Homing and homeostasis of T-lymphocytes in CD18_1hn-_1hn/_1hn- mice [Elektronische Ressource] / vorgelegt von Tsvetelina Veleva-Oreshkova

K.Orechkov

Sujets

• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2008
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Universität Ulm
Universitätsklinik für Dermatologie and Allergologie
Ärztliche Direktorin: Prof. Dr. Karin Scharffetter-Kochanek







-/-Homing and homeostasis of T lymphocytes in CD18 mice






Dissertation
zur Erlangung des Doktorgrades der Humanbiologie
der Medizinischen Fakultät der Universität Ulm











vorgelegt von
Tsvetelina Veleva-Oreshkova
geb. in Sofia, Bulgaria

Ulm, 2008 Tsvetelina Veleva-Oreshkova Prefix























Dekan: Herr Prof. Dr. med. Klaus-Michael Debatin
1. Berichterstatter: Frau Prof. Dr. med. Karin Scharffetter-Kochanek
2. Berichterstatter: Herr Prof. Dr. med. Jörg Reimann

Tag der mündlichen Prüfung: 17.10.2008
2Tsvetelina Veleva-Oreshkova Prefix
I. TABLE OF CONTENTS

1. INTRODUCTION..................................................................................................... 9
1.1. Beta integrin adhesion receptors and their role in inflammation ....................... 9 2
1.1.1. Structure and function of β integrins ..................................................................................... 9 2
1.1.2. Reduced expression or loss of β integrin function leads to a rare disease termed leukocyte 2
adhesion deficiency 1 (LAD1).............................................................................................. 11
1.2. Organization of the immune system...................................................................... 12
1.2.1. Primary immune organs........................................................................................................ 13
1.2.2. Secondary immune organs .................................................................................................... 13
1.2.3. Non-lymphoid organs ........................................................................................................... 16
1.3. Lymphocyte recirculation....................................................................................... 16
1.3.1. Chemokines are upstream regulators of lymphocyte migration............................................ 18
1.3.2. Naïve and central memory lymphocytes recirculate through pLN ....................................... 19
1.3.3. Effector/memory lymphocytes recirculate through non-lymphoid organs ........................... 20
1.3.4. Lymphocyte homeostasis...................................................................................................... 21
1.4. T cell development and activation ......................................................................... 22
1.4.1. Conventional T cells ............................................................................................................. 22
1.4.1.1. Intrathymic development of conventional T cells....................................................... 22
1.4.1.2. Activation of naïve and antigen-experienced conventional T cells............................. 25
1.4.1.3. Induction of T cell anergy and regulatory function of anergic cells ........................... 25
1.4.2. Unconventional T cells form a link between adaptive and innate immunity....................... 26
1.4.2.1. nal DN T cells participate in innate immunity............................................ 28
1.4.2.1.1 TCRγδ DN T cells: Origin and function................................................................. 28
1.4.2.1.2. NKT cells: origin, recirculation and function ......................................................... 30
1.4.2.1.3. Non-NKT TCRαβ DN T cells: origin and function ............................................... 33
1.4.2.2. Unconventional TCRαβ DN T cells as a part of adaptive immunity .............................. 34
+1.4.2.2.1. B220 TCRαβ DN T cells in gld and lpr mice: origin and function....................... 34
1.4.2.2.2. TCRαβ DN T cells from transgenic and mutant mice: origin and function ........... 35
1.5. Aim of the work....................................................................................................... 36

3Tsvetelina Veleva-Oreshkova Prefix
2. MATERIALS AND METHODS............................................................................ 37
2.1. Materials .................................................................................................................. 37
2.1.1. Mouse strains ........................................................................................................................ 37
2.1.2. Antibodies ............................................................................................................................. 37
2.1.3. Chemicals.............................................................................................................................. 38
2.1.4. Buffers and solutions ............................................................................................................ 40
2.1.5. Laboratory devices................................................................................................................ 40
2.2. Methods................................................................................................................... 41
2.2.1. Mice ..................................................................................................................................... 41
2.2.2. FACS staining...................................................................................................................... 41
2.2.3. Intracellular staining for Foxp3 and TGF- β......................................................................... 41
2.2.4. Adoptive transfer experiments ............................................................................................. 41
2.2.5. Lymphocyte isolation from non-lymphoid organs............................................................... 42
2.2.6. BrdU in vivo incorporation .................................................................................................. 42
2.2.7. CFSE proliferation assays.................................................................................................... 43
2.2.8. Enrichment of total T cells by magnetic bead sorting.......................................................... 43
2.2.9. Coculture suppression assays............................................................................................... 44
2.2.10. Mixed lymphocyte reaction with BM-derived allogeneic DC............................................. 44
2.2.11. T cell reconstitution assays in vivo ...................................................................................... 45
2.2.12. Homeostatic proliferation assays in vivo ............................................................................. 45
2.2.13. Culture assays with IL-2, IL-7 and IL-15 ............................................................................ 45
32.2.14. H thymidin proliferation assays.......................................................................................... 46
2.2.15. Cytometric bead array determination of IFN- γ, IL-2, TNF- α, IL-4 and IL-5...................... 46
2.2.16. Immunofluorescence histology............................................................................................ 46
2.2.17. Generation of transgenic animals......................................................................................... 47
2.2.18. Statistical analysis................................................................................................................ 48
3. RESULTS................................................................................................................. 49
3.1. Murine CD18 deficiency results in a disturbed lymphocyte trafficking............ 49
-/-3.1.1. CD18 mice reveal a severe peripheral lymphadenopathy and hypoplastic pLN in parallel49
3.1.2. β integrins are required for homing of naïve lymphocytes to pLN...................................... 51 2
-/-3.1.2.1. Lymphocytes from CD18 mice cannot enter pLN but recirculate through non-lymphoid
organs .............................................................................................................................. 51
-/-3.1.2.2. Impaired homing of CD18 lymphocytes to pLN is not due to an altered chemokine
receptor expression or activation..................................................................................... 53
4Tsvetelina Veleva-Oreshkova Prefix
-/-3.2. CD18 mice harbor unconventional DN T cells................................................... 55
+ - - -/-3.2.1. Unconventional CD3 CD4 CD8 DN T cells occur in cLN from CD18 mice ................... 55
-/-3.2.2. CD18 TCRαβ and TCR γδ DN T cells recirculate through liver and lungs........................ 56
-/-3.2.3. CD18 TCRαβ γδ DN T cells show an antigen experienced-like phenotype and
recirculation pattern .............................................................................................................. 58
3.2.4. CD18 deficiency does not affect DETC migration to epidermis during mouse ontogeny.... 59
-/-3.3. Role of thymus in the generation of CD18 TCR αβ and TCRγδ DN T cells..