Evaluating the role of CD200 signalling in renal cell carcinoma immune evasion Elise Rees Thesis submitted for the award of PhD 31 st March 2020 Abstract CD200 is a transmembrane protein with known immunosuppressive properties. This ligand functions by engaging its receptor, CD200R, found on many immune cell populations. Overexpression of CD200 has been linked to a number of cancer types as a mechanism of immune evasion. An in-human study has demonstrated a clinical benefit of targeting this ligand, which has led to the interest in identifying other cancer types that may also benefit from CD200 checkpoint inhibition. The main aim of this project was to determine whether CD200 expression was a relevant therapeutic target in renal cell carcinoma (RCC). We demonstrated that CD200 is expressed at protein level across RCC subtypes, which we suggest originates from the cell of origin for this cancer type, the renal tubules. We characterised the impact of CD200 expression level on the immune infiltrate and demonstrated a significant reduction in the presence of helper T cells, whilst NK cell presence remained relatively unaffected and suggestive of NK cell dysfunction. Using cell culture assays, we demonstrate that tumour CD200 expression inhibited CD200R+ NK cell function, both the cytotoxic and effector activity, which led to a diminished ability to kill CD200 expressing target cells. Blocking the CD200 signal was sufficient to restore NK cell activity in vitro and restore tumour cell killing. In addition to providing an inhibitory signal to interacting NK cells, the engagement of CD200R by its ligand induced the up-regulation of the Fas death receptor pathway, which resulted in apoptosis of the NK cell via induction of the caspase 8-dependent extrinsic pathway. These findings reveal a novel immunosuppressive mechanism by which both NK cell activity and presence at the tumour bed is inhibited. This study provides evidence that suggests that some RCC patients may benefit from CD200 immune checkpoint inhibition. ii Acknowledgements Firstly, I would express my heartfelt thanks to my supervisor, Dr Girish Patel, for giving me this opportunity and for his continual help, encouragement and guidance throughout my PhD. Secondly, many thanks go to my second supervisor, Dr Richard Clarkson, for his support and advice. I am very grateful to all the members of the GKP lab group, both past and present, for their help and advice as well as their friendship and support. I am also thankful the support I received from members of The European Cancer Stem Cell Research Institute. I would also like to thank KESSII and the European Social Fund, who made this project possible as well as member of the local health board that gave me an insight into the clinical side of cancer. Finally, I would like to thank my family and friends for their ongoing support throughout my time at university. iii List of Abbreviations AD - Alzheimer's disease ADAMs - Membrane-anchored proteins disintegrin and metalloprotease domains AJCC - American joint committee on cancer AML - Acute myeloid leukaemia AP-3 - Adaptor protein 3 APC - Antigen presenting cells BAP1 - BRCA1 associated protein-1 BCC - Basal cell carcinoma ccRCC - Clear cell renal cell carcinoma CD200 - Cluster of differentiation 200 CD200R - CD200 receptor CD200tg - CD200 transgene cFLIP - caspase 8-like inhibitory protein chRCC - Chromophobe renal cell carcinoma CIA - Collagen-induced arthritis CLL - Chronic lymphocytic leukaemia CLP - Common lymphoid progenitor CNS - Central nervous system CRs - Completed responses CSCC - Cutaneous squamous cell carcinoma CSCs - Cancer stem cells cSMAC - Central supramolecular activation complex CTL - Cytotoxic T cell CTLA-4 - Cytotoxic T lymphocyte-associated Antigen 4 DCs - Dendritic cells DD - Death domain DP - Double positive thymocytes DR - Death receptor dSMAC - Distal supramolecular activation complex ERKs - Extracellular signal-regulated kinases F-actin - Filamentous actin v FADD - FAS-associated death domain FasL - Fas ligand FHL3 - Familial haemophagocytic lymphohistiocytosis subset 3 FoxP3 - Forkhead box P3 GATA3 - GATA-binding protein HCC - Hepatocellular carcinoma HIF1-a - Hypoxia-inducible factor 1 alpha HIF2-a - Hypoxia-inducible factor 2 alpha HNSCC - Head and neck squamous cell carcinoma HPS2 - Hermansky-Pudlak syndrome subset 2 HSCs - Hematopoietic stem cells IFNg - Interferon gamma I - Immunoglobulin ISF - Ig superfamily IL- - Interleukin iNK - Immature NK cell IPEX - Immune Dysregulation Polyendocrinopathy, Enteropathy, X-linked disease IS - Immunological synapse ITAM - Immunoreceptor tyrosine-based activation motif ITIM - Immunoreceptor tyrosine-based inhibition motif iTres - Induced Tregs KIRs - Killer immunoglobulin-like receptors LAG-3 - Lymphocyte activation gene-3 MAPKs - Mitogen-activated protein kinases MDP - Macrophage-dendritic cell progenitor MHC - Major histocompatibility complex MPPs - Multipotent progenitors mRCC - Metastatic renal cell carcinoma MTOC - Microtubule organisation centre mTOR - Mammalian target of rapamycin mTORC - Mammalian target of rapamycin complex MZ - Marginal zone B cell vi NCR - Natural cytotoxicity receptor NK - Natural killer cells NKG2D - Natural-killer group 2, member D NSCLC - Non-small-cell lung cancer nTreg - Natural Tregs ORR - Overall response rate OS - Overall survival PBRM1 - Polybromo 1 PD-1 - Programmed cell death protein 1 PDGFB - Platelet-derived growth factor beta PFS - Progression free survival PI3K - Phosphatidylinositol-3-kinase PMBCL - Primary mediastinal B-cell lymphoma pRCC - Papillary renal cell carcinoma pSMAC - Peripheral supramolecular activation complex PTB - Phosphotyriosine-binding RCC - Renal cell carcinoma sCD200 - soluble form CD200 sFasL - Soluble fas ligand SHIP 1 - Lipid phosphatase SH2 domain-containing inositol-5-phosphatase SHP-1 - SRC homology-containing tyrosine phosphatase 1 SMAC - Supramolecular activation cluster SNAREs - Soluble N-ethylmaleimide-sensitive factor attachment protein receptors SP - Single positive thymocytes TCR - T cell receptor TGF - Transforming growth factor TIL – Tumour infiltrating lymphocyte TIM-3 - T cell immunoglobulin and mucin domain 3 TKI - Tyrosine kinase inhibitor TME - Tumour microenvironment TNF - Tumour necrosis factor TNFR - TNF-receptor vii TNM - Tumour-node-metastasis TRADD - TNFR-associated death domain TRAIL - TNF-related apoptosis-inducing ligand TRAIL-R - TNF-related apoptosis-inducing ligand receptor Tregs - Regulatory T cells VEGF - Vascular endothelial growth factor VEGFR - Vascular endothelial growth factor receptor VHL - Von Hippel-Lindau VISTA - V-domain immunoglobulin-containing suppressor of T-cell activation WAS - Wiskott-Aldrich syndrome WASp - Wiskott-Aldrich syndrome protein WT - Wild-type viii Table of Contents Chapter 1: Introduction 1.1 Hallmarks of cancer ……………………………………………………………………………..………….. 1 1.1.1 The tumour microenvironment …………………………………………………………..…………. 1 1.2 Cells of the immune system …………………………………………………………………….………. 2 1.2.1 Myeloid Lineage …………………………….………………………………………………………….…... 3 1.2.1.1 Granulocytes …………..……………………………...……………………………….…………..… 4 1.2.1.1.1 Neutrophils …………………………………………………...……………..……………...… 4 1.2.1.1.2 Basophils ……………….……………………………………………....………………….…… 4 1.2.1.1.3 Eosinophils ………………………………..………………….……….………………….……. 5 1.2.1.1.4 Mast Cells ……………………………………………………………...……………………..… 5 1.2.1.2 Macrophages …………………………………………………………………….….……………. 5 1.2.1.3 Dendritic Cells ……………………………………..………………………………...………….. 6 1.2.2 Lymphoid Lineage …………………………………………………………………………………….…….. 7 1.2.2.1 B Lymphocytes ……………………………………………………………………………………...... 7 1.2.2.2 T Lymphocytes ……………………………………………………………………………………..…. 8 1.2.2.2.1 CD4+ T cells ………………………………………………………………………….………….. 8 1.2.2.2.1a Th1 Differentiation ……………………………………………………………………….… 9 1.2.2.2.1b Th2 Differentiation ……………………………………………………………………..….. 9 1.2.2.2.1c Regulatory T cells ………………………………………………………………….………... 9 1.2.2.2.2 CD8+ T cells ………………………………………………………………………………..…... 10 1.2.2.2.3 Memory T cells ………………………………………………………………………………... 11 1.2.2.3 T cell Activation …………………………………………………………………………………….... 11 1.2.2.3.1 T cell receptor ……………………………………………………………………………….... 14 1.2.2.3.1a Stimulatory Receptors ……………………………………………………………….….. 14 1.2.2.3.1b Inhibitory Receptors ……………………………………………………………………... 14 1.2.2.4 Natural Killer Cells …………………………………………………………………………………... 17 1.2.2.4.1 NK cell development and maturation …………………………………………….... 17 1.2.2.4.2 NK cell subsets ……………………………………………………………………………….... 18 1.2.2.4.3 Regulation of NK cell activation …………………………………………………….….. 18 1.2.2.4.3a Inhibitory Receptors on NK cells …………………………………………………….. 19 1.2.2.4.3b Activating Receptors on NK cells ………………………………………..………….. 20 ix 1.2.3 NK cell activity ………………………………………………………………………………………….. 22 1.2.3.1 Cytotoxic Immune Response of NK cells ……………………………………………. 22 I. Formation of immunological synapse (IS) between target and NK cell and the reorganisation of the actin skeleton II. Polarisation of microtubule organisation centre (MTOC) and secretory lysosome toward lytic synapse III. Docking of secretory lysosome with the plasma membrane of NK cells IV. Fusion of secretory lysosome with the plasma membrane of target cells 1.2.3.1.1 Lytic Granules ……………………………………………………………………………….... 25 1.2.2.5.2a Perforin/Granzyme Apoptosis Pathway ……………………….....…….……… 25 1.2.2.5.2b Other contents of lytic granules ………………………………..………………….. 26 1.2.2.5.3 Death receptor-mediated apoptosis pathways ………………..……………… 26 1.2.2.5.4 Death receptor signalling