Natural Killer Cell Subsets and Multiple Sclerosis Alison Jane Harris Imperial College London Department of Medicine Thesis submitted for the degree of Doctor of Philosophy 1 Declaration of Originality I hereby declare that the work presented here is my own and that all references to the work of others are accompanied by appropriate citations. 2 ABSTRACT This thesis addresses the hypothesis that multiple sclerosis (MS) patients have an imbalance in the frequencies of natural killer cell subsets, with fewer immunoregulatory and more proinflammatory NK cells compared to healthy controls. Ten NK cell subsets were defined according to expression of CD56, CD8, CD27 and CD57. Subset frequencies were compared by performing multiparameter flow cytometry on PBMCs from healthy controls and patients with clinically isolated syndrome (CIS) or relapsing-remitting MS. CD56dim CD8+ CD57+ CD27- NK cells had a higher frequency in untreated relapsing-remitting MS patients compared to healthy controls and their frequency was decreased in patients on interferon-β treatment. CD56bright NK cells, particularly the CD8- CD27- subset, had a significantly lower frequency in untreated MS and CIS patients. CD8- CD57- CD27- subsets of both CD56dim and CD56bright NK cells were expanded in interferon-β-treated patients in remission but not relapse. Characteristics of two CD56dim subsets displaying an apparent reciprocal relationship (CD8+ CD57+ CD27- and CD8- CD57- CD27-) were compared by qRT-PCR, flow cytometry and killing assays. CD56dim CD8+ CD57+ CD27- NK cells were more cytotoxic and expressed higher levels of transcripts encoding IFNγ, T-bet and other factors associated with IFNγ signalling. This is consistent with the hypothesis that there is a shift towards a more pro-inflammatory NK cell phenotype in MS patients, which is reversed by interferon-β treatment. In order to determine whether these NK cells exert their effects mainly in the blood or in the central nervous system (CNS), mononuclear cells were extracted from the CNS of Line 7 mice, which develop spontaneous autoimmune demyelination due to expression of a human MS-associated HLA/TCR combination. NK cells were detected in spleen, but not CNS, of these mice, suggesting that their role in this particular disease model is restricted to regulation of other leukocytes outside the CNS. 3 Acknowledgements I would like to thank my supervisors Prof. Danny Altmann and Dr. Rosemary Boyton for their feedback on this thesis. I am grateful to Danny for giving me the opportunity to work on this project and giving me the freedom to pursue my own ideas and to Rosemary for helping me to focus them in the latter stages of my project. A big thank you to everyone on the 8th floor who has helped me over the years, to all those who have taught me techniques, collected blood, kept the place running smoothly, discussed ideas and made the department a more fun place to be. In particular, I would like to thank Dr. Fatemah Kamel for her tireless work in collecting patient blood samples. Thank you also to Dr. Richard Nicholas, Dr. Omar Malik and Dr. Paolo Giannetti for providing access to MS and CIS pateints, the nurses at Charing Cross who helped to take the samples and to all the people that donated blood, both patients and controls, without whom this study would not have been possible. I would also like to extend my gratitude to Anna Ettorre for looking after the FACS Aria II, which was essential for virtually all the experiments I performed, and for helping to set up the machine for cell sorting. I am grateful to Dr. Dan Lowther who optimised and taught me the Line 7 CNS extraction procedure and to Marie-Laure Aknin, Dr. Debs Chong, Dr. Karen Chu and Kathryn Quigley who assisted in the scoring and processing of these mice. Finally I would like to thank my friends, especially Nishkala Thiru and Richard Browning, for distracting me when experiments weren’t working and the Medical Research Council for funding me throughout my Masters and PhD. 4 Table of Contents 1. INTRODUCTION ................................................................................................ 18 1.1 Natural Killer Cells ....................................................................................... 18 1.1.1 NK Cell Development ............................................................................ 18 1.1.2 NK Cell Function is Determined by the Balance of Signals from Activating and Inhibitory Receptors .................................................................... 21 1.1.3 NK Cell Education Determines Responsiveness to Activating Signals 27 1.1.4 NK Cells Form Synapses with Target Cells and Kill Susceptible Targets via Granzyme Release and Other Mechanisms ................................................... 