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 wildtype CD45.1 recipients ...... 61
7.7 Generation of bone marrow chimeras ...... 61
7.8 Immunization with NP29-KLH ...... 62
7.9 Flow cytometric analyses ...... 62
7.10 EdU staining ...... 62
7.11 Active Caspase 3 labeling ...... 63
7.12 Flow cytometric cell sorting ...... 63
7.13 RNA isolation and cDNA synthesis of sorted germinal center B cells ...... 63
7.14 Next-generation sequencing of germinal center B cells ...... 64
7.15 Immunofluorescent staining of frozen tissue...... 65
7.16 Molecular Dynamics ...... 66
7.17 Mouse genotyping ...... 66
8 Attachment ...... 68
8.1 Sequence of the hB-varia construct ...... 68
III Table of contents
8.2 Sequence of the synthetic Bvaria2 construct ...... 75
9 References ...... 78
10 List of abbreviations ...... 88
10.1 Abbreviations ...... 88
10.2 Units ...... 92
10.3 Prefix symbols ...... 93
10.4 Greek letters ...... 93
11 List of publications ...... 94
12 Danksagung ...... 95
IV Zusammenfassung
1 Zusammenfassung
Die Keimzentrumsreaktion ist ein hochkompetitver Prozess, in welchem B Zellen selektiert werden, die hochaffine Antikörper (Ak) produzieren. Ziel dieser Arbeit war es zu untersuchen was dabei mit solchen B Zellen passiert, die durch eine niedrige Affinität nicht selektiert werden. Dafür wurde ein Transfersystem mit Mäusen genutzt, welche eine transgene schwere Kette exprimieren, die, zusammen mit einer λ1 leichten Kette, einen niedrigaffinen anti-NP Ak bilden. Transferiert man deren Splenozyten in Wt-Rezipienten, werden B1-8lo Keimzentrums (Kz) B Zellen zwischen Tag 6 und Tag 9 der anti-NP Antwort reproduzierbar auskompetitiert. Dies ging mit einer Akkumulation von B1-8lo Kz B Zellen in der LZ und einer verringerten Frequenz proliferativer B1-8lo Zellen in der DZ einher. Weiterhin konnten wir die molekularen Mechanismen dieser Beobachtung entschlüsseln. Eine W33L Mutation, welche kanonisch in der anti-NP Antwort von C57BL/6 Mäusen selektiert wird, hat eine 10-fache Affinitätssteigerung zur Folge. Wie erwartet, führte die Affinitätsreifung endogener Kz Zellen zu einer positiven Selektion dieser Mutation. Im Gegensatz dazu zeigten B1-8lo Kz B Zellen keine Anzeichen einer positiven Selektion von W33L. Die dadurch fehlende Affinitätssteigerung erklärt somit den Nachteil der B1-8lo Zellen in der Kompetition. Die Herstellung rekombinanter Ak und die anschließende Messung der Affinität für NP ermöglichte uns zu zeigen, dass eine der vier bereits vorhandenen Aminosäurenaustausche innerhalb der B1-8lo schweren Kette ausreicht um die Affinität eines unmutierten B1-8 Ak 10-fach zu reduzieren. Dieser Austausch alleine erklärt somit die niedrige Affinität des B1-8lo Ak. Derselbe Austausch verhindert außerdem eine W33L- vermittelte Affinitätssteigerung, was zu einer reduzierten Konkurrenzfähigkeit von B1-8lo Kz B Zellen führt. Um das Schicksal dieser B1-8lo Kz B Zellen näher zu analysieren, wurden Apoptose und die Differenzierung zu Memory B Zellen (MBZ) untersucht. Obwohl die Frequenz apoptotischer B1-8lo Kz B Zellen im Vergleich zu endogenen Zellen erhöht war, hat der Großteil dieser Zellen keine NP-Bindung gezeigt. Es bleibt unklar ob dies an einer anderen Spezifität liegt oder an der Tatsache, dass apoptotische Zellen Oberflächenmoleküle herunterregulieren, was zu einer beeinträchtigten NP-Bindung führen könnte. Überraschenderweise fanden wir eine hohe Frequenz an CD38hiFas-NP+ B1-8lo B Zellen, wobei es sich dabei vermutlich um MBZ handelt. Interessanterweise blieb die Frequenz dieser Population zu allen analysierten Zeitpunkten relativ konstant, obwohl zur selben Zeit NP-spezifische B1-8lo B Zellen im Kz auskompetitiert werden. Eine massive Differenzierung niedrigaffiner Kz B Zellen zu MBZ muss durch weitere Experimente bestätigt werden, würde aber weiter zum Verständnis der kompetitiven Prozesse im Kz beitragen.
1 Summary
1 Summary
The GC reaction is a highly competitive process in which high affinity B cells are favored to survive and proliferate in order to generate Abs of high affinity. To further analyze this competition, focusing on the fate of low affinity B cells, we made use of a transgenic transfer system. The mice used for this model express a transgenic heavy chain which, when paired with a λ1 LC, generate NP-specific Abs of low affinity. When splenocytes of such mice were transferred into wt recipients, B1-8lo GC B cells are reproducibly outcompeted between day 6 and 9 of the anti-NP response. This competitive phase was accompanied by an accumulation of B1-8lo GC B cells in the LZ together with a decreased frequency of proliferative B1-8lo cells in the DZ. We were further able to shed light on the molecular mechanisms leading to the disadvantage of B1-8lo GC B cells. A W33L mutation which is canonically found to be selected in C57BL/6 mice in response to NP is known to enhance affinity 10-fold. As expected, affinity maturation of endogenous GC cells led to a positive selection of cells bearing this mutation. In contrast, B1-8lo GC B cells did not show any selection of W33L, resulting in a selective disadvantage due to the absence of an increased affinity. Through generating recombinant Abs and measuring their affinity to NP, we were able to show that one of the four pre-existing amino acid exchanges within the B1-8lo heavy chain sequence was sufficient to reduce the affinity of an unmutated B1-8 Ab 10-fold. Hence, this amino acid change solely explains the low affinity of the B1-8lo Ab. Furthermore, the same amino acid exchange was found to interfere with an affinity enhancing effect of W33L, preventing B1-8lo B cells to stay competitive, being outcompeted by endogenous cells. To shed light on the fate of B1-8lo GC B cells during competition, we analyzed apoptosis and memory B cell (MBC) differentiation. Although the frequency of apoptotic B1-8lo GC B cells was increased compared to endogenous cells, the majority of these cells did not bind NP. It remains unclear whether this is due to increased apoptosis of B1-8lo GC B cells that exhibit another specificity than NP or due to the fact that surface molecules are downregulated on apoptotic cells, which might lead to impaired Ag binding. Surprisingly, we found B1-8lo cells to largely contribute to CD38hiFas-NP+ B cells, which are supposed to mainly contain MBCs. Interestingly, the frequency of this population remained fairly constant at all time points analyzed, although NP-specific B1-8lo B cells were concurrently outcompeted in the GC. Although it needs to be proven that this population indeed contains MBCs, a contribution of low affinity B cells to the MBC pool has been described before. Nevertheless, the extent of this contribution might give new insights into the fate of low affinity B cells which are unable to compete in the GC reaction.
2 Introduction
2 Introduction
2.1 B cell development
2.1.1 Stages of B cell development are defined by recombination events of the B cell receptor in the bone marrow
Hematopoietic stem cells (HSCs) first appear in the dorsal aorta of the mesoderm-derived aorta-gonad-mesonephros region of murine embryos at day 10 after gestation (E10). After migrating to the fetal liver, these HSCs seed the bone marrow (BM), giving rise to B cells and other cells of the immune system (A. Muller et al. 1994)(Medvinsky and Dzierzak 1996)(de Bruijn 2000). Within the BM, B cells undergo several developmental stages that are defined by the rearrangement processes of their immunoglobulin (Ig) gene segments forming the membrane-bound B cell receptor (BCR)(reviewed in Yancopoulos and Alt 1986). The BCR is composed of two heavy and light chains, connected via disulfide bonds. There exist two different types of light chains, κ and λ, which are expressed at a ratio of 95:5 in C57BL/6 mice, respectively (Arakawa, Shimizu, and Takeda 1996). Both, heavy and light chains contain a variable and a constant region. The variable region of the heavy chain is composed of a variable (VH), a diversity (DH) and a joining (JH) segment, whereas the variable region of the light chain is only built up by a variable (VL) and a joining (JL) segment (Tonegawa 1983) (reviewed in Pieper, Grimbacher, and Eibel 2013). In the mouse genome, there exist around
150 VH segments, 9 DH segments and 4 JH segments. For the kappa light chain there exist
140 VL and 4 JL segments (Schatz and Ji 2011). These segments are encoded in separate clusters in the genome, which can be joined in every possible combination, being one source of antibody diversity. The process being responsible for the joining of the different segments is called V(D)J-recombination, happening at the pro-B cell stage in the BM (reviewed in Yancopoulos and Alt 1986). Each segment is flanked by recombination signal sequences (RSSs), which are composed of conserved nonamer and heptamer sequences. These sequences, in turn, flank a non-conserved spacer sequence being either 12 or 23 base pairs (bps) long. The RSSs are recognized by two enzymes RAG-1 and RAG-2, encoded by the Recombination activating gene. These enzymes act by first introducing double-strand (ds) DNA breaks and subsequently rejoining the DNA by excision of the interspersed DNA sequence, whereas only RSSs with a 12 base pair (bp) linker can join to RSSs with a 23 bp linker. This ensures recombination of the different segments in a correct order (D→J and V→DJ joining for the heavy chain and V→J joining for the light chain) (Alt et al. 1984)(Grawunder et al. 1995)(reviewed in Schatz and Ji 2011). After successful
3 Introduction rearrangement of the heavy chain variable region, the constant µ heavy chain gene segment is joined to be, together with a surrogate light chain composed of lambda5 and VpreB, transiently expressed as the pre-BCR on the B cell surface (Figure 1) (Sakaguchi and Melchers 1986)(Kudo and Melchers 1987). The expression of the pre-BCR initiates the next developmental stage known as pre-B cell. Only expression of a functional membrane bound BCR leads to proliferation of pre-B cells (Kitamura et al. 1991)(Kitamura et al. 1992). Furthermore, the presence of a pre-BCR prevents the formation of a second heavy chain with a different specificity, known as allelic exclusion, happening at the V→DJ recombination stage (Kitamura and Rajewsky 1992)(Ehlich et al. 1994)(Schlissel and Morrow 1994)(Loffert et al. 1996)(reviewed in Pieper, Grimbacher, and Eibel 2013). If the rearrangement of the heavy chain has been successful and results in functional signaling events at the pre-BCR, the V→J rearrangement of the light chain gene segments is initiated, leading to a functional membrane-bound IgM molecule expressed on immature B cells (Figure 1) (Tsubata, Tsubata, and Reth 1992)(Schlissel and Morrow 1994)(Constantinescu and Schlissel 1997) (reviewed in Parkin and Cohen 2001).
Figure 1: Developmental stages of B cells in the BM are defined by recombination events of the BCR. Upon seeding the BM, HSCs give, among others, rise to B cells. The developmental stages of B cells in the BM are defined by recombination events of the BCR. At the pro-B cell stage, VDJ recombination of the heavy chain sequence takes place. Functional recombination leads to expression of the heavy chain, together with the surrogate light chain, on the surface of pre-B cells. Effective signaling of the pre-BCR induces recombination of a light chain sequence, giving rise to the mature BCR expressed on immature B cells. The figure is adapted from (Cambier et al. 2007).
2.1.2 GOD – Generation of diversity
As already mentioned, there exist several V, (D) and J segments that can be combined in any possible combination to generate unique B cell receptors (Tonegawa 1983), leading to a high number of possibilities to form Abs. The joining of the segments is an imprecise mechanism, which leads to random addition of nucleotides by the terminal deoxynucleotidyl transferase (Tdt) between the joined ends (Desiderio et al. 1984). This in turn leads to an even higher Ab diversity. The repertoire is further increased by the combinatorial diversity of the different heavy and light chains. Another mechanism promoting Ab diversification
4 Introduction happens in germinal centers (GCs), where activated B cells undergo somatic hypermutation (SHM). SHM leads to random mutations of the variable gene segments of heavy and light chains to shape the antibody response and enhance affinity (Li et al. 2004)[review]. With this diversification, the number of possible Abs becomes unimaginably high.
2.1.3 Migration of B cells to secondary lymphoid organs
After successful completion of their development, immature B cells leave the BM, while alternative RNA processing events result in fully matured B cells co-expressing IgM and IgD isotypes (Geisberger, Lamers, and Achatz 2006). Mature B cells migrate to secondary lymphoid organs, such as the spleen, lymph nodes, Peyer´s patches, tonsils and mucosal tissues via the blood system (reviewed in Pieper, Grimbacher, and Eibel 2013). The migration into these organs is triggered by stromal cells, such as follicular dendritic cells (FDCs) or fibroblastic reticular cells, attracting B cells by producing CXCL13, a ligand for the receptor CXCR5, expressed on B cells (reviewed in Gonzalez et al. 2011). As a consequence, B cells “seed” secondary lymphoid organs and form organized structures called B cell follicles (G. Muller, Hopken, and Lipp 2003)(Cyster 2005). These follicles are located adjacent to T cell zones, but are clearly separated from those (Howard, Hunt, and Gowans 1972)(Nieuwenhuis and Ford 1976). Follicular B cells were shown to be highly motile, moving at a speed of around 6 µm/min (Victora et al. 2010). Upon infection or immunizations, GCs are formed within B cell follicles to initiate the humoral immune response.
2.2 The germinal center reaction
2.2.1 Establishment of germinal centers
Activation of B cells can be induced by direct binding of Ag in the periphery, or within B cell follicles of secondary lymphoid organs. Within follicles, B cell activation is facilitated by the presence of FDCs. These cells are stromal cells of non-hematopoietic origin and use complement receptors (CD21, CD35) (reviewed in Gonzalez et al. 2011) as well as Fc receptors (FcɣRIIb) (Qin et al. 2000) to capture immune complexes (ICs) on their surface (Kunkl and Klaus 1981). The Ag being bound in ICs can, in turn, be presented to B cells, enhancing the probability of B cell activation by local Ag concentration (Figure 2). Indeed, naïve B cells were found to be highly motile while scanning FDCs for Ag. However, if B cells
5 Introduction are not activated after a certain time, they leave the lymphoid organ via the blood (spleen) or the lymph (lymph node) to re-enter the circulation (Cyster 2005). Furthermore, the interaction of B cells and FDCs is important for affinity selection in established GCs, as will be explained later.
Figure 2: FDCs present Ag to B cells within follicles. FDCs are able to capture ICs on their surface in order to present Ag to follicular B cells. Besides Ag-specific signals and co-stimulatory signals, cytokines and adhesion molecules are crucial for this interaction. The figure is adapted from (El Shikh et al. 2010)
Once B cells become activated, they upregulate CCR7 and head towards the T/B cell boundary. The migration of B cells towards the boundary was shown to be random when far away from the border, but becomes directional when reaching a distance of around 140 µm (Okada et al. 2005). The directed movement was shown to be dependent on the presence of a CCL21 gradient within the follicle and the chemokine receptor CCR7 expressed on B cells (Reif et al. 2002)(Okada et al. 2005). Once reaching the T / B cell border, B cells present Ag bound to MHC class II molecules to the T cell receptor (TCR) of Ag-primed T helper cells (Mempel, Henrickson, and Von Andrian 2004)(reviewed in Batista and Harwood 2009). These T-B cell conjugates are highly motile (~9 µm/min) and interact for about 10-60 min, whereas non-cognate conjugates interact for only a few minutes (Okada et al. 2005). Furthermore, one B cell is able to contact more than one T cell, but not vice versa. Thereby, B cells determine the directionality of the T-B cell conjugates. These early processes result in cytokine secretion by the T cells to induce survival, proliferation and differentiation of B cells (Mills and Cambier 2003)(Okada et al. 2005). The initiation of the GC response starts after migration of fully activated B cells from the T/B border to the center of the follicle. One protein that was shown to be crucial for this locomotion is the G protein-coupled receptor EBI2 (Gatto et al. 2009)(Pereira et al. 2009). Already at day 3 of the immune response, small GCs are detectable, whereas most proliferating B cells are located outside of the GC (Kerfoot et al. 2011)(F. J. Weisel et al. 2016). However at day 4, the majority of proliferating B cells are located within GCs (reviewed in De Silva and Klein 2015)(F. J. Weisel et al. 2016). Around the same time, short- lived plasmablasts arise within the splenic red pulp (Liu et al. 1991).
6 Introduction
As the size of the GC increases due to proliferation, naive IgM+ IgD+ B cells are displaced and form the mantle zone surrounding the GC. Compared to their naïve counterparts, GC B cells were found to be larger in size and more irregularly shaped due to filopodia formation (Hauser et al. 2007). However, naïve B cells were found to be also able to make contacts to FDCs in the light zone and be consequently activated (C. D. C. Allen et al. 2007)(Schwickert et al. 2007). This might ensure activation of high affinity B cells that have not initially been activated at the onset of the immune response to a certain Ag. Furthermore, this might be an efficient way of responding to epitope shifting as observed for some pathogens, such as Influenza viruses.
2.2.2 The germinal center – structure and function
GCs are defined histologically visible areas within B cell follicles of secondary lymphoid organs and are mainly composed of activated, highly motile (6.6 µm/min) B cells (Hauser et al. 2007)(Shulman et al. 2014). A major purpose of GCs is the production of high affinity antibodies produced by plasma cells. The underlying mechanisms originate in the GC reaction and are highly compartmentalized. Already since 1930 it is known that a fully established GC is composed of two distinguishable zones, which are named after their appearance in histological sections: the dark zone (DZ) and the light zone (LZ). The DZ is composed of densely packed proliferating B cell blasts, whereas B cells in the LZ appear more dispersed due to the presence of interspersing networks of FDCs (Rohlich 1930)(Nieuwenhuis 1984)(reviewed in “Germinal centers”, McLennan 1994)(reviewed in De Silva and Klein 2015). The two zones can further be distinguished by the differential expression of several surface markers. While DZ B cells are CXCR4hiCD83loCD86lo, LZ B cells are CXCR4loCD83hiCD86hi (C. D. C. Allen et al. 2004)(Victora et al. 2010). Especially CXCR4 was shown to be crucial for the structural organization of GCs. Hence, in CXCR4-deficient mice, GC B cells fail to form DZs. Instead, the GCs of these mice are only composed of a LZ compartment, shown by CXCL13 expression, a chemokine highly expressed in the LZ, accompanied by the presence of FDCs throughout the GCs (C. D. C. Allen et al. 2004). In addition, SDF-1, a ligand for CXCR4 was found to be solely expressed in DZ. Regarding the LZ, it was shown that CXCR5 mRNA is concentrated in this zone (Cyster et al. 2000). Indeed, later studies proofed CXCR5 to be important to guide GC cells to the LZ (C. D. C. Allen et al. 2004). Hence, CXCR4 and CXCR5 are important chemokines that direct GC B cells to the DZ and LZ, respectively, whereas only CXCR4 was found to be essential for the structural segregation of these zones.
7 Introduction
Besides their differential expression of surface molecules, DZ and LZ B cells also differ in their expression profiles. Hence, microarray analyses of sorted cells revealed that B cells in the LZ upregulate cell surface molecules and genes linked to lymphocyte activation (e.g. BCR and CD40 signaling) and apoptosis, while B cells in the DZ show enhanced expression of mitosis-related genes and genes related to SHM (Victora et al. 2010). These clearly depicts that the segregation of the two zones is linked to separation of different processes within the GC. Moreover, highly motile (9 µm/min) follicular T helper (Tfh) cells (Shulman et al. 2014) and Macrophages were shown to be present in the LZ (reviewed in Victora and Nussenzweig 2012). Tfh cells are defined by the expression of CXCR5, ICOS, PD-1, Bcl-6, IL-4 and IL-21, whereas CXCR5 and PD-1 show the highest expression on T cells located in the GC (Shulman et al. 2013). Using the OVA-specific OT-II system, Ag-specific T cells were found to be present in T cell zones of LNs 3 days post-immunization and 2 days later also in GCs (Shulman et al. 2013). Thereby, the GC is initially colonized by a polyclonal population of T cells. In contrast to B cells, Tfh cells are not clonally restricted to individual GCs, but are able to enter adjacent GCs to enhance the probability of T cell help to GC B cells, favoring the generation of high affinity antibodies (Shulman et al. 2013). Movement of activated B cells into different GCs would decrease diversity as the probability of a single clone dominating the whole immune response would increase herewith (Küppers et al. 1993)(Shulman et al. 2013). Furthermore, newly activated Tfh cells were able to enter existing GCs and participate in the GC reaction (Shulman et al. 2013). This is thought to serve as a way to keep GCs going and to be able to react on pathogens that change their antigenic epitopes during infection (Shulman et al. 2013).
