Role of Lymphotoxin Beta and Cell Adhesion Molecule (CEACAM1) in Innate and Adaptive Immune Activation Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie an der Universität Duisburg-Essen Germany vorgelegt von MR. VISHAL S. KHAIRNAR Aus Satana, Maharashtra, Indien Februar, 2017 The experiments on which this work is based have been carried out at the Institute of Immunology, Faculty of Medicine, University Hospital Essen, at the University of Duisburg-Essen. 1. Examiner: Prof. Dr. Karl S. Lang 2. Examiner: Prof. Dr. Matthias Gunzer Chairman of the Audit Committee: Prof. Dr. Ulf Dittmer Day of the oral exam: 23rd of June 2017 Die der vorliegenden Arbeit zugrunde liegenden Experimente 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. Dr. Matthias Gunzer Vorsitzender des Prüfungsausschusses: Prof. Dr. Ulf Dittmer Tag der mündlichen Prüfung: 23rd June 2017 Dedicated to my Parents (Aai and Aappu) … Should have faith in God but more trust in yourself… -Aappu Table of Contents Summary ............................................................................................................................................................... 1 Zusammenfassung…………………………………………………………………………………………………………………………………………..2 1. Chapter I: Introduction ............................................................................................................................... 3 1.1 Immune System .................................................................................................................................... 4 1.2 Types of Immunity ............................................................................................................................... 5 1.2.1 Innate Immunity ........................................................................................................................... 5 1.2.1.1 Macrophages ........................................................................................................................... 5 1.2.1.2 Granulocytes .......................................................................................................................... 6 1.2.1.3 Dendritic Cells (DC’s)............................................................................................................. 6 1.2.2 Adaptive Immunity ...................................................................................................................... 7 1.2.2.1 B cells ...................................................................................................................................... 7 1.2.2.1.a Development of B cells ............................................................................................... 8 1.2.2.1.b Functional role of B cells .......................................................................................... 10 1.2.2.2 T cells ................................................................................................................................... 11 1.2.2.2.a CD4 T cells ................................................................................................................ 12 1.2.2.2.b CD8 T cells ................................................................................................................ 12 1.2.2.2.c Memory T cells .......................................................................................................... 13 1.2.2.2.d Effector T cells .......................................................................................................... 14 1.3 Lymphotoxins ..................................................................................................................................... 14 1.4 Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)………………………... 16 1.5 Viruses ................................................................................................................................................ 18 1.5.1 Lymphocytic choriomeningitis virus (LCMV) .......................................................................... 18 1.5.2 Vesicular Stomatitis Indiana Virus (VSV) ................................................................................. 19 1.6 Mouse models used ............................................................................................................................ 20 1.7 References .......................................................................................................................................... 24 2. Chapter II: Two separate mechanisms of enforced viral replication balance innate and adaptive immune activation ................................................................................................. 30 2.1 Abstract.............................................................................................................................................. 31 2.2 Introduction ....................................................................................................................................... 32 2.3 Methods ............................................................................................................................................. 33 2.3.1 Mice ........................................................................................................................................... 33 2.3.2 Virus and plaque assays ............................................................................................................. 33 2.3.3 Lymphocyte transfer .................................................................................................................. 33 I 2.3.4 Diphtheria toxin ......................................................................................................................... 33 2.3.5 Cell culture and generation of bone rr deri ed cr es ........................................ 34 2.3.6 Flow cytometry .......................................................................................................................... 34 2.3.7 ELISA ........................................................................................................................................ 34 2.3.8 Histology .................................................................................................................................... 34 2.4 Results ............................................................................................................................................... 35 2.4.1 Viral amplification is suppressed in peripheral organs but is allowed in spleen and lymph nodes ............................................................................................................. 35 2.4.2 Lack of lymphotoxin beta limits the flow along the marginal zone ........................................... 35 2.4.3 Usp18 and lymphotoxin beta allow viral replication in the spleen……………………………..36 2.4.4 Usp18 and lymphotoxin beta are essential for inducing systemic type I interferon production, but only Usp18 influences CD8+ T-cell priming ..................................................... 37 2.4.5 Extracellular spread of virus is essential for inducing systemic type I interferon production but not for inducing CD8+ T-cell responses ............................................................. 37 2.5 Discussion.......................................................................................................................................... 38 2.6 Ethics Statement ................................................................................................................................ 40 2.7 Acknowledgments ............................................................................................................................. 40 2.8 Figure Legends .................................................................................................................................. 40 2.8.1 Figure 1: Viral amplification is suppressed in peripheral organs but allowed in spleen and lymph nodes......................................................................................................................... 40 2.8.2 Figure 2: Lack of lymphotoxin beta limits flow along the marginal zone. ................................ 41 2.8.3 Figure 3: Ubiquitin-specific peptidase 18 and lymphotoxin beta allow viral replication in the spleen. ............................................................................................................................... 41 2.8.4 Figure 4: Ubiquitin-specific peptidase 18 and lymphotoxin beta are essential for inducing systemic type I interferon, but only ubiquitin-specific peptidase 18 influences the priming of CD8+ T cells. ...................................................................................................... 42 2.8.5 Figure 5: Extracellular spread of virus is essential for inducing systemic type I interferon but not for inducing a CD8+ T-cell response. ............................................................................. 42 2.8.6 Supplementary Fig. 1: Lymphocytic choriomeningitis virus replicates in the absence of CD169+ macrophages. ................................................................................................................ 43 2.8.7 Supplementary Fig. 2: Usp18 deficient T cells have higher MHC-I expression after LCMV infection. .......................................................................................................................