Immunological and translational consequences of an + altered CD8 T cell cytolytic activity Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Anthea Wirges 2018 Die vorliegende Arbeit wurde in der Zeit vom Juli 2014 bis Dezember 2018 in der Arbeitsgruppe „Translationale Tumorimmunologie“ unter der Leitung von Dr. Armin Rehm am „Max-Delbrück-Centrum für Molekulare Medizin“ in der Helmholtz- Gemeinschaft in Berlin angefertigt. 1. Gutachter: Dr. Armin Rehm 2. Gutachter: Prof. Dr. Oliver Daumke Disputation: 06.05.2019 4 Index I ABBREVATIONS ..................................................................................................... V II TABLES .................................................................................................................. XI III FIGURES .............................................................................................................. XIII IV ZUSAMMENFASSUNG ............................................................................................ 1 V ABSTRACT ............................................................................................................... 3 1. INTRODUCTION ....................................................................................................... 5 1.1 The immune system .......................................................................................... 5 1.2 T cell-mediated immunity ................................................................................... 6 1.2.1 T lymphocytes ................................................................................................ 6 1.2.2 Effector and memory CD8+ T cell differentiation ............................................ 7 1.2.2.1 The course of a CD8+ T cell response ................................................... 7 1.2.2.2 Memory CD8+ T cell subsets .................................................................. 8 1.2.2.3 Models of CD8+ T cell diversification ...................................................... 8 1.2.2.4 Transcriptional regulation of CD8+ T cell differentiation ......................... 9 1.2.3 Effector molecules of CD8+ T cells ................................................................ 9 1.2.3.1 Granzymes ........................................................................................... 10 1.2.3.2 Cytokines .............................................................................................. 11 1.2.4 The secretory pathway of CD8+ T cells ........................................................ 12 1.2.4.1 Secretion of effector molecules ............................................................ 12 1.2.4.2 EBAG9 and its role in the regulated effector molecule secretion ......... 14 1.2.5 T cell exhaustion .......................................................................................... 15 1.3 Cancer immunotherapies ................................................................................. 16 1.3.1 Checkpoint inhibitors .................................................................................... 16 1.3.2 Adoptive T cell therapy (ATT) ...................................................................... 17 1.3.2.1 Tumor-infiltrating lymphocytes (TILs) ................................................... 18 1.3.2.2 TCR-modified T cells ............................................................................ 18 1.3.2.3 CAR-modified T cells ............................................................................ 19 1.3.3 RNA interference (RNAi) .............................................................................. 19 1.3.3.1 The RNA interference pathway ............................................................ 19 1.3.3.2 Applications of the RNAi pathway to T cell engineering ....................... 21 2. AIM OF THE THESIS .............................................................................................. 23 3. MATERIAL .............................................................................................................. 25 3.1 Plasmids and retroviral vectors ........................................................................ 25 3.2 Oligonucleotides .............................................................................................. 26 3.3 Antibodies and MHC multimers ....................................................................... 26 I Index 3.3.1 Conjugated antibodies specific for mouse surface antigens ....................... 26 3.3.2 Conjugated antibodies specific for human surface antigens ....................... 27 3.3.3 MHC Multimers ............................................................................................ 28 3.3.4 Unconjugated primary antibodies for Western Blot ..................................... 28 3.3.5 Secondary antibodies for Western Blot ....................................................... 28 3.4 Cell lines .......................................................................................................... 29 3.5 Mice ................................................................................................................. 29 3.6 Chemicals and consumables .......................................................................... 30 3.7 Kits .................................................................................................................. 31 3.8 Software .......................................................................................................... 31 4. METHODS .............................................................................................................. 33 4.1 Molecular biology ............................................................................................ 33 4.2 Cell culture ...................................................................................................... 35 4.3 Functional assays ............................................................................................ 38 4.4 Protein biochemistry ........................................................................................ 40 4.5 In vivo experiments ......................................................................................... 41 4.6 Statistics .......................................................................................................... 43 5. RESULTS ............................................................................................................... 45 5.1 EBAG9 regulates CD8+ T cell memory differentiation ..................................... 45 5.1.1 Loss of EBAG9 leads to an enhanced antigen-specific memory CD8+ T cell development after immunization with the strong Tag neoantigen ............... 45 5.1.2 Deletion of EBAG9 confers mice with a selective advantage for the development of a larger HY-specific CD8+ memory pool ............................ 48 5.2 Target site validation for EBAG9 knockdown .................................................. 53 5.3 Analysis of RNAi-modified mouse T cells ........................................................ 55 5.3.1 Efficient RNAi-mediated EBAG9 downregulation in primary mouse T cells 55 5.3.2 The engineered knockdown of EBAG9 amplifies antigen-specific killing by cytotoxic mouse T cells in vivo .................................................................... 57 5.4 RNAi-mediated silencing of EBAG9 in human T cells ..................................... 60 5.4.1 Development of a g-retroviral vector for RNAi-mediated EBAG9 knockdown and the expression of an antigen-specific CAR ........................................... 60 5.4.2 Downregulation of EBAG9 increases granzyme A release whereas cytokine secretion is not affected ............................................................................... 63 5.4.3 Downregulation of EBAG9 confers CAR T cells with enhanced cytolytic activity .......................................................................................................... 66 5.4.4 No link between enhanced cytolytic activity and exhaustion of RNAi-modified CAR T cells upon repetitive antigen stimulation .......................................... 69 II Index 5.4.5 T cell differentiation upon repetitive antigen stimulation is not altered due to the loss of EBAG9 ........................................................................................ 73 5.5 EBAG9 silencing amplifies the cytolytic activity of CAR T cells at low effector frequencies in a multiple myeloma xenograft model .......................................... 75 6. DISCUSSION .......................................................................................................... 81 6.1 EBAG9 links cytolytic strength to CD8+ memory formation ............................. 81 6.1.1 Loss of EBAG9 allows for the preferential formation of CD8+ memory T cells ..................................................................................................................... 81 6.1.2 Transcriptional regulation of memory formation depends on the antigenic challenge ...................................................................................................... 84 6.2 RNAi-mediated targeting of the secretory pathway increases the cytolytic activity of mouse CD8+ T cells ........................................................................................ 86 6.2.1 EBAG9 is a suitable target for RNAi-mediated T cell engineering ..............