NKT cells between innate and acquired immunity [Elektronische Ressource] : function and specificity / von Marcus Niemeyer

Niemeyer

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2005
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

NKT cells between innate and acquired immunity:
function and specificity




D i s s e r t a t i o n



zur Erlangung des akademischen Grades


d o c t o r r e r u m n a t u r a l i u m
( Dr. rer. nat.)


im Fach Biologie



eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin


von

Diplom-Biologe Marcus Niemeyer
geboren 28.04.1975 in Leverkusen



Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD




Gutachter:

1. Prof. Dr. Richard Lucius

2. Prof. Dr. Stefan H. E. Kaufmann

3. PD Dr. Ulrich E. Schaible


Tag der mündlichen Prüfung: 29.08.2005




Content I

Content I

Abbreviations IV

1 Introduction 1
1.1 Cells comprising the immune system......................................................................2
1.2 Innate immunity ......................................................................................................3
1.2.1 Cells of the innate immune system 3
1.2.2 Receptors for innate immune response 4
1.3 Linking innate and adaptive immunity .....................................................................5
1.4 The adaptive immune system .................................................................................5
1.4.1 Cells of the adaptive immune response 5
1.4.2 Receptors for adaptive immune responses 6
1.4.3 Different T cell populations produce different adaptive responses 6
1.5 Antigen presentation...............................................................................................8
1.6 CD1 molecules .....................................................................................................10
1.6.1 CD1d 11
1.7 Infection with mycobacteria...................................................................................12
1.7.1 The mycobacterial cell wall 13
1.7.2 Immune responses to mycobacterial infection 13
1.8 CD1 lipid antigens and CD1 reactive T cells .........................................................14
1.9 NKT cells ..............................................................................................................16

2 Aims of this thesis 19

3 Material 20
3.1 Cells, Cell lines .....................................................................................................20
3.2 Cell culture medium ..............................................................................................20
3.3 Bacteria ................................................................................................................20
3.4 Bacteria Culture medium ......................................................................................20
3.5 Mice......................................................................................................................21
3.6 Human samples....................................................................................................21
3.7 Antibodies.............................................................................................................21
3.8 Primers .................................................................................................................23
3.9 Plasmids...............................................................................................................24
3.10 Chemicals.............................................................................................................24
3.11 Buffers and solutions ............................................................................................25 Content II

3.12 Enzymes...............................................................................................................26
3.13 Equipment ............................................................................................................26
3.14 Other material.......................................................................................................27
3.15 Kits .......................................................................................................................27
3.16 Software ...............................................................................................................27
3.17 Web resources......................................................................................................28

4 Methods 29
4.1 Immunological methods........................................................................................29
4.1.1 Cell culture 29
4.1.2 Bacterial cell culture 29
4.1.3 Infection of cells with M. bovis BCG 29
4.1.4 Infection of mice with M. bovis BCG 30
4.1.5 Generation of murine macrophages and dendritic cells from bone marrow 30
4.1.6 Generation of human DCs from peripheral blood 30
4.1.7 Generation and restimulation of human NKT cell lines and clones 31
4.1.8 Single cell isolation from different tissues 31
4.1.9 Red blood cell lysis 31
4.1.10 Flow cytometry 31
4.1.11 Isolation and purification of cells by MACS and FACS sorting 32
4.1.12 Isolation of lysosomes from fibroblast 33
4.1.13 T cell stimulation assay 33
4.1.14 Dual luciferase reporter assays for NF-kB activation. 34
4.2 Biochemical methods............................................................................................35
4.2.1 ELISA 35
4.2.2 Detection of [3H] thymidine incorporation 35
4.2.3 Isolation of lipids from M. bovis BCG 35
4.2.4 SDS polyacrylamide gel electrophoresis 36
4.2.5 Silver staining 36
4.2.6 High performance thin layer chromatography (HPTLC) analysis 37
4.2.7 Matrix assisted laser disorption ionization – mass spectrometry (MALDI-MS) 37
4.2.8 Protein quantification by Bradford 37
4.2.9 Generation of tetrameric mCD1d - lipid complexes 37
4.3 Molecular biological methods................................................................................38
4.3.1 Isolation and purification of RNA from single cell suspensions 38
4.3.2 Generation of cDNA and RT-PCR 38
4.3.3 Real-time RT-PCR 39
4.3.4 RNA microarray 39

Content III

5 Results 41
5.1 Characterization of NKT cells................................................................................41
+5.1.1 Transcriptome analysis of NKT cells compared with NK cells, conventional CD4 T cells
and Treg cells 41
5.1.2 Unique gene expression of NKT cells 46
+5.1.3 Genes with expression levels shared by NK cells, conventional CD4 T cells or Treg cells
and NKT cells 49
5.1.4 Transcriptome analysis of naïve NKT cells vs. activated NKT cells 52
5.2 Endogenous NKT cell lipid ligands........................................................................56
5.2.1 iGb3: the endogenous NKT cell antigen 57
5.2.2 iGb3 and iGb3 analogues 59
5.3 Mycobacterial PIM is a natural NKT cell ligand .....................................................63
5.4 TCR vs. TLR induced NKT cell activation .............................................................67
5.4.1 Mycobacterial PIMs induce NKT cell activation via TCR and TLR 67
5.4.2 Mass-spectrometry analysis of TLR and TCR activating lipid fractions 71
5.4.3 Toll-like receptor expression by NKT cells 74

6 Discussion 76
6.1 Defining NKT cells through their gene expression pattern.....................................76
6.1.1 The NKT cell: portrayal by means of gene expression profiling 77
6.1.2 Activated NKT cells: self-control, effector functions and immuno-regulation 80
6.2 NKT cell activity: induction by iGb3 is controlled through antigen availability ........82
6.3 Characterization of PIM-reactive NKT cells...........................................................87
6.4 Toll like receptor vs. T cell receptor mediated recognition of mycobacterial lipids .89
6.5 Towards an understanding of NKT cells ...............................................................92

7 Summary 93
8 Zusammenfassung 95
9 Literature 98
Publications 114
Erklärung 115
Danksagung 116
Suppliers 117

Abbreviations IV

Abbreviations
Ab antibody
aGalCer alpha-galactosyl-ceramide
aGalA alpha-galactosidase A
Ag antigen
APC antigen presenting cell
-APC allophycocy, fluorescent dye for FACS analysis
b2m beta-2-microglobulin
BCG bacillus Calmette et Guérin
BCR B cell receptor
BMMf bone marrow macrophage
BSA bovine serum albumin
C57BL/6 C57BL/6 = widely used inbred mouse strain.
CD# "cluster of differentiation": international nomenclature for cell surface
molecules (CD number)
CD1d tetramer tetrameric complex of murine soluble CD1d molecules used for FACS
analysis of NKT cells
ConA concanavalin A
CTL cytolytic (cytotoxic) T lymphocyte
Cy5 Cy-Chrome 5, fluorescent dye for flow cytometry and microarray analysis
Cy3 Cy-Chrome 3, fluorescent dye for flow cytometry and microarray analysis
Da Dalton
DAPI 4, 6-diamidino-