Production of Recombinant Human Soluble CD83 in an Eukaryotic System and Generation of Tissue-Specific CD83 Knock-out Mice [Elektronische Ressource] / Christine Staab. Betreuer: Lars Nitschke

Production of Recombinant Human Soluble CD83 in an Eukaryotic System and Generation of Tissue-Specific CD83 Knock-out Mice Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Christine Staab aus Tübingen Production of Recombinant Human Soluble CD83 in an Eukaryotic System and Generation of Tissue-Specific CD83 Knock-out Mice Herstellung von rekombinantem humanen löslichem CD83 in Eukaryoten und Generierung von gewebespezifischen CD83 Knock-out Mäusen Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Christine Staab aus Tübingen Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 21.07.2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Lars Nitschke Zweitberichterstatter: Prof. Dr. Eckhart Kämpgen 1 Summary/Zusammenfassung _____________________________________________ 1 1.1 Summary _______________________________________________________________ 1 1.
Publié le : samedi 1 janvier 2011
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Source : D-NB.INFO/1015475329/34
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Production of Recombinant Human Soluble CD83 in an
Eukaryotic System
Generation of Tissue-Specific CD83 Knock-out Mice
Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von Christine Staab
aus Tübingen
Production of Recombinant Human Soluble CD83 in an
Eukaryotic System and
Generation of Tissue-Specific CD83 Knock-out Mice
Herstellung von rekombinantem humanen löslichem CD83 in
Eukaryoten und
Generierung von gewebespezifischen CD83 Knock-out Mäusen
Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg
Erlangung des Do
ktorgrades Dr. rer. nat.
vorgelegt von Christine Staab
aus Tübingen
Vorsitzender der Promotionskommission: Erstberichterstatter: Zweitberichterstatter:
Prof. Dr. Eckhart Kämpgen
Prof. Dr. Lars Nitschke
Prof. Dr. Rainer Fink
Tag der mündlichen Prüfung:
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität
Summary/Zusammenfassung _____________________________________________ 11.1Summary 1_______________________________________________________________ sssung ______________________________________________________ 1.2Zusammenfa 3Introduction 5___________________________________________________________ 2.1 _____________________________________________________ ystThe Immune S em 5__________________________________________________ 2.2Characteristics of CD83 62.2.1 __________CD83 Gene Expression and Processing 6__________________________________ of the Human and the Murine GeneCD83 7_________________________ 7Human and Murine CD83 Promoter and its Regulation ___________________________2.2.1.3CD83 mRNA Transport _______________________________________________ 8_____ Variants of CD83 mRNA _____________________________________________ 92.2.2The Membrane Bound CD83 (mCD83) 9 ____________________________________________2.2.2.1mCD83 Expressed on DCs Plays an Important Role for T Cell Activation ___________ 11 sRole of mCD83 on Activated ReThe gulatory T Cell ___________________________ 11 p ___________________________________The Role of mCD83 in T Cell Develo ment Role of mCD83 in B Cell Regulation and Function 13 __________________________2.2.2.5The Role of mCD83 in B- and CD4+T Cell Lo ev 14ng ity___________________________ of the Potential Ligand of mCD83 ______________________________ is a Target for Viral Immune Escape __________________________________ 152.2.3 and Functions sticsThe Role of Soluble CD83 (sCD83), Ch _____________________  aracteri 16.4Biolo 172.2 gical and Clinical Relevance of sCD83 _______________________________________ 2.3 18Formulation of the Thesis________________________________________________ Results 19_______________________________________________________________ 3.1 19Isolation of Functional sCD83 from HEK 293T Cells by HPLC_________________ 3.1.1 19Cloning and Expression of the Plasmid pZipH-sCD83________________________________3.1.2Purification of sCD83 Using HPLC 19______________________________________________ 3.1.3Electrophoretic Analysis of sCD83 Applying Coomassie Staining, Immunoblotting and Determination of Protein Glycosylation ________________ 21_ _________________________________ 3.1.4Functionalin vitro 23Test for sCD83 in the Murine System_____________________________ 3.2Generation of Tissue-Specific CD83 Knock-out Mice 25_________________________ 3.2.1Generating of CD83 Tissue-Specific Knock-out Mice Using the Cre/loxPStrategy