Stereotyped B Cell Receptors in Chronic Lymphocytic Leukaemia

Transcription

1 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 405 Stereotyped B Cell Receptors in Chronic Lymphocytic Leukaemia Implications for Antigen Selection in Leukemogenesis FIONA MURRAY ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2008 ISSN ISBN urn:nbn:se:uu:diva-9438

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3 Arthur, you have no historical perspective. Science in those days worked in broad strokes. They got right to the point. Nowadays, it's all just molecule, molecule, molecule. Nothing ever happens big. The Tick (to his sidekick Mothman)

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5 List of Papers This thesis is based on the following papers, referred to in the text by their roman numerals; Paper I Gerard Tobin, Ulf Thunberg, Karin Karlsson, Fiona Murray, Anna Laurell, Kerstin Willander, Gunilla Enblad, Mats Merup, Juhani Vilpo, Gunnar Juliusson, Christer Sundström, Ola Söderberg, Göran Roos, Richard Rosenquist. Subsets with Restricted Immunoglobulin Gene Rearrangement Features Indicate a Role for Antigen Selection in the Development of Chronic Lymphocytic Leukemia. Blood 2004 Nov 1;104(9): Paper II Mia Thorsélius*, Alexander Kröber*, Fiona Murray, Ulf Thunberg, Gerard Tobin, Andreas Bühler, Dirk Kienle, Emilia Albesiano, Lan-Phuong Dao- Ung, James Wiley, Juhani Vilpo, Anna Laurell, Göran Roos, Karin Karlsson, Nicholas Chiorazzi, Roberto Marasca, Hartmut Döhner, Stephan Stilgenbauer, Richard Rosenquist. Strikingly Homologous Immunoglobulin Gene Rearrangements and Poor Outcome in V H 3-21-utilizing Chronic Lymphocytic Leukemia Independent of Geographical Origin and Mutational Status. Blood 2006 Apr 1;107(7): *MT and AK contributed equally to this work. Paper III Fiona Murray*, Nikos Darzentas*, Anastasia Hadzidimitriou 2 *, Gerard Tobin, Myriam Boudjograh, Cristina Scielzo, Nikolaos Laoutaris, Karin Karlsson, Fanny Baran-Marzsak, Athanasios Tsaftaris, Carol Moreno, Achilles Anagnostopoulos, Federico Caligaris-Cappio, Dominique Vaur, Christos Ouzounis, Chrysoula Belessi, Paolo Ghia, Fred Davi, Richard Rosenquist and Kostas Stamatopoulos. Stereotyped Patterns of Somatic Hypermutation in Subsets of Patients with Chronic Lymphocytic Leukaemia: Implications for the Role of Antigen Selection in Leukemogenesis. Blood 2008 Feb 1;111(3): *FM, ND and AH contributed equally to this work

6 Paper IV Anastasia Hadzidimitriou*, Nikos Darzentas*, Fiona Murray*, Tanja Smilevska, Eleni Arvaniti 4, Athanasios Tsaftaris, Nikolaos Laoutaris, Achilles Anagnostopoulos, Fred Davi, Paolo Ghia, Richard Rosenquist, Kostas Stamatopoulos, and Chrysoula Belessi. Evidence for the Significant Role of Immunoglobulin Light Chains in Antigen Recognition and Selection in Chronic Lymphocytic Leukaemia. Pre-published online. Blood 23 Oct 2008, doi: /blood *AH, ND and FM contributed equally to this work Reprints were made with permission from the publishers.

7 Contents INTRODUCTION The B cell immunoglobulin Structure of the immunoglobulin Organisation of the immunoglobulin loci B cell development and generation of antibody diversity Stem-cell to pro-b cell Pro-B cell to pre-b cell to mature B cell B cell interaction with antigen The germinal centre reaction Mechanisms of IG diversity and IG gene rearrangements as clonal markers IG gene usage in normal B cells Marginal zone B cells Chronic lymphocytic leukaemia Background Treatment options Prognostic markers Early evidence of antigen selection in CLL The origin of CLL Somatic hypermutation patterns in CLL The potential role of self-antigens, exogenous antigens and superantigens in CLL AIMS PATIENT MATERIAL AND METHODS Patient material PCR amplification and nucleotide sequence analysis Sequence analysis and data mining Statistical analysis RESULTS & DISCUSSION Characterisation of new CLL subsets (Paper I) Further characterisation of the IGHV3-21 subset (Paper II) Stereotyped subsets and clinical correlations Light chain gene usage in CLL... 47

8 Stereotyped patterns of somatic hypermutation in CLL (Paper III) Examination of the role of light chains in antigen recognition in CLL (Paper IV) What are the culprit antigens in CLL? CONCLUDING REMARKS APPENDIX I ACKNOWLEDGEMENTS REFERENCES... 65

9 ABBREVIATIONS AID APE BCR C CA CDR CD40L CLL CSR D FR FDC FM GC HC IDC IG IGH IGK IGL J LC KDE MZ MALT mir MMR NAL NHEJ N-regions ORF PCR Pro-B cell Pre-B cell P-segments R Activation induced cytidine deaminase Apurinic endonuclease B cell receptor Constant Cold agglutinin Complementarity determining region CD40 ligand Chronic lymphocytic leukaemia Class switch recombination Diversity Framework region Follicular dendritic cells Follicular mantel Germinal centre Heavy chain Interdigitating dendritic cells Immunoglobulin Immunoglobulin heavy chain Immunoglobulin kappa chain Immunoglobulin lambda chain Joining Light chain Kappa deleting element Marginal zone Mucosa associated lymphoid tissue Micro-RNA Mismatch repair N-acetyllactosamine Non-homologous end joining Nucleotide additions Open reading frame Polymerase chain reaction Progenitor B cell Precursor B cell Palindromic duplications Replacement

