Immunological recognition of the SV40 T antigen in a mouse model [Elektronische Ressource] / Sonja Seewaldt

I. Förster

Sujets

• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene
der Technischen Universität München



Immunological recognition of the SV40 T antigen in a mouse model


Sonja Seewaldt


Vollständiger Abdruck der von der Fakultät für Chemie
der Technischen Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzende: Univ.-Prof. Dr. S. Weinkauf

Prüfer der Dissertation:
1. Univ.-Prof. Dr. J. Buchner
2. I.A. Förster
3. Dr. H. Kessler



Die Dissertation wurde am 30.04.2003 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 20.01.2004 angenommen.





Die Ergebnisse dieser Arbeit sind zum Teil veröffentlicht:
Seewaldt S., Alferink J., Förster I. (2002). Interleukin-10 is crucial for maintenance but
not for developmental induction of peripheral T cell tolerance. Eur. J. Immunol. 32,
3607-3616.

















Content I
TABLE OF CONTENTS

Contents I
Index of figures III
Abbreviations IV

1 INTRODUCTION ....................................................................................................... 1
1.1 Central tolerance 2
1.2 Peripheral tolerance ................................................................................................. 3
1.2.1 Clonal anergy........................................................................................................... 3
1.2.2 Clonal deletion......................................................................................................... 4
+ + 1.2.3 Regulatory CD25 CD4 T cells ............................................................................... 4
1.3 Role of cytokines in self-tolerance ........................................................................... 7
1.4 Tumor antigens 8
1.5 The model: RT2/TCR1 mice.................................................................................... 9
1.6 Aim of this PhD work ............................................................................................. 12
2 MATERIALS AND METHODS .............................................................................. 14
2.1 Materials.................................................................................................................. 14
2.1.1 Chemicals, radioactive substances, media............................................................. 14
2.1.2 Reagents and laboratory supplies .......................................................................... 15
2.1.3 Buffers. 15
2.1.4 Peptides and primers.............................................................................................. 16
2.1.5 Antibodies and second step reagents ..................................................................... 17
2.1.6 Mice ....................................................................................................................... 19

2.2 Methods.................................................................................................................... 20
2.2.1 Genotyping............................................................................................................. 20
2.2.2 Ab production, purification and in vitro testing .................................................... 21
2.2.3 Peptide/SEB treatment........................................................................................... 22
2.2.4 Organ removal ....................................................................................................... 22
2.2.5 APC isolation......................................................................................................... 22
2.2.6 Magnetic activated cell sorting (MACS) ............................................................... 22
2.2.7 Flow cytometry...................................................................................................... 23
2.2.8 Proliferation assays................................................................................................ 24
2.2.9 Cytokine detection ................................................................................................. 26
2.2.10 Histology and immunohistology.......................................................................... 26

Content II
3 RESULTS ................................................................................................................... 28
3.1 Role of IL-10 in peripheral tolerance induction .................................................. 28
3.1.1 Developmental induction of tolerance in the absence of IL-10............................. 28
KO3.1.2 Breakage of tolerance in RT2/TCR1/IL-10 mice by single antigenic challenge34
KO3.1.3 Lack of peptide-induced T cell tolerance in TCR1/IL-10 mice......................... 38
KO3.1.4 Increased sensitivity to bacterial superantigens in IL-10 mice.......................... 42
KO3.1.5 Combined treatment of RT2/TCR1/IL-10 mice with peptide and CpG-ODN to
induce tumor immunity ......................................................................................... 44
+ + +3.2 CD25 Id CD4 T cells of RT2/TCR1 mice have regulatory function in vitro... 50
3.3 Role of peripheral lymph nodes and LTβR in peripheral tolerance induction 53
3.3.1 LTβR deficient mice on the C3HeB/FeJ background contain rudimentary MLN
structures ............................................................................................................... 53
KO 3.3.2 Lack of SV40 T Ag-specific T cell tolerance in RT2/TCR1/LTβR mice.......... 56
+ + +3.3.3 Role of the thymus in the generation of regulatory CD25 Id CD4 T cells in
KORT2/TCR1 and RT2/TCR1/LTβR mice............................................................ 61
3.3.5 Additional findings: LN metastasis found in animals bearing the RT2 transgene 65
4 DISCUSSION............................................................................................................. 67
4.1 Role of IL-10 in peripheral tolerance induction .................................................. 67
4.2 Regulatory T cells in the RT2/TCR1 model ......................................................... 73
4.3 Dependency of peripheral tolerance induction on LTβR signaling ................... 77
4.4 Outlook / Future perspective ................................................................................. 82
5 SUMMARY ................................................................................................................ 84
6 REFERENCES........................................................................................................... 86
7 Acknowledgements...................................................................................................... 96
Index of Figures III
INDEX OF FIGURES
+ + KOFigure 1: Frequency of Id CD4 in thymus and periphery of wt and IL-10 mice...... 28
+Figure 2: SV40 T Ag-specific CD4 T cell tolerance in RT2/TCR1 and RT2/TCR1/IL-
KO10 mice.......................................................................................................... 30
+ + KOFigure 3: Cell division rates of Id CD4 T cells from wt and IL-10 mice. ................ 31
+ +Figure 4: Expression of activation/memory markers on Id CD4 T cells in RT2/TCR1
KOand RT2/TCR1/IL-10 mice........................................................................... 33
Figure 5: Loss of SV40 T Ag-specific tolerance upon antigenic challenge ................... 35
Figure 6: Expresssion of MHC class II and costimulatory molecules on APC of wt
KOversus IL-10 mice ......................................................................................... 37
Figure 7: Lack of peptide induced T cell tolerance in IL-10 deficient mice. ................. 39
+ +Figure 8: Quantification of cell division rates of Id CD4 T cells from TCR1 and
KOTCR1/IL-10 mice following in vivo peptide treatment. ............................... 41
Figure 9: SEB induced tolerance in wt animals.............................................................. 43
+ + KOFigure 10: Sustained responsiveness of Id CD4 T cells from RT2/TCR1/IL-10 mice
following longterm in vivo CpG-ODN/peptide treatment................................ 46
Figure 11: Blocking of IL-10 during in vivo peptide stimulation................................... 49
+ + +Figure 12: CD25 Id CD4 T cells of tolerant RT2/TCR1 mice show regulatory function
in vitro............................................................................................................... 51
KOFigure 13: LTβR mice possess lymph node-like mesenteric structures. .................... 54
KOFigure 14: Cellular composition of MLN and rudimentary MLN of wt versus LTβR
mice .................................................................................................................. 55
+ + KOFigure 15: Frequency of Id CD4 T cells in thymus and spleen of wt and LTβR mice.
.......................................................................................................................... 57
+ + KOFigure 16: Id CD4 T cel