Role of complement receptors in the induction of retroviral- specific CTL responses by dendritic cells Doctoral thesis submitted to the Medical University of Innsbruck for obtaining the academic degree Doctor of Philosophy (PhD) by Muneeb Ahmad Idrees Supervisor: Univ.-Prof. Dr. Heribert Stoiber Department of Hygiene, Microbiology and Social Medicine Division of Virology Medical University of Innsbruck Innsbruck, March 2017 DEDICATION This thesis is dedicated to my wonderful parents, who have raised me to be the person I am today. You have been with me every step of the way, through good times and bad. Thank you for all the unconditional love, guidance, and support that you have always given me, helping me to succeed and instilling in me the confidence that I am capable of doing anything I put my mind to. Thank you for everything. I love you! Table of Contents SUMMARY ............................................................................................................................... 1 1. INTRODUCTION ............................................................................................................................ 2 1.1. Complement System ........................................................................................................ 2 1.1.1. Complement activation pathways ............................................................................. 2 1.1.1.1. Classical pathway ............................................................................................... 3 1.1.1.2. Lectin pathway ................................................................................................... 4 1.1.1.3. Alternative pathway ........................................................................................... 5 1.1.2. Effector functions mediated by complement system ................................................ 5 1.1.3. Protection of host by regulation of complement system ........................................... 7 1.1.4. Complement evasion by pathogens .......................................................................... 9 1.1.5. Complement receptors ............................................................................................ 10 1.1.5.1. Complement receptor type 1 (CR1) ................................................................. 10 1.1.5.2. Complement receptor type 2 (CR2) ................................................................. 12 1.1.5.3. Complement receptor type 3 (CR3) ................................................................. 12 1.1.5.4. Complement receptor type 4 (CR4) ................................................................. 14 1.1.6. Complement in regulation of adaptive immunity ................................................... 14 1.2. Dendritic cells ................................................................................................................ 14 1.2.1. Maturation of DCs .................................................................................................. 15 1.2.2. Role of DCs in the induction of T cell responses ................................................... 16 1.2.2.1. MHC-I restricted antigen presentation (Endogenous pathway) ....................... 16 1.2.2.2. MHC-II restricted antigen presentation (Exogenous pathway) ........................ 16 1.2.2.3. Cross-presentation ............................................................................................ 17 1.3. Friend Virus ................................................................................................................... 18 1.3.1. Friend Virus as a platform to investigate immune response in retroviral infections ........................................................................................................................................... 19 1.3.2. Friend virus induced erythroleukemia .................................................................... 19 I 1.3.3. Advantages of FV as retroviral infection model ..................................................... 20 1.3.4. Comparison of FV and HIV-1 ................................................................................ 21 1.3.5. Morphology of FV .................................................................................................. 22 1.3.5.1. Replication cycle of FV .................................................................................... 23 1.3.5.2. Maturation of FV virion ................................................................................... 24 1.3.5.3. Genome of FV .................................................................................................. 25 1.3.5.4. Proteins of FV .................................................................................................. 26 1.3.5.4.1. Gag ............................................................................................................. 26 1.3.5.4.2. Pol .............................................................................................................. 27 1.3.5.4.3. Env ............................................................................................................. 27 1.3.6. Pathology of Friend disease: EpoR and gp55 Molecular interactions .................... 29 1.3.7. Resistance to FV infection: role of SF-Stk ............................................................. 30 1.3.8. Immune responses in FV infection ......................................................................... 32 1.3.8.1. Role of CD8+ T cells in FV-specific immunity ................................................ 32 1.3.8.2. Role of CD4+ Regulatory T cells in FV-specific immunity ............................. 32 1.3.8.3. Role of CD4+ Helper T cells in FV-specific immunity .................................... 33 1.3.8.4. Role of antibodies and B cells in FV-specific immunity ................................. 34 1.3.8.5. Role of Natural Killer T (NKT) cells in FV-specific immunity....................... 34 2. AIMS OF THE STUDY ................................................................................................................ 36 3. MATERIALS AND METHODS ................................................................................................. 37 3.1. Materials ........................................................................................................................ 37 3.1.1. Equipment ............................................................................................................... 37 3.1.2. Chemicals and media .............................................................................................. 38 3.1.3. Antibodies ............................................................................................................... 38 3.1.4. Buffers and solutions .............................................................................................. 39 3.1.5. Animals ................................................................................................................... 40 3.2. Methods ......................................................................................................................... 40 II 3.2.1. Mice ........................................................................................................................ 40 3.2.2. Cultivation of Mus dunni cells ................................................................................ 40 3.2.2.1. Storage of Mus dunni cells ............................................................................... 40 3.2.3. Virus stocks production .......................................................................................... 41 3.2.4. Quantification of virus by infectious centers (IC) assay ......................................... 41 3.2.5. Flow cytometry ....................................................................................................... 42 3.2.6. Generation of mouse bone marrow-derived DCs (bmDCs).................................... 42 3.2.7. Opsonization of FMuLV ......................................................................................... 43 3.2.8. Real-time PCR ........................................................................................................ 43 3.2.9. Virus capture assay ................................................................................................. 44 3.2.10. In vitro productive infection of bmDCs for IC assay............................................ 44 3.2.11. In vitro infection of bmDCs for FACS analysis ................................................... 44 3.2.12. Isolation of CD8+ T cells ...................................................................................... 45 3.2.13. Co-culture of FMuLV loaded bmDCs with FV-specific CD8+ TCRtg T cells .... 45 3.2.14. Statistical analysis ................................................................................................. 45 4. RESULTS ........................................................................................................................................ 46 4.1. FMuLV propagation and IC assay ................................................................................. 46 4.2. Loading dunni cells with FMuLV- Viability and Infection level .................................. 47 4.3. Generation of bmDCs and their phenotypical characterization ..................................... 49 4.4. Flow cytometry analysis of FMuLV-loaded bmDCs .................................................... 51 4.5. Viability and infection