Biological role of the expression of tumor necrosis factor receptor ligand family molecules on acute leukemia cells [Elektronische Ressource] / vorgelegt von Ludmila Glouchkova

Sujets

• Gesundheit

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2003
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Biological role of the expression of tumor necrosis
factor receptor-ligand family molecules on acute
lymphoblastic leukemia cells
Inaugural - Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakult¨at
der Heinrich-Heine-Universit¨at Dusseldor¨ f
vorgelegt von
Ludmila Glouchkova
aus Podolsk
Dusseldorf¨
2003










































Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine Universität Düsseldrof

1. Referent: Prof. Dr. Ulrich Göbel
2. Korreferent: Prof. Dr. Heinz Mehlhorn
3. Korreferent: PD Dr. med. Dagmar Dilloo

Tag der mündlichen Prüfung: 1. Dezember 2003 Contents ii
Contents
1 Introduction 1
1.1 Leukemia development, biology and classification . . . . . . . . . . . . . . 1
1.2 Stimulation of anti-leukemia immune response . . . . . . . . . . . . . . . . 3
1.2.1 Costimulatory molecules CD80 and CD86 . . . . . . . . . . . . . . 4
1.2.2 CD27-CD70 costimulatory pathway . . . . . . . . . . . . . . . . . 6
1.2.3 Costimulatory molecule CD40 . . . . . . . . . . . . . . . . . . . . . 8
1.3 Vaccine strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 CD95-mediated programmed cell death . . . . . . . . . . . . . . . . . . . . 10
1.5 Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.6 Plan of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2 Patients, materials and methods 18
2.1 Patients selection and characteristic . . . . . . . . . . . . . . . . . . . . . . 18
2.2 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.4 Antibodies and Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.5 Apoptosis-detection kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.6 Enzyme Linked Immuno Sorbent Assay (ELISA) . . . . . . . . . . . . . . . 27
2.7 Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.8 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.9 Isolation of mononuclear cells and leukemia cells . . . . . . . . . . . . . . . 27
2.10 Isolation of T lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.10.1 Production of neuraminidase-treated sheep erythrocytes . . . . . . 28
2.10.2 T lymphocytes isolation . . . . . . . . . . . . . . . . . . . . . . . . 28
2.11 Isolation of ultrapure B-lymphocytes using positive Magnetic Activated Cell
Sorter (MACS)-selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.11.1 Cell staining with antibodies-coupling magnetic beads . . . . . . . . 30
2.11.2 Magnetic cell separation . . . . . . . . . . . . . . . . . . . . . . . . 30
2.12 Surface and intracellular marker analysis by flow cytometry using a Fluo-
rescence Activated Cell Sorter (FACScan) . . . . . . . . . . . . . . . . . . . 31Contents iii
2.12.1 Staining of surface molecules . . . . . . . . . . . . . . . . . . . . . . 31
2.13 Detection of cytokine secretion by using Enzyme Linked Immuno Sorbent
Assay (ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.14 Cultivation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.14.1 Cultivation of cell lines . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.14.2 Cryoconserving and thawing of cells . . . . . . . . . . . . . . . . . . 33
2.14.3 Stimulation of leukemia cells by CD40 cross-linking . . . . . . . . . 33
2.15 Primary Mixed Lymphocytes Reaction (MLR) . . . . . . . . . . . . . . . . 34
2.16 Generation of anti-leukemia-specific T-cell lines . . . . . . . . . . . . . . . 34
2.17 Cytotoxic assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.18 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.18.1 Induction of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.18.2 Apoptosis detection . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.19 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3 Results 38
3.1 CD40 expression on ALL blasts . . . . . . . . . . . . . . . . . . . . . . . . 38
3.2 Expression of costimulatory molecules and CD27 on ALL blasts . . . . . . 39
3.3 Stimulation of ALL blasts by CD40-cross-linking . . . . . . . . . . . . . . 42
3.4 Analysisofthecorrelationbetweentheexpressionofcostimulatorymolecules
