INVOLVEIMN THYMIC NEGA'' ckJL^du'nà oiXAS h\'^(WU 2_ l/\cU ;U it/v +0 cU fcxU iiU 'w ^ J2^r^aH A ^ pn? hex/v^s submitted by ARIANE VOLKMANN for the degree of Doctor of Philosophy University of London Department of Molecular Immunology National Institute for Medical Research London 1996 ProQuest Number: 10018495 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10018495 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 TABLE OF CONTENTS ABSTRACT.................................................................................................................... 5 ACKNOWLED(3EMENTS................................................................................................... 7 ABBREVIATIONS.......................................................................................................... 9 1. INTRODUCTION.....................................................................................................11 1.1 Function of the Immune System ...............................................................................11 1.2 T cells..........................................................................................................................12 1.3 Thymic architecture and microenvironment ............................................................16 1.4 T cell development and selection ..............................................................................18 1.5 Antigen processing and presentation .......................................................................22 1.6 Professional antigen presenting cells .......................................................................24 1.6.1 B cells..................................................................................................................24 1.6.2 Macrophages ...................................................................................................... 25 1.6.3 Dendritic cells .................................................................................................... 26 1.7 C5 as model antigen ..................................................................................................29 1.8 T cell receptor transgenic mice ................................................................................ 29 1.9 Aims of the project .................................................................................................... 35 2. MATERIALS AND METHODS..............................................................................38 2.1 Monoclonal antibodies ..............................................................................................38 2.2 Animals ......................................................................................................................39 2.3 M edia.........................................................................................................................39 2.4 Determination of cell viability and number ............................................................ 39 2.5 FACS analysis ........................................................................................................... 40 2.6 Magnetic cell sorting .................................................................................................40 2.7 Bone marrow cultures ...............................................................................................41 2.8 Generation of a dendritic cell line ............................................................................41 2.9 Antigen presentation assays ..................................................................................... 42 2.10 Fetal thymic organ culture (FTOC) .........................................................................42 2.11 Thymic reaggregation culture .................................................................................. 43 2.12 Nonspecific esterase staining ................................................................................... 43 2.13 Phagocytosis assay ....................................................................................................43 2.14 Mixed leukocyte reaction (MLR) ............................................................................44 2.15 Cytokine treatment of tsD C ......................................................................................44 2.16 Preparation of skin cells ............................................................................................44 2.17 Confocal microscopy ................................................................................................ 45 2.18 Electron microscopy ................................................................................................. 45 2.19 Treatment with brefeldin A and cycloheximide .....................................................46 2.20 Biochemical analysis ................................................................................................. 46 2.20.1 Subcellular fractionation by density gradient electrophoresis ........................46 2.20.2 SDS-PAGE and Western blotting .....................................................................47 2.20.3 Biochemical analysis of MHC class II surface molecules ..............................48 3. RESULTS........................................................................................................................ 49 3.1 Tolerance induction in early T cell development .................................................... 49 3.1.1 T cell development in fetal thymic organ culture ........................................... 49 3.1.2 Status of prenatal tolerance in vivo ...................................................................56 3.1.3 C5 specific double positive thymocytes are susceptible to deletion ...............................................................................................................61 3.1.4 Efficiency of thymic APC in T cell activation and thymocyte deletion ...............................................................................................................64 3.1.5 What are the reasons underlying the failure of macrophages to present C5 for negative selection? ...................................................................75 3.2 Characterization of a conditionally immortalized dendritic cell line ......................82 3.2.1 Morphology and surface marker expression ....................................................82 3.2.2 TsDC differ from macrophages in their phagocytosis abilities and in their enzyme content .....................................................................................86 3.2.3 Antigen presentation capacity of tsD C ............................................................ 86 3.2.4 The influence of cytokines on surface molecule expression ...........................89 3.2.5 Antigen capture ..................................................................................................98 3.2.6 The intracellular distribution of molecules involved in antigen processing and presentation ..............................................................................103 3.2.7 The influence of cytokines on functional activity ............................................109 4. DISCUSSION.................................................................................................................. 113 5. REFERENCES............................................................................................................... 136 TABLES AND FIGURES Table 1 Cell recovery from thymus lobes ....................................................................... 62 Fig. 1A Increase of cell number in FTOC ...................................................................... 50 Fig. IB Non-transgenic thymocytes in FTOC ............................................................... 52 Fig. 1C C5 TCR transgenic thymocytes in FTOC ........................................................53 Fig. ID C5+/- TCR transgenic thymocytes in FTOC .....................................................54 Fig. IE C5+ TCR transgenic thymocytes in FTOC .......................................................55 Fig. 2 C5 specific response of thymocytes .................................................................. 57 Fig. 3 Thymocytes on day 19 of gestation .................................................................. 59 Fig. 4 Thymocytes from dl9 tg C5+ embryos are not C5 reactive ........................... 60 Fig. 5 C5 peptide induced deletion of DP in FTOC ....................................................63 Fig. 6 Purification of thymic APC by MACS .............................................................66 Fig. 7 C5 presentation by thymic A f c ........................................................................ 67 Fig. 8 DC, mEC and cEC but not macrophages can induce negative selection in thymus reaggregation culture ........................................................ 70 Fig. 9 C5 specific T cells in thymus reaggregates without C5+ APC or with