Monitoring and characterization of T-lymphocyte reconstitution after allogeneic stem cell transplantation [Elektronische Ressource] / vorgelegt von Ahmad Abu-Khader

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 5 Mo

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Monitoring and Characterization of
T-Lymphocyte Reconstitution after
Allogeneic Stem Cell Transplantation
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER
NATURWISSENSCHAFTEN (DR.RER.NAT.) DER FAKULTÄT III
- BIOLOGIE UND VORKLINISCHE MEDIZIN -
DER UNIVERSITÄT REGENSBURG
vorgelegt von
Ahmad Abu-Khader
aus
Amman – Jordanien
Februar / 2006The work presented in this thesis was carried out in the Department of Hematology and
Oncology at the University Hospital Regensburg from February 2004 to March 2006.
Parts of this work will be presented in:
nd32 Annual Meeting of the European Group for Blood and Marrow Transplantation,
Hamburg, Germany, March 19-22 2006.
Promotionsgesuch eingereicht am: 16.02.2006
Die Arbeit wurde angeleitet von: Prof. Dr. med. Ernst Holler
Prüfungsausschuss:
Vorsitzender: Prof. Dr. Richard Warth
1. Gutachter: Prof. Dr. Hans Kalbitzer
2. Gutachter: Prof. Dr. Ernst Holler
3. Prüfer: Prof. Dr. Ralph WitzgallTo
my parents,
my wife Reem,
my twins Jana and Mutaz
and
for the coming twins.____________________________________________________________Table of Contents
Table of Contents
Page
1 Introduction........................................................................................... 1
1.1 Stem cell transplantation (SCT)........................................................... 1
1.1.1 Stem Cell Sources................................................................................... 2
1.1.2 Autologous SCT...................................................................................... 2
1.1.3 Allogeneic SCT....................................................................................... 2
1.2 Cellular reconstitution after SCT........................................................ 4
Cytomegalovirus (CMV) infection after allogeneic SCT...................1.3 5
1.4 Graft-versus-host disease (GvHD)....................................................... 7
1.5 Graft-versus-leukemia (GvL) effect..................................................... 9
Minor histocompatibility antigens (mHAgs)......................................1.6 11
1.7 Immune monitoring approaches.......................................................... 12
1.7.1 Cytokines................................................................................................. 12
1.7.2 Mixed lymphocytes reaction (MLR)....................................................... 13
1.7.3 Tetramer (TM) staining........................................................................... 13
1.7.4 Intracellular cytokines (ICC) flow cytometry......................................... 14
1.7.5 Enzyme-linked immunospot (ELISPOT) ............................................... 14
1.7.6 Real time-polymerase chain reaction (RT-PCR)..................................... 15
2 Materials and Methods......................................................................... 17
Materials................................................................................................2.1 17
2.1.1 Equipments.............................................................................................. 17
2.1.2 Chemicals and disposable goods............................................................. 17
2.1.3 Monoclonal antibodies............................................................................ 19
2.1.4 CD8 and IFN-γ primers........................................................................... 19
2.1.5 CD8 and IFN-γ probes............................................................................ 19
2.1.6 Kits.......................................................................................................... 20
2.1.7 Media....................................................................................................... 20
2.1.8 Cell lines.................................................................................................. 20
2.2 Patients................................................................................................... 20
2.2.1 Analysis of T-cell reactivity to CMV in patients.................................... 20
2.2.2 Analysis of a potential GvL effect (e.g. WT1) in patients...................... 21
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2.2.3 Analysis of T-cell reactivity to mHAg (e.g. HY) in patients.................. 22
2.2.4 Analysis of patients for GvHD................................................................ 22
2.3 Methods.................................................................................................. 23
2.3.1 Cells processing....................................................................................... 23
2.3.1.1 Isolation of PBMCs................................................................................. 23
2.3.1.2 Freezing and storage....... 23
2.3.1.3 Thawing and resting................................................................................ 23
2.3.2 MLR................................ 23
2.3.3 Flow cytometry....................................................................................... 24
2.3.3.1 Cell surface immunophenotyping........................................................... 24
2.3.3.2 ICC staining............................................................................................. 25
2.3.3.3 TM staining..................... 25
2.3.3.4 PKH staining......................................................................................... 2626
2.3.3.5 Flow cytometric analysis......................................................................... 26
2.3.4 RT-PCR.......................... 26
2.3.4.1 Cell isolation........................................................................................... 26
2.3.4.2 RNA isolation.......................................................................................... 26
2.3.4.3 cDNA synthesis....................................................................................... 27
®2.3.4.4 RT-PCR (TaqMan ) procedure............................................................... 27
2.3.4.5 Results analysis....................................................................................... 28
2.3.5 ELISPOT................................................................................................. 28
2.4 Data evaluation and statistics............................................................... 29
2.4.1 Flow cytometry............... 29
2.4.2 Classical MLR......................................................................................... 29
2.4.3 ELISPOT................................................................................................. 29
2.4.4 RT-PCR.......................... 29
2.4.5 Linear regression and correlations.......................................................... 29
3 Results.................................................................................................... 30
3.1 Reconstitution of CMV-reactive T cells.............................................. 30
3.1.1 TM detection assay was able to detect CMV-reactive T cells................ 31
3.1.2 IC IFN-γ assay can detect CMV-reactive CTLs..... 34
3.1.3 RT-PCR detection assay can monitor CMV-reactive T cells.................. 40
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3.1.4 ELISPOT assay detected CMV-reactive CTLs....................................... 44
3.1.5 Comparison of CMV-monitoring methods in transplanted patients....... 49
3.1.5.1 Sensitivity comparisons between monitoring assays.............................. 49
3.1.5.2 Impact of serotyping on reconstitution of CMV-reactive CTLs............. 50
3.1.5.3 Correlation of CMV monitoring assays.................................................. 51
3.2 Monitoring of WT1-specific T cells..................................................... 54
3.2.1 RT-PCR assay can detect WT1-reactive T cells..................................... 55
3.2.2 ELISPOT assay can not assess WT1-reactive CTLs............................... 59
3.3 Monitoring of mHAg-reactive T cells.................................................. 63
3.3.1 RT-PCR assay can detect HY-reactive T cells........................................ 64
3.3.2 ELISPOT assay can not detect HY-reactive CTLs................................. 66
3.4 Monitoring of GvHD............................................................................. 71
3.4.1 Detection of IFN-γ by IC flow cytometry assay..................................... 71
3.4.1.1 Establishment of GvHD model............................................................... 71
3.4.1.2 IC flow cytometry can not detect alloreactive T cell in GvHD patients. 76
3.4.2 RT-PCR can detect IFN-γ mRNA in the healthy MLR settings............. 80
3.4.3 RT-PCR can assess alloreactive T cells in GvHD patients..................... 80
3.5 Impact of clinical parameters on T cell functionality........................ 81
4 Discussion............................................................................................... 87
Monitoring of CMV-reactive T cells....................................................4.1 87
4.2 Monitoring of WT1-reactive T cells..................................................... 93
4.3 Monitoring of HY-reactive T cells....................................................... 96
Monitoring of alloreactive T cells..........................