A model for interclonal competition in the germinal center: Dynamic selection processes by-pass the affinity dead-end of low affinity anti-NP specific B cells Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer nat. vorgelegt von Andreas Acs aus Timisoara Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 15. Februar 2019 Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer Gutachter/in: Prof. Dr. Thomas Winkler PD Dr. rer. nat. Dr. habil. med. Dirk Mielenz Table of contents Table of contents 1 Zusammenfassung ............................................................................................................ 1 1 Summary ............................................................................................................................ 2 2 Introduction ....................................................................................................................... 3 2.1 B cell development ........................................................................................................ 3 2.1.1 Stages of B cell development are defined by recombination events of the B cell receptor in the bone marrow ........................................................................................... 3 2.12 GOD – Generation of diversity ................................................................................ 4 2.1.3 Migration of B cells to secondary lymphoid organs ................................................ 5 2.2 The germinal center reaction ......................................................................................... 5 2.2.1 Establishment of germinal centers ......................................................................... 5 2.2.2 The germinal center – structure and function ......................................................... 7 2.2.3 The dark zone: Site of proliferation, somatic hypermutation and class switch recombination ................................................................................................................. 8 2.2.4 The light zone: Site of affinity selection ................................................................ 10 2.2.5 Germinal center B cells constantly migrate between the dark zone and light zone to ensure affinity selection ............................................................................................ 11 2.2.6 Output of Germinal Centers: Memory B cells and plasma cells ........................... 12 2.2.7 Apoptosis in germinal centers .............................................................................. 13 2.3 The NP system ........................................................................................................... 14 2.3.1 The NP system and B1-8 mice ............................................................................ 14 3 Results ............................................................................................................................. 17 3.1 Frequency of NP-specific B1-8lo GC B cells decreases at early time points of the anti- NP response in competition to endogenous wildtype cells ............................................... 17 3.2 Competitive processes lead to a shifted DZ/LZ ratio of B1-8lo GC B cells, accompanied with a decreased proliferative capacity in the DZ ............................................................. 19 3.3 Analysis of Ig heavy chain repertoires using flow cytometric sorted GC B cells in the B1-8lo transfer model ........................................................................................................ 21 3.4 B1-8lo GC B cells fail to select the characteristic affinity enhancing W33L mutation in response to NP ................................................................................................................ 22 I Table of contents 3.5 As B1-8lo GC B cells fail to select the characteristic affinity enhancing W33L mutation, another mutation (S104G) in the CDR3 was found to be strongly selected in response to NP ................................................................................................................................... 23 3.6 Generation of recombinant antibodies to assess the influence of relevant mutations on their affinity to NP ............................................................................................................. 24 3.7 Analysis of pre-existing amino acid changes within the heavy chain of B1-8lo mice and their influence on affinity to NP using ELISA and Surface Plasmon Resonance ............... 26 3.8 The W33L mutation, known to enhance affinity of anti-NP antibodies 10-fold, does not improve the affinity in the context of B1-8lo ....................................................................... 26 3.9 Pre-existing amino acid changes in the sequence of the B1-8lo heavy chain interfere with the affinity enhancing effect of W33L ........................................................................ 28 3.10 The S104G mutation found to be positively selected in B1-8lo GC B cells enhances affinity 6-fold .................................................................................................................... 29 3.11 B1-8lo GC B cells participate in the GC reaction for an extended period of time when competition is reduced ..................................................................................................... 29 3.12 The frequency of apoptotic NP-specific B1-8lo GC B cells is increased over time, but is not significantly increased compared to endogenous cells .............................................. 31 3.13 High frequencies of B1-8lo B cells are found within the NP-specific CD38hiFas-IgG1- B cell population, despite of B1-8lo B cells being outcompeted in the GC ............................ 33 3.14 Molecular Dynamics of B1-8-related antibodies ....................................................... 34 4 Addendum ....................................................................................................................... 37 4.1 Establishment of hB-varia mice .................................................................................. 37 5 Discussion and outlook .................................................................................................. 39 5.1 Discussion ................................................................................................................. 39 5.2 Outlook ...................................................................................................................... 46 6 Materials ........................................................................................................................... 48 6.1 Antibodies and reagents for flow cytometry ................................................................ 48 6.2 Antibodies and reagents for immunofluorescence ...................................................... 49 6.3 Antigens / Immunization ............................................................................................. 49 6.4 Buffers ....................................................................................................................... 49 6.5 Chemicals and reagents ............................................................................................ 50 6.6 Consumables ............................................................................................................. 53 II Table of contents 6.7 Devices ...................................................................................................................... 52 6.8 Kits ............................................................................................................................. 53 6.9 Mice ........................................................................................................................... 54 6.10 PCR and Cloning ..................................................................................................... 54 6.11 Plasmids .................................................................................................................. 55 6.12 Primer ...................................................................................................................... 55 6.13 Software ................................................................................................................... 56 6.14 Others ...................................................................................................................... 56 6.15 Antibody panels ....................................................................................................... 57 7 Methods ........................................................................................................................... 58 7.1 Restriction digests ...................................................................................................... 58 7.2 Synthesis of the Bvaria2 plasmid ............................................................................... 58 7.3 Subcloning of the human CD19 promoter .................................................................. 58 7.4 Ligation of Bvaria2 with the human CD19 promoter .................................................. 59 7.5 Testing descendants from pronucleus injections of the hB-varia construct ................. 60 7.6 Transfer of splenocytes from transgenic B1-8lo or B1-8hi mice into