Identifying lineage relationships in human T cell populations by Celia Lara Menckeberg A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Immunity and Infection College of Medical and Dental Sciences The University of Birmingham December 2010 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ii ABSTRACT CD4+ and CD8+ T cell populations can be divided into subpopulations based on expression of surface markers CCR7 and CD45RA. The resulting populations are referred to as naive, central memory, effector memory and effector memory RA+ (EMRA). The aim of this study was to identify potential lineage relationships between these subpopulations for both CD4+ and CD8+ T cells through microarray analysis. The genes found to distinguish between these subpopulations include many molecules with known functions in T cell differentiation, including CCR7, CD45RA, granzymes, L-selectin and TNF receptors. Several genes from the tetraspanin family of proteins were found to be differentially expressed at mRNA and protein level; suggesting a possible role for these genes in CD4+ and CD8+ T cell activation, migration and lysosomal function. Other genes identified, such as LRRN3 and CXCR5 which were expressed highest on naive and CM T cells respectively, provide interesting gene targets to follow up on their function in these T cell populations. Microarray data was validated through Real Time PCR and suggests that both CD4+ and CD8+ T cells differentiate along a linear pathway of naive to central memory to effector memory. The transcriptional programmes responsible for these differentiation steps were distinct between CD4+ and CD8+ T cells, although additional elements were common to both subsets. iii Dedicated to Mama en Papa iv ACKNOWLEDGEMENTS First I would like to thank my supervisor Dr. John Curnow for guiding me through this project and for adopting me when it was very much needed. The encouragement and advice he has given have helped me grow as a scientist and as a person. I would also like to thank Professor Mike Salmon, without whom this project would not have been possible. Huge thanks to Steve (Stev) Kissane for all his help throughout these years, not only was he my microarray specialist but also a source of knowledge in many different topics. Thanks for always being there to help with any questions I had and for the countless lab bench and lunch time chats. I would like to thank Dr. Graham Wallace for all his support throughout and helpful discussions along the way and Dr. Ewan Ross for being a source of knowledge and always ready to help. Also I want to thank all my lovely friends that I made working on the 3rd floor IBR. You were my family away from home and made living in Brum for four years so very special. Fern (Barry) Barrington, thank you for being my Harborne and vacation buddy; I loved our movie and Sunday cooking nights! Dr. Sarah (Chuckles) Essex thank you so much for being the smiling face I saw sitting across from me for almost four years, as well as for introducing me to the wonder that is a lemon drizzle cake. Katherine (Kaaath) Howlett for being my gym and pub buddy and subsequent fellow Harbonite! And for the many, many, many excellent cakes in these past four years. Lorraine (Lore) Yeo thank you for taking over from Sarah when it was needed and of course for the excellent white chocolate and raspberry muffins. To all my other friends, Dr. Peter Hampson, Dr. Jason Lee, Dr. Sian Lax, Siobhan Restorick, Hema Chahal, Emma Yates and many others, thank you for being a huge part in why the department of Rheumatology was such a nice place to work. I would like to acknowledge Professor Janet Lord and Professor Chris Buckley in their roles as head of the Marie Curie TRIFID project and head of the Department of Rheumatology respectively. To my lovely friends and family back home, Ranjeeta Bendter, Francis Saridin and Serge Parabirsing, thank you for always supporting me and for always being only a phone call away. Jeeta, thank you for sticking with me for almost 25 years (playground buddies for life!) and Francis, my study-buddy, 10 years and counting! Serrie thanks for staying positive throughout everything that has happened. I want to thank my sister Tanja and her husband Rodney for all their help along the way and for giving me the best gift ever by making me the proud auntie of Rafaël. Mostly I would like to thank my parents for always believing in me and encouraging me; for instilling in me a thirst for knowledge from a very