10 RAG 1&2 Recombination activating gene 1&2 RSS Recombination signal sequence S Silent SCT Stem cell transplantation SHM Somatic hypermutation SLE Systemic lupus erythematosus SpA Staphylococcus superantigen TdT Terminal deoxynucleotidyl transferase T H T helper UNG Uracil DNA glycosylase V Variable Kappa Lambda

11 INTRODUCTION Chronic lymphocytic leukaemia (CLL) is an accumulative disease of neoplastic CD5 + B cells that occurs predominantly in the elderly population. It is a heterogeneous disorder with respect to both its biologic and clinical features. Many patients are asymptomatic and follow a relatively benign disease course, whilst others have a rapidly fatal condition, despite prompt initiation of treatment. However, the exact biological reason(s) for the existence of these alternative prognoses has not yet been fully clarified. Thus, much effort has been invested in the identification of reliable and practical prognostic markers that can identify aggressive cases at an early stage of disease. To date, the mutation status of the immunoglobulin heavy variable (IGHV) gene rearrangements in leukaemic cells has been found to be one of the most reliable prognostic markers in CLL. Furthermore, many studies have focused on the structure of the IG gene rearrangements in CLL cells in an attempt to gain a greater understanding of the nature of the disease. Much evidence has been presented supporting the idea that CLL tumours carrying certain IG gene rearrangements may have recognised a common antigen, which possibly conferred a growth advantage to the clone by means of ongoing antigenic stimulation, at least in certain subsets of cases. In this thesis I will focus on the IG gene rearrangements of CLL cells and investigate the prognostic value of IG features in subgroups of CLL patients, with the aim of gaining information on how the IG structure might relate to the disease development at a biological level. To understand the significance of the B cell receptor (BCR) in CLL, it is necessary to know the basics of normal B cell development, the IG gene rearrangement process and how B cells interact with antigen. These topics will be outlined in the following sections. The B cell immunoglobulin All B cells carry multiple identical copies of IG on their cell surface. The IGs, together with accessory proteins, constitute the surface complexes known as BCRs, by which the cell recognises and binds foreign antigen. The B cell plays a crucial role in the adaptive immune response, its chief functions being antigen presentation and antibody production in order to eliminate foreign antigen 1. Prior to contact with antigen, a B cell with a functional BCR is described as a naïve B cell. The specificity of each IG is unique to 11

12 that B cell; when antigen is encountered to which the BCR adequately binds, affinity maturation of the IG occurs in specialised structures of the secondary lymphoid organs. This mature B cell can then differentiate into either an antibody producing plasma cell or a long-lived memory cell 2. However, this is not the only role of the BCR; far from being an inert molecule, it is also an active and dynamic signal transmitter. It is through the IG that the cell receives external signals which can induce it to proliferate, become anergic (nonresponsive to further antigen stimulation), edit its BCR or, under certain circumstances, undergo apoptosis. The outcome of antigen stimulation depends on multiple factors, such as the cells in the surrounding microenviroment, co-receptor interaction, and the type and concentration of antigen 3. Structure of the immunoglobulin Each IG molecule is composed of four polypeptide chains; two identical heavy chains (HCs) and two identical light chains (LCs), each consisting of a variable (V) and constant (C) region 4 (Figure 1). The V region of the HC IG of each B cell is generated by the joining of distinct variable (IGHV), diversity (IGHD) and joining (IGHJ) genes at the IGH locus and the V and J LC genes at the immunoglobulin kappa (IGK) and immunoglobulin lambda (IGL) loci 4. The V region is the part of the molecule that binds antigen, while the C region determines the isotype of the molecule and thus confers its effector function. The isotype of the IG can be altered via class switch recombination after antigen encounter (further described below) 5. Each V region is comprised of evolutionarily conserved framework regions (FRs) interspersed with hypervariable regions, known as complementarity determining regions (CDRs) 6,7. The FRs maintain the structural integrity of the IG molecule, while it is the CDRs which generate the huge diversity of the antigen binding pocket. In particular, the CDR3 is the most hypervariable region of the molecule and, unlike the CDR1 and 2 which are encoded by the IGHV gene, it is generated by the process of VDJ joining (described in detail below). Organisation of the immunoglobulin loci The IGH locus is encoded on chromosome 14, at band 14q32.33, very close to the telomere At the IGH locus, there are IGHV genes in total (depending on the haplotypes analysed), of which are functional genes 4,11. The 23 functional IGHD genes and 6 functional IGHJ genes are situated downstream of the IGHV genes. A series of 9 constant (IGHC) genes are also encoded in this region. A gene is described as functional if the coding region has an open reading frame without a stop codon. Besides functional genes, the IGH locus contains numerous pseudogenes, which are non- 12

13 functional due to detrimental point mutations or premature stop codons. Several genes have been found that are in frame, yet carry alterations which may affect the protein folding and have not yet been found to be transcribed. These genes are described as having an open reading frame (ORF) 4,11. Figure 1. Antibody structure and V(D)J rearrangement of the IG genes. The IGHV genes are divided into seven different homology subgroups with at least 80% homology within each group 4. The IGHV3 subgroup is the largest, consisting of 21 potentially functional genes, followed by the IGHV4 and IGHV1 subgroups which have 10 and 9 functional members, respectively. The remaining IGHV subgroups (IGHV2, IGHV5, IGHV6, IGHV7) are 13

