UNIVERSITE LOUIS PASTEUR ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE

De
Publié par

Niveau: Supérieur, Doctorat, Bac+8
UNIVERSITE LOUIS PASTEUR ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE THESE Présentée pour obtenir le grade de DOCTEUR DE L'UNIVERSITE LOUIS PASTEUR – STRASBOURG I Discipline : Sciences du Vivant ? Aspects Moléculaires et Cellulaires de la Biologie Spécialité : Immunologie Par Sébastien WIECKOWSKI Synthetic CD40L mimetics: biological effects and potential applications in immunotherapy Directeur de Thèse Prof. Dr. Sylvie FOURNEL Co-directeur de Thèse Dr. Gilles GUICHARD Soutenue publiquement le 30 mai 2007 Membres du jury : Rapporteur interne Prof. Dr. Jean-Louis PASQUALI Rapporteur externe Dr. Hugues LORTAT-JACOB Rapporteur externe Prof. Dr. Winfried WELS Examinateur Dr. Jean-Yves BONNEFOY Invité Dr. Sylviane MULLER

  • sciences du vivant ?

  • docteur de l'universite louis

  • cd40

  • pasteur ecole doctorale des sciences de la vie et de la sante

  • fondation de la recherche médicale

  • cd40l

  • signaling


Publié le : mardi 1 mai 2007
Lecture(s) : 81
Source : scd-theses.u-strasbg.fr
Nombre de pages : 225
Voir plus Voir moins

