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

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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 st1 Referee: Prof. Dr. Günter J. Hämmerling nd2 Referee: Prof. Dr. Lutz Gissmann 5 TABLE OF CONTENTS TABLE OF CONTENTS...................................................................................................................... 1 FIGURE AND TABLE INDEX ........................................................................................................... 5 ACKNOWLEDGEMENTS.................................................................................................................. 7 ABBREVIATIONS ............................................................................................................................... 8 1 SUMMARY ...................................................................................................................................

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Publié le 01 janvier 2008
Nombre de lectures 12
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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

5
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
1
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
$/$
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
'/'5.5.3 DKK3 CD8 T CELLS DISPLAY NO CHANGES IN THE P38 MAPK AND PKCΘ/NF' ΚΒ SIGNALING
87
5.6 DKK3 IS SECRETED BY IMMUNE PRIVILEGED TISSUES AND CONTRIBUTES TO THE CONTROL
OF T CELL REACTIVITY ....................................................................................................................... 88
5.6.1 DKK3 IS EXPRESSED BY IMMUNE PRIVILEGED TISSUES............................................................. 88
5.6.2 SECRETORY DKK3 PROTEIN SUPPRESSES CD4 AND CD8 T CELL PROLIFERATION................... 90
5.7 DKK3 REGULATES T CELL RESPONSES IN EXPERIMENTAL AUTOIMMUNE ENCEPHALITIS .. 94
'/' 5.7.1 DKK3 MICE DISPLAY MORE SEVERE AND PERSISTENT EAE.................................................... 95
5.7.2....................................................................................................................................................... 96
5.7.3....................................................................................................................................................... 96
'/'5.7.4 THE INCREASED EAE CHRONICITY IN THE DKK3 MICE IS ACCOMPANIED BY PERSISTENCE OF
ACTIVATED CD8 T CELLS IN THE CNS.................................................................................................. 96
5.7.5 NEURON' DERIVED DKK3 IS CRUCIAL FOR THE CONTROL OF EAE ........................................... 97
6 DISCUSSION .............................................................................................................................. 102
6.1 CHARACTERIZATION OF THE GENE EXPRESSION PROFILE OF THE DES$TCR REGULATORY
CD8 T CELLS ...................................................................................................................................... 102
6.2 DKK3 IS UP$REGULATED IN TOLERANT CD8 T CELLS AND IS CRUCIAL FOR THEIR
REGULATORY FUNCTION ................................................................................................................... 105
6.3 DKK3 AFFECTS POLYCLONAL T CELL REACTIVITY ............................................................... 106
$/$
6.4 CELLULAR MECHANISMS OF DKK3 T CELL HYPERPROLIFERATION.................................. 108
$/$
6.5 MOLECULAR MECHANISMS OF DKK3 T CELL HYPERPROLIFERATION .............................. 109
6.6 DKK3 IS SECRETED BY IMMUNE PRIVILEGED TISSUES AND CONTRIBUTES TO THE CONTROL
OF T CELL REACTIVITY ..................................................................................................................... 110
6.6.1 DKK3 IS EXPRESSED BY IMMUNE PRIVILEGED TISSUES........................................................... 110
6.6.2 SECRETORY DKK3 PROTEIN SUPPRESSES CD4 AND CD8 T CELL PROLIFERATION................. 111
6.7 DKK3 REGULATES T CELL RESPONSES IN EXPERIMENTAL AUTOIMMUNE ENCEPHALITIS
(EAE).................................................................................................................................................. 112
'/' 6.7.1 DKK3 MICE DISPLAY MORE SEVERE AND PERSISTENT EAE.................................................. 112
'/'6.7.2 THE INCREASED EAE CHRONICITY IN THE DKK3 MICE IS ACCOMPANIED BY ACTIVATED CD8
T CELL' PERSISTENCE IN THE CNS....................................................................................................... 113
6.7.3 NEURON' DERIVED BUT NOT T CELL' DERIVED DKK3 IS CRUCIAL FOR THE CONTROL OF EAE115
6.8 CONCLUSIONS AND OUTLOOK................................................................................................. 117
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7 REFERENCES............................................................................................................................ 119
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FIGURE AND TABLE INDEX
Figure 31. Schematic representation of T cell development in the thymus.____________________________ 17
Figure 32. Development of naturally arising regulatory T cells in the thymus. ________________________ 19
Figure 33. Schematic representation of the signalling pathways in T cell activation and T cell anergy
induction. ______________________________________________________________________________ 22
Figure 34. Schematic representation of the molecular mechanisms reported to contribute to the regulatory role
of nTregs. ______________________________________________________________________________ 24
Figure 35. CD8 T cell tolerance induction by keratinocytes during the neonatal phase. _________________ 28
Figure 36. Domain structure of the Dkk family. ________________________________________________ 31
Figure 37. The Wnt signalling pathway. ______________________________________________________ 33
Figure 51 Isolation of a highly pure naïve DesTCR CD8 T cell population.__________________________ 53
Figure 52. Quality control of the isolated totalRNA in Agilent Bioanalyzer 2100._____________________ 55
Figure 53. Summary of the mRNA amplification protocol. _______________________________________ 56
Figure 54. Estimation of the efficiency of the reverse transcription in Agilent Bioanalyser 2100. _________ 57
Figure 55. Quality control of the labelled fragmented aRNA.______________________________________ 57
Figure 56. CEL picture of the hybridized microarrays.___________________________________________ 58
Figure 57. Quality control of the microarrays experiments based on probe intensity distribution. _________ 59
Figure 58. Box plots of gene intensity ranges in the nine arrays before and after data normalization. ______ 60
Figure 59. The naïve, activated and tolerant CD8 T cells display differential expression only of a limited
number of genes. _________________________________________________________________________ 61
+ + +
Figure 510. Upregulation of CD4 CD25 FoxP3 natural Treg associated molecules in the tolerant CD8 T
cells. __________________________________________________________________________________ 62
Figure 511. Upregulation of the Dkk3 mRNA in the tolerant CD8 T cells. ___________________________ 64
Figure 512. Characterization of the antimouse Dkk3 monoclonal antibody.__________________________ 65
Figure 513. Dkk3 protein expression in tolerant DesTCR CD8 T cells. _____________________________ 66
bFigure 514. Dkk3 is essential for K – positive tumor graft acceptance mediated by the tolerant DesTCR CD8
T cells._________________________________________________________________________________ 67
b
Figure 515. Dkk3 is indispensable for the suppression of antiK reactivity of CD8 T cells in the DesTCR x
b
KerK mice. _____________________________________________________________________________ 68
Figure 516. Blocking of the secreted Dkk3 protein abolishes T cell tolerance to autologous skin. Des
b mice underwent autologous skin transplantation.______________________________________ 69 TCRxKerK
Figure 517. No presence of Dkk3 can be traced on the tolerant DesTCR CD8 T cellsurface by flow cytometry.
______________________________________________________________________________________ 69
Figure 518. No Dkk3 protein expression can be traced in lymphocytes and NK cells by flow cytometry. ____ 71
Figure 519. No Dkk3 expression can be traced in monocytes, dendritic cells and splenic stromal cells by flow
cytometry. ______________________________________________________________________________ 71
Figure 520. T cell proliferation in vitro is increased in the absence of Dkk3. _________________________ 72
Figure 521. Absence of Dkk3 expression in the spleen results in increased CD4 and CD8 T cell proliferation. 73
Figure 522. Increased Dkk3/ T cell proliferation in vitro is not a result of preactivation in vivo..________ 74
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