A Novel Murine Myelin Oligodendrocyte Glycoprotein Fusion Protein, MOGtag, Induces Appropriate Autoimmune B Cell Germinal Center Responses And Central Nervous System Autoimmune Disease

.............................................................................................................................. ii Co-Authorship Statement ................................................................................................... iii Acknowledgments .............................................................................................................. iv Table of Contents ................................................................................................................ v List of Figures .................................................................................................................. viii List of Abbreviations .......................................................................................................... x Chapter 1 ............................................................................................................................. 1 1 Introduction and literature review .................................................................................. 1 1.1 Multiple sclerosis .................................................................................................... 1 1.2 Genetics in Multiple Sclerosis ................................................................................ 2 1.2.1 Familial genetic predisposition ................................................................... 2 1.2.2 Immune-mediated genetic predisposition ................................................... 3 1.3 Environmental Factors in Multiple Sclerosis .......................................................... 3 1.3.1 Viral infections ............................................................................................ 4 1.3.2 Sunlight exposure ........................................................................................ 4 1.4 Immunology of Multiple Sclerosis ......................................................................... 5 1.4.1 Immune response against foreign antigens ................................................. 5 1.4.2 Immune response in Multiple Sclerosis ...................................................... 8 1.5 Pathology of Multiple Sclerosis .............................................................................. 8 1.5.1 Diagnosis and clinical monitoring .............................................................. 8 1.5.2 White and gray matter pathology ................................................................ 9 1.5.3 Available clinical therapies ....................................................................... 10 1.6 Animal models of Multiple Sclerosis ................................................................... 11 1.6.1 Experimental Autoimmune Encephalomyelitis ........................................ 11 vi 1.6.2 Lessons learned from EAE: good and bad ................................................ 13 1.7 Thesis hypothesis and objectives .......................................................................... 15 Chapter 2 ........................................................................................................................... 16 2 Materials and Methods ................................................................................................. 16 2.1 Mice ...................................................................................................................... 16 2.2 Antibodies for flow cytometry and histology ....................................................... 16 2.3 Recombinant mouse MOGtag ................................................................................ 16 2.3.1 Vector design ............................................................................................ 16 2.3.2 Protein expression and purification .......................................................... 17 2.3.3 TEV protease cleavage of MOGtag protein ............................................... 17 2.3.4 MOGtag Alexa Fluor 647 fluorophore conjugation ................................... 18 2.4 Adoptive transfer and immunization .................................................................... 18 2.5 Induction of experimental autoimmune encephalomyelitis .................................. 18 2.6 Flow cytometry ..................................................................................................... 19 2.7 Immunofluorescent histology ............................................................................... 19 2.8 Image and statistical analyses ............................................................................... 19 Chapter 3 ........................................................................................................................... 20 3 Results .......................................................................................................................... 20 3.1 Design of the mouse MOG1-125 thioredoxin tag expression vector ....................... 20 3.2 Generation and purification of the mouse MOG1-125 thioredoxin tag expression vector ..................................................................................................................... 20 3.3 MOG-specific B cells recognize MOGtag ............................................................. 23 3.4 Effective generation of a T cell-dependent B cell germinal center response with MOGtag .................................................................................................................. 26 3.5 MOGtag and MOG35-55 peptide activate different B cells to induce a germinal center response ...................................................................................................... 29 3.6 Characterization of MOGtag-induced EAE ........................................................... 29