Genetic and physical modification of human monocyte derived dendritic cells in order to improve vaccination protocols [Elektronische Ressource] / vorgelegt von Ilka Knippertz

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Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Ilka Knippertz aus Mönchengladbach Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 01.07.2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Lars Nitschke Zweitberichterstatter: Prof. Dr. Eckhart Kämpgen Table of contents Abbreviations 1. Summary - English .......................................................................................... 1 - German ......................................................................................... 3 2. Introduction.......................................................................................... 5 2.1. The biology of dendritic cells ................................................................ 5 2.1.1. Dendritic cell subsets ............................................................................ 5 2.1.2. Intimate link between antigen capture/-processing, maturation, activation and migration of DC.............................................................. 7 2.1.2.1. Antigen capture ........................................
Publié le : mardi 1 janvier 2008
Lecture(s) : 21
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2008/1016/PDF/ILKAKNIPPERTZDISSERTATION.PDF
Nombre de pages : 189
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Genetic and physical modification of human
monocyte-derived dendritic cells in order to improve
vaccination protocols






Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades






vorgelegt von
Ilka Knippertz
aus Mönchengladbach Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg












Tag der mündlichen Prüfung: 01.07.2008

Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Lars Nitschke

Zweitberichterstatter: Prof. Dr. Eckhart Kämpgen Table of contents

Abbreviations

1. Summary - English .......................................................................................... 1
- German ......................................................................................... 3

2. Introduction.......................................................................................... 5

2.1. The biology of dendritic cells ................................................................ 5
2.1.1. Dendritic cell subsets ............................................................................ 5
2.1.2. Intimate link between antigen capture/-processing, maturation,
activation and migration of DC.............................................................. 7
2.1.2.1. Antigen capture ..................................................................................... 7
2.1.2.2. Antigen processing ................................................................................ 7
2.1.2.3. Maturation of DC ................................................................................... 9
2.1.2.4. The cell surface molecule CD83.......................................................... 10
2.1.2.4.1. Functions of membrane bound CD83 and soluble CD83 .................... 10
2.1.2.4.2. The CD83 promoter............................................................................. 11
2.1.2.5. Activation of DC................................................................................... 12
2.1.2.5.1. Innate immunity activation signals ....................................................... 12
2.1.2.5.2. Adaptive immunity activation signals ................................................... 13
2.1.2.6. Migration of DC.................................................................................... 14
2.1.3. Control of the type of T cell response by DC ....................................... 15
2.1.4. The role of heat shock proteins in DC-mediated immunity .................. 17
2.1.4.1. The human heat shock protein 70 family............................................. 18
2.1.4.2. Regulation of the heat shock response................................................ 20
2.1.4.3. The hsp-APC interaction as an inducer of adaptive and innate
immune events .................................................................................... 21
2.1.4.4. Heat shock factor 1-independent activation of dendritic cells by
heat shock ........................................................................................... 22
2.1.5. DC in cancer immunotherapy .............................................................. 23
2.1.5.1. Immune escape mechanisms of cancer .............................................. 24
2.1.5.2. Genetic modification of DC for therapeutic vaccination ....................... 25
2.2. Adenovirus and its use as a vector for cancer gene therapy and
genetic DC-mediated vaccination ........................................................ 26
2.2.1. Adenoviruses and gene therapy.......................................................... 26
2.2.2. Virus structure ..................................................................................... 27
2.2.3. The viral life cycle ................................................................................ 28
2.2.3.1 Binding and entry................................................................................. 28
2.2.3.2. Expression of viral genes..................................................................... 29
2.2.3.3. Virus assembly and release................................................................. 30
2.2.4. Use of adenovirus vectors in gene therapy.......................................... 31
2.2.4.1. Targeting of adenovirus vectors .......................................................... 31
2.2.4.2. Adenovirus vectors .............................................................................. 32
2.2.4.2.1. First- and second-generation adenovirus vectors................................ 32
2.2.4.2.2. Helper-dependent (gutless; high capacity) adenovirus vectors ........... 33
2.2.4.3. DC-based adenoviral vaccines in gene therapy .................................. 33

