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) ...... 23 Recombinant CLL antibody (paper I & III) ...... 23 Test subject material (Paper III) ...... 23 PN‐MGUS cell line and patient material (Paper IV) ...... 24 IG gene sequencing ...... 24 Immunohistochemistry ...... 24 Immunofluorescence and confocal microscopy ...... 25 Receptor clustering and co‐localization ...... 25 Western Blot ...... 26 Mass spectrometry (MALDI‐TOF MS, ESI‐MS/MS) ...... 26 Enzyme‐Linked ImmunoSorbent Assay (ELISA) ...... 27 Elispot ...... 27 Flow cytometry ...... 27 Flow cytometric multiplex array ...... 28 Fluorescence in situ hybridization (FISH) analysis ...... 28 High resolution short tandem repeat (STR) analysis ...... 29 Affinity purification of proteins and MHC class II bound peptides ...... 29 High resolution MHC class I and class II genotyping ...... 29 dResults an discussion ...... 31 Identification of new antigens in CLL (Paper I) ...... 31 CLL cell‐of‐origin ...... 32 Importance of the recognized antigen in CLL ...... 32 Characterization of EBV transformed CLL cell lines (Paper II) ...... 33 Further investigation of IGHV3‐21 CLL subset‐2 antigen specificity (Paper III) ...... 35 Ability of myelin reactive IgM MGUS B cells to present antigen and activate T cells (Paper IV) ...... 36 Anti‐myelin Abs ...... 37 IgM MGUS B cells as APCs and T helper cell activation...... 37 Initial trigger of B cell expansion ...... 38 Future aspects ...... 39 Conclusions ...... 41
Acknowledgements ...... 43 References ...... 47
LIST OF PUBLICATIONS
This thesis is based on the following papers, referred to in the text by their Roman numerals (I-IV).
I. Lanemo Myhrinder A*, Hellqvist E*, Sidorova E, Söderberg A, Baxendale H, Dahle C, Willander K, Tobin G, Bäckman E, Söderberg O, Rosenquist R, Hörkkö S, Rosén A. A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies. Blood 2008 Apr 1; 111(7):3838-48.
*EH and ALM contributed equally to this work
II. Lanemo Myhrinder A, Hellqvist E, Jansson M, Nilsson K, Hultman P, Jonasson J, Klein E, Weit N, Herling M, Rosenquist R, Rosén A. Molecular authenticity of neoplastic and normal “sister” cell lines established from chronic lymphocytic leukemia patients. Submitted 2009
III. Hellqvist E, Morad V, Borch K, Rosén A. IGHV3-21 stereotyped subset 2 chronic lymphocytic leukemia cells make autoantibodies that bind to a 11.5 kDa gastric mucosal antigen. Manuscript 2009
IV. Hellqvist E, Kvarnström M, Söderberg A, Vrethem M, Ernerudh J, Rosén A. Myelin protein zero is naturally processed in IgM MGUS B cells: Aberrant triggering of patient T cells. Accepted for publication, Haematologica 2009
Reprints were made with permission from the publishers.
ABBREVIATIONS
Ab Antibody FO Follicular
AEC 3-amino-9-ethyl cabazole FR Framework
AID Activating-induced cytidine GBS Guillain-Barré syndrome deaminase GC Germinal centre ALP Alkaline phosphatase HC Heavy chain APC Antigen-presenting cell HCDR Heavy chain ATM Ataxia telangiectasia complementarity mutated determining region
BCR B cell receptor HLA Human leukocyte antigen
C Constant HRP Horseradish peroxidase
CDR Complementarity IFNγ Interferon gamma determining region Ig Immunoglobulin CLL Chronic lymphocytic IGH Immunoglobulin heavy leukemia chain CLLU1 CLL up-regulated gene 1 IGHV Immunoglobulin heavy D Diversity chain variable
DAB Diaminobenzidine IGK Immunoglobulin kappa chain DC Dendritic cell IGKV Immunoglobulin kappa EBV Epstein-Barr virus chain variable ELISA Enzyme-linked immuno- IGL Immunoglobulin lambda sorbent assay chain Elispot Enzyme-linked immunospot IGLV Immunoglobulin lambda ESI Electrospray ionization chain variable
FDC Follicular dendritic cell IL Interleukin
FISH Fluorescence in situ ITAM Immunoreceptor tyrosine hybridization based activation motif
J Joining oxLDL Oxidized low density lipoprotein KCDR3 Kappa chain complementarity PBMC Peripheral blood determining region 3 mononuclear cells
LC Light chain PN Polyneuropathy
LCDR3 Lambda chain PN-MGUS Polyneuropathy associated complementarity with monoclonal determining region 3 gammopathy of undetermined significance LCL Lymphoblastoid cell line P0 Myelin protein zero LPS Lipopolysaccharide PRAP-1 Proline rich acidic protein 1 mAb Monoclonal antibody Pre-B cell Precursor B cell MAG Myelin associated glycoprotein Pre-BCR Precursor B cell receptor
MALDI Matrix-assisted laser Pro-B cell Progenitor B cell desorption/ionization RAG1, RAG2 Recombination activating MALT Mucosal-associated gene 1 and 2 lymphoid tissue RSS Recombination signal M-CLL Mutated (IGHV gene) sequence chronic lymphocytic SGPG Sulphate-3-glucoronyl leukemia paragloboside MDA-LDL Malondialdehyde modified SHM Somatic hypermutation low density lipoprotein sIgM Surface IgM MGUS Monoclonal gammopathy of undetermined significance SpA Staphylococcal protein A
MHCII Major histocompatibility STR Short tandem repeat complex class II TCR T cell receptor MS Mass spectrometry TdT Terminal deoxynucleotidyl MZ Marginal zone transferase m/z Mass-to-charge ratio TH T helper
TI T cell independent
TOF Time-of-flight V Variable
UM-CLL Unmutated (IGHV gene) ZAP-70 Zeta-associated protein 70 chronic lymphocytic
leukemia
BACKGROUND
B CELLS AND THEIR ROLE IN THE IMMUNE SYSTEM
B cells originate in the bone marrow and are part of the adaptive immune system. Each B cell expresses a unique B cell receptor (BCR) with the ability to recognize and bind a specific antigen, e.g. protein, lipid or carbohydrate usually found on bacteria or viruses. Prior to antigen encounter the B cell circulates throughout the body and is called a naïve B cell. Upon antigen encounter and BCR binding of the specific antigen with proper binding strength and co-stimulation, the naïve B cell undergoes antibody (Ab) affinity maturation and differentiates to either a memory B cell or an Ab-secreting plasma cell. The secreted Abs or immunoglobulins (Igs) as they are also called have the same binding specificity as the BCR. The Abs opsonize or neutralize the antigen and trigger elimination processes. B cells are also professional antigen presenting cells (APCs) able to internalize the antigen bound to its BCR, process it in endosomes, present the resulting peptides in major histocompatibility complex class II (MHCII) receptors and activate T cells for even more efficient pathogen elimination. In this way the BCR repertoire enables the immune system to recognize and fight off a wide array of pathogens.
THE B CELL RECEPTOR
The BCR is composed of an immunoglobulin molecule and a noncovalently associated complex of the two co-receptors CD79a (Igα) and CD79b (Igβ)1. The CD79a/CD79b co- receptors contain immunoreceptor tyrosine based activation motifs (ITAMs) which are responsible for intracellular signaling from the BCR.
Immunoglobulin structure
The immunoglobulin or antibody is made up from two identical heavy chains (HC) and two identical light chains (LC) connected by disulfide bonds (Figure 1), giving the IG two identical antigen binding sites1,2. In humans, there are five different HC (α, δ, ε, γ, µ) making up the different Ig isotypes (IgA, IgD, IgE, IgG, IgM). Each HC consists of one variable (V) region and three to four constant (C) regions. Two different kinds of LC exist (κ and λ) and each LC has one V domain and one C domain. The C-terminal C-region of the immunoglobulin heavy chain (IGH) performs the effector function of the Ig whereas the N-terminal V-regions of the HC and LC are responsible for antigen binding. The V region of
| 1 BACKGROUND
each HC and LC can be divided into four framework (FR) regions and three complementarity-determining regions (CDR1, CDR2, CDR3), also called hypervariable regions due to the immense amino acid variability in these regions. The six CDRs from both the HC and LC make up the three-dimensional structure of the antigen-binding site. The heavy chain complementarity determining region 3 (HCDR3) is the most variable of all CDRs since it spans the V/D/J gene junctions (described below).
IMMUNOGLOBULIN STRUCTURE
Ig heavy chain variable region (IGHV) V D J C
CDR1 CDR2 CDR3 FR1 FR2 FR3 FR4 Variable region
-S-S- Constant region -S-S-
Light chain (κ, λ) Ig kappa or lambda variable region (IGKV/IGLV)
V J C Constant region
CDR1 CDR2 CDR3 FR1 FR2 FR3 FR4
Heavy chain (γ, μ, δ, α, ε) Figure 1. Immunoglobulin structure and detailed view of the heavy chain and light chain variable regions. V = variable gene, D = diversity gene, J = joining gene, C = constant, FR = framework region, CDR = complementarity determining region.
Immunoglobulin gene rearrangement
The extensive variability of the immunoglobulin V region is generated by the rearrangement of gene segments in the immunoglobulin heavy chain variable (IGHV) and immunoglobulin kappa chain variable (IGKV) or immunoglobulin lambda chain variable (IGLV) region loci during B cell development1-4. The IGHV region is coded by diversity (D), joining (J) and variable (V) gene segments (Figure 2A). In humans, there are 23 D gene segments, 6 J gene segments and 38-46 V gene segments that are functional and they are located on chromosome 143. During gene rearrangement, one of the D gene segments is first joined with one of the J gene segments and this combined D/J segment is then joined with one V gene segment giving rise to the rearranged V/D/J gene. During IGKV/IGLV gene
2 | BACKGROUND
recombination one V gene is joined to one J gene creating the complete LC V region gene (Figure 2B)1-4. The genes encoding the IGKV/IGLV region are located on two different chromosomes. The functional 34-38 V genes and 5 J genes encoding the κ LC are located on chromosome 2, and the 29-33 V genes and 4-5 J genes for the λ LC on chromosome 223. The rearranged V/(D)/J gene is finally joined with a C gene by RNA splicing to give rise to functional heavy and light chains1-4. IGHV and IGKV/IGLV gene rearrangement is regulated by a protein complex containing recombination activating gene 1 (RAG1) and 2 (RAG2) encoded proteins. RAG1 and RAG2 recognize and cleave the DNA strands at recombination signal sequences (RSSs) flanking the V, D and J gene segments. The RSSs are conserved heptamer or nonamer sequences separarated by 12 or 23 long nucleotide sequences. The gene segments are then brought together and ligated according to the 12/23 rule. The IGV region diversity is further extended by the random nucleotide insertions or deletions introduced by the enzyme terminal deoxynucleotidyl transferase (TdT) in the V/(D)/J gene segment junctions and by the somatic hypermutation that takes place after B cell antigen encounter and activation.
A B HEAVY CHAIN GENES LIGHT CHAIN GENES
IGHV genes IGHD genes IGHJ genes IGKV or IGLV genes IGKJ or IGLJ genes
D-J recombination V-J recombination
V-DJ recombination V J C CDR1 CDR2 CDR3 FR1 FR2 FR3 FR4 V D J C
CDR1 CDR2 CDR3 FR1 FR2 FR3 FR4
Figure 2. Gene rearrangement of A) immunoglobulin heavy chain variable gene loci B) immunoglobulin kappa or lambda chain gene loci
| 3 BACKGROUND
B CELL DEVELOPMENT
B cells originate from hematopoietic stem cells in the bone marrow and to some extent from the fetal liver where their development is dependent upon stromal cell interaction and signals5-7. B cell development is divided into different stages depending on the rearrangement of the Ig genes and expression of the finished HC and LC as well as other cell surface markers (Figure 3). All Ig gene recombination events take place in the bone marrow. If a B cell is unable to move to the next stage in the development process that particular B cell will be anergized or eliminated through apoptosis.
Stem cell to Pre-B cell
The hematopoietic stem cell expresses all Ig genes in germline configuration. The first B committed cell is the progenitor B cell (pro-B cell) and this is also the B cell developmental stage where IGHV gene rearrangement starts with joining of the D and J gene segments (Figure 3)3,5-7. Pro-B cells express CD79a, CD79b and calnexin on their surface as well as surface markers CD19 and CD43. The IGHV joining to the IGHD/J takes place in the late pro-B cell. When a productive heavy chain V/D/J rearrangement is achieved, the pro-B cell grows and becomes a large precursor B cell (pre-B cell). The successfully rearranged Ig heavy chain is expressed intracellularly in the large pre-B cell in combination with a surrogate light chain to form a pre-B cell receptor (pre-BCR). The pre- BCR is also expressed to some extent on the surface of the large pre-B cell providing signals for the large pre-B cell to divide and differentiate into a small pre-B cell. The pre- BCR also provides signals for cell survival and inhibition of further heavy chain V to D/J rearrangement, a process called allelic exclusion.
Pre-B cell to mature B cell
The small pre-B cell loses the expression of surface marker CD43 but retains CD19 expression7. The IG light chain V-J gene rearrangement starts in the small pre-B cell and when it is successful and a productive IG light chain, either κ or λ, is expressed the cell will start to express IgM on the cell surface in combination with co-receptors CD79a and CD79b and become an immature B cell3,5-7. Rearrangement of IGKV loci always occur first (in humans) and IGLV loci rearrangement only takes place if the first rearrangement was unsuccessful. The immature B cell subsequently undergoes negative selection where cells expressing functional BCR receive survival signals whereas a non-functional or strongly autoreactive BCR leads to deletion or anergy. B cells expressing non-functional or strongly autoreactive BCRs can however undergo multiple rounds of Ig light chain gene
4 | BACKGROUND
rearrangement and IGHV changes in an attempt to rescue the B cell; a process known as receptor editing and IGHV replacement. The surviving immature B cells exit the bone marrow as a transitional B cells. The transitional B cells undergo further development, start to produce IgD in addition to IgM and become mature naïve B cells that start to circulate through the peripheral blood and lymphoid organs in search for foreign antigen. The mature naïve B cells can be divided into three types: marginal zone (MZ) B cells which mainly reside in the spleen, B-1 B cells which are mainly characterized in mouse and follicular (FO) B cells. Differentiation of transitional B cells into these mature naïve B cell subsets is dependent on Notch2, B cell-activating factor (BAFF) and level of BCR stimulation7,8.
IGH gene IGK/L gene rearrangement rearrangement preBCR BCR CD79a/b
Hematopoietic Antigen encounter Pre-B cell Stem cell Pro-B cell Immature B cell Mature B cell
CD19+ CD19+ CD43+ CD43- CD79a/b Apoptosis Apoptosis IgM+ IgD+ CD19+ CD19+ CD43+ CD43- preBCR IgM+ BM CD79a/b CD79a/b T cell FDC Activated B cell
Plasma cell
Centrocytes
GC LIGHT ZONE Centroblasts Apoptosis Proliferation DARK ZONE Memory B cell SHM Class switching
Figure 3. B cell development and germinal centre reaction. BM = bone marrow, GC = germinal centre, IGH = Immunoglobulin heavy, IGK/L = Immunoglobulin kappa or lambda, FDC = Follicular dendritic cell, SHM = Somatic hypermutation, BCR = B cell receptor, preBCR = precursor B cell receptor
| 5 BACKGROUND
NORMAL B CELL INTERACTION WITH ANTIGEN
Naïve B cells mainly come in contact with antigen in the secondary lymphoid organs, such as lymph nodes and, where the tissue architecture is optimal for antigen presentation and activation9. The B cell can interact with both soluble antigens and antigens presented by macrophages, dendritic cells (DC) or follicular dendritic cells (FDC). Antigen is often presented in immune complexes coated with complement or antibodies. Upon binding antigen to its BCR, the antigen-BCR complex is endocytosed by the B cell, degraded and returned to the cell surface as antigenic peptides presented in MHCII molecules. Upon antigen encounter the B cell can either go through T-cell dependent activation or T-cell independent activation.
T-cell dependent B-cell activation
During T-cell dependent activation, the naïve FO B cell recognizes and binds an antigen in the primary follicle, but needs T-cell help to be able to respond5,9,10. Before acquiring T-cell help however, the FO B cell starts to proliferate and some cells differentiate into short- lived IgM-secreting plasma cells that enable a rapid first-line response to the pathogen. Although quick, this initial IgM response is usually of low affinity to the protein antigen. Further fine tuning of the Ab is required for more effective pathogen elimination. Therefore, other cells enter the germinal centre reaction in the secondary follicle (Figure 3), where affinity maturation and class switch recombination occurs with the assistance of CD4+ T helper (TH) cells.
Germinal centre reaction
During the germinal centre reaction, the activated B cell first enters the dark zone, starts to proliferate and differentiates into centroblasts (Figure 3)3,5,10. This is also the phase where somatic hypermutations (SHM) occur in the IGV region, a process known as affinity maturation. SHMs are single base replacements that are initiated by the enzyme activating-induced cytidine deaminase (AID). The three CDRs are hotspots for SHM although FR regions are also targeted. No SHMs take place in the C region. In the next phase, the centroblasts move to the light zone of the secondary follicle where antigen presenting FDCs and TH cells reside. The centrocytes are then selected by their BCR affinity for antigen presented on FDCs. Centrocytes with high affinity for antigen receive survival signals and the ones with low affinity are eliminated. TH cell T-cell receptor (TCR) binding and recognition of antigenic peptide presented in MHCII on the centrocytes and results in cytokine secretion. This provides a second signal for proliferation and
6 | BACKGROUND
differentiation. Binding of B cell co-receptor CD40 to CD40L on TH cells is also important for B cell activation since it stimulates the B cell to proliferation and expression of co- stimulatory molecules such as B7. The centrocyte goes through class switch recombination, during which the Ab isotype is changed from IgM to IgG, IgA or IgE depending on the required immune response. Class switching is also initiated by the enzyme AID. The result of the germinal centre reaction is long-lived plasma cells producing high-affinity antigen-specific Abs and memory B cells. As exemplified by MZ B cells, SHM can also take place outside of the germinal centre.
T-cell independent B-cell activation
T-cell independent (TI) antigens can activate B cells directly without the need for T-cell help. There are two different kinds of T-cell independent antigens, TI-1 and TI-2. TI-1 antigens have the ability to activate many B cell clones independent of their antigen specificity if the TI-1 antigen is present in very high concentration3,9. Other receptors on the B cell such as Toll-like receptors (TLR) bind the TI-1 antigen. At low concentrations of TI-1 antigen only the B cell with a BCR specific for the antigen will be able to respond. Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is an example of a TI-1 antigen. TI-1 antigens do not efficiently give rise to memory B cells. TI-2 antigens are usually made up from repetitive units present on some pathogens, for example polysaccharides found on the surface of encapsulated bacteria or viruses11. The repetitive nature of these antigens can extensively cross-link the BCRs and thus activate the B cell. Streptococcus pneumoniae (S. pneumoniae) capsular polysaccharide is an example of a TI-2 antigen11. The T-cell independent B-cell activation induces a rapid early immune response against invading pathogens with B cell proliferation and IgM secretion from plasmablasts, but also generation of memory B cells in the case of TI-2 antigens.
B CELLS AS ANTIGEN-PRESENTING CELLS
In addition to their important role in Ab production B cells are also professional APCs with the capacity to activate naïve T cells12,13. B cells constantly express high levels of MHCII molecules but are only induced to express co-stimulatory molecules under certain conditions such as when stimulated and activated by bacteria. B cells present peptide antigens in their MHCII molecules from endocytosed extracellular pathogens and toxins, or pathogens that multiply in intracellular vesicles. Peptides are loaded onto the MHCII molecules inside the late endosomal compartment of the B cell and are subsequently transported to the cell surface where they are presented to CD4+ naïve T cells (Figure 4).
| 7 BACKGROUND
Activation of T cells
Naïve CD4+ T cells continuously sample peptides presented in the MHCII on APCs. A naïve T cell with a TCR specific for the antigenic peptide becomes activated upon binding the peptide-MHCII complex and the necessary co-stimulatory molecules12,14. B7 (CD80, CD86), and CD40 are co-stimulatory molecules expressed by APCs which bind CD28 and CD40 ligand respectively on the naïve T cell (Figure 4). Upon activation, the T cell starts producing interleukin-2 (IL-2), an autocrine cytokine that drives naïve T cell proliferation and differentiation into effector T cells. T cells recognizing antigen presented in MHC II in the absence of co-stimulatory signals enter a state of inactivity or anergy.
Upon recognition of a specific antigenic peptide-MHC II complex and with the appropriate co-stimulatory signals, the naïve CD4+ T cell can differentiate into various types of effector cells; often divided into TH1 and TH2 based on their secreted cytokines (Figure 4)15,16. These effector T cells skew the immune response depending on the pathogen recognized. Extracellular pathogens, in particular parasites, mainly induce a TH2 response while intracellular pathogens tend to give a TH1 response. In addition, IL-17 secreting cells, termed TH17, were recently described as another TH subpopulation being involved mainly in combating extra-cellular pathogens15.
The TH1/TH2 concept
TH1 cells induce a cell-mediated immune response with the activation of macrophages15,16. They also and stimulate B cells to class switch and produce highly effective opsonizing IgG Abs (IgG1, IgG3 in humans) that bind to the extracellular pathogen. This makes it easier for phagocytes to eliminate them. Typical TH1-cytokines include the macrophage- activating interferon gamma (IFN-γ) and lymphotoxin-α that recruits macrophages to the site of infection. IFN-γ, the hallmark TH1 cytokine, also has a TH2 inhibitory effect. TH2 cells on the other hand secrete B-cell activating cytokines IL-4, IL-5, IL-9 and IL-13. The TH2 cells induce humoral immunity and stimulate B cells to produce neutralizing Abs, mainly IgE and IgG4, which interact with mast cells and eosinophils through their constant regions. Both TH1 and TH2 cells can stimulate IgM production from B cells.
8 | BACKGROUND
Antigen
BCR
CD79 a/b
TH1 IFN-γ Lymphotoxin α Endosome IL-2 B B7 CD28 IL-2R MHC II
TCR
CD40 CD4 CD40L T TH2 IL-4 IL-5 IL-9 IL-13
Figure 4. B-cell activation of T cells. BCR = B cell receptor, MHC II = Major histocompatibility complex II, IL-2R = Interleukin 2 receptor, TCR = T cell receptor, TH = T helper cell
NATURAL ANTIBODIES AND B-1 B CELLS
Natural Abs can be considered to be part of the innate immunity. They are germline- encoded polyspecific IgM Abs involved in the first line of defense against blood borne encapsulated bacteria by reacting with TI antigens such as bacterial capsular polysaccharides and phosphorylcholine in the cell wall17. In mice, these natural Abs have an important role in the early immune response against S. pneumoniae18 as well as a suggested protective role against formation of atherosclerotic plaques by binding to oxLDL17,19. Natural Abs also bind autoantigens on apoptotic cells and have been suggested to have an important “house-keeping” role by removing dead or dying cells from the circulation17,20. Natural Abs have been described to be of low affinity but high avidity making them especially effective at binding solid-phase antigens such as cell surfaces. Noteworthy, monospecific natural antibodies have also been found21,22. Although this type of Ab has been best described in mouse, where they are produced by the B-1a B cell population17, they can also be found in humans. The human B cell counterpart is not very well defined, but is often assumed to be a CD5+ B cell mainly expressing IgM Abs with unmutated or minimally mutated V(D)J gene
| 9 BACKGROUND
rearrangements22,23. The human natural IgM Abs have in addition to binding encapsulated bacteria also shown cross-reactivity to blood-group antigens and intermediate filament proteins.
Parallels are often drawn between the human MZ B cells and the much studied B-1 B cell compartment in mouse. There are many similarities between these two B cell populations but also some differences. B-1 B cells express IgMhigh, IgDlow, CD21high, CD23low, CD1d+ phenotype much like human MZ B cells. B-1a B cells also express surface marker CD5 while human MZ B cells are CD5-. Similar to human MZ B cells mouse B-1a B cells also mount a quick IgM response to blood borne antigens and especially towards encapsulated bacteria17,24. Some differences include that a majority of the MZ B cells express mutated Ig genes and as the name suggests they are mainly localized in the marginal zone of the spleen25. The mutational frequency is however lower in MZ B cells compared to isotype switched B cells. Mouse B-1a B cells on the other hand express unmutated Ig genes and are primarily found in the pleural and peritoneal cavities17.
MARGINAL ZONE B CELLS
MZ B cells mainly reside in the spleen, in the marginal zone that separates the white pulp from the red pulp, but are also found peripheral blood26. They generally express a polyreactive BCR of low affinity/avidity and have an IgMhigh, IgDlow, CD21high, CD23low, CD1c+, CD27+ phenotype9,26. MZ B cells often carry mutations in their IGHV genes but they can also have unmutated IGHV genes11,26. The IGHV mutational frequency is however lower in IgM MZ B cells compared to isotype-switched B cells26. Three important functions are generally described to these B cells.
The first is to continuously sample the blood for foreign antigens and swiftly respond with IgM production upon antigen recognition9,11. The MZ B cells are therefore considered to be important in the early immune response to both T cell dependent and independent antigens. DCs also residing in the marginal zone of the spleen can present antigen to the MZ B cells and quickly induce MZ B cell differentiation to IgM producing plasma cells without the need for T cell help9. MZ B cells have been shown to be especially important in mounting immune responses against capsular polysaccharide antigens (TI-2 antigens) on encapsulated bacteria such as S. pneumoniae, Neisseria meningitides and Haemophilus. influenzae11. Capsular polysaccharide antigens bind complement factors, especially C3d, even in the absence of specific IG11. The ability of the MZ B cell to mount TI immune responses is thought to be because of their high CD21 surface expression. CD21, or complement receptor 2 (CR2), is part of a BCR-modulating complex capable of lowering
10 | BACKGROUND
the activation threshold of the B cell11. The CD21high surface expression on MZ B cells has led to the speculation that these cells have a lower activation threshold for antigens coated with complement that in addition to binding the BCR (even with low affinity) also bind to CD2111.
The second implied function is that MZ B cells in humans have an important role as antigen providers to the FDCs by binding immune complexes to their complement receptors9,24. Upon binding of blood-derived immune complexes, the MZ B cells migrate to the follicles, deliver the immune complex to FDCs and then move back to the marginal zone again9.
The third suggested function of MZ B cells is to present lipid antigens to natural killer T (NKT) cells via the CD1d receptor, similar to what has been seen in mouse24,26. It is also possible that the NKT cells then activate MZ B cells through CD40-CD40L interaction and induce both class switch recombination and somatic hypermutations. MZ B-cell-like cell populations have also been found in human lymph nodes, tonsils and Peyer’s patches although the exact role of these compared to the splenic MZ B cells is not known9,26.
CHRONIC LYMPHOCYTIC LEUKEMIA
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small neoplastic B cells with small cytoplasm and surface marker phenotype sIgweak, CD5+, CD23+, FMC7-, CD22weak/- 27-29. Diagnosis of CLL is based on a B lymphocyte count of at least 5x109/L, morphology of the leukemic cells and a phenotype scoring criteria proposed by Matutes et al.27 using the surface markers described above30. CLL cells also typically express high levels of CD43 and CD19 while CD79b, CD20 and IgD are downregulated27. 400-500 new CLL cases are diagnosed annually in Sweden making it the most common adult leukemia. Men are affected twice as often as women and the median age at diagnosis is 70 years, but many are under 50 at diagnosis. Looking at the geographic occurrence of CLL it becomes apparent that this disease is more frequently found in North America and Western Europe whereas it is uncommon in Asia31. This could reflect exposure to environmental factors, a genetic predisposition or a combination of both. A genetic susceptibility associated with CLL is known to exist since relatives to CLL patients have a higher risk of developing CLL compared to the general population32,33. The exact gene(s) involved in this susceptibility is nevertheless unknown.
Infiltration of lymph nodes, spleen and bone marrow is common and a hallmark sign of CLL is the proliferative centers, also called pseudofollicles, found in these compartments30. CLL was for a long time considered to be a disease where the malignant
| 11 BACKGROUND
cells accumulated in the circulation due to increased resistance to apoptosis. Recent studies however have shown that CLL cells both proliferate and die. About 0.1-1.75% of the CLL clone is renewed each day, which was shown by DNA incorporation of deuterium in vivo in CLL patients34. When looking at the shorter telomere length of CLL cells it is apparent that these cells have undergone more rounds of cell division than normal age matched B cells35. Even though a majority of CLL cells found in the circulation are resting cells in the G0/early G1 phase of the cell cycle, they have the phenotype of activated antigen-experienced B cells with high expression of CD69, CD23, CD25 and CD71 and a low expression of CD22, CD79b and IgD36. Gene expression studies of CLL cells show profiles similar to memory B cells with upregulation of CD2737.
CLL is a heterogeneous disease both when it comes to clinical behavior and molecular expression. Patients can be divided into two subgroups based on the degree of somatic hypermutations present in the IGHV genes expressed by the malignant cells. Patients whose malignant clone expresses mutated IGHV genes generally have a more indolent course of disease with longer survival times than patients with unmutated IGHV genes, who exhibited a more aggressive disease with shorter survival38,39. There are exceptions to this rule, however, which is exemplified by the mutated IGHV3-21 CLL cases.
Prognostic markers in CLL
Due to the heterogeneous disease progression seen among CLL patients much work has been focused on finding prognostic markers. Two clinical staging systems, Rai40 and Binet41, were developed to predict and monitor disease progression. Both of these staging systems estimate prognosis based on the clinical symptoms of the patient such as lymphocytosis in blood and bone marrow, lymphadenopathy, splenomegaly, hepatosplenomegaly, thrombocytopenia and anemia. Recently suggested prognostic markers include chromosomal aberrations, IGHV gene mutational status38,39 and 38,42 43 expression of CD38 , zeta-associated protein 70 (ZAP-70) and CLL up-regulated gene 1 (CLLU1)44,45.
Chromosomal aberrations are frequent in CLL cells (~80%), but there is no single deletion or translocation common for all CLL cases46. The most frequent cytogenetic abnormalities found in CLL are del 13q14, del 11q22-23, trisomy 12 and del 17p1330. Del 13q14 is found in 40- 50% of CLL patients and it is associated with a more indolent course of disease when found on one allele only. The 13q locus encodes for the two micro-RNA genes miR-15 and miR-16 that have been shown to target Bcl-247,48. Del 11q22-23 present in 20% of patients encodes the ataxia telangiectasia mutated (ATM) gene30. The ATM gene is involved in the regulation of apoptosis and presence of this deletion in CLL patients is linked to a worse
12 | BACKGROUND
prognosis49. 15% of patients exhibit trisomy 12 and 15% del17p1350. 17p is the location for the p53 locus and deletion of this apoptosis-regulating gene is associated with an aggressive clinical course and resistance to chemotherapy46.
As mentioned above, the mutational status of the IGHV genes used by CLL cells can be correlated to prognosis, where CLL patients with mutated IGHV genes generally have a better prognosis and longer survival (293 months median survival) than patients with unmutated IGHV genes (95 months median survival)38,39. Presence of CD3838, ZAP-7043 and upregulation of gene CLLU144,45,51 in the malignant cells is also associated with a more rapid disease progression. Markers ZAP-70 and CLLU1 are expressed to a higher extent in unmutated CLL cases than mutated CLL cases. High expression of CD38 has also been associated with mutated IGHV genes in several studies while other studies show a weaker correlation. CD38 is reported to be associated to BCR signaling52,53 and is expressed in CLL cells that have recently undergone cell division54. ZAP-70 is a T-cell-associated tyrosine kinase that is expressed in human normal mature B lymphocytes after activation55,56. CLLU1, a gene with unknown function mapped to chromosome 12q22, is considered CLL specific and is overexpressed in CLL cells compared to other normal B cells and other B cell malignancies44,45,51.
Utilization of IGHV and IGLV genes in CLL
CLL cells display a biased usage of certain IGHV and IGLV genes compared to normal B cells39,57-59. The use of IGHV1-69 and IGHV1-2 genes is overrepresented in the unmutated CLL cases (100% identity to germline gene) while IGHV4-34, IGHV3-7 and IGHV3-23 are the most common in mutated CLL (<98% identity to germline gene). A group of borderline- mutated IGHV genes (98-98.9% identity to germline gene) utilized in CLL has also been identified. In this borderline-mutated CLL group IGHV3-21 gene usage is predominant.
Highly homologous (>60% sequence identity) and in some cases even identical CDR3 amino acid sequences in the heavy chains have been identified in groups of CLL patients, so called “stereotyped” HCDR3s57,60,61. Based on the stereotyped HCDR3 expressed, CLL cases are often divided into subsets. Members of the same subset often utilize the same IGHV/D/J gene combination, but use of the same IGHV gene is not a necessity as was shown for one of the largest defined subsets, subset-1. Members of this subset used IGHV genes of the same clan; IGHV1-2/IGHV1-3/IGHV1-18/IGHV5-a or IGHV7-4-157. Almost 30% of CLL patients belong to one of the 110 different subsets defined to date and the subsets are found among both mutated and unmutated cases, although they are present to a higher degree in unmutated cases (43% UM vs. 16%M)57.
| 13 BACKGROUND
Members of the same HCDR3 subset also show a restricted light chain usage and subset biased kappa chain CDR3 (KCDR3) and lambda chain CDR3 (LCDR3) regions60-65. Utilization of these homologous HCDR3 and K/LCDR3 within the same subset leads to stereotyped BCRs with presumably very similar antigen binding sites between members of the subset. Use of these stereotyped BCRs is considered unique for CLL and was either absent or occurred at a much lower frequency in non-CLL sequences. Noteworthy is also the association of certain CLL subsets to clinical characteristics such as age at diagnosis or prognosis, e.g. IGHV4-34/IGKV2-30-expressing CLL patients tend to be IgG switched, are diagnosed at a younger age and show an indolent course of disease60.
Looking at the somatically mutated CLL cases belonging to the same subset, specific CLL biased and CLL subset biased mutations leading to the same amino acid replacement can be seen in the CDR regions of both heavy chains and light chains as compared to non-CLL sequences57. These mutation patterns presumably result in a changed BCR antigen binding capacity. For example, subset-2 (IGHV3-21 group) has a serine deletion within the HCDR2 that is subset biased57,66.
IGHV3-21 CLL
CLL cases expressing IGHV3-21 genes were first shown by Tobin et al. in 200258 to differ from the general classification of prognosis based on IGHV mutational status. Despite the majority of these patients expressing mutated IGHV3-21 genes they display a more aggressive course of disease and a shorter survival time compared to other mutated CLL cases. As previously mentioned, the IGHV3-21 mutated cases show a lower degree of mutations compared to other CLL cases that are considered somatically mutated57,67. IGHV3-21 gene usage in CLL seems to be more common in northern European countries (Sweden68,69, Finland69, UK70) than in the Mediterranean countries60,71 (9% of all CLL cases in Sweden68 vs. 1.5-3.5% in other countries60,67,71). The expression of CD38 and ZAP-70, two markers associated with poor prognosis and not usually expressed by mutated CLL cases, have been observed in mutated IGHV3-21 cases60,63.
The IGHV3-21 subgroup of CLL patients can be further divided into groups depending on the presence or absence of HCDR3 amino acid motifs. Many IGHV3-21 CLL cases expressed stereotyped BCRs belonging to subset-2, characterized by the recombination of IGHV3-21, IGHJ6 and a very short IGHD region resulting in 9 amino acid long HCDR3 region with very similar, and in many cases identical, amino acid motif (ARDANGMDV)60,61,63,68. This HCDR3 motif, called motif-1 in a recent report67 can be seen in 40-56% of IGHV3-21 CLL cases60,63,67. A restricted use of light chain gene IGLV3-21 and IGLJ3 gene is also observed leading to a LCDR3 motif QVWDS(S/G)DHHPWV in 40-70% of IGHV3-21 cases60,63. Whether these
14 | BACKGROUND
stereotyped subset-2 BCRs are associated with a more aggressive disease than non- stereotyped IGHV3-21 cases is uncertain with some studies showing an association60,72 while others do not63. A second HCDR3 motif, DPSFYSSSWTLFDY, also called motif-2 was recently identified in a small group of IGHV3-21 CLL patients67. This motif is associated with IGKV3-20 light chain usage. In the IGHV3-21 group with heterogeneous HCDR3 region, a diverse use of IGHJ gene and IGV light chain genes can be seen as well as a variable clinical course72.
The IGHV3-21 subgroup of CLL patients also contain disease-biased amino acid deletions and substitutions as was first shown by Belessi et al.66. A deletion of one amino acid (serine) in the HCDR2 is observed in 25% of IGHV3-21 cases belonging to HCDR3 homology subset-257,66. This deletion was only observed in 0.78% of mutated non-CLL IGHV3-21 sequences (normal, autoimmune or other B cell malignancies) and is thus CLL biased. Many of the IGHV3-21 sequences carrying the serine deletion also contain a serine to threonine substitution in the CDR1 region57. The highly homologous BCRs found in many of the IGHV3-21 CLL cases and the presence of subset specific somatically introduced mutations suggest that an antigen, foreign or autoantigen, play a role in the selection and development of some IGHV3-21 cases.
Cellular origin of CLL
The identity of the CLL cell-of-origin has been and still remains a mystery, although certain biological features of CLL cells provide us with important clues. The majority of CLL patients have a malignant clone expressing IgM. Approximately half of CLL patients express somatically mutated IGHV genes indicating that at least 50% of CLL cells have entered a germinal centre and undergone SHM in response to antigenic stimuli38,39. These observations originally lead to the hypothesis that unmutated CLL (UM-CLL) cells are derived from naïve CD5+ pre-germinal centre B cells and the mutated CLL (M-CLL) cells from a memory CD5+ B cell compartment. This model has since been modified. CLL cells, both with mutated and unmutated IGHV genes, express surface markers that are typical for antigen-activated B cells36. The hallmark surface molecule is CD5 and much focus has been aimed at this molecule when trying to determine the cell of origin. Gene expression profiling studies show that CLL cells resemble normal memory B cells regardless of IGHV mutational status37,73. Combining these molecular features, a model was proposed that both UM- and M-CLL cells derive from antigen-experienced B cells. One cell of origin candidate is the presumed human counterpart of the murine CD5+ B-1 B cells. Although not an established counterpart in humans, CD5+ B cells are thought to produce polyreactive, autoreactive natural antibodies also reacting with bacteria74. Mouse B-1a B cells express CD5 and produce germline encoded Abs that are polyreactive and
| 15 BACKGROUND
autoreactive17. B-1 B cells can also be seen as clonal expansions in older mice75. However, CD5 can also be upregulated on normal B cells after activation76,77, which makes it difficult to determine if the CD5 expression on CLL cells is caused by activation or if it is a B-cell lineage-specific marker.
MZ B cells have more recently been suggested as possible cells of origin. The human MZ B cell, although CD5-, shares many similarities with B-1 B cells such as the production of autoreactive polyreactive Abs that mainly react with TI antigens9,26. They also share many attributes with CLL cells. MZ B cells mainly express IgM just like CLL cells and they have been shown to mainly express somatically mutated IGHV genes but unmutated IGHV genes are also found11,26. MZ B cells also express memory B cell surface marker CD27 and have been found in the peripheral blood similar to CLL cells.
Antigens in CLL
Gene expression studies37,73, phenotype analysis36 and the presence of stereotyped BCRs in CLL57,60,61 all indicate a role for antigen in the pathogenesis of CLL. Since the antigenic specificity of CLL cells could provide clues to the cell of origin and the antigen itself might send survival signals to the CLL cells or even constitute a potential proliferation trigger in the disease, we wanted to study which antigen(s) CLL cells bind to (Paper I). The antigenic specificity of CLL cells is largely unknown. Some of the stereotyped receptors found in CLL are homologous to the HCDR3 found in autoreactive Abs with known antigen specificity for example anti-rheumatoid factor and anti-cardiolipin Abs60. CLL Abs have been reported as polyreactive and autoreactive, reacting with two or more of the following: the Fc part of IgG, ssDNA, dsDNA, histones, cardiolipin, myoglobin and cytoskeletal components78-81. However, some monospecific CLL Abs have also been observed, reacting with only one of the above mentioned antigens78. A later study on recombinant CLL Abs also concluded that CLL Abs were auto- and polyreactive, adding insulin and LPS to the list of antigens82. The same study also showed this polyreactivity to be found mainly in UM-CLL, although M-CLL displayed a higher polyreactivity compared to normal naïve B cells82. Superantigens could also play a role, especially in the IGHV3 CLL subgroup of patients since IG encoded by the IGHV3 gene is known to bind the superantigen staphylococcal protein A (SpA) by regions outside of the antigen binding site (FR1, FR3, CDR2)83. The antigen specificity of CLL cells recently described by us in paper I and by others will be further discussed in the RESULTS AND DISCUSSION section.
16 | BACKGROUND
POLYNEUROPATHY ASSOCIATED WITH MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE
Monoclonal gammopathy of undetermined significance
Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant monoclonal B cell expansion with associated monoclonal immunoglobulin production (serum M-component) found in 3.2% of individuals over the age of 5084. The incidence of MGUS increases with age and can after 70 years of age be found in 5.3% of the population, and in 7.5%-10% of 85-year olds84,85. MGUS occurs more frequently in men than in women84 and MGUS incidence is higher in African-Americans than Caucasians which might suggest a genetic predisposition86. MGUS is diagnosed by a concentration of serum paraprotein <3 g/dl, <10% plasma cells in the bone marrow and absence of symptoms due to a malignant B cell proliferation e.g. lytic bone lesions, anemia, hypocalcaemia or renal failure87,88. It is a relatively stable condition in most patients, however the term “undetermined significance” included in the name is due to the fact that 11% of MGUS cases progress to malignant disorders within 25 years follow-up89. Approximately 70% of MGUS cases are IgG, 17% IgM, 11% IgA and 2% are biclonal84,89. Depending on the immunoglobulin isotype used by the MGUS clone, progression to different malignant B cell disorders is seen. IgG and IgA MGUS develops into multiple myeloma at a rate of 1% per year90. IgM MGUS on the other hand mainly gives rise to Waldenströms macroglobulinemia and lymphoma at a rate of 1.5% per year90-92. There seem to be geographic differences in IgM MGUS with higher prevalence in France93 and the US84 compared to Asia, Ghana94, Sweden and the Mediterranean region95.
Polyneuropathy associated with monoclonal gammopathy
In polyneuropathy, there is a systemic destruction of peripheral nerves, primarily affecting the myelin or the axons. There are many mechanisms leading to polyneuropathy, including nutritional, toxic and metabolic. Immune-mediated mechanisms constitute a large group of polyneuropathies, of which one is linked to the occurrence of MGUS. Although polyneuropathy has been reported in a few cases of IgG and IgA MGUS96-99, it is mainly observed in IgM MGUS where it can be seen in approximately half of the patients99-101. The neuropathy is often of a symmetrical slowly progressive sensory or sensory/motor demyelinating character and is considered to be immune-mediated although the exact mechanisms remain to be identified100,102. Monoclonal antibodies (mAb) produced by the expanding clone have been suggested to have a direct pathogenic role in the neuropathy but the involvement of T cells has also
| 17 BACKGROUND
been implicated. Both of these pathogenic entities will be discussed further in the following sections.
Role of B cells and antibodies
The monoclonal IgM Abs produced by the expanding B cell clone in polyneuropathy associated with MGUS (PN-MGUS) have been shown to bind peripheral nerve myelin components expressing the HNK-1/CD57 trisaccharide epitope103,104. Myelin protein zero (P0), one of the components frequently targeted, is a 28kDa glycoprotein present on the membrane of the myelin-producing peripheral-nerve Schwann cells105. P0 is an adhesion protein that plays an important role in the compaction of peripheral nerve myelin. Other molecules expressing the HNK-1 epitope include myelin-associated glycoprotein (MAG)106- 110, gangliosides111,112 and sulphate-3-glucoronyl paragloboside (SGPG)113,114. A directly pathogenic role of the anti-myelin Abs found in PN-MGUS patients have been suggested since they are found deposited at sites of peripheral nerve demyelination115-117. Intraneural injection with patient anti-MAG Abs in feline sciatic nerve have also been shown to cause demyelination118 and chickens injected with anti-MAG IgM Abs develop demyelination119. Anti-peripheral nerve myelin Abs can however also be found in healthy blood donors120.
Role of T cells
In addition to IgM Abs, infiltrating T cells can be found at sites of nerve lesions121,122. This observation has lead to the hypothesis that T cells might start an inflammatory reaction which leads to the breakdown of the peripheral-nerve myelin and the subsequent nerve degeneration. Supporting this theory is the finding that both CD4+ and CD8+ circulating T cells have an activated phenotype in PN-MGUS patients123. Increased levels of soluble IL-2 receptors, a sign of T cell activation, are also found124. Peripheral myelin protein peptides induce a TH1-like response with IFN-γ secretion in PN-MGUS patient peripheral blood mononuclear cells (PBMCs) in vitro104. A genetic component predisposing certain individuals to PN-MGUS also seems to exist, as an association between human leukocyte antigen (HLA)-DR haplotypes carrying a non-polar tryptophan residue at position 9 in the DRβ chain and PN-MGUS can be seen125. T-cell regulation of anti-MAG producing B cells has been observed in vitro126 but not much was known about the antigen-presenting capacity of clonal B cells in PN-MGUS. It was therefore of interest to study the myelin specific B cell clone in PN-MGUS to see if the cells could bind, process and present myelin and P0 in MHCII molecules and subsequently activate specific T cells (Paper IV).
18 | BACKGROUND
Autoimmunity and B cells
The ability of the immune system to recognize and discern self from non-self is imperative127. Healthy self tissues or antigens should be tolerated while non-self agents e.g. bacteria and viruses and damaged or apoptotic cells, should be eliminated. This is not an easy task since many pathogens try to evade immune system detection by mimicking self-tissues in the body. During the development of B and T cells, strongly auto-reactive cells are deleted in a process called clonal deletion, while weakly autoreactive cells receive survival signals (Figure 3)13. These weakly autoreactive B and T cells are strictly regulated in the periphery. They are part of the normal natural immunity and do not necessarily cause autoimmune disease with breakdown of self-tissues. Instead, the natural antibodies, which are usually polyreactive and of IgM isotype, have been suggested to make up the first-line of defense against invading pathogens, enabling a quick response until a specific immune response develop17. It is apparent that the regulation of auto-reactive cells can fail in some situations, leading to the development of autoimmune disease13,128. Autoimmune diseases are generally not believed to be caused by a single factor. Instead they are viewed as multifaceted conditions with several factors and molecular pathways known to take part in the autoimmune responses. Certain HLA alleles are known to predispose to autoimmunity129. Self-proteins altered by mutations, misfolding, degree of expression, posttranslational modification130,131 or oxidation132 could represent neo-antigens and be perceived as foreign. Examples of this include citrullination of arginine in vimentin133 and oxidation of type II collagen134 which both have been implicated as autoantigens in rheumatoid arthritis. Defects in the clearance of apoptotic cells and changes in the expression levels or activity of regulatory proteins have also been shown to be involved in autoimmunity. Exactly how these factors and other possible mechanisms, contribute to the development of autoimmune disease is still not clear, but several models for the initial trigger have been proposed.
Molecular mimicry model
One model often used to explain the starting point of autoimmunity is the molecular mimicry model128,135. In this model, autoimmunity is preceded by a microbial infection that induces a normal immune response with generation of antibodies and/or activation of T cells. However, due to structural similarity of pathogen to epitopes on self-tissues these antibodies and T cells in addition to clearing the infection could (under certain conditions yet to be defined) also start attacking self-tissues, leading e.g. in the case of PN-MGUS to the breakdown of peripheral nerve myelin. This initial cross-reactive Ab immune response could also, after antigen degradation, lead to exposure of other parts of the same self- protein in MHC molecules and subsequent development of new auto-antibodies or
| 19 BACKGROUND
activation of autoreactive T cells, a process known as epitope-spreading136,137. Interestingly a recent study showed an increased risk of developing MGUS after respiratory infections138. Cross reactivity of anti-MAG Abs with bacteria has also been noted139. Molecular mimicry between microbes and self-tissues has been suggested to be involved in several autoimmune diseases such as systemic lupus erythematosus (SLE)137 and Guillain-Barré syndrome (GBS)140. Molecular mimicry was recently demonstrated in a local form of glomerulonephritis associated with anti-LAMP2 Abs that display cross- reactivity with bacterial adhesin FimH and neutrophil cytoplasma141.
20 | AIMS OF THE THESIS
The overall aim of this thesis was to assess B cell interaction with antigen in the two B cell expansions CLL and PN-MGUS. The more specific aims for each paper were as follows:
Paper I To characterize the antigen-binding specificity of CLL antibodies from Epstein-Barr virus (EBV)-transformed cell lines and ex vivo patient cell cultures
Paper II To classify EBV-transformed CLL and normal lymphoblastoid cell lines with regard to phenotype, genotype and authentic neoplastic origin.
Paper III To further investigate the antigen binding specificity of CLL antibodies belonging to HCDR3 homology subset-2 by analyzing their binding pattern to human gastric mucosa tissue sections, with various degrees of inflammation and Helicobacter pylori (H. pylori) infection status.
Paper IV To establish the antigen processing- and presenting capacity of a myelin protein-zero-specific B cell line established from a PN-MGUS patient.
| 21
22 | MATERIALS AND METHODS
CLL CELL LINES AND PATIENT MATERIAL (PAPER I & II)
Seventeen EBV transformed cell lines established from seven CLL patients were used in paper I and II142-149. These cell lines were either previously established in our laboratory or collected from other laboratories. The names of the CLL cell lines are I83-E95 (CLL), I83- LCL (LCL), CII (CLL), CI (LCL), WaC3CD5+ (CLL), Wa-osel (LCL), 232B4 (CLL), 232A4 (LCL), PGA1 (CLL), PG-EBV (LCL), AI-60 (CLL), AII (LCL), AIII (LCL), CLL-HG3 (CLL), EHEB (CLL), MEC-1 (CLL) and MEC-2 (CLL). Eight of these cell lines were analyzed for their antigen specificity in paper I and all of the seventeen CLL cell lines were characterized for authentic CLL origin in paper II, i.e. based on their molecular profile compared to the respective patient malignant cells and the CLL population as a whole. The antigen specificity of 23 primary CLL cultures established from CLL patients attending the Hematology Clinic of Linköping University Hospital were also analyzed in paper I. Detailed information on the CLL cell lines and clinical characteristics of the CLL patients can be found in paper I and II.
RECOMBINANT CLL ANTIBODY (PAPER I & III)
A recombinant mAb (rVH3-21) was established from a CLL patient (case 7 in Tobin et al.200361) expressing stereotyped BCRs belonging to HCDR3 homology subset-2 (IGHV3- 21/IGHJ6/very short IGHD region) characterized by a short 9 amino acid HCDR3 amino acid sequence ARDANGMDV and the utilization of IGLV3-21/IGLJ3 light chain genes and LCDR3 sequence QVWDGSSDHP. The establishment of this recombinant Ab was described in paper I and the antigen specificity was analyzed in paper I and III.
TEST SUBJECT MATERIAL (PAPER III)
The ability of CLL Ab, rVH3-21, to bind antigen present in gastric mucosa corpus tissue sections from 29 volunteers attending a gastroduodenoscopy as part of a population survey in the community of Linköping was analyzed in paper III150. The test subjects were both H. pylori+ and H. pylori- with non-atrophic- and atrophic gastritis with varying degrees of inflammation. Control subjects without gastritis were also tested. More information on test subject characteristics can be seen in paper III. The IG specificity of a
| 23 MATERIALS AND METHODS
primary CLL culture established from a CLL patient (ID-23) belonging to HCDR3 homology subset-2 was also investigated.
PN-MGUS CELL LINE AND PATIENT MATERIAL (PAPER IV)
An EBV transformed B cell line, TJ2, established in our laboratory from a PN-MGUS patient with chronic progressive sensory-motor polyneuropathy attending the Neurology Clinic of Linköping University Hospital was used in paper IV together with PBMCs from the same patient151. Molecular characteristics of the TJ2 cell line can be found in paper IV.
The following section includes an overview of the methods used in this thesis. The methods are described in detail in paper I, II, III and IV.
IG GENE SEQUENCING
Sequencing of the rearranged IGHV and IGKV/IGLV genes was performed in paper I, II and IV to characterize the cell lines and patient CLL cells. It was also used to verify an authentic neoplastic origin of the CLL cell lines (I and II).
Principle of method: The IGHV gene mutational status is a prognostic factor in CLL3. Genomic DNA or cDNA is extracted from the cells and used to amplify the rearranged IGHV and IGKV/IGLV genes in a PCR with gene specific primer pairs. The resulting PCR products are separated by gel electrophoresis and positive products are then sequenced using a DNA sequencer. The acquired IGHV gene sequence is submitted and aligned against germline gene sequences in the IMGT database (http:/imgt.cines.fr)152.
IMMUNOHISTOCHEMISTRY
Immunohistochemistry was used for screening CLL Ab specificity against paraffin embedded human tissue micro arrays in paper I. The technique was used for further investigation of subset-2 CLL Ab specificity towards paraffin embedded tissue sections of human gastric mucosa in paper III.
Principle of method: Immunohistochemistry is a method to localize specific molecules, in this case proteins, in tissue sections153,154. Formalin-fixed or frozen tissue sections are incubated with an antibody against the protein of interest. An enzyme conjugated
24 | MATERIALS AND METHODS
secondary Ab is then applied in combination with a chromophore to stain the protein of interest. Commonly used enzyme conjugates include horseradish peroxidase (HRP) and alkaline phosphatase (ALP). Diaminobenzidine (DAB) and 3-amino-9-ethyl cabazole (AEC) are examples of chromophores. DAB results in a brown stain and AEC a red stain. The tissue section, commonly counterstained with hematoxylin which gives a blue color to the cell nucleus, is examined in a bright field microscope.
IMMUNOFLUORESCENCE AND CONFOCAL MICROSCOPY
Immunofluorescence was used in paper I for investigating the antigen specificity of CLL Abs in more detail and in paper IV to examine the co-localization of P0 protein or peptides with surface IgM (sIgM) or MHCII respectively on TJ2 cells. Vimentin expression on the surface of CLL cells was also investigated with immunofluorescence in paper I. The presence of P0 antigen in the endosomal compartment of TJ2 cells was studied with immunofluorescence and confocal microscopy in paper IV.
Principle of method: Immunofluorescence is used to detect specific molecules on the surface of or inside cells and tissues153. Tissue sections or cells are stained for a protein of interest using a primary Ab aimed at the investigated protein and a fluorescent dye labeled secondary Ab specific for the primary Ab. When the fluorescent dye is excited by light of one wavelength it will emit light of a different wavelength, causing the positive sections of the tissue or cell to light up. This light can be detected in a fluorescence microscope. Co-localization of two specific proteins can be investigated by using Abs conjugated with different fluorescent dyes, for example Alexa 594 (red) and Alexa 488 (green). Co-localized proteins will in this example appear as yellow areas of light.
Principle of method: Confocal microscopy generates an image with higher resolution compared to regular immunofluorescent microscopy155. A laser beam scans the sample with point illumination, only allowing light emitted from a certain focal plane to pass through a pin-hole and to be detected. In this way, light emitted from out-of-focus planes of the sample is eliminated. Confocal microscopy enables the user to determine the location of labeled molecules (e.g. a protein) inside a cell and is often used to render three dimensional images of cells and tissues.
RECEPTOR CLUSTERING AND CO-LOCALIZATION
Receptor clustering was used in paper IV for investigating the association of protein P0 or P0 peptides to sIgM or MHCII respectively.
| 25 MATERIALS AND METHODS
Principle of method: Ab induced receptor clustering is used to investigate if two cell surface receptors or a cell surface receptor and its ligand are associated to each other156,157. Receptor clustering/capping is induced by Abs specific for the first receptor of interest. The second receptor/ligand is then stained using specific Abs and the degree of co-localization between the two receptors are evaluated microscopically.
WESTERN BLOT
Western blot was used in paper I and III to study the antigen specificity of CLL Abs in more detail. Reconfirmation of CLL antigen specificity was performed with western blot in paper I.
Principle of method: Western blot is a method used to detect proteins in a sample153. Proteins are separated by size using gel electrophoresis and transferred to a membrane. Primary Abs aimed at the protein of interest is used in combination with a secondary enzyme conjugated Ab and a substrate which generate a luminescent signal.
MASS SPECTROMETRY (MALDI-TOF MS, ESI-MS/MS)
Mass spectrometry was used to identify the CLL specific protein antigens in paper I and III.
Principle of method: Mass spectrometry (MS) is used to obtain information about a protein such as peptide masses, amino acid sequence, post translational modifications and identity158. One commonly used application when studying complex protein samples is separation by 1- or 2D gel electrophoresis. The protein of interest is excised from the gel and subjected to an in-gel enzyme digestion (usually by trypsin) to generate peptides that are analyzed by MS. Two commonly used ionization MS techniques are matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). In both of these techniques the peptides are dried and ionized. In MALDI the peptides are dissolved in a matrix which acts as a proton donor/captor when exposed to laser irradiation. In ESI the peptide ionization is achieved by evaporating the liquid of the sample in an electric field. The mass of the ionized peptides is then analyzed in a mass analyzer. In the time-of flight (TOF) mass analyzer, ionized peptides are accelerated in an electric field, allowed to fly through a vacuum tube and are separated according to their mass-to-charge ratio (m/z). The quadrupole mass analyzer also analyzes the m/z ratio of the peptides but in addition allows the selection of a particular peptide for sequencing in a collision chamber (MS/MS). The peptide masses can be used to identify proteins by searching in databases.
26 | MATERIALS AND METHODS
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
ELISA was used for IG isotype determination and quantification of the secreted IG from CLL cell line culture supernatants and primary CLL cultures in paper I, II and III. CLL Ab specificity was also investigated with ELISA in paper I.
Principle of method: ELISA is used for detection of an antigen or antibody in solution159-161. A microtiter plate is coated with antigen or a capture Ab. Samples are added to the plate and the Ab/antigen to be measured binds to the antigen/capture Ab on the bottom of the plate. An enzyme labeled detection Ab specific for the measured Ab/antigen is used in combination with a substrate to generate a signal (colored product, fluorescence, chemoluminescence) that can be measured in a plate reader.
ELISPOT
Elispot was used for measuring T cell activation, in the form of IFN-γ secretion after P0 peptide stimulation, in paper IV.
Principle of method: Elispot allows the detection of secreted substances from individual cells, and is often used to detect cytokines secreted from T cells162. Cytokine specific Abs are bound to the bottom of microtiter plate wells. A specific amount of cells is added to the well and sink down to the Ab coated bottom where the secreted cytokine is captured. The captured cytokine is detected by a secondary cytokine specific Ab conjugated with an enzyme such as HRP. A substrate, which is insoluble in contrast to the soluble substrates used in ELISA, is then added and the resulting enzymatic reaction gives rise to a circle/spot around each cell secreting the specific cytokine. The spots are then counted and the frequency of cells secreting the specific cytokine can be calculated.
FLOW CYTOMETRY
Flow cytometry was used for further characterization of the CLL Ab binding specificities to apoptotic Jurkat cells and for evaluating the presence of vimentin on serum starved macrophages in paper I and for phenotyping of the CLL cell lines in paper II. Activation of autologous T cells with IFN-γ production after stimulation with P0 incubated TJ2 cells was analyzed by flow cytometry in paper IV.
Principle of method: Flow cytometry is a method for evaluating cell surface markers, cytokine production and cell viability at a single cell level153. It can also be used for cell cycle analysis and cell sorting. The cells are stained with fluorescent dye conjugated Abs
| 27 MATERIALS AND METHODS
specific for the markers of interest and are subsequently passed one by one in front of a light source that excites the fluorescent dye on the cell. Light hitting the cell is also scattered and reveals information about the size (forward scatter) and granularity (side scatter) of the cell. The scattered and emitted light is detected by the flow cytometer and can be used to analyze cell populations expressing different cell surface markers or to sort out a specific cell population for further experiments. The method also enables detection of intracellular proteins by permeabilizing the cell membrane allowing Abs to bind intracellularly, while at the same time Abs against cell surface antigens are preserved. By adding brefeldin the proteins remain in the Golgi compartment.
FLOW CYTOMETRIC MULTIPLEX ARRAY
Multiplex arrays were used in paper I to investigate the CLL Ab specificity towards a panel of S. pneumoniae capsular polysaccharides and in paper IV to determine P0 antigen specific IL-2 secretion from T cells in a PN-MGUS patient.
Principle of method: Flow cytometry multiplex arrays can measure the presence of several cytokines or Ab reactivities at the same time in a sample159,163. Beads containing a combination of red and infrared fluorescent dyes are coated with capture Abs, antigen, oligonucleotides, receptors or peptides depending on the analyte of interest. Varying levels of fluorescent dye inside the micro beads makes it possible to differentiate between different bead sets. The bead sets are added to the sample and bind their respective molecule. A biotin conjugated detection Ab and a fluorochrome conjugated streptavidin are added followed by flow cytometric detection in a two-laser flow cytometer, which is designed for this particular purpose. When the bead passes through the flow cytometer the red and infrared fluorescent dyes are excited by one laser (for detection of specific bead sets) at the same time as the fluorochrome conjugated streptavidin is excited by a different laser (for detection of the bound analyte).
FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ANALYSIS
FISH was used for detecting chromosomal aberrations of the CLL cell lines in paper II.
Principle of method: FISH is used to detect and quantify chromosomal abnormalities such as deletions, trisomies or translocations in cell preparations153,164,165. Fluorochrome conjugated DNA probes specific for the chromosome region of interest are hybridized to interphase or metaphase chromosome preparations attached to microscope slides. Fluorsescent signal is evaluated and quantified in a microscope.
28 | MATERIALS AND METHODS
HIGH RESOLUTION SHORT TANDEM REPEAT (STR) ANALYSIS
STR analysis was used in paper II to verify true patient origin of the CLL cell lines and normal counterparts (LCL).
Principle of method: Short tandem repeats (STRs) are 2 to approximately 7 nucleotide long repetitive DNA sequences, e.g. (CATG)n in a particular locus166. Interallelic variation of the number of repeats (n), which is induced by slipped strand mispairing, results in large numbers of alleles of different sizes being present in the population. Therefore, a majority of individuals will be heterozygous in most of their STR loci. By analyzing the size of several STR loci simultaneously, a DNA profile unique to a certain individual can be obtained. The STRs are amplified by PCR and the product is separated by size using capillary- or gel electrophoresis. The fluorochrome conjugated primers used in the PCR reaction renders a fluorescently labeled amplified STR product that can be detected, separated from other STRs analyzed and compared to a size standard.
AFFINITY PURIFICATION OF PROTEINS AND MHC CLASS II BOUND PEPTIDES
Affinity purification was used for extracting CLL specific antigens from cell lysates in paper I & III and for concentrating CLL Abs in paper III. MHCII bound peptides were also extracted by affinity purification in paper IV.
Principle of method: Affinity purification uses the interaction between antibody and antigen or other biological interactions for binding and purifying a protein of interest167,168. The capture Ab is immobilized on a matrix and the sample containing the specific antigen is passed over the column. The capture Abs bind to the antigen while all other proteins are washed away. By lowering the pH, the antibody-antigen affinity is changed leading to the elution of the antigen.
HIGH RESOLUTION MHC CLASS I AND CLASS II GENOTYPING
Genotyping of MHC class I and class II was performed for the TJ2 cell line in paper IV in order to enable bioinformatic prediction of MHC specific protein P0 derived peptides.
| 29 MATERIALS AND METHODS
Principle of method: MHC typing is commonly used in the clinical setting for matching organ donors and recipients since the compatibility will influence the outcome of the transplant14,169,170. The MHCI and MHCII genes are amplified from DNA using commercially available PCR and high resolution MHC specific primer sets. The PCR product is separated by gel electrophoresis and identified.
30 | RESULTS AND DISCUSSION
IDENTIFICATION OF NEW ANTIGENS IN CLL (PAPER I)
In recent years there has been an extensive increase of knowledge about the IGHV gene repertoire in CLL cells. The identification of groups of patients using stereotyped BCRs57,60,62 as well as genotypic and phenotypic studies showing an antigen experienced profile of the CLL cells,36,37,73 lead to the theory that CLL cells are selected by their antigen binding properties at some point during the malignant transformation process. In paper I we asked ourselves the question: Which are the antigen(s) recognized by CLL cells? mAbs with various IGHV gene usage and HCDR3 homology subset belonging were isolated from EBV transformed CLL cell lines or patient peripheral blood CLL cells and were found to bind proteins vimentin, cofilin-1, filamin B and proline rich acidic protein 1 (PRAP-1). Cardiolipin, oxidized low density lipoprotein (oxLDL) and S. pneumoniae capsular polysaccharides were other antigen targets found. Interestingly many of these antigens are found on apoptotic cells and bacteria (Figure 5)19,171-182. Binding to apoptotic Jurkat cells was also noted for some of the CLL mAbs. Of particular interest; two of the CLL mAbs tested belonged to HCDR3 homology subset-1 and bound to malondialdehyde modified LDL (MDA-LDL), a commonly used LDL oxidation model. An oligo- or polyreactive antigenbinding pattern was observed for most of the CLL Abs tested, and many also displayed cross-reactions between autoantigens and epitopes exposed on bacteria. The observed antigen recognition patterns, with preferential binding of autoantigens on apoptotic cells and encapsulated bacteria, is reminiscent of natural antibodies and indicate that CLL cells originate from a natural antibody producing B cell population.
IgM Wa/IGHV3-30.3UM/subset-32 vimentin I83/IGHV3-30M
p241/IGHV3-21UM Bacteria e.g. oxLDL p457/IGHV1-18UM/subset-1 IgM p497/IGHV1-2UM/subset-1 oxLDL Streptococcus Wa/IGHV3-30.3UM/subset-32 PC, cardiolipin Apoptotic HG3/IGHV1-2UM pneumoniae I83/IGHV3-30M cell PnC polysaccharides filamin B HG3/IGHV1-2UM ID-6/IGHV1-69UM PRAP-1 CII/IGHV1-69UM/subset-5 Wa/IGHV3-30.3UM/subset-32 ID-20/IGHV4-34M cofilin-1 232B4/IGHV3-48M (IgG) rVH3-21M/subset-2 IgG/IgM (monovalent rec. IgM)
Figure 5. Summary of the CLL Ab specificities found in paper I.
| 31 RESULTS AND DISCUSSION
CLL cell-of-origin
In both mice and human, although better defined in mice, natural antibodies are described as autoreactive unmutated polyspecific IgM Abs of low affinity17,20. They have been described to make up an important first line of defense against blood borne bacteria, especially encapsulated bacteria that display T cell independent antigens. Natural antibodies also have a role in the clearance of apoptotic cells. Looking at the antigen-specificity of CLL cells defined in paper I in addition to the hallmark expression of cell surface molecule CD5 on CLL cells it is tempting to draw parallels to the major natural antibody producing CD5+ B1-a cell population in mouse. CLL cells, at least the unmutated cases, have previously been suggested to originate from a CD5+ B1-a-like B cell population in humans. However, conclusive evidence for a B1-a B cell population in humans is lacking, and a B1-a B cell-like origin would not explain the mutated Ig genes found in CLL. The splenic MZ B cell compartment has also been implicated as the cell-of-origin. These cells express both mutated and unmutated IGHV genes, same as CLL cells11,26. MZ B cells produce IgM Abs that are weakly autoreactive and react rapidly to blood borne antigens as well as apoptotic cells24, very similar to the CLL antibody specificities found by us. MZ B cells also express memory surface marker CD2726 which was shown to be expressed on CLL cells36. One discrepancy between CLL cells and MZ B cells is the expression of CD5 for which MZ B cells are negative26. This could speak against a MZ origin of CLL cells. However, apart from being a B cell lineage marker, CD5 is also upregulated on activated B cells76,77. In addition one must not forget that CLL cells are cancer cells and that certain observations in gene expression and phenotypic profile could be a consequence of the malignant transformation and not associated to the cell-of-origin which complicates the picture further. Transitional B cells, which in humans express surface marker CD5183, is another potential precursor to CLL as was recently suggested in some cases of CLL with unmutated IGHV genes184.
Importance of the recognized antigen in CLL
The ability of CLL cells to bind apoptotic cells and oxidized epitopes have since the publication of paper I been reconfirmed by Catera et al. who showed that recombinant CLL Abs bound intracellular structures of live- and surface structures of apoptotic Jurkat cells as well as oxidatively generated epitopes185. Additional CLL specific antigens have also been identified. The cytoplasmic structures showed by Herve et al.82 to be recognized by CLL HCDR3 subset-6 CLL (IGHV1-69/IGHD3-16/IGHJ3 with restricted IGHV3- 20 usage) was later characterized as non-muscle myosin heavy chain IIA186. Interestingly, non-muscle myosin heavy chain IIA is also exposed on apoptotic cells just like many of the cytoskeletal CLL specific protein antigens found in our study.
32 | RESULTS AND DISCUSSION
The existence of stereotyped receptors in CLL indicates that there is an antigen selection occurring at some point in the malignant development of at least some CLL cases. It is also possible that this antigen continue to drive the proliferation and progression of the leukemic clone. Infectious microbes have been implicated in the pathogenesis of other leukemias and lymphomas for example H. pylori and its association to gastric mucosal- associated lymphoid tissue (MALT) lymphoma187. Interestingly, Landgren et al. recently showed that repeated episodes of pneumonia were associated with an increased risk of developing CLL188. Capsular bacteria S. pneumoniae and H. influenzae are major causes of pneumonia. This combined with our results that some CLL Abs bind to S. pneumoniae capsular polysaccharides makes it tempting to speculate about an infectious background to at least some cases of CLL. That CLL cells react with microbial antigens has also been shown by Hatzi et al189. It is possible that an initial microbial infection with activation of the CLL progenitor cell could, due to molecular mimicry, lead to cross-reactivity to autoantigens. Alternatively, the initial bacterial infection leads to activation of a natural Ab producing B cell, perhaps a MZ B cell. For some reasons, yet to be identified, this B cell is allowed to continuously proliferate making it more susceptible to tumor transforming events. BCR binding to autoantigens naturally generated during apoptosis and oxidation could provide the CLL cell with chronic BCR stimulation and survival signals; perhaps even proliferation signals. Antigen stimulation of the BCR without the necessary co-stimulatory signals could however also render the B cell anergic. Indeed, studies of the BCR signaling capacity of CLL cells show that the BCR is unresponsive in approximately half of the CLL cases, and that this unresponsiveness is more common among CLL expressing mutated IGHV genes compared to unmutated IGHV genes190,191.
Although malignant, the CLL cells are dependent on survival signals from the in vivo microenvironment. CLL cells taken from the in vivo compartment and cultured in vitro rapidly undergo apoptosis. It is possible that antigen binding to the BCR and subsequent signaling could provide a survival and proliferation signal in at least the unmutated CLL cases with responsive BCRs. UM-CLL also show shorter telomere lengths compared to M- CLL indicating a higher proliferation rate in these patients192. Antigenic stimulation to increased proliferation could also be responsible for the worse prognosis associated with the unmutated BCRs38,39.
CHARACTERIZATION OF EBV TRANSFORMED CLL CELL LINES (PAPER II)
The establishment of EBV transformed CLL cell lines could be a useful tool to study molecular mechanisms behind the disease since it is very difficult to keep ex vivo CLL cells
| 33 RESULTS AND DISCUSSION
alive in culture for any extended period of time. However, CLL cells have proven quite resistant to EBV transformation often resulting in the outgrowth of a normal EBV transformed B cell instead of the malignant clone. This in combination with the information that 15-30% of all cell lines in use today are cross-contaminations193 motivated us to perform genetic and molecular characterization of the EBV transformed CLL cell lines used in paper I as well as additional CLL cell lines collected from our and other laboratories (n=10) in order to investigate the true malignant origin. Normal EBV transformed LCL cell lines established from the same patient as the CLL cell lines were available in some cases and also included in the study (n=6). A conclusively authentic neoplastic origin where the CLL cell line could be compared to patient blood samples was seen for CLL cell lines I83-E95, CLL-HG3, WaC3CD5+, 232B4, MEC-1 and MEC-2. For I83-E95, CLL-HG3, MEC-1 and MEC-1 identical IGHV gene rearrangement with patient in vivo CLL cells was shown. Two functional IGHV gene rearrangements were found in the WaC3CD5+ cell line, one of which was identical to the patient sample. The second, and dominant, IGHV rearrangement showed HCDR3 homology to CLL subset 32 which in itself does not conclusively prove a CLL origin since this subset contain very few patients and is only classified as a potential CLL homology subset. The presence of two IGHV rearrangements in the WaC3CD5+ cell line could however indicate a biclonal CLL in the patient, which has previously been reported in CLL. The Wa-osel cell line which was originally reported to be a normal LCL counterpart to WaC3CD5+ was shown to carry identical IGHV rearrangement to the patient in vivo CLL clone and therefore represented the neoplastic clone. A biclonal CLL was also indicated in the 232B4 patient in vivo CLL cells. The 232B4 CLL cell line showed identical IGHV gene rearrangements as one of these in vivo CLL clones and was thus considered of truly authentic CLL origin.
Strong molecular evidence supporting CLL neoplastic origin but no available patient blood samples for comparison was shown for cell lines CII and PGA1. For CLL cell line CII however, IGHV gene analysis revealed IGHV rearrangement with CLL subset-5 stereotyped HCDR3 sequence. The HCDR3 homology subsets observed in CLL patients are considered unique for this disease, and the CII cell line was therefore considered verified. The cell line also showed CD5 expression and trisomy 12, two markers that was reported in the original reference. The presumably normal LCL counterpart CI expressed identical IGHV gene rearrangements (subset-5) as the CII cell line, expressed surface marker CD5 and trisomy 12. This suggests that the CI cell line also represent the malignant CLL clone present in the patient. For CLL cell line PGA1 very little information is reported in the original reference and no patient material was available for comparison rendering it difficult to conclusively verify a true CLL origin of this cell line. The PGA1 cell line has retained trisomy 12 as was reported in the original reference, but have lost ring rearrangement of chromosome 15. Trisomy 12 is a common chromosomal aberration found in 15% of CLL patients but it is not exclusive for CLL. It does however suggest a CLL
34 | RESULTS AND DISCUSSION
origin for this cell line. For two of the cell lines tested, EHEB and AI-60, no conclusive evidence for true CLL origin could be found. The EHEB cell line had lost expression of CD5, no other CLL specific markers were found, and patient in vivo cell material was not available. AI-60 proved difficult to culture rendering it problematic to perform phenotypic and genotypic analysis and thus to verify a true CLL origin. IGHV gene analysis did not show HCDR3 identity to any of the CLL homology subsets identified up to date. STR analysis did show a common patient origin for AI-60 and the originally described normal LCLs AII and AIII. For LCL cell lines I83-LCL, 232A4 and PG/B95-8, a verified CLL patient origin with identical STR-pattern between LCL cell line and respective CLL cell line and a lack of CLL markers was observed indicating EBV transformation of a normal B cell.
FURTHER INVESTIGATION OF IGHV3-21 CLL SUBSET-2 ANTIGEN SPECIFICITY (PAPER III)
Our previous studies in paper I showed that the recombinant antibody, rVH3-21, with IGHV3-21/IGHD-/IGHJ6 rearrangement belonging to HCDR3 homology subset-2 bound to human gastric mucosa corpus glandular tissue. The aim of paper III was to further characterize this binding specificity and include another CLL HCDR3 homology subset-2 antibody, ID-23. The binding pattern of rVH3-21 was analyzed against a panel of gastric mucosa corpus tissue sections from test subjects without gastritis as well as test subjects with non-atrophic gastritis and atrophic gastritis with various degree of inflammation and H. pylori infection status. CLL antibody ID-23 was tested against three of the tissue sections from test subjects without gastritis, but did not show any specific binding to glandular tissue. Some binding to connective tissue was observed. The rVH3-21 antibody on the other hand bound to glands connective tissue in a majority of gastric mucosa tissue sections tested both H. pylori positive and negative, indicating that the antigen recognized is an autoantigen. Affinity purification revealed the rVH3-21 specific antigen as an 11.5kDa protein. The identity of this protein and whether it is present in the glands or connective tissue of the gastric mucosa remains to be determined. Selection by a common antigenic epitope has been suggested for CLL patients expressing stereotyped BCRs with homologous HCDR3 sequences and restricted light chain usage and in particular for the IGHV3-21 subgroup of patients. The ability of CLL cells and recombinant CLL Abs to recognize autoantigens and oxidized epitopes have been shown previously by us and others.
The different binding patterns of the two CLL Abs, both of which belong to HCDR3 homology subset-2, differ with regards to specificity to glandular tissue. This finding was somewhat surprising, since CLL Abs with homologous HCDR3 sequences presumably bind
| 35 RESULTS AND DISCUSSION
the same antigen. However, the two CLL subset-2 antibodies utilize different IGHD and IGHJ genes and have slightly different HCDR3 amino acid sequences which could explain the difference in antigen binding. In addition, the IGLV/IGKV gene usage of ID-23 was not available and a difference in the light chain antigen binding site could also contribute to the different antigen binding patterns observed.
From or results in paper I it appears that most CLL Abs are auto- and poly- or oligoreactive, but the rVH3-21 subset-2 Ab seem to be an exception. It did not react with apoptotic Jurkat cells or bacteria. A study by Herve et al. on a similar recombinant IGHV3- 21 subset-2 CLL Ab showed that this Ab was neither auto- nor polyreactive82. However, although subset-2 CLL appears to have a more restricted antigen binding pattern the results presented in Paper III suggest that this CLL subset is autoreactive, similar to what was shown by us in Paper I.
ABILITY OF MYELIN REACTIVE IGM MGUS B CELLS TO PRESENT ANTIGEN AND ACTIVATE T CELLS (PAPER IV)
That normal B cells are professional APCs is well established, but the knowledge of whether this antigen presenting and T cell activating capacity is retained in pre-malignant and malignant B cell expanding disorders is scarce. Activation of inflammatory mediating T cells has been suggested previously in IgM PN-MGUS104,121,123-125. In paper IV we investigated the ability of an EBV transformed P0 specific, IgM producing PN-MGUS B cell line to bind, process and present antigen in the form of myelin and P0. We also investigated if this B cell line could activate autologous T cells. We found that the PN- MGUS B cell line (TJ2) effectively bound both myelin and P0 to its BCR. The antigen-BCR complex was also endocytosed and myelin/P0 peptides were presented in MHCII molecules on the surface of TJ2 cells. Isolated naturally processed MHCII peptides induced IFN-γ secretion from patient autologous PBMCs but not from control PBMCs, suggesting the presence of previously activated P0-specific T cells in the PN-MGUS patient. P0 incubated TJ2 cells were also able to activate patient autologous T cells with resulting IFN-γ and IL-2 cytokine production. The T cell activation observed in our study supports the theory of T cell involvement in the pathogenesis of PN-MGUS.
Although considered to be immune mediated the exact mechanisms behind the observed peripheral nerve demyelination and degeneration in IgM PN-MGUS patients is still not clear.
36 | RESULTS AND DISCUSSION
Anti-myelin Abs
The myelin specific IgM mAbs produced by the expanding B cell clone are considered pathogenic and most likely contribute to the demyelination since deposits of mAbs are noted on sites of nerve degeneration116,117. It is possible that these mAbs bind to HNK-1 expressing proteins such as MAG and P0 in the myelin sheath and induce demyelination by activation of complement factors. Indeed, deposits of complement C1q, C3d, and C5 can also be seen at sites of demyelination194,195. Binding of mAbs to the damaged nerve myelin could also inhibit regeneration of the myelin sheath similarly to what has been shown for anti-cerebroside Abs in vitro196. It is however also important to note that anti- myelin Abs have been observed in healthy blood donors without clinical signs of neuropathy120 indicating that they are part of our normal immune system. The anti-myelin IgM Ab produced by the B cell clone used in our study expressed somatically mutated IGHV genes suggesting that the B cell has entered a germinal centre and gone through affinity maturation at some point during disease development. Whether the anti-myelin Abs found in healthy blood donors also express mutated IGHV genes is not known but in the case of unmutated IGHV genes, the lower affinity might explain why these Abs do not cause clinical symptoms of neuropathy.
IgM MGUS B cells as APCs and T helper cell activation
In paper IV we show that the myelin-specific B cell clone in an IgM MGUS patient with polyneuropathy can act as APCs and activate autologous T helper cells with resulting IFN- γ and IL-2 production. Production of IFN-γ indicate a TH1 response to P0 present in the investigated PN-MGUS patient which could contribute to the breakdown of peripheral nerve myelin similar to what was recently observed in the NOD-B72-2KO mouse model of autoimmune peripheral neuropathy197. IFN-γ is also a macrophage activating cytokine and it is possible that macrophages contribute to the nerve damage. Antigen presenting capacity was recently shown in neoplastic B cells with the malignant clone presenting tumor antigen to CD4+ T helper cells, presumably with anti-tumor effect as a result198. However, due to presentation of the same antigen by tolerogenic bone marrow-derived APCs this tumor-specific T cell response was rendered anergic. One should be open to a similar scenario occurring in PN-MGUS, however, the presence of both CD4+ and CD8+ T cells in the nerve lesions121 and the significantly higher levels of IFN-γ compared to IL-4 in PN-MGUS patients in response to P0 peptides speak against these T cells being anergic104.
| 37 RESULTS AND DISCUSSION
Initial trigger of B cell expansion
Exactly what the trigger is for the myelin reactive B cell expansion seen in PN-MGUS patients is unknown although there are many possible causes. Acquired cytogenetic changes or other tumor transforming events could lead to B cell expansions. Loss of T cell suppression or persistent antigen stimulation is other potential causes. Looking at all MGUS cases (IgG, IgA, IgM with or without PN) an association between Hepatitis C virus infection199 as well as immunosuppression after organ transplants is observed101. Molecular mimicry after an initial bacterial infection has been suggested in PN-MGUS after noting that anti-MAG Ab from several patients cross reacted with bacterial polypeptides139. P0 and MAG reactive mAb produced by the TJ2 B cell clone used in paper IV were previously investigated by us for their possible bacterial cross-binding properties against a panel of common bacterial antigens but no specific binding was noted151. The IGHV3-15 TJ2 Ab did however bind B cell superantigen SpA which is known to bind framework regions of IG belonging to the IGHV3 family. Recently, an association between prior infectious disorders particularly pneumonia and poliomyelitis and risk for development of MGUS and MM was revealed138. Another study also showed an association between mycobacterial infections and MGUS200.
38 | FUTURE ASPECTS
The antigen specificity of CLL cells presented in this thesis has added one piece to the CLL cell-of-origin-puzzle. The exact nature of the CLL progenitor still needs to be elucidated and remains one of the important questions in this field of research. The antigen specificity is not known for all CLL homology subsets, and further studies are needed in this area. For the CLL homology subsets with known antigen specificity, e g subset-1, the next step would be to perform functional studies to see whether the antigen can influence CLL cell survival and proliferation. It would also be interesting to investigate if CLL patients where the antigen is identified have a T cell population with the same antigen specificity as the malignant B cell clone. Further studies on T cells in PN-MGUS are also needed, in particular to see how the activated P0 specific T helper cells affect the P0 specific B cells with regards to stimulation and regulation. B cell receptor signaling and proliferation of the myelin specific B cell clone after antigen stimulation with P0 in PN- MGUS patients is another interesting area to study.
| 39
| 40 CONCLUSIONS
We have characterized the antigen specificity of CLL cells with diverse IGHV gene usage and IGHV gene mutational status, some expressing stereotyped BCRs. EBV transformed CLL cell lines were also scrutinized with regard to genotype and phenotype in order to investigate a true neoplastic origin. In PN-MGUS we explored the antigen presenting and T cell activating capacity of a P0 specific B cell line established from a PN-MGUS patient. The results of Paper I-IV are summarized below.
CLL Ab binding specificities found includes vimentin, cofilin-1, filamin B, PRAP-1, cardiolipin, oxLDL and S. pneumoniae capsular polysaccharides. Interestingly many of these antigens are found exposed on apoptotic cells and bacteria. The antigen binding specificities of CLL Abs found is similar to that of natural Abs, indicating that CLL cells arise from a B cell population with an important role in the removal of apoptotic cells and encapsulated bacteria. (Paper I)
Six of the EBV transformed CLL cell lines were found to be of truly authentic neoplastic origin (I83-E95, CLL-HG3, WaC3CD5+, 232B4, MEC-1 and MEC-2). Indications of a biclonal CLL was found in two of the cell lines (WaC3CD5+ and 232B4). For two of the cell lines, a strong support for CLL neoplastic origin was found (CII and PGA1), whereas no conclusive evidence could be found for two other cell lines (EHEB and AI-60). Two of the “normal” LCLs showed signs of neoplastic origin (Wa-osel and CI), pointing out the importance of verifying cell lines used in research. (Paper II)
A recombinant CLL Ab belonging to HCDR3 homology subset-2, was found to bind an 11.5kDa antigen present in either connective tissue or glands of human gastric mucosa. This binding specificity was present irrespective of H. pylori infection status of gastric mucosa test subject suggesting that the recognized structure is an autoantigen. (Paper III)
The autoreactive P0 specific monoclonal B cell line established from an IgM MGUS patient with polyneuropathy could act as an APC and trigger IL-2 and IFN-γ cytokine production from autologous P0 specific T cells. This study shows the importance of clonal B cells not only as antibody producers, but also as APCs and adds further evidence to the involvement of T cells in the pathogenesis of PN-MGUS. (Paper IV)
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| 42 ACKNOWLEDGEMENTS
I would like to express a warm gratitude to all the people who have made this thesis possible and who have encouraged and supported me during my PhD studies, especially the following people:
A special thank you to my supervisor Anders Rosén for giving me the opportunity to do my PhD studies in your lab. Thank you for your guidance, support and your great knowledge during these years. Your sixth sense for knowing the identity of the CLL antigens never stops surprising me. Thank you also for all the unexpected adventures and laughs during our conference trips and visits to other research groups, making them fun and memorable. Thank you!
Many thanks to my co-supervisor Jan Ernerudh for your creativity, expertise in immunology and neurology and for the input to the IgM MGUS project and especially for your support when I was stressed out and moral was low.
Richard Rosenquist Brandell for your immense knowledge in the field of CLL and for always taking time to answer my questions, quickly and straight to the point.
Maria Kvarnström, my ”original MGUS expert” who introduced me to the lab. If I ever need to pulverize more myelin I will give you and Ahmad a call
My closest co-worker and dear friend Anna Lanemo Myhrinder. Thank you for all the interesting discussions both about science and life and for always being there. Lab work was always fun with your self-composed songs and practical jokes. Not to mention our much deserved spa-weekends. I could not have finished this thesis without you!
Ann-Charlotte Bergh for being a good friend and for sharing chocolate elephants with me during our very late night flow cytometry lab. Thank you also for all the fun conference trips and student movie nights. I will beat you in guitar hero one day.
Anita Söderberg for your kindness, excellent laboratory skills, microscopy advice and wise views on various subjects.
Eva Bäckman for your expertise in the lab and valuable input to my research-projects during lab meetings.
Johan Akter Hossain for always having a kind word to everyone, for sharing funny stories from growing up in Bangladesh and for excellent dinners with just the perfect amount of chili.
| 43 ACKNOWLEDGEMENTS
Vivian Morad for very good and fun laboratory work. It is with great confidence I hand over “my” VH3-21 project to you.
Margaretha Lindroth for your sense of humor and jokes and also for the beautiful electron microscopy images of cells that decorate the cover of my thesis.
Chamilly Evaldsson my computer support. Although you are newest member of our group we have had many, many interesting discussions about CLL. Thank you solving my many problems with jumping letters and pictures while writing this thesis. Not to mention your crash course in thesis writing!
My student Ulrik Lindquist for your laboratory work on my project and for lots of fun in the lab.
All my co-authors for great collaboration; Helen Baxendale, Kenneth Nilsson, Per Hultman, Jon Jonasson, Eva Klein, Charlotte Dahle, Nicole Weit, Marco Herling. Sohvi Hörkkö for inviting us into your lab in Oulu and sharing your knowledge on OxLDL. Ekaterina Sidorova for the early work on CLL antigens and knowledge on cell lines, Magnus Vrethem for brining the clinical views into research, your extensive knowledge in neurology and for showing me how/where to get bovine nerves, and Kurt Borch for your enthusiasm about our project and always taking time to discuss science. I always leave our meetings full of inspiration.
The Uppsala crew: Fiona Murray, Lesley Sutton, Gerard Tobin, Larry Mansouri, Mattias Jansson, Meena Kanduri, Ingrid Thörn, Marie Sevov and Ola Söderberg for excellent laboratory skills and for all the fun times at CLL meetings.
Anette Molbaek for your laboratory work always performed perfect and with a smile.
Kerstin Willander and Anita Lönn for being the source of all practical knowledge in the lab and for your always cheerful mood.
Alexander Vener and Maria Turkina for excellent advice on mass spectrometry
The people at floor 12 in the Cell Biology building, Mats, Siri, Elisabeth and Tobias but also Kathryn Tonissen for excellent advise on recombinant proteins.
The people at AIR and the blood bank: Marie-Anne, Christina, Jenny, Petra, Karin, Jeanette for laboratory support and interpretation advice.
Everyone else at floor 13 in the Cell Biology building for making work a lot of fun and for nice company during coffee breaks: Björn “the second” , Richard, Hanna, Cilla, Anna G, Annelie, Ingemar, Thomas, Maria H, Maria W, Birgitta, Lorena, Lan, Alexey, Sophie, Anna-Lotta, Angelika och Martin.
44 | ACKNOWLEDGEMENTS
The administrate staff Monika Hardmark, Ingrid Nordh, Anette Wiklund and Viveca Axén for solving all my practical questions. A special thanks to Viveca for finding a room for my dissertation during university exam week.
Emelie and Elisavet, my two vampire maniac friends for the fun movie nights. My travel companions Elisabeth and Siri for all the travel-fun and pep-talks. Björn “the first” and Mia for being good friends, computer support, fashion advice and for making tasty cocktails.
My “old” friends Matilda, Anders, Elinor, Mattias, Robert and Leif for giving me a perspective on life outside of research. Thank you for all the fun pizza and poker nights, scuba diving, discussions over a latte, suggestions on where to publish my papers, board game weekends and much more.
Sist men inte minst vill jag tacka min familj: Min älskade mamma och pappa som alltid har ställt upp och stöttat mig och trott på det jag gör. Carina den bästa systern man kan ha, Håkan och solstrålarna Majken och Vera för att ni alltid får mig att le.
This work was kindly supported by grants from the Swedish Cancer Society and the Swedish Research Council.
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