On the origin of giant multinuclear Reed-Sternberg cells and the role of CD4 T cells in Hodgkin lymphoma

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1 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)

2 Table of contents 1 Summary 1 2 Zusammenfassung 4 3 Introduction The immune system Innate immunity Adaptive immunity T lymphocytes CD8 Tcells CD4T cells The immunological synapse : B lymphocytes Immunological memory Self-tolerance and tumor immunology Lymphoid malignancies Lymphomas Non-Hodgkin lymphoma (NHL) Hodgkin lymphoma Diagnosis of HL HL therapy and prognosis HL research - state of the art In situ research The cellular origin of HL HL microenvironment In vitro research Development of giant RS cells Co-culture with lymphocytes HL stem cells In vivo research HL mouse models Immunotherapy concepts 29 4 Specific Aims 30 5 Material and Methods Material Chemicals 31 I

3 5.1.2 Buffers and solutions Cell culture media Bacteria Enzymes and ladders Lentiviral plasmids Cell lines Antibodies Commercial kits Plastic material Analytical software Technical equipment Methods Molecular biology 38 V Lentiviral particles Lentiviral plasmids Transfer vectors Transformation of bacteria Analytical DNA preparation Preparative DNA preparation DNA restriction analysis Agarose gel electrophoresis Preservation of bacterial clones Cell culture Cultivation of adherent cell lines Cultivation of suspension cell lines Cultivation of human primary cells Isolation of peripheral blood mononuclear cells Magnetic activated cell sorting (MACS) Negative isolation Depletion Fluorescence activated cell sorting (FACS) Sample preparation Cell sorting Flow cytometry Size exclusion of HRS cells Co-cultivation of HRS cells and PBMC/T cells Growth curves 43 II

4 .. Table of contents Cluster formation assay Blocking antibodies Conventional cell counting FACS-based cell counting Preservation of cell lines and primary cells Thawing of cell lines Production of lentiviral particles Transfection DNA precipitation mixture Titration Transduction of HRS cells Colony formation assay Gene expression profiling Time-lapse' imaging and single cell tracking Time-lapse imaging Single cell tracking Animal experiments Animal experiment permission Animal husbandry conditions Immunodeficient mice Generation of ex vivo L428 cells HL-like mouse model Weight curves Tumor growth curves Blood collection from mice Endpoint analysis of in vivo experiments Necropsy of mice Processing of organ samples for histological analysis 50 6 Results Subpopulations of HL cell line L CD20 expressing L428 cells L428 subpopulations sorted via cell size or CD15/CD30 expression Clonal growth and proliferation potential of L428 subpopulations L428 subpopulations generated by size-exclusion Time-lapse microscopy and single cell tracking of HRS cells Giant HRS cells show a tremendous increase in lifetime RS cell formation is based on re-fusion of daughter cells 60 III

5 6.2.3 Re-fusion of HRS cells is based on incomplete cytokinesis RS cells preserve a residual proliferation capacity Giant HRS cell development and fate is committed in the ancestor generation Long-term co-culture of CD4 T cells and HL cell line L Spontaneous rosetting of L428 cells by human T cells CD4 T cells develop anti-hrs cell cytotoxicity in long-term co-culture Dexamethasone prevents CD4 T cell-mediated HRS cell killing Mechanistic insight into CD4 T cell-mediated HRS cell killing Strong binding to MHC-II leads to death of HRS cells CD4 T-cell anti-hrs cell cytotoxicity is based on MHC-II incompatibility In vivo interaction studies of CD4 T cells and HL cell line L Co-injection of L428 cells and PBMC into NSG mice T cells are able to kill HRS cells in vivo 83 f HL tumor rejection by purified CD4 T cells Ex vivo L428 cells show in vivo adaptation-induced behavior 87 7 Discussion Characterization of RS cell dynamics Subpopulations of the HL cell line L Enrichment of CD20 positive L428 cells Giant L428 cells express CD 15 and CD RS cells evolve from re-fusion of daughter cells Giant and long-living cells are rare within HL cell lines Giant HRS cell progenitors show re-fusion and trichotomy Only multinucleated giant cells are able to proliferate RS cell development is an impaired behavior The role of CD4 T cells in HL MHC-II mismatched CD4 T cells are reactive against L428 cells in co-culture Rosetting of L428 cells by T cells displays anti-tumor activity HRS cells induce cytotoxic CD4 T cells HRS cells and CD4 T cells build reactive immunological synapses Anti-HRS cell reaction ofcd4 T cells is based on MHC-II incompatibility MHC incompatible T cells reject solid HL tumors in vivo MHC incompatible T cells reject solid HL tumors in vivo HRS cells are resistant to MHC-II compatible CD4 T cells in vivo Characterization of HL tumors ex vivo Currently ongoing experiments - Outlook 105 IV

6 8 References Abbreviations Publications and Presentations Curriculum Vitae Acknowledgments - Danksagung 124

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