Regulation of Monocyte/Macrophage Activation by Leukocyte Immunoglobulin-Like Receptor B4

Regulation of monocyte/macrophage activation by Leukocyte Immunoglobulin-Like Receptor B4

(LILRB4)

Mijeong (May) Park

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

School of Medical Sciences Faculty of Medicine April 2016

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Park First name: Mijeong Other name/s: May Abbreviation for degree as given in the University calendar: PhD School: Medical Sciences Faculty: Medicine Title: Modulation of monocyte/macrophage activation by leukocyte immunoglobulin-like receptor B4 (LILRB4)

Abstract The leukocyte immunoglobulin-like receptor B4 (LILRB4) belongs to a family of cell surface receptors, primarily expressed on mono-myeloid cells. LILRB4 has been shown to inhibit FcγRI- mediated pro-inflammatory cytokine production by monocytes and induce tolerogenic dendritic cells in vitro. It is believed that LILRB4 regulates monocyte/macrophage activation through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) by paring with activating receptor, bearing ITAM, and by dephosphorylation of non-receptor tyrosine kinases via recruitment of Src homology phosphatase-1 (SHP-1). However, the exact mechanism and the functions depending on its structure and stimuli are still unclear. In addition, regulatory functions of LILRB4 paring with non- ITAM associated activating receptors, including TLR4 is less researched. Thus, this thesis investigates, for the first time, the functions of LILRB4 in regulation of monocyte/macrophage activation depending on the position of the tyrosine residues of its ITIMs, and stimuli. Here it is shown for the first time that LILRB4 is a complex immuno-regulatory receptor that exerts dual inhibitory and activating functions in FcγRI and/or TLR4-mediated monocyte/macrophage activation including receptor-ligand internalisation, endocytosis, cytokine production, phagocytosis and bactericidal activity. Most importantly, each ITIM of LILRB4 has a different function in monocyte/macrophage activation depending on the position of its tyrosine residue. Thus, the tight regulation of these ITAM-containing and non-ITAM receptors by each LILRB4 ITIM may play a key role in fine-tuning immune responses. In addition, the results for the regulatory function of LILRB4 using various ITIM mutants in response to different stimuli provide better understanding of the signalling pathways of LILRB4 that may contribute to use LILRB4 as a potential therapeutic agent for an appropriate immune response using selective LILRB4 ITIM/s. Although no LILRB4 ligand is identified, new methodologies and the potential candidate ligands of LILRB4 which are provided here may contribute to identifying the nature of LILRB4 ligand/s in the near future.

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I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

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‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

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TABLE OF CONTENTS

TABLE OF CONTENTS ...... i LIST OF TABLES ...... vi ABBREVIATIONS ...... x PUBLISHED WORK FROM THIS THESIS ...... xvi PUBLISHED WORK DURING PHD ...... xvi ABSTRACTS (AS PRESENTING AUTHOR) ...... xvi ACKNOWLEDGEMENTS ...... xvii ABSTRACT ...... xix

CHAPTER 1. INTRODUCTION ...... 1 1.1 Leukocyte immunoglobulin-like receptors (LILRs) ...... 4 1.1.1 Nomenclature of leukocyte immunoglobulin-like receptors ...... 5 1.1.2 Genetic organisation of LILRs and related receptors ...... 6 1.1.3 LILR homologues and orthologues ...... 8 1.1.4 Classification of LILRs ...... 9 1.1.5 Ligands for LILRs ...... 17 1.1.6 Rodent orthologues of LILRB1, B3 and B4 ...... 23 1.1.7 LILR expression, gene regulation and functions ...... 25 1.1.1 Clinical association between disease and LILR expression ...... 30 1.1.2 Expression, functions and clinical significance of LILRB4 ...... 31 1.1.3 The role of LILRs in the regulation of innate immune responses ...... 33 1.1.4 Coupling of activating and inhibitory signals by receptors containing ITAMs and ITIMs ...... 47 1.2 Statement of aims ...... 60

CHAPTER 2. GENERAL METHODS ...... 62 2.1 Antibodies, buffers and reagents ...... 62 2.2 Isolation of Peripheral blood mononuclear cells (PBMCs) ...... 66 2.3 Cell lines and tissue culture ...... 67 2.4 Quality controlling of cell lines and antibodies for endotoxin contamination ...... 68 2.5 Quality controlling of cell surface receptor expression of THP-1 cell lines ...... 71 2.6 In vitro differentiation of THP-1 cell lines ...... 71 2.7 Establishing activation protocols for PBMCs and THP-1 cell lines ...... 72 i

2.7.1 Stimulation of THP-1 cells and primary monocytes by LPS ...... 72 2.7.2 Cross-linking of FcγRI and co-ligation of LILRB4 on THP-1 cells and primary monocytes ...... 73 2.7.3 Optimization of cross-linking and co-ligation antibody concentrations for cells transfected CD25-LILRB4 ITIMs ...... 76 2.8 Detection of TNF-α by Enzyme-linked immunosorbent assay (ELISA) ...... 79 2.9 Detection of multiple cytokine production by Luminex MAGPIX ...... 79 2.10 Silver staining, mass spectrometry, Western blotting and Immunoprecipitation ...... 80 2.10.1 Silver staining ...... 81 2.10.2 Mass spectrometry ...... 81 2.10.3 Western blotting ...... 82 2.10.4 Immunoprecipitation ...... 83 2.11 Molecular biology methods ...... 84 2.11.1 RNA extraction, reverse transcription and quantitative RT-PCR ...... 84 2.11.2 Sub-cloning of LILRB4 into mammalian expression vectors ...... 87 2.11.3 Site-directed mutagenesis of the LILRB4 ITIMs in the pMH-Neo-CD25 LILRB4 construct ...... 90 2.11.4 Plasmid DNA transfection to mammalian cells ...... 91 2.12 Flow cytometry ...... 93 2.13 Quality controls of CD25-LILRB4 transfected THP-1 cells ...... 94 2.13.1 Cell viability using LIVE/DEAD® Viability/Cytotoxicity Kit...... 94 2.13.2 Cell proliferation using 3H-thymidine incorporation assay ...... 95 2.13.3 Apoptosis using annexin V-FITC and propidium iodine (PI) ...... 95 2.14 Measurement of bacterial phagocytosis using pHrodo ...... 96 2.15 Assessment of bead phagocytosis ...... 96 2.16 Measurement of bactericidal activity ...... 97 2.17 Synthesis of peptides containing LILRB4 ITIMs ...... 98 2.17.1 High-performance liquid chromatography (HPLC) to determine purity of synthetic peptides containing LILRB4 ITIMs ...... 99 2.17.2 Intracellular staining to determine internalisation of synthetic peptides containing LILRB4 ITIM(s) ...... 99 2.18 Statistical analysis ...... 100

CHAPTER 3. RESULTS ...... 101 3.1 Regulation of Tyr phosphorylation of multiple proteins after FcγRI cross-linking on THP-1 cells by LILRB4 ...... 101

3.1.1 Silver staining of SDS-PAGE gel loaded with immunoprecipitates from FcγRI cross- linked THP-1 cells showed enrichment of multiple Tyr phosphorylated proteins .... 101 3.1.2 Identification of the Tyr phosphorylated proteins from FcγRI cross-linked THP-1 cells by Nano LC-MS/MS ...... 103 3.1.3 Ingenuity Pathway Analysis of Tyr phosphorylated proteins from FcγRI cross-linked THP-1 cells identified by Nano LC-MS/MS ...... 109 3.1.4 Validation of mass spectrometry results of Tyr phosphorylated proteins involved in clathrin-mediated endocytosis by Western blotting ...... 111 3.1.5 Co-ligation of FcγRI with LILRB4 inhibited global Tyr phosphorylation of multiple proteins ...... 113 3.1.6 LILRB4 significantly inhibited Tyr phosphorylation of multiple signalling proteins related to clathrin-mediated endocytosis ...... 115 3.1.7 LILRB4 signals dephosphorylation of FcγRs, Cbl and HGS via its intracellular Tyr based inhibitory motifs (ITIMs) ...... 120 3.1.8 Summary and conclusions ...... 122 3.2 Regulation of FcγRI-mediated monocyte activation by LILRB4 depends on the position of the Tyr residues in its ITIMs ...... 126 3.2.1 Immunophenotyphic profiling of THP-1 cell lines ...... 126 3.2.2 Quality controls of stable CD25-LILRB4 ITIM over-expressing THP-1 cells ...... 128

3.2.3 Cell proliferation was supressed by over-expressing the middle Tyr389 but enhanced by

the distal Tyr419 of LILRB4 ITIM...... 133

3.2.4 Over-expression of the middle Tyr389 of LILRB4 ITIMs increased early apoptosis . 135 3.2.5 Over-expression of CD25-LILRB4 ITIMs differentially modulated FcγRI-mediated TNF-α production ...... 137 3.2.6 Over-expression of CD25-LILRB4 ITIMs suppressed FcγRI-mediated bacterial phagocytosis ...... 141 3.2.7 All transfected cells except for cells over-expressing LILRB4 ITIMs with the middle

Tyr389 (FYF) showed increased phagocytosis of IgG-coated beads ...... 143 3.2.8 Over-expression of CD25-LILRB4 ITIMs differentially regulated bactericidal activity ...... 145 3.2.9 Preliminary results exploring the potential use of synthetic LILRB4 ITIM peptides to modulate FcγRI-mediated TNF-α production ...... 147 3.2.10 Summary and conclusions ...... 149 3.3 Regulation of LPS-mediated monocyte activation by LILRB4 ...... 152 3.3.1 Over-expression of CD25-LILRB4 ITIMs differentially modulated LPS-mediated TNF-α production ...... 152 3.3.2 Two different signalling pathways are involved in the regulation of LPS-mediated cell activation by LILRB4 ...... 157 3.3.3 Summary and conclusions ...... 159

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CHAPTER 4. PRELIMINARY SCREENING FOR LILRB4 LIGANDS ...... 163 4.1 Introduction ...... 163 4.2 Materials and Methods...... 166 4.2.1 Sub-cloning of the extracellular domains of LILRB4 into pAPtag-5 mammalian expression vector ...... 166 4.2.2 Transfection of LILRB4-APtag-His, LILRB4-His in pAPtag-5 and pAPtag-5 vector alone into 293T cells ...... 172 4.2.3 Measurement of alkaline phosphatase activity for quantification of recombinant LILRB4-APtag-His and APtag-His in culture supernatants ...... 172 4.2.4 Purification of LILRB4-APtag-His, LILRB4-His and APtag-His proteins ...... 173 4.2.5 Binding of LILRB4-APtag-His to the surface of peripheral blood mononuclear cells and Jurkat T cell line ...... 174 4.2.6 Preparation of Jurkat cell lysates and cell membrane proteins ...... 175 4.2.7 Detection of LILRB4-APtag-His binding proteins by Far-Western blotting of membrane proteins derived from Jurkat cells ...... 177 4.2.8 Immunoprecipitation of LILRB4 ligand/s from Jurkat cells and mass spectrometric peptide sequencing of precipitates ...... 178 4.2.9 Detection of N-glycosylation sites in recombinant LILRB4-His ...... 181 4.3 Results...... 182 4.3.1 Placental alkaline phosphatase-tagged and untagged extracellular domain of LILRB4 proteins were successfully produced by transfected 293T cells...... 182 4.3.2 LILRB4-APtag-His bound to the surface of PBMCs ...... 185 4.3.3 Passage and activation dependent binding of recombinant LILRB4 on Jurkat cells . 187 4.3.4 Competitive blocking LILRB4-APtag-His binding to Jurkat cells ...... 189 4.3.5 Far Western blotting showed binding of LILRB4-APtag-His to proteins derived from Jurkat cells...... 190 4.3.6 Five candidate ligands for LILRB4 were identified using a combination of immunoprecipitation and mass spectrometry ...... 192 4.3.7 Potential post-translational modification of the recombinant LILRB4 proteins produced in 293T cells ...... 196 4.4 Summary and conclusions ...... 199

CHAPTER 5. GENERAL DISCUSSION ...... 201 5.1 LILRB4 suppresses Tyrosine phosphorylation of multiple signalling proteins involved in clathrin-mediated endocytosis...... 201 5.2 LILRB4 exerts dual inhibitory and activating functions on FcγRI-mediated TNF-α production in THP-1 cells depending on the position of the Tyr residues of its ITIMs. ... 208 5.3 CD25-LILRB4 over-expressing cells differentially regulate phagocytosis of IgG opsonised polystyrene beads and bactericidal activity...... 216 iv

5.4 LILRB4 differentially regulates LPS-mediated THP-1 cell activation depending on the presence of the Tyr residues of its ITIMs...... 222 5.5 Potential interaction of LILRB4 with multiple cell surface ligands on T cells ...... 226 5.6 Conclusion...... 232

REFERENCES ...... 236 APPENDIXES ...... 264 Appendix I. Common chemicals and reagents ...... 264 Appendix II. MASCOT search results of mass spectrometric peptides sequencing of LILRB4- AP-His immunoprecipitated proteins as candidate ligands for LILRB4* ...... 267 Appendix III. Screening of mRNA expressions ...... 269

LIST OF TABLES

Table 1-1. Nomenclature of LILRs ...... 6 Table 1-2. LILR Ligands ...... 18 Table 1-3. Expression of human LILRs ...... 25 Table 1-4. Functions of LILRs ...... 27 Table 1-5. Expression of LILRs in diseases ...... 31 Table 1-6. ITAM-bearing adaptor proteins on immune cells ...... 50 Table 1-7. ITIM-containing receptors...... 52 Table 1-8. ITAM bearing inhibitory receptors (ITAMi)...... 57

Table 2-1. List of primary and secondary antibodies used throughout this thesis ...... 63 Table 2-2. List of buffers and their components used throughout this thesis ...... 65 Table 2-3. Cell line culture conditions ...... 70 Table 2-4. Forward and reverse primer sequence sets used for quantitative RT-PCR ...... 86 Table 2-5. Primer sets used for construction of CD25-LILRB4 chimeric DNA construct ... 89 Table 2-6. Primer sets used for site-directed mutagenesis of the LILRB4 ITIMs in the pMH- Neo-CD25 LILRB4 construct ...... 91 Table 2-7. Synthetic peptides containing LILRB4 ITIM(s) and controls ...... 99

Table 3.1-1. Mascot search results of mass spectrometric peptides sequencing of tyrosine phosphorylated proteins upon FcγRI cross linking of THP-1 cells* ...... 105 Table 3.2-1. Proportions of viable cells in stably transfected THP-1 cells as determined by LIVE/DEAD® assay (n=2) ...... 128 Table 3.2-2. Effect of CD25-LILRB4 ITIMs in monocyte functions relative to mock transfected cells ...... 150

Table 4-1. Primer sets used for sub-cloning of LILRB4 into pAPtag-5 mammalian vector 168 Table 4-2. PCR reaction used for sub-cloning of LILRB4 into pAPtag-5 mammalian vector ...... 169 Table 4-3. LILRB4 candidate ligands repeatedly detected by Nano LC-MS/MS peptide mass sequencing ...... 195 Table 4-4. Amino acid sequences of deglycosylated LILRB4-His using PNGase F ...... 198

LIST OF FIGURES

Figure 1-1 The leukocyte receptor complex (LRC) on human chromosome 19q13.4...... 7 Figure 1-2 Classification of LILRs on the basis of their cytoplasmic and transmembrane domains...... 11 Figure 1-3 Classification of LILRs on the basis of their extracellular domains with their interaction with MHC class I...... 13 Figure 1-4 Schematic diagram of the intracellular domains of LILRB...... 17 Figure 1-5 Structure of LILRB1-HLA-A2 complex and LILRB4...... 21 Figure 1-6 Alignment of the amino acid sequences of extracellular domains of LILRB4 and LILRB1...... 22 Figure 1-7 Phylogenetic trees of LILR Ig domains...... 23 Figure 1-8 FcγRI-mediated signalling pathways...... 36 Figure 1-9 ITAM bearing FcγRI signalling...... 39 Figure 1-10 Clathrin-mediated endocytosis upon FcγRI-mediated cell activation...... 43 Figure 1-11 TLR4-mediated cell activation...... 46 Figure 1-12 Schematic diagram of phosphorylation and the structure of Src, Syk/Zap70 and SHP-1/SHP-2...... 47 Figure 1-13 LILRB4-mediated inhibition of FcγRI-mediated monocyte activation...... 54 Figure 1-14 Different phosphatases are recruited by different receptors that induce different signalling pathways...... 55 Figure 1-15 ITAM-mediated cellular activation and inhibition (ITAMi) ...... 58

Figure 2-1 Cross-linking of FcγRI...... 75 Figure 2-2 Cross-linking of FcγRI and co-ligation with LILRB4...... 76 Figure 2-3 Optimization of plate-immobilised antibody concentration for FcγRI cross- linking and CD25 co-ligation...... 78 Figure 2-4 Quantitative RT-PCR products using 27 multi-gene primers...... 87 Figure 2-5 Schematic diagram of CD25-LILRB4 ITIM plasmids...... 93

Figure 3.1-1 Expression of FcγRI and LILRB4 on the surface of THP-1 cells...... 101 Figure 3.1-2 Representative silver staining of Tyr phosphorylated proteins in THP-1 cells upon cross-linking of FcγRI...... 102 Figure 3.1-3 Predicted signalling pathways upon cross-linking of FcγRI...... 110 Figure 3.1-4 Validation of mass spectrometry data by Western blotting...... 112 Figure 3.1-5 Detection of Tyr phosphorylated proteins in THP-1 cells by Western blotting using anti-Tyr polyclonal antibody after FcγRI cross-linking or co-ligation vii

with anti-LILRB4 mAb...... 114 Figure 3.1-6 Detection of Tyr phosphorylated proteins in THP-1 cells by Western blotting using specific antibodies after FcγRI cross-linking or co-ligation with anti- LILRB4 mAb...... 116 Figure 3.1-7 Detection of total proteins in THP-1 cells by Western blotting using specific antibodies after FcγRI cross-linking or co-ligation with anti-LILRB4 mA...118 Figure 3.1-8 Detection of Tyr phosphorylated proteins in THP-1 cells over-expressing LILRB4 ITIMs (YYY) and a mock control after FcγRI cross-linking...... 121 Figure 3.1-9 Schematic diagram demonstrating possible roles of Tyr phosphorylation of key molecules involved in clathrin-mediated endocytosis of FcγRI and ligands, and their regulation by LILRB4...... 124

Figure 3.2-1 Expression of relevant receptors on the cell surface of THP-1 cells ...... 126 Figure 3.2-2 Expression of CD25-LILRB4 ITIMs on the cell surface of stably transfected THP-1 cells...... 130 Figure 3.2-3 Expression of FcγRI on the cell surface of stably transfected THP-1 cells....131 Figure 3.2-4 Expression of native LILRB4 on the cell surface of stably transfected THP-1 cells...... 132 Figure 3.2-5 Expression of cytosolic SHP-1, SHP-2 and SHIP in stably transfected THP-1 cells...... 133 Figure 3.2-6 Baseline cell proliferation in stably transfected THP-1 cells...... 134 Figure 3.2-7 Baseline apoptosis in stably transfected THP-1 cells...... 136 Figure 3.2-8 Detection of TNF-α production after co-ligation of FcγRI with CD25 in stably transfected THP-1 cells...... 138 Figure 3.2-9 Detection of TNF-α production after FcγRI cross-linking in stably transfected THP-1 cells...... 140 Figure 3.2-10 FcγRI-mediated bacterial phagocytosis in PMA-differentiated stably transfected THP-1 cells...... 142 Figure 3.2-11 Phagocytosis of IgG-coated polystyrene beads by CD25-LILRB4 ITIMs transfected PMA-differentiated THP-1 cells...... 144 Figure 3.2-12 Bactericidal activity by PMA-differentiated-stably transfected THP-1 cells...... 146 Figure 3.2-13 Detection of FcγRI-mediated TNF-α production in THP-1 and PBMCs after treatment of synthetic LILRB4 ITIM peptides...... 148

Figure 3.3-1 Expression of TLR4 on the surface of stably transfected THP-1 cells...... 152

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Figure 3.3-2 LPS-mediated cytokine production in stably transfected THP-1 cells...... 154 Figure 3.3-3 Detection of LPS-mediated TNF-α production in stably transfected cells.....156 Figure 3.3-4 Detection of the level of intracellular signalling proteins involved in NFκB and MAPK pathways in transfected cells...... 158 Figure 3.3-5 Schematic diagram demonstrating TLR4-mediated cell activation and its regulation by LILRB4...... 162

Figure 4-1 Sub-cloning and production of soluble LILRB4-APtag-His and LILRB4-His in stably transfected HEK 293T cells...... 167 Figure 4-2 Membrane protein preparation using Ficoll gradient and Coomassie blue staining...... 177 Figure 4-3 A schematic representation of the proteomic approaches to identify LILRB4 ligands from Jurkat cells...... 180 Figure 4-4 Purification of LILRB4-APtag-His, LILRB4-His and APtag-His and validation by silver stain and Western blotting...... 184 Figure 4-5 A representative binding of LILRB4-APtag-His on PBMCs...... 186 Figure 4-6 Jurkat cell passage and activation dependent binding of LILRB4-APtag- His...... 188 Figure 4-7 Competitive binding blockage by LILRB4-His on binding of LILRB4-APtag- His to Jurkat cells...... 189 Figure 4-8 Representative Far-Western blot of protein fractions isolated from Jurkat cells...... 191 Figure 4-9 Representative silver staining of LILRB4-APtag-His or APtag-His immunoprecipitated membrane proteins from Jurkat cells...... 194 Figure 4-10 Representative silver staining of deglycosylated LILRB4-His using PNGase F...... 197

Figure 5-1 Schematic diagram of the intracellular domains of LILRB4 and LILR...... 212 Figure 5-2 Schematic diagram demonstrating the interaction of CD47 with SIRP-α and αvβ3, and prediction of the interaction with native and/or recombinant LILRB4...... 230 Figure 5-3 Schematic representation demonstrating the regulation of monocyte/ macrophage activation by LILRB4 via its intracellular domains and binding of its unknown ligand/s...... 234

ABBREVIATIONS

°C degree Celsius 3H-Thymidine tritiated thymidine Ab antibody ACD acid-citrate-dextrose ADCC antibody-dependent cellular cytotoxicity ANGPTL angiopoietin-like protein ANOVA analysis of variance AP alkaline phosphatase AP-1 activator protein 1 APC antigen presenting cell Asp (D) aspartic acid BCA bicinchoninic Acid Protein Assay BCP bromo-3-chloropropane BCR B cell receptor BLAST basic local alignment search tool BLV bovine leukemia virus Bmax maximum specific binding bp base pair BSA bovine serum albumin BST2 bone marrow stromal cell antigen 2 Ca2+ calcium ions CBL casitas B-lineage Lymphoma c-Cbl cytoplasmic Casitas B-lineage lymphoma protein CD cluster of differentiation cDNA complementary deoxyribonucleic acid CFU colony forming unit CK creatine kinase CLB cross-linking buffer CTLA-4 cytotoxic T-lymphocyte antigen-4 DAG diacylglycerol DAPI 4’,6’-diamidino-2-phenylindole DCs dendritic cells

DMEM Dulbecco’s Modified Eagle’s Medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP Deoxynucleotide D-PBS Dulbecco’s phosphate-buffered saline DTT dithiothreitol E. coli Escherichia coli EBV Epstein-Barr virus EDN eosinophil-derived neurotoxin EDTA ethylenediaminetetraacetic acid EGFR epidermal growth factor receptor ELISA enzyme-linked immunosorbent assay Eps epsin ErbB1 epidermal growth factor receptor 1 Erk extracellular-signal-regulated kinase F(ab΄)2 fragment antigen-binding FBS fetal bovine serum Fc fragment crystallisable region of an antibody molecule FCAR Fc receptor for IgA FcR Fc receptor FcRγ common γ-chain of Fc receptor FcαR Fc-alpha receptor FcγRI Fc gamma receptor I FcεRI Fc-epsilon receptor I FITC fluorescein isothiocyanate FRET fluorescence resonance energy transfer FTICR fourier transform ion cyclotron resonance G418 geneticin GAPDH glyceraldehyde 3-phosphate dehydrogenase GFP green fluorescence protein Grb2 growth factor receptor-bound protein 2 HBHA Hank buffer saline solution with MgCl2 and CaCl2 HBSS Hanks’ Balanced Salt Solution HCMV human cytomegalovirus HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HGS hepatocyte-growth-factor regulated tyrosine kinase substrate

His histidine HIV human immunodeficiency virus HLA human leukocyte antigen HLA human leukocyte antigen HMGB1 high-mobility group protein B1 HPLC high-performance liquid chromatography hr hour HRP horseradish peroxidase hrs hours HSC heat shock cognate protein HSP heat shock protein IAP integrin associated protein IFN interferon Ig immunoglobulin IL interleukin ILT immunoglobulin like transcript IP immunoprecipitation IP3 inositol trisphosphate IPA Ingenuity pathway analysis IREM-1 immune receptor expressed by myeloid cell 1 ITAMs immunoreceptor tyrosine-based activating motifs ITIMs immunoreceptor tyrosine-based inhibitory motifs JNK C-Jun N-terminal kinase K binding affinity constant KD dissociation constant kDa kilo Dalton KIR killer immunoglobulin-like receptor LAIR leukocyte-associated Immunoglobulin-like Receptors LAL limulus amebocyte lysate LAR leukocyte common antigen-related receptor LAT linker of activated T cells LBP LPS binding protein Lck lymphocyte-specific protein tyrosine kinase LC-MS/MS liquid chromatography tandem mass spectrometry LILRA leukocyte immunoglobulin-like receptors subfamily A

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LILRB leukocyte immunoglobulin-like receptors subfamily B LILRs leukocytes immunoglobulin-like receptors LPS lipopolysaccharide LRC leukocyte receptor complex LRR leucine-rich-repeats LTC4 leukotriene C4 LTR long terminal repeat Lyn Lyn kinase Mak MyD88 adapter-like MAPK mitogen activated protein kinases MCP-1 monocyte chemotactic protein-1 MgCl2 magnesium chloride MHC major histocompatibility complex min minute MIR myeloid inhibitory receptor MMTV mammary tumor virus Mn2+ Manganous ion MOI multiplicity of infection MPA microscopic polyangiitis mRNA messenger ribonucleic acid MS multiple sclerosis MΦs macrophages NaCl sodium chloride NCR1 natural cytotoxicity receptor 1 NFAT nuclear factor of activated T-cells NFκB nuclear factor kappa betta N-glycosylation asparagine (N)-linked glycosylation NK cell natural killer cell OD405 optical density (Absorbance at 405 nm) PBMCs peripheral blood mononuclear cells PBS phosphate buffered saline PCR polymerase chain reaction PD-1 programmed death-1 PE phycoerythrin fluorochrome

xiii pH power of hydrogen Phe (F) phenylalanine PI propidium iodine PI3K phosphatidylinositol 3-kinases PIP2 phosphatidylinositol 4,5-bisphosphate PIP3 phosphatidylinositol 3,4,5-trisphosphate PIRs paired immunoglobulin-like receptors PKC protein kinase c PLC-γ phospholipase C-γ PMA phorbol 12-myristate 13-acetate PTKs protein tyrosine kinase PTPs protein tyrosine phosphatases pTyr phosphorylated tyrosine PVDF polyvinylidene difluoride RA rheumatoid arthritis RhD rhesus D immunoglobulin RSV respiratory syncytial virus RT room temperature S. typhimurium Salmonella typhimurium SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEAP secreted placental alkaline phosphatase sec second SFFV spleen focus-forming virus SH2 Src homology 2 SH3 Src homology 3 SHIP Src homology 2 domain-containing Inositol 5-Phosphatase SHP-1 Src homology 2 domain-containing phosphatase-1 SHP-2 Src homology 2 domain-containing phosphatase-2 SIGLECs sialic acid-binding Ig-like lectins SIRP-α signal regulatory protein-α SLE systemic lupus erythematosus SLP76 Src homology 2 domain-containing leukocyte protein of 76 kDa SNPs single nucleotide polymorphisms SPR surface plasmon resonance

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SPY75 hematopoietic cell-specific Lyn substrate STAM signal transducing adapter molecule STAT signal transduction and transcription Syk spleen tyrosine kinase TAT transactivator of transcription TBS tris buffer saline TCR T cell receptor TGF-β1 transforming growth factor-β TIR Toll interleukin- receptor TIRFM internal reflection fluorescence microscopy TLR Toll-like receptor TLT-1 TREM-like transcript 1 TM transmembrane TMB 3,3',5,5'-Tetramethylbenzidine TNF-α tumour necrosis factor-α TREM triggering receptor expressed by myeloid cell TRIM21 tripartite motif-containing protein 21 TSP-1 thrombospondin-1 Tween20 polyoxyethylenesorbitan monolaurate Tyr (Y) tyrosine UIM ubiquitin interacting motif Zap70 ζ-associated protein of 70 kDa β2m beta-2-microglobulin

PUBLISHED WORK FROM THIS THESIS

 Park M, Lui R, An H, Hsu K, Arm J, Thomas P, Geczy C, Tedla N. Dual positive and negative regulation of monocyte activation by leukocyte immunoglobulin-like receptor B4 depends on the position of the tyrosine residues in its ITIMs and the nature of stimuli. Submitted to PLOS ONE, [submitted]

 Park M, Raftery M, Thomas P, Geczy C, Bryant K, Tedla N. Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis. Accepted in Scientific Reports, [Accepted]

PUBLISHED WORK DURING PHD

 Loke WS, Freeman A, Garthwaite L, Prazakova S, Park M, Hsu K, Thomas PS, Herbert C: T-bet and interleukin-27: possible TH1 immunomodulators of sarcoidosis. Inflammopharmacology 2015, 23(5):283-290.

 Park M, Boys EL, Yan M, Bryant K, Cameron B, Desai A, Thomas PS, Tedla1 NT: Hypersensitivity pneumonitis caused by house cricket, Acheta domesticus. Journal of Clinical & Cellular Immunology 2014, 5(4).

ABSTRACTS (AS PRESENTING AUTHOR)

 Park M, Tedla N. CCR3P.211. The middle tyrosine residue of tyrosine-based inhibitory signalling motifs is a key molecule in inhibitory functions of the leukocyte immunoglobulin-like receptor B4 in FcγRI mediated cytokine production and bacterial phagocytosis. Presented poster in American Association of Immunologists (AAI), in New Orleans, America

 Park M, An H, Hameed A,Thomas P, Tedla N. 77. The leukocyte immunoglobulin-like receptor B4 differentially regulates FcγRI cytokine production, phagocytosis and bactericidal activity via its three intracellular immunoreceptor tyrosine-based inhibitory signalling motifs. Presented poster in Australian Society for Medical Research (ASMR), in Sydney, Australia

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ACKNOWLEDGEMENTS

There are many individuals to whom I owe my warmest and deepest gratitude. These acknowledgements are only a small token of my appreciation for their contribution, guidance, support and encouragement. To my supervisor, Associate Professor Nicodemus Tedla, I am very grateful for the opportunity to undertake this project. You have been very patient and understanding, encouraging, and have helped me through these years of discovery and learning, and I thank you for many years of guidance and financial support. Your perseverance, determination and passion for science are awe-inspiring. To my joint supervisor, Professor Paul Tomas, I would like to thank you for your continuous support and insightful comments. I wouldn’t be here if you did not give me the opportunity to work with you at the beginning of my stay in Australia. I am extremely lucky to have met you and to do my research here. Both my supervisors have made the completion of my PhD possible. I am forever grateful for all the support and encouragement you have given me. To Professor Carolyn Geczy, Dr. Kenneth Hsu, Dr. Katherine Bryant and Dr. Cameron Barbara and Associate Professor Mark Raftery, your help with experimental procedures and advice were critical to the success and the publications from my thesis. To David Agapiou, Penelope Ralph, Shayne Beckham and Sean Young, I really appreciate your help in proofreading my thesis. I am grateful to all the members at the Inflammation and Infection Research Centre, especially to Dr. Hongyan An, Dr. Terry Lee, Dr. Barry Ken, Ms. Alice Wong, Dr. Yuen Ming Chung and Dr. Lincoln Gomes. Your support, encouragement, assistance and company made my time here so much more fun, memorable and enjoyable. To my family, who have shown unconditional and unlimited love and support throughout my seemingly endless years of study, I am forever indebted to them. I am extremely fortunate and very proud to be a member of this family. Lastly, my PhD would not have been possible without the scholarships, University International Postgraduate Award (UIPA) from UNSW, SoMS Academic Excellence (Top-Up) scholarship from School of Medical Sciences and UIPA continuation scholarship from my supervisor Nicodemus Tedla. Thank you.

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DEVOUTLY THANKFUL

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ABSTRACT

A balance between activating and inhibitory signals is critical in the immune system in order to respond to pathogens. Excessive immune responses can induce host tissue damage as well as trigger autoimmune diseases. Therefore, inhibition or appropriate regulation of cell activation is essential to achieve immune balance by termination of excessive immune responses, and to prevent autoimmune diseases by enhancing tolerance. Immune cells express multiple inhibitory receptors for adequate response, diverse levels of negative regulation, and appropriate stages. Over past decades, many inhibitory receptors have been discovered, and most of them have one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs). These receptors contain consensus sequences (I/V/L/S)xYxx(L/V) in their intracellular domain, which regulate cell activation by the recruitment of phosphatases such as SHPs and SHIP.

Tyrosine phosphorylation is reversible by kinases and phosphatases, and is critical in eukaryotes. This modulates intracellular signalling pathways to response stimuli, and is also important for signal transduction, cell growth, proliferation and differentiation.

LILRB4 is an inhibitory cell surface receptor and expresses on myeloid cells.

Clinically, the increased expression of LILRB4 is associated with better clinical outcomes in renal transplant recipients. By contrast, increased expression in some cancers, particularly in pancreatic cancer, has been associated with impaired immune effector responses and poorer outcomes. LILRB4 contains two C2-type extracellular Ig domains and a long cytoplasmic tail containing three ITIMs with sequences

358VTYAKY363, 410VTYAQL415 and 440SVYATL445, respectively. Co-ligation of

LILRB4 to FcγRI in monocytes has been shown to inhibit Ca2+ mobilization, tyrosine phosphorylation through SHP-1 recruitment that resulted in the inhibition of the pro-

xix inflammatory cytokine, TNF-α, production. In addition, Salmonella-infected macrophages up-regulated LILRB4 has been shown to prevent excessive immune responses. However, the regulation of monocyte and macrophage functions by LILRB4 including phagocytosis/endocytosis and bactericidal activity in FcγRI-mediated cell activation, and the signalling pathways by LILRB4 in LPS-mediated inflammatory cytokine production have yet to be described. More importantly, the exact contribution of each ITIM of LILRB4 to its regulatory function is still unclear, although three ITIMs of LILRB4 could differentially regulate leukocyte activation.

This thesis presents new functions for LILRB4 in FcγRI- and LPS-mediated monocyte and macrophage activation. Firstly, LILRB4 is a potent inhibitor of FcγRI- mediated Tyr phosphorylation of the multiple signalling proteins, including clathrin,

Cbl, HGS and TRIM21, involved in clathrin-mediated endocytosis. Secondly, LILRB4

ITIMs exert dual inhibitory and activating functions in FcγRI-mediated cytokine production, phagocytosis and bactericidal activity depending on the position of the tyrosine residues. In addition, each LILRB4 ITIMs differentially regulates cell basal proliferation and apoptosis. Thirdly, intracellular LILRB4 inhibits LPS-mediated TNF-α production through IKK- or PI3K-dependent pathways depending on the presence of the tyrosine residues. Lastly, new methodological approaches to identify and to characterise

LILRB4 ligand(s) will be focused. This thesis provides new findings about LILRB4 functions and provides a better understanding of how its signalling pathways may contribute to the use of LILRB4 as a potential therapeutic agent for appropriate immune response regulation of over-exuberant or unregulated immune responses.

CHAPTER 1. INTRODUCTION

Excessive unregulated immune activation or inadequate responses can be detrimental to the host. Thus, a tight balance between excessive activation and quiescence by activating receptors is required for effective and proportionate immune responses [1, 2]. Although mechanisms that activate the immune system are widely studied, the processes that terminate immune activation have not been fully elucidated.

In recent years, an increasing number of cell surface receptors have been identified as immune inhibitory molecules on the basis of a consensus amino acid sequence, the immunoreceptor tyrosine-based inhibitory motif (ITIM), found in their intracellular domains, and by their ability to suppress activation signals initiated by other receptors

[2, 3]. These inhibitory receptors are believed to mediate this function only upon their cell surface co-clustering with activating counterparts that often contain an intracellular immunoreceptor tyrosine-based activating motif (ITAM) [4, 5]. Briefly, ligation of an

ITAM-containing activating receptor on the cell surface leads to tyrosine phosphorylation of the ITAMs and activation of a cascade of protein tyrosine kinases, subsequently resulting in cellular activation [6-8]. Co-engagement of the activating receptor with an ITIM-containing inhibitory receptor via shared or different ligand/s induces clustering and tyrosine phosphorylation of the inhibitory receptor, providing substrates to phosphatases that dephosphorylate (deactivate) numerous intracellular substrates, including tyrosine kinases activated by the ITAM-containing receptor [4, 9,

10]. Hence, the pairing of activating receptors that trigger swift immune activation and inhibitory receptors that efficiently terminates responses has been proposed as one of the mechanisms that maintains healthy immune function [1, 2]. Although this paradigm of activation: inhibition coupling between ITAM and ITIM-bearing receptors is

1 necessary to modulate immune responses, ITIM-bearing inhibitory receptors may also regulate ITAM independent immune cell activation pathways, such as Toll-like receptors (TLRs), to mitigate the potentially dire biological consequences of unregulated innate immune activation.

The main focus of this thesis is to identify new mechanisms that terminate responses to two of the most important activation pathways of the innate immune system: ITAM-dependent FcγRI-mediated activation [11, 12], and ITAM-independent, but protein tyrosine kinase dependent, Toll-like receptor 4 (TLR4)-mediated stimulation

[13, 14]. The leukocyte immunoglobulin-like receptor B4 (LILRB4), an ITIM- containing cell surface receptor, represents a newly recognised and potentially important inhibitory molecule that may tightly regulate these processes [15-17].

LILRB4 is highly expressed on monocytes and macrophages and potently inhibits

FcγRI-mediated cytokine production by recruiting phosphatases that dephosphorylate

(deactivate) a number of protein tyrosine kinases [15]. Consistent with its immune regulatory functions, LILRB4 has been shown to play a key role in the rejection/acceptance of organ transplantation, and its up-regulation is strongly associated with immunosuppression in cancers and severe bacterial infections [18, 19].

Moreover, genetic deletion of the mouse LILRB4 (gp49B1), the murine orthologue of human LILRB4, generated FcεRI-induced unchecked inflammatory responses in vivo

[20, 21].

The first part of this project will therefore investigate the effects of LILRB4 ligation on FcγRI-mediated receptor/ligand endocytosis, phagocytosis and bactericidal activity, as well as lipopolysaccharide (LPS)/TLR4-mediated monocyte activation.

Hence, the current literature on these relevant topics will be comprehensively reviewed.

These topics include detailed analysis of LILR structure, expression, function and their

2 clinical associations, with special focus on LILRB4, and current insights into FcγRI- and TLR4-mediated monocyte and macrophage activation and functions, including mediator production, phagocytosis, endocytosis, bacterial killing, and their associated signalling pathways.

The second and third part of the project will focus on dissecting mechanisms of

LILRB4-mediated regulation of monocyte activation in response to FcγRI or LPS stimulation. It is possible that the balance between the ITAM-bearing FcγRI and the

ITIM-containing LILRB4 may regulate the threshold and amplitude of monocyte activation, in which the net outcome is determined by the relative strength of these opposing signals. However, the concept of ITAM-mediated immune activation and

ITIM-mediated immune termination is being challenged by recent findings which have demonstrated that under certain circumstances, receptors containing the activation motifs can inhibit cellular activation (termed as ITAMi), and that receptors with the canonical inhibitory motifs were able to trigger cellular activation (termed ITIMa) [22], suggesting a greater degree of complexity. ITIM-bearing receptors may transduce inhibitory or paradoxically, activating signals, depending on the nature and/or strength of stimuli, that may trigger selective partial or complete (de) phosphorylation of different tyrosine residues within their ITIM-consensus sequences. Moreover, while

ITIM-like motifs, immunoreceptor tyrosine-based switch motifs (ITSMs) [23, 24], SH3 binding domains [25, 26], and proline-rich regions [27, 28] in the intracellular domains of such immune receptors may further contribute to their overall functions, this has not been fully investigated. Of particular interest in this project is whether LILRB4 that contains three unique ITIM-consensus sequences is capable of transducing both activating and inhibitory signals depending on the position of the tyrosine residues within its ITIMs. Therefore, current knowledge and insight into ITAM- and ITIM-based

3 immune signalling in the context of LILRB4 will be thoroughly reviewed. Current controversies with regards to ITAMi and ITIMa and the potential role of ITSM will be discussed.

Ligands for LILRB4 are unknown, severely hindering characterisation of its functions in vivo, despite in vitro evidence for its immune-regulatory functions and its strong links to disease [29-31]. Thus, the last part of this project involves the development of methods and tools for the identification and characterisation of LILRB4 ligands. As a prelude to this, the current literature on LILR ligands and the limitations, strengths and controversies of methodologies used to identify ligands to some LILR members will be thoroughly reviewed and discussed. Moreover, LILRs, including

LILRB4, are selectively conserved in humans and primates, and since there are no rodent homologues, in vivo studies are restricted. However, the paired immunoglobulin-like receptors (PIRs), PIRA and PIRB, and the mouse LILRB4 (also known as gp49B1), are considered murine orthologues that regulate leukocyte functions through similar signalling cascades [20, 32]. These will be briefly reviewed with special emphasis on the ligands and functions of the mouse LILRB4, which is considered to be the orthologue to the human LILRB4.

1.1 Leukocyte immunoglobulin-like receptors (LILRs)

The expanding families of regulatory receptors containing intracellular immunoreceptor tyrosine-based activating motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that transduce coordinated opposing signals have been identified, and the immunoglobulin G Fc receptors represent one of the best characterised [5, 33, 34]. Recently, the leukocyte immunoglobulin-like receptors

(LILRs) that comprise a family of ITAM-containing activating and ITIM-containing 4 inhibitory receptors that have highly homologous “ligand binding” extracellular domains have been identified as key receptors that may modulate innate immune responses through the activation: inhibition coupling paradigm [35-38].

1.1.1 Nomenclature of leukocyte immunoglobulin-like receptors

Leukocyte immunoglobulin-like receptors (LILRs) were first identified in 1997 by several groups at approximately the same time, leading to divergent and confusing synonyms, including leukocyte immunoglobulin-like receptors (LIR or LILR), immunoglobulin-like transcript (ILT), myeloid inhibitory receptor (MIR) and CD85a-m

[39, 40] (Table 1-1).

LILRB4 (also known as ILT3, LIR5, CD85k) was first cloned from monocytes and human lung cDNA libraries by Arm et al (1997), termed as HM18, and described as a human orthologue to the mouse gp49B1 (mLILRB4) [21]. At the same time, Cella et al (1997) identified a full length LILRB4 cDNA from EBV-B cell lines and designated it as ILT3 [41], with Borges et al (1997) confirming its expression on monocytes, B cells, NK cells and dendritic cells and termed it LIR-5 [42]. To avoid these inconsistencies, the HUGO Gene Nomenclature Committee in 2000

(http://www.genenames.org/genefamilies/LILR) approved LILRs as the official name, with further sub-classification to LILRAs “A” symbolising activating subfamily, and

LILRBs “B” indicating the inhibitory subgroup (Table 1-1).

Table 1-1. Nomenclature of LILRs (modified from HGNC and the following reference [39]) Classification Name/Gene ID Alternative names (synonyms) Determinant (CD) LILRA1 CD85i LIR6, LIR-6 LILRA2 CD85h LIR7, LIR-7, ILT1, Subfamily A LILRA3 CD85e LIR4, LIR-4, ILT6, HM43, HM31 (LILRAs) LILRA4 CD85g ILT7 LILRA5 CD85f LIR9, ILT11 LILRA6 CD85b ILT8 LILRB1 CD85j LIR1, LIR-1, ILT2, MIR-7 LILRB2 CD85d LIR2, LIR-2, ILT4, MIR10, MIR-10 Subfamily B LILRB3 CD85a LIR3, LIR-3, ILT5, HL9 (LILRBs) LILRB4 CD85k LIR5, LIR-5, ILT3, HM18 LLIRB5 CD85c LIR8, LIR-8 LILRP1 CD85l ILT9, LILRA6P Pseudo Genes LILRP2 CD85m ILT10

1.1.2 Genetic organisation of LILRs and related receptors

Thirteen LILR genes and two pseudogenes are encoded on the leukocyte receptor complex (LRC) locus on human chromosome 19q13.4 [43]. LRC has a total size of at least 1Mb, and encodes numerous other related multi-gene families including killer inhibitory receptors (KIRs), the Fc receptor for IgA (FCAR), the leukocyte-associated immunoglobulin-like receptors (LAIR), and the natural cytotoxicity receptor 1 (NCR1)

(Figure 1-1) [39, 44]. The genes of these receptors encode structurally related transmembrane proteins containing one to six extracellular immunoglobulins (Ig)-like domains that are divided into two subgroups comprising constant (C) type or variant (V) type, based on the structure of their Ig domains [45]. Most of these receptors belong to

Ig C2-type receptors that have evolved to produce multiple polymorphic forms of proteins, perhaps due to selection pressure from exposure to rapidly diversifying pathogens [45, 46]. The polymorphism of receptors can affect their expression, function, 6 and binding to endogenous ligands [47]. The LILR gene cluster has two haplotypes and limited allelic variations compared to the closely related KIRs that display a high level of genetic variation, allelic diversity, and have multiple haplotypes [46, 48]. LILRB4 shows a high degree of gene polymorphism, encoding up to 15 unique variants among individuals [49], although the functional significance of this remains unknown.