3. Tasks .................................................................................................. 36

4. Material and Methods........................................................................ 38

4.1. Material................................................................................................ 38
4.1.1. Chemicals............................................................................................ 38
4.1.2. Buffers and cell culture media ............................................................. 39
4.1.2.1. General buffers.................................................................................... 39
4.1.2.2. Reagents for transfection of cells ........................................................ 40
4.1.2.3. ELISA buffer ........................................................................................ 41
4.1.2.4. Buffer for SDS-PAGE and Western blotting......................................... 41
4.1.2.5. Buffer for ChIP-chip microarray ........................................................... 42
4.1.2.6. Cell lines and cell culture media .......................................................... 43
4.1.2.7. Further cell media................................................................................ 44
4.1.3. Weight markers for gel electrophoresis ............................................... 45
4.1.3.1. Weight markers for DNA gel electrophoresis....................................... 45
4.1.3.2. Weight marker for protein gel electrophoresis ..................................... 45
4.1.4. Bacteria ............................................................................................... 46
4.1.5. Plasmid vectors ................................................................................... 46 4.1.6. Primers ................................................................................................ 46
4.1.6.1. Primers used for screening of human- and adenoviral genome .......... 47
4.1.6.2. Primers used for screening of BAC-transgenic mice ........................... 47
4.1.6.3. Primers used for Real Time PCR ........................................................ 48
4.1.6.4. Primers used for cloning...................................................................... 48
4.1.6.4.1. Primers used for cloning of CD83 promoter sequences ...................... 48
4.1.6.4.2. Further primers used for cloning from the human genome.................. 50
4.1.7. Adenoviruses....................................................................................... 50
4.1.8. Human cytokines ................................................................................. 51
4.1.9. Antibodies............................................................................................ 51
4.1.9.1. Antibodies used for FACS ................................................................... 52
4.1.9.2. Antibodies used for ELISA................................................................... 53
4.1.9.3. Antibodies used for Western blotting and immunoprecipitation ........... 53
4.1.9.4. Antibodies used for MACS................................................................... 53
4.1.10. Purchased kits for RNA and DNA purification...................................... 54
4.1.11. Mice..................................................................................................... 54

4.2. Methods............................................................................................... 54
4.2.1. Generation of transformation-competent bacteria and
transformation...................................................................................... 54
4.2.1.1. Generation of chemical-competent E.coli and transformation
by heat................................................................................................. 54
4.2.1.2. Generation of electro-competent E. coli and transformation by
electroporation..................................................................................... 55
4.2.2. Molecular biology methods.................................................................. 56
4.2.2.1. Isolation of DNA................................................................................... 56
4.2.2.1.1. Isolation of small plasmids (< 12 kb).................................................... 56
4.2.2.1.2. Isolation of large plasmids (> 12 kb).................................................... 56
4.2.2.1.3. Isolation of bacterial artificial chromosome (BAC)- DNA
(Alkaline lysis)...................................................................................... 57
4.2.2.1.4. Isolation of human chromosomal DNA from cells ................................ 58
4.2.2.1.5. Isolation of total DNA from tissue ........................................................ 58
4.2.2.1.6. Preparation of DNA fragments from PCR reactions and
enzymatic digestions ........................................................................... 59 4.2.2.1.7. Preparation of DNA fragments from gel electrophoresis ..................... 59
4.2.2.2. Preparation of DNA for ChIP-chip microarray...................................... 59
4.2.2.3. Isolation of total RNA from animal cells ............................................... 60
4.2.2.4. Nucleic acid quantification ................................................................... 60
4.2.2.5. Separation of DNA or RNA using agarose gel electrophoresis............ 61
4.2.2.6. Pulse-field gel electrophoresis (PFGE)................................................ 61
4.2.2.7. Reverse transcription........................................................................... 61
4.2.2.8. Polymerase chain reaction (PCR) ....................................................... 62
4.2.2.9. Two-step PCR ..................................................................................... 63
4.2.2.10. Quantitative real time- PCR (qPCR) .................................................... 64
4.2.2.11. Cloning of DNA fragments or PCR products........................................ 65
4.2.2.11.1. Dephosphorylation of cleaved DNA..................................................... 65
4.2.2.11.2. Conversion of DNA overhangs ............................................................ 65
4.2.2.11.3. Ligation................................................................................................ 65
4.2.2.12. Sequencing of DNA ............................................................................. 66
4.2.3. Protein biochemistry methods ............................................................. 66
4.2.3.1. Preparation of whole cell extracts for SDS-PAGE ............................... 66
4.2.3.2. Bradford assay .................................................................................... 66
4.2.2.3. Denaturing (SDS) discontinuous polyacrylamide gel
electrophoresis (PAGE): Laemmli method........................................... 67
4.2.2.4. Western blotting................................................................................... 67
4.2.4. Cell culture........................................................................................... 68
4.2.4.1. Generation of DC................................................................................. 68
4.2.4.2. Cryopreservation of primary cells ........................................................ 68
4.2.4.3. Cryopreservation of cell lines............................................................... 69
4.2.5. Heat shock........................................................................................... 69
4.2.5.1. Heat shock of DC ................................................................................ 69
4.2.5.2. Heat shock of cell lines........................................................................ 69
4.2.6. Flow cytometric analysis (FACS)......................................................... 70
4.2.7. Cytometric bead array (CBA)............................................................... 70
4.2.8. Transient transfection methods and luciferase reporter assay ............ 70
4.2.8.1. Transfection of DNA with the DEAE-Dextran method.......................... 70
TM
4.2.8.2. Lipofection of DNA with Lipofektamin (Invitrogen) or
TM Lipofektamin PLUS reagent (Invitrogen) .......................................... 71 4.2.8.3. Transfection of RNA with Transmessenger transfection
reagent (Qiagen) ................................................................................. 72
4.2.8.4. Luciferase reporter assay .................................................................... 72
4.2.9. Cytokine enzyme linked immunosorbent assay (ELISA) ..................... 72
4.2.10. Transwell migration assay ................................................................... 73
4.2.11. Cytotoxic T cell induction assay........................................................... 74
4.2.12. Tetramer staining and phenotyping of antigen-specific
+ CD8 T cells......................................................................................... 74
4.2.13. Recombinant adenoviruses ................................................................. 75
4.2.13.1. Cloning of plasmids containing the recombinant adenoviral
genome................................................................................................ 75
4.2.13.2. Preparation of recombinant adenoviruses ........................................... 75
4.2.13.3. Determination of the physical particle concentration ........................... 76
4.2.13.4. Determination of the infectious particle concentration ......................... 76
4.2.13.5. Adenoviral transduction of cells........................................................... 77

