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T cell infiltration into tumours is essential for tumour antigen recognition and tumour cell elimination. The aim of this study was to develop a better understanding of T cell infiltration into tumours, focusing on two opposing arms of an immune response, anti-tumour CD8 Tcells and Regulatory T cells (Tregs). Activated CD4 T helper cells are also of importance but could not be studied due to the time constraints of the project. The effect of T cell signalling at the immunological synapse following interactions between T cells and APCs presenting cognate antigen have been well studied [1]. The endothelium is neither a stereotypical APC nor simply a passive filter barrier for non-cognate infiltrating T cells. The endothelium can activel influence the development of an inflammatory response depending on the functional state of both the endothelium and interacting T cells (resting versus recently activated T cells)and the type of interactions (cognate versus non-cognate). The hypothesis was that recognition of antigens presented in the context of major histocompatibility complex (MHC)molecules by endothelium aids T cell transmigration and hence infiltration into tissues, including into tumours. In this study, the data highlights that high avidity TRP-2 specific CD8 T cell transmigration across murine lung endothelium requires recognition of TRP-2 peptide presented by the endothelium, aiding recruitment of antigen-specific T cells into tissues in the absence of endothelial cell killing. In order for antigen specific T cells to migrate into the tumour, the tumour endothelium therefore needs to present tumour antigens. In addition to CD8 T cells, high numbers of Tregs have been found within tumours but the key mediators for this recruitment remain uncertain. The data shows a novel mechanism for Treg transmigration where cognate antigen-specific recognition of self-peptides was required for transmigration with preferential transmigration of Tregs across syngeneic rather than allogeneic endothelium. Upregulation of major histocompatibility complex (MHC) class II and adhesion molecules, by IFN-γ and TNF-α, together with a gradient of the tumour associated chemokine CXCL12 were also pre-requisites for efficient Treg transmigration. Previous studies have shown that high CXCL12 expression can induce fugetaxis of tumour cells leading to efficient metastatic spread and a poor prognosis. These results would suggest that low levels of CXCL12 and recognition of self peptides presented by self MHC on endothelial cells allows efficient migration of Tregs whereas higher levels of CXCL12 may promote tumour metastases and lead to fugetaxis of the Tregs leading to an even worse prognosis. In conclusion recognition of cognate antigen presented by endothelium enhances antigenspecific transmigration of CD8 and Regulatory T cells. This study therefore reports a novel mechanism for T cell subset infiltration into tumours where high avidity CD8 T cells require recognition of cognate tumour antigen presented on tumour endothelium in the context of MHC class I and conversely regulatory T cell infiltration into tumours depends on the repertoire of self-peptides presented on tumour endothelium in the context of MHC class II. Alteration of antigen presentation or MHC expression on tumour endothelium therefore represents a mechanism whereby T cell infiltration can be altered to re-direct anti-tumour immune responses.

