The function of Krüppel-like factor 2 in B cell development [Elektronische Ressource] = Die Funktion von Krüppel-like Faktor 2 in der B-Zellentwicklung / vorgelegt von Rebecca Winkelmann geb. Heidbüchel

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The function of Krüppel-like factor 2 in B cell development Die Funktion von Krüppel-like Faktor 2 in der B-Zellentwicklung Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Rebecca Winkelmann geb. Heidbüchel aus Kleve Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 17. 12. 2010 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Hans-Martin Jäck Zweitberichterstatter: Prof. Dr. Falk Nimmerjahn 2 Table of contents Table of contents 1 Zusammenfassung ............................................................................. 1 2 Summary ............................................................................................. 2 3 Introduction......................................................................................... 3 3.1 B cell development and homeostasis................................................................ 3 3.1.1 Central B cell development................................................................................3 3.1.2 Peripheral B cell development...........................................................................6 3.1.3 Plasma cell generation and migration..
Publié le : vendredi 1 janvier 2010
Lecture(s) : 24
Source : D-NB.INFO/1010641980/34
Nombre de pages : 100
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The function of Krüppel-like factor 2 in B cell development

Die Funktion von Krüppel-like Faktor 2 in der B-Zellentwicklung










Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.











vorgelegt von
Rebecca Winkelmann geb. Heidbüchel

aus Kleve





Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg

















Tag der mündlichen Prüfung: 17. 12. 2010

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Hans-Martin Jäck
Zweitberichterstatter: Prof. Dr. Falk Nimmerjahn
2 Table of contents
Table of contents
1 Zusammenfassung ............................................................................. 1
2 Summary ............................................................................................. 2
3 Introduction......................................................................................... 3
3.1 B cell development and homeostasis................................................................ 3
3.1.1 Central B cell development................................................................................3
3.1.2 Peripheral B cell development...........................................................................6
3.1.3 Plasma cell generation and migration................................................................9
3.2 Krüppel-like factor 2 (KLF2) ............................................................................ 11
3.2.1 Krüppel-like transcription factors .....................................................................11
3.2.2 KLF2 in T lymphocytes....................................................................................12
3.2.3 Regulation of KLF2..........................................................................................13
4 Specific Aims .................................................................................... 15
5 Results............................................................................................... 16
5.1 B cell speficic KLF2 deletion in vivo ................................................................ 16
5.1.1 KLF2 expression profile during B cell development .........................................16
5.1.2 Normal B cell development in the bone marrow of KLF2-deficient animals......18
5.1.3 Enlarged spleen size and increased numbers of splenic B cell populations
in KLF2-deficient animals ................................................................................20
5.1.4 Decreased frequencies of B cells in the peritoneal cavity, the blood and
peyers patches of KLF2-deficient mice............................................................25
5.1.5 Reduced numbers of plasma cells in the bone marrow after boost
immunization with TD antigen..........................................................................28
5.1.6 Distribution of peripheral B cell subsets is controlled by KLF2 regulating
L-selectin and α β integrin but not S1P1 ........................................................32 4 7
5.1.7 KLF2 determines Fo B cell identity ..................................................................35
5.2 Biochemical analyses of KLF2 ........................................................................ 37
5.2.1 KLF2 protein modifications in splenic B cells and non B cells..........................37
5.2.2 In silico phosphorylation analysis of KLF2 amino acid sequence.....................38
5.2.3 KLF2 is posttranslationally modified by phosphorylation..................................40
I Table of contents
6 Discussion......................................................................................... 42
6.1 KLF2-deficiency results in impaired B cells homeostasis and plasma cell
homing ............................................................................................................ 42
6.2 Biochemical analyses of KLF2 ........................................................................ 47
7 Material and Methods ....................................................................... 50
7.1 Material ........................................................................................................... 50
7.1.1 Chemicals .......................................................................................................50
7.1.2 Antibodies .......................................................................................................50
7.1.3 PCR primer .....................................................................................................53
7.1.4 Plasmids..........................................................................................................54
7.1.5 Cell lines..........................................................................................................55
7.1.6 Mouse strains..................................................................................................55
7.1.7 Bacteria...........................................................................................................56
7.1.8 Technical Equipment.......................................................................................56
7.1.9 Plastic material................................................................................................57
7.1.10 Consumable material.......................................................................................58
7.2 Methods .......................................................................................................... 59
7.2.1 Cell culture ......................................................................................................59
7.2.1.1 Cultivation and harvest of vertebrate cell lines.................................................59
7.2.1.2 Cryoconservation ............................................................................................59
