Target recognition by natural killer cells [Elektronische Ressource] : identification and characterization of inhibiting and activating factors / presented by Sabrina Hoffmann

Sabrina Hoffmann

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences












presented by

Diplom-Biologin
Sabrina Hoffmann
born in: Heidelberg

Oral-examination: ................................................





Target Recognition by Natural Killer Cells
-
Identification and Characterization of Inhibiting and Activating Factors





















Referees: Prof. Dr. Günter J. Hämmerling
Prof. Dr. Carsten Watzl

1


Science may set limits to knowledge, but should not set limits to imagination.
(Bertrand Russell)

2 Table of Contents
Table of Contents

Acknowledgments........................................................................................................5

Summary (English).......................................................................................................6

Zusammenfassung (Deutsch)......................................................................................7

I.Introduction.................................................................................................................8
I.1 Natural killer cell biology......................................................................................................8
I.2 Inhibitory NK cell receptors................................................................................................12
I.3 Activating NK cell receptors...............................................................................................14
I.4 The lectin like family of receptors......................................................................................16
I.5 The family of Natural Cytotoxicity Receptors.....................................................................17
I.6 A game of hide and seek – NK cells and cytomegalovirus................................................20

Aim of the thesis.........................................................................................................24

M. Materials and Methods..........................................................................................25
M.1 Material............................................................................................................................25
M.1.1 Antibodies.........................................................................................................25
M.1.2 Bacteria.............................................................................................................26
M.1.3 Buffers, Chemicals and reagents......................................................................26
M.1.4 Cells..................................................................................................................28
M.1.5 Virus strains......................................................................................................29
M.1.6 Constructs.........................................................................................................29
M.1.7 Enzymes...........................................................................................................30
M.1.8 Oligonucleotides................................................................................................30
M.2 Methods...........................................................................................................................31
M.2.1 Molecular Biology..............................................................................................31
M.2.2 Cell Biology.......................................................................................................32
M.2.3 Protein Biochemistry.........................................................................................37

R. Results...................................................................................................................39
R.1 Characterization of CLEC12B..........................................................................................39
R.1.1 Identification of CLEC12B.................................................................................39
3 Table of Contents
R.1.2 NKG2D and CLEC12B do not recognize the same ligands..............................41
R.1.3 CLEC12B is expressed on myeloid cells...........................................................42
R.1.4 CLEC12B can function as an inhibitory receptor...............................................43
R.1.5 CLEC12B signals through recruitment of SHP-1 and SHP-2............................44
R.2 NKp30 ligands are downregulated upon cytomegalovirus infection.................................46
R.2.1 Downregulation of NKp30L................................................................................46
R.2.2 An immediate early/early HCMV gene product is responsible for NKp30L
downregulation............................................................................................................47
R.2.3 Reduced NKp30-dependent lysis of HCMV infected fibroblasts.......................49
R.3 Identification of NKp30 ligands.........................................................................................52
R.3.1 Heparan sulfate is not the ligand for NKp30......................................................52
R.3.2 Characterization of NKp30L..............................................................................54
R.3.3 Identification of NKp30L through an siRNA library............................................56

D. Discussion.............................................................................................................61
D.1 Identification of CLEC12B................................................................................................61
D1.1 CLEC12B functions............................................................................................61
D.1.2 Endogenous CLEC12B expression...................................................................62
D.2 Human Cytomegalovirus downregulated NKp30L...........................................................62
D.2.1 Downmodulation of NKp30L might be a new immune evasion mechanism......62
D.2.2 How is NKp30L downmodulated?.....................................................................63
D.3 Characteristics and Identification of NKp30L...................................................................65
D.3.1 Heparan sulfate is not the ligand for NKp30L....................................................65
D.3.2 NKp30L involves a cell cycle regulated protein component..............................66
D.3.3 Identification of NKp30L through an siRNA library............................................66

References.................................................................................................................71

Abbreviations..............................................................................................................82

Publications................................................................................................................84

4 Acknowledgments
Acknowledgments

At first, I’d like to thank Prof. Dr. Carsten Watzl. Thank you not only for supervision,
helpful discussions and recommendations but for your constant support. Your good
spirits and your persistence have always been an example to me. Thank you for
leaving your door open at all times.
Furthermore, I highly appreciate that Prof. Dr. Günter J. Hämmerling agreed to
represent this scientific work in the Faculty of Natural Sciences of the University of
Heidelberg.
Of course I won’t forget all the past and present members of the Watzl lab: Philipp
Eißmann, Johanna Endt, Rüdiger Flaig, Stephan Meinke, Doris Urlaub, Maren Claus,
Stefanie Margraf, Kristine Kohl, Birgitta Messmer, Sabine Wingert, Patrick Rämer
and Mina Sandusky. There were times when I wanted to leave this whole mess and
just stayed because of you. Thank you my friends for bearing my temper, bolstering
me up and for coming to me for advice.
I’d also like to thank all members of the Institute for Immunology. Wherever I went for
advice I’ve been welcomed and helped without hesitation. The friendly atmosphere in
this institute is an outstanding example. A special thank you goes to Dr. Guido
Wabnitz who never accepts mediocre experiments and always pushed me to do my
best.
Without help and support from the persons in my private life this thesis would never
have been possible. My parents and family have always been a safe haven to return
to and they always believed in me. Last but certainly not least, I want to thank
Marcus. I don’t find words to express my love and gratitude to you. Without you I
wouldn’t be the person I am today and I’d certainly not be that happy.

Thank you all!

5 Summary
Summary
Natural killer (NK) cells are able to attack and destroy tumorous or virally infected
c