stereotyped b cell receptors in chronic lymphocytic leukaemia173003/fulltext01.pdf · gene...

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 405

Stereotyped B Cell Receptors inChronic Lymphocytic Leukaemia

Implications for Antigen Selection in Leukemogenesis

FIONA MURRAY

ISSN 1651-6206ISBN 978-91-554-7367-9urn:nbn:se:uu:diva-9438

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“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)

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 Ju-

liusson, Christer Sundström, Ola Söderberg, Göran Roos, Richard Rosen-

quist. Subsets with Restricted Immunoglobulin Gene Rearrangement Fea-tures Indicate a Role for Antigen Selection in the Development of Chronic Lymphocytic Leukemia. Blood 2004 Nov 1;104(9):2879-85.

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 VH3-21-utilizing Chronic Lym-phocytic Leukemia Independent of Geographical Origin and Mutational Status. Blood 2006 Apr 1;107(7):2889-94

*MT and AK contributed equally to this work.

Paper III Fiona Murray*, Nikos Darzentas*, Anastasia Hadzidimitriou2*, 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 Ro-

senquist 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):1524-33

*FM, ND and AH contributed equally to this work

Paper IV Anastasia Hadzidimitriou*, Nikos Darzentas*, Fiona Murray*, Tanja Smi-

levska, Eleni Arvaniti4, Athanasios Tsaftaris, Nikolaos Laoutaris, Achilles

Anagnostopoulos, Fred Davi, Paolo Ghia, Richard Rosenquist, Kostas Sta-

matopoulos, 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:10.1182/blood-2008-07-166868

*AH, ND and FM contributed equally to this work

Reprints were made with permission from the publishers.

Contents

INTRODUCTION ........................................................................................ 11 The B cell immunoglobulin ...................................................................... 11

Structure of the immunoglobulin ......................................................... 12 Organisation of the immunoglobulin loci ............................................ 12

B cell development and generation of antibody diversity ........................ 15 Stem-cell to pro-B cell ......................................................................... 16 Pro-B cell to pre-B cell to mature B cell ............................................ 17

B cell interaction with antigen.................................................................. 20 The germinal centre reaction ............................................................... 20 Mechanisms of IG diversity and IG gene rearrangements as clonal

markers ................................................................................................ 24 IG gene usage in normal B cells .......................................................... 24 Marginal zone B cells .......................................................................... 26

Chronic lymphocytic leukaemia ............................................................... 27 Background .......................................................................................... 27 Treatment options ................................................................................ 28 Prognostic markers .............................................................................. 28 Early evidence of antigen selection in CLL ......................................... 30 The origin of CLL ................................................................................ 32 Somatic hypermutation patterns in CLL .............................................. 33 The potential role of self-antigens, exogenous antigens and

superantigens in CLL ........................................................................... 33

AIMS ............................................................................................................ 35

PATIENT MATERIAL AND METHODS .................................................. 37 Patient material ......................................................................................... 37 PCR amplification and nucleotide sequence analysis .............................. 37 Sequence analysis and data mining .......................................................... 39 Statistical analysis .................................................................................... 39

RESULTS & DISCUSSION ......................................................................... 41 Characterisation of new CLL subsets (Paper I) ........................................ 41 Further characterisation of the IGHV3-21 subset (Paper II) ......................... 43 Stereotyped subsets and clinical correlations ........................................... 46 Light chain gene usage in CLL ................................................................ 47

Stereotyped patterns of somatic hypermutation in CLL (Paper III) ......... 48 Examination of the role of light chains in antigen recognition in CLL

(Paper IV) ................................................................................................. 52 What are the culprit antigens in CLL? ..................................................... 55

CONCLUDING REMARKS ........................................................................ 57

APPENDIX I ................................................................................................ 59

ACKNOWLEDGEMENTS .......................................................................... 61

REFERENCES ............................................................................................. 65

ABBREVIATIONS

AID Activation induced cytidine deaminase

APE Apurinic endonuclease

BCR B cell receptor

C Constant

CA Cold agglutinin

CDR Complementarity determining region

CD40L CD40 ligand

CLL Chronic lymphocytic leukaemia

CSR Class switch recombination

D Diversity

FR Framework region

FDC Follicular dendritic cells

FM Follicular mantel

GC Germinal centre

HC Heavy chain

IDC Interdigitating dendritic cells

IG Immunoglobulin

IGH Immunoglobulin heavy chain

IGK Immunoglobulin kappa chain

IGL Immunoglobulin lambda chain

J Joining

LC Light chain

KDE Kappa deleting element

MZ Marginal zone

MALT Mucosa associated lymphoid tissue

miR Micro-RNA

MMR Mismatch repair

NAL N-acetyllactosamine

NHEJ Non-homologous end joining

N-regions Nucleotide additions

ORF Open reading frame

PCR Polymerase chain reaction

Pro-B cell Progenitor B cell

Pre-B cell Precursor B cell

P-segments Palindromic duplications

R Replacement

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

TH T helper

UNG Uracil DNA glycosylase

V Variable

� Kappa

� Lambda

11

INTRODUCTION

Chronic lymphocytic leukaemia (CLL) is an accumulative disease of neop-

lastic 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 exis-

tence 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 ongo-

ing 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 inte-

ract 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 func-

tions being antigen presentation and antibody production in order to elimi-

nate foreign antigen1. 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

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 cell2. 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 rece-

ives external signals which can induce it to proliferate, become anergic (non-

responsive to further antigen stimulation), edit its BCR or, under certain

circumstances, undergo apoptosis. The outcome of antigen stimulation de-

pends on multiple factors, such as the cells in the surrounding microenviro-

ment, co-receptor interaction, and the type and concentration of antigen3.

