A thesis presented for the degree of Doctor of Philosophy by Rhea F Cornely Faculty of Medicine Centre for Vascular Research 2013 ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed: ________________________________ Rhea F Cornely Date: ________________________________ ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International. I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed: ________________________________ Rhea F Cornely Date: ________________________________ ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed: ________________________________ Rhea F Cornely Date: ________________________________ The activation of T cells by an antigen presenting cell is one of the essential first steps for an effective immune response. Activation of the T cell receptor (TCR) leads to fundamental changes to T cell morphology, cytoskeletal rearrangement, membrane order and the formation of TCR microclusters which then propagate downstream signalling. TCR signalling eventually induces T cell proliferation as well as the production and secretion of interleukin 2 (IL-2) which binds to the IL-2 receptor and further stimulates T cell proliferation in an autocrine and paracrine fashion. Both signalling processes are dependent on specialised membrane domains enriched in cholesterol also termed lipid rafts and were found to sustain transient interactions with the cortical actin meshwork. Annexin A6 (AnxA6) is a calcium-activated cytosolic phospholipid membrane binding protein which has been suspected to play a role in T cell development and is upregulated in a wide range of immune cells including T cells. AnxA6 is involved in receptor endocytosis, vesicle budding and cholesterol homeostasis. It has been found to preferentially bind cholesterol-rich phospholipid membranes and to interact with the cortical cytoskeleton to allow endocytosis and vesicle budding. The aim of this study was to show that AnxA6 is crucial for an effective T cell mediated immune response. It was hypothesised that AnxA6 influences receptor signalling directly by mediating a link between the cortical cytoskeleton and cholesterol-rich membrane domains and thus AnxA6 might be part of a mechanism to target receptors to a specific membrane environment. Alternatively, AnxA6 influences signalling processes indirectly through maintaining the optimal composition of the plasma membrane due to its involvement in cholesterol homeostasis – as AnxA6 plays a role in cholesterol transport from intracellular membranes to the plasma membrane as well as low-density lipoprotein endocytosis. In this study a delayed-type contact hypersensitivity was elicited in AnxA6 knock-out mice to generate a T cell mediated immune response in vivo. The results showed that AnxA6 mice did mount a T cell mediated immune response but the levels of proliferating CD4+ T cells were significantly lower than in wild type mice. Investigating the source of this proliferation defect, western blot, fluorescence microscopy, qPCR and flow cytometry approaches were used to measure the response of primary murine T cells to T cell receptor as well as IL-2 receptor stimulation. Neither the early nor the late response of TCR signalling was affected in AnxA6-/- T cells. Instead, activated AnxA6-/- T cells secreted more IL-2 and the response to IL-2 stimulation was found to be impaired in AnxA6-/- T cells. In parallel, it was investigated if and how the membrane composition of primary AnxA6-/- T cells was different from wild type T cells. The membrane order of TCR stimulated as well as naïve cells was measured with polarity-sensitive dye Laurdan. To characterise the cholesterol content and phospho- and sphingolipid composition the lipid phase was extracted from whole T cell lysates and analysed by mass spectrometry. AnxA6-/- T cells were found to have a lower degree of plasma membrane order which implies that these membranes are more fluid. In agreement with this, AnxA6-/- T cells were found to have an altered membrane lipid composition. Some of these changes potentially affect the fluidity of the T cell plasma membrane: Cholesterol, which decreases plasma membrane fluidity, is less abundant in the T cells of AnxA6-/- mice. Levels of phosphatidylethanolamine with arachidonic acid, a polyunsaturated fatty acid which increases membrane fluidity, were more abundant in AnxA6-/- T cells. In conclusion, it could be shown that AnxA6 does play an important role in T cells and is required for efficient proliferation in a T cell mediated immune response. It is likely that the lower degree of IL-2 signalling efficiency in vitro and proliferation defect of CD4+ T cells observed in AnxA6-/- mice in vivo were a result of the changes in membrane composition. I thank the University of NSW for supporting me with a University International Postgraduate Award (UIPA) scholarship and an Australian Postgraduate Award (APA) A big thanks goes also to all the collaborators and university facilities that made this work possible: Todd Mitchell from the University of Wollongong for providing me with new and improved protocol for lipid extraction and for data acquisition of same lipid extracts. Sarah Norris for MS sample acquisition and patiently answering my questions. The various sunlight deprived facilities of the Mark Wainwright Analytical Centre: The majority of the microscopy data was acquired in the Biomedical Imaging Facility (BMIF). I am very grateful for their microscopes and knowledgable & sociable staff, in particular Alex Macmillan and Michael Carnell. Russell Pickford from the Bioanalytical Mass Spectrometry Facility (BMSF) provided Abbie and me with mass spec advice and new & intact phospholipid standards. I am just as grateful for the Flow Cytometry Facility resources with ALL the colours and Chris Brownlee. When it comes to flow cytometry the support of BD Biosciences in the person of Andrew Lim and Martin Baker deserves a mention as well. Thanks to Sophie Pageon for reading a statistically significant amount of thesis with data that was not significantly different. Thanks to Margaret Fennen for reading all of the thesis and add many missing commas. Thanks to my co-supervisor Thomas Grewal for reading corrections and a lot of helpful discussions and annexin advice over the years. Thanks also to my supervisor Katharina Gaus for reading corrections and helpful suggestions and for showing me what it means to be successful researcher. Thanks to my family for all the love and support despite the diameter of a whole planet between us! Thanks to Simon for all the love and support and putting up with me the last few years despite the close proximity. Thanks to Abbie for help, advice and for doing the PhD experience with me. Thanks for being there near and far: Reut, Lies, Alison. Figure 1-1: TCR signalling. .......................................................................................................................... 4 Figure 1-2: Immune synapse structure. .................................................................................................. 6 Figure 1-3: IL-2 receptor consists of three subunits – CD25, CD122 and ɣc. .............................. 9 Figure 1-4: Structure of membrane lipids (1). ................................................................................... 11 Figure 1-5: Structure of membrane lipids (2). ................................................................................... 12 Figure 1-6: The effect of polar head group and chain length of fatty acid moieties on the transition temperature of phospholipids. ........................................................................................... 13 Figure 1-7: Models of membrane organisation.................................................................................. 19 Figure 1-8: AnxA6 expression is particularly high in immune cells...........................................