UNIVERSITE LOUIS PASTEUR
ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE
THESE
Présentée pour obtenir le grade de
DOCTEUR DE L’UNIVERSITE LOUIS PASTEUR – STRASBOURG I
Discipline : Sciences du Vivant Aspects Moléculaires et Cellulaires de la Biologie
Spécialité : Immunologie
Par
Sébastien WIECKOWSKI
Synthetic CD40L mimetics: biological effects
and potential applications in immunotherapy
Directeur de Thèse Prof. Dr. Sylvie FOURNEL
Co-directeur de Thèse Dr. Gilles GUICHARD
Soutenue publiquement le 30 mai 2007
Membres du jury :
Rapporteur interne Prof. Dr. Jean-Louis PASQUALI
Rapporteur externe Dr. Hugues LORTAT-JACOB
Rapporteur externe Prof. Dr. Winfried WELS
Examinateur Dr. Jean-Yves BONNEFOY
Invité Dr. Sylviane MULLER
>Remerciements
Je tiens ici à exprimer ma sincère reconnaissance à toutes les personnes qui ont contribué à ce travail de
thèse, réalisé au Laboratoire d’Immunologie et Chimie Thérapeutiques (UPR 9021).
Je remercie les membres de mon jury, le Professeur Jean-Louis Pasquali (Université de Strasbourg, Hôpital
Civil), le Docteur Hugues Lortat-Jacob (Institut de Biologie Structurale, Grenoble), ainsi que le Docteur
Jean-Yves Bonnefoy (Transgene, Strasbourg), d’avoir accepté de juger mon travail.
I am grateful to Professor Winfried Wels (Georg-Speyer-Haus, Frankfurt am Main) for accepting to evaluate
this work.
Je remercie le Docteur Sylviane Muller, directrice de l’UPR 9021, de m’avoir accueilli dans son unité et
d’avoir accepté de participer à mon jury.
Je tiens à exprimer mes remerciements au Professeur Sylvie Fournel, ma directrice de thèse, qui m’a supervisé
avec beaucoup d’optimisme. Son enthousiasme pour la recherche, son appui constant et sa grande
disponibilité m’ont apporté les meilleures conditions possible pour accomplir ce travail. Sylvie, tu as su me
faire confiance, me donner beaucoup de liberté dans ce projet et me recadrer lorsque c’était nécessaire.
Je remercie le Docteur Gilles Guichard, mon co-directeur de thèse, de m’avoir continuellement motivé
durant mon doctorat. Gilles, merci pour ta rigueur et ta précision qui m’ont beaucoup apporté.
Je remercie le Docteur Nathalie Trouche, sans qui le projet n’aurait pas été ce qu’il est.
Un grand merci au Docteur Johan Hoebeke, pour m’avoir initié à la résonance plasmonique de surface, mais
aussi pour m’avoir montré sa vision de la recherche.
J’ai une pensée particulière pour mes anciens maîtres de stage, en particulier le Docteur Robert Feil grâce à
qui tout a réellement commencé.
Merci à tous les collègues actuels et anciens de l’ICT pour leur gentillesse, leurs encouragements et leur aide.
Travailler à vos côtés fut un réel plaisir. Un merci particulier à Hayet, Sara, Sylvie « CpG », Jürgen et Pierre.
Special thanks to Julien Beyrath for his careful reading of the manuscript.
Merci à toutes les personnes qui ont été impliquées dans ce projet, en particulier à Alberto Bianco, Olivier
Chaloin et Hélène Dumortier.
Je remercie le Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, ainsi que
la Fondation pour la Recherche Médicale pour leur support financier.
Je remercie ma famille, en particulier mes parents et ma p’tite frangine Steph, pour leur soutien sans faille.
Maman, Papa, merci d’avoir cru en moi et de m’avoir aidé de tout votre possible dans mes projets. Je vous en
serai éternellement reconnaissant.
Je termine ces remerciements en exprimant toute mon estime à ma compagne Béatrice, qui a su me soutenir
au quotidien et supporter mes quelques variations d’humeur… Merci Béa pour avoir partagé avec moi tous
les mauvais et surtout les bons moments durant ces années, et pour m’avoir continuellement encouragé. Je
n’oublierai jamais combien tu t’es investie dans tout cela. Cette thèse est aussi un peu la tienne. List of Headings
Abbreviations ................................................................................................................... vii
List of Figures ........................................................................................................................... viii
List of Tables ............................................................................................................................. ix
INTRODUCTION ................................................................................................................. 1
CHAPTER 1
BIOLOGICAL OUTCOMES OF THE CD40 CD40L INTERACTION
1.1 Discovery of CD40 (also named CDw40 and Bp50) ....................................................... 4
1.1.1 Identification of CD40 ............................................................................................................... 4
1.1.2 Cellular expression of CD40 ...................................................................................................... 4
1.2 Discovery of CD40 ligand (also named CD40L, CD154, gp39 and T-BAM) ..................... 6
1.2.1 Cloning of CD40L ....................................................................................................................... 6
1.2.2 Cellular expression of CD40L ..................................................................................................... 7
1.3 Other proteins interact with CD40 ............................................................................... 9
1.4 CD40L binds to other receptors .................................................................................... 9
1.5 Level of CD40 crosslinking is important for activation of biological responses ............ 10
1.5.1 Engagement of CD40 with anti-CD40 Ab ................................................................................ 10
1.5.2 Engagement of CD40 with CD40L ........................................................................................... 11
1.6 CD40 plays a central role in immune response ........................................................... 12
1.6.1 Discovery of the X linked hyper IgM syndrome ..................................................................... 12
1.6.2 Role of CD40 in B lymphocytes ............................................................................................... 12
1.6.2.1 Effect of anti-CD40 mAb on B cell proliferation .............................................................................. 13
1.6.2.2 CD40 engagement and B cell activation ......................................................................................... 13
1.6.2.3 CD40 is important for isotype switching ......................................................................................... 14
