong/consistent signals versus weak/intermittent signals. Finally we find evidence that the quality of the positive selection signal not only controls the CD4/CD8 lineage decision, but also impacts on the future homeostatic survival potential of T-cells by influencing levels of the prosurvival interleukin-7 receptor alpha chain (IL7rα).

The contemporary hygiene hypothesis suggests that certain microorganisms that were present throughout human evolution modulate the host immune system to reduce allergy associated T helper 2 (Th2) responses and inflammatory diseases by augmenting regulatory T cells. The prototypic environmental mycobacterium, M. vaccae has been used in mouse models of asthma to support this hypothesis, but data from human models and possible mechanisms are very limited. In view of the role of dendritic cells (DCs) in shaping adaptive T cell responses, the effect of innate immune interactions between human DCs and M. vaccae on allogeneic and antigen specific DC-dependent polarisation of T cells was tested. M. vaccae can stimulate cellular activation via Toll-like receptor 2 (TLR2) and therefore was compared to a specific TLR2 ligand (Pam3CSK4) and alternative stimulation with a TLR4 ligand (LPS). M. vaccae alone induced DC-dependent inhibition of Th2 responses, in contrast to Pam3CSK4, which had the opposite effect and LPS, which had no polarising effect. Comparison of DC maturation, genome-wide transcriptional response, and cytokine production in response to each stimulus did not correlate with the specific functional effects. In particular, directly comparable DC transcriptional responses to M. vaccae and Pam3CSK4 suggested that TLR2-mediated transcriptional regulation was not sufficient for inhibition of Th2 responses. Exclusive transcriptional responses to M. vaccae implicated a role for CREB1-dependent gene expression and analysis of signalling events confirmed selective early activation of the CREB pathway by M. vaccae. Collectively, this work has established that M. vaccae interaction with DCs does inhibit human Th2 responses and that further study of the CREB pathway in this model may provide novel insight into the molecular mechanisms of DC-dependent T cell polarisation. The final chapter of results presents development and validation of a novel approach for using short interspersed elements (SINEs) as a tool for normalisation of RT-qPCR data.

The mechanisms of maintenance of humoral immunity to infectious pathogens, particularly the contributions of memory B cells and long-lived plasma cells in maintaining specific serum antibody titres, are not well understood. Furthermore, it is not clear whether sequential heterologous humoral immune responses and disease pathology can result in the dysregulation and loss of previously acquired antibody-mediated immune responses to unrelated antigens. Here, depletion of memory B cells using anti-hCD20 monoclonal antibodies in hCD20 transgenic mice was used to dissect the role of memory B cells and long-lived plasma cells in maintaining long-term serum antibodies after intranasal Influenza A infection. Next, an experimental model of sequential infections with Influenza A/PR/8/34 and Plasmodium chabaudi chabaudi (AS) was set up, with a 15-20 week interval between the infections, in order to investigate whether sequential infection with P. chabaudi would affect pre-established humoral immunity to Influenza A. This study demonstrates that memory B cells are essential for the maintenance of long-lived serum Ab titres to Influenza A, as depletion of memory B cells results in the eventual loss of long-lived plasma cells and serum antibodies. Sequential infection with P. chabaudi results in the loss of pre-established serum antibodies to Influenza A by inducing the loss of long-lived plasma cells in an FcγRI,II,III-dependent manner, and this renders mice susceptible to secondary infection with Influenza A. However, this loss of pre-established humoral immunity is temporary, as serum antibodies do eventually return to normal levels. These findings demonstrate a mechanism shared by memory B cells and long-lived plasma cells which ensures that serum antibodies are maintained for long periods of time in the face of continuous generation and incorporation of new specificities throughout the lifetime of the host. A more complete understanding of the parameters that affect the longevity of immunological memory and how heterologous infections influence this will be vital in our understanding of the effect of continuous exposure to infectious pathogens on the efficacy and longevity of previously established immune memory.