14 much less frequently rearranged and comprise only 6 functional genes in total 4,11. Based on nucleotide sequence similarity, IGHV subgroups are in turn assigned into broader categories, known as clans. Clan I is comprised of IGHV1, IGHV5 and IGHV7 genes, clan II contains IGHV2, IGHV4 and IGHV6 genes, while clan III is made up of IGHV3 genes only 4. The LC can be one of two isotypes; a kappa ( ), or lambda ( ), although, in general, only one specificity will be expressed on the cell. This is known as isotype exclusion. IGK genes are located on the short arm of chromosome 2 at 2p11.2 and the locus spans 1800kb in total 12,13. The IGK locus is comprised of functional IG kappa variable (IGKV) genes, 5 IG kappa joining (IGKJ) genes and 1 IG kappa constant (IGKC) gene. IGKV genes belong to seven subgroups; IGKV1 (clan I), IGKV2, IGKV3, IGKV4, IGKV6 (clan II) and IGKV5 and IGKV7 (clan III). The IGKV6 and IGKV7 gene subgroups consist only of non-functional genes 4. The genomic organisation of the IGK genes is rather unique, in that all genes are organised into two cassettes, the proximal cassette lying immediately upstream of the IGKJ cluster, and the distal cassette which is separated from the proximal cassette by 800kb, and therefore situated furthest from the IGKJ cluster 14 (Figure 2). The distal cassette is in fact a duplication of the proximal cassette, yet it lies in an inverted orientation. Consequently, the IGKV genes of the distal cluster are almost mirror images of their counterpart genes located downstream and are denoted by the letter D in the gene name. In some cases, the genes from the proximal and distal cluster cannot be distinguished from each other in terms of nucleotide sequence and are accordingly described as a gene pair e.g. IGKV1-12/IGKV1D

15 Figure 2. The human IGK locus. From IMGT, the international ImMunoGeneTics information system, with kind permission from Marie-Paule Lefranc. Functional genes are represented in grey, ORF genes are white, pseudogenes are represented in black. Similarly, the IGL genes are encoded on the long arm of chromosome 22 at position 22q ,16. The locus consists of functional IG lambda variable (IGLV) genes, belonging to 10 functional subgroups (IGLV1-10), 4 functional IG lambda joining (IGLJ) genes and 4-5 functional IG lambda constant (IGLC) genes 4,11. In contrast to the IGH locus, there are no D segments at the IGK/L loci. Consequently, the degree of LC diversity is much more limited than that of the heavy chain. B cell development and generation of antibody diversity The extraordinary diversity of the human antibody repertoire is dependent upon a combinatorial association of IG gene segments. This process, known as V(D)J recombination, is initiated during the antigen-independent phase of B cell development in the bone marrow and is characterised by ordered gene rearrangements leading to the assembly of V, D (for heavy chains only) and J genes into a V(D)J gene complex 17,18. 15

16 Stem-cell to pro-b cell B cell development occurs via a stepwise process in the bone marrow 19,20. Progenitor B cells (pro-b cells) differentiate from lymphoid stem cells in response to stimulation from neighbouring cells in the bone marrow. Pro-B cells typically express CD43, CD19 and CD It is at this point of B cell development that rearrangement of the IGH locus begins. V(D)J recombination Pro-B cells begin IGH rearrangements by the joining of one IGHD gene to one IGHJ gene on the first IGH allele. If successful, this is followed by the joining of a IGHV gene to the IGHD-J rearranged complex to form the whole variable region of the IG molecule 18,22 (Figure 1). The process of VDJ recombination is mediated by the enzymes encoded by the recombination activating genes 1 (RAG 1) and RAG 2, which target recombination signal sequences (RSSs) flanking either side of each IGHV, IGHD and IGHJ gene 18,23. Each RSS consists of a conserved heptamer and nonamer separated by a non-conserved spacer of 12 or 23 nucleotides in length 24. RAG 1 and 2 introduce nicks into the DNA strand at the heptamer-rsss. All genes of a particular type, e.g. IGHVs, are flanked by RSSs with the same spacer length (Figure 3). However, only genes that are flanked by dissimilar spacer lengths can recombine with each other. This is known as the 12/23 rule and prevents IGHV and IGJV genes, which both have 23 nucleotide spacers, from rearranging with each other. Instead this mechanism allows for rearrangement of an IGHV gene to an IGHD gene, which are flanked by RSSs bearing spacers of dissimilar length 24. Similarly, IGK/LV genes are flanked by RSSs with 12 bp spacers, whereas all rearrangeable IGK/LJ genes are flanked by 23-bp RSSs and thus fulfil the 12/23 recombination rule. Once cleavage of the heptamer-rss junction at the IGH/K/L loci has occurred, the intervening DNA is excised, forming a circular strand of noncoding sequence, and the respective genes are joined, e.g. the IGHV gene is joined with the IGHD-J complex or the IGVK gene is joined with the IGJK gene (Figure 3). The repair of the double strand breaks introduced by the RAG enzymes is carried out by the non-homologous end joining proteins (NHEJ) Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PK and Artemis However, this process of joining is imprecise and can contain short deletions, due to exonuclease activity, palindromic duplications (P-segments) or nucleotide additions (N-regions), the latter introduced by terminal deoxynucleotidyl transferase (TdT) While exonuclease activity and introduction of N nucleotides into the junctional regions create higher diversity in the CDR3 of the IG molecule, these processes are not risk-free. Random introduction or deletion of nucleotides can shift the reading frame so that it no longer encodes for the correct amino acid sequence on translation of the 16

