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.

Asthma is a chronic disease characterised by variable airflow obstruction, bronchial hyperresponsiveness and airways inflammation. At an immunological level Th2 inflammation and the presence of activated eosinophils and mast cells are key features of asthma. ST2, the receptor for the novel cytokine IL-33, is expressed upon Th2 lymphocytes and mast cells but its role in clinical and experimental asthma remains unclear. IL-33 has been shown to induce local and systemic eosinophilia when administered to the peritoneum of mice. In this thesis I have set out to test the hypothesis that the activation of mast cells by IL-33 acting on cell surface ST2 plays a critical role in allergic airways inflammation. I began by studying the function of ST2 on mast cells in vitro. I found that ST2 was expressed at an early stage of development, and correlated closely with the expression of the stem cell factor receptor (c-kit), a marker present on mast cells from a progenitor stage. Despite this mast cells generated form ST2 gene deleted mice proliferated and matured normally. When mast cells were activated by IL-33, acting in an ST2-dependent manner, pro-inflammatory cytokines and chemokines were released that have potential roles in asthma, specifically IL-6, IL-13, MIP-1α and MCP-1. To extend these findings I looked at the role of ST2 in allergic airways inflammation. I first optimised and validated an ovalbumin and adjuvant based ‘short’ twelve day model of murine asthma and demonstrated that ST2 gene deletion results in attenuated eosinophilic inflammation. In addition to being ST2 dependent it is possible that this adjuvant based short model is mast cell dependent, unlike longer adjuvant based models which are mast cell and ST2 independent. Therefore I went on to study an adjuvant-free model of asthma which has been demonstrated to be mast cell dependent. In this adjuvant-free model of asthma the airway inflammation was attenuated in ST2 gene deficient mice compared with wild type mice, while AHR was unaffected. There was an associated reduction in IgE production and thoracic lymph node recall Th2 cytokine responses. I then examined the effect of ST2 activation in the lungs. When IL-33 was administered directly to the airways of naïve mice it induced the features of experimental asthma. There was extensive eosinophilic inflammation within the lung tissue and airspaces. The Th2 cytokines IL-5 and IL-13, and the eosinophil chemoattractant chemokines eotaxin-1 and eotaxin-2 were detected at increased concentrations. Significant airways hyperresponsiveness was also generated. Using ST2 gene deleted mice I demonstrated that these effects were ST2 specific. Although I have shown that mast cells are activated by IL-33 in vitro, I used mast cell deficient mice to demonstrate that the eosinophilic inflammation generated by IL-33 is unaffected by the absence of mast cells. These data show that IL-33 can induce in the lungs the cardinal pathological characteristics of asthma, and that it appears to act upstream of other important mediators such as IL-13 and the eotaxins. Furthermore the IL-33 receptor ST2 is required in an adjuvant free model of asthma, which is more akin to human disease. Placing these findings in the context of recent evidence that IL-33 is released by structural cells in response to damage or injury suggests that IL-33 may play a key role in initiating the immunological features of clinical asthma. As a consequence of this position in the hierarchy of inflammation IL-33 offers a promising direct target for novel biological therapies in asthma.

The transcription factors STAT3 and NF-κB play key roles in inflammation, immunity and cell fate. In the liver, they are responsible for transcribing hundreds of genes in response to combinations of IL-6, TNFα and IL-1β, and so together co-ordinate the acute phase response to infection. Dysregulated STAT3 and NF-κB signalling leads to chronic inflammation and is implicated in the development of many cancers. A variety of highly context-dependent intercellular and intracellular mechanisms have been discovered which facilitate both positive and negative cross-talk between STAT3 and NF-κB. Whilst the long-term signalling dynamics of NF-κB have been characterised in single cells, and were found to be oscillatory, imaging studies on STAT3 have focused upon the short-term mechanisms of nuclear transport rather than the long-term dynamics. STAT3 has been shown to oscillate in a population of synchronised cells so it is possible that STAT3 will exhibit oscillatory spatio-temporal signalling dynamics in response to cytokine stimulation. The primary aim of this thesis was to characterise the long-term signalling dynamics of STAT3 in response to IL-6, using fluorescent fusion protein reporters for STAT3 and its inhibitor SOCS3, in conjunction with live single cell fluorescence microscopy. Towards these aims, STAT3 and SOCS3 fluorescent fusion proteins were constructed. The responses of a candidate cell line to IL-6 and TNFα were investigated, and then the fluorescent reporters were characterised in that cell line. The N-terminal tagged EGFP-STAT3 reporter was found to be the most accurate reporter of IL-6 signalling. The EGFP-STAT3 was then used to investigate the single cell spatio-temporal dynamics of STAT3 in response to differently timed lengths of IL-6 stimulation. STAT3 was found to oscillate with a period of approximately 90 min in response to continuous IL-6 stimulation, but only underwent a transient nuclear translocation in response to a 30 min IL-6 pulse. Furthermore, the patterns of gene expression were characterised for the timed IL-6 treatments. The quantified single cell dynamics were used to constrain an existing generic model of STAT:SOCS signalling; the model was able to capture the observed single cell dynamics using a minimal ordinary differential equation approach. The secondary aim of the thesis was to study cross-talk between STAT3 and NF-κB using live cell microscopy techniques. The effects of co-stimulation of NF-κB and STAT3 were investigated using combinations of TNFα and IL-6 stimuli. Combinations of single or dual transfections, and single or dual stimulation were performed as controls in order to tease apart the effects of co-expression and co-stimulation. The importance of the timing of cytokine stimulation was also investigated. Finally, the effects of IL-1β upon IL-6 induction of STAT3 were investigated, as this was shown elsewhere to inhibit STAT3 signalling and so was expected to produce interesting spatio-temporal signalling effects. This preliminary study revealed distinct subpopulations of cells with different p65 and STAT3 response patterns. The STAT3 response was knocked down or significantly delayed in many cells but a small subset exhibited atypical oscillatory dynamics. Interestingly, the p65 dynamics were also significantly perturbed by IL-6 and IL-1β co-stimulation, indicating that there are cross-talk events occurring in both directions. Consequently these studies represent a very important area for future investigation.