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) region4 (Figure 1). The V region of the HC IG

of each B cell is generated by the joining of distinct variable (IGHV), diver-

sity (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) loci4. 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 telomere8-10. At the IGH locus, there are 123-129 IGHV genes in total

(depending on the haplotypes analysed), of which 38-46 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-

13

functional due to detrimental point mutations or premature stop codons. Sev-

eral 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 group4. The IGHV3 subgroup is the larg-

est, consisting of 21 potentially functional genes, followed by the IGHV4

and IGHV1 subgroups which have 10 and 9 functional members, respective-

ly. The remaining IGHV subgroups (IGHV2, IGHV5, IGHV6, IGHV7) are

14

much less frequently rearranged and comprise only 6 functional genes in

total4,11. 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 only4.

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 total12,13. The IGK locus is com-

prised of 31-35 functional IG kappa variable (IGKV) genes, 5 IG kappa join-

ing (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 sub-

groups consist only of non-functional genes4. The genomic organisation of

the IGK genes is rather unique, in that all genes are organised into two cas-

settes, 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 cluster14 (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 clus-

ter 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-12.

15

Figure 2. The human IGK locus. From IMGT®, the international ImMunoGeneTics

information system®, http://imgt.cines.fr, with kind permission from Marie-Paule

Lefranc. Functional genes are represented in grey, ORF genes are white, pseudo-

genes are represented in black.

Similarly, the IGL genes are encoded on the long arm of chromosome 22 at

position 22q11.215,16. The locus consists of 29-33 functional IG lambda vari-

able (IGLV) genes, belonging to 10 functional subgroups (IGLV1-10), 4

functional IG lambda joining (IGLJ) genes and 4-5 functional IG lambda

constant (IGLC) genes4,11. In contrast to the IGH locus, there are no D seg-

ments 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 complex17,18.

16

Stem-cell to pro-B cell

B cell development occurs via a stepwise process in the bone marrow19,20.

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 CD1021. 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 molecule18,22 (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

gene18,23. Each RSS consists of a conserved heptamer and nonamer separated

by a non-conserved spacer of 12 or 23 nucleotides in length24. 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 rear-

ranging 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 length24. 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 oc-

curred, the intervening DNA is excised, forming a circular strand of non-

coding 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 Artemis25-28.

However, this process of joining is imprecise and can contain short dele-

tions, due to exonuclease activity, palindromic duplications (P-segments) or

nucleotide additions (N-regions), the latter introduced by terminal deoxynuc-

leotidyl transferase (TdT)29-31. 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 intro-

duction 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

17

nucleotide sequence, thereby making it non-functional. Consequently, a

functional V(D)J gene combination will usually only arise in 1 of 3 rear-

rangements. Once a successful VDJ recombination has occurred at the first

locus, recombination is down-regulated, which prevents further recombina-

tion of the second IGH allele32. This feedback mechanism promotes allelic

exclusion, in order that only one of the IGH loci is expressed on each B

cell33.

Figure 3. Representation of the cleavage of RSSs during VDJ recombination, and

the process of nucleotide addition and deletion by TdT and exonuclease, respective-

ly.

Pro-B cell to pre-B cell to mature B cell

Stromal cells in the bone marrow secrete cytokines and promote the matura-

tion of pro-B cells into precursor B cells (pre B cells). This stage of matura-

tion 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 surface34,35. 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-BCR34,35. Signaling through this re-

ceptor complex prompts the pre B cell to undergo several rounds of prolife-

ration36. This proliferative burst is followed by arrest of the cell cycle and

loss of expression of the surrogate LC. Rearrangement of one of the

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 recombina-

tion, rearrangement of the IGKV and IGKJ gene segments will first occur on

one IGK allele in an attempt to create a functional kappa chain37,38. This

level of allelic exclusion exists due to the fact that the recombinase machi-

nery 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 LC39,40. 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 re-

gion, both kappa enhancers and an IGKV-IGKJ joint. The second alternative

involves rearrangement of an RSS in the IGKJ-IGKC intron to the down-

stream KDE which results in deletion of the gene coding for the C region of

the kappa chain39-41 (Figure 4). Both of these alternatives render the IGK

rearrangement irreversibly non-productive since a complete kappa protein

will not be produced42. Although it is most frequently non-functional IGK

rearrangements that undergo this process of deletion, functional IGKV-IGKJ

joints are also deleted by this process43. Only if creation of a functional rear-

rangement fails on both IGK alleles will rearrangement of the IGL locus

proceed.

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 self-

antigens undergo apoptosis. However, these cells can be given a second

chance and avoid this fate by continued RAG expression in the cell44. 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 reac-

tivity to self-antigenic epitopes and allow the cell to continue its develop-

ment. This process of alteration of the specificity of immature BCRs is

known as receptor editing44-46. In rare instances, this kind of editing to im-

prove tolerance of the cell can result in the creation of cells that carry mul-

tiple receptors. This is known as allelic inclusion, or receptor “dilution”,

where the original autoreactive specificity is diluted out by the new ‘safe’

BCR47,48.

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 surface49. 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 follicu-

lar 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 characteris-

tics 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).

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, anti-

gen-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 place50. 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 antigen50,51. Following antigen contact, IG genes are further mod-

ified 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 den-

dritic 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 recog-

nise antigens in the follicle are activated and begin to migrate out of the fol-

licles 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 (TH) 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 cell52. 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 follicle50.

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 appear-

ance)53. IG expression of these cells is down-regulated and the process of

SHM begins (described in detail below). SHM involves the random intro-

duction of mutations to the rearranged IGHV gene at a rate of 1 mutation per

1000bp per generation54, 106 times higher than spontaneous mutation

21

rate55,56. 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

TH cells. Within the light zone, the centrocytes are exposed to a range of

antigens presented via the immune complexes on the surface of the FDCs53.