17 nucleotide sequence, thereby making it non-functional. Consequently, a functional V(D)J gene combination will usually only arise in 1 of 3 rearrangements. Once a successful VDJ recombination has occurred at the first locus, recombination is down-regulated, which prevents further recombination of the second IGH allele 32. This feedback mechanism promotes allelic exclusion, in order that only one of the IGH loci is expressed on each B cell 33. Figure 3. Representation of the cleavage of RSSs during VDJ recombination, and the process of nucleotide addition and deletion by TdT and exonuclease, respectively. Pro-B cell to pre-b cell to mature B cell Stromal cells in the bone marrow secrete cytokines and promote the maturation of pro-b cells into precursor B cells (pre B cells). This stage of maturation is marked by the loss of CD43 expression along with the expression of a heavy chain with a μ constant region first in the cytoplasm and, then, on the cell surface 34,35. The μ heavy chain is linked to the VpreB protein which in association with 5 (which has an IG C domain like structure) is known as the surrogate LC. The surrogate LC associates with the signal transduction molecules Ig and Ig, to form the pre-bcr 34,35. Signaling through this receptor complex prompts the pre B cell to undergo several rounds of proliferation 36. This proliferative burst is followed by arrest of the cell cycle and loss of expression of the surrogate LC. Rearrangement of one of the 17

18 IGK/IGL loci must occur in order for the pre B cell to become an immature B cell. IGK and IGL gene rearrangement The process of LC gene rearrangement is hierarchical and involves both allelic and isotypic exclusion. According to the ordered model of recombination, rearrangement of the IGKV and IGKJ gene segments will first occur on one IGK allele in an attempt to create a functional kappa chain 37,38. This level of allelic exclusion exists due to the fact that the recombinase machinery can only gain access to one allele at a time. However, in the case that the initial rearrangement produces a non-functional IGK gene rearrangement due to, for example, the introduction of a stop codon or loss of the reading frame, the second IGK allele will be rearranged in the next attempt to create a functional LC 39,40. Non-functional (or unacceptable/potentially dangerous see below) IGKV-IGKJ rearrangements can be deleted by means of two alternatives. Firstly, rearrangement of the kappa deleting element (KDE), which is located 3 to the IGKC gene, to an upstream IGKV gene segment can occur, thereby deleting the entire intervening region, i.e. the IGKC region, both kappa enhancers and an IGKV-IGKJ joint. The second alternative involves rearrangement of an RSS in the IGKJ-IGKC intron to the downstream KDE which results in deletion of the gene coding for the C region of the kappa chain (Figure 4). Both of these alternatives render the IGK rearrangement irreversibly non-productive since a complete kappa protein will not be produced 42. Although it is most frequently non-functional IGK rearrangements that undergo this process of deletion, functional IGKV-IGKJ joints are also deleted by this process 43. Only if creation of a functional rearrangement fails on both IGK alleles will rearrangement of the IGL locus proceed. 18

19 Figure 4. The mechanism of KDE rearrangement. The upper part of the diagram represents the IGK locus pre-rearrangement. The lower part of the diagram represents the two alternative products, post-rearrangement. During maturation in the bone marrow, B cells undergo a process of negative selection whereby those cells bearing BCRs with high affinity against selfantigens undergo apoptosis. However, these cells can be given a second chance and avoid this fate by continued RAG expression in the cell 44. The primary IG gene rearrangement can be modified so that it gains a different specificity, by undergoing a secondary LC rearrangement at the IGK or IGL locus. In some cases, this secondary rearrangement will cancel out the reactivity to self-antigenic epitopes and allow the cell to continue its development. This process of alteration of the specificity of immature BCRs is known as receptor editing In rare instances, this kind of editing to improve tolerance of the cell can result in the creation of cells that carry multiple receptors. This is known as allelic inclusion, or receptor dilution, where the original autoreactive specificity is diluted out by the new safe BCR 47,48. Once the cell carries an acceptable, functional LC gene rearrangement, the B cell ceases to express TdT or the RAG 1 and 2 enzymes and expresses a complete IG molecule along with accessory molecules on its surface 49. It is this structure that is known as the BCR. After maturation, B cells re-circulate through secondary lymphoid organs as part of the long lived pool as follicular mantle (FM) cells or join more static compartments at specific locations such as the marginal zone (MZ) of the spleen as MZ B cells. The characteristics of these cell groups differ in a number of ways and it appears that they play alternative roles in the immune response (further described below). 19

20 B cell interaction with antigen Foreign antigens enter the body by a number of ways, such as via the blood, the airways or the intestinal tract. The site of entry dictates which lymphoid tissue they will first encounter; the lymph nodes, spleen, mucosa-associated lymphoid tissue (MALT) or tonsilar tissue. Traditionally, the second, antigen-dependent, phase of B cell development is thought to begin when the naïve B cell exiting the bone marrow enters into the primary follicles of the secondary lymphoid organs where contact with, and selection by antigen takes place 50. When a mature FM B cell encounters antigen that it is specific for, and binds it with adequate affinity, it will undergo a process of affinity maturation, as previously mentioned. This process occurs in the germinal centre (GC) of lymph nodes and ultimately results in the production of B cells carrying BCRs with a considerably higher degree of affinity to their cognate antigen 50,51. Following antigen contact, IG genes are further modified by two distinct processes: the V region is diversified by somatic hypermutation (SHM) while the C region may be changed by class-switch recombination (CSR) 5. The germinal centre reaction As lymph filters through the lymph nodes, blood borne antigens are caught by the network of interdigitating dendritic cells (IDDs) and follicular dendritic cells (FDCs) which make up the primary lymphoid follicles of the lymph node (or other secondary lymphoid tissue) 50,51. The FDCs and IDDs present this trapped antigen to the B cells in the follicle. B cells that recognise antigens in the follicle are activated and begin to migrate out of the follicles towards the T cell zones. The initial interaction between B cells and T cells occurs at the interface of the follicle and the T cell zone. The activated B cells can then present antigen to CD4 + helper T (T H ) cells. If the T cell recognises the peptide presented by the B cell, it synthesises CD40 ligand (CD40L). Binding of CD40L to CD40 on the B cell surface causes activation of the B cell 52. This B cell-t cell interaction predominantly occurs in the extra-follicular areas of the lymph node. The activated B cell is now known as a centroblast and will migrate into the follicle 50. The antigen-activated follicle, described as a secondary follicle, is comprised of three zones; the follicular mantle zone, which is made up of the ring of B cells surrounding the GC, and the dark and light zones of the GC. In the dark zone, the centroblast rapidly divides (thus creating a dark, dense appearance) 53. IG expression of these cells is down-regulated and the process of SHM begins (described in detail below). SHM involves the random introduction of mutations to the rearranged IGHV gene at a rate of 1 mutation per 1000bp per generation 54, 10 6 times higher than spontaneous mutation 20