of ion yst _______________________________________________________________ 25Recombinat S em 3.2.2In Vitro ______________Experiments to Establish CD83 Tissue-Specific Knock-out Mice 26__ of the T g ting Vector ___________________________________________ 26ar e of Three ES Cell Lines Transfected with pRapidflirt-CD83 Applying Different PCR Strategies _______________ 27____________________________________________________ of the Nested PCR Analyses of Transfected Bruce4, V6.5 and JM8A3 ES Cell Lines 303´loxP PCR Analysis of Bruce4, V6.5 and JM8A3 ES Cell Clones _______________3.2.2.3Screening of Three ES Cell Lines Transfected with pRapidflirt-CD83 Applying Two Different Southern Blot Strategies ____ ________________________________ 31________________ Bruce4, V6.5 and JM8A3 ES Cell Clones ___________ 315´Southern Blot Analyses Bruce4, V6.5 and JM8A3 ES Cell Clones ___________ 333´Southern Blot Analyses 3.2.3In Vivo 35 _________________Experiments to Establish CD83 Tissue-Specific Knock-out Mice3.2.3.1 astocytes or Mor ________________Microinjection of Transfected ES Cells into Bl ulae 353.2.3.2Genetic ly ______________________________________________ Ana ses of Chimeras 373.2.3.3Genetic Analyses of the Offspring from the 40% and 70% V6.5 Chimera ____________ 383.2.3.4 39Genetic Analyses of the JM8A3 Offspring ____________________________________ Discussion 41____________________________________________________________ 4.1 41 ______________________________Purifying Human sCD83 from HEK 293T Cells4.1.1Cloning and Expression of Eukaryotic Recombinant sCD83 ___________________________ 414.1.2Qualitative Ana y Recombinant sCD83______________________________ yotic l ses of Eukar 424.1.3 __________________________________________ 43Glycosylation Sites of Eukaryotic sCD834.1.4Eukaryotic Recombinant sCD83 Inhibits DC-Mediated T Cell Proliferation 44 _______________
p cific CD83 Knock-out Mice _________________________ 4.2 45Generation of Tissue-S e4.2.1The Cre-loxP Recombination System _____________________________________________ 454.2.2 46Selection of ES Cell Lines_____________________________________________________ 4.2.3 46Nested PCR and 3´and 5´Southern Blotting________________________________________ 4.2.4Genetic Analyses of Chimeras and Of p ing _______________________________________ fs r 484.2.5 ________Tissue-Specific CD8 Ongoing Experi : 3 Knock-out Mice ments and Further Outlook 49
___________________________________________________ Materials & Methods 51
5.1Materials 51______________________________________________________________ 5.1.1Laboratory Equipmen _________________________________________________________ t 515.1.2Expendable Materials 52_________________________________________________________ 5.1.3Chemicals 52__________________________________________________________________ 5.1.4Radioactive Chemicals 53________________________________________________________ 5.1.5Plasmids 53___________________________________________________________________ 5.1.6 ____________________________________Primers for DNA Amplifications 53____________ 53Cloning Fragments for Tissue-Specific CD83 Knock-out Mice____________________ 53ES Cell Screening for Nested PCR __________________________________________5.1.6.3 545´ Southern Blot Probes___________________________________________________´ Southern Blot Probes 54___________________________________________________ issue-S3´loxP PC 54 C ecificR Screening of T p D83 Knock-out Mice __________________ 5.1.7 ___________________________________________________________________E ym nz es 545.1.8Western Blot Antibodies 54_______________________________________________________ 5.1.9 55Weight Marker for DNA and Protein Gel Electrophoresis _____________________________ 5.1.10 55Strain of Bacteria__________________________________________________________ 5.1.11Mice 55____________________________________________________________________ 5.1.12 yo ____________________________________________Murine Em br nic Stem Cell Lines 555.1.13Human Cell Line 55__________________________________________________________ 5.1.14 56Cell Medium and Additives__________________________________________________ 5.1.15Media for Primary Cells and Cell Lines _____________________ 56Specific Composition of 5.1.16 57Buffers and Solutions for DNA Purification and Detection__________________________ 5.1.17Buffers for Protein Expression and Detection 58 ____________________________________5.1.18 ________________________________________________ 59Commercially Purchased Kits5.1.19Computer Programs ________________ 59 ________ ________________________________