and the biological course of leukemia . . . . . . . . . . . . . . . . . . . . . 47
3.4.1 Patients’ characteristics . . . . . . . . . . . . . . . . . . . . . . . . 47
3.4.2 Prognostic factors and expression of costimulatory molecules on pri-
mary ALL blasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.4.3 Association between expression of CD80 and CD86 on primary ALL
blasts and prognostic factors . . . . . . . . . . . . . . . . . . . . . . 51
3.4.4 Association between expression of CD27 and CD70 on primary ALL
blasts and prognostic factors . . . . . . . . . . . . . . . . . . . . . . 53
3.4.5 PrognosticfactorsandexpressionofcostimulatorymoleculesonCD40-
stimulated ALL blasts . . . . . . . . . . . . . . . . . . . . . . . . . 55Contents iv
3.4.6 Expression of costimulatory molecules and CD27 and CNS disease
on ALL patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.4.7 Prognosticfactorsandexpressionofcostimulatorymolecules: summary 57
3.5 T-cell stimulatory capacity of CD40-stimulated ALL blasts . . . . . . . . . 59
3.6 Generation of anti-leukemia specific cytotoxic T-cells . . . . . . . . . . . . 65
3.6.1 Characteristicsofcellsusedforgenerationofanti-leukemiacytotoxic
T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.6.2 RoleofCD70fortheexpansionofT-celllinesusingCD40-stimulated
or unstimulated ALL blasts. . . . . . . . . . . . . . . . . . . . . . . 67
3.6.3 Anti-leukemia cytotoxicity of T-cell lines generated using unstimu-
lated or CD40-stimulated ALL blasts. . . . . . . . . . . . . . . . . . 71
3.6.4 Role of CD70 for the anti-leukemia cytotoxic activity of T-cell lines
generated with unstimulated or CD40-stimulated ALL blasts. . . . 75
3.7 CD95 system in ALL blasts . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.7.1 CD95 expression on primary ALL blasts . . . . . . . . . . . . . . . 77
3.7.2 CD95-mediated apoptosis in primary ALL blasts . . . . . . . . . . 77
3.8 Survival and CD95 expression on ALL blasts after CD40 engagement . . . 79
3.8.1 Survival of ALL blasts during cultivation on fibroblasts . . . . . . . 79
3.8.2 DynamicofCD95expressiononALLblastsduringtheCD40-stimulation 80
3.8.3 CD95 expression on CD40-stimulated ALL blasts . . . . . . . . . . 81
3.9 Functional activity of CD95 expression induced by CD40-stimulation on
ALL blasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.10 Effect of protein synthesis inhibitor to CD95-mediated apoptosis of CD40-
stimulated ALL blasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.11 Clinical relevance of the expression and function of CD95 on ALL blasts . 87
3.11.1 Patients’ characteristics . . . . . . . . . . . . . . . . . . . . . . . . 87
3.11.2 Association between CD95 expression and function on primary and
CD40-stimulated ALL blasts and prognosis factors . . . . . . . . . 91
4 Discussion 93Contents v
4.1 Expression of CD70 and CD27 on ALL cells can be modified via in vitro
cocultivation of primary ALL cells with CD40L-expressing fibroblasts . . . 93
4.2 The CD70-CD27 pathway synergistically enhances the CD80 and CD86-
CD28 pathway of antigen presentation by ALL cells . . . . . . . . . . . . . 94
4.3 The CD70-CD27 pathway is necessary for the generation of anti-leukemia
cytotoxic T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.4 A high CD27 expression rate correlates with patients belonging to the low
risk group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.5 Induction of CD95 expression does not completely sensitize ALL cells for
CD95-mediated apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.6 Anti-apoptoticproteinsareinvolvedinlowsensitivityofALLcellstoCD95-
mediated apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5 Summary 104
6 References 105vi
Abbreviation
Ag Antigen
Acute lymphoblastic leukemiaALL
Acute myelogenous leukemiaAML
Antigen-presenting cellAPC
BM Bone marrow
Bone marrow transplantationBMT
BSA Bovine serum albumin
◦ Celsius degreeC
Chronic lymphocytic leukemiaCLL
Chronic myelogenous leukemiaCML
Central nervous systemCNS
CycloheximideCHX
cpm Count per minute
Cytotoxic T lymphocyteCTL
Cytotoxic T lymphocyte Ag-4CTLA-4
DimethylsulfochloridDMSO
Deoxyribonucleic acidDNA
EthylendiamintetraacetatEDTA
Enzyme linked immuno sorbent assayELISA
FACS Fluorescence activa