Ageing is accompanied by impairments in immune responses. In this thesis, an age associated numerical and functional deficit in a novel subset of immunosuppressive CD19\(^+\)\(^v\)\(^e\) CD24\(^h\)\(^i\) CD38\(^h\)\(^i\) B cells is reported which might be an additional factor contributing towards increased risk of systemic autoimmunity with advancing age. The immune system is profoundly affected by environmental factors such as stress. This thesis also examined the synergistic effect of the physical stress of a hip fracture and the psychological stress of depressive symptoms on immune function of older adults. It was observed that 37% of our hip fracture patients had developed depressive symptoms 6 weeks post-surgery. A significant decline in neutrophil, monocyte and NK cell functioning was reported in hip fracture patients with depressive symptoms, but not in those with hip fracture alone. Additionally, an increased cortisol: DHEAS ratio was also only found in hip fracture patients with depressive symptoms. An elevated level of pro-inflammatory (IL6, TNFα) and anti-inflammatory (IL10) cytokines were observed in hip fracture patients with depressive symptoms. In the adaptive immune system, a reduction in circulating T cells and an accumulation of senescent and activated T cells was also found in the hip fracture patients who developed depressive symptoms. Finally, the age associated numerical and functional deficit in IL10 production by CD19\(^+\)\(^v\)\(^e\) CD24\(^h\)\(^i\) CD38\(^h\)\(^i\) B cells was further suppressed on exposure to chronic stress in hip fracture patients with depressive symptoms.

The thymus is organised into functionally distinct microenvironments that facilitates development of a diverse and self-tolerant T cell repertoire. Following positive selection, thymocytes undergo chemotactic migration from the cortex to the medulla, a site that mediates negative selection of potentially autoreactive CD4+ and CD8+ single positive (SP) thymocytes. Importantly, current models suggest that the medulla also fosters the continued maturation of SP thymocytes, post-selection. However, the mechanisms of thymic medulla function remain unclear. Using a novel approach based on chemokine receptor expression, we have mapped stages in the positive selection process, and developed a model to study αβT cell development in the absence of medullary thymic epithelial cells (mTEC), but in the presence of an otherwise intact immune system. We show that mTEC are dispensible for the continued development of newly selected CD4+CD69+ SP thymocytes, yet are essential for the generation of FoxP3+ regulatory T cells and their FoxP3-CD25+ progenitors. In addition, although CCR4 represents a marker of early stage CD4+ SP positive selection, it is dispensable for SP medullary accumulation and intrathymic development. Collectively these findings highlight differences in the developmental requirements of conventional and regulatory CD4+ T cells, and rule out a role for CCR4 in cortex to medulla migration.

Mesenchymal stem cells (MSCs) are a subset of multipotent cells with a variety of trophic and immunosuppressive functions. Isolation of murine MSCs has traditionally been hampered by the presence of contaminating cells in culture. In this study, I prospectively isolate murine MSCs based on the co-expression of platelet-derived growth factor (PDGF) receptor alpha and stem cell antigen‐1 (PαS MSCs) and present novel data regarding their in vitro phenotype, karyotype and immunomodulatory functions. However, PαS MSCs undergo senescence after extended culture, resulting in a loss of function. Addition of fibroblast growth factor 2 (FGF2), PDGF‐BB or transforming growth factor-beta 1 (TGF-β1) was able to overcome senescence in MSC cultures. These factors also ‘lineage primed’ MSCs down specific fates at the genetic and phenotypic levels, with un‐supplemented MSCs primed towards bone, FGF2 or PDGF‐BB supplemented cells primed towards fat, and FGF2 supplemented cells primed towards cartilage. TGF‐β1 supplementation attenuated tri‐lineage differentiation of PαS MSCs but maintained their immunosuppressive functions. These findings were confirmed in a mouse model of inflammatory liver injury, with late‐passage TGF‐β1 MSCs improving liver injury compared to controls. In summary, these results have significant translational relevance as I reveal that culture conditions can functionally ‘prime’ MSCs down specific fates.