7.2.1.3 Thawing of cryoconserved cells.......................................................................59
7.2.1.4 Cell counting ...................................................................................................60
7.2.2 Isolation of primary B and T lymphocytes from different organs.......................60
7.2.2.1 Erythrocyte depletion.......................................................................................60
- +
7.2.2.2 MACS-sorting of CD43 splenic and CD19 bone marrow B cells ....................60
7.2.3 Flow cytometry ................................................................................................61
7.2.3.1 Analyses of surface expression and intracellular proteins................................61
TM
7.2.3.2 MoFlo cell sorting .........................................................................................61
7.2.4 Histology .........................................................................................................62
7.2.5 Molecular biological methods ..........................................................................62
7.2.5.1 Plasmid DNA and RNA preparation.................................................................62
7.2.5.2 DNA restriction analyses .................................................................................62
7.2.5.3 DNA fragment agarose gel electrophoresis .....................................................63
II Table of contents
7.2.5.4 Isolation of genomic tail DNA...........................................................................63
7.2.5.5 DNA/RNA concentration..................................................................................63
7.2.5.6 Polymerase chain reaction (PCR)....................................................................63
7.2.5.7 cDNA synthesis...............................................................................................64
7.2.5.8 Qualitative RT-PCR.........................................................................................64
®
7.2.5.9 Quantitative TaqMan -RT-PCR.......................................................................65
7.2.5.10 Affymetrix microarray analysis.........................................................................65
7.2.6 Antibody isotype detection using ELISA ..........................................................65
7.2.7 IgG specific Elispot..........................................................................................66
7.2.8 Proteinbiochemistry.........................................................................................66
7.2.8.1 Cell lysates......................................................................................................66
7.2.8.2 Nuclear extracts ..............................................................................................66
7.2.8.3 BCA test..........................................................................................................67
7.2.8.4 Phosphatase inhibitor and Ly294002 stimulation.............................................67
7.2.8.5 Immunoprecipitation ........................................................................................67
7.2.8.6 SDS PAGE......................................................................................................68
7.2.8.7 Western blot analysis ......................................................................................69
7.2.9 Calcium measurements...................................................................................69
7.2.10 Transfection and infection of vertebrate cells...................................................70
7.2.10.1 Transient transfection of adherent cells using calcium phosphate ...................70
7.2.10.2 Infection of NIH3T3 cells .................................................................................70
7.2.11 In vivo methods ...............................................................................................70
7.2.11.1 Immunization...................................................................................................70
7.2.11.2 FTY720-treatment ...........................................................................................71
7.2.11.3 BrdU-treatment................................................................................................71
7.2.12 Statistics..........................................................................................................71
8 References ........................................................................................ 72
9 Abbreviations.................................................................................... 87
10 Publications....................................................................................... 91
11 Vita ..................................................................................................... 92
12 Acknowledgment .............................................................................. 93
III Figures and Tables
Figures
Figure 1. Schematic view of B cell development. 5
Figure 2. Formation of plasma cells. 10
Figure 3. Role of KLF2 in programming T cell quiescence. 12
Figure 4. Analyses of KLF2 expression profile in different B cell lines. 16
Figure 5. Analyses of KLF2 expression profile in B cells. 17
Figure 6. Schematic view of KLF2 expression profile during B cell development. 18
Figure 7. KLF2 conditional knockout strategy. 19
Figure 8. Analysis of B cell precursors and recirculating B cells in the bone marrow from
wildtype and KLF2-deficient mice. 20
Figure 9. Analysis of of spleen physiognomy from wildtype and KLF2-deficient mice. 21
Figure 10. Analysis of B cells in spleen from wildtype and KLF2-deficient mice. 22
Figure 11. KLF2 deficiency does not affect in vitro survival capacity and proliferation of
Fo and MZ B cells. 23
Figure 12. Analysis of spleen architecture from wildtype and KLF2-deficient mice. 24
Figure 13. Analysis of B cells in lymph node, liver and blood from wildtype and
KLF2-deficient mice. 25
Figure 14. Analysis of thymi from wildtype and KLF2-deficient mice. 26
Figure 15. Analysis of peyers patches from wildtype and KLF2-deficient mice. 26
Figure 16. Analysis of peritoneal B cell subsets from wildtype and KLF2-deficient mice. 27
Figure 17. Analysis of antibody titers in non-immunized wildtype and KLF2-deficient mice. 27
Figure 18. Analyses of Ig-titers after immunization of wildtype and KLF2-deficient mice
with T-independent antigens. 28
Figure 19. Analyses of Ig-titers after immunization of wildtype and KLF2-deficient mice
with the T-dependent antigen TNP-KLH. 30
Figure 20. Analyses of plasma cells after immunization of wildtype and KLF2-deficient
mice with the T-dependent antigen TNP-KLH. 31
Figure 21. Identification of potential KLF2 target genes. 33
Figure 22. Verification of KLF2 target genes. 35
Figure 23. Analyses of complement receptor (CD21/35) expression on spleen and
lymph node B cells from wildtype and KLF2-deficient mice. 36
Figure 24. KLF2 expression pattern after different sorting strategies and fractionizing
into nuclear and cytosolic proteins. 38
Figure 25. In silico analysis of potential KLF2 phosphorylation sites. 39
IV Figures and Tables
Figure 26. KLF2 is posttranslationally modified by phosphorylation. 41
Figure 27. KLF2 phosphorylation via PI3K signalling leads to its degradation. 48