B cells with enhanced binding affinity for the initial stimulating antigen re-

ceive survival signals from TH 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 eliminated57. The centrocyte will then differentiate into a

re-circulating memory B cell or an IG secreting plasma cell50 (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 SHM50. 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

help52,58,59. As previously mentioned, lymphoid tissues are divided into folli-

cular 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 zone60. Splenic MZ cells and their

functional equivalents, e.g. SE tonsilar cells, have been intensely studied

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 specificity51. 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)61-64. These motifs have been

defined as RYGW and WRCY (R=A/G Y=C/T W=A/T), or the more com-

prehensive DGYW/WRCH (D=A/G/T, H=T/C/A), where the mutation hots-

pot exists at the G or C residues (underlined)65-68. Two types of substitution

mutations can occur in SHM; transition and transversion mutations. A transi-

tion mutation is change of a purine to another purine (e.g. A to G) or a pyri-

midine 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 single-

stranded DNA resulting in a U-G mismatch68. 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 (transi-

tion) 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 back-

bone 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 cytosine69,70. Alternatively the MSH2/MSH6

heterodimer, excises base pairs surrounding the initially targeted C nucleo-

tide. Subsequent replication over this abasic site by the DNA mismatch re-

pair machinery (MMR) and error prone polymerases will result in random

incorporation of any of the four nucleotides71 (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 mu-

tations indicates a preference for transitions over transversions (at an approx-

imate ratio of 60:40), with purines targeted more frequently than pyrimi-

dines; (2) mutations are concentrated mainly in the CDRs and most often are

single nucleotide substitutions rather than deletions or insertions; (3) certain

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 mutations70,72.

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, result-

ing in switching of antibody isotype from IgM to IgG, IgE, or IgA, respec-

tively, without changing antigen specificity. This process also involves AID

enzymatic activity and occurs by the joining of two switch regions and si-

multaneous excision of the intervening loop of IGHC regions73. The DNA

sticky ends are then ligated by the NHEJ proteins which are also active dur-

ing VDJ recombination74. The isotype of an antibody determines the manner

24

in which captured antigens are eliminated or the location where the IG is

first encountered73,75. 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 epi-

topes, 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 antigens76. In combination with antigen-dependent activation,

cytokine-induced signalling provides specificity to CSR77.

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 recom-

bination, 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 neverthe-

less possible. Thus, when considering both the HC and the LC there is a

potential for 2 x 106 combinations in total. In addition to this, the introduc-

tion 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 genes7. Therefore, the

chance of two unselected B cells carrying exactly the same BCR is approx-

imately 1 in 2.3 x 1012.

All cells that have passed the pre B stage of development will have under-

gone 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 use-

ful hints about the cell population from which the lymphoma or leukaemia

first arose, since the IGHV mutation status can indicate if the cell has under-

gone the SHM process78. (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

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. demonstrat-

ed 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 in-

dividual 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 fre-

quently used IGHV3 genes, whereas the IGHV4-59, IGHV4-34 and

IGHV4-39 were the most over-represented genes of the IGHV4 subgroup79.

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 repertoire80. There

also appeared to be no preferential pairing between IGHV and particular

IGKV genes81. 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 repertoires82.

The processe of V(D)J recombination occurs before exposure to antigen and

thus creates the pre-immune repertoire. Exposure to auto-antigen or exogen-

ous 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 rearranged80,83-85. Additionally, there is most likely

evolutionary selection for genes that are effective against prominent patho-

gens possibly making them more efficient at rearrangement in the pre-

immune 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 there-

fore 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. Apro-

pos 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

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 indi-

viduals86,87. 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 com-

prised of both naïve and memory B cells, in that some carry mutations in the

IGHV genes while others are unmutated88-92. This population may be sus-

tained by stimulation by T cell independent antigens, such as carbohydrate

antigens on encapsulated bacteria or viruses93,94. In fact, it has been demon-

strated that in vitro, MZ cells are the only B cells capable of mounting T

cell-independent responses95,96.

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, respectively97. After

encounter with foreign antigen, all mature B cell subsets are capable of gene-

rating plasma cells, although MZ and B1 cells are faster and more efficient94.

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

compartment100,101. B1 cells also appear to have a limited ability to undergo

isotype switching and are therefore most often IgM secreting cells102. These

restrictions also imply that the ability of B1 cells to form a germinal centre

reaction is limited103. 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 res-

ponses against gut/peritoneum and blood borne antigens94. 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 com-

mon pathogens such as S. pneumoniae and filariae104,105. Additionally, these

cells appear to serve ‘housekeeping’ functions in the removal of cell debris

and apoptotic bodies, hence their autoreactive specificities60,105. In contrast,

mature re-circulating FM cells are a more diverse pool containing antigen

27

specific B cells that are recruited for long term T-dependent antigen res-

ponses and high affinity memory generation via SHM.

Chronic lymphocytic leukaemia

Background

CLL is the most frequently occurring adult leukaemia, with approximately

400-500 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 65-70 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 109/L 106.

CLL arises due to a monoclonal expansion of B cells which express the CD5

molecule on the cell surface107. 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 lymphocytes106. 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 rates108.

As previously mentioned, a proportion of CLL patients will follow an ag-

gressive course while the remaining patients have a relatively indolent dis-

ease. 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.

28

Treatment options

Since many patients will follow an indolent disease course, a ‘watch and

wait’ approach is generally employed for CLL patients. Asymptomatic pa-

tients are not treated and if it does appear that there is disease progression of

a previously indolent case, fludarabine in combination with cyclophospha-

mide is the first line treatment, with the aim of long-term remission111,112. If

however, the patient is older or unable to tolerate such a regimen, chloram-

bucil can be used, with the aim of keeping the patient symptom free113. Cer-

tain monoclonal antibodies such as anti-CD20 (Rituximab) and anti-CD52

(Campath 1H/Alemtuzumab) have been incorporated with earlier treatment

regimes and have improved response rates114-116. Rituximab, although inef-

fective as a single treatment, can be given in combination with fludarabine

and/or cyclophosphamide114,115,117. In general, stem cell transplantation

(SCT) is reserved for younger patients with unfavourable risk factors. Cur-

rently, autologous SCT is rarely undertaken since it has not been shown to

be curative, while allogeneic SCT may be a treatment option in young pa-

tients with poor prognostic markers118,119. Non-myeloablative, or reduced-

intensity conditioning (RIC) allogeneic transplants, because of their gentler

chemotherapy and radiation regimes, are associated with a lower risk of

transplant-related mortality and minimal toxicity120-122.

