Human cytomegalovirus (HCMV) infection in newborns and immunocompromised individuals with immature or deficient immune systems can cause life-threatening diseases. The clinical and subsequent economical burden of HCMV infection led the US Institute of Medicine designating a vaccine for HCMV as the highest level of priority. Complex virus-host interactions have developed over millions of years of co-evolution, making the understanding of the pathogenesis of HCMV disease particularly challenging. Consequently, a crucial factor in aiding the development of effective vaccinations and therapies to significantly reduce morbidity and mortality associated with HCMV infection is elucidating what immune mechanisms contribute to/impede protection against infection. For example, is the induction of immunomodulatory agents such as cytokines beneficial or harmful to the host during infection? Given the known immunosuppressive properties of one such cytokine, interleukin-10 (IL-10), in conjunction with the evolutionary acquisition by HCMV of its own IL-10 homologue, I hypothesised that mammalian- and viral-IL-10 suppress protective immunity during acute CMV infection. Utilising a mouse model of CMV infection, I revealed a surprising antiviral role for IL-10 during acute infection in vivo, which was achieved via limitation of activation-induced death of NK cells. The IL-10-related cytokine interleukin-22 (IL-22) provides critical protection against certain infectious agents and I therefore hypothesised that IL-22 provides protective immunity during acute CMV infection. Utilising the murine infection model once more, I discovered a tissue-specific antiviral role for IL-22 during acute infection in vivo and made the surprising finding that neutrophils play a protective role during infection. I also demonstrated that neutrophils can directly inhibit viral replication in vitro. Thus, novel insights into cytokine biology in the context of viral infections in vivo revealed by these studies highlighted important considerations for future research into herpesvirus infections, and has major implications for the treatment of this important infectious disease.

Initially, prostaglandins (PGs) were considered to only exist as free acid mediators. Although, formation of PG glycerol esters and PG ethanolamides by cellular cyclooxygenase (COX)-2 has been reported, generation of complex oxidised lipids via COX-1 has not been considered. In this study, formation of sixteen unique PG-containing phospholipids generated by agonist-activated human platelets is demonstrated using lipidomic approaches. Precursor scanning-tandem mass spectrometry identified a group of specific lipids comprising PGE2, PGD2 and two previously undescribed PG-like molecules (named PGb and PGc), attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/ and 18:0a/). PGb and PGc were also detected as free eicosanoids and their structures remain to be characterised. These novel lipids formed within 2-5 minutes of platelet activation by thrombin, collagen or ionophore and required activation of several intracellular signalling intermediates, including cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (MAPK), src tyrosine kinases, phospholipase C (PLC) and cytosolic calcium. Unlike free PGs that are secreted, PG-PEs remain cell associated, suggesting an autocrine mode of action. Aspirin supplementation in vivo (75 mg/day) or in vitro (1 mM) blocked their generation, indicating that COX-1 is required. Pharmacological studies using inhibitors of fatty acyl re-esterification significantly reduced formation of PG-PEs. Furthermore, purified COX-1 was unable to directly oxidise PE in vitro. Collectively, these indicate that PG-PEs are initially formed as free PGs via COX-1, and then rapidly esterified into PEs. In summary, this is the first demonstration of acute generation of PG-PEs in agonist-activated human platelets from endogenous substrate via COX-1. These unique lipids may represent additional bioactive molecules from this key platelet enzyme.

Hepatitis C virus (HCV) infects 3% of the global population and HCV-related liver inflammation is a major cause of liver failure and hepatocellular carcinoma. Current treatments are based upon long courses of interferon-α (IFNα) injections, which have significant side effects and are only effective in 40-80% of individuals depending on viral genotype. Natural killer (NK) cells are innate lymphocytes, which can kill virally infected cells and are stimulated by IFNα. To establish a chronic infection HCV must evade immune responses. I hypothesised that NK cells are important for the successful eradication of HCV and that chronic HCV infection impinges upon NK cell function to prevent viral clearance. I found that NK cell function was reduced in chronic HCV and correlated with the proportion of NKp46+ NK cells in vitro. In keeping with these findings NKp46-rich intrahepatic NK cell populations were more activated and the proportion of these cells correlated with liver inflammation. During interferon-α treatment individuals who had the greatest increase in NK cell function in response to increasing stimulation had the fastest rate of viral clearance and were most likely to successfully clear the virus. Using a novel adenovirus vector expressing HCV proteins I have discovered that NS5B protein reduces NK cell cytotoxicity and cytokine production. Therefore, in this thesis I have described novel insights into the mechanisms of HCV immunoevasion, HCV-related disease pathogenesis with implications for viral eradication therapy.

