Characterization of tolerogenic rat bone marrow-derived dendritic cells and regulatory T cells [Elektronische Ressource] = Charakterisierung tolerogener dendritischer Knochenmarkszellen und regulatorischer T-Lymphozyten aus der Ratte / submitted by Anja Matuschek

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Characterization of tolerogenic rat bone marrowderived dendritic
cells and regulatory T cells

Charakterisierung tolerogener dendritischer Knochenmarkszellen
und regulatorischer TLymphozyten aus der Ratte



Doctoral thesis for a doctoral degree
at the Graduate School of Life Sciences,
JuliusMaximiliansUniversität Würzburg,
Section Infection and Immunity


submitted by
Anja Matuschek
from
Lauchhammer


Würzburg, 2010







Submitted on:




Members of the Promotionskomitee:

Chairperson:
Primary Supervisor: Priv.Doz. Dr. rer. nat. Christoph Otto
Supervisor (Second): Priv.Doz. Dr. rer. hum. biol. Ulrike Kämmerer
Supervisor (Third): Prof. Dr. rer. nat. Roland Benz








Date of Public Defence:
Date of Receipt of Certificates:
















Für meine
Eltern, Großeltern und Clemens




Die größte Tragödie in der Wissenschaft überhaupt
ist der Tod einer wunderschönen Hypothese
durch die Hand einer hässlichen Tatsache.

T. H. Huxley (1825 - 1895)
Table of Contents
Table of Contents

1 Abbrevations 1

2 Summary 3

3 Zusammenfassung 6

4 Introduction 9

4.1 The immune system 9

4.1.1 The innate immune system 10

4.1.2 The adaptive immune system 10

4.2 Dendritic cells 11

4.2.1 Dendritic cells: differentiation from precursor 12
populations

4.2.1.1 Plasmacytoidderived DC (PDC) 12

4.2.1.2 Myeloidderived DC (MDC) 13

Dendritic cell maturation stages 14 4.2.2

Immature DC 4.2.2.1 14

4.2.2.2 Semimature DC 15

Mature DC 15 4.2.2.3

Dendritic cellmediated T cell activation 16 4.2.3

4.3 T cells 19

Differentiation of effector T cells 4.3.1 19

4.3.1.1 T 1 cells 19 H

4.3.1.2 T 2 cells 19 H

4.3.1.3 T 17 cells 20 H

4.4 Central and peripheral T cell tolerance 21

4.4.1 Central tolerance 21

4.4.2 Peripheral tolerance 22

4.4.3 Regulatory T (T ) cells 23 reg

4.4.3.1 Naturally occurring T cells 23 reg

4.4.3.2 Induced T cells 25 reg

4.5 Tolerogenic DC and T cells 26 reg

Table of Contents

Tolerogenic DC and T cells in autoimmunity, 29 4.5.1 reg
transplantation and tumorimmunity

