The role of HLA-G-expressing regulatory T cells in multiple sclerosis [Elektronische Ressource] : a perspective of beneficial inflammation in the central nervous system inflammation / vorgelegt von Yu-Hwa, Huang

Yu-Hwa Huang

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• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 15 Mo

Extrait

The Role of HLA-G-expressing Regulatory T cells in Multiple
Sclerosis
: A Perspective of Beneficial Inflammation in the Central Nervous
System Inflammation



Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg



Vorgelegt von
Yu-Hwa, Huang
Aus Taiwan




Würzburg, 2009











Eingereicht am:
Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. M. J. Müller
Gutachter: Prof. Dr. H. Wiendl
Gutachter: Prof. Dr. Buchner

Tag des Promotionskolloquiums:
Doktorurkunde ausgehändigt am:





Erklärung



Hiermit erkläre ich ehrenwörtlich, die vorliegende Arbeit selbständig angefertigt und keine
anderen als die angegebenen Quellen und Hilfsmittel verwendet zu haben.
Diese Arbeit hat weder in gleicher noch in ähnlicher Form in einem anderen
Prüfungsverfahren vorgelegen.
Ich habe in keinem früheren Verfahren einen akademischen Titel erworben oder zu erwerben
versucht.






……………………………
Yu-Hwa, Huang
Würzburg, July 2009
 







To my parents and mz sister Contents
CONTENTS
1. ZUSAMMENFASSUNG ..................................................................................................... 7
2. SUMMARY OF THE STUDY............................ 9
3. INTRODUCTION.............................................................................................................. 11
3.1 THE TOLEROGENIC NON-CLASSICAL MHC CLASS I MOLECULE HLA-G ....................... 11
3.1.1. Classical major histocompatibility complex class I molecules.............................. 11
3.1.2 Non-classical HLA class Ib molecule: HLA-G .................................................... 11
3.1.3 HLA-G and its firstly described biological role in pregnancy ............................. 12
3.1.4 Receptors for HLA-G ........................................................... 12
3.1.5 HLA-G and T-cell responses................................................. 14
3.1.6 HLA-G expression under pathological conditions.............. 14
3.2 MULTIPLE SCLEROSIS ................................................................................................... 15
3.2.1 Pathology............. 15
3.2.2 HLA-G in MS....... 18
3.3 REGULATORY T CELLS................................................................................................. 18
3.3.1 Immunological tolerance..................... 18
3.3.2 Distinct subsets of regulatory T cells and their mechanisms of suppression ....... 18
3.3.3 Regulatory T cells in multiple sclerosis ................................................................ 21
4. AIMS OF THE STUDY..................................... 24
5. MATERIAL AND METHODS......................................................... 26
5.1 MATERIAL....................................................................................... 26
5.1.1 Reagent kits............................................. 26
5.1.2 Primary antibody..................................................................... 26
5.1.3 Secondary antibody................................. 27
5.1.4 Chemical reagents... 27
5.1.5 Media, Buffers and Solutions:................................................................................. 28
5.1.6 Consumables ........................................... 30
5.1.7 Instruments and Software........................ 31
5.1.8 Human Subjects....................................................................... 31
5.2 METHODS ........................................................................................................................ 34
5.2.1 Immunohistochemical studies of CNS samples....................... 34
5.2.2 Cell culture.............................................................................................................. 35
5.2.3 Cell purification...... 36
5.2.4 CFSE labeling......... 37 Contents
5.2.5 Analysis of T cell suppression ................................................................................. 37
5.2.6 Flow cytometric analysis......................... 39
5.2.7 Detection of intracellular proteins.......... 39
5.2.8 Transmigration assay.............................................................................................. 39
5.2.9 Chemotaxis assays................................... 39
