Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL): prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL
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| Sprache: | English |
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2008
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| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-9423 http://d-nb.info/989353001/34 Inhaltsverzeichnis |
| Beschreibung: | 147 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) |b prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL |c von Sabine Schreck |
| 264 | 1 | |c 2008 | |
| 300 | |a 147 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2008 | ||
| 583 | 0 | |a Langzeitarchivierung Nationalbibliothek gewährleistet | |
| 650 | 0 | 7 | |a Tumorimmunologie |0 (DE-588)4128018-0 |2 gnd |9 rswk-swf |
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Datensatz im Suchindex
| _version_ | 1804137714377818112 |
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| adam_text | Contents 4
Contents
1 Summary 13
1.1 Background 13
1.2 Hypothesis I 13
1.3 Experimental design I 13
1.4 Results I 14
1.5 Conclusions I 14
1.6 Background II 15
1.7 Hypothesis II 15
1.8 Experimental design II 15
1.9 Results II 16
1.10 Conclusions II 16
2 Introduction 22
2.1 The Epstein Barr virus (EBV) 22
2.1.1 History and biology 22
2.1.2 Structure 22
2.1.3 Primary infection 23
2.2 Hodgkin lymphoma (HL) 24
2.2.1 Epidemiology 24
2.2.2 Classification and histopathology of HL 24
2.2.3 Genotype and phenotype of neoplastic Hodgkin and Reed-Stemberg 26
(HRS) cells
2.2.4 EBV-associated Hodgkin lymphoma 26
2.2.4.1 EBV genes expressed in latency II and tumourigenesis 26
2.2.4.2 EBV-infection of HRS cells 27
2.2.5 Tumour-infiltrating lymphocytes (TIL) in HL 28
2.3 Immune cells 29
2.3.1 T-lymphocytes 30
2.3.2 B-lymphocytes 30
2.3.3 Activation of T-cells after contact with foreign antigen 31
2.3.4 Regulatory T-cells (Treg) 32
2.3.4.1 Characterisation of Treg 33
2.3.4.2 The forkhead transcription factor FoxP3 34
2.3.4.3 Suppression of effector T-cells by Treg 34
Contents 5
2.4 Tumour-infiltrating lymphocytes and their prognostic influence 35
2.4.1 T-lymphocytes in tumours 35
2.4.1.1 CD8+cytotoxic T-lymphocytes (CTL) and Granzyme B 35
in the prognosis of tumours
2.4.1.2 CD4+T-cells 36
2.4.1.2.1 CD4+Thl-ce!ls 37
2.4.1.2.2. CD4+Th2-cells 37
2.4.1.2.3 Treg in malignant diseases 38
2.4.1.2.4 Depletion of Treg 39
2.4.2 B-lymphocytes in tumours 40
2.5 Secondary malignancies after HL 41
2.6 Immunotherapeutic approaches in the treatment of HL using EBV- 41
specific CTL
2.7 Chemokines critical for the recruitment of lymphocytes 42
2.7.1 Structural classification 42
2.7.2 Functional classification 43
2.7.3 Examples of chemokines 43
2.7.3.1 RANTES/CCL5 43
2.7.3.2 TARC/CCL17 44
2.7.3.3 MDC/CCL22 44
2.8 Hypotheses 45
2.8.2 Hypothesis I 45
2.8.3 Hypothesis II 45
2.9 AID project 46
2.9.1 Activation induced cytidine deaminase (AID) 46
2.9.2 AID in the development of tumours 47
2.9.3 Testicular cancer 47
2.9.4 Spermatogenesis 47
2.9.5 Hypothesis 48
3 Results 49
3.1 Prognostic impact of tumour-infiltrating T-cells and EBV in HL 49
3.1.1 Validation of antibodies directed against FoxP3, c-Maf and T-bet 49
3.1.2 Expression of FoxP3, T-bet, c-Maf and Epstein-Barr viral transcripts 52
in Hodgkin lymphoma
3.1.3 CD20+and CD21+lymphoid follicles in HL 54
3.1.4 Definition of cut-offs used for Kaplan Meier analyses 55
3.1.5 T-cell subsets, EBV-status and prognosis of HL 58
3.1.6 CD20+ and CD21+ lymphoid follicles in HL and prognosis 61
3.2 Tumour cell induced leukocyte migration in HL 64
3.2.1 Recruitment of TIL through production of chemokines and cytokines 64
by HRS cells
3.2.1.1 Production of chemokines and cytokines in HRS cells of cHL 64
biopsies
Contents 6
3.2.1.2 Detection of chemokines and cytokines in HL derived cell 66
lines with RNAse protection assay (RPA)
3.2.1.3 Detection of chemokines in HL derived cell lines with 68
Sandwich ELBA
3.2.2 Determination ofEBV-status in EBV-infected KHM-2 cells 69
3.2.3 Chemotaxis assay with peripheral blood mononuclear cells (PBMC) 70
from healthy individuals and HL patients in comparison
3.2.4 Composition of PBMC from healthy individuals and HL patients 71
3.2.5 Analyses of PBMC composition after chemotaxis assay 73