Introduction: Alloimmunity is a major contributor to chronic allograft injury. There are currently no routine clinical cell-based assays that allow quantification of the recipients' alloimmune response towards a graft. Previous work from our group identified indirect alloimmune responses to non-polymorphic regions of HLA Class 1. The aim of this thesis was to assess the alloimmune response in renal transplant recipients (RTRs) by using synthetic peptides to non­polymorphic regions ofHLA Class 2. Methods: Responses to newly synthesized HLA Class 2 peptides were tested in RTRs via any­ interferon ELISPOT assay. Cell surface staining techniques and Luminex technology were used to identify the T-cell subsets driving the immune responses and subsequent cytokine production respectively. Results: Increased responses to HLA Class 2 derived peptides were detected in renal transplant recipients compared to healthy controls. The activated effector memory subset ofT-cells was expanded in RTRs compared to healthy controls and generated these responses. T effector memory cell dependent TNF-a and IL-2 and T regulatory dependent IL-10 synthesis in the presence of specific peptide antigen was detected. Conclusion: A potential reproducible assay ofT cell alloreactivity has been identified to help stratify RTRs at risk of an ongoing alloimmune response but needs further testing in a larger multicentre study.

The Pet Autotransporter protein was engineered and recently proposed as recombinant protein production (RPP) system. This system allows targeting the protein of interest in the culture supernatant fraction. The reduction of diversity and quantity of process impurities and size and number of downstream steps required, increase the overall process robustness and speed-up the process development time for RPP. In the context of this study the platform was investigated for the production of a “difficult” E.coli protein with commercial relevance, C1275. Pet autotransporter was suitable for the production and one step-purification of a protein with comparable purity, thermal stability and immunogenicity to that produced using conventional technology. Additionally, the autotransporter platform was tested for the first time in scaled-up production conditions. The system was compatible with fermentation and the scaled-up conditions resulted in high yield of antigen production even though a further system optimization would be required for a one step-purification process.

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.

Resistance to infection is essential to ensure survival and thus maximise offspring potential. However, resistance is not ubiquitous across the animal kingdom, or even within a population from the same species. It is thought that this is due, in part, to the costs involved in producing and maintaining a competent immune system and a corresponding decrease in other fitness-related characteristics. The focus of this research was to determine how immune resistance can impact upon mechanisms relating to ageing, resistance to oxidative stress and motor function. In order to do this a Drosophila melanogaster model system was implemented, selected for resistance to larval parasitism by the parasitoid wasp, Asobara tabida. Firstly, it was necessary to gain a greater understanding of the immune mechanisms within the Drosophila model. This included how aspects of the immune system changed over time, in order to determine how these might act upon other processes at different stages of the ageing system. Resistance to larval parasitism corresponded to an increased number of circulating immune cells during the larval phase. This difference was no longer apparent in the adult Drosophila. Young resistant adult females revealed increased levels of overall cell metabolism, measured by the production of intracellular reactive oxygen species (ROS), a finding not seen in males or in the developing larvae. Lifespan was reduced in the resistant female, but not male, Drosophila. It was hypothesised that this reduction may in part be due to the augmented production of intracellular ROS in the young adult female, which at high concentrations can cause oxidative stress with known cytotoxic effects. However, differences in resistance did not translate to altered survival under acute oxidative stress, induced by the consumption of the toxin paraquat. Other factors may regulate these changes in longevity in the resistant females, such as genetic or resource-based trade-offs. Functional assays were performed to assess motor function in the larvae and adult Drosophila. Resistant larvae showed less turning behaviour on a non-food background than their control counterparts, a trait generally linked to more proficient motor function. Differences in motor function continued into the adult females, where increased climbing velocities were found irrespective of age. This implies that changes in motor function may be determined during development, thus variations in resistance during this phase can cause life-long impacts on the individual, presumably by altering the development of other physiological systems.

