Development of regulatory T-cells and induction of mucosa specific homing [Elektronische Ressource] / Christiane Siewert

Siewert, Christiane

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Development of regulatory T cells and induction
of mucosa-specific homing
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Christiane Siewert
Master of Science (Immunology)
(geb. 03.01.1966 in Bergheim)
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I:
Prof. Dr. Christian Limberg


Gutachter: 1. Prof. Dr. Alf Hamann
2. Prof. Dr. Richard Lucius
3. Prof. Dr. Hans-Dieter Volk


Tag der mündlichen Prüfung: 16. Mai 2007 Table of Contents
Zusammenfassung
Abstract
List of Abbreviations
1 Introduction ............................................................................................................................................. 1
+1.1 CD4 regulatory T cells....................................................................................................................... 2
+1.1.1 Features of CD4 Tregs .................................................................................................................... 2
1.1.2 Mode of action .................................................................................................................................. 3
1.1.3 Foxp3................................................................................................................................................. 4
+
1.1.4 Origin of CD4 Tregs ....................................................................................................................... 5
+1.1.5 Integrin α β expression on CD4 Tregs ........................................................................................ 6 E 7
+1.1.6 Peripheral maintenance of CD4 Tregs........................................................................................... 8
1.1.7 Mucosal immune homeostasis ......................................................................................................... 8
1.2 Migration of T cells ...........................................................................................................................10
1.2.1 The multistep model .......................................................................................................................10
1.2.2 Molecules involved in the transmigration cascade.......................................................................10
1.2.3 Organ-selective migration..............................................................................................................12
1.2.3.1 Migration and homing of naïve T cells to lymphoid tissue ......................................................12
1.2.3.2 Migration and homing of effector/memory T cells to non-lympoid tissue..............................13
1.2.4 Regulation of organ-specific migration.........................................................................................14
1.3 Aims and objectives...........................................................................................................................16
2 Materials and Methods .........................................................................................................................17
2.1 Material...............................................................................................................................................17
2.1.1 Material and Reagents ....................................................................................................................17
2.1.2 Buffers and Media ..........................................................................................................................18
2.1.3 Instruments......................................................................................................................................18
2.1.4 Staining reagents and antibodies for flow cytometry ...................................................................19
2.1.5 Mice .................................................................................................................................................19
2.2 Methods ..............................................................................................................................................20
2.2.1 Isolation of lymphocytes from mouse tissue.................................................................................20
2.2.2 Isolation of lymphocyte subsets by MACS and FACS ................................................................20
+2.2.2.1 Isolation of naïve CD4 T cells...................................................................................................21
-2.2.2.2 Isolation of α lymphocytes for adoptive transfer....................................................................22 E
+2.2.2.3 Isolation of naïve CD4 T cell subsets for homing receptor induction in vitro.......................22
2.2.2.4 Isolation of tissue-specific DCs for homing receptor induction in vitro..................................22
2.2.3 Flow cytometric analysis of lymphocytes.....................................................................................23
2.2.3.1 Fluorescent labelling of surface molecules on lymphocytes ....................................................25
2.2.3.2 Intracellular labelling of Foxp3 ..................................................................................................25
2.2.3.3 Intracellular staining of BrdU .....................................................................................................26
2.2.3.4 Enumeration of lymphocyte numbers by flow cytometry ........................................................26
2.2.3.5 Labelling of T cells with CFSE ..................................................................................................27
2.2.4 In vitro culture and activation of T cells .......................................................................................28
2.2.4.1 Generation of Th1 cells...............................................................................................................28
2.2.4.2 In vitro proliferation assay ..........................................................................................................28
2.2.4.3 Induction of homing receptors on Tregs by tissue-specific DCs..............................................28
2.2.4.4 Induction of homing receptors on Tregs by soluble factors .....................................................29
2.2.5 In vivo experiments and adoptive transfer ....................................................................................29
2.2.5.1 In vivo labelling with BrdU.........................................................................................................29
2.2.5.2 Depletion of commensal microflora...........................................................................................30
2.2.5.3 Antigen-specific activation in vivo.............................................................................................30
2.2.5.4 Th1-mediated DTH model ..........................................................................................................31
2.2.5.5 Homing of adoptively transferred Tregs ....................................................................................31
2.2.5.6 Data management and statistical analysis..................................................................................32
3 Results....................................................................................................................................................33
3.1 Origin and development of Tregs .....................................................................................................33
3.1.1 Phenotype and in vivo proliferation of effector/memory-like Tregs ...........................................33
+ +3.1.1.1 α -expression on CD4 Foxp3 Tregs correlates with an effector/memory phenotype...........33 E
+3.1.1.2 α Tregs show high proliferation under homeostatic conditions............................................34 E
+3.1.1.3 α Treg numbers are stable in the absence of thymic output..................................................36 E
+ +3.1.1.4 Removal of intestinal microflora affects in vivo proliferation of CD4 Foxp3 Tregs ............37
+3.1.1.5 Lack of microbial stimuli results in reduced cell numbers of Foxp3 Tregs...........................39
+3.1.2 Generation of effector/memory-like Foxp3 Tregs by antigen-specific activation in vivo ......42
+ +3.1.2.1 In vivo conversion of naïve-like CD25 Tregs into α Tregs..................................................42 E
+ +3.1.2.2 In vivo generation of α Foxp3 cells from conventional naïve T cells ..................................44 E
+3.2 Induction of gut-specific homing receptor expression on Foxp3 Tregs .......................................48
3.2.1 Induction of gut-specific homing receptors by tissue-specific DCs............................................48
3.2.2 Induction of gut-specific homing receptors by soluble factors....................................................50
3.2.3 Phenotype and function of in vitro modulated Tregs ...................................................................52
3.2.4 Migratory behav