Figure 1-1. The leukocyte receptor complex (LRC) on human chromosome 19q13.4. LRC is comprised of multi gene families encoding LILRs, LAIRs, LENGs, KIRs, FCAR and NCR1. LILRs are encoded by two gene clusters surrounding LAIR1 and LAIRA2. LILRA2, A1, B1, B4, P1 and P2 are translated to centromere and the other LILRs are translated to telomere. The LILRB4 gene is located on LRC from 54,643,889 - 54,670,359 (26,471 bp and 448 aa) and translated to the telomere. Figure modified from the following references [16, 50, 51].

1.1.3 LILR homologues and orthologues

LILRs are highly conserved in humans and primates, and closely related to Ig (- like) receptors, including Fc receptors, KIRs and other LRC-encoded immune receptors

[39, 52, 53]. These Ig-domain proteins are known as paired receptors that positively and negatively regulate the immune system through ITAM and ITIM motifs [54]. In addition, many of them have been shown to interact with major histocompatibility complex (MHC) molecules, suggesting that these receptors may play a key role in immune regulation [52].

There is a lack of LILR homologues in rodents that limits the use of animal models for functional analysis of LILRs in vivo, and most functional studies on LILRs have been performed in vitro. However, murine paired immunoglobulin-like receptors

(PIRs) are known as an orthologues of LILRs [18, 55-57]. PIRs were firstly identified as homologous to human FcαRI, an IgA specific receptor, but they are now proposed as orthologues of LILRs due to their structural similarity, genomic localization, ligands, and expression profiles [56, 58, 59]. PIRs are encoded by the mouse chromosome 7

(syntenic to LRC on human chromosome 19q13.4), and expressed on various haematopoietic cell lineages including B cells, mast cells, macrophages, granulocytes and dendritic cells [60]. Similar to human LILRs, PIRs are classified into activating

PIRAs and inhibitory PIRBs, depending on their structure of transmembrane and intracellular domains [20], but only one PIRB and at least six PIRAs exist [48]. PIRAs are also associated with the FcRγ chain for the delivery of activating signals, while engagement of PIRB with activating receptors inhibits cellular activation [52]. PIRs contain six Ig-like domains, and have been shown to bind murine MHC class I molecules as well [61].

Alignment (ALIGN) analysis of extracellular domains of LILRB4 showed a high amino acid sequence identity with a immunoglobulin receptor family including mouse gp49A, gp49B, bovine Fcγ2R, human KIRs, human FcαR and human NK cell inhibitory receptors [21]. Among them, gp49B1 is murine orthologue of LILRB4 (also termed as mLILRB4) that was initially described as a family of homologues of LILRs, and subsequently renamed mLILRB4 due to the close extracellular domain homology to human LILRB4 [18, 21]. Unlike human LILRB4, gp49B1 contains only two ITIMs and has been shown to suppress FcεRI-mediated mast cell activation [20]. In addition, gp49B1 inhibits LPS-induced intravascular neutrophil adhesion, indicating that its regulatory function is not limited to ITAM-containing receptors [32]. This suggests that human LILRB4 may modulate both ITAM-containing receptors such as FcRs and non-

ITAM receptors such as TLR4 (the best known LPS receptor).

1.1.4 Classification of LILRs

LILRs are classified into activating, inhibitory and soluble receptors based on the structure of their transmembrane and cytoplasmic domains (Figure 1-2) [39], or into two groups based on the predicted MHC-I binding (Group 1) and MHC-I non-binding ability (Group 2)(Figure 1-2) [29-31].

Activating LILRs (LILRAs) have a short cytoplasmic domain and positively charged arginine residue in their transmembrane domain which is associated with a negatively charged adapter protein containing immunoreceptor tyrosine-based activating motifs (ITAMs) that transduce activating signals (Figure 1-2A) [36, 38]. To date, the known adaptor protein to LILRAs is FcRγ, but other possible adapter proteins, such as DAP12, will be discussed in Section 1.1.4.1. Interestingly, FcγRI (a high

9 affinity receptor for IgG, see details in Section 1.1.3.1) uses the same adaptor as

LILRAs, hence it may signal in the same way.

In contrast, inhibitory LILRs (LILRBs) do not have a positively charged arginine or lysine residue within their transmembrane; rather, they have a long cytoplasmic domain containing two to four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that transduces inhibitory signals (Figure 1-2B) [35-37].

Soluble LILRs have no transmembrane and cytoplasmic domains, hence they are secreted (Figure 1-2C) [42]. Although other LILRs may have soluble components due to proteolytic cleavage of their membrane counterparts or alternately spliced variants,

LILRA3 and sLILRA5 are the only members that encode genes for soluble proteins [42,

62]. It is noteworthy that LILRA3 and sLILRA5 are classified as activating receptors based solely on the structural similarities of their extracellular domains to activating

LILRs, although it is likely that they act as soluble antagonists [63]. Recent studies have shown that alternative mRNA splicing can produce soluble proteins for other LILRs, including LILRA1, 2, 5 and LILRB 1, 4 [64-66].

A B C

Figure 1-2. Classification of LILRs on the basis of their cytoplasmic and transmembrane domains (A) Schematic representation of a prototypic activating LILR contains a positively charged arginine residue in its transmembrane domain which is associated with the negatively charged aspartic residue of ITAM-containing a common γ adaptor protein. (B) Represents a typical inhibitory LILR with a non-charged TM domain and a long cytoplasmic tail containing four ITIMs. (C) Soluble LILRs contain no cytoplasmic or TM domains. TM= transmembrane; x denotes any amino acid in the ITAM or ITIM; adapted and modified from the following reference [39].

The extracellular immunoglobulin (Ig) domains of LILRs are responsible for their ligand binding on the cell surface. Recent studies regarding LILRB1 crystal structures enable LILRs to be classified into two groups based on their residues in extracellular ligand binding domains [67]. Group 1 including LILRB1 is predicted to interact with 11

MHC class I, while no members of Group 2 may interact with MHC class I [39] (Figure

1-3). Among the members of Group 1, LILRB1, B2, A1 and A3 have been confirmed to bind MHC class I, but the interactions of MHC class I and the ligand for LILRA2 are still unknown (see binding details in Section 1.1.5). Although this classification may provide insights into potential ligands for LILRs, there are limitations on the data from the crystal structure of LILRs produced in non-eukaryotic systems. The use of such truncated extracellular domains would inefficiently fold and inappropriately post- translationally modify the proteins. This may affect the interaction of the receptor with the ligands, which may be important when interpreting the results.

Figure 1-3. Classification of LILRs on the basis of their extracellular domains with their interaction with MHC class I All LILRs have two to four highly homologous extracellular C2-type Ig-like domains and from none to seven multiple N-linked glycosylation sites. (A) Schematic representation of Group 1 which is predicted to interact with MHC class I. (B) Schematic representation of Group 2 which may have no interaction with MHC class I. Only LILRB4 has no predicted N-glycosylation site. Figure adapted [39] and modified based on current proteomic database in 2015 (www.uniprot.org). The percentages represent the amino acid identity of the extracellular domain compared to LILRB1. The homology search was performed using LALIGN from ExPASy (www.ch.embnet.org).

In general, the extracellular Ig and Ig-like molecules contain a tetramer of two light chains and two heavy chains that are linked by disulphide bonds and are classified into variable (V), constant 1 and 2 (C1 and C2), and intermediate (I) types [68]. Ig-like domains are associated with various functions, but the most important role is ligand recognition at the cell surface [69]. All LILRs contain two or four C2-type Ig-like molecules in their extra cellular domains, which are highly homologous (ranging from

56 to 84% similarity in identity to LIR1, LILRB1), and responsible for binding their ligand/s at the cell surface (Figure 1-3) [39, 70]. This suggests that LILRs could recognise similar ligands or share the same ligands, which may lead to competitive or synergistic effects. For example, a high degree of homology was recorded for the extracellular domains of LILRB2 at 82% identity to LILRB1; both receptors have been shown to bind the same MHC-I class molecules; and both induced inhibitory signals [71,

72].

The different number of Ig-like domains may affect the ligand binding affinity and specificity of receptors. For instance, three Ig-like domains of FcγRI are able to bind to monomeric IgG and IgG2a with high binding affinity, while two Ig-like domains of FcγRII and FcγRIII bind to various IgG including IgG1, IgG2 and IgG2a with low binding affinity [73-75]. Interestingly, without the third Ig-like domains of

FcγRI, the other two domains were able to bind IgG1, IgG2a and IgG2b, although the binding affinity was low, suggesting that this third domain has an important role in determining the high affinity of FcγRI for its ligand [76].

The extra cellular domains of LILRs contain two to seven multiple potential N- linked glycosylation sites, with the exception of LILRB4 [38]. Glycosylation is a post- translational modification and plays a critical role in protein structure, function, and stability, as well as protein-protein interaction [77, 78]. In particular, three-dimensional

14 configurations of receptors can be affected by glycosylation in modulating the interaction between receptors and their ligands, resulting in distinct effects [79]. There are five classes of glycosylation, including N-glycosylation, O-glycosylation, C- mannosylation, phosphoglycation, and glypiation, depending on the site glycan linkage

[80]. Among these, N-linked glycosylation is the most common type (about 90%) of glycisidic bonds, and is the attachment of oligosaccharides (glycans) to a nitrogen atom

(usually asparagine residues) [79]. This glycosylation is important for some eukaryotic protein folding and for preventing non-specific protein-protein interactions, or for protection from proteases [81]. Furthermore, it influences their ligand binding affinity

[17, 82]. Most LILRs, excluding LILRB4, are predicted to contain N-linked glycosylation sites on their extracellular Ig-like domains. Recombinant LILRA3 protein produced in mammalian 293T cells showed five N-glycosylation sites including N140,

N281, N302, N341 and N431, molecular weight 70 kDa (de-glycosylated LILRA3 being 50 kDa) [83]. Importantly, the appropriate glycosylation of recombinant LILRA3 was critical to high binding affinity on U937 cells, as well as for inhibition of LPS-mediated

TNF-α production on PBMCs, when compared to non-glycosylated recombinant

LILRA3 from E. coli [83]. In addition, LILRA2 containing seven N-glycosylations was

20 kDa larger than the 49 kDa non-glycosylated LILRA2, while the effect of glycosylation on binding affinity remains unknown [38]. To date, while only LILRA3 glycosylation sites have been experimentally mapped, the glycosylation of other LILRs is only predicted, and their modulation in ligand binding affinity/avidity remains to be elucidated. Also, not many studies have used a mammalian system to produce recombinant proteins for the identification of ligands and functions due to the difficulties encountered in these cells. It is therefore important to note that mammalian

15 systems for producing LILRs will be required for optimal glycosylation and post- translational modification in order to identify potential ligands.

LILRB4 belongs to the group of inhibitory LILRs and has a long cytoplasmic tail containing three ITIMs with sequences 358VTYAKY363, 410VTYAQL415 and

440SVYATL445, respectively. LILRB4 also has two SH3 binding motifs (PxxP) between three ITIMs and proline-rich regions in its intracellular domains (Figure 1-4). However, the contributions of these intracellular consensus sequences to LILRB4 functions are unclear. LILRB4 contains two C2-type Ig-like extracellular domains, in contrast to other inhibitory LILRs (Figure 1-3). No N-linked glycosylation site and no ligands for

LILRB4 have been identified [84]. Soluble LILRB4 has been shown to contain two Ig- like domains and an intracellular domain with three ITIMs, but it lacks the stalk and a transmembrane region [65]. This soluble LILRB4 is 10 kDa smaller than the 55 kDa membrane of LILRB4. Cortesini et al (2007) found the LILRB4 gene transcript was lacking exon 7 in some malignancies which corresponds to the transmembrane domain, and suggested that soluble LILRB4 may be produced [85]. The functional significance of LILRB4 in these structural changes remains unknown.

Figure 1-4. Schematic diagram of the intracellular domains of LILRB4 LILRB4 contains two C2-type Ig-like extracellular domains, three ITIMs, and two SH3 domains in the intracellular doamin. The ITIM consensus sequences, (I/V/L/S)xYxx(L/V), are highlighted; the SH3 binding motifs, PxxP, are underlined; and proline rich regions are in bold (where x denotes any amino acid).

1.1.5 Ligands for LILRs

To date, it remains unclear how LILRs influence immune cell activation in vivo due to insufficient knowledge of the nature and characteristics of the LILR ligands.

Although some LILR ligands have been identified (Table 1-2), most ligands of LILRs are poorly defined. Methods including binding assay [86], flow cytometry [70, 87], immunoprecipitation [88], and sensitive surface plasmon resonance [29] are currently used for ligand identification with truncated recombinant LILRs. Some of the 17 difficulties encountered in these processes include the large amount of purified proteins that is required for screening, and that the receptors are highly glycosylated, which limits the use of prokaryotic systems. Another obstacle to identifying ligands is inconsistent identification of proteins, deriving from the complex nature of the interaction of receptors and their ligands.

Table 1-2. LILR Ligands [39, 89] Name/Gene ID Ligands Binding affinity References A1 HLA-B27, HLA-C - [30, 86] A2 Unknown - - HLA-C - A3 [86, 90] LILRAs Nogo66 A4 BST2 6 μM [91] A5 Unknown - - A6 Unknown - - HLA-A,B,C,E,F,G 6.5~100 μM B1 [70-72, 87, 92, 93] UL18 2~61 nM HLA-A,B,C,F,G 14~46 μM [70, 72, 87, 88, 92, B2 UL18 12 nM 93] LILRBs ANGPTLs, CD1d, Nogo66 - B3 Unknown - - B4 Unknown - - B5 Unknown - - BST2; Bone marrow stromal cell antigen 2, UL18; a human cytomegalovirus class MHC-I homolog, Nogo66; a neurite outgrowth inhibitor (also known as Reticulon-4)

Some LILRs have been shown to bind to classical and non-classical MHC-I molecules at a low affinity [39]. Thus, known ligands for LILRs can be divided into two groups: MHC-I and non-MHC-I molecules, although ligands for some LILRs are still unknown.

1.1.5.1 Major histocompatibility (MHC) LILR ligands

MHC is a set of cell surface molecules which has a major function in immune activation and autoimmunity in binding peptide antigens derived from pathogens, and by presenting them on the cell surface to be recognised by appropriate T cells [94].

Human MHC is also named as human leukocyte antigens (HLA) and grouped into classical HLA (A, B and C) and non-classical HLA (E, F and G) [95]. MHC-I molecules are expressed on all nucleated cells and platelets, and classified into three classes: I, II and III. Among them, MHC-I and II are associated with antigen presentation [96].

UL18, a homologue of MHC class I antigens, is encoded by the UL18 gene of human cytomegalovirus (HCMV), and the first identified ligand for LILRB1 using immunoprecipitation in 1997 [71]. Later, LILRB2 was also shown to bind to UL18 with a lower binding affinity when compared to LILRB1, although LILRB2 has 82% amino acid identity to LILRB1 [97]. The interaction of LILRB1 and LILRB2 with HLA molecules has been extensively studied. LILRB1 was shown to engage HLA-A, B, C, E,

F and G with a weak binding affinity at Kd ≈6.5-100 μM [29, 31, 71, 72, 92], and

LILRB2 was shown to bind HLA- A, B, C, F and G at a low affinity Kd ≈14-46 μM [39,

92, 98, 99]. This affinity is considerably lower than with UL18 (nM range), and other receptors with their ligands such as FcγRI with IgG1 (nM range) [100].

Following the first identification of LILR ligands, other LILRs e.g. LILRA1,

LILRA3, LILRB1 and LILRB2 have been shown to bind to MHC molecules (Table 1-2)

[29-31]. It is important to note that the extracellular domains of these receptors share conserved residues with LILRB1 to at least 79% identity (Figure 1-3). HLA-B27 is clinically significant, due to its strong association with inflammatory diseases such as the spondyloarthropathies [30], and has been identified as a ligand for LILRA1.

However, the functional significance of the interaction between LILRA1 and HLA-B27 remains unclear.

MHC-I contains three α domains and one β2 microglobulin (β2m) domain (Figure

1-5) [101]. Together, α1 and α2 provide a peptide binding groove, α3 provides a cell binding site, and β2m is required for the stability of the peptide binding groove [102].

For instance, KIR and TCR bind to α1 and α2 domains of MHC-I, which contrasts with the fact that the extracellular 1 and 2 domains of LILRB1 interact with α3 and β2m of

MHC-I respectively [67], and there is no binding activity on 3 and 4 domains of

LILRB1 with MHC-I (Figure 1-5). Although domain 3 and 4 of LILRB1 do not participate in its MHC-I binding activity, these domains may contribute to co-ligation that may enhance the binding affinity and avidity of the receptor-ligand. It is worth noting that the binding site and affinity may be affected by receptor polymorphisms.

Likewise, the MHC class molecules, α1 and α2 domains of MHC-I and KIR are highly polymorphic, while in contrast α3 and LILRs are conserved [45] (Section 1.1.2).

Recently, the crystal structure of LILRB4 showed that the extracellular domains of LILRB4 are unsuitable for the binding of MHC-I, and the D2 domain of LILRB4 was closely related to the D4 domain of other LILRs, which have no interaction with

MHC-I (Section 1.1.5.3) [103].

Figure 1-5. Structure of LILRB1-HLA-A2 complex and LILRB4 KIR2DL1 and TCR bind to α1 and α2 of MHC-I, while the extracellular domain 1 and 2 of LILRB1 bind to α3 and β2m of MHC-I. LILRB4 has two extracellular Ig domains but has no interaction with MHC-I and no ligand for LILRB4 identified. Modified [67, 103].

1.1.5.2 Non-MHC LILR ligands

LILRA4 was shown to bind bone marrow stromal cell antigen 2 (BST2), and inhibited TLR-7, 9 and HIV-mediated interferon on plasmacytoid DCs [91, 104]. BST2 known as tetherin, inhibits the release of enveloped viruses such as HIV-1 [105], and is up-regulated by high levels of IFN-I on activated pDCs [104, 105], which suggest that

LILRA4 may act as a homeostatic regulatory molecule through the engagement with

BST2. Angiopoietin-like proteins (ANGPTLs) have a regulatory role in angiogenesis and metabolism [106], and were reported as a ligand for LILRB2 using co-expression of these proteins on 293T cells [88]. LILRB2 and LILRA3 also has been shown to bind

Nogo66, a neurite outgrowth inhibitory protein found in the central nervous system

(CNS) [90, 107], suggesting that the blockade of LILRB2 could be a potential therapeutic approach to inducing axonal regeneration in damaged CNS tissues.

1.1.5.3 Prediction of LILRB4 ligand binding sites

LILRB4 has two C2-type Ig-like extracellular domains and no potential N- glycosylation sites, in contrast to other inhibitory LILRs (Figure 1-3) [84]. In addition, while all LILRs have highly homologous extracellular domains, a lower degree of homology is found for LILRB4 at 63% (identity to LILRB1, the LILRs most studied for their ligands) compared to other LILRs [108]. This suggests that LILRB4 may have unique ligand/s and distinct functions. New analysis using the LALIGN from ExPASy

(www.ch.embnet.org) indicated that LILRB4 has 53.4% identity and 72.1% similarity to LILRB1 (Figure 1-6).

10 20 30 40 50 60 LILRB4 MIPTFTALLCLGLSLGPRTHMQAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRL : : .:.:.:::::::::::.::: ::::::::::::::. :. ::. ::: :..:::: LILRB1 MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRL 10 20 30 40 50 60

70 80 90 100 110 120 LILRB4 DKEESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPV-GWSQPSDPLELVMTG .:.. : : . : : .:..: :::.: ..:::::::: : . : :. :::::::.:: LILRB1 YREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTG 70 80 90 100 110 120

130 140 150 160 170 180 LILRB4 AYSKPTLSALPSPLVTSGKSVTLLCQSRSPMDTFLLIKE-RAAHPLLHLRSEHGAQQHQA :: :::::: :::.:.:: .: : :.:. .: : : :: . :: . :. . .: LILRB1 AYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRA 130 140 150 160 170 180

190 200 210 LILRB4 EFPMSPVTSVHGGTYRCFSSHGFSHYLLSHPSDPLELIV : ..::. . :::.. . : : : ::: :::.: LILRB1 IFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLV 190 200 210

Figure 1-6. Alignment of the amino acid sequences of extracellular domains of LILRB4 and LILRB1 LILRB4 has 53.4% identity (double dot points) and 72.1% similarity (single dot point) in 219 amino acids overlap with LILRB1.

Although a ligand for LILRB4 has yet to be identified, recombinant LILRB4 was shown to bind activated CD4+ T cells [65, 109]. Recently, the crystal structure of

LILRB4 showed that the D2 domain of LILRB4 was closely related to the D4 domain of other LILRs (Figure 1-7). Also LILRB4 was conformationally and electrostatically unsuitable for the binding of MHC-I, suggesting its ligand binding site is at the D1 and

D2 hinge region [103].

A B

Figure 1-7. Phylogenetic trees of LILR Ig domains (A) Comparison of D1 and D2 of LILRs shows that LILRB4 is not close to the group of LILRs with MHC-I binding capacity (LILRB1, A3, A1, A2 and B2). (B) In contrast, D2 of LILRB4 is close to D2 of LILRA5 and D4 of other LILRs. The distances are measured in centimorgans. Adapted [103].

1.1.6 Rodent orthologues of LILRB1, B3 and B4

Paired Ig-like receptor B (PIRB), known as the murine orthologue of LILRB1 and

B3, was shown to bind a ligand expressed on Staphylococcus aureus, and inhibit TLR- mediated inflammatory responses on bone marrow-derived macrophages [110]. Also, 23 the mouse orthologue of LILRB4, gp49B1, was reported to bind both mouse and human integrin αvβ3, and inhibit IgE-mediated mast cell activation; the binding affinity and kinetics, however, were not determined [111, 112]. Integrin αvβ3 is a receptor for vitronectin, and expressed on a variety cell types including osteoclasts, activated endothelial cells and differentiated macrophages [113, 114], and the blockade of this receptor has been shown to inhibit endothelial cell growth and induce cell apoptosis

[115]. However, αvβ3 also binds fibroblast growth factor 1 (FGF1) [116], endothelial adhesion molecule 1 (CD31/PECAM-1) [117] and CD47 [118]. CD47 is a member of an IgV-like superfamily and also binds to SIRP-α [119]. Thus, the interaction of mouse

LILRB4 and αvβ3 may need in-depth investigation due to the multiple binding capabilities of αvβ3 which could lead to difficulties in interpretation. Although the experimental data are not complete, αvβ3 and other associated proteins, including CD47 and SIRP-α, should be considered and investigated as potential ligands of human

LILRB4.

Although some LILRs and their orthologues have been shown to bind with MHC and non-MHC class I molecules, their binding affinity is low, suggesting the presence of other ligands and co-ligands with high affinity. Currently, there are technical issues with the methods used to identify LILR ligands, namely, using truncated recombinant proteins that are produced in non-mammalian systems, which can affect post- translational modification and receptor/ligand interactions. Therefore, new methods, including the expression of full-length LILRs in eukaryotic systems, and identification of co-ligands using co-immunoprecipitation will be required to identify LILR ligands.

1.1.7 LILR expression, gene regulation and functions

LILRs are widely expressed in immune cells, including myeloid cells (such as monocytes, macrophages, dendritic cells and granulocytes), B lymphocytes, NK cells and T lymphocytes, which are closely associated with innate and acquired immune systems [35, 39]. In contrast, other receptors, including KIRs, are encoded by LRC and have relatively narrow expression patterns on leukocytes. Thus, this extensive expression of LILRs on immune cells could indicate their functional diversity or alternate expression. The expression patterns of activating and inhibitory LILRs are summarised in Table 1-3.

Table 1-3. Expression of human LILRs Name/Gene ID Expression References A1 monocytes, MΦs, DCs, B cells [86] A2 monocytes, MΦs, DCs, eosinophils, basophils, subsets [120] of T cells, subsets of NK cells A3 monocytes, macrophages, dendritic cells, B cells, LILRAs [121] subsets of T cells, NK cells A4 plasmacytoid DCs [122] A5 monocytes, synovial tissue macrophages, neutrophils [64] A6 monocytes, monocyte derived osteoclasts [123] B1 monocytes, monocyte derived osteoclasts, macrophages, dendritic cells, eosinophils, B cells, T [42, 71, 72] cells, subsets of NK, and placental stromal cells B2 monocytes, monocyte derived osteoclasts macrophages, dendritic cells, neutrophils, and [70, 87]

eosinophils LILRBs B3 monocytes, monocyte derived osteoclasts, dendritic [124, 125] cells, neutrophils, eosinophils, basophils B4 monocytes, monocyte derived osteoclasts, MΦs, [41, 126] DCs, endothelial cells B5 monocytes, NK cells, mast cell granules [42] Macrophages; MΦs, Dendritic cells; DCs, Natural killer cells; NK cells

A variety of factors can influence expression of LILRs and their mRNA. For instance, interleukin-10 and interferon-α up-regulate LILRB2, B3 and B4 on APCs

[127-129]. In addition, the expression of LILRB4 on APCs was enhanced by treatment with corticosteroids, aspirin, niflumic acid, and vitamin D3 [130-132]. In contrast, the immunosuppressive agent, rapamycin, induced down-regulation of LILRB1, B2 and B4 on DCs [133]. Also, DC activation by inflammatory stimuli such as tumour necrosis factor-α (TNF-α) can induce down-regulation of LILRB2 and B4 [36]. The expression of LILRA3 is down-regulated by TNF-α, but is up-regulated by IL-10 and IFN-γ [121].

The function of LILRs in vivo is not yet fully elucidated, owing to their ligands remaining unidentified. However, antibody cross-linking in vitro is a useful and convenient method for identifying functions of cell surface receptors when their natural receptor ligands are unknown. Specific antibodies can facilitate cross-linking to cells and co-ligation with other receptors. In addition, in vitro cross-linking has been shown to be highly comparable to functional outcomes in vivo [134, 135]. For instance, similar cell signalling patterns were shown to exist between T cell receptors cross-linked with anti-CD3/28 antibodies, and the receptors with their natural ligand, such as MHC-I and relevant antigen [134]. In addition, the cross-linking of LILRA4 on DCs showed effects similar to those shown when activated with its ligand, BST2 [135].

It is important to note that most functional studies for LILRs have been performed using antibody cross-linking, and show that LILRs can modulate the immune responses via their transmembrane associated with adaptor protein containing ITAMs (LILRAs), or intracellular motifs containing ITIMs (LILRBs). Nevertheless, the engagement between LILRs and their natural ligands is critical to understanding their biological functions. The function of LILRs are summarised in Table 1-4.

Table 1-4. Functions of LILRs Name/Gene ID Effect of LILR engagement in vitro

A1 Unknown

Induction of monocyte calcium influx, eosinophil EDN, LTC4, IL-12

release, basophilic histamine, LTC4&IL-4 secretion, RBL cell A2 serotonin release [38, 124, 125]

Inhibition of dendritic cell maturation and anti-microbial activity [136]

LILRAs A3 Unknown Induction of intracellular calcium influx and IFN-α production [122] A4 Inhibition of IFN-α and TNF-α productions on co-stimulation of LILRA4 with TLR7 or TLR9 in plasmacytoid DCs [122]

A5 Induction of TNF-α, IL-1β, IL-6, IL-10 [62]

A6 Unknown

Inhibition of T and NK cell activation B1 Blocking of BCR and FCR signals in B cells and monocytes [37, 70]

Inhibition of PTK-mediated phosphorylation and calcium mobilisation B2 on cross-linking of LILRB2 with FcγRI [70]

LILRBs B3 Inhibition of histamine release and IL-4 production upon cross-linking of LILRB3 with FcεRI in basophils [124] Inhibition of cellular calcium influx and cytokine production [15, B4 41]

B5 Unknown

LILRA1 has been shown to bind the HLA-C free heavy chain but with a lower affinity when compared to LILRB1 [86], and also to bind HLA-B27, which is associated with inflammatory diseases such as the spondyloarthropathies [30]. However, the functional significance of LILRA1 binding to HLA-B27 as well as the clinical significance of this receptor have remained unclear. LILRA2 has unique functional single nucleotide polymorphisms (SNPs) that generate an alternative splicing isoform

27 and is associated with systemic lupus erythematosus (SLE), and microscopic polyangiitis (MPA) [137]. Cross-linking of LILRA2 was shown to inhibit dendritic cell maturation and anti-microbial activity [136], but to induce calcium influx on monocytes

[38]. In addition, this cross-linking induced eosinophil-derived neurotoxin (EDN), leukotriene C4 (LTC4) and IL-12 release from eosinophils [125], histamine, LTC4 and

IL-4 secretion from basophils [124], and serotonin release from RBL cells [38].

LILRA3 contains no transmembrane and intracellular domains, and has high structure homology with LILRA1 and LILRB1, suggesting a role as a soluble agonist or antagonist to these receptors [42, 121]. Although LILRA3 has been shown to bind

HLA-B27, this functional significance is not clear [30]. LILRA4 recognises bone marrow stromal cell antigen 2 (BST2) and suppresses plasmacytoid dendritic cell activation [91]. Cross-linking of LILRA4 on the cell surface of plasmacytoid dendritic cells induces intracellular calcium influx and IFN-α production [122]. However, costimulation of LILRA4 with TLR7 or TLR9 in plasmacytoid DCs significantly inhibited

IFN-α and TNF-α production [122]. LILRA5 is expressed on monocytes and neutrophils as membrane-bound and soluble forms [64]. The function of soluble

LILRA5 is unknown, but membrane LILRA5 has been shown to be associated with the adaptor protein, FcRγ, which induces cell activation. Cross-linking of LILRA5 on monocytes has induced pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and immune-regulatory cytokine, IL-10 [62]. LILRA6 is expressed in monocytes and monocyte-derived osteoclasts at the mRNA level, but the function and ligand for this receptor remains unknown [123]. LILRA6 and LILRB3 are paired receptors which transduce opposing signals into cells, as activating and inhibitory receptors respectively

[123]. LILRA6 shows high levels of alleic polymorphism [138] that may affect

28 regulatory functions on the cell surface by engaging with other receptors, including

LILRB3.

LILRB1 recognises UL18, encoded by human cytomegalovirus (HCMV) [139].

LILRB1 also binds to a broad range of human MHC class I; HLA class I, including

HLA-A, B, C, E and G [39]; inhibits T and NK cell activation; and could block BCR and FCR signals in B cells and monocytes [37, 70]. LILRB2 binds to various HLA class

I molecules as well as UL18, similarly to LILRB1, but the binding affinity of LILRB2 with UL18 is much lower (<1500-fold) than LILRB1 with UL18 [97]. Cross-linking of

LILRB2 with FcγRI inhibited PTK-mediated phosphorylation and calcium mobilisation

[70]. LILRB3 is highly polymorphic [123] and cross-linking of LILRB3 with FcεRI inhibited histamine release and IL-4 production in basophils [124]. LILRB4 has been shown to bind activated CD4+ T cells, but no ligand for LILRB4 has been identified

[109, 140]. Co-ligation of LILRB4 on macrophages induced anti-inflammatory cytokines, such as IL-10 [19]. The up-regulation of LILRB4 and LILRB2 is associated with tolerogenic antigen presenting cells [130]. Membrane and soluble LILRB4, produced by alternative splicing, induces CD8+ T suppressor cells, reducing transplant rejection by promoting tolerance [129, 130]. Cross-linking of LILRB4 with FcγRI,

FcγRIII, CD11b, and HLA-DR inhibited cellular calcium influx, and cytokine production in monocytes and macrophages [15, 41]. LILRB5 was shown to be expressed in cytoplasmic granule membranes in mast cells, but to release it onto the cell surface upon cross-linking of FcεRI [141]. A genome wide association study conducted in a cohort of Canadians has been shown that a SNP (rs2361797) located upstream of the LILRB5 gene is significantly associated with serum creatine kinase (CK) levels in users of statins (drugs which treat cardiovascular diseases), suggesting a regulatory

29 function of LILRB5 in serum CK clearance [142]. LILRB5 has been reported to bind to

HLA-B27 and HLA-B27 heavy chains as ligands [143].

1.1.1 Clinical association between disease and LILR expression

The expression level of LILRs is also closely associated with autoimmune and other inflammatory diseases (Table 1-5). For instance, LILRA2, A3, A5, B2 and B3 have been shown to be over-expressed in patients with active rheumatoid arthritis (RA).

LILRA2 is also over-expressed in lepromatous leprosy lesions [136], and in synovial tissue of patients with RA [125]. LILRA3 is found in the serum of healthy individuals but LILRA3 deficiency is a risk factor for multiple sclerosis (MS) in some populations, such as Germans and Spanish, but not in Polish patients [144, 145]. LILRA5 is extensively expressed by synovial tissue macrophages in patients with RA [62].

LILRB1 is up-regulated in patients with human immunodeficiency virus (HIV) infection, and suppresses HIV-1 replication [146]. In addition, the level of LILRB1 up- regulation is correlated with the reactivation of human cytomegalovirus (HCMV), suggesting a potential use in the early diagnosis of HCMV in lung transplant patients

[139]. LIRB2 is up-regulated in patients in early stage RA [147], HIV [148], and sepsis

[149]. LILRB3 is abundantly expressed in synovial tissue in patients with RA (80%)

[150]. An allele that increases the risk of Takayasu arteritis was shown to inhibit mRNA expression of LILRB3 [151]. LILRB4 is up-regulated in patients with pancreatic cancer with poor prognosis [85], on B cells in patients with chronic lymphocytic leukaemia

[152], and Salmonella infection [19]. In addition, the level of LILRB4 is increased in patients with MS during remission or type I IFN treatment [153].

Table 1-5. Expression of LILRs in diseases Name/Gene ID Expression Effect of LILR engagement in vitro Lepromatous leprosy [136] A2 Over-expression RA [136] LILRAs Deficiency MS [144, 145] A3 Over-expression RA [121] A5 Over-expression RA [62] HIV infection [146] B1 Over-expression Human cytomegalovirus (HCMV) [139] RA [147] B2 Over-expression HIV [148] Sepsis [149] LILRBs B3 Over-expression RA [150]

Pancreatic cancer with poor prognosis [85] Chronic lymphocytic leukaemia [152] Salmonella B4 Over-expression infection [19] MS during remission or type I IFN treatment [153] Rheumatoid arthritis; RA, multiple sclerosis; MS, human immunodeficiency virus; HIV

1.1.2 Expression, functions and clinical significance of LILRB4

LILRB4 is expressed on monocytes, monocyte derived osteoclasts, macrophages, and dendritic cells [41]. This broad expression pattern of LILRB4 indicates its major role in the innate immune responses. LILRB4 is highly polymorphic, with more than 15

SNPs having been identified [49]. Although the significance of LILRB4 polymorphisms has not been elucidated, it is associated with functional defects and may induce perturbed immune responses and diseases. The expression of soluble LILRB4 is unusual in healthy individuals, but patients with colorectal carcinoma express soluble

LILRB4 [65]. Only 6% of healthy donors produce soluble LILRB4, compared to more than 40% of patients with melanoma, colorectal cancer, or pancreatic cancer [65]. The

31 function of soluble LILRB4 is unknown, but it may have a function as an antagonist or agonist for the typical LILRB4.

Clinically, increased expression of LILRB4 is associated with impaired immune responses leading to cancer progression, but has been linked to a better outcome in organ transplantation. CD8+CD28-T suppressor cells from heart transplant recipients without allograft rejection were able to induce up-regulation of LILRB4 and B2, but a down-regulation of the costimulatory molecule, CD86, on donor antigen presenting cells [130]. However, this was not seen in patients with unsuccessful transplants, and a high level of CD86 was presented [130]. Alloantigen-specific suppressor T cells up- regulate LILRB4 on APCs, which may regulate the effector functions of suppressor and regulatory T cells that lead to immune tolerance [154]. Treatment with soluble LILRB4 was also shown to prevent rejection of human pancreatic islets transplanted in humanized NOD/SCID mice [155].

Up-regulation of LILRB4 may play a key role in enhancing immune tolerance.

LILRB4 is also up-regulated in Salmonella typhimurium (S. typhimurium) infection in macrophages and lipopolysaccharide in DCs, suggesting an important role in responsing to bacterial infection [19]. LILRB4 ligation induced up-regulation of anti-inflammatory cytokine, IL-10 production, but down-regulation of pro-inflammatory chemokine IL-8 on macrophages [19]. This may suggest that innate immune responses induced by bacterial infection could be inhibited by LILRB4 through up-regulation of anti- inflammatory cytokines, including IL-10. Furthermore, up-regulation of LILRB4 in patients with chronic lymphocytic leukaemia may indicate a mechanism of immune evasion by cancer cells [152].

LILRB4 inhibits monocyte and macrophage activation in response to stimuli including CD11b, HLA-DR and FcγRIII, and has a function in antigen uptake,

32 suggesting a dual function as an inhibitory receptor and antigen-capturing molecule [41].

Co-ligation of LILRB4 on monocytes in FcγRI-mediated cell activation significantly inhibited TNF-α production, calcium mobilisation and phosphorylation of multiple intracellular protein kinases, including Syk [15].

A natural ligand for LILRB4 has not been identified, but recombinant LILRB4 was shown to bind activated CD4+ T cells [65, 109]. Mutants over-expressing the extracellular domains of LILRB4 (i.e. soluble LILRB4 without a transmembrane or intracellular domain) also inhibited T cell proliferation and cytotoxicity. These data suggest that LILRB4 may bind to a ligand/s which is expressed on the T cell surface, and induce an inhibitory effect upon T cell alloreactivity [140]. In addition, soluble

LILRB4-treated T cells significantly over-expressed mRNA of the transcriptional repressor BCL6, which inhibits the differentiation of Th1, 2 and 17 cells [156].

Both membrane and soluble LILRB4 have regulatory functions and induce immune tolerance. Therefore, to enhance understanding of the function of LILRB4, its ligand/s will require identification and the intracellular domains of LILRB4 containing

ITIMs will also need to be investigated.

1.1.3 The role of LILRs in the regulation of innate immune responses

LILRs are mainly expressed on leukocytes, especially in myeloid cells such as monocytes, macrophages and dendritic cells, suggesting a role in the innate immune response [89]. The cells of the innate immune system play a key role in the recognition and clearance of pathogens, as well as in the initiation of adaptive immune responses.

Macrophages, derived from blood monocytes, exert powerful effects in the elimination of micro-organisms by recognition, engulfment, and release of oxygen species, cytokine

33 and chemokines through their surface receptors, including FcRs and TLRs [157].

LILRB4 regulates FcγRI, for example, monocyte LILRB4 reduces the production of

FcγRI-mediated pro-inflammatory cytokines such as TNF-α via de-phosphorylation of tyrosine kinases, and multiple signalling proteins which include Syk, Lck, LAT and ErK.

This is secondary to the recruitment of the tyrosine phosphatase, SHP-1, via its three

ITIMs, and results in inhibition of ITAM-associated cell activation [15, 18, 41].

Although TLR receptors contain no ITAM motifs, LILRs suppress TLR activity [89,

122]. For instance, SHP-1 recruited by phosphorylated ITIM of LILRs inhibits TNF-α production by IRAK1 de-phosphorylation in TLR4-mediated cell activation [158], and

SHP-2 and SHIP inhibit downstream signalling by de-phosphorylation of TANK- binding kinase 1 (TBK-1) and phosphoinositide 3-kinase (PI3K), respectively [159].

Thus, LILRB4 ITIMs may play a regulatory role in TLR-mediated cell activation via tyrosine de-phosphorylation of adaptor proteins, including MyD88 adapter-like (Mak) and Myd88.

A greater understanding of the signalling pathways of the LILRB4-related receptors, including FcγRI and TLR4, will assist in the eventual determination of the function of LILRB4.

1.1.3.1 Mechanisms of FcγRI-mediated signalling pathway

Fc receptors (FcRs) are key molecules for recognising and eliminating foreign antigens through the induction of multiple inflammatory mediators and antigen presentation [160]. FcRs recognize the Fc portion of antibodies [11, 160], specifically,

FcγRs (FcγRI, FcγRIIA, FcγRIIIA and FcγRV) bind IgG; FcαRI binds IgA; and FcεRI binds IgE [11, 160, 161]. Cross-linking of FcγRs by immune complexes initiates

34 activation of cellular responses as well as internalisation of receptor/ligand/s (Figure 1-8)

[160, 162]. FcγR-mediated cell activation has been well-studied, and its association with

ITAM-dependent multiple signalling cascades resulting in cytokine release and an oxidative burst is established [163]. In contrast, the pathway of FcγR-mediated receptor/ligand internalisation has been less extensively investigated.

FcγRI, also known as CD64, is a high affinity receptor that binds monomeric IgG

(IgG1, IgG3 and IgG4 but not IgG2), and is expressed on the surface of monocytes, macrophages, neutrophils, eosinophils and dendritic cells [161, 164]. While FcγRI plays a key role in protection against bacterial infection, it is also able to trigger certain autoimmune diseases, such as arthritis and thrombocytopenia [165]. FcγRI contains a ligand binding α chain containing three C2-type Ig domains, a hydrophobic transmembrane domain and a short cytoplasmic tail, and is associated with the adaptor protein, FcRγ, which contain ITAMs [166]. Upon cross-linking of FcγRI, tyrosine phosphorylation and de-phosphorylation occur in specific proteins, such as Src and Syk, resulting in FcγRI-mediated cell activation, including cytokine production and phagocytosis or receptor/ligand internalisation (Figure 1-8) [160, 163].

Figure 1-8. FcγRI-mediated signalling pathways When IgG-opsonised pathogens bind to FcγRI, Src family kinases that phosphorylate tyrosine residues of ITAM are activated. Phosphorylated ITAM becomes a docking site for tyrosine protein kinase, Syk, and initiates further downstream signalling pathways such as cytokine production, differentiation and proliferation through NFκB or MAPK translocation. Phosphorylated Syk also induces phagocytosis by actin polymerisation and results in internalisation of particles by the formation of phagosomes for foreign antigen clearance. Clathrin-mediated endocytosis can occur upon FcγRI cross-linking, and depending on the size and solubility of the immune complexes, this cross-linking triggers phagocytosis by forming phagocytic-cups, or triggers clathrin-mediated endocytosis by forming clathrin-coated pits for antigen clearance. Both of these processes lead to antigen presentation.

1.1.3.1.1 FcγRI-mediated cytokine production

Upon activation of FcγRI, Src family kinases such as Src and Lck are activated, including rapid and transient phosphorylation of ITAMs [167] (Figure 1-9). The phosphorylated ITAMs create docking sites for the SH2 domain-containing protein tyrosine kinases (PKT), such as spleen tyrosine kinase (Syk) [168]. PKTs bind doubly- phosphorylated ITAMs, leading to the activation of downstream pathways by phosphorylation of numerous signalling and adaptor proteins [169]. For instance, activated phospholipase C-γ (PLC-γ) induces calcium flux, leading to the nuclear factor of activated T cells (NFAT) translocation to the nucleus and activate diacylglycerol

(DAG) [170, 171]. Activated DAG induces mitogen-activated protein kinases (MAPK), including extracellular signal regulated kinases (ERK) and c-Jun N-terminal kinase

(JNK), in turn leading to the translocation of activator protein 1 (AP-1) to the nucleus.

In addition, DAG activates protein kinase C (PKC) leading to the translocation of nuclear factor kappa light chain enhancer of activated B cells (NFκB) to the nucleus

[172]. These transcription factors induce cytokine production, calcium mobilisation, phagocytosis, migration, proliferation and increased cell survival [173].

NFκB is known as a master transcriptional regulator in mammalian immune systems that stimulates transcription of more than 150 genes to induce phagocytosis, cytokine and chemokine production, oxidative burst, and antibody-dependent cellular cytotoxicity (ADCC) [174]. NFκB is rapidly activated in response to infection, inflammation, and stressful conditions such as ionizing radiation [175]. In resting cells, the heterodimeric transcription factor, including p50 and p65, is anchored by I-κBα or I-

κBβ (inhibitors) in the cytoplasm (Figure 1-9). Within minutes of stimulation, I-κB kinase is activated and phosphorylates serine residues of I-κBα, and the phosphoserines are degraded by E3-ubiquitin ligase such as c-Cbl [176]. The proteasomal degradation

37 of I-κBα leads to dissociation of I-κBα from NFκB (p50/p65), resulting in translocation of NFκB to the nucleus for gene transcription [177]. Phosphorylation of p65 by protein kinase A (PKA) also increases the transcriptional activity of NFκB [178-180] .