young age and for being my rock to fall back on. You are my heroes. v ABBREVIATIONS ADD2 Beta-adducin AEBP1 Adipocyte Enhancer Binding Protein 1 ANOVA Analysis of Variance APC Antigen Presenting Cell BCR B Cell Receptor BHLHB2 Basic Helix-Loop-Helix protein B2 BM Bone Marrow BSA Bovine Serum Albumin CLIP Class-II-associated Ii peptide CLP Common Lymphoid Progenitor CM Central Memory CMP Common Myeloid Progenitor Cn Calcineurin CTLA-4 Cytotoxic T lymphocyte antigen-4 Cy3/Cy5 Cyanine 3 or 5 DAG Diacylglycerol DC Dendritic Cells DN Double Negative DP Double Positive EAE Experimental Autoimmune Encephalomyelitis EBV Epstein-Barr virus EDTA Ethylenediaminetetraacetic acid EM(RA) Effector Memory (RA) ER Endoplasmic Reticulum vi ETP Early Thymic Progenitor FACS Fluorescent Activated Cell Sort FDC Follicular Dendritic Cell FDR False Discovery Rate GPS Glutamine Penicilin Streptomycin GZM Granzyme HCL Hierarchical Clustering HEV High Endothelial Venules HLA Human Leukocyte Antigen HSC Hematopoietic Stem Cell HVEM Herpesvirus entry mediator ICAM1 Intracellular Cell Adhesion Molecule 1 IFNγ Interferon γ Ig Immunoglobulin Ii MHC-II specific chaperone invariant chain IL Interleukin IP3 Inositol 1,4,5-triphospahate IS Immunological synapse KLRB1 Killer Cell Lectin-like Receptor subfamily B member 1 KLRC4 Killer Cell Lectin-like Receptor subfamily C member 4 KLRF1 Killer Cell Lectin-like Receptor subfamily F member 1 LAT Linker for T cell Activation LFA-1 Lymphocyte Function-associated Antigen-1 LIGHT homologous to LTs, exhibits inducible expression, and competes with HSV gD for herpesvirus entry mediator, a receptor expressed by T lymphocytes or TNSF14 (p)LN (peripheral) Lymph Node Lo/Ld Liquid order/Liquid disorder vii LRRN3 Leucine Rich Repeat Neuronal protein 3 LTα1β2 Lymphotoxinα1β2 LTβR Lymphotoxinβ Receptor LTi Lymphotoxin inducer LTo Lymphotoxin organiser MAdCAM-1 Mucosal Addressin Cell Adhesion Molecule-1 MALT Mucosal-Associated Lymphoid Tissue MHC Major Histocompatibility Complex MLP Multilymphoid Progenitor MTMR8 Myotubularin related protein 8 MVB Multivesicular Body MTUS1 Mitochondrial Tumor Suppressor 1 isoform 3 MYBL1 Myb-related protein A MYO1F Myosin-If NFAT Nuclear factor of activated T cells NGFRAP1 Nerve Growth Factor Receptor (TNFRSF16) Associated Protein 1 NK cell Natural Killer cell NKG7 Natural Killer Cell protein 7 PALS Periarteriolar lymphoid sheath PAMP Pathogen Associated Molecular Patterns PBMC Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline PCA Principal Component Analysis PCR Polymerase Chain Reaction PF Primary Follicles PIP2 Phospholipid phosphatidylinositol-4,5-biphosphate PLEKHK1 Pleckstrin homology domain containing family K member 1 viii PKC Protein Kinase C PLC1 Phospholipase C-1 PNAd Peripheral Lymph Node Addressin PP Peyer’s Patches PRDM8 PR domain zinc finger protein 8 PRR Pattern Recognition Receptor RORγt RAR-related orphan receptor gamma RT Reverse Transcription SAM Significance Analysis of Microarrays SH3BP5 SH3 domain-Binding Protein 5 SLC22A17 Solute carrier family 22 member 17 SLO Secondary Lymphoid Organs SLP-76 SH2 domain containing leukocyte protein of 76 kDa SMAC (c/p-) Central/peripheral supramolecular activation cluster SP Single Positive TAP Transporter associated with antigen processing TCR T Cell Receptor TEM Tetraspanin Enriched Membrane TFH T follicular helper Th T helper TLR Toll like Receptor TNFα Tumour Necrosis Factor α TRANCE(R) TNF-related activation-induced cytokine (receptor) (n/i) Treg (Natural/inducible) T regulatory cell VCAM1 Vascular Cell Adhesion Molecule 1 ZAP70 -associated protein of 70kDa ZNF Zinc Finger ix TABLE OF CONTENTS ABSTRACT............................................................................................................................................ii ACKNOWLEDGEMENTS...................................................................................................................iv ABBREVIATIONS ................................................................................................................................v TABLE OF CONTENTS.......................................................................................................................ix LIST OF FIGURES ..............................................................................................................................xii LIST OF TABLES..............................................................................................................................xvii