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 cas-

es. Since CLL is known to show a high degree of clinical heterogeneity be-

tween 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 sub-

groups in CLL. In 1999, two independent groups reported that the mutation

status of the IGHV genes divided CLL into two clinical entities which car-

ried markedly divergent prognoses123,124. 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 outcome125. The prog-

nostic usage of the IGHV gene mutational status was subsequently verified

29

in numerous studies and is considered to be one of the strongest independent

prognostic markers in CLL126-131.

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 aberration132,133. 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 CLL134. These non-coding micro-RNA

genes reportedly target the BCL2 oncogene, giving a clear link to their pa-

thogenic effect in CLL126. Other common genomic alterations include dele-

tion of chromosome 17p13 (within which the TP53 gene is located) and de-

letion of chromosome 11q22-23 (which harbors the ataxia telangiectasia

mutated gene)135-137. Both of these genes are involved in apoptosis regulation

pathways and their deletion in CLL cells is associated with resistance to

chemotherapy and poor outcome126,138. In addition, trisomy of chromosome

12 is associated with an intermediate outcome126,139. Interestingly, one gene,

which is involved in the pathogenesis of T cell pro-lymphocytic leukaemia,

is also over-expressed in CLL140,141. 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 CLL142. 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 eluci-

dated.

CD38 CD38 is a transmembrane protein which upon antibody ligation, catalyses

the conversion of NAD+ to cADPR, causing Ca2+ flux into the cell. It has

been reported to augment signaling of B cell receptors and thereby regulate

apoptosis123,143-147. 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 active148. 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 sur-

vival in CLL149,150. A number of studies then demonstrated that CD38 posi-

tive/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 unmu-

tated IGHV genes123,129,146,151,152. Thus, CD38 was considered as a potential

surrogate marker for IGHV gene mutational status. However, according to

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. Initial-

ly a 30% cut-off was proposed, however later studies suggested that lower

cut-off margins between 5-20% could be employed123,129,145,146,153. Further-

more, 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 disease129,145,146,153.

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 cells154-157. In CLL, intra-

cellular ZAP-70 expression has been found to correlate with IGHV gene

mutation status, with high ZAP-70 levels being mostly observed in unmu-

tated cases158. 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 basis159,160. However, a number of different methods (direct and indi-

rect antibody assays) which were employed showed discordant results in up

to a third of cases159-163. 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 repertoire124,128,164-166. 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 genes86,124,128,164-169. Moreover, on examination of

HCDR3 characteristics, amino acid composition and charge, Fais et al. iden-

tified sets of BCRs with highly restricted HCDR3s164. They proposed three

prototypic BCRs using the IGHV1-69, IGHV3-7 and IGHV4-34 genes164.

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 IGHV4-34

31

sequences used either an IGHJ4 or IGHJ6 gene, the HCDR3 could be short

and basic or longer and acidic. The finding of IG rearrangements from indi-

vidual patients with highly similar HCDR3s led to the hypothesis that a

common antigen could be selecting out clones leading to the restricted recep-

tors observed.

Tobin et al. first reported a subset of patients using the IGHV3-21 gene,

which had a poor prognosis despite the fact that two-thirds of these patients

carried mutated IGHV genes128. In addition, it was observed that they dis-

played distinctive, short HCDR3s and a predominant lambda LC expression.

A follow-up study confirmed and expanded these findings170. Firstly, a large

proportion of the IGHV3-21+ cases (70%, 21/30 cases) displayed a HCDR3

comprised of the IGHJ6 gene and had no easily identifiable IGHD region.

Examination of the 9 codon long HCDR3s revealed a highly conserved ami-

no acid motif (ARDANGMDV) in 40% (12/30) of cases. Again, a predomi-

nant lambda expression was evident, with 90% of IGHV3-21+ cases carrying

an IGLV3-21 (V�2-14) gene170. This implicated that tumour cells from dif-

ferent patients were possibly recognising a common antigenic epitope. Fur-

thermore, 68% of patients, despite expressing mutated IGHV genes, had a

poor overall survival (83 months) and thus appeared to be an exception to

the rule with regards to the prognostic classification of IGHV gene mutation

status125,128. It was also noteworthy that the frequency of IGHV3-21+ cases in

these cohorts (11-12%) was similar to that reported in some British co-

horts161,171, yet was much higher compared to other European cohorts 131,172,173

Subsequently, Ghiotto et al. examined 25 isotype-switched CLL samples and

found 5 IgG-expressing cases with remarkably similar BCRs174. These latter

cases rearranged the same IGHV4-39, IGHD6-13 and IGHJ5 genes and four

of these cases employed the IGKV1-39/ID-39 (V�O12/2) gene. Consequent-

ly, the HCDR3s and LCDR3s were virtually identical at the amino acid lev-

el174. In addition, these five patients displayed several clinical characteristics.

Atypically for CLL, there was a male:female ratio of 1:4. Also patients had

aggressive disease, recurrent infections and a high occurrence of secondary

solid tumours.

Considering the huge potential for variation in the BCR structure; 2.3 x 1012

different combinations, it is extremely unlikely that tumour cells from dis-

tinct patients would display such similar IG gene rearrangements unless

there was some selective force for certain antibody structures. These obser-

vations have therefore strongly implicated antigen recognition and antigen

selection in the development of CLL, by triggering B cells carrying certain

BCRs. The potential antigens, either autoantigens or foreign antigens, could

32

possibly sustain stimulation of the B cell proliferation, thereby allowing an

increased susceptibility to a transformation event.

The origin of CLL

If it is proposed that antigen selection and stimulation leads to the clonal

expansion of CLL cells, then which cell population does the original CLL

clone arise from? Much uncertainty remains as to the cellular origin of CLL

and the true normal counterpart of the CLL cell has as yet not been eluci-

dated. In the late 1980’s it was first hypothesised that CLL originated from a

pre-GC repertoire, with no evidence of mutations within the IG genes167,168.

