Lysosomal alpha-galactosidase A controls the generation of self lipid antigens for NKT cells [Elektronische Ressource] / Alexandre F. Darmoise. Gutachter: Stefan H.E. Kaufmann ; Arturo Zychlinsky ; Florian Winau

Lysosomal α-galactosidase A controls the generation of
self lipid antigens for NKT cells

Dissertation
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingerichtet an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt Universität zu Berlin
von
Diplom-Biologe
Alexandre F. Darmoise
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön
Gutachter/in: 1. Prof. Dr. Dr. h.c. Stefan H.E. Kaufmann
2. Prof. Dr. Arturo Zychlinsky
3. Prof. Dr. Florian Winau

Tag der mündlichen Prüfung: 13. Juli 2010



























Contents

List of Figures ................................................................................................. V
Abstract ......... VII
Zusammenfassung ........................................................................................ IX
Abbreviations ................................ XI
1 Introduction ............................................................................................... 1
1.1 Components of the immune system ........ 1
1.2 Concepts of immunity ............................................................. 4
1.3 Innate immunity ...................................................................... 6
1.3.1 Components of the innate immune system ...................................................... 7
1.3.2 Receptors of innate immune cells .... 7
1.4 Adaptive Immunity .................................................................................................. 9
1.4.1 Cells of the adaptive immune system ............................... 9
1.4.2 The effector mechanisms of adaptive immunity ............................................... 9
1.4.3 Subset commitment by helper T lymphocytes ................ 11
1.5 Antigen presentation to T lymphocytes ................................................................. 13
1.5.1 Major histocompatibility complex molecules ................... 14
1.5.2 MHC class I molecules .................................................................................. 14
1.5.3 MHC class II molecules ................. 15
1.6 The family of CD1 molecules ................ 16
1.6.1 The CD1 complex .......................................................................................... 16
1.6.2 Architecture of CD1 molecules ....................................... 18
1.6.3 Cellular expression of CD1 molecules............................ 19
1.6.4 Intracellular trafficking and lipid-loading of CD1 molecules ............................. 20
1.7 Group 1 CD1-restricted lipid-specific T-cell responses .......................................... 22
1.7.1 Effector functions of lipid-reactive T cells ....................... 22
1.7.2 Self and foreign antigens ............................................... 22
1.8 CD1d-restricted NKT cells ..................................................... 24
1.8.1 Definition of NKT cells .................... 24
1.8.2 Phenotype and tissue distribution of NKT cells .............. 25
I
1.8.3 Development of iNKT cells ............................................................................. 26
1.8.4 Effector functions of activated iNKT cells ....................... 28
1.8.5 The activation pathways of iNKT cells in infectious diseases ......................... 30
1.8.5.1 Direct activation of iNKT cells by microbial ligands ..................................... 30
1.8.5.2 Indirect activation of iNKT cells by self GSLs .............. 31
2 Aims of this work .................................................................................... 35
3 Material ..................................................................................................... 37
3.1 Cell culture medium .............................. 37
3.2 Bacterial culture media ......................................................... 37
3.3 Mice ...................................................................................... 37
3.4 Antibodies ............................................. 38
3.5 Reagents, chemicals, and cytokines ..................................... 39
3.6 Buffers .................................................. 40
3.7 Equipment ............................................ 41
3.8 Kits ....................................................... 42
3.9 Other materials ..................................... 42
3.10 Software ............................................................................... 43
4 Methods .................................... 45
4.1 Isolation of bone marrow cells and generation of dendritic cells ............................ 45
4.2 Preparation of single-cell suspensions from organs .............................................. 45
4.3 Isolation of T lymphocytes ..................................................... 46
4.4 T-cell assays ......................................... 46
4.5 Serum isolation from murine blood ........................................................................ 47
4.6 ELISA ................................................... 47
4.7 Generation of αGalCer-loaded CD1d tetramers .................................................... 48
4.8 Adoptive T-cell transfer ......................................................... 48
4.9 Adoptive DC transfer ............................. 49
4.10 Flow cytometry ...................................................................................................... 49
4.11 Bacterial cell culture .............................. 50
4.12 Maturation of DCs with bacteria or TLR agonists .................................................. 50
4.13 Gb3 staining in DCs ................................ 51
4.14 Protein quantification ............................................................ 51
4.15 Lysosomal enzyme assays ................................................... 52
II
4.16 GSL extraction from DCs ...................................................................................... 52
4.17 Thin layer chromatography (TLC) ......... 53
5 Results ..................................................................................................... 55
5.1 α-Gal A-deficient DCs spontaneously activate iNKT cells in vitro .......................... 55
5.2 NKT-cell activation depends on α-Gal A enzymatic activity in DCs ....................... 59
5.3 Deficiency in α-Gal A activates iNKT cells in vivo .................................................. 59
-/-5.4 Homeostasis of iNKT cells is impaired in α-Gal A mice ....... 65
5.5 Deficiency of α-Gal A induces iNKT-cell tolerance ................ 69
5.6 TLR engagement of DCs stimulates iNKT-cell responses ..................................... 71
5.7 Infection inhibits α-Gal A activity downstream of TLRs in DCs .............................. 72
5.8 α-Gal A is a central regulator of TLR-induced GSL accumulation ......................... 74
5.9 Invariant NKT cells react with TLR-inducible galabiaosylceramide ........................ 75
6 Discussion ............................................................................................... 79
6.1 Lysosomal α-Gal A deficiency in DCs promotes iNKT-cell activation .................... 79
6.2 Expansion of iNKT cells in α-Gal A-deficient hosts ................................................ 81
6.3 Deficiency of α-Gal A induces peripheral iNKT-cell tolerance ................................ 82
6.4 Infection inhibits α-Gal A activity in DCs through TLR engagement ....................... 85
6.5 Lysosomal α-Gal A is a central effector of TLR-induced GSL induction ................ 87
6.6 DC-inducible iNKT-cell self GSL antigen(s) ........................................................... 89
6.7 Pathways of galabiaosylceramide synthesis and degradation ............................... 93
6.8 Potential regulation of α-Gal A in tumor cells ........................ 94
6.9 Potential involvement of iNKT cells in Fabry disease ............................................ 94
Literature ........................................................................ 97
Appendix ...................................... 119
Publications ................................................................. 123
Danksagung ................................. 125
Erklärung ...................................................................... 127

III


























IV
List of Figures
Figure 1. The two-signal theory. ............................................................................................ 5
Figure 2. Structural organization of antibody and TCR. ....................... 10
Figure 3. T-cell effector functions. 11
Figure 4. Polarization of T-cell functions depends on the immunological milieu and APC-
derived factors. .........................................................................