3.3 AID project 75
3.3.1 AID expression in normal spermatogenesis 75
3.3.2 AID expression in intratubular germ cell neoplasia unclassified 77
(IGCNU) and testicular germ cell tumours
4 Discussion 79
4.1 Prognostic impact of tumour-infiltrating T-cells and EBV in HL 79
4.1.1 Validation of antibodies directed against FoxP3, c-Maf and T-bet 80
4.1.2 Expression of GrB, T-bet, c-Maf and FoxP3 in Hodgkin lymphoma 82
4.1.3 T-cell subsets and prognosis of HL 82
4.1.4 EBV and prognosis of HL 84
4.1.5 Influence of B-lymphocytes on the prognosis of HL patients 85
4.2 Tumour cell induced leukocyte migration in HL 87
4.2.1 Detection of chemokines in HL derived cell lines and HL biopsies 87
4.2.2 Comparison of KMH-2 cells and KMH2-EBV cells 89
4.2.3 Migration of PBMC from healthy donors 90
4.2.4 Migration ofHL patient PBMC 91
43 AID project 94
5 Material and Methods 96
5.1 Material 96
5.1.1 Reagents and devices 96
5.1.2 Tissue samples 96
5.1.3 Cell lines 97
5.2 Methods 98
5.2.1 Immunohistochemistry (IHC) and immunofluorescence 98
5.2.2 In situ hybridisation (IsH) 99
5.2.3 Computer assisted microscopic analysis and Kaplan Meier analysis 101
5.2.4 RNAse protection assay (RPA) 102
5.2.5 Chemoattractants 102
5.2.5.1 Generation of cell culture supernatants 102
5.2.5.2 Sandwich ELISA 103
5.2.6 Human PBMC 103
Contents 7
5.2.7 Flow cytometry 104
5.2.8 Chemotaxis assay 105
5.2.9 Cytospins 105
5.3 AID project 106
5.3.1 Tissue samples
5.3.2 Immunohistochemistry and immunofluorescence 106
5.3.3 Cell lines 107
5.3.4 Intracellular staining and flow cytometry 107
5.3.5 Reverse transcriptase (RT-) polymerase chain reaction (PCR) 108
5 Bibliography 110
5.1 List of abbreviation 110
5.2 List of tables 112
5.3 List of figures 113
5.4 List of literature 114
6 Supplement to material and methods 126
6.1 Supplement to material 126
7.1.1 Reagents IHC 126
7.1.2 Antibodies 127
7.1.3 Mixtures for IHC 128
7.1.4 Probes, vectors, cloning and kits 128
7.1.5 Reagents IsH 128
7.1.6 Mixtures for IsH 130
7.1.7 Primer for AID RT-PCR 130
7.1.8 Reagents RT-PCR 130
7.1.9 Cell culture, flow cytometry, chemotaxis assay and buffy coat 131
7.1.10 Sandwich ELISA 132
7.1.11 Mixtures fur ELISA 133
7.1.12 Antibodies for flow cytometry 133
7.1.13 Mixtures for flow cytometry 134
7.1.14 Reagents for RPA 134
7.1.15 RunGelforRPA 135
7.1.16 Equipment 135
7.1.17 Companies 136
7.2 Supplement to methods 137
7.2.1 IHC 137
7.2.1.1 Immunohistochemical staining 137
7.2.1.2 Immunohistochemical double staining 138
7.2.1.3 Immunufluorescent double staining 139
7.2.2 IsH 140
7.2.2.1 Probe synthesis 140
7.2.2.2 IsH with radioactive labelled probes 141
7.2.3 RNAse protection assay 142
Inhaltsübersicht 8
Inhaltsübersicht
1 Zusammenfassung 17
1.1 Hintergrund I 17
1.2 Hypothese I 17
1.3 Methoden I lg
1.4 Ergebnisse I 18
1.5 Schlussfolgerung I 19
1.6 Hintergrund II 19
1.7 Hypothese II 19
1.8 Methoden II 20
1.9 Ergebnisse II 20
1.10 Schlussfolgerung II 21
2 Einleituns 22
2.1 Das Epstein-Barr Virus (EBV) 22
2.1.1 Historie und Biologie 22
2.1.2 Struktur 22
2.1.3 Primärinfektion und Persistenz 23
2.2 Hodgkin lymphoma (HL) 24
2.2.1 Epidemiologie 24
2.2.2 Klassifikation und Histopathologie 24
2.2.3 Genotyp und Phänotyp neoplastischer Hodgkin und Reed-Sternberg 26
(HRS) Zellen
2.2.4 EBV-assoziiertes Hodgkin Lymphom 26
2.2 4.1 EBV-Gene während Latenzphase II und Tumorgenese 26
2.2.4.2 EBV-Infektion in HRS Zellen 27
2.2.5 Tumor-infiltrierende Lymphozyten (TIL) im HL 28
2.3 Immunzellen 29
2.3.1 T-Lymphozyten 30
2.3.2 B-Lymphozyten 30
2.3.3 Aktivierung von T-Zellen nach Antigenkontakt 31
2.3.4 Regulatorische T-Zellen (Treg) 32
2.3.4.1 Charakterisierung von Treg 33
2.3.4.2 Forkhead Transcriptionsfaktor FoxP3 34
2.3.5.3 Unterdrückung von Effektor Zellen durch Treg 34
Inhaltsübersicht 9
2.4 Tumor-infiltrierende Lymphozyten und ihre prognostische Bedeutung 35
2.4.1 T-Lymphozyten in Tumoren 35
2.4.1.1 CD8+ zytotoxische T-Zellen (CTL) und Granzyme B für die 35
Prognose von Tumoren
2.4.1.2 CD4+T-Zellen 36
2.4.1.2.1 CD4+Thl-Zellen 37
2.4.1.2.2 CD4+Th2-Zellen 37
2.4.1.2.3 Treg in malignen Erkrankungen 38
2.4.1.2.4 Depletion von Treg 39
2.4.2 B-Lymphozyten in Tumoren 40
2.5 Zweittnmoren nach HL 41
2.6 Immunotherapeutische Ansätze für HL Therapie mittels EBV- 41
