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Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva. The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva-infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria-specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK was shown to inhibit killing by CD8+ T cell clones.

LAG-3 is an immune inhibitory marker. These immune inhibitory markers function to reduce the capability of immune cells to elicit a proper immune response and to help maintain tolerance. During an acute infection, these markers assist in controlling the immune system, however, during a chronic infection these markers prevent the immune response from persisting to effectively fight the disease. Contrary to other immune inhibitory markers, LAG-3 is not highly expressed on T cells during chronic viral infections, such as HIV. The majority of information available on LAG-3 has been gained from murine models and cell lines. This thesis uses primary human T cells in order to observe rapid expression of surface LAG-3 from a pre-formed intracellular store and cleavage of these surface molecules into soluble variants by matrix metalloprotease cleavage. LAG-3 and sLAG-3 expression is compared with CD69 and cytokine expression to help understand the early immune response. / October 2015

HIV-1 chronically infected individuals represent a large disease burden, making the development of a therapeutic vaccine for use in chronic infection a priority. However, therapeutic vaccination has not been successful to date. Most approaches have employed viral vectored vaccines encoding full length viral proteins, which aimed to boost pre-existing CD8+ T cell responses by mimicking natural HIV-1 infection. Simply boosting these pre-existing CD8+ T cell responses which have previously failed to control the virus may be insufficient. Although HIV-1 has a huge capacity to diversify, certain regions are less tolerant of mutations due to structural and functional constraints. We therefore hypothesised that it would be necessary to redirect the immune response to more vulnerable regions of the proteome, such as conserved regions. HIVconsv is a rationally designed conserved region vaccine. Vaccination of 19 chronically HIV-1 infected HAART treated patients with MVA.HIVconsv was safe and well tolerated. There was a significant increase in the magnitude of HIVconsv-specific T cell response following vaccination (p = 0.001), as measured by IFN-γ Elispot assay, but changes observed in vaccinees did not reach statistical significance when compared with placebo recipients (p = 0.48). The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro is highly predictive of virus control in vivo and is thus a possible surrogate of vaccine efficacy. There was a trend towards increased CD8+ T cell mediated inhibition following vaccination with 2x10⁸pfu MVA.HIVconsv (17% inhibition pre- vs 54% inhibition post-vaccination, p = 0.06). However, measurement of the latent HIV-1 reservoir by quantification of total HIV-1 DNA in circulating CD4+ T cells by droplet digital PCR showed no reduction in size upon vaccination, indicating CD8+ T cells induced by vaccination with MVA.HIVconsv were not of sufficient potency to impact the reservoir. In a second cohort of HIV-1 infected individuals, antiviral inhibitory activity was measured in 36 HIV-1-infected STEP and Phambili trial participants. Sustained potent CD8+ T cell antiviral inhibitory responses were rare but were strongly correlated with IFN-γ responses to so-called ‘beneficial’ low entropy regions in HIV-1 Gag and Pol, that had been reported previously to be associated with HIV-1 control, (r = 0.69, p = 0.0001). This correlation was still significant after controlling for protective HLA alleles, whereas responses to conserved elements were only weakly correlated with viral inhibition (r = 0.41, p = 0.04). These data indicate that immunogens that are based on the selection of regions within the viral proteome by conservation score alone may not induce the most effective HIV-1-specific T cell responses and they highlight the importance of systematically selecting specific regions associated with HIV-1 control, together with exclusion of immunodominant decoy epitopes.

Influenza A virus (IAV) infection is a highly contagious respiratory disease, which can cause substantial morbidity and occasionally death. Invariant natural killer T (iNKT) cells, a subset of CD1d-restricted T lymphocytes, have been identified as important modulators of immunity, mediating both pro- and anti-inflammatory responses. We show that iNKTs play an important role for the generation of protective innate and adaptive immune responses to IAV, and enhance heterotypic immunity to influenza virus following vaccination with a novel pseudotyped virus, S-FLU, which lacks HA expression. iNKT-deficient mice (Jα18^-/-) showed increased susceptibility and lung pathology following IAV infection, which correlated with an exaggerated accumulation of inflammatory monocytes and neutrophils in the lung. Consistent with these findings, we demonstrated in IAV-infected CD1d^-/-:CD1d^{+/+} mixed bone marrow chimeric mice, that the lack of CD1d expression on myeloid cells purified from the lungs of IAV-infected mice significantly increased expression of genes linked to cell activation, survival and polarisation between pro- and antiinflammatory responses. We extended these results by examining the role of chemokine signalling during IAV infection, and identified a novel role for fractalkine (CX3CL1) and its receptor (CX3CR1) in innate immune cell recruitment. The use of CX3CR1-iNKT cell double knockout mice revealed that, although upregulated in Jα18^-/- mice, CX3CR1-CX3CL1 signalling is not required for cell migration during exacerbated IAV-responses. Finally, we showed that iNKT-deficient mice displayed reduced longevity of peripheral IAVspecific CD8^+ T cells following S-FLU vaccination, compared with wild-type mice. S-FLU vaccination protected mice following 5 day heterotypic challenge, however vaccinated mice exhibited reduced viral clearance, and importantly a significant reduction in IAV specific memory T cell response, suggesting a possible role of iNKT cells during T cell priming in modulating the lifespan of antigen-specific T cell responses. Although additional experiments are warranted, these results suggest that harnessing iNKT cells should be considered to modulate the innate and adaptive immune response to optimise heterotypic vaccine design and for therapeutic intervention against acute influenza infection.