5. Results................................................................................................ 78

5.1. Generation of human dendritic cells that simultaneously secrete
IL-12 and have migratory capacity mediated by adenoviral gene
transfer of human CD40L in combination with IFN-γ treatment ........... 78
5.1.1. Transduction of moDC with Ad5hCD40L leads to an intracellular,
but not to a surface expression of the CD40L protein.......................... 79
5.1.2. Transduction with Ad5hCD40L induces partial maturation of iDC ....... 81
5.1.3. Timing of IFN-γ-treatment after transduction of iDC with
Ad5hCD40L influences IL-12p70-expression ...................................... 83
5.1.4. Kinetics of CD40L- and IL-12p70 expression after modulation
of DC with Ad5hCD40L and IFN-γ....................................................... 84
5.1.5. Dendritic cells transduced with Ad5hCD40L and stimulated with
IFN-γ are able to simultaneously migrate and secrete IL-12p70......... 85
5.1.6. moDC modified with Ad5hCD40L in combination with IFN-γ
+
efficiently prime naive CD8 T cells in vitro.......................................... 90 5.2. Analysis of the human CD83 promoter for cell type- as well as
status-specificity and development of a promoter-responsive
system for multiple transgene expression in human dendritic cells ..... 91
5.2.1. Approaches to identify CD83 promoter sequences which are
specifically active in mature human dendritic cells .............................. 91
5.2.1.1. Analysis of up-/downstream or internal gene fragments of the human
CD83 gene in order to identify additional regulatory elements ............ 92
5.2.1.2. Generation of CD83-BAC transgenic animals ..................................... 94
TM5.2.1.3. ChIP-chip microarray analysis of the human CD83-gene ................ 96
5.2.2. Development of a vector system for the expression of multiple
therapeutic transgenes ........................................................................ 99
5.2.2.1. Development of the Multiple Transgene Switch system .................... 100
5.2.2.1.1. A hsp70B` promoter fragment can be efficiently induced by heat
shock in HeLa cells............................................................................ 100
5.2.2.1.2. Specific expression of the tumor antigen MelanA, the
Th1-cytokine IL-12 and the survival protein Bcl-xL under the
control of the hsp70B` promoter in HeLa cells................................... 102
5.2.2.1.3. Generation of a plasmid containing a triple hsp70B`-bclxL-hsp70B`-
MelA-hsp70B`-IL12 cassette (p3xhsp70B`)....................................... 104
5.2.2.1.4. HeLa cells lipofected with p3xhsp70B` express MelanA, Bcl-xL
and IL-12 specifically after transduction with mHSF1 or after
exposure to heat................................................................................ 104
5.2.2.1.5. The multiple switch system is functional in Ad vectors after
transduction of DC............................................................................. 106
5.2.2.1.6. DC transduced with Ad5-3xhsp70B`are efficient in priming
+
naive CD8 T cells in vitro.................................................................. 107
5.2.2.2. Effects on DC caused by the overexpression of HSF1...................... 109
5.2.2.2.1. Upregulation of heat shock proteins 40, 70A and 70B`
in moDC after transduction with Ad5mHSF1 ..................................... 109
5.2.2.2.2. Ad5mHSF1 only minimally affects MC-induced upregulation
of DC-surface maturation markers..................................................... 111
5.2.2.2.3. DC transduced with Ad5mHSF1 showed enhanced secretion
of the anti-inflammatory cytokine IL-10 and the pro-inflammatory
cytokine TNF-α .................................................................................. 112 5.3. Mild thermal stress modulates dendritic cell function resulting in
+ an enhanced capacity to prime naive CD8 T cells to differentiate
into CTL in vitro ................................................................................. 114
5.3.1. moDC exposed to heat show an enhanced expression of hsp70A.... 114
5.3.2. Heat treatment induces an upregulation of maturation markers
on the surface of moDC..................................................................... 115
5.3.3. Mild thermal stress induces the simultaneous upregulation of the
anti-inflammatory cytokine IL-10 and the pro-inflammatory
cytokine TNF-α .................................................................................. 117
5.3.4. Stimulation by mild thermal heat does not influence the migratory
capacity of moDC in vitro................................................................... 118
5.3.5. Heat treatment enhances the capacity of human moDC to prime
+
naive CD8 T cells to differentiate into melanoma antigen-specific
CTL in vitro ........................................................................................ 119