21 rate 55,56. As the cells enter the light zone, surface IG is up-regulated and the cells, now called centrocytes, become smaller. The light zone is a less dense region of the GC and is made up of these centrocytes, along with FDCs and T H cells. Within the light zone, the centrocytes are exposed to a range of antigens presented via the immune complexes on the surface of the FDCs 53. B cells with enhanced binding affinity for the initial stimulating antigen receive survival signals from T H cells and proliferate in the presence of the antigen.. Meanwhile, centrocytes that no longer bind the antigen, exhibit decreased affinity to their cognate antigen or recognise auto-antigens, die by apoptosis and are eliminated 57. The centrocyte will then differentiate into a re-circulating memory B cell or an IG secreting plasma cell 50 (Figure 5). Once selected, memory B cells no longer require surface immunoglobulin or antigen for continued long-term survival. Figure 5. The germinal centre reaction GCs have long been considered as the only sites capable of sustaining a high rate of SHM 50. However, it has been shown that it is possible that B cells can gain mutations outside of the GC reaction and independently of T cell help 52,58,59. As previously mentioned, lymphoid tissues are divided into follicular and extrafollicular areas. MZ B cells can be found in extrafollicular areas such as the MZ of the spleen, the subepithelial layer in the tonsils and the MALT. Some MZ B cells can also be found in small quantities in the lymph nodes just outside of the mantel zone 60. Splenic MZ cells and their functional equivalents, e.g. SE tonsilar cells, have been intensely studied 21

22 with regard to their ontogenesis, functional status and IG gene characteristics and will be described in more detail below. Somatic hypermutation SHM of IGV genes creates a second cycle of diversification after V(D)J recombination, which increases antibody diversity and produces antibodies with higher specificity 51. During this process, mainly base substitutions and occasionally insertions or deletions are introduced into a region of 1-2 kb surrounding the antibody-coding sequence. In normal B cells, replacement mutations are preferentially clustered within the CDRs rather than the FRs, which are enriched with certain hotspot motifs recognised by the enzyme activation induced cytidine deaminase (AID) These motifs have been defined as RYGW and WRCY (R=A/G Y=C/T W=A/T), or the more comprehensive DGYW/WRCH (D=A/G/T, H=T/C/A), where the mutation hotspot exists at the G or C residues (underlined) Two types of substitution mutations can occur in SHM; transition and transversion mutations. A transition mutation is change of a purine to another purine (e.g. A to G) or a pyrimidine to another pyrimidine (e.g. C to T); while a transversion mutation involves a change from a purine to a pyridimine or vice versa (C to G or T to A). During the process of SHM, AID deaminates the cytosine residues in singlestranded DNA resulting in a U-G mismatch 68. Uracils are not normally present in DNA, so when the DNA strand is replicated, the newly introduced uracil is recognised as a T and consequently two daughter species are created; one that remains unmutated and one that undergoes a C-T (transition) change. Alternatively, the uracil is excised by uracil-dna glycosylase (UNG), creating a site which lacks a nucleotide (an abasic site) (Figure6). By the base excision repair (BER) system, cleavage of the abasic site by apurinic endonuclease (APE) causes a break in the ribose phosphate backbone of the DNA sequence. This break then leads to normal DNA repair by error-prone DNA polymerases, which frequently introduce mutations at the position of the deaminated cytosine 69,70. Alternatively the MSH2/MSH6 heterodimer, excises base pairs surrounding the initially targeted C nucleotide. Subsequent replication over this abasic site by the DNA mismatch repair machinery (MMR) and error prone polymerases will result in random incorporation of any of the four nucleotides 71 (Figure 3). While the exact mechanism of SHM has not yet been completely clarified, the process is characterised by certain unique features: (1) the nature of mutations indicates a preference for transitions over transversions (at an approximate ratio of 60:40), with purines targeted more frequently than pyrimidines; (2) mutations are concentrated mainly in the CDRs and most often are single nucleotide substitutions rather than deletions or insertions; (3) certain 22

23 codons are targeted more often by the mutational process, while others are less likely to undergo changes and (4) a striking bias exists for G and C over A and T nucleotide mutations 70,72. Figure 6. The mechanism of SHM. Class switch recombination The IGH locus consists of an ordered array of five C (IGHC) genes: mu, delta, gamma, epsilon and alpha. Class switch recombination (CSR) replaces the IGHC gene to be expressed from mu to gamma, epsilon or alpha, resulting in switching of antibody isotype from IgM to IgG, IgE, or IgA, respectively, without changing antigen specificity. This process also involves AID enzymatic activity and occurs by the joining of two switch regions and simultaneous excision of the intervening loop of IGHC regions 73. The DNA sticky ends are then ligated by the NHEJ proteins which are also active during VDJ recombination 74. The isotype of an antibody determines the manner 23

