Novel Mechanisms of Innate Immunity
Total Page：16
File Type：pdf, Size：1020Kb
Novel mechanisms of innate immunity Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Björn Bulitta aus Wolfenbüttel 1. Referent: Professor Dr. Lothar Jänsch 2. Referentin: Professorin Dr. Dunja Bruder 3. Referent: Professor Dr. Stephan Dübel eingereicht am: 3.8.2015 mündliche Prüfung (Disputation) am: 15.12.2015 Druckjahr 2016 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Abschlussarbeiten Bulitta, B. (2011). Modulation of NK cell effector function by kinase inhibitors, Masterarbeit, Technische Universität Braunschweig Publikationen Scheiter, M. & Bulitta, B., Van Ham, M., Klawonn, F., König, S., & Jänsch, L. (2013). Protein kinase inhibitors CK59 and CID755673 alter primary human NK cell effector functions. Front Immunol, 4(MAR). Tagungsbeiträge Bulitta B., Scheiter M., Garritsen H., Klawonn F., & Jänsch L.: Novel kinases involved in NK cell effector responses and microtubule dynamics. (Poster) 14th Meeting of the Society for Natural Immunity, Heidelberg (2013) Bulitta B., Scheiter M., Gröbe L., & Jänsch L.: Proteomic definition of primary human MAIT cells. (Poster) 44th Annual Meeting of the German Society for Immunology, Bonn (2014) I II Abstract Over decades, one of the central dogmas of immunity has been that immune cells can be classified as parts of either the adaptive or innate immune system. In recent years, however, the lines between these two systems have become increasingly blurred. For example, subsets of innate natural killer (NK) cells have been demonstrated to display memory phenotypes, while recently identified mucosal-associated invariant CD8 T cells (MAIT cells) realize innate cytotoxicity. MAIT and NK cells both constitute predominant populations in human blood and other organs and share a high expression of CD161. NK cells detect virus infected, and abnormally transformed cells, and MAIT cells are able to identify and directly lyse bacterial-infected cells. While NK activation is controlled by a multitude of receptors, MAIT responses essentially depend on a TCRa chain that is invariant, as opposed to what is found in conventional T cells. Although NK cell activation is well defined, the underlying signaling networks are still obscure. In the first part of this thesis, the role of several kinases in NK cell activity was inves- tigated. It could be shown that chemical inhibition of CaMKII and PKD kinases, respectively, leads to a significant decrease of NK cell cytotoxicity and cytokine release, underscoring the importance of kinases CaMKII and PKDs in NK cell activation. Notably, these experiments also indicate significant donor-variations, inhibitory pathways and further involved cell types, which concertedly bias drug-modulated NK responses. In the second part, new mechanisms of innate immunity were investigated for the first time in primary human MAIT cells. Currently, the exact role of MAIT cells in health and disease remains widely undefined, and proteomic data about this innate-like T cell subset is missing. Proteomes of primary human MAIT were quantitatively compared donor-dependently with innate NK cells and conventional CD8 T cells by mass spectrometry. In total, 5500 proteins could be identified from five representative human donors. These data reveal a functional inventory of MAIT cells that integrates phenotypes from both NK and CD8 cells. Donor-dependent analyses then defined a MAIT-specific profile of effector molecules, and regulators of exocytosis and proliferation, respectively. Furthermore, this thesis provides candidates that can now be considered as novel MAIT markers and/or likely contribute to their anti-bacterial unique phenotype. In this context, the first observation of III the immunological synapse (IS) of a MAIT cell interacting with a bacterial infected target cell is presented and recruitment of candidate protein S100A4 to the site of anti-microbial activity is confirmed. IV Contents 1 Introduction 1 1.1 Functions and components of innate and adaptive immunity . .1 1.2 Function and activation of innate natural killer cells . .4 1.3 The biology of adaptive cytotoxic T cells . .6 1.3.1 Priming and activation of CD8 T cells . .6 1.3.2 Formation of the cytotoxic T cell immunological synapse, and CD8+ T cell effector functions . .9 1.4 Mucosal-associated invariant T cells . 10 1.4.1 Phenotype of MAIT cells . 10 1.4.2 Activation and function of MAIT cells . 12 1.4.3 Development of MAIT cells . 15 1.4.4 MAIT cells and diseases . 16 1.5 Aim of thesis . 18 2 Material and Methods 19 2.1 Equipment and software . 19 2.1.1 Equipment . 19 2.1.2 Software . 19 2.2 Chemicals, buffers and media . 20 2.2.1 Buffers . 20 2.2.2 Media . 