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Rho Gtpase Cdc42 Controls Invariant Chain Processing and MHC II Loading in Dendritic Cells

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Rho Gtpase Cdc42 Controls Invariant Chain Processing and MHC II Loading in Dendritic Cells Aus dem Institut für Immunologie der Ludwig-Maximilians-Universität München Vorstand: Prof. Dr. Thomas Brocker Rho GTPase Cdc42 controls invariant chain processing and MHC II loading in Dendritic Cells Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Anna Schulz, geb. Wähe aus Darmstadt 2013 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuer: Prof. Dr. Thomas Brocker Zweitgutachter: Prof. Dr. Patrick A. Baeuerle Dekan: Prof. Dr. med Dr.h.c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 06.11.2013 ii AUTHOR’S DECLARATION Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation „Rho GTPase Cdc42 controls invariant chain processing and MHC II loading in Dendritic Cells“ von mir selbständig und ohne unerlaubte Hilfe angefertigt wurde. Ich habe mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Hilfen und Quellen bedient. Die Dissertation wurde in der jetzigen oder ähnlichen Form bei keiner anderen Hochschule eingereicht und hat auch noch keinen anderen Prüfungszwecken gedient. Anna Schulz iii This work contains results presented in the following publication: Schulz, A. et al. Rho GTPase Cdc42 controls invariant chain processing and MHC II loading in Dendritic Cells. Manuscript in preparation. This publication was also achieved and will be cited as “Paper I”: Luckashenak, N.*, Wähe, A.*, Breit, K., Brakebusch, C., Brocker, T. 2013. Rho- family GTPase Cdc42 controls migration of Langerhans cells in vivo. J Immunol. 190:27-35. *equal contribution iv ABBREVIATIONS ABBREVIATIONS SI Units were applied as described elsewhere. APC 1. antigen presenting cell 2. allophycocyanin BDP F-Bar domain protein BMDC bone marrow-derived dendritic cell BSA bovine serum albumin CIITA MHC class II transactivator ca constitutively active CBA cytometric bead array CCR7 chemokine (C-C motif) receptor 7 CCV clathrin coated vesicle CD cluster of differentiation cDC conventional dendritic cell Cdc42 cell division cycle 42 CDE clathrin dependent endocytosis cDNA complementary DNA cTEC cortical epithelial cell CDP common DC precursor CFSE carboxyfluorescein-diacetate-succinimidylester CIE clathrin independent endocytosis CLIP class II-associated Ii peptide CMP common myeloid precursor ConB concanamycin B CP crossing point CTL cytotoxic T lymphocyte Ctsl cathepsin L Ctss cathepsin S CytD cytochalasin D DAPI 4',6-diamidino-2-phenylindole dNTP desoxyribonucleotidtriphosphate DC dendritic cell DMSO dimethyl sulfoxide v ABBREVIATIONS dn nominant negative DNA desoxyribonucleic acid DTT dithiothreitol DQ-OVA non-fluorescent self-quenched conjugate of OVA dsRNA double stranded RNA ECL enhanced chemiluminescense EDAC 1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide EDTA ethylenediamintetraacetic acid ER endoplasmic reticulum FACS fluorescence activated cell sorter F-actin filamentous actin FCS fetal calf serum FITC fluoresceinisothiocyanate flx floxed Flt3L Flt3 Ligand for forward FSC forward scatter GAP GTPase-activating protein Gapdh glyceraldehyde-3-phosphate dehydrogenase GDI guanine nucleotide-dissociation inhibitor GEF guanine exchange factor GM-CSF granulocyte-macrophage colony stimulating factor GO Gene Onthology GTPase GTP-binding protein H2-M H2-M beta 2 chain HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hip1r Huntingtin interacting protein 1 related HIV human immunodeficiency virus HPRT hypoxanthine-guanine phosphoribosyltransferase HRP horseradish peroxidase IFN interferon Ig Immunglobuline Ii invariant chain IL interleukin kb kilobase vi ABBREVIATIONS kDa kilo Dalton ko knockout LAMP1 lysosome-associated membrane glycoprotein 1 LatB latrunculin B LC Langerhans cell LCA leucocyte common antigen LC-MSMS liquid chromatography-tandem mass spectrometry LPS lipopolysaccharide MIIC MHC II loading compartment M6pr mannose-6-phosphate receptor MACS magnetic cell sorting MAPK Mitogen-activated protein kinase M-CSF macrophage colony stimulating factor mDC migratory DC MES compound 2-(N-morpholino)ethanesulfonic acid MFI mean fluorescent intensity MHC major histocompatibility complex MLN mesenteric lymph node MT microtubule MTOC microtubule organizing center MVB multi vesicular bodies mRNA messenger RNA NF-κB nuclear factor kappaB NK cell natural killer cell Noco nocodazole NP-40 Nonidet (octyl phenoxypolyethoxylethanol) P-40 OVA ovalbumin PBS phosphate buffered saline PBMC peripheral blood mononuclear cell PCR polymerase chain reaction pDC plasmacytoid dendritic cell PE phycoerythrin PerCP peridinin-chlorophyll-a protein qPCR quantitative PCR PKC protein kinase C vii ABBREVIATIONS PMSF phenylmethylsulfonyl fluoride