Small heat-shock proteins are conserved molecular entities present in all mammalian cells. They have historically been studied in the context of being intracellular molecular chaperones that are constitutively expressed, with a capacity to be induced by cellular stress, in order to promote and remediate protein folding. More recently however, growing evidence suggests that the action of small heat-shock proteins is not limited to protein folding but also extends to a wider range of important cellular roles. Of the 11 small heat-shock proteins that are expressed in mammalian cells, only 4, HSPB1, HSPB5, HSPB6 and HSPB8, are expressed in the central nervous system (CNS). The role of these small heat-shock proteins in the CNS is thought to be protective as they show widespread upregulation during several neurological conditions. Confoundingly however, studies from R6/2 animal models of Huntington’s disease show a selective reduced expression of HSPB5 in these animals, raising pertinent questions as to whether this reduction is cause or effect of the condition. Here, we have investigated whether reduced expression of HSPB5 has a detrimental effect, focussing specifically on HSPB5’s proposed immune modulatory role. Using mice inoculated with S. typhimurium, we found that, in our hands, mice lacking HSPB5 did not appear to be phenotypically different from wild type animals and equally, the reduced expression of HSPB5 did not exacerbate systemic inflammation or potentiate disease progression. Furthermore, to investigate HSPB5’s role in the CNS, we inoculated animals with ME7 Prion and also found that deficiency in HSPB5 did not alter phenotype or behaviour and did not negatively influence disease progression. Lastly, we investigated whether the reduced expression of HSPB5 as observed in R6/2 animals was reciprocated in humans. Our findings show that in humans disease, there is no reduction of HSPB5. Our findings suggests that in C57BL/6 animals, HSPB5 does not appear to have an immune modulatory role; they also highlight how data obtained from animal models should be taken tentatively.

Allergic contact dermatitis (ACD) is a prevalent skin condition caused by chemical haptens, which enter the epidermis, and by modifying self-proteins, render them immunogenic via the activation of hapten-specific T-cells. It is currently not known which specific protein modifications are responsible for sensitization. This work investigates the extreme sensitizer DNCB, which confers a dinitrophenyl (DNP) protein modification. Immortalised keratinocytes (HaCaT), incubated with DNCB were compared to ex vivo human skin using immunofluorescence and western blot detection of DNP-proteins, showing widespread protein modification and similarities between tissue and cells when using a clinical dose. Proliferation assays using lymphocytes from DNCB-sensitive donors showed responses to DNP proteins isolated from DNCB-treated HaCaT cells and primary keratinocytes. While western blot analysis of pH gradient separated fractions identified a number of DNP-proteins in the DNP-HaCaT cell lysates, these were not immunogenic in lymphocyte assays. The model protein human serum albumin (HSA) was used to investigate the modification kinetics of DNCB by identifying which amino acid residues were changed more readily using a range of DNCB concentrations and incubation times. Cysteine residues, including those in disulphide bonds and particularly cysteine 34 are more readily modified than lysine in HSA, suggesting that DNCB is able to alter the structure of proteins. A novel process of hapten-reversal by a process termed ‘thiolysis’ was found to remove DNP groups from the cysteine residues of synthetic peptides derived from the sequence of HSA containing cysteine 34 using the reducing agent dithiothreitol. Identical peptides with C34>K34 showed no such hapten-reversal. This corresponds to the unexpected immunogenicity of the cysteinyl peptide and also the DNP modified tripeptide glutathione. Anti-DNP western blots show that the DNP group is transferred to other proteins during incubation with human monocytes in culture. This suggests a cellular process of removing DNP groups and GILT, a thiol reductase present in the endosomes is presented as a candidate for this process. This work demonstrates that DNCB can generate a wide variety of DNP protein adducts and that cysteinyl moieties are able to stimulate lymphocyte proliferation by way of hapten transfer. This highlights a potentially novel process involved in the mechanism of contact allergy.

