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Interleukin-12 Signalling Pathways in Human T Lymphocytes

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Interleukin-12 Signalling Pathways in Human T Lymphocytes Interleukin-12 signalling pathways in human T lymphocytes Veronica Athié-Morales A thesis submitted to University College London at the University of London for the degree of Doctor of Philosophy April 2001 Lymphocyte Activation Laboratory Imperial Cancer Research Fund 44 Lincoln's Inn Fields London WC2A 3PX ProQuest Number: U642645 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642645 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The aim of the work that constitutes the present PhD thesis was to elucidate the intracellular signalling pathways downstream of the cytokine Interleukin-12 (IL-12) in human T lymphocytes. IL-12 plays a key role in the onset of the cellular immune response by driving the differentiation of naive Th cells into Thl cells. The important role of IL-12 in human immune responses has been emphasised by recent reports that mutations of the IL-12 receptor (IL-12R) are found in immunodeficient patients suffering from recurrent Mycoplasma sp. and Salmonella sp. infections. IL- 12 exerts its function through interaction with the IL-12R that leads to the activation of the Janus kinases Tyk2 and Jak2. IL-12 also induces tyrosine phosphorylation and DNA binding of STAT4 (Signal Transducer and Activator of Transcription 4), an essential response for Thl cell differentiation. We have extensively studied the regulation of the transcription factor STAT4 in response to IL-12, using PHA-activated human T cells isolated from healthy donors as a model. These studies include: a) characterisation of other possible signals that induced in response to IL-12, could regulate the activation of STAT4; b) a comparison between the characteristics of STAT4 activation in response to IL-12 and to Interferon-a (IFN-a); and c) study of the mechanism by which IL-12 induced STAT4 activation is potentiated by IL-2. Acknowledgements I would like to thank the people that made this PhD possible. Firstly Dr. Doreen Cantrell for close supervision and teaching. Most of this work was generously supported by Roche Discovery Welwyn Ltd., from where I like to thank Dr. Kate Hilyard and Dr. Horst Flotow for continued support and encouragement. This work could not have been realised without the technical support of the ICRF Oligonucleotide Synthesis Service; Nicola O'Reilly and Elisabeth Li at the ICRF Oligopeptide Synthesis Service; Derek Davis and Aaron Rae in the FACS laboratory; and Kim for excellent secretarial work. I would like to thank the past and present members of the Lymphocyte Activation Laboratory for tolerating my constant "anti-noise" and "tidiness" hysteria. Stefan and Julian for "philosophy Fridays". Helen for teaching me "fine protocols". Paul for being a very good teacher, "psychiatrist" and "landlord" and for fruitful scientific discussions. Sally for very enjoyable chats when lunching in the park and teaching me molecular biology. Steve for "computer support" and "car purchasing advice". Virginie for incredible fun moments in the tube and scientific discussions. Patrick for his songs and "English irony". Emmanuel for her good humour and whistling. Arian for "history lessons" and good laughs. Jane, for always cheering me up and understanding. Anita for making me feel that I was not the only one "turning machines and hoods o ff. Ulrica for wonderful chats. Maighread for "five more minutes at the computer"... and pleasant conversations. Isa and Manolo for simply be such good friends. I could not have gone through all these years without the love and support of my family (too many to make a list...) and fiiends: Aixa, Miguel Angel, Cat, Vera, Julia, Kerstin, Boija, Susana, Desi, Della, and all my friends in Mexico (for keeping my incoming email box always full....). Finally, I would like to dedicate this thesis to the three people I love the most: Gloria, José and Fabiân, for their love and everlasting support even during my most capricious decisions .... m Publication Athié, M. V., Flotow, H., Hilyard, K. L., Cantrell, D. A. 2000. IL-12 selectively regulates STAT4 via phosphatidylinositol 3-kinase and Ras-independent signal transduction pathways. EJI 30 (5), 1425-34. IV Table of Contents ABSTRACT II PUBLICATION IV TABLE OF CONTENTS V LIST OF FIGURES X LIST OF TABLES XII ABBREVIATIONS XIII CHAPTER 1 1 INTRODUCTION 1 1.1 Immune responses to pathogen infections 2 1.2 Some cells o f the immune response: T lymphocytes and NK cells 2 1.2.1 T lymphocytes 2 