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

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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..........................................................................................
Publié le : mercredi 1 janvier 2003
Lecture(s) : 36
Tags :
Source : W210.UB.UNI-TUEBINGEN.DE/DBT/VOLLTEXTE/2003/820/PDF/STEFAN_IRION_THESIS.PDF
Nombre de pages : 144
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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 .................................................................................................... 112
This Thesis in Context with Current Theories of Positive Selection............. 112
Mechanisms of Positive Selection ............................................................... 118
Outlook.......................................................................................................... 120
SUMMARY......................................................................................123
REFERENCES .................................................................................124
ACKNOWLEDGEMENTS....................................................................142
CURRICULUM VITAE........................................................................144

6 INDEX OF FIGURES

+Figure 1: Peripheral Blood Stain of the C10.4 TCR mouse....................... 51 trans
+/+ -/-Figure 2: Phenotype of the C10.4 RAG2 mouse......................................... 53
+
Figure 3: The ND1/9mer is a Partial Agonist to Peripheral C10.4 TCR T trans
Cells .......................................................................................................... 54
+/+ -/-Figure 4: Phenotype of the C10.4 TAP1 mouse ......................................... 56
+
Figure 5: C10.4 TCR cytolytic cells from different H-2 backgrounds can trans
lyse targets coated with the cognate peptide............................................. 58
Figure 6: Microsatellite Mapping to determine the H2-M3 genotype................. 60
+
Figure 7: C10.4 TCR T cells are selected on H2-M3 ................................. 61 trans
Figure 8: Efficiency of the TAP knockout system.............................................. 63
Figure 9: Positive selection on the naturally occuring self-peptide ND1/9mer .. 66
+/+ -/-
Figure 10: Functional Characteristics of C10.4 TAP FTOC Derived
Thymocytes Incubated with the ND1/9mer, the AttM/9mer Peptide, or the
AttM/5mer Peptide..................................................................................... 68
Figure 11: Agonistic activity of the mitochondrial fM peptides .......................... 73
Figure 12: Comparison of Positive Selection Abilities of the ND1/9mer, the
AttM/9mer Peptide, and the AttM/5mer Peptide ........................................ 75
Figure 13: The effects of serum peptidases in a FTOC with the AttM/6mer ..... 77
Figure 14: Potency of the used peptidase inhibitor cocktail.............................. 79
Figure 15: The ND1/9mer is Stable in FTOC Media......................................... 81
Figure 16: The AttM/9mer Peptide is Stable in FTOC Media............................ 82
Figure 17: The AttM/6mer Peptide is Degraded in FTOC Media ...................... 83
+/+ -/-
Figure 18: Control C10.4 TAP FTOC Stained for CD4, CD8, TCR, and CD24
.................................................................................................................. 85
Figure 19: Peptide binding to H2-M3................................................................ 91
Figure 20: Overview of the binding characteristics of the ND1/9mer, the
AttM/9mer and the AttM/5mer ................................................................... 92
Figure 21: Dependence of Positive Selection on the Concentrations of the
Peptide Mutants ........................................................................................ 98
Figure 22: FTOCs Using Different ND1 Variations ......................................... 101
7 Figure 23: Functionality of the Matured Thymocytes from FTOCs ................. 103
+/+ -/-Figure 24: Purity of C10.4 RAG2 lymph node cells................................... 106
Figure 25: Physiological peptide concentrations presented by H2-M3 ........... 108
Figure 26: Agonistic activity of the different peptide mutants.......................... 110

INDEX OF TABLES

Table 1: Nomenclature, Sequence and Binding of the Peptides Used in This
Study ......................................................................................................... 95
8 ABBREVIATIONS USED IN THIS THESIS

RAG Recombination Activating M -Microglobulin 2 2
Gene APC Antigen Presenting Cell
SP Single Positive APL Altered Peptide Ligand
TAP Transporter Associated BCR B Cell Receptor
with Antigen Processing BSA Bovine Serum Albumin
TCR T Cell Receptor CD Cluster of Differentiation
+
TCR TCR Transgenic CLIP Class II-Associated trans
TN Triple Negative Invariant Chain Peptide
CTL Cytotoxic Lymphocytes
DN Double Negative
DP Double Positive
FCS Fetal Calf Serum
fM Formylated Methionine
FTOC Fetal Thymic Organ
Culture
HSC Hematopoietic Stem Cell
mAb Monoclonal Antibody
MHC Major Histocompatibility
Complex
MMTV Mouse Mammary Tumor
Virus
Mta Maternally Transmitted
Antigen
OVA Ovalbumin
PBL Peripheral Blood
Lymphocytes

PCR Polymerase Chain
Reaction

9
bbLIST OF PUBLICATIONS

Irion, S., Berg, E. R., and Staerz, U. D. (2000). A Physiological Ligand of
Positive Selection is Seen with High Specificity. J Immunol 164, 4601-06.

Berg, R. E., Irion, S., Kattman, S., Princiotta, M. F., and Staerz, U. D. (2000). A
Physiological Ligand of Positive Selection Is Recognized as a Weak Agonist. J
Immunol 165, 4209-16.

Berg, R. E., Princiotta, M. F., Irion, S., Moticka, J. A., Dahl, K. R., and Staerz, U.
D. (1999b). Positive Selection of an H2-M3 Restricted T Cell Receptor.
Immunity 11, 33-43.
10

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