spezifischer CTL
2.7 Chemokine für die Rekrutierung von Lymphozyten 42
2.7.1 Strukturelle Klassifizierung 42
2.7.2 Funktionelle Klassifizierung 43
2.7.3 Beispiele für Chemokine 43
2.7.3.1 RANTES/CCL5 43
2.7.3.2 TARC/CCL17 44
2.7.3.3 MDC/CCL22 44
2.8 Formulierung von Hypothesen 45
2.8.1 Hypothese I 45
2.8.2 Hypothese II 45
2.9 ABD Projekt 46
2.9.1 Aktivitätsinduzierte Cytidindeaminase (AID) 46
2.9.2 AID in der Entwicklung von Tumoren 47
2.9.3 Hodentumore 47
2.9.4 Spermatogenese 47
2.9.5 Hypothese 48
3 Ergebnisse 49
3.1 Prognostische Bedeutung von Tumor-infiltrierenden T-Zellen und EBV 49
im HL
3.1.1 Validierung der gegen FoxP3, c-Maf und T-bet gerichteten 49
Antikörper
3.1.2 Expression von FoxP3, T-bet, c-Maf und Epstein-Barr viralen 52
Transkripten im HL
3.1.3 CD20+undCD21+LymphfollikelimHL 54
3.1.4 Definition von cut-offs für Kaplan Meier Analysen 55
3.1.5 T-Zell Untergruppen, EBV-Status und Prognose bei HL 58
3.1.6 CD20+und CD21+Lymphfollikel im HL und Prognose 61
Inhaltsübersicht 10
3.2 Tumorzcll-induzierte Leukozyten Migration im HL 64
3.2.1 Rekrutierung von TIL durch die Expression von Chemokinen und 64
Zytokinen durch HRS Zellen
3.2.1.1 Produktion von Chemokinen und Zytokinen in HRS Zellen 64
von cHL Biopsien
3.2.1.2 RNAse Protection assay zur Detektion von Chemokinen und 66
Zytokinen in HL abgeleiteten Zelllinien
3.2.1.3 Detektion von Chemokinen in HL abgeleiteten Zelllinien 68
mittels Sandwich ELISAs
3.2.2 Bestimmung des EBV-Status in EBV-infizierten KMH2 Zellen 69
3.2.3 Chemotaxis Assay mit periphären mononukleären Blutzellen 70
(PBMC) von gesunden Spendern und HL Patienten im Vergleich
3.2.4 Zusammensetzung von PBMC gesunder Spender und HL Patienten 71
3.2.5 Analyse der PBMC Zusammensetzung nach Chemotaxis assay 73
3.3 AID Projekt 75
3.3.4 AID-Expression während der normalen Spermatogenese 75
3.3.5 AID-Expression in intratubulären Keimzell neoplasien (IGCNU) 77
und in Keimzelltumoren des Hodens
4 Diskussion 79
4.1 Prognostische Bedeutung von Tumor-infiltrierenden T-Zellen und 79
EBV im HL
4.1.1 Validierung der gegen FoxP3, c-Maf und T-bet gerichteten 80
Antikörper
4.1.2 Expression von GrB, T-bet, c-Maf und FoxP3 im HL 82
4.1.3 T-Zell Untergruppen und Prognose bei HL 82
4.1.4 EBV und Prognose bei HL 84
4.1.5 Einfluss von B-Lymphozyten auf die Prognose von HL-Patienten 85
4.2 Tumorzcll-induzierte Leukozyten Migration im HL 87
4.2.1 Nachweis von Chemokinen in HL-abgeleiteten Zelllinien und in HL 87
Biopsien
4.2.2 Vergleich von KMH-2 Zellen mit KMH2-EBV Zellen 89
4 2.3 Wanderung von PBMC gesunder Spender 90
4.2.4 Wanderung von PBMC von HL-Patienten 91
4.3 AID Projekt 94
5 Material and Methoden 96
5.1 Material 96
5.1.1 Reagenzien und Geräte 96
5.1.2 Gewebeproben 96
5.1.3 Zelllinien 97
5.2 Methoden 98
5.2.1 Immunhistochemie (ICH) und Immunfluoreszenz 98
5.2.2 In situ Hybridisierung (IsH) 99
5.2.3 Computer unterstützte mikroskopische Analyse und Kaplan Meier 101
Inhaltsübersicht 11
Analyse
5.2.4 RNAse protection assay (RPA) 102
5.2.5 Chemische Lockstoffe 102
5.2.5.1 Generierung von Zellkulturilberständen 102
5.2.5.2 Sandwich ELISA 103
5.2.6 Humane PBMC 103
5.2.7 Durchflusszytometrie 104
5.2.8 Chemotaxis Assay 105
5.2.9 Zytospins 105
5.3 AID project 106
5.3.1 Gewebeproben 106
5.3.2 Immunhistochemie und Immunfluoreszenz 106
5.3.3 Zelllinien 107
5.3.4 Intrazelluläre Färbung und Durchflusszytometrie 107
5.3.5 Reverse Transkriptase (RT-) Polymerase Kettenreaktion (PCR) 108
6 Ouellenverzeichnis 110
6.1 Abkürzungsverzeichnis 110
6.2 Tabellenverzeichnis 112
6.3 Abbildungsverzeichnis 113
6.4 Literaturverzeichnis 114
7 Emänzuns zu Material und Methoden 126
7.1 Ergänzung zu Material 126
7.1.1 Reagenzien IHC 126
7.1.2 Antikörper 127
7.1.3 Ansätze IHC 128
7.1.4 Sonden, Vektoren, Klonierung und Kits 128
7.1.5 Reagenzien IsH 128
7.1.6 Ansätze IsH 130
7.1.7 Primer für AID RT-PCR 130
7.1.8 Reagenzien RT-PCR 130
7.1.9 Zellkultur, Durchflusszytometrie, Chemotaxis Assay und Bufiy coat 131
7.1.10 Sandwich ELISA 132
7.1.11 Ansätze für ELISA 133
7.1.12 Antikörper für Durchflusszytometrie 133
7.1.13 Ansätze für Durchflusszytometrie 134
7.1.14 Reagenzien für RPA 134
7.1.15 Gelansatz für RPA 135
7.1.16 Geräte 35
7.1.17 Firmen 136
Inhaltsübersicht 12
7.2 Ergänzung zu Methoden 137
7.2.1 IHC 137