24 in which captured antigens are eliminated or the location where the IG is first encountered 73,75. For example, IgM is secreted in pentameric form and thus has 10 antigen binding sites, giving it a very high valency. This makes the molecule more efficient at binding antigens with many repeating epitopes, such as viral particles. However due to its large size, IgM does not diffuse well through membranes. Conversely, IgA is predominantly found in external secretions, such as saliva, since it has a monomeric form and is more easily secreted. CSR is induced in vivo by both T-dependent and T- independent antigens 76. In combination with antigen-dependent activation, cytokine-induced signalling provides specificity to CSR 77. Mechanisms of IG diversity and IG gene rearrangements as clonal markers The considerable number of functional IG germline genes, along with the mechanisms involved in IG diversification, generate a huge potential for variation in BCR structure. If one first considers the process of VDJ recombination, it creates the potential for 6348 (46 IGHV x 23 IGHD x 6 IGHJ) possible functional gene combinations on the HC alone. While the LC does not have the same potential for diversity due to the absence of D genes, 365 gene combinations (40 IGKV x 5 IGKJ + 33 IGLV x 5 IGLJ) are nevertheless possible. Thus, when considering both the HC and the LC there is a potential for 2 x 10 6 combinations in total. In addition to this, the introduction of somatic hypermutations and N nucleotide addition/exomuclease trimming at V(D)J junctions has been estimated to increase the potential for variation 1000 fold for both the IGH and IGK/L genes 7. Therefore, the chance of two unselected B cells carrying exactly the same BCR is approximately 1 in 2.3 x All cells that have passed the pre B stage of development will have undergone VDJ recombination and will carry a particular IGH gene rearrangement on one or both alleles. When a B cell undergoes malignant transformation and clonal proliferation, each daughter cell will carry exactly the same IG gene rearrangement. This makes IG gene rearrangements a very specific clonal marker of B cell tumours. Analysis of IG genes can also provide useful hints about the cell population from which the lymphoma or leukaemia first arose, since the IGHV mutation status can indicate if the cell has undergone the SHM process 78. (See The origin of CLL below). IG gene usage in normal B cells While the number of potential IGHV-D-J rearrangements is enormous, there does appear to be a natural over-representation of certain IGHV genes in the 24

25 repertoire of normal B cells. Analysis of peripheral blood cells by single cell PCR can give some idea about the frequency of IG gene usage since the selection of cells should be unbiased and representative of the population of cells in circulation at that time. One study by Brezinschek et al. demonstrated that certain IGHV gene subgroups are observed at a higher than expected frequency in the periphery (when considering the total number of IGHV genes per subgroup) 79. The IGHV3, IGHV1 and IGHV4 gene subgroups predominated both in the productive and non-productive repertoires. At individual gene level, just nine IGHV genes were expressed by over 50% of B cells. The IGHV3-23, IGHV3-30 and IGHV3-7 genes were the most frequently used IGHV3 genes, whereas the IGHV4-59, IGHV4-34 and IGHV4-39 were the most over-represented genes of the IGHV4 subgroup 79. The same group performed a similar analysis on IGKV and IGLV gene usage in peripheral blood B cells. The IGKV3-20 (A27), IGKV3-15 (L2), IGKV3-11 (L6), IGKV1-5 (L12a), IGKV2-30 (A17) and IGKV1-39/ID-39 (O12/O2) genes were preferentially used in the functional repertoire 80. There also appeared to be no preferential pairing between IGHV and particular IGKV genes 81. Analysis of lambda gene usage revealed that the IGLV2-14 (2A2), IGLV2-23 (2B2) and IGLV1-47 (1G) genes were predominant in both the productive and non-productive repertoires 82. The processe of V(D)J recombination occurs before exposure to antigen and thus creates the pre-immune repertoire. Exposure to auto-antigen or exogenous antigen then leads to processes such as SHM and receptor editing which create further IG diversity. Hence, the biases in gene usage reported in the aforementioned studies are most likely due not only to selection by antigen but also by inherent bias in the pre-immune repertoire due to genetic and epigenetic elements. Factors such as recombination efficiency due to RSS composition, RAG enzyme cleavage efficiency, gene location, and in the case of LC genes, transcriptional orientation may all affect the frequency at which certain genes are rearranged 80, Additionally, there is most likely evolutionary selection for genes that are effective against prominent pathogens possibly making them more efficient at rearrangement in the preimmune repertoire. These types of studies give a general idea of inherent biases with the IGH/K/L repertoires; however, it should be noted that they were performed on a sampling of B cells from only two donors. It is therefore questionable how representative these gene frequencies are on a larger scale, particularly in terms of potential racial differences in gene usage (due to shared genetic background) and age related biases in the repertoire. Apropos the latter, these studies were performed on relatively young individuals and therefore may not be representative of the IG repertoire in the elderly population. The Stevenson group aimed to identify alterations in the IGHV repertoire with age in normal, healthy individuals. They showed that while 25