However, subsequent reports describing that the IGHV genes were mutated

in roughly half of all CLL cases forced re-evaluation of this

theory123,124,164,165. It instead appeared that there may be two separate entities

of CLL; those cases with unmutated IGHV genes originating from a naïve

CD5+ pre-GC compartment, the other, displaying mutated genes originating

from a post-GC compartment of antigen experienced memory B cells. How-

ever, subsequent gene expression profiling illustrated that both mutated and

unmutated CLL displayed a profile more similar to that of antigen-

experienced cells, supporting the idea that both cell groups had encountered

antigen, regardless of whether they carried IGHV gene mutations or

not175,176. Extensive characterisation of cell surface markers also confirmed

the idea that CLL arose from antigen-activated cell populations177. Further-

more, the consistent reports that one subgroup showing unmutated IGHV

genes had a more aggressive disease course highlighted the relevance of

understanding the difference between these groups at a biological level.

More recently, it has been proposed that the unmutated/low mutated subset

of CLL and perhaps even the mutated subset may arise from a marginal zone

cell population178. This hypothesis arose as similarities between the popula-

tion of MZ cells and CLL cells became evident. Both populations are most

frequently not isotype switched and therefore predominantly express

IgM98,99,102. CLL cells, particularly those of the unmutated subtype, display

polyreactivity/autoreactivity similar to the reactivity of natural antibodies

produced by MZ cells in a T cell-independent response94,179-181. Reflecting

this, the IGHV gene repertoire in MZ cell populations and CLL cells is high-

ly restricted47,94. The one incongruity in this speculation is that CLL cells by

definition are always CD5+, whereas MZ cells most often are CD5-. Never-

theless, this may be related to cell activation states, since MZ cells have been

shown to express CD5 when activated182-184. It is therefore possible that CLL

arises from a MZ cell population rather than from the FM cell population

initially proposed. Following this line of reasoning, CLL could result from a

33

clonal response of MZ cells to T-independent antigens such as polysaccha-

ride structures expressed on capsular bacteria. Extending this idea, it is also

conceivable that the dual reactivity of MZ cells to self-epitopes on apoptotic

cells could allow for chronic stimulation of this cell population.

Somatic hypermutation patterns in CLL

In the analysis of IGHV repertoires it became evident that certain genes

tended to be predominantly mutated (e.g. IGHV3-21, IGHV4-34, IGHV3-7),

while others were most often unmutated (IGHV1-69)124,128,164,166,167. Since

there appeared to be biases both in the IGHV gene usage in CLL and the

mutation targeting of these genes, it was of interest to determine if the muta-

tion patterns in CLL were typical of that of the canonical SHM process. This

was first addressed by Messmer et al., who observed that CLL mutations did

display a targeting preference for RGYW motifs, a base change bias for tran-

sition mutations and a focusing of replacement mutations away from the FRs

to the CDRs of the IGHV gene185. Otherwise, relatively little is known about

the pattern of SHM in CLL using certain IGHV genes, particularly in rela-

tion to that of normal B cells from healthy individuals.

The potential role of self-antigens, exogenous antigens and

superantigens in CLL

Currently, the exact binding specificities of CLL IGs are largely unknown. It

is possible that both self-antigens and exogenous environmental antigens

could provide either chronic or transient stimulus and confer a proliferative

advantage to the (still elusive) CLL B cell progenitors. As previously

mentioned, studies of B cell reactivity in CLL have revealed that 50% of

cells display autoreactivity180,186. In particular, unmutated BCRs have been

shown to be associated with autoreactivity and polyreactivity against mole-

cules such as DNA, insulin and LPS, while BCRs in mutated CLL do not

show such polyreactive properties186. In addition, it was demonstrated that

when somatically mutated sequences were reverted to their germline confi-

guration they acquired a greater degree of polyreactivity186. It is therefore

possible that mutated CLL cells are selected by singular antigens for which

they are specific, while unmutated CLL cells could possibly bind a wider

range of epitopes and thus be stimulated by a number of distinct antigens.

Furthermore, while it is self-evident that the CDR3 plays a crucial role in

antigen recognition, there is also evidence highlighting that antigens can

interact with the IG molecule outside of the hypervariable regions. Bacterial

superantigens have been found to interact with FR1, FR3 and the CDR2 of

the IGHV3 subgroup genes. Likewise, certain self-antigens bind framework

34

regions of IGHV3 and IGHV4 subgroup genes186-188. Specifically, it has been

demonstrated in mice that binding by the staphylococcus protein A (SpA), a

prototypical superantigen, can result in clonal suppression and deletion of B

cell clones, yet in vitro stimulation with SpA can contribute to selection of B

cells and possibly support cell growth189-194.

In cold agglutinin (CA) disease, auto-antibodies are directed against the N-

acetyllactosamine structures (NAL) within I/i antigens on foetal and adult

red blood cells195,196. The heavy chain of these antibodies appears to be en-

coded solely by the IGHV4-34 gene197. The interaction of IGHV4-34 antibo-

dies with NAL epitopes, also present on various other exogenous and self-

antigens is largely independent of the conventional antigen binding site and

mainly involves FRs, especially HFR1198,199. The IGHV4-34 gene is very

frequent in normal individuals, however, B cells expressing this gene are

censored at multiple checkpoints during B cell development to alleviate their

inherent autoreactivity200. In this context, it is not contradictory that the titers

of IGHV4-34 antibodies are at very low levels in sera from healthy individu-

als, despite the fact that the IGHV4-34 gene is frequent in the normal reper-

toire201. In contrast, IGHV4-34 antibodies are up-regulated in patients with

systemic lupus erythematosus (SLE), indicating that breakdown in the con-

trol of IGHV4-34+ B cells could be a factor in the development of autoim-

munity200,202. The IGHV4-34 gene is very also very frequent in the repertoire

of CLL, prompting speculation about the possible antigenic specificity of the

leukemic IGHV4-34 BCRs as well as the mechanisms of leukemogenesis in

this group of patients.

