Dysfunctional plasmacytoid dendritic cells but not NK cells in the peripheral blood of stage IV melanoma patients [Elektronische Ressource] / presented by Geok Choo, Sim

Vladimir Nicolas

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 38
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

INAUGURAL-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
Master of Science: Geok Choo, Sim
born in Johor, Malaysia









Dysfunctional Plasmacytoid Dendritic Cells but not NK cells in
the Peripheral Blood of Stage IV Melanoma Patients

























Referees: Prof. Dr. Rainer Zawatzky
PD. Dr. Annette Paschen





The present work was performed in the research group led by PD. Dr.Annette Paschen,
clinical cooperation unit for Dermato-Oncology, German Cancer Research Center
(DKFZ) and University Hospital Mannheim.

Publications during PhD work:

1. Annette Paschen, Antje Sucker, Bettina Hill, Iris Moll, Marc Zapatka, Xuan Duc
Nguyen, Geok Choo Sim, Isabelle Gutmann, Jessica Hassel, Jürgen C. Becker,
Alexander Steinle, Dirk Schadendorf and Selma Ugurel. Differential clinical
significance of individual NKG2D ligands in melanoma: soluble ULBP2 as an
indicator of poor prognosis superior to S100B. Clin Cancer Res 2009;15(16):5208–
15

2. Geok Choo Sim, Xuan Duc Nguyen, Christine Falk, Drik Boorken, Dirk
Schadendorf and Annette Paschen. Regulation of TLR9 responses in plasmacytoid
dendritic cells of melanoma patients. 2010 (Maunuscript in preparation).

Work presented in conferences:

3. G.C. Sim, D. Schadendorf, A. Paschen. The influence of tumour cells on the
crosstalk between activated or resting natural killer and dendritic cell. Abstract in
the proceedings of DC2007; P080, 2007.

4. Geok Choo Sim, Dirk Schadendorf and Annette Paschen. Differential Influence of
Tumor Cells and IFN- α on the Crosstalk between Activated or Resting Natural Killer
Cells and Dendritic Cells. Abstract in the proceedings of Natural Killer cells
symposium 2008; C23, 2008.

5. Geok Choo Sim, Dirk Schadendorf, Christine Falk, Annette Paschen. Serum
Cytokine, Chemokine and Growth Factor Profiles of Stage IV Melanoma Patient:
Defining Combinatorial Patterns. Abstract in the Keystone symposia: Mobilizing
Cellular Immunity for Cancer therapy, Utah; Jan 2009.

6. Geok Choo Sim, Dirk Schadendorf, Christine Falk, Annette Paschen. Defining
combinatorial patterns of chemokines, cytokines and growth factors in sera from
stage IV melanoma patients. Abstract in the proceedings of Cancer Imunotherapy
CIMT, P75; 2009.




Table of contents

Table of contents

Acknowledgement…………………………………………………… IX
Abstract………………………………………………………………. X
Zusammenfassung.................................................................................XII
List of abbreviations…………………………………………………. XIV
1.0 INTRODUCTION
1.1 Cancer…………………………………………………………………….. 1
1.1.1 Melanoma…………………………………………………………. 1
1.1.2 Melanoma therapies……………………………………………….. 2
1.2 The immune system………………………………………………………. 4
1.2.1 Innate immunity……………………………………………………. 5
1.2.2 Adaptive immunity……………………………………… 6
1.3 Dendritic Cells (DCs)…………………………………………………….. 7
1.3.1 DC development…………………………………………………… 8
1.3.2 Antigen uptake and processing by DC…………………………….. 9
1.3.3 DC as Antigen Presenting Cell……………………………………. 9
1.3.4 Human DCs……………………………………………………….. 10
1.3.5 mDC and NK interaction…………………………………………. 12
1.3.6 Plasmacytoid dendritic cells (pDCs)……………………………… 12
1.3.7 DCs in cancer……………………………………………………… 16
1.4 Natural Killer (NK) cells and their subsets…………………………….. 17
1.4.1 NK cell receptors…………………………………………………. 18
1.4.2 NK cells killing…………………………………………………….. 21
1.4.3 NK cells in cancer…………………………………………………. 21
1.5 TLR family and signalling………………………………………………. 22
1.5.1 TLR family………………………………………………………… 22
1.5.2 TLR signalling………………………………………………………24
1.5.3 TLRs expression on DC subsets…………………………………… 24
1.5.4 TLR9 ligands………………………………………………………. 25
1.6 Regulation of type I IFN production……………………………………. 26
1.6.1 Interferons…………………………………………………………. 26
1.6.2 The regulation of IFN-α/β production……………………………... 27
1.6.3 The role of transcription factors IRF3 and IRF7………………….. 29
1.6.4 ISGF3 signalling pathway…………………………………………. 31
1.7 Chemokines……………………………………………………………….. 31
1.7.1 Chemokine classification………………………………………….. 31
1.7.2 Chemokines in leucocytes recruitment…………………………….. 31
1.8 Aims of study ………………………………………………………………32


