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Macrophages and Adaptive Immune System + Crosstalk between Splenic CD169 Macrophages and Adaptive Immune System Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie an der Universität Duisburg-Essen vorgelegt von Vikas Duhan aus Katwal, Haryana, Indien Essen, April 2017 All the experiments for this study have been carried out at the Institute of Immunology, Faculty of Medicine, University Hospital Essen, at the University of Duisburg-Essen or at another equivalent facility. 1. Examiner: Prof. Dr. Karl S. Lang 2. Examiner: Prof. Dr. Wiebke Hansen 3. Examiner: Prof. Dr. Lukas Flatz Chairman of the Audit Committee: Prof. Dr. Sven Brandau Day of the Oral Examination: September 4, 2017 Die Experimente der vorliegenden Arbeit wurden am Institut für Immunology, Medizinische Fakultät, der Universität Duisburg-Essen oder an einer anderen gleichwertigen Einrichtung durchgeführt. 1. Gutachter: Prof. Dr. Karl S. Lang 2. Gutachter: Prof.’in Dr. Wiebke Hansen 3. Gutachter: Prof. Dr. Lukas Flatz Vorsitzender des Prüfungsausschusses: Prof. Dr. Sven Brandau Tag der mündlichen Prüfung: September 4, 2017 Table of contents Summary ..................................................................................................................... 1 Zusammenfassung ....................................................................................................... 2 Introduction ................................................................................................................ 3 1.1 Immune System ........................................................................................................... 4 1.1.1 Structure of Immune System ....................................................................................... 5 1.1.1a Spleen ................................................................................................................................... 6 1.1.2 Innate immune system ................................................................................................ 8 1.1.2a Dendritic Cells ..................................................................................................................... 8 1.1.2b Macrophages ....................................................................................................................... 9 1.1.2b1 Metallophilic macrophages/MZ CD169+ macrophages .............................................. 10 1.1.2b2 Liver macrophage ........................................................................................................ 10 1.1.2c Interferons .......................................................................................................................... 11 1.1.3 Adaptive immune system .......................................................................................... 11 1.1.3a T cells ................................................................................................................................. 12 1.1.3a1 T cell activation ............................................................................................................ 12 1.1.3a2 Effector T cells ............................................................................................................. 13 1.1.3a3 Memory T cells ............................................................................................................ 15 1.1.3a4 Exhausted T cells ......................................................................................................... 15 1.1.3b B cells ................................................................................................................................. 16 1.1.3b1 B cell activation ........................................................................................................... 17 1.1.3b2 Functions of B cells ...................................................................................................... 19 1.2 Pathogens .................................................................................................................. 20 1.2.1 Lymphocytic choriomeningitis virus (LCMV) ............................................................. 20 1.2.2 Listeria Monocytogenes (LM) ................................................................................... 21 1.3 Mouse models ............................................................................................................ 21 Chapter 2 ................................................................................................................... 24 CD169+ macrophages regulate PD-L1 expression via type I interferon and thereby prevent severe immunopathology after LCMV infection ................................... 24 2.1 Abstract ..................................................................................................................... 25 2.2 Introduction .............................................................................................................. 26 2.3 Results ....................................................................................................................... 27 2.3.1 Depletion of CD169+ macrophages affects a subtype of F4/80+ macrophages in the liver and viral control .............................................................................................................. 27 i 2.3.2 CD169+ macrophages in the spleen and lymph nodes contribute to the production of IFN-I…………………………………………………………………………………………………………………………….28 2.3.3 CD169+ macrophages have limited impact on CD8+ T cell priming but are essential for controlling acute viral infection and prevention of immunopathology ............................ 29 2.3.4 CD169+ macrophages induce PD-L1 expression which prevents immunopathology 30 2.3.5 CD169+ macrophages prevent severe immunopathology during chronic viral infection .................................................................................................................................. 31 2.4 Discussion .................................................................................................................. 31 2.5 Methods ..................................................................................................................... 33 2.5.1 Mice ........................................................................................................................... 33 2.5.2 Depletion of the cells ................................................................................................. 33 2.5.3 Generation of bone marrow–derived macrophages and transfer experiment ......... 33 2.5.4 Plaque assay .............................................................................................................. 34 2.5.5 Real-time polymerase chain reaction ........................................................................ 34 2.5.6 Lymphocyte transfer .................................................................................................. 34 2.5.7 Flow cytometry .......................................................................................................... 34 2.5.8 Enzyme-linked immunofluorescent assays ................................................................ 34 2.5.9 Histology ................................................................................................................... 34 2.5.10 Alanine aminotransferase .......................................................................................... 35 2.5.11 Statistical analysis ..................................................................................................... 35 2.6 Ethics Statement ........................................................................................................ 35 2.7 Acknowledgments ..................................................................................................... 35 2.8 Figure Legends .......................................................................................................... 36 2.8.1 Figure 1: Depletion of CD169+ macrophages affects a subtype of F4/80+ macrophages in the liver and viral control ............................................................................. 36 2.8.2 Figure 2: CD169+ macrophages in the spleen and lymph nodes contribute to the production of IFN-I .................................................................................................................. 36 2.8.3 CD169+ macrophages have limited impact on CD8+ T cell priming but are essential for controlling acute viral infection and prevention immunopathology ................................ 37 2.8.4 Figure 4 CD169+ macrophages induce PD-L1 expression which inhibits immunopathology .................................................................................................................. 37 2.8.5 Figure 5: CD169+ macrophages prevent severe immunopathology during chronic viral infection .......................................................................................................................... 38 2.9 Supplementary Figures ............................................................................................. 39 2.9.1 Figure 1: Gating strategy of CD169+ macrophages .................................................... 39 ii 2.9.2 Figure 2: Expression of CD169 on immune cells ........................................................ 39 Chapter 3 ................................................................................................................... 50 Virus-specific antibodies allow viral replication in the marginal zone,
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