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In this study, we hypothesized that T cells differentiate in the skin as a consequence of localised proliferation at the site of secondary immune challenge in humans. One consequence of cellular proliferation is telomere shortening, which can be counteracted by telomerase. It is recognised that telomerase is up-regulated in activated T cells that are resident in lymphoid tissues or the blood. We have used the Mantoux Test (MT), a secondary immune response, to determine the degree of cellular differentiation in the skin and its consequences during an episode of cutaneous inflammation. We have established a skin suction blister technique to isolate lymphocytes from MTs up to 19 days after induction for flow cytometric analysis, heteroduplex analysis, cell culture and measurement of telomerase activity (TRAP assay). Skin biopsies were also collected for immunohistochemical staining. Marked antigen-specific CD4+ T cell expansion with preserved clonality was observed in the skin during the MT. In contrast to recent murine studies, we found that this expansion was mediated in part by the extensive proliferation of CD4+ T cells in the skin. This was associated with substantial telomeric shortening in the antigen-specific CD4+ T cells in the skin, indicating accelerated cellular differentiation. Despite the development of a highly differentiated population of CD4+ T cells during the MT, there appeared to be no increase in the proportion of anergic/suppressive CD4+CD25+ T cells, which might mediate the down-regulation of inflammation during resolution. The erosion of telomeres appeared to be mediated by the reversible inhibition of telomerase by type 1 interferons in vivo. This reversible inhibition was distinct from the irreversible down-regulation of telomerase that was observed when MT skin T cells were repeatedly activated in vitro culminating in cell senescence. The inhibition of telomerase activity in T cells during secondary immune responses in the skin represents a possible control checkpoint that may limit uncontrolled T cell expansion in non-lymphoid tissues in vivo.

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This project investigates the potential of Natural Killer (NK) cells to respond to pathogen associated molecular patterns (PAMP) as model adjuvants and establishes the potential of PAMP to aid the recovery of antigen specific T-cell responses in chronic HIV-1 infection. Innate immunity is mediated by through the recognition of conserved PAMP recognised by receptors such as the toll-like receptors (TLR), expressed by accessory cells including blood DC, which are responsible for initiating adaptive immune responses, but also activate NK cells. NK cells are no longer seen solely as killers, but instead produce cytokines, including IFN-γ, and participate in the generation of adaptive immune responses with DC. HIV-1 infection potentially chronically activates TLR pathways, via recognition of the viral genome and as a result of damage to mucosal surfaces leading to microbial translocation and systemic recognition of PAMP including bacterial lipopolysaccharides. NK cell activation, cytokine production, differentiation and proliferation in response to PAMP have therefore been examined. NK cell responsiveness to certain TLR agonists was refractory in HIV-1 infected individuals although responses were maintained within CD56+CD16- NK cells, potentially providing help for antigen specific T-cell responses. The effects of HIV-1 infection on NK cell maturation were also investigated. T-cell activation by TLR agonists was also refractory in HIV-1 infected individuals. The impact of TLR ligation on antigen specific T-cell responses was investigated, combining TLR agonists with peptide pools (influenza, Epstein-Barr virus and cytomegalovirus or HIV-1 gag antigens). Of the TLR agonists tested, only CpG DNA resulted in enhanced frequencies of IFN-γ producing T-cells in HIV-1 infected individuals, whereas LPS demonstrated consistently reduced IFN-γ production. In conclusion, maintenance of responsiveness of CD56+CD16- NK cells and enhancement of HIV-1 antigen specific T-cell responses by CpG DNA have implications for both repopulation of the NK cell compartment and reconstitution of acquired immunity during HIV-1 infection.

T cell migration to sites of inflammation is an essential step of an effective immune response. T cell recruitment is enhanced by the recognition of their cognate antigen presented by the endothelium and their retention in the tissue is controlled by resident antigen presenting cells. Vav1 is a guanidine exchange factor for RhoGTPases that is activated through TCR signalling and is important for the cytoskeletal re-arrangements occurring during T cell activation and migration. In this study we have investigated the role of Vav1 in the recruitment and retention of antigen-specific T cells to sites of inflammation. HY-specific Ab-restricted WT and Vav1-/- T cells were generated and fully characterised for specificity and expression of migration-related markers. Vav1-/- T cells showed defects in adhesion, migration in response to ICAM-1, CXCL12 and CXCL10 and increased migratory speed in in vitro assays. Despite displaying defective motility in vitro, both constitutive migration and recruitment of Vav1-/- T cells to antigenic sites occurred normally. However, retention of Vav-1-/- T cells into antigenic tissue was profoundly impaired. This may be due to the inability by Vav-1-/- T cells to respond to ‘stop’ signals delivered by co-engagement of TCR and CD28. In contrast to recent reports on wild type T cells, Vav1-/- T cell migration and retention to sites of inflammation was not affected by engagement of the co-stimulatory molecule CD28, suggesting that Vav-1 recruitment during CD28-mediated signalling is instrumental for Vav-1-mediated regulation of T cell traffic

Natural Killer (NK) cells are lymphocytes that identify and kill virus-infected and tumour cells dependent on recognition via numerous activating and inhibitory surface receptors. The mechanisms by which NK cells can integrate activating and inhibitory signals at the immune synapse remain unclear, although a correlation has been established between a cell’s response and the organisation of receptors into specific molecular patterns at the synapse. Here, I used three novel imaging techniques to investigate the spatial and temporal dynamics of molecular recognition by human NK cells. The inhibitory NK cell receptor KIR2DL1 was tagged with the photoswitchable protein tdEosFP, which can be irreversibly switched from a green to red fluorescent state using ultraviolet light. First, tdEosFP was used to visualise the localisation and organisation of KIR2DL1 at the synapse at unprecedented resolution using photoactivated localisation microscopy. KIR2DL1 was organised in pre-existing protein clusters with a diameter of 84.2 ± 5.4 nm. Upon receptor ligation, clusters became smaller by 15% and denser by 27%, in an actin-independent manner. Surprisingly, the ligation of other receptors such as NKG2D and CD94/NKG2A affected the nanoscale organisation of KIR2DL1 by increasing the cluster density by 18-19% and decreasing the average cluster diameter by 20%. Thus, the distribution of KIR2DL1 is directly influenced by other receptors. Second, the movement of photoswitched molecules was temporally and spatially resolved and the diffusion coefficient of KIR2DL1 in the membrane of NK cells determined to be 0.167 ± 0.077 μm2.s-1. KIR2DL1 was continuously recruited to an immune synapse, where it remained stable, unless a second synapse was formed, in which case KIR2DL1 was able to traffic between synapses. Finally, a system using a photocleavable peptide to temporally control NK cell inhibition was established. This enables the timing of inhibitory signalling mediated by KIR2DL1 to be controlled. Together, these complementary approaches provide important new insights on how the nanoscale organisation of KIR2DL1 at the synapse can reflect different stimulatory conditions and novel spatio-temporal information on KIR2DL1 recruitment to the synapse.

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