7.2.1.1 Immunohistochemische Färbung 137
7.2.1.2 Immunohistochemische Doppelfarbung 138
7.2.1.3 Immunufluoreszenz DoppelfUrbung 139
7.2.2 IsH 140
7.2.2.1 Probensynthese 140
7.2.2.2 IsH mit radioaktiv-markierten Proben 141
7.2.3 RNAse protection assay 142
Bibliography 112
6.2 List of tables
Table 1: Pattern of EBV latency programs in different EBV-associated diseases 23
Table 2: Microscopic quantification of double labelled cells 49
Table 3: Thresholds for T-cell subsets defined for the discrimination of subgroups 58
Table 4: Kaplan Meier analyses 58
Table 5: Characterisation of 2nd malignancies 60
Table 6: RPA results for the expression of chemokines and cytokines in cHL 67
derived cell lines
Table7: ELISA results for the expression of chemokines in cHL derived cell lines 68
Table 8: Composition of PBMC in healthy individuals and HL patients 71
Table 9: Composition of PBMC after migration towards cell culture 73
supematants
Table 10: Clinical background of HL and second malignancies 97
Table 11: List of reagents used for immunohistochemistry 126
Table 12: List of primary and secondary antibodies used for IHC and 127
immunofluorescence
Table 13: List of sonds and vectors used for IsH 128
Table 14. List of reagents used for in situ hybridisation 128
Table 15: Primer used for RT-PCR 130
Table 16 List of reagents used for RT-PCR 130
Table 17: List of cell culture reagents 131
Table 18. Reagents used for ELISA 132
Table 19: Antibodies used for flow cytometry 133
Table 20: List of reagents used for RPA 134
Table 21: Equipment 135
Bibliography 113
6.3 List of figures
Figure 1: Morphology of classical Hodgkin lymphoma 25
Figure 2: Three classes of effector T-cells, specialised to deal with three 32
classes of pathogen
Figure 3: Central and peripheral tolerance 33
Figure 4: Overview of effector T-cells and Treg 35
Figure 5: Accumulation of Treg in the tumour cell environment 39
Figure 6: Double immunostaining of tonsils or HL 51
Figure 7: Immunohistochemical detection of FoxP3, T-bet and c-Maf in HL 53
Figure 8: EBER-specific in situ hybridisation on HL biopsies 54
Figure 9: Immunohistochemical staining against CD20 in HL 55
Figure 10. Thresholds for CD20 and CD21 56
Figure 11: Thresholds for T-cell subsets 57
Figure 12: Results of survival rates in relation to immunohistochemical variables 59
Figure 13: Results of survival rates in relation to EBV status 60
Figure 14: Results of survival rates in relation to interfollicular CD20* 61
B-lymphocytes
Figure 15: Results of survival rates in relation to CD20+lymphoid follicles 62
Figure 16: Resultsof survival rates in relation to CD21+ lymphoid follicles 63
Figure 17: Detection and quantification of MDC, TARC and RANTES in 65
HL biopsies
Figure 18: RPA with total RNA of HL derived cell lines 67
Figure 19: EBER-specific in situ hybridisation on KMH2-EBV cells 69
Figure 20: Chemotaxis assay with PBMC from healthy persons and HL patients 70
Figure 21: Flow cytometry for the detection of Treg in PBMC of healthy donors 72
and HL patients
Figure 22: Migration of CD4+ lymphocytes towards cell culture supematants 73
Figure 23: Migration of CD8+ lymphocytes towards cell culture supematants 74
Figure 24. Migration of FoxP3+ lymphocytes towards cell culture supematants 74
Figure 25: Immunohistochemistry and immunofluorescence on testicular germ cell 76
tumours
Figure 26. Reverse transcriptase polymerase chain reaction (RT-PCR) 76
Figure 27: IHC on testicular germ cell tumours 77
|
| adam_txt |
Contents 4
Contents
1 Summary 13
1.1 Background 13
1.2 Hypothesis I 13
1.3 Experimental design I 13
1.4 Results I 14
1.5 Conclusions I 14
1.6 Background II 15
1.7 Hypothesis II 15
1.8 Experimental design II 15
1.9 Results II 16
1.10 Conclusions II 16
2 Introduction 22
2.1 The Epstein Barr virus (EBV) 22
2.1.1 History and biology 22
2.1.2 Structure 22
2.1.3 Primary infection 23
2.2 Hodgkin lymphoma (HL) 24
2.2.1 Epidemiology 24
2.2.2 Classification and histopathology of HL 24
2.2.3 Genotype and phenotype of neoplastic Hodgkin and Reed-Stemberg 26
(HRS) cells
2.2.4 EBV-associated Hodgkin lymphoma 26
2.2.4.1 EBV genes expressed in latency II and tumourigenesis 26
2.2.4.2 EBV-infection of HRS cells 27
2.2.5 Tumour-infiltrating lymphocytes (TIL) in HL 28