Indirectly, activation of FcγRI leads to modulation of the caspases. Apoptosis is programmed cell death that is highly regulated and controlled by multiple intracellular signalling proteins [181]. Caspases are intracellular proteases and have an important role in maintaining homeostasis via regulation of cell death and inflammation. Caspase -

3, 6, 7, 8 and 9 in mammalians are associated with apoptosis, while other members of caspases such as caspase -1, 4, 5, 12 are associated either with inflammation, or with functions that are still unclear [182]. Among them, active caspase 9 initiates apoptosis; however, phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt could suppress apoptosis by inhibiting a caspase-9 cascade [183].

Figure 1-9. ITAM bearing FcγRI signalling Upon ligand binding to FcγRI bearing ITAMs, the Src family kinase is activated and phosphorylates tyrosine residue (s) of ITAMs. The phosphorylated ITAMs recruit Syk which binds doubly phosphorylated ITAMs, leading to activation of downstream signalling pathways by phosphorylation of multiple signalling proteins. PLC-γ induces calcium flux leading to NFAT translocation to nucleus and activates DAG. Activated DAG induces MAPK activation including ERK and JNK leading to AP-1 translocation to nucleus, and PKC activation leading to NFκB translocation to nucleus. These transcription factors induce cytokine and chemokine production, phagocytosis, calcium mobilisation, migration, proliferation and cell survival. In contrast, activated PI3K suppresses apoptosis by inhibiting caspase 9. 39

1.1.3.1.2 FcγRI-mediated receptor/ligand internalization

Depending on the size and solubility of immune complexes, cross-linking of

FcγRI (and other FcγRs) triggers phagocytosis by the formation of phagocytic-cups through actin-polymerization, or it triggers clathrin-mediated endocytosis by forming clathrin-coated pits [184-187]. Although there is controversy regarding the size of the immune complex required to initiate FcγRI-mediated phagocytosis or clathrin-mediated endocytosis, importantly, these two distinct signalling pathways occur either simultaneously or alternatively, and lead to receptor/ligand internalisation, antigen degradation and presentation [162].

1.1.3.1.2.1 FcγRI-mediated phagocytosis

Phagocytosis is a process characterised by the uptake of particles such as bacteria, dead cells and small mineral particles by monomyeloid cells [188]. The function of phagocytosis is to remove cellular debris, eliminate pathogens, and to break down antigens for processing and presentation as peptides that prime the adaptive immune response [189].

The opsonisation of antigens by complement and immunoglobulins, leads to recognition by the complement receptors [190], and the Fc receptors, respectively [34], thus allowing efficient phagocytosis. The interaction of these receptors with their ligands allows uptake of the pathogen into the phagosome, that then fuses with lysosomes or endosomes which contain large amounts of hydrolases and other enzymes, thus leading to the destruction of the invading pathogen [191]. Simultaneously, the binding of the IgG-coated pathogen to FcγRI receptors will trigger tyrosine phosphorylation of multiple signalling molecules, leading to the production of

40 mediators, such as TNF-α, IL-1β, IL-6 and IL-8, that further elaborate responses to pathogens [192] (Section 0 ).

When large IgG opsonized antigens (> 0.5μm in diameter) bind to extracellular Fc binding domains of FcγRI, the Src family kinases that phosphorylate tyrosine residues of the immunoreceptor tyrosine-based activating motifs (ITAMs) are activated in the associated FcγRI common γ-chain [185, 188]. Subsequently, phosphorylated ITAMs recruit and phosphorylate Syk family kinases that recruit additional signalling proteins such as LAT and growth factor receptor-bound protein 2 (Grb2) [193]. Grb2 activates

PI3K and phospholiphase C which induces actin polymerisation and internalisation of particles by the formation of phagosomes [188]. The early phagosome (~pH 5.4) matures into an increasingly acidified form, or late phagosome (~pH 5.0), and phagolysosome (~pH 4.5) for antigen clearance [188, 189].

1.1.3.1.2.2 Clathrin-mediated endocytosis

Clathrin-mediated endocytosis is associated with the internalisation of small immune complexes (<0.2 μm in diameter), and of soluble aggregated molecules into cells that results in negative regulation by ubiquitination and degradation (Figure 1-10)

[184, 187, 194]. Clathrin-coated pits are formed by multiple accessories and adaptor proteins such as dynamin, adaptor protein-2 (AP2), epsin, and Eps15-containing ubiquitin interacting motif (UIM) that induce receptor ubiquitination by ubiquitin ligase,

Cbl [195-197]. Heat shock protein (HSP) 70 is associated with the disassembly of clathrin/AP2-coated pit [198]. In addition, a heat shock cognate protein (HSC) 70, a constitutive member of a heat shock protein family, is a key molecule with auxilin for the clathrin pit to fuse with the endosome by the disassembly of clathrin-coated pits

[199, 200]. Receptors are sorted in early endosomes and can be sent back to the plasma 41 membranes to be recycled within a few minutes, or they can be degraded by Cbl directly or delivered to lysosomes to be degraded [195]. Ubiquitinated proteins are recognised by hepatocyte-growth-factor regulated tyrosine kinase substrate (HGS or HRS) via its

UIM, and sorted from early endosomes (~pH 6.8) to late endosomes and endolysosomes

(~ pH 4.5) for degradation [201, 202]. Cytoplasmic antibodies or immune complexes which escape from endosomes, recognisable by a cytosolic intracellular Fc receptor, tripartite motif-containing protein 21 (TRIM21, also known as E3 ubiquitin-protein ligase), result in proteasomal degradation [203].

Figure 1-10. Clathrin-mediated endocytosis upon FcγRI-mediated cell activation Upon IgG binding to FcγRI, multiple adaptor proteins such as clathrin, dynamin and epsin form clathrin-coated pits. Eps15 contains a ubiquitin motif that recruits ubiquitin ligase, Cbl, inducing receptor ubiquitination. HSP70 dissembles the clathrin pits. The ubiquitinated proteins are recognised by HGS (or HRS) and transferred to endolysosomes for degradation. Cytoplasmic antibodies or immune complexes that escape from endosomes can be recognized by a cytosolic intracellular Fc receptor, TRIM21, also known as E3 ubiquitin-protein ligase, and result in proteasomal degradation.

Pathogen clearance by clathrin-mediated endocytosis requires less energy compared to phagocytosis, and the internalised receptors can be degraded or recycled within a few minutes and retain a consistent level of receptor expression [204]. In contrast, several hours are required for gene expression and mRNA transcription to keep the consistency of receptor expression levels after receptor degradation by phagocytosis.

In addition, phagocytosis is PI3K-dependent, while clathrin-mediated endocytosis is ubiquitination-dependent [188, 195].

1.1.3.2 Mechanisms of TLR4 signalling pathway

Toll-like receptors (TLRs) are type I transmembrane glycoproteins and are expressed on plasma membranes (TLR1, 2, 4, 5, 6 and 10), and endosomal membranes

(TLR3, 4, 7, 8, 9, 11, 12 and 13) of myeloid lineage such as macrophages and dendritic cells [205]. TLRs contain ectodomain-containing a leucine-rich-repeats (LRR) extracellular domain for the ligand (pathogens) recognition, a TM domain, and a short cytoplasmic region containing Toll/interleukin-1 receptor (TIR) domain which is associated with cytoplasmic adaptor proteins such as MyD88 and TRIF for cell signal transduction (Figure 1-11). During Toll-like receptor (TLR)-mediated signal transduction in response to microorganisms, several serine, threonine and tyrosine proteins kinases are activated and lead to the activation of NFkB and IRFs [206].

Toll-like receptor-4 (TLR4) is expressed on monocytes and macrophages and plays a key role in innate immune responses by recognising, for example, gram negative bacterial lipopolysaccharide (LPS), respiratory syncytial virus (RSV) fusion protein, envelope protein from mammary tumour virus (MMTV), high-mobility group protein

B1 (HMGB1), heat shock proteins (HSP), hyaluronic acid, and β-defensin 2 [205, 207].

TLR4 is expressed on both plasma and endosomal membranes and multiple tyrosine

44 kinases are involved in its intracellular signalling pathways, including Syk, Lyn, Btk and Hck [205, 208, 209].

LPS recognition is facilitated firstly by LPS binding protein (LBP) and CD14, then secondly by TLR4 and MD-2 which induce MyD88-dependent and MyD88- independent pathways to activate pro-inflammatory cytokines, and Type I interferon genes respectively (Figure 1-11) [210]. Both pathways activate transcription factors,

NFkB and AP-1 via MyD88, IRF-5, IRAKs and TRAF6 or via TRIF, TRAF3 and RIP1

[211]. On the other hand, CD11b stimulation also leads to activation of the tyrosine kinase family members such as Src and Syk, without requiring the binding of integrin ligands as in TLR stimulation [212]. CD11b is an integrin family member which regulates leukocyte adhesion and migration to mediate the inflammatory response [213].

Activated Syk leads to the tyrosine phosphorylation of My88 and TRIF that promotes degradation of these adaptor molecules by the E3 ubiquitin ligase, Cbl-b, which, as a consequence, inhibits TLR signalling pathways [214].

Figure 1-11. TLR4-mediated cell activation Upon LPS binding to TLR4, TRIF-dependent and MyD88-dependent pathways are activated and induce inflammatory cytokines by activation of IRF3, MAPKs and NFκB translocation. In contrast, CD11b is activated by P13K activation, and induces negative feedback by activation of E3 ubiquitin ligase, Cbl-b, thus degrading the adaptor molecules. ℗ (yellow), tyrosine phosphorylation; ℗ (yellow-green), serine or threonine phosphorylation

1.1.4 Coupling of activating and inhibitory signals by receptors containing

ITAMs and ITIMs

Intracellular signalling pathways of both activating and inhibitory LILRs are closely associated with phosphorylation and/or de-phosphorylation of tyrosine residues in ITAM and/or ITIM [108]. Protein phosphorylation is a post-translational modification and has a key role in signal transduction, cell growth, proliferation, differentiation, migration and gene transcription [215]. After the appropriate stimulus, proteins containing specific residues such as serine, threonine and tyrosine are phosphorylated or dephosphorylated by protein kinases or phosphatases (Figure 1-12)

[216].

Figure 1-12. Schematic diagram of phosphorylation and the structure of Src, Syk/Zap70 and SHP-1/SHP-2 Protein phosphorylation is reversible by kinase and phosphatases. Src, Syk and Zap70 are tyrosine kinases which contain SH2 and/or SH3 domains and a catalytic domain (kinase). In contrast, SHP-1 and SHP-2 are tyrosine phosphatases which contain two SH2 domains and a catalytic domain (phosphatase).

Tyrosine phosphorylation was discovered in 1979, and has emerged as an important mechanism of signal transduction and regulation in eukaryotic cells [216].

Although the level of phosphotyrosine composition (< 1%) is low, compared to serine

(< 90%), and threonine (< 10%), SH2 domain binding affinity for phosphorylated tyrosine residues is stronger than phosphorylated serine and threonine [216]. Also, the tyrosine phospho-turnover is very rapid in that phosphotyrosine residues on the epidermal growth factor receptor 1 (ErbB1) has been shown to turn over up to 1000 times in a few seconds [217]. In addition, transmembrane receptors containing ITAMs,

ITIMs or groups related to tyrosine phosphorylation may control down-stream signalling pathways by phosphorylation or dephosphorylation of tyrosine resides at the beginning of cell signalling cascades.

The reversible tyrosine phosphorylation in eukaryotes is critical in intracellular signalling pathways which are associated with multiple substrates [216, 218]. Tyrosine kinases and tyrosine phosphatases are encoded by 90 and 107 genes respectively in the human genome [219]. Among the 90 genes for PTKs, 85 encode for catalytically active kinases, but only 81 are catalytically active in 107 genes for PTPs [219].

There are two types of PTKs. These comprise non-receptor PTKs such as Src and

Syk, and receptor PTKs, e.g. the insulin receptor and epidermal growth factor receptor

(EGFR) [220, 221]. Both types have a function in cellular responses, but receptor PTKs contain ligand binding sites in their extracellular domains [221]. Many non-receptor

PTKs have been shown to play a role in cellular signalling. Particular PTKs, such as Src family kinases, are directly associated with transmembrane signalling pathways. PTKs contain protein-protein interaction domains such as Src homology 2 (SH2) and/or 3

(SH3) domains, and a catalytic domain (Figure 1-12) [222]. The SH2 domain recognises phospho-tyrosine residues in a specific sequence, and the SH3 domain

48 interacts with the proline-containing motif (PxxP), but is tyrosine-phosphorylation independent [216, 222].

PTPs are likewise sub-categorised as non-receptor PTPs such as SHP-1, and as receptor PTPs e.g. the leukocyte common antigen-related receptor (LAR) [223]. PTPs contain a catalytic domain which is closely associated with cell signalling by the dephosphorylation of tyrosine residues in multiple signalling proteins [223, 224].

Among PTPs, SHP-1 contains two SH2 domains and a central catalytic domain, and is one of the key molecules in the regulatory function of LILRB4.

Intracellular signalling pathways of both activating and inhibitory LILRs are closely related to tyrosine phosphorylation in association with ITAM and its ITIM respectively [108]. Receptor engagement to its ligand/s induces a rapid and transient phosphorylation of tyrosine residues within ITAMs or ITIMs. The phosphotyrosine in

ITAM (Yxx(L/I)(6-8)Yxx(L/I)), or ITIM ((I/V)xYxx(L/V)), creates appropriate binding sites for the SH2 domain containing tyrosine kinases or tyrosine phosphatases, and induces activating or inhibitory signals into cells respectively [4].

1.1.4.1 Immunoreceptor tyrosine-based activating motifs (ITAMs)

Activating cell surface receptors, including LILRAs, are non-covalently associated with one or more transmembrane adaptor molecules which contain a short extracellular region, a conserved aspartic residue within their TM, and a varying length cytoplasmic tail containing ITAM(s) (Figure 1-2) [4]. The ITAM contains consensus sequences which comprise Yxx(L/I)(6-8)Yxx(L/I) (where x denotes any amino acid).

These conserved sequences were first identified in the antigen receptors on T cells and

B cells, and FcεRI on mast cells by Michael in 1989 [225]. Later, ITAMs were observed upon many cell surface receptors including TCR, BCR, NK cell receptors, and several 49

Fc receptors, while LILRAs and other receptors were identified on hematopoietic cells

[4].

LILRA2 [38], LILRA4 [122] and LILRA5 [62] have been shown to be associated with the FcR common γ chain (FcRγ) that is shared between various Fc receptors, including FcαR, FcεR and FcγR [121]. FcRγ contains a short extracellular domain, a negatively charged transmembrane aspartic residue and an immunoreceptor tyrosine- based activating motif (ITAM) in the cytoplasmic regions [62]. The association of activating receptors and adaptor molecules containing ITAMs allows transmission of stimulatory signals into cells from the extracellular domains of the receptor through its ligands’ interaction [4, 226]. Different activating immune receptors can share the same adaptor protein containing ITAM(s), as listed in Table 1-6. For instance, FcRγ is expressed on myeloid cells and associated with a variety of receptors, including LILRs and FCRs.

Table 1-6. ITAM-bearing adaptor proteins on immune cells. Adapted from [22, 227] Adaptor ITAM sequences Associated Cell expression proteins Yxx(L/I)(6-8)Yxx(L/I) receptors

FcRγ YTGLSTRNQETYETL Monocytes, MΦs, DCs, B DCAR, LILRs, cells, T cells, NK cells, hOSCAR, FCRs, FcεRIβ YEELNIYSATYSEL mast cells, osteoclasts KIR2DLY, NKp46 Monocytes, MΦs, DCs, NK cells, mast cells, TREMs, MDL-1 , DAP12 YQELQGQRSDVYSDL osteoclasts, subsets of B CD94 cells and T cells CD3γ YQPLKDREDDQYSHL T cells CD3δ YQPLRDRDDAQYSHL T cells TCR, CXCR4 CD3ε YEPIRKGQRDLYSGL T cells, activated NK cells CD3ζ (1,2,3) YxxLxxxxxxxYxxL/I T cells, NK cells Igα (CD79a) YEGLNLDDCSMYEDI B cells BCR, MHC-II Igβ (CD79b) YEGLDIDQTATYEDI Macrophage s; MΦs, Dendritic cells; DCs, Natural killer; NK cells

Although activating receptors on immune cells are engaged with the same adaptor molecule containing ITAMs to transduce stimulatory signals, the cells can induce markedly different functional outcomes.

The consensus sequences of ITAM are also found in a few viral proteins including

Epstein-Barr virus (EBV), Bovine leukaemia virus (BLV) gp30, mammary tumour virus

(MMTV) env gp52, SIV Nef, Hantavirus-G1, and KSHV K1, suggesting an association of viral pathology, such as virus proliferation, disease pathogenesis and oncogene transformation, with ITAM-mediated activating signalling cascades in infected cells

[228].

1.1.4.2 Immunoreceptor tyrosine-based inhibitory motifs (ITIMs)

A recent proteomic study showed more than 100 membrane receptors contain one or more ITIMs [229]. Although some inhibitory surface receptors do not have ITIMs such as CTLA-4 and CD200R, many other inhibitory receptors contain tyrosine-based

ITIM motif(s) to recruit effector molecules [2, 3]. ITIMs with consensus sequences

(I/V/L/S)xYxx(L/V) are crucial for the inhibitory function, which is phosphotyrosine dependent [230, 231]. The inhibitory function of intracellular domains containing ITIM was firstly observed in FcγRIIB, a low affinity IgG receptor, on B cells by Sebastian et al. in 1992 [232]. Later, 13 amino acids (AENTITYSLLKHP) containing a tyrosine residue in

FcγRIIB were identified by Tatsushi et al in 1994, as a key motif to modulate B cell activation [233]. The provisional (I/V)xYxx(L/V) sequence was then proposed by Marc in 1996, based on the sequence alignment of ITIM-bearing receptors such as FcγRIIB and KIR [234].

ITIM-containing receptors are listed in Table 1-7. Several ITIM-containing receptors have activating counterparts which are not alternatively spliced proteins from genes encoding ITIMs containing receptors, but encoded by a separated gene. On the other hand, ITIM-containing receptors can be modulated by alternative splicing such as soluble LILRB4.

Table 1-7. ITIM-containing receptors. Adapted from [235] ITIM-containing Activating Cell expression Known ligands receptors counterparts Herpes virus entry Human B cell, T cell mediator - BTLA (HVEM) Epithelial cells, granulocytes, CD66a CD66, CD62E - subsets B, T cells, NK cells FcγRIIB Myeloid, B , mast cells IgG FcγRIIA (CD32B) Myeloid, B cells, NK cells, Activating Inhibitory Siglecs Sialic acid cytotrophoblasts Siglecs Inhibitory KIRs Activating T cells, NK cells HLA-A,B,C (CD158) KIRs KIR2, 3DL T cells, NK HLA-A,B,C KIR2, 3DS DCs, MΦs, B cells, T cells, NK LAIR Unknown - cells Myeloid, B cell, subsets of T cells, MHC-I for B1, LILRBs (CD85) LILRAs NK cells B2 PD-1 B, T, NK PD-1 ligand - PECAM-1 Neutrophils, MΦs, mast cells, B CD31 - (CD31) cell, T cells, NK cells Herpes simplex PILR-α Myeloid PILR-β virus-I SIRP-α Neutrophils, DCs, MΦs, mast cells CD47 SIRP-β TREMLs (TLTs) Monocytes, MΦs, DCs, neutrophils Unknown TREMs Macrophage s; MΦs, Dendritic cells; DCs, Natural killer; NK cells

Many studies have shown co-aggregation of ITIM-containing receptors with

ITAM-associating receptors through their shared ligand to induce inhibitory signals into cells [15, 37, 236]. However, little is known about the regulatory function of ITIM- containing receptors in engagement with activating receptors without ITAM motifs, such as Toll-like receptors.

Inhibitory cell surface receptors containing ITIMs have a key role in counter- regulation of excessive ITAM-mediated cell activation. The tyrosine residues of ITIMs are phosphorylated by the Src family of kinases that creates docking sites for Src homology 2 domain (SH2)-containing phosphatases (SHP) 1, 2, and SH2 domain- containing inositol phosphatase (SHIP), and inhibits or terminates the activating signal through dephosphorylation of protein kinases involved in activating cascades (Figure

1-13) [9].

Figure 1-13. LILRB4-mediated inhibition of FcγRI-mediated monocyte activation Cross-linking of the high affinity IgG receptor, FcγRI, with LILRB4 induces activating signals through tyrosine phosphorylation of ITAMs by phosphorylation of protein kinase, Syk. However, phosphorylated ITIMs of LILRB4 recruit tyrosine phosphatase, SHP-1, that terminates activating signals by dephosphorylation of protein kinases involved in activating signal cascades.

Different immune receptors containing ITIMs may recruit different phosphatases

(Figure 1-14). For instance, the ITIMs of KIR interact with SHP-1, while the ITIM of

FcγRIIB interacts with both SHP-1 and SHIP [237]. In addition, SHPs and SHIP use different mechanisms for cellular inhibition. SHIP uses its catalytic domain to hydrolyse the phosphatidylinositol-trisphosphate (PIP3) to phosphatidylinositol-diphosphate

(PIP2), thus preventing the PIP3 association with PLC-γ that suppresses calcium mobilisation following cellular activation [238]. While SHP-1 dephosphorylates the

54 tyrosine residues of signalling molecules, including the phosphorylated ITAM, Src kinases, ZAP70/Syk, PLC-γ, Vav1 and PI3K in the earliest signalling cascades, this then leads to termination of the activating cascade [239-242]. Although the effects of

SHP-2 are less well-described, it may have functions similar to SHP-1 [243, 244].

Figure 1-14. Different phosphatases are recruited by different receptors that induce different signalling pathways SHP-1 is recruited by LILRB4 and inhibits activation of PKTs, while SHIP is recruited by FcγRIIB, hydrolyses phosphatidylinositol-trisphosphate (PIP3) with resultant inhibition of calcium mobilisation.

There is evidence that the inhibitory function of the ITIMs of CD158d, a KIR family member, was phosphotyrosine-independent, in that the inhibitory function of a single ITIM with a tyrosine mutation was not completely abolished and could still

55 weakly bind SHP-2 [3]. This suggests that not only the tyrosine residue of ITIMs, but also the conserved ITIM sequences without tyrosine are important in recruiting tyrosine phosphatases to induce appropriate immune responses.

The presence of ITIMs within the intracellular domain of all LILRBs suggests that these receptors abrogate cellular activation through the recruitment of protein tyrosine phosphatases such as SHP-1, SHP-2 and SHIP [39]. The significance of the quantitative effect of many ITIMs and the molecular mechanisms in inhibitory LILRs has yet to be described. It is possible that the number of ITIMs may affect the intensity of the inhibitory functions through recruitment of different types and amounts of phosphatases to the ITIMs. In previous studies of LILRB1 using site-targeted mutagenesis of each tyrosine residue within all four ITIMs present in this receptor, it was demonstrated that only three of the four are needed for SHP-1 recruitment and cellular inhibition [245]. In addition, only SHP-1 was involved in the LILRB4-mediated regulation, but no recruitment of SHP-2 was detected by immunoprecipitation on LILRB4 cross-linking on mouse bone marrow-derived mast cells and human monocytes [18, 41].

1.1.4.3 Inhibitory ITAM (ITAMi)

ITAM-mediated cellular activation (and ITIM-mediated inhibition) in cellular responses is well established. However, this paradigm has been challenged recently by evidence that some receptors containing ITAM transduce inhibitory signals under certain circumstances; these are termed inhibitory ITAM, or ITAMi (Table 1-8). For instance, the FcαRI associated with the adaptor protein, FcRγ-containing ITAM motifs, has been shown to inhibit IgG-mediated phagocytosis in monocytes, and IgE-mediated degranulation in mast cells [246]. In addition, FcRγ-bearing FcγRIII inhibited PI3K

56 phosphorylation as well as phagocytosis [247]. Also, TLR-mediated cytokine production in macrophages was enhanced in DAP12-deficient mice, suggesting an inhibitory function of DAP12 which contains ITAM motifs [248].

Table 1-8. ITAM bearing inhibitory receptors (ITAMi). Adapted from [249] Associated Recruited Receptors adaptor Negative regulation phosphatases protein Induction of calcium mobilization and BCR Igα Unknown tyrosine phosphorylation in cytoplasmic truncated mutant mice [250] Inhibition of IgG-mediated phagocytosis, IgE FcαRI FcRγ SHP-1 mediated degranulation [246] none Suppression of total cellular tyrosine FcγRIIa (contain SHP-1, SHIP phosphorylation, down-regulation of NFκB- Yxx(I/L) dependent gene transcription [251] Inhibition of PI3K phosphorylation, Inhibition FcγRIII FcRγ SHP-1 of E. coli phagocytosis [247] MHC-II FcRγ SHP-1 Suppression of dendritic cell maturation [252] TREM2 DAP12 Unknown Inhibition of TNF-α production [253]

ITAM-mediated cell activation via Syk or Zap70 requires double phosphorylated

ITAM, which is dependent on the interaction between an ITAM containing a receptor and its ligand/s (high affinity and avidity). For instance, ligation of FcαRI by multimeric

IgA induces activating signals via strong phosphorylation of ITAMs, which enhances recruitment of Syk [22, 254]. In contrast, ligand binding at low affinity, avidity or valency, such as monomeric IgA or anti-FcαRI Fab fragments, induces inhibitory signals via weak (partial) phosphorylation of ITAMs which could simultaneously recruit Syk and Src homology 2 domain (SH2) containing phosphatases, SHP-1 (Figure

1-15) [255].

Figure 1-15. ITAM-mediated cellular activation and inhibition (ITAMi) An activated receptor with low ligand binding affinity induces partial phosphorylation of ITAM which recruits PTPs such as SHP-1 and SHIP, resulting in inhibitory signals within cells. However, double phosphorylated ITAM induces activating signals into cells by recruitment of PTKs such as Syk.

1.1.4.4 Activating ITIM (ITIMa)

Several ITIM-containing receptors have been shown to transduce activating signals under certain circumstances, and are termed activating ITIM, or ITIMa [22]. For instance, TREM-like transcript 1 (TLT-1) is expressed on the cell surface of platelets and has two ITIMs on its intracellular domain that enhance FcεRI-mediated intracellular calcium flux, although it was shown to recruit SHP-2 [256]. In addition, signal

58 regulatory protein-α (SIRP-α) expressed on myeloid and neuronal cells has two ITIMs, but was shown to enhance nitric oxide production in macrophages [257].

1.1.4.5 ITIM-like motifs and immunoreceptor tyrosine-based switch motifs

(ITSMs)

Recently, it has been shown that ITIM-like motifs (YVKM) and immunoreceptor tyrosine-based switch motifs (ITSMs, TxYxxI/V) also play a key role in the regulatory functions of cellular activation by recruitment of SH2 domain containing phosphatases

[23, 24]. For instances, cytotoxic T lymphocyte antigen-4 (CTLA-4) has an ITIM-like motif and a capacity to bind SHP-2 with an inhibitory function [258]. ITSMs have been identified on a few immune receptors such as inhibitory KIRs [259], CD150 [260], sialic acid-binding Ig-like lectins (SIGLECs) [229], and programmed cell death 1 (PD-1)

[24]. While the function of these motifs is less well understood, the sequence similarities between ITSM and those of ITAMs and ITIMs suggest moderation of

ITAM/ITIM effects.

1.2 Statement of aims

1. Important consequences of surface cross-linking of FcγRI by immune

complexes on monocytes are the transduction of activating signals and

internalisation of the receptor. LILRB4 has been shown to potently inhibit one

of these activating signals leading to the suppression of pro-inflammatory

cytokine production through tyrosine dephosphorylation of multiple proteins.

However, its role in the immune complex-mediated FcγRI internalisation

remains unknown. Moreover, little is known about the identities of the

tyrosine phosphorylated proteins that may be involved in FcγRI internalisation

that may be targeted by LILRB4. The first aim of this project was therefore to

globally map tyrosine phosphorylated proteins in response to antibody cross-

linking of FcγRI on monocytes, identify potential phosphorylated signalling

molecules involved in receptor internalisation and investigate their regulation

by LILRB4.

2. LILRB4 is believed to inhibit monocyte/macrophage activation through

recruitment of phosphatases to the phosphorylated tyrosine residues in its

three unique ITIMs but the relative contribution of each ITIM to its functions,

and whether one or more of these ITIMs have paradoxical activating functions,

remain unknown. This will be addressed in the second aim of this project

using extensive site-targeted mutagenesis and stable over-expression

monocyte cell model systems.

3. ITIM-containing LILRB4 can inhibit activating receptors that signal via

ITAM-bearing adaptor proteins such as the common γ-chain of the FcγRI and

FcεRI. However, human LILRB4 and its mouse orthologue have been shown

to modulate innate immune functions in response to bacterial infections,

suggesting it may regulate non-ITAM but tyrosine phosphorylation-dependent

TLR-mediated monocyte activation. In this aim, the effects of LILRB4

ligation on LPS-mediated cytokine production and underlying mechanisms

will be investigated.

4. Ligands for LILRB4 are unknown; hence most of the functional studies to

date are performed by artificial (non-physiological) ligation of the receptor

using antibodies. This has hindered a definitive analysis of the functions of

LILRB4 in vitro and in vivo. Previous attempts to identify ligands have failed,

primarily due to the unique structure of LILRB4 as compared to other LILRs

and due to a lack of suitable reagents and methods. This aim will generate new

tools and develop an improved ligand screening method in an attempt to

identify and characterise candidate LILRB4 ligand/s.

CHAPTER 2. GENERAL METHODS

This chapter presents details of the commonly used primary cells, cell lines, antibodies, reagents and buffers, and common methods used in this thesis. Optimisation steps and associated quality control results for the key methods are briefly outlined.

2.1 Antibodies, buffers and reagents

Primary and secondary antibodies are used for receptor cross-linking and/or co- ligation experiment, assessment of protein expression by flow cytometry, Western blot and/or immunoprecipitation, and listed in Table 2-1. Buffers and their components used throughout this thesis are listed in Table 2-2, and specific chemicals used to prepare these buffers are catalogued in Appendix I.

Table 2-1. List of primary and secondary antibodies used throughout this thesis MW Antibodies (Abs) Dilution Applications Company (kDa) 500 ng/ml or anti-FcγRI (clone: 276426) - CL, F R&D System 5 µg/ml 750 ng/ml or kindly donated by Dr. Luis anti-LILRB4 - CL, F 5 µg/ml Borges, Amgen Inc 750 ng/ml or Affinity Bioreagents anti-CD25 (clone: 143-13) - CL, F 5 µg/ml Thermo Scientific

IgG1 mAbs (clone: MOPC 21) - Control CL, F, IP Sigma-Aldrich Mouse anti-pTyr-100 mAb Multiple 1:2000 WB Cell Signalling anti-human clathrin 180 1:500 WB Therrmo Scientic anti-human TRIM21 mAb* 50 1 µg/ml WB R&D System Primary anti-HSP70 70 1:1000 WB StressGen Abs anti-β-actin 45 1 µg/ml WB Sigma-Aldrich anti-c-cbl 120 1:1000 WB Sigma-Aldrich anti-HGS (Hepathocyte growth factor- 100 1:1000 WB Therrmo Scientific regulated tyrosine kinase substrate) anti-FcγRs 15 1:1000 WB Upstate Rabbit anti-SHP-2 67 1 µg/ml WB Millipore anti-SHIP 145 1 µg/ml WB Millipore anti-MEK1/2 45 1:1000 WB Cell Signalling anti-phospho MEK1/2 (p-MEK1/2) 45 1:1000 WB Cell Signalling CL; cross-linking and co-ligation, F; flow cytometry, WB; Western blot, IP; immunoprecipitation * mouse anti-human TRIM21 mAb (R&D System) was biotin labelled using lightning-linkTM biotin conjugation kit (Innova Biosciences) according to manufacturer’s instruction.

Table 2-1 continued. List of primary and secondary antibodies used throughout this thesis MW Antibodies (Abs) Dilution Applications Company (kDa) anti-P38 45 1:1000 WB Cell Signalling anti-phospho P38 (p-P38) 38 1:1000 WB Cell Signalling anti-phospho Erk (p-Erk) 38 1:1000 WB Cell Signalling Primary Rabbit anti-Akt 42 & 44 1:1000 WB Cell Signalling Abs anti- Akt (p-Akt) 60 1:1000 WB Cell Signalling anti-p50/p105 (NFκB) 60 1:1000 WB Cell Signalling anti-p65 (NFκB) 50&105 1:1000 WB Santa Cruz Goat anti-SHP-1 65 1 µg/ml WB R&D System AffinitPure F (ab’) fragment goat anti-mouse IgG 2 - 50 µg/ml CL Jackson Immunology (Fcγ-specific) (115-006-071) AffiniPure Goat anti-mouse IgG (Fcγ-specific) (115-005-071) - 15 µg/ml CL Jackson Immunology FITC conjugated mouse anti-TLR4 - 5 µg/ml F BD Biosciences

FITC conjugated mouse IgG1 mAbs - 5 µg/ml F BD Biosciences PE conjugated CD25 (IL-2 α chain) - 5 µg/ml F BD Biosciences PE conjugated CD122 (IL-2 β chain) - 5 µg/ml F BD Biosciences Secondary PE conjugated CD132 (IL-2 γ chain) - 5 µg/ml F BD Biosciences Abs PE conjugated CD14 - 5 µg/ml F BD Biosciences PE conjugated CXCR4 - 5 µg/ml F BD Biosciences PE or FITC conjugated goat anti-mouse secondary Abs - 3 µg/ml F BD Biosciences HRP conjugated goat anti-mouse Ab HRP conjugated goat anti-rabbit Ab - 1: 5000 WB Bio-Rad HRP conjugated rabbit anti-goat Ab HRP conjugated streptavidin - 1: 5000 WB Dako CL; cross-linking and co-ligation, F; flow cytometry, WB; Western blot, IP; immunoprecipitation

Table 2-2. List of buffers and their components used throughout this thesis Buffers pH components Solvent

Cross-linking buffer (CLB)** 7.2 ~ 7.4 10 mM HEPES, 1 mM MgCl2, 0.1 mM CaCl2 and 0.1% bovine serum albumin μI PBST buffer* 7.2 ~ 7.4 0.05% Tween-20 PBS TBST buffer* 7.2 ~ 7.4 0.1% Tween-20 TBS ELISA Dilution buffer 7.2 ~ 7.4 1% BSA PBS Immuno- 0.1% Triton X-100 Dilution buffer 8.0 TSA precipitation (TSA buffer: 0.01 M Tri-Cl, 0.14 M NaCl, 0.025% NaNa3) Wash buffer I 8.0 TSA buffer alone TSA (IP) Wash buffer II 6.9 50 mM Tri-Cl MQ Silver stain Fix I - 50% (v/v) methanol, 5% (v/v) acetic acid MQ Sensitizing sol. - 0.05% sodium thiosulfate solution MQ Silver - 0.2% silver nitrate MQ Developing sol. - 2% sodium carbonate , 1.74 mM formaldehyde MQ Fix II - 0.5% (v/v) acetic acid MQ Western blot lysis buffer* 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA and 1% NP-40 MQ 2x tyrosine sample buffer 6.8 200 mM Tris-HCl, 40% (v/v) glycerol, 2% (w/v) SDS, 0.04 % (w/v) coomassie blue G MQ

Pervanadate stock sol. (x100) - 10 mM of sodium orthovanadate, 3.36% H2O2 PBS

Coupling buffer 5.5 0.01 M K2HPO4, 0.15 M NaCl MQ Phagocytosis Wash buffer 7.4 0.01 M Tris, 0.15 M NaCl, 1 mM EDTA, 0.1% BSA, 0.1% NaN3 MQ *filtered using 0.22 μm polyethersulfone (PES) membranes (Corning Inc., NY, USA), ** filtered using sing 0.2 µm Zetapore membranes (Cuno, Blacktown, NSW, Australia), Milli-Q water (MQ, filtered using an ion exchange cartridge, Millipore)

2.2 Isolation of Peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated from whole venous blood of healthy volunteers or buffy coats obtained from Australian Red Cross Blood Service (Sydney, NSW) by a standard density gradient centrifugation (Sigma-Aldrich) [125, 261]. In brief, 20 ml of venous blood collected in acid-citrate-dextrose (ACD) anti-coagulated tubes was resuspended in D-PBS in a 1:1 ratio and overlayed onto 20 ml Ficoll-PaqueTM PLUS in 50 ml falcon tubes (Thermo Fisher Scientific). PBMCs were then separated by gradient centrifugation at 800xg for 30 min without a break. The PBMCs fraction (upper band) was collected and centrifuged twice in D-PBS for 10 min at 400xg. Cell numbers and proportions of leukocyte subsets were analysed using a haemocytometer (Beckman

Coulter Inc., NSW, Australia), resuspended and seeded at 2 x 106 - 2 x 107 cells/ml in appropriate media and immediately used for subsequent experiments.

Alternatively, large amounts of PBMCs (~109 cells) were purified from 100 ml buffy coats obtained from Red Cross. Briefly, buffy coats were mixed with 4.5% dextran T-500 in D-PBS in a ratio of 2:1 in 50 ml Falcon tubes and incubated for 30 min at 37ºC, 5% CO2 leading to red blood cell sedimentation. The red blood cell- depleted buffy coat was then layered onto multiple 20 ml Ficoll containing tubes and

PBMCs purified by gradient centrifugation as above. Some cells derived from the buffy coats were immediately used for experiments and multiple aliquots of 2 x 107 cells were cryopreserved in liquid nitrogen for future use. Generally, the purified mononuclear cells contained 10-15% monocytes, 85-90% lymphocytes and < 1.5% contaminating granulocytes with > 95% viability as assessed by 0.4% trypan blue staining and microscopic visualisation/counting.

The collection of venous blood from healthy subjects or the procurement of buffy coat from the Australian Red Cross Blood Service was approved by the University of

NSW Human Research Ethics Committee.

2.3 Cell lines and tissue culture

Table 2-3 shows a summary of the various cell lines used and the specific reagents and conditions required for culturing and maintaining each cell line. The following standard in vitro cell culture protocol was used for all the cell lines. In brief, 3-5 x 106 cryopreserved cells in 1.2 ml Cryovials (Corning, Sigma-Aldrich), were taken from vapour-phase liquid nitrogen tank, thawed at 37ºC in the water bath and washed in 10 ml pre-warmed complete medium before centrifugation at 300xg for 5 min at RT . Cell pellets were then resuspended at 2 x 104 – 4 x 105 cell/ml in appropriate complete media with supplements (Table 2-3) and seeded in 25-75 cm2 cell culture flasks (Corning,

Sigma-Aldrich) and incubated in an endotoxin minimised humidified tissue culture incubator at 37ºC and 5% CO2 (Panasonic) for two or three days. For adherent cells, media were aspirated and cells gently rinsed with 10-30 ml of D-PBS followed by 1-

2min incubation at 37ºC with 5% CO2 in 1-2 ml of 0.25% Trypsin-EDTA (ethylene- diamine-tetra-acetate) followed by trypsin neutralisation with base media containing 10% of FBS. Cells were then transferred to 50 ml Falcon tubes and centrifuged at 300xg for

10 min. After two washes with D-PBS, cells were counted and resuspended in appropriate media at desired concentrations. Suspensions of cells were directly collected to 50 ml falcon tubes, pelleted by centrifugation, washed twice with D-PBS, counted and resuspended in appropriate media at desired concentrations. Cells were passaged every two or three days and used for experiments between passages 5-20 (Table 2-3).

Aliquots of early passage cells were also cryopreserved for future use. In brief, cells (3- 67

5 x 106) were suspended in 1 ml complete medium containing 10% Dimethyl sulfoxide

(DMSO) in a 1.2 ml Cryovials and placed at -80ºC overnight, using an isopropyl alcohol-insulated freezing container (Thermo Fisher Scientific, MA, USA). The following day, tubes were transferred to a vapour-phase liquid nitrogen tank for long- term storage.

2.4 Quality controlling of cell lines and antibodies for endotoxin contamination

Monocytes and THP-1 are extremely sensitive to LPS and mycoplasma contamination that could lead to false results [262, 263]. To minimise endotoxin contamination, cell media and buffers were filtered using 0.2 µm Zetapore membranes

(Cuno, Blacktown, NSW, Australia), and cells were incubated in an endotoxin minimised cell culture incubator. The levels of endotoxin in cross-linking antibodies and cultured cells were regularly tested using Limulus Amebocyte Lysate (LAL) Gel

Clot method according to manufacturer’s kinetic protocol (Associates of Cape Cod Inc.

Falmouth, MA, USA). Mycoplasma contamination in cultured cells was tested every six months using MycoAlertTM mycoplasma detection kit according to manufacturer’s instruction [264]. In brief, cells were cultured for three days under standard cell culture condition, and then cell culture supernatant was collected by centrifugation at 200xg for

5 min at RT. To wells of a white 96 well plate (Porvair Sciences, Norfolk, UK) 100 μl of samples was mixed with 100 μl of MycoAlertTM reagent and incubated for 5 min at

RT. The EnSpire Multimode Plate Reader (PerkinElmer) was used to measure the luminescence of the 1st reaction (Reading A). For the 2nd reaction (Reading B), 100 μl of MycoAlertTM substrate was added to the 1st reaction and incubated for 10 min at RT, followed by luminescence measurement. The ratio of Reading B to Reading A was used

68 to determine whether cells was contaminated by mycoplasma (<0.9, negative; 0.9-1.2, borderline; >1.2, contamination).

Table 2-3. Cell line culture conditions

THP-1 HEK 293T Jurkat Organism Homo sapiens, human Homo sapiens, human Homo sapiens, human Origin ATCC® TIB-202 Invitrogen R700-07 ATCC® TIB-152

Epithelial cell Monocyte T lymphocyte Cell type (Human embryonic kidney, HEK, cells (Acute monocytic leukemia) (Acute T cell leukemia) transformed with SV40 T antigen)

Culture properties Suspension Adherent Suspension

DMEM (Dulbecco’s Modified Eagle’s Base culture medium RPMI 1640 (Life Techologies) RPMI 1640 (Life Techologies) Medium) (Life Techologies)

Base supplements 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg /ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES (all from Life Technologies) and 20 mM sodium bicarbonate (Sigma-Aldrich)

0.1% -mercaptoethanol 10 mM non-essential amino acid (Life Additional supplements - (Life Techologies) Techologies)

Starting cell number 2-4 x 105 cells/ml 2-5 x 104 cells/cm2 1 x 105 cells/ml for subculture Optimal passage number Passage 5-20 Passage 5-20 Passage 7-10 for experimental use

2.5 Quality controlling of cell surface receptor expression of THP-1 cell lines

Flow cytometry was used to identify expression of relevant cell surface receptors including CD25 (IL-2Rα chain), CD122 (IL-2Rβ chain), CD132 (IL-2Rγ chain), CD14,

CXCR4, TLR4, FcγRI and LILRB4 on THP-1 cells. In brief, 3 x 105 cells were washed twice in PBS and resuspended in 50 µl PBS. PE-conjugated primary antibody or isotype matched negative control (5 µg/ml each) was incubated with cells for 30 min at RT.

After incubation with antibodies, cells were washed twice in cold PBS containing 0.5%

BSA, then fixed in 1% paraformaldehyde in 350 μl PBS. The endogenous CD25 was determined by using activating cells with 100 ng/ml LPS at 37ºC in 5% CO2 for 6 hrs.

Cells were acquired on FACScanTM flow cytometry (BD Biosciences) and data analysed using FlowJo software (Tree star Inc.). The antibodies for flow cytometry used throughout this thesis are listed in Table 2-1.

2.6 In vitro differentiation of THP-1 cell lines

Phorbol 12-myristate 13-acetate (PMA) was used to differentiate monocytoid

THP-1 cells to macrophages as described [265-267]. In brief, desired concentrations of

THP-1 or CD25-LILRB4 ITIMs chimeric protein expressing THP-1 mutants were cultured in complete RPMI medium containing 100 ng/ml PMA at 37ºC in 5% CO2 for three days. Differentiation of cells was confirmed by assessing morphological changes and cell counts. Differentiated cells were larger, partially adherent, vesicular with ruffled edges and non-replicating, as contrasted to the round, non-adherent and rapidly dividing undifferentiated cells. These differentiated cells were thoroughly washed and immediately used for phagocytosis and bactericidal activity assays (Sections 2.15 and

2.16).

2.7 Establishing activation protocols for PBMCs and THP-1 cell lines

The primary aim of this project was to investigate the regulation of innate immune cell activation by LILRB4. Monocyte and macrophages are professional phagocytic cells and have a critical role in innate immunity through their cell surface receptors

[268]. Among numerous receptors on monocytes/macrophages, FcγRI and TLR4 are key receptors in immune response (Section 1.1.3) [157]. FcγRI cross-linking on monocytes has been shown to induce cytokine production, phagocytosis and endocytosis [15, 160, 163]. It has been well-defined that in the signalling pathways in

TLR4-mediated monocyte activation, TLR4 recognises LPS, a major surface membrane components of gram negative bacteria, also known as lipoglycans and endotoxin [269], and induces pro-inflammatory cytokines such as TNF-α, [210].

THP-1 cells are a human monocytic cell line derived from a one-year infant with acute monocytic leukaemia, and have been used extensively to investigate signalling cascades involved in FcRs and TLRs-mediated monocyte activation [270]. Therefore,

THP-1 cells have been used throughout this thesis to identify the regulatory function of

LILRB4 in FcγRI and TLR4-mediated cell activation.

