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The Role of Cflip in Regulatory T Cell Homeostasis

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The Role of Cflip in Regulatory T Cell Homeostasis The role of cFLIP in regulatory T cell homeostasis Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von M. Sc. Carlos Plaza Sirvent geb. am 31.10.1982 in Elche (Alicante), Spanien Gutachter: Prof. Dr. Ingo Schmitz Prof. Dr. Andreas Krueger eingereicht am 23. Juni 2015 verteidigt am 23. September 2015 Table of contents 1. Abstract ....................................................................................................................... 1 2. Introduction ................................................................................................................ 2 2.1. Cell death ............................................................................................................... 2 2.2. Apoptosis ............................................................................................................... 5 2.2.1. Morphological features ................................................................................... 5 2.2.2. Caspases .......................................................................................................... 6 2.2.3. BCL-2 family .................................................................................................. 8 2.2.4. Death receptors ................................................................................................ 8 2.2.5. Signaling pathways of apoptosis ..................................................................... 9 2.2.5.1. The intrinsic pathway ............................................................................... 9 2.2.5.2. The extrinsic pathway ............................................................................. 10 2.2.5.3. The crosstalk between the pathways ...................................................... 12 2.2.5.4. The granzyme B pathway ....................................................................... 12 2.2.6. The FLICE-inhibitory proteins ..................................................................... 14 2.2.7. The role of the FLICE-inhibitory proteins (cFLIP) in the immune system .. 16 2.3. Apoptosis and immune homeostasis .................................................................... 17 2.3.1. Function of immune system .......................................................................... 19 2.3.2. Lymphocyte development ............................................................................. 19 2.3.3. Treg development .......................................................................................... 20 2.3.4. Treg function ................................................................................................. 22 2.3.5. Treg homeostasis ........................................................................................... 23 2.4 Aims of the thesis ................................................................................................. 25 3. Materials .................................................................................................................... 26 3.1. Chemicals ............................................................................................................. 26 3.2. Cell culture material and devices ......................................................................... 26 3.2.1. Cell culture media and supplements.............................................................. 27 3.2.2. Medium for cell culture ................................................................................. 27 3.2.3. Reagents, antibodies and cytokines used for cell stimulation ....................... 28 3.3. Materials, devices and reagents for flow cytometry ............................................ 28 3.3.1. Devices .......................................................................................................... 28 3.3.2. Fluorescent dyes ............................................................................................ 29 3.3.3. Flow cytometry antibodies ............................................................................ 29 i 3.4. Reagents and materials used for Western blotting ............................................... 30 3.4.1. Primary antibodies......................................................................................... 30 3.4.2. Horseradish peroxidase-conjugated secondary antibodies ............................ 30 3.5. Reagents and devices for confocal microscopy ................................................... 31 3.5.1. Reagents for microscopy ............................................................................... 31 3.5.1.1. Fluorescent dyes ..................................................................................... 31 3.5.1.2. Fluorochrome-labeled antibodies ........................................................... 31 3.6. Reagents for in vivo treatments ............................................................................ 31 3.7. Frequently used buffers ....................................................................................... 32 3.8. Oligonucleotides .................................................................................................. 33 3.8.1. Oligonucleotides for quantitative RT-PCR ................................................... 33 3.8.2. Oligonucleotides for mouse genotyping ....................................................... 33 3.9. Mouse strains ....................................................................................................... 34 4. Experimental procedures ......................................................................................... 34 4.1. Molecular biology methods ................................................................................. 34 4.1.1. RNA isolation ................................................................................................ 34 4.1.2. Photometric determination of DNA/RNA concentration .............................. 34 4.1.3. Reverse transcription ..................................................................................... 35 4.1.4. Polymerase chain reaction (PCR) ................................................................. 35 4.1.5. Analytic agarose gels .................................................................................... 36 4.1.6. Quantitative real-time polymerase chain reaction (qRT-PCR) ..................... 36 4.2. Protein biochemical methods ............................................................................... 37 4.2.1. Cell lysis ........................................................................................................ 37 4.2.2. Determination of protein concentration ........................................................ 37 4.2.3. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) ............................... 37 4.2.4. Western blotting ............................................................................................ 37 4.3. Mouse surgery and cellular methods ................................................................... 38 4.3.1. Isolation of organs from mice ....................................................................... 38 4.3.2. Flow cytometry analysis................................................................................ 38 4.3.3. Cell isolation by flow cytometry ................................................................... 39 4.3.4. Viability studies in Tcon and Treg cells........................................................ 39 4.3.5. Active caspase 3 detection by Western blot .................................................. 39 4.3.6. In vitro stimulation with death ligands .......................................................... 39 ii 4.3.7. Active caspase 3/7 determination by confocal microscopy .......................... 40 4.3.8. Histology and immunohistochemistry .......................................................... 40 4.3.9. Determination of cytokines in serum ............................................................ 40 4.3.10. Autoantibodies determination ..................................................................... 41 4.3.11. In vivo treatments ........................................................................................ 41 4.4. Statistical Analysis ............................................................................................... 41 5. Results ........................................................................................................................ 42 5.1 Regulatory T cells manifest a higher apoptosis rate than conventional T cells .... 42 5.2 Pan-caspase inhibitor and IL-2 improves Treg viability....................................... 45 5.3 cFLIP deficiency in Treg cells caused autoimmune disease in mice.................... 51 5.4 Treg cells are sensitive to CD95-, but not DR5- or TNFR1-induced cell death ... 66 5.5 Tcon and Treg cells show identical death receptor profile expression ................. 68 5.6 Neither anti-CD95L nor QVD could protect the Treg cells of CflarFoxp3 mice in vivo .............................................................................................................................. 69 5.7 cFLIPL protects Treg cells from CD95-mediated cell death in vitro .................... 71 6. Discussion .................................................................................................................. 74 6.1. Treg cells show an elevated apoptosis rate under steady-state
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