2.2.3 The dark zone: Site of proliferation, somatic hypermutation and class switch recombination
The initiation of the GC response is established by proliferation of activated B cells (Jacob et al. 1993), whereas proliferation is still evident in the DZ of a fully established GC (Victora et al. 2010) (Figure 3). Furthermore, SHM takes place in the DZ to alter BCR affinity by randomly inserting mutations into the variable segments of the heavy and the light chain gene segments (Jacob, Kassir, and Kelsoe 1991). It was predicted that the frequency is about one somatic mutation per 103 base pairs per division (McKean et al. 1984). Rajewski et al showed that the mutation rate of GC B cells that undergo SHM is 106-fold higher compared to spontaneous mutation events. Hence, the mutation rate of GC B cells turned out to be around 10-5 – 10-3 mutations per base pair per generation in mice and humans (Rajewsky, Forster, and Cumano 1987). The molecule being responsible for the increased mutation
8 Introduction rates in GC B cells is the activation-induced cytidine deaminase (AID) that was discovered in 1999 (Muramatsu et al. 1999). The same group found AID to be specifically expressed in activated B cells located in the DZ of the GC. Furthermore, AID-deficient mice were shown to lack SHM and class switch recombination (CSR) (Muramatsu et al. 2000). AID was shown to act on both DNA strands (Milstein, Neuberger, and Staden 1998) via proteolytic deamination of cytosine to uracil (reviewed in Li et al. 2004). The presence of uracil in the DNA induces one of two possible repair mechanisms: base excision repair (BER), which induces C:G mutations or mismatch repair (MMR), which accounts for A:T mutations. When BER is initialized, an uracil DNA glycosylase excises the uracil, resulting in an abasic site within the DNA backbone (reviewed in Teng and Papavasiliou 2007). Hence, error-prone polymerases (polymerase ɵ and Rev1) can induce transitions or transversions which will result in point mutations (reviewed in Teng and Papavasiliou 2007). The MMR also works via deamination of cytosine to uracil by AID. However, The MSH2/MSH6 complex, together with the exonuclease I, is excising the uracil and adjacent nucleotides. The error prone Polymerase ɳ fills in the gaps and tends to introduce mutations at A:T base pairs, changing the nucleotide content of the variable region (reviewed in Di Noia and Neuberger 2007). The goal of SHM is to enhance Ab affinity. BCRs that have acquired frame shifts or stop codons will be eliminated by apoptosis in the DZ (Mayer et al. 2017). B cells expressing functional BCRs will migrate to the LZ in order to test their affinity, whereas high affinity B cells will be positively selected. Another event happening in the DZ of the GC is the class switch recombination. As already mentioned, immature B cells that migrate into peripheral lymphoid organs co-express unmutated IgD and IgM on their surface. However upon infection, other Ig isotypes dominate the immune response (Rajewsky, Forster, and Cumano 1987). In general, the constant region of the IgM heavy chain (Cµ) is replaced by the Cɣ, Cɛ or Cɑ segment. These segments are located downstream of Cµ and result in the expression of an IgG, IgE or IgA isotype, all of which have special effector functions, depending on the type of infection (reviewed in Li et al. 2004). Together with SHM, CSR is also induced by AID (Muramatsu et al. 2000). However, proteolytic deamination of cytosine is not happening in the V regions, but within the switch regions, located upstream of each constant gene segment, respectively. As already described for the BER upon SHM, UNG generates an abasic site where the deamination by AID took place. APE1 introduces a single strand (ss) nick by cutting the DNA backbone. When such ss nicks cluster within the switch region, ds breaks are induced leading to recruitment of proteins of the NHEJ complex. This in turn results in recombination with a switch region of a downstream constant segment (reviewed in Di Noia and Neuberger 2007)(reviewed in Li et al. 2004).
9 Introduction
As AID is initiating both, SHM and CSR, it seems that it is acting in different ways, respectively. Studies on AID mutants showed that the N-terminus is competent in inducing CSR, while the C-terminus is relevant for SHM. Different co-factors might further play a role for the different processes (Barreto et al. 2003)(Shinkura et al. 2004).
2.2.4 The light zone: Site of affinity selection
Already in early publications, increasing ratios if replacement/silent mutations in the CDR1 and CDR2 accompanied by a decreased CDR3 diversity, led to the idea of a directed selection mechanism in the GC (Jacob et al. 1993). Nowadays, it is known that these selection processes take place in the LZ of the GC. Hence, DZ B cells in the LZ interact with FDCs, which present Ag in form of immune complexes on their surface (Qin et al. 2000)(reviewed in Kosco-Vilbois 2003)(reviewed in Gonzalez et al. 2011). As B cells were found to rapidly undergo apoptosis when cultured in vitro, but have a prolonged survival upon cross-linking of the BCR and induced CD40 signaling (Liu et al. 1989)(reviewed in MacLennan 1994), competitive binding of B cells to a limited amount of Ag presented on FDCs and subsequent BCR crosslinking was thought to be the limiting step for affinity selection (Brink et al. 2007)(Liu et al. 1989). Although BCR crosslinking might still play a role for affinity selection, recent publications showed that T cell help provided by Tfh cells is the limiting factor to allow expansion of high affinity clones (Victora et al. 2010)(Gitlin 2014). Hence, Tfh cells trigger DZ re-entry along with affinity-dependent proliferation. With this and the notion that the BCR is an endocytotic receptor (Rock, Benacerraf, and Abbas 1984)(Lanzavecchia 2007), the role of the BCR within the selection process is capturing and internalizing Ag presented on FDCs selection (Batista, Iber, and Neuberger 2001)(Suzuki et al. 2009) in order to permit Ag presentation to Tfh cells (Victora et al. 2010)(Gitlin 2014) (Figure 3). Hence, high affinity GC B cells will be able to capture and internalize more Ag from FDCs resulting in a higher density of peptide:MHCII complexes displayed on the surface of the B cell. As a result, presentation of high amounts of Ag to Tfh cells leads to prolonged T-B interactions and thus increased T cell help inducing higher proliferation rates of GC B cells. T cell help was shown to include increased intracellular Ca2+ as well as IL-4 and IL-21 levels (Shulman et al. 2014). The number of divisions induced by Tfh cells is predicted to range from 1-6 (Gitlin 2014). GC B cells that underwent the most divisions had a significantly higher frequency of high affinity mutations (Gitlin 2014).
10 Introduction
2.2.5 Germinal center B cells constantly migrate between the dark zone and light zone to ensure affinity selection
As already mentioned, the purpose of GCs is to generate B cells expressing high affinity BCRs. To reach this, B cells constantly migrate between the two zones, known as cyclic re- entry (M Meyer-Hermann, Deutsch, and Or-Guil 2001)(C. D. C. Allen et al. 2007)(Hauser et al. 2007)(Schwickert et al. 2007)(Victora et al. 2010). This model suggests that affinity maturation is achieved by repeated cycles of proliferation in the DZ, with subsequent affinity selection in the LZ, in which GC B cells with the highest affinity are selected to recycle back to the DZ (Figure 3). Using mice expressing photoactivatable GFP to specifically trace cells in the LZ or DZ, a net flow of cells moving from DZ to the LZ was observed. This led to the conclusion that selection takes place in the LZ, allowing only a part of the GC B cells to re- enter the DZ (Victora et al. 2010). Other experiments further supported this idea as B cell proliferation, due to artificial AG delivery, was first observed in the DZ, before becoming visible in the LZ (Gitlin 2014). Although S-phase labeled cells have also been described to be present in the LZ (C. D. C. Allen et al. 2007)(Hauser et al. 2007), the actual division might occur exclusively in the DZ (Victora et al. 2010)(Gitlin 2014). Another publication further suggested that the interzonal movement is not dependent on extrinsic factors, but due to an intrinsic cellular “timer” (Bannard et al. 2013). This idea is a result of experiments using mixed BM chimeras in which a major part of the cells arose from wildtype (wt) precursors while a smaller portion lacked CXCR4. CXCR4-deficient GC B cells were found in the LZ of the GC, however interestingly a proportion of these cells were found to exhibit a “DZ-like” phenotype. The model suggests that once activated, possibly by signals delivered by Tfh cells in the LZ, this timer is thought to induce a change in the expression pattern of surface molecules (e.g. downregulation of CD83 and CD86 and upregulation of CXCR4), resulting in interzonal movement. The necessity to gradually re-cycle between the two compartments can be displayed by the notion that CXCR4-deficient B cells, which are restricted to the LZ, are outcompeted by wt B cells, as they fail to accumulate mutations and to proliferate in the DZ (Bannard et al. 2013).
11 Introduction
Adapted from Victora and Nussenzweig 2012
Figure 3: The Germinal center reaction. Activated B cells enter the GC and undergo proliferation in the DZ. Furthermore, SHM induces random mutations into the variable gene segments of the heavy and light chain. B cells which acquire mutations leading to non- functional BCRs undergo apoptosis, while B cells bearing functional BCRs migrate to the LZ. There, they interact with FDCs to bind and internalize Ag presented on FDCs, whereas high affinity B cells will internalize more Ag. Following this, B cells interact with Tfh cells in order to receive T cell help. This step was showing to be rate limiting, as high affinity B cells are able to present more Ag to Tfh cells, receiving more T cell help. Selected B cells re-enter the DZ to undergo further rounds of proliferation, depending on the strength of T cell help. The figure is adapted from (Victora and Nussenzweig 2012).
2.2.6 Output of germinal centers: Memory B cells and plasma cells
B cells that are selected to exit the GC can either differentiate into memory B cells (MBCs), to enable quick immune responses upon re-infection or into Ab-secreting plasma cells (PCs) (reviewed in De Silva and Klein 2015). Already 4 days post-immunization, first extrafollicular plasmablasts can be found in the splenic red pulp (Liu et al. 1991). A recent publication even reported GC-derived plasmablasts as early as day 5 post-immunization, arising at the interface of the GC and the T cell zone (Zhang et al. 2018). Weisel et al. showed that the generation of both MBCs and long-lived plasma cells (LLPCs) underlies a “temporal switch” in which MBCs are formed early in the GC reaction, while LLPCs are formed at later time points (F. J. Weisel et al. 2016). These findings disprove the idea that MBCs and LLPCs are
12 Introduction formed simultaneously by asymmetric division (Barnett et al. 2012). According to Weisel et al., very early MBCs are already formed, mostly at the T-B border, at d2 of the immune response, even before GC and plasmacyte formation (F. J. Weisel et al. 2016). The purpose of these early extra-GC derived and mostly unswitched and unmutated MBCs is to enable prompt generation of affinity selected antibodies to altered variants of pathogens upon re- infection (F. J. Weisel et al. 2016)(Zuccarino-Catania et al. 2014). Another “wave” of MBCs is produced during the GC reaction. The generation of these IgM+ MBCs was found to peak at day 6-8. Also formation of IgG1+ MBCs distinctly increased and peaked at day 6-8 followed by a decrease with only half of these cells being left at day 12-14 (Shinnakasu et al. 2016)(F. J. Weisel et al. 2016). In contrast to the early MBCs derived prior to the GC, these cells lack CD73 expression. As frequencies of GC-derived MBCs start to decrease at day 9-11, LLPCs start to expand between day 12-15, peaking at day 15-32 and staying constant until day 41. These high affinity long-lived cells that appear late in the immune response are thought to replace low affinity cells that have been generated earlier in the response (F. J. Weisel et al. 2016).
2.2.7 Apoptosis in germinal centers
In 1989, in vitro studies using human tonsillar B cells showed that the rate of apoptosis of sorted GC B cells (IgD/CD39 negative fraction) is enhanced compared to sorted naïve B cells (IgD/CD39 positive fraction) when cultured at 37°C. After 16 h, 75% of the cultured GC cells underwent apoptosis with no living cells being left after 40 h. The “lifespan” of the GC cells was able to be prolonged by BCR- and CD40-dependent activation (Liu et al. 1989). Furthermore, microarray analyses of sorted cells revealed that especially B cells in the LZ upregulate genes linked to apoptosis (Victora et al. 2010). The general notion suggested that apoptosis in the LZ emerges from low affinity B cells which have not been positively selected and consequently die. Early studies by DalPorto et al. supported this by analyzing apoptosis in transgenic H50Gµa mice expressing low affinity anti-(4-hydroxy-3-nitropheny1)acetyl (NP) Abs, when paired with λ1 LC (Joseph M Dal Porto et al. 2002). Immunization of these mice using NP-CGG resulted in GC formation containing transgenic B cells. In late GCs (day 16- 20 post-immunization), the frequency of GC B cells containing endogenous BCRs, which escaped from allelic exclusion, increased. At these time points, H50Gµa mice showed enhanced frequencies of apoptotic cells as demonstrated by TUNEL staining, most probably due to competition of endogenous cells (Joseph M Dal Porto et al. 2002). A recent publication however, suggests similar rates of apoptosis, independent of BCR affinity as indicated from flow cytometric analyses using an anti-active Caspase 3 Ab or active Caspase 3 reporter mice (Mayer et al. 2017). Although apoptotic markers were shown to be
13 Introduction predominantly upregulated in GC B cells of the LZ (Victora et al. 2010), Mayer et al. found an equal frequency of apoptotic GC cells in the LZ and the DZ. However, the causes of apoptosis in both zones were reported to be different. While apoptosis in the DZ is mainly due to deleterious mutations, acquired by SHM, apoptosis in the LZ was suggested to happen by default. Hence, B cells in the LZ undergo apoptosis, despite of the BCR affinity, and will die if no positive survival signals by Tfh cells will be induced. The notion that apoptosis in the LZ is not linked to BCR affinity remains controversial and needs further investigations to be fully proven.
2.3 The NP system
2.3.1 The NP system and B1-8 mice
The immune response of C57BL/6 mice to the hapten NP has been studied extensively in the past decades (Imanishi and Makela 1973)(Jack, Imanishi-Kari, and Rajewsky 1977)(Jacob, Kassir, and Kelsoe 1991). A reason for this is the special feature that NP, in contrast to most other Ags, induces a restricted Ab response: In the primary response, anti- NP antibodies are dominantly composed of a λ1 LC, which is only used in about 5% of the total Ab repertoire under steady-state conditions (Arakawa, Shimizu, and Takeda 1996), together with the V186.2 (VH1-72), DFl16.1 (DH1) and most commonly JH2 segment of the heavy chain (Jack, Imanishi-Kari, and Rajewsky 1977)(Cumano and Rajewsky 1985). During the secondary response, the frequency of κ-bearing LCs is increasing, but λ1 LCs are still present (Reth, Hammerling, and Rajewsky 1978). An analysis of anti-NP Abs recovered from GCs of immunized mice showed a 10-fold range in affinity, although built up by the same VDJ-segments. This nicely demonstrates how the CDR3 region contributes to antibody diversity (Joseph M Dal Porto et al. 2002). Another feature of anti-NP antibodies is their heteroclicity, meaning that they bind the NP derivate NIP with higher affinity than NP itself, even when NP was used as immunogen (Imanishi and Makela 1974)(Imanishi and Makela 1975). As already mentioned, the anti-NP response has already been studied since the 70s, when hybridomas of spleens from NP-immunized C57BL/6 mice have been used to analyze SHM and Ab affinities (see references above). One such clone (B1-8) was found to express the germline VH1-72 together with the DH1 and JH2 segment and has been called the “prototype primary response antibody” (Cumano and Rajewsky 1985). Its Ka-value was found to be 5x105 M-1 (D. Allen et al. 1988). This unmutated sequence was used to produce a transgenic heavy-chain knock-in mouse, called the B1-8 mouse (Sonoda et al. 1997).
14 Introduction
The NP system is perfectly suited to investigate antibody selection processes as a mutation at position 33 within the CDR1 of the heavy chain (W33L) is canonically found to be selected starting at day 8 of the immune response. This amino acid change is known to enhance the affinity of the anti-NP antibody 10- to 102-fold (Cumano and Rajewsky 1986)(Furukawa et al. 1999). Another pathway of affinity maturation in response to NP emerges at later time points (~after 6 weeks) (Furukawa et al. 1999) and secondary immunizations (Tashiro et al. 2015), enhancing the affinity even 1-3 orders of magnitude compared to the W33L mutation. This effect is due to the presence of Y99G, arising from randomly inserted nucleotides during the process of VDJ recombination and mediated by TdT. Interestingly, the affinity enhancement only occurs in the absence of W33L (Furukawa et al. 1999). Hence, the W33L mutation might be a fast affinity-enhancing mutation in the early phase of the response, as this single mutation is sufficient to enhance the affinity 10-fold. However, additional mutations were not able to further enhance the affinity of W33L bearing Abs, suggesting that kind of a “ceiling affinity” is reached for this pathway (Furukawa et al. 1999). However, the Y99G pathway includes a broader range of affinities, as additional mutations within the CDRs have an influence on the affinity. This might explain the “delayed” appearance, as affinity maturation needs more time to select those mutations, in combination with Y99G, that contribute to an even higher affinity compared to W33L bearing Abs (Furukawa et al. 1999). With W33L known to enhance affinity 10-fold, transgenic B1-8hi mice have been generated by targeting the pre-recombined B1-8 heavy chain, containing this mutation (Shih, Roederer,
6 -1 and Nussenzweig 2002). Paired with a λ1 light chain, this antibody has an affinity of 5x10 M (Shih, Roederer, and Nussenzweig 2002). Furthermore, the sequence of a pre-recombined heavy chain of a low affinity antibody found in an anti-NP hybridoma (3C52) (Cumano and Rajewsky 1986) was used to generate B1-8lo mice (Shih, Roederer, and Nussenzweig 2002).
5 -1 When paired with a λ1 light chain, the affinity of this antibody is ~4x lower (Ka = 1.25x10 M ) compared to the unmutated B1-8 sequence (Shih, Roederer, and Nussenzweig 2002). It should be appreciated that this 40-fold difference in affinity, regarding B1-8lo and B1-8hi, is achieved although the V, D and J segments used are identical. Hence, single amino acid changes are capable of significantly changing the affinity of an antibody. As only 3-5% of the B1-8 mice express the λ light chain, 95-97% of these B cells do not bind to NP as they express the κ LC (Shih, Roederer, and Nussenzweig 2002)(Shih et al. 2002). To avoid VH replacements in the transgenic B1-8lo and B1-8hi mice, a silent mutation was introduced into the heavy chain sequence used for targeting. Hence, most B cells express the targeted allele together with a normal proportion of B cells expressing the λ1 light chain (3-5%) and bind to NP (Shih, Roederer, and Nussenzweig 2002). The residual pool of B cells expresses the κ LC and does not bind to NP (Shih, Roederer, and Nussenzweig 2002). B1-8lo and B1-8hi mice were shown to have normal proportions of different B cell subsets. Furthermore,
15 Introduction immunization of the mice resulted in a similar increase in anti-NP IgM and IgG levels, although IgM levels in immunized B1-8hi mice reached their maximum earlier. Compared to wt mice immunized with NP, the amount of produced antibody was slightly lower in the transgenic mice (Shih, Roederer, and Nussenzweig 2002). Both B1-8lo and B1-8hi transgenic mice showed similar frequencies of GC B cells compared to wt mice. PNA+ B cells were visible in histological sections at day 7 of the response. Taken together, B1-8lo and B1-8hi mice are able to produce GCs with frequencies comparable to wt mice and independent of affinity (Shih, Roederer, and Nussenzweig 2002). However, the number of replacement (R) mutations due to SHM was lower in B1-8hi mice, presumably due to different selection processes of high affinity cells (Shih, Roederer, and Nussenzweig 2002).
In this thesis, a transfer model using transgenic B1-8lo mice was used to shed further light on the competitive processes of the germinal center reaction. Especially the fate of low affinity GC B cells, which are outcompeted by cells of higher affinity, has been aim of this study. Therefore, we wanted to analyze the features of low affinity GC B cells regarding their zonal distribution and proliferation and if the competitive disadvantage results in increased apoptosis or preferential differentiation into MBCs. On a molecular level, analyses of SHM and affinity selection are used to complement the understanding of molecular mechanisms leading to a competitive disadvantage in this transfer model. Hence, this model will be a useful tool to investigate competition in GCs and especially the fate of low affinity GC B cells.
16 Results
3 Results
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
In order to generate an environment in which GC competition can be studied, we transferred
lo 5 -1 splenocytes of B1-8 mice (CD45.2), containing low affinity BCRs (Ka = 1.25 x 10 M ) (Shih, Roederer, and Nussenzweig 2002) for the hapten NP, into wildtype (CD45.1) recipients. Upon immunization with 100 µg NP-KLH in Alum, B1-8lo GC B cells expand, as can be seen by flow cytometry at day 6 post-immunization (Figure 5A and B). However, between day 6 and day 9 of the anti-NP response, the frequency of total B1-8lo GC B cells drastically decreases (Figure 4 and 5B), while frequencies of transferred T cells and naïve B cells remain stable (Figure 5E), showing that the loss of cells is specific for B1-8lo GC B cells. The decreased frequency especially becomes evident when looking at NP-specific B1-8lo GC cells (Figure 5D). In contrast, B1-8hi GC B cells participate in the GC reaction for an extended period, of time (Figure 5C). Hence, our model system is perfectly suited to study competitive processes in the GC as low affinity GC B cells are reproducibly outcompeted between day 6 and 8 of the anti-NP response.
Figure 4: Loss of B1-8lo GC B cells can be seen in histological sections. Histological analyses of frozen splenic sections confirm the loss of B1-8lo GC B cells between day 6 and day 9 in flow cytometric analyses. Depicted are CD45+ B1-8lo cells in IgD- GCs. Most GCs at day 8 post-immunization are composed of endogenous cells (lower panel). GCs containing higher frequencies of B1-8lo B cells are rarely found, compared to GCs at day 6 post-immunization (upper panel).
17 Results
Figure 5: B1-8lo GC B cells are outcompeted by endogenous wildtype GC B cells, while B1-8hi GC B cells participate in the GC reaction for an extended period of time. (A) Gating strategy used to distinguish endogenous and donor GC B cells. A competitive environment was produced by i.v. injection of 8x106 B1-8lo or B1-8hi splenocytes (CD45.2+/+) into wildtype (CD45.1+/+) recipients. The following day, mice were immunized with 100 µg NP-KLH in alum. Mice were sacrificed at different time points post-immunization. B) B1-8lo GC cells start to form GCs, however a drastic decrease in their frequency is evident between day 6 and day 9 of the anti-NP response. Depicted are flow cytometric analyses of four different experiments (n = 2-5). C) In contrast to B1-8lo, B1-8hi GC B cells expand until day 12 post-immunization. Shown are two different experiments (n = 2-4). Dots resemble individual mice and error bars indicate the mean value with standard deviation (SD) for each time point. D) The decreased frequency is also evident for NP-specific B1-8lo GC B cells. Depicted is the mean value of three different experiments (n =1-5) with error bars indicating the standard deviation (SD) for each time point. E) Frequencies of T cells (blue) and naïve B cells (red) do not change in the course of the immune response. T cells are gated as B220- cells and naïve B cells as B220+GL7-Fas- cells, followed by CD45.1 and CD45.2 discrimination. Depicted are the mean value with SD for each time point of three different experiments (n = 2-5).