1.6.2.4 CD40 is important for rescue from apoptosis of mature B lymphocytes ........................................ 16
1.6.3 Role of CD40 in other immune cells ........................................................................................ 16
1.7 Function of CD40 in malignant cells ............................................................................ 17
CHAPTER 2
CD40 AND CD40L: GENES AND PROTEINS
2.1 Characteristics of the CD40 protein sequence ............................................................ 19
2.1.1 The human sequence .............................................................................................................. 19
2.1.2 The mouse sequence .............................................................................................................. 20
- iii -
>>>List of Headings
2.2 Regulation of CD40 expression ................................................................................... 21
2.2.1 Organization of the CD40 gene ............................................................................................... 21
2.2.1.1 The mouse gene .............................................................................................................................. 21
2.2.1.2 The human gene ............................................................................................................................. 21
2.2.2 Transcriptional regulation of CD40 expression ....................................................................... 22
2.2.3 Post-transcriptional regulation of CD40 expression ............................................................... 23
2.2.3.1 Alternative splicing of the CD40 mRNA .......................................................................................... 23
2.2.3.2 Stability of the CD40 mRNA ............................................................................................................ 24
2.2.4 Post-translational regulation of CD40 expression .................................................................. 25
2.3 Characteristics of the CD40L protein sequence ........................................................... 26
2.3.1 The mouse sequence .............................................................................................................. 26
2.3.2 The human sequence .............................................................................................................. 27
2.4 Regulation of CD40L expression ................................................................................. 28
2.4.1 Organization of the CD40L gene ............................................................................................. 28
2.4.2 Transcriptional regulation of CD40L expression ..................................................................... 28
2.4.3 Post-transcriptional regulation of CD40L expression ............................................................. 30
2.4.4 Post-translational regulation of CD40L expression ................................................................. 30
2.5 Three-dimensional view of the CD40 CD40L interaction ............................................ 32
2.5.1 The TNF/TNF-R superfamily .................................................................................................... 32
2.5.2 The TNF family proteins .......................................................................................................... 36
2.5.3 The TNF-R family proteins ....................................................................................................... 37
2.5.4 The three-dimensional structure of CD40L ............................................................................. 39
2.5.5 Three-dimensional model of the CD40–CD40L interaction .................................................... 40
2.5.5.1 A three-dimensional model for CD40 .............................................................................................. 40
2.5.5.2 Residues important for the CD40–CD40L interaction ..................................................................... 41
2.5.5.3 Construction of a CD40–CD40L interaction model ......................................................................... 42
2.5.6 Usefulness of the three-dimensional model of the CD40–CD40L interaction ........................ 45
CHAPTER 3
CELL SIGNALING INITIATED FROM CD40
3.1 CD40 clustering and downstream signaling ................................................................ 46
3.1.1 Consequences of membrane receptor crosslinking in cell signaling ...................................... 46
3.1.2 Minimal CD40 oligomerization requirement for activation of cell signaling .......................... 46
3.1.2.1 Extracellular oligomerization .......................................................................................................... 46
3.1.2.2 Intracellular oligomerization .......................................................................................................... 47
3.1.3 Structural mechanisms governing initiation of intracellular signals ....................................... 48
3.2 Role of pre-ligand binding assembly domain in CD40 ................................................. 49
3.2.1 The PLAD domain .................................................................................................................... 49
- iv -
>List of Headings
3.2.2 PLAD requirement for CD40 signaling..................................................................................... 50
3.3 CD40 signaling is initiated within membrane rafts ..................................................... 51
3.3.1 Membrane rafts ...................................................................................................................... 51
3.3.2 CD40 is localized into lipid raft microdomains ........................................................................ 52
3.3.3 Caveolae are implicated in CD40 signaling ............................................................................. 52
3.3.4 Translocation of CD40 into lipid rafts ..................................................................................... 53
3.4 CD40 induced cell signaling ....................................................................................... 54
3.4.1 CD40–activated signaling pathways ....................................................................................... 54
3.4.1.1 Intermediates implicated in CD40 signaling ................................................................................... 54
