Aus dem Forschungszentrum Borstel Leibniz-Zentrum für Medizin und Biowissenschaften Programmbereich Asthma & Allergie Forschungsgruppe Immunbiologie THE ROLE OF THE ACTIN-REGULATORY PROTEINS CORONIN 1A AND CORONIN 1B IN THE REGULATION OF NATURAL KILLER CELL DEVELOPMENT AND FUNCTION DISSERTATION zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftliche Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von André Jenckel Bad Segeberg, 2013 Erste/r Gutachter/in: Prof. Dr. Dr. Silvia Bulfone-Paus Zweite/r Gutachter/in: Prof. Dr. Thomas Roeder Tag der mündlichen Prüfung: 09.01.2014 Zum Druck genehmigt: 09.01.2014 gez. Prof. Dr. Wolfgang J. Duschl, Dekan Table of contents II Table of contents Table of contents ................................................................................................................. II Abbreviations ..................................................................................................................... VI List of figures...................................................................................................................... IX List of tables ....................................................................................................................... XI 1 Introduction .............................................................................................................. 1 1.1 The immune system ................................................................................................... 1 1.2 Natural killer cells ....................................................................................................... 2 1.2.1 Stages of NK cell development .................................................................................. 5 1.2.2 NK cell cytotoxicity and its regulation ......................................................................... 7 1.3 The actin cytoskeleton and its function ......................................................................10 1.4 Coronins ...................................................................................................................11 1.4.1 Coronins: Structure, classification and expression in mammalian tissues/cells .........12 1.4.2 Coronins and the actin cytoskeleton ..........................................................................14 1.4.3 Regulation of coronins ..............................................................................................16 1.4.4 Functions of coronins in the mammalian system .......................................................17 2 Aim of the study ......................................................................................................19 3 Materials ..................................................................................................................20 3.1 Mice and cell lines .....................................................................................................20 3.2 Chemicals and reagents ...........................................................................................20 3.3 Molecular and cell biology kits and reagents .............................................................22 3.4 Primer and UPL probes .............................................................................................22 3.5 Antibodies .................................................................................................................23 3.5.1 Antibodies for flow cytometry and immunofluorescence microscopy .........................23 3.5.2 Antibodies used for stimulation .................................................................................25 3.5.3 Antibodies for Western blotting .................................................................................25 3.6 Laboratory equipment ...............................................................................................26 3.7 Laboratory supplies ...................................................................................................27 Table of contents III 3.8 Software ....................................................................................................................27 4 Methods ...................................................................................................................28 4.1 Cell isolation, cultivation and stimulation ...................................................................28 4.1.1 Isolation of cells from spleen, lymph nodes, bone marrow and liver ..........................28 4.1.2 Cell cultivation ...........................................................................................................29 4.1.3 Purification and expansion of NK cells ......................................................................29 4.1.4 Purification of CD4+ and CD8+ T cells .......................................................................29 4.1.5 Purification of B cells .................................................................................................30 4.1.6 Generation of bone marrow-derived mast cells .........................................................30 4.1.7 Generation of bone marrow-derived macrophages ...................................................30 4.1.8 Generation of bone marrow-derived dendritic cells ...................................................31 4.1.9 Culture of YAC-1 and A20 cells .................................................................................31 4.1.10 NK cell stimulation ....................................................................................................31 4.2 Quantitative real-time PCR analysis ..........................................................................32 4.2.1 Total RNA isolation ...................................................................................................32 4.2.2 cDNA synthesis .........................................................................................................32 4.2.3 Quantitative real-time PCR ........................................................................................33 4.3 Flow cytometry and intracellular staining ...................................................................34 4.3.1 Analysis of surface molecules ...................................................................................34 4.3.2 Intracellular FACS staining ........................................................................................34 4.4 SDS polyacrylamide gel elektrophoresis (SDS-PAGE) and Western blot (WB) .........35 4.4.1 Sample preparation ...................................................................................................35 4.4.2 SDS-PAGE ...............................................................................................................35 4.5 Western blotting ........................................................................................................36 4.6 Immunofluorescence microscopy ..............................................................................36 4.7 Transwell migration assay .........................................................................................37 4.8 In vitro lactate dehydrogenase (LDH)-based killing assay .........................................37 4.9 Flow cytometric conjugate assay...............................................................................38 4.10 Cytoskeleton-rich fraction isolation ............................................................................38 4.11 CD107a-degranulation assay ....................................................................................39 Table of contents IV 4.12 Cytokine measurement by ELISA ..............................................................................39 4.13 Statistical analysis .....................................................................................................40 5 Results .....................................................................................................................41 5.1 Coro1a and Coro1b are co-expressed in NK cells .....................................................41 5.2 Subcellular localization of Coro1a and Coro1b in NK Cells .......................................44 5.3 Increased cellular F-actin levels in Coro1a-deficient NK cells ....................................46 5.4 CXCL10 and CXCL12- induced migration of Coro1a-/- and Coro1a-/-Coro1b-/- NK cells is impaired by the loss of Coro1a ..............................................................................47 5.5 Coro1a-deficiency affects absolute numbers of naïve NK cells in the bone marrow but not in the spleen ........................................................................................................48 5.6 Impaired NK cell maturation in Coro1a- and Coro1aCoro1b-deficient mice ...............50 5.7 IL-15-expanded wild type, Coro1a-/- and/or Coro1b-/- NK cells show a similar phenotype .................................................................................................................56 5.8 Coro1a-/- and Coro1a-/-Coro1b-/- NK cells show decreased cytotoxicity ......................58 5.9 The impact of Coro1a- and Coro1b-deficency on NK cell activities and their modulators ................................................................................................................59 5.9.1 Intercellular conjugate formation is not affected by the loss of Coro1a or Coro1b .....59 5.9.2 Akt and Erk phosphorylation in Coro1a-/- and/or Coro1b-/- NK cells upon cellular activation ...................................................................................................................62