18 Results
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
Next, we investigated if the decreased frequency of B1-8lo GC B cells is associated with a change in their DZ/LZ distribution (Figure 6A). At day 6 post-immunization, the DZ/LZ ratio of B1-8lo and endogenous GC B cells is >1, indicating that more cells are localized in the DZ of the GC (Figure 6B). However, accompanied with the decrease of B1-8lo GC B cell numbers, the DZ/LZ ratio shifts, resulting in more B1-8lo GC B cells being localized in the LZ. Especially at day 7 and 8, the decreased DZ/LZ ratio is significant when compared to endogenous GC B cells. Latter ones show a constant DZ/LZ ratio at all time points. Hence, competition drives low affinity B cells to be preferentially localized in the LZ.
Figure 6: The DZ/LZ ratio of B1-8lo GC B cells changes in the course of the GC response. A) Gating strategy to distinguish DZ and LZ GC B cells. B) At day 6 of the anti- NP response, the DZ/LZ ratio of both endogenous and B1-8lo GC B cells is >1, meaning that more cells are localized in the DZ. However, with the progress of the response, the DZ/LZ ratio of B1-8lo GC B cells decreases below 1. The DZ/LZ ratio of B1-8lo GC B cells is significantly lower at day 7 and 8 of the immune response, compared to endogenous cells. To test significance, a Mann-Whitney test (at day 7 p = 0.0492 and day 8 p < 0.0001) was used. The figure shows flow cytometric analyses of three different experiments (n =3-5). Dots resemble individual mice and bars indicate the mean value with SD for each time point.
To find out if the preferential localization of B1-8lo GC B cells in the LZ is accompanied with decreased proliferation in the DZ, 5-ethynyl-2´-deoxyuridine (EdU)-labeling was performed (Figure 7A). EdU is a thymidine analogue that gets incorporated into the DNA of dividing cells traversing S phase of the cell cycle. Hence, preferentially DZ GC B cells will be labeled as proliferation takes place in this compartment. In addition, also a subset of some LZ GC B
19 Results cells might be labeled. However, these cells are thought to be on the way to the DZ, preparing for division (Victora et al. 2010)(Gitlin 2014). The proliferative capacity of both endogenous and B1-8lo GC B cells peaks at d6 post-immunization and decreases in the course of the response (Figure 7B). At the onset of the immune response, the EdU uptake of B1-8lo GC B cells equals that of endogenous GC B cells. However, at day 8, the frequency of proliferative B1-8lo GC B cells is significantly lower compared to endogenous cells. Interestingly, this coincides with the shifted DZ/LZ ratio of B1-8lo GC B cells (Figure 6B). Taken together, these findings show that competition affects the zonal distribution as well as proliferation of low affinity GC B cells starting at day 7 – 8 of the anti-NP response and in parallel to the loss of low affinity GC B cells.
Figure 7: B1-8lo GC B cells show a decreased frequency of proliferative GC B cells at day 8 of the immune response, compared to endogenous cells. A) Gating strategy used to determine EdU+ GC B cells. To measure the proliferative capacity of GC B cells, 0.8 mg EdU was injected intraperitoneally two hours before the mice were sacrificed. As proliferation is a hallmark of GC B cells (red asterisks), Fas-PNA- naïve B cells (blue asterisk) do not incorporate EdU into their DNA. B) At day 6 and 7, the frequency of EdU+ GC B cells is comparable among endogenous and B1-8lo GC B cells, peaking at day 6 of the immune response. However, EdU uptake is clearly decreased in B1-8lo GC B cells at day 8, compared to endogenous GC B cells. Significance was tested using a Mann-Whitney test (p=0.0017). The figure shows flow cytometric analyses of two different experiments (n =2-5). Dots resemble individual mice and bars indicate the mean value with SD for each time point.
20 Results
3.3 Analysis of Ig heavy chain repertoires using flow cytometric sorted GC B cells in the B1-8lo transfer model
To check whether B1-8lo GC B cells are able to select the characteristic affinity enhancing W33L mutation of an anti-NP response (D. Allen et al. 1988), immunoglobulin heavy chain (IgH) sequences of CD19+CD38loFas+GL7+ GC B cells sorted by flow cytometry were sequenced using an Illumina MiSeq platform. Therefore, RNA was isolated and transcribed into cDNA. Subsequently, immunoglobulin heavy chain sequences were PCR amplified using a VH1-72-specific forward primer located in the leader sequence together with an universal Cɣ reverse primer. Due to specific molecular markers within the sequence of B1-8lo B cells, it was possible to filter endogenous and B1-8lo GC B cells for NGS analysis without pre-sorting on the flow cytometer (Figure 8A). At first, unproductive sequences were sorted out. Within the FR3 of the B1-8lo heavy chain, a silent mutation within Cys96 has been to prevent V replacement (Shih et al. 2002). After filtering the correct sizes of the FRs and CDRs, B1-8lo sequences were further characterized by the presence of Gly24 within the FR1, whereas endogenous sequences contain Ala24. Furthermore, B1-8lo sequences have a Thr98 within CDR3, which is unusual in endogenous sequences as Arg98 is found to be highly conserved
lo among the majority of murine VH segments. The R98T in the B1-8 sequence has most probably resulted as a consequence of VDJ-rearrangement rather than SHM within the original clone 3C52 (Cumano and Rajewsky 1986). Although R98T is generating a hotspot motif, two nucleotides within this codon are changed, decreasing the probability of back mutation to Arg98. The amino acids at position 24 and 98 are used for filtering to get rid of chimeric sequences resulting from “mis-annealing” during the process of cDNA amplification. Such chimeric sequences are composed of both B1-8lo and endogenous segments, whereas the “chimeric joint” was found to mainly occur between the FR2 and Cys96 of the FR3 in the respective sequences. Using this approach, the total number of filtered B1-8lo IgH sequences ranged from 5.6x104 to 8.6x103. Endogenous sequences ranged from 2.5x103 to 5x104. Although the number of B1-8lo sequences was higher compared to endogenous sequences at the early phase of the response, the numbers decreased with time (Figure 8B). The decrease of B1-8lo-derived IgH sequences confirms the decrease of B1-8lo GC B cell frequencies observed in flow cytometry (Figure 5B).
21 Results
Figure 8: Frequencies of filtered B1-8lo and endogenous IgH sequences confirm the decrease of B1-8lo GC B cells in flow cytometric analyses. A) Filter strategy to distinguish IgH sequences of B1-8lo and endogenous GC B cells due to the molecular markers, described in the text. B) Frequency of filtered sequences. The majority of sequences at day 5 to day 7 are derived from B1-8lo cells. However, this changes at day 8 and confirms the flow cytometric analyses (Figure 5B). Depicted are the mean values with SD of a single experiment (n = 2-7) for each time point.
3.4 B1-8lo GC B cells fail to select the characteristic affinity enhancing W33L mutation in response to NP
The NP system is ideally suited to study selection processes in vivo, as a W33L mutation within the heavy chain of C57BL/6 mice, immunized with NP linked to a carrier protein, is known to enhance affinity 10-fold and hence is found to be positively selected as early as day 8 (D. Allen et al. 1988). As expected, first signs of positive selection in endogenous GC B cells were evident at day 8 post-immunization (Figure 9A). Already at day 9, around 40% of all endogenous heavy chain sequences contained the W33L mutation, confirming that these cells are positively selected within the GC reaction. The ratio of replacement to silent (R/S) mutations within the different CDRs and FRs clearly demonstrates that the selection process, indicated by a high R/S ratio, is focused on the CDR1 of the heavy chain, containing the W33L mutation (Figure 9B, left panel). In contrast, the W33L mutation was almost completely absent in B1-8lo GC B cells, only found at negligible frequencies (Figure 9A). Although B1-8lo heavy chains do undergo somatic hypermutation, selection in CDR1 or CDR2 is not evident (Figure 9B, right panel). The lack of W33L selection is very surprising as it is a hallmark of the anti-NP response in C57BL/6 mice, used as an evidence of positive selection. Hence, the absence of W33L serves as a molecular explanation of why B1-8lo GC B cell frequencies drastically decrease early in the anti-NP response. As the W33L mutation
22 Results is not selected, the 10-fold increase in affinity, which is evident in endogenous GC B cells, is missing. This results in a competitive disadvantage of B1-8lo cells within the GC reaction.
Figure 9: Frequency of W33L mutations and R/S ratios of IgH sequences of B1-8lo and endogenous GC B cells. A) The selection of W33L, within the CDR1 of the heavy chain, is canonically found to be selected in C57BL/6 mice in response to NP. Hence, around 10% of endogenous IgH sequences contain this mutation at day 8 post- immunization. At day 9 the frequency even increases up to 40%. However, this mutation is not found to be selected in B1-8lo GC B cells. B) R/S ratio of CDR1-2 and FR1-3 of endogenous and B1-8lo IgH sequences. Endogenous cells show positive selection within the CDR1, as the R/S ratio increases with time. This is due to the positive selection of W33L, located within the CDR1. In contrast, B1-8lo sequences do not show any signs of positive selection for the CDR1 or CDR2, as the R/S ratios do not increase with time.
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
The absence of W33L selection in B1-8lo GC B cells (Figure 9) was surprising, as this single mutation is known to enhance affinity of anti-NP Abs 10-fold, ensuring to stay competitive. However, we found another mutation with in the CDR3 of the heavy chain (exchange of Ser104 to Gly104) selected in B1-8lo GC B cells (Figure 10). At day 8 of the anti-NP response, around 30% of all remaining B1-8lo heavy chain sequences contain the S104G mutation, increasing to 50% at day 9. Hence, the observation that B1-8lo GC B cells do not select the affinity enhancing W33L mutation, being characteristic for an anti-NP response, is not due to an intrinsic defect of these cells to undergo positive selection. Instead, we show that B1-8lo GC B cells are directed into an alternative way of selection.
23 Results
Figure 10: The S104 G mutation within the CDR3 of B1-8lo heavy chain sequences was found to be highly selected. Although B1-8lo GC B cells show no selection of W33L (Figure 9), another mutation (S104G) within the CDR3 was found to be strongly selected. Already at day 8 post-immunization, around 30% of all B1-8lo heavy chain sequences bear this mutation. At day 9, the frequency even increases up to 50%. Nevertheless, it has to be taken into account that at these time points, the total frequency of B1-8low GC B cells is already very low (Figure 5B).
3.6 Generation of recombinant antibodies to assess the influence of relevant mutations on their affinity to NP
To find out why B1-8lo GC B cells fail to select the W33L mutation, we examined if pre- existing amino acid exchanges in the heavy chain sequence of B1-8lo mice might interfere with this process. As already described, B1-8lo transgenic mice were generated by knock-in of a heavy chain sequence, which is based on a clone (3C52) isolated from an anti-NP hybridoma (Cumano and Rajewsky 1986). The sequence of this heavy chain differs from the unmutated (B1-8) sequence in four amino acids. At position 24, located in FR1, Alanine is changed to Glycine. As this position is known not to be involved in NP binding, together with the fact that Alanine and Glycine are both unpolar and neutral, it can be assumed that this amino acid change is not interfering with the selection of W33L. This is also true for the exchange at position 31, containing a change from Serine to Threonine, which can be considered as a change that has little structural consequences as this exchange has a positive value in the BLOSUM62 matrix. However, the influence of the residual two amino acid changes (H35Q and R98T) on the structure and affinity of B1-8lo antibodies might be more relevant. At position 35, a Histidine (positively charged) is changed to a Glutamine (polar, uncharged), altering the physical properties in very close proximity to position 33, which might interfere with an affinity enhancing effect of Leu33. Additionally, His35 is known to form H-bonds with the nitro group of NP (based on the crystal structure of B1-8 generated by Dr. T. Simon (PDB ID: 1A6W; http://www.rcsb.org) and personal discussion with Dr. T. Simon, Diarect AG). Hence, a change to Glu35 might abolish this interaction. Also the change from Arginine to Threonine at position 98 might change the properties of an anti-NP Ab. This is because Arg98 is highly conserved among different V segments of murine heavy chains, suggesting an important role for Ab integrity and stability. Changing the Arg98 residue to Thr98 might result in destabilization or decreased affinity. To test whether these two amino acid changes are the reason for the low affinity of B1-8lo antibodies and whether they interfere with a positive selection of Leu33, recombinant Abs were generated, bearing
24 Results different mutational combinations of the relevant amino acid positions in the heavy chain (Figure 11). These recombinant Abs were used to determine relative affinities to NP using ELISA and more detailed binding properties using SPR. Furthermore, it was analyzed if acquisition of S104G, found to be positively selected in B1-8lo GC B cells (Figure 10), results in enhanced affinity and hence might compensate the absence of the canonical W33L mutation. These results have been acquired as part of Miriam Bittel´s Master´s thesis.
Figure 11: Schematic representation of the heavy chain sequences of recombinant anti-NP antibodies generated to test affinities of different mutational combinations found to be relevant in our transfer model. The heavy chain sequences for the B1-8lo, B1-8 and B1-8hi antibodies were used as control antibodies, as their affinities to NP have already been described in literature. As W33L was not found to be selected in B1-8lo GC B cells, we generated this combination recombinantly to determine if W33L results in increased affinity in the context of B1-8lo. To test if the amino acid exchange H35Q, present in the B1-8lo sequence, interferes with an affinity enhancing effect of the close-by position 33, we designed a B1-8lo antibody bearing the W33L mutation together with a back mutated Q35H. Furthermore, we analyzed if the H35Q mutation, in the context of the (unmutated) B1-8 sequence, negatively affects its affinity. As described above, the exchange of Arg98 to Threonine might contribute to the low affinity of B1-8lo antibodies, as Arg98 is highly conserved among different murine V segments. To check this, the R98T mutation was also analyzed in the context of the unmutated B1-8 sequence. Finally, to elucidate if the S104G mutation, found to be strongly selected in B1-8lo GC B cells, enhances the affinity of anti-NP antibodies, this mutation was analyzed in the context of B1-8lo, B1-8 and B1-8hi.
25 Results
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
To produce recombinant anti-NP antibodies, HEK293T cells were co-transfected with a plasmid for the lambda1 light chain together with one of the ten different heavy chain plasmids (Figure 11). The fully assembled antibodies were purified from the supernatant and their relative affinities for NP were tested using ELISAs. To exclude that the heavy chain sequences obtained from NGS derive from BCRs specific for the carrier KLH, NP-BSA was used as antigen for ELISAs. To determine relative affinities, each antibody was tested in an
ELISA using NP8-BSA and another using NP30-BSA as antigen. EC50 values obtained from
ELISAs using NP30-BSA serve as “base line”, as differences in the affinity of the anti-NP antibodies do not become visible in this approach. This is due to the fact that high avidity, resulting from the high molecular NP30, compensates for low affinity. Differences in affinities are only visible when using the low molecular NP8-BSA. An approved method to determine relative affinities is provided by plotting the ratio of the EC50-values of NP8-BSA / NP30-BSA obtained from ELISAs. To measure absolute affinities and gain further insight into binding properties, Surface Plasmon Resonance (SPR) measurements have been performed. Therefore, a goat anti-mouse IgG1 capture Ab has been immobilized to a CM5 sensor chip. In a next step, recombinant Abs have been used as ligand, to finally measure binding events to NP8-BSA, used as analyte.
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
The missing selection for W33L in B1-8lo GC B cells (Figure 9) raises the question if this mutation has an effect on the affinity of the B1-8lo antibody at all. Therefore, a recombinant B1-8lo antibody containing the W33L mutation (B1-8lo W33L) was generated and its affinity for NP was tested using ELISA and SPR. Indeed, the W33L mutation that is known to enhance the affinity of anti-NP antibodies 10-fold does not improve the affinity of B1-8lo GC B cells (Figure 12, B1-8lo W33L). The affinity of this antibody is comparable to the affinity of B1- 8lo in both, ELISA and SPR. Hence, the lack of increased affinity explains the absence of B1- 8lo GC B cells selected for W33L, leading to disadvantage in competition to endogenous, W33L bearing cells.
26 Results
Figure 12: Affinity measurements of recombinant anti-NP antibodies using ELISA and SPR. ELISA was used to measure relative affinities of the ten recombinant anti-NP antibodies, shown in Figure 11. For measuring absolute affinities, SPR analyses have been performed. Therefore, a goat anti-mouse IgG1 antibody has been immobilized to capture the recombinant antibodies used as ligands. NP8-BSA has then been used as analyte. (A) The ratios of the EC50 – values obtained from ELISAs using NP8-BSA and NP30-BSA have been plotted to visualize relative affinities. (B) Ka – values are defined by calculating the ratio of kon / koff to obtain absolute values for affinities of the recombinant anti-NP Abs. The results show that the H35Q mutation, being present in the B1-8lo heavy chain, does not contribute to the low affinity (compare B1-8 with B1-8 H35Q). Only the ELISA measurements, but not SPR, suggest that H35Q might affect affinity if W33L is present (compare B1-8lo W33L with B1-8lo W33L Q35H in Panel A). Furthermore, the amino acid exchange 98T was shown to be the reason for the low affinity of the B1-8lo antibody (compare B1-8, B1-8lo and B1-8 R98T) in both, ELISA and SPR. Both methods also show that the S104G mutation, found to be selected in B1-8lo GC B cells, is able to enhance the affinity 6-fold (compare B1-8lo and B1-8lo S104G). However, it does not reach the level of B1-8hi. In the context hi of B1-8 and B1-8 , S104G had no significant effect, neither in ELISA, nor in SPR.
27 Results
-1 -1 -1 kon (M s ) koff (s-1) KA (M ) KD (M) B1-8lo 4.31 x 103 1.02 x 10-2 4.23 x 105 2.37 x 10-6 B1-8 1.90 x 104 6.91 x 10-3 2.75 x 106 3.64 x 10-7 B1-8hi 1.65 x 105 2.37 x 10-3 6.96 x 107 1.44 x 10-8 B1-8lo W33L 1.00 x 104 2.09 x 10-2 4.78 x 105 2.09 x 10-6 B1-8lo W33L Q35H 5.01 x 104 7.12 x 10-3 7.04 x 106 1.42 x 10-7 B1-8 H35Q 3.01 x 104 7.23 x 10-3 4.16 x 106 2.40 x 10-7 B1-8 R98T 6.69 x 103 1.55 x 10-2 4.32 x 105 2.32 x 10-6 B1-8lo S104G 2.02 x 104 8.12 x 10-3 2.49 x 106 4.02 x 10-7 B1-8 S104G 4.70 x 104 7.16 x 10-3 6.56 x 106 1.52 x 10-7 B1-8hi S104G 2.23 x 105 4.22 x 10-3 5.28 x 107 1.89 x 10-8
Table 1: List of values obtained from SPR analyses of the ten recombinant anti-NP Abs. The list has been adapted from Miriam Bittel (Master´s thesis).
3.9 Pre-existing amino acid changes in the sequence of the B1-8lo heavy chain interfere with the affinity enhancing effect of W33L
To analyze if the H35Q mutation, within the B1-8lo heavy chain is responsible for the absent selection of the W33L mutation, we designed two different recombinant antibodies addressing this. The first antibody was generated introducing the H35Q mutation into the context of the unmutated B1-8 sequence (Figure 11, B1-8 H35Q). With this, we wanted to test if this mutation might be the reason for the low affinity of the B1-8lo antibody. In fact, the affinity of the B1-8 antibody was not decreased by the H35Q mutation (Figure 12, B1-8 H35Q), concluding that this mutation does not contribute to the low affinity of B1-8lo. This was true for ELISA and SPR measurements. However, this mutation seems to affect affinity once the Trp33 is mutated to Leu33. This can be seen as the W33L in the B1-8lo background has an affinity enhancing effect, once Glu35 is back mutated to His35 (Figure 12A, B1-8lo W33L Q35H). But still, the affinity does not reach the level of B1-8h. However, this finding is only true for the data obtained by ELISA (Figure 12A). Still, this shows that the pre-existing H35Q mutation in the B1-8lo heavy chain is not the reason for its low affinity, but might negatively affect the affinity enhancing effect of W33L. Hence, there has to be another mutation preventing the B1-8lo W33L and B1-8lo W33L Q35H antibodies to reach higher affinity. Indeed, we found that the amino acid change R98T in the CDR3 of the B1-8lo heavy chain sequence is the actual cause of the low affinity of B1-8lo antibodies. Once this mutation was inserted into the unmutated B1-8 background (Figure 11, B1-8 R98T), the affinity of this antibody dropped to the levels of B1-8lo (Figure 12, B1-8 R98T). This also explains why the B1-8lo W33L Q35H antibody does not reach the affinity of B1-8hi, as the presence of R98T still abrogates the effect of W33L. With this, the R98T amino acid exchange in the B1-8lo 28 Results heavy chain sequence is solely responsible for a) its low affinity and b) abrogates the affinity increasing potency of W33L.
3.10 The S104G mutation found to be positively selected in B1-8lo GC B cells enhances affinity 6-fold
To analyze the effect of the S104G mutation found to be selected in B1-8lo GC B cells (Figure 10), this mutation was used in different recombinant Abs containing the unmutated B1-8, B1- 8lo and B1-8hi backbone. As expected, this mutation enhances the affinity in the context of the B1-8lo sequence 6-fold (compare B1-8lo with B1-8lo S104G in Figure 12), explaining the strong selection of this mutation in response to NP (Figure 10). However, the affinity level of B1-8hi is not reached. S104G results in a slight increase of affinity when introduced into the unmutated B1-8 sequence. However, this is only seen in ELISA measurements (compare B1-8 and B1-8 S104G in Figure 12A). In the context of B1-8hi, the S104G mutation has no effect (compare B1-8hi with B1-8hi S104G). Hence, acquiring the S104G mutation seems to be a potent alternative way of increasing BCR affinity, independent of the W33L pathway, explaining the positive selection of B1-8lo GC B cell clones bearing this mutation.