26 the frequency of IGHV1-69 gene usage does not increase with age, there was a notable over-representation of IGHV4-34 expressing cells in elderly individuals 86,87. This illustrates that caution is warrented when interpreting IG gene frequencies obtained from just one age-group or ethnic background. Marginal zone B cells In the spleen, the marginal zone is located at the junction of the red and white pulp. It is populated by macrophages, dendritic cells and B cells. Splenic MZ and tonsilar subepithelial (MZ-like) B cells appear to be comprised of both naïve and memory B cells, in that some carry mutations in the IGHV genes while others are unmutated This population may be sustained by stimulation by T cell independent antigens, such as carbohydrate antigens on encapsulated bacteria or viruses 93,94. In fact, it has been demonstrated that in vitro, MZ cells are the only B cells capable of mounting T cell-independent responses 95,96. Much study has been focused on B1 and B2 cell populations in mice, which appear to be similar to human MZ cells and FM B cells, respectively 97. After encounter with foreign antigen, all mature B cell subsets are capable of generating plasma cells, although MZ and B1 cells are faster and more efficient 94. It appears that B1 cells (which are usually CD5 + ) 98,99 use a limited number of IGHV germline genes, generally carry less mutations and have restricted N region diversity and exonuclease activity compared to B2 cells. This kind of restriction in IGHV gene repertoire has also been reported in the human MZ compartment 100,101. B1 cells also appear to have a limited ability to undergo isotype switching and are therefore most often IgM secreting cells 102. These restrictions also imply that the ability of B1 cells to form a germinal centre reaction is limited 103. So, whilst re-circulating FM B cells are recruited into GCs and undergo affinity maturation, it is unknown to what extent MZ B cells can be recruited into GCs and interact with T cells. It may be that B1 and MZ B cells have evolved to provide first line responses against gut/peritoneum and blood borne antigens 94. The observed restriction in IG gene usage may allow for the rapid development of short term responses to a limited number of conserved antigens; thus creating a bridge between natural and adaptive immunity. It has also been shown that many MZ cells carry BCRs with autoreactive specificities, yet are allowed to persist in the B cell population due to their effective binding of certain common pathogens such as S. pneumoniae and filariae 104,105. Additionally, these cells appear to serve housekeeping functions in the removal of cell debris and apoptotic bodies, hence their autoreactive specificities 60,105. In contrast, mature re-circulating FM cells are a more diverse pool containing antigen 26

27 specific B cells that are recruited for long term T-dependent antigen responses and high affinity memory generation via SHM. Chronic lymphocytic leukaemia Background CLL is the most frequently occurring adult leukaemia, with approximately cases diagnosed annually in Sweden. Its incidence in men is twice that reported for women and, in general, CLL most frequently occurs in individuals over the age of 60 with a median age at diagnosis of years. The disease has also been found to be more frequent in certain geographic areas, particularly Western Europe and Northern America, and is much rarer in, for instance, Asia. Some of the more common sites of involvement are the bone marrow, lymph nodes, and spleen, however CLL is often first identified by routine blood count, its most characteristic feature being a lymphocytosis of higher than 5 x 10 9 /L 106. CLL arises due to a monoclonal expansion of B cells which express the CD5 molecule on the cell surface 107. This clonal population of cells also typically express CD19 and CD23 with reduced levels of IgM, IgD and CD79b, representing the phenotype typical of mature activated B lymphocytes 106. In clinical practice, immunophenotyping is frequently used as the primary basis for CLL diagnosis. It was originally thought that the clonal expansion is associated with increased cell survival due to defective apoptosis mechanisms. However, more recent studies have shown that the rate of proliferation in CLL could be quite high, and thus the clone most probably results due to a combination of both increased rate of cell proliferation and reduced apoptotic rates 108. As previously mentioned, a proportion of CLL patients will follow an aggressive course while the remaining patients have a relatively indolent disease. In order to estimate the clinical outcome of CLL patients, two staging methods were developed; the Rai and Binet staging systems. Both systems are based on clinical investigation of the degree of physical symptoms such lymphadenopathy, hepatosplenomegaly and cytopenias (anaemia and/or thrombocytopenia) 109,110. These systems continue to be routinely used in disease evaluation, however they do not accurately predict prognosis in early stage patients. 27

28 Treatment options Since many patients will follow an indolent disease course, a watch and wait approach is generally employed for CLL patients. Asymptomatic patients are not treated and if it does appear that there is disease progression of a previously indolent case, fludarabine in combination with cyclophosphamide is the first line treatment, with the aim of long-term remission 111,112. If however, the patient is older or unable to tolerate such a regimen, chlorambucil can be used, with the aim of keeping the patient symptom free 113. Certain monoclonal antibodies such as anti-cd20 (Rituximab) and anti-cd52 (Campath 1H/Alemtuzumab) have been incorporated with earlier treatment regimes and have improved response rates Rituximab, although ineffective as a single treatment, can be given in combination with fludarabine and/or cyclophosphamide 114,115,117. In general, stem cell transplantation (SCT) is reserved for younger patients with unfavourable risk factors. Currently, autologous SCT is rarely undertaken since it has not been shown to be curative, while allogeneic SCT may be a treatment option in young patients with poor prognostic markers 118,119. Non-myeloablative, or reducedintensity conditioning (RIC) allogeneic transplants, because of their gentler chemotherapy and radiation regimes, are associated with a lower risk of transplant-related mortality and minimal toxicity Prognostic markers The understanding of the disease pathogenesis of CLL is complicated by the fact that no single mutation or genomic aberration is present in all CLL cases. Since CLL is known to show a high degree of clinical heterogeneity between individual patients, it is of great importance to develop prognostic markers that are both reliable and practical. IGHV gene analysis IGHV gene analysis has proved to be instrumental in defining clinical subgroups in CLL. In 1999, two independent groups reported that the mutation status of the IGHV genes divided CLL into two clinical entities which carried markedly divergent prognoses 123,124. The IGHV gene mutation status distinguishes between these clinical subsets, where those with mutated genes have a much longer survival than those with unmutated genes (in the initial studies; 293 months median survival v s 95 months and unreached v s 108 months) 123,124. To define unmutated cases, a 2% mutation cut-off level (i.e. deviation from the germline) has become standard, where genes with <98% identity to the germline classified as mutated, and those with 98% germline identity considered as unmutated. This division has been shown to give the best discrimination between cases with good and poor outcome 125. The prognostic usage of the IGHV gene mutational status was subsequently verified 28

