NKT cells are a population of T cells that have a broad range of regulatory effects on the immune system. Somewhat paradoxically, they can both suppress and potentiate cell-mediated immune responses; for example, while they can suppress some autoimmune diseases, they can also promote potent tumour rejection. There is accumulating evidence to suggest that this functional dichotomy can be explained by the existence of functionally distinct NKT cell subsets, which can differentially regulate the behaviour of other immune cells and drive remarkably different outcomes in disease settings. / Studies in vivo and in vitro have demonstrated remarkable functional diversity between NKT cell subsets of different phenotypes, and deriving from different tissues. This thesis examined functional differences between NKT cell subsets in the context of type 1 diabetes in NOD mice, identified a phenotypically and developmentally distinct IL-17 producing NKT cell subset, and investigated the functional effects of thymic NKT cells on the development and maintenance of conventional T cells and thymic stromal cells. The data presented in this thesis adds to the accumulating evidence that NKT cells are a functionally heterogeneous population, and reiterates the important point that subsets of NKT cells should be studied separately in order to properly understand the biological function of this important regulatory T cell population, and to maximise their clinical potential.

One of the major challenges facing clinicians is how to effectively manage excessive host immune response to pathogenic insults resulting in sepsis. This is demonstrated by the fact that despite over half-century research efforts, sepsis and its spectrum of diseases (severe sepsis and septic shock) are still associated with poor clinical outcome. Currently, sepsis is a leading cause of death in intensive care units. The immune system protects the host against pathogens and is therefore armed with an arsenal of deadly ammunitions (including chemicals, cells and proteins) necessary for the elimination of microbes. It is therefore paramount that the immune system must develop mechanisms necessary to prevent destruction of the host it is designed to protect. A good example of such a mechanism is found in the subset of lymphocytes known as regulatory T cells (Tregs). There is unequivocal experimental evidence of the role of Tregs in the maintenance of immune homeostasis and self tolerance and aberrant Treg function has been linked with several inflammatory diseases. Since sepsis is a disease marked by a hyper-inflammatory state, I investigated the possible role of Tregs in dampening sepsis-induced excessive inflammation. Using a murine model of lipopolysaccharide (LPS) infusion and bacterial infection, I show that Tregs are essential for survival during sepsis because their depletion leads to acute death to an otherwise non-lethal dose of LPS. This enhanced susceptibility to LPS following Treg depletion was also observed using live E. coli infection. Next, I probed the mechanism by which Tregs protect against LPS challenge. I found that defective Treg function leads to exaggerated activity of two immune cells – CD4+ effector T cells and neutrophils in response to LPS, leading to severe inflammatory response. Hence, this work successfully illustrates the critical role of Tregs in regulating other immune cells and the catastrophic consequences of defective Treg function during an immune response. Overall, this work highlights the significant role of Tregs in the regulation of bacteria associated inflammatory processes. The findings hold implications for the successful management of sepsis and have potential for use in development of adequate therapeutic intervention for sepsis. / October 2016

Ambient air pollution, including airborne particulate matter (PM) derived from combustion of fossil fuels (FF) or biomass (BM), has detrimental inflammatory effects on human health. Myeloid antigen presenting cells, including dendritic cells (DCs) regulate immune responses in the airway and sample inhaled PM. This study tests the hypothesis that PM interacts with multiple environmental sensing pathways in DCs with outcomes that depend on particle size and composition as determined by combustion source. The effects of different sized PM (< 10μm, PM10; < 2.5μm, PM2.5), derived from the combustion of FF or BM, on human monocyte-derived or ex vivo sputum DCs, were examined. DC activation status, cytokine production and aryl hydrocarbon receptor (AhR) signalling were assessed by flow-cytometry, multiplex ELISA and qRT-PCR, following exposure to PM. Pathway-specific antagonists were used to explore underlying mechanisms. Particle size and combustion source influenced the effects of PM on DCs. Irrespective of combustion source, PM10 but not PM2.5, induced MoDC maturation and stimulated production of inflammatory cytokines, including IL-1β and IL-18, indicative of inflammasome activation. These responses were dependent, at least in part, on TLR4 as was the induction of IDO by PM10. AhR signalling was induced by PM in both MoDC and ex vivo sputum DC. It was stimulated by both PM10 and PM2.5 and was induced more strongly by BM-derived PM. AhR activation was independent of DC maturation and TLR4 signalling. Additionally, BM- but not FF-derived PM increased NADH levels in DC suggestive of altered metabolism. Thus, PM induces a complex programme of DC activation, influenced by size and combustion source, which includes classical maturation, inflammasome dependent cytokine release and AhR signalling as well as potential metabolic changes. In the airway, exposure to different PM and the changes in DCs induced by them may lead to altered responses to inhaled antigen.