2.7.1 Stimulation of THP-1 cells and primary monocytes by LPS

The optimal concentrations and treatment duration of LPS-mediated activation of

THP-1 cells and primary monocytes are widely and routinely used in vitro [120, 271,

272]. In brief, 3.5 x 105 cells in 200 µl of CLB (Table 2-2) containing 100 nM LPS in

PBS (E. coli serotype 005:B5) or containing PBS alone as a negative control were plated onto a flat-bottomed 96 well plate and incubated at 37°C, 5% CO2 for 3 hrs (for 72

RNA extraction) or 15 hrs (for ELISA). After cell activation, cells were lysed using

TRIzol reagent for RNA extraction (Section 2.11.1) or 100 µl of cell-free supernatants were collected for ELISA (Section 2.8).

2.7.2 Cross-linking of FcγRI and co-ligation of LILRB4 on THP-1 cells and

primary monocytes

Cross-linking is a convenient method to stimulate cell surface receptors to transduce signals, and co-ligation is a useful tool to identify functions of specific receptors, particularly when the nature of their ligand/s is unknown. Antibody cross- linking of receptors in cell suspension is generally used to study the phosphorylated state of signalling proteins (Figure 2-1A), and plate-immobilised antibodies are used to assess secretory functions such as cytokine production (Figure 2-1B). Thus, this study used anti-FcγRI and/or anti-LILRB4 in cell suspension to cross-link the receptor and identify intracellular phosphorylated proteins, using Western blot and mass spectrometry; and plate-immobilised anti-FcγRI and/or anti-LILRB4 to determine cytokine production, using ELISA. The concentration and the time to stimulate normal

THP-1 cells using anti-FcγRI are well-optimised, as described [15].

For antibody cross-linking of receptors in cell suspension, in brief, cells (2 x 107 cells for IP and 2 x 106 cells for total lysate) were washed twice in D-PBS and resuspended in CLB, followed by incubation with 5 µg/ml of unconjugated anti-FcγRI or IgG1 for 15 min at RT with gentle mixing several times. Cells were then washed once in 1 ml of CLB and resuspended in 100 µl of CLB, followed by cross-linking with

15 µg/ml of AffiniPure goat anti-mouse IgG (Fcγ-specific) for 90 sec. Cell activation was stopped by adding cold PBS, and cells were harvested by centrifugation at 4 °C for

73 further experiment including Western blot and immunoprecipitation (Sections 2.10.3 and 2.10.4).

For cross-linking of plate-immobilised antibodies, in brief, a flat-bottomed 96

® well plate (Costar 3596, Corning, NY) was coated with 50 µl of 50 µg/ml F(ab’)2 fragment goat anti-mouse IgG (Fcγ-specific) in PBS at 4°C overnight in duplicates.

Wells were then washed once with 0.9% NaCl and coated with 500 ng/ml of anti-FcγRI in PBS containing 2.5% BSA at 37°C, 5% CO2 for 2 hrs. Unbound antibodies were aspirated and washed once with 0.9% NaCl. A total of 3.5 x 105 THP-1 cells in 100µl

CLB were then added to each well and incubated for 30min at 37°C, 5% CO2, followed by the addition 100 µl of CLB containing 20% FBS. After incubation for 15 hrs at 37°C,

5% CO2, 100 µl of cell-free supernatants were collected for ELISA (Section 2.8).

It is important to note that the secondary antibody is Fcγ-specific, and was used to minimize cross-reactivity to other immunoglobulins such as IgM and IgA, and F(ab’)2 fragment was used to avoid binding to other Fc receptors

(https://www.jacksonimmuno.com). Thus, AffiniPure goat anti-mouse IgG (Fcγ- specific) was used for cross-linking of antibodies in cell suspension to enhance cell aggregation (Figure 2-1A), while F(ab’)2 fragment goat anti-mouse IgG (Fcγ-specific) was used for immobilised antibody cross-linking, due to the simultaneous incubation of both primary and secondary antibodies (Figure 2-1B).

A B

Figure 2-1. Cross-linking of FcγRI (A) THP-1 cells (2 x 106 cells for total lysate and 2 x 107 cells for IP) in suspension were cross- linked for 90sec with anti-FcγRI, used to induce cell activation and detection of phosphorylated signalling proteins. (B) Cross-linking of immobilised anti-FcγRI on a 96 well plate was used to activate 3.5 x 105 cells leading to TNF-α production.

Co-ligation of LILRB4 on THP-1 cells and CD25-LILRB4 ITIMs transfected

THP-1 cells (Section 2.11.2) was used, by adding specific antibodies including anti-

LILRB4 and anti-CD25, to simultaneously engage with anti-FcγRI and/or directly secondary antibodies, including AffiniPure goat anti-mouse IgG (Fcγ-specific) or F

(ab’)2 fragment goat anti-mouse IgG (Fcγ-specific) (Figure 2-2). Unconjugated primary antibodies and secondary antibodies used for the cross-linking and co-ligation experiment are listed in Table 2-1.

A B

Figure 2-2. Cross-linking of FcγRI and co-ligation with LILRB4 (A) THP-1 cells (2 x 106 cells for total lysate and 2 x 107 cells for IP) in suspension were cross- linked and co-ligated for 90 sec with anti-FcγRI and anti-LILRB4, used to determine the regulatory function of LILRB4 in protein phosphorylation. (B) Cross-linking and co-ligation of immobilised anti-FcγRI and anti-LILRB4 on a 96 well plate and incubation with 3.5 x 105 cells was used to determine the regulatory function of LILRB4 in TNF-α production.

2.7.3 Optimization of cross-linking and co-ligation antibody concentrations for

cells transfected CD25-LILRB4 ITIMs

Since the concentration of antibodies, and the time to activate normal THP-1 cells using cross-linking/co-ligation of anti-FcγRI in suspension and plate immobilisation have been well-established [15], we followed the protocol as described in Section 2.7.2.

However, the concentrations of anti-FcγRI and anti-CD25 for activating THP-1 cells transfected with CD25-LILRB4 ITIMs (Section 2.11.2) were optimised as described below.

To establish the optimal antibody concentration to be used for transfectants, a dose response analysis was performed. The vector-only transfected cells, a representative negative control (Mock), was used to determine the optimal concentration of anti-FcγRI. Mock and the cells transfected with a wild type of LILRB4

ITIMs (YYY), as a representative positive control, were used to determine the optimal concentration of anti-CD25. In brief, serial dilutions of anti-FcγRI or IgG1 control alone were immobilised on a flat-bottomed 96 well plate (Costar®3596, Corning, NY) and used for the cross-linking of FcγRI, followed by TNF-α ELISA, as described in Sections

2.7.2 and 2.8. The results showed that 75 ng/ml of anti-FcγRI was sufficient for detectable mock cell activation, but 500 ng/ml of anti-FcγRI was chosen as the optimal concentration for further experiment based on our results (Figure 2-3A), and the optimal concentration for normal THP-1 as described [15]. The same concentration of 500 ng/ml of anti-FcγRI and serial dilutions of anti-CD25 were used for anti-CD25 optimisation, and the concentration at 750 ng/ml was chosen for further experiment

(Figure 2-3B).

Figure 2-3. Optimization of plate-immobilised antibody concentration for FcγRI cross- linking and CD25 co-ligation (A) 3.5 x 105 cells in 200 µl of CLB were dispensed in duplicates onto 96-well flat bottom plates coated with increasing concentrations of anti-FcγRI mAb which was immobilised onto 15 µg/ml of goat anti-mouse secondary antibody. This was incubated for 15 hrs and culture supernatants were used for TNF-α production (n = 2). (B) 3.5 x 105 cells in 200µl of CLB were dispensed in duplicates onto 96-well flat bottom plates coated with increasing concentrations of anti-CD25 mAb, 500 ng/ml of anti-FcγRI mAb which was immobilised onto 15 µg/ml of goat anti-mouse secondary antibody. This was incubated for 15 hrs and culture supernatants were used for TNF-α production (n= 2). The optimal concentration of each antibody red dotted lined.

2.8 Detection of TNF-α by Enzyme-linked immunosorbent assay (ELISA)

A DuoSet ELISA kit was used to detect TNF-α production in culture supernatants following standard laboratory methods [15, 62, 120] and according to the manufacturer’s instructions. In brief, flat-bottom 96 well plates (Maxisorp, NUNC) were coated with capture antibody diluted in 100 μl PBS per well in duplicates and incubated overnight at RT. The plate was then washed three times with PBST buffer

(Table 2-2) and blocked with 1% BSA in PBS for 1 hr at RT. After three more washes with PBST buffer, the plate was incubated with samples or standards diluted in 100 μl appropriate reagent dilution buffer (Table 2-2), then incubated for 2 hrs at RT. A standard curve of 8 standard 2-fold serial dilutions (ranging from 1000 – 0 pg/ml) was used. After three washes, biotinylated detection antibody was added in 100 μl reagent dilution buffer and incubated for 2 hrs at RT, followed by incubation with pre-warmed

100 μl substrate 3,3’,5,5’-tetramethybenzidine (TMB) for 20 min at RT in the dark. The reaction was stopped by adding 50 μl 2 N H2SO4, and the absorbance measured at 450 nm using a microplate reader.

2.9 Detection of multiple cytokine production by Luminex MAGPIX

The levels of IL-6, IL-10 and TNF-α were analysed using Luminex MAGPIX according to manufacturer’s instruction (Millipore, Billerica, MA, USA). Briefly,

200 μl of assay buffer was added to each well of a 96-well plate and removed by vacuum, then 25 μl of standard or cell culture supernatant was added to each appropriate well (assay buffer was used as a blank), followed by incubation with 25 μl

Premixed Beads and 25 μl PBS in each well overnight with shaking at 4°C. Following incubation, the plate was washed twice by adding 200 μl wash buffer and vacuumed.

50 μl of detection Abs were added and incubated for 1 hr at RT with shaking and then 79 conjugated with 50 μl streptavidin–PE. Following the extended incubation for 30 min and washing, the samples were mixed with 100 μl sheath fluid and then run on a

Luminex MAGPIX instrument.

2.10 Silver staining, mass spectrometry, Western blotting and

Immunoprecipitation

Silver staining, mass spectrometry, Western blotting and immunoprecipitation were mainly used in this study for protein visualisation, detection, isolation and identification. Protein samples used for these methods were generally extracted from

PBS-washed cells using Western lysis buffer containing freshly made 2 mg/ml protease inhibitors and 10 µM pervanadate (see in Table 2-2). After vortexing for 1 min, cell lysate was incubated on ice for 30 min and the supernatant collected by centrifugation at

20,000xg for 10 min at 4°C.

A BCA protein assay kit was used to quantify protein in the cell lysates according to the manufacturer’s instructions [273]. In brief, 10 μl protein samples and standards

(BSA ranged from 2-0 mg/ml) were incubated with 200 μl working buffer (50 parts A +

1 part B) in a 96 well plate and incubated at 37ºC for 30 min, followed by measurement of the absorbance at 562 nm using SpectraMax®M2 Microplate Reader (Molecular

Devices, CA, USA).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins under reducing or non-reducing conditions [274-276]. In most experiments 10% polyacrylamide resolving and stacking gels were freshly prepared.

Once the gels were set at room temperature, wells were rinsed, loaded with protein samples and electrophoresed at constant 40 volts for 30 min followed by 100 volts for

70 min (Bio-Rad PowerPackTM). Proteins resolved in the SDS-PAGE were then used for silver staining or transferred onto membranes for Western blotting (Section 2.10.3).

Generally, 20µg of protein containing 2x (v/v) Tricine sample buffer with DTT (~25µl) was pre-heated at 100ºC for 5 min for reducing gels; proteins contained Tricine sample buffer without DTT, and were not pre-heated for non-reducing gels.

2.10.1 Silver staining

Silver stain is highly sensitive compared to other chemical stains in detecting

proteins, hence it was used to visualise the purified protein to confirm purity and

protein bands prior to their identification using mass spectrometry [277, 278]. In brief,

acrylamide gels containing proteins were fixed with Fix I (Table 2-2) for 10 min

followed by 50% (v/v) methanol in water for 10 min. Gels were then washed twice in

water for 10 min each, incubated in 0.05% sodium thiosulfate solution in water for 1

min, rinsed in water and incubated in 0.2% silver nitrate in water for 1 hr. The silver

nitrate was rinsed in water and developing solution was added (Table 2-2), until the

desired protein bands were visible. The reaction was stopped and fixed with Fix II

(Table 2-2) at 4ºC overnight. All steps were performed with constant agitation at 70

rpm on a platform shaker.

2.10.2 Mass spectrometry

Silver-stained bands were excised and proteins were identified by Nano Liquid

Chromatography tandem Mass Spectrometry (Nano LC-MS/MS), as described [83, 276].

In-gel proteins were digested with 2 ng/µl trypsin and incubated in 1% formic acid and

3x 100µl acetonitrile at RT. Pooled eluents from each digest were dried, resuspended in

15µl 0.05% heptafluorobutyric anhydride in 1% formic acid and injected into a fritless

Nano column (75 µm x 10cm), using an Ultimate 3000 HPLC with an auto-sampler system (Dionex, Amsterdam, Netherlands) [277, 279]. Peptides were eluted using a linear gradient from mobile phase A (0.1% formic acid in H2O) to mobile phase B (0.1% formic acid in 80% acetonitrile) over 35 min at a flow rate of 0.3 µl/min. Positive ions of tryptic digests were generated by electrospray and a survey scan m/z 350-1750 acquired in the Fourier transform ion cyclotron resonance (FTICR) cell of a LTQ-FT

Ultra mass analyser (Thermo Electron, Bremen, Germany). Peak lists of LC-MS/MS data were generated using MASCOT Daemon/extract_msn (Matrix Science, London,

England, Thermo) and interrogated using MASCOT version 2.1

(http://www.matrixscience.com) followed by searching against Homo sapiens proteins in the Swissprot protein database (version 80). Precursor tolerances were 4.0 ppm and product ion tolerances were ± 0.4 Da, and acceptable cut-off scores for individual LC-

MS/MS spectra were set to 20.

2.10.3 Western blotting

Western blotting was used for detection of Tyr phosphorylated proteins using anti-phospho Tyr, and multiple intracellular signalling proteins including clathrin, Cbl,

HGS, HSP, TRIM 21, SHP-1, SHP-2 and SHIP. In brief, cell lysates (20 µg) were loaded onto 10% SDS-PAGE gels, separated under reducing conditions, transferred onto methanol-activated polyvinylidene difluoride membranes (PVDF, 0.2 µm pore size) at 4ºC at constant 75 volts for 45 min. The membranes were blocked for 2 hrs at RT in

TBST buffer (Table 2-2) containing 5% non-fat dry skim milk for standard Western blots or 5% BSA for Western blots designed to detect phosphorylated proteins. After a short rinse with TBST buffer, membranes were incubated at 4ºC overnight with an 82 appropriate concentration of primary antibody in TBST buffer. This was followed with three washes (10 min each) with TBST buffer, incubation for 90 min at RT with desired concentrations of relevant secondary antibody diluted in TBS, and a further three washes in TBST. Lastly, immunoreactivity was detected using a chemiluminescent reagent (Western Lighting) and images visualised by ImageQuant™ LAS 4000 (GE

Healthcare Life Sciences). The entirety of primary and secondary antibodies used for

Western blotting are listed in Table 2-1.

In some experiments, membranes were stripped for 30 min at 65°C using 62.5 mM Tris-HCl (pH 6.8) containing 2% SDS and 100 mM β-mercaptoethanol, followed by five washes in TBS, blocking in TBST containing 5% non-fat dry skim milk or 5%

BSA for 2 hrs at RT, and re-probed with new sets of primary and secondary antibody pairs.

2.10.4 Immunoprecipitation

Immunoprecipitation was used to isolate and concentrate Tyr-phosphorylated proteins using anti-pTyr mAb (clone 4G10). In brief, total 2 x 107 cells were lysed with cold Western lysis buffer. After vortexing for 1 min, samples were incubated on ice for

30 min with the supernatants collected by centrifugation at 20,000xg for 10 min at 4°C.

Specific proteins were immunoprecipitated at 4ºC overnight with 5 µg/ml of primary antibody, followed by incubation for 2 hrs at 4°C with 10 µg/ml of goat anti-mouse Ab conjugated Sepharose beads (Zymed Laboratories Inc.). The incubation with both primary and secondary antibody was performed with gentle rotation. Bead-bound proteins were washed once with 1 ml cold dilution buffer (Table 2-2), twice with TSA

(Table 2-2) and once with 50 mM Tris buffer pH 6.9. Beads were then resuspended in

Tricine gel loading buffer containing 10 mM DTT, heated for 5 min at 100°C and supernatants resolved in 10% Tris-Tricine SDS-PAGE gels under reducing conditions, then silver-stained for protein identification using mass spectrometry (Section 2.10.1) or

Western blotted for protein detection (Section 2.10.3).

2.11 Molecular biology methods

A number of standard molecular biology methods, including RNA extraction, multigene quantitative RT-PCR, plasmid DNA extraction, sub-cloning of LILRB4 DNA constructs to different plasmid vectors, site-directed mutagenesis of LILRB4-ITIMs and

DNA sequencing were used in this project.

2.11.1 RNA extraction, reverse transcription and quantitative RT-PCR

2.11.1.1 RNA extraction, reverse transcription

Total RNA was extracted from PBMCs or THP-1 cells using TRIzol® reagent as described. In brief, 5 x 106 cells were lysed in 1 ml TRIzol reagent followed by the addition of 200 μl bromo-3-chloropropane (BCP) and high speed centrifugation at 4°C.

The RNA in the aqueous phase was transferred to a new Eppendorf tube and precipitated by adding 2 μl of glycogen (RNase free) and 500 μl isoprophyl alcohol. The

RNA pellet was then washed with 1 ml of fresh 75% ethanol by gentle vortexing, followed by brief centrifugation at 4°C, supernatant removed and pellet air dried for 5-

10 min. Followed by addition of 35 μl DEPC treated water incubated for 10 min at 60°C to elute the RNA. The extracted RNA was quantified using a nanodrop (Thermo

Scientific), with integrity examined by running a small aliquot in 0.8% agarose gel and either immediately used to synthesise cDNA by reverse transcription or stored at -70°C.

For reverse transcription 1 - 1.5 µg of RNA incubated at 37°C for 30 min with 1

μl Turbo DNase and 1 μl of 10x Turbo DNase buffer in 8 μl DEPC water to remove contaminating genomic DNA. After deactivation of the DNase using 1 μl of 50 mM

EDTA at 75°C for 10 min, the reverse transcription reaction was started by adding 1 μg of DNase treated RNA to SuperScriptTM III First strand Synthesis SuperMix as per the manufacturer’s instructions. The mixture was then incubated at 25°C for 10 min, at

50°C for 30 min and at 85°C for 5 min followed by addition of 1 μl of 2U RNase H and further incubation at 37°C for 20 min. The synthesized cDNA was either used for quantitative real time RT-PCR or stored at - 20°C.

2.11.1.2 Multigene quantitative real time RT-PCR

An in-house 27 gene multiplex real time PCR array was established to determine mRNA levels of selected pro-inflammatory and immune-regulatory cytokines, redox and cell signalling molecules (Table 2-4) in PBMCs, THP-1 cells and THP-1 cell mutants. First specific primer sets for each molecule were designed using OLIGO 7

Primer design, Analysis Software (http://www.oligo.net/index.html) and Primer3

Programme (http://frodo.wi.mit.edu), and primers for RT-PCR purchased from Life

Technologies (Table 2-4). The following quantitative RT-PCR protocol was established after extensive optimisation experiments aimed at finding a single optimal RT-PCR condition that could be used for all sequences simultaneously. In brief, 20 ng cDNA in

TE buffer (pH 8.0) (1 µl) was added in duplicates into 384-well plates containing 3 µl of SYBR Green select master mix and 1.25 µM (2.5 µl) of each primer set. Plates were then sealed with MicroAmp optical adhesive film, and products amplified using

QuantStudioTM 12K Flex Real-Time PCR System (Applied Biosystems). The PCR cycling parameters were set as 50°C for 2 min initial holding, 95°C for 2 min initial 85 denaturing and 40 cycles of PCR (comprising 95°C for 1 sec and 60°C for 30 sec annealing, and 95°C for 15 sec), 60°C for 1 min and 95°C for 15 sec continuous melting curve stage. Amplified mRNAs were normalized using the mean values of HPRT and

GAPDH housekeeping genes and the cut-off threshold was set to 37 Cp (crossing point) or CT (threshold values) value. Amplification of the correct amplicon was further confirmed by agarose gel electrophoresis of the quantitative real time RT-PCR product and visualisation of the correct size (Figure 2-4).

Table 2-4. Forward and reverse primer sequence sets used for quantitative RT-PCR Genes Forward (5’→ 3’) Reverse (5’→3’) HPRT TCAGGCAGTATAATCCAAAGATGGT AGTCTGGCTTATATCCAACACTTCG GAPDH CATGAGAAGTATGACAACAGCCT AGTCCTTCCACGATACCAAAGT TNF-α ATGAGCACTGAAAGCATGATCC GAGGGCTGATTAGAGAGAGGTC NFκB1 ATTTGAAACACTGGAAGCACGA GCGGATTAGCTCTTTTTCCCG IKKB CACCATCCACACCTACCCTG CTTATCGGGGATCAACGCCAG INF-γ TCGGTAACTGACTTGAATGTCCA TCCTTTTTCGCTTCCCTGTTTT SHP-1 GGTGTCCACGGTAGCTTCC ACAGGTCATAGAAATCCCCTGAG IL-1β CACGATGCACCTGTACGATCA GTTGCTCCATATCCTGTCCCT IL-5 CTGCCTACGTGTATGCCATCC CATTGGCTATCAGCAGAGTTCG IL-6 AACCTGAACCTTCCAAAGATGG TCTGGCTTGTTCCTCACTACT IL-10 TCAAGGCGCATGTGAACTCC GATGTCAAACTCACTCATGGCT IL-12β TGCCCATTGAGGTCATGGTG CTTGGGTGGGTCAGGTTTGA IL-13 GAAGGCTCCGCTCTGCAAT TCTGGGTCTTCTCGATGGCA CCL-2 CAGCCAGATGCAATCAATGCC TGGAATCCTGAACCCACTTCT CXCL-10 GTGGCATTCAAGGAGTACCTC GCCTTCGATTCTGGATTCAGACA TGF-β1 CAAGCAGAGTACACACAGCAT TGCTCCACTTTTAACTTGAGCC FGF-2 AAGCGGCTGTACTGCAAAAAC TGAGGGTCGCTCTTCTCCC NOS2 TCATCCGCTATGCTGGCTAC CTCAGGGTCACGGCCATTG NOS3 TGGTACATGAGCACTGAGATCG CCACGTTGATTTCCACTGCTG Arginase1 AGTGGACAGACTAGGAATTGGC TCCAGTCCGTCAACATCAAAAC Arginase2 CGCGAGTGCATTCCATCCT CCTTTTCATCAAGCCAGCTTCTC NOX-1 TTTTCCTGGTTCAACAACCTGT CCACTTTGCCTAATTCCTCCATC NOX-2 TGGAGTGGCACCCTTTTACAC CCACAAGCATTGAACAGCCC ACOX1 TCTTTCCACTCCTGGCCACTG AAGCCATCCGACATGCTTCAA CAT TCAGGTGCGGGCATTCTATGT TCCAGAAGAGCCTGGATGTGG PRDX2 CAGCAACCGTGCAGAGGACTT TCTGGTCACGTCAGCAAGCAG IDO TCATCTCACAGACCACAAGTCA GCAAGACCTTACGGACATCTCC

Figure 2-4. Quantitative RT-PCR products using 27 multi-gene primers Quantitative RT-PCR on mRNA extracted from PBMCs and/or cells transfected with LILRB4 ITIMs (YYY) that were activated with 100 nM LPS showed multi gene mRNA expression. As a quality control of RT-PCR and primer sets, the PCR products were loaded onto 0.8% agarose gel and visualised.

2.11.2 Sub-cloning of LILRB4 into mammalian expression vectors

One of the aims of this study was to investigate the relative roles of each LILRB4

ITIM to the inhibitory functions of LILRB4. To elucidate this, chimeric DNA constructs containing the extracellular and transmembrane domains of IL-2 receptor α- chain and the intracellular domains of LILRB4 ITIMs comprising site-directed mutations of each functional tyrosine residues (Y) to phenylalanine (F) were cloned into a pMH-Neo expression vector (CD25-LILRB4 ITIMs). A total of nine constructs, comprising three single mutants, three double mutants, one triple mutant, one wild type and one vector only (mock) construct, were stably transfected into the human monocytic cell line, THP-1. It is important to note that the investigation of ITIM function by the 87 site targeted-mutation of tyrosine to phenylalanine (Y-to-F) has been successfully utilised in many similar previous studies [230, 280, 281].

2.11.2.1 Generation of chimeric DNA construct containing the extracellular

domains of CD25 and the intracellular domains of LILRB4

The extracellular domain and transmembrane domain of human CD25 (starting from the leader peptide at -21Met to 238Leu) were fused in frame and upstream to the intracellular domain of LILRB4 (258Gln to 425His) by three-step overlap extension polymerase chain reaction. A high-fidelity Platinum Pfx DNA polymerase was used in all PCR amplifications. The pMLSV N1/N4-S plasmid (American Type Culture

Collection, ATCC), containing complete coding sequence of human CD25 [282], was used as the template for the first-round PCR amplification using the forward primer A and the reverse primer B (Table 2-5). The PCR cycling parameters were initial denaturation at 94°C for 2 min, followed by 28 cycles of denaturation at 94°C for 1 min, annealing at 64°C for 40 sec, and extension at 68°C for 1 min and 10 sec, and a final elongation step at 68°C for 8 min. The resulting PCR product contained an XbaI site at the 5’ end for directional cloning into vector and a Kozak sequence as a ribosomal binding site directly upstream of the translation initiation codon (ATG) for efficient translation [283]. The 0.8-kb PCR product was purified from a 1.5% Nusieve GTG agarose gel.

The full-length LILRB4 cDNA in pcDNA3 mammalian expression vector, kindly donated by Dr. Luis Borges, Amgen Inc., [21] was used as the template for PCR amplification of the intracellular domains of LILRB4 using C and D as forward and reverse primers respectively (Table 2-5). The PCR cycling parameters were 1 cycle of

94°C for 4 min, followed by 28 cycles of 94°C for 1 min, 58°C for 40 sec, and 68°C for

1 min and 10 sec, and a final extension step at 68°C for 8 min. The 0.5-kb PCR product was purified from a 1.5% Nusieve GTG agarose gel.

Equimolar amounts of gel-purified products from the first-round and second- round PCR amplifications were used as the templates in the third-round PCR amplification using the forward primer A and reverse primer D (Table 2-5). The PCR cycling parameters were 1 cycle of 94°C for 4 min, followed by 28 cycles of 94°C for 1 min, 58°C for 40 sec, and 68°C for 2 min and 10 sec, and a final extension step at 68°C for 8 min. The resulting 1.3 kb-PCR fragment was gel-purified, sequentially digested with XbaI and BamHI, gel-purified again, and ligated into the XbaI-BamHI-digested and gel-purified pMH-Neo mammalian expression vector [284]. The resulting expression plasmid was designated pMH-Neo-CD25-LILRB4.

The long terminal repeat (LTR) of Friend spleen focus-forming virus (SFFV) on the vector drives the gene expression. The entire insert containing CD25-LILRB4 fusion gene (1.3 kb) was subjected to sequence analysis using the Sanger method [285] and confirmed a correct chimeric DNA sequence in-frame.

Table 2-5. Primer sets used for construction of CD25-LILRB4 chimeric DNA construct Primers Primer sequences (5’ to 3’)

GACCTGTCTAGAGCCACCATGGATTCATACCTGCTGATGTGG A (XbaI site underlined followed by a Kozak sequence in italic and double underlined)

B GTTTTCCCTGACGCCAGTGTTGGAGCCCACTCAGGAGGAGGACGC

C GCGTCCTCCTCCTGAGTGGGCTCCAACACTGGCGTCAGGGAAAAC

ATTCGCGGATCCCCTGGATTAGTGGATGGCCAGAGTG D (BamHI site underlined and the complementary natural translational stop codon of LILRB4 double underlined)

2.11.3 Site-directed mutagenesis of the LILRB4 ITIMs in the pMH-Neo-CD25

LILRB4 construct

Transformer Site-directed Mutagenesis kit, which was based on the method of

Deng and Nickoloff (1992) was used to generate multiple site-directed mutants [286]. In brief, the tyrosine residue (Y) at positions 337, 389 and 419, located on the ITIM motif of I, II and III in the intracellular domains of LILRB4, was replaced by phenylalanine

(F). Three single mutants (Y337F, Y389F and Y419F), three double mutants

(Y337F/Y389F, Y337F/Y419F and Y389F/Y419F), and one triple mutant

(Y337F/Y389F/Y419F) were made using primers listed in Table 2-6.

A unique XmnI site was present in the region of ampicillin resistance gene in the pMH-Neo vector, but absent in the insert containing CD25-LILRB4 chimeric gene.

Elimination of the unique XmnI site was a useful selection strategy because the parental template can be removed by digestion with the XmnI restriction enzyme. Therefore, a primer was designed for the purposed of replacing the unique XmnI site with a Psh AI site. The sequence of the selection primer was 5’-

AAAGTGCTCATCATTGGACAACGGTCTTCGG-3’ (Table 2-6). The bases of the

Psh AI site are double underlined with the mutated nucleotide (italicised and boldfaced) for converting the unique XmnI site into a Psh AI. These minor mutations within the ampicillin resistance gene did not affect function. T4 DNA polymerase was used for synthesis of all mutant plasmid DNAs. This enzyme possesses 3’  5’ exonuclease activity, which allows the incorrect base pairs to be excised and re-inserted with the corrected ones. Therefore, this enzyme has proofreading ability and extreme fidelity.

Complete sequencing of the mutants was performed using the Sanger method.

Table 2-6. Primer sets used for site-directed mutagenesis of the LILRB4 ITIMs in the pMH-Neo-CD25 LILRB4 construct Primers Primer sequences (5’ to 3’)

GCAGTGACGTTCGCCAAGGTGAAACACTCCAGAC Y337F (the mutagenic primers with mutated bases underlined)

GGATGTGACCTTCGCCCAGCTGCACAG Y389F (the mutagenic primers with mutated bases underlined) GCCTCTCCAGCTGAGCCCAGTGTCTTTGCCAC Y419F (the mutagenic primers with mutated bases underlined) AAAGTGCTCATCATTGGACAACGGTCTTCGG Selection (Psh AI site double underlined and the two mutated nucleotides for converting the unique XmnI site into a Psh AI site are in italics and boldfaces)

2.11.4 Plasmid DNA transfection to mammalian cells

Lipofectamine® LTX DNA transfection reagent was used to express specific protein according to manufacturer’s instructions (Life Technologies). In brief, suspension cells (5 x 105) were resuspended in 2 ml of appropriate complete medium without antibiotics and settled onto a 6 well plate followed by incubation at 37°C, 5%

CO2 for 1 hr. DNA-lipid complex was then added drop by drop to the cells. Adherent cells (3 x 105) were cultured in 2 ml of appropriate complete medium in 6 well plate at

37°C, 5% CO2 overnight to be 70-90% confluent. Culture medium was aspirated and cells were then gently washed once in D-PBS and pre-incubated in 2 ml complete medium without antibiotics (penicillin and streptomycin) at 37°C, 5% CO2 for 1 hr.

DNA-lipid complex was then added drop by drop to the cells. Total 6 μl Lipofectamine

LTX was diluted in 119 μl Opti-MEM medium and incubated with 2.5 μg DNA in

122.5 μl Opti-MEM containing 2.5 μl PLUS reagent for 5 min at RT. To each well 250

μl of the DNA-lipid complex was added. Transient transfectants were generated by incubation at 37°C, 5% CO2 for 48 hrs. Appropriate selection antibiotic (details are 91 described in appropriate chapters) was added 48 hrs post-transfection to generate stable transfectants and single cells with high transgene expression were further selected within three weeks by limiting dilution.

2.11.4.1 Generation of stably pMH-Neo-CD25-LILRB4 transfected THP-1 cells

Seven constructs of pMH-Neo-CD25-LILRB4 (with one or more Tyr to Phe mutation(s) at positions 337, 389 and 419 of the LILRB4 cytoplasmic domain), along with the pMH-Neo-CD25-LILRB4 wild type construct and the pMH-Neo empty vector were stably transfected into THP-1 cells using Lipofectamine® 2000 (Life

Technologies) [287]. These included one wild-type ITIM (YYY), one triple mutant without any functional ITIMs (FFF), three single mutants that had two functional ITIMs

(YYF, YFY, FYY), three double mutants with one functional ITIM (YFF, FYF, FFY)

(Figure 2-5) and one vector only (negative control). Selection antibiotic (0.8 mg/ml of

G418) was added 48 hrs post-transfection and single cells with high transgene expression were further selected within three weeks by limiting dilution. The successful stable transfection was confirmed by surface expression of CD25 using flow cytometry, as described in Section 2.12.

Figure 2-5. Schematic diagram of CD25-LILRB4 ITIM plasmids Plasmid constructs of the extracellular and transmembrane domains of human CD25 α-chain fused to the intracellular domain of human LILRB4 containing various tyrosine to phenylalanine site-directed mutants were cloned into pMH-Neo vector and transfected into THP-1 cells.

2.12 Flow cytometry

Two step flow cytometry was used to confirm surface expression of CD25-

LILRB4 ITIMs and expression of relevant cell surface markers including FcγRI, native

LILRB4 and TLR4. In brief, 3 x 105 cells were washed twice in PBS and resuspended in

50 µl PBS. Unconjugated primary antibody or isotype matched negative control (5

µg/ml each) was incubated with cells for 45 min at RT. Cells were then washed once in

PBS containing 0.5% BSA, followed by incubation with 3 µg/ml of PE or FITC conjugated secondary antibodies for 1 hr on ice. Alternatively, PE or FITC conjugated

93 primary antibody or isotype matched negative control (5 µg/ml each) was incubated with cells for 30 min at RT. After incubation with antibodies, cells were washed twice in cold PBS containing 0.5% BSA, then fixed in 1% paraformaldehyde in 350 μl PBS.

2.13 Quality controls of CD25-LILRB4 transfected THP-1 cells

To accurately assess whether transfection with the CD25-LILRB4 ITIM mutants affected cell viability, 0.4% trypan blue staining for microscopic visualisation/counting and a LIVE/DEAD® viability assay kit were used before each experiment [288]. Effects of stable transfection on apoptosis and cell proliferation were assessed using Annexin-

V/Propidium iodide (PI) apoptosis kit, and by standard 3H-thymidine (1 µCi/ml) incorporation assay respectively, according to the manufacturer’s instructions [289-291].

2.13.1 Cell viability using LIVE/DEAD® Viability/Cytotoxicity Kit

A total of 3.5 x 104 transfected cells were cultured in 100 µl of RPMI complete media containing G418 in a flat-bottomed 96 well plate in duplicates at 37ºC, 5% CO2.

After two days, 100µl of PBS containing 2 µM Calcein AM and 4 µM EthD-1

(LIVE/DEAD® viability/Cytotoxicity Kit) was added to the cells and incubated for 30 min at 37°C, 5% CO2. Proportions of live cells that converted non-fluorescent Calcein

AM to green fluorescent dye and dead cells that took up EthD-1 and fluoresced red were measured using SpectraMax®M2 Microplate Reader with 530 nm excitation and

645 nm emission spectra.

2.13.2 Cell proliferation using 3H-thymidine incorporation assay

A total of 2 x 105 transfected cells were cultured in 200 µl of RPMI complete media containing G418 in a 96 well filtration plate (Millipore) in duplicates at 37ºC, 5%

3 CO2 for two days. Cells were then pulsed with 1 µCi of H-thymidine followed by incubation for additional 24 hrs. Cells in filtration wells were washed three times with

200µl of PBS using a vacuum manifold (Millipore). Plates were dried at 37ºC for 30 min and membrane filters in each well harvested to scintillation vials using a Brandel cell harvester (Gaithersburg). Finally, 4 ml of scintillation fluid was added to each vial, incubated for 1 hr at RT with intermittent vortexing and incorporated 3H-thymidine measured using a liquid scintillation β-counter (Perkin Elmer).

2.13.3 Apoptosis using annexin V-FITC and propidium iodine (PI)

To compare the proportion of cell death by apoptosis among the different transfectants, an Annexin V-FITC and propidium iodine (PI) apoptosis detection kit was used according to the manufacturer’s instructions. In brief, cells were washed twice in cold PBS and then resuspended in 1 x Binding buffer at a concentration of 3 x 106 cells/ml. 100 µl of the cell suspension (3 x 105 cells) was stained with 5 µl of annexin

V-FITC and 1 µg/ml of PI for 15 min at RT in the dark. This was followed by addition with 400 µl of 1 x Binding buffer and immediate data acquisition using FACScanTM flow cytometry. Unstained cells and single stained cells (Annexin V or PI alone) were used as controls. A total of 1 x 104 events were acquired for each sample and all data was acquired within one hour.

2.14 Measurement of bacterial phagocytosis using pHrodo

Each transfectant (5 x 104 cells) was in vitro differentiated for three days using

100 ng/ml of PMA in complete RPMI media containing G418 in a 5 ml FACS tube. For bacteria-pHrodo labelling, Salmonella typhimurium (strain LT2) were labelled using the pHrodo phagocytosis particle labelling kit, lyophilised at 1 mg aliquots and stored at -

80°C according to the manufacturer’s instructions [120]. Frozen S. typhimurium particles were thawed on ice, sequentially washed with 1 ml of 100% methanol and 1 ml of Buffer C. Bacterial particles were then thoroughly resuspended in 200 µl of

Buffer B (Molecular probes) by repeated vortex and sonication (three times for 30 sec at

50% power cycle, Kontes, NJ, USA) followed by incubation with 10% of human IgG for 10 min at RT and left on ice until use. The pHrodo labelled opsonised particles in

100 µl of CLB were added onto 4 x 105 cells of PMA-differentiated transfectants in 100

µl of CLB in 5 ml FACS tubes at a ratio of 20:1 and incubated for 90 min at 37°C, 5%

CO2. This was followed by incubation of tubes on ice for 15 min, two washes with 1 ml of Buffer B and re-suspension in 300 µl of Buffer C. The pH dependent conversion of the non-fluorescent pHrodo to a red fluorescence dye upon uptake by the transfectants and fusion to low pH lysosome was detected by FACScan flow cytometry. A total of 5 x 104 events were acquired for each transfectants, and proportions of positive cells in 4 independent experiments determined using FlowJo software.

2.15 Assessment of bead phagocytosis

To compare phagocytosis among the different stably transfected mutants, assessment of 2 µm carboxylate polybeads (Polysciens, Warrington, PA) uptake was used. Briefly, 50 mg carboxylate polybeads were washed three times in 5 ml coupling buffer (Table 2-2) by centrifugation with 4000xg for 10 min and resuspended in 2.5 ml 96 coupling buffer. After, 1 ml of 3.7 mM 1-ethy-3 (3-dimethyaminoprophy) carbodimide was added to the beads, followed by the addition of 2.5 mg to human IgG diluted in 2 ml Baxter water (pH 5.3) adjusting pH between 4.5 and 6.0. Beads were then washed three times in wash buffer (Table 2-2) followed by biotinylation with 3 mg/ml of Sulfo-

NHS-LC-Biotin in PBS for 30 min at 4°C with gentle rotation. Excess unbound biotin was quenched with 3 ml RPMI media for 10 min at 4°C with gentle rotation, washed twice in PBS, and labelled with streptavidin-conjugated Alexa488 in a 1:1000 dilution in PBS for 15 min at RT. Beads were washed once in PBS, resuspended in RPMI medium containing 10 mM HEPES and 0.1% BSA and then stored at 4°C until use.

PMA differentiated transfectants (0.5 x 106 each) in a sterile 5 ml FACS tube were washed once using CLB and resuspended in 200 µl of CLB containing Alexa488 and human IgG labelled beads at a ratio of 10:1 (1 x 107 beads) and incubated for 60 min at

37°C, 5% CO2. Cells were then washed 5 times in cold PBS and fixed using 4% paraformaldehyde for 10 min at RT. The proportion of cells that ingested beads were detected by FACScan flow cytometry. A total of 5 x 104 events were acquired for each transfectant and data analysed using FlowJo software. Phagocytic index was calculated as a ratio of % phagocytosis relative to mock-transfected cells.

2.16 Measurement of bactericidal activity

A total of 5 x 104 cells of transfectants were PMA-differentiated and resuspended in CLB in round-bottom 5 ml FACS tubes and infected with freshly-grown human serum opsonised live S. typhimurium at a 50 multiplicity of infection (MOI).

After 90 min infection at 37°C and 5% CO2, 200 µg/ml of gentamicin was added to kill extracellular bacteria by gentle shaking of tubes at 100 rpm for 30 min at 37°C. Cells

97 were then washed three times with 3 ml of PBS and lysed with 100 µl of 2% saponin in

PBS at RT for 15 min with constant shaking at 100 rpm and intermittent vortexing.

Serially-diluted lysates were plated onto 10 cm LB agar plates and incubated at 37°C overnight and bacterial colonies for each sample counted by a blinded observer in five independent experiments. Number of colonies from each transfectant was then adjusted to their corresponding phagocytic index and data presented as mean colony numbers ± standard error of mean (SEM) in which higher counts represented poor bactericidal activity.

2.17 Synthesis of peptides containing LILRB4 ITIMs

The ultimate aim of LILRB4 research is the possible use of LILRB4 as a potential therapeutic agent. To address this, we custom-designed and synthesized TAT containing fusion LILRB4 ITIM peptides (Peptron Inc. Korea, http://www.peptron.co.kr/) to regulate immune responses based on our results (Section 3.2). It is important to note that HIV-TAT48-57 facilitates the internalisation of peptides, proteins and nucleic acids, and is commonly used as a drug delivery tool [292].

Four synthetic peptides were synthesised and included: TAT conjugated wild type of three LILRB4 ITIM motifs (TAT-YYY); TAT conjugated three LILRB4 ITIM motifs without tyrosine residues (TAT-FFF); TAT conjugated the middle LILRB4 ITIM motif (TAT-FYF); and TAT peptides alone (TAT) as a control (Table 2-7). In brief,

THP-1 cells or PBMCs were pre-incubated with serial dilutions of the peptides (0, 5, 10,

20, 50 and 100 μg/ml) for 30 min, and FcγRI-mediated TNF-α production was analysed as described in 2.7.2 and 2.8. The quality of peptides and the property of peptide internalisation were investigated using HPLC and intracellular staining as described in

2.17.1 and 2.17.2 respectively. 98

Table 2-7. Synthetic peptides containing LILRB4 ITIM(s) and controls Peptide Sequences Length Source/use

LILRB4 TAT-YYY GRKKRRQRRRAVTYAKVDVTYAQLSVYATL 30 aa (three ITIMs) Control for TAT-FFF GRKKRRQRRRAVTFAKVDVTFAQLSVFATL 30 aa TAT-YYY LILRB4 TAT-FYF GRKKRRQRRRQDVTYAQLHS 20 aa (aa408 –417)

TAT GRKKRRQRRR 10 aa Control for all

*The sequences of TAT peptides are underlined and Tyrosine(Y) or Phenylalanine (F)in red

2.17.1 High-performance liquid chromatography (HPLC) to determine purity of

synthetic peptides containing LILRB4 ITIMs

The quality and quantity of peptides were analysed by HPLC (600S Delivery

System, Waters) using C4 purification hydrophobicity column (300Å, 5μm particle size,

250 x 4.6 mm, Vydac, Hesperia, CA, USA). The column was equilibrated using 100% buffer A (containing 95% water, 5% acetonitrile, 0.01% Trifluoroacetic acid) for 10 min.

The program was set to gradient mode starting at 100% buffer A with increasing buffer

B (containing 99.9% acetonitrile, 0.095% Trifluoracetic acid). Each peptide sample was injected into the column with maximum pressure at 2500 psi and flow rate at 1 ml/min.

2.17.2 Intracellular staining to determine internalisation of synthetic peptides

containing LILRB4 ITIM(s)

A total of 5 x 106 PBMCs in CLB were incubated with/without 5 μM peptide for

30 min at RT, and then washed once with PBS. Cells (2 x 105) in PBS were then fixed using 4% paraformaldehyde for 10 min, washed three times with PBS, and

99 permeabilised with 0.5% saponin, 0.1% BSA and 0.01% NaN3 for 10 min. After washing, 0.2 mg/ml mouse anti-TAT or mouse IgG was added and incubated for 30 min at RT, and then cells were washed once with permeable buffer. After, cells were incubated with 2 μl goat anti-mouse-PE for 45 min on ice. After washing twice in PBS,

4% paraformaldehyde was added into cells, which were then analysed by FACScan flow cytometry. A total of 5 x 104 events were acquired for each sample and proportions of positive cells in 4 independent experiments determined using FlowJo software.

2.18 Statistical analysis

A pathway analysis was performed using QIAGEN’s Ingenuity®Pathway Analysis

(IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). P values were determined with the right-tailed Fisher exact test.