1.2.2 NK cells 3 1.3 T lymphocyte differentiation 4 1.3.1 T helper cell differentiation 4 1.3.2 T cytotoxic cell differentiation 8 1.4 Interleukin-12 9 1.4.1 Structure of IL-12 9 1.4.2 Regulation of IL-12 production 11 1.4.3 IL-12 receptor 12 1.4.4 Regulation of IL-12R expression 15 1.4.5 Biological functions of IL-12 18 1.5 Interleukin-2 20 1.5.1 The IL-2 receptor 20 1.5.2 IL-2-induced signalling pathways 21 1.5.2.1 The Ras/ Mitogen Activated Protein Kinase pathway 22 1.5.2.2 The PI-3K pathway 27 1.5.3 Bilogical functions of IL-2 29 1.6 Inteferons 30 1.6.1 Type I Interferons 30 1.6.2 The IFN-ot/p receptor 31 1.6.3 Bilogical functions of IFN-o/p 32 1.6.4 Interferon type II 33 1.6.5 The IFN-y receptor 33 1.6.6 Biological function of IFN-y 34 1.7 The Jak/STATsignalling pathway 35 1.7.1 The Jak family of tyrosine kinases 35 1.7.2 Biological functions of Jaks 39 1.7.3 STATs 41 1.7.4 Prototypical model for Jak/ STAT activation 41 1.7.5 STATs serine phosphorylation 42 1.7.6 Biological functions of STATs 47 1.7.7 STATs and oncogenesis 50 1.7.8 Association of STATs with other proteins 51 1.7.9 Negative regulation of the Jak/ STAT signalling cascade 54 1.8 IFN-a/p signalling through the Jak/STAT pathway 56 1.9 IFN-y signalling through the Jak/STAT pathway 57 1.10 IL-2 signalling through the Jak/ ST A T pathway 5 8 1.11 The IL-12/STAT4 response and its role in Thl cell differentiation 59 1.11.1 The link between IL-12, STAT4 and Thl cell differentiation 59 1.11.2 STAT4 protein expression 60 1.11.3 Factors that influence Th cell differentiation 60 1.11.4 The IL-12/ STAT4 response and Thl cell differentiation 62 1.11.5 The IFN-a / STAT4 response 64 1.11.6 Genes that are commonly activated by IL-12 and IFN-a 64 1.11.7 IFN-a and Thl cell differentiation 65 1.11.8 Other activators of STAT4 66 1.11.9 Cytokines that synergise with IL-12 for Thl cell differentiation 66 1.11.10 Regulation of IFN-y production during Th 1 cell differentiation 67 1.12 Aims 68 CHAPTER 2 71 Materials and Methods 71 2.1 Chemicals, Cells, Cytokines and Inhibitors 71 2.2 Total cell lysate 72 2.3 Cell fractionation 72 2.4 Affinity precipitation o f DNA binding proteins 73 2.5 Affinity precipitation ofpeptide binding proteins 74 2.6 Affinity precipitation using GST-fusion proteins 75 2.7 Protein standardisation 76 VI 2.8 Western blotting 11 2.9 Densitometric analysis o f bands obtained by Western blotting 78 2.10 Plasmids, Transient transfections and CA T-assays 81 2.11 Staining o f cell surface molecules for flow cytometry analysis 79 2.12 Cell viability analysis 80 2.13 Acid wash for cytokine removal 80 CHAPTER 3 81 IL-12 selectively regulates STAT4 in human T lymphocytes 81 3.1 Introduction 81 3.2 Results 83 3.2.1 IL-12 activates STAT4 in both PBL-T cells and KIT225 cells 83 3.2.2 IL-12 only activates STAT4 in activated PBL-T cells 83 3.2.3 IL-12 activation of STAT4: Dose response and comparison of the effectiveness of different oligonucleotides as reagents to affinity precipitate active STAT4 89 3.2.4 IL-12 does not induce DNA binding, tyrosine and serine phosphorylation of STATl, STAT3 or STATS in human T lymphocytes 91 3.2.5 The constant presence of IL-12 is required for sustained activation of STAT4 93 3.2.6 IL-12 selectively regulates the cellular localisation of STAT4 95 3.2.7 IL-12 induces the transcriptional activity of the GRR-CAT reporter gene 100 3.3 Discussion 102 CHAPTER 4 108 IL-12 regulates STAT4 serine phosphorylation via Phosphatidylinositol 3-kinase, Protein kinase C and Ras-independent signal transduction pathways 108 4.1 Introduction 108 4.2 Results 110 4.2.1 STAT4 is not regulated by the Ras/ MAPK pathway during IL-12 responses 110 4.2.2 Role of PI3-K in the IL-12-induced STAT4 response 115 4.2.3 The IL-12/ STAT4 response is PKC-independent 119 4.3 Discussion 122 CHAPTERS 128 The IFN-cc/ STAT4 response in human T lymphocytes: Characteristics and comparison with the IL-12/ STAT4 response 128 5.1 Introduction 128 vn 5.2 Results 130 5.2.1 IFN-a activates STAT4 in both PBL-T cells and Kit225 cells 130 5.2.2 IFN-a activates STATl, STAT3 and STAT4 with different kinetics in human T lymphocytes 130 5.2.3 IFN-a regulates the cellular localisation of STAT4 131 5.2.4 The IFN-a/STAT4 response is more transient than the IL-12/ STAT4 response 135 5.2.5 IFN-a does not have a negative effect on PBL-T cell survival 136 5.2.6 IFN-a negatively regulates its own STAT responses without altering the IL-12/ STAT4 response 139 5.2.7 IFN-a down-regulates the cell surface expression of the IFN-a receptor 140 5.2.8 IL-2 does not regulate the transient IFN-a/ STAT4 responses
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