35

AIMS

The main objective of this thesis was to detail IGH and LC gene rearrange-

ment features as well as SHM patterns in order to reveal evidence for antigen

selection in CLL pathogenesis. More specifically the aims were as follows;

I To identify new CLL subsets defined according to HCDR3 homology.

We will simultaneously examine for biases in light chain gene usage and

evidence of restricted LCDR3 characteristics within these subsets.

II To determine if the restricted HCDR3 features and biased usage of the

IGLV3-21 gene previously observed in the IGHV3-21 CLL subgroup is

limited to Scandinavian cohorts, or if the distinctive features of this clin-

ically important subgroup are independent of geographical origin.

III To analyse the SHM patterns of a large cohort of IGH and IGK/L CLL

rearrangements and ascertain if patterns differ from that of ‘normal’

SHM.

IV To examine the features of secondary rearrangements of the LC loci in

order to explore the possible role of LCs as editors to heavy chains in

malignant CLL cells and determine the contribution of LCs in antigen

recognition.

37

PATIENT MATERIAL AND METHODS

Patient material

In paper I, 346 CLL patient samples were obtained from the University Hos-

pitals of Uppsala, Umeå, Linköping and Huddinge, Sweden, and Tampere,

Finland between 1981 and 2001. All IGHV3-21+ patient samples (n=32)

were then collected from the same Swedish/Finnish cohort for analysis in

paper II. To extend this study, a further 58 CLL samples, all carrying a rear-

ranged IGHV3-21 gene, were amassed from Germany, Italy, USA and Aus-

tralia. In paper III, all the Swedish and Finnish samples from the previous

studies were included. The Scandinavian material was then extended by an

additional 148 cases making up a total of 494 cases. This cohort was then

combined with a further 1445 cases, acquired from collaborating institutions

in France, Greece, Italy, Spain to make a total of 1939 CLL cases. In paper

IV 725 patients from the aforementioned institutions were included for ex-

tensive analysis of their clonal LC rearrangements.

Tumour material was obtained mostly from peripheral blood and bone mar-

row, although a proportion of tumor samples were obtained from lymph

nodes, spleen and ascites. CLL diagnosis was based on morphologic and

immunophenotypic features according to the World Health Organisation

classification203 (papers I & II) or the criteria of the National Cancer Institute

Working Group (papers III & IV)129,164,170,174,204-208. In paper IV LC isotype

restriction (kappa or lambda) was determined in the majority of cases

(709/725) by means of flow cytometry with the ratio limits of kappa:lambda

expression set at >3 or <0.3.

PCR amplification and nucleotide sequence analysis

Polymerase chain reaction (PCR) amplification of all IGHV-IGHD-IGHJ,

IGKV-IGKJ, IGLV-IGLJ and KDE rearrangements was performed on either

genomic DNA (gDNA) or cDNA. Subgroup specific IGHV, IGLV, IGKV

primers or an IGJK intron-specific primer were employed along with con-

sensus IGHJ, IGLJ and IGKJ primers or a KDE-specific primer, respective-

ly. The primer sets and amplification conditions employed in these analyses

are described in detail in the respective papers of this thesis and have pre-

38

viously been described elsewhere209,210. Direct sequencing of both the for-

ward and reverse strands was performed for the majority of cases although,

where required, PCR products were cloned using the Zero Blunt Topo PCR

cloning kit. In papers I and II, sequencing reactions were performed using

the BigDye Terminator Kit Cycle Sequencing Reaction Kit or the DYEnam-

ic ET dye Terminator Kit and analysed by automated DNA sequencer (ABI

377 or ABI3700).

In papers I and II, in order to define IGHV/IGHD/IGHJ, IGLV/IGLJ gene

usage and IGH/IGL mutation status, sequences were submitted to 3 different

databases (IMGT, GenBank/IgBlast, and V-BASE)7,211,212 and aligned to the

closest matching germline genes. In the third and fourth studies, the IMGT

database (http://imgt.cines.fr) was used for both germline gene alignments

and mutation analysis11. In all four papers, sequences with less than 98%

identity to germline were classified as mutated. In papers III and IV howev-

er, we created two further mutation categories; those with 98-98.9% identity

to germline and those with 99-99.9% identity to germline. These groups

were designated as ‘borderline mutated’ or ‘minimally mutated’, respective-

ly. Finally, those sequences with 100% germline identity were named ‘truly

unmutated’. In papers I, III and IV both functional and non-functional se-

quences were included in the analyses, however in paper III only potentially

functional sequences (in frame and with no stop codons) were analysed.

In paper I in frame rearrangements were converted to amino acid sequences

and aligned using the multiple sequence alignment software Clustal X (1.83)

for Windows. In that study, to be classified as a ‘homologous HCDR3

group’, a number of criteria were required. Firstly, common usage of the

same IGHV gene between all sequences, secondly, a HCDR3 homology of

at least 60% between HCDR3s, and finally, each group required a minimum

of at least 3 cases. In paper II, the same sequence alignment software was

used for H/LCDR3 analysis as in paper I. ‘High CDR homology’ was de-

fined as one or fewer amino acid deviations from the most common CDR3.

Those sequences with 2-3 amino acid deviations were classified as having

‘moderate homology’. In paper III, all sequences were analysed for their

HCDR3 composition and batched according to their HCDR3 homology.

Each subset was required to have at least 60% homology between the CDRs,

however unlike paper I, it was not required that all sequences used the same

IGHV gene and a subset was created if two or more sequences had sufficient

CDR3 homology.

In paper IV, germline analysis was performed on two cases utilising the

IGLV3-21 gene to determine if one particular stereotyped mutation was a

genuine mutation rather than an as yet unidentified allele. In one case T cells

were isolated from patient peripheral blood by depletion of CD19 B cells,

39

followed by positive CD4 selection, and in the second case granulocytes

were isolated by gradient ficoll separation.