A balanced immune response is required to mediate clearance of a virus infection without immune-mediated disease. CD4 and CD8 T cells are capable of both exerting antiviral effector functions and regulating the immune response. The regulatory T cell (Treg) subset of CD4 T cells helps to modulate immune activation and inflammation. During respiratory syncytial virus (RSV) infection in mice, conventional CD4 T-cell-mediated cytokine production has been shown to contribute to immune-mediated pathology. I demonstrate that Tregs are critical to control immunopathology during RSV infection. This was demonstrated through diphtheria toxin (DT)-mediated Treg elimination in a mouse strain expressing the DT receptor (DTR) under the control of the Foxp3 promoter. However, these mice were unable to maintain extended Treg depletion limiting the effectiveness of this model. In addition, DT-treated wild-type (WT) mice were found to be a necessary control for adverse DT-induced disease. In humans, I have shown that activated Tregs are reduced in the peripheral blood of RSV-infected infants compared to controls. RSV-infected infants also exhibited an increased proinflammatory cytokine response in nasal aspirates. However, the alarmin cytokine IL-33, which has been shown to mediate Treg homeostasis, was the only cytokine that exhibited reduced protein levels in RSV-infected infants compared to controls. Thus, severe RSV infection in infants may be due to lack of proper Treg-mediated immune regulation. Similar to RSV, regulation of the T cell response during chronic viral infection with lymphocytic choriomeningitis virus (LCMV) is vital to prevent immune-mediated pathology. During LCMV and human chronic viral infections, CD4 and CD8 T cells exhibit T cell exhaustion where they lose the ability to exert effector functions. However, a functional CD4 and CD8 T cell response is required for viral clearance. During human chronic viral infection, an association between increased CD4 and CD8 T cell function and enhanced viral control has been identified that can be influenced by genetic factors. I aimed to identify the contribution of the host genetic factors that contribute to enhanced CD8 T cell function and viral control using the LCMV model. I found that increasing the major histocompatibility complex (MHC) diversity resulted in enhanced viral control in both a C57BL and BALB genetic background. Thus, induction of a broader T cell response was associated with enhanced viral control. However, mice expressing a heterozygous MHC on the C57BL background also exhibited mortality following chronic viral infection. Both CD4 and CD8 T cells were shown to contribute to this mortality and exhibited reduced T cell exhaustion during LCMV infection in these mice. Heterozygous MHC expression on the C57BL mouse background was also associated with an increased T helper (Th)-1 skewed CD4 T cell response compared to mice on the BALB background. Furthermore, CD4 T-cell-mediated IFN-γ production contributed to both CD8 T cell effector activity and mortality during chronic LCMV infection. Thus, both T cell epitope diversity and host genetics contribute to LCMV-induced mortality. Collectively, my data highlight both the need for effective immune-meditated viral control and regulation of T-cell-mediated pathology during both acute and chronic viral infections.

The Leukocyte Immunoglobulin-like Receptors (LILRs) are a family of receptors that is broadly expressed on all leukocytes and have the ability to regulate their function. A substantial amount of evidence suggests that LILRs may be involved in immune homeostasis but also immune dysregulation. We therefore studied the role of LILRs in relation to the autoimmune disease, rheumatoid arthritis (RA). RA is a chronic and systemic inflammatory disease involving inflammation of the joints affecting the synovial membrane, cartilage and bone. Much of the tissue damage is a result of an inappropriate immune response within the joint space caused by the unwarranted activation of leukocytes. Here were report that LILRA2 (an activating receptor) that has been previously shown to be highly expressed in the rheumatoid synovium, induces the production of pro-inflammatory cytokines TNF-α, IL-1, IL-6, IFN-γ and IL-10 in primary monocytes. These cytokines are known to have an important role in the pathogenesis of RA indicating a pathway by which LILRA2 exacerbates RA. Co-ligation of LILRB4 (an inhibitory receptor) with LILRA2 abolishes cytokine production suggesting that LILRB4 is able to suppress the function of LILRA2. Expression of both LILRA2 and LILRB4 are regulated by inflammatory cytokines and LPS, indicative of a feedback mechanism. There is also cross-talk between LILRs and TLR4 as co-stimulation with LPS and either LILRA2 or LILRB4 inhibits cytokine production. A differential expression of LILRs has also been identified on lymphocytes of patients with RA whereby an increase of LILRA1 (activating) and LILRB1 (inhibitory) expressing circulating lymphocytes is present in RA patients when compared to healthy control subjects. From these studies, we propose that LILRs have a functional role in RA by regulating local and systemic inflammation. The presence of LILRA2 in the RA joint is detrimental since its potent ability to induce inflammatory cytokines, particularly TNF-α, can initiate leukocyte recruitment and activation of proteases. Along with TLR4, LILRA2 and LILRB4 have the potential to moderate the innate immune system via regulation of cytokine production. Furthermore, suppression of LILRA2 function may serve as a therapeutic tool in many inflammatory diseases.