2.3 Immune cells 29
2.3.1 T-lymphocytes 30
2.3.2 B-lymphocytes 30
2.3.3 Activation of T-cells after contact with foreign antigen 31
2.3.4 Regulatory T-cells (Treg) 32
2.3.4.1 Characterisation of Treg 33
2.3.4.2 The forkhead transcription factor FoxP3 34
2.3.4.3 Suppression of effector T-cells by Treg 34
Contents 5
2.4 Tumour-infiltrating lymphocytes and their prognostic influence 35
2.4.1 T-lymphocytes in tumours 35
2.4.1.1 CD8+cytotoxic T-lymphocytes (CTL) and Granzyme B 35
in the prognosis of tumours
2.4.1.2 CD4+T-cells 36
2.4.1.2.1 CD4+Thl-ce!ls 37
2.4.1.2.2. CD4+Th2-cells 37
2.4.1.2.3 Treg in malignant diseases 38
2.4.1.2.4 Depletion of Treg 39
2.4.2 B-lymphocytes in tumours 40
2.5 Secondary malignancies after HL 41
2.6 Immunotherapeutic approaches in the treatment of HL using EBV- 41
specific CTL
2.7 Chemokines critical for the recruitment of lymphocytes 42
2.7.1 Structural classification 42
2.7.2 Functional classification 43
2.7.3 Examples of chemokines 43
2.7.3.1 RANTES/CCL5 43
2.7.3.2 TARC/CCL17 44
2.7.3.3 MDC/CCL22 44
2.8 Hypotheses 45
2.8.2 Hypothesis I 45
2.8.3 Hypothesis II 45
2.9 AID project 46
2.9.1 Activation induced cytidine deaminase (AID) 46
2.9.2 AID in the development of tumours 47
2.9.3 Testicular cancer 47
2.9.4 Spermatogenesis 47
2.9.5 Hypothesis 48
3 Results 49
3.1 Prognostic impact of tumour-infiltrating T-cells and EBV in HL 49
3.1.1 Validation of antibodies directed against FoxP3, c-Maf and T-bet 49
3.1.2 Expression of FoxP3, T-bet, c-Maf and Epstein-Barr viral transcripts 52
in Hodgkin lymphoma
3.1.3 CD20+and CD21+lymphoid follicles in HL 54
3.1.4 Definition of cut-offs used for Kaplan Meier analyses 55
3.1.5 T-cell subsets, EBV-status and prognosis of HL 58
3.1.6 CD20+ and CD21+ lymphoid follicles in HL and prognosis 61
3.2 Tumour cell induced leukocyte migration in HL 64
3.2.1 Recruitment of TIL through production of chemokines and cytokines 64
by HRS cells
3.2.1.1 Production of chemokines and cytokines in HRS cells of cHL 64
biopsies
Contents 6
3.2.1.2 Detection of chemokines and cytokines in HL derived cell 66
lines with RNAse protection assay (RPA)
3.2.1.3 Detection of chemokines in HL derived cell lines with 68
Sandwich ELBA
3.2.2 Determination ofEBV-status in EBV-infected KHM-2 cells 69
3.2.3 Chemotaxis assay with peripheral blood mononuclear cells (PBMC) 70
from healthy individuals and HL patients in comparison
3.2.4 Composition of PBMC from healthy individuals and HL patients 71
3.2.5 Analyses of PBMC composition after chemotaxis assay 73
3.3 AID project 75
3.3.1 AID expression in normal spermatogenesis 75
3.3.2 AID expression in intratubular germ cell neoplasia unclassified 77
(IGCNU) and testicular germ cell tumours
4 Discussion 79
4.1 Prognostic impact of tumour-infiltrating T-cells and EBV in HL 79
4.1.1 Validation of antibodies directed against FoxP3, c-Maf and T-bet 80
4.1.2 Expression of GrB, T-bet, c-Maf and FoxP3 in Hodgkin lymphoma 82
4.1.3 T-cell subsets and prognosis of HL 82
4.1.4 EBV and prognosis of HL 84
4.1.5 Influence of B-lymphocytes on the prognosis of HL patients 85
4.2 Tumour cell induced leukocyte migration in HL 87
4.2.1 Detection of chemokines in HL derived cell lines and HL biopsies 87
4.2.2 Comparison of KMH-2 cells and KMH2-EBV cells 89
4.2.3 Migration of PBMC from healthy donors 90
4.2.4 Migration ofHL patient PBMC 91
43 AID project 94
5 Material and Methods 96
5.1 Material 96
5.1.1 Reagents and devices 96
5.1.2 Tissue samples 96
5.1.3 Cell lines 97
5.2 Methods 98
5.2.1 Immunohistochemistry (IHC) and immunofluorescence 98
5.2.2 In situ hybridisation (IsH) 99
5.2.3 Computer assisted microscopic analysis and Kaplan Meier analysis 101
5.2.4 RNAse protection assay (RPA) 102
5.2.5 Chemoattractants 102
5.2.5.1 Generation of cell culture supernatants 102
5.2.5.2 Sandwich ELISA 103
5.2.6 Human PBMC 103
Contents 7
5.2.7 Flow cytometry 104
5.2.8 Chemotaxis assay 105
5.2.9 Cytospins 105
5.3 AID project 106
5.3.1 Tissue samples
5.3.2 Immunohistochemistry and immunofluorescence 106
5.3.3 Cell lines 107
5.3.4 Intracellular staining and flow cytometry 107
5.3.5 Reverse transcriptase (RT-) polymerase chain reaction (PCR) 108
5 Bibliography 110
5.1 List of abbreviation 110
5.2 List of tables 112
5.3 List of figures 113
5.4 List of literature 114