3.11 B1-8lo GC B cells participate in the GC reaction for an extended period of time when competition is reduced
To generate recipient mice, which do not respond to NP, CD45.1 mice were irradiated sub-
-/- -/- lethally and reconstituted with bone marrow cells from 33C9 x Rag1 and CD8 x JHT mice the following day. 33C9 mice express a human antibody and due to the Rag1-/- background, BM cells will exclusively give rise to B cells expressing the transgene as V replacement is
-/- prevented. BM cells of CD8 x JHT mice are the source of CD4 T cells in order to provide T cell help for GC formation. Two months after reconstitution, the transfer of B1-8lo splenocytes was performed as described above. Although recipient mice have been irradiated sub- lethally, a small fraction of irradiation resistant endogenous CD45.1 B cells was detectable (Figure 13A, left panel). Nevertheless, B1-8lo B cells are still found to be present in GCs 14 days post-immunization, making up to 80% of all GC B cells (Figure 13A, right panel). The 33C9-derived GC B cells are most probable specific for the carrier KLH, as they express a human kappa light chain. The presence of B1-8lo GC B cells at later time points reconfirms that the decrease in B1-8lo GC frequencies in our transfer model described above, is due to the presence of endogenous competitors and not a consequence of allogeneic rejection. Although the frequency of B1-8lo B cells is only slightly higher compared to endogenous B
lo cells, the frequency of VH1-72 heavy chains is significantly higher in the transgenic B1-8 B
29 Results cell pool, which might be the explanation for this advantage. However, this experiment needs to be repeated. In another experiment, 33C9 Rag competent (KK33C9) mice have been directly used as recipients for B1-8lo splenocytes. As B cells of these mice express a human heavy chain and
κ light chain, the frequency of endogenous heavy chains built up by a VH1-72 segment paired with a λ1 light chain should be low enough to represent a low competitive environment. Indeed at day 14 post-immunization, B1-8lo cells still make up 30-70% of all NP-specific GC B cells (Figure 13B). For further confirmation, this experiment needs to be repeated. Taken together, B1-8lo GC B cells have the potential to participate in the GC reaction for an extended period of time if competition is reduced.
Figure 13: When competition is reduced, B1-8lo GC B cells participate in the GC reaction for an extended period of time. A) In this experimental setup reduced competition was achieved by sub-lethal irradiation of Ly5.1 mice and reconstitution with bone marrow cells from 33C9 -/- -/- x Rag and CD8 x JHT mice, the following day. This combination lead to reconstitution of lymphocyte compartments by B cells mainly expressing a human antibody (33C9) and CD4 T cells. A small fraction of Ly5.1 B cells was irradiation-resistant and repopulated the B cell compartments with a frequency of 1.5% – 8.5% (left panel). Due to the low number of Ly5.1 competitors, the frequency of B1-8lo GC B cells reaches around 80% at day 14 post-immunization (right panel). Shown is the flow cytometric analysis of one experiment (n = 1-3). Dots represent single mice and lines show the mean with SD for each day of analysis. B) In another setting, reduced competition was achieved by using KK33C9 mice as recipients. These mice are heterozygous for a human heavy chain and κ light chain knock-in, reducing the frequency of endogenous heavy and light chains due to allelic exclusion. Shown is the flow cytometric analysis of one experiment (n = 3). Dots represent single mice and lines show the mean with SD.
30 Results
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
To assess if the fate of outcompeted B1-8lo GC B cells is enhanced apoptosis, we used a FITC-labeled inhibitor specific for active Caspase 3 (FITC-DEVD-FMK) to stain apoptotic events. Using this compound, 2-7% of all GC B cells are apoptotic at all time points analyzed (Figure 14A). In contrast, only 0.1 – 1.2 % of non GC B cells are apoptotic. This confirms that GCs contain enhanced frequencies of apoptotic cells (Mayer et al. 2017). Our frequencies are in agreement with those published by Mayer et al. using an Ab specific for active Caspase 3. Compared to endogenous GC B cells, the frequency of apoptotic B1-8lo cells is increased (Figure 14B). However, when focusing on Ag-specificity, the frequency of NP-specific apoptotic B1-8lo GC cells is not significantly increased compared to endogenous cells, although it increases over time (Figure 14C). It has to be taken into account that the analysis of apoptotic NP-specific B1-8lo GC B cells is challenging as their numbers are very low. Moreover, apoptotic GC B cells show decreased expression of surface markers (Figure 14D) and very weak NP binding compared to their living counterparts (Figure 14E). This impedes a closer analysis of Ag-specific apoptotic cells, as late apoptotic Ag-specific cells might have completely lost their ability to bind NP. Furthermore, apoptotic cells show signs of unspecific NP binding, which becomes especially evident in apoptotic B220 negative cells, compared to their non-apoptotic counterparts (Figure 14E). This might be due to membrane leakiness, resulting in unspecific intracellular binding. Although we did not detect increased apoptosis in low affinity GC B cells, this system might be optimized for further and more detailed analysis.
31 Results
Figure 14: Influence of competition on apoptosis in GC B cells. A) GC B cells depict increased frequencies of apoptotic cells compared to non GC B cells. Bars resemble the mean value of the single mice (dots). SD is depicted by error bars. Shown are 2 experiments with n=2-6. B) In a competitive setting, B1-8lo GC B cells show increased frequencies of apoptotic cells, compared to endogenous cells. However, this difference becomes less prominent for NP-specific GC B cells (C). Nevertheless, the frequency of apoptotic NP-specific B1- 8lo GC B cells tends to increase in course of the GC response. D) Apoptotic GC B cells show down-regulation of the surface markers B220, GL7 and Fas. E) Apoptotic GC B cells exhibit weak binding capacities for NP, compared to their living counterparts. In addition, apoptotic cells show signs of unspecific NP binding most probably due to the leakiness of their membranes. This can especially be observed in B220 negative cells. Depicted is a representative analysis of a mouse analyzed day 6 post-immunization.
32 Results
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
Next, we wanted to analyze if B1-8lo GC B cells have the potential to differentiate into memory B cells. Due to the lack of specific memory B cell markers, we focused on the analysis of NP-specific non GC B cells (CD19+CD38hiFas-NP+), subdivided into IgG1- and IgG1+ cells. It has to be taken into account that the IgG1- population also contains naïve NP- specific B cells. However, two observations strongly suggest that a significant proportion of these CD38hiFas-NP+IgG1- B cells are in fact memory B cells. First, the frequency of this population is clearly increased day 8 post-immunization compared to a non-immunized mouse analyzed day 9 after transfer (corresponding to day 8 post-immunization) (Figure 15, left panel). Second, the capacity of this population to bind NP is enhanced in immunized mice, compared to an unimmunized mouse. However, this difference is only observed for B1- 8lo cells and less prominent for endogenous cells (Figure 15B). Finally, further experiments are necessary to confirm these observations. Nevertheless, it is interesting that B1-8lo B cells make up at least 40% of the CD38hiFas-NP+IgG1- population at all time points analyzed, but only 9% without immunization. It should be appreciated that the frequency of this population is by far less drastically decreased as the frequency of NP-specific B1-8lo GC B cells during the same time points (~26% vs ~70% decrease from day 6 to day 10 post-immunization) (Figure 5D). However, CD38hiFas-NP+IgG1+ B1-8lo B cells showed a more severe decrease in frequency (~60% decrease from day 6 to day 8 post-immunization). Taken together, these findings suggest the need of a more detailed analysis regarding the memory B cell response of B1-8lo B cells in this transfer model. This additional analyses might support our hypothesis of a high frequency of B1-8lo B cells differentiating into memory B cells at early time points of the anti-NP response, independent of the competitive disadvantage of B1-8lo GC B cells evident around day 7 post-immunization.
33 Results
Figure 15: Analysis of NP-specific CD38hiFas- B cells A) The frequency of both, IgG1- and IgG1+ NP-specific CD38hiFas- B1-8lo B cells is increased upon immunization. Most probably, a significant part of these populations contain MBCs. Interestingly, the frequency of NP-specific CD38hiFas-IgG1- B1-8lo B cells stays relatively constant at all time points analyzed (left panel), although B1-8lo GC B cells are rapidly outcompeted (Figure 5). In contrast, the frequency of NP-specific CD38hiFas-IgG1+ B1-8lo B cells is clearly decreasing in the course of the immune response (right panel). Depicted are the mean values of single mice (dots). SD is exhibited by error bars. Shown are 3 experiments with n=3-5. B) NP-specific CD38hiFas- B1-8lo B cells of immunized mice depict an increased NP binding capacity compared to an unimmunized mouse. This enhanced NP binding might be indicative for memory B cells. However, this effect is less prominent in endogenous cells. Depicted is a representative analysis of a mouse analyzed day 6 post-immunization.
3.14 Molecular Dynamics of B1-8-related antibodies
To get more detailed insight into binding properties of B1-8-derived Abs to its Ag, molecular dynamics simulations have been performed in cooperation with the Bioinformatics from the Institute of Biochemistry in Erlangen. These simulations were performed on the basis of the crystal structure of a B1-8 Fv with a resolution of 1.77 Å (PDB: 1A6W; available at http://www.rcsb.org), binding to NIP. The measurements have been performed by substituting the amino acid positions of interest based on the crystal structure mentioned
34 Results above. Interestingly, the affinity enhancement of W33L was found not to be due to an increased binding energy (Figure 16).
System Energy SD SE B1-8hi (run1) -32.96 kcal/mol ±2.8 0.13 B1-8hi (run2) -33.25 kcal/mol ±2.9 0.13 B1-8 (run1) -32.59 kcal/mol ±3.2 0.14 B1-8 (run2) -33.14 kcal/mol ±2.9 0.13
hi Figure 16: Binding energy of a B1-8 Fv fragment compared to a B1-8 Fv using molecular dynamics.
Molecular dynamics analysis was used to calculate binding energies for a B1-8 Fv fragment to NIP, obtained from hi the crystal structure 1A6W (PDB), compared to the same fragment containing a W33L substitution (B1-8 Fv). Two independent runs have been performed, both showing that W33L does not enhance affinity to NP by increasing the binding energy due to forming additional molecular bonds (data from Benedikt Diewald and Prof. Dr. Heinrich Sticht, unpublished).
To gain further insight in the influence of W33L on NIP binding, the conformation of the binding pocket has been analyzed. For this, Y101 of the heavy chain (Y101H) was of special interest as this amino acid is the most flexible part of the CDR3 loop and gets immobilized upon binding to NIP. Furthermore, W93 of the lambda light chain (W93L) is decisive for NIP binding and has an influence on the orientation of the CDR3 loop of the heavy chain, making NP more accessible. This is also the reason for the lambda1 LC being essential for Abs to properly bind to NIP/NP. Thus, the distance between Cζ of Y101H and Cβ of W93L, which is located on the opposite side of the binding pocket, was measured over time. The distance
hi was found to shift between ~8 and ~18 Å for both Abs. However, the B1-8 Fv exhibited a prolonged open conformation of the binding pocket, increasing the probability of Ag binding
hi (Figure 17A). This fits to the higher kon-rate found for the B1-8 Ab using SPR (Table 1). Hence, the advantage of acquiring and selecting the W33L mutation within the NP system is all about higher chances to form immune complexes rather than formation of additional molecular bonds to enhance binding energies to the Ag.
When measuring the dihedral angle of Y101H, a ”binding competent“ conformation was found to be present at an angle of around 60° (Figure 17B). In B1-8hi and B1-8lo W33L, this angle is almost exclusively found to be at this position. However, for B1-8 and B1-8lo there exists an alternative, twisted conformation at around -60°, which is stabilized by NIP-binding. The change between the two conformations (60° and -60°) proceeds slower in B1-8lo, compared to B1-8, indicating a higher energy barrier between both conformations for this Ab. This means, Ag binding to the unfavorable conformation of B1-8lo costs more energy compared to the unfavorable conformation of B1-8, further decreasing Ag binding of B1-8lo. This further
lo explains the low kon-rate found for B1-8 in the SPR analysis. Consequently, the simulations
35 Results using molecular dynamics, so far complement our findings regarding affinity and binding rates from SPR analyses.
Figure 17: Molecular dynamic simulations of B1-8-derived Abs based on a B1-8 crystal structure.
A) The conformation of the binding pocket can be analyzed by measuring the distance between Cζ of Y101H and
Cβ of W93L as both amino acids are essential for NIP/NP binding. Especially Y101H is the most flexible part of the CDR3 and gets immobilized once immune complex formation occurs. A small distance indicates a closed pocket confirmation, while an enlarged distance indicates an open pocket. With this approach, we were able to show that hi a simulated B1-8 Fv results in prolonged open pocket conformations compared to the unmutated B1-8 Fv. This increases the probability of an immune complex formation. For both Fv analyzed, two independent measurements have been performed (red and blue lines, respectively). B) Alternative dihedral angles in simulated B1-8-derived Abs indicate a decreased Ag binding potential of B1-8 and B1-8lo Abs (data from Benedikt Diewald and Prof. Dr. Heinrich Sticht, unpublished).
36 Addendum
4 Addendum
4.1 Establishment of hB-varia mice
In another project, a transgenic mouse was established to generate a tool in which B cells are individually labeled by differential expression of fluorescent protein combinations. These mice express the Brainbow3.0 reporter construct (without farnesyl tags) under the control of a human CD19 promoter, interspersed with a rabbit beta-globin intron. To generate transgenic mice expressing multiple copies of the transgene in order to generate a broad panel of different color combinations, PNI was used as a method of choice. Expression of the transgene was assessed by PCR and flow cytometric analyses of blood samples, as mOrange2 is expressed by default if the transgene is present and expressed. mOrange expression was hardly detectable in blood-derived B cells, most probably due to fixation of whole blood samples. Two positive founder mice were mated witch C57BL/6 mice. Transgenes of both founder mice were germline transmitted. Organs of offspring mice were further analyzed using flow cytometric analyses. To check whether recombination of lox- flanked fluorescent protein sequences generates expression of different “colors”, transgene positive mice were crossed to Mx-Cre or Cre-ERT2 mice. Flow cytometric analysis of splenocytes show that expression of the transgene is B cell specific, and expressed by ~90% of all B cells (Figure 18A). When crossed to Mx-Cre mice, recombination results in co- expression of mOrange2 and GFP (Figure 18B). However, mKate2 was only marginally detectable. First analyses suggest that hB-varia mice might serve as a powerful tool to trace single B cells and study B cells clonality. This might be of special interest for GC studies. As a next step, these mice have to be analyzed using 2-photon microscopy in which also mKate2 can be excited more accurate. The hB-varia 1441 line has not been further investigated because of weak breeding.
37 Addendum
Figure 18: Analysis of the founder hB-varia 482. A) Flow cytometric analysis of splenocytes from hB-varia 482. mOrange2 expression is B cell specific (left panel). ~90% of the B cells express the transgene (right panel). B) Flow cytometric analysis of hB-varia 482 crossed to Mx-Cre to check recombination events resulting in expression of all fluorescent proteins (mOrange 2, GFP, mKate2).
38 Discussion and outlook
5 Discussion and outlook
5.1 Discussion
In this thesis, a transfer model has been established which enables investigation of transgenic low affinity GC B cells which are reproducibly outcompeted by endogenous competitors early in the GC reaction. The decrease of B1-8lo GC B cells came along with an accumulation in the LZ and a decreased proliferative capacity in the DZ. Aa expected, a W33L mutation, which is canonically found in the CDR1 of the heavy chain in C57BL/6 mice after NP immunization, was found to be selected in endogenous GC cells, enhancing the affinity of their BCRs 10-fold. Surprisingly, this mutation was almost completely absent in B1- 8lo GC B cells at all time points analyzed. A pre-existing amino acid change at position 98 within the heavy chain sequence of B1-8lo mice was shown to interfere with an affinity increasing effect of W33L, explaining the absent selection. However, an alternative way of affinity selection was found in B1-8lo GC B cells, leading to an accumulation of a S104G mutation within the CDR3 of the heavy chain, increasing the affinity 6x. Furthermore, B1-8lo GC B cells exhibited an increased frequency of apoptotic cells compared to endogenous cells, most of which did not or only very weakly bind NP. Most striking has been the increase of NP-specific CD38hiFas- B1-8lo B cells upon immunization, staying fairly constant at all time points analyzed, independent of the decreasing B1-8lo GC B cell frequency. Hence, this thesis might shed new light on the fate of low affinity B cells, which seem to massively differentiate into MBCs, although unable to compete in the GC reaction. The extent to what this differentiation occurs was especially striking. However, the final proof that these cells are indeed GC-derived long-lived MBCs remains to be provided. Our recent knowledge about GC selection processes is based on experiments using high affinity B1-8hi B cells in combination with the DEC205 system to artificially increase Ag presentation to Tfh cells (Victora et al. 2010)(Gitlin 2014). Hence, the focus has been set on mechanisms which underlie positive selection of high affinity GC B cells, whereas the fate of low affinity cells is still not solved satisfactorily. Especially in the early phase of the anti-NP response, low affinity antibodies are involved in the establishment of GCs (J M Dal Porto et al. 1998) and contribute to the formation of MBCs and plasma cells (Smith et al. 1997)(J M Dal Porto et al. 1998)(Joseph M Dal Porto et al. 2002). During affinity maturation, enhanced competition leads to positive selection of high affinity cells (Victora et al. 2010)(Gitlin 2014)(Amitai et al. 2017), reducing the frequency of low affinity variants (Weiss and Rajewsky 1990)(Ziegner, Steinhauser, and Berek 1994). However, when competition is reduced or absent, low affinity GC B cells do not only support formation of GCs, but even participate in the GC reaction for an extended period of time (Joseph M Dal Porto et al.
