Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten des Klinikums der Ludwig-Maximilians-Universität München Direktor: Univ.-Prof. Dr. med. Christian Weber The role of the co-stimulatory CD27/CD70 dyad in atherosclerosis Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Holger Winkels aus Geilenkirchen, Nordrhein Westfalen, Deutschland 2016 Mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Erstgutachter: Prof. Dr. rer. nat. Alexander Faussner Zweitgutachter: Prof. Dr. rer. nat. Ludger Klein Dekan: Prof. Dr. med. dent. Reinhard Hickel Abgabe der Dissertation: 21.06.2016 Datum der Disputation: 10.01.2017 Eidesstaatliche Versicherung Winkels, Holger Name, Vorname Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema: The role of the co-stimulatory CD27/CD70 dyad in atherosclerosis selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. Ort, Datum Unterschrift Doktorandin/Doktorand THE RESULTS OF THIS WORK WILL BE PARTLY PUBLISHED IN: Winkels H*, Meiler S*, Smeets E, Lievens D, Engel D, Spitz C, Buerger C, Beckers L, Dandl A, Reim S, Ahmadsei M, Hartwig H, Holdt LM, Hristov M, Megens RTA, Schmitt MM, Biessen EA, Borst J, Faussner A, Weber C, Lutgens E#, Gerdes N#. CD27 co- stimulation increases frequency of regulatory T cells and reduces atherosclerosis in hyperlipidemic mice. European Heart Journal. In Revision Winkels H*, Meiler S*, Smeets E*, Lievens D, Engel D, Spitz C, Buerger C, Rinne P, Beckers L, Dandl A, Reim S, Ahmadsei M, Van den Bossche J, Holdt LM, Megens RTA, Schmitt MM, de Winther M, Biessen EA, Borst J, Faussner A, Weber C, Lutgens E#, Gerdes N#. CD70 limits atherosclerosis and promotes macrophage function. Thrombosis and Haemostasis 2017 117 1: 164-175 THE RESULTS OF THIS WORK WERE PRESENTED AT THE FOLLOWING CONFERENCES AS POSTER AND ORAL PRESENTATIONS: Oral presentation 84th EAS Congress, Innsbruck, Austria, 29 May-1 June 2016 Title: ‘CD27 co-stimulation fosters regulatory T cell survival and ameliorated progression of atherosclerosis’ Gordon Research Conference Atherosclerosis, Newry, Maine, USA, 21-26 June 2015 Title: ‘Co-stimulation via CD27 increases frequency of regulatory T cells and ameliorates atherogenesis in hyperlipidemic mice’ Poster presentation Cardiovascular Research Workshop @ Bayer, Heiligenhaus, Germany 14-16 April, 2016 Title: ‘Deficiency of the co-stimulatory molecule CD27 impairs Treg survival and exacerbates atherosclerosis’ Annual Meeting of the German Atherosclerosis Society (DGAF), Rauischholzhausen, Germany, 7-9 April, 2016 Title: ‘Deficiency of the co-stimulatory dyad CD27/CD70 exacerbates atherosclerosis’ Gordon Research Conference Atherosclerosis, Newry, Maine, USA, 21-26 June 2015 Title: ‘Co-stimulation via CD27 increases frequency of regulatory T cells and ameliorates atherogenesis in hyperlipidemic mice’ 17th International Symposium on Atherosclerosis, Amsterdam, The Netherlands, 23-26 May 2015 Title: ‘Deficiency of the co-stimulatory molecule CD27 impairs Treg survival and exacerbates atherosclerosis’ ATVB/PVD Arteriosclerosis, Thrombosis and Vascular Biology/Peripheral Vascular Disease, San Francisco, California, USA, 7-9 May 2015 Title: ‘Deficiency of the co-stimulatory molecule CD27 impairs Treg development and exacerbates atherosclerosis’ Gordon Research Conference Atherosclerosis, Stowe, Vermont, USA, 16-21 June 2013 Title: ‘Pharmacological inhibition of CD70 hampers progression of atherosclerosis’ World Immune Regulation Meeting (WIRM) VI, Davos, Switzerland, 18-21 March 2012 Title: ‘T cell homeostasis in atherosclerosis‘ TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................ i LIST OF ABBREVIATIONS ..................................................................................... v 1 INTRODUCTION ........................................................................................ 1 1.1 Atherosclerosis – a chronic inflammatory disease ........................................... 1 1.2 The role of T cells in atherosclerosis ................................................................ 3 1.3 Tregs in atherosclerosis ................................................................................... 6 1.4 Macrophages in atherosclerosis ...................................................................... 9 1.5 Macrophage polarization in atherosclerosis ................................................... 11 1.6 B cells and Immunoglobulins in atherosclerosis ............................................. 12 1.7 An introduction to the CD27/CD70 dyad ........................................................ 14 1.8 The CD27/CD70 dyad in T cell responses ..................................................... 15 1.9 The CD27/CD70 dyad in B cell responses ..................................................... 16 1.10 Implications of the CD27/CD70 dyad in autoimmune disorders ..................... 17 1.11 The role of CD27/CD70 interactions in tumor immunology............................. 18 1.12 The role of CD27/CD70 costimulation in atherosclerosis ............................... 19 1.13 Rationale ....................................................................................................... 20 2 MATERIALS AND METHODS ................................................................. 23 2.1 General equipment ........................................................................................ 23 2.1.1 Table 1: General equipment used for this thesis ....................................... 23 2.2 Human specimen ........................................................................................... 24 2.2.1 Gene expression of CD27 and CD70 in human plaques ........................... 24 2.2.2 Human carotid endarterectomy specimens and tissue processing ............ 24 2.2.3 Histological staining of CEA sections ........................................................ 25 2.3 Mice ............................................................................................................... 25 2.3.1 Genotyping................................................................................................ 25 2.3.2 Surgical procedure .................................................................................... 27 2.3.3 Bone marrow transplantation .................................................................... 28 2.4 Protein assays ............................................................................................... 28 2.4.1 Flow cytometry .......................................................................................... 28 2.4.2 Table 2: Antibodies used for flow cytometry .............................................. 30 2.4.3 Plasma preparation and lipid analysis ....................................................... 30 2.4.4 Plasma analysis ........................................................................................ 31 2.4.4.1 Anti-oxLDL-Ig ELISA ....................................................................... 31 2.4.4.2 TGF1 ELISA.................................................................................. 32 i TABLE OF CONTENTS 2.4.4.3 Bead Arrays .................................................................................... 32 2.4.5 Histochemistry (morphometry and histology) ............................................. 34 2.4.6 Immunohistochemistry .............................................................................. 34 2.4.7 Confocal microscopy ................................................................................. 35 2.4.8. Table 3: Primary antibodies used in immunohistochemistry ...................... 36 2.4.9. Table 4: Secondary antibodies used in immunohistochemistry .................. 37 2.4.10 Western blot .............................................................................................. 37 2.5 Cell culture and functional assays .................................................................. 37 2.5.1 CD4+ T cell isolation .................................................................................. 37 2.5.2 Treg suppression assay ............................................................................ 38 2.5.3 Treg chemotaxis assay ............................................................................. 38 2.5.4 L929-conditioned medium ......................................................................... 38 2.5.5 Bone marrow-derived macrophages .......................................................... 39 2.5.6 Metabolic analysis ..................................................................................... 39 2.5.7 Nitric oxide production ............................................................................... 40 2.5.8 Reactive oxygen species production ......................................................... 40 2.5.9 Uptake of fluorescent E. coli particle ......................................................... 40 2.5.10 Uptake of Dil-conjugated