6 Supplement to material and methods 126
6.1 Supplement to material 126
7.1.1 Reagents IHC 126
7.1.2 Antibodies 127
7.1.3 Mixtures for IHC 128
7.1.4 Probes, vectors, cloning and kits 128
7.1.5 Reagents IsH 128
7.1.6 Mixtures for IsH 130
7.1.7 Primer for AID RT-PCR 130
7.1.8 Reagents RT-PCR 130
7.1.9 Cell culture, flow cytometry, chemotaxis assay and buffy coat 131
7.1.10 Sandwich ELISA 132
7.1.11 Mixtures fur ELISA 133
7.1.12 Antibodies for flow cytometry 133
7.1.13 Mixtures for flow cytometry 134
7.1.14 Reagents for RPA 134
7.1.15 RunGelforRPA 135
7.1.16 Equipment 135
7.1.17 Companies 136
7.2 Supplement to methods 137
7.2.1 IHC 137
7.2.1.1 Immunohistochemical staining 137
7.2.1.2 Immunohistochemical double staining 138
7.2.1.3 Immunufluorescent double staining 139
7.2.2 IsH 140
7.2.2.1 Probe synthesis 140
7.2.2.2 IsH with radioactive labelled probes 141
7.2.3 RNAse protection assay 142
Inhaltsübersicht 8
Inhaltsübersicht
1 Zusammenfassung 17
1.1 Hintergrund I 17
1.2 Hypothese I 17
1.3 Methoden I lg
1.4 Ergebnisse I 18
1.5 Schlussfolgerung I 19
1.6 Hintergrund II 19
1.7 Hypothese II 19
1.8 Methoden II 20
1.9 Ergebnisse II 20
1.10 Schlussfolgerung II 21
2 Einleituns 22
2.1 Das Epstein-Barr Virus (EBV) 22
2.1.1 Historie und Biologie 22
2.1.2 Struktur 22
2.1.3 Primärinfektion und Persistenz 23
2.2 Hodgkin lymphoma (HL) 24
2.2.1 Epidemiologie 24
2.2.2 Klassifikation und Histopathologie 24
2.2.3 Genotyp und Phänotyp neoplastischer Hodgkin und Reed-Sternberg 26
(HRS) Zellen
2.2.4 EBV-assoziiertes Hodgkin Lymphom 26
2.2 4.1 EBV-Gene während Latenzphase II und Tumorgenese 26
2.2.4.2 EBV-Infektion in HRS Zellen 27
2.2.5 Tumor-infiltrierende Lymphozyten (TIL) im HL 28
2.3 Immunzellen 29
2.3.1 T-Lymphozyten 30
2.3.2 B-Lymphozyten 30
2.3.3 Aktivierung von T-Zellen nach Antigenkontakt 31
2.3.4 Regulatorische T-Zellen (Treg) 32
2.3.4.1 Charakterisierung von Treg 33
2.3.4.2 Forkhead Transcriptionsfaktor FoxP3 34
2.3.5.3 Unterdrückung von Effektor Zellen durch Treg 34
Inhaltsübersicht 9
2.4 Tumor-infiltrierende Lymphozyten und ihre prognostische Bedeutung 35
2.4.1 T-Lymphozyten in Tumoren 35
2.4.1.1 CD8+ zytotoxische T-Zellen (CTL) und Granzyme B für die 35
Prognose von Tumoren
2.4.1.2 CD4+T-Zellen 36
2.4.1.2.1 CD4+Thl-Zellen 37
2.4.1.2.2 CD4+Th2-Zellen 37
2.4.1.2.3 Treg in malignen Erkrankungen 38
2.4.1.2.4 Depletion von Treg 39
2.4.2 B-Lymphozyten in Tumoren 40
2.5 Zweittnmoren nach HL 41
2.6 Immunotherapeutische Ansätze für HL Therapie mittels EBV- 41
spezifischer CTL
2.7 Chemokine für die Rekrutierung von Lymphozyten 42
2.7.1 Strukturelle Klassifizierung 42
2.7.2 Funktionelle Klassifizierung 43
2.7.3 Beispiele für Chemokine 43
2.7.3.1 RANTES/CCL5 43
2.7.3.2 TARC/CCL17 44
2.7.3.3 MDC/CCL22 44
2.8 Formulierung von Hypothesen 45
2.8.1 Hypothese I 45
2.8.2 Hypothese II 45
2.9 ABD Projekt 46
2.9.1 Aktivitätsinduzierte Cytidindeaminase (AID) 46
2.9.2 AID in der Entwicklung von Tumoren 47
2.9.3 Hodentumore 47
2.9.4 Spermatogenese 47
2.9.5 Hypothese 48
3 Ergebnisse 49
3.1 Prognostische Bedeutung von Tumor-infiltrierenden T-Zellen und EBV 49
im HL
3.1.1 Validierung der gegen FoxP3, c-Maf und T-bet gerichteten 49
Antikörper
3.1.2 Expression von FoxP3, T-bet, c-Maf und Epstein-Barr viralen 52
Transkripten im HL
3.1.3 CD20+undCD21+LymphfollikelimHL 54
3.1.4 Definition von cut-offs für Kaplan Meier Analysen 55
3.1.5 T-Zell Untergruppen, EBV-Status und Prognose bei HL 58
3.1.6 CD20+und CD21+Lymphfollikel im HL und Prognose 61
Inhaltsübersicht 10
3.2 Tumorzcll-induzierte Leukozyten Migration im HL 64
3.2.1 Rekrutierung von TIL durch die Expression von Chemokinen und 64
Zytokinen durch HRS Zellen
3.2.1.1 Produktion von Chemokinen und Zytokinen in HRS Zellen 64
von cHL Biopsien
3.2.1.2 RNAse Protection assay zur Detektion von Chemokinen und 66
Zytokinen in HL abgeleiteten Zelllinien
3.2.1.3 Detektion von Chemokinen in HL abgeleiteten Zelllinien 68
mittels Sandwich ELISAs
3.2.2 Bestimmung des EBV-Status in EBV-infizierten KMH2 Zellen 69
3.2.3 Chemotaxis Assay mit periphären mononukleären Blutzellen 70
(PBMC) von gesunden Spendern und HL Patienten im Vergleich
3.2.4 Zusammensetzung von PBMC gesunder Spender und HL Patienten 71
3.2.5 Analyse der PBMC Zusammensetzung nach Chemotaxis assay 73
