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Publié par | johannes_gutenberg-universitat_mainz |
Publié le | 01 janvier 2011 |
Nombre de lectures | 16 |
Langue | English |
Poids de l'ouvrage | 71 Mo |
Extrait
Exploring the function of IL -10, BTLA and
DCs as regulators of immune responses
Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaf t
Am Fachbereich Biolog ie
der Johannes Gutenberg -‐Universität Mainz
Nir Yogev
geb. am 31. Dezember 1973 in Kibutz Kineret, Isr ael
Mainz, 2010 ABBREVIATIONS................................ ................................ ................................ ................................ ..IV
1 INTRODUCTION ............................... 1
1.1 INTERLEUKIN -‐10 ........... 4
1.1. 1 IL-10 protein, gene and expression4
1.1. 2 IL-10 receptor and signaling ...........................................................................5
1.1. 3 IL-10R signal transduction...............6
1.1. 4 IL-10 Function........6
1.1.4.1 Antigen presenting cells ............ 6
1.1.4.2 T cell................................s ................. 7
1.1. 5 IL-10 in infectious disease8
1.1. 6 IL-10 in autoimmunity.......................9
1.1. 7 IL-10 and experimental autoimmune encephalomyelitis.................10
1.2 B AND T L YMPHOCYTE ATTENUATOR ................................ ................................ ................................ ....11
1.2. 1 Co -receptors of the CD28 and TNFR superfamilies.............................12
1.2. 2 BTLA structure, expression and signaling...............13
1.3 DENDRITIC CELLS ................................ ........15
2 MATERIALS AND METHOD S................................ ................................ ................................ .....17
2.1 CHEMICALS AND BIOLOGI CAL MATERIAL 17
2.2 M OLECULAR BIOLOGY 19
2.2. 1 Competent cels and isolation of plasmid DNA......19
2.2. 2 Isolation of genomic DNA from ES cells and mouse organs.............19
2.2. 3 RT -PCR and quantitative real -time PCR ...................................................20
2.2. 4 Agarose gel electrophoresis and DNA gel extraction.........................20
2.2. 5 DNA sequencing ..................................................................20
2.2. 6 Quantification of DNA......................21
2.2. 7 Polymerase Chain Reaction (PCR)..............................21
2.2. 8 Southern blot analysis22
2.3 CELL BIOLOGY .............. 23
2.3. 1 Embryonic stem cel culture ..........................................23
2.3. 2 Tat -Cre protein (HTNC) treatment.............................................................24
2.3. 3 EL-4 cell line culture and electroporation...............24
2.3. 4 Preparation of Mouse Embryonic Fibroblasts (MEFs).......................25
2.3. 5 Preparation of c ells from lymphoid organs............25
2.3. 6 Culture of ex vivo lymphocytes.....26
2.3. 7 Cel counting .........................................26
2.3. 8 Adoptive T cel transfer and CFSE labeling.............26
2.3. 9 Flow Cytometry...................................27
2.3.10 Flow Cytometry and Intracelular Cytokine Staining (ICS)...........28
2.3.11 Magnetic cel sorting and FACS sorting.................29
2.3.12 Isolation of CNS infiltrates29
2.3.13 Isolation of splenic DCs..................30
2.3.14 Induction of CD11c -CreER activity in vivo..........................................30 T
2.3.15 In vitro 2D2 -iTreg differentiation and analysis..30
2.3.16 T and iTreg in vitro Differentiation ......................................................30 eff
2.4 IN VIV DOEPLETION OF CD25+ CELLS .....31
2.5 IN VIV DOEPLETION OF DENDRITIC CELLS .............................. 31
2.6 TISSUE PREPARATION FOR IMMUNOH ISTOCHEMISTRY ........32
2.6. 1 Flow -Cytomix ........................................32
2.6. 2 ELISA........................32
II 2.7 M OUSE E XPERIMENTS ................................ ................................ ................................ ............................... 32
2.7. 1 Induction and assessment of EAE................................32
2.7. 2 Induction and assessment of Colitis............................33
2.7. 3 MCMV and MHV Infection ...............................................33
2.8 STATISTICS ................... 34
2.9 M ICE ................................ .............................. 34
3 RESULTS .......... 35
3.1 INTERLEUKIN -‐10 ................................ ........35
3.2 BTLA ............................ 57
3.2. 1 Generation of BTLA over expressing mouse............................................57
3.2. 2 BTLA over expression by dendritic cels...................62
3.2. 3 BTLA over expression by T cels ...................................70
3.3 DENDRITIC CELLS ........82
4 DISCUSSIO................................N ...99
4.1 INTERLEUKIN -‐10 ........99
4.2 B AND T L YMPHOCYTE ATTENUATOR ................................ .101
4.3 DENDRITIC CELLS ................................ .....105
5 REFERENCES ................................ ................................ 109
6 SUMMARY ................................ .....130
7 ZUSAMMENFASSUNG ................ 131
8 ACKNOWLEDGEMENTS ................................ ................................ ............ 132
9 LEBENSLAUF 133
10 PUBLICATIONS ................................ ......................... 134
11 ERKL ÄRUNG ................................ ................................ .............................. 135
III Abbreviations
Ab antibody
ALT al a ni ne transaminase
APC antigen presenting cell or allophycocyanin
approx. approximately
BAC bacterial artificial chromosome
Bio biotinylated
β-‐ME β-‐mercaptoethanol
bp base pair
BMDCs bone marrow derived dendritic cells
BSA bovine serum albumin
°C temperature in celsius degrees
CD cluster of differentiation
CFA Complete Freund’s Adjuvant
CFSE c arboxyfluorescein diacetate succinimidyl ester
cDNA complementary DNA
CNS central nervous system
cpm counts per