39 Discussion and outlook
2002)(Shih et al. 2002). This has been shown by immunization experiments of mice expressing transgenic heavy chain sequences, resulting in low affinity anti-NP Abs when paired with a λ1 light chain. To further analyze the fate of low affinity GC B cells as a consequence of competition in GCs, we made use of a transgenic transfer model, in which B1-8lo splenocytes are transferred into wildtype recipients. Using this approach, NP-specific low affinity (B1-8lo) GC B cells are reproducibly outcompeted by NP-specific endogenous competitors early in the GC response. The notion that B1-8lo GC B cells are rapidly outcompeted has already been described (Shih et al. 2002). However, a co-transfer of B1-8lo and B1-8hi mice into wildtype recipients has been used in this publication. Although B1-8lo B cells do express a BCR of 40-fold lower affinity, when paired with a λ1 light chain, this rapid loss in competition to B1-8hi is surprising when taking into account that SHM acts randomly (Weiss, Zoebelein, and Rajewsky 1992) and efficiently generates Abs of higher affinities. Especially for the NP system, a single mutation selected within the CDR1 of the heavy chain (W33L) is known to be sufficient to enhance affinity 10-fold (D. Allen et al. 1988). Furthermore, Shih et al. showed that B cells of B1-8lo mice are able to mutate and even showed a higher number of replacement mutations compared to B1-8hi mice in a non-competitive environment (Shih et al. 2002), making the loss of B1-8lo GC B cells even more remarkable. This observation raises the question why not even a small fraction of mutated B1-8lo GC B cells is able to compete with B1-8hi cells in this co-transfer model. As a possible explanation for the loss of B1-8lo GC B cells, it was hypothesized that these cells unsuccessfully bind and internalize Ag bound on FDCs in the presence of competitors. A recent publication stated that especially GC entry is dependent on MHC-dependent T cell help, which is less stringent for selection processes of established GCs (Yeh et al. 2018). As B1-8lo and B1-8hi BCRs extremely differ in their starting affinity and hence in peptide:MHC density, such pre-GC interactions with T cells might predominantly drive B1-8hi B cells into the GC, explaining the much lower frequency of B1-8lo GC B cells compared to our system. Instead of co-transferring B1-8lo and B1-8hi splenocytes, we decided to solely transfer B1-8lo splenocytes (CD45.2) into wildtype (CD45.1) recipients. Using this approach, we excluded competitors already having an advantage by expressing a BCR of at least 40-fold higher affinity. With this approach, a higher frequency of B1-8lo GC B cells is found, compared to Shih et al., as early high affinity competitors for GC entry are absent. All the more, the rapid decrease of B1-8lo GC B cells in competition to endogenous wildtype cells was even more
lo surprising. As the transgenic rearranged B1-8 heavy chain already expresses the VH1-72 segment needed for proper NP binding, and the notion that these cells are present at high frequencies upon transfer, it could be assumed that, although exhibiting low affinity, these cells should have an advantage compared to the highly diverse endogenous B cell pool (J M
40 Discussion and outlook
Dal Porto et al. 1998)(Jacob et al. 1993). Surprisingly, NGS analysis revealed that the loss of B1-8lo GC B cells is based on the absence of the affinity increasing W33L mutation in these cells, even at time points when the competitors have accumulated the affinity increasing mutation. This absence probably results in a disadvantage of B1-8lo GC B cells to stay competitive, explaining the drastic decrease of B1-8lo GC B cells. The reason for the absence of W33L selection was found to arise from a pre-existing amino acid change within the sequence of the B1-8lo heavy chain. This change from R98 to T98 is sufficient to decrease the affinity of an unmutated B1-8 antibody 10-fold, explaining the low affinity of B1-8lo Abs. When a B1-8lo Ab containing W33L, which was almost completely absent in vivo, was generated recombinantly, W33L did not exhibit any increase in affinity. This clearly shows that the pre-existing R98T exchange interferes with an affinity increase due to W33L. This in turn explains the absence of B1-8lo GC B cells selected for W33L, while endogenous competitors were selected for this mutation. Hence, W33L is not able to restore the low affinity of B1-8lo Abs. However, another mutation (S104G) within the CDR3 of B1-8lo GC B
lo cells was found to be highly selected. Although its affinity enhancing effect on a B1-8 Ab (KA
6 -1 = 2.5 x 10 M ) does not reach the level of a W33L mutation on a B1-8 background (KA = 7.0 x 107 M-1), this mutation enables an alternative way to reach higher affinities. As S104G is found in around 60% of all B1-8lo heavy chain sequences at day 9 post-immunization, it prolongs the survival of a small fraction of B1-8lo GC B. This nicely demonstrates the flexibility of the GC reaction to produce higher affinity, by-passing the affinity dead-end of a canonic mutation by subsequent selection of alternative mutations. It would be interesting to find out whether W33L mutations of the B1-8lo heavy chain can be found in other compartments of the GC response, particularly among plasma cells or memory B cells. Efficient affinity selection requires interzonal migration of GC B cells to undergo different subsequent GC programs in the LZ and the DZ (M Meyer-Hermann, Deutsch, and Or-Guil 2001)(C. D. C. Allen et al. 2007)(Hauser et al. 2007)(Schwickert et al. 2007)(Victora et al. 2010). Early in the GC reaction, both B1-8lo and endogenous GC B cells showed a similar DZ/LZ ratio at day 6 of the anti-NP response, with more cells being located in the DZ. This is not surprising as the DZ is known to be the site of B cell proliferation (Victora et al. 2010)(Gitlin 2014). Furthermore, initial B cell proliferation, prior to the onset of selection processes, ensures the generation of an adequate pool of B cells which have the potential to achieve higher affinity once selection takes place (Jacob et al. 1993). This pool was also shown to contain B cells of low affinity and, regarding to NP-immunization, even antibodies that do not contain the canonical VH1-72 heavy chain segment (J M Dal Porto et al. 1998) (Jacob et al. 1993). In addition, it was proposed that during this early establishment phase newly activated B cells can further join the GC (Jacob et al. 1993). However, at day 8 post- immunization, when the number of Tfh (Shulman et al. 2013) cells and thus T cell help
41 Discussion and outlook
(Dominguez-Sola et al. 2012) peaks, selection processes begin to restrict clonal diversity (Jacob et al. 1993)(Tas et al. 2016) to select high affinity B cells. Especially for the NP- system, this was shown to result in a focused GC repertoire towards B cells expressing a
VH1-72 bearing heavy chain and accumulation of B cells selected for the W33L mutation (D. Allen et al. 1988)(Jacob et al. 1993). The onset of selection at day 8 post-immunization coincides with a shift of the DZ/LZ ratio of B1-8lo GC B cells, such that they are preferentially found to be present in the LZ, while the DZ/LZ ratio of endogenous cells is still unchanged. Such a shift in the zonal distribution has already been described for endogenous GC B cells which are outcompeted by transferred B1-8hi cells (Victora et al. 2010). An explanation for this accumulation in the LZ might be that only a small proportion of B1-8lo GC B cells receive signals from Tfh cells inducing re-direction into the DZ, as this process was shown to be controlled by competition in the LZ (Victora et al. 2010). Also a mathematical model predicted that the DZ/LZ ratio decreases, when recycling is reduced (Michael Meyer-Hermann et al. 2012). Furthermore, it was shown that the frequency of LZ cells migrating to the DZ is in general much lower compared to DZ cells migrating to the LZ (Victora et al. 2010). This net flux into the LZ might further contribute to an accumulation of B1-8lo cells in this zone. Regarding endogenous GC B cells, this net flux is most probably compensated by increased proliferation in the DZ due to preceding selection processes within the LZ. In addition to the shifted DZ/LZ ratio of B1-8lo GC B cells, the frequency of EdU+ B1-8lo GC B cells was found to be lower compared to endogenous cells at day 8 post-immunization. This observation fits to the fact that the number of divisions in the DZ is linked to the amount of Ag presented to Tfh cells in the LZ and hence, to BCR affinity (Gitlin 2014). In competition to B1- 8hi cells, endogenous cells were also found to exhibit lower proliferation rates in addition to a shifted DZ/LZ ratio (Victora et al. 2010), showing that competition controls zonal migration and proliferation. Hence, the decreased proliferative capacity of B1-8lo GC B cells might further contribute to the shifted DZ/LZ ratio and is clearly disadvantageous in competition to highly proliferative endogenous cells. The proliferative B1-8lo GC B cell pool still present at day 9 of the anti-NP response might be composed of positively selected cells which have acquired the S104G mutation, as this mutation was present in approximately 60% of all B1- 8lo derived heavy chain sequences day 9 post-immunization. Next, we wanted to analyze the fate of the B1-8lo B cells being outcompeted in the GC. Increased apoptosis was shown to be evident in GCs (Mayer et al. 2017), driving B cells which are not positively selected by Tfh cells to undergo programmed cell death. Experimental evidence leading to this conclusion is based on a transfer model, in which endogenous GC B cells are outcompeted by transferred B1-8hi B cells. The outcompeted cells accumulate in the LZ, in which the expression of apoptotic markers was found to be increased (Victora et al. 2010). Mathematical models supported these findings (Michael
42 Discussion and outlook
Meyer-Hermann et al. 2012) and suggested that apoptotic events in the GC LZ are dependent on BCR affinity (M. E. Meyer-Hermann, Maini, and Iber 2006). Hence, apoptosis of low affinity GC B cells would be dependent on the presence of high affinity competitors. This model might also explain the increase of TUNEL+ GC B cells in transgenic mice expressing a low affinity BCR, once endogenous competitors (escaping allelic exclusion) join the late GC response (Joseph M Dal Porto et al. 2002). However, Mayer et al. recently proposed the model “death by default” in which GC B cells undergo apoptosis independent of BCR affinity. With this, high and low affinity B cells upregulate apoptotic markers when recycling into the LZ and only those B cells that do interact with Tfh cells are being “rescued” from apoptosis. If apoptosis in the GC is dependent on BCR affinity or not still remains controversial. In our transfer model, we were able to reconfirm the frequency of apoptotic GC B cells published by Mayer et al.. While they were using an anti-active Caspase 3 Ab, we were making use of a FITC-labeled Caspase 3 inhibitor, labeling apoptotic cells without the need of fixation and permeabilization. Furthermore, we found an increased frequency of apoptotic B1-8lo GC B cells compared to endogenous cells at day 8 and day 10 post-immunization. However, when focusing on Ag-specificity, NP+ B1-8lo GC B cells only tend to have a slightly increased frequency of apoptotic cells at day 8 and 10 post-immunization compared to endogenous cells. With this, the majority of apoptotic B1-8lo GC B cells does not bind NP. As a significant proportion of B1-8lo GC B cells is NP specific (70-80%) and the notion that apoptotic cells exhibit downregulation of surface molecules (Mayer et al. 2017) and decreased NP binding capacity, it remains to be elucidated if apoptotic B1-8lo GC B cells that do not bind NP indeed exhibit another specificity or if the majority of these cells has been NP-specific, but lost the potential to bind NP due to apoptosis. A down-regulation of the BCR due to apoptosis was recently also demonstrated for B cells bearing deleterious mutations leading to programmed cell death in the DZ (Stewart et al. 2018). Moreover, it has to be taken into account that the total number of apoptotic NP-specific B1-8lo GC B cells measured in our setting is generally low, which might skew the actual results. Finally, a co-transfer model of B1-8lo and B1-8hi splenocytes into wt recipients might be better suited to shed further light on apoptosis in GCs. This is because both B1-8lo and B1-8hi B cells express the same variable heavy chain gene segments and solely differ in few amino acid positions and thus in their binding affinity. In contrast, wt recipients express a variety of different combinations of variable segments, before their repertoire is focused on VH1-72 bearing
BCRs in the GC (Jacob et al. 1993). This restriction to VH1-72 segments is most probably achieved by apoptosis of NP-specific B cells expressing other variable segments, possibly skewing the frequency of apoptotic NP-specific endogenous GC B cells.
43 Discussion and outlook
Finally, the function of GCs is the generation of Ab secreting PCs and MBCs. In absence of competition, low affinity B cells were shown to differentiate into plasmacytes (J M Dal Porto et al. 1998) and MBCs (Joseph M Dal Porto et al. 2002). To our surprise, we found that NP- specific B1-8lo B cells are found to a great extend (26-60%) within the CD38hiFas- compartment at all time points analyzed. As the frequency of this population is increased 2-3- fold upon immunization, we expect that this population contains a significant proportion of memory B cells. However, this has to be confirmed using specific markers, such as CD73, CD80 and PD-L2 (Tomayko et al. 2010)(Zuccarino-Catania et al. 2014). With this, the composition of MBC subsets regarding CD80 and PD-L2 expression can be elucidated. Double-negative (DN) MBCs are formed before and early in the GC response, followed by PD-L2 single-positive (SP) and finally double-positive (DP) cells. Furthermore, DNs are mainly IgM and carry very few mutations, while DPs are mainly IgG-switched and carry many more mutations. SP MBCs were shown to be intermediate regarding mutational load and the frequency of IgG-switched cells. In addition, DNs were shown to have the highest potential to re-enter GCs while DPs efficiently give rise to IgG-expressing antibody-forming cells (AFCs) (Zuccarino-Catania et al. 2014). As B1-8lo GC B cells are drastically outcompeted in the GC, it is even more surprising that the frequency of CD38hiFas- B1-8lo B cells remains fairly constant. It has to be taken into account that MBCs can also be generated outside of the GC (reviewed in Kurosaki, Kometani, and Ise 2015)(reviewed in Weisel and Shlomchik 2017). These extra- GC MBCs are thought to be a common way to keep the initial repertoire untouched from SHM as B cells potentially reactive against similar, but distinct Ags especially upon epitope shifting. Although extra-GC MBCs were shown to be generated at least until d6 post- immunization and even contribute to the majority of MBCs generated until d3 post- immunization, the vast majority of early MBCs formed at d4 and later was shown to be of GC origin (F. J. Weisel et al. 2016). After the peak of IgM MBC generation within the first days after immunization, IgG1-switched MBCs start to occur, peaking at day 6-8 (F. J. Weisel et al. 2016). Regarding CD38hiFas-NP+ B1-8lo B cells in our transfer model, a small proportion was found to be switched to IgG1. In contrast to IgG1- cells, the frequency of this population decreases with progress of the anti- NP response. It will be of high interest to compare the mutational load of IgG1- and IgG1+ CD38hiFas-NP+ B1-8lo B cells. These two populations might further contribute to different MBC subsets, regarding CD80 and PD-L2 expression and hence, might have different functions. As already mentioned, DN IgM+ MBCs have the potential to re-enter GCs, while SPs can also and DPs exclusively give rise to IgG-expressing AFCs (Zuccarino-Catania et al. 2014).
44 Discussion and outlook
Furthermore, it would be interesting to assess the frequency of S104G and W33L in the two different populations. A massive differentiation of B1-8lo B cells into MBCs, although being outcompeted in GC would be an interesting new finding, complementing the knowledge about the fate of low affinity GC B cells. This is all the more surprising given that Shih et al. only found B1-8hi but not B1-8lo B cells giving rise to a MBC pool. However, it has to be mentioned that Shih et al. solely defined MBCs as B cells seeding the marginal zone in the course of the anti-NP response (Shih et al. 2002). The extent to what B1-8lo B cells were found to contribute to the CD38hiFas-NP+ B cell population is particularly striking and suggests that low affinity B cells might predominantly differentiate to MBCs early in the GC response, maintaining the diversity of the early reactive B cell pool. This enables adaption of the immune response to altered versions of the Ag. Shinnakasu et al. have already shown that especially low affinity IgG1+ LZ cells, mainly generated between day 9-11 post-immunization, are favored to differentiate into MBCs (Shinnakasu et al. 2016), while high affinity GC cells preferentially differentiate into PCs. Furthermore, Bach2 expression was shown to be increased in cells which have only received weak T cell help. This increased Bach2 expression was driving low affinity NP+IgG1+ LZ cells to become MBCs. This notion might serve as an alternative explanation for B1-8lo GC B cells accumulating in the LZ in competition to endogenous cells. These low affinity cells might receive T cell help insufficient to re-direct them into the DZ, but prolonging survival within the LZ to enable differentiation into MBCs. Taken together, our findings suggest that not only IgG1+, but also IgG1- MBCs might have the potential to preferentially differentiate into MBCs. It would be interesting to asses if Bach2 might also play a role in this process. To our knowledge, this is the first analysis of the competitive processes in the GC reaction combining processes on a cellular and molecular level. As affinity selection requires binding and internalization of Ag presented on FDCs, B1-8lo GC B cells might be disadvantaged
lo because recombinant B1-8 Abs exhibit a low kon - and high koff – value. Hence, the generation of Ab:Ag complexes is inefficient and Ag is not retained properly. This is all the more problematic as W33L, shown to prolong the open pocket conformation of a B1-8 Ab, has no effect on a B1-8lo Ab. Hence, during the interaction of B1-8lo GC B cells with FDCs, a low amount of Ag captured by B1-8lo GC B cells might lead to inefficient Ag presentation. The subsequent help from Tfh cells results in insufficient recycling events and weak proliferation signals indicated by a shifted DZ/LZ ratio and a decreased frequency of Edu+ B1-8lo GC B cells. In contrast, high affinity endogenous competitors, select the canonic W33L mutation leading to affinity selection. To our knowledge, this is the first time showing that W33L is leading to a prolonged open binding pocket and not to formation of additional molecular bonds. Hence, once NP is bound by a B1-8 or B1-8hi Ab, the binding forces are similar. A
45 Discussion and outlook prolonged open pocket conformation increases the probability of Ag binding, enabling efficient Ag capturing and subsequent presentation to Tfh cells in order to be selected for DZ re-entry. The R98T amino acid exchange pre-existing in the heavy chain of B1-8lo mice most probably resulted from recombination events rather than somatic mutation. This event led to an exchange of two neighboring nucleotides in the same codon. As both nucleotides need to be mutated to enable an exchange to R98, back-mutation is highly improbable. The decrease in affinity due to R98T is not surprising when taking into account that an arginine at position 98 is highly conserved among different murine V segments. Hence, a change from a basic to a polar/neutral amino acid can readily change the properties of the CDR3 loop. MD analyses of a B1-8 Ab revealed that the CDR3 loop of the heavy chain is relatively tensed. This might explain why many crucial amino acid changes, such as R98T, S104G, Y99G and W33L (which is directly influencing Y101) are located in this region. It would be interesting to analyze if R98T also interferes with Y99G, found to be selected in late anti-NP responses (Furukawa et al. 1999) and secondary immunizations (Tashiro et al. 2015), reaching affinities in the nanomolar range if W33L is absent (Furukawa et al. 1999). As already mentioned, W33L influences Y101 making it more flexible in B1-8hi Abs, because L33 removes the tension arising from W33. A reason for that is the formation of a pi-stack resulting in “stickiness” of W33L and Y101. In general, Y101 was found to be the most flexible amino acid, which can be viewed as the hinge of the CDR3 loop (personal communication with Benedikt Diewald and Prof. Dr. H. Sticht, Bioinformatics, Institute for Biochemistry, Friedrich- Alexander University Erlangen-Nuremberg). Combining the data from our cellular and molecular analyses, we were able to link the presence of specific mutations to their influence regarding competition processes in the GC. This revealed both, the flexibility of the GC reaction, but also the complexity of affinity maturation even in response to a simple Ag as NP.
5.2 Outlook
To complete the picture of the B1-8lo B cell fate in this competition model, ELISPot analysis have to be conducted in order to analyze PC differentiation of B1-8lo cells. Regarding the experiments on MBCs performed in this thesis, further control mice are necessary to confirm the increase of NP-specific CD38hiFas- B1-8lo B cells after immunization. Additional experiments need to be conducted to support the hypothesis that the majority of NP-specific CD38hiFas- B1-8lo B cells are indeed MBCs and to asses if they have been formed GC- dependent or –independent. As longevity is a hallmark of MBCs, it should be checked if CD38hiFas- B1-8lo B cells can also be found at later time points, at least day 30, in our
46 Discussion and outlook transfer model. Analysis of unimmunized control mice, which have received B1-8lo splenocytes would exclude an unexpected longevity of transferred naïve donor B cells. Especially EdU-labeling at distinct timeframes after transfer of B1-8lo cells would enable identification of cells that have stayed quiescent since EdU-incorporation. Such cells could then be sorted (and pooled from different mice, if necessary) to analyze SHM. This would indicate if these MBCs are GC-derived or might be of extra-GC origin. Sequencing of both IgM- and IgG-bearing heavy chains would reveal maximal information. Furthermore, it would be highly interesting to assess if the W33L mutation, which is not selected in B1-8lo GC B cells, can be found in the B1-8lo MBC pool. In addition, isolated MBCs could be used as donor cells for another transfer to prove their potential to quickly react upon being re- challenged with Ag. Therefore, using a system of reduced competition would be advantageous to analyze the GC reaction and PC formation (e.g. using ELISpot). Furthermore, CD38hiFas- B1-8lo B cells could be characterized as MBCs using new markers which are recently under investigation (Weisel, unpublished). In addition, it would be interesting to analyze the contribution of B1-8lo and B1-8hi B cells to the MBC pool when co-transferred into recipient mice. Especially if it holds true that B1-8lo B cells hardly make it into the GC, because competitors of 40-fold higher affinity successfully compete for GC entry upon interaction with T cells. If, in this setting, the frequency of both NP-specific B1-8lo GC B cells and CD38hiFas- B1-8lo B cells is clearly reduced compared to our transfer model, this would indirectly show that NP-specific CD38hiFas- B1-8lo B cells described in this thesis are indeed GC-derived. In contrast, if GC entry of B1-8lo B cells in competition to B1-8hi B cells is reduced, extra-GC-derived MBCs would still be emerging. However, if the NP-specific CD38hiFas- B cell pool turns out to be mainly composed of B1-8lo cells, a massive differentiation into MBCs might be an alternative explanation for the low frequency of B1-8lo GC B cells by Shih et al.. This might further be supported by the notion that high affinity cells preferentially enter the GC to differentiate into plasma cells (Shinnakasu et al. 2016). However, to analyze this, a more comprehensive kinetic analysis is, compared to Shih et al., is necessary. Further interesting experiments regarding our transfer model would be transfer of B1-8lo and/or B1-8hi splenocytes into ongoing GCs of immunized recipient mice. Also the influence of different numbers of transferred B1-8lo splenocytes or the Ag used for immunization (e.g.
NP8-BSA vs. NP30-BSA) might be interesting to analyze. Additional ELISPot analyses might reveal if B1-8lo B cells differentiate into PCs when competition is reduced.
