Peptide specificity during positive selection [Elektronische Ressource] / vorgelegt von Stefan Irion

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Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2003
• Nombre de lectures: 38

Extrait

Aus dem Interfakultären Institut für Zellbiologie der Universität Tübingen
Abteilung Immunologie
Abteilungsleiter: Professor Dr. H. - G. Rammensee

Und dem National Jewish Medical and Research Center
Denver, Colorado
Professor Dr. U. D. Staerz



Peptide Specificity During Positive Selection



Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Medizin

der Medizinischen Fakultät
der Eberhard - Karls - Universität
zu Tübingen

vorgelegt von
Stefan Irion
aus
Balingen
2003


























Dekan: Professor Dr. C. D. Claussen

1. Berichterstatter: Professor Dr. H. - G. Rammensee
2. Berichterstatter: Frau Professor Dr. C. Müller

TABLE OF CONTENTS
TABLE OF CONTENTS..........................................................................3
INDEX OF FIGURES..............................................................................7
INDEX OF TABLES...............................................................................8
ABBREVIATIONS USED IN THIS THESIS ...................................................9
LIST OF PUBLICATIONS .....................................................................10
ZUSAMMENFASSUNG ........................................................................11
CHAPTER I.......................................................................................13
Introduction..................................................................................................... 13
Alpha/Beta T Cell Development in the Thymus ............................................. 15
Positive Selection of Thymocytes .................................................................. 16
Antagonist Theory ................................................................................... 17
Differential Avidity Theory........................................................................ 19
Gemisch Model........................................................................................ 21
Non-interference Model ........................................................................... 24
Altered Peptide Hypothesis ..................................................................... 24
Cell Types Involved in Positive Selection ................................................... 25
CD4 and CD8 Co-receptor Involvement in Positive Selection .................... 26
Other Molecules Involved in Positive Selection .......................................... 26
Negative Selection in the Thymus ................................................................. 27
Clonal Deletion ........................................................................................... 27
Cell Types Involved in Clonal Deletion .................................................... 28
Clonal Inactivation ...................................................................................... 29
Cell Types Involved in Clonal Inactivation ............................................... 30
3 CD4 and CD8 Co-receptor Involvement in Negative Selection................... 30
Other Molecules Involved in Negative Selection......................................... 31
The Experimental System.............................................................................. 33
The Non-Classical MHC Class Ib Molecule, H2-M3 ...................................... 33
Discovery of H2-M3 .................................................................................... 33
Requirement of Formyl Methionine for Peptide Binding to H2-M3.............. 34
Presentation of Self-Peptides by H2-M3..................................................... 34
Presentation of Listeria Monocytogenes Antigens by H2-M3 ..................... 35
Expression of H2-M3 .................................................................................. 35
Importance of H2-M3 Following Bacterial Infection .................................... 36
+
The C10.4 TCR Mouse........................................................................... 36 trans
Experimental Rationale.................................................................................. 37
CHAPTER II......................................................................................39
Materials and Methods................................................................................... 39
Mice............................................................................................................ 39
Cell Culture Medium ................................................................................... 39
Rat Concanavalin A Supernatants........................................................... 40
Cells and Cell Lines.................................................................................... 40
Peptides...................................................................................................... 40
Cytotoxicity Assays..................................................................................... 42
Antibodies and Flow Cytometric Analyses.................................................. 42
Staining of Peripheral Blood Lymphocytes (PBL) to Determine Phenotype43
Screening of Tail DNA Using Microsatellite Mapping ................................. 44
Fetal Thymic Organ Cultures...................................................................... 44
Expansion of FTOC Thymocytes and Their Use in a CTL Assay ............... 45
H2-M3 Up-regulation Assay........................................................................ 46
Naïve Activation Assay............................................................................... 46
Preparation of T cells:.............................................................................. 46
Preparation of APCs:............................................................................... 46
4 CHAPTER III.....................................................................................48
+
Characteristics of the C10.4 TCR Mouse .............................................. 48 trans
+
Staining and Specificity of T Cells Isolated from a C10.4 TCR Mouse on a trans
RAG2 Deficient Background.......................................................................... 52
+
Staining of T Cells Isolated from a C10.4 TCR Mouse on a TAP1 trans
Deficient Background .................................................................................... 55
+Positive Selection of C10.4 TCR Thymocytes on Different MHC trans
Backgrounds.................................................................................................. 57
wt +
Requirement of H2-M3 for Positive Selection of C10.4 TCR Thymocytestrans
...................................................................................................................... 59
Development of a Fetal Thymic Organ Culture (FTOC) System to Analyze
Positive Selection .......................................................................................... 62
Efficiency of the TAP knockout system.......................................................... 62
+ND1 is the Physiological Ligand of Positive Selection for C10.4 TCR trans
Thymocytes ................................................................................................... 64
The ND1/9mer Induces Positive Selection of Functionally Mature Thymocytes
...................................................................................................................... 67
Summary ......................................................................................................... 69
CHAPTER IV ....................................................................................70
The ND1/9mer Functions as a Partial Agonist ............................................. 70
Positive Selection Induced by the ND1/9mer and Cognate AttM Peptides.... 74
Degradation of Certain Cognate AttM Peptides in FTOC Media.................... 80
Positive Selection Induced by the ND1/9mer and Cognate AttM Peptides.... 84
+
Functionality of C10.4 TCR T Cells after Selection with the ND1/9mer or trans
the Cognate AttM Peptides............................................................................ 87
The Ability of the ND1/9mer and Cognate AttM Peptides to Bind H2-M3 ...... 88
Summary ......................................................................................................... 93
CHAPTER V .....................................................................................94
5 Peptide Specificity During Positive Selection.............................................. 94
Design of ND1 Peptide Mutants .................................................................... 94
Binding of the Peptide Mutants to H2-M3 ...................................................... 96
Recognition of ND1 Mutant Peptides in an FTOC system............................. 96
Summary ....................................................................................................... 111
CHAPTER VI ..................................................................................112
Discussion ................................................