Relative contributions of the intracellular domains of LILRB4 to its regulatory functions were assessed by comparing data from each clone expressing CD25-LILRB4

ITIM mutants to vector only (mock)-transfected cells. One-way ANOVA with

Dunnett’s multiple comparison was used to evaluate expression levels of CD25, FcγRI,

LILRB4 and TLR4 on the surface of transfectants and/or THP-1 cells, effects on TNF-α production, bacterial phagocytosis, bactericidal activity, apoptosis, cell proliferation and gene regulation using GraphPad Prism 6.0 software (GraphPad Software Inc.), and protein expression quantified by densitometry analysis using ImageJ software

(http://rsbweb.nih.gov/ij) and analysed by Student’s t-test. P values < 0.05 were considered statistically significant.

100

CHAPTER 3. RESULTS

3.1 Regulation of Tyr phosphorylation of multiple proteins after FcγRI cross-

linking on THP-1 cells by LILRB4

3.1.1 Silver staining of SDS-PAGE gel loaded with immunoprecipitates from

FcγRI cross-linked THP-1 cells showed enrichment of multiple Tyr

phosphorylated proteins

To determine the identities of the Tyr phosphorylated signalling proteins by peptide mass spectrometry sequencing, THP-1 cells that express high levels of surface

FcγRI (Figure 3.1-1) were activated by anti-FcγRI antibody cross-linking and lysates immunoprecipitated using anti-pTyr mAb.

A B

Figure 3.1-1. Expression of FcγRI and LILRB4 on the surface of THP-1 cells (A) Representative flow cytometry analysis showing surface expression of FcγRI and LILRB4 on THP-1 cells, hence ideal model of FcγRI cross-linking and FcγRI-LILRB4 co-ligation studies. The mean fluorescence intensities of FcγRI and LILRB4 expression on THP-1 cells were 35.6 ± 4.6 and 75.4 ± 12.4 respectively, compared to 3.3 ± 0.2 for isotype matched negative control (B) Data from 3 independent experiments presented as mean fluorescence intensity (MFI) ± SEM (*p<0.05; **p<0.01; ***p<0.001).

101

Silver staining of SDS-PAGE gels loaded with anti-pTyr mAb-precipitated lysates from FcγRI cross-linked cells showed enrichment of eight bands at approximately 100, 70, 50, 47, 43, 35, 30 and 14 kDa, compared to precipitated lysates from control IgG1 treated cells, confirming specificity (Figure 3.1-2).

Figure 3.1-2. Representative silver staining of Tyr phosphorylated proteins in THP-1 cells upon cross-linking of FcγRI Multiple proteins were Tyr phosphorylated in THP-1 cells incubated with anti-FcγRI mAb followed by 90 sec cross-linking with secondary antibody in suspension and immunoprecipitation with anti-pTyr4G10 mAb (numbered arrows in lane 2). There was minimal Tyr phosphorylation in precipitates from cells treated with control IgG1 (lane 1) (n=3). The two strong bands at ~50 kDa and ~25 kDa in lanes 1 and 2 (asterisk) are the heavy and light chains of the immunoglobulins used for immunoprecipitation.

102

3.1.2 Identification of the Tyr phosphorylated proteins from FcγRI cross-linked

THP-1 cells by Nano LC-MS/MS

The silver stained bands (Figure 3.1-2) were excised, and peptides mass sequenced by Nano LC-MS/MS (method described in Sections 2.10.1 and 2.10.2).

MASCOT search output from 3 combined experiments identified 80 hits of 25 Tyr phosphorylated candidate proteins in peptides sequenced from pTyr immunoprecipitated lysates of anti-FcγRI cross-linked cells, but not from IgG1 control treated cells (Mowse score > 50, p 0.05; >3 peptide matches) (Table 3.1-1). A large number of these peptides were Tyr kinases including Syk and mitogen-activated protein kinase 9, cytoskeletal proteins or cytoskeletal actin binding proteins including actinin and tubulin, and ubiquitin-related proteins including Cbl, some of which have been previously shown to be Tyr phosphorylated in response to FcγRI cross-linking [15].

However, several high hit peptides corresponded to proteins that have not been previously reported to be phosphorylated in response to FcγRI cross-linking. These include: TRIM21 (a ubiquitin related protein reported to be a high affinity intracellular receptor of Fc portion of immunoglobulins [203]); Twinfilin-1 (ubiquitous actin binding protein that may regulate actin filament turnover [293]); hematopoietic cell specific Lyn substrate 1 (actin binding protein that may play a role in regulating T cell immune synapses [294, 295]); SH2 domain containing leukocyte protein of 76 kDa (that may regulate ERK-mediated activation [296]); a linker for activation of T cell family member 2 (adaptor protein associated with lipid rafts for AKT signalling [297]); Crk- like protein (adapter protein in signal transduction [298]); Coatomer subunit epsilon

(cytosolic protein complex that associates with vesicle assembly [299]); docking protein

2 (adaptor or scaffolding protein that associates with EGF receptor signalling [300]); protein Tyr phosphatase 18 (may regulate MEK/ERK-mediated activation [301]);

103

Phosphatidylionositol 3, 4, 5-triphosphate 5-phosphatase 1 (SHIP) (associated with Shc in response to multiple cytokines [302]); protein phosphatase 1 gamma (may regulate a variety of cellular functions by dephosphorylation of multiple proteins [303]); 1- phosphatidylionositol 4, 5-biphosphate phosphodiesterase gamma 2 (bind to EG-1 and may regulate transmembrane signalling [304]); Toll-like receptor 6 (recognition of mycoplasmal macrophage-activating lipopeptide cooperating with TLR2 [305]); and

Ubiquitn-40S ribosomal protein s27a (a carboxyl extension of ubiquitin and a function in protein degradation [306]).

104

Table 3.1-1. Mascot search results of mass spectrometric peptides sequencing of tyrosine phosphorylated proteins upon FcγRI cross linking of THP-1 cells* Band Phosphoproteins detected in anti-FcγRI cross-linked cells only Score Peptide matched MW alpha actinin 1, sarcomeric (F-actin cross-linking protein) 5321 249 102 alpha actinin 4 1927 77 102 E3 ubiquitin-protein ligase CBL 964 71 99 hepatocyte growth factor-regulated tyrosine kinase substrate (isoform CRA_c/d) 231 17 86 1 Toll-like receptor 6 116 12 92 microtuble-associated protein 2 123 8 199 ALG-2 interacting protein 1 (hp95/ program cell death 6 interacting protein) 112 3 96 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1 (SHIP1) 98 3 133 protein Tyr kinase Syk 951 67 72 heat shock cognate 71 kDa protein 831 46 70 hepatocyte growth factor-regulated tyrosine kinsae substrate 209 31 86 actinin, alpha 1 654 22 103 2 protein SPY75 (hematopoietic cell-specific Lyn substrate 1) 289 11 54 lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of 76 kDa) 287 11 60 E3 ubiquitin-protein ligase CBL (proto-oncogene c-CBL, RING finger protein 55) 146 6 100 Fc gamma receptor type I/ Fc fragment of IgG receptor, CD64) 76 4 42 ATP-dependent DNA helicase II, 70 kDa subunit (G22P1) 64 3 70 elongation factor 1 alpha 1 402 30 50 lymphocyte cytosolic protein 2 (SLP76) 293 27 60 3 tripartite motif containing 21(TRIM21) 277 21 54 hematopoietic lineage cell-specific protein 261 13 53

105

Table 3.1-1 continued: Mascot search results of mass spectrometric peptides sequencing of Tyr phosphorylated proteins upon FcγRI cross linking of THP-1 cells*

Band Phosphoproteins detected in anti-FcγRI cross-linked cells only Score Peptide matched MW beta-actin 254 10 42 tubulin beta chain 235 10 50 signal transducing adaptor molecule 2B (STAM2) 232 8 58 coronin, actin binding protein, 1C variant 230 7 53 alpha actinin (4) 227 7 103 docking protein 2 210 6 45 3 protein Tyr kinase or Syk 175 7 72 ARP3 actin-related protein 3 homolog 154 7 47 signal transducing adaptor molecule 1 (STAM1) 83 4 59 heat shock protein 70 kDa 93 3 70 E3 ubiquitin-protein ligase CBL (Ring finger protein 55) 97 5 99 unnamed human protein (IgG receptor Fc region II precursor) 78 3 35 alpha-tubulin 74 3 50 ARP3 actin-related protein 3 1338 61 47 actin non-muscle 6.2 504 19 48 mitogen-activated protein kinase 9 103 4 41 4 alpha actinin 4 95 3 102 actin 7 91 3 37 docking protein 2 79 3 45 signal transducing adaptor molecule 2B (STAM2) 75 3 58

106

Table 3.1-1continued: Mascot search results of mass spectrometric peptides sequencing of Tyr phosphorylated proteins upon FcγRI cross linking of THP-1 cells*

Band Phosphoproteins detected in anti-FcγRI cross-linked cells only Score Peptide matched MW POTE ankyrin domain family member E 835 38 35 actin related protein 2/3 complex subunit 1B (p41-ARC) 577 35 40 F-actin capping protein alpha-1 subunit 480 29 32 F-actin capping protein beta subunit (actin filament muscle Z-line) 208 4 30 clathrin light chain (LCB3) 170 8 23 histone cluster1, H1 83 8 23 5 capping protein alpha (actin filament muscle Z-line, alpha 2) 141 6 33 actin related protein 2/3 complex subunit 2 126 5 34 EF-hand domain family, member D2 100 5 27 protein tyrosine kinase (PTK9 or Twinfilin-1) 99 4 40 protein phosphatase 1 gamma 97 3 37 protein Tyr kinase (Syk) 80 4 72 E3 ubiquitin-protein ligase CBL 70 3 100 actin related protein 2/3 complex subunit 2 515 38 34 actin related protein 2/3 complex subunit 1B (p41-ARC) 434 17 41 F-actin capping protein beta subunit (actin filament muscle Z-line, beta) 307 15 31 EF-hand domain family, member D2 279 10 27 6 linker for activation of T cells family member 2 133 6 31 F-actin capping protein alpha-1 subunit 122 5 33 high affinity immunoglobulin gamma Fc receptor I 97 5 32 actin related protein 2/3 complex subunit 4 isoform a 96 4 20

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Table 3.1-1continued: Mascot search results of mass spectrometric peptides sequencing of Tyr phosphorylated proteins upon FcγRI cross linking of THP-1 cells*

Band Phosphoproteins detected in anti-FcγRI cross-linked cells only Score Peptide matched MW Crk-like protein 58 3 33 6 coatomer subunit epsilon 56 3 34 spectrin beta chain, non-erythrocyte 4 221 14 28 EF hand domain containing protein D2 311 17 26 7 clathrin light chain B 106 13 25 clathrin light chain A 91 8 27 protein tyrosine phosphatase, non-receptor type 18 81 3 50 alpha actinin 4 2158 101 102 actinin, alpha 1 1607 78 103 clathrin heavy chain 1 (or KIAA0034) 375 25 191 E3 ubiquitin-protein ligase CBL 354 13 100 splicing factor proline/glutamine rich 288 16 76 8 ubiquitin-40S ribosomal protein s27a 219 17 17 hepatocyte growth factor-regulated tyrosine kinase substrate 186 6 62 high affinity immunoglobulin epsilon receptor subunit gamma 179 5 10 protein Tyr kinase (p72 Syk) 95 4 96 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCG2) 82 3 14 *Peptides repeatedly detected/enriched in FcγRI cross-linked but not control IgG1 treatedTHP-1 cell lysates immunoprecipitated using anti-pTyr mAb (n=3)

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3.1.3 Ingenuity Pathway Analysis of Tyr phosphorylated proteins from FcγRI

cross-linked THP-1 cells identified by Nano LC-MS/MS

To examine potential signalling pathways enriched by antibody cross-linking of

FcγRI, all 80 hits were imported into the IPA software (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). The analysis predicted the clathrin-mediated endocytosis pathway as the most enriched signalling pathway (p = 2.19 x 10-13) (Figure 3.1-3A). The second most enriched pathway was Fc receptor-mediated phagocytosis in macrophage and monocytes (p = 4.11 x 10-13) (Figure 3.1-3A). The enriched Tyr-phosphorylated proteins associated with each pathway are listed in Figure 3.1-3B.

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Figure 3.1-3. Predicted signalling pathways upon cross-linking of FcγRI (A) Ingenuity pathway analysis of all 80 specific hits of the three experiments combined showed that clathrin-mediated endocytosis is the most enriched signalling pathway (p = 2.19 x 10-13), followed by FcγRI-mediated phagocytosis and integrin signalling with p= 4.11 x 10-13, and p = 1.88 x 10-9 respectively. (B) The Tyr phosphorylated and identified proteins associated with each pathway are listed.

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3.1.4 Validation of mass spectrometry results of Tyr phosphorylated proteins

involved in clathrin-mediated endocytosis by Western blotting

Most of the proteins identified are known to be involved in phagocytosis and/or clathrin-mediated endocytosis pathways including: Fc receptors γ chain, Syk, clathrin,

Cbl, HGS, STAM1/2, HSP70, TRIM21, actin, actin-related proteins, actinin-4, tubulin and actin binding proteins (Table 3.1-1 and Figure 3.1-3B). [184-187]. In particular, four of these including clathrin, Cbl, HGS and HSP70 have been reported to be involved in clathrin-mediated receptor endocytosis [195, 199, 200], strongly validating our data (Figure 3.1-3). The mass spectrometry result of Tyr phosphorylated proteins involved in clathrin-mediated endocytosis, the most enriched signalling pathway, was validated by Western blotting using appropriate antibodies including anti-clathrin, anti-

Cbl, anti-HGS and anti-HSP70. The presence of TRIM21 and FcγRs were also validated to serve the particular interest of this study and as a positive control respectively (Figure 3.1-4).

111

Figure 3.1-4. Validation of mass spectrometry data by Western blotting The presence of six Tyr phosphorylated proteins identified by mass spectrometry was validated by Western blot using specific antibodies including anti- clathrin, anti-Cbl, anti-HGS, anti-HSP70, anti-TRIM21 and anti-FcγRs. THP-1 cells cross-linked with anti-FcγRI or negative control mAb followed by immunoprecipitation using anti-pTyr showing enriched Tyr phosphorylation of clathrin, Cbl, HGS, HSP70, TRIM21 and FcγRs and strongly validating the mass spectrometry results. 112

3.1.5 Co-ligation of FcγRI with LILRB4 inhibited global Tyr phosphorylation of

multiple proteins

A 15 minute incubation with anti-FcγRI mAb followed by a 90 second cross- linking with secondary antibody in suspension (method described in Section 2.7.2 and

2.10.4) resulted in substantial Tyr phosphorylation of multiple proteins in total lysates of THP-1 cells, compared to cells treated with an isotype matched a negative control,

IgG1 (Figure 3.1-5A). The phosphorylation was further enriched in lysates immunoprecipitated using anti-pTyr mAb (Figure 3.1-5B). These results were consistent with those from the silver staining (Figure 3.1-2). Importantly, co-ligation of

FcγRI with LILRB4 markedly inhibited Tyr phosphorylation in total lysates (Figure

3.1-5A) and pTyr enriched immunoprecipitates (Figure 3.1-5B). Total lysates and immunoprecipitates from anti-LIRB4-only ligated cells showed no increase in Tyr phosphorylated proteins (Figure 3.1-5B).

113

A B

Figure 3.1-5. Detection of Tyr phosphorylated proteins in THP-1 cells by Western blotting using anti-Tyr polyclonal antibody after FcγRI cross-linking or co-ligation with anti- LILRB4 mAb (A) Total lysates from THP-1 cells cross-linked with anti-FcγRI and negative control mAb showed multiple strong immunoreactive bands in proteins ranging from 25 kDa to 150 kDa. Phosphorylation of most of these proteins (except for a 100 kDa protein(s)) was markedly reduced upon co-ligation with anti-LILRB4. Cells ligated with negative control or anti-LILRB4 mAb alone showed minimal Tyr-phosphorylation (n=2). (B) Immunoprecipitation of lysates with anti-pTyr mAb further enriched the immunoreactive bands in FcγRI and negative control co-ligated cells, but not in the FcγRI and LILRB4 co-ligated cells confirming specific regulation of Tyr phosphorylated proteins by LILRB4 (n=1). The bottom panels in A and the first half of B show the same membranes re-blotted with anti-β-actin antibody to confirm equal protein loading from the total cell lysates.

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3.1.6 LILRB4 significantly inhibited Tyr phosphorylation of multiple signalling

proteins related to clathrin-mediated endocytosis

The Tyr phosphorylated proteins that were markedly suppressed by LILRB4 were those newly identified by mass spectrometry namely: clathrin, HSP70, Cbl, HGS and

TRIM21 (Figure 3.1-3). This was shown firstly by pTyr immunoprecipitation of total lysates from the anti-FcγRI and anti-LILRB4 or anti-FcγRI and control IgG co-ligated cells, and followed by Western blotting using specific antibodies (method described in

Section 2.10.3). Western blot incubating using the common γ chain of the Fc receptors was used as a positive control for Tyr phosphorylation in response to FcγRI and its suppression after co-ligation with anti-LILRB4 [15]. Immunoprecipitates from anti-

FcγRI and control IgG co-ligated cells showed strong immunoreactive bands when blotted with anti-clathrin, anti-HSP70, anti-Cbl, anti-HGS and anti-TRIM21antibodies

(Figure 3.1-6A), further validating the mass spectrometry data. Importantly, co-ligation of FcγRI with LILRB4 markedly inhibited Tyr phosphorylation of all proteins except

HSP70 (Figure 3.1-6A). Semi-quantitative analysis indicated that LILRB4 significantly reduced Tyr phosphorylation of HGS by an average of 80.2%, Cbl by 51.3%, clathrin by 42.5% and TRIM21 by 36.9% (Figure 3.1-6B). In contrast, LILRB4 co-ligation significantly enhanced FcγRI-mediated Tyr phosphorylation of HSP70 by 36.4%, suggesting selective effects (Figure 3.1-6B).

115

116

Figure 3.1-6. Detection of Tyr phosphorylated proteins in THP-1 cells by Western blotting using specific antibodies after FcγRI cross-linking or co-ligation with anti-LILRB4 mAb (A) THP-1 cells co-ligated with anti-FcγRI and negative control IgG1 mAb or with anti-FcγRI and anti-LILRB4 followed by immunoprecipitation using anti-pTyr showed that LILRB4 significantly reduced Tyr phosphorylation of clathrin, Cbl, HGS and TRIM21, but enhanced HSP70. (B) Densitometry values from three independent experiments analysed by Student’s t- test (*p<0.05; **p<0.01; ***p<0.001). FcγRs was used as a positive control for Tyr phosphorylation in response to FcγRI and its inhibition after co-ligation with anti-LILRB4.

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As expected, the brief cross-linking/co-ligation protocol did not affect the total amounts of any of the above proteins (Figure 3.1-7A). Densitometry analysis from three independent experiments showed no significant difference between cells co-ligated with anti-FcγRI and IgG1, and anti-FcγRI and anti-LILRB4 (Figure 3.1-7B).

118

Figure 3.1-7. Detection of total proteins in THP-1 cells by Western blotting using specific antibodies after FcγRI cross-linking or co-ligation with anti-LILRB4 mAb (A) Total lysate from THP-1 cells co-ligated with anti-FcγRI and negative control IgG1 mAb or with anti-FcγRI and anti-LILRB4 showed no changes in total amounts of FcγRs, clathrin, HSP70, Cbl, HGS and TRIM21. The bottom panel shows the same membrane re-blotted with anti-β-actin antibody to confirm equal protein loading (B) Densitometry values from three independent experiments normalised to the level of β-actin.

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3.1.7 LILRB4 signals dephosphorylation of FcγRs, Cbl and HGS via its

intracellular Tyr based inhibitory motifs (ITIMs)

To determine whether the intracellular ITIM motifs of LILRB4 were involved in the regulation of Tyr phosphorylation of the above proteins, the wild type DNA construct of the LILRB4 ITIMs containing the three active Tyr residues (YYY) was stably transfected and expressed in THP-1 cells as described in Section 2.11.2. Cell lysates from FcγRI cross-linked transfected cells (YYY and a mock control) were immunoprecipitated with anti-Tyr mAb and Western blotted with antibodies specific to

FcγRs, clathrin, Cbl, HGS, HSP70 and TRIM21 (Section 2.10.3).

THP-1 cells over-expressing LILRB4 ITIMs (YYY) markedly inhibited Tyr phosphorylation of FcγRs, Cbl and HGS (Figure 3.1-8A). Semi-quantitative analysis indicated that YYY significantly reduced Tyr phosphorylation of FcγRs by 58.4%, Cbl by 39.4% and HGS by 30.7% compared to cells transfected vector alone however, there was no differences in Tyr phosphorylation of clathrin, HSP70 and TRIM21 (Figure

3.1-8B).

120

Figure 3.1-8. Detection of Tyr phosphorylated proteins in THP-1 cells over-expressing LILRB4 ITIMs (YYY) and a mock control after FcγRI cross-linking (A) THP-1 cells stably transfected with LILRB4 ITIMs (YYY) and a mock control cross-linked with anti-FcγRI followed by immunoprecipitation using anti-pTyr mAb showed that over- expressing LILRB4 ITIMs significantly reduced Tyr phosphorylation of FcγRs, Cbl and HGS, but had less effect on Tyr phosphorylation of clathrin, HSP70 and TRIM21. (B) Densitometry values from three independent experiments analysed by Student’s t-test (ns: no significant; *p<0.05; **p<0.01; ***p<0.001).

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3.1.8 Summary and conclusions

Fc receptors (FcRs) are key molecules in the recognition and elimination of foreign antigens through induction of multiple inflammatory mediators and antigen presentation [160]. FcγRI, also known as CD64, is a high affinity receptor that binds monomeric IgG and is expressed on mono-myeloid cells [161, 164]. Cross-linking of

FcγRI by immune complexes initiates activation of cellular responses as well as internalization of the receptor/ ligand/s [160, 162]. Tyr phospho-signalling mechanisms involved in FcγRI-mediated cell activation is known to lead to cytokine release and/or induction of oxidative bursts [163]. Importantly, it has been shown that LILRB4 potently inhibits FcγRI-mediated cytokine production by Tyr dephosphorylating

(deactivating) these key signalling kinases [2, 307]. In contrast, the signalling involved in FcγRI receptor internalization following cross-linking by immune complexes is less well defined. Clathrin-mediated receptor endocytosis is believed to be involved [160,

162, 184, 185], and likely controlled by phosphorylation and dephosphorylation mechanisms [184, 308, 309]. However, the identity of the signalling molecules controlled by Tyr phosphorylation, and what regulates their phosphorylation state remains poorly understood. Therefore, the first aim of this thesis was to globally identify Tyr phosphorylated proteins following FcγRI cross-linking, and to investigate whether their phosphorylation is regulated by LILRB4.

MASCOT search output identified 25 high confidence candidate phosphorylated proteins (Mowse score > 50, p 0.05), and input into the Ingenuity Pathway Analysis

(IPA) suggested that the most significant confidence candidate, Tyr phosphorylated proteins, are associated with the clathrin-mediated endocytosis pathway. The identities of six key proteins and their phosphorylation state were validated by a combination of

Western blotting and immunoprecipitation. It was confirmed that the common γ-chain

122 of the Fc receptor, clathrin, E3 ubiquitin protein ligase Cbl, hepatocyte growth factor- regulated tyrosine kinase substrate (HGS), heat shock protein 70 (HSP70) and tripartite motif-containing 21(TRIM21) were strongly Tyr phosphorylated in response to FcγRI cross-linking. Importantly, co-ligation of LILRB4 with FcγRI caused significant Tyr dephosphorylation of these proteins, with the exception of HSP70.

In conclusion, these results demonstrated that FcγRI cross-linking on monocytes causes Tyr phosphorylation of multiple proteins involved in clathrin-mediated endocytosis and importantly, that co-ligation of LILRB4 dephosphorylates these potential key molecules. LILRB4 may therefore regulate Fc-receptor-dependent monocyte functions such as immune-complex endocytosis and/or antibody opsonised pathogen phagocytosis, by modulating internalisation of the Fc receptor/ligand complexes via the clathrin-dependent pathway (Figure 3.1-9).

123

124

Figure 3.1-9. Schematic diagram demonstrating possible roles of Tyr phosphorylation of key molecules involved in clathrin-mediated endocytosis of FcγRI and ligands, and their regulation by LILRB4 Cross-linking of FcγRI by immune-complexes causes Tyr phosphorylation of the ITAMs of its common γ chain and Syk, and transduces activating signals. This simultaneously initiates phosphorylation of clathrin, which causes lateral diffusion of receptor-ligand complexes to clathrin-coated pits, membrane invagination and generation of clathrin-coated vesicles, and/or initiates phosphorylation of Cbl that may directly ubiquitinate the receptor. Phosphorylated Cbl triggers phosphorylation of HSP70 that facilitates un-coating of the vesicles, a precondition for vesicles to fuse with early endosomes and release ligands. The released receptors are transported to either the late endosome and/or lysosome for proteosomal and/or lysosomal degradation or are recycled to the cell surface. The immune complexes in the endosome are either directly degraded by Cbl, or are delivered to the lysosome by phosphorylated HGS-STAM 1/2 complex for final degradation. During transfer, the immune complexes may escape the endosome and are recognised by phosphorylated TRIM21 for proteasomal degradation. Co-ligation of FcγRI with LILRB4 may recruit phosphatases such as SHP-1 to its ITIMs that dephosphorylate (deactivate) the key molecules including clathrin ①, FcγRI and Syk ②, Cbl ③, HGS and STAM 1/2 ④ and TRIM21⑤. These effects may lead to inhibition of cellular activation and/or receptor- ligand endocytosis and degradation with potential clinical consequences.

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3.2 Regulation of FcγRI-mediated monocyte activation by LILRB4 depends on

the position of the Tyr residues in its ITIMs

3.2.1 Immunophenotyphic profiling of THP-1 cell lines

The relevant cell surface receptors including CD25 (IL-2Rα chain), CD122 (IL-

2Rβ chain), CD132 (IL-2Rγ chain), CD14, FcγRI, CXCR4, LILRB4 and TLR4 on

THP-1 cells were determined by flow cytometry. THP-1 cells do not express CD25 and

CD122, while express CD132, CD14, CXCR4, TLR4, FcγRI and LILRB4 (Figure

3.2-1). In addition, no endogenous CD25 in THP-1 cells was expressed upon activation of cells with LPS. THP-1 cells are known to signal via the common γ-chain of the Fc receptors and LILRB4 suppress activation through the recruitment of SHP-like phosphatase [15].

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Figure 3.2-1 Expression of receptors on the cell surface of THP-1 cells that are relevant to the functional assays and exclude bystander effects used in CD25-LILRB4 ITIMs chimeric constructs Representative flow cytometry analysis showing surface expression of CD25 (IL-2Rα), CD122 (IL-2Rβ), CD132 (IL-2Rγ), CD14, CXCR4, TLR4, FcγRI, LILRB4 on THP-1 cells in resting states, and endogenous CD25 on cells in activated states by LPS. The IL-2Rα, β and γ, and endogenous IL-2Rα in THP-1 cells were determined to prove that a THP-1 model is suitable for the study of CD25-LILRB4 ITIMs chimeric constructs. Although THP-1 cells express IL-2Rγ, the γ chain requires both IL-2Rα and β for signalling [310]. CD14 [311] and TLR4 [312] are known as LPS receptors, and CXCR4 [313] is known as HIV-1 co-receptor. These receptors were determined as relevant cell surface markers in functional assays regarding the regulation of LPS-induced TNF-α production by LILRB4 ITIMs and the regulation of FcγRI-induced TNF-α production by TAT-conjugated synthetic LILRB4 ITIMs respectively. Additionally, the presence of FcγRI and LILRB4 was determined to confirm that THP-1 cells have the signalling machinery required for identifying the function of the three ITIMs. Data from single experiment presented as mean fluorescence intensity (n=1).

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3.2.2 Quality controls of stable CD25-LILRB4 ITIM over-expressing THP-1 cells

THP-1 cells were stably transfected with plasmid DNA encoding all eight CD25-

LILRB4 ITIM chimeric receptors and one vector alone as a control, generating nine different transfected cell lines (method described in Sections 2.11.2 - 2.11.4). All the stably transfected cells consistently showed >94% viability (ranging from 94.5 ± 1.6 to

95.5 ± 1.2) with little or no difference among the different ITIM mutants (Table 3.2-1).

Table 3.2-1. Proportions of viable cells in stably transfected THP-1 cells as determined by LIVE/DEAD® assay (n=2) CD25-LILRB4 ITIMs Mean ± SEM (%)

FFF 95.5 ± 1.2

YYY 95.4 ± 1.2 YYF 95.4 ± 1.0

YFY 95.1 ± 1.0

FYY 95.5 ± 0.7

YFF 95.3 ± 1.3

FYF 94.5 ± 1.6

FFY 95.2 ± 1.1

Expression of the CD25-LILRB4 chimeras on the surface of transfected cells and a control was confirmed by flow cytometry prior to use. All CD25-LILRB4 ITIM- transfected cells expressed high levels of surface CD25 with comparable mean fluorescence intensities (MFI). These were 512.0 ± 80.3 (YYY), 781.7 ± 172.3 (FFF),

546.7 ± 158.1 (YYF), 647.3 ± 95.4 (YFY), 404.0 ± 74.5 (FYY), 802.7 ± 165.8 (YFF),

904.3 ± 223.5 (FYF) and 454.7 ± 32.4 (FFY), confirming successful transfection

(Figure 3.2-2). As expected, vector only transfected (mock) or non-transfected THP-1

128 cells did not express CD25-LILRB4 chimeras compared to an isotype-matched negative control, IgG1 (Figure 3.2-2).

Cell surface expression of FcγRI and native LILRB4 was determined by flow cytometry to ensure they were not affected by transfection. Stable over-expression of

CD25-LILRB4 ITIM chimeric proteins did not significantly alter surface expression of native FcγRI (Figure 3.2-3) or native LILRB4 (Figure 3.2-4), when compared to mock transfected or non-transfected cells. Similarly, Western blot analysis of SHP-1, SHP-2 and SHIP (known as key molecules in ITIMs-mediated regulation, although only SHP-1 has been shown to be involved in the inhibitory function of LILRB4) indicated that none of the construct(s) considerably altered the total amounts of these phosphatases, although the levels were variable: less monomer form of SHP-2 in cells over-expressing

YYF and high amounts of SHIP in cells over-expressing FYF (Figure 3.2-5).

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Figure 3.2-2. Expression of CD25-LILRB4 ITIMs on the cell surface of stably transfected THP-1 cells (A) Representative flow cytometry analysis showing surface expression of CD25-LILRB4 ITIMs on transfected cells. Similar levels of surface expression of CD25 were shown in the various CD25-LILRB4 ITIM mutant over-expressing cells; as expected no CD25 was expressed on vector only (mock)-transfected cells. (B) Data from four independent experiments presented as mean fluorescence intensity (MFI) ± SEM.

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Figure 3.2-3. Expression of FcγRI on the cell surface of stably transfected THP-1 cells (A) Representative flow cytometry analysis showing surface expression of FcγRI on transfected cells. Similar levels of surface expression of FcγRI in the various CD25-LILRB4 ITIM mutant over-expressing cells compared to vector only (mock)-transfected cells were shown indicating that stable over-expression of CD25-LILRB4 ITIM chimeric proteins did not significantly alter surface expression of native FcγRI. (B) Data from four independent experiments presented as mean fluorescence intensity (MFI) ± SEM.

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Figure 3.2-4. Expression of native LILRB4 on the cell surface of stably transfected THP-1 cells (A) Representative flow cytometry analysis showing surface expression of native LILRB4 on transfected cells. Similar levels of surface expression of native LILRB4 in the various CD25- LILRB4 ITIM mutant over-expressing cells compared to vector only-transfected cells indicating that none of the construct(s) significantly altered surface expression of native LILRB4. (B) Data from three independent experiments presented as mean fluorescence intensity (MFI) ± SEM.

132

Figure 3.2-5. Expression of cytosolic SHP-1, SHP-2 and SHIP in stably transfected THP-1 cells Representative Western blots of SHP-1, SHP-2 and SHIP in total cell lysates from CD25- LILRB4 ITIM mutants and vector only-transfected cells. Over-expression of CD25-LILRB4 ITIM chimeric proteins did not alter the total amount of these phosphatases. In particular, less difference was shown in the level of SHP-1 among transfected cells. The bottom panels show the same membranes re-blotted with anti-β-actin antibody to confirm equal protein loading from the total cell lysates (n=2).

3.2.3 Cell proliferation was supressed by over-expressing the middle Tyr389 but

enhanced by the distal Tyr419 of LILRB4 ITIM

All transfected cells exhibited robust baseline proliferation reflected by high 3H- thymidine incorporation ranging from 1 to 2 x 105 cpm allowing valid comparisons

(Figure 3.2-6A). Cells transfected with FYY and FYF exhibited significantly reduced proliferation by 35.2 ± 0.8% and 37.5 ± 3.9% compared to mock transfected cells

(Figure 3.2-6B). In contrast, cells over-expressing FFY were 28.0 ± 9.2% higher than mock transfected cells (Figure 3.2-6B) Proliferation of other cells over-expressing

CD25-LILRB4 ITIM including FFF, YYY, YYF, YFY and YFF was variably reduced by 3.7% compared to mock transfected cells (method described in Section 2.13.2).

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Figure 3.2-6. Baseline cell proliferation in stably transfected THP-1 cells (A) 3H-thymidine incorporation assay in mock and CD25-LILRB4 ITIM transfected cells. There was a significantly lower baseline proliferation of cells over-expressing the single intact middle Tyr389 (FYF) and the combination of middle Tyr389 and distal419 (FYY). A higher proliferation was seen in cells over-expressing the single distal Tyr419 (FFY) when compared to mock transfected cells (n=4; *p<0.05; **p<0.01). (B) A summary of percentage inhibition or enhancement of baseline cell proliferation by various CD25-LILRB4 ITIM over-expressing cells relative to mock transfected cells; YYY and FFY showed increased proliferation while all the other mutants showed variable increase in cell proliferation.

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3.2.4 Over-expression of the middle Tyr389 of LILRB4 ITIMs increased early

apoptosis

Transfected cells were stained with Annexin V-FITC and propidium iodine (PI) to ascertain the proportion of cells undergoing early apoptotic cell death, in addition to detecting cells that were already undergoing apoptosis or necrosis, (method described in

Section 2.13.3) allowing valid comparisons (Figure 3.2-7A). Different stages of apoptosis were apparent in all CD25-LILRB4 ITIM over-expressing cells, with early apoptotic cells (Annexin V positive, PI negative) ranging from 1.8-7.7% and late apoptotic cells (Annexin V positive, PI positive) ranging from 3.7-5% (Figure 3.2-7A and B). These values were 2 to 8 and 1.4- to 2-fold higher than mock transfected cells for early and late apoptosis respectively (Figure 3.2-7B). There was a significantly higher percentage of early apoptosis in cells over-expressing FFF, YYF and FYF mutants when compared to mock transfected cells (Figure 3.2-7B). However, increases in the proportion of late apoptosis was significant only in FFF mutant over-expressing cells when compared to mock transfected cells (Figure 3.2-7B). The level of necrotic cells (≤1.5%) in all transfected cells was consistent with data generated using the

LIVE/DEAD® assay kit (Table 3.2-1).

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Figure 3.2-7. Baseline apoptosis in stably transfected THP-1 cells (A) Representative flow cytometry of transfected cells co-stained with Annexin V-FITC and Propidium iodide (PI). (B) Quantification of cells undergoing early or late apoptosis showed variable increase in apoptosis in various CD25-LILRB4 ITIM mutants compared to mock transfected cells; the most significant increase was observed in cells over-expressing the middle

Tyr389, FYF (n=4; *p<0.05; **p<0.01; ***p<0.001, # p<0.05 compared to corresponding mock).

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3.2.5 Over-expression of CD25-LILRB4 ITIMs differentially modulated FcγRI-

mediated TNF-α production

FcγRI cross-linking on THP-1 cells and monocytes from peripheral blood induces

TNF-α production, whilst co-ligation of FcγRI with LILRB4 supresses TNF-α production in both cells by 50-60% and 40-70% respectively [15]. To investigate the effects of ITIM mutations on LILRB4-mediated inhibition of TNF-α production, FcγRI on transfected cells was co-ligated with CD25-LILRB4 ITIM chimeric receptors

(method described in Section 2.7.3), and TNF-α production was determined. All CD25-

LILRB4 ITIM expressing cells and vector only transfected cells produced TNF-α, ranging from 3.8 ± 1.1 pg/ml (FYF) to 150.1 ± 27.6 pg/ml (YFF) upon co-ligation of

FcγRI and CD25, but produced ≤ 2.3 pg/ml when co-ligated with control IgG1

(background control, not shown) (Figure 3.2-7A) (Figure 3.2-8A). As expected, FcγRI- mediated TNF-α production upon co-ligation with CD25 was strongly reduced in cells over-expressing ITIMs with three functional Tyr residues (YYY) by 92.4 ± 4.7% compared to mock transfected cells (Figure 3.2-8B). Interestingly, cells over-expressing the middle Tyr389 (FYF) maximally inhibited TNF--α production by 96.7 ± 3.3%

(Figure 3.2-8B). The single intact distal Tyr419 (FFY) was also involved in limiting

TNF-α production by 80.5 ± 5.2% albeit somewhat less than that achieved with the mutant FYF or YYY (Figure 3.2-8B). In contrast, cells over-expressing the proximal

Tyr317 (YFF) exhibited higher FcγRI-mediated TNF-α production by 50.4 ± 14.2% compared to mock transfected cells (Figure 3.2-8B). Consistent with the opposing effects of the proximal Tyr317 and the middle Tyr389, a mutant with both functional Tyr residues (YYF) showed TNF-α inhibition of 19.7 ± 21.7%, markedly lower than the

96.7% suppression observed when only the middle Tyr389 was intact, but displayed less activation than the proximal Tyr317 alone (Figure 3.2-8B). Surprisingly, co-ligation of

137

FcγRI and CD25 on cells transfected with LILRB4 ITIMs without three Tyr residues

(FFF) partially reduced TNF-α production by up to 38.0 ± 12.9% compared to mock transfected cells (Figure 3.2-8B).

Figure 3.2-8. Detection of TNF-α production after co-ligation of FcγRI with CD25 in stably transfected THP-1 cells (A) TNF-α production in transfected cells over-expressing various CD25-LILRB4 ITIM mutants co-ligated with FcγRI and CD25 was differentially regulated compared to mock- transfected cells (n=3; *p<0.05; **p<0.01 compared to corresponding mock). (B) A summary of percentage inhibition or enhancement of TNF-α production by CD25-LILRB4 ITIM over- expressing cells relative to mock-transfected cells; YYY and FYF showed the most inhibition while YFF showed marked enhancement. 138

Interestingly, similar effects were observed after FcγRI cross-linking alone, although co-ligation with CD25 substantially amplified the inhibitory or activating effects of the LILRB4 ITIMs (Figure 3.2-9). All CD25-LILRB4 ITIM over-expressing cells and vector only (mock) transfected cells produced TNF-α ranging from 3.2 ± 6.2 pg/ml (FYF) to 75.9 ± 17.8 pg/ml (YFF) upon FcγRI cross-linking, but produced ≤ 2.3 pg/ml when co-ligated with control IgG1 (background control, not shown) (Figure

3.2-9A). Consistently, cross-linking of FcγRI showed strongly reduced TNF-α production in cells over-expressing LILRB4 ITIMs with three Tyr residues (YYY), by

85.4 ± 4.4% compared to mock (Figure 3.2-9B). In addition, cross-linking of FcγRI on transfected cells also showed that the middle Tyr389 (FYF) maximally suppressed TNF-

α by 95.8 ± 1.1%, while cells expressing the proximal Tyr317 (YFF) and the proximal

Tyr317 with the middle Tyr389 (YYF) enhanced FcγRI-mediated TNF-α production by

8.4 ± 18.3% and 6.2 ± 31.2% respectively compared to mock transfected cells (Figure

3.2-9B). Similarly, the transfected cells YFY, FYY and FFY were also involved in limiting TNF-α production by 82.9 ± 3.5% , 76.9 ± 8.1% and 55.0 ± 2.9% respectively

(Figure 3.2-9B). Although substantially amplifying their inhibitory effects, this cross- linking of FcγRI-induced TNF-α was consistent with the effect in co-ligation of FcγRI and CD25, with the exception of YYF (Figure 3.2-8 and Figure 3.2-9).

139

Figure 3.2-9. Detection of TNF-α production after FcγRI cross-linking in stably transfected THP-1 cells (A) Transfected cells over-expressing various CD25-LILRB4 ITIM mutants cross-linked with FcγRI differentially regulated TNF-α production compared to mock-transfected cells (n=3; *p<0.05; **p<0.01compared to corresponding mock). (B) A summary of percentage inhibition or enhancement of TNF-α production by CD25-LILRB4 ITIM over-expressing cells relative to mock transfected cells; YYY and FYF showed highest inhibition, while YYF and YFF showed marked enhancement.

140

3.2.6 Over-expression of CD25-LILRB4 ITIMs suppressed FcγRI-mediated

bacterial phagocytosis

Phagocytosis is a process characterized by uptake of particles such as bacteria, and FcγRI is one of the key receptors for recoging IgG opsonised pathogens. To determine the effects of ITIM mutations on LILRB4-mediated regulation of FcγRI- mediated bacterial phagocytosis, human serum opsonised and pHrodo labelled S. typhimurium were incubated with each transfected cell line, and the levels of cells taking up bacteria were analysed using flow cytometry (Figure 3.2-10A) (method described in Section 2.14).

Flow cytometry analysis showed that 4.3 ± 0.1% of mock-transfected-PMA- differentiated THP-1 cells phagocytised serum opsonised bacteria (Figure 3.2-10B).

Phagocytosis was significantly reduced in all transfected cells over-expressing CD25-

LILRB4 ITIM mutants compared to vector alone transfected cells (Figure 3.2-10C and

D). Bacterial phagocytosis in cells over-expressing FYY was 72.8 ± 6.7% less than that observed in mock transfected cells, whereas cells containing the YFF mutant were relatively more responsive but exhibited 45.9 ± 12.1% of the uptake seen in mock transfected cells (Figure 3.2-10C and D). Unexpectedly, the ITIM backbone alone with the three Tyr mutated cells (FFF) also maximally suppressed phagocytosis (by 73.9 ±

9.5% compared to mock) (Figure 3.2-10C and D).

141

A B

C D

142

Figure 3.2-10. FcγRI-mediated bacterial phagocytosis in PMA-differentiated stably transfected THP-1 cells (A) Lyophilized S. typhimurium was labelled with pHrodo dye followed by IgG opsonisation and incubation with macrophage-like transfected cell for 90 min at 37ºC. (B) Representative flow cytometry of PMA-differentiated mock or CD25-LILRB4-FFF transfected cells incubated with media only (upper panels) or IgG-opsonised-pHrodo labelled S. typhi (lower panels); upper left quadrants show percentage of bacteria positive cells; insets show red fluorescent phagocytised bacteria inside the cells. (C) Quantification of cells taking up opsonised bacteria by phagocytosis showed significantly lower phagocytosis in the various CD25-LILRB4 ITIM mutants compared to mock transfected cells (n=4; *p<0.05; **p<0.01; ***p<0.001). (D) A summary of percentage inhibition of bacterial phagocytosis in CD25-LILRB4 ITIM over- expressing cells relative to mock transfected cells showing similar inhibition by all clones including FFF; YFF showed the least inhibition.

3.2.7 All transfected cells except for cells over-expressing LILRB4 ITIMs with

the middle Tyr389 (FYF) showed increased phagocytosis of IgG-coated

beads

To determine the effects of ITIM mutations on LILRB4-mediated regulation of

FcγRI-mediated phagocytosis, IgG-coated-Alexa488-labelled beads were incubated with each transfected cell line, and the levels of cells taking up the beads were analysed using flow cytometry (Figure 3.2-11A) (method described in Section 2.15). The mock- transfected-PMA-differentiated THP-1 cells took up beads at 7.9 ± 0.7% (Figure

3.2-11B). This was lower than the 10.9 ± 0.8% found in wild type ITIMs (YYY) transfected cells, or the 10-13% observed in cells transfected with ITIMs containing two intact Tyr residues (YYF, YFY or FYY), or cells transfected with ITIMs containing the single intact proximal Tyr337 (YFF), or the single intact distal Tyr419 (FFY) (Figure

3.2-11B). The uptake of beads by these transfected cells was 1.3- to 1.6-fold higher than the mock transfected cells (Figure 3.2-11C). In contrast, only 6.3 ± 1.3% of cells transfected with ITIMs containing the single middle Tyr389 (FYF) phagocytosed beads 143

(Figure 3.2-11B) which is 1.4-fold lower than the bead uptake by mock transfected cells

(Figure 3.2-11C).

