T cell transcriptomes: Uncovering the mechanisms for T cell effector function through gene profiling Tatyana Chtanova December 2004 A thesis submitted for the degree of Doctor of Philosophy Garvan Institute of Medical Research, St Vincent’s Hospital and the Faculty of Science, University of New South Wales Sydney, NSW, Australia Manuscripts • Chtanova T., R.A. Newton, S. M. Liu, L. Weininger, D. G. Silva, M. Hughes, F. Bertoni, F. Sallusto, M. S. Rolph and C. R. Mackay. Comprehensive leukocyte microarray datasets allow identification of T cell subset restricted genes, and signatures for different types of T cell responses. Manuscript in preparation. • Liu, S. M., T. Chtanova, R. Newton, M. Sisavanh, K. L. Good, D.G. Silva, S. Zimmer, M. J. Frost, S. G. Tangye, M. S. Rolph and C. R. Mackay. Comprehensive microarray datasets of immune cells reveal novel leukocyte subset-specific genes, as well as genes associated with different types of immune response. Submitted to Blood. • Chtanova, T., S. G. Tangye, R. A. Newton, N. Frank, M. R. Hodge, M. S. Rolph, and C. R. Mackay. 2004. T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol 173:68. • Ng, L.G., A.P.R. Sutherland, R. Newton, F. Qian, T. G. Cachero, M. Scott, J. S. Thompson, J. Wheway, T. Chtanova, C. Xin, J. Groom, S. G. Tangye, S. L. Kalled, F. Mackay, and C. R. Mackay. 2004. BAFF-R is the principal BAFF receptor facilitating BAFF co-stimulation of B and T cells. J Immunol 174:807. • Chtanova, T., and C. R. Mackay. 2001. T cell effector subsets: extending the Th1/Th2 paradigm. Adv Immunol 78:233. • Chtanova, T., R. A. Kemp, A. P. Sutherland, F. Ronchese, and C. R. Mackay. 2001. Gene microarrays reveal extensive differential gene expression in both CD4+ and CD8+ type 1 and type 2 T cells. J Immunol 167:3057. Presentations • Keystone Symposium. T cell development, 2004, Banff, Canada Chtanova, T., S. G. Tangye, R. A. Newton, M. R. Hodge, M. S. Rolph, and C. R. Mackay. T follicular helper cells (TFH) express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. Oral and poster presentations. • The 13th St. Vincent’s campus Research Symposium, 2003, Sydney Australia Chtanova, T., M. J. Frost, S. G. Tangye, R. A. Newton, M. R. Hodge , M. S. Rolph, and C. R. Mackay. T follicular helper cells distinguished from Th1 and Th2 subsets by their expression of certain cytokines, cell surface molecules and the transcription factor BCL-6. Poster presentation. • 32nd Annual Meeting for the Australasian Society for Immunology, 2002, Brisbane, Australia Chtanova, T., R. A. Kemp, M. J. Frost, A. P. R. Sutherland, F. Ronchese†, M. S. Rolph, and C. R. Mackay. Distinguishing genes critical for T cell development and effector function. Poster presentation. • 11th International Congress of Immunology, 2001, Stockholm, Sweden Chtanova, T., R. A. Kemp, A. P. Sutherland, F. Ronchese, and C. R. Mackay. i Gene microarrays reveal extensive differential gene expression in both CD4(+) and CD8(+) type 1 and type 2 T cells. Oral and poster presentations. • The 10th St. Vincent’s campus Research Symposium, 2000, Sydney Australia Chtanova, T., A. Sutherland, A. Quan and C.R. Mackay. Identification of genes for asthma pathogenesis. Oral presentation. ii Acknowledgements Firstly, a huge thank you to my supervisor Professor Charles Mackay for his guidance, support and encouragement. Thank you for teaching me a great many things about immunology and what science is all about. I would also like to thank my co- supervisor Dr Michael Rolph for his thoughtful advice and support. I would like to thank many collaborators for generously sharing data and reagents with me. In particular, thank you to Dr Roslyn Kemp (Malaghan Institute, New Zealand) for providing polarized mouse cells; Dr Stuart Tangye (Centenary Institute, Australia) for all the advice and generous sharing of many reagents; Dr Martin Hodge and Nita Frank for kindly providing IL-21R antibody; Dr Diego Silva (ANU Medical School, Australia) for bioinformatics help; Dr David Lowinger for providing tonsil tissue. I am very grateful to the following people for providing their microarray data: Dr Federica Sallusto (Institute for Research in Biomedicine, Switzerland), Dr Rebecca Newton, Sue Liu, Dr Sabine Zimmer and Mary Sisavanh (Garvan Institute, Australia). Thank you also to Lilach Weininger, Melinda Frost and Trina So for their help with the Affymetrix system. I have been very lucky to work with an amazing group of people and I am very grateful to everyone in the Arthritis and Inflammation Research Program for all their help and support over these years. It has been a lot of fun to be a part of this dynamic and exciting group of people. In particular, I had