The innate immune system has a variety of ways of recognizing infection from pathogens such as viruses and bacteria. One of its first lines of defence is to detect Pattern Associated Molecular Patterns (PAMPs) using Pattern Recognition Receptors (PRRs) such as the Toll-Like Receptors (TLRs), the RIG-Like Helicases (RLHs) and the Nod-Like Receptors (NLRs). These receptors recognize certain molecular structures from the pathogens and lead to first line of defence which includes increased cytokines and IFNs. This study delineate the human body’s innate immune responses to human respiratory viruses such as HRV (Human Rhinovirus), RSV (Respiratory Syncytial Virus) and IAV (Influenza A virus). In Vitro experiments carried out on various kinds of lung tissues suggest that respiratory disease pathogenesis is related to inflammatory mediators including interleukins and cytokines produced by the host’s innate immune system. Virus induced respiratory tract infection are known to trigger bronchiolitis, wheezing and acute asthma exacerbations, as a result of inflammation of lung tissues and excessive release of pro- inflammatory cytokines such as IL-1β. This study identifies that intracellular macromolecular complexes called inflammasomes assemble as a result of viral trigger. Inflammasomes convert the inactive forms of the pro-inflammatory cytokines to their active forms. Although the exact mechanism of activations of these complexes was unknown. This study identified that Virus encoded proteins such as the 2B protein of HRV, the SH protein from RSV and the Influenza M2 which are also termed viroporins can activate the inflammasome by causing ion imbalance (across cells membranes and organelles). Thus providing a trigger for inflammasome assemblage. v Drugs which act as Ion channel blockers have been shown to block viroporin activity and hence reduce IL-1β production. Therefore in the future the use of ion inhibitors could be a possible therapeutic intervention in order to reduce lung inflammation and prevent associated respiratory disease such as COPD and Asthma.

Bacterial infection is a feature of long-term peritoneal dialysis (PD) and a cause of loss of peritoneal function and treatment failure. Understanding how local inflammation is initiated and peritoneal host defence mechanisms are activated in PD patients is key to reducing the detrimental consequences of peritonitis. The capacity of human peritoneal mesothelial cells (HPMC) to ingest bacteria has been described, and the ability of the Toll-like family of innate immune receptors (TLR) to trigger inflammatory responses to pathogens has been demonstrated. However, the pathogen recognition ability, the potential role of TLRs, the specific role in the early inflammatory response, and the regulation of HPMC’ putative ability for pathogen recognition have not been fully investigated. To address these issues, the present study aimed at characterising TLR expression and responses in HPMC and evaluating the capacity of modulators of pro-inflammatory responses, namely soluble TLR2 (sTLR2) and the IL-6/sIL-6R complex, to regulate TLR-mediated HPMC and peritoneal responses in vitro and in vivo. HPMC were found to respond to an array of bacterial pathogens via expression and function of a specific set of TLRs. HPMC responses were susceptible to modulation by sTLR2 and sIL-6R, resulting in inhibition of TLR2-driven HPMC responses. In vivo, sTLR2 and IL-6/sIL- 6R reduced neutrophil influx partly by inhibiting NF-κB activation in stromal cells of the peritoneum. IL-6 signalling counteracted TLR2-mediated responses by reducing peritoneal leukocyte recruitment and chemokine production. Notably, following the establishment of a mouse model of peritoneal bacterial infection, IL-6 signalling was confirmed to be beneficial to bacterial clearance. The results of this thesis confirm and extend the knowledge of the pivotal role that HPMC play in peritoneal responses to infection. The capacity of sTLR2 and IL-6/sIL-6R to modulate peritoneal responses demonstrated in this study may inform the design of new therapeutic strategies to reduce PD-associated peritonitis and thus improve treatment outcomes.