Tables
Table 1. Summary of transitional B cell surface marker expression 6
Table 2. Antibodies in alphabetical order 51
Table 3. Oligonucleotides 53
Table 4. Established plasmids 54
Table 5. Mouse strain overview 56
Table 6. Technical equipment in alphabetical order 56
Table 7. Plastic material in alphabetical order 57
Table 8. Consumable material in alphabetiacl order 58
Table 9. PCR component overwiew. 64
Table 10. Overwiew of components for SDS PAGE 68

V Zusammenfassung
1 Zusammenfassung
Der Transkriptionsfaktor Krüppel-like Faktor 2 (KLF2) kontrolliert die Auswanderung von
T-Lymphozyten aus lymphatischen Organen über die Regulation des Sphingosin 1-
Phosphat Rezeptors 1 (S1P1).
In Mäusen mit einer B Zell-spezifischen Deletion von KLF2 konnten wir im ersten Teil
der vorliegenden Doktorarbeit zeigen, dass KLF2 die Homöostase von B-Zellen in
peripheren lymphatischen Organen und die Migration von Plasma-Zellen ins
Knochenmark kontrolliert. Im Gegensatz zu T-Zellen, geschieht dies vermutlich über die
Expressionskontrolle von α β Integrin und L-Selektin, aber nicht durch Regulation von 4 7
S1P1, da die Expression von S1P1 in KLF2-defizienten B Zellen kaum verändert war.
Alle Milz B-Zell Populationen waren in KLF2-defizienten Mäusen mit erhöhter Anzahl
nachweisbar mit überproportionaler Selektion (5 fach) für Marginal Zonen (MZ) B-Zellen.
Im Gegensatz dazu wurden weniger Peyersche Plaques mit stark reduzierten B-Zellen,
weniger peritoneale B1 Zellen und eine reduzierte Anzahl rezirkulierender B-Zellen im
Knochenmark detektiert.
Nach sekundärer Thymus-abhängiger Immunisierung waren die IgG Serum-titer
erniedrigt und antigen-spezifische Plasma-Zellen im Knochenmark nicht mehr
nachzuweisen, obwohl die Anzahl von antigen-spezifischen Plasma-Blasten in der Milz
unverändert war. Damit konnten wir erstmals zeigen, dass KLF2 wichtig für das Homing
von Plasmazellen in das Knochenmark ist.
KLF2 ist stark in Fo und nicht in MZ B Zellen detektierbar. Wir konnten zeigen, dass
KLF2 eine Rolle in der Festlegung der follikulären (Fo) B Zell Identität spielt. KLF2-
defiziente Fo B-Zellen induzierten nach IgM Stimulation eine Kalziumantwort ähnlich der
von MZ B-Zellen und MZ B-Zell Signaturgene wie CD21 und CxCR7 wurden in KLF2-
defizienten Fo B-Zellen nicht herunterreguliert.
Phosphorylierungskaskaden sind ein wichtiger Bestandteil in lymphozytären
Signalwegen. Im zweiten Teil der Doktorarbeit konnten wir zeigen, dass KLF2 unter
direkter oder indirekter Beteiligung des PI3-Kinase Signalweges posttanslational
phosphoryliert wird.
1 Summary
2 Summary
The zinc finger transcription factor Krüppel-like factor 2 (KLF2) controls T lymphocyte
egress from lymphoid organs by regulating sphingosin 1-phosphate receptor 1 (S1P1).
In the first part of this thesis we could show that this is not the case for B cells. Instead,
KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of
plasma cells to the bone marrow, presumably by controlling the expression of β 7
integrin. We conclude this from mice with a B cell-specific deletion of KLF2. In such
mice, S1P1 expression on B cells was only slightly affected.
