On the origin of giant multinuclear Reed-Sternberg cells and the role of CD4 T cells in Hodgkin lymphoma Uber die Entstehung von multinuklearen Reed-Sternberg Riesenzellen und die Rolle von CD4 T-Zellen im Hodgkin Lymphom ' Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie der Goethe-Universitat in Frankfurt am Main von Benjamin Rengstl aus Langen (Hessen) Frankfurt am Main, Dezember 2013 (D30) http://d-nb.info/1052196497
Table of contents 1 Summary 1 2 Zusammenfassung 4 3 Introduction 9 3.1 The immune system 9 3.1.1 Innate immunity 9 3.1.2 Adaptive immunity 10 3.1.2.1 T lymphocytes 11 3.1.2.1.1 CD8 Tcells 11 3.1.2.1.2 CD4T cells 12 3.1.2.1.3 The immunological synapse : 13 3.1.2.2 B lymphocytes 14 3.1.2.3 Immunological memory 16 3.1.2.4 Self-tolerance and tumor immunology 16 3.1.3 Lymphoid malignancies 17 3.2 Lymphomas 17 3.2.1 Non-Hodgkin lymphoma (NHL) 19 3.2.2 Hodgkin lymphoma 20 3.2.2.1 Diagnosis of HL 20 3.2.2.2 HL therapy and prognosis 21 3.3 HL research - state of the art 21 3.3.1 In situ research 22 3.3.1.1 The cellular origin of HL 22 3.3.1.2 HL microenvironment 23 3.3.2 In vitro research 25 3.3.2.1 Development of giant RS cells 25 3.3.2.2 Co-culture with lymphocytes 26 3.3.2.3 HL stem cells 27 3.3.3 In vivo research 28 3.3.3.1 HL mouse models 28 3.3.3.2 Immunotherapy concepts 29 4 Specific Aims 30 5 Material and Methods 31 5.1 Material 31 5.1.1 Chemicals 31 I
5.1.2 Buffers and solutions 31 5.1.3 Cell culture media 32 5.1.4 Bacteria 32 5.1.5 Enzymes and ladders 32 5.1.6 Lentiviral plasmids 33 5.1.7 Cell lines 33 5.1.8 Antibodies 34 5.1.9 Commercial kits 35 5.1.10 Plastic material 36 5.1.11 Analytical software 36 5.1.12 Technical equipment 37 5.2 Methods 38 5.2.1 Molecular biology 38 V 5.2.1.1 Lentiviral particles 38 5.2.1.1.1 Lentiviral plasmids 38 5.2.1.1.2 Transfer vectors 38 5.2.1.2 Transformation of bacteria 38 5.2.1.3 Analytical DNA preparation 39 5.2.1.4 Preparative DNA preparation 39 5.2.1.5 DNA restriction analysis 39 5.2.1.6 Agarose gel electrophoresis 40 5.2.1.7 Preservation of bacterial clones 40 5.2.2 Cell culture 40 5.2.2.1 Cultivation of adherent cell lines 40 5.2.2.2 Cultivation of suspension cell lines 40 5.2.2.3 Cultivation of human primary cells 41 5.2.2.4 Isolation of peripheral blood mononuclear cells 41 5.2.2.5 Magnetic activated cell sorting (MACS) 41 5.2.2.5.1 Negative isolation 41 5.2.2.5.2 Depletion 42 5.2.2.6 Fluorescence activated cell sorting (FACS) 42 5.2.2.6.1 Sample preparation 42 5.2.2.6.2 Cell sorting. 42 5.2.2.6.3 Flow cytometry 43 5.2.2.7 Size exclusion of HRS cells 43 5.2.2.8 Co-cultivation of HRS cells and PBMC/T cells 43 5.2.2.8.1 Growth curves 43 II
.. Table of contents 5.2.2.8.2 Cluster formation assay 43 5.2.2.8.3 Blocking antibodies 44 5.2.2.9 Conventional cell counting 44 5.2.2.10 FACS-based cell counting 44 5.2.2.11 Preservation of cell lines and primary cells 44 5.2.2.12 Thawing of cell lines 45 5.2.2.13 Production of lentiviral particles 45 5.2.2.13.1 Transfection 45 5.2.2.13.2 DNA precipitation mixture 45 5.2.2.13.3 Titration 45 5.2.2.14 Transduction of HRS cells 46 5.2.2.15 Colony formation assay 46 5.2.2.16 Gene expression profiling 46 5.2.2.17 Time-lapse' imaging and single cell tracking 46 5.2.2.17.1 Time-lapse imaging 47 5.2.2.17.2 Single cell tracking 47 5.2.3 Animal experiments 48 5.2.3.1 