The impact of rat cytomegalovirus gamma chemokine on dendritic cells DISSERTATION Zur Erlangung des akademischen Grades Do c to r r er um na tur a l ium (Dr. rer. nat.) im Fach Biologie Eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von Julia Cecilia Madela-Mönchinger Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Bernhard Grimm Gutachter: 1. Prof. Dr. Detlev H. Krüger 2. PD Dr. Sebastian Voigt 3. Prof. Dr. Martin Messerle Tag der mündlichen Prüfung: 23.04.2021 The experiments described in this dissertation have been conducted from January 2017 to July 2020 at the Robert Koch Institute under the supervision of Prof. Dr. Detlev H. Krüger and PD Dr. Sebastian Voigt. Für Stephan und meine Schwestern Nauka jest jak niezmierne morze, im więcej jej pijesz, tym bardziej jesteś spragniony. Science is like an immeasurable sea, the more you drink it, the thirstier you are. Stefan Żeromski Table of contents Table of contents Table of contents ................................................................................................................... 1 Index of figures and tables .................................................................................................... 5 Abbreviations ........................................................................................................................ 7 1. Introduction ...................................................................................................................12 1.1 Cytomegalovirus ...................................................................................................12 1.1.1 Classification of Cytomegaloviruses ..................................................................12 1.1.2 Rat cytomegalovirus ..........................................................................................13 1.1.3 CMV structure and replication ...........................................................................14 1.2 Immune system and response to CMV infection ...................................................15 1.2.1 Dendritic cells and antigen presentation ............................................................15 1.2.2 CMV host entry, spread and immune response .................................................18 1.2.3 Immune evasion by CMV ..................................................................................19 1.3 Chemokines, chemokine receptors and viral mimicry ............................................20 1.3.1 Classification and characterization of chemokines and chemokine receptors ....20 1.3.2 Viral mimicry .....................................................................................................21 1.3.3 XCL1 and XCR1................................................................................................23 1.3.4 The RCMV-E homolog vXCL1 (e156.5) ............................................................24 1.4 Objectives .............................................................................................................25 2. Materials .......................................................................................................................26 2.1 Media, buffers and solutions .................................................................................26 2.2 Equipment .............................................................................................................28 2.3 Consumables ........................................................................................................30 2.4 Software................................................................................................................31 2.5 Chemicals .............................................................................................................31 2.6 Reagents ..............................................................................................................32 2.7 Kits ........................................................................................................................32 2.8 Primers and Probes ..............................................................................................33 1 Table of contents 2.9 Plasmids ...............................................................................................................34 2.10 Antibodies and beads............................................................................................34 2.11 Viruses ..................................................................................................................35 2.12 Eukaryotic cells .....................................................................................................36 2.13 Rat strains .............................................................................................................36 3. Methods ........................................................................................................................37 3.1 Molecular biology ..................................................................................................37 3.1.1 Polymerase Chain Reaction (PCR) ...................................................................37 3.1.2 Genotyping of Xcr1 knockout rats .....................................................................37 3.1.3 Quantitative real-time PCR (qRT-PCR) .............................................................38 3.1.4 DNA isolation from tissue ..................................................................................38 3.1.5 Isolation of virion DNA .......................................................................................39 3.1.6 DNA Sequencing ...............................................................................................39 3.1.7 RNA isolation and sequencing ..........................................................................40 3.1.8 Detection of differentially expressed genes .......................................................42 3.2 Protein biochemistry and flow cytometry ...............................................................42 3.2.1 ELISA for vXCL1 detection ................................................................................42 3.2.2 Antibody coupling ..............................................................................................43 3.2.3 Antigen staining with fluorophore-coupled antibodies ........................................43 3.2.4 Fixable dead staining and intracellular staining .................................................43 3.2.5 Chemokine binding ...........................................................................................44 3.2.6 Flow cytometry analysis ....................................................................................44 3.3 Virology .................................................................................................................44 3.3.1 Virus infection ...................................................................................................44 3.3.2 Generation of high titer virus stocks ..................................................................44 3.3.3 Virus titration by plaque assay ...........................................................................45 3.3.4 Generation of RCMV-B vxcl1 virus ..................................................................45 3.4 Cell biology ...........................................................................................................46 3.4.1 Propagating and counting cells .........................................................................46 3.4.2 Freezing and thawing cells ................................................................................46 2 Table of contents 3.4.3 Preparation of single cell suspensions from spleens .........................................46 3.4.4 Magnetically isolation of rat DC .........................................................................46 3.4.5 Isolation of leukocytes from salivary glands .......................................................47 3.4.6 Preparation of single cell suspensions from lymph nodes .................................47 3.4.7 Collection of blood for cell enrichment ...............................................................47 3.4.8 Chemotaxis assay .............................................................................................47 3.4.9 Stimulation of spleen cells with PMA and ionomycin .........................................48 3.5 Animal experiments ..............................................................................................48 3.5.1 Animal infection and sample collection ..............................................................48 3.5.2 Organ homogenization and titration...................................................................49 3.6 Statistics ...............................................................................................................49 4. Results ..........................................................................................................................50 4.1 Analysis of XCR1 expression on DC and XCL1 expression on lymphocytes .........50 4.1.1 Two major DC subsets are identified in rat spleens ...........................................50 4.1.2 Identification of XCR1 and XCL1 expression in Xcr1+/+ and Xcr1-/- rats..............53 4.1.3 RCMV-B attracts DC from Xcr1+/+ and Xcr1-/- rats..............................................54 4.1.4 DC, T