_______________________________________________________________ 5.2Methods 605.2.1 60Eukaryotic Expression of Recombinant Soluble CD83 (sCD83) ________________________Culture for Human Embryonic Kidney (HEK) ____________________ 605.2.1.1 CellsCell 293T ______________________________________ 60rvation of HEK 293T Cells 60Thawing HEK 293T Cells_______________________________________________5. of HEK 293T Cells ________________________________ 60__ __________ of the Extracellular Domain of Human CD83 61___________________________ 61Cell Transfection Using Calcium-Phosphate ___________________________________ 61of Recombinant sCD83 from HEK 293T CellsIsolation _________________________ Measurement Using the Bradford Assay________________________________ 625.2.1.6 is _________________________________________________Immunoblotting Analy s 625.2.1.7Co Staining ______________________________________________________ omassie 635.2.1.8 ______ hyGlycosylation Sites of sCD83 Using Reductive CarboxymAnalysis of et lation 635.2.1.9Generation of Murine Bone Marrow-Derived Dendritic Cells 63_____________________ ____Allogeneic T cell Proliferation 64_________________________________________ 5.2.2Generation of CD83 Conditional Knock-out Mice 64___________________________________ Chain Reaction (PCR) __________________________________________ 645.2.2.2Cloning of the Vector CD83 pRapidflirt ______________________________________ 655. 65 _________________________________Storage of Bacteria for Plasmid Preparation5. 65Transformation of Chemically Competent Bacteria ___________________________5. for Ana 65Plasmid Pre tionpara lytic Purposes__________________________________ Preparation for Quantitative Purposes_______________________________ 665. of DNA Using Restriction Enzymes 66_______________________________ 6Ligation of DNA Fragments into pRapidf ________________________________ lirt 665. of DNA from Agarose Gels ______________________________________ 665. Gel Electrophoresis_____________________________________________ 665. of DNA Concentrations 67_____________________________________ 67for Cryopreserved Embryonic Stem (ES) Cells ________________Working Procedures
8 67Cryopreservation of Embryonic Fibroblast (EMFIs) and (ES) Embryonic Stem Cells5. 67 __________________Thawing of Embryonic Fibroblast Cells (EMFIs) and ES Cells5. of Embryonic Fibroblast Cells (EMFIs)___________________________ 685. of EMFIs 68__________________________________________________ of Embryonic Stem Cells (ES cells)______________________________ 685.2.2.4Transfection of ES Cells 68__________________________________________________ 68Linearization and Precipitation of Sterile DNA______________________________ rop ________________________________ 695. of ES Cells via Elect oration of Transfected ES Cell Clones 69____________________________________ 69Purification of DNA from ES Cell Suspension_______________________________ Positive Homologue Recombinated ES Cells __________________________ 705. Polymerase Chain Reaction (PCR) __________________________________ 705. 71Southern Blot________________________________________________________ 71Isolation of Genomic DNA from ES Cell Clones_________________________ g_______________________________Gel Electrophoresis for Southern Blo ttin 715. Blotting_ 71_________________________________________________ 72Amplification of the Hybridization Probes______________________________ and Purification of the Hybridization Probes 72_________________ 72Hybridization of the Probes with the Membrane__________________________ ________________________________ 72Washing the Membrane______ _______ aging __________ to Visualize DNA Hybridiza hoim tion on the Membrane 735.2.2.6 73Blastocyte and Morula Injection ____________________________________________5. 73Preparation of the ES Cells for Blastocyte Injection___________________________ .2Blastocyt d Morula Injecti __________________________________________ e an on 735. _______Screening of Floxed Chimeras and Their Offspring ____ 73________________
Ref _________________________________________________________ erence List 74
_______________________________________________________________ Indices 82
___________________________________________________ Lists of Abbreviations 82
of g __________________________________________________________ List Fi ures 84
List of Tables 85__________________________________________________________
Acknowledgements _____________________________________________________ 86