Sequence analysis and data mining

For the SHM analysis in paper III and paper IV, non-CLL IG sequences

were retrieved from the IMGT/LIGM-DB database (http://imgt.cines.fr/cgi-

bin/IMGTlect.jv?) and any partial, out-of frame or clonally related sequences

were excluded from the analysis. The final collection of 5303 unique IGHV-

D-J and 4709 IGK/L-J sequences included sequences from B cell lympho-

proliferative disorders, normal B cells, ‘immune dysregulation’ disorders

and autoreactive cells.

All 1967 CLL and 5303 non-CLL IGHV sequences and all 612 IGKV-J and

279 IGLV-J CLL and 4709 non-CLL IG LC sequences were submitted to

the IMGT/VQUEST analysis software to obtain IGH/K/L gene and allele

usage, percentage of nucleotide identity to the germline, CDR3 length, and

somatic hypermutation characteristics. In addition, each nucleotide mutation

in every sequence was recorded, as was the change or preservation of the

corresponding amino acid, identified as replacement (R) or silent (S), respec-

tively. In order to compare the degree of change due to a mutation in a par-

ticular codon, amino acids were grouped into 1 of 5 categories, compiled

according to standardised biochemical criteria11. Also, to account for the fact

that a mutation is more likely to occur in an HFR than a HCDR due to its

greater length, amino acid changes were ‘weighted’ (normalised) so that it

was possible to compare mutation frequencies between regions and between

sequence groups (i.e. IGH/K/LV groups/subsets). Finally, analysis of muta-

tion targeting to the tetranucleotide hotspot (4-NTP) motifs RGYW/WRCY

(R = A/G, Y = C/T, and W = A/T)66 and DGYW/WRCH (D = A/G/T, H =

T/C/A)65 was performed for all mutated sequences.

Statistical analysis

Kaplain-Meier survival analysis and log rank tests were employed in paper II

to determine any differences in survival between selected groups. Overall

survival was defined as the time from diagnosis to last follow-up or death.

For paper I and II, all analysis was performed using Statistica v6.0. In paper

III and IV, descriptive statistics were used for mutation frequency counts and

distributions. For comparisons of mutation frequencies between groups Chi-

square and Fisher’s exact test were used. All analyses were performed using

the Statistical Package SPSS Version 12.0 in the final two papers.

41

RESULTS & DISCUSSION

Characterisation of new CLL subsets (Paper I)

A subset of CLL patients had previously been identified, all of whom carried

IGHV3-21 rearrangements with distinguishing IG features such as restricted

usage of the IGLV3-21 gene, short HCDR3s with a distinctive amino acid

composition and inferior outcome128,170. It was consequently of interest to

determine if other such subsets sharing specific HCDR3 features existed.

Thus, in paper I, we set out to further investigate the degree to which BCRs

from distinct CLL tumours were similar to each other, and examined what

proportion of patient sequences displayed restricted amino acid motifs in the

HCDR3 and LCDR3, particularly in patients using IGHV genes other than

the IGHV3-21 gene. In total, 368 functional rearrangements, from 346 CLL

cases, were analyzed for their IGHV/IGHD/IGHJ usage and HCDR3 com-

position. IGK and IGL usage was also investigated to determine if there

were any notable LC associations with IGH genes displaying homologous

HCDR3s. Multiple sequence alignment was performed for all HCDR3 se-

quences to determine the degree of homology between sequences.

Subsets were created according to shared IGHV/D/J gene usage, a HCDR3

homology of at least 60% between HCDR3s, with a minimum 3 cases per

subset. In total, seven subsets were defined according to these criteria. Three

subsets using the IGHV1-69 and IGHV1-2 genes carried virtually identical

HCDR3s (>75%amino acid identity). The first IGHV1-69 subset was com-

prised of 4 cases using an IGHD3-16 gene, employing the same D reading

frame, and an IGHJ3 gene. The second IGHV1-69 subset had 3 cases which

carried an IGHD3-3 gene and an IGHJ6 gene and finally, the IGHV1-2 sub-

set was comprised of 5 cases using the IGHD6-19 and IGHJ6 genes. Thus,

these subsets were almost identical in terms of HCDR3 length and composi-

tion and even displayed shared N regions. Remarkably, the restriction of IG

gene usage even extended to the light chain gene; in the first IGHV1-69

subset, all four cases employed an IGKV3-20 (V�A27) gene rearrangement,

while in the second IGHV1-69 subset the IGLV3-9 (V�2-6) gene was rear-

ranged in two of three cases. Similarly, three of five IGHV1-2 cases were

paired with an IGKV4-1 (V�B3) gene rearrangement. Furthermore, we again

identified a subset of 22 cases utilising the IGHV3-21 and IGHJ6 genes,

with an extremely short, indefinable IGHD region resulting in HCDR3s

42

which were highly similar in amino acid composition and length (see also

paper II).

A further three subsets were found to display moderately restricted HCDR3s

(60-75% amino acid identity). These subsets employed the IGHV1-3,

IGHV1-18 and IGHV4-39 genes and also displayed a remarkable restriction

in IGHD, IGHJ and LC usage. In general, the HCDR3s were of similar

length, although the N regions were less well conserved. In the first subset,

nine cases showed rearrangements using the IGHV1-3/IGHD6-19/IGHJ4

gene combination; the HCDR3s in this subset could differ in length by one

amino acid residue due to variability at the IGHD/IGHJ junction. In the

second of the moderate homology groups, the IGHV1-18 gene was rear-

ranged with an IGHD6-19 or IGHD3-22 gene and an IGHJ4 gene. The final

group used a combination of the IGHV4-39/IGHD6-13 or IGHD6-29/IGHJ5

genes and once again had similar HCDR3 lengths, despite junctional amino

acid differences. In addition all three of these subsets predominantly rear-

ranged the IGKV1-39/1D-39 (V�O2) gene in combination with common

IGKJ genes.