47 Materials
6 Materials
6.1 Antibodies and reagents for flow cytometry
Product / Antigen Fluorochrome Clone Company B220 PerCP-Cy5.5 RA3-6B2 Biolegend, San Diego (USA) CD16/32 (Fc Block) 2.4G2 Hölzel Diagnostika, Köln CD19 BV421 6D5 Biolegend, San Diego (USA) eFluor660 ebio1D3 ebioscience, Frankfurt CD38 PE 90 Biolegend, San Diego (USA) bio n.a. BD Pharmingen, Heidelberg CD45.1 PE-CF594 (ECD) A20 BD Bioscience, Frankfurt PE A20 BD Pharmingen, Heidelberg CD45.2 FITC 104 BD Bioscience, Frankfurt APC 104 Biolegend, San Diego (USA) BV421 104 Biolegend, San Diego (USA) CD86 FITC GL1 ebioscience, Frankfurt CXCR4 BV421 L276F12 Biolegend, San Diego (USA) Fas (CD95) PE-Cy7 Jo2 BD Bioscience, Frankfurt GL7 AF647 GL7 Biolegend, San Diego (USA) Biosearch Technologies, NP36 PE n.a. Novato (USA) Vector Laboratories, PNA FITC n.a. Burlingame (USA) Molecular Probes, Eugene Streptavidin Pacific Orange n.a. (USA) BD FACS™ Lysing BD Bioscience, Frankfurt Solution BD Truecount™ Tubes BD Bioscience, Frankfurt VersaComp Antibody Beckman Coulter, Brea
Capture beads (USA)
48 Materials
6.2 Antibodies and reagents for immunofluorescence
Product / Antigen Fluorochrome Clone Company
Biolegend, San Diego CD45.2 AF647 104 (USA) IgD bio 11-26 ebioscience, Frankfurt Biolegend, San Diego Ki67 PE 16A8 (USA) Molecular Probes, Streptavidin AF350 n.a. Eugene (USA) Roti®-Liquid Barrier Roth, Karlsruhe Marker Roti®-Mount FluorCare Roth, Karlsruhe Tissue-Tek® O.C.T™ Sakura, Alphen aan den
Compound Rijn (NL)
6.3 Antigens / Immunization
Product Company Imject® Alum Thermo Fisher Scientific, Rockford (USA) NP-KLH Biosearch Technologies, Novato (USA)
6.4 Buffers
50x TAE buffer
242 g Tris + 60.5 ml Acetic acid + 100 ml 0.5 M EDTA, pH 8.0 → add up to 1ml with H2O
Antibiotics drinking water 1.1 g/l Neomycin sulfate + 106 U/l Polymixin B sulfate
Buffer QF 1.25 M NaCl + 50mM Tris-Cl, pH 8.5 + 15% isopropanol
49 Materials
Blocking solution (IF) PBS + 10% FCS + 0.1% BSA + Fc-block (1:100)
Erythrocyte Lysis Buffer
0.15 M NH4Cl + 0.02 M HEPES + 0.1 mM EDTA
FACS buffer PBS + 2% FCS
Injection buffer 5 mM Tris + 0.1 mM EDTA, pH 7.5 in Aqua ad injectabilia
MACS buffer PBS + 2% FCS + 0.2 mM EDTA
PBS
137 mM NaCl + 2.7 mM KCl + 4.3 mM Na2HPO4 + 1.4 mM KH2PO4 , pH 7.3
R10 Medium RPMI (1640 A) + 10% FCS + 2mM L-Glutamine + 100 U/ml Penicillin + 100 µg/ml Streptomycin + 500 µM β-mercaptoethanol
Staining Solution (IF) PBS + 2% FCS + 0.05% Tween20
Washing buffer (IF) PBS + 0.05% Tween20
6.5 Chemicals and reagents
Reagent Company 100 bp DNA ladder New England Biolabs, Ipswich (USA) 1 kbp DNA ladder New England Biolabs, Ipswich (USA) 2-propanol Roth, Karlsruhe 5-ethynyl-2-deoxyuridine (EdU) Carbosynth, Berkshire (UK) 6x loading dye New England Biolabs, Ipswich (USA)
50 Materials
Acetic acid Roth, Karlsruhe Acetone Roth, Karlsruhe Agarose Bio and Sell, Feucht Ampicilin Sodium Salt Applichem, Darmstadt Ammonium chloride (NH4Cl) Roth, Karlsruhe Aqua ad injectabilia Braun, Melsungen Bovine serum albumin Sigma, Steinheim β-mercaptoethanol (for RNA isolation) Sigma, Steinheim β-mercaptoethanol (for R10 medium) PAN Biotech, Aidenbach Dipotassium hydrogen phosphate (K2HPO4) Sigma, Steinheim Disodium hydrogen phosphate (Na2HPO4) Roth, Karlsruhe PAN Biotech, Aidenbach or Anprotec, DPBS Bruckberg EDTA Sigma, Steinheim Ethanol Roth, Karlsruhe FCS PAN Biotech, Aidenbach HCl (hydrogen chloride) Roth, Karlsruhe HEPES Roth, Karlsruhe Kanamycin Sigma, Steinheim LB agar Sigma, Steinheim LB broth Sigma, Steinheim L-Glutamine (200 mM) PAN Biotech, Aidenbach Neomycin sulfate Sigma, Steinheim Penicillin (10.000 U/ml) PAN Biotech, Aidenbach Polymixin B sulfate Thermo Fisher Scientific, Rockford (USA) RPMI (1640) PAN Biotech, Aidenbach Sodium hydroxide (NaOH) Roth, Karlsruhe Streptomycin (10 mg/ml) PAN Biotech, Aidenbach SYBR™ Safe DNA Gel Stain Invitrogen, Karlsruhe Tris (Trizma base) Sigma, Steinheim Tween® 20 Sigma, Steinheim
51 Materials
6.6 Consumables
Name Company
1 ml Syringe (Injekt-F) Braun, Melsungen CELLSTAR® Polypropylen tubes 15 ml Greiner Bio-One, Frickenhausen CELLSTAR® Polypropylen tubes 50 ml Greiner Bio-One, Frickenhausen Cellstar® Serological pipettes, 1/2 mL, Greiner Bio-One, Frickenhausen sterile Cell strainer (70 μm) VWR,Radnor (USA) CellTricsR 50 μm Partec,Gorlitz Dialysis Tubing NeoLab, Heidelberg FACS tubes (5 ml, Polystyrol) Becton Dickinson,Franklin Lakes (USA) Kimtech Wipe Kimberly-Clark, Irving (USA) PCR tubes Bio and Sell, Feucht Petri dish, Ø 100/20 mm, PS, sterile Greiner Bio-One, Frickenhausen Reaction tubes 1.5 ml, 2 ml Nerbe, Winsen Reaction tubes 1.5 ml, 2 ml, 5 ml (safe lock) Eppendorf, Hamburg Syringes BD Bioscience, Frankfurt UVette® cuvettes, sterile Eppendorf, Hamburg
6.7 Device
Device Company
Avanti centrifuge Beckman Coulter, Brea (USA) Axioplan 2 Zeiss, Jena Axiovert 200 Fluorescence microscope Zeiss, Jena BB17-6 irradiation box Gamma-Service Medical, Leipzig Biobeam 2000 irradiator Gamma-Service Medical, Leipzig Biofuge fresco Heraeus, Hanau Biofuge Megafuge 1.0R Heraeus, Hanau Biofuge pico Heraeus, Hanau Bio Photometer Eppendorf, Hamburg Camera Controler + Digital Camera C4742- Hamamatsu, Shizuoka (JPN) 95 CM3050 S Cryostat Leica, Wetzlar
52 Materials
Cytoflex S Flow Cytometer Beckman Coulter, Brea (USA) E-Gel® Safe Imager™ Invitrogen, Karlsruhe HI3220 pH/ ORP Meter Hanna Instruments, Woonsocket (USA) HXP 120 V Compact Light Source Kübler Codix Mastercycler Pro S Eppendorf, Hamburg MiSeq Illumina, San Diego (USA) MoFlo Astrios Beckman Coulter, Brea (USA) UVsolo TS Imaging System Biometra, Göttingen
6.8 Kits
Kit Company
Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Invitrogen, Karlsruhe Cytometry Assay Kit CaspGLOW™ Fluorescein Active Caspase-3 Thermo Fisher Scientific, Rockford (USA) Staining Kit DNeasy Blood and Tissue Kit Qiagen, Venlo (NL) EndoFree Plasmid Maxi Kit Qiagen, Venlo (NL) OneStep RT-PCR Kit Qiagen, Venlo (NL) QIAGEN-tip 20 Qiagen, Venlo (NL) QIAprep Spin Miniprep Kit Qiagen, Venlo (NL) QIAquick Gel Extraction Kit Qiagen, Venlo (NL) QIAquick PCR Purification Kit Qiagen, Venlo (NL) QIAshredder spin columns Qiagen, Venlo (NL) RNeasy Mini Kit Qiagen, Venlo (NL) StrataClone PCR Cloning Kit Agilent Technologies, Santa Clara (USA)
53 Materials
6.9 Mice
B1-8lo mice (Stock No. 007776 at www.jax.org) and B1-8hi mice (Stock No.007594 at www.jax.org) (Shih, Roederer, and Nussenzweig 2002) were kindly provided by Prof. Dr. F. Nimmerjahn (Department of Biology, Division of Genetics at the Friedrich-Alexander University Erlangen-Nürnberg). For all experiments, heterozygous B1-8lo/+ (defined as B1-8lo) or heterozygous B1-8hi/+ (defined as B1-8hi) mice were used.
BALB/cJ mice have been kindly provided by Prof. Dr. L. Nitschke (Department of Biology, Division of Genetics at the Friedrich-Alexander University Erlangen-Nürnberg).
C57BL/6 mice have been purchased from Chanvier and are bred at the Biotechnologisches Entwicklungslabor of the Division of Genetics, Friedrich-Alexander University Erlangen- Nürnberg.
-/- -/- -/- CD8 x JHT mice were obtained by crossing CD8α (CD8 ) mice, purchased from The
Jackson Laboratory (Stock No. 002665) (Fung-Leung et al. 1991), with JHT mice, which have been a gift from Prof. Dr. HM Jäck (Division of Molecular Immunology, University Hospital Erlangen)(Chen et al. 1993).
CD45.1 (Pepboy) mice were purchased from The Jackson Laboratory (Stock No. 002014).
KK33C9 mice were established by Dr. Kristin Kruse, a former PhD student in the working group of Prof. Dr. Thomas H. Winkler (Department of Biology, Division of Genetics at the Friedrich-Alexander University Erlangen-Nürnberg). 33C9 x Rag-/- mice were kindly provided by Thomas Weisenburger (Department of Biology, Division of Genetics at the Friedrich- Alexander University Erlangen-Nürnberg).. hB-varia 482 and hB-varia 1441 mice have been established in this thesis.
Mx-Cre mice have been kindly provided by Prof. Dr. R. Slany (Department of Biology, Division of Genetics, Friedrich-Alexander University Erlangen-Nürnberg)
For transfer experiments into BM chimeras, B1-8lo mice were crossed with CD45.1 mice to obtain heterozygous B1-8lo mice expressing both allelic variants of CD45 (CD45.1 and CD45.2).
6.10 PCR and Cloning
Product Company
5X PCR Mastermix Bio and Sell, Feucht Agentcourt® AMPure® PCR Purification Beckman Coulter, Brea (USA) dNTPs New England Biolabs, Ipswich (USA) LongAmp®Taq Polymerase New England Biolabs, Ipswich (USA) Nextera XT Index Kit v2 Set D Illumina, San Diego (USA)
54 Materials
One Shot TOP10 Chemically Competent E. Thermo Fisher Scientific, Rockford (USA) coli Restriction buffers New England Biolabs, Ipswich (USA) Restriction enzymes New England Biolabs, Ipswich (USA) SOC Outgrowth Medium New England Biolabs, Ipswich (USA) T4 DNA Ligase New England Biolabs, Ipswich (USA) Taq 2X Master Mix New England Biolabs, Ipswich (USA) Taq DNA Polymerase Bio and Sell, Feucht
6.11 Plasmids
Name Description Source rabbit β-globin intron and Brainbow3.0 cassette Synthesized by Thermo Bvaria2 without farnesyl tag Fisher Scientific hCD19Prom Human CD19 Promoter cloned via T/A cloning in Strata Prepared in this Thesis using the StrataClone PCR Cloning Kit Klon6 pCAG- www.addgene.org (ID: Brainbow3.0 cassette Brainbow3.0 45176) (Cai et al. 2013) pUHG10-3 source for rabbit β-globin intron (BamHI – EcoRI) Winkler lab
6.12 Primer
Sequence (Overhang, Restriction site, Primer TW Company sequence) hCD19 5´-TAA GCA CCG CGG CTT GCG TTA 1650 Biomers, Ulm Prom_fwd CCG CGA ATT GTG-3´ ; (SacII) hCD19 Integrated DNA 5´-TGC TTA GGA TCC CAC CCA GCT Prom_rev 1668 Technologies, Coralville TCG CGC AG-3´ ; (BamHI) 2_BamH1 (USA) Integrated DNA 186.2 5´-TTC TTG GCA GCA ACA GCT ACA- 1686 Technologies, Coralville forw 3´ (USA)
55 Materials
Integrated DNA Cmu rev 5´-AAA TGG TGC TGG GCA GGA AG- 1689 Technologies, Coralville outer 3´ (USA) 3-Cg1 5´-GGA AGG TGT GCA CAC CGC TGG Eurofins Genomics GmbH, 1856 outer AC-3´ Ebersberg VH186_ 5´-TCG TCG GCA GCG TCA GAT GTG Integrated DNA Raj_Illu- 1711 TAT AAG AGA CAG TTC TTG GCA Technologies, Coralville mina_forw GCA ACA GCT ACA-3´ (USA) C- 5´-GTC TCG TGG GCT CGG AGA TGT Integrated DNA gamma_ 1713 GTA TAA GAG ACA GCC ARK GGA Technologies, Coralville Univ_Illu- TAG ACH GAT GGG-3´ (USA) mina_rev B-VARIA_ 5´-AAC TAC ATC CTG GTA ATC ATC 1643 Biomers, Ulm 2_FWD C-3´ B-VARIA 5´-CCT TAG GAC CGT TAT AGT TAC 1644 Biomers, Ulm 2_REV G-3´
6.13 Software
Software/Programs Source
FlowJo v10 https://www.flowjo.com/ Geneious6.0 https://www.geneious.com/ GraphPad Prism 6 https://www.graphpad.com/ (Safonova et al. 2015); http://yana- Ig Repertoire Constructor safonova.github.io/ig_repertoire_constructor/ ImageJ https://imagej.net/Welcome IMGT® (ImMunoGeneTics) http://www.imgt.org/ Microsoft Office Microsoft
6.14 Others
Sanger Sequencing of plasmids has been performed by LGC Genomics, Berlin. gBlocks® gene fragments for generation of recombinant Abs has been purchased at Integrated DNA Technologies®.
56 Materials
The Bvaria2 plasmid has been synthesized by Thermo Fisher Scientific.
Molecular Dynamics analyses have been performed by B. Diewald and Prof. Dr. H. Sticht from the Department of Biochemistry, Division of Bioinformatics, Friedrich-Alexander University Erlangen-Nuremberg
Next-generation sequencing has been performed in collaboration with Dr. A. Ensser from the Division of Clinical and Molecular Virology, University Hospital Erlangen
6.15 Antibody panels
To stain GCs, following combination has been used: CD19 BV421, CD38 PE, Fas PE-Cy7, GL7 AF647, CD45.1 ECD, CD45.2 FITC. To analyze NP-specific GCs, CD38 PE was replaced by NP PE. (Template: “Kompetition_GC_neu”)
To stain apoptosis in GC, following combination has been used: B220 PerCP-Cy5.5, Fas PE- Cy7, GL7 AF647, CD45.1 ECD, CD45.2 BV421, NP PE, DEVD FITC. (Template: “Apo”)
To distinguish between DZ and LZ, following combination has been used: B220 PerCP- Cy5.5, CD38 PE, Fas PE-Cy7, CD45.1 ECD, CD45.2 APC, CD86 FITC, CXCR4 BV421. (Template: “Kompetition_DZ-LZ”)
To analyze proliferation, following panel has been used: B220 PerCP-Cy5.5, CD38 PE, PNA FITC, Fas PE-Cy7, CD45.1 ECD, CD45.2 BV421, EdU AF647. (Template: “Kompetition_EdU”) → Caution!! : EdU is overcompensated and has to be set to 0% with FlowJo!
MBCs have been analyzed using CD19 BV421, CD38 bio + SA-PacificOrange, Fas PE-Cy7, NP PE, CD45.2 APC, IgG1 FITC. (Template: “Bmem_Florian Weisel”)
57 Methods
7 Methods
7.1 Restriction digests
All restriction digests were performed with 0.5 -1.5 µg plasmid DNA in a total volume of 20 µl and conditions were adjusted dependent on the enzymes used. Analytic digests were incubated for 40-60 min and analyzed on a 1% agarose gel.
7.2 Synthesis of the Bvaria2 plasmid
A plasmid was synthesized by Thermo Fisher Scientific including a rabbit β-globin intron followed by the Brainbow3.0 cassette into a pMK-RQ backbone. The sequence of the rabbit β-globin intron corresponds to the BamHI – EcoRI fragment of the plasmid pUHG10-3. The sequence of the Brainbow3.0 cassette corresponds to the EcoRI – HindIII fragment of the initial pCAGBrainbow3.0 vector, purchased by www.addgene.org (ID: 45176). The sequence of Brainbow3.0 was changed to get rid of the farnesyl tags, to ensure cytoplasmic expression. Furthermore, a NotI site was added at the end of the cassette, while all other NotI sites were eliminated by adding silent mutations. With this, the NotI site became unique to isolate the fragment from the vector. Codon optimization was performed by Thermo Fisher Scientific. The sequence map of Bvaria2 can be found on the CD delivered by Thermo Fisher Scientific and attached to this thesis.
7.3 Subcloning of the human CD19 promoter
DNA of human PBMCs from a buffy coat was isolated using the DNeasy Blood and Tissue Kit. The human CD19 promoter was PCR-amplified, leading to a 6.4 kbp band (see picture of agarose gel below). The PCR product was purified using the QIAquick PCR Purification Kit. The purified DNA was used for T-A cloning using the Strataclone PCR Cloning Kit. Plasmids were isolated using the QIAprep Spin Miniprep Kit. Restriction digests and Sanger Sequencing (LGC Genomics) were used to verify positive clones. Clone 6 was used for further preparations as it was inserted in the correct orientation during T-A cloning.
58 Methods
Final Component Volume concentration ddH2O 15 µl 5x LongAmp Taq Reaction Buffer 5 µl 1X 10 mM dNTPs 0.75 µl 300 µM 10 µM Forward Primer (TW1650) 1 µl 0.4 µM 10 µM Reverse Primer (TW1668) 1 µl 0.4 µM DNA 1 µl LongAmp Taq DNA Polymerase 1 µl 0.25 U
Step Temperature Time 1 94 °C 30 sec 2 94 °C 30 sec 3 55 °C 1 min 4 65 °C 6 min 5 Back to step 2 30 x 6 65 °C 10 min 7 4 °C ∞
Table 2 and 3: Reagents and PCR conditions used for human CD19 promoter amplification.
7.4 Ligation of Bvaria2 with the human CD19 promoter
1 µg of the Bvaria2 plasmid as well as the plasmid containing the hCD19 promoter was cut with SpeI and NotI-HF in CutSmart Buffer, respectively. The digest was incubated for 1 h at 37 °C. The fragments were cut out from the agarose gel (4.4 kbp and 10.5 kbp, respectively) and purified using the QIAquick Gel Extraction Kit. 33 ng of the Bvaria2 fragment was ligated to 20 ng of the linearized vector containing the hCD19 promoter (vector : insert = 1 : 4), using T4 DNA Ligase and Ligation Buffer. The ligation reaction was incubated for 2 h at room temperature (RT). The reaction was heat inactivated for 10 min at 65 °C. 2 µl of the reaction was used for transformation of One Shot TOP10 chemically competent cells and spread on LB agar plates containing 50 µg/ml kanamycin. The plates were incubated at RT for three days. 15 – 20 colonies grew on each of the four plates. Two clones were used for O/N culture at 30 °C in LB medium containing 100 µg/ml ampicillin. Diagnostic digests and Sanger sequencing confirmed the successful ligation of the hB-varia construct. The plasmid was transformed into One Shot TOP10 chemically competent cells and DNA was isolated using the EndoFree Plasmid Maxi Kit and eluted in 25 µl Endotoxin-free Buffer TE. The concentration was measured using a photometer. 50 µg of the plasmid DNA was digested in CutSmart Buffer using ClaI and NotI-HF (3 U/µg DNA, respectively) in a total volume of 300 µl. After 90 min incubation at 37 °C, the DNA was loaded on a 1% agarose gel. Therefore, fresh and filtered buffers and clean electrophoresis chambers and combs (mid- size; 5 wells) were used. For 100 ml of 1% agarose, 1 µl SYBR™ Safe DNA Gel Stain was
59 Methods used. The sample was distributed over three gel pockets without the use of a DNA ladder, as this might be a source of contamination. In the meanwhile, a dialysis tube was first washed with 100% ethanol, then with water and subsequently stored in 1x TAE buffer. Furthermore, a Qiagen Tip20 column was equilibrated with 1 ml Buffer QBT. The band, containing the hB- varia fragment was cut out from the gel using a scalpel and a blue light lamp and transferred into the dialysis tube (closed with a clipper at one side). Fresh 1x TAE buffer was added and the tube was closed with a second clipper, avoiding air bubbles. The dialysis tube, containing the gel slice, was placed into an electrophoresis chamber, filled with fresh 1x TAE buffer. The gel slice has to be aligned parallel to the chamber, in order to assure an equal elution. The elution was performed at 80 V for ~ 1 h. Afterwards, the polarity of the chamber was reversed for ~ 30 sec, in order to direct the DNA away from the dialysis tube. The elution was checked at blue light. If not at least 80 – 90% of the DNA is eluted, the elution has to be continued. A 5 ml syringe and a 0.9 x 40 cannula were used to transfer the DNA from the tube into a 15 ml tube. For purification, the DNA sample was applied on the equilibrated Qiagen Tip20 column and washed twice with Buffer QC. Then, the fragment was eluted into a 2 ml reaction tube using 800 µl buffer QF and precipitated at full speed in a table centrifuge for 30 min at 4°C using 560 µl (0.7 volumes) of isopropanol. Afterwards, the pellet was washed with 1 ml of 70% ethanol for 10 min at 4°C at full speed and subsequently air-dried for 5 – 10 min. Finally, the pellet was resuspended in 20 µl of sterile injection buffer. The concentration was measured using a photometer. The result has been a yield of 5 µg/µl with A260/280 = 1.59 and A260/230 = 1.94. The purity and concentration of the DNA was verified on a 1% agarose gel. Therefore, 50 and 100 ng of the DNA was loaded and compared to a standardized DNA (here: 1 kb DNA ladder). The concentration of the fragment was compared to the 10 kbp band of the DNA standard. 6 µl of the ladder contain 42 ng of the 10 kbp band. The DNA was used for pronucleus injection to generate transgenic mice expressing multiple copies of the hB-varia construct.
7.5 Testing descendants from pronucleus injections of the hB-varia construct
Biopsies of mice were tested for the inserted transgene using a PCR (see below). Next, blood of positive mice was tested for mOrange2 expression. Therefore, ~50 µl of blood has been stained with the same amount of double-concentrated CD19 Ab (e.g. eFluor660 or BV421) and incubated for 20 min at RT. Next, 1 ml of a 1:10 dilution of the BD FACS™ Lysing
Solution in ddH2O has been added and incubated for 15 min at RT. Samples have been washed at 1200 rpm for 2 minutes and washed another time with 1 ml MACS buffer. Cells have been resuspended in 200 µl MACS buffer and analysed on a flow cytometer. It has to be taken into account that the expression of mOrange2 is significantly decreased due to
60 Methods fixation. Transgenic mice were crossed to C57BL/6 or Mx-Cre mice. Two founders have been established: hB-varia 482 and hB-varia 1441.