3.3 AID Projekt 75
3.3.4 AID-Expression während der normalen Spermatogenese 75
3.3.5 AID-Expression in intratubulären Keimzell neoplasien (IGCNU) 77
und in Keimzelltumoren des Hodens
4 Diskussion 79
4.1 Prognostische Bedeutung von Tumor-infiltrierenden T-Zellen und 79
EBV im HL
4.1.1 Validierung der gegen FoxP3, c-Maf und T-bet gerichteten 80
Antikörper
4.1.2 Expression von GrB, T-bet, c-Maf und FoxP3 im HL 82
4.1.3 T-Zell Untergruppen und Prognose bei HL 82
4.1.4 EBV und Prognose bei HL 84
4.1.5 Einfluss von B-Lymphozyten auf die Prognose von HL-Patienten 85
4.2 Tumorzcll-induzierte Leukozyten Migration im HL 87
4.2.1 Nachweis von Chemokinen in HL-abgeleiteten Zelllinien und in HL 87
Biopsien
4.2.2 Vergleich von KMH-2 Zellen mit KMH2-EBV Zellen 89
4 2.3 Wanderung von PBMC gesunder Spender 90
4.2.4 Wanderung von PBMC von HL-Patienten 91
4.3 AID Projekt 94
5 Material and Methoden 96
5.1 Material 96
5.1.1 Reagenzien und Geräte 96
5.1.2 Gewebeproben 96
5.1.3 Zelllinien 97
5.2 Methoden 98
5.2.1 Immunhistochemie (ICH) und Immunfluoreszenz 98
5.2.2 In situ Hybridisierung (IsH) 99
5.2.3 Computer unterstützte mikroskopische Analyse und Kaplan Meier 101
Inhaltsübersicht 11
Analyse
5.2.4 RNAse protection assay (RPA) 102
5.2.5 Chemische Lockstoffe 102
5.2.5.1 Generierung von Zellkulturilberständen 102
5.2.5.2 Sandwich ELISA 103
5.2.6 Humane PBMC 103
5.2.7 Durchflusszytometrie 104
5.2.8 Chemotaxis Assay 105
5.2.9 Zytospins 105
5.3 AID project 106
5.3.1 Gewebeproben 106
5.3.2 Immunhistochemie und Immunfluoreszenz 106
5.3.3 Zelllinien 107
5.3.4 Intrazelluläre Färbung und Durchflusszytometrie 107
5.3.5 Reverse Transkriptase (RT-) Polymerase Kettenreaktion (PCR) 108
6 Ouellenverzeichnis 110
6.1 Abkürzungsverzeichnis 110
6.2 Tabellenverzeichnis 112
6.3 Abbildungsverzeichnis 113
6.4 Literaturverzeichnis 114
7 Emänzuns zu Material und Methoden 126
7.1 Ergänzung zu Material 126
7.1.1 Reagenzien IHC 126
7.1.2 Antikörper 127
7.1.3 Ansätze IHC 128
7.1.4 Sonden, Vektoren, Klonierung und Kits 128
7.1.5 Reagenzien IsH 128
7.1.6 Ansätze IsH 130
7.1.7 Primer für AID RT-PCR 130
7.1.8 Reagenzien RT-PCR 130
7.1.9 Zellkultur, Durchflusszytometrie, Chemotaxis Assay und Bufiy coat 131
7.1.10 Sandwich ELISA 132
7.1.11 Ansätze für ELISA 133
7.1.12 Antikörper für Durchflusszytometrie 133
7.1.13 Ansätze für Durchflusszytometrie 134
7.1.14 Reagenzien für RPA 134
7.1.15 Gelansatz für RPA 135
7.1.16 Geräte '35
7.1.17 Firmen 136
Inhaltsübersicht 12
7.2 Ergänzung zu Methoden 137
7.2.1 IHC 137
7.2.1.1 Immunohistochemische Färbung 137
7.2.1.2 Immunohistochemische Doppelfarbung 138
7.2.1.3 Immunufluoreszenz DoppelfUrbung 139
7.2.2 IsH 140
7.2.2.1 Probensynthese 140
7.2.2.2 IsH mit radioaktiv-markierten Proben 141
7.2.3 RNAse protection assay 142
Bibliography 112
6.2 List of tables
Table 1: Pattern of EBV latency programs in different EBV-associated diseases 23
Table 2: Microscopic quantification of double labelled cells 49
Table 3: Thresholds for T-cell subsets defined for the discrimination of subgroups 58
Table 4: Kaplan Meier analyses 58
Table 5: Characterisation of 2nd malignancies 60
Table 6: RPA results for the expression of chemokines and cytokines in cHL 67
derived cell lines
Table7: ELISA results for the expression of chemokines in cHL derived cell lines 68
Table 8: Composition of PBMC in healthy individuals and HL patients 71
Table 9: Composition of PBMC after migration towards cell culture 73
supematants
Table 10: Clinical background of HL and second malignancies 97
Table 11: List of reagents used for immunohistochemistry 126
Table 12: List of primary and secondary antibodies used for IHC and 127
immunofluorescence
Table 13: List of sonds and vectors used for IsH 128
Table 14. List of reagents used for in situ hybridisation 128
Table 15: Primer used for RT-PCR 130
Table 16 List of reagents used for RT-PCR 130
Table 17: List of cell culture reagents 131
Table 18. Reagents used for ELISA 132
Table 19: Antibodies used for flow cytometry 133
Table 20: List of reagents used for RPA 134
Table 21: Equipment 135
Bibliography 113
6.3 List of figures
Figure 1: Morphology of classical Hodgkin lymphoma 25
Figure 2: Three classes of effector T-cells, specialised to deal with three 32
classes of pathogen
Figure 3: Central and peripheral tolerance 33