The development and function of T helper (Th) cells and regulatory T cells (Tregs) are plastic processes that are regulated by cytokines. In our project we first analyzed the effect of different cytokines on the development of induced (i) Tregs. It has been demonstrated that iTregs arise from CD4+ CD25- T cells upon stimulation with alloantigen in the presence of transforming growth factor β (TGF-β). The development of these Tregs and their proliferation were inhibited by interleukin (IL)-4 and IL-12. The aquired results also demonstrated distinct responses of naturally occuring (n) Tregs and iTregs to the regulatory action of IL-4 and an opposite role of IL-4 in maintenance of nTregs and iTregs phenotype. An important role in the induction of T cell subsets may play also mesenchymal stem cells (MSCs) which can, under specific conditions, produce TGF-β and IL-6. Depending on the current production of TGF-β or IL-6, MSCs can qualitatively regulate the ration between Tregs and Th17 cells. Anti-inflammatory Tregs and pro-inflammatory Th17 cells are induced upon stimulation in the presence of TGF-β and TGF-β and IL-6, respectively. In addition to our previous work we studied the role of IL-12 in the development of Tregs and Th17 cells. It was shown that Treg and also Th17 cell differentiation was...

Regulatory T cells (Treg) are critical to the maintenance of immune tolerance, partly by controlling the unwanted activation of effector T cells (Teff) and thereby enhancing the resolution of autoimmune and allergic inflammation. Recent data suggest that Treg can specialize to better control different types of inflammation by using transcriptional machinery which controls differentiation and function of Teff. This thesis addresses questions related to the efficacious use of Treg, notably their ability to adopt distinct phenotypic profiles under different inflammatory contexts and their need to recognize antigen in the inflamed organ. Two differentially mediated mouse disease models were used in this project, namely Th1/Th17-mediated experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis and Th2-mediated allergic airways inflammation (AAI) as a model of asthma. A new model of rMOG-induced AAI was developed to specifically answer the questions on the importance of cell phenotype versus antigen-reactivity for the effective Treg-mediated suppression. It was demonstrated that Treg from the inflamed CNS in EAE had an upregulated expression of Th1 master regulator T-bet and Th1-associated chemokine receptor CXCR3, whereas Treg derived from the inflamed lung in AAI had an increased expression of Th2 master regulator GATA-3, lacked expression of T-bet and displayed decreased levels of CXCR3. This specialized and activated phenotype was restricted to tissue-derived Treg. The importance of appropriate Treg phenotype for effective suppression was suggested by the observed inability of CNS-derived Treg to inhibit AAI. A different Treg subset, TGF-β-induced Treg (iTreg), was shown to express high levels of T-bet and CXCR3, but not GATA-3 upon induction in vitro. iTreg effectively suppressed both Th1 and Th2 types of inflammation and the antigenreactivity was key to this. This thesis demonstrates that Treg are capable of acquiring a distinct phenotype corresponding with a CD4+ T cell response driving inflammatory disease and identifies antigen-reactivity as key to the efficacious suppression of inflammation. It also highlights substantial phenotypic differences between iTreg and naturally-occurring Treg which could be associated with different modes of suppression.

<p>Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.</p><p>It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs. </p><p>In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.</p><p>Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.</p> / Dissertation

Infection with hepatitis C virus (HCV) can cause liver damage known as fibrosis, which often leads to liver disease and hepatocellular carcinoma. The impairment of circulating, bulk (non-specific and specific) CD8+ T cells within HCV-infection, characterized by an altered phenotype and the increased expression of pro-apoptotic genes, is observed when compared to uninfected controls. The relationship between bulk CD8+ T cell function and the extent of liver damage has not been demonstrated. In this study, widespread immune alterations were observed in untreated HCV infection with advanced liver fibrosis. Untreated HCV-infected individuals with advanced fibrosis possessed a significantly decreased proportion of naïve CD8+ T cells and an increased proportion of late effector memory CD8+ T cells compared to uninfected controls. Upon T cell receptor (TCR) stimulation, these individuals also had an increased intracellular IFN-γ expression for four CD8+ T cell subsets, a decreased CD107a expression for central memory CD8+ T cells, and a decreased perforin induction for naïve and central memory CD8+ T cells. These immune alterations did not reverse 24 weeks after viral cure. This study indicates there is a relationship between the differentiation and function of bulk CD8+ T cells and the extent of liver damage within HCV infection.

A highly effective vaccine against malaria is urgently needed, with leading vaccination strategies involving the induction of protective antigen-specific CD8⁺ T-cells via heterologous prime-boost viral vector immunization, targeting primarily the pre- erythrocytic liver stages of the Plasmodium falciparum lifecycle. To date, the greatest immunogenicity has been obtained through a heterologous prime boost regimen, where vaccination with an Adenoviral vector is followed 8 weeks later by a Modified Vaccinia Ankara virus (MVA) boost. Experimental work directed at providing a greater understanding of CD8⁺ T-cell memory responses induced by Ad-MVA vaccinations lead to the development of a novel vaccine strategy aimed at priming CD8⁺ T-cells in the periphery and subsequently targeting them to hepatic tissue with protein loaded poly(lactic- co-glycolic acid) nanoparticles or recombinant viral vectors. Durable Ag-specific CD8+ T- cells exhibiting a phenotype of tissue-resident memory T-cells were generated in the liver, with a ten-fold increase over the conventional heterologous vector regimen. Importantly, in P. berghei sporozoite challenge models of liver-stage malaria, this strategy was found to result in unprecedented levels of sterile protection across multiple clinically relevant antigens and mouse strains. This prime and target immunization strategy for liver-stage malaria may provide a novel general approach for prevention or immunotherapy against other hepato-trophic pathogens.