Accordingly, all splenic B cell subsets were present, but their numbers were increased
with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches
harboring less B cells were found, and fewer B1 cells in the peritoneal cavity as well as
recirculating B cells in the bone marrow were detected.
Upon thymus-dependent immunization, IgG titers were diminished and antigen-specific
plasma cells were absent in the bone marrow, although numbers of antigen-specific
splenic plasmablasts were normal.
KLF2 is abundant in follicular (Fo) B cells but barely detectable in MZ B cells. We could
show that KLF2 plays a role in determining the identity of follicular B cells, since KLF2-
deficient follicular B cells showed calcium responses similar to MZ B cells and failed to
downregulate MZ B cell signature genes, such as CD21 and CxCR7.
Phosphorylation cascades are indispensable for lymphocyte signaling. In the second
part of my thesis we could show that KLF2 is phosphorylated directly or indirectly by the
PI3 kinase signaling pathway.

2 Introduction
3 Introduction
3.1 B cell development and homeostasis
B lymphocytes play a prominent role in the combat of infectious diseases. The effector B
cells, called plasma cells (PCs) or antibody secreting cells (ASCs), secret antibodies
which specifically bind, neutralize and eliminate pathogens or toxins by recruitment of
phagocytotic cells.
Antibodies consist of two identical heavy and light immunoglobulin chains. Since
antibodies produced by a single plasma cell can only detect one specific antigen,
diversity is obtained during B cell development by gene segment (V, D, J-segment)
rearrangements at the heavy and light chain locus and differentiation is controlled by
several checkpoints.
3.1.1 Central B cell development
B cell development in vertebrates takes place in the bone marrow (Fig. 1). B lymphoid
cells originate from a hematopoietic stem cell (HSC) along a highly ordered but flexible
pathway. HSCs are pluripotent stem cells giving rise to all lymphoid (B cells, T cells,
natural killer cells) and myeloid cells (erythrocytes, megacaryocytes). Development into
B or T cells segregates after the common lymphoid progenitor (CLP) stage by
upregulation of transcription factors like E2A, EBF-1, PU.1, Ikaros leading to the
differentiation into precursor progenitor (prepro-) B cells (reviewed in Matthias and
Rolink, 2005). In the next stage, the progenitor (pro-) B cell, D to J rearrangement is H H
initiated at the immunoglobulin heavy chain (IgH) locus by upregulation of the
recombination complex consisting mainly of Rag1 and Rag2 (recombination activating
gene 1 and 2) followed by rearrangement of V to DJ in the late pro-B stage H H
(Mombaerts et al., 1992; Shinkai et al., 1992; Tonegawa, 1983; Willerford et al., 1996).
After productive rearrangement, two heavy chains (μHC) can assemble with the
surrogate light chain components VpreB and λ5 as well as the signalling molecules Igα
(encoded by the mb1 gene) and Igβ, (encoded by the B29 gene) to form the pre-B cell
receptor (Hombach et al., 1990; Karasuyama et al., 1990; Tsubata and Reth, 1990).
3

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