Myeloid cells express a plethora of C-type lectin receptors (CLR) that can regulate inflammatory responses. Dectin-1 belongs to a sub-family of CLRs that possesses an extracellular C-type lectin domain (CTLD) and a single YxxL intracellular motif (hemITAM) that allows signalling via Syk kinase and induction of downstream functions. Based on consensus sequences for the CTLD and hemITAM, we identified CLEC-2 as a dectin-1-like receptor. CLEC-2 was previously characterised as a Syk-coupled platelet receptor able to induce platelet aggregation when targeted by the snake venom rhodocytin and by cells expressing the endogenous protein podoplanin. I generated monoclonal antibodies against mouse CLEC-2 and found that CLEC-2 is also expressed on lymphoid and myeloid cells, including dendritic cells (DC). Notably, treatment with LPS increases CLEC-2 expression by myeloid cells and synergises with CLEC-2 signaling to induce increased secretion of IL-10 but not IL-12. This increased IL-10 production is also observed in the serum of mice administered with anti-CLEC-2 mAb and LPS, and is dependent on the presence of macrophages and DCs. Furthermore, I generated a CLEC-2 conditional KO mouse line that will provide a tool to study CLEC-2 function in myeloid cells in vivo. Collectively, these data indicate that CLEC-2 expression is not restricted to platelets and that it plays a role on the vascular development and modulation of TLR responses.

Vascular disease in systemic sclerosis (SSc) leads to significant morbidity and mortality. No robust animal model of vasculopathy in SSc has been described. The hypothesis underpinning work described in this thesis is that a primary defect in TGFβ signalling is sufficient to generate the fibrotic, vascular and inflammatory phenotype of this condition. This is explored using a novel transgenic mouse model of SSc (TβRIIΔk-fib). The transgenic mouse strain TβRIIΔk-fib carries a kinase-deficient type II TGFβ receptor which is expressed under the control of a fibroblast specific promoter leading to balanced ligand-dependent upregulation of TGFβ signalling. Consequent increased TGFβ ligand in the peri-fibroblast microenvironment modulates other cell types. The phenotype is one of ubiquitous skin and gut fibrosis and increased susceptibility to severe and persistent fibrosis in response to epithelial lung injury. In this thesis, a cardiovascular phenotype is characterised for the first time, with adventitial fibrosis and medial attenuation within the large elastic arteries of the systemic circulation resulting in systemic hypertension with cardiac fibrosis. Within the pulmonary arterial circulation, there is ubiquitous medial hypertrophy, perivascular inflammation and mild pulmonary hypertension. In both circulations, the phenotype can be exaggerated additional vascular stress: NO synthase inhibition results hypertensive renal stress and VEGFR2 inhibition results in obliterative vascular changes representative of pulmonary arterial hypertension. This thesis demonstrates a unique phenotype that is strikingly relevant to that of human SSc vasculopathy, providing compelling evidence for the role of altered TGFβ signaling in systemic and pulmonary vasculopathy and for the role of altered cell interactions and responses to injury in the development of vascular consequences. A paradigm in which a background TGFβ dependent vasculopathy renders mice susceptible to injury leading to hallmark features of SSc vasculopathy is suggested. This model provides mechanistic insight and a potential platform for preclinical interventional studies in these important complications of SSc.

Initial sequencing of the human and mouse genomes revealed that substantial fractions were composed of retroelements (REs) and endogenous retroviruses (ERVs), the latter being relics of ancestral retroviral infection. Further study revealed ERVs constitute up to 10% of many mammalian genomes. Despite this abundance, comparatively little is known about their interactions, beneficial or detrimental, with the host. This thesis details two distinct sets of interactions with the immune system. Firstly, the presentation of ERV-derived peptides to developing lymphocytes was shown to exert a control on the immune response to infection with Friend Virus (FV). A self peptide encoded by an ERV negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and resulted in an impaired immune response. However, CD4+ T cell-mediated antiviral activity was fully preserved and repertoire analysis revealed a deletional bias according to peptide affinity, resulting in an effective enrichment of high-affinity CD4+ T cells. Thus, ERVs exerted a significant influence on the immune response, a mechanism that may partially contribute to the heterogeneity seen in human immune responses to retroviral infections. Secondly, a requirement for specific antibodies was shown in the control of ERVs. In a range of mice displaying distinct deficiencies in antibody production, products from the intestinal microbiota potentially induce ERV expression. Subsequent recombinational correction of a defective murine leukaemia virus (MLV) results in the emergence of infectious virus. In the long term, this leads to retrovirus-induced lymphomas and morbidity. ERVs, therefore, provide a potential link between the intestinal microbiota and a range of pathologies, including cancer. Finally, a new computational tool, REquest, was developed for use in the above studies. REquest allows the mining of retroelement (RE) and ERV expression data from the majority of commercially available human and murine microarray platforms and allows rapid hypothesis testing with publicly available data.