Human ageing is associated with a progressive decline in the function of the immune system, commonly referred as immunosenescence. This is characterized by the shrinkage of the naïve T-cell repertoire and a concomitant accumulation of highly differentiated effector-memory cells and dysfunctional senescent T-cells. These systemic immune alterations have clinical implications and have been associated with increased morbidity and mortality in the elderly. However chronological ageing may not be the only factor influencing immunosenescence and certain lifestyle factors may moderate or potentiate the rate at which immune alterations occur. The studies comprised within this thesis investigated the effects of lifestyle factors such as physical activity or obesity, along with latent viral infections on the proportions of highly differentiated and senescent T-cells. Data were gathered from individuals of various age, physical activity, body composition and latent viral infection status to assess the effects of a wide range of lifestyle factors on T-cell proportions. The different levels of T-cell differentiation were assessed by four-colour flow cytometry using monoclonal antibodies specific to cell surface markers associated with T-cell phenotypes. The role played by leptin on T-cell activation was assessed by in vitro stimulation assays followed by cell surface phenotype and gene expression analysis. The effects of acute bouts of exercise on T-cells subsets were characterized using a submaximal cycling protocol. Aerobic fitness was associated with lower proportions of senescent and higher proportions of naïve T-cells, particularly within the CD8+ T-cell compartment in healthy adult men. The beneficial impact of aerobic fitness, and consequently of regular physical activity, on the ageing immune system was independent of age, latent viral infection status and body composition. Furthermore, the moderating effect of higher estimated VO2max on the proportions of senescent T-cells suggested that a transition from low physical activity, characterized as having an estimated VO2max below average, to regular physical activity, characterized as having a VO2max above average, could prevent the age-associated accumulation of senescent T-cells during decades. Obesity and excess serum leptin in adolescents were shown to be associated with changes in T-cell subsets associated with immunosenescence, such as reduced proportions of naïve and early T-cell and increased proportions of effector-memory and senescent T-cells. In addition, high physiological concentrations of leptin enhanced the mitogen-induced T-cell activation in vitro suggesting a potential role in the accumulation of senescent T-cells observed in obese individuals. Latent CMV infection was also associated with similar reductions in naïve T-cell proportions and increased proportions of highly differentiated and senescent T-cells in young adults. Although CMV infection appeared to be associated with an amplified exercise-induced preferential mobilization of highly differentiated and senescent T-cells in blood, those cells may not have been specific for CMV. It is concluded from this work, therefore, that chronological ageing is not the only factor associated with the accumulation of senescent T-cells in the elderly. By preventing obesity, and by potentially inducing senescent T-cells frequent mobilization and subsequent deletion via apoptosis, regular physical activity may prevent the accumulation of highly differentiated and senescent T-cells in the elderly, and consequently reduce morbidity and mortality in later life.

CD8+ T-cell antigen sensitivity is critical for optimal control of persistent viral infections, including HIV-1. The devastating HIV-1 pandemic may be countered by development of a cytotoxic T lymphocyte based vaccine if qualities associated with protection can be defined at the molecular level. However, the heterogeneity of the total viral-specific CTL response confounds identification of protective correlates and T-cell sensitivity is no exception and remains controversial. To address this issue we reduced the heterogeneity of the HIV-1 CTL response to single units, generating 19 CTL clones that recognise the same HIV-1 derived epitope restricted by HLA B*08. Correlation of functional assays directly with the ability of each clone to control HIV-1 replication in vitro, the “viral suppression assay,” identified antigen sensitivity as a key quality for anti-viral efficacy. Remarkably, four clones from this panel, isolated from one individual, a long-term non-progressor, all used an identical TCR yet had distinct antigen sensitivity and suppressive activity. Two of these clones were characterised in detail, and had distinct cytokine profiles, regulated by epigenetic mechanisms, and differential expression of a group of cell surface receptors with the potential to modulate the signalling threshold to antigen. Expression of the TNFα locus of the high sensitivity clone with “Good” suppression was repressed by DNA methylation. Understanding how CTL qualities required for optimal control of HIV-1 replication differentiate and are then enriched in the total CTL response, and if repression of TNFα contributes to this process, will contribute to rational vaccine design. This is the first evidence that avidity maturation in CD8+ T cells with the same TCR affinity occurs in viral infections in humans as reported in the mouse. This suggests the induction of high sensitivity CTL will be critical for an effective HIV-1 vaccine, but offers hope that this can be achieved even in individuals without protective HLA alleles, by further exploration of peripheral avidity maturation and epigenetic regulation of the HIV-1 specific CD8+ T-cell response.

The migration of T lymphocytes is a vital component of the immune system, with roles in immunosurveillance and inflammation. The role of Phosphoinositide 3-kinase within T lymphocyte migration is unclear, with some evidence that it may be a disposable signal. Here, using Staphylococcal Enterotoxin B activated peripheral blood mononuclear cells and the T cell line CEM cells, the role of Phosphoinositide 3-kinase and its downstream kinases was investigated. CCL22 mediated CEM cell migration and CXCL12 mediated peripheral blood mononuclear cell migration were shown to be independent of Phosphoinositide 3-kinase using several different broad-spectrum Phosphoinositide 3-kinase inhibitors. However, these cells were Akt-dependent, as demonstrated by incubation with the Akt inhibitor Akti-1/2. Differences in the effect of the inhibitors on Akt activity were discovered, indicating that either Akt can be activated in the absence of Phosphoinositide 3-kinase, or differences exist regarding the relative abundance of each protein within the cell. Th17 cells are a subtype of the T helper cell family and have been shown to be involved in inflammation and immune diseases. Mouse splenocytes were polarised to a Th17 phenotype and analysed for the surface expression of chemokine receptors. CCR2, CCR6 and CCR9 were shown to be expressed on Th17 cells and upregulated under Th17 polarising conditions. However, only CCR2 and CCR6 induced migration of Th17 cells. This migration was sensitive to Phosphoinositide 3-kinase and Akt inhibitors. This data reveals a model for the migration of Th17 cells to areas of inflammation, and sheds light on the role of Phosphoinositide 3-kinase during this process.