Although it is evident that physiological ageing is accompanied by marked alterations in the function of innate immune cells, little is known regarding the underlying mechanism(s). Furthermore, the effect of age on many novel aspects of innate immunity is unknown. This thesis has identified the mechanism(s) behind the well-documented age-related decline in natural killer (NK) cytotoxicity (NKCC) and demonstrated for the first time that human ageing is accompanied by a significant reduction in the generation of neutrophil extracellular traps (NETs). Following target cell recognition, it was found that NK cells from older adults secreted into the immunological synapse (IS) significantly lower levels of perforin, a pore-forming protein that plays a non-redundant role in NKCC. This impairment led to reduced perforin binding to the target cell surface, an event that correlated strongly with NKCC. Underlying the reduction in perforin secretion was defective polarisation of lytic granules to the IS, which was associated with delayed activation of extracellular signal-regulated kinase 1/2. Whilst no age-related difference was observed in NET production triggered by phorbol 12-myristate 13-acetate (PMA), neutrophils from older adults generated significantly fewer NETs when challenged with interleukin-8 or lipopolysaccharide, which was accompanied by a reduction in reactive oxygen species generation. As PMA activates cells independent of membrane receptors, aberrant intracellular signalling proximal to the neutrophil membrane may underlie the age-related impairment in NET production.

Physiological ageing is accompanied with an increase in systemic inflammatory mediators (inflammageing), a functional decline of the immune system (immunesenescence), altered endocrine function (adrenopause) and reduced physical activity which predisposes the elderly to increased risk of disease. Little is known about the interplay between physical activity, inflammageing, adrenopause and immunesenescence and what impact interventions may have in the elderly. This thesis identified the consequences of inflammageing and its association with immunesenescence and the impact physical activity plays on limiting the severity of inflammageing. Cytomegalovirus drives immunesenescence but was not associated with inflammageing. Instead inflammageing was associated with reduced physical activity and increased body fat. Furthermore, inflammageing and adrenopause was associated with increased frailty and mortality risk over a ten-year period. Accelerometer defined physical activity levels in healthy elders revealed a reduced inflammatory profile and improved neutrophil migration towards interleukin-8. Acute exercise revealed an enhanced inflammatory profile indicative of positive tissue adaptation. Furthermore there was a reduced ratio of cortisol to dehydroepiandrosterone-sulphate which was accompanied by enhanced neutrophil and monocyte bactericidal function. Subsequently ten-weeks of high-intensity interval training, which was more than half the time commitment of regular aerobic training, revealed similar reduced inflammation and improved neutrophil and monocyte bactericidal capacity.

Until recently, CD4\(^+\)THelper (T\(_H\)) cells were thought to be permanently committed to a single lineage (e.g. T\(_H\)1, T\(_H\)17, T\(_H\)2 etc.). However there is now increasing evidence that T\(_H\) cells are plastic in nature and can gain phenotypic feature of other TH cells. Within this study I have investigated the overlap and plasticity of T\(_H\)1 cells and their presence in health and in the inflammatory setting of multiple sclerosis. Although CCR6 is considered a T\(_H\)17 marker there are other T\(_H\)cell subsets that express CCR6. I have identified a novel subset of T\(_H\)1 cells that express functional CCR6. CCR6\(^+\)T\(_H\)1 cells transcriptionally express 'T\(_H\)17'-related genes (e.g. RORC, IL-23R and IL4I1) but are distinct from IFN\(\gamma\)\(^+\)IL-17\(^+\) cells that also express CCR6, RORC and T-bet. Additionally I have identified candidate miRNAs that may play a role in controlling phenotypic features of these cells. T\(_H\)17 cells have been implicated in the pathogenesis of multiple sclerosis and enter the cerebrospinal fluid through CCR6-dependent migration. CCR6\(^+\)IFN\(\gamma\)\(^+\) cells were increased within the cerebrospinal fluid of patients with multiple sclerosis, suggesting a possible role in disease pathogenesis.