The immune system must be controlled to prevent damage caused by inappropriate responses and extended inflammation. Regulatory T cells (Tregs), known to be generated by the thymus, must be maintained in the face of an ever-increasing human lifespan and associated thymic atrophy in order to protect the host, but whether they are maintained by expansion of pre-existing Tregs or conversion of conventional T cells is not yet known. There are known to be two subsets of FOXP3+ regulatory T cells: naive and memory cells, expressing CD45RA and CD45RO respectively. In this work the characteristics of CD45RA+ and CD45RO+ regulatory T cells were investigated in healthy adults. We found proliferative and phenotypic differences between the two subsets, and evidence that CD45RA+ Tregs can replenish the memory Treg pool on activation. It is, however, becoming more accepted that CD45RO+ Tregs are also likely to be composed of many cells that were converted externally to the thymus from conventional T cells, and our work suggests a mechanism for this conversion: anergy induction. We also found that the two Treg subsets are able to migrate to disparate tissues. Investigation of cutaneous immune responses in vivo revealed the presence of a significant proportion of Tregs, their numbers rising and falling in concordance with the number of conventional T cells. Finally, these investigations of Treg subsets were extended to investigate atopic dermatitis (AD), a hypersensitivity condition in which Tregs are implicated. We found significantly fewer CD45RA+ Tregs among AD patients, with unexpectedly low rates of turnover of these cells in AD skin, despite the presence of high proportions of CD4+FOXP3+ cells. Overall, the findings from this study imply disparate roles for CD45RA+ and CD45RO+ Tregs, and provide further evidence supporting a role for dysregulated regulatory T cell function in the pathogenesis of atopic dermatitis.

Conditional mutagenesis allows the introduction of tissue specific mutations in the mouse and is of crucial importance in converting genome sequence information into functional data for biomedical research. Mice expressing the Cre recombinase in a spatially controlled manner are essential in creating such conditional knock-outs. A wide variety of Cre mice have been generated, but there is a distinct lack of models expressing the recombinase faithfully and at high levels in cells of the innate immune system. To address this need, three target genes, Itgb2l, Marco and Msr1, were chosen to create novel neutrophil and macrophage specific knock-in models harbouring iCre2, a recombinase engineered for increased expression levels. Two strategies were employed. Initially gene specific bacterial artificial chromosomes in which the iCre2 fragment replaced the endogenous translation start codon were created by Red/ET recombineering. Utilization of these BAC vectors for embryonic stem cell targeting successfully created knock-ins but the identification of homologous recombinants was complicated by the vectors’ large size. As the discovery of mutations impeding iCre2 functionality in the knock-in lines necessitated repeating the vector creation process, novel shorter vectors were designed. These vectors achieved targeting frequencies of around 10% and facilitated the isolation and verification of 9 Itgb2l and Marco specific iCre2 knock-in murine embryonic stem cell lines on the 129 genetic background. To determine tissue specific iCre2 expression before generating mouse models, an in vitro haematopoietic differentiation system, utilising three-dimensional embryoid body formation and selective expansion of progenitors in the presence of IL-3 and MCSF, was adapted. Embryonic stem cells were successfully differentiated into macrophages as assessed by CD11b and F4/80 marker expression. Collectively, this work has established the foundations for obtaining viable myeloid specific Cre producer mouse strains and discusses the potential of their future application in elucidating the role of macrophages and neutrophils in innate immune function.