Immune regulation through homotypic T cell, T cell interaction [Elektronische Ressource] = Immunregulation durch homotypische T-Zell-T-Zell-Wechselwirkungen / vorgelegt von Katja Thümmler

Sujets

• Naturwissenschaften

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Immune regulation through
homotypic T cell/T cell interaction

Immunregulation durch
homotypische T-Zell/T-Zell-Wechselwirkungen








Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander Universität
Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.



vorgelegt von
Katja Thümmler
aus
Oelsnitz/Vogtland


Als Dissertation genehmigt
von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander Universität Erlangen-Nürnberg










Tag der mündlichen Prüfung: 22.04.2010

Vorsitzender der
Promotionskommision: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Thomas Winkler
Zweitberichterstatter: Prof. Dr. Dres. h.c. Joachim R. Kalden










Meinen Eltern und Schwestern


„Kein Fortschritt ist möglich
ohne Erweiterung des Herzens.“
Hans KruppaTable of contents
TABLE OF CONTENTS
1 SUMMARY ........................................................................................................... 1
2 ZUSAMMENFASSUNG ........................................................................................ 3
3 INTRODUCTION.................................................................................................. 5
3.1 T cell subsets ........................................................................................................................................................5
3.1.1 Th1/Th2 paradigm ........................................................................................................................................7
3.1.2 Expanding T cell effector subsets – Th17, Th22, Th9 cells.......................................................................7
3.1.3 Regulatory CD4 T cells ................................................................................................................................9
3.2 Homotypic T cell/T cell interaction................................................................................................................11
4 AIMS OF THE THESIS....................................................................................... 15
5 MATERIALS AND METHODS........................................................................... 16
5.1 Materials.............................................................................................................................................................16
5.1.1 Reagents, enzymes, cytokines....................................................................................................................16
5.1.2 Antibodies ...................................................................................................................................................17
5.1.3 TaqMan Gene Expression Assays for human genome.............................................................................20
5.1.4 Kits...............................................................................................................................................................20
5.1.5 Human cells.................................................................................................................................................21
5.1.6 Mice .............................................................................................................................................................21
5.2 Methods ..............................................................................................................................................................22
5.2.1 Purification of human cells.........................................................................................................................22
5.2.2 Mice and purification of murine cells........................................................................................................23
5.2.3 Flow cytometry ...........................................................................................................................................25
5.2.4 Cell cultures.................................................................................................................................................26
5.2.5 Generation of activated stimulator T cells ................................................................................................26
5.2.5.1 Human T cells .....................................................................................................................................26
5.2.5.2 Murine T cells .....................................................................................................................................26
5.2.6 Co-culture experiments of activated T cells with responder T cells .......................................................27
5.2.7 In vivo expansion of OVA-specific T cells ...............................................................................................27
5.2.8 CFSE labeling for the analysis of cell division.........................................................................................28
5.2.9 Fluorescence microscopy ...........................................................................................................................28
5.2.10 Preparation of total RNA, and amplification of cDNA by real-time PCR............................................29
5.2.11 Cytokine Enzyme-linked Immunosorbent Assay (ELISA)....................................................................30
5.2.12 Proliferation assay.....................................................................................................................................31
5.2.13 Statistical analysis.....................................................................................................................................31
6 RESULTS............................................................................................................ 32
6.1 Prerequisite for homotypic T cell/T cell interaction....................................................................................32
6.1.1 Generation of stimulator CD4 T cells........................................................................................................32
6.1.2 Activated CD4 T cells express surface receptors characteristic for APCs..............................................33
6.1.3 Cytokine secretion after T cell activation under different polarizing conditions ...................................34
6.2 Consequences of homotypic T cell/T cell interaction ..................................................................................35
6.2.1 Proliferation.................................................................................................................................................35
6.2.2 Cytokine production ...................................................................................................................................36
6.3 Phenotypical characterization of T cells resulting from T cell/T cell interaction...................................39 Table of contents
6.3.1 T cells acquire a mild activated phenotype after T cell/T cell interaction ..............................................39
6.4 Mechanisms underlying homotypic T cell/T cell interaction .....................................................................41
6.4.1 T cell/T cell interaction is dependent on cell/cell contact ........................................................................41
6.4.2 Blocking APC-characteristic receptor/ligand interactions.......................................................................43
6.4.3 Allogeneic T cell/T cell interaction ...........................................................................................................44
6.4.4 The role of adhesion receptors in T cell/T cell interaction.......................................................................45
6.4.4.1 The adhesion receptor LFA-1 and its ligand.....................................................................................45
6.4.4.2 Expression of LFA-1 and its ligands on T cells upon homotypic T cell/T cell interaction ...........47
6.4.4.3 The adhesion receptor CEACAM-1 ..................................................................................................47
6.5 Functional characterization of T cells resulting from T cell/T cell interaction ......................................48
6.5.1 Cytokines secreted after T cell contacts and their effect on proliferation of CD25 negative T cells....48
6.5.2 Suppressor phenotype of T cells upon homotypic T cell/T cell interaction............................................49
6.6 Mechanisms underlying the inhibitory effect of Tregs generated by T cell interaction........................51
6.6.1 Inhibition through IL-2 deprivation, cell contact and immuno-modulatory cytokines ..........................51
6.7 Murine T cell/T cell interaction ......................................................................................................................52
6.7.1 Proliferation and cytokine production .......................................................................................................52
6.7.2 Suppressor phenotype.....................