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To understand how immune balance is affected in immunoscnescencc and discover opportunities for correction with immunotherapy, a cross-sectional study of pro- and anti-inflammatory CD4+ T cell subsets in the peripheral blood of donors of different, ages was conducted. A whole blood assay using extra- and intracellular flow cytometry was designed to enumerate anti-inflammatory natural CD127lowCD25highFoxp3+ T regulatory cells (nTreg) and inducible IL-10+IFNy- T regulatory cells (iTreg) versus proinflammatory T helper 17 cells (Thl7) and T helper 1 cells (Thl), both IL-10+ and IL-10- . The frequency of these cell types was also linked to functional measurements of IL-17, IL-10 and IFN7 in whole blood supernatants after an overnight stimulation. We tested the hypothesis that the balance between these subsets changes with older age. This work shows for the first time that iTreg increase relative to nTreg and Thl7, but not Thl, with healthy ageing in man. We also identify that the previously reported increases in IL-10 levels relate to changes in the iTreg population. However, they did not increase relative to the Thl subset. During this project, scope for improvement in multicolour flow cytometry data analysis was identified, in order to minimise subjectivity and maximise efficiency of nTreg enumeration. The hypothesis that automating nTreg analysis with the SamSPECTRAL algorithm is superior to baseline C means clustering and traditional manual gating was investigated. SamSPECTRAL was qualitatively better than /c-means in clustering nTreg from flow cytometry data containing overlapping, non-spherical clusters with different densities, and was more objective than traditional manual gating. However, across 90 data files, an optimal solution was not always achieved, and statistical measures of cluster validity did not support the visual evidence that SamSPECTRAL better captured the natural structure of the data. A novel extension of SamSPECTRAL to include an automated elliptical gating step allowed for comparison of test and control datasets to correct nTreg frequency measurements for false positive events. As manual inspection of each solution was required, however, the ability to entirely automate nTreg analysis was prevented. We hope that this work will encourage further collaboration between the disciplines of immunology and computer science to advance the study of cancer and ageing.

Dendritic cells (DCs) are key members of the immune system as they detect danger and reflect this during presentation of antigen to T cells. DC produce cytokines that influence both the innate and adaptive immune system. Notably they produce the IL- 12-family of cytokines known for their influence on T helper (Th) cell priming. Interleukin-23 is one such cytokine that is recognised for its roles in supporting Thl7 cell differentiation characterised by IL-17 and IL-22 production, in driving IL-17 secretion by y6 T cells, and in its contribution to anti-bacterial immunity and autoimmunity. A body of evidence exists that Dectin-l/TLR-2 Syk- CARD9-NFkB signalling pathways, p38 MAPK/Erk- NFk-B signalling pathways and PI3K/Akt pathways are involved in IL-23 production in their respective settings. However, little is known at the molecular level regarding the regulation of IL-23 secretion in human DC. Here we show for first time that the Ataxia Telangiectasia Mutated (ATM) pathway and DNA-Protein kinase catalytic subunit (DNA-PKcs), involved in DNA-damage-sensing and DNA double strand break (DSB) repair, act as repressors of IL-23 in DC. Inhibition of ATM with the highly-selective antagonist KU55933, or DNA-PKcs with NU7441, markedly increased IL-23 secretion human monocyte-derived DC (moDC) and freshly isolated CDlc+ myeloid DC (myDC). In contrast, inhibiting the closely related mammalian target of rapamycin (mTOR) had no effect on IL-23. Priming naive CD4+ T- cells in the presence of supernatant from ATM or DNA-PKcs-inhibited DC resulted in increased Thl7 responses. Whilst ATM or DNA-PKcs-blockade increased the abundance of pl9, p35 and p40 mRNA, IL- 12p70 secretion was much less affected. In order to further examine a role for ATM and DNA-PKcs in IL-23 regulation we exposed DC to low doses of ionizing radiation. Exposure of DC to X-rays resulted in a rapid increase (15min) in ATM phosphorylation and a corresponding depression of IL-23 production. Importantly, ATM-inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of X-rays to suppress IL-23. Interestingly, the viability of DC exposed to X-rays after DNA-Pkcs inhibition was substantially affected but unaffected in DC exposed to IR after ATM inhibition, suggesting an important role for DNA-PKcs in the resistance of DC to DNA-damage. To explore how IL-23 was repressed by ATM or DNA-PKcs we examined the unfolded protein response (UPR) pathways by measuring generation of the spliced form of X-box protein-1 (XBPls), a key ER-stress transcription factor. When ATM or DNA-Pkcs were inhibited the abundance of XBPls mRNA increased and was followed 3hr later by increased p l9 transcription and subsequent IL-23 release. Unexpectedly, in DC deficient for the UPR-related CHOP transcription factor, inhibition of ATM or DNA-PKcs continued to secrete high levels of IL-23. However, inhibition of the PERK arm of the UPR reduced IL-23 production by matured DC, and importantly partially suppressed ATM- or DNA-PKcs-dependent IL-23. In summary, in addition to their canonical roles in DNA-repair, ATM and DNA-PKcs also regulate immune-activation by DC. Whilst their activation further represses IL-23-dependent responses, their inhibition permits markedly high levels of IL-23 to be generated. These pathways thus represent new therapeutic targets in autoimmunity and vaccine development.

The technique of magnetic resonance imaging (MRI) is widely used in both structural and functional imaging. This thesis explores some important developments and applications in high field MRI. The work in this thesis has been undertaken by the author except where indicated by reference. Initially, a purpose-built dynamic functional phantom was devised and used for quality control and testing imaging pulse sequences. A multi-echo GEBPI sequence was assessed and the most efficient ways to combine multiecho data taking account of the effect of bandwidth were explored by using this phantom. Subsequently, this multi-echo sequence was applied for T-i mapping of the whole brain and cerebellum with different slice thicknesses and orientations and used for characterization of the physiological noise in the resting-state brain on 3.0 and 7.0 T systems. Furthermore, physiological noise was also investigated by using a spin echo imaging method. The physiological noise in. gradient and spin echo images was compared. The results support the hypothesis that physiological noise has similar origins as the BOLD signal. Contrast-enhanced MR angiography provides accurate information about vascular structure. The field strength dependence of Rl and R2 relaxivities of a blood pool contrast agent (Gadofosveset) was measured ex vivo in human blood. A computer simulation was performed to investigate quantitatively the performance of contrast-to-noise (CNR) ratio of Gadofosveset-enhanced MRA at different field strengths. Finally, highresolution contrast-enhanced MR angiographic imaging was performed on human subjects at 7.0 T. The initial results demonstrate that, despite posHible technical problems, high quality imaging is feasible at 7.0 T. These H)sults also show that the ultra-high field strength has the potential to offer tllgllificant improvements in CNR and spatial resolution.

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