Regulatory T cells (Tregs) are a distinct lymphocyte lineage, functionally defined as T cells that inhibit an immune response, which is crucial for maintaining peripheral tolerance and the prevention of autoimmunity. Tregs have been implicated in tumour immune evasion, suppressing the anti-tumour immune response, resulting in tumour progression. The aim of this PhD was to recognise how Tregs function and understand how they are able to modulate tumour immunology. In order to research their proposed role in cancer, it was necessary to be able to phenotype, sort and expand functional Tregs. In preliminary research, strategies were designed to phenotype and test the functionality of ex vivo Tregs and commercial isolation kits were tested and compared in terms of cost efficiency and effectiveness. The mechanisms of Treg-mediated suppression were investigated, including the necessity for cell-to-cell contact and the involvement of key cytokines. The importance of particular cytokines in Treg-mediated suppression was not clear; but cell contact appears to be required for optimal suppression. The project then aimed to address whether or not the importance of Tregs highlighted in the literature was reflected in a clinical setting. The significance of immune cell orientation within the tumour microenvironment was researched, investigating the co- localisation between key immune cell subtypes and their correlation with areas of tumour proliferation, apoptosis, hypoxia and vasculature coverage. In order to achieve this successfully, immunohistochemical techniques were optimised and image analysis algorithms were constructed to facilitate rigorous and systematic quantification of immune cell infiltrates in primary colorectal and metastatic liver cancer patients. Significant increases in the prevalence of Tregs, CD8 cells, macrophages and natural killer (NK) cells were observed within the stroma, compared with the tumour. A metastatic phenotype was alluded to; encompassing elevated Tregs and reduced numbers of CD8 cells. Further to this, the level of Tregs in the peripheral blood and tumour tissue of liver- metastatic patients were assessed to investigate whether any correlation existed between circulating and tumour-infiltrating Tregs. It was shown that within peripheral blood, patients exhibited a TreghighCD8lowNKlow phenotype, when compared with healthy volunteers. Finally, the project aimed to build an in vitro human Treg tumour-killing suppression model to examine the ability of Tregs to inhibit NK cell-mediated cytotoxicity. The mechanism for this was investigated, evaluating the importance of the NK group 2 member D (NKG2D) receptor ligand interaction, to mediate cell killing in a transforming growth factor-β dependent manner. This study adds to the ongoing discussion on the role of immune cells in metastatic development. Accumulating evidence points to a critical role of immune infiltrates in allowing metastatic development, where Tregs support tumour growth by suppressing the host anti-tumour immune response.

Alloresponses to blood group antigens result from antigen mismatch between donor and recipient during blood transfusion or transplantation and between mother and fetus during pregnancy. During pregnancy, antigen mismatch can result in haemolytic disease of the newborn (HDN), a disease characterised by the development of potentially harmful alloantibodies, which cross the placenta and mediate the destruction of fetal erythrocytes. This project investigates examples of clinically important alloresponses to blood group antigens and, more specifically, characterises the ymphocytes that either drive or regulate these responses. The main aims or this project were to first map alioreactive T-helper cell epitopes and secondly to clone using a novel method, IL-10 secreting blood group specific regulatory cells. The work focussed on two major antigens, the kell (K) 1 and Rhesus (Rh) D antigens.

