La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres
Télécharger Lire

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

De
140 pages
Publié par :
Ajouté le : 01 janvier 2008
Lecture(s) : 31
Signaler un abus

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.. 61
-/-3.3.1. TCRαβ DN thymocytes are only slightly increased in thymi from CD18 mice ................ 62
3.3.2. Thymus is indispensable for the development of unconventional TCR αβ and TCRγδ DN T
-/-cells from CD18 BM .......................................................................................................... 64
3.3.3. Presence of a functional thymus alone may be insufficient for the generation of
-/-unconventional TCRαβ and TCRγδ DN T cells from CD18 BM ...................................... 66
-/-3.4. Phenotype and function of CD18 TCRαβ DN T cells....................................... 68
-/-3.4.1. CD18 TCRαβ DN T cells are not type I NKT cells ........................................................... 68
-/-3.4.2. CD18 TCRαβ DN T cells are not regulatory in vitro........................................................ 71
-/-3.4.2.1. CD18 TCRαβ T cells cannot suppress polyclonally activated T cells in vitro......... 71
-/- 3.4.2.2. CD18 TCRαβ DN T cells do not reveal a classical regulatory phenotype ............... 74
-/-3.5. CD18 DN T cells expand in vivo but have activation mechanisms different
from conventional T cells........................................................................................ 75
-/-3.5.1. DN T cells proliferate in cLN of native CD18 mice.......................................................... 75
-/-3.5.2. CD18 DN T cells are highly responsive to IL-2 in vitro resembling antigen-experienced
conventional T cells .............................................................................................................. 77
-/-3.5.3. CD18 TCRαβ and TCR γδ DN T cells secrete Th1 cytokines only at low amounts .......... 81
-/-3.5.4. CD18 TCRαβ DN T cells are unresponsive in MLR suggesting no participation of
classical MHC complexes in their activation........................................................................ 83
3.5.5. Maximal proliferation of DN T cells can be achieved independently of costimulatory
signaling by CD28 ................................................................................................................ 84
-/-3.6. CD18 TCRαβ and TCRγδ DN T cells expand by homeostatic proliferation. 86
-/-3.6.1. CD18 TCRαβ and TCR γδ DN T cells proliferate homeostatically in lymphopenic
environment .......................................................................................................................... 87
-/-3.6.2. TCRαβ DN T cells from CD18 mice proliferate upon the homeostatic cytokine IL-7 in
vitro....................................................................................................................................... 90
-/-3.6.3. CD18 TCRγδ DN T cells and TCR αβ DN T cells proliferate upon IL-15 in vitro............ 92
5Tsvetelina Veleva-Oreshkova Prefix
-/- trans -/-3.7. TCRαβ DN T cells are generated in male CD18 H-Y RAG2 mice........... 94
-/- trans -/-3.7.1. Male CD18 H-Y RAG2 mice reveal increased percentages of peripheral TCR αβ DN T
cells carrying the autoreactive transgenic V β6 TCR ............................................................ 95
-/- trans -/-3.7.2. TCRαβ DN T cells from male CD18 H-Y RAG2 mice show functional features
-/-identical to those from CD18 mice..................................................................................... 99
4. DISCUSSION ........................................................................................................ 101
5. PERSPECTIVES................................................................................................... 114
6. REFERENCES...................................................................................................... 116
7. SUMMARY............................................................................................................ 135


Appendix

CURRICULUM VITAE 137
PUBLICATION LIST.......................................................................................................... 138
ACKNOWLEDGEMENTS................................................................................................. 140
6Tsvetelina Veleva-Oreshkova Prefix
II. ABBREVIATIONS

APC antigen presenting cell
axLN axillary lymph node
BALT bronchial-associated lymphoid tissue
BCA-1 B cell-attracting chemokine 1 (CXCL13)
BM bone marrow
BrdU 5-bromo-2’-deoxyuridine
ELC EBV-induced molecule1 ligand chemokine (CCL19)
CFSE 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester
CMFDA 5-chloromethylfluorescein diacetate - green cell tracker
CMRA rhodol-based fluorophore - orange cell tracker
ConA concanavalin A
DC dendritic cells
DETC dendritic epidermal T cells
DLI donor lymphocyte infusion
DN double negative
FACS fluorescence-activated cell sorter
GALT gut-associated lymphoid tissue
GVHD graft versus host disease
HEV high endothelial venules
ICAM intracellular adhesion molecule
IFN interferon
IL interleukin
ingLN inguinal lymph node
KO knock-out
LAD1 leukocyte adhesion deficiency 1
LCMV lymphocytic choriomeningitis virus
LFA-1 lymphocyte associated antigen-1
LN lymph node
MAdCAM-1 mucosal addressin cell adhesion molecule-1
MALT mucosa-associated lymphoid tissue
MFI mean fluorescence intensity
MHC major histocompatibility complex
7Tsvetelina Veleva-Oreshkova Prefix
MLR mixed lymphocyte reaction
pLN peripheral lymph nodes
Plt paucity of lymph node T cells
PMA phorbol myristat acetat
PMC peripheral mononuclear cells
PP Peyer’s patches
RAG recombination activating genes
SDF stromal cell-derived factor
SP single positive (CD4 and CD8 T cells)
SLC secondary lymphoid tissue chemokine CCL21
TARC Thymus and activation-regulated chemokine
TCR T cell receptor
TECK Thymus-expressed chemokine
VLA-4 very late antigen-4
VCAM-1 vascular cell adhesion molecule-1
WT wild type