21 2.2.3 Kinase inhibitors . 22 2.3 Cells . 22 2.4 Antibodies . 22 2.5 Methods of Microbiology . 24 2.5.1 Used bacterial strains . 24 2.5.2 Cultivation and fixation of bacteria . 24 2.6 Methods of Cell Biology . 25 2.6.1 Culturing of cells . 25 V Contents 2.6.2 Isolation of PBMCs from human peripheral blood . 25 2.6.3 Fluorescence activated cells sorting of primary human immune cells cells . 25 2.6.4 Immunofluorescence analysis of MAIT cell activation . 26 2.6.5 Flow cytometric analysis of MAIT cell activation . 27 2.6.6 Magnetic activated sorting of human NK cells . 27 2.6.7 NK cell degranulation assay . 28 2.6.8 NK cell cytokine release assay . 28 2.6.9 Flow cytometric analysis of NK cell degranulation and cytokine release 29 2.7 Semi-quantitative proteomic analysis . 29 2.7.1 Cell lysis and digestion (urea lysis) . 29 2.7.2 Reverse-phase peptide clean up and desalting . 30 2.7.3 Peptide labeling with iTRAQTM ........................ 30 2.7.4 Subfractionation of complex peptide samples through SCX chromatog- raphy . 31 2.7.5 Mass spectrometric analyses and data interpretation . 31 2.8 Statistical evaluation and determination of significantly regulated proteins . 32 3 Results 35 3.1 The role of Src kinases, CamKII and PKD kinases in NK cell activation . 35 3.1.1 Inhibition of Src kinases, CaMKII and PKD kinases decreases NK cell degranulation and cytokine release in primary human PBMCs . 35 3.1.2 Donor-specific responses of pure NK cells treated with Dasatinib, CK59 and CID755673 . 37 3.2 MAIT cells isolated from human donor blood are suitable material for proteomic analyses . 41 3.3 First insights into the proteome of primary human MAIT cells reveal T cell specific proteins and pathways . 44 3.4 Mass spectrometry generates highly reproducible data from small cell numbers 52 3.5 Conserved and novel functions in innate immunity in MAIT cells . 56 3.5.1 Flow cytometry allows the generation of highly pure MAIT cells, CD8+ T cell and NK cell samples . 56 3.5.2 Protein identification in MAIT, NK and cCD8+ T cells shows large overlap of proteins in different donors . 59 3.5.3 Differential protein expression in MAIT, NK and cCD8+ T cells . 62 VI Contents 3.5.3.1 MAITs cells are a cell type distinct from NK and cCD8+ T cells on the proteomic level . 62 3.5.3.2 Robust identification of regulated proteins in MAIT, NK and cCD8+ T cells indicates distinct proteomic profiles . 67 3.5.3.3 Proteomic data is validated by flow cytometry . 71 3.5.4 Immunological effector proteins show distinct abundance in MAIT cells 72 3.5.4.1 MAIT cells possess a unique set set of effector molecules . 72 3.5.4.2 Proteins regulating exocytosis and proliferation are upregu- lated in MAIT cells . 73 3.6 First-time characterization of the MAIT cell immunological synapse . 77 3.6.1 Formation of the MAIT immunological synapses . 77 3.6.2 S100A4 is associated with microtubules in MAIT cells . 79 3.6.3 S100A4 localizes at the MAIT IS in a time-dependent manner . 81 4 Discussion 87 4.1 Chemical inhibition of NK cell effector functions . 87 4.1.1 Specificity of used kinase inhibitors . 87 4.1.2 Role of PKD and CaMKII in NK cell activation . 88 4.1.3 Perspectives for kinase inhibitors in cancer and NK cell therapy . 89 4.2 Novel mechanisms of innate immunity in MAIT cells . 91 4.2.1 Proteomic analysis of low-abundant primary immune cells . 91 4.2.2 Proteomic results are in accordance with current knowledge . 91 4.2.3 Is the definition of a “typical” MAIT cell possible with proteomics? . 93 4.2.4 Best of both worlds: Typical MAIT cells between NK cells and CD8 T cells 94 4.2.5 Defining the typical MAIT cell phenotype with proteomics . 95 4.2.5.1 A new set of markers for human MAIT cells . 95 4.2.5.2 The arsenal of MAIT cells - innate immunity requires a specific pattern of effector molecules . 96 4.2.5.3 Increased capacity for exocytosis in typical human MAIT cells 101 4.2.5.4 Controlling the killer: MAIT cells display a unique set of proteins that regulate proliferation . 103 4.2.6 First insights into the molecular mechanisms of MAIT cytotoxicity . 105 5 Summary and outlook 109 5.1 Coordination of NK effector function by kinases Fyn, CaMKII and PKD . 109 5.2 Proteomic analysis reveals novel mechanisms of innate immunity in MAIT cells110 5.3 First-time characterization of the MAIT cell immunological synapse . 112 VII Contents Appendix 115 List of Figures 179 List of Tables 181 List of Abbreviations 183 Bibliography 187 Acknowledgements 209 VIII 1 Introduction 1.1 Functions and components of innate and adaptive immunity During the course of evolution, even the smallest of life forms have developed rudimentary protection mechanisms to shield themselves from the potential harmful influence of infectious agents.