Rac1 Ras-related C3 botulinum toxin substrate 1 rDC resident DC rev reverse RhoA transforming protein RhoA RNA ribonucleic acid SA streptavidin SDS sodium dodecyl sulfate SEM standard error of the mean SIINFEKL OVA257-264 SSC side scatter TAP transporter associated with antigen processing TCR T cell receptor TH cell T helper cell TEMED tetramethylethylendiamin TGF-β transforming growth factor beta TGN trans-Golgi network TLR toll like receptor TNF tumor necrosis factor vs. versus WASP Wiskott-Aldrich syndrome protein wt wild type viii TABLE OF CONTENTS TABLE OF CONTENTS AUTHOR’S DECLARATION .......................................................................................... iii ABBREVIATIONS .............................................................................................................. v TABLE OF CONTENTS .................................................................................................... ix 1 SUMMARY .................................................................................................................... 1 2 ZUSAMMENFASSUNG .............................................................................................. 2 3 INTRODUCTION ......................................................................................................... 3 3.1 Dendritic cells as antigen presenting cells .......................................................... 3 3.1.1 DCs – sentinels of the immune system ........................................................ 3 3.1.2 DC maturation ................................................................................................ 4 3.1.3 DC antigen presentation ................................................................................ 6 3.1.3.1 Uptake of soluble and particulate antigens ......................................... 6 3.1.3.2 Antigen cross-presentation .................................................................... 8 3.1.3.3 MHC II antigen presentation: A role for invariant chain .................. 9 3.1.3.4 DC - T cell interaction ........................................................................... 10 3.2 Small Rho GTPase Cdc42 and its cellular functions ........................................ 11 3.2.1 Small Rho GTPases ....................................................................................... 11 3.2.2 Constraints in studying Rho GTPases ....................................................... 12 3.2.3 Cdc42 contributes to actin dynamics ......................................................... 12 3.2.4 Rho GTPases are targets of bacterial toxins .............................................. 14 3.2.5 Functions of Cdc42 in DCs .......................................................................... 14 3.3 Aims of the study .................................................................................................. 17 3.3.1 Characterization of a DC-specific knockout for Cdc42 ........................... 17 3.3.2 Elucidation of the mechanism by which Cdc42 controls MHC II antigen presentation in DCs ...................................................................................... 17 3.3.3 Examination of MHC II loading in DC subsets ........................................ 17 4 MATERIALS AND METHODS ................................................................................ 18 4.1 Materials ................................................................................................................. 18 4.1.1 Antibodies ...................................................................................................... 18 4.1.2 Chemicals ....................................................................................................... 20 4.1.3 Consumables ................................................................................................. 20 4.1.4 Devices ........................................................................................................... 20 4.1.5 Inhibitors ........................................................................................................ 21 4.1.6 Media and solutions ..................................................................................... 21 4.1.7 Mouse strains ................................................................................................ 24 ix TABLE OF CONTENTS 4.1.8 Fluorescent probes, proteins, peptides and primers ............................... 24 4.2 Methods .................................................................................................................. 25 4.2.1 Cellular and immunological methods ....................................................... 25 4.2.1.1 Generation of bone marrow-derived dendritic cells (BMDCs) ....... 25 4.2.1.2 Flow cytometry – Fluorescence activated
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