8Tsvetelina Veleva-Oreshkova Introduction
1. INTRODUCTION

1.1. Beta integrin adhesion receptors and their role in inflammation 2

Integrins control leukocyte development and maturation in the bone marrow (BM), recircul-
ation of naïve cells in secondary lymphoid tissue and leukocyte responses to inflammatory
signals emanating from the injured tissues (1) by integrating extracellular environment with
the cytoskeleton (2). The group of integrins is represented by 24 distinct members with β and 2
β expressed only on leukocytes (1). Noteworthy, β integrins (CD11/CD18) are crucially in-7 2
volved in regulating the recirculation pattern of leukocytes from innate and adaptive immun-
ity to tissues. For example, neutrophils emigration to the sites of infection (3) and naïve lym-
phocyte extravasation through high endothelial venules (HEV) in peripheral lymph nodes
(LN) (4) are severly impaired in β integrins deficient mice. Apart from that, they importantly 2
participate in cellular adhesion and signaling in cytotoxicity function of NK and CD8 T cells
(5-7), phagocytosis and cytokine secretion of macrophages (8) and lymphocytes (9, 10) or
cellular adhesion and costimulatory signaling controling lymphocyte activation (11) stimula-
ted by antigen presenting cells (APC).

1.1.1. Structure and function of β integrins 2

Beta2 integrins are heterodimeric receptors, composed of a common β chain (CD18) non-2
covalently bound with one out of four different alpha chains termed α β (LFA-1), α β L 2 M 2
(MAC-1), α β (p150/95) and α β (CD11d/CD18) (Figure 1A). They are differentially X 2 D 2
expressed by the diverse leukocytes subpopulations (12-14), and bind to a variety of ligands
including blood coagulation Factor X, fibronectin, fibrinogen, complement fragment iC3b,
polysaccharides, bacterial and fungal products (15), CD90 (Thy1) (16), and, importantly,
immunoglobulin superfamily members of intercellular adhesion molecules (ICAM-1, ICAM-
2, ICAM-3) (12, 14, 17), and others. LFA-1 is the only β integrin that is expressed on cells of 2
the lymphoid lineage (7, 12, 14). It critically contributes to important T and B lymphocyte
functions like transmigration, cytokine production, proliferation and cytotoxic activity (4, 6,
14, 18, 19). These occur during immune and other inflammatory responses. Furthermore,
lymphocyte function can be regulated by surface receptors (for examples of the ICAM family)
that bind to β integrins expressed on the interacting counterpart immune cells. Such may be 2
of lymphoid or myeloid origin (20).
9Tsvetelina Veleva-Oreshkova Introduction
A B
p-SMAC c-SMAC
extracellular
intracellular
α β LFA-1 TCR/CD3
CD28LFA-1 CD11a CD18
Mac-1 CD11b c-SMAC central supramolecular activation clusters
p150/95 CD11c p-SMAC peripheral supramolecular activation clusters
CD11d
Modified from Monks et al. Nature 1998


Figure 1. Structure of β integrins. (A) The common β chain (CD18) associates with one out of four 2
different α subunits, α , α , α and α , forming the distinct functional heterodimers LFA-1 (CD11a/CD18), L M X D
Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or CD11d/ CD18. CD11/CD18 heterodimeric molecules are
involved in cell-cell and cell-matrix adhesion during leukocyte recirculation and immune response formation.
(B) Structural organization of supramolecular activation clusters (SMAC) formed at the site of contact between
the surface of a T cell with an APC (cross-section through the centre of a T cell-APC contact) showing a segre-
gation of TCR/CD3 and CD28 in the central (c-) SMAC and LFA-1 in peripheral (p-) SMAC.

ICAM-1 is inducible on leukocytes and endothelial cells by inflammatory mediators like IL-1,
TNF or LPS, while ICAM-2 is constitutively expressed on endothelium (12). To avoid an
unwanted, permanent random attachment of flowing blood leukocytes to vessel walls, circula-
ting peripheral blood lymphocytes express constant levels of LFA-1 in its inactive form (1,
21). Change of LFA-1 into its active conformation can be obtained by triggering distinct
chemokine receptors or antigen receptors (T cell receptor, TCR; B cell receptor, BCR) on
lymphocytes that lead to a transient change of affinity and avidity (clustering) of LFA-1 re-
ferred to as inside-out signaling (2, 22). Ligand binding to ICAM-1 induces intracellular sig-
nals affecting apoptosis, cytotoxicity, proliferation, cytokine production and antigen present-
ation termed outside-in signaling (21). Interestingly, the role of β integrin ligands for acti-2
vation of T cells is demonstrated by a costimulatory function of ICAM-1 inducing full T cell
activation in addition to TCR-mediated stimulation (23). In contrast, activated Mac-1 ex-
pressed on interacting dendritic cells (DC) can alternatively prevent complete activation of T
cells (20).
10

Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin