Linköping University Medical Dissertations No. 1158 Antigen interaction with B cells in two proliferative disorders - CLL and MGUS Eva Hellqvist Akademisk avhandling som för avläggande av medicine doktorsexamen offentligen försvaras i Aulan, Katastrofmedicinskt Centrum, Linköping, fredagen den 15 januari 2010 kl. 9.00 Fakultetsopponent Dr Anne Mette Buhl Department of Hematology, Rigshospitalet Copenhagen, Denmark Division of Cell Biology Department of Clinical and Experimental Medicine Linköping University, SE-581 85 Linköping, Sweden Linköping 2010 All text, tables and illustrations are © Eva Hellqvist, 2010 The original papers included in this thesis have been reprinted with the permission of respective copyright holder: Paper I: © the American Society of Hematology Paper IV: © the Ferrata Storti Foundation ISSN: 0345-0082 ISBN: 978-91-7393-475-6 Printed in Sweden by LiU-Tryck, Linköping 2010 “This world, after all our science and sciences, is still a miracle; wonderful, inscrutable, magical and more, to whosoever will think of it. ” – Thomas Carlyle TO MY GREAT SURPRISE SUPERVISOR Anders Rosén, Professor Division of Cell Biology Department of Clinical and Experimental Medicine Linköping University, Sweden CO-SUPERVISOR Jan Ernerudh, Professor Division of Clinical Immunology Department of Clinical and Experimental Medicine Linköping University, Sweden OPPONENT Anne Mette Buhl, Associate Professor Department of Hematology Rigshospitalet Copenhagen, Denmark COMMITTEE BOARD Hodjattalah Rabbani, Associate Professor Immune and Gene Therapy Lab Cancer Center Karolinska Karolinska University Hospital, Sweden Mikael Sigvardsson, Professor Experimental Hematology unit Department of Clinical and Experimental Medicine Linköping University, Sweden Sven Hammarström, Professor Division of Cell Biology Department of Clinical and Experimental Medicine Linköping University, Sweden ABSTRACT The aim of the work presented in this thesis was to elucidate B cell interaction with antigen in the two B cell proliferative disorders chronic lymphocytic leukemia (CLL) and monoclonal gammopathy of undetermined significance (MGUS). In the first part we investigated the antigen specificity of CLL cells and characterized Epstein-Barr virus (EBV)-transformed CLL cell lines with regard to phenotype and genotype. The second part consists of studies on the antigen presenting capacity of myelin protein zero (P0) specific MGUS B cells and their relation to T cells and development of polyneuropathy. CLL cells are considered antigen experienced and different patient-derived CLL cells expressing B cell receptors (BCR) with highly homologous antigen binding sites are believed to have been selected by a common antigen at some point during the leukemogenesis. In paper I, we investigated the antigen specificity of CLL-cell derived antibodies (Abs) with various IGHV gene usage and stereotyped BCR subset belonging. Identified CLL antigens included vimentin, filamin B, cofilin-1, proline rich acidic protein-1, cardiolipin, oxidized low density lipoprotein and Streptococcus pneumoniae capsular polysaccharides. Many of the CLL Abs studied displayed an oligo- or polyreactive antigen binding pattern and the identified antigens were either associated with apoptotic cells or microbial infection. This is similar to what has been described for innate natural antibodies, possibly indicating that CLL cells are derived from a natural-antibody- producing B cell population. Further characterization of CLL homology subset-2 antigen specificity showed binding to glands in human gastric mucosa for a CLL homology subset-2 Ab with HCDR3 motif-1, suggesting that this CLL subset recognize an autoantigen much like the CLL Abs tested in Paper I. Characterization of EBV-transformed CLL and normal lymphoblastoid cell lines (LCLs) in paper II showed that eight of the CLL cell lines were verified to be of authentic neoplastic origin. Indication for a biclonal CLL was found in two of the cell lines and two of the presumably normal LCLs turned out to represent the malignant CLL clone. For three cell lines no conclusive evidence for CLL origin could be found emphasizing the importance of verifying the identity of cell lines used in research. In contrast to CLL, the antigen specificity of B cells in polyneuropathy associated with MGUS (PN- MGUS) is well characterized. Pathogenic autoantibodies produced by the expanding B cell clone are aimed at myelin proteins and PN-MGUS is generally regarded an autoimmune-like disorder. The role of T cells is less well defined but T cell activation has been suggested. We investigated if the myelin-P0-specific B cell clone in a PN-MGUS patient could act as antigen presenting cells (APCs) and activate autologous T cells. We found that PN-MGUS B cells efficiently bound P0 to BCRs and presented P0peptides in MHC class II molecules. P0-specific activation of T cells with increased secretion of IFN-γ and IL-2 was observed indicating a role for Po-specific B cells as APCs in PN-MGUS. TABLE OF CONTENTS Background .............................................................................................................................................. 1 B cells and their role in the immune system ....................................................................................... 1 The B cell receptor............................................................................................................................... 1 Immunoglobulin structure .............................................................................................................. 1 Immunoglobulin gene rearrangement ............................................................................................ 2 B cell development .............................................................................................................................. 4 Stem cell to Pre‐B cell ...................................................................................................................... 4 Pre‐B cell to mature B cell ............................................................................................................... 4 Normal B cell interaction with antigen ............................................................................................... 6 T‐cell dependent B‐cell activation ................................................................................................... 6 Germinal centre reaction ................................................................................................................ 6 T‐cell independent B‐cell activation ................................................................................................ 7 B cells as antigen‐presenting cells ....................................................................................................... 7 Activation of T cells ......................................................................................................................... 8 The TH1/TH2 concept ...................................................................................................................... 8 Natural antibodies and B‐1 B cells....................................................................................................... 9 Marginal zone B cells ......................................................................................................................... 10 Chronic lymphocytic leukemia .......................................................................................................... 11 Prognostic markers in CLL ............................................................................................................. 12 Utilization of IGHV and IGLV genes in CLL ..................................................................................... 13 IGHV3‐21 CLL ................................................................................................................................. 14 Cellular origin of CLL ...................................................................................................................... 15 Antigens in CLL .............................................................................................................................. 16 Polyneuropathy associated with Monoclonal gammopathy of undetermined significance ............ 17 Monoclonal gammopathy of undetermined significance ............................................................. 17 Polyneuropathy associated with monoclonal gammopathy ......................................................... 17 Role of B cells and antibodies ........................................................................................................ 18 Role of T cells ................................................................................................................................. 18 Autoimmunity and B cells ............................................................................................................. 19 Molecular mimicry model ............................................................................................................. 19 Aims of the thesis .................................................................................................................................. 21 Materials and methods ......................................................................................................................... 23 CLL cell lines and patient material (Paper I & II) ..............................................................................