31 1.1.5 NK Cells Produce a Range of Cytokines and Chemokines ................... 38 1.1.6 NK Cells Interact with Other Leukocytes and Regulate their Function 48 1.1.7 NK Cells can be Divided into Subsets with Distinct Functions ............ 55 1.2 The Role of T cells and NK Cells in Multiple Sclerosis ............................... 66 1.2.1 Genetic and Environmental Risk Factors are Associated with Development of MS ............................................................................................. 67 1.2.2 CD4+ T cells Infiltrate the CNS and Promote Recruitment and Activation of Other Leukocytes, leading to Myelin and Axonal Damage .......... 70 1.2.3 Disease Initiation Requires Activation of Peripheral T Cells – Potential Role of Viruses and Impaired Immunoregulatory Mechanisms .......................... 78 1.2.4 Current Therapies for MS have Benefits and Limitations ..................... 80 1.2.5 Evidence of a Role for NK Cells in Multiple Sclerosis ......................... 86 1.3 Aims .............................................................................................................. 96 2. METHODS ........................................................................................................... 97 2.1 Human PBMC Isolation ................................................................................ 97 2.2 Flow Cytometry Evaluation of Human PBMC (Surface Staining) ............... 98 2.3 PBMC Stimulation and Intracellular Cytokine Staining ............................... 98 2.4 Cell Sorting for RNA .................................................................................... 99 5 2.5 RNA Extraction ........................................................................................... 100 2.6 Reverse Transcription ................................................................................. 101 2.7 Quantitative RT-PCR Arrays ...................................................................... 101 2.8 Individual qRT-PCR Assays ....................................................................... 103 2.9 K562 Killing Assays ................................................................................... 103 2.10 Preparation and Staining of Cells from Line 7 mice ............................... 104 2.11 Buffers and Medium: ............................................................................... 107 3. RRMS PATIENTS EXHIBIT DIFFERENCES IN FREQUENCIES OF SPECIFIC NK CELL SUBSETS ............................................................................... 125 3.1 Expression and Co-expression of CD8, CD27 and CD57 on CD56dim and CD56bright NK Cells ................................................................................................ 127 3.2 Confirmation of Differences in Frequency of NK Cell Subsets in RRMS Patients and the Effects of IFNβ Treatment ........................................................... 129 3.3 Multiparameter Analysis Reveals Differences in NK Cell Subset Frequencies that are not Restricted to the CD56bright Population ............................................... 133 3.4 Subset Frequencies in Healthy Adults do not Differ Significantly in Relation to Age or Gender .................................................................................................... 138 3.5 Discussion ................................................................................................... 140 4. TRANSCRIPTIONAL AND FUNCTIONAL DIFFERENCES BETWEEN CD56DIM CD8- CD27- CD57- AND CD56DIM CD8+ CD27- CD57+ NK CELLS ..... 146 4.1 Identification of Transcriptional Differences between CD8- CD27- CD57- and CD8+ CD57+ CD56dim NK Cell Subsets ......................................................... 147 4.2 Cell Surface Expression of Cytokine Receptors ......................................... 157 4.3 Differences in Expression of Activating and Inhibitory Receptors ............ 159 4.4 CD56dim CD8- CD27- CD57- NK Cells Produce Similar Levels of IFNγ to CD8+ CD57+ NK Cells and do not make IL-17 or IL-22 ...................................... 162 4.5 CD56dim CD8+ CD57+ NK cells are more cytotoxic than CD56dim CD8- CD27- CD57- NK Cells .......................................................................................... 165 4.6 Discussion ................................................................................................... 167 6 5. NK CELLS DO NOT INFILTRATE THE CNS IN THE LINE7 MURINE MODEL OF MULTIPLE SCLEROSIS .................................................................... 173 5.1.1 NKp46+ NK Cells were Not Detected in the CNS of Line 7 Mice ...... 175 5.1.2 Lack of NKp46 Detection was not due to Antibody Sequestration ..... 178 5.1.3 NKG2D was also Not Detected on CNS-derived Lymphocytes ......... 179 5.1.4 Discussion