Molecular mechanisms of peripheral T cell tolerance [Elektronische Ressource] : identification of Dickkopf 3 as a novel immune modulator / presented by Maria Papatriantafyllou

Molecular Mechanisms of Peripheral T cell
Tolerance: Identification of Dickkopf 3 as a Novel
Immune Modulator












INAUGURAL!DISSERTATION



Maria Papatriantafyllou


Heidelberg, October 2008
INAUGURAL!DISSERTATION



Submitted to the
Fakultät für Biowissenschaften of
Ruprecht Karl Universität
Heidelberg






















Presented by
Maria Papatriantafyllou
Born in Athens, Greece
Oral!examination: 31st of October 2008
Molecular mechanisms of peripheral T cell
tolerance: Identification of Dickkopf 3 as a novel
immune modulator






















Supervisor:
Prof. Dr. Bernd Arnold

st
1 Referee: Prof. Dr. Günter J. Hämmerling
nd2 Referee: Prof. Dr. Lutz Gissmann

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TABLE OF CONTENTS
TABLE OF CONTENTS...................................................................................................................... 1
FIGURE AND TABLE INDEX ........................................................................................................... 5
ACKNOWLEDGEMENTS.................................................................................................................. 7
ABBREVIATIONS ............................................................................................................................... 8
1 SUMMARY ................................................................................................................................... 11
2 ZUSAMENFASSUNG.................................................................................................................. 13
3 INTRODUCTION......................................................................................................................... 15
3.1 INTRODUCTION TO THE IMMUNE SYSTEM................................................................................. 15
3.2 IMMUNOLOGICAL TOLERANCE ................................................................................................. 16
3.2.1 THE ROLE OF IMMUNOLOGICAL TOLERANCE ............................................................................ 16
3.2.2 CENTRAL T CELL TOLERANCE .................................................................................................. 17
3.2.3 PERIPHERAL T CELL TOLERANCE.............................................................................................. 19
3.2.4 IMMUNOLOGICAL PRIVILEGE .................................................................................................... 28
3.2.5 FAILURE OF PERIPHERAL T CELL TOLERANCE: THE EXAMPLE OF EXPERIMENTAL AUTOIMMUNE
ENCEPHALITIS........................................................................................................................................ 30
3.3 DICKKOPF 3................................................................................................................................. 30
3.3.1 THE DICKKOPF FAMILY OF PROTEINS AND DICKKOPF3............................................................ 30
3.3.2 FUNCTION OF THE DKK PROTEINS............................................................................................. 31
3.3.3 DKK3 IS A DIVERGENT MEMBER OF THE DICKKOPF FAMILY..................................................... 33
3.3.4 THE FUNCTIONS OF DKK3 ......................................................................................................... 34
3.4 AIM OF THE STUDY ..................................................................................................................... 36
4 MATERIALS AND METHODS ................................................................................................. 37
4.1 MATERIALS ................................................................................................................................. 37
4.1.1 CHEMICALS ............................................................................................................................... 37
4.1.2 ANTIBODIES .............................................................................................................................. 37
4.1.3 MICROARRAYS.......................................................................................................................... 39
4.1.4 CELL LINES................................................................................................................................ 39
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4.1.5 BUFFERS.................................................................................................................................... 40
4.1.6 CELL CULTURE MEDIA.............................................................................................................. 41
4.2 METHODS .................................................................................................................................... 42
4.2.1 TRANSFECTED CELL LINES ....................................................................................................... 42
4.2.2 MICE.......................................................................................................................................... 42
4.2.3 T CELL AND DENDRITIC CELL PURIFICATION EX VIVO .............................................................. 43
4.2.4 FLOW CYTOMETRY ................................................................................................................... 45
4.2.5 MOLECULAR BIOLOGY.............................................................................................................. 46
4.2.6 PROTEIN BIOCHEMISTRY........................................................................................................... 48
4.2.7 IN VITRO ASSESSMENT OF THE T CELL FUNCTION...................................................................... 49
4.2.8 IN VIVO EXPERIMENTS ............................................................................................................... 50
5 RESULTS ...................................................................................................................................... 52
5.1 GENE EXPRESSION PROFILE OF REGULATORY DES$TCR CD8 T CELLS ................................ 52
5.1.1 ISOLATION OF NAÏVE, ACTIVATED AND TOLERANT CD8 T CELLS FOR GENE EXPRESSION
ANALYSIS............................................................................................................................................... 52
5.1.2 TOTAL'RNA ISOLATION AND MRNA AMPLIFICATION FOR THE MICROARRAY ANALYSIS ....... 53
5.1.3 GENE EXPRESSION ANALYSIS WITH THE AFFYMETRIX MOUSE GENECHIP 430.2 MICROARRAYS
58
5.2 DKK3 IS UPREGULATED IN REGULATORY DES$TCR CD8 T CELLS AND IS CRUCIAL FOR
THEIR REGULATORY FUNCTION.......................................................................................................... 62
5.2.1 UPREGULATION OF DKK3 MRNA IN THE TOLERANT CD8 T CELLS ......................................... 63
5.2.2 GENERATION OF THE MOLECULAR TOOLS FOR THE DETECTION OF DKK3 PROTEIN IN THE
TOLERANT CD8 T CELLS ....................................................................................................................... 64
5.2.3 DKK3 PROTEIN EXPRESSION BY THE TOLERANT CD8 T CELLS................................................. 65
5.2.4 THE ROLE OF DKK3 IN THE MAINTENANCE CD8 TOLERANCE .................................................. 66
5.3 DKK3 AFFECTS POLYCLONAL T CELL REACTIVITY ................................................................. 70
5.3.1 DKK3 EXPRESSION IN POLYCLONAL T CELLS............................................................................ 70
'/'5.3.2 DKK3 SPLENOCYTES DISPLAY INCREASED PROLIFERATION IN VITRO..................................... 72
'/'5.3.3 DKK3 T CELL HYPERPROLIFERATION IS NOT AN EFFECT OF INCREASED T CELL ACTIVATION
STATUS OR NATURAL TREG DEFICIENCY............................................................................................... 74
'/'5.3.4 ISOLATED DKK3 CD8 BUT NOT CD4 T CELLS DISPLAY INCREASED PROLIFERATION IN
COMPARISON TO WILD TYPE CONTROL T CELLS.................................................................................... 75
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5.4 ATTRIBUTES OF DKK3 T CELL HYPERPROLIFERATION ........................................................ 76
'/'5.4.1 THYMIC SELECTION IS UNALTERED IN DKK3 MICE................................................................. 76
'/'5.4.2 T CELL' EXTRINSIC FACTORS PREDOMINANTLY CONTRIBUTE TO DKK3 T CELL
HYPERPROLIFERATION........................................................................................................................... 77
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5.5 MOLECULAR MECHANISMS OF DKK3 T CELL HYPERPROLIFERATION ................................ 82
5.5.1 DKK3 DOES NOT INTERFERE WITH THE TGF' Β PATHWAY IN T CELLS...................................... 82
5.5.2 LACK OF DKK3 LEADS TO AN ALTERED ERK PATHWAY ACTIVITY UPON CD8 T CELL
STIMULATION......................................................................................................................................... 84
'/'5.5