Component Volume Final concentration ddH2O 7 µl 2x Bio and Sell Mastermix 10 µl 1X 5 µM Forward Primer (TW1643) 1 µl 0.25 µM 5 µM Reverse Primer (TW1644) 1 µl 0.25 µM DNA 1 µl
Step Temperature Time Table 4 and 5: PCR Screening for the hB-varia 1 94 °C 3 min transgene. 2 94 °C 45 sec 3 52 °C 1 min
4 72 °C 1 min 5 Back to step 2 34 x 6 72 °C 5 min 7 4 °C ∞
7.6 Transfer of splenocytes from transgenic B1-8lo or B1-8hi mice into wildtype CD45.1 recipients
Transfer experiments were conducted under sterile conditions. Therefore, spleens from heterozygous B1-8lo or B1-8hi mice were isolated and suspended in FACS buffer using the stamp of a 2 ml syringe and a 70 µm cell strainer. Splenocytes were centrifuged at 1300 rpm for 7 min. Supernatant was discarded and the cells were resuspended in 5 ml erythrocyte lysis buffer. For efficient erythrocyte lysis, cells were incubated at RT for 5 min. Afterwards, samples were filled up with FACS buffer and filtered using a sterile 50 µm filter. Cell numbers were determined using a Neubauer chamber while the samples were centrifuged at 1300 rpm for 8 min. Pellet was resuspended at a concentration of 8 x 106 cells per 100 µl with PBS. 8 x 106 splenocytes were transferred intravenously into wildtype CD45.1 recipients (Pepboys).
7.7 Generation of bone marrow chimeras
Antibiotics were added to the drinking water of CD45.1 (Pepboy) mice 3-4 days before sub-
-/- lethal irradiation (9 Gy). The following day, BM cells were isolated from CD8 x JHT and 33C9 x Rag-/- mice under sterile conditions. Therefore, BM cells have been washed out from both femurs using a syringe. After suspending the cells, erythrocyte lysis and filtering have
61 Methods been performed as described for splenic cells. BM cells from both strains have been mixed 50:50 to a concentration of 5 x 106 cells / 100 µl. Finally, 100 µl cell suspension was injected i.v. into irradiated CD45.1 (Pepboy) recipients. Antibiotics treatment was continued for another 10 days. 8 weeks after BM transfer, chimeric mice have been used for transfer experiments.
7.8 Immunization with NP29-KLH
NP29-KLH was diluted with PBS to a final concentration of 1 mg/ml and stored at -20°C.
Before immunization, 100 µl (≙ 100 µg) NP29-KLH per mouse were mixed with an equal volume of Alum and incubated at 37°C for 30 min. 200 µl (≙ 100 µg) NP29-KLH in Alum were injected intraperitoneally, one day after transfer.
7.9 Flow cytometric analyses
For flow cytometric analyses, spleens from transferred mice were isolated and suspended using the stamp of a 2 ml syringe and a 70 µm cell strainer. Splenocytes were centrifuged for 7 min. at 1300 rpm and resuspended in 5 ml erythrocyte lysis buffer. For efficient erythrocyte lysis, cells were incubated at RT for 5 min. Cells were washed with FACS buffer, filtered using a 50 µm filter and centrifuged. 2-4 x 106 cells have been resuspended in 100 µl Fc- blocking solution (1:100 in FACS buffer) and incubated for 30 min at 4°C. 100 µl double- concentrated Ab mix was added to the samples and incubated for at least 30 min at 4°C. Afterwards, cells were washed and resuspended in FACS buffer for analysis. Samples were filtered before acquisition on a Beckman Coulter Cytoflex S. Compensation has been performed using Beckman Coulter VersaComp Antibody Capture beads and quality control was performed according to the manufacturer´s protocol.
7.10 EdU staining
To analyze the proliferative capacity of GC B cells, EdU staining was performed to label cells being in S phase of the cell cycle. Therefore, 0.8 mg EdU (stock: 0.8 mg/200 µl) was injected i.p. at the day of analysis. After 2 h, mice have been sacrificed and EdU staining was performed according to the Click-iT™ Plus EdU Alexa Fluor™647 Flow Cytometry Assay Kit. Therefore, splenic cell suspensions have been prepared as described above. After erythrocyte lysis and filtering, staining was performed according to the manufacturer´s protocol. For Ab staining, the Fc-blocking solution was directly added (1:100) to the Ab Mix.
62 Methods
7.11 Active Caspase 3 labeling
Isolation of splenic cells in R10 medium and erythrocyte lysis has been performed as described above. ~3-5 x 106 cells have been resuspended in 300 µl R10 medium. 1 µl FITC- DEVD-FMK from the CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit was added and cells were incubated for 30 min at 37°C and 5%CO2. 200 µl cold R10 medium has been added and cells have been centrifuged at 1400 rpm for 5 minutes. Supernatant was discarded and cells were washed with 500 µl Wash Buffer. Washing step was repeated and cells were resuspended in 100 µl Ab solution containing Fc-block and stained as described above.
7.12 Flow cytometric cell sorting
Spleens from transferred mice were isolated and suspended using the stamp of a 2 ml syringe and a 70 µm cell strainer. Splenocytes were centrifuged for 7 min at 1300 rpm and resuspended in 5 ml erythrocyte lysis buffer. For efficient erythrocyte lysis, cells were incubated at RT for 5 min. Cells were washed with MACS buffer, filtered using a 50 µm filter and centrifuged. Up to 70 x 106 cells were resuspended in 500 µl Fc-blocking solution (1:100 in MACS buffer) and incubated for at least 30 min. 500 µl double concentrated Ab mix was added and incubated at least for another 30 min (note: for sorting, Abs have been used in higher concentrations compared to standard analyses). Afterwards, cells were washed with MACS buffer and resuspended in MACS buffer at a concentration of ~ 40 x 106 cells per ml. Before sorting, cells were filtered again. GC B cells were sorted according to following parameters: CD19+ CD38lo GL7+ Fas+. 105 – 2 x 105 cells were sorted into 350 µl RLT buffer containing 1% β-mercaptoethanol. Cells were stored at -80°C until RNA was isolated.
7.13 RNA isolation and cDNA synthesis of sorted germinal center B cells
RNA of sorted GC B cells was isolated using the RNeasy Mini Kit. RNA was eluted in 80 µl RNase-free water and stored at -80°C. The OneStep RT-PCR Kit was used for cDNA synthesis, following PCR amplification. The forward primer (TW1686) is located in the leader sequence of the VH1-72 segment. Two outer reverse primers were used, which bound to the Cµ (TW1689) and Cɣ (TW1856) region, respectively. Following mix and PCR program was used.
63 Methods
Component Volume Final concentration 5x QIAGEN OneStep RT-PCR Buffer 10 µl 1X 10 mM dNTPs 2 µl 400 µM of each dNTP
10 µM VH1-72 Forward Primer (TW1686) 6 µl 0.6 µM
10 µM Cµ Reverse Primer (TW1689) 3 µl 0.3 µM
10 µM Cɣ Reverse Primer (TW1856) 3 µl 0.3 µM QIAGEN OneStep RT-PCR Enzyme Mix 2 µl Template RNA 24 µl
Step Temperature Time Table 6 and 7: Transcription of RNA from flow 1 50 °C 30 min cytometric sorted GC B cells into cDNA and
2 95 °C 15 min subsequent amplification of VH1-72 expressing 3 94 °C 1 min heavy chains using the One-Step RT-PCR Kit. 4 54 °C 1 min
5 72 °C 1 min
6 Go to step 3 35x
7 72 °C 10 min 8 4 °C ∞
7.14 Next-generation sequencing of germinal center B cells
For next-generation sequencing, PCR-amplified cDNA of heavy chain sequences has been used to add Illumina-specific sequences using the Illumina 5’ (TW1711) and 3’ (TW1713) primer.
Component Volume Final concentration ddH2O 14 µl 5 µM Illumina Forward Primer (TW1711) 2 µl 0.25 µM 5 µM Illumina Reverse Primer (TW1713) 2 µl 0.25 µM NEB 2x Mastermix 20 µl 1X Template DNA 2 µl
Step Temperature Time Table 8 and 9: PCR for adding Illumina-specific 1 94 °C 3 min sequences at the 5´- and 3´- ends of PCR amplified
2 94 °C 1 min VH1-72 sequence. 3 56 °C 1 min 4 72 °C 1 min 5 Go to step 2 10x
6 72 °C 10 min
7 4 °C ∞
PCR products were purified using Agentcourt® AMPure® PCR Purification and eluted in 40 µl Buffer EB. Before pooling the samples for the sequencing run, distinct combinations of
64 Methods index-primer using the Nextera XT Index Kit v2 Set D have been added to the different PCR products via a second PCR to be able to distinguish the different mouse samples in the pool.
Component Volume Final concentration ddH2O 13 µl 5 µM Index Primer A 1 µl 0.25 µM 5 µM Index Primer B 1 µl 0.25 µM Bio and Sell 5x PCR 4 µl 1X Mastermix Template DNA 1 µl
Step Temperature Time Table 10 and 11: PCR for adding index primer to 1 94 °C 3 min distinguish different samples in the pooled samples. 2 94 °C 1 min 3 55 °C 1 min 4 72 °C 1 min 5 Go to step 2 8x 6 72 °C 10 min 7 4 °C ∞
5 µl of the PCR products were analyzed on a 1.5% agarose gel. According to the intensity of the PCR product on the agarose gel, the samples were pooled, adjusting the amount of DNA for each sample to be equally contributing to the total pool. Finally, the samples were purified using Agentcourt® AMPure® PCR Purification, eluted in 40 µl Buffer EB and analyzed using an Illumina MiSeq Platform. Reads were merged using Ig Repertoire Constructor. Cleaned reads were uploaded to IMGT High V-Quest. Filters were used to distinguish sequences derived from B1-8lo and endogenous GC B cells (Figure 8A).
7.15 Immunofluorescent staining of frozen tissue
After spleens have been isolated from transferred mice, ~ one third has been embedded in Tissue-Tek® O.C.T™ Compound and stored at -80°C. For immunofluorescent analyses, the spleens were sliced into 8 µm thick sections. Tissue sections have been fixed in acetone for 10 min at -20°C. Afterwards, the samples have been air-dried for 15 min, marked with Roti®- Liquid Barrier Marker and stored at -20°C. For staining, slides have been warmed-up at RT for 15 min and rehydrated for 5 min in PBS. To prevent unspecific signals, each section has been incubated with 60 µl blocking solution for 30 min at RT in a dark wet chamber. The blocking solution was discarded and 60 µl Ab mix in staining solution was added. After 30 min incubation at RT in a dark wet chamber, the slides have been dipped in PBS, followed by two washing steps in washing buffer for 5 min, respectively. Streptavidin conjugated to a
65 Methods fluorescent dye has been added and incubated for another 30 min to stain biotinylated primary Abs. Washing steps have been repeated as described, adding a final 5 min PBS wash. The slides have been carefully wiped with Kimtech wipes and embedded in 2-3 drops of Roti®-Mount FluorCare. Sections have been analyzed using a Zeiss Axioplan 2.0 microscope and ImageJ software.
7.16 Molecular Dynamics
Simulations and analyses of Molecular Dynamics have been performed by B. Diewald and Prof. Dr. H. Sticht from the Department of Biochemistry, Division of Bioinformatics, Friedrich- Alexander University Erlangen-Nürnberg.
7.17 Mouse genotyping
Component Volume Final concentration ddH2O 7 µl 5 µM Fwd. Primer 1 µl 0.25 µM 5 µM Rev. Primer 1 µl 0.25 µM Bio and Sell 2x PCR 10 µl 1X Mastermix Bio and Sell Taq DNA 0.1 µl 0.025 U/µl Polymerase Template DNA 1 µl
Table 12: PCR mix used for all mouse genotypings.
Strain Primer TW number Length B1-8lo and B1-8hi B1-8 forward TW1614 500 bp wildtype forward TW1616 337 bp common reverse TW1615 hB-varia 482 and hB-varia 1441 hB-varia forward TW1643 265 bp h-Bvaria reverse TW1644 Mx-Cre Mx-Cre forward TW1636 500 bp wildtype reverse TW1510 Rosa26 Cre-ERT2 Cre-ERT2 forward TW1509 265 bp Cre-ERT2 reverse TW1510 Rosa26 Rainbow2.1 Rainbow2.1 forward TW1596 300 bp Rosa26 forward TW1635 386 bp Rosa26 reverse TW1597
Table 13: Primers used for genotyping of different mouse strains.
66 Methods
B1-8lo Temp. h- Temp. Mx- Temp. Step Time Time Time B1-8hi [°C] Bvaria [°C] Cre [°C] 1 95 2 min 94 3 min 95 2 min 2 95 15 sec 94 45 sec 95 15 sec 3 65 1 min 52 1 min 54 1 min 4 72 30 sec 72 1 min 72 1 min 5 Go to 2 35x Go to 2 35x Go to 2 35x 6 72 2 min 72 5 min 72 5 min 7 4 ∞ 4 ∞ 4 ∞ Cre- Temp. Rain- Temp. Step Time Time ERT2 [°C] bow2.1 [°C] 1 95 2 min 94 3 min 2 95 15 sec 94 30 sec 3 51 45 sec 54 1 min 4 72 45 sec 72 1 min 5 Go to 2 33x Go to 2 35x 6 72 5 min 72 5 min 7 4 ∞ 4 ∞
Table 14: PCR programs used for genotyping of different mouse strains.
67 Attachment
8 Attachment
8.1 Sequence of the h-Bvaria construct
Following color code is used: hCD19 Promoter / rabbit β-globin intron / Brainbow3.0
GGCTTGCGTTACCGCGAATTGTGGCGGGGCCCTCCCTGCACCTCCGCCCTCTTGGAGC GTCCCTCGGGGCAGGGCCGGGTCGGCTTCATTTTTCTGAGCGCCTCGGAGTCCCCGC GTAGTTAGTCGGAGTCTGAAACTTGCACGTCCCCACCCCCATTTTGTTACTCAGCCAGT CACCGCTTCCTGCTGGCCCCCCTTTCCTCCTCCCAACGCCCTCAAATCCCTCCTCTCCT CTCCGCCTGTGTCTAGATCAGGCTCTTTATCTGTGACCTCCACCCGGACGGTGACAGG CAATTCAGATTTATGTCCAGAGCCTTGGTTCCCCTTCCCTCAAACCTTTTTTCCCTCTGT AAAGGACACGCCAGGAACCTGGAAGTCCTTGATTCTACGTGTCCTCCTGCAAGTCCCGT AAACTCTACTTCCAATGTGCTGACTATTCCAGAATGTGCCCCTTTTTCTCCATCTCCAGC GCTAACACTCCGTCCCAGCTCCCATCACTTCTCCTGAACTTCCGCAGTGTCGACTCCAC CCTTTAATTAATTAATTCTCCGTAAAGCAGCTGGAGCGATTTTTGTAAAACAGATAATGCA GTTTGAAAATGGCCAAAGGTAGAACGCTTTTACACTGTTGGTGGGAATGTAAATTAGTTC AACCATTGTGGAAGACAGTGTGGTGATTCCTCAAAGACCTAGAACCAGAAATACCATTT GACCCAGAAATCCCATTACTGGGTATATACCTAAAGGAATATAAATCATTCTATTACAAA GATACATGCACGAGTATGTTCATTGAAGCACTATTCAAAATAGCAAAGACATGGACTCAA CCCAAATGCCCATCAATGATAGACTGGATAAAGAAAATGTGGTACATATACACCATGGAA TACTATACAGCCATAAAAAGGAACGAGATCATGTCCTTTGCAGGGACATGGATGAAGCT AGAAGCTATTATCCTCAGCAAATTAATGCAGCAACAGAAAGCTAAACATGCATGTTCTCA CTTATAAGTGGGAGCTGAACAATGAGAACACATGGACACAGGGAGGGGACCAACACAC ATGGGGGCCTGTGGGGTGGGTGTTGGGAGGGGGGAGGGAGAGCATCAGCAAGAGTA GCTAATGCATCCTGGGCTTGATACCTAGGTGATGGGTTAATGGGTGCGGTAAACTACCA TGGCAGATGTTTCCCTGTGTAACAAACCTGCACATCGTGCACATGTACCCCAGAACTTG AAATAAAGAAAATGGACAAAGGATATGAACGGATATTTCACTGCTGTCACAGGTGGCAA TATGTGCATGAAAAGATGTTAAACATCATTAGCTATTTATTCGGGAAATACAAATTAAAAC CACAATTGCTTTCATCACTACGCACTTCTTGGAATGGCTAAAATAAGAATGCTAACACCA AATGCTGGTGAGTATGTGGAGAAACTAGGTTTCTCATATTTTGTTGATGGGAATGTGAAA TGGTATAGCCACTCTGCAAAAAGAGTATGGCAGTTTTGAACAAAACTAAACACGCACTTA CTTTTTCATCAAGTATAAAACCAAGTGTTTTCCACCAAGCTTGGAGCTGAGGTCTCGGCT CAAATGTTACCTCCTCAGTGAGGCCTTCCCTGATCACCCTAACTAAAATAACCACCTCTG GCTGAGTGTGGTGGCTTATGCCTATAATGCTAGCACTTTAGGAGGCCAATGGCAGGAG GATCACTTGAGCCCAGGGGTTCGAGACCAGCCTGAACAACATAGTGAGACCTCATCTCT ACAAAAAATTAAAAAATTAGCTGGGCATGGCCGGACACGGTGGCTCACGCCTGTAATCC CAGCACTTTGGGAGGCTGAGGAGGGCGGATCACGAGGTTAGGAGATCAAGACCATCCT
68 Attachment
GGCTAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCGGGCATGGTG GCATGCGCCTATAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCC GGGAGGCAGAGGTTGCAGTGAGCTGAGATCATGCCATTACACTCCAGCCTGGGCAGCA GAGCAAGACTCTGTCTCAAAAAAAAAAAAAAATTAGCTGGGCATGGTGATGTGCATCTG TAGTCCAAGCTACTCAAGAGGCTGAGGCGGGAAGATCTACTTGAGCCCTGGAGGCTGA GGCTGCAGTGAGCATGATTGTGCCACTGCACTCCAGCCTGAGGGAGAGAAAGACCCTG TCTAAAAAAAAAAAAAAAAAATTAAATCCAGACTCCCTGTGCATGCAGCAGGGTTTCTGG GGAAGGGGCTGAAATAATGACTAGAAATCAGCCTCCAGTCAGTTTGGGGAGGGCCTTC CTGACAGCTCTCAGGTTTGAGTTTATCCTGCAGGCCCTGGGAGCCACTGAAGGCCATT GAACAGGGGAAGCACATGACTAACATCAAAGAAGTTCTCTTGACATGCGGAGTGTAGAT GGGAACCAAAAGATATAGGGGACAGGGAGCCCAGGAGAGAGGCTGTTGAATTGCTTCT GATTGTGAAGGAAGGATTCAGAGGGGAAAATTGATTCCATCAATATTTAGGATATAAAAT GGACTGGCCTTGATGATAGAGTGCATGTGGGGGAAGAGAAAAGAAAATTTCGACCAGA TGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGTTGGGTGGATCACC TGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCTTGTCTCTACTAAAA ATAGAAAAATTAGCTGGGCGTGGTGGTGCGCACCTGTAATCCCAGCTACTCAGGTGGC TGAGGCAGGAGAATCACTTGAACCTGGAAGGCAGATGTTGCAGTGAGCCGAGATCGCA TTACTGTACTCCAGCCTGGGCAACAAGAGTGAAACTCTGTCTCAAAAAAAAAAAAAGAAA AGAAAATTTTCTTTCAGCTAGGTACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGA GGCTGAGGCAGGAAGATTGCCTGGGCCCAGGAGTTCAAGACCAGCCTGGGCAATATAG TGAGAAAGGAGAATAAAAGGAGCTTTCCTCGCCTCTACAAAAAAAGGGAGAGAGTTTTC TTCCTAACTTCTTTTCCTTTTTTTTTTTTTTTGAAATGCTCTTGTCTCCCAGGCTGGAGTG CAATGGCGCGATCTTCACTCACTGCAACCTCCGCCTTTCAAGTTCAAGCGATTCTCCTG CTTCAGCCTCCCAAGTAGCTGGGATTACAAGCGCGCACCACCGCACCAAGCTAATTTTT GTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGCCTTGAACTCCTGAC CTCAAATGATCCGCCCCGCTCAGTCTCTCAAAGTGCTGGGATTACATGAGCTACCTTGC CTGGCCCCTAACTTCTTTTCTTTCCTTTATCCAGCAAATACCTATTGAGTGTCTACTAGAT GCCTGTGCACATCTAGGGATGGTAAAGATTTCTGTTCTCAAAGAACATTCTGGTGCAGG AGGCAGATGAGAAGACTAGCAAATACACATGCATAATTTGAGATTGGGGCAAGTGCTGG GAATGAAATAGGGGCATGCGATCAAAAAAGCATGTGAGTAAAGGGAATTTGAGAAGGTT TTCTTTTTCTTTTTTCTTTTCTTTTCTTTCTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGACT CTGTCATCCAGGCTGGAGTGCAAAGGTGCAATCTCGGCTCACTGCAACCTCTGCCTCC CAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTATAGGCATGAGT TAATTTTTTTGTATTTTTAGTAGATACTGGGTTTCACCATGTTGGCCAGGCTGGTCTCATT CTCCTGACCTCAAGTAATCCACCCGCCTCAGCCTCCCAAAGTGCTGAGATTACAGGCAT GAACCACTGTGCCCAGTCTTTTTTTCATATTTTTTGTAGAGATGGTGTTTCACCATGTTGC CCAGGCTAGACTTGAACTCCTGGGCTCAAGTGATCCTCCCACCTCAGCCTCCTGAGTAG
69 Attachment
CTGAGACTACAGGTATGCACCATCATGCCCAGATACATTTTTTTTGGTATTTTTAGTAAA GACGGATTTCTCCATTTTGCCCAGGCTGGTTTCGAACTCCTGGGCTCAAATGATCTGCC CACCTGGGCTTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCCGAG ATATATATATATATATATATATATATATATATATATATATATATATATAGAGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGAGAGAGAAAGAAAGAGAGCAGGCTCTTGTTGCCCAGG CTGGAGTGCAGTGGTGCCATCTTGGCTCACTGCAACCTCCATCCCCCTGATCCTCCCG CCTCAGCCTCCCAAGTAGCTGGGACCACAGGCATGTGCCACCACACCCAGCTAATTTTT TGTATTTTTGGTAGAGATGGGTTTTGCCATATTGCCCAGGCTGGTCTCCAACTCCTGAG CACAAGCAATCTGCCTGCCTTGGCCCCACAAAGTGCTGGGAGGTGTGAGTCACCTCGC CTGGCCTGAGATATATTTTTTTAAATAATAATATATAGATTTTTGAAGCACTGGGGGGCA CAGGGAAGGCCTCTTTGAAGAAGTACCATTTGAACTGAGACCTAAGTGATGAAAAGAAC CAGTCCAGCAAAGAACCAGAGCCCCCAGCAGAGATCCAGGAAAAAGGGCTGCAAGTGC AAGGGCCCTGAGGCAGGGAAGCACTTGGCAAGGAGAGTGGTGGAGGCACGAGGTGGA AAATGTAGGTAGGTCAGCAACACTCGGCCTACTAGGCCTTGGGTTGGAGTTTTTATTTTA GCTAGATGAAAAGCAACTGACATTTTTTGTTTTTTAAAAAATTTCTATAGAGATGGGTTCT CGCTGTGTTGCACAGGCTGGTCTCAAATTCCTGTCCTCAAAGGATCCTCTCGCCTCGGC CTCCTAAAGTATTGGGATTACAGGCATGAGCCTCTGTGCCTGGCTGTAACTGACATGTT TTAAGCAGGGGAATGACATGCTCTAGTGAAAGCCAGTCTGGGCAGCTGGGTAGCTAAT GAGGGGATTAGAGAGATTTTGTTGAATGAAAGGCAGATTGAGTCCTGCTACTCGCCCCC TTCATTCCCCTTCATTCATGCCTCATTCTTCCGCCTCCCAGCCGCCTCAACTGGCCAAA GGGAAGTGGAGGCCCTGCCACCTGTAGGGAGGGTCCCCTGGGGCTTGCCCACAGCAA ACAGGAAGTCACAGCCTGGTGAGATGGGCCTGGGAATCAGCCACTGAGAAAGTGGGTC TCTTGGGTCCCTGAATTCTTTTTCTGAGTCCCTGCAGCAGTGAAAAAGACACAGAGGCA CATAGAGAGTGACAGAGAAAGAGAGAGACAGAGAGGAGAGGCATGGGGCAGAATAAG AACAGATTTAGGAGTTAGAACTCCTGGGTTCTTTTAAAACAATTTTTCTTTTAGAGACAGG GTCTTGTTGTGTTGCCCGGACTGGAGCACAGTGGCTATTCCCAGGCATAATCATGGTGC ACTGCAGCCTTGAACTCCTGGGCTCAAGCGATCCTTCTACCTCAGCCTCCCAAGGACCT GGGACCATAGGCGTGTACCACTGTGCCTGGCTTTTGCCTGGTTTTAAACTGAGGCAGTA TGACTTGAGCTCTTAGGCATTAATTGAAGCTGTATCTCATTAACTGAGGGCTTATGATGT GCTGGACACTGGGCTAATAGTGCTGAACATATTGTCATTTTTAATCTTCACAAACAATATT TGTATAGGACTGTTTTCTTTTCTTTTTTTTTTTTGAAACAGAGTCTCACTCTGGTGCCCAG GCTGGAGTGCAGTGGTGTGATCTCGGCTCACTGCAACCTCCGCCTCCTGGTTTCCAGT GATTCTCCTGCCTCAGCCTCCTAAGTAGCTGGGATTACAGGTGTGCGCCACCATGCCC GGCTAATTTTTTTTTTTTTTTTTGAGAAGGAGTCTATGTGCCCAGCATTGTTCTAGAGCAC TTGCAATTAGTGGTGAACAACACGGTCTCTACTCCAAGGGGCTCACATTCTTGTGCAGA AAACAGAAATGAACAAATAAACACACAAGATCATTTCCCGTGGTAGTGAGAGCTGGGAT GAAAATAAAACAGCGTGGCAGGGAGGAGGCAAGTGTTGTGAGTCTGGAGGGTTCCTGG
70 Attachment