CTLA-4 is an important inhibitor of T cell immune responses. The location of CTLA-4 in intracellular vesicles is the most dominating aspect of its biology, yet the significance of this at the functional level remains to be completely understood. I have therefore investigated the role of the CTLA-4 cytoplasmic domain in the intracellular trafficking of the receptor with particular emphasis on sorting signals encoded within this domain. We found that CTLA-4 was located in punctate intracellular vesicles in transfected cells, activated T cells and in regulatory T cells. CTLA-4 internalisation from the cell surface was clathrin dependent and was driven by the YVKM motif encoded within the cytoplasmic domain. Post-internalisation CTLA-4 colocalised with markers of late endosomes. Since the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression we investigated this further and found that ubiquitination of intracellular lysine residues targets CTLA-4 to lysosomes. The ability of CTLA-4 to recycle was dependent on the YVKM motif and subtle changes in this motif reduced recycling efficiency. Moreover, in the absence of lysine residues CTLA-4 recycling was enhanced. CTLA-4 transendocytosis was conserved through evolution but the exact sorting signals required for this function remain to be identified. Overall this thesis emphasises the importance of the CTLA-4 cytoplasmic domain in regulating its intracellular trafficking.

TNF-α, a pro-inflammatory cytokine, is implicated in the immune response in chronic obstructive pulmonary disease (COPD) secondary to Alpha-One Antitrypsin Deficiency (A1ATD). This thesis firstly describes studies in monocytes from A1ATD-related COPD subjects, examining the effect of the rs361525 TNF-A single nucleotide polymorphism, previously associated with 100-fold greater TNF-α concentration in the sputum of affected patients. Secondly, the autocrine effects of TNF-α on monocytes from healthy subjects are considered, in particular the differential roles of its two receptors, TNF-α receptor 1 (TNFR1) and 2 (TNFR2), an important topic given recent interest in selective TNFR1 blockade in TNF-α associated diseases. Unexpectedly, TNF-α mRNA expression and secreted protein was not greater in A1ATD-related COPD subjects with the rs361525 polymorphism when compared to matched wild-type subjects. Reasons may include the cell type and stimulus used or inadequate power. In monocytes from healthy subjects, autocrine binding of TNF-α increased production of pro-and anti-inflammatory cytokines. Trends were observed for TNFR1 blockade to reduce both types of cytokine, for IL-10 to be reduced by TNFR2 blockade and for TNFR1 expression at the monocyte surface to be up-regulated by TNF-α-TNFR2 binding. Further studies are required to fully characterise the relative roles of TNFR1 and TNFR2 in monocytes.

A large body of evidence supports the role of activated stromal cells in the persistence of inflammation. The switch from resting to pathogenic stroma appears to be associated with the development of tertiary lymphoid organs (TLOs) within sites of chronic inflammation. However little is known about the immunological function of the stromal component. We utilised a murine model of inducible TLO formation in inflamed salivary glands to investigate the role of activated stromal cells characterised by the expression of gp38 and FAP during TLO development. We demonstrated that during inflammation, stroma-derived ICOSL engages ICOS on T cells, necessary for the release of lymphotoxin α and consequent TLO formation. Whilst dissecting the role of stromal cells in this context, we demonstrate that gp38 expression is required for the upregulation of adhesion molecules involved in cell clustering. Depletion of gp38+FAP+ stromal cells led to a significant reduction in lymphoid chemokine production, a decreased number of infiltrating lymphocytes and severely compromised TLO formation. Collectively, we provide evidence that activated stromal cells express FAP, provide co-stimulatory signals, and are necessary for the establishment of viral-induced TLOs, highlighting a potential novel therapeutic target in TLO-associated autoimmune diseases.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. In COPD, an imbalance is believed to exist between the activities of neutrophil serine proteinases (NSPs) such as neutrophil elastase (NE) and proteinase 3 (PR3), and their endogenous inhibitors such as alpha-1-antitrypsin (A1AT). Hence, a deficiency of A1AT predisposes to the development of COPD. Following their release from neutrophils, NSPs may bind to local inhibitors depending on their concentrations and affinities, to substrate such as lung elastin or to the neutrophil cell membrane where they remain active. This work has demonstrated that; NSPs bound to the proteinase “inhibitor” alpha-2-macroglobulin (A2M) remain active, and A2M:NE complexes are able to degrade elastin in vitro; NE bound to elastin is poorly inhibited by A1AT; and PR3 binding to the neutrophil cell membrane is greater when the local concentration of A1AT is reduced. The role of PR3 has not previously been studied in detail. However, PR3 activity was found to be present in sputum from clinically stable subjects with COPD or A1AT deficiency and was greater than NE activity. Hence, PR3 is likely to be important in the pathogenesis of COPD and could potentially be a target for therapeutic inhibitors.