A B

Figure 3.2-11. Phagocytosis of IgG-coated polystyrene beads by CD25-LILRB4 ITIMs transfected PMA-differentiated THP-1 cells (A)PMA-differentiated transfected cells took up IgG-coated polystyrene beads after 60 min incubation at 37°C, 5% CO2 (B) Flow cytometry showing proportion of beads phagocytosis in stably transfected cell (n=2, mean ± SEM). (C) A summary comparison of bead phagocytosis (phagocytic index) showing 1.3- to 1.6-fold increase of CD25-LILRB4 ITIM mutants, except for FYF; no effects were observed in cells transfected with FFF relative to mock transfected cells. 144

3.2.8 Over-expression of CD25-LILRB4 ITIMs differentially regulated

bactericidal activity

To determine the effects of ITIM mutations on LILRB4-mediated regulation in

FcγRI-mediated bactericidal activity, serum-opsonised S. typhi was incubated with each transfected cell line and killing activity was analysed using colony forming units

(Figure 3.2-12A) (method described in Section 2.16). Cell lysates from mock- transfected-PMA-differentiated cells that were infected with serum-opsonised S. typhi formed an average of 117.5 ± 10 colonies (Figure 3.2-12B). Transfected cells over- expressing YYY, FYY, YFY or FYF formed 87.8 ± 6.1, 78.4 ± 5.4, 86.1 ± 17.1 and

66.9 ± 2.1 colonies respectively, but these were not statistically significant when compared to mock transfected cells (Figure 3.2-12B). However, cells transfected with

YYF or FFY formed 40.6 ± 0.4 and 33.4 ± 0.9 colonies respectively that represented significant increases in bactericidal activity compared to mock transfected cells (Figure

3.2-12B). In contrast, cells over-expressing a single proximal Tyr337 (YFF) significantly suppressed bactericidal activity as reflected by the formation of higher number colonies compared to mock transfected cells (143.6 ± 17 versus 117.5 ± 10) (Figure 3.2-12B).

These demonstrated that cells over-expressing YYY, YYF, FYY, YFY, FYF or FFY variably increased bactericidal activity by up to 70% (range 24.5-71.3%) as compared to mock transfected cells (Figure 3.2-12C). In contrast, cells over-expressing YFF significantly suppressed bactericidal activity by up to 40% (Figure 3.2-12C).

Interestingly, cells over-expressing the LILRB4 intracellular domains without the three

Tyr residues (FFF) also substantially enhanced bactericidal activity by 30.4 ± 0.4%, but this was not statistically significant (Figure 3.2-12B and C).

145

B C

146

Figure 3.2-12. Bactericidal activity by PMA-differentiated-stably transfected THP-1 cells (A) S. typhi was opsonised using human serum and incubated with PMA-differentiated transfected cells for 90 min at 37ºC. Extracellular bacteria were killed by adding gentamycin, and bacterial colonies formed in LB agar plate were counted. (B) Quantification of intracellular viable S. typhi was measured, and the number of colonies was adjusted to the phagocytic index of each transfected cell line. The lower number of colony formations compared to mock represented increased bactericidal activity (n=5; *p<0.05; **p<0.01). (C) A summary of percentage enhancement or inhibition of bacteria killed by CD25-LILRB4 ITIM over- expressing cells relative to mock transfected cells showing that all transfected cells except for YFF increased bactericidal activity up to 70%.

3.2.9 Preliminary results exploring the potential use of synthetic LILRB4 ITIM

peptides to modulate FcγRI-mediated TNF-α production

Four synthetic peptides including a wild type of three LILRB4 ITIM (TAT-YYY), three LILRB4 ITIM motifs without tyrosine residues (TAT-FFF), the middle LILRB4

ITIM motif (TAT-FYF), and TAT peptides alone (TAT) as a control were used to identify the function of LILRB4 ITIM(s), and to determine its potential as a therapeutic agent. Synthetic LILRB4 ITIM peptides marginally inhibited TNF-α production in

FcγRI-mediated THP-1 and PBMCs activation. The peptide containing the middle ITIM of LILRB4 pre-treated THP-1 cells maximally inhibited TNF-α production up to 40.35% compared to non-peptide treated cells (Figure 3.2-13A). However, the control (TAT alone) was also able to inhibit TNF-α production at 10μM concentration of peptide treatment (Figure 3.2-13A). Similarly, the middle LILRB4 ITIM was able to suppress

TNF-α production in FcγRI-activated PBMCs, but the TAT-peptide, a negative control, also inhibited TNF-α production (Figure 3.2-13B). Also, FcγRI-mediated TNF-α production in THP-1 cells and PBMCs pre-incubated with 20 μM of each peptide showed no difference in TNF-α production between cells treated with peptides

147 containing LILRB4 ITIM(s) and a negative control (TAT) (Figure 3.2-13C) (method described in Sections 2.17 and 2.8).

148

Figure 3.2-13. Detection of FcγRI-mediated TNF-α production in THP-1 and PBMCs after treatment of synthetic LILRB4 ITIM peptides THP-1 cells and PBMCs cross-linked with FcγRI after incubation with each peptide for 30 min induced different levels of TNF-α production. (A) THP-1 cells cross-linked with FcγRI after incubation with different concentrations of peptides showed that cells incubated with 20 μM synthetic LILRB4 ITIM peptides suppressed TNF-α production compared to cells incubated with TAT peptide or without peptide (n=1). (B) PBMCs cross-linked with FcγRI after incubation with different concentrations of peptides showing that all cells incubated with each peptide suppressed TNF-α production compared to cells incubated without peptide (n=1). (C) FcγRI-mediated TNF-α production in THP-1 cells and PBMCs pre-incubated with 20 μM peptides showed no significant effect of LILRB4 ITIM(s) on TNF-α production compared to a control, TAT (n=2).

3.2.10 Summary and conclusions

Pairing of immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoreceptor tyrosine-based activating motifs (ITAM)-containing receptors is postulated as a central paradigm that regulates the threshold and amplitude of leukocyte activation [4, 9, 22]. Upon co-ligation with an ITAM-containing activating receptor, Tyr residues in one or more ITIMs of an inhibitory counterpart are phosphorylated by Src family protein Tyr kinases recruited to the ITAMs [2, 15, 108]. Phosphorylated ITIMs act as substrates for Src homology 2 domain (SH2)-containing phosphatases (SHPs,

SHIP) that terminate signals by dephosphorylating various Tyr kinases involved in activation [2, 15, 41, 108]. However, this paradigm is challenged by recent findings demonstrating that under certain circumstances, ITIMs can mediate activation (termed

ITIMa) and ITAMs can mediate inhibition (ITAMi) [22, 256, 257, 314]. Moreover,

ITIM-like motifs (YVKM) [23], immunoreceptor Tyr-based switch motifs (ITSMs,

TxYxxI/V) [24], SH3 binding domains [25, 26] and proline-rich regions in the intracellular domains of ITIM-containing proteins [27, 28], contribute to the overall

149 function of these receptors. This indicates that receptors bearing these consensus motifs may have the capacity to transduce complex regulatory signals.

LILRB4 uniquely contains three ITIMs with sequences 335VTYAKY340,

387VTYAQL392 and 417SVYATL422 [307]. However, it is not clear which of these motifs and their Tyr residues are responsible for the inhibitory function of LILRB4. Thus, the second aim of this thesis was to identify the contribution of these three LILRB4 ITIMs depending on the position of Tyr residues and stimuli in FcγRI-mediated TNF-α production, bacterial phagocytosis and bactericidal activity. To address this, Tyr residues at positions 337, 389 and 419 were single, double or triple mutated to phenylalanine and stably transfected into THP-1 cells. Interestingly, transfected cells over-expressing a mutant with single intact middle Tyr389 (FYF) significantly reduced baseline cell proliferation, but markedly enhanced early apoptosis. Additionally, functional assays including FcγRI-mediated TNF-α production, phagocytosis and bactericidal activity indicate that LILRB4 has complex immune regulatory roles depending on the position of the functional tyrosine residues within the three ITIMs

(Table 3.2-2).

Table 3.2-2. Effect of CD25-LILRB4 ITIMs in monocyte functions relative to mock transfected cells CD25-LILRB4 TNF-α production Bead phagocytosis Bactericidal activity ITIM construct FFF ↓↓ - ↑ YYY ↓↓↓ ↑↑ ↑ YYF - ↑ ↑↑ YFY ↓↓↓ ↑↑ ↑↑ FYY ↓↓↓ ↑↑ ↑ YFF ↑↑ ↑↑ ↓↓ FYF ↓↓↓ ↓ ↑ FFY ↓↓↓ ↑↑ ↑↑↑ ↑, < 33.3%; ↑↑, 33.3 to 66.7%; ↑↑↑, > 66.7% (↑: increase) ↓, < -33.3%; ↓↓, -33.3 to -66.7%; ↓↓↓, > -66.7% (↓: decrease) 150

Intact middle Tyr389 (FYF) alone was sufficient to maximally inhibit FcγRI- mediated TNF-α production, but paradoxically, proximal Tyr337 (YFF) significantly enhanced TNF-α production. In contrast, bactericidal activity was significantly enhanced in transfected cells over-expressing LILRB4 ITIM with the middle Tyr389 or distal Tyr419, while intact proximal Tyr337 (YFF) markedly inhibited bacteria killing.

Interestingly, all transfected cells containing one or more intact Tyr residues variably increased IgG-coated polystyrene beads phagocytosis, except for the transfected cells containing single intact middle Tyr389 (FYF), which marginally suppressed it.

In conclusion, results presented here suggested that LILRB4 has dual inhibitory and activating functions in monocytes and macrophages depending on the nature of the stimuli and the position of the functional Tyr residues in its ITIMs.

151

3.3 Regulation of LPS-mediated monocyte activation by LILRB4

3.3.1 Over-expression of CD25-LILRB4 ITIMs differentially modulated LPS-

mediated TNF-α production

All transfected cells stably over-expressing CD25-LILRB4 ITIM chimeric proteins did not demonstrate significantly altered surface expression of native TLR4 when compared to mock transfected cells (Figure 3.3-1A and B).

Figure 3.3-1. Expression of TLR4 on the surface of stably transfected THP-1 cells (A) Representative flow cytometry analysis showing similar levels of surface expression of TLR4 on transfected cells over-expressing various CD25-LILRB4 ITIM mutants. (B) Data from three independent experiments presented as mean fluorescence intensity (MFI ± SEM) (ns: no significance). 152

To investigate the effects of ITIM mutations on LILRB4-mediated regulation in

LPS-mediated monocyte activation, cytokine production including IL-6, IL-10 and

TNF-α were firstly screened using Luminex analyser (MAGPIX®), and the TNF-α concentration was further confirmed using ELISA. All CD25-LILRB4 ITIM over- expressing cells and vector only (mock) transfected cells produced TNF-α ranging from

51.49 pg/ml (YYY) to 2371 pg/ml (YYF) upon LPS stimulation (Figure 3.3-2A). In contrast, low levels of IL-6 and IL-10 were produced in all transfected cells (Figure

3.3-2A). A consistent result was shown in LPS-mediated TNF-α production using

ELISA (Figure 3.3-2B).

All transfected cells except for YYF inhibited LPS-mediated TNF-α production compared to mock transfected cells. The lowest level of TNF-α was shown in cells over-expressing ITIMs with three functional Tyr residues (YYY) at 32.1 pg/ml upon

IgG+LPS and 28.7 pg/ml upon CD25+LPS. Contrastingly, minimal inhibition was shown in cells over-expressing the middle Tyr389 (FYF) at 433.5 pg/ml upon IgG+LPS, and 216 pg/ml upon CD25+LPS compared to mock transfected cells at 657.7 pg/ml upon IgG+LPS and 546.7 pg/ml upon CD25+LPS. Cells over-expressing both the proximal Tyr317 and the middle Tyr389 (YYF) exhibited enhanced TNF-α production at

1522.3 pg/ml upon IgG+LPS and at 955.8 pg/ml upon CD25+LPS compared to mock transfected cells.

Interestingly, cells transfected with LILRB4 intracellular domains without three

Tyr residues (FFF) produced TNF-α at 40.3 pg/ml upon IgG+LPS and at 39.8 pg/ml upon CD25+LPS. Similar to the results of FcγRI-mediated TNF-α (Section 3.2.5), stably transfected cells over-expressed CD25-LILRB4 ITIMs, and although inducing similar effects on cell activation without co-ligation of CD25, in the results showed

153 substantially inhibited TNF-α in certain mutants, including YYF and FYF (Figure 3.3-2)

(method described in Sections 2.7.1 - 2.8).

Figure 3.3-2. LPS-mediated cytokine production in stably transfected THP-1 cells (A) LPS-mediated IL-6, IL-10 and TNF-α production in transfected cells after CD25 cross- linking showing that only the production of TNF-α was highly regulated by transfected cells (n=1). (B) TNF-α production was confirmed by ELISA showing that all transfected cells, except YYF, inhibited LPS-mediated TNF-α production compared to mock transfected cells. Although co-ligation of CD25 substantially amplified effects (black histograms), in this setting, CD25 co- ligation had no significant impact on LILRB4 ITIM-mediated regulation (white histograms) (n=2, mean ± SEM).

154

Cells transfected with various CD25-LILRB4 ITIM mutants differentially regulated LPS-mediated TNF-α production without CD25 co-ligation (Figure 3.3-3A).

All CD25-LILRB4 ITIM over-expressing cells and vector only (mock) transfected cells produced LPS-mediated TNF-α ranging from 51.6 ± 11.7 pg/ml (FFF) to 1139 ± 219 pg/ml (YYF) upon LPS stimulation (Figure 3.3-3A). Cells over-expressing LILRB4

ITIM with the single intact proximal Tyr317 (YFF) and the middle Tyr389 (FYF) showed less inhibition of TNF-α production, but with both functional Tyr residues (YYF) present TNF-α production was enhanced by up to 80.8 ± 28.5% (Figure 3.3-3B).

Interestingly, cells transfected with non-functional Tyr residues (FFF) significantly reduced TNF-α production by up to 92.0 ± 1.4% compared to mock-transfected cells

(Figure 3.3-3B).

155

Figure 3.3-3. Detection of LPS-mediated TNF-α production in stably transfected cells (A) Cells over-expressing various CD25-LILRB4 ITIM mutants differentially regulated LPS- mediated TNF-α production depending on the position and the presence of the Tyr residues (n=4; *p<0.05; **p<0.01 compared to corresponding mock). (B) A summary of percentage inhibition or enhancement of TNF-α production by CD25-LILRB4 ITIM over-expressing cells relative to mock transfected cells; cells transfected with three functional Tyr residues (YYY) and with non-functional Tyr residues (FFF) showed the most inhibition, while YYF showed enhancement. 156

3.3.2 Two different signalling pathways are involved in the regulation of LPS-

mediated cell activation by LILRB4

Western blotting was used to identify whether the downstream signalling pathways which induce TNF-α, such as NFκB and MAPK pathways, were regulated by

LILRB4 depending on the presence of the Tyr residue of its ITIMs. There were no differences in the levels of phosphorylated and total proteins of Mek1/2, p38 and Erk upon LPS stimulation between transfected cells over-expressing FFF and YYY, or mock transfected cells (Figure 3.3-4A). However, increased p105 was shown in cells over-expressing LILRB4 ITIM without Tyr residues (FFF), while NFκB including p65 and p50 was not affected (Figure 3.3-4B). In addition, phosphorylation of Akt was inhibited in cells over-expressing LILRB4 ITIMs with three intact Tyr residues (YYY)

(Figure 3.3-4C).

157

B C

D E

158

Figure 3.3-4. Detection of the level of intracellular signalling proteins involved in NFκB and MAPK pathways in transfected cells Transfected cells over-expressing YYY, FFF and Mock were stimulated with 1 μg/ml of LPS for 1 hr and key molecules including Mek1/2, p38 and Erk in MAPK pathway, and NFκB, p105 and Akt in NFκB pathway were determined by Western blotting. (A) No differences in the levels of phosphorylated and total Mek1/2, P38 and Erk were shown among transfected cells. (B) Cells over-expressing FFF increased p105, while no differences in total amount of p65 and p50 among transfected cells. The bottom panel shows the same membrane re-blotted with anti-β- actin antibody to confirm equal protein loading. (C) Densitometry analysis of three independent experiments of B showing significant enhancement of p105 in cells over-expressing FFF (***p<0.001). (D) Cells over-expressing YYY inhibited phosphorylation of Akt, but no differences in total amount of Akt compared to transfected cells with FFF and mock. (E) Densitometry analysis of three independent experiments of D showing inhibition of pAkt in cells over-expressing YYY.

3.3.3 Summary and conclusions

Toll-like receptors (TLRs) are type I transmembrane proteins, which play a key role in host defence against pathogens through activation of the innate immune systems

[315, 316]. Ligation of individual or aggregated TLRs, induce NFκB or MAPK activation resulting in gene transcription is associated with inflammatory cytokines, nitric oxide, co-stimulatory molecules and MHC classes to defend against pathogens

[159, 317]. TLR4 is expressed on monocytes and macrophages [159], and plays a key role in innate immune responses by recognizing gram negative bacterial lipopolysaccharides (LPS) [205, 207]. It is well known that multiple Tyr kinases including Syk, Lyn, Btk and Hck, are involved in TLR4-mediated intracellular signalling pathways [205, 208, 209].

A recent study showed that Salmonella-infected macrophages up-regulated

LILRB4, and co-ligation of LILRB4 on macrophages induced anti-inflammatory cytokine, such as IL-10 [19]. However, the exact mechanism of this regulatory function

159 of LILRB4 has yet to be fully elucidated. In addition, the regulatory functions of

LILRB4 paring with non-ITAM associated activating receptors, including TLR4, is less researched. It is, however, well known that NFκB and MAPK pathways are involved in

TLR4-mediated signalling pathways [315], which suggests that TLR4 is also a suitable model for identifying the regulatory function of LILRB4 in the ITAM independent pathway. Thus, the third aim of this thesis was to identify the regulatory function of

LILRB4 in LPS (or TLR4)-mediated TNF-α production, and its signalling pathways depending on the Tyr residues in its ITIMs.

The results indicate that cells over-expressing various CD25-LILRB4 ITIM mutants differentially regulated LPS-mediated TNF-α production depending on the position of Tyr residues of its ITIMs. The three ITIMs of LILRB4 (YYY) were a potent inhibitor of TLR4-mediated TNF-α production and surprisingly, LILRB4 ITIM backbones without functional Tyr residues (FFF) also significantly inhibited TNF-α production in response to LPS. However, these two transfected cell lines differentially regulated mRNA expression of multiple genes, including IL-1β and IL-12β (Appendix

III C), suggesting that distinct signalling pathways are involved in inhibiting TNF-α production. This result regarding differential regulations depending on the presence of

Tyr residues in ITIMs of LILRB4 was validated by Western blotting, using multiple antibodies to detect specific proteins associated with the pathway involved in TNF-α production. Cells over-expressing three intact ITIMs of LILRB4 (YYY) inhibited phosphorylation of Akt, which is activated by PI3K and induces TNF-α production

(Figure 3.3-5). In contrast, cells over-expressing LILRB4 ITIMs without Tyr residues

(FFF) may inhibit proteolysis of p105 to be p50, which is translocated for gene transcription and TNF-α production. Therefore, further study will be required to

160 determine how the intracellular domains of LILRB4 without Tyr resides inhibit p105 proteolysis in LPS-mediated cell activation.

In conclusion, the results presented here suggest that intracellular LILRB4 inhibits

LPS-mediated TNF-α production through IKK- or PI3K-dependent pathways, depending on the presence of the Tyr residues of its ITIMs.

161

Figure 3.3-5. Schematic diagram demonstrating TLR4-mediated cell activation and its regulation by LILRB4 Upon LPS binding to TLR4, TRIF-dependent and MyD88-dependent pathways are activated and induce inflammatory cytokines by activation of IRF3, MAPKs and NFκB translocation. However, the three ITIMs of LILRB4 may recruit Tyr phosphatase, especially SHP-1, upon TLR4-mediacted cellular activation that supresses TNF-α production via de-phosphorylation of PI3K, Akt and other down-stream signalling proteins. In contrast, LILRB4 ITIMs without Tyr resides may inhibit p105 degradation to generate p50 and lead to inhibition of gene transcription and TNF-α production.

162

CHAPTER 4. PRELIMINARY SCREENING FOR LILRB4

LIGANDS

4.1 Introduction

LILRB4 is an immune regulatory cell surface receptor, primarily expressed on mono-myeloid cells [19, 85]. Expression of LILRB4 has strong clinical associations with diseases where fine control of the immune response is vital to prevent harm to the host while mounting effective responses, including infections and cancers [19, 85, 318,

319]. Interestingly, and consistent with its immune regulatory role, in situ increased expression of LILRB4 prevents rejection in recipients of organ transplants [18, 19].

However, in vivo functions of LILRB4 have not been fully elucidated, primarily due to a lack of knowledge of its natural ligands. Some LILRs including LILRA1, LILRA3,

LILRB1 and LILRB2 have been shown to interact with MHC-I molecules, albeit with low affinities [39], but LILRB4, which is predicted to be conformationally and electrostatically and structurally unique, is not expected to bind MHC-I [103]. Moreover,

LILRB4 has low amino acid identity to the prototypic MHC-I binding LILRB1, and unlike LILRB1 and other LILRBs, it is predicted to have no N-glycosylation sites that can affect ligand binding [108]. Of interest are recent studies which have shown that some LILRs can also functionally interact with non-MHC molecules, including binding of LILRA4 to bone marrow stromal cell antigen 2 (BST2) [91, 104] and interaction of

LILRB2 with Angiopoietin-like proteins (ANGPTLs) [106, 320] β-amyloid [321] and

Nogo protein [322]. It is therefore reasonable to postulate that LILRB4 may have unique non-MHC-I ligand/s.

Recently, the mouse orthologue of LILRB4 (gp49B1) was shown to bind the integrin αvβ3, and the interaction between these proteins to inhibit IgE-mediated mast 163 cell activation [111]. Given that extracellular domains of LILRB4 and gp49B1 have ~62% amino acid sequence identities [21], and there is ~90% amino acid sequence homology between human and mouse αvβ3 [111], it is conceivable that human LILRB4 may interact with αvβ3. However, unlike the human LILRB4, gp49B1 has two potential N- glycan sites [323] that may affect its ability to selectively bind this integrin [324, 325].

It is also noteworthy that the method used for the identification of αvβ3 as a ligand for gp49B1 was established solely on cell based assays that did not take into the consideration the contribution of other proteins, including fibroblast growth factor 1

(FGF1) [116], endothelial adhesion molecule 1 (CD31/PECAM-1) [117] and CD47

[118] that are known to associate with αvβ3. Therefore it is not clear whether the interaction of αvβ3 with gp49B1 is direct or through one or more of these proteins.

Moreover, this study lacked some gold standard experiments for ligand receptor interactions, such as surface plasmon resonance, ligand binding kinetics, affinity, avidity and competition studies.

The first attempt to characterise and identify LILRB4 ligands was made by Suciu-

Foca and colleagues [109, 155]. Although this group did not identify LILRB4 ligand/s, some of their original observations were highly relevant to this project. They discovered that high affinity LILRB4 ligand/s is/are expressed on the surface of T cells, and that binding of LILRB4 increased upon allogenic activation of these cells [109], which has allowed this study to focus its search for LILRB4 ligand/s on T cells. Suciu-Foca et al also found that soluble recombinant LILRB4 containing only the extracellular domains of the protein-inhibited CD4+T cell proliferation, despite lacking its intracellular inhibitory motifs [109]. Indicating that the soluble recombinant LILRB4 may be ligating a cell surface receptor on T cells that transduces inhibitory signals, this clue was

164 a useful additional tool for selecting likely candidates following the identification of a number of LILRB4 binding proteins by peptide mass spectrometry sequencing.

Both Castells and Suciu-Foca and their colleagues used gp49B1 or LILRB4 Fc fusion proteins for their ligand studies [109, 111]. Although Fc fusion proteins are commonly used for ligand screening [326-328], there is evidence that adding Fc fusion can affect the quaternary structure of proteins, which can affect ligand binding [329-

331]. For example, Fc fusion recombinant ephrins form a dimeric protein that dramatically increases binding affinity to its receptors by 6000-fold compared to monomeric ephrins [332]. Moreover, the predicted LILRB4 ligand/s is/are expressed on immune cells are likely to express endogenous Fc-receptors that may non-specifically interact with the Fc portion of the fusion protein [109], even after abolishing N-glycan sites of the Fc fusion by targeted mutations.

This project presents a new approach to characterise and identify LILRB4 ligands using a method that utilises the specificity and high affinity of ligand-receptor interaction developed by Flanagan and Leder [333]. The construct was made by inserting the 5 end of LILRB4 cDNA, and encoding the extracellular domain into a mammalian expression vector pAPtag-5, producing a fusion of the C-terminal end of

LILRB4 with secreted placental alkaline phosphatase. This provided the fusion protein with an intrinsic enzyme activity that can be used to trace the protein with high sensitivity and accurate quantification. The availability of a variety of indicator substrates for alkaline phosphatase, the high stability to heat of the placental isozyme, the high specific activity of the mammalian enzyme, and the availability of an isozyme- specific inhibitor that can be used to reduce background phosphatase activities [334] made this an attractive method. Moreover, the additional downstream C-terminal 6- histidine tag allowed for simple purification of the recombinant fusion protein using

165 metal affinity resins under mild conditions which preserves the biological condition of the proteins without disrupting its conformation [335]. Importantly, the alkaline phosphatase tag fused to LILRB4 can be bound to the available Sepharose-conjugated monoclonal antibody for pulldown of potential ligands.

Here, the production and purification of recombinant LILRB4-APtag-His, untagged recombinant LILRB4 (LILRB4-His) and APtag-His protein control are described. The LILRB4-APtag-His was used to perform experiments to analyse its ligand binding characteristics on T cells; the untagged LILRB4-His was used for competition assays; and the APtag-His control protein was used to exclude non-specific binding. Furthermore, LILRB4-APtag-His was used as bait for pulldown of potential ligands using specific Sepharose-conjugated anti-APtag monoclonal antibody from the surface of T cells and sequenced in-gel trypsin digested peptides using highly sensitive

Nano liquid chromatography tandem mass spectrometry (LC-MS/MS).

4.2 Materials and Methods

4.2.1 Sub-cloning of the extracellular domains of LILRB4 into pAPtag-5

mammalian expression vector

The entire extracellular domains of LILRB4 were sub-cloned into a pAPtag-5 mammalian expression vector and stably expressed in human embryotic kidney epithelial 293T cells, leading to the production of recombinant LILRB4 protein- containing C-terminal placental alkaline phosphatase and 6 histidine tags (LILRB4-

APtag-His) (Figure 4-1). A recombinant LILRB4 protein without the alkaline phosphatase tag was produced by introducing a stop codon in front of the AP-tag and the reintroduction of C-terminal His-tag (LILRB4-His). A control recombinant

166 placental alkaline phosphatase protein was produced by stable transfection of 293T cells with the empty vector (APtag-His).

Figure 4-1. Sub-cloning and production of soluble LILRB4-APtag-His and LILRB4-His in stably transfected HEK 293T cells pAPtag-5 vectors were linearized using HindIII or HindIII/XhoI and the extracellular domains of human LILRB4 gene (starting from the leader peptide at 0Gln to 236Glu) or human LILRB4 gene with 6xHis and stop codon (TGA) were inserted, and stably transfected into 293T cells. Zeocin was used for selection of stable transfected cells which may secrete ~ 97 kDa of LILRB4-APtag-His and ~30 kDa of LILRB4-His respectively (the protein size calculated based on their amino acid sequences).

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4.2.1.1 PCR amplification of LILRB4 plasmid DNA

Briefly, the extracellular domains of human LILRB4 (starting from the leader peptide at 0Gln to 236Glu) was amplified from a full length of LILRB4 DNA in pCR2.1 cloning vector (kindly donated by A/Professor Jonathan Arm) using Pfu DNA polymerase and primers sets listed in Table 4-1. Two DNA constructs were generated using primer sets A and B for LILRB4-APtag-His and primers A and C for LILRB4-His

(Figure 4-1).

Table 4-1. Primer sets used for sub-cloning of LILRB4 into pAPtag-5 mammalian vector Primers Primer sequences (5’ to 3’)

Forward for GCCAAGCTTTGCAGGCAGGGCCCCTCCCCAAA A LILRB4-APtag-His and (HindIII site underlined) LILRB4-His

Reverse for GCCAAGCTTCCTCCCAGTGCCTTCTCAGACC B LILRB4-APtag-His (HindIII site underlined) GCCCTCGAGTCAATGATGATGATGATGATGCT CCCAGTGCCTTCTCAG C Reverse for LILRB4-His (XhoI site underlined followed by a stop codon in italic and double underlined and 6x histidine double underlined) Forward for D CCCAAGAACA AGGCCAGATT internal LILRB4 Reverse for E AGTAACAGCG GTATCTCCCT internal LILRB4

Each PCR reaction contained a reaction mix listed on Table 4-2 and the PCR cycling parameters for LILRB4-APtag-His were an initial denaturation at 94°C for 2 min, followed by 35 cycles of PCR stage at 94°C for 30 sec (denaturation), 63°C for 45 sec (annealing) and 72°C for 60 sec (extension). The cycles were finished with a final extension step at 72°C for 10 min. The PCR cycling parameters for LILRB4-His were

168 an initial denaturation at 94°C for 2 min, followed by 35 cycles of PCR stage at 94 °C for 30 sec (denaturation), 53°C for 45 sec (annealing) and 72°C for 45 sec (extension).

The cycles were finished with a final extension step at 72°C for 10 min. PCR products were visualised on 1% (w/v) agarose gel under UV (Gel doc. Bio-Rad), and the products excised for gel purification.

Table 4-2. PCR reaction used for sub-cloning of LILRB4 into pAPtag-5 mammalian vector Reagent Volume (μl) Company 10x PCR buffer 2.5 Life Technologies 10 mM dNTP 0.5 Life Technologies 25 ng/μl forward primer 5 Sigma-Aldrich 25 ng/μl reverse primer 5 Sigma-Aldrich DNA polymerase (pfu ultra) 1 Agilent Technologies 50 ng/μl DNA template 1 - PCR water 10 Life Technologies Final volume 25 -

4.2.1.2 Gel purification of PCR products and ligation to pAPtag-5 vector

The PCR products (1.9 kb for LILRB4-APtag-His and 0.76 kb for LILRB4-His) were gel-purified using QIAquick Gel Extraction Kit (QIAGEN). In brief, DNA bands were excised from an agarose gel and transferred into clean Eppendorf tubes and gels dissolved in 3 volumes of buffer QG to 1 gel volume at 50 °C for 5-10 min. One gel volume of isopropanol was then added, samples mixed, and transferred to a QIAquick spin column and centrifuged for 1min at 10,000xg at RT. Columns were washed with

0.75 ml wash buffer and spun at 10,000xg at RT for 1 min and finally, purified DNA eluted in 30 μl UltraPure DNase/RNase-free distilled water. The concentration and quality of the purified DNA were measured using NanoDrop (Thermo Scientific). The purified PCR product for LILRB4-APtag-His was digested with HindIII, LILRB4-His

169 was digested with HindIII and XhoI, and circular pAPtag-5 vector was linearized with

HindIII or HindIII and XhoI at 37 oC for 2 hrs followed by heat activation at 65 oC for

15 min. A total of 1 μg HindIII linearized pAPtag-5 vector was then dephosphorylated using the Rapid DNA Dephosphorylation and Ligation kit according to the manufacturer’s instruction (Roche). The vector linearized using the two restriction enzymes did not require dephosphorylating. Ligation reactions containing 1:3 vector to insert molar ratio in total volumes of 8 μl were mixed with 2 μl of 5x DNA ligation buffer, 10 μl T4 ligation buffer and 1 μl T4 DNA ligase and incubated at 22 °C for 5 min as per the manufacturer’s instructions (Roche).

4.2.1.3 Transformation LILRB4-APtag-His, LILRB4-His and pAPtag-5 plasmid

constructs into competent E-coli

LILRB4 containing pAPtag-5 constructs were transformed into competent E. coli

(JM109) using the standard heat-shock transformation method. In brief, DNA plasmid

(50 ng) was gently mixed with 50 μl of competent E. coli and incubated for 10 min on ice. Bacteria were then heat-shocked at exactly 42oC for 45 sec, and immediately incubated on ice for 2 min. Cold SOC media (500 μl) was added to the bacteria suspension followed by incubation at 37oC with shaking at 220 rpm for 1 hr. Aliquots

(50 and 200 μl) of the bacterial suspension were spread and seeded onto Luria-Bertani

(LB) agar containing 100 μg/ml of ampicillin, and incubated at 37°C overnight. The following day, a few colonies were selected for plasmid DNA isolation using a plasmid mini kit, and the presence of LILRB4 DNA inserts and the orientation of their insertion was confirmed by digestion using EcoRI-XhoI for LILRB4-APtag-His, and HindIII and

XhoI for LILRB4-His respectively.

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4.2.1.4 Sequencing of LILRB4 plasmid DNA constructs

Plasmid DNA constructs with the correct insertion were sent to the Ramaciotti

Centre (UNSW, Australia) for Sanger sequencing using L-AP forward, R-AP reverse primers (Gene Hunter) and an internal LILRB4 primer (Table 4-1). In brief, DNA sequencing reaction was prepared in a final volume of 20 μl UltraPure DNase/RNase- free distilled water containing 500 ng DNA template, 1 μl of Big Dye terminator, 4 pmol of primer (forward and reverse primers separately) and 1.5 μl of 5x sequencing buffer. The PCR cycling parameters were 25 cycles at 96oC for 10sec, 50oC for 5sec and 60oC for 4min. The PCR products were then purified by adding 5 µl of 125 mM

EDTA followed with 60 µl of 100% ethanol, vortexed briefly and incubated at RT for

15 min to precipitate the extension products. The products were transferred to 1.5 ml

Eppendorf tube and spun for 20min at 16,000xg. Supernatants were carefully aspirated and 250 µl 70% (w/v) ethanol was added to the tube, followed by a brief vortex, then centrifugation at 16,000xg for 10min at 4oC. Supernatants were aspirated and DNA was dried at 90oC for 1 min. The purified DNA was then sent to the Ramaciotti Centre for sequencing using AB13730 capillary sequencer (Applied Biosystems). DNA sequencing results were analysed using the nucleotide Basic Local Alignment Search

Tool (BLAST) on the website of the National Centre for Biotechnology Information

(NCBI, www.ncbi.nlm.nih.gov), and the European Molecular Biology Laboratory

European Bioinformatics Institute (EMBL-EBI) (www.ebi.ac.uk).

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4.2.2 Transfection of LILRB4-APtag-His, LILRB4-His in pAPtag-5 and pAPtag-

5 vector alone into 293T cells

HEK 293T cells were stably transfected with LILRB4-APtag-His or LILRB4-His plasmid DNA or pAPtag-5 vector alone using Lipofectamine® 2000 [287] (see also

Section 2.11.4). Selection antibiotic (0.3 mg/ml of Zeocin) was added 48 hrs post- transfection and single cells with high production of alkaline phosphatase (AP) activity

(as described in Section 4.2.3) were further selected by limiting dilution. The single stably transfected cell clone with the highest AP activity in LILRB4-APtag-His or

APtag-His was expanded and aliquots frozen in liquid nitrogen. The identity of recombinant proteins was confirmed by Western blotting using anti-AP polyclonal antibody. To detect AP untagged LILRB4-His, stably transfected single clones were expanded in serum minimised media and supernatants were Western blotted using anti-

LILRB4 monoclonal antibody. The clone that produced the highest amounts of recombinant protein was further expanded and aliquots frozen in liquid nitrogen. The identity of each recombinant protein was further confirmed by peptide mass spectrometry sequencing.

4.2.3 Measurement of alkaline phosphatase activity for quantification of

recombinant LILRB4-APtag-His and APtag-His in culture supernatants

Cell-free culture supernatants were heated at 65°C for 15 min to inactivate endogenous phosphatase activity and chilled on ice. Heat inactivated samples and placental alkaline phosphatase standards (Sigma-Aldrich) were added into 96 well flat bottom plates in duplicates and pre-incubated at 37oC for 10 min. Pre-warmed AP substrate containing 6.8 mg/mL p-nitrophenol phosphate in 2x AP buffer (2M diethanolamine, 1 mM MgCl2, 20 mM L-homoarginine, 1 mg/mL BSA and pH 9.8) 172 were added into each well. The optical density (O.D) was measured using

SpectraMax®M2 Microplate Reader (Molecular Devices, CA, USA) at 405 nm at time points ranging from 0 to 60 min. The concentration of each protein was calculated based on the units of AP activity per 1 mg of protein. The placental alkaline phosphatase with a molecular mass of 67 kDa was calculated to have 1500 units of AP activity per 1 mg of protein and 1 mg of LILRB4-APtag-His (molecular mass of 97 kDa) had 915 units of

AP activity.

4.2.4 Purification of LILRB4-APtag-His, LILRB4-His and APtag-His proteins

Stably transfected 293T cells expressing LILRB4-APtag-His, LILRB4-His, or

APtag-His protein were cultured in a T175 tissue culture flask containing 100 ml complete DMEM media with 10% FBS at 37°C, 5% CO2 until the cells became 80% confluent (two or three days). Cells were then gently washed using 30 ml D-PBS, cultured in 100 ml serum-free complete DMEM media for two days, cell free supernatant harvested, spun at 300xg for 10 min and filtered using 0.22 μm pore filters.

The filtered supernatants were simultaneously buffer exchanged and eight times concentrated into 50 mM Tris, 300 mM NaCl, pH 8.0 using YM-100 kDa cut-off

(Millipore) membrane for LILRB4-APtag-His, YM-10 kDa cut-off membrane for

LILRB4-His and YM-30 kDa cut-off membrane for APtag-His in a stirred ultrafiltration nitrogen gas pressure chamber (model 8400, Amicon, Inc. Beverly, MA). The concentrated proteins were loaded onto a 1 ml of BD TalonTM Metal Affinity Resin column (BD Biosciences) that was equilibrated with exchange buffer at 4°C with 1 ml/min of flow rate using close circuit Econo pump (Bio-Rad) for binding via their respective histidine tags. The flowthrough buffers were recirculated 3 times through the

173 binding column. Unbound proteins were then washed with 30 ml of exchange buffer followed by 10 ml of exchange buffer containing 0.5 mM imidazole. Finally, bound proteins were eluted using 50 mM imidazole in exchange buffer into 8 x 1 ml fractions.

The purity and enrichment of each protein were confirmed by AP activity assay, silver staining and/or Western blotting. Protein fractions were stored at 4°C for use within two weeks.

4.2.5 Binding of LILRB4-APtag-His to the surface of peripheral blood

mononuclear cells and Jurkat T cell line

First, the optimal concentration of LILRB4-APtag-His required for the maximal binding to the surface of leukocytes was determined using peripheral blood mononuclear cells (PBMCs) derived from a healthy subject (Section 2.2). This was followed by binding assays on PBMCs from multiple healthy donors and on activated and non-activated Jurkat CD4 T cell lines using the optimal concentration of LILRB4-

APtag-His protein. Briefly, 5 x 106 PBMCs were washed twice with D-PBS and once with HBHA buffer (Hank’s balanced salt solution, 0.5 mg/ml BSA, 20 mM HEPES, 0.1%

(w/v) NaN3 and pH 7.0). Cells were then resuspended in 1 ml HBHA buffer containing increasing concentrations of purified LILRB4-APtag-His or APtag-His control (rang 0-

32 nM) and incubated for 90 min at RT on a rotating wheel. Cells were then washed five times with 1 ml cold HBHA buffer with gentle vortex and centrifugation at 200xg for 10 min at 4°C. Cell pellets were lysed with 350 μl of 1% (v/v) Triton X-100 in Tris-

HCl (10 mM, pH 8.0) and vortexed for 30sec. The lysates were centrifuged at 20,000xg at 4°C for 10min and the supernatant collected and analysed for AP activity (Section

4.2.3). For all subsequent binding experiments using PBMCs or activated and non-

174 activated Jurkat cells, single fixed optimal concentrations of LILRB4-APtag-His and

APtag-His protein (16 nM each) were used.

To assess whether activation alters ligand binding, Jurkat cells were activated with anti-CD3/CD28 beads as described [134, 336]. In brief, 10 x 106 Jurkat cells in 5 ml complete RPMI in a 6 well plate were stimulated with anti-human CD3/CD28 coated beads at 1:1 bead to cell ratio at 37°C, 5% CO2 overnight according to the manufacturer’s instructions (Dynabeads® Human T-Activator CD3/CD28). Cells were then washed twice with D-PBS by centrifugation and beads removed using a magnetic rack (Life technologies). Cell numbers were counted and resuspended in 1 ml HBHA buffer at 5 x 106 cells and incubated with 16 nM of LILRB4-APtag-His or APtag-His proteins as above. The effect of passage numbers on ligand binding was investigated using Jurkat cells.

The specificity of LILRB4 binding to the surface of Jurkat cells was determined by pre-incubating cells with untagged purified LILRB4-His protein. Briefly, 5 x 106 cells in 1 ml HBHA buffer were pre-incubated with or without 100 nM LILRB4-His for

30min at RT on a rotating wheel, washed once in HBHA and incubated in 16 nM

LILRB4-APtag-His or APtag-His protein for 90min at RT. This was followed by washing five times in cold HBHA buffer, cell lysis, collection of supernatant, and measurement of AP activity as described (Section 4.2.3).

4.2.6 Preparation of Jurkat cell lysates and cell membrane proteins

A total of 2 x 107 Jurkat cells that showed maximum specific binding to LILRB4-

APtag-His (passage 9 cells) were washed twice in D-PBS, and protein extracted using either detergent lysis buffer (150 mM NaCl, 10 mM Tris-HCl, 2 mM EDTA, 1% NP-40 and 2 mg/mL of freshly prepared protease inhibitor) and non-detergent lysis. For non- 175 detergent lysis, cells were washed twice with D-PBS and resuspended in homogenisation buffer (0.25 M sucrose, 20 mM HEPES and 0.5 mM EDTA) and sonicated using an ultrasonic cell disruptor 3 times for 10sec at 40% power cycle followed by 30sec on ice (Kontes, NJ, USA). Lysates prepared using detergent or sonication were then incubated on ice for 30min in Eppendorf tubes and insoluble proteins removed by micro-centrifugation at 20,000xg for 15 min at 4°C.

In some experiments, membrane proteins were isolated from the non-detergent lysates by density gradient centrifugation as described [337, 338]. In brief, 4 x 107

Jurkat cells (passage 9) were washed twice with D-PBS and homogenised in 2 ml homogenisation buffer (0.25 M sucrose, 20 mM HEPES and 0.5 mM EDTA) using an ultrasonic cell disruptor. Cell homogenate (1 ml x 2) was overlayed on the top 3.4 ml of

1-22% Ficoll gradient prepared in gradient buffer (0.25 M sucrose, 10 mM HEPES and

1 mM EDTA) and 1 ml of 45% Nycodenz in gradient buffer at the bottom of 5.5 ml ultracentrifuge tube (Beckman). The tubes were heat sealed and spun at 50,000 rpm for

90min at 10°C using an Optima ultracentrifuge with a Vti 65 rotor (Beckman Coulter).

Protein fractions were collected using a 21G needle (BD) (200 μl per one fraction

(Figure 4-2A) and proteins detected by SDS-PAGE gel and coomassie staining (Figure

4-2B). Amounts were quantified using a standard Bradford assay as per the manufacturer’s instructions (Bio-Rad). Fraction 1-5 (45% Nycodenz), fraction 6-11

(lysosome), fraction 12-14 (endosome), fraction 15-20 (plasma membrane), and fraction

21-26 (cytoplasm) had an average of 0.09 mg/ml, 0.12 mg/ml, 0.17 mg/ml, 1.05 mg/ml, and 1.25 mg/ml of protein respectively (n=2).

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A B

Figure 4-2. Membrane protein preparation using Ficoll gradient and Coomassie blue staining Jurkat cells from cell passage 9 (4 x 107 cells) were prepared and homogenised in non-detergent buffer containing sucrose using ultrasonic cell disruptor. Cell homogenate was underwent Ficoll gradient purification using ultracentrifugation. Protein fractions were then collected and 10 μl purified proteins were separated by SDS-PAGE, and stained using Coomassie blue (n=2).

4.2.7 Detection of LILRB4-APtag-His binding proteins by Far-Western blotting

of membrane proteins derived from Jurkat cells

Far-Western blotting is a method used to detect protein-protein interaction after resolving proteins in SDS-PAGE and transferred onto membranes [339], unlike standard Western blotting in which no antibodies are used to immunoblot the membranes. Briefly, membrane proteins isolated from Jurkat cells using non-detergent and density gradient centrifugation (as described in 4.2.6) were heated at 100°C for

5min with DTT, loaded onto a 10% SDS-PAGE gel and transferred onto a PVDF membrane (0.2 µm pore size). The membrane was then blocked with 2.5% BSA in

TBST buffer for 2 hrs at RT, washed once with TBST buffer and then incubated with 16

177 nM LILRB4-APtag-His or APtag-His in HBHA binding buffer at 4°C overnight. After

5 washes with HBHA buffer, the membrane was rinsed once with AP buffer (100 mM

Tris-HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl2) and incubated in AP buffer containing 0.33 mg/ml NBT and 0.17 mg/ml BCIP substrate for overnight at 4°C in the dark.

