Influenza A virus infection is a major cause of human morbidity and mortality. T cell immunity is believed to play critical roles for host defenses against influenza A infection. Once intracellular influenza A infection is established, viral clearance is mainly dependent on virus-specific CD8+ T cells. CD4+ T cells are important for adaptive immunity to natural influenza A infection or vaccination by providing help to B cells for antibody production and also providing help to CD8+ T cells for the generation of cytotoxicity. In addition, virusspecific CD4+ and CD8+ T cells are rich sources of effector cytokines, such as IFN-and TNF-, which can promote the function of antigen presenting cells and have direct antiviral activity. Cross-subtype reactive CD4+ and CD8+ memory T cells also affect the clearance of virus infection even in those who lack virus-specific antibodies. Therefore, the aim of our study is to assess the influenza virus-specific T cell responses and define their possible protective role in pandemic H1N1 virus and seasonal influenza infection in human. First we determined whether healthy adults have the cross-reactivity of memory CD4+ and CD8+ T cells against pandemic virus. In April of 2009, 7 pandemic H1N1 infected patients and 17 their healthy contacts who had no pandemic influenza infection were recruited in this study. By using intracellular IFN-staining and flow cytometry, we examined their pandemic H1N1 virus and seasonal influenza H1N1-specific CD4+ and CD8+ T cell responses. Healthy contacts did have measurable but low frequencies of cross-reactive influenza-specific CD4+ and CD8+ T cells, though the frequencies of these T cells specific to pandemic H1N1 virus were slightly lower than that specific to seasonal H1N1 virus. Furthermore, when compared the pandemic H1N1-specific T cell responses between healthy contacts and patients with pandemic H1N1 infection, we can found that the healthy contacts have higher pandemic H1N1 specific-T cell responses than patients, suggesting these pre-existing pandemic H1N1 specific-T cells may have protection from pandemic influenza virus infection. In addition, we conducted a prospective T cell immunity and influenza surveillance study in a cohort of more than 200 healthy volunteers before the influenza season and investigated whether the pre-existing T cell immunity is related to the protection from influenza infection in the next coming influenza season. Using intracellular IFN-staining assay, we examined their pre-existing seasonal influenza H1N1, H3N2, seasonal influenza B virus-specific CD4+ and CD8+ T cell responses. Due to the small number of cases of influenza infection in the coming influenza season, the results only showed a trend that the subjects who have higher frequency of influenza virus strain-specific T cells may have lower chance to suffer from same strain of influenza infection, which to some extent, reflect the pre-exist memory T cells have association with the protection in the coming influenza season. In conclusion, T cells play an important role in defensing against influenza infection. The higher influenza virus specific-T cells response activity in healthy adults may have a protection against influenza virus infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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Thapsigargin is a sesquiterpene lactone which was isolated from the plant Thapsia gargantica. It has been used in studies investigating intracellular calcium pools and calcium signalling as it causes a rise in intracellular calcium concentration. It inhibits the endoplasmic/sarcoplasmic reticulum family of calcium-ATPases and allows calcium to leak out of intracellular stores followed by a sustained influx of extracellular calcium. Porcine lymphocytes are used throughout this thesis due to their ready availability and as they have been used in this laboratory for many years as a model for human lymphocyte activation. In this study, the effects of thapsigargin on the activation of porcine and human peripheral blood mononuclear cells in vitro was studied and compared with those of the calcium ionophore, ionomycin and an alternative endoplasmic/sarcoplasmic reticulum calcium-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone. The drugs were also used in combination with the phorbol ester, phorbol myristate acetate, used to activate protein kinase C, and the immunosuppressive drug, FK506, which inhibits calcium dependent T cell activation. Thapsigargin was found to raise intracellular calcium concentration in both porcine and human cells in a concentration-dependent manner but was more effective in human than in porcine cells. In porcine cells, thapsigargin raised intracellular calcium concentration to a similar level as the mitogenic lectin conconavalin A but not to as high a level as ionomycin or 2,5-Di-(tent-butyl)-1,4-benzohydroquinone. In human cells, thapsigargin was as effective as both ionotnycin and 2,5-Di-(tert butyl)-1,4-benzohydroquinone. Neither phorbol myristate acetate nor FK506 had any effect on the rise in cytoplasmic calcium concentration induced by thapsigargin in porcine cells. As expected, all three of the drugs used to raise intracellular calcium concentration in combination with phorbol myristate acetate were found to be mitogenic for both human and porcine cells. They induced DNA synthesis at 48 hours post stimulation in a concentration dependent manner. Thapsigargin was found to induce DNA synthesis at a lower cytoplasmic calcium concentration than either of the alternative calcium mobilisers in porcine cells. FK506 inhibited the DNA synthesis induced by ionomycin/ phorbol myristate acetate and 2,5-Di-(tert-butyl)-1,4-benzohydroquinone/phorbol myristate acetate in both human and porcine cells and DNA synthesis induced by thapsigargin/phorbol myristate acetate in human cells. However, FK506 enhanced DNA synthesis in porcine cells that had been exposed to an antagonistic combination of thapsigargin and phorbol myristate acetate. This result was surprising as FK506 is expected to inhibit calciumdependent activation via the inhibition of the calcium-dependent protein phosphatase, calcineurin. On further investigation it was found that the porcine cells incubated with thapsigargin, phorbol myristate acetate and FK506 were able to proliferate in the absence of measurable levels of the cytokine interleukin 2. Furthermore, calcineurin activation was inhibited in these cells. It was also found that combinations of thapsigargin, phorbol myristate acetate and/or FK506 did not affect activation of some of the protein kinases believed to be involved in T cell activation in any different way from the alternative mitogenic combinations used in this study. The effects of thapsigargin and other mitogens on apoptosis in porcine and human peripheral blood mononuclear cells were also investigated. lt was found that a very high proportion of resting porcine cells in culture were apoptotic and that there were fewer apoptotic cells in proliferating cultures. There were fewer apoptotic cells in resting human cell cultures and all combinations of mitogenic or immunosuppressive drugs increased the proportion of apoptotic human cells. In addition, DNA synthesis was occurring in only a small proportion of the population of stimulated human cells regardless of the mitogen(s) used. The data presented show that the effects of thapsigargin on cytoplasmic calcium concentration and/or the effects of phorbol myristate acetate on protein kinase C activation cannot entirely account for the results and that the activation of porcine peripheral blood mononuclear cells does not represent an entirely accurate model of human lymphocyte activation.

