The impact of transforming growth Factor-b1 on dendritic cell development and subset specification [Elektronische Ressource] / Piritta Felker

Titta

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 28 Mo

Extrait

The Impact of Transforming Growth Factor-β1 on
Dendritic Cell Development and Subset Specification


Von der Fakultät für Mathematik, Informatik und
Naturwissenschaften der RWTH Aachen University zur Erlangung
des akademischen Grades einer Doktorin der Naturwissenschaften
genehmigte Dissertation


vorgelegt von
M.Sc. Piritta Felker (geb. Jäntti)
aus Rovaniemi, Finnland




Berichter: Universitätsprofessor Dr. Martin Zenke
Universitätsprofessor Dr. Lothar Elling
Universitätsprofessor Dr. Ralf Weiskirchen
Tag der mündlichen Prüfung: 21.04.2011

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
TABLE OF CONTENTS

Table of Contents 
Zusammenfassung............................................................................................ I 
Summary ......................................................................................................... III 
1 Introduction. 1 
1.1 The Immune System.......................... 1 
1.2 Antigen Presenting Cells.................................................................................... 1 
1.3 Dendritic Cell Biology......................... 2 
1.4 Dendritic Cell Subsets........................ 3 
1.4.1 Tissue DC.....................................................................................3 
1.4.2 Lymphoid Organ Resident cDC....................................................4 
1.4.3 Circulatory pDC............4 
1.4.4 Inflammatory DC...........................................................................5 
1.4.5 Human DC....................5 
1.5 Hematopoiesis................................... 5 
1.6 Dendritic Cell Development................................................ 7 
1.6.1 DC Development in Steady State.................7 
1.6.2 DC Development in Inflammation................................8 
1.6.3 LC Development.........................................................................10 
1.7 Molecular Mechanisms in DC Development.................... 10 
1.7.1 External Cues in DC Development.............................................11 
1.7.2 Internal Cues in DC Development ..............................................12 
1.8 Transforming Growth Factor-β1....... 14 
1.8.1 TGF-β1 Signalling Pathway........................................................14 
1.8.2 TGF-β1 Signalling in Hematopoiesis ..........................................16 
1.8.3 TGF-β1 Signalling in Immunity...................18 
1.8.4 TGF-β1 Signalling in DC Development......................................19 
1.9 AIMS AND OBJECTIVES ....................................................................................... 20 
2 Materials and Methods............. 21 
2.1 Materials........................................................................................................... 21 
2.2 Mice................. 21 
2.3 Bone Marrow Transplantations........ 21 
2.4 Cell Culture ...................................................................................................... 22 
2.4.1 Cell Culture Supplements...........................22 
+2.4.2 Culture of Human CD34 Progenitors and DC................................23 
2.4.3 Culture of Mouse Bone Marrow Progenitors..............................23 
2.4.4 Culture of Mouse DC ..................................................................24 
2.4.5 Culture of B Cells........................................24 
2.4.6 Culture of Cell Lines...24 
2.4.7 TGF-β1 Treatments....25 
2.5 Flow Cytometry ................................................................................................ 25 
2.6 Cell Sorting...... 27  TABLE OF CONTENTS

2.7 Colony Forming Cell Assay ..............................................................................27 
2.8 Cell Proliferation with CFSE Labelling..............................................................28 
2.9 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).......................28 
2.10 DNA Microarrays ............................................................29 
2.11 Western Immunoblotting.................................................31 
2.11.1 Cell Lysate Preparation for Proteins .........................................31 
2.11.2 SDS Polyacrylamid Electrophoresis (SDS-PAGE)...................31 
2.11.3 Western Immunoblotting...........................................................31 
2.12 ELISA..............................................................................32 
2.13 Adenovirus Infections.....................32 
2.14 Statistical Analysis..........................................................32 
3 Results ....................................................................... 33 
3.1 Steady State vs. Inflammatory DC Development in vitro..33 
3.2 Amplification of Multipotent Progenitors from Bone Marrow.............................35 
3.2.1 Morphology and Growth of Amplified Progenitors......................35 
3.2.2 In vivo Differentiation Potential of Amplified Progenitors ............................................37 
3.2.3 DC Differentiation In vitro from Amplified Progenitors................40 
3.3 Common Dendritic Cell Progenitor ...................................41 
3.3.1 Amplified Progenitor Culture Comprises Multipotent Progenitors (MPP) and Common
DC Progenitors (CDP) .........................................................................41 
3.3.2 CDP Have Restricted Developmental Potential..........................................................43 
3.3.3 CDP Readily Differentiate into Multiple DC Subsets in vitro.......50 
3.4 Molecular Mechanisms Underlying the DC-Priming of CDP.............................53 
3.4.1 CDP Have a DC-Primed Transcription Profile ............................................................53 
3.4.2 The Transcription Profile of Amplified CDP Correlates with the in vivo CDP .............57 
3.5 TGF-β1 Signalling in DC Development.............................59 
3.5.1 TGF-β1 Induces IRF-8 Expression in Human DCs.....................................................59 
3.5.2 TGF-β1 Accelerates DC Development and Directs Subset Specification Towards cDC
.............................................................................................................62 
3.6 E2A/Id Antagonism in DC Development...........................70 
3.6.1 Impaired pDC Development from E2A Deficient Bone Marrow Cells .........................70 
3.7 Inflammatory DC Development is TGF-β1 Insensitive .....................................73 
4 Discussion ................................................................. 78 
4.1 DC Development from Common DC Progenitors.............78 
4.1.1 In Vitro Generated CDP..............................................................79 
4.1.2 DC-Priming of CDP.....................................................................80 
4.2 Impact of TGF-β1 on DC Development............................82 
4.2.1 TGF-β1 Accelerates DC Differentiation and Directs DC Subset Specification Towards
cDC......................................................................................................................................82 
4.2.2 E2A in DC Differentiation............................85 
4.2.3 TGF-β1 in DC Development During Steady State and Inflammation .........................85 
5 References................................................................................................. 88 
6 Appendix.. 103 TABLE OF CONTENTS

6.1 Abbreviations ................................................................................................. 103 
6.2 Table of figures.............................. 105 
6.3 Supplementary Tables ................................................................................... 107 
Publications.................................. 108 
Declaration of Authorship............. 109 
Eidesstattliche Erklärung.............................................................................. 109 
Acknowledgements...................... 110 

ZUSAMMENFASSUNG I

Zusammenfassung
Dendritische Zellen (dendritic cells, DC) sind professionelle
antigenpräsentierende Zellen mit einer Schlüsselrolle bei antigenspezifischen
Immunreaktionen und der Erhaltung von T Zelltoleranz. DC entwickeln sich
aus hämatopoetischen Stammzellen des Knochenmarks und heute bietet sich
ein zunehmend präziseres Bild ihrer Entwicklung über verschiedene
hämatopoetische Vorläuferzellstadien. So wurde kürzlich eine gemeinsame
DC Vorläuferzelle (common dendritic cell progenitor, CDP) für „klassische“ DC
(conventional DC, cDC) und plasmazellähnliche DC (plasmacytoid DC, pDC)
beschrieben. Die molekularen Mechanismen, die die Entwicklung von CDP
aus hämatopoetischen Stammzellen und die CDP Spezifizierung in die