In vitro generation and characterization of acute myeloid leukemia-reactive CD8_1hn+ cytotoxic T-lymphocyte clones from healthy donors [Elektronische Ressource] / Eva Distler

Eva Lecomte

Sujets

• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 34
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

In vitro generation and characterization
of acute myeloid leukemia-reactive
+CD8 cytotoxic T-lymphocyte clones
from healthy donors






Dissertation
zur Erlangung des Grades
“Doktor der Naturwissenschaften“

am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz





Eva Distler
geb. am 03.05.1976 in Gross-Gerau





Mainz, 2007























„Ich erkläre, dass ich die vorgelegte Thesis selbständig, ohne unerlaubte fremde Hilfe und
nur mit den Hilfen angefertigt habe, die ich in der Thesis angegeben habe. Alle Textstellen,
die wörtlich oder sinngemäß aus veröffentlichten oder nicht veröffentlichten Schriften
entnommen sind, und alle Angaben, die auf mündlichen Auskünften beruhen, sind als
solche kenntlich gemacht. Bei den von mir durchgeführten Untersuchungen habe ich die
Grundsätze guter wissenschaftlicher Praxis, wie sie in der Satzung der Johannes
Gutenberg-Universität Mainz zur Sicherung guter wissenschaftlicher Praxis niedergelegt
sind, eingehalten.“ CONTENTS
1. Abstract.................................................................................................................................................. 5
2. Introduction.......... 7
2.1 Acute myeloid leukemia (AML)............................................................................................ 7
2.1.1 General characteristics and subgroups of leukemias..................................... 7
2.1.2 Diagnosis of AML ......................................................................................................... 8
2.1.3 Classification of AML...................................................................................................8
2.1.4 Prognosis of AML.. 10
2.1.5 Treatment of AML. 11
2.2 Immunological background12
2.2.1 The Immune System.................................................................................................... 12
2.2.2 Innate Immunity........................................................................................................... 12
2.2.3 Specific Immunity......................................................................................................... 14
2.2.3.1 T and B cell receptor .................................................................................... 14
2.2.3.2 Development of αβ-T cells in the thymus............................................ 15
2.2.3.3 Antigen processing and presentation ................................................... 17
2.2.3.4 T lymphocyte activation.............................................................................. 19
2.2.3.5 Effector mechanisms of T lymphocytes ................................................ 20
+ 2.2.3.6 Differentiation of CD8 T cells.................................................................. 22
2.3 Immunotherapy of malignancies ........................................................................................ 26
2.3.1 Immunity to tumors .................................................................................................... 26
2.3.2 Approaches for cancer immunotherapy.............................................................. 27
2.3.3 Hematopoietic stem cell transplantation (HSCT) as immunotherapy...... 28
2.3.4 Immune reactions in allogeneic HSCT: Graft-versus-leukemia (GVL)
effect and graft versus-host disease (GVHD) .................................................... 29
2.3.5 Separation of GVL from GVHD: Potential target antigens for specific
allogeneic T cell therapy............................................................................................ 30
2.3.6 Adoptive T cell therapy.............................................................................................. 31
2.3.7 Minor histocompatibility antigens......................................................................... 32
2.3.8 Leukemia-associated antigens................................................................................35
2.3.9 Approaches for antigen identification ................................................................. 37
2.3.9.1 Biochemical approach using HPLC and MS ....................................... 37
2.3.9.2 cDNA expression cloning ........................................................................... 37
2.3.9.3 Genetic linkage analysis.............................................................................. 37
2.3.9.4 „Reverse Immunology“................................................................................38
2.3.9.5 SEREX (serological screening of cDNA expression libraries)......... 38 CONTENTS 2
2.4 Motivation and aim of this work ......................................................................................... 39
3. Materials & Methods....................................................................................................................... 41
3.1 Primary cells of patients and healthy donors................................................................. 41
3.2 Cell lines........................................................................................................................................ 42
3.2.1 K562 cells......................................................................................................................... 42
3.2.2 Tumor cell lines............................................................................................................. 42
3.2.3 CD40L-transfected murine fibroblasts ................................................................. 42
3.3 Cell culture................................................................................................................................... 43
3.3.1 Cell culture material.................................................................................................... 43
3.3.1.1 Substances used for cell culture .............................................................. 43
3.3.1.2 Cell culture media .........................................................................................44
3.3.1.3 Cytokines..........................................................................................................44
3.3.2 Freezing and thawing of cells.................................................................................. 45
3.3.3 Cultivation of cell lines in suspension culture ................................................... 45
3.3.4 of adherent-growing cell lines ......................................................... 45
3.3.5 Isolation of PBMCs from buffy coats by Ficoll density centrifugation..... 45
3.3.6 Cryo-preservation of leukaphereses..................................................................... 46
3.3.7 Overnight pre-culture of leukemic blasts ........................................................... 46
3.3.8 Maturation of leukemic blasts................................................................................. 46
3.3.9 Generation of EBV-transformed B cell lines....................................................... 47
3.3.10 Generation of PHA-activated PBMC blasts ........................................................ 47
3.3.11 Generation of dendritic cells („Fast DCs“) 47
3.3.12 Generation of stromal fibroblasts.......................................................................... 48
3.4 Immunological methods........................................................................................................ 49
3.4.1 Magnetic cell separation (MACS)........................................................................... 49
3.4.1.1 Principle............................................................................................................. 49
3.4.1.2 Materials for magnetic cell separation.................................................. 49
+ + 3.4.1.3 Isolation of CD8 CD62L T cells from healthy donor PBMCs...... 49
+ 3.4.1.4 Positive isolation of CD14 monocytes from PBMCs....................... 50
+ 3.4.1.5 9 B cells from PBMCs ................................ 51
+ 3.4.1.6 Isolation of CD15 granulocytes from whole blood ....................... 51
3.4.2 Flow cytometry.............................................................................................................. 51
3.4.2.1 Principle............................................................................................................. 51
3.4.2.2 Monoclonal antibodies used for flow cytometry .............................. 52
3.4.2.3 Further materials and equipment for flow cytometry analysis .... 53
3.4.2.4 Immunofluorescent staining ..................................................................... 54
3.4.2.5 T cell receptor V beta chain analysis...................................................... 54 CONTENTS 3 3.4.3 Mixed lymphocyte/leukemia culture (MLLC)..................................................... 56 3.4.3.1 Mini-MLLC........................................................................................................56
3.4.3.2 Maxi-MLLC (bulk cultures) ..............