the great pleasure of working with Alison Saunders and Lilach Weininger, both of whom have made my time here so much more enjoyable. Of course, a special mention goes to Dr Frederic Sierro (a.k.a. Swiss Fred) – there is no one I would have rather shared an office with! Thank you for your sense of humour, our many discussions and my lessons in Swiss geography. Many thanks go to my wonderful friends who have kept me sane during these years. A special thank you goes to Jerry Frenkel for being a wonderful friend, colleague and bridesmaid extraordinaire. Throughout my life my parents have always been a huge inspiration to me. I thank you wholeheartedly for your unfailing support through every step of my life. And finally, an enormous thank you to my future husband, James. No words can describe how much your love and support over all these years have meant to me. I could not have done it without you. This thesis is dedicated to you. iii Abstract T cells are at the heart of the adaptive immune response. They mediate many important immunological processes that provide protection against viruses, bacteria and other pathogens. T cell functions include cytotoxicity, cytokine production, immunosuppression, recruitment of other effector cells to the sites of infection, and help for antibody production. The aim of the work described in this thesis was to use gene expression profiling to gain insights into different aspects of T cell biology. In particular we wanted to examine the mechanisms of effector function and identify the genes that underlie the functions of different effector subsets. One of the fundamental divisions in effector T cell responses is based on two distinct patterns of cytokine production by CD4+ T cells. IFN-γ -producing Th1 cells are a major effector subset that protects against intracellular pathogens, while Th2 cells produce IL-4, IL-5 and IL-13 and mediate protection against large extracellular pathogens. We conducted a comprehensive analysis of T cell polarization by profiling gene expression in both mouse and human Th1 and Th2 cells polarized using different experimental protocols, as well as mouse CD8+ type 1 and type 2 T cells. One of the major outcomes of this extensive analysis was the identification of a number of novel markers for Th1 and Th2 cells which may have important roles in Th2 differentiation/function. The other was the delineation of some of the major influences on gene expression profiles during polarization such as the effects of different T cell types, species and differentiation conditions. Providing help to B cells for antibody production is the major function of the + third effector subset of CD4 T cells termed T follicular homing or TFH cells. These cells express the chemokine receptor CXCR5+ and home to B cell follicles where they interact with B cells. However, relatively little is known about the generation of these cells, and the mechanisms of their effector function are yet to be determined. Using oligonucleotide microarrays we identified a TFH-specific gene expression signature. This signature included many novel genes which will undoubtedly enable better identification and characterization of this novel subset. For instance, IL-21 and CD84, which were intimately associated with TFH cells and have already been shown to affect B cell differentiation and function, provide a possible mechanism for TFH help to B cells. The transcription factor BCL6 which was also preferentially expressed by TFH cells may act as a key regulator of T cell fate. iv A comprehensive study profiling all the major leukocyte subsets revealed distinct gene expression signatures for every leukocyte subset, and enabled identification of numerous leukocyte subset specific genes, and in particular many novel T cell-specific genes. A detailed examination of most major T cell subsets identified distinguishing features of each subset together with gene expression changes associated with T cell activation and exposure to cell culture conditions. In addition, we described a distinctive transcriptional profile for γδ T cells and examined the differences between central and effector memory T cells. We also showed that specific gene expression signatures provide a powerful tool for subset classification. Taken together this work provides important insights into T cell differentiation and effector function, and provides a basis for future work examining numerous novel genes relevant to T cell biology. v Contributions of collaborators I would like to acknowledge the contributions of the following people to the work presented in this thesis: • Dr Roslyn Kemp (Malaghan Institute, New Zealand) provided polarized Th1 and Th1 and