3.4.1.2 Common signaling pathways activated by CD40 ........................................................................... 55
3.4.2 Implication of TRAF adaptor molecules .................................................................................. 56
3.4.2.1 CD40 signals through adaptor molecules ....................................................................................... 56
3.4.2.2 The TNF-R–associated factors ........................................................................................................ 56
3.4.2.2.1 The TRAF family ...................................................................................................................... 56
3.4.2.2.2 TRAFs are located into lipid rafts ............................................................................................ 57
3.4.2.2.3 Regulation of TRAF functions .................................................................................................. 57
3.4.2.2.4 TRAF protein structure ........................................................................................................... 58
3.4.2.3 The TRAF–CD40 interaction ............................................................................................................ 59
3.4.2.3.1 TRAF–binding sites .................................................................................................................. 59
3.4.2.3.2 CD40–mediated activation of TRAFs ...................................................................................... 60
3.4.2.3.3 The TRAF–CD40 interaction viewed at the three-dimensional level ...................................... 61
3.4.2.4 TRAF–specific signaling .................................................................................................................. 62
3.4.2.4.1 TRAF1 ...................................................................................................................................... 62
3.4.2.4.2 TRAF2 ...................................................................................................................................... 63
3.4.2.4.3 TRAF3 ...................................................................................................................................... 63
3.4.2.4.4 TRAF4 ...................................................................................................................................... 64
3.4.2.4.5 TRAF5 ...................................................................................................................................... 65
3.4.2.4.6 TRAF6 ...................................................................................................................................... 65
3.4.3 TRAF–independent CD40 signaling ......................................................................................... 66
Poster 1: summary of the signaling pathways engaged by CD40 ...................................... 67
CHAPTER 4
THE CD40 CD40L INTERACTION AS A TARGET FOR IMMUNOTHERAPY
4.1 Blockade of the CD40–CD40L interaction ................................................................... 70
4.1.1 CD40-based treatment of autoimmune diseases ................................................................... 71
4.1.1.1 Systemic Lupus Erythematosus ....................................................................................................... 72
4.1.1.2 An anti-CD40L therapy for other autoimmune diseases? ............................................................... 74
4.1.2 Chronic inflammation ............................................................................................................. 75
4.1.3 Transplantation ....................................................................................................................... 76
4.2 Boosting the immune response by activation of the CD40 signaling ........................... 77
4.2.1 Hyper-IgM syndrome and immunodeficiencies ...................................................................... 77
4.2.2 Infections ................................................................................................................................. 78
4.2.3 Vaccination .............................................................................................................................. 80
- v -
>>List of Headings
4.2.4 Cancer ..................................................................................................................................... 82
CHAPTER 5 ....................................................................................................................... 85
C3-SYMMETRIC PEPTIDE SCAFFOLDS
ARE FUNCTIONAL MIMETICS OF TRIMERIC CD40L
Nat Chem Biol 1, 377-382 (2005)
Fournel, S., Wieckowski, S., Sun, W., Trouche, N., Dumortier, H., Bianco, A., Chaloin, O., Habib, M., Peter, J. C.,
Schneider, P., Vray, B., Toes, R. E., Offringa, R., Melief, C. J., Hoebeke, J., and Guichard, G.
CHAPTER 6 ...................................................................................................................... 109
SOLID PHASE SYNTHESIS OF CD40L MIMETICS
Org Biomol Chem 4, 1461-1463 (2006)
Bianco, A., Fournel, S., Wieckowski, S., Hoebeke, J., and Guichard, G.
CHAPTER 7 ...................................................................................................................... 119
SMALL TRIVALENT ARCHITECTURES MIMICKING
HOMOTRIMERS OF THE TNF SUPERFAMILY MEMBER CD40L:
DELINEATING THE RELATIONSHIP BETWEEN STRUCTURE AND EFFECTOR FUNCTION
(submitted to JACS)
Sun, W., Wieckowski, S., Trouche, N., Chaloin, O., Briand, J.-P., Hoebeke, J., Fournel, S., and Guichard, G.
CHAPTER 8 ...................................................................................................................... 156
COOPERATIVITY IN THE INTERACTION OF SYNTHETIC CD40L MIMETICS
WITH CD40 AND ITS IMPLICATION IN CELL SIGNALING
Biochemistry 46, 3482-3493 (2007)
Wieckowski, S., Trouche, N., Chaloin, O., Guichard, G., Fournel, S., and Hoebeke, J.
CHAPTER 9 ...................................................................................................................... 177
CELL-SPECIFIC SIGNALING INDUCED BY SYNTHETIC CD40L MIMETICS
IN MALIGNANT AND NORMAL CELLS
(in preparation)
Wieckowski, S., Trouche, N., Guichard, G., and Fournel, S.
CHAPTER 10 .................................................................................................................... 214
SMALL MOLECULE CD40L MIMETICS PROMOTE CONTROL OF PARASITEMIA
AND ENHANCE T CELLS PRODUCING INTERFERON-