T cells are believed to be important in the control of varicella zoster virus (VZV) replication but little is known of T cell epitopes and the relationships between T cell responses, viral load and clinical disease during primary infection. I initially set to investigate the immune responses to two of the main VZV glycoproteins (gE and gI) using ex vivo and cultured IFNγ ELISpot assays. I identified several novel CD4+ T cell epitopes within gE and gI and characterized the phenotype of gE DRB1*1501 tetramer specific responses in healthy immune donors. I then set out to investigate the function and phenotype of VZV specific T cells in primary infection and their relationship to viral loads and clinical disease severity by using glycoprotein E/DRB1*1501 specific MHC class II tetramers, ex vivo IFNγ ELISpot assays and quantitative real time PCR assays. I compared the frequency and phenotype of specific T cells with virological and clinical outcomes in 32 adult individuals with primary VZV infection. In healthy immune donors, the gE specific T cells showed a early intermediate stage of differentiation with evidence of recent activation. Patients with acute primary infection had higher VZV/DRB1*1501 tetramer specific T cell responses and expressed markers of activation and effector differentiation. Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of the patients (p<0.05, r=-0.64). These data would be compatible with a role for gE and gl-specific T cells in the control of viral replication during both primary infection and re-activation.

Protein-protein recognition plays a central role in the surveillance of self and non-self in the mammalian immune system and ultimately in cellular survival within the organism. Two systems of fundamental importance to the immune system are the Tumour Necrosis Factor (TNF) and the T cell receptor (TCR) families. High-throughput methods developed within the Oxford Protein Production Facility have been successfully applied to the production of members of the TNF receptor and ligand superfamilies for structural characterisation. The TNF receptor DR6 was successfully refolded from E.coli inclusion bodies using a rapid-dilution technique and yielded diffraction quality crystals. Data collected from these crystals will be used to obtain an x-ray crystallographic model of DR6. Vascular Endothelial Growth Inhibitor (VEGI) was produced as a soluble recombinant protein in E.coli, and formed a number of poorly diffracting crystals, it is hoped that further trials and optimization of conditions will lead to improved data quality. Lymphotoxin β receptor was produced in a Eukaryotic system. This has shed light on the complications posed by signal peptide cleavage and glycosylation on the production of protein for crystallization trials. TNF superfamily proteins are ideal targets for the design of novel therapeutic agents due to their involvement in a number of disease pathologies. Various methods of molecular docking and small molecule design were applied to the search for potential inhibitors of receptor binding for the TNF ligand proteins TRAIL and BAFF. A number of potential drug leads were identified from the National Cancer Institute drug database. The Natural Killer (NK) T cell restricted TCRs recognise CD1d-presented glycolipid. Determination of the crystal structures of the invariant NK TCR and the NK restricted TCRs 5E and 5B shows that these proteins adopt the canonical structures of class I MHC restricted TCRs. This suggests that the binding of CD1d-glycolipid by these receptors will conform to the same model of binding seen for the class I MHC restricted TCRs.

Tc17 cells and the semi-invariant human mucosal associated invariant T (MAIT) cells are important CD8+ tissue-homing cell populations. Both are characterized by high expression of CD161 (++) and type-17 differentiation, yet their origins and relationships remain poorly defined. By transcriptional and functional analyses it is demonstrated that a pool of polyclonal, pre-committed type-17 CD161++CD8αβ+ T cells exists in cord blood, from which a prominent MAIT cell (TCR Vα7.2+/Vβ2 or 13.2) population emerges post-natally. During this expansion, CD8αα T-cells appear exclusively within CD161++CD8+/MAIT subset, sharing cytokine production (IL17, IL-22 and IFN-γ), chemokine-receptor expression (CCR2, CCR6 and CXCR6), TCR-usage and transcriptional profiles with their CD161++CD8αβ+ counterparts. These data demonstrate the origin and differentiation pathway of MAIT cells from a naïve type-17 pre-committed CD161++CD8+ T cell pool and the distinct phenotype and function of CD8αα cells in man. The CD161++CD8αβ and CD8αα T cell subsets are reduced in the peripheral circulation in chronic hepatitis B and C and are enriched in the liver in chronic hepatitis C. Their potential role in immunity to chronic viral hepatitis B and C is demonstrated by their expression of activation/exhaustion markers CD69, CD25, HLA-DR and PD-1. In addition a substantial distinct CD161-CD8β^low population is demonstrated in chronic hepatitis B, co-characterised by a CD28^low, HLA-DR^high phenotype and high expression of IFN-γ, with important implications for the development of immunotherapy and vaccination.