Leishmania spp. are protozoan parasites that cause a spectrum of pathologies in humans and other vertebrates, ranging symptomatically from cutaneous ulceration to visceral dissemination. In order to survive within the host, Leishmania are able to evade and modulate the host immune responses through the actions of their virulence factors; however, few putative virulence factors have been characterised during in vivo infection with Leishmania. The Leishmania major genome has revealed the presence of three peptide inhibitors of S1A family serine peptidases (ISPs), which are orthologues of a bacterial protease inhibitor, ecotin. Serine peptidases of the S1A family are absent in Leishmania; therefore, the ISPs have been proposed to inhibit the activity of host serine peptidases, such as those expressed by cells of the innate immune response. ISP2, which is expressed in the mammalian-infective metacyclic promastigote and amastigote stages, has previously been shown to inhibit neutrophil elastase (NE), a serine peptidase expressed by neutrophils, monocytes, and macrophages. This inhibition prevents the activation of a Toll-like receptor 4 (TLR4)-NE pathway during Leishmania-macrophage interaction promoting Leishmania survival and growth in macrophages in vitro. The aims of this project were to assess whether the presence or absence of ISP2 in L. major affects parasite survival in vivo, and to investigate the effects of ISP2 on immune cell dynamics in vivo, specifically with regards to cell recruitment and activation, using the C57BL/6 mouse model. Parasite burdens were performed in mice infected with L. major wild-type (WT) parasites, a cell line deficient in ISP2/3 (Δisp2/3), and a cell line re-expressing ISP2/3 (Δisp2/3:ISP2/3). L. major Δisp2/3 parasites could not be detected at the site of inoculation by 5 wk post-infection compared with WT and Δisp2/3:ISP2/3 parasites, but parasites of all three cell lines were detected in the draining lymph nodes (dLNs) throughout the course of infection. These data were corroborated using in vivo bioluminescence imaging (BLI) of luciferase-expressing (LUC2) versions of these cell lines, in which only a low bioluminescent signal was observed at the site of inoculation with the LUC2-expressing Δisp2/3 cell line over the course of infection, compared with the LUC2-expressing L. major and Δisp2/3:ISP2/3 cell lines. These results suggest that ISP2 may be important in the establishment and persistence of Leishmania infection by conferring parasite survival, particularly at the site of infection. Serine peptidases of innate immune cells, such as NE, function in the proteolytic cleavage of cytokines, chemokines, and cell receptors, to regulate immune cell recruitment and activation. Flow cytometric analysis of innate cell populations at the site of inoculation, in response to L. major WT and Δisp2/3 parasites over 5 wk of infection, was conducted. This line of investigation revealed significantly higher numbers of monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells (moDCs) at 2 wk in Δisp2/3 infection. MoDCs have crucial functions in the induction of antigen-specific T helper 1 responses, which are considered to be important for parasite clearance. MoDCs at the site of Δisp2/3 infection showed an upregulation of the DC co-stimulatory molecule CD80 compared with those from WT infection suggesting an upregulation of DC maturation. MoDCs have also been shown to be the major producers of inducible nitric oxide synthase (iNOS) during L. major infection, which catalyses the production of nitric oxide that is responsible for the killing of Leishmania. Intracellular staining of iNOS through flow cytometric techniques showed that iNOS expression in moDCs was not affected by the presence or absence of ISP2; there was, however, an increase in iNOS expression in other innate cell types, the resident macrophages and DCs, monocytes, and monocyte-derived macrophages, at the site of Δisp2/3 infection compared with those from WT and Δisp2/3:ISP2/3 infections. At the 2 wk time-point, there was also a significant increase in the concentration of IFN-γ, a cytokine that induces iNOS expression, in response to Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as determined by ELISA. Quantitative in vivo BLI of myeloperoxidase (MPO) activity of activated phagocytes was determined over a period of 7 wk, which, also, indicated differences in phagocyte activation at the site of inoculation in L. major WT and Δisp2/3 infections. Taken together, these results indicate that the immune response is more primed towards Leishmania killing in Δisp2/3 infection compared with WT infection, which suggests that ISP2 modulates these immune responses to facilitate Leishmania survival. Infections in transgenic mice deficient in NE, Ela-/-, showed similar monocyte recruitment and moDC activation responses in Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as those observed in the C57BL/6 mice. This indicates that NE may not be the major target for ISP2 in vivo, or that there may be compensations for the loss of NE by other serine peptidases in this model. Although the exact mechanism by which ISP2 modulates the recruitment and activation of the innate immune cells in vivo remains to be determined, this study has, for the first time, shown numerous differences in the innate immune responses induced following infection with either L. major WT or a mutant deficient in a putative virulence factor using in vivo techniques, such as in vivo imaging (IVIS) and flow cytometry, to compare the infections.