Functional comparison of cord and adult blood-derived dendritic cells. Title. Author(s) Wong, On-hang.; 黃安恆. Citation.

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1 Title Functional comparison of cord and adult blood-derived dendritic cells Advisor(s) Chiang, AKS; Huang, F Author(s) Wong, On-hang.; 黃安恆. Citation Issued Date 2004 URL Rights The author retains all proprietary rights, (such as patent rights) and the right to use in future works.

2 TABLE OF CONTENTS Declaration...i Acknowledgments...ii Table of Contents...iii List of Figures...viii List of Tables...x Abbreviations...xi CHAPTER I. INTRODUCTION 1.1. Neonatal Immunity in Human B Cells T Cells Dendritic Cells Dendritic Cells (DCs) DC Heterogeneity Antigen Capture by DCs... 10

3 Receptor-Mediated Endocytosis Phagocytosis Macropinocytosis DC Maturation and Activation Signals for DC Maturation and Activation Receptors for DC Activation Antigen Presentation DC Immunity vs. Tolerance Central Tolerance Peripheral Tolerance DC Capture of Dying Cells Cell Death Apoptosis Necrosis DC Recognition of Dying Cells DC Clearance of Dying Cells Modulation of Immune Responses by Dying Cells Hypothesis and Project Objectives

4 CHAPTER II. MATERIALS AND METHODS 2.1. Study Samples Isolation of Mononuclear Cells Isolation of CD14 + Monocytes Generation of Monocyte-Derived Dendritic Cells in vitro Generation of Immature Dendritic Cells Generation of Mature Dendritic Cells Isolation of CD3 + T cells Lymphocyte Transformation by Epstein-Barr Virus Preparation of EBV from B95-8 Laboratory Strain Establishment of EBV Transformed Lymphoblastoid Cell Lines (LCLs) EBV Nuclear Antigen (EBNA) Staining Preparation of Cell Smears Anti-Complement Immunofluorescence (ACIF) Test Induction and Detection of Early Apoptosis and Necrosis of LCLs Phagocytosis Assay

5 2.10. Immunophenotyping Microscopy Confocal Microscopy Cytokine Determination T Cell Proliferation Assay Statistical Analysis.. 38 CHAPTER III. RESULTS 3.1. Establishment of Fetal or Adult Lymphoblastoid Cell Lines (LCLs) Kinetic Study for the Induction of Early Apoptotic LCLs Effects of LPS on Cord and Adult DC Maturation Phagocytosis of Dying Cells by Immature Cord and Adult DCs Effects of Dying Cells on Cord and Adult DC Maturation Production of IL-10, IL-12p70 and TNF-α by Cord and Adult DCs after LPS Stimulation Modulation of IL-10, IL-12p70 and TNF-α Production by Cord and Adult Dcsupon Ingestion of Dying Cell 59

6 3.8. Following Ingestion of Necrotic but not Early Apoptotic LCLs, DCs showed an Enhanced Capacity in Stimulating allogeneic T-Cell Proliferation. 61 CHAPTER IV. DISCUSSION 4.1. Time Dependent Apoptotic Cell Death induced by UV-irradiation Uptake of Dying Cells by Adult and Cord Blood-Derived DCs Phenotypic Changes of Adult and Cord Blood-Derived DCs upon LPS Stimulation Phenotypic Changes of Adult and Cord Blood-Derived DCs triggered by Apoptotic and Necrotic Cells Cytokine Expression Profiles of Adult and Cord Blood-Derived DCs following Interaction with Dying Cells Efficient Selection of T Cells with Magnetic Cell Sortor (MACS) Antigen Presenting Activities of Adult and Cord Blood-Derived DCs in Response to Endogenous and Exogenous Dangr Signals Further Studies Study of T Cell Cytokine Profiles in the DC-T Cell Co-culture...77

7 4.8.2 Investigation of the Cellular Mechansims of Cord and Adult DCs Induction of T Cell Deletion or Anergy by Neonatal DCs upon Ingestion of Dying Cells Conclusions...80 REFERENCES.81

8 LIST OF FIGURES Figure 1. The developmental features of DCs....9 Figure 2. EBNA staining of LCLs 41 Figure 3. AV and PI staining of LCLs before and after UV-irradiation...43 Figure 4. Kinetics of apoptotic death induction...44 Figure 5. Representative photographs of LCLs at different time points after UV-irradiation...45 Figure 6. Phenotypic analysis of adult and cord DCs after LPS stimulation...47 Figure 7. Surface markers expression of adult and cord DCs after LPS stimulation. 48 Figure 8. Fluorescent dye labeling of DCs and LCLs.. 50 Figure 9. Phagocyosis of early apoptotic or necrotic LCLs by immature cord DCs Figure 10. Phagocyosis of early apoptotic or necrotic LCLs by immature adult DCs Figure 11. Confocal microscopy of adult DCs phagocytose early apoptotic or necrotic LCLs 53

9 Figure 12. Surface marker expression on cord and adult DCs after exposure to early apoptotic LCLs, necrotic LCLs or in culture medium.55 Figure 13. Production of IL-10, IL-12p70 and TNF-α by cord and adult DCs after LPS stimulation.58 Figure 14. Modulation of IL-10, IL-12p70 and TNF-α by cord and adult DCs upon ingestion of dying LCLs Figure 15. T cell stimulatory capacity of cord and adult DCs after cultured with medium, LPS, early apoptotic or necrotic LCLs for 36 h...62

10 LIST OF TABLES Table 1. List of monoclonal antibodies used in flow cytometry 39 Table 2. A summary of cord and adult DCs in response to endogenous and exogenous signals..65 LIST OF TABLES

11 Page 1 ABBREVIATIONS ACIF APCs AV BrdU CD40L CMBC CR CTLs DCs DNA EBNA EBV ELISA FBS FcR GM-CSF HBSS Anti-complement immunofluorescence Antigen-presenting cells Annexin V Bromodeoxyuridine CD40 Ligand Cord blood mononuclear cells Complement receptor Cytototoxic T lymphocytes Dendritic cells Deoxyribonucleic acid Epstein-Barr virus nuclear antigen Epstein-Barr virus Enzyme-linked immunosorbent assay Fetal bovine serum Receptor of Fc portion of Immunogloblins Granulocyte/macrophage colony stimulating factor Hanks balanced salt solution HMGB1 High mobility group box 1 hsps IFN-γ Ig IL- Heat shock proteins Interferon-gamma Immunoglobulin Interleukin- CHAPTER I. INTRODUCTION

12 Page 2 LCLs MFI MHC MMR MR NaH 3 NF-κB PBMCs PBS PFA PI PtdSer SEM SLE SRs TCRs Th TLR TNF-α Lymphoblastoid cell lines Mean fluorescence intensity Major histocompatibility complex Macrophage-mannose receptor Mannose Receptor Sodium azide Nuclear factor κb Peripheral blood mononuclear cells Phosphate buffer saline Paraformaldehyde Propidium iodide Phosphatidylserine Standard error mean Systemic lupus erthematosous Scavenger receptors T cell receptors T helper Toll-like receptor Tumor necrosis factor α CHAPTER I. INTRODUCTION

TABLE OF CONTENT. Page ACKNOWLEDGEMENTS. iii ENGLISH ABSTRACT THAI ABSTRACT. vii LIST OF TABLES LIST OF FIGURES. xvi ABBREVIATIONS.

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