CD4+CD25+Foxp3+ T regulatory (Treg) cells are critical in maintaining self tolerance and promoting the acceptance of allogeneic tissue/organ grafts. To be widely applied in clinical settings, there needs to be a readily available source of Treg cells, a requirement that is better met if non-histocompatible donor cells could be utilized in adoptive therapy. Therefore, to investigate the therapeutic potential of fully allogeneic Treg cells to control autoimmune disease or allograft rejection, we utilized IL-2R beta-deficient mice that exhibit rapid lethal autoimmunity due to low production of an ineffective population of Treg cells. We show that adoptive transfer of MHC-mismatched Treg cells into IL-2R beta-/- mice resulted in life-long engraftment of the donor cells, which exhibited skewed reactivity toward host alloantigens, and prevented autoimmunity. When such animals received skin grafts, they exhibited tolerance to those grafts that expressed MHC molecules from which the donor Treg cells were derived. Collectively, these data provide proof-of-principle that effective engraftment by allogeneic Treg cells controls autoimmunity and leads to favorable conditions for long-term acceptance of allografts. Current data indicates that CD4+CD25+Foxp3+ Treg cells exhibit a broad TCR repertoire. However, the relationship between this diversity and capacity to control a similarly diverse population of potentially autoreactive T cells remains to be defined. To investigate this issue, we assessed the TCR repertoire of chimeric donor Treg cells in IL-2R beta-/- mice that were adoptively treated with a diverse polyclonal Treg inoculums. We demonstrate that autoimmune disease was fully prevented by engrafted donor Treg cells in spite of a TCR repertoire that is less diverse than the input cells. However, in settings where the input TCR repertoire is limited by utilizing donor Treg cells that express a single TCR beta chain, control of disease was hampered, correlating with a limited TCR alpha repertoire within the engrafting donor Treg cells. Collectively, these findings suggest that for adoptive therapy, a diverse TCR repertoire of input Treg cell inoculums is an essential requirement for effective control of polyclonal autoreactive T cells but perturbations in the repertoire that results in significant limitation to this diversity may compromise Treg cell efficacy at fully keeping autoaggressive cells in check.

We aimed to prospectively investigate the paternal antigen-induced cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to hormone treatment in women undergoing in vitro fertilisation (IVF) and to examine the predictive value of the cytokine secretion profile in the outcome of IVF treatment, in a pilot study. Twenty-five women were included and IVF treatment was successful for six and unsuccessful for 19 women. Blood samples were collected before IVF treatment, on four occasions during IVF and four weeks after embryo transfer. The numbers of Th1-, Th2- and Th17-associated cytokine-secreting cells and cytokine levels in cell supernatants were analysed by enzyme-linked immunospot-forming (ELISpot), enzyme-linked immune-sorbent (ELISA) or Luminex assay. None of the cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, TNF and GM-CSF) had any predictive value regarding IVF outcome. The majority of the cytokines reached their peak levels at ovum pick-up, suggesting an enhancing influence of the hormonal stimulation. Pregnancy was associated with a high number of IL-4-, IL-5- and IL-13-secreting cells four weeks after ET. In conclusion, the results do not support our hypothesis of a more pronounced peripheral Th1 and Th17 deviation towards paternal antigens in infertile women with an unsuccessful IVF outcome, although this is based on a small number of observations. A larger study is required to confirm this conclusion. Higher numbers of Th2-associated cytokine-secreting cells in pregnant women four weeks after ET do corroborate the hypothesis of a Th2 deviation during pregnancy.

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases. / May 2005

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.