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A double immunolabelling technique was developed to study microscopically the interactions between CD4, CD45 and the T cell receptor on the surface of rat T cells induced by the phenomenon of co-capping. It was found that both CD4 and CD45 passively co-cap with the actively capped T cell receptor, that the T cell receptor and CD45 passively co-cap with CD4, but that neither CD4 nor the T cell receptor co-cap with CD45. Co-crosslinking and active capping of CD45 with either the T cell receptor or CD4 prevented CD4 or the T cell receptor respectively, from passively co-capping. These experiments were extended to study the effects of particular antibody crosslinking conditions on T cell proliferation and tyrosine phosphorylation. A correlation was found to exist between receptor distribution and the effects of particular antibody combinations on proliferation and tyrosine phosphorylation. The significance of this with respect to T cell activation is discussed. Finally, an observation is reported concerning the failure of some cell lines to cap antibody-crosslinked surface molecules. Preliminary investgations into the nature and extent of the phenomenon are described.

Amendments page is pasted onto the front end paper. / Includes bibliographical references (28 leaves). / 1v. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Shows that the NKT cells and the thymus-independent ?ga?s/?gb?s T cells present in athymic rats are phenotypically and functionally related. Raises the possibility that thymus-independent ?ga?s/?gb?s T cells are distinct from conventional T cells and that their functions in normal individuals are regulatory, as has been suggested for NKT cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999

Copies of author's previously published articles inserted. / Errata pasted onto verso of back end-paper. / Bibliography: leaves 163-189. / xviii, 203 leaves, [26] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the results of an investigation to determine the molecular basis of an uncharacterised (4;11)(q21;p15) translocation in a patient with T-cell lymphocytic leukemia. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2000