1.1 Summary The glycosylated cell surface protein CD83 with a molecular mass of 45kDa belongs to the
immunoglobulin super family. CD83 is expressed on Langerhans-, interdigitating reticulum
cells and activated lymphocytes. Moreover, CD83 is the best known surface marker for
human mature dendritic cells (DCs). CD83 exists in two forms, the membrane bound CD83
(mCD83) and the soluble form (sCD83). The mCD83 consists of a transmembrane, an
intracellular region and an extracellular part with a single V-type immunoglobulin domain
and also exhibits N-glycan binding sites. In contrast, sCD83 exclusively consists of the
extracellular part. While mCD83 shows immune stimulatory capacities, sCD83 displays inhibitory functions affecting immune processes. For instance, mCD83 influences the CD4+T
cell and B cell development and function. sCD83 has inhibitory properties, affecting DCs
maturation and DC-mediated T cell stimulation as well as diminishing clinical symptoms in
the experimental autoimmune encephalomyelitis and immunosuppression in transplantation
models, preventing rejection of transplanted skin, kidney and heart. Recently, the induction of
tolerogenic DCs by sCD83 was observed which increase the number of regulatory T cellsin
vivo. Hence CD83 is a highly interesting molecule for prospective clinical investigations and
the need to elucidate the specific functions on different cell types for mCD83 and sCD83 is a
prerequisite for its clinical application. Two projects were addressed in this PhD thesis.
The first project dealt with the generation, purification and functional characterization of
recombinant sCD83 molecules expressed in human embryonic kidney (HEK) 293T cells. In
this eukaryotic expression system, sCD83 can be purified without the co-isolation of
lipopolysaccharide. Moreover the protein is glycosylated in contrast to sCD83 produced from
a prokaryotic expression system. In addition, production of sCD83 in the eukaryotic system
was less time consuming and provided a higher protein yield in comparison to the previously
reported prokaryotic expression system.
In the second project, CD83 tissue-specific knock-out mice were generated using the Cre/loxP
recombination system. Therefore,loxPwere inserted within exon 1 and 2 of the five  sites
exons of CD83 (floxed) in embryonic stem (ES) cells. Three different ES cell lines -JM8A3,
V6.5 and Bruce4- were therefore used. Positive ES cell clones were then injected in murine
embryonic blastocytes or morulae. A JM8A3 chimera exhibited germline transmission and
generated floxed offspring which will now be mated with CD11c-Cre (DCs), mb1-Cre (B
cells), CD4-Cre (T cells) and foxp3-Cre (regulatory T cells) mice, hence deleting CD83 only
on a specific cell type.
With these two interesting projects, specific functions and fundamental mechanisms of CD83
can be elucidated in different cell types. Gaining such new information, clinical applications
of CD83 are in reach for the future.
1.2 amusZgnusssafnem Das glykosylierte Transmembranprotein CD83 mit einer Molekülmasse von 45kDa gehört zur
Immunoglobulin-Superfamilie. CD83 wird von Langerhans- und Retikulumzellen, sowie von
aktivierten Lymphozyten exprimiert. Darüber hinaus ist CD83 vor allem als eines der besten
Oberflächenmarkerproteine auf humanen, reifen dendritischen Zellen (DZ) bekannt. CD83
liegt sowohl als membrangebundene (mCD83) als auch in löslicher Form (sCD83) vor. Dabei
besteht mCD83 aus einer transmembranen-, einer zytosolischen und einer extrazellulären
Domäne, welche eine V-Typ ähnliche Immunoglobulindomäne sowie N-glycan
Bindungstellen aufweist. Dagegen besteht sCD83 alleinig aus dem extrazellulären Teil des
Proteins. Während mCD83 immunstimulatorisches Potential besitzt, wirkt sCD83 inhibitorisch auf das Immunsystem, zum Beispiel beeinflusst mCD83 die CD4+ und B- T-Zellentwicklung und funktion. sCD83 dagegen hat einen inhibitorischen Effekt auf DZ und
T-Zellen, welcher sich sowohl auf die Reifung von DZ und die DZ-vermittelte T-
Zellstimulation auswirkt. Darüber hinaus unterdrückt sCD83 die klinischen Symptome der
experimentellen autoimmunen Enzephalomyelitis und verhindert die Abstoßung von Haut,
Nieren und Herztransplantaten. Zudem wurde kürzlich nachgewiesen, dass sCD83 tolerogene
DZ induziert undin vivodie Anzahl der sogenannten regulatorischen T-Zellen erhöht. CD83 ist somit ein hoch interessantes Molekül für zukünftige klinische Anwendungen, das
allerdings weitere Aufklärung von Funktionen und Mechanismen voraussetzt. Zwei
Forschungsansätze wurden in dieser Doktorarbeit realisiert.