Figure 4: Overview of effector T-cells and Treg 35
Figure 5: Accumulation of Treg in the tumour cell environment 39
Figure 6: Double immunostaining of tonsils or HL 51
Figure 7: Immunohistochemical detection of FoxP3, T-bet and c-Maf in HL 53
Figure 8: EBER-specific in situ hybridisation on HL biopsies 54
Figure 9: Immunohistochemical staining against CD20 in HL 55
Figure 10. Thresholds for CD20 and CD21 56
Figure 11: Thresholds for T-cell subsets 57
Figure 12: Results of survival rates in relation to immunohistochemical variables 59
Figure 13: Results of survival rates in relation to EBV status 60
Figure 14: Results of survival rates in relation to interfollicular CD20* 61
B-lymphocytes
Figure 15: Results of survival rates in relation to CD20+lymphoid follicles 62
Figure 16: Resultsof survival rates in relation to CD21+ lymphoid follicles 63
Figure 17: Detection and quantification of MDC, TARC and RANTES in 65
HL biopsies
Figure 18: RPA with total RNA of HL derived cell lines 67
Figure 19: EBER-specific in situ hybridisation on KMH2-EBV cells 69
Figure 20: Chemotaxis assay with PBMC from healthy persons and HL patients 70
Figure 21: Flow cytometry for the detection of Treg in PBMC of healthy donors 72
and HL patients
Figure 22: Migration of CD4+ lymphocytes towards cell culture supematants 73
Figure 23: Migration of CD8+ lymphocytes towards cell culture supematants 74
Figure 24. Migration of FoxP3+ lymphocytes towards cell culture supematants 74
Figure 25: Immunohistochemistry and immunofluorescence on testicular germ cell 76
tumours
Figure 26. Reverse transcriptase polymerase chain reaction (RT-PCR) 76
Figure 27: IHC on testicular germ cell tumours 77 |
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| spelling | Schreck, Sabine Verfasser aut Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL von Sabine Schreck 2008 147 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2008 Langzeitarchivierung Nationalbibliothek gewährleistet Tumorimmunologie (DE-588)4128018-0 gnd rswk-swf Lymphogranulomatose (DE-588)4127860-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Lymphogranulomatose (DE-588)4127860-4 s Tumorimmunologie (DE-588)4128018-0 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:29-opus-9423 https://open.fau.de/handle/openfau/641 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-9423 Resolvingsystem http://d-nb.info/989353001/34 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016538335&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schreck, Sabine Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL Tumorimmunologie (DE-588)4128018-0 gnd Lymphogranulomatose (DE-588)4127860-4 gnd |
| subject_GND | (DE-588)4128018-0 (DE-588)4127860-4 (DE-588)4113937-9 |
| title | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL |
| title_auth | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL |
| title_exact_search | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL |
| title_exact_search_txtP | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL |
| title_full | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL von Sabine Schreck |
| title_fullStr | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL von Sabine Schreck |
| title_full_unstemmed | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL von Sabine Schreck |
| title_short | Tumour cell induced leukocyte migration in Hodgkin Lymphoma (HL) |
| title_sort | tumour cell induced leukocyte migration in hodgkin lymphoma hl prognostic impact of t cell subsets b cells and epstein barr virus ebv infection in hl |
| title_sub | prognostic impact of T-cell subsets, B-cells and Epstein-Barr virus (EBV)-infection in HL |
| topic | Tumorimmunologie (DE-588)4128018-0 gnd Lymphogranulomatose (DE-588)4127860-4 gnd |
| topic_facet | Tumorimmunologie Lymphogranulomatose Hochschulschrift |
| url | https://open.fau.de/handle/openfau/641 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-9423 http://d-nb.info/989353001/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016538335&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schrecksabine tumourcellinducedleukocytemigrationinhodgkinlymphomahlprognosticimpactoftcellsubsetsbcellsandepsteinbarrvirusebvinfectioninhl |