Direct ex vivo identification of individual antigen specific T-Cells with optimal avidity for protection [Elektronische Ressource] / Robert A. Knall

Robert.Knall

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

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



Direct ex vivo identification of individual antigen-specific T cells
with optimal avidity for protection


Robert A. Knall




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. S. Scherer
Prüfer der Dissertation: 1. Univ.-Prof. Dr. D. Busch
2. Univ.-Prof. Dr. D. Haller



Die Dissertation wurde am 07.08.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 23.10.2007 angenommen. I TABLE OF CONTENTS
I TABLE OF CONTENTS


I TABLE OF CONTENTS .................................................................................................... 2
II INDEX OF FIGURES ........................................................................................................ 4
III ABBREVIATIONS.............................................................................................................. 6
1 INTRODUCTION ............................................................................................................... 9
1.1 THE IMMUNE SYSTEM ............................................................................................................. 9
1.2 ADAPTIVE IMMUNITY 9
1.3 T CELLS .................................................................................................................................. 10
1.4 MOLECULAR STRUCTURE OF THE T CELL RECEPTOR ........................................................ 13
1.5 T CELL DEVELOPMENT.......................................................................................................... 14
1.6 T CELL RECEPTOR – LIGAND INTERACTIONS....................................................................... 18
1.7 ASSESSING T CELL AVIDITY.................................................................................................. 23
1.7.1 Functional T cell assays ............................................................................................................... 25
1.7.1.1 ELISPOT assay.......... 25
1.7.1.2 Intracellular cytokine staining............................................................................................. 26
511.7.1.3 Chromium release assay ................................................................................................... 26
1.7.2 Measuring structural avidity ...................................................................................................... 27
1.7.2.1 MHC tetramer-based techniques ......................................................................................... 27
1.7.2.2 Surface plasmon resonance ................................................................................................. 29
1.8 T CELL THERAPY ................................................................................................................... 32
1.9 THE LISTERIA MONOCYTOGENES INFECTION MODEL .......................................................... 35
1.10 AIM OF THIS PHD WORK ....................................................................................................... 36
2 MATERIAL AND METHODS 38
2.1 MATERIAL.............................................................................................................................. 38
2.1.1 Chemicals and reagents ............................................................................................................... 38
2.1.2 Buffers and media........................................................................................................................ 39
2.1.3 Peptides......................................................................................................................................... 41
2.1.4 Antibodies..................................................................................................................................... 41
2.1.5 MHC tetramers............................................................................................................................ 42
2.1.6 MHC Streptamers........................................................................................................................ 42
2.1.7 Gels................................................................................................................................................ 42
2.1.8 Mice.......................... 43
2.1.9 Microscope and equipment for bulk TCR avidity measurements........................................... 43
2.1.10 Evotec Cytocon400 System........................................................................................................ 44
2.1.11 Equipment .................................................................................................................................. 44
2.1.12 Software...................................................................................................................................... 45
2I TABLE OF CONTENTS
2.2 METHODS ............................................................................................................................... 45
2.2.1 Generation of T cell lines............................................................................................................. 45
2.2.2 T cell staining................................................................................................................................ 46
2.2.2.1 Antibody and MHC multimer staining for FACS analysis................................................... 46
2.2.2.2 FACS acquisition and analysis ............................................................................................ 47
2.2.3 Streptamers .................................................................................................................................. 47
2.2.3.1 Protein production.......... 47
2.2.3.2 Refolding and fluorescence conjugation of MHC class I molecules.................................... 47
2.2.3.3 Multimerization ................................................................................................................... 48
2.2.4 Functional avidity assays............................................................................................................. 48
2.2.4.1 Intracellular cytokine staining............................................................................................. 48
512.2.4.2 Chromium release assay ................................................................................................... 49
2.2.4.3 Listeria monocytogenes infection and adoptive cell transfer .............................................. 49
2.2.4.4 Measurement of bacterial load ............................................................................................ 50
2.2.5 Measuring structural avidity ...................................................................................................... 50
2.2.5.1 Streptamer staining for structural avidity assays ................................................................ 50
2.2.5.2 Bulk analysis of T cell receptor avidity................................................................................51
2.2.5.3 T cell receptor avidity assay and subsequent single cell sorting ......................................... 51
2.2.5.4 Data analysis ....................................................................................................................... 52
3 RESULTS .......................................................................................................................... 53
3.1 T CELLS WITH HIGHER FUNCTIONAL AVIDITY CONFER BETTER PROTECTION................. 53
3.1.1 Two different LLO -specific T cell lines show differing functional avidities..................... 53 91-99
3.1.2 Differing functional avidities translate into differing protective capacities............................ 55
3.2 A NOVEL ASSAY SYSTEM FOR THE ASSESSMENT OF STRUCTURAL TCR AVIDITY ............ 57
3.2.1 Principle........................................................................................................................................ 57
3.2.2 T cell receptor avidity assay for bulk measurements................................................................ 60
3.2.3 Data analysis................................................................................................................................. 62
3.2.4 Evaluation and validation of the assay....................................................................................... 67
3.3 HIGHER FUNCTIONAL AVIDITY CORRELATES WITH HIGHER STRUCTURAL AVIDITY ......... 70
3.4 TCR AVIDITY ASSAY FOR SINGLE CELL MEASUREMENTS AND SUBSEQUENT SORTING...... 74
4 DISCUSSION.................................................................................................................... 77
4.1 A NEW WAY TO DETERMINE STRUCTURAL T CELL AVIDITY .............................................. 77
4.2 STRUCTURAL AVIDITY AS A MAJOR DETERMINANT OF T CELL FUNCTION ..........