Zum einen wurde rekombinantes sCD83 in humanen embryonalen Nieren (HEK) 293T-
Zellen hergestellt, aufgereinigt und funktionell getestet. In diesem eukaryotischen System
kann sCD83 ohne die Ko-Isolierung von Lipopolysaccharid aufgereinigt werden und ist im
Gegensatz zu Protein aus einem prokaryotischen Expressionssystem, wie in der Natur
vorkommend, glykosyliert. Zudem erwies sich die Aufreinigung in Eukaryoten zeitsparender
und die Ausbeute des Proteins lag höher als bei einer entsprechenden Aufreinigung aus
Im zweiten Forschungsansatz wurden gewebespezifische CD83 Knock-out Mäuse mit Hilfe
des Cre/loxP Systems generiert. Hierfür wurdenloxP um Exon 1 und 2 der fünf Sequenzen
Exons von CD83 ins Genom von embryonalen Stammzellen (ES-Zellen) eingefügt
(floxed). Als ES Zelllinien dienten JM8A3, V6.5 und Bruce4. Positive ES-Zellen wurden
daraufhin in murine Blastozysten und Morulae injeziert. Eine JM8A3 Chimäre zeigte
Keimbahntransmission und erzeugte gefloxte Nachkommen, welche nun mit CD11c-Cre
(DZ), CD4-Cre (T-Zellen), mb1-Cre (B-Zellen) und foxp3-Cre (regulatorische T-Zellen)
Mäusen verpaart werden, um CD83 auf einem spezifischen Zelltyp zu depletieren.
Hilfe dieser Ergebnisse
können nun spezifische Funktionen und grundlegende
Mechanismen von CD83 in den verschiedenen Zelltypen untersucht werden und tragen dazu
bei, CD83 einen Schritt näher an klinische Anwendungen zu bringen.
2.1 The Immune System
The immune system in mammals is a complex organization of different lymphatic organs and
immune cells which help to successfully defend the mammalian organism from pathogens
like viruses, bacteria, fungi and parasites. The immune cells are derived from pluripotent,
hematological progenitors located in the bone marrow. The cells are classified into myeloid
and lymphatic precursors. Macrophages, mast cells, granulocytes, neutrophils and dendritic
cells (DCs) arise from the myeloid precursor. In contrast, B and T cells derive from the
lymphatic precursor (Janeway CA et al., 2002).
The immune cells have different functions in respect to defense mechanisms commonly
known as the innate and adaptive immune response. In case of the innate immune response,
invasion of microorganisms is battled by macrophages and neutrophils as a first line of
defense. These cells can detect a variety of surface molecules from microorganisms and
eliminate them by incorporation into their cell body followed by degradation. Their defense
ability however is constrained as they only express highly conserved surface receptors to
corresponding surface molecules of the microorganisms. Through evolution however,
microorganisms found escape mechanisms to circumvent the innate immune response
(Janeway CA et al., 2002).
Therefore, as a second line of defense, the adaptive immune response functions in a much
more flexible way in mammals. Dendritic cells (DCs) play a major role in this mechanism as
they incorporate pathogenic materials without the need of a specific receptor for detection.
Once DCs incorporated pathogenic material they get activated, mature and migrate to the
draining lymph nodes. There, they present the incorporated and processed materials on
histocompatibility complex (MHC) class I and II molecules to T cells. A T cell which
recognizes the presented antigen via its specific T cell receptor gets activated and starts to
proliferate (theory of clonal selection) and then can specifically eliminate the immunogenic
pathogen (Hodgkin et al., 2007). In addition, T cells can also activate B cells engaging to their
specifically determined B cell receptor and hereupon B cells secrete antibodies to form
complexes with the pathogen (Janeway CA et al., 2002).
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