Investigations into Human Cytomegalovirus (HCMV) pathogenesis should be based on strains that closely reflect the causative agent of disease, however HCMV invariably mutates in vitro, generating phenotypically distinct laboratory-adapted strains. In particular, mutations are selected in the HCMV genome UL128 locus (UL128L) that encodes sub-units of a virion envelope pentameric complex that impedes virus propagation in fibroblasts (the cell type most commonly used in vitro), but is required for infection of several naturally targeted cell-types (e.g. epithelial, endothelial, and myeloid cells). Addressing this issue, the genome of wildtype HCMV strain Merlin was cloned as a stable bacterial artificial chromosome (BAC), however similarly to clinical isolates, viruses reconstituted from the Merlin-BAC grow poorly and are prone to de novo mutation in cell culture. Direct comparison to viruses from the Merlin-BAC revealed that viruses from the BACcloned versions of strains TR (TR-BAC), TB40E (TB40-BAC4) and VR1814 (FIX-BAC) could be propagated more efficiently in fibroblasts, despite containing intact UL128L ORFs. Unique nucleotide variations identified in TB40-BAC4 and FIX-BAC UL128L ORFs were transferred into the Merlin-BAC, generating variants that produced greater titres of cell-free virus following reconstitution. Virions from these novel Merlin variants displayed reduced pentameric complex content, but remained able to infect epithelial cells, albeit with slightly compromised efficiency. The greater fitness of viruses from these novel Merlin-BAC variants alleviated the selective pressures for the selection of de novo UL128L mutations in fibroblasts. The Merlin virion proteome was determined, with up to 30 novel components identified to provide a more comprehensive picture of wildtype HCMV virion composition. Comparison of virions from several Merlin variants demonstrated that varying pentameric complex content impacted the incorporation of other components, however virions from the novel Merlin variants produced in this work closely matched those from the parental Merlin variant containing wildtype UL128L ORFs. Thus, the novel Merlin-BAC variants produced in this work represent valuable reagents for future HCMV research.

Effective immunity is fundamental to life on a dirty planet. Appropriate immune responses control infections and protect against cancer. Inappropriate immune responses lead to autoimmunity and allergy. A fine balance between aggression and tolerance is therefore central to effective immune function at the system level. This is a particular problem for T cells, which recognize peptide antigens bound to host major histocompatibility complex (MHC) molecules. Faced with a composite antigenic structure, the distinction between “foreign and dangerous” and “self and harmless” becomes both difficult and imperative, especially when the antigen persists. In this thesis, antigen-specific T cell responses were investigated under conditions of chronic antigenic stimulation to inform our understanding of this process. In T cell receptor transgenic mice, continuous antigenic stimulation without adjuvant lead to increased in vivo turnover of antigen-specific CD4+ T cells but “aborted” immune activation, characterized by depletion of these cells from the circulation and spleen. Full immune activation and expansion of antigen-specific memory/effector CD4+ T cells required the presence of adjuvant, in this case IL-1β, which induces an inflammatory environment. Further isotope labelling studies in human immunodeficiency virus-infected subjects suggested that the surface marker CD57 demarcates a “steady state” within the CD8+ T cell memory compartment, whereby CD57+ cells have lower in vivo turnover rates compared to their CD57- counterparts. These observations provide a potential mechanistic explanation for the preferential accumulation of CD57+CD8+ cells under conditions of chronic antigenic stimulation. Another persistent pathogen, cytomegalovirus (CMV), expresses a viral interleukin (IL)-10 homologue. Memory T cell inflation and antiviral cytokine production in murine CMV(MCMV)-infected mice were suppressed by IL-10. Conversely, IL-10 blockade or deficiency lead to the inflation of certain antigenspecific T cell populations and reduced viral load, most likely as a consequence of the enhanced immune response. Reactivation of human CMV was also apparent in subjects with dasatinib-associated large granular lymphocyte expansions. Consistent with a causative association, the expanded T cell and NK cell populations in these subjects were oligoclonal and exhibited a late differentiated (CD27-CD57+) phenotype, indicative of chronic antigenic stimulation. In addition, CD8high and CD8low T cells were observed within both the total and CMV-specific CD8+ T cell compartments, consistent with CMVdriven activation. In summary, these data show that antigen alone is not sufficient to induce full immune activation, even under conditions of chronic stimulation. Additional signals, such as those provided by an inflammatory environment, are required to trigger full T cell activation and expansion. Persistent viruses attempt to undermine this process, for example by the expression of homologues that mimic host immune regulators. Even in the presence of viral reactivation and immune system perturbations, however, the T cell compartment can demonstrate remarkable resilience in its ability to generate fully differentiated and functional expansions. The persistence of certain memory T cell subsets under such conditions appears to play an important role in the immune response to chronic “dangerous” antigens.

