PKM2 regulation of metabolism but not transcription is important for natural killer cell responses A dissertation submitted to Trinity College Dublin in candidature for the degree of Doctor of Philosophy School of Biochemistry and Immunology Trinity College, Dublin 2020 By Jessica Walls Under the supervision of Dr. David Finlay, Dr. Clair Gardiner and Dr. Daniel McVicar Declaration I declare that this thesis has not been submitted as an exercise for a degree at this or any other university and it is entirely my own work. I agree to deposit this thesis in the University’s open access institutional repository or allow the library to do so on my behalf, subject to Irish Copyright Legislation and Trinity College Library conditions of use and acknowledgement ii For my Mam and Dad iii Publications Nutrient sensing, signal transduction and immune responses Seminars in Immunology Vol. 29, Issue 5 October 2016 Jessica Walls, Linda Sinclair and David Finlay Glucose represses dendritic cell-induced T cell responses Nature Communications Vol. 8, 30 May 2017 Simon J. Lawless*, Nidhi Kedia-Mehta*, Jessica F. Walls, Ryan McGarrigle, Orla Convery, Linda V. Sinclair, Maria N. Navarro, James Murray ,David K. Finlay *These authors contributed equally to this work iv Table of Contents Declaration ................................................................................................................ ii Publications .............................................................................................................. iv Summary ................................................................................................................ xiii Abbreviations .......................................................................................................... xv Acknowledgements ............................................................................................... xvii Chapter 1: Introduction .......................................................................................... xxi 1.1. Natural Killer cells ....................................................................................... 1 1.2. NK cell development ................................................................................... 2 1.3. NK cell functions ......................................................................................... 4 1.4. NK cell mediated cytotoxicity ...................................................................... 6 1.5. Death Ligands ............................................................................................. 6 1.6. NK cell cytokine and chemokine production ................................................ 7 1.7. NK cell receptor signalling ........................................................................... 8 1.8. NK cell cytokine signal transduction .......................................................... 10 1.9. NK cell control of cytomegalovirus ............................................................ 13 1.10. Human NK cell biology and immunotherapies ........................................... 15 1.11. Immunometabolism ................................................................................. 17 1.11.1. Metabolism for energy production ...................................................... 17 1.11.2. Metabolism for biosynthesis ................................................................ 19 1.11.3. Metabolic configurations of immune cells ........................................... 22 1.11.4. Direct metabolic regulation of immune function ................................. 25 1.12. Natural Killer cell metabolism ................................................................... 26 1.13. Regulation of NK cell metabolic reprogramming ....................................... 27 1.13.1. mTORC1 ................................................................................................. 27 1.13.2. c-Myc ..................................................................................................... 28 1.13.3. SREBP ..................................................................................................... 29 1.14. Glycolytic regulation of immune function ................................................. 31 1.15. Pyruvate Kinase and Glycolysis ................................................................. 35 1.15.1. Isoforms ................................................................................................. 35 1.15.2. Conformations ....................................................................................... 36 1.16. Regulation of PKM2 conformation ............................................................ 38 1.16.1 Allostery ................................................................................................ 38 1.16.2. Post-translational modifications ............................................................ 38 1.17. Non-canonical functions of PKM2 ............................................................. 40 v Aims ....................................................................................................................... 42 Chapter 2: Materials and Methods ........................................................................... 43 2.1. Materials ................................................................................................. 44 2.1.1. Chemicals ................................................................................................... 44 2.1.2. Equipment ................................................................................................. 45 2.1.3. Animal Husbandry ..................................................................................... 46 2.1.4. Antibodies .................................................................................................. 47 2.1.5. Miscellaneous buffers ................................................................................ 49 2.2. Methods ................................................................................................. 50 2.2.1. Cell Culture ................................................................................................ 50 2.2.2. RNA Analysis .............................................................................................. 52 2.2.3. Flow Cytometry ......................................................................................... 59 2.2.4. Protein Analysis ......................................................................................... 62 2.2.5. Seahorse analysis ....................................................................................... 67 2.2.6. Metabolomics ............................................................................................ 72 2.2.7. in vivo experiments .................................................................................... 73 2.2.8. Statistical analysis ...................................................................................... 74 Chapter 3: PKM2 is not required for NK cell activation in vivo ................................. 75 Introduction ........................................................................................................ 76 3.1. PKM2 is induced in splenic NK cells by in vivo administration of poly(I:C) ....... 78 3.2. Generation of an NK cell specific Pkm2 knock out mouse .............................. 80 3.3. PKM2KO Splenic NK cell numbers, frequencies and maturity are normal .......... 85 3.4. PKM2KO NK cells are efficiently activated following Poly I:C injection ............. 88 3.5. PKM2WT and PKM2KO NK cells increase IFNγ production comparably in response to poly(I:C) in vivo ................................................................................................ 90 3.6. Deletion of PKM2 in splenic NK cells does not affect poly(I:C) induced expression of CD98 in vivo ................................................................................... 92 3.7. Deletion of PKM2 in splenic NK cells does not affect poly(I:C) induced proliferation in vivo ............................................................................................. 94 3.8. Splenic PKM2WT and PKM2KO NK cells respond similarly to MCMV infection 4 days post infection .............................................................................................. 96 3.9. Splenic PKM2WT and PKM2KO NK cells express similar levels of CD69 after MCMV infection .................................................................................................. 98 3.10. PKM2WT and PKM2KO splenic NK cells demonstrate a similar frequency of IFNγ production post-MCMV infection ........................................................................ 100 3.11. Frequency of Ly49H+ cells is comparable between PKM2WT and PKM2KO mice infected with MCMV ........................................................................................... 102 3.12. PKM2WT and PKM2KO Ly49H+ cells incorporate the same amount of BrDU after MCMV infection ................................................................................................. 104 vi 3.13. Serum cytokines are comparable between mice with PKM2WT and PKM2KO NK cells 4 days post MCMV infection ....................................................................... 106 3.14. Relative splenic viral load of MCMV was comparable between mice with PKM2WT or PKM2KO NK cells ............................................................................... 108 Discussion .........................................................................................................