4.2.8 Immunoprecipitation of LILRB4 ligand/s from Jurkat cells and mass

spectrometric peptide sequencing of precipitates

Total cell lysates prepared using the detergent or non-detergent cell lysis methods or membrane proteins isolated using gradient density centrifugation were incubated with 10 μg of LILRB4-APtag-His as bait, or APtag-His as a negative control overnight at 4°C with gentle rotation. The following day, 10 µg of anti-placental AP monoclonal antibody was directly conjugated to 50µl of Sepharose bead slurry (Gene Hunter) and further incubated for 4 hrs with gentle rotation at 4°C. The bead-bound proteins were washed once with 1 ml of dilution buffer followed by two washes with TSA buffer pH

8.0 (see Table 2-2) and a final wash with 50 mM Tri-Cl pH 6.9 by centrifugation at

200xg for 4min at 4°C between each wash and 20,000xg for 10 sec at 4°C after the final wash. Bead containing bound proteins were resuspended in 30 µl of Tricine sample buffer (Bio-Rad) containing 10 mM DTT and proteins eluted by 5 min heating at

100 °C followed by a quick spin and loading of supernatants onto 10% SDS-PAGE gels sliver staining (Section 2.10.1 for silver staining protocol). Visible bands in the lanes loaded with the LILRB4-APtag baited samples and corresponding lanes from the control APtag-His baited samples (mostly with no visible bands) were excised and sent to the UNSW Biomedical Mass Spectrometry Core Facility for in-gel tryptic digestion

178 and Nano LC-MS/MS peptide sequencing (Figure 4-3) (sample preparation and

MASCOT search of peptide matches are described in Section 2.10.2). Proteins with a high MASCOT score and ≥3 peptide matches found in the LILRB4-APtag-His baited, but not in the control APtag baited samples, in four independent experiments were considered to be positive candidate LILRB4 binding proteins. Membrane proteins that are known to be involved in immune-regulation were marked as priority candidate ligands.

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180

Figure 4-3. A schematic representation of the proteomic approaches to identify LILRB4 ligands from Jurkat cells (1) Jurkat cells from cell passage 9 were prepared and lysed using lysis buffer or sonication. Following this total cell lysates were used directly or underwent Ficoll gradient purification for membrane protein isolation. (2) Proteins (total protein or membrane protein) were then incubated with either LILRB4-APtag-His or AP-His overnight at 4 °C. (3) Anti-AP conjugated Sepharose beads were then added to pull-down LILRB4-APtag-His or AP-His baited proteins. (4) The proteins-AP beads complexes were washed and (5) separated by SDS-PAGE and visualised using silver stain. (6) The protein bands were excised and sent to BMSF for protein identification using mass spectrometry (LC-MS/MS).

4.2.9 Detection of N-glycosylation sites in recombinant LILRB4-His

Potential N-glycosylation sites in LILRB4 were determined by enzyme de- glycosylation of the recombinant protein using PNGase F kit (BioLabs) followed by tryptic digest and peptide mass spectrometry. In brief, two sets of 1 μg of recombinant

LILRB4-His were resuspended in 2 μl of 10x denaturing buffer heated for 10 min at

100°C. This was followed by addition of 2 μl 10x G7 reaction buffer, 2 μl 10% NP-40, with or without 1 μl PNGase F and H2O, to a final total volume of 20μl. After 1 hr at

37°C de-glycosylation/control reaction, samples were loaded onto 10% SDS-PAGE gel and silver stained. Protein bands were excised and sent to the UNSW Biomedical Mass

Spectrometry Core Facility for in-gel tryptic digestion and Nano LC-MS/MS peptide sequencing and analysis [83].

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4.3 Results

4.3.1 Placental alkaline phosphatase-tagged and untagged extracellular domain

of LILRB4 proteins were successfully produced by transfected 293T cells

Two soluble recombinant human LILRB4 proteins containing the entire extracellular domains of LILRB4, with or without C-terminal placental alkaline phosphatase and histidine tags (LILRB4-APtag-His or LILRB4-His), or a recombinant

AP protein with a His tag (APtag-His), were secreted by stably transfected 293T cells into serum minimised culture supernatants. The production and release of these recombinant proteins to culture supernatants was first determined by using an alkaline phosphatase activity assay and/or by Western blotting using anti-LILRB4, and clones with high transgene expression were further selected by limiting dilution and clonally expanded for large scale protein purification.

Recombinant proteins were purified from 3-400 ml of LILRB4-APtag-His and

LILRB4-His and 50-100 ml of APtag-His of confluent culture supernatants using columns loaded with cobalt ion TALON. The purity of the different eluted fractions of each protein was determined by silver staining, and identities confirmed by Western blots (Figure 4-4). The purified LILRB4-APtag-His elusion fractions (1 ml each) showed a single ~110 kDa band and LILRB4-His was a single 40 kDa protein, indicating highly purified protein (Figure 4-4A and B). Most of the column-bound

LILRB4-APtag-His was eluted in fractions 1-4 (Figure 4-4A) and LILRB4-His was eluted in fractions 2-3 (Figure 4-4B). Interestingly, the size of the LILRB4-APtag-His fusion protein was ~10 kDa larger than the expected combined size of the extracellular domain of LILRB4 (30 kDa) and the APtag (67 kDa) (Figure 4-1), and similarly,

LILRB4-His was slightly larger than the calculated size of the extracellular domains of

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LILRB4, suggesting some posttranslational modification or experimental artefact, such as the nature of the buffers used. Notably, there were large amounts of APtag-His in all fractions of the purified protein and this was derived from amounts of culture supernatants five times less than LILRB4 (Figure 4-4C), indicating high efficiency of production of 293T cells of the APtag alone, but not of the LILRB4 fusion proteins.

Based on the AP activity, it was estimated that LILRB4-APtag-His from fraction 2 was

230 nM and APtag protein from fraction 6 was 1765 nM. All purified recombinant proteins were also quantified using BCA assay; 0.04 mg/ml of LILRB4-APtag-His

(fraction 2), 0.07 mg/ml of LILRB4-His (faction 2) and 0.35 mg/ml of APtag-His

(fraction 6). A total of ~50 μg of purified proteins was obtained from 3-400 ml of cell supernatant from LILRB4-APtag-His and LILRB4-His and 50-100 ml of cell supernatant from APtag-His transfected cells. LILRB4-APtag-His and LILRB4-His were freshly purified prior to use and stored at 4°C for a maximum of two weeks.

To further confirm the identities of all three recombinant proteins, bands from the silver stained gels were tryptically in-gel digested and mass spectrometry peptide sequenced. A MASCOT search for each peptide identified the expected recombinant fusion protein with very high scores; LILRB4-APtag-His, MASCOT score 827, peptide match 34; LILRB4-His MASCOT score 1167, peptide match 87, and APtag-His

MASCOT score 2108, peptide match 109.

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184

Figure 4-4. Purification of LILRB4-APtag-His, LILRB4-His and APtag-His and validation by silver stain and Western blotting Recombinant soluble proteins were loaded onto BD TalonTM Metal Affinity Resin column for His tag protein purification. Unbound proteins were then washed twice (wash 1 and wash 2), and then His tag proteins were eluted using Exchange buffer containing 50 mM imidazole in 8 x 1 ml fractions (F1~8), followed by silver stain and Western blot for each fraction. ~110 kDa of LILRB4-APtag-His, ~ 40 kDa of LILRB4-His and ~70 kDa of APtag-His were predominantly detected. The fractions with high concentration and purity, F2 and 3 of LILRB4-APtag-His (A) and LILRB4-His (B) and F 6, 7 and 8 of APtag-His (C) were used for further studies. A total of ~50 μg of purified proteins were obtained from 3-400 ml of serum minimised cell supernatant from LILRB4-APtag-His or LILRB4-His transfected cells, and 50-100 ml of serum minimised cell supernatant from APtag-His transfected cells (n=7).

4.3.2 LILRB4-APtag-His bound to the surface of PBMCs

The binding specificity and affinity of LILRB4-APtag-His to PBMCs were determined using ligand binding assay (method described in Section 4.2.5). The apparent dissociation constant of interaction was: equilibrium dissociation constant (KD)

= 16.06 ± 3.23 nM and maximum specific binding (Bmax), 1.1 ± 0.11 nM (Figure

4-5A). PBMCs incubated with LILRB4-APtag-His had AP activity, whereas little activity was observed in PBMCs incubated with APtag-His control (Figure 4-5B).

Interestingly, differential binding of LILRB4-APtag-His shown between individuals ranged from 0.27-1.04 at OD405 (Figure 4-5B), and the binding average of LILRB4-

APtag-His to PBMCs from three donors was significant when compared to APtag-His control – an average of OD405 at 0.67 ± 0.14 and 0.06 ± 0.02 respectively (Figure 4-5B).

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Figure 4-5. A representative binding of LILRB4-APtag-His on PBMCs (A) Binding of freshly purified LILRB4-APtag-His with different concentrations at 0, 8, 16 and 2 32 nM on PBMCs showing that KD = 16.06 ± 3.23 nM, Bmax = 1.1 ± 0.11 nM and R = 0.97 determined using Graphpad Prism. Error bars represent standard error of one experiment in duplicate. The optimal concentration of LILRB4-APtag-His red dotted lined. (B) Binding of 16 nM purified LILRB4-APtag-His to PBMCs from 3 donors was determined confirming binding specificity. (n=3 from donor 1, n=2 from donor 2 and n=1from donor 3). Significant binding of LILRB4-APtag-His compared to APtag-His control to PBMCs red dotted squared (*p=0.001; One way ANOVA). Error bars represent standard error of the mean.

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4.3.3 Passage and activation dependent binding of recombinant LILRB4 on

Jurkat cells

Significant binding of LILRB4-APtag-His compared to APtag-His control to

Jurkat cells was observed – an average of OD405 at 1.14 ± 0.22 and 0.08 ± 0.01 respectively (p<0.001). Interestingly, differential binding of LILRB4-APtag-His was shown between cell passages ranged from 0.60-2.33 at OD405; passage 5 at 0.60 ± 0.11

(n=1), passage 6 at 0.62 ± 0.01 (n=2), passage 7 at 1.38 ± 0.17 (n=3), passage 8 and 9 at

1.95 ± 0.12 (n=5), passage 10 at 1.86 ± 0.47 (n=3), passage 11 at 0.63 ± 0.13 (n=2) and passage 12 at 0.97 ± 0.12 (n=1). The binding of LILRB4-APtag-His to Jurkat cells was

2- to 3- higher for cells in passages 7-10, compared to other passage numbers, and dramatically decreased from cell passage 11 onwards (Figure 4-6A). The binding of

Jurkat cell passages 8 and 9 was 3.14-fold higher when compared to cells in passage 5.

The binding of LILRB4-APtag-His on CD3/CD28 activated Jurkat cells was increased compared to non-activated cells (Figure 4-6B) However, only marginal activation of binding was shown in activated cells compared to non-activated cells for cells at passage 9 (Figure 4-6B), with 1.6-fold increase at passage 4, 1.4-fold increase at passage 6, and 1.1-fold increase at passage 9 respectively. In addition, the binding on activated cells from three experiments was not significant when compared to non- activated cells.

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Figure 4-6. Jurkat cell passage and activation dependent binding of LILRB4-APtag-His (A) Jurkat cells from cell passage 5-12 were prepared and binding of LILRB4-APtag-His to cells was determined using 16 nM of purified LILRB4-APtag-His and APtag-His as a control. High binding of LILRB4-APtag-His to Jurkat cells was shown when cells were passage 7-10 suggesting that LILRB4 binding was cell passage number dependent. Error bars represent standard error of the mean. Significant binding of LILRB4-APtag-His compared to APtag-His control (P7, n=3; P8 and 9, n=5; and P10, n=3; *p<0.001; One way ANOVA). (B) Jurkat cells were stimulated with CD3/CD28 beads for overnight and binding of LILRB4-APtag-His to Jurkat cells was determined (n=3). Binding of LILRB4-APtag-His was increased in CD3/CD28 activated cells compared to non-activated cells. But only marginal activation of LILRB4-APtag- His binding was shown in activated cells compared to non-activated cells when cells were passage 9. Non-significant binding of activated cells compared to non-activated cells red dotted squared. Error bars represent standard error of the mean (ns: no significant; One way ANOVA).

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4.3.4 Competitive blocking LILRB4-APtag-His binding to Jurkat cells

LILRB4-APtag-His binding to Jurkat cells was competitively blocked using 6.25 times in excess of untagged (cold) LILRB4-His (100 nM) by 81.8% (Figure 4-7) (n=2).

The binding of LILRB4-APtag-His after pre-incubation with cold LILRB4-His was

0.42 at OD405 as compared to the 0.14 at OD405 in cells incubated with APtag control, indicating that the blocking was near complete (Figure 4-7). These results suggest specific binding of LILRB4 to the surface of Jurkat cells.

Figure 4-7. Competitive binding blockage by LILRB4-His on binding of LILRB4-APtag- His to Jurkat cells Jurkat cells from cells passage 9 were prepared and competitive binding to cells was determined. Cells were pre-incubated with/without 100 nM of LILRB4-His for 30 min, and subsequently incubated with 16 nM of purified LILRB4-APtag-His. Cells were incubated with APtag-His as a control. Jurkat cells were competitively blocked with pre-incubation of cells with LILRB4-His confirming binding specificity (n=2).

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4.3.5 Far Western blotting showed binding of LILRB4-APtag-His to proteins

derived from Jurkat cells

Plasma membrane, cytoplasmic, endosomal and lysosomal fractions of Jurkat cell lysate were separated using SDS-PAGE, transferred onto a PVDF membrane and blotted with 16 nM of LILRB4-APtag-His or APtag-His control (Figure 4-8). Blotting with LILRB4-APtag-His, but not with APtag-His control, showed interaction with distinct bands at ~15, 20 and a strong interaction with a 40 kDa in lanes loaded with plasma membrane protein fraction, and there was a single 40 kDa band on the cytoplasmic fraction (Figure 4-8A and B). A very faint reactive band was also evident at

~45 kDa in the endosomal fraction in membrane blotted with LILRB4-APtag-His but not in APtag-His control (Figure 4-8A and B).

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Figure 4-8. Representative Far-Western blot of protein fractions isolated from Jurkat cells A total of 10 μg each protein fraction was loaded onto a 10% SDS-PAGE gel, transferred onto a PVDF membrane and Far-Western blotted using 16 nM LILRB4-APtag-His (A) or APtag-His (B). Far-Western blot showed binding of LILRB4-APtag-His with a ~15, 20 and 40 kDa proteins in the plasma membrane fraction, a 40 kDa protein in the cytoplasmic fraction and a very faint band was evident at ~45 kDa in the protein isolated from the endoplasmic compartment. In contrast, there was no binding of any protein to APtag-His control, confirming specificity (n=1).

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4.3.6 Five candidate ligands for LILRB4 were identified using a combination of

immunoprecipitation and mass spectrometry

To identify LILRB4 ligand/s, membrane proteins isolated from Jurkat cells using non-detergent lysis buffer and gradient separation were incubated with LILRB4-APtag-

His as a bait or APtag-His as control, and proteins that bound to the LILRB4 immunoprecipitated using anti-AP monoclonal antibody were directly conjugated to

Sepharose beads. Silver staining of 10% SDS-PAGE gels showed seven unique protein bands in lanes loaded with precipitated pulled down using LILRB4-APtag-His but not

APtag-His control (bands 1, 2, 4, 5, 7, 8 and 9) in three independent experiments

(Figure 4-9). Additional two bands were visible in both lanes but appeared stronger in the lane loaded with proteins pulled down by LILRB4-APtag-His as bait (bands 3 and 6)

(Figure 4-9).

All the nine protein bands from the LILRB4-APtag-His lane and corresponding size gel fragments from the APtag control lane with or without visible bands were excised (Figure 4-9), and peptide mass sequenced by Nano LC-MS/MS (see Sections

4.2.8 for sample preparation, and 2.10.2 for mass spectrometry). Six membrane proteins were consistently identified using LILRB4-APtag-His as bait (Mowse score > 45, peptide matches ≥3, p < 0.05) (Table 4-3). Interestingly, all these proteins have molecular masses ranging from 28-40 kDa, sizes similar to the proteins shown to interact with LILRB4-APtag-His by Far Western blot (Figure 4-8). Although the

MASCOT scores for these proteins were modest (Mowse score 45-115), none was detected when APtag control was used as bait, suggesting specific interaction with

LILRB4. Moreover, in suitable accord with their expected functions upon interaction with LILRB4, these candidate ligands are all involved in immune-regulatory functions

(Table 4-3). The strongest candidate ligand, CD47 also known as integrin-associated

192 protein (IAP) is a membrane protein belonging to the immunoglobulin superfamily [119,

340]. It is ubiquitously expressed in immune cells, erythrocytes and cancer cells, and interacts with several proteins including thrombospondin-1 (TSP-1) [341, 342], signal- regulatory protein alpha (SIRP-α) [119, 340] several membrane integrins, most commonly αvβ3 [343]. CD47/SIRP-α interaction leads to bidirectional signalling, resulting in different cell-to-cell responses, including inhibition of phagocytosis in macrophages [119, 340] and T cell activation [344]. Interestingly, SIRP-α is an immunoglobulin-like receptor expressed on macrophage and contains ITIMs similar to

LILRB4, although the sequence homology of their extracellular domains is 29.3% identity and 51.7% similarity (analysed using the LALIGN from ExPASy

(www.ch.embnet.org)). CD47 is known to stably associate with αvβ3 and regulate cell adhesion, spreading and migration [345]. It is intriguing whether the binding of the mouse orthologue of LILRB4 (gp49B1) to αvβ3 reported by Castells et al [111] involved its interaction with CD47. The second strongest candidate is CD1b, a member of the

CD1 family of glycoproteins (CD1a-e) that are related to the class I MHC molecules

[346, 347]. CD1b is expressed in cells specialised for antigen presentation and is involved in the presentation of lipid antigens to T cell [348, 349]. Recently, it was reported that inhibitory LILRB2 interacted with CD1c and d and blocked its recognition by natural killer T (NKT) cells [99, 350]. CD2, also known as LFA-2, is a member of the immunoglobulin superfamily and acts as a co-stimulatory molecule in T cells and

NK cells [351, 352] and is involved in cell adhesion through interaction with LFA-3

[353] or CD48 [354]. CD48 is a CD2 subfamily molecule, highly expressed on T cells,

B cells, monocytes and granulocytes [355-357]. CD48/CD2 coupling together with other pairs such as CD28/CD80 and MHC-I/LFA-1 contributes to the formation of immunological synapses between T cells and antigen presenting cells [358]. Its

193 interaction with CD2 has also been shown to promote lipid raft formation [359]. CD82 is a membrane glycoprotein expressed in various leukocytes and through interaction with MHC-II molecules inhibits cell growth and cancer metastasis [360, 361].

As expected, the ~110 kDa band in the lane loaded with precipitates from the

LILRB4-APtag-His baited samples contained multiple peptides matching LILRB4-

APtag-His fusion protein (Figure 4-9, lane 2, band 2), and in the control lane where

APtag-His control was used most of the protein corresponding to the 70 kDa was placental alkaline phosphatase (Figure 4-9, lane 1, band 3). Other membrane proteins that showed 1-3 specific peptide matches, and detected in one or more of the three independent experiments, are listed in Appendix II.

Figure 4-9. Representative silver staining of LILRB4-APtag-His or APtag-His immunoprecipitated membrane proteins from Jurkat cells Membrane proteins isolated from Jurkat cells were incubated with 10 μg of LILRB4-APtag-His or APtag-His as a control followed by immunoprecipitation with anti-AP Sepharose beads. Immunoprecipitated proteins were silver stained, and the protein bands (as indicated by arrows from 1 to 9) from each sample were excised and analysed by mass spectrometry (n=3). The ~25 kDa strong band visible in both lanes was the light chain of the anti-AP immunoglobulin used for immunoprecipitation. 194

Table 4-3. LILRB4 candidate ligands repeatedly detected by Nano LC-MS/MS peptide mass sequencing (n=3)

MW Name* Alternative names Expression Known ligands Functions References (kDa) Inhibition of phagocytosis, most human cell type, [119, 340, CD47 Integrin-associated protein 35 SIRP-α, TSP-1, αvβ3 stimulation of cell-cell fusion, erythrocytes, cancer stem cells 341, 343] T cell activation

CD1b T cell surface glycoprotein 37 antigen presenting cells Glycolipids, LILRB2 Lipid antigen presentation [99, 350] CD1b Co-stimulatory molecule for T T cells, [351, 352, CD2 LFA-2 39 LFA3, CD48 cells and NK cells, Cell natural killer cells 354] adhesion B-lymphocyte activation Contributes to the formation of marker BLAST-1 immunological synapses T and B lymphocytes, [355-357, CD48 or signalling lymphocytic 45 LFA-2, CD244 (2B4) between T cells and antigen monocytes, granulocytes 362, 363] activation molecule 2 presenting cells, may promote (SLAMF2) lipid raft formation MHC class II, CD19, monocytes, granulocytes, Metastasis suppressor CD63, Duffy antigen Inhibition of cell growth and [360, 361, CD82 178 lymphocytes, Kangai-1 receptor for chemokine metastasis 364-366] epithelial cells, fibroblasts (DARC, CD234) *None of these proteins were detected when APtag-His protein was used as bait.

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4.3.7 Potential post-translational modification of the recombinant LILRB4

proteins produced in 293T cells

One interesting observation made during the production of the recombinant

LILRB4 proteins in the mammalian expression system (293T cells) was that both

LILRB4-APtag-His and the LILRB4-His were ~10 kDa larger than their calculated masses (Figure 4-1 and Figure 4-4), which suggests that these mammalian-produced proteins may have post-translationally modified [274]. N-glycosylation is the most common (about 90%) post-translational modification of mammalian proteins – the attachment of glycan to asparagine residues [79]. Each N-linked glycosylation increases molecular weight of a protein by approximately 2.5 kDa [367, 368], and contributes to the thermodynamic stability and solubility of proteins [369] that may influence ligand binding and functions [17, 82]. Although LILRB4 is not predicted to N-glycosylate

(www.cbs.dtu.dk/services/NetNGlyc-1.0), its extracellular domain contains three asparagine residues at N33, N73 and N79 that could potentially glycosylate, adding the observed extra ~10 kDa (Figure 4-4 To address this, purified LILRB4-His protein was de-glycosylated using PNGase F, resolved in SDS-PAGE gel and sliver stained, and excised bands were sent for mass sequencing. Results show that treatment with PNGase

F did not change the molecular mass of LILRB4-His (Figure 4-10), strongly indicating that the additional mass was not due to N-glycosylation. Nano LC-MS/MS analysis after trypsin peptide digestion detected only one (N73) of the three aspiring residues and found that this residue was not glycosylated (Table 4-4). However, due to the poor peptide coverage which failed to detect the other two asparagine residues (N33 and N79), it is premature to conclude that LILRB4 was not N-gylcoylated based on this incomplete data and the silver staining results (Figure 4-10). Hence future repeat

196 experiments using other digestion enzymes that may improve coverage, such as chymotrypsin [370] and Glu-C [371], are required.

Figure 4-10. Representative silver staining of deglycosylated LILRB4-His using PNGase F Purified 1 μg of LILRB4-His alone or LILRB4-His digested with PNGase F were loaded on to a SDS-PAGE gel and silver stained (n=1). The same size of LILRB4-His proteins about 40 kDa between PNGase F treated and non-treated samples was shown, suggesting no N-glycosylation in recombinant LILRB4 from 293T cells.

197

Table 4-4. Amino acid sequences of deglycosylated LILRB4-His using PNGase F

* N (asparagine)  D (aspartic acid) if modification occurred; sequence coverage is underlined

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4.4 Summary and conclusions

Notwithstanding data from this and previous studies showing that LILRB4 has a critical role in immune regulation, a lack of LILRB4 ligand/s has to date limited elucidation of its function to in vivo studies [19, 85, 318, 319]. In addition, the current method for identifying ligands necessitates updating for the following reasons

Firstly, although some LILRs have been reported to bind classical and non- classical MHC molecules [39, 91, 104], there are technical issues in the methods used to identify LILR ligands. These methods have used truncated recombinant proteins, produced in non-mammalian systems that potentially affect post-translational modification and receptor/ligand interaction. Secondly, LILRB4-Fc fusion proteins recently produced from a mammalian system were shown to bind to T cells [109]. This binding, however, could be caused by non-specific binding from its Fc portion, which easily interacts with FcRs on innate immune cells. Thirdly, the determined binding affinity of LILRs with their ligands was considerably low [29, 31, 71, 72, 92], which would suggest the presence of other ligands and co-ligands with higher affinity. Lastly, a large amount of purified protein is required for candidate ligand/s screening. But there are few mammalian systems for production of recombinant LILRs including LILRB4.

Thus, the last aim of this thesis was to generate and optimise new tools for an improved ligand screening method in an attempt to identify candidate LILRB4 ligand/s.

To overcome these problems, extracellular full-length recombinant LILRB4 including LILRB4-APtag-His and LILRB4-His, and APtag-His as a control were produced from a mammalian cell line (293T cells). The recombinant LILRB4 had the expected molecular mass and no N-glycosylation site, suggesting its correct protein folding and post-translational modification. The purified LILRB4-APtag-His was able to bind PBMCs and the Jurkat T cell line. Interestingly, the binding on Jurkat cells was 199 cell activation-independent and approximately three-fold higher when cells were passage 8 and 9 compared to other passages. Also, the binding was competitively blocked by LILRB4-His by 81.8%, confirming its binding specificity. Importantly,

LILRB4-APtag-His showed strong interaction with ~ 40 kDa of isolated membrane proteins using Far-Western blot, and the potential ligands were pulled down using

LILRB4-APtag-His as bait, and identified by Nano LC-MS/MS (Mowse score > 45, peptide matches ≥ 3, p < 0.05) (Appendix II). The top five proteins, which have molecular masses ranging from 28-40 kDa, and are involved in immune-regulatory functions, are considered as LILRB4 candidate ligands. These include CD47, CD1b,

CD2, CD48 and CD82 (Table 4-3). Among them, CD47 is the strongest candidate ligand, due to its interaction with αvβ3 (known as a ligand for the mouse orthologue of

LILRB4, gp49B1 [111]). Thus, further experimentation will be required to characterise and confirm its interaction with LILRB4 and to define their functional interactions.

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CHAPTER 5. GENERAL DISCUSSION

5.1 LILRB4 suppresses Tyrosine phosphorylation of multiple signalling proteins

involved in clathrin-mediated endocytosis.

Phosphorylation of intracellular proteins is a key post translational modification involved in many biological processes, including signal transduction, cell growth, proliferation, differentiation, migration and gene transcription [183, 215, 372]. In response to external or internal stimuli, proteins containing specific amino acid residues, mainly serine, threonine and tyrosine (Tyr), are phosphorylated or dephosphorylated by protein kinases or phosphatases respectively [216, 373]. Phosphorylation is addition of a phosphate onto proteins which induces conformational changes of proteins and generally results in cellular activation, while dephosphorylation is, in general, a negative regulator of cellular activation [374] (Section 1.1.4).

FcγRI is one of the most important activating receptors in the innate immune system which, upon its cross-linking by immune complexes engages the ITAM containing common γ [166]. This causes Tyr phosphorylation of a cascade of signalling molecules including upstream Src kinases and Syk, and downstream MAPKs leading to effector functions such as cytokine production, phagocytosis and/or ligand internalization [160, 163] (Section 1.1.3.1). Although FcγRI-mediated innate immune activation is critical in protecting against pathogens, sustained and uncontrolled immune activation can cause host tissue damage in diseases such as rheumatoid arthritis and thrombocytopenia [12, 165]. One potential mechanism for regulating excessive activation through FcγRI is clathrin-mediated endocytosis, wherein ubiquitination and degradation of the receptors and its ligands occurs without compromising its vital functions of efficient pathogen clearance and inflammatory mediator production. How 201 this fine balance between maintaining sufficient surface receptors and their degradation by endocytosis is regulated is, however, not well established. Therefore, this study proposed that clathrin-mediated endocytosis of FcγRI following antibody cross-linking may be regulated by Tyr phosphorylation/dephosphorylation of one or more signalling molecules. Moreover, LILRB4, which is known to Tyr dephosphorylate signalling molecules in FcγRI-mediated monocyte activation [15], may play a key role in maintaining a homeostatic balance.

In this study, a global proteomic approach was used for the first time to identify simultaneous Tyr phosphorylation of multiple proteins following antibody cross-linking of FcγRI on the monocytic cell line THP-1 (Table 3.1-1 and Figure 3.1-3). Ingenuity

Pathway Analysis (IPA) revealed that several of these proteins were involved in clathrin-mediated endocytosis and, as expected, in Fc-receptor mediated phagocytosis

(Figure 3.1-3), including the common γ-chain of the Fc receptors, Syk, clathrin, Cbl,

HGS, STAM1/2, HSP70, actin, actin-related proteins, actinin-4, tubulin and actin binding proteins (Table 3.1-1). Interestingly, several peptides matching to proteins that have not been previously reported to be phosphorylated in response to FcγRI cross- linking, and are not known to be involved in endocytosis/phagocytosis, were also identified with high confidence. These included TRIM21, mitogen-activated protein kinase 9, Twinfilin-1, hematopoietic cell specific Lyn substrate 1, SH2 domain containing leukocyte protein of 76 kDa, linker for activation of T cell family member 2,

Crk-like protein, Coatomer subunit epsilon, docking protein 2, protein Tyr phosphatase

18, Phosphatidylionositol 3, 4, 5-triphosphate, Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP-1), protein phosphatase 1 gamma, 1- phosphatidylionositol 4, 5-biphosphate phosphodiesterase gamma 2, Toll-like receptor 6 and Ubiquitn-40S ribosomal protein s27a. Although some of these proteins are known

202 to be involved in a wide variety of cellular processes such as apoptosis, proliferation, differentiation, transcription regulation and phagosome development [315, 375-377], the main focus of this project was to further investigate proteins that were recognised by the IPA analysis to be involved in clathrin-mediated endocytosis.

The top five of these molecules were clathrin, Cbl, HGS, HSP70 and the common

γ chain of the Fc receptors; thus, their phosphorylation after FcγRI cross-linking was thoroughly validated by Western blotting (Figure 3.1-4). Although TRIM21 was not recognised as a molecule involved in endocytosis by the IPA analysis, it was included for further validation because of its recently discovered functions as an intracellular high affinity Fc receptor [378, 379]. While phosphorylation of the common γ chain and

Syk upon antibody cross-linking of FcγRI is well-known [195, 199, 200], this study is the first to report Tyr phosphorylation of some key molecules involved in the clathrin- mediated endocytosis, namely clathrin, Cbl, HGS, HSP70 and TRIM21. The results suggest that Tyr phosphorylation of multiple upstream and downstream molecules may be important for FcγRI-mediated functions which are critical in host immune defence including phagocytosis [188, 189], endocytosis [186, 380] and mediator production

[184, 192] (Section 1.1.3.1). There is sufficient evidence to suggest that Tyr phosphorylation of the upstream signalling molecules, such as the common γ chain of the Fc-receptors and Syk, is closely involved in Fc-dependent phagocytosis and mediator production [188, 189, 381]. Furthermore, it has been shown that Fc-receptors are also required in antibody-mediated neutralization of toxins [382, 383] and venoms

[384] possibly through clathrin-mediated endocytosis for degradation [382].

Clathrin has a key role in receptor-ligand endocytosis by formation of clathrin pits containing multiple accessories and adaptor proteins such as dynamin, adaptor protein-2

(AP2), epsin and Eps15 [195-197] (Section 1.1.3.1.2.2 and Figure 1-10). There is

203 limited evidence showing that Tyr phosphorylation of the clathrin heavy chain following bacterial infection of epithelial cells was required for the formation of clathrin pits [385]. Tyr phosphorylation of clathrin is induced by activation of Src family kinases such as Src, Lyn and Lck, and results in internalisation of membrane receptors such as epidermal growth factor receptor (EGFR) [386], BCR [387] and TCR [388]. It is well known that cross-linking of FcγRI on monocyte, macrophage and dendritic cells triggers Tyr phosphorylation of multiple upstream protein kinases including Src family kinases and Syk [160, 163]. However, to date there is no data on the phosphorylation state of clathrin in monocytes/macrophages in response to FcγRI antibody cross-linking.

In contrast, Tyr phosphorylation of Cbl can occur by ligand mediated clustering of FcγR and other membrane receptors [15, 389], including its phosphorylation in myeloid cells upon FcγRI cross-linking [390], and in macrophages after Shigella infection [391]. It is also well established that binding of Cbl to key signalling kinases such as Syk causes their ubiquitination and degradation leading to termination of cellular activation [392-

394], such as in the inhibition of FcγR-mediated phagocytosis of IgG-coated sheep RBC by COS cells [184]. However, there is no direct evidence indicating that Tyr phosphorylation of Cbl is required for its binding to signalling kinases such as Syk.

Interestingly, in the most widely studied model of Cbl dependent endocytosis, ubiquitination and degradation of the Epidermal growth factor receptor (EGFR) [185,

395], cells over-expressing Tyr-deleted Cbl mutants lost their ability to promote receptor polyubiquitination and led to slowed-down receptor internalisation [396]. This suggests that Tyr phosphorylation of Cbl possibly enhances its functions. Evidence increasingly suggests that hepatocyte growth factor-regulated tyrosine kinase substrate

(HGS) is a key downstream regulator of endosomal trafficking of ubiquitinated- membrane proteins [202, 397], and that it is readily Tyr phosphorylated on cells

204 activated through FcεRI ligation [392] or upon treatment with EGF [398], platelet- derived growth factor [398]or IL-2 [399]. While it is noteworthy that Tyr phosphorylation of HGS was Syk dependent during FcεRI-mediated activation of RBL cells, [400], the functional significance of Tyr phosphorylation of HGS to its functions is still to be fully elucidated.

Taken altogether, this direct and circumstantial evidence indicates that Tyr phosphorylation of clathrin, Cbl and HGS likely promotes endocytosis and endosomal and/or lysosomal degradation of receptors. It is, therefore, plausible that the strong phosphorylation of these molecules in response to FcγRI antibody cross-linking may enhance its internalisation leading to its down-regulation on monocytes and macrophages. However this proposal requires further investigation, down-regulation of surface FcγR1 may cause signalling through this receptor to rapidly terminate, preventing unregulated monocyte activation. Given that excessive phosphorylation- dependent loss of FcγRI surface expression may be detrimental to the host, it must therefore be tightly regulated. This study has, for the first time, demonstrated that co- ligation of LILRB4 with FcγRI significantly suppressed Tyr phosphorylation of clathrin,

Cbl and HGS (Figure 3.1-6), suggesting that LILRB4 may regulate clathrin-Cbl-HGS dependent internalisation of this important receptor leading to cell surface expression of sufficient receptors and retention of selected functions. Similarly, results show that cells over-expressing a wild-type intracellular LILRB4 selectively enhanced FcγR1-mediated phagocytosis of particles and increased bactericidal activity, while strongly down- regulating pro-inflammatory cytokine production (see Section 3.2; also Section 5.2 of this chapter).

Moreover, mice with genetic disruption of gp49B had more pronounced inflammatory responses, but poorer clinical outcomes when compared to wild-type

205 animals [112, 401], indicating unregulated inflammation and inefficient bacterial clearance. Although the functional significance is not fully understood, a recent study showed strong up-regulation of LILRB4 on in vitro-cultured human blood macrophages during Salmonella infection, and ligation of LILRB4 in these macrophages up-regulated

IL-10 to selectively suppress pro-inflammatory chemokine IL-8 [19]. IL-10 is a complex cytokine that displays both strong anti-inflammatory [402, 403] and pro- bacterial phagocytosis properties [404, 405], and it strongly up-regulates FcγRI expression on monocytes [406]. Thus up-regulation of IL-10 in response to LILRB4 ligation gives further support to the selective anti-inflammatory and pro-phagocytosis functions of LILRB4, and to the notion that LILRB4 ligation may retain higher surface expression of FcγRI, through decreased internalisation and by enhancing expression via

IL-10. This proposal does, however, require experimental validation by comparing the expression levels of FcγRI in LILRB4 ligated and non-ligated monocytes, and by investigating the dynamic internalisation of the receptor upon LILRB4 ligation in live cells using advanced microscopy. Syk is a key kinase in FcγR-mediated phagocytosis via binding to PI3K promoting phagocytosis [407], and there is some evidence that Tyr phosphorylated Cbl competitively binds to Syk and block its interaction with PI3K, leading to subsequent inhibition of phagocytosis [184]. Thus, the promotion of phagocytosis by LILRB4 may also be partly explained by its ability to dephosphorylate

(deactivate) Cbl and enhance Syk binding to PI3K, which could activate the upstream signalling pathway of FcγRI-mediated phagocytosis. Interestingly, site-directed mutagenesis studies have indicated that LILRB4-mediated dephosphorylation of Cbl

(Figure 3.1-8) is likely through recruitment of phosphatases such as SHP-1 [408, 409].

Whether SHP-1, or similar phosphatases such as SHP-2 and SHIP, modulates LILRB4- mediated dephosphorylation of clathrin, Cbl and/or HGS remains to be elucidated.

206

One molecule that has not been previously reported in FcγRI-mediated functions and/or clathrin-mediated receptor endocytosis, but was found to be significantly Tyr phosphorylated upon FcγRI cross-linking, and to be strongly suppressed by LILRB4, is the E3 ubiquitin ligase, TRIM21 (Figure 3.1-6). This novel finding was of particular interest, since TRIM21 has been recently reported to be a high affinity intracellular Fc receptor [378] implicated in the elimination of antibody-bound intracellular viruses

[203], and decreased type I IFN production in response to viral infection [410]. Tyr phosphorylation of TRIM21 was shown to be critical for its function as a negative regulator of INF-β promotor activity [411]; whether phosphorylation enhances or diminishes its ubiquitin ligase activity that leads to the elimination of antibody-bound intracellular viruses remains poorly understood. Although TRIM21 was reported to be an intracellular high affinity Fc receptor responsible for the elimination of antibody- bound viruses that escaped neutralising antibodies and/or cell-mediated immunity [378], the upstream mechanisms that link the extracellular antibody-bound viruses to the intracellular TRIM21 are not known. The results presented in this project suggest that binding of the antibody bound viruses to FcγRI of the cell surface might be the first stage of internalisation of the virus-antibody complex that would be presented to an activated (phosphorylated) TRIM21 for subsequent endosomal degradation, and that this may be regulated by LILRB4. Although there is no data that associates LILRB4 with viral diseases, there is accumulative evidence indicating that various members of the LILRB family have multifaceted regulatory functions, including: modulation of T cell activation, cytokine production and/or antigen presentation in viral infections, such as HIV [148, 412-414]; dengue virus [415]; Epstein–Barr virus [416]; cytomegalovirus

[139, 417]; human immunodeficiency virus [148]; and hepatitis B and C viruses [418].

Results presented here may therefore represent a new alternate mechanism of FcγRI-

207

TRIM21-LILRB4 mediated control of viral infections. Future studies which investigate the functional relationships between FcγR1, TRIM21 and LILRB4 in innate immune responses defending against viral infection, using in vitro models and by establishing clinical in vivo associations, will provide deeper insight.

Interestingly, HSP70, which is important in the disassembly of clathrin coated pits

[198] and functions as a molecular chaperon [419, 420], was also strongly phosphorylated by FcγRI cross-linking, but contrary to all the other molecules in the clathrin-mediated endocytosis pathway, was further phosphorylated by LILRB4, indicating selective LILRB4 effects (Figure 3.1-6). C-terminal Tyr phosphorylation of

HSP70 has been described as a switch for regulating co-chaperon binding in cancer cells, in that it either facilitates protein folding, or directs proteins for ubiquitin- mediated degradation [421]. However, its phosphorylation in the context of FcγRI cross-linking and LILRB4 ligation in monocytes is novel, and the functional consequence of this modification is not yet clear.

5.2 LILRB4 exerts dual inhibitory and activating functions on FcγRI-mediated

TNF-α production in THP-1 cells depending on the position of the Tyr

residues of its ITIMs.

Natural ligand/s for LILRB4 are unknown, hence studies aimed at defining its functions are performed using specific LILRB4 ligating antibodies. However, cells expressing this receptor (monocytes and monocytic cell lines) also express numerous inhibitory LILRs that have high structural homology to LILRB4 [39, 108] (Section

1.1.4). This may confound results because of the potential cross-reaction of anti-

LILRB4 antibodies with other inhibitory LILRs. This problem was overcome in this study by transgenic expression of a chimeric protein containing surface CD25 and 208 intracellular LILRB4 ITIMs on THP-1 cells that normally do not express CD25 as well as endogenous CD25 allowing specific antibody-based ligation experiments. The main focus of the project was to investigate functional contribution of the three intracellular

ITIMs of LILRB4; hence we deliberately excluded the transmembrane domains of

LILRB4 in our chimeric constructs as they can potentially confound results. THP-1 cells do not express the CD25 (IL-2Rα and IL-2Rβ chains (CD122) but contain the IL-

2Rγ-chain (CD132) (Figure 3.2-1). The γ-chain cannot signal in the absence the first two [310]; hence the CD25 α-LILRB4 chimeric surface expression used in our study completely excludes a confounding signalling via the IL-2 receptor complex. Therefore, the CD25α-LILRB4 ITIM chimeric protein overexpression without the LILRB4 transmembrane domain we believe provided a specific model that will detect effects related only to the LILRB4 ITIM mutants. Moreover, new experiments show that treatment of the CD25-LILRB4 ITIM chimeric cells with LPS did not affect expression of CD25 (Figure 3.2-1).

Generation of these CD25-LILRB4 ITIM chimeras also ensured stringent selection of stably-transfected cells that expressed similar amounts of each ITIM mutant, using comparable CD25 expression levels as a selection criteria (Figure 3.2-2), and thereby removing another major variable that could confound comparisons. Moreover, the possibility of variant effects of stable transfection of THP-1 cells with the different chimeric constructs on cell viability, and on expression of key relevant molecules including FcγRI, SHP-1, SHP-2, SHIP and native LILRB4, were minimised because all transfected cells expressed comparable levels of these proteins (Figure 3.2-3, Figure

3.2-4 and Figure 3.2-5). Consideration of these parameters permitted unbiased and more valid comparisons of functions among the various mutants. Furthermore, the THP-1 cell line was selected because it constitutively expresses high levels of functional native 209

LILRB4, which indicates that this cell line contains all the signalling machinery regulated by LILRB4 [15]. Although it would have been simpler to use a cell line that does not express endogenous LILRB4, we reasoned that such cell line will also likely lack the intracellular signalling machinery related to native LILRB4, hence we chose a more challenging but biologically relevant model by using a cell line that have all the

LILRB4 signalling machinery and designed a chimeric constructs that overcame the limitation of having endogenous LILRB4. This thoroughly quality-controlled approach such as immunophenotypic profiling of THP-1 cells by determining the relevant cell surface markers including CD14, CD64, CXCR4, LILRB4 and TLR4 as well as relevant intracellular signalling proteins including Syk and SHP-1 (Figure 3.2-1) was used to extensively define the role of each of the Tyr residues in the LILRB4 ITIMs in relation to its expected inhibitory functions.

It is well established that Src family kinases are activated by FcγRI cross-linking

[167], and phosphorylate (activate) downstream protein kinases such as Syk and MAPK leading to TNF-α production [168] (Section 1.1.3.1.1); and LILRB4 is described as a receptor that transduces only inhibitory signals by recruiting SHP-1 to its three ITIMs, leading to termination of protein Tyr kinases mediated activation signals [15, 18, 39, 41,

422] (Section 1.1.4.2). This is consistent with previous study showing inhibition of

FcγRI-mediated Tyr phosphorylation of Syk through recruitment of SHP-1-like phosphatase to LILRB4 ITIMs [15]. Using a site-directed mutagenesis approach, this study made the first-time finding that LILRB4 is a complex immuno-regulatory receptor that may suppress or enhance FcγRI-mediated monocyte activation through differential activation of its three ITIMs. Results showed that THP-1 cells over-expressing the three intact Tyr residues (YYY), and those that express single intact middle Tyr residue

(FYF), maximally reduced FcγRI-mediated TNF-α production, while, paradoxically, 210 cells over-expressing YYF and YFF enhanced their activation (Figure 3.2-8 and Figure

3.2-9). The middle Tyr389 was found to be critical to inhibitory functions whereas the proximal Tyr337 consistently enhanced FcγRI-mediated TNF-α production. Consistent with these opposing effects, the inhibition triggered by the middle Tyr389 is markedly blunted, but not abolished, by the presence of the proximal Tyr337 in mutants containing both tyrosine residues (YYF). This blunting effect is, in turn, counter-negated (reversed) by the re-introduction of the distal Tyr389 residue in mutants containing all three tyrosine residues (YYY). The distal Tyr419 alone (FFY), or in combination with the middle (FYY) or proximal (YFY) Tyr, has intermediate inhibitory effects, suggesting it may play a role in fine-tuning responses. Similar Tyr site-specific activating or inhibitory effects of

LILRB4 were observed on analysis of mRNAs of multiple cytokines and immune regulatory molecules in monocytes stimulated through FcγRI cross-linking, or after

Salmonella challenge in vitro, further validating our results (Appendix III A and B).

However, the mRNA data requires further investigation, since experimental replicates were generally limited, and results were not confirmed at protein levels. Interestingly, in

LILRB1, an inhibitory receptor closely related to LILRB4, only the two distal ITIMs are required to inhibit FcεR-mediated serotonin release whereas the two proximal

ITIMs have no apparent inhibitory functions and their roles are undefined [230].