Throughout our life cycle, the human body is exposed to harmful microorganisms. The innate immune system is a fundamental factor in the human body, which helps eliminate foreign organisms through specific signalling pathways with the involvement of immune receptors and signalling molecules. Viruses have evolved to infect the host and bypass the host immune responses,however a plethora of Pattern recognition receptors exist in the cell that are capable of detecting viral pathogens and mounting an innate immune response. Herpes simplex Virus Type 2 and Cytomegalovirus are common human pathogens that cause genital ulcerations, organ failure and mental health problems like encephalitis. In this study, we have aimed to identify the host’s innate immune response to HSV2 and HCMV infection in primary vaginal cells as well as Hela cells. Our data have shown that these viruses are recognized by TLR2 on the cell surface followed by intracellular PRRs such as TLR9, DAI, and IFI16, which trigger cytokine activation and release. Confocal imaging has revealed that these PRRs are located in different cell compartments and during viral cell entry and replication they can identify viral presence at specific parts of the cell. Therefore it seems that different PRRs are strategically placed in different cell locations to detect virus invasion and replication in order to activate cytokine secretion and protect the host. When different agonists for PRRs were used it was revealed that they were effective against Herpes virus infection thus indicating that a combination of PRRs agonists especially ones that trigger different cytokines could provide a wider spectrum prophylaxis to the host and they can be used to generate efficient treatment against HSV2 and HCMV infection.

The aetiology of Common Variable Immunodeficiency (CVID) has fascinated immunologists since Dr. Janeway reported the first case in 1953. While the advances in molecular biology have enlightened us on the aetiology in some patients, the majority is not caused by inherited genetic disorders. A convincing mechanism accounting for the intrinsic variable and partial nature of the condition has yet been proposed. CVID separates itself from other primary antibody deficiencies by the procurement of an abnormal T-cell compartment. Data from this study support that both T-cells and B-cells are subjected to similar deficiency. Investigation of the T-cell receptor repertoire by next-generation sequencing and multi-parametric flow cytometry suggests a severe reduction in naïve T-cell output from the thymus. Similarly, the study of long-lived plasma cell generation and survival highlighted the greatest functional deficits in the naïve B-cell pool, altogether supporting an acquired arrest in lymphogenesis. Using DNA methylation as a surrogate marker for pre-VDJ clonality, this study further shows that some CVID patients exhibited clonal haematopoiesis, adjoining CVID to other clonal haematopoiesis related acquired haematological disorders. Further work is being focused on using high resolution techniques to confirm this association and mechanistically define the development of antibody deficiency in adulthood.

CD4\(^+\) T cells modulate an immune response through the production of effector cytokines. In some circumstances the effector function of CD4\(^+\) T cells is diminished, which may have beneficial (peripheral tolerance) or detrimental (exhaustion, senescence) consequences. Here I characterise a population of CD4\(^+\) cells in human peripheral blood which exhibit complete hyporesponsiveness to in vitro stimulation, as indicated by an absence of CD69 upregulation and the failure to secrete any of thirteen candidate cytokines. These T cells had an effector memory phenotype (CD45RA\(^-\)CCR7\(^-\)\(^/\)\(^+\)CD62L\(^{lo}\)CD27\(^{lo}\)), but their intermediate expression of PD-1 did not suggest a state of exhaustion. Although regulatory T cells (CD25\(^{hi}\)CD127\(^{lo}\)) contributed to the hyporesponsive population it was not predominated by this phenotype. However, the possibility that these hyporesponsive cells represent a non-classical regulatory subset could not be excluded. CD4\(^+\) T cells can enter the central nervous system (CNS) via the blood cerebrospinal fluid barrier, but their biological activity and recruitment pathways are under-defined. Preliminary studies had suggested that hyporesponsive CD4\(^+\) T cells were enriched in uninflamed human cerebrospinal fluid (CSF). However, this investigation found that CSF and brain-derived CD4\(^+\) T cells readily upregulate CD69 upon activation (mouse, human) and have robust IFNγ responses (rat). This evidence supports a role for CD4\(^+\) T cells in CNS immune surveillance and immunity. This investigation also showed that the proportion of CCR7\(^+\) and CCR7\(^-\) memory CD4\(^+\) T cells in the CSF was a direct reflection of the distribution in the peripheral blood in both mouse and man. This suggests that CSF recruitment is not CCR7-dependent as is previously described, and shows effector memory cells enter the CSF space in the absence of neuropathology. Such findings have implications for the understanding of normal immune function in the CNS, and the protective or pathogenic contribution of CD4\(^+\) T cells to neuroinflammatory disorders such as multiple sclerosis.