Table of contents

2.0 MATERIALS
2.1 Lab Equipments…………………………………………………………. 35
2.2 Chemicals…………………………………………………………………. 35
2.3 Consumables……………………………………………………………… 36
2.4 Enzyme/Reagents……………………………………………………….. 37
2.5 Antibodies for flow ctyometry and immunofluorescence staining…… 37
2.5.1 Primary Antibody and Fc fusion proteins…………………………. 37
2.5.2 Secondary antibodies………………………………………………. 38
2.6 Cell culture media and supplements…………………………………….. 38
2.6.1 Media………………………………………………………………. 38
2.6.2 Media supplements………………………………………………….38
2.7 Cells ……………………………………………………………………….. 38
2.7.1 Primary cells……………………………………………………….. 38
2.7.2 Human cell lines…………………………………………………… 38
2.8 Oligonucleotides…………………………………………………………. 39
2.8.1 Oligonucleotides used for standard PCR……………………………39
2.8.2 Labelled oligonucleotides used for quantitative real-time PCR…… 39
2.8.3 Oligonucleotides used for cell stimulation………………………… 39
2.9 Kits and standards………………………………………………………. 39
2.10 Multiple protein bead array (Luminex) cytokine assay ………………39
2.11 Buffers…………………………………………………………………… 39
2.12 Software………………………………………………………………….. 40

3.0 METHODS
3.1 Cell biology methods ………………………………………………………40
3.1.1 Sample collection…………………………………………………... 40
3.1.2 Cell culture method………………………………………………… 40
3.1.3 Preparation of human PBMC from buffy coats……………………. 41
3.1.4 Cryopreservation and thawing of cells……………………………...41
3.1.5 Dead cells removal………………………………………………… 42
+ + mDCs and BDCA4 pDCs 3.1.6 Isolation of BDCA1
from cryopreserved PBMCs using MACS eparation ………………42
+ +3.1.7 Co-culture of BDCA1 mDCs with BDCA4 pDCs………………. 42
3.1.8 PBMC stimulation for cytokines, chemokines and
growth factor analysis……………………………………………… 43
3.1.9 Immunofluorescence Staining………………………………………43
3.1.10 Confocal microscopy analysis and quantification of nuclear
translocation……………………………………………………….. 43
3.1.11 Analysis on NK and DC interactions……………………………… 44
3.1.11.1 Generation of monocyte-derived DC…………………… 44
3.1.11.2 Interaction of IL-2 activated NK cells with moDCs…….. 44
3.1.11.3 Interaction of IL-2 activated NK cells with mDC1……... 45
3.2 Immunological methods………………………………………………….. 45
3.2.1 Determination of cell surface antigen expression using flow
cytometry ………………………………………………………….. 45
3.2.2 Whole blood enumeration of mDCs and pDCs……………………. 46
Table of contents

3.2.3 Whole blood enumeration of NK cells…………………………….46
3.2.4 Cell sorting using flow cytometry…………………………………. 47
3.2.5 Detection of intracellular TLR9, MyD88, p4E-BP1 and IRF7
by flow cytometry analysis………………………………………… 47
3.2.6 Enzyme-link immunosorbent sandwich assay (ELISA)
for cytokine detection in cell culture supernatants………………… 48
3.2.7 ELISA for soluble NKG2DL detection in sera from
melanoma patients…………………………………………………..49
3.2.8 Degranulation assay………………………………………………... 49
3.2.9 Detection of intracellular IFN-γ and perforin by flow cytometry….. 49
3.2.10 Multiplex protein bead array assays for cytokines, chemokines and
growth factors detection……………………………………………. 50
3.3 Molecular biological method……………………………………………...50
3.3.1 RNA isolation from PBMCs……………………………………… 50
3.3.2 Quantitation of nucleic acids concentration using the 51
spectrophotometer…………………………………………………. 51
3.3.3 cDNA synthesis from total RNA………………………………….. 51
3.3.4 Polymerase chain reaction (PCR)………………………………….. 51
3.3.5 Quantitative real-time RT-PCR…………………………………… 52
3.4 Protein chemical methods……………………………………………….. 52
3.4.1 Generation of cell lysates………………………………………….. 52
3.4.2 Bradford protein assay…………………………………………….. 53
3.4.3 SDS poly-acrylamide gel electrophoresis and Western Blot……… 53
3.5 Statistical analysis 54

4.0 RESULTS
4.1 Absolute number and balance of circulating DC subsets in
advanced melanoma patients
4.1.1 Reduction of pDC and mDC2 but not mDC1 numbers in
melanoma patients………………………………………………… 55
4.1.2 Imbalance in blood DC compartments of melanoma patients……. 56

4.2 mDC1/pDC interaction upon TLR9 engagement in stage IV
melanoma patients
4.2.1 TLR9 triggering on pDCs leads to an up-regulation of CD40 on
co-cultured mDC1 from healthy donors but not from melanoma
patients…………