AGAATGGGGCCTGAGGCGTGACCACCGCCTTCCTCTCTGGGGGGACTGCCTGCCGCC CCCGCAGACACCCATGGTTGAGTGCCCTCCAGGCCCCTGCCTGCCCCAGCATCCCCTG CGCGAAGCTGGGTGGGATCCTAAGCAAAGGGCGAATTCCACATTGGGCTGCAGCCCG GGGGATCCACTAGTTCTAGAGCGGCCGCACCGCGGTGGCGGCCGCTCTAGAACTAGT GGATCCTCTTGACTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTT TCGCTATTGAAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAA TGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCT ACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAA ACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTCGTTTATTTGTCAGATTGTAAG TACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTAATTATATTGTACTTCAGCACA GTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCT GGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACATCCTGGTAATCATCCTGCCT TTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCA AACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGC AACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAGGATATCACA AGTTTGTACAAAAAAGCAGGCTTTAAAGGAACGCACGTAACTATAACGGTCCTAAGGTA GCGAACCTAGGATAACTTCGTATAGCATACATTATACGAAGTTATCGGCGCGCATAACTT CGTATAGGATACTTTATACGAAGTTATCAAGCGCTGCCACCATGGTGAGCAAGGGCGAG GAGAATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCT CCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAG GGCTTTCAGACCGCTAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGG ACATCCTGTCCCCTCATTTCACCTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGA CATCCCCGACTACTTCAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATG AACTACGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGC GAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTGA TGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACG GTGCCCTGAAGGGCAAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACA CCTCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCT ACATCGTCGACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAA CAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG AAGCTGAACCCTCCTGATGAGAGTGGCCCCGGCTGCATGAGCTGCTGTGTGCTCTCCT GAGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGC TTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGT TGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTC ACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTAT CTTAAGGCGTGCTAGCATAACTTCGTATAGCATACATTATACGAAGTTATCTAACGTTGC CACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA
71 Attachment
GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGA TGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAG TTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACG GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGA CCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGAT CACTCTCGGCATGGACGAGCTGTACAAGAAGCTGAACCCTCCTGATGAGAGTGGCCCC GGCTGCATGAGCTGCTGTGTGCTCTCCTGAGCGGCCGCGACTCTAGATCATAATCAGC CATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAAC CTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTT ACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAG TTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGTTTAAACATATAACTTCGTAT AGGATACTTTATACGAAGTTATCGCGTACGGCCACCATGGTGAGCGAGCTGATTAAGGA GAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGC ACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGCG GTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACG GCAGCAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTC CCCGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACC GCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAG GGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGC CTCCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGC CCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCC AAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGG AAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGG CCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAGAAAGCTGAACCCTCCTGATGA GAGTGGCCCCGGCTGCATGAGCTGCTGTGTGCTCTCCTGAGCGGCCGCGACTCTAGAT CATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCT CCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGC TTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTACTAGTGAAG TTCCTATTCTCTAGAAAGTATAGGAACTTCCGTAACTATAACGGTCCTAAGGTAGCGAAT GCATCTAGACCCAGCTTTCTTGTACAAAGTGGTGATTCGCGGCCGCACTCCTCAGGTGC
72 Attachment
AGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCA CTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCT GACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGT CTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTT GGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAA AGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCC TTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTA AAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCA TAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTATCGATACC GTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATT GCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG TGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTG TCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG GGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCT CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATC GCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTAT CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGAT CCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCT GACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATA GTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT
73 Attachment
GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC GGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAG AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCAT CTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCG TTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAG GCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGT TGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGC GAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTT TTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTA GAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAG GAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACAC CCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAA CTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGT TGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGC TCCACCGC
This sequence is based on an assembly product using Geneious6.0. The complete sequence can also be found in Geneious6.0 using following directory:. This sequence is NOT based on sequencing of the construct. The construct has been partially sequenced, which can be found using the following directory in Genious6.0: “Andi” → “Klonierung und Zwischenprodukte hCD19Bvaria” → “5 Complete Sequence (hCD19Prom + betaglobin intron + BB3.0 Reference Assembly). The files from the sequencing results are named: “hCD19PromBVARIA_Lig8_Klon20.Seq-neu-fwd2”, “hCD19PromBVARIA_Lig8_Klon20.Seq- neu-rev2”, “hCD19PromBVARIA_Lig8_Klon20.Seq-neu-rev3”, “Lig8_Klon20.TW1644”, “Lig8_Klon20.Seq-new-fwd”
74 Attachment
8.2 Sequence of the synthetic Bvaria2 construct
This plasmid has been purchased from Thermo Fisher Scientific. Following color code is used: rabbit β-globin intron / mOrange2 / EGFP / mKate2]
CACTATAGGGCGAATTGAAGGAAGGCCGTCAAGGCCGCATTAAGCAACTAGTGGATCC TCTTGACTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTA TTGAAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAA GATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTG TTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTT AGCTTGCATTTGTAACGAATTTTTAAATTCACTTTCGTTTATTTGTCAGATTGTAAGTAC TTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTAATTATATTGTACTTCAGCACAGT TTTAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTG GCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACATCCTGGTAATCATCCTGCCTT TCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCA AACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGC AACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAGGATATCACA AGTTTGTACAAAAAAGCAGGCTTTAAAGGAACGCACGTAACTATAACGGTCCTAAGGTA GCGAACCTAGGATAACTTCGTATAGCATACATTATACGAAGTTATCGGCGCGCATAACTT CGTATAGGATACTTTATACGAAGTTATCAAGCGCTGCCACCATGGTTTCCAAGGGCGAA GAGAACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAAGTGCGGATGGAAGGCA GCGTGAACGGCCACGAGTTTGAGATCGAAGGCGAAGGCGAGGGCAGACCCTACGAGG GATTTCAGACCGCCAAGCTGAAAGTGACCAAAGGCGGCCCTCTGCCTTTCGCTTGGGA CATCCTGTCTCCACACTTCACCTACGGCAGCAAGGCCTACGTGAAGCACCCTGCTGACA TCCCCGACTACTTCAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGAGAGTGATGAA CTACGAGGACGGCGGCGTGGTCACAGTGACACAGGATAGTTCTCTGCAGGACGGCGA GTTCATCTACAAAGTGAAGCTGAGGGGCACAAACTTCCCCAGCGACGGCCCTGTGATG CAGAAAAAGACCATGGGCTGGGAAGCCAGCAGCGAGAGAATGTACCCTGAGGATGGC GCCCTGAAGGGCAAGATCAAGATGAGACTGAAGCTGAAGGATGGCGGCCACTACACCA GCGAAGTGAAAACCACCTACAAGGCCAAGAAACCCGTGCAGCTGCCTGGCGCCTACAT CGTGGATATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAACAG TACGAGAGAGCCGAGGGGAGACACTCTACAGGCGGAATGGACGAGCTGTACAAATGAG CTGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTA AAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTT AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA AGGCGTGCTAGCATAACTTCGTATAGCATACATTATACGAAGTTATCTAACGTTGCCACC ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG
75 Attachment
GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT GGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCG AGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACG GCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATG GCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCC CAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACT CTCGGCATGGACGAGCTGTACAAGTGAGCTGCCGCGACTCTAGATCATAATCAGCCATA CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGA AACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAA ATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTGTTTAAACATATAACTTCGTATAGG ATACTTTATACGAAGTTATCGCGTACGGCCACCATGGTGAGCGAGCTGATTAAGGAGAA CATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACAT CCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGCGGTCG AGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAG CAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCCG AGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTA CCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGT GAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTCC ACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTG AAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCCAAGA AACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGGAAAG AATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAG ATACTGCGACCTCCCTAGCAAACTGGGGCACAGATGAGCTGCCGCGACTCTAGATCAT AATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCC CCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGC ATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTACGAGTGAAGTTCC TATTCTCTAGAAAGTATAGGAACTTCCGTAACTATAACGGTCCTAAGGTAGCGAATGCAT CTAGACCCAGCTTTCTTGTACAAAGTGGTGATTCGCTGCCGCACTCCTCAGGTGCAGGC TGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGA GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACT
76 Attachment
TCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCT CACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTT AGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGG TCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGAC TTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATT TTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCT GTCCCTCTTCTCGCGGCCGCTAAGCACTGGGCCTCATGGGCCTTCCTTTCACTGCCCG CTTTCCAG
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87 List of abbreviations
10 List of abbreviations
10.1 Abbreviations
A Adenine AA Amino acid Ab Antibody AF Alexa Fluor AFC Antibody-forming cell Ag Antigen AID Activation-induced cytidine deaminase Ala Alanine APC Allophycocyanine APE1 Apurinic / apyrimidinic endonuclease 1 Arg / R Arginine BBS BES-buffered saline Bcl6 B cell lymphoma 6 BCR B cell receptor BER Base excision repair BES N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid bio biotin BM Bone marrow bp / bps base pair / base pairs BSA Bovine serum albumin BV Brilliant Violet™ C Cytosine
Cα IgA constant region CCL C-C motif ligand CCR C-C motif chemokine receptor CD Cluster of differentiation
Cδ IgD constant region cDNA copy DNA
CDR Complementarity-determining region
Cγ IgG constant region
CGG Chicken gamma globulin
Cε IgE constant region
Cµ / Cmu IgM constant region
88 List of abbreviations
CSR Class switch recombination CXCL C-X-C motif ligand CXCR C-X-C chemokine receptor Cy Cyanine Cys Cystein ddH2O Double-distilled water
DH Diversity segment heavy chain DMEM Dulbecco´s modified eagle medium DMSO Dimethyl sulfoxide DN Double-negative DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate DP Double-positive DPBS Dulbecco´s phosphate-buffered saline ds Double-stranded DZ Dark zone EBI2 Epstein-Barr virus-induced G-protein coupled receptor 2
EC50 Half-maximal effective concentration EDC N-ethyl-N’-(3-diethylaminopropyl) carbodiimide EDTA Ethylenediaminetetraacetic acid EdU 5-ethynyl-2'-deoxyuridine ELISA Enzyme-linked immunosorbent assay ELISpot Enzyme-linked immunospot ERT Triple-mutant estrogen receptor FACS Fluorescence-activated cell sorting Fc Fragment crystallizable FCS Fetal calve serum FDC Follicular dendritic cell FITC Fluorescein isothiocyanate FMK Fluoromethyl ketone FR Framework region FSC Froward scatter Fv Fragment variable fwd Forward G Guanine GC Germinal center GFP Green fluorescent protein
89 List of abbreviations
Glu / Q Glutamine Gly / G Glycine h / hu Human H-bond Hydrogen bond HEK Human embryonic kidney HEPES Hydroxyethyl piperazineethanesulfonic acid His / H Histidine HRP Horseradish peroxidase IC Immune complex ICOS Inducible T cell costimulatory IF Immunofluorescence Ig Immunoglobulin IgH Immunoglobulin heavy chain IL Interleukin IMGT Immunogenetics i.p. Intraperitoneally i.v. Intravenously
JH Joining segment heavy chain
JL Joining segment light chain
KA Association constant Kbp / kbps Kilobasepairs
KD Dissociation constant KLH Keyhole limpet hemocyanine LB Lysogeny broth LC Light chain Leu / L Leucine LLPC Long-lived plasma cell LN Lymph node LZ Light zone m / mu murine MACS Magnetic associated cell sorting MBC Memory B cell MHC Major histocompatibility complex MMR Mismatch repair mRNA messenger RNA MSH 2/6 MutS protein homolog 2/6 NGS Next-generation sequencing
90 List of abbreviations
NHEJ Non-homologous end-joining NHS N-hydroxysuccinimide NIP 4-Hydroxy-3-iodo-5-nitrophenylacetyl hapten NP 4-Hydroxy-3-nitrophenylacetyl hapten ODP o-Phenylenediamine dihydrochloride O/N Overnight OVA Ovalbumin p Plasmid PBMC Peripheral blood mononucleated cell PBS Phosphate-buffered saline PC Plasma cell PCR Polymerase chain reaction PDB Protein data bank PE Phycoerythrin PerCP Peridinin chlorophyll protein PD-L2 Programmed cell death ligand 2 PNA Peanut agglutinin PNI Pronucleus injection Prom Promoter PS Polystyrol Q / Glu Glutamine R Replacement R / Arg Arginine RAG 1/2 Recombination activating gene 1/2 Raj Rajewski rev Reverse RNA Ribonucleic acid rpm Rounds per minute RPMI medium Roswell memorial park institute medium RSS Recombination signal sequence RT Room temperature RT-PCR Reverse transcription PCR S Silent SA Streptavidine SD Standard deviation SDF-1 Stromal cell-derived factor 1 SDS Sodium dodecyl sulfate
91 List of abbreviations
SE Standard error Ser / S Serine SHM Somatic hypermutation SOC Super optimal broth with catabolite repression SP Single positive SPR Surface plasmon resonance SSC Side scatter Strata Strataclone ss Single-stranded T Thymine TAE Tris acetate EDTA TCR T cell receptor TdT Terminal deoxynucleotidyl transferase Temp. Temperature Tfh T follicular helper cell Thr / T Threonine TMB 3,3',5,5'-Tetramethylbenzidine Trp / W Tryptophane TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling UNG Uracil-DNA glycosylase Univ Universal
VH Variable segment heavy chain
VL Variable segment light chain W / Trp Tryptophane Y Tyrosine
10.2 Units
Å Ångström °C Degree Celsius cal Calory g Gramm Gy Gray h Hour l Liter m Meter M Molar min Minute
92 List of abbreviations mol Mol sec Second U Units V Volt
10.3 Prefix symbol
Cc / 2 cubic K Kilo µ Micro m Milli n Nano
10.4 Greek letters
α Alpha β Beta γ Gamma δ Delta ε Epsilon ζ Zeta η Eta θ Theta κ Kappa λ Lambda
93 List of publications
11 List of publications
Weisenburger T, von Neubeck B, Schneider A, Ebert N, Schreyer D, Acs A, Winkler TH. Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA. Front Immunol. 2018 Jul 5;9:1551. doi: 10.3389/fimmu.2018.01551. eCollection 2018.
Zhang Y, Tech L, George LA, Acs A, Durrett RE, Hess H, Walker LSK, Tarlinton DM, Fletcher AL, Hauser AE, Toellner KM. Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells. J Exp Med. 2018 Apr 2;215(4):1227-1243.
Rakhymzhan A, Leben R, Zimmermann H, Günther R, Mex P, Reismann D, Ulbricht C, Acs A, Brandt AU, Lindquist RL, Winkler TH, Hauser AE, Niesner RA. Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo. Sci Rep. 2017 Aug 2;7(1):7101.
Müller J, Lunz B, Schwab I, Acs A, Nimmerjahn F, Daniel C, Nitschke L. Siglec-G Deficiency Leads to Autoimmunity in Aging C57BL/6 Mice. J Immunol. 2015 Jul 1;195(1):51-60.
94 Danksagung
12 Danksagung
An dieser Stelle möchte ich mich ganz herzlich bei allen bedanken, die mich in den letzten Jahren beim Erstellen dieser Arbeit unterstützt und dabei mitgewirkt haben.
Ganz besonders danken möchte ich...
… Herrn Prof. Dr. Thomas Winkler für die Betreuung dieser Doktorarbeit, welche meine Kenntnisse der Immunologie, insbesondere der Affinitätsreifung von Antikörpern, um ein Vielfaches bereichert hat. Ganz besonders möchte ich mich für die vielen fruchtvollen Diskussionen, Anregungen und Ideen bedanken, die mich immer wieder motiviert haben.
… Herrn Prof. Dr. Lars Nitschke, der mir als Zweitbetreuer ebenfalls ermöglicht hat meine Promotion am Lehrstuhl für Genetik zu bearbeiten und mich in meinem Vorhaben unterstützt hat.
… der AG Winkler, welche ich sehr vermissen werde. Vielen Dank für die so angenehme Arbeitsatmosphäre, die Hilfsbereitschaft und die Möglichkeit das manchmal frustrierende Doktorandenleben mit viel Humor erleben zu dürfen.
… meinen fleißigen Bachelor-/Masterstudenten Miriam Bittel und Paul Haase, die einen großen Beitrag geleistet und somit zu meiner Arbeit beigetragen haben.
… Prof. Dr. Heinrich Sticht und Benedikt Diewald vom Institut für Biochemie, welche sich, unter anderem, mit den „Molecular Dynamics“ Daten zum B1-8 System befassen und uns einen interessanten und sehr wichtigen Einblick in das molekulare Verständnis von anti-NP Antikörpern gegeben haben.
… Dr. Thomas Simon, der ein unfassbar großes Wissen über die murine anti-NP Immunantwort hat und uns in Diskussionen wichtige Denkanstöße geliefert hat.
… allen weiteren Mitarbeitern aus anderen Arbeitsgruppen, insbesondere der AG Nitschke, mit denen ich nicht nur labortechnisch viel Zeit verbracht habe und der AG Jäck, mit denen wir das ein oder andere Fest geschmissen und (mehr oder weniger) tiefsinnige wissenschaftliche Diskussionen geführt haben.
… meiner Familie, insbesondere meinen Eltern, die mir mein Studium ermöglicht haben, meiner Schwester, die mir schon in frühen Jahren vieles beigebracht hat und meiner Nichte, die es immer schafft mir Freude zu bereiten.
… meiner Verlobten, die stets an mich glaubt und mir die Kraft gibt auch schwierige Situationen zu meistern. Danke.
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