4.5.1.1 Autoimmunity 29

4.5.1.2 Transplantation 30

4.5.1.3 Tumorimmunity 32

5 Aims of the study 34

5.1 Questions 35

6 Materials 36

6.1 Animals 36

6.2 Antibodies 36

6.3 Primer for Reverse transcriptasepolymerase chain 38
reaction (RTPCR)

6.4 Chemicals, reagents, cytokines and commercial kits 39
6.5 Culture medium, buffer und stock solution 41

6.6 Equipment and aids 43

7 Methods 44
7.1 Preparation of bone marrowderived immature dendritic 44
cells

7.2 Isolation of mature splenic dendritic cell population 44

7.3 Isolation of lymph node T cells 45

pos 7.4 Depletion of CD25 T cells from lymph node cell 45
suspension

7.5 Cell yield evaluation 45

7.6 Flow cytometric analysis 45

7.6.1 Phenotyping 45

7.6.2 Apoptosis assay 46

7.6.3 Quantitation of antigen uptake by dendritic cells 46

7.7 Cytokine analysis by ELISA (enzymelinked 46
immunosorbent assay)

7.8 Nitric Oxide (NO) assay 47

7.9 Proliferation assay for naϊve T cells 47

7.10 Inhibition assay 48

7.10.1 IL4 DC mediated inhibition of T cell proliferation assay 48
Table of Contents

7.10.2 T cellmediated inhibition after incubation with 48
immature IL4 DC

7.11 Restimulation assay 48

7.12 Reverse trancriptasepolymerase chain reaction (RTPCR) 49

7.13 Real time PCR 49

7.14 Western Blotting 50

7.15 Signal transduction analysis 50

7.16 Statistical analysis 51

8 Results 52

8.1 Generation of bone marrowderived immature IL4 DC 52

8.1.2 Evaluation of growth conditions required for the 52
generation of IL4 DC

8.2 Characterization of the phenotype of immature IL4 DC 56
generated with 5 ng/ml GMCSF and 5 ng/ml IL4

8.2.1 Cluster formation and changes in cell size during the 56
development of IL4 DC from bone marrow progenitors

8.2.2 Phenotypic changes during the development of 58
immature IL4 DC from bone marrow progenitors

8.2.3 IL4 DC demonstrate an immature phenotype: A 60
comparison of the surface expression of different
molecules by immature IL4 DC and mature SDC

8.2.4 RTPCR analysis revealed specific mRNA for MHC 64
class II molecules, costimulatory molecules, immune
regulatory and suppressive cytokines and iNOS

8.2.5 Bone marrowderived IL4 DC demonstrate endocytic 66
activity

8.3 Characterization of the IL4 DCmediated inhibition of T cell 68
proliferation

8.3.1 IL4 DC are unable to activate naïve T cells 68
8.3.2 IL4 DCmediated inhibition of T cell activation is 69
associated with increased levels of TGFβ and reduced
levels of IFNγ and IL2

8.3.3 IL4 DCmeditated inhibition of preactivated T cell 71
proliferation

8.3.4 IL4 DCmediated inhibition of mature SDCinduced T 72
cell proliferation in cocultures


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8.3.5 T cell proliferation stopped in the presence of IL4 DC 74
within 24 hours

8.3.6 IL4 DC are phenotypically and functionally resistant to 76
maturation induced by TNFα, LPS and CD40L

8.4 Why IL4 DC inhibit T cell proliferation so fast? Some 80
experiments for explanation

8.4.1 Supernatant of IL4 DC cultures inhibits SDCinduced 80
T cell proliferation

8.4.2 82 IL4 DC secrete TGFβ, not IL10 and NO

8.4.3 84 TGFβ, not IL10, is detectable in competition assays
with IL4 DC

8.4.4 LPSstimulated IL4 DC secrete NO 85

8.4.5 Inhibition of LPSinduced NO secretion does not 86
influence IL4 DC mediatedinhibition of T cell
proliferation

8.4.6 IL4 DC do not induce T cell apoptosis 89

8.4.7 IL4 DC express the inhibitory molecules PDL1 and 90
PDL2
8.4.8 PDL1 and PDL2 surface expression is decreased by 92
inhibition of MEKpathway but does not influence IL4
DCmediated inhibition of T cell proliferation

8.5 Immature IL4 DC induce T cells with regulatory properties 94
and T celllike phenotype reg
8.5.1 IL4 DCinduced expansion of naturally occurring T 94 reg
cells

8.5.2 IL4 DCmediated induction of anergic T cells 97
8.5.3 IL4 DCinduced anergic T cells demonstrate immune 99
inhibitory properties
9 Discussion 103


9.1 The outcome of a specific IL4 DC population depends on the 103
combination of the cytokines GMCSF and IL4

9.2 Immature IL4 DC are resistant to different maturation stimuli 108
9.3 Implication for a soluble factor responsible for the IL4 DC 110
mediated suppressive effect

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9.4 PDL1 and PDL2 signalling on IL4 DC: a possible role in 113
tolerance induction?

9.5 IL4 DC are able to induce anergic T cells with immune inhibitory 114
properties: implications for the generation of T cells reg

10 Conclusions 118

11 References 122

12 Appendix 146

13 Index of Figures 155

14 Index of Tables 157

Original scientific publications 159

Poster presentations 159

Oral scientific talks 160

National and international congresses and workshops 161

Awards and scholarships 161

Curriculum Vitae 162

Education 162

Danksagung 163

Affidavit 164



















1 Abbrevations
1 Abbrevations


APC Antigenpresenting cell

BMDC Bone marrowderived dendritic cell

BN Brown Norway

CD Cluster of differentiation

cpm Counts per minute

CTL Cytotoxic T cell

DC Dendritic cell

FACS Fluorescence activated cell sorting

FCS Fetal calf serum

FITC Fluorescein Isothiocyanate

FSC Forward scatter

IFNγ Interferongamma

IL2 Interleukine2 <