5.2.10 Statistics ................................................................................ 40
6. RESULTS............................................................ 41
6.1 SUPPRESSIVE MECHANISM MEDIATED BY NATURALLY OCCURRING HLA-G EXPRESSING
+
REGULATORY CD4 T CELLS................................................................. 41
+6.1.1 Identification of naturally occurring HLA-G-expressing regulatory CD4 T cells
in human peripheral blood............................................................... 41
pos6.1.2 HLA-G T –mediated suppression is independent of cell-cell contact and is reg
through T-T interaction.................................................................... 43
pos6.1.3 TCR-engagement facilitates HLA-G T –mediated suppression...................... 45 reg
pos6.1.4 HLA-G T –mediated suppression starts from day two of co-culture and is reg
reversible.......................................................................................................................... 47
pos6.1.5 The HLA-G T -mediated suppression does not entirely depend on sHLA-G-reg
ILT-2 interaction .............................................................................................................. 48
pos6.1.6 HLA-G T -mediated suppression critically depends on IL-10........................ 50 reg
6.2 SPECIFIC CNS RECRUITMENT AND SUPPRESSIVE FUNCTION OF HLA-G-EXPRESSING
REGULATORY T CELLS IN MS PATIENTS ................................................................................ 54
pos6.2.1 HLA-G T are enriched in the cerebrospinal fluid (CSF) and found in brain reg
lesions from MS patients .................................................................................................. 54
pos6.2.2 Phenotypes of CSF-derived HLA-G T in MS patients... 56 reg
pos6.2.3 --G T in MS patients reflects their ability to reg
migrate ............................................................................................................................. 58
pos6.2.4 HLA-G T preferentially express CCR5.......................... 61 reg
pos6.2.5 Inflammatory chemokines facilitate HLA-G T trafficking in a model of the reg
human blood-brain barrier .............................................................................................. 63
6.2.6 Inflammatory chemokine-mediated trafficking across the blood-brain barrier
posincreases the suppressive capacity of HLA-G T....................... 65 reg
pos6.2.7 HLA-G T are functionally active in the periphery from MS patients............... 66 reg
pos6.2.8 HLA-G T are functionally competent to suppress autologous T-cell activation reg
at sites of inflammation in MS.......................................................................................... 67
7. DISCUSSION ..................................................... 69 Contents
7.1 THE MECHANISM OF SUPPRESSION MEDIATED BY NATURALLY OCCURRING HLA-G
+EXPRESSING REGULATORY CD4 T CELLS............................................................................. 69
7.2 SPECIFIC CNS RECRUITMENT AND SUPPRESSIVE FUNCTION OF HLA-G-EXPRESSING
REGULATORY T CELLS IN MS PATIENTS................ 72
7.3 FINAL ASSESSMENT ......................................................................................................... 75
8. REFERENCE..................... 77
9. APPENDIX ......................................................................................................................... 92
9.1 ABBREVIATIONS.............. 92
9.2 PUBLICATION LIST........................................................................................................... 94
9.3 PERFORMANCES RELATED TO THIS STUDY....... 95
9.4 ACKNOWLEDGEMENT...... 97
9.5 CURRICULUM VITAE........................................................................................................ 98

1. Zusammenfasung 7
1. Zusammenfassung
Die Regulation von Effektor-T-Zellen ist ein wichtiger Mechanismus zur Kontrolle organ-
spezifischer Entzündungen. Dabei sind regulatorische T-Zellen (T ) maßgeblich an der reg
Aufrechterhaltung peripherer Immuntoleranz und parenchymaler Immunhomöostase beteiligt.
Eine neue Population von humanen, natürlich vorkommenden T Zellen wurde durch ihre reg
konstitutive Expression des immuntolerogenen Moleküls HLA-G identifiziert.
+ pos Im ersten Teil dieser Arbeit wurden die Mechanismen, durch die CD4 HLA-G T Zellen reg
negihre Zielzellen (autologe HLA-G T-Zellen) modulieren, aufgeklärt. Unter Verwendung
eines Suppressionsansatzes in Abwesenheit von antigenpräsentierenden Zellen (APC) wurden
negT-T-Zell-Interaktionen, die die Proliferation von HLA-G T-Zel