29 in numerous studies and is considered to be one of the strongest independent prognostic markers in CLL Genomic aberrations While there is no singular aberration found in all CLL, a number of genomic aberrations have been identified that are of prognostic value. One of these, the chromosome 13q14.3 deletion, which occurs in over 50% of cases, is considered to be a marker of a relatively indolent disease, if present as a single aberration 132,133. The deleted region has more recently been found to encode 2 micro-rna genes (mir-15a and mir-16-1), which were found to be deleted or down-regulated in CLL 134. These non-coding micro-rna genes reportedly target the BCL2 oncogene, giving a clear link to their pathogenic effect in CLL 126. Other common genomic alterations include deletion of chromosome 17p13 (within which the TP53 gene is located) and deletion of chromosome 11q22-23 (which harbors the ataxia telangiectasia mutated gene) Both of these genes are involved in apoptosis regulation pathways and their deletion in CLL cells is associated with resistance to chemotherapy and poor outcome 126,138. In addition, trisomy of chromosome 12 is associated with an intermediate outcome 126,139. Interestingly, one gene, which is involved in the pathogenesis of T cell pro-lymphocytic leukaemia, is also over-expressed in CLL 140,141. It is known as TCL-1 and is located at 14q32. Mice that over-express Tcl-1 in B cells, develop a lymphoma of CD5 + B cells that is very similar to CLL 142. It was therefore of much interest to determine if abnormalities of TCL-1 were to be found in CLL. However, to date, the reason for the over-expression of this gene has not been elucidated. CD38 CD38 is a transmembrane protein which upon antibody ligation, catalyses the conversion of NAD + to cadpr, causing Ca 2+ flux into the cell. It has been reported to augment signaling of B cell receptors and thereby regulate apoptosis 123, Furthermore, a relationship has been revealed between BCR cross-linking and CD38 expression. In cells that were found to be CD38 negative, there was minimal or no activation of the signal transduction pathway following surface IG cross-linking. Conversely, in CD38 positive cells, the signaling pathway was found to be active 148. CD38 expression has been of much interest in CLL, since it has been shown to have prognostic value and positivity was associated with disease progression or shorter survival in CLL 149,150. A number of studies then demonstrated that CD38 positive/cd38 negative subgroups correlated inversely with the IGVH mutation status; where low CD38 expression occurred more frequently in mutated cases, while high CD38 expression correlated with the presence of unmutated IGHV genes 123,129,146,151,152. Thus, CD38 was considered as a potential surrogate marker for IGHV gene mutational status. However, according to 29

30 other studies, this relationship has not appeared to be consistently strong and the best clinical cut-off level to define positivity is still under debate. Initially a 30% cut-off was proposed, however later studies suggested that lower cut-off margins between 5-20% could be employed 123,129,145,146,153. Furthermore, while CD38 expression does carry an independent prognostic value, it is evident that CD38 levels can change over time in some patients and may not therefore be an ideal prognostic marker in early stage disease 129,145,146,153. ZAP-70 Zap-70 (70-kDa zeta-associated protein) is an intracellular tyrosine kinase. ZAP-70 is normally expressed in T cells and natural killer cells and has a critical role in the initiation of T cell signalling. It is also expressed to some extent in normal B cells, particularly activated B cells In CLL, intracellular ZAP-70 expression has been found to correlate with IGHV gene mutation status, with high ZAP-70 levels being mostly observed in unmutated cases 158. It was earlier proposed that ZAP-70 could act as a surrogate marker for IGHV gene mutation status, since analysis by flow cytometric methods and\or RNA expression levels, would be easier to perform on a routine basis 159,160. However, a number of different methods (direct and indirect antibody assays) which were employed showed discordant results in up to a third of cases Moreover, as ZAP-70 can be expressed in normal activated B cells it hampers the use of normal B cells as negative control in flow cytometric analysis. In short, while it still has value as an independent marker, many issues regarding standardisation of both the protocols and techniques employed in ZAP-70 analysis remain to be resolved before it can universally used as a prognostic marker. Early evidence of antigen selection in CLL Intriguingly, it has consistently been observed that CLL is characterised by a particularly skewed usage of IGHV genes. Not only are certain genes, such as IGHV1-69, IGHV3-21, IGHV4-34, and IGHV3-7, over-represented in CLL, but also the combined usage of IGVH/IGHD/IGHJ genes is distinct from the normal B cell repertoire 124,128, It was initially observed that IGHV1-69-using CLL cases were predominantly unmutated, had particularly long heavy chain CDR3s (HCDR3) and displayed preferential rearrangement of certain IGHD and IGHJ genes 86,124,128, Moreover, on examination of HCDR3 characteristics, amino acid composition and charge, Fais et al. identified sets of BCRs with highly restricted HCDR3s 164. They proposed three prototypic BCRs using the IGHV1-69, IGHV3-7 and IGHV4-34 genes 164. The IGHV1-69 BCR was predominantly unmutated, used an IGHD3-3 gene and an IGHJ6 gene, encoding a long, tyrosine- rich highly acidic HCDR3. In contrast, the IGHV3-7 IG were mutated and associated with IGHD3 and IGHJ4, resulting in a shorter less acidic HCDR3 structure. The IGHV