Dendritic cell vaccines in tumor immunotherapy [Elektronische Ressource] : immune activation strategies with ligands for the Toll-like receptors 7 and 9 / Cornelia Wurzenberger

Cornelia Wurzenberger

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München








Dendritic cell vaccines in tumor immunotherapy:
Immune activation strategies with ligands for the
Toll-like receptors 7 and 9





Cornelia Wurzenberger
aus München
2008



Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Professor Dr. Stefan Endres und Frau Dr. Dr. Carole
Bourquin betreut und von Frau Professor Dr. Angelika M. Vollmar vor der Fakultät
für Chemie und Pharmazie vertreten.







Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 09.12.2008




Cornelia Wurzenberger







Dissertation eingereicht am: 09.12.2008
1. Gutachter: Prof. Dr. Stefan Endres
2. Gutachter: Prof. Dr. Angelika M. Vollmar
Mündliche Prüfung am: 21.01.2009



















Dedicated to my family



INDEX
1 SUMMARY......................................................................................................................1
2 INTRODUCTION .............................................................................................................5
2.1 Dendritic cells .........................................................................................................7
2.1.1 Functions of dendritic cells.................................................................................7
2.1.2 Dendritic cell subsets .........................................................................................9
2.1.3 Isolation or generation of dendritic cells ........................................................... 10
2.1.4 Dendritic cell vaccines ..................................................................................... 11
2.2 Toll-like receptors ................................................................................................ 13
2.2.1 The Toll-like receptors and their ligands .......................................................... 13
2.2.2 Signaling pathways of Toll-like receptors ......................................................... 16
2.3 Objectives ............................................................................................................. 18
3 MATERIALS AND METHODS ...................................................................................... 19
3.1 Materials ............................................................................................................... 21
3.1.1 Technical equipment........................................................................................ 21
3.1.2 Chemicals, reagents and buffers ..................................................................... 21
3.1.3 Kits .................................................................................................................. 23
3.1.4 Cell culture reagents and media ...................................................................... 23
3.1.5 FACS antibodies and MHC class I pentamers ................................................. 25
3.1.6 Software .......................................................................................................... 26
3.2 Cell culture ........................................................................................................... 26
3.2.1 General culture conditions ............................................................................... 26
3.2.2 C26 colon carcinoma cell line .......................................................................... 26
3.2.3 Dendritic cells .................................................................................................. 27
3.2.3.1 Generation of bone-marrow-derived dendritic cells ...................................... 27
3.2.3.2 Antigen loading of dendritic cells.................................................................. 29
3.2.3.3 In vitro activation of dendritic cells ............................................................... 29
3.2.4 Isolation of DC and T-cell subtypes by magnetic cell separation (MACS) ........ 29
3.2.5 Assessment of T-cell responses in vitro ........................................................... 30
3.2.6 Analysis of DC-induced suppression of regulatory T-cell function .................... 31
3.2.7 Transwell migration assays.............................................................................. 31
3.3 Immunological methods ...................................................................................... 32
3.3.1 Enzyme-linked immunosorbent assay (ELISA) ................................................ 32
3.3.1.1 Cytokine ELISA ........................................................................................... 32

3.3.1.2 Measurement of immunoglobulin concentrations in mouse sera .................. 32
3.3.2 Flow cytometry ................................................................................................ 33
3.3.2.1 Analysis of surface molecule expression ..................................................... 33
3.3.2.2 MHC pentamer staining ............................................................................... 34
3.3.2.3 CFSE staining .............................................................................................. 34
3.4 Animal experimentation ....................................................................................... 34
3.4.1 Animals............................................................................................................ 34
3.4.2 Organ and single cell preparation .................................................................... 35
3.4.2.1 Preparation of serum and isolation of blood cells ......................................... 35
3.4.2.2 Spleen cell isolation ..................................................................................... 35
3.4.2.3 Preparation of bone marrow cells ................................................................ 35
3.4.2.4 Isolation of lymph node cells ........................................................................ 36
3.4.3 Immunization of mice ....................................................................................... 36
3.4.3.1 Dendritic cell immunizations ........................................................................ 36
3.4.3.2 Detection of specific immune responses ...................................................... 36
3.4.4 C26 tumor experiments ................................................................................... 37
3.4.4.1 Tumor challenge .......................................................................................... 37
3.4.4.2 Dendritic cell therapy ................................................................................... 37
3.4.4.3 Rechallenge of mice with tumor cells ........................................................... 38
3.4.5 Analysis of in vivo migration of dendritic cells .................................................. 38
3.5 Statistical analysis ............................................................................................... 38
3.6 Molecular biology methods ................................................................................. 39
3.6.1 RNA isolation ................................................................................................... 39
3.6.2 Analysis of gene expression profiles by microarray ......................................... 39
3.6.2.1 Generation and hybridization of cRNA ......................................................... 40
3.6.2.2 Data readout and analysis ........................................................................... 40
4 RESULTS ..................................................................................................................... 41
4.1 Kinetics of dendritic cell activation by TLR9 in tumor therapy ......................... 43
4.1.1 Cytokine production ......................................................................................... 43
4.1.2 Expression of costimulatory molecules ............................................................ 45
4.1.3 Induction of T helper cell responses ................................................................ 47
4.1.4 Initiation of cytotoxic T-cell responses ............................................................. 48
4.1.5 Inhibition of regulatory T-cell function .............................................................. 51
4.1.6 Migratory capacity of short-term activated dendritic cells ................................. 52
4.1.7 In vivo migration to draining lymph nodes ........................................................ 54
4.1.8 Immunization with short-term activated dendritic cells ..................................... 55

4.1.9 Tumor therapy with short-term activated dendritic cells ................................... 58
4.2 Distinctive features of TLR7 stimulation .................