6. Discussion ...................................................................................... 121

6.1. Modification of human monocyte-derived DC by adenoviral gene
transfer of CD40L ............................................................................. 121
6.1.1. After transduction of DC with Ad5hCD40L, CD40L-protein is
expressed intracellularly but not on the cell surface .......................... 121
6.1.2. Efficacious induction of IL-12p70-production is dependent on the
time point of IFN-γ-addition................................................................ 123
6.1.3. Linking migratory- and IL-12 secreting capacity of moDC ................. 123
+6.1.4. Improved priming of naive autologous CD8 T cells to develop
into MelanA antigen-specific CTL by DC treated with both
Ad5hCD40L and IFN-γ ...................................................................... 126
6.1.5. Possible benefits for clinical applications........................................... 126
6.1.6. Future developments......................................................................... 128

6.2. Identification of transcriptional regulatory sequences of the human
CD83 gene ........................................................................................ 129
6.2.1. Analysis of CD83 genomic fragments................................................ 129
6.2.2. Generation of transgenic animals to study the human CD83
promoter in vivo ................................................................................. 130
6.2.3. Analysis of the human CD83 gene chromatin activation status......... 131

6.3. Controlled induction of multiple therapeutic transgenes by
mHSF1 using the hsp70B`promoter .................................................. 132
6.3.1. The pGL3-3xhsp70B` vector system ................................................. 133
6.3.2. Ad5-3xhsp70B`-mediated gene transfer in human
monocyte-derived dendritic cells ....................................................... 134
6.3.3. Influence of overexpression of HSF1 on DC...................................... 136
6.3.4. Future prospects................................................................................ 136

6.4. Modulation of human monocyte-derived dendritic cells by
physiologically relevant thermal stress .............................................. 137
6.4.1. Thermal regulation of DC maturation................................................. 138
6.4.2. Thermal regulation of cytokine release.............................................. 138
6.4.3. Thermal regulation of migration......................................................... 139
6.4.4. Thermal regulation of DC-mediated T cell stimulation ....................... 140
6.4.5. Challenges for clinical development .................................................. 141

6.5. Concluding remarks........................................................................... 142

7. Literature.......................................................................................... 143

Acknowledgements/ Danksagung

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