Generation of Autologous and Allorestricted Cytotoxic T Cell Clones Directed Against Ewing Tumor Antigens

TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Entwicklungsgenetik Generation of autologous and allorestricted cytotoxic T cell clones directed against Ewing Tumor antigens Stefan Pirson Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. S. Scherer Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst 2. Univ.-Prof. Dr. St. Burdach Die Dissertation wurde am 18.05.2009 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 02.12.2009 angenommen. Table of Contents Table of Contents 1 Introduction ................................................................................................. 1 1.1 Ewing Family Tumor (EFT) ................................................................... 1 1.1.1 History ............................................................................................ 2 1.1.2 Incidence and symptomatology ...................................................... 3 1.1.3 Diagnosis ....................................................................................... 3 1.1.4 Prognostic factors .......................................................................... 4 1.1.5 Treatment ....................................................................................... 5 1.2 Immunotherapy ..................................................................................... 7 1.2.1 Dendritic Cells ................................................................................ 8 1.2.2 T cell therapy ................................................................................ 10 1.3 Aim of the study .................................................................................. 11 2 Materials ................................................................................................... 13 2.1 Instruments and Equipment ................................................................ 13 2.2 Commonly used materials .................................................................. 14 2.3 Chemicals and biological reagents ..................................................... 15 2.4 Kits ...................................................................................................... 17 2.5 Cell Culture media and solutions ........................................................ 17 2.6 Cells .................................................................................................... 19 2.7 ELISpot Reagents ............................................................................... 20 2.7.1 Antibodies .................................................................................... 20 2.7.2 Enzymes, Buffers, Plates ............................................................. 20 2.8 Flow cytometry reagents ..................................................................... 20 2.8.1 Reagents ...................................................................................... 20 2.8.2 Antibodies .................................................................................... 21 2.9 Peptides .............................................................................................. 22 2.10 Plasmids .......................................................................................... 23 2.11 List of manufacturers ....................................................................... 27 3 Methods .................................................................................................... 29 i Table of Contents 3.1 Cell culture .......................................................................................... 29 3.1.1 Cell counting ................................................................................. 29 3.1.2 Cryopreservation of cells .............................................................. 30 3.1.3 Thawing of cryopreserved cells .................................................... 30 3.1.4 Culture of adherent tumor cell lines .............................................. 30 3.1.5 Culture of suspension tumor cell lines .......................................... 31 3.2 Isolation of peripheral blood mononuclear cells .................................. 31 3.2.1 Isolation of PBMC from blood donor unit (500ml) ......................... 31 3.2.2 Isolation of PBMC from whole blood ............................................. 32 3.3 Isolation of CD14+ cells ....................................................................... 32 3.4 Isolation of CD8+ cells ......................................................................... 32 3.5 Generation of Dendritic cells ............................................................... 33 3.6 Lipofection ........................................................................................... 34 3.7 Flow cytometry .................................................................................... 35 3.8 Cell sorting with Pentamer-staining ..................................................... 35 3.9 RNA isolation ...................................................................................... 36 3.10 cDNA synthesis................................................................................ 37 3.11 Real-time PCR ................................................................................. 38 3.12 Minipreparation of plasmid DNA ...................................................... 39 3.13 Restriction analysis .......................................................................... 40 3.14 DNA agarose gel electrophoresis .................................................... 40 3.15 Maxipreparation of plasmid DNA ..................................................... 40 3.16 In vitro Priming ................................................................................. 41 3.17 Limiting Dilution................................................................................ 41 3.18 ELISpot ............................................................................................ 42 3.19 CTL expansion ................................................................................. 44 3.20 In silico prediction of peptide epitopes ............................................. 44 3.21 HLA-A*0201 / peptide-binding assay ............................................... 45 3.22 Vβ analysis of TCR repertoire .......................................................... 46 3.23 Generation of EBV-immortalized B cells (LCLs) .............................. 48 3.24 Up-regulation of HLA expression in EFT cell lines by IFNγ .............. 49 3.25 Peptide titration assay ...................................................................... 50 ii Table of Contents 3.26 Statistical analysis ........................................................................... 50 4 Results ...................................................................................................... 51 4.1 mRNA Chip ......................................................................................... 51 4.1.1 CHM1 ........................................................................................... 51 4.1.2 GPR64 ......................................................................................... 51 4.1.3 EZH2 ............................................................................................ 51 4.1.4 STEAP ......................................................................................... 52 4.1.5 ITM2A .......................................................................................... 52 4.1.6 LIPI ............................................................................................... 52 4.2 Real-Time PCR ................................................................................... 54 4.2.1 CHM1 ........................................................................................... 54 4.2.2 GPR64 ......................................................................................... 54 4.2.3 EZH2 ............................................................................................ 54 4.2.4 STEAP ......................................................................................... 54 4.2.5 ITM2A .......................................................................................... 54 4.2.6 LIPI ............................................................................................... 54 4.3 HLA-A*0201 status of EFT cell lines ................................................... 56 4.3.1 A673 ............................................................................................. 57 4.3.2 TC-71 ........................................................................................... 57 4.3.3 SBSR-AKS ................................................................................... 57 4.4 Peptide-binding assay ........................................................................ 58 4.4.1 CHM1 ........................................................................................... 61 4.4.2 GPR64 ......................................................................................... 61 4.4.3 EZH2 ............................................................................................ 61 4.4.4 STEAP and EWS-FLI1 ................................................................. 61 4.4.5 ITM2A .........................................................................................

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