Animal experiment permission 48 5.2.3.2 Animal husbandry conditions 48 5.2.3.3 Immunodeficient mice 48 5.2.3.4 Generation of ex vivo L428 cells 48 5.2.3.5 HL-like mouse model 49 5.2.3.5.1 Weight curves 49 5.2.3.5.2 Tumor growth curves 49 5.2.3.5.3 Blood collection from mice 49 5.2.3.6 Endpoint analysis of in vivo experiments 50 5.2.3.6.1 Necropsy of mice 50 5.2.3.6.2 Processing of organ samples for histological analysis 50 6 Results 51 6.1 Subpopulations of HL cell line L428 51 6.1.1 CD20 expressing L428 cells 51 6.1.2 L428 subpopulations sorted via cell size or CD15/CD30 expression 52 6.1.3 Clonal growth and proliferation potential of L428 subpopulations 55 6.1.4 L428 subpopulations generated by size-exclusion 56 6.2 Time-lapse microscopy and single cell tracking of HRS cells 57 6.2.1 Giant HRS cells show a tremendous increase in lifetime 57 6.2.2 RS cell formation is based on re-fusion of daughter cells 60 III
6.2.3 Re-fusion of HRS cells is based on incomplete cytokinesis 63 6.2.4 RS cells preserve a residual proliferation capacity 64 6.2.5 Giant HRS cell development and fate is committed in the ancestor generation 66 6.3 Long-term co-culture of CD4 T cells and HL cell line L428 68 6.3.1 Spontaneous rosetting of L428 cells by human T cells 68 6.3.2 CD4 T cells develop anti-hrs cell cytotoxicity in long-term co-culture 70 6.3.3 Dexamethasone prevents CD4 T cell-mediated HRS cell killing 71 6.3.4 Mechanistic insight into CD4 T cell-mediated HRS cell killing 73 6.3.5 Strong binding to MHC-II leads to death of HRS cells 76 6.3.6 CD4 T-cell anti-hrs cell cytotoxicity is based on MHC-II incompatibility 77 6.4 In vivo interaction studies of CD4 T cells and HL cell line L428 80 6.4.1 Co-injection of L428 cells and PBMC into NSG mice 81 6.4.2 T cells are able to kill HRS cells in vivo 83 f 6.4.3 HL tumor rejection by purified CD4 T cells 85 6.4.4 Ex vivo L428 cells show in vivo adaptation-induced behavior 87 7 Discussion 90 7.1 Characterization of RS cell dynamics 90 7.1.1 Subpopulations of the HL cell line L428 91 7.1.1.1 Enrichment of CD20 positive L428 cells 91 7.1.1.2 Giant L428 cells express CD 15 and CD30 92 7.1.2 RS cells evolve from re-fusion of daughter cells 93 7.1.2.1 Giant and long-living cells are rare within HL cell lines 94 7.1.2.2 Giant HRS cell progenitors show re-fusion and trichotomy 94 7.1.2.3 Only multinucleated giant cells are able to proliferate 96 7.1.2.4 RS cell development is an impaired behavior 96 7.2 The role of CD4 T cells in HL 98 7.2.1 MHC-II mismatched CD4 T cells are reactive against L428 cells in co-culture 98 7.2.1.1 Rosetting of L428 cells by T cells displays anti-tumor activity 98 7.2.1.2 HRS cells induce cytotoxic CD4 T cells 99 7.2.1.3 HRS cells and CD4 T cells build reactive immunological synapses 100 7.2.1.4 Anti-HRS cell reaction ofcd4 T cells is based on MHC-II incompatibility 101 7.2.2 MHC incompatible T cells reject solid HL tumors in vivo 103 7.2.2.1 MHC incompatible T cells reject solid HL tumors in vivo 103 7.2.2.2 HRS cells are resistant to MHC-II compatible CD4 T cells in vivo 104 7.3 Characterization of HL tumors ex vivo 104 7.4 Currently ongoing experiments - Outlook 105 IV
8 References 107 9 Abbreviations 115 10 Publications and Presentations 119 11 Curriculum Vitae 122 12 Acknowledgments - Danksagung 124