DURING EXPERIMENTAL TRYPANOSOMA CRUZI INFECTION
J Immunol 178, 6700-6704 (2007)
Habib, M., Rivas, M. N., Chamekh, M., Wieckowski, S., Sun, W., Bianco, A., Trouche, N., Chaloin, O.,

Dumortier, H., Goldman, M., Guichard, G., Fournel, S., and Vray, B. [ collaboration]
CONCLUSION AND PERSPECTIVES ................................................................................... 220
- vi -
>vAbbreviations
Ab antibody MEK MAPK/ERK kinase
ADP adenosine diphosphate MHC major histocompatibility
Ag antigen complex
APC antigen–presenting cells mini-CD40L small synthetic CD40L mimetic
ASM acid sphingomyelinase mRNA messenger RNA
bp base pair MW molecular weight
BCR B cell receptor NF-AT nuclear factor of activated T
C three-fold axial cells 3
NF- B nuclear factor BC4BP alpha chain of C4b binding
protein NIK NF- B–inducing kinase
cAMP cyclic adenosine N-ter N terminus
monophosphate ODN oligodeoxynucleotide
CD40-B anti-CD40 activated B cells OmpC outer membrane protein C
PDB Protein Data Bank CD40-Ig recombinant human CD40
coupled to human IgG1 Fc PHA phyto-hemagglutinin
domain PI phosphoinositide
CRD cysteine rich domain PI3K phosphatidyl inositol-3 kinase
C-ter C terminus PKC protein kinase C
Da Dalton PLAD pre–ligand binding assembly
DC dendritic cell domain
DISC death inducing signaling PLC 2 phospholipase C 2
complex PMA phorbol 12-myristate 13-
dsDNA double strand DNA acetate [also named 12-O-
EC extracellular tetradecanoyl phorbol-13-
EGR-1 early growth response-1 acetate (TPA)]
EMSA electrophoretic mobility shift PBMC peripheral blood mononuclear
assay cells
ERK extracellular signal-regulated PNA peptide nucleic acid
kinase PTEN phosphatase and tensin
F(ab') two Fab units linked by homolog deleted on 2
disulfide bridges chromosome 10
Fab fragment antigen binding sCD40L soluble CD40L
Fc fragment crystallizable scFv single chain fragment variable
GAS gamma activated sequences SDS PAGE sodium dodecyl
GC germinal center sulfate polyacrylamide gel
HIGM X linked hyper IgM electrophoresis
HSP70 unrelated 70 kDa sIgM surface IgM
mycobacterial heat shock SLE systemic lupus erythematosus
protein SPR surface plasmon resonance
IC intracytoplasmic STAT signal transducer and activator
IFN interferon of transcription
Ig immunoglobulin TACE metalloprotease-disintegrin
I B inhibitor of B TNF converting enzyme
IKK I B kinase T-BAM T cell-B cell activating molecule
IL interleukin T T cytotoxic C
IP inositol (1,4,5)-triphosphate TCR T-cell receptor 3
IS immune system TGF tumor growth factor
JAK3 Janus kinase 3 T T helper H
kb kilobase TM transmembrane
LMP1 latent membrane protein 1 TNF tumor necrosis factor
LPS lipopolysaccharide TNF-R tumor necrosis factor receptor
LT lymphotoxin TRAF TNF-R–associated factor
mAb monoclonal antibody T regulatory T cells reg
MAPK mitogen activated protein UTR untranslated region
kinase
- vii -
rv?v??>r>>>?>>>>?>>?List of Figures
Figure Title Page
Figure A Design of mini-CD40Ls 3
Figure 1 Nucleic acid and predicted protein sequences of human CD40 19
Figure 2 Alignment of the protein sequences of mouse and human CD40 20
Figure 3 Organization of the mouse CD40 gene 21
Figure 4 Organization of the human CD40 gene 22
Figure 5 Potential putative cis-regulatory elements located within the human CD40
gene promoter 23
Figure 6 Alternative splicing variants of the mouse CD40 mRNA 24
Figure 7 Alignment of the protein sequences of human and mouse CD40L 27
Figure 8 Organization of the human CD40L gene 28
Figure 9 Putative cis-regulatory elements located within the human and mouse
CD40L promoters 29
Figure 10 Interaction network between proteins of the TNF and TNF-R families 33
Figure 11 Structure of the TNF homology domain of human CD40L 36
Figure 12 Topology of the TNF homology domain of human CD40L 37
Figure 13 Three-dimensional structure of human TNF-R1 38
Figure 14 Structure of human CD40L 40
Figure 15 Three-dimensional model of human CD40 41
Figure 16 Residues implicated in the CD40–CD40L interaction 43
Figure 17 Residues involved in stabilization of the CD40–CD40L interaction 44
Figure 18 Summary of the residues implicated in the CD40–CD40L interaction 45
Figure 19 Implication of PLAD in TNF-R and CD40 signaling 50
Figure 20 CD40 reorganization into rafts is dependent on ASM 53
Figure 21 The TRAF family members 58
Figure 22 Three-dimensional structure of the human TRAF2’s TRAF domain 59
Figure 23 Three-dimensional structure of the TRAF-C domain of human TRAF6
complexed with a peptide derived from the CD40 intracytoplasmic domain 61
Figure 24 Three-dimensional model of the TRAF2–CD40 interaction 62
Figure 25 JAK and TRAF binding motifs on the intracytoplasmic sequence of CD40 66
Figure 26 CD40L controls immune cell functions and inflammatory reaction 71
Figure 27 Molecular defects leading to hyper-IgM syndromes 78
Figure 28 CD40 Troy and Vaccibody 81
- viii - List of Tables
Table Title Page
Table 1 List of other CD40 expressing cells 5
Table 2 List of other CD40L expressing cells 8
Table 3 General data on the TNF/TNF-R superfamily members 34
Table 4 Summary of phase 1 anti-CD40 targeted therapy studies 83
- ix -
>>>INTRODUCTION

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.