Because the second proximal LILRB1 ITIM has 98% sequence homology to the proximal LILRB4 ITIM (Figure 5-1), it may possess activating functions similar to

LILRB4.

211

Figure 5-1. Schematic diagram of the intracellular domains of LILRB4 and LILRB1 The ITIM consensus sequences are highlighted, the SH3 binding motifs are underlined and proline rich regions are in bold. In LILRB1, the two distal ITIMs with SH3 binding motif in their N-terminal but not the two proximal ITIMs that do not possess the N-terminal SH3 binding motifs have been reported to inhibit Fc-receptor mediated secretory function [230]. This is consistent with this study’s finding which shows that the middle and distal LILRB4 ITIMs contain N-Terminal SH3 binding motifs.

These results demonstrated that the duality of activating and inhibitory functions of LILRB4 that depends on the position of the Tyr residues within its ITIMs, challenges the current paradigm which proposes ITIM containing receptors are exclusively inhibitory [22, 256, 257, 314]. Similar, though limited, results showing receptors containing ITIMs transducing activating signalling have been reported, termed in this

212 study and by others as immunoreceptor tyrosine-based inhibitory motif with activation properties (ITIMa) (Section 1.1.4.4). One of such receptors is a triggering receptor expressed on myeloid cells-like protein 1 (TREM-like transcript 1, also known as TLT-

1). This is an Ig-like cell surface receptor with two ITIM sequences within its intracellular domain [256, 423], one of which enhanced FcεRI-mediated intracellular calcium flux through recruitment of SHP-2 [256]. Other ITIM-containing receptors have subsequently been shown to transduce activating signals, including induction of nitric oxide in macrophages upon ligation of Signal regulatory protein-α (SIRP-α) [257], activation of transfected COS-7 cells upon ligation of the ITIM-containing chemokine receptor CCK2R [424-426], and dual activating and inhibitory roles of the paired immunoglobulin-like receptor B (PIRB) on murine eosinophils treated with eotaxin and leukotriene B4, respectively [314]. The mechanisms which underlie ITIMs transducing activating signals are ill-defined. However, interestingly, there is a large body of evidence showing the reverse effect, whereby ITAM-containing receptors can transduce inhibitory signals [22, 256, 257, 314], which may provide some insight into the mechanisms involved in the dual activating and inhibitory effects of LILRB4. A typical example is ligation of FcαRI by monomeric IgA eliciting anti-inflammatory effects, with IgA oligomers mediating strong pro-inflammatory effects [427].

Monomeric IgA induces partial, “weaker” phosphorylation of the ITAMs that simultaneously recruit Syk and SHP-1, resulting in net inhibition [246]. By contrast, multimeric IgA induces greater phosphorylation with enhanced recruitment of Syk family kinases, but not SHP-1, leading to net cell activation [246]. Moreover, O'Neill et al. (2011) discovered that constitutive phosphorylation of SHIP-1 and its adaptor protein Dok-1 in anergic B cells is due to BCR ITAM monophosphorylation [428]. By contrast, engagement with foreign antigens causes dual phosphorylation of BCR ITAMs

213 leading to potent B cell activation [428]. This phenomenon has recently been postulated as a therapeutic mechanism following the administration of intravenous immunoglobulin for inflammatory disorders [429]. It is likely that similar mechanisms modulate the ability of the proximal LILRB4 ITIM to effect activating signalling, and the middle tyrosine transducing inhibitory signals. It is possible that partial (weak) ligation of LILRB4 by a low affinity and/or low concentration of ligand/s may cause mono-phosphorylation of the proximal Tyr leading to net activating function, while phosphorylation of all Tyr residues and/or middle Tyr residue may preferentially occur upon ligation by alternate high affinity and/or high concentration of ligand/s (Section

5.5).

Surprisingly, over-expression of plasmid constructs containing the dominant inhibitory functions such as the construct with the three intact Tyr residues (YYY), or with the one that contained an intact middle Tyr residue (FYF), were able to suppress

FcγRI-mediated TNF-α production without co-ligation with anti-CD25, albeit less significantly (Figure 3.2-8). Similar results were observed by Bellon et al (2002) in

LILRB1 site-directed mutant over-expressing rat basophil leukemic cells (RBL) activated through the high affinity IgE receptor, in which co-ligation with LILRB1 was found to be unnecessary for inhibition [230]. This was likely due to the forced over- expression of large amounts of ITIM/s which may have constitutively recruited more phosphatases in close proximity to FcγRI ITAMs, leading to spontaneous suppression without ligation of the inhibitory transgenic receptor. Future studies using clones with low levels of transgenic expression may resolve these somewhat unexpected results.

Moreover, cells over-expressing the LILRB4 ITIM without functional Tyr residues

(FFF) substantially reduced FcγRI-mediated TNF-α production (Table 3.2-2), indicating the possibility that additional structural elements within the LILRB4 intracellular

214 domain may also have regulatory functions. Consistent with this, similar inhibitory effects were observed when the Tyr residue within the single ITIM of the killer inhibitory receptor CD158d was mutated [3]. The structural domains mediating these phospho-Tyr-independent effects are unknown; reports have indicated that proline-rich regions [27, 28, 229], and SH3-binding motifs within ITIM backbones may functionally interact with several protein kinases or adaptor proteins and regulate cellular activation

[25-28, 229]. LILRB4 contains two SH3 binding motifs and a proline-rich region

(Figure 5-1), but it remains unclear whether these structures contribute to its immune regulatory functions.

Interestingly, cells that over-expressed single middle Tyr389 also significantly inhibited cell proliferation and enhanced apoptosis, which in part may explain some of its potent inhibitory effects (Figure 3.2-6 and Figure 3.2-7). Although there is no extant data pertaining to the effect of over-expressing ITIMs motifs in cell proliferation and apoptosis, there is evidence showing that downstream over-expression of inactive SHP-

1 mutant in myelo-monocytic cell line, U937 cells increased proliferation, but inhibited apoptosis [430]. In addition, it has been shown that inhibition of Syk phosphorylation enhanced apoptosis, but blocked cell proliferation in B cell [431], and T cell lymphoma

[432]. Hence, it is plausible to suggest that over-expression of the functional middle

Tyr389 of LILRB4 ITIM may have enhanced the recruitment of activated SHP-1 [18, 41], or SHP-like phosphatases [15], contributing to the suppression of proliferation and increased cell death which would, in turn, amplify its inhibitory effects on TNF-α production. It is noteworthy that these proliferation and apoptosis studies were performed in the absence of any cell stimulation; it would be interesting therefore to investigate the effects of each ITIM/s on proliferation and/or apoptosis following FcγRI cross-linking, or in response to bacterial infection.

215

5.3 CD25-LILRB4 over-expressing cells differentially regulate phagocytosis of

IgG opsonised polystyrene beads and bactericidal activity.

In addition to mediator production, FcγRI is a key receptor in phagocytosis of IgG opsonised pathogens/particles [34, 433]. Phagocytosis is a process characterised by uptake of large particles/pathogens (> 0.5 μm in diameter), mostly by phagocytes such as macrophages [188] (Section 1.1.3.1.2.1). Opsonisation of antigens by complement and immunoglobulins leads to recognition by the complement receptors [190], and the

Fc receptors, respectively [34], thus allowing efficient phagocytosis. Although FcγRI- dependent phagocytosis of antibody-opsonised particles/pathogens by macrophages has a different downstream signalling pathway [184-187], its upstream signalling has several overlaps with endocytosis, including with phosphorylation of Syk and Cbl [392-

394]. Briefly, when large IgG opsonized antigens bind to extracellular Fc binding domains of FcγRI, the Src family kinases that phosphorylate Tyr residues of ITAMs are activated in the associated FcγRI common γ-chain [185, 188]. Subsequently, phosphorylated ITAMs recruit and phosphorylate Syk family kinases that activate downstream signalling proteins distinct from the molecules involved in the downstream endocytosis pathway [186, 380]. These include phosphorylation of LAT and growth factor receptor-bound protein 2 (Grb2) [193] and the subsequent activation of PI3K and phospholiphase C that induces actin polymerisation and the formation of phagosomes which mature into phagolysosomes, and clear the offending pathogens [188, 189].

Binding of the IgG-coated pathogen to FcγRI receptors may also concurrently trigger

Tyr phosphorylation of multiple signalling molecules that are involved in the production of mediators, such as TNF-α, IL-1β, IL-6 and IL-8, and which further elaborate responses to pathogens [192] (Section 1.1.3.1.1 ). It has been previously demonstrated that LILRB4 dephosphorylated FcγRI-mediated Syk, an important upstream kinase

216 common to phagocytosis and mediator production, and suppressed TNF-α production

[15], which suggests that LILRB4 may also suppress phagocytosis. However, LILRB4 also dephosphorylates Cbl [15] that, upon phosphorylation, is a negative regulator of phagocytosis/endocytosis through its promoting the degradation of Syk [392-394] and increasing receptor internalisation [15, 389], which suggests that LILRB4 may enhance phagocytosis by reversing the negative effects of Cbl. One plausible explanation for these seemingly contradictory observations is that LILRB4 may have a dual pro- and anti-phagocytosis effect depending on the position of the Tyr residues in its ITIMs, similar to its effects on the FcγRI-mediated TNF-α production (Section 5.2, above). To ascertain this, phagocytosis of IgG-coated 2 µm polystyrene beads was assessed in

THP-1 cells over-expressing the different LILRB4 ITIM mutants. The results showed a modest enhancement of bead phagocytosis in all cells over-expressing LILRB4 ITIM mutants. The effects on TNF-α production (Figure 3.2-8 and Figure 3.2-9) were more complex with two exceptions: FYF, which caused marked suppression of bead uptake and significantly inhibited TNF-α production, suggestive of its dominant inhibitory effects; and FFF, which showed no effect. An inhibitory receptor containing an ITIM,

FcγRIIB, was shown to inhibit FcγRIIA-mediated IgG-coated sheep-RBCs in COS-1 cells via Tyr phosphorylation of its ITIM [434]. In addition, over-expressing SHP-1 or

SHIP further increased the inhibitory function of FcγRIIB in phagocytosis [435].

Interestingly, the higher binding strength of CD47 to SIRP-α containing ITIMs increased the effect of its inhibitory function in phagocytosis of IgG-opsonised sheep-

RBCs in THP-1 macrophages [436]. Although the direct function of SHP-1 activation in phagocytosis was not studied, the indication is that the activated SHP-1, by interaction of CD47 and SIRP-α, may inhibit Fc receptor-dependent phagocytosis. In contrast, Cbl- deficient bone marrow-derived macrophages inhibited FcγR-mediated phagocytosis,

217 and increased binding to immune complexes on their cell surface [437]. Thus, cells over-expressing FYF may recruit SHP-1 which may inhibit bead uptake and phagocytosis, while other mutants may enhance phagocytosis by preventing Cbl- dependent ubiquitination of Syk leading to phagosome formation.

Notably, results presented in this study showed a significantly reduced uptake of human serum opsonised S. typhimurium in all transfected cells over-expressing LILRB4

ITIM mutants, when compared to vector alone transfected cells (Figure 3.2-10). These suppressive effects on bacteria uptake, that were contrary to the results that showed modest enhancement of bead phagocytosis by most LILRB4 ITIMs, suggest that

LILRB4 may differentially regulate endocytosis, which is the major mechanism for bacteria internalisation and the uptake of large beads that occurs primarily through phagocytosis [160, 162, 184, 185]. This proposal is consistent with those results presented in Section 3.1 showing that ligation of LILRB4 significantly suppressed Tyr phosphorylation of key proteins involved in clathrin-mediated endocytosis (Figure

3.1-6). The opposing effects of LILRB4 on Syk and Cbl phosphorylation and/or the dominant inhibitory effects of the middle Tyr residue may explain the modest net enhancement of bead phagocytosis observed in most of the cells expressing one or more intact Tyr residue(s).

Notwithstanding that the suppression of bacteria endocytosis was universal, significant differences were observed in bactericidal activities among the different

LILRB4 ITIM mutants (Figure 3.2-12). Dissimilar to their mainly inhibitory effects on

TNF-α production, most of the LILRB4 ITIM mutants enhanced bactericidal activity against S. typhimurium when compared to mock transfected cells (Table 3.2-2). This may indicate that LILRB4 primarily inhibits the secretory functions of monocytes, but promotes bacterial clearance. Remarkably, the single proximal Tyr337 (YFF) over-

218 expressing cells significantly enhanced TNF-α production, but markedly suppressed bactericidal activity in contrast to the profound inhibition of TNF-α production and significant increase of bacterial killing in cells over-expressing the single distal Tyr419

(FFY) residue (Table 3.2-2). There is evidence which shows that leukocytes from ITIM- containing sialic acid-binding Ig-like lectins (SIGLECs)-deficient mice are more efficient in killing Streptococcus by producing more pro-inflammatory cytokines, and are able to clear infection more quickly than a normal mouse upon a low dose infection.

An overwhelming dose of bacteria will, however, exaggerate cell activation and trigger shock and more rapid death [438]. Profound immune defects result from insufficiency of inhibitory signalling pathways suppressing activation thresholds and hyper-responses which lead to autoimmunity and chronic inflammation [439, 440], suggesting that innate immune responses sufficient to eliminate pathogens should be generated, while the level of inflammatory activation should not be so high as to cause widespread host tissue damage. Thus, the tight regulation of immune activation by various LILRB4

ITIMs may play a role in fine-tuning immune responses (Table 3.2-2).

The aim of this study was to systematically determine the role of each of the three

ITIMs, in the intracellular domain of LILRB4 in its regulatory functions of monocyte activation. This was only possible by using site-targeted mutagenesis and generation of stable transfectants, hence the rational for the use of a monocytic cell line (THP-1) that expressed LILRB4 and its relevant signalling machinery as the closest model representing primary monocytes. The use of primary cells, especially monocytes for such complex studies were not feasible. We recognised the use of cell lines as a major limitation of this project hence we attempted to replicate data generated in the transfected THP-1cells in primary monocytes cells using synthetic ITIM peptides with limited success, as these experiments required more extensive optimisation that were

219 not carried out due to time constrains. One potential clinical application of the results which demonstrated selective regulation of TNF-α production, phagocytosis and/or bactericidal activity by the different LILRB4 ITIM mutants is the design of synthetic

ITIM-containing peptides with specified functions. For instance, a synthetic peptide containing intact middle Tyr may down-modulate excessive, potentially harmful TNF-α production, but effect more efficient bacterial clearance. One research group has recently shown synthetic peptides containing the ITIM sequences of the immune receptor (IREM-1, immune receptor expressed by myeloid cell, or known as CD300F) expressed on myeloid cells-1 to suppress B cell activating factor (BAFF)-mediated IL-8 production and phagocytosis [441]. The ITIM motif with Tyr205 was shown to be a major docking site for SHP-1 [442], and the synthetic peptide-containing the ITIM sequences of IREM-1 (amino acid 201-210) to mimick its inhibitory function in BAFF- mediated THP-1 activation [443]. In the current study, based on the finding that the

YYY and FYF displayed potent inhibition of TNF-α production while promoting bacterial clearance, two functional peptides containing YYY and FYF fused with HIV- transactivator of transcription (HIV-TAT48-57) were synthesised to facilitate peptide internalisation into cells [441, 443]. Two additional peptides, as controls, were also synthesised, one containing LILRB4 ITIMs without Tyr (FFF-TAT), and the other being a TAT alone. The synthetic YYY-TAT and FYF-TAT partially inhibited FcγRI- mediated TNF-α production in THP-1 and primary peripheral blood mononuclear cells

(Figure 3.2-13), results which were quite modest and were only marginally better than the control peptides. This was primarily due to the inefficient uptake of the peptide, both by THP-1 cells and the primary cells, as determined by intracellular staining using an antibody against the TAT peptide (data not shown), however that the TAT peptide is commonly used for protein delivery into cells [444]. It has been shown that TAT bound

220

CXCR4 and increased its expression [445], and THP-1 cells (Figure 3.2-1) and PBMCs

[445] expressed CXCR4. In addition, tat peptide was able to stimulate pro-inflammatory cytokines including TNF-α, IL-6 and INF-γ [446, 447] suggesting the expression of

CXCR4 and the use of TAT may or may not contribute the readout of TNF-α production. These encouraging very preliminary results provide a proof of concept and feasibility, but extensive future optimisation studies, incorporating improved delivery using nanoparticles [448], are required.

In summary, results presented in this section suggest that preferential activation of the single proximal Tyr residue may lead to enhanced pro-inflammatory mediator production, at the expense of limited bacterial killing, whereas selective activation of the distal Tyr may polarise monocytes towards efficient bacterial clearance, with minimal inflammation. A similar polarisation of effector cells occurs following activation of related receptor, LILRA2 [120, 136, 449]. Interestingly, the effect of the single middle Tyr389 (FYF) on phagocytosis and bactericidal functions was minimal, despite its exhibiting the most dominant inhibitory functions with respect to cytokine production (Table 3.2-2), indicating specific regulatory functions. Taken altogether, the evidence provided here is sufficient to indicate the complex inhibitory and activating functions of LILRB4 in monocytes/macrophages, depending on the position of the functional Tyr residues in its ITIMs and the nature of the stimuli. Additional experiments will be required in order to fully illuminate LILRB4’s functions and to delineate the contribution of each of its ITIMs. Comparative co-immunoprecipitation studies of the amounts of phosphorylated phosphatases including SHP-1, 2 and SHIP recruited to the ITIMs of each mutant at baseline and post-co-ligation with FcγRI, and investigation of identities of the phosphatases involved in LILRB4-mediated regulation of monocyte activation by specific gene silencing sh/siRNAs in each mutant would 221 strengthen and/or validate our results, and provide insight into the underlying mechanisms of LILRB4-mediated fine-tuning of the innate immune system. Further studies could include comparing the relative levels of phosphorylated Src, Syk, clathrin,

Cbl, HGS, HSP70, and TRIM21 upon FcγRI-cross linking and/or CD25-LILRB4 ITIMs co-ligation; defining the regulatory function of the additional elements including SH3 binding motifs and a proline-rich regions; and optimising delivery systems for synthetic inhibitory peptide, to further strengthen our understanding of the immune regulatory functions of LILRB4 and its potential use as a therapeutic agent.

5.4 LILRB4 differentially regulates LPS-mediated THP-1 cell activation

depending on the presence of the Tyr residues of its ITIMs.

Essential to appropriate immune response to infection, are various receptors on the cell surface that receive and transmit signals to activate the immune system. Both

TLRs and LILRs are widely expressed on immune cells, predominantly on myelomonocytic cells [89]. TLRs are non-ITAM containing activating receptors important in initiating innate immune responses via activation of NFκB and/or MAPK pathways in response to microbes [450, 451] (Section 1.1.3.2), while LILRs have been shown to provide inhibitory or balance effects on TLR activity [159].

The expression of gp49B1 (a mouse orthologue of human LILRB4) was up- regulated in response to LPS, and disruption of gp49B1 elevated sensitivity to LPS [32].

LILRB4 was also shown to be up-regulated on in vitro-cultured human blood macrophages following Salmonella infection as well as Salmonella LPS [19], suggesting that LILRB4 may play a key role in the regulation of TLR4-mediated cell activation. This project thus elucidates the immune-regulatory functions of LILRB4 through its three intracellular ITIMs in LPS (TLR4)-mediated monocyte activation. 222

Possibly, it may represent the function of LILRB4 in non-ITAM activating receptor- mediated cellular activation.

Here, data showed that LPS-mediated TNF-α production was inhibited by cells over-expressing CD25-LILRB4 ITIM mutants except for YYF, which enhanced TNF-α production (Figure 3.3-2). Interestingly, cells over-expressing three Tyr residues (YYY) and those over-expressing without Tyr residues (FFF) both showed the most significant inhibition of LPS-mediated TNF-α production (Figure 3.3-2 and Figure 3.3-3).

Consistently, mRNA expression of TNF-α and NFκB1, upon LPS stimulation, was down-regulated by cells over-expressing YYY or FFF compared to vector only transfected cells (Appendix III C). Although further mRNA analysis is required for experimental replicates and confirmation of protein levels, the results from cytokine production and mRNA expression suggest that LILRB4 regulates LPS-mediated monocyte activation through Tyr residues in its ITIMs both dependently and independently. Unexpectedly, however, a low level of IL-6 and IL-10 was determined in all cells. LILRB4 ligation was recently observed to change the surface phenotype of

LPS-matured dendritic cells in vitro leading to up-regulation of IL-10 in in vitro cultured macrophages in response to Salmonella infection [19]. IL-10 is known to inhibit several inflammatory cytokines, including TNF-α, showing that LPS-mediated

TNF-α production was abrogated by IL-10 in alveolar macrophages and peripheral blood monocytes in vitro [452]. Although LPS-activated THP-1 macrophages have been reported to secrete IL-6 and IL-10, there is evidence that THP-1 monocytes

(undifferentiated form) do not secrete IL-6 and IL-10 [453]. Thus, further study will be required to determine IL-10 production using differentiated cells over-expressing

CD25-LILRB4 ITIM mutants in response to LPS, or performing time-dependent

223 cytokine secretion, such as an early time-point for TNF-α and a late time-point for IL-10

[271].

Upon LPS binding to TLR4, TRIF-dependent and MyD88-dependent pathways are activated, inducing inflammatory cytokines, including TNF-α by activation of IKK and Akt, and translocation of NFκB [210] (Section 1.1.3.2). The NFκB family includes

RelA (p65), RelB, c-Rel, NFκB1 (p50/p105) and NFκB2 (p52/p100) [454]. The form p50/p65 is the most abundant, and while inactive when it forms a complex with IkBα within the cytoplasm, it is translocates into the nucleus upon cell activation by proteasome-mediated IkBα degradation [455]. The p105 is a precursor of p50 subunit, and requires proteolysis for translocation into the nucleus for gene transcriptions [456,

457], and the 26S proteasome is responsible for the processing of p50 from p105 [458].

Interestingly, cells over-expressing FFF significantly increased amounts of p105 compared to cells over-expressing YYY and vector only transfected cells (Figure 3.3-4).

It is possible that cells over-expressing FFF may inhibit 26S proteasome activity, which may be associated with its inhibitive function in LPS-induced TNF-α production.

Inhibition of 26S proteasome was shown to induce apoptosis in pancreatic cancer cells in vitro [459], and here, data in Section 3.2.4 showed that cells over-expressing FFF enhanced early apoptosis (Figure 3.2-7). Up-regulation of LILRB4 in pancreatic cancer has been linked to poor outcomes [85], therefore an investigation of whether a blockage of three Tyr of LILRB4 ITIMs, or over-expressing intracellular LILRB4 ITIMs without

Tyr resides, induces pancreatic cancer cell apoptosis with minimal production of pro- inflammatory cytokines is of interest. The inhibitory function of LILRB4 ITIMs without functional Tyr had a result similar to that for the previous FcγRI-mediated TNF-α production and pHrodo-labelled S. typhimurium phagocytosis in Section 3.2. Evidence exists which shows that mutation of Tyr within ITIM of KIR in NK cells did not

224 completely abolish its inhibitory function, and still weakly recruited SHP-2 [3]. Thus, it is possible that specific Tyr phosphatases are able to bind to LILRB4 ITIMs without

Tyr, possibly down-regulating LPS- and FcγRI-mediated TNF-α production, or that additional structural elements within its intracellular domain, such as SH3 binding motifs or proline-rich regions, may be involved in this regulation.

In contrast, cells over-expressing YYY suppressed phosphorylation of Akt (Figure

3.3-4). Depending on the stimuli and cell type, Thr308 and/or Ser473 of Akt are phosphorylated upon cell activation [460, 461]. The phosphorylation of Akt is induced by activation of the phosphoinositide 3-kinase (PI3K), which is activated by Tyr kinases such as Src and Syk [462]. In turn, de-phosphorylation of Akt could be induced by the termination of PI3K signalling via Tyr phosphatases such as SHPs and SHIP [463, 464].

There is evidence showing that Akt phosphorylation was enhanced in SHIP-deficient mouse macrophages in response to LPS [465]. In addition, IREM-1 ITIM-containing synthetic peptides inhibited LPS-induced TNF-α in THP-1 cells via activation of SHP-1 and SHP-2 [441]. This evidence indicates that the three ITIMs of LILRB4 may recruit

Tyr phosphatase, especially SHP-1 [41], upon TLR4-mediacted cellular activation, resulting in the suppression of TNF-α production via de-phosphorylation of PI3K, Akt and other downstream signalling proteins.

Both p105 and Akt are important downstream effector molecules producing TNF-

α via activation of IKK and PI3K respectively [206, 466], which suggests that LILRB4 may inhibit LPS-mediated TNF-α production through IKK or PI3K dependent pathways, depending on the presence of the tyrosine residues (Figure 5-3).

225

5.5 Potential interaction of LILRB4 with multiple cell surface ligands on T cells

The purpose of this chapter was to identify native LILRB4 ligands from the plasma membrane of T cells that were previously shown to bind to soluble LILRB4-Fc fusion protein [109]. To achieve this, placental alkaline phosphatase tagged and untagged recombinant LILRB4 were successfully produced in a mammalian expression system, and purified to a very high quality protein using Metal Affinity Resin. However, the amounts of the recombinant LILRB4 proteins produced, 125 μg per litre of culture supernatant for LILRB4-APtag-His, and 200 μg per litre of culture supernatant for

LILRB4-His, were significantly lower than that produced for recombinant APtag-His control protein (1000 μg/litre) using the same expression system. Interestingly, this study’s laboratory was able to produce > 750 μg/litre of LILRA3-APtag-His protein using the same vector and mammalian cells and culture conditions [83]. LILRA3 is heavily N-glycosylated and larger [83] than LILRB4, which is likely not to have N- glycans, and this significant difference between these two members of the LILR family may have contributed to differences in the stability and yield of these proteins. The crystal structure of LILRB4 has recently been shown to contain an unstable form of a

310 α helix in the D2 domains compared to other α helixes, suggesting an unstable tertiary structure [103]. Indeed, the purified LILRB4-APtag-His protein had lost its ability to bind membrane proteins on the surface of peripheral blood leukocytes and

Jurkat T cells within two weeks of storage at 4oC, in contrast with LILRA3-APtag-His, stable at 4oC for over six months. It is therefore possible that its lack of N-glycans may increase LILRB4 protein’s susceptibly to degradation/aggregation. Hence, the mammalian-produced recombinant LILRB4 proteins involved in all pertinent experiments in this project were used within two weeks of their production.

226

LILRB4-APtag-His was used as a convenient tool to determine its surface binding to PBMCs and Jurkat cells and to identify potential ligands. Binding of LILRB4 to

PBMCs was high affinity, saturable (Figure 4-5) and highly reproducible among different donors. Similarly, there was significant binding of LILRB4 to the surface of

Jurkat T cell lines. However, the affinity constant (K), dissociation constant (KD) and maximum specific binding (Bmax) were not established on this cell lines due to time constrains. These readily available cells were however used to screen LILRB4 binding proteins, which was one of the most important objectives of this project. Six membrane proteins were consistently identified using LILRB4-APtag-His as bait (Mowse score >

45, peptide matches ≥3, p < 0.05) (Table 4-3). Interestingly, all these proteins have molecular masses ranging from 28-40 kDa, sizes similar to those proteins shown to interact LILRB4-APtag-His by Far-Western blot (Figure 4-8). Although the MASCOT scores for these proteins were modest (Mowse score 45-115), none was detected when the APtag control was used as bait, suggesting specific interaction with LILRB4.

Moreover, in strong alignment with their expected functions upon interaction with

LILRB4, these candidate ligands are all involved in immune-regulatory functions

(Table 4-3). The strongest candidate ligand, CD47, also known as integrin associated protein (IAP), is a membrane protein belonging to the immunoglobulin superfamily

[119, 340]. It is ubiquitously expressed in immune cells, erythrocytes and cancer cells and interacts with several proteins, including thrombospondin-1 (TSP-1) [341, 342], signal-regulatory protein alpha (SIRP-α) [119, 340], and several membrane integrins, most commonly αvβ3 [343]. CD47/SIRP-α interaction leads to bidirectional signaling, resulting in different cell-to-cell responses, including inhibition of phagocytosis in macrophages [119, 340] and T cell activation (Figure 5-2) [344]. Interestingly, SIRP-α is an immunoglobulin-like receptor expressed on macrophage and contains ITIMs

227 similar to LILRB4, although the sequence homology of their extracellular domains is

29.3% identity and 51.7% similarity (analysed using the LALIGN from ExPASy

(www.ch.embnet.org)). CD47 is known to stably associate with αvβ3 and to regulate cell adhesion, spreading and migration [345]. It is intriguing whether binding of the mouse orthologue of LILRB4 (gp49B1) to αvβ3 reported by Castells et al [111] involves its interaction with CD47. The second strongest candidate ligand is CD1b, a member of the

CD1 family of glycoproteins (CD1a-e) that are related to the class I MHC molecules

[346, 347]. CD1b is expressed in cells specialized for antigen presentation, and is involved in the presentation of lipid antigens to T cell [348, 349]. Recently, it was reported that inhibitory LILRB2 interacted with CD1c and d, and blocked its recognition by natural killer T (NKT) cells [99, 350]. CD2, also known as LFA-2, is a member of the immunoglobulin superfamily, acts as a co-stimulatory molecule in T cells and NK cells [351, 352], and is involved in cell adhesion through interaction with

LFA-3 [353] or CD48 [354]. CD48 is a CD2 subfamily molecule, highly expressed on

T cells, B cells, monocytes and granulocytes [355-357]. CD48/CD2 coupling together with other pairs such as CD28/CD80 and MHC-I/LFA-1 contributes to the formation of immunological synapses between T cells and antigen presenting cells [358]. Its interaction with CD2 has also been shown to promote lipid raft formation [359]. CD82 is a membrane glycoprotein expressed in various leukocytes, and through interaction with MHC-II molecules it inhibits cell growth and cancer metastasis [360, 361]. Unlike traditional methods of ligand screening, such as expression cloning, where screening for binding of pre-selected recombinant proteins does not take into account the possibilities of multiple LILRB4 ligand/s or co-ligands, the simple but powerful proteomic approach allowed unbiased global screening of native cell surface ligands and found results suggestive of multiple LILRB4 binding partners. However, whether these are

228 independent ligands of various affinities, or whether some act as ligand-co-ligand pair/s or are bystander proteins that bind to a common protein shared by LILRB4 and the candidate ligand remains to be elucidated. Consistent with the proposal that LILRs have more than one ligand, there is increasing evidence that LILRs, including LILRB2 [29-

31, 88] and LILRA3 [86, 90], may possess multiple non-MHC and MHC ligand/s with various affinities and which display diverse functions (Sections 1.1.5.1 and 1.1.7).

229

Figure 5-2. Schematic diagram demonstrating the interaction of CD47 with SIRP-α and αvβ3, and prediction of the interaction with native and/or recombinant LILRB4 Activation of CD47 (by the stress-released matricellular protein thrombospondin-1, TSP1) is a potent inhibitor of Ca2+, ROS and Akt [467], and the interaction of CD47 and SIRP-α inhibits phagocytosis [119, 340]. CD47 has also been shown to bind αvβ3 which is a ligand for mouse LILRB4, gp49B1. The ~62% amino acid sequence identities between human and mouse

LILRB4, and the ~90% amino acid sequence homology between human and mouse αvβ3 suggest that human native and/or recombinant LILRB4 may interact with αvβ3 or interact with αvβ3 related proteins such as CD47and SIRP-α.

230

Although some exciting preliminary results were presented, this part of the project was not completed, owing to the time constraints of my PhD candidature. The following recommendations are made for future experiments that will be required for further characterisation and confirmation of the identities of LILRB4 ligand/s, and definiton of their functional interactions. First, improved yield of LILRB4 protein production can be obtained by minimising degradation i.e. by keeping constituents cold and using protease inhibitors) [468, 469], and by scaling up the quantities of culture supernatants by using

293F cells (cells are adapted to suspension culture in serum free medium) that can be transiently transfected with the LILRB4 plasmid DNA constructs in bulk and grown in suspension in volumes of up to ten litres [470-472]. Second, extensive characterisation of LILRB4 binding to the surface of Jurkat cells can be performed, include determination of the binding affinity constant (K), dissociation constant (KD) and maximum specific binding (Bmax), using LILRB4-APtag-His [90, 473]. Third, the generation of full length plasmid DNA constructs in mammalian expression vectors for each of the five strongest candidate ligands should be conducted, over-expressing them individually or in combination and performing comparative ligand binding studies and in situ staining using LILRB4-APtag-His [83, 90]. Fourth, generate recombinant proteins for each of the candidate ligands from the most likely (CD47) to the least likely

(CD82), and undertake comparative binding affinity to recombinant LILRB4 using surface plasmon resonance (SPR) [90, 474]; this will provide the relative binding affinities of each ligand to LILRB4, or indicate the requirement of more than one particular ligand for high affinity binding [475, 476]. Fifth, advanced microscopy, such as fluorescence resonance energy transfer (FRET) [477], or total internal reflection fluorescence microscopy (TIRFM) [478], is required to investigate whether one or more of these proteins are independent ligands, or act as co-ligands by generating various

231 fluorescence-tagged plasmid DNA constructs of each protein and of LILRB4 which can be individually co-expressed with LILRB4, or multiple ligands co-expressed with

LILRB4 in appropriate mammalian cell line(s) [109, 479, 480]. If a ligand/s is confirmed by one or more of the above measures, co-immunoprecipitation studies using native LILRB4 and the ligands can be performed.

5.6 Conclusion

The regulation of monocyte/macrophage activation by LILRB4 is extensively studied and presented in this thesis. Here, it has been shown for the first time that

LILRB4 is a complex immuno-regulatory receptor that exerts dual inhibitory and activating functions in FcγRI and/or TLR4-mediated monocyte/macrophage activation, including receptor-ligand internalisation, endocytosis, cytokine production, phagocytosis and bactericidal activity. Although FcγRI and TLR4 are important for the recognition and elimination of foreign antigens, the lack of inhibitory receptors in their family group can induce over-exuberant or unregulated immune responses, such as sepsis by bacterial and viral infection, leading to failed homeostasis and profound immune defects caused by suppressed activation thresholds and hyper-responses leading to autoimmunity and chronic inflammation. Thus, the tight regulation of these activating receptors, and possibly other ITAM-containing and non-ITAM receptors, by LILRB4 may play a key role in fine-tuning immune responses. Most importantly, the findings for the regulatory function of LILRB4 using various ITIM mutants provide a clearer understanding of the signalling pathways of LILRB4 that may contribute to LILRB4’s application as a potential therapeutic agent for an appropriate immune response using selective LILRB4 ITIM/s. While the data presented in this thesis showed that LILRB4 has a critical role in immune regulation in-vitro, the lack of knowledge of LILRB4

232 ligand/s continues to limit elucidation of its function for in vivo studies. In-vivo and/or ex-vivo studies would strengthen some of the novel and interesting results presented in this project. However, lack of rodent homologue of human LILRB4 precluded animal based in-vivo work. The identification of native LILRB4 ligands for which this project has made a significant progress would make future patient based ex-vivo study very exciting, otherwise current ex-vivo studies are/will be limited to descriptive observational studies. The potential candidate ligands of LILRB4 which have been provided here will contribute to the identification of the nature of LILRB4 ligand/s in the near future. In particular, further experimentation will focus on characterising and confirming the interaction of the strong candidate CD47 with LILRB4, and defining their functional interactions. Should CD47 be confirmed as a ligand of LILRB4, co- immunoprecipitation studies using native LILRB4 will follow.

233

234

Figure 5-3. Schematic representation demonstrating the regulation of monocyte/ macrophage activation by LILRB4 via its intracellular domains and binding of its unknown ligand/s Co-ligation of FcγRI with LILRB4 recruits phosphatases such as SHP-1 to its ITIMs that supress cytokine production by inhibiting Tyr phosphorylation of Syk, and dephosphorylate the key molecules, including clathrin, FcγRI and Syk, Cbl, HGS and TRIM21, involved in clathrin- mediated endocytosis of FcγRI and ligands. The inhibition of receptor endocytosis by LILRB4 may enhance phagocytosis. Upon TLR4 activation, MyD88-dependent pathways are activated and induce inflammatory cytokines by activation of NFκB (p50/p65) translocation. However, the three ITIMs of LILRB4 supresses TNF-α production via de-phosphorylation of PI3K, Akt and other downstream signalling proteins. In contrast, LILRB4 ITIMs without Tyr resides inhibit p105 degradation to generate p50 and lead to inhibition of gene transcription and TNF-α production.

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APPENDIXES

Appendix I. Common chemicals and reagents

1-ethy-3 (3-dimethyaminoprophy) carbodimide Thermo Fisher Scientific 3,3,5,5-tetramethybenzidine (TMB) Panbio 3H-thymidine PerkinElmer 4’,6’-diamidino-2-phenylindole (DAPI) Life technologies 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) Roche 5x sequencing buffer Ramaciotti Centre, UNSW β-mercaptoethanol Life technologies Acetic acid Ajax Finechem Acetonitrile Ajax Finechem Agarose Sigma-Aldrich Ampicillin Sigma-Aldrich Annexin-V/Propidium iodide (PI) apoptosis kit BD Biosciences anti-AP-conjugated sepharose beads Gene hunter BCA protein assay kit Thermo Scientific Pierce BD TalonTM Metal Affinity Resin column BD Biosciences Big Dye terminator Ramaciotti Centre, UNSW Bovine serum albumin Sigma-Aldrich Bromo-3-chloropropane (BCP) Sigma-Aldrich Carboxylate polybeads Polysciens CD3/CD28 beads (Human T-Activator CD3/CD28) Dynabeads® Chemiluminescent reagent (Western Lighting) PerkinElmer Coomassie Brilliant Blue R-250 Sigma-Aldrich Diethanolamine Sigma-Aldrich Diethylpyrocarbonate (DEPC) Sigma-Aldrich Dimethyl sulfoxide (DMSO) Sigma-Aldrich Dithiothreitol (DTT) Bio-Rad Laboratories Dynabeads® Human T-Activator CD3/CD28 Life technologies E. coli (JM109) Promega EcoRI Roche Ethanol, absolute Ajax Finechem Ethylenediaminetetraacetic acid disodium salt (EDTA) Sigma-Aldrich Ficoll gradient (Phamacia) Sigma-Aldrich Ficoll-PaqueTM PLUS (GE Healthcare Life Sciences) Sigma-Aldrich Formaldehyde Sigma-Aldrich Formic acid Riedel-de Haen G418 Life Technologies Gel extraction kit QIAGEN Gentamycin Invitrogen 264

Glycogen (RNAs free) Thermo Scientific Goat anti-mouse antibody conjugated sepharose beads Zymed Laboratories Inc. Hank’s balanced salt solution Sigma-Aldrich Heptafluorobutyric anhydride Roche HindIII Roche Human IgG Sigma-Aldrich Human TNF-α ELISA (DuoSet ELISA kit) R&D system

Hydrogen peroxide 30% (H2O2 ) Ajax Finechem Imidazole Sigma-Aldrich Isoprophyl alcohol Sigma-Aldrich

K2HPO4 Sigma-Aldrich Kanamycin Sigma-Aldrich L-AP and R-AP primers Gene Hunter L-homoarginine Sigma-Aldrich Lightning-linkTM biotin conjugation kit Innova Biosciences Lipofectamine® 2000 Life Technologies Lipofectamine® LTX DNA transfection reagent Life Techologies LIVE/DEAD® viability assay Kit Molecular Probes LPSE.coli Serotype 005:B5 Sigma-Aldrich Methanol Fronine Pty. Ltd.

MgCl2 Sigma-Aldrich MycoAlertTM mycoplasma detection kit Lonza NaCl Sigma-Aldrich Neutral red Sigma-Aldrich Nitro blue tetrazolium (NBT) Roche Nusieve GTG agarose gel Lonza Nycodenz Nycodenz Pharma Opti-MEM medium Life Techologies pAPtag-5 expression vector Gene Hunter Paraformaldehyde Electron Microscopy Sciences Pfu DNA polymerase Life technologies Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich pHrodo phagocytosis particle labelling kit Molecular Probes Plasmid mini kit QIAGEN Platinum Pfx DNA polymerase Life Technologies pMLSV N1/N4-S plasmid American Typed Culture Collection PNGase F kit BioLabs p-nitrophenol phosphate Sigma-Aldrich Polyacrylamide resolving and stacking gels Life Techologies Poly-L-Lysine (PLL) Sigma-Aldrich Polyvinyldifluoride membranes (PVDF, 0.2 µm pore size) Millipore Protease inhibitors Roche Applied Science

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QIAquick Gel Extraction Kit QIAGEN Quick Start Bradford Protein assay Bio-Rad Rapid DNA Dephos & Ligation Kit Roche RNase H Invitrogen Saponin Sigma-Aldrich Scintillation fluid Perkin Elmer Selection antibiotic (0.3 mg /ml of Zeocin) Life Technologies Skim milk Coles SOC media Life technologies

Sodium azide (NaN3) Sigma-Aldrich Sodium azide Sigma-Aldrich Sodium bicarbonate Sigma-Aldrich Sodium carbonate Sigma-Aldrich Sodium chloride Sigma-Aldrich Sodium orthovanadate Sigma-Aldrich Sodium thiosulfate Sigma-Aldrich Stirred ultrafiltration chamber Amicon Streptavidin-conjugated Alexa488 Invitrogen Sucrose Sigma-Aldrich Sulfo-NHS-LC-Biotin Pierece

Sulphuric acid (H2SO4) Sigma-Aldrich SuperScriptTM III First strand Synthesis SuperMix Invitrogen SYBR Green select master mix (cat # 4472908) Applied Biosystems Transformer Site-directed Mutagenesis kit Clontech Tricine, purity 99% titration Sigma-Aldrich Trifluoroacetic acid Sigma-Aldrich Tris (hydroxymethyl) aminomethane MP Biomedicals Triton X-100 Sigma-Aldrich TRIzol reagent Invitrogen Trypan blue Life technologies Trypsin Promega Trypsin-EDTA (ethylene-diamine-tetra-acetate) Life Technologies Turbo DNase Ambion UltraPure DNase/RNase free distilled water Life technologies Water for irrigation Baxter Healthcare XhoI Roche YM-30, 50 and 100kDa membranes Millipore

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Appendix II. MASCOT search results of mass spectrometric peptides sequencing of LILRB4-AP-His immunoprecipitated proteins as candidate ligands for LILRB4* candidate ligands MW(kDa) 1 2 3 1 Hsp90 co-chaperone Cdc37 44 0 1 5 2 Isoform 2 of BRCA2 and CDKN1A-interacting protein 36 0 0 3 3 Vesicular integral-membrane protein VIP36 40 0 0 2 4 Cluster of Vesicle-associated membrane protein 3 (IPI00549343) 11 0 0 5 5 Vesicle-associated membrane protein 3 11 0 0 5 6 Basement membrane-specific heparan sulfate proteoglycan core protein 469 1 10 1 7 Transmembrane protein 109 26 0 1 0 8 Translocating chain-associated membrane protein 1 43 0 2 0 9 Transmembrane emp24 domain-containing protein 10 25 0 2 0 10 Isoform 1 of Transmembrane and coiled-coil domain-containing protein 1 21 0 2 0 11 Isoform 1 of Adipocyte plasma membrane-associated protein 46 0 0 2 12 Vesicle-associated membrane protein 8 11 0 0 2 13 Isoform 1 of Vesicle-associated membrane protein-associated protein A 28 0 1 0 14 Erythrocyte band 7 integral membrane protein 32 0 1 0 15 Isoform 1 of Transmembrane protein 63B 95 0 0 0 16 Isoform 4 of Multiple C2 and transmembrane domain-containing protein 2 35 0 0 1 17 Isoform 1 of Transmembrane protein 132A 110 0 1 2 18 Isoform XD of Plasma membrane calcium-transporting ATPase 4 138 0 1 0 19 Transmembrane 9 superfamily member 4 75 0 0 1 20 Isoform 3 of UPF0663 transmembrane protein C17orf28 63 0 0 1 21 leucine-rich repeat transmembrane protein FLRT1 74 0 0 1 22 Seven transmembrane helix receptor 39 0 1 1 * Peptides identified in LILRB4-AP-His immunoprecipitated proteins, but not in AP-His immunoprecipitated proteins from four independent experiments

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Appendix II continued. MASCOT search results of mass spectrometric peptides sequencing of LILRB4-AP-His immunoprecipitated proteins as candidate ligands for LILRB4* candidate ligands MW(kDa) 1 2 3 23 Isoform 2 of Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 157 0 0 1 24 Peroxisomal membrane protein 11B 28 1 1 0 25 Isoform 1 of Tumour necrosis factor alpha-induced protein 8 23 0 1 0 26 Tumour necrosis factor alpha-induced protein 3 90 1 0 0 * Peptides identified in LILRB4-AP-His immunoprecipitated proteins, but not in AP-His immunoprecipitated proteins from four independent experiments

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Appendix III. Screening of mRNA expressions

A. Screening of mRNA expression in transfectants after FcγRI stimulation

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B. Screening of mRNA expression in transfectants after Salmonella stimulation

C. Screening of mRNA expression in transfectants after LPS stimulation

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