Table1. Subsets defined in paper I as of 2004. (See also appendix I)

IGHV No. of IGHD gene IGHJ HCDR3 IGK/IGL Identified in gene cases gene lenght (AAs) gene* other materials

IGHV3-21 22 none identified IGHJ6 9 IGLV3-21 Several; see introduction

IGHV1-69 4 IGHD3-16 IGHJ3 20 IGKV3-20 Widhopf & Messmer et al.

IGHV1-69 3 IGHD3-3 IGHJ6 23 IGKV3-20 Widhopf et al.

IGHV1-2 5 IGHD1-26 IGHJ6 16 IGKV4-1IGHV1-3 9 IGHD6-19 IGHJ4 13-14 IGKV1D-39 Messmer et al.

IGHV1-18 3 IGHD6-19/3-22 IGHJ4 12 IGKV1D-39 Messmer et al.

IGHV4-39 5 IGHD6-13 IGHJ5 18 IGKV1D-39 * Represents the most frequently used within that subset.

**Please refer to references 183 and 184.

In summary, in this study we identified several new IG subsets, consisting of

51 sequences in total (14% of 368 functional IGHV rearrangements), which

carried highly homologous or virtually identical HCDR3s. This was asto-

nishing considering the extremely low chance of one CLL patient randomly

exhibiting the same IGH rearrangement as another unrelated patient (approx-

imately one in 2.3 x 1012). The HCDR3 stereotypy observed indicates that

there must be a selective pressure and recognition of similar antigenic epi-

topes within these subsets. As suggested earlier, it is therefore possible that

continued antigenic stimulation could result in clonal expansion and conse-

quently proliferating B cells would have a greater chance of undergoing a

transformation event.

In parallel, two independent groups also identified a number of subsets de-

fined according to restricted HCDR3 characteristics. Widhopf et al. analysed

43

a total of 1220 CLL cases and identified the same IGHV1-69/IGD3-

16/IGHJ3 subset as that reported in our study in a total of 15 cases. This IGH

gene rearrangement was also paired with an IGKV3-20 (V�A27) LC gene.

Thus, 1.3% (15/1220) of their cohort carried a virtually identical IGH gene

rearrangements209. A further 15 of their cases also belonged to the IGHV1-

69/IGD3-3/IGHJ6 subset. Messmer et al. also observed the IGHV1-

69/IGD3-16/IGHJ3 rearrangement paired with the IGKV3-20 gene in five of

255 CLL cases (~2%)210. They also identified the IGHV1-3/IGHD6-

19/IGHJ4 and IGHV1-18/IGHD6-19orIGHD3-22/IGHJ4 subsets, both of

which were preferentially paired with an IGKV1D-39 rearrangement. (See

table above.) This group identified a further four HCDR3 subsets not identi-

fied in our material210. It is therefore possible that there are many more sub-

sets that were not identified due to their low frequency in our material. In

conclusion, the findings by us and others further strengthen the concept of

antigen selection as a factor in the development of CLL, particularly in sub-

sets displaying stereotyped HCDR3s.

Further characterisation of the IGHV3-21 subset (Paper II)

In Sweden, the frequency of IGHV3-21 cases reported was relatively high

(9%) compared to studies from other countries (0-3%) (See paper I)131,173,174.

Besides this, it became apparent that there was a marked restriction in

HCDR3 structure, along with biased usage of the IGLV3-21 gene amongst

these cases128,170. Moreover, IGHV3-21+ patients from the Swedish/Finnish

cohort had inferior prognosis despite the fact that almost 2/3’s of patients

had mutated IGHV genes128,170. To determine if these findings were merely a

regional phenomenon due to a shared genetic background, we extended the

analysis of IGHV3-21 CLL in paper II to include cases from more diverse

geographical locations such as Germany, Italy, USA, Australia as well as

Sweden and Finland. Analysis of the IGH genes of 90 IGHV3-21+ CLL cas-

es revealed that 57 (63%) patients carried somatically hypermutated IGHV

genes while 33 (37%) patients had unmutated genes. In support of our pre-

vious findings, it was evident that many of the HCDR3s in these patients

were highly restricted in their structure and composition; they were typically

short, had no recognisable D gene, used the IGHJ6 gene and were composed

of the conserved amino acid motif; ARDANGMDV. Fifteen of 90 patients

carried this exact HCDR3 motif, while 21 patients carried the motif with one

amino acid deviation, and in 14 patients the motif differed by 2-3 amino

acids. (Figure 7)

44

Figure 7. Alignments of the H/LCDR3s of IGHV3-21 cases. Each dot represents the

same amino acid as that indicated in the uppermost sequence.

Thus in total, as many as 56% of IGHV3-21 patients displayed stereotyped

HCDR3s. In addition, there was a strikingly restricted usage of one particu-

lar LC gene; the IGLV3-21 (V�2-14) in 72% of all patients, which was most

often joined with the IGLJ3 gene. Seventy percent (63/90) of patients also

displayed moderate to high homology of the motif QVWDS(S/G)DHHPWV

in the LCDR3. Moreover, these highly restricted IG HCDR3s and LCDR3s

were found in patients regardless of their geographical origin.

Our analyses of KDE rearrangements in 42 lambda expressing cases re-

vealed that 98% carried an IGKV-KDE or an IGJK intron-KDE rearrange-

ment on at least one allele. Thus, the LC rearrangement followed the tradi-

tional ordered model (i.e. kappa, kappa, lambda). This finding further sup-

ports the concept of a selective pressure for a particular rearrangement at the

LC loci, counter-selecting IGKV-J rearrangements and favouring an

IGLV3-21 rearrangement.

Survival analysis was performed on 64 patients and revealed no difference in

overall survival between mutated (median survival 79 months) and unmu-

tated IGHV3-21 cases (median survival unreached, p=0.17). Furthermore, no

difference in overall survival was observed between IGHV3-21 patients

using homologous and non-homologous HCDR3s or between cases carrying

short versus long HCDR3s. Similarly, no survival differences were apparent

between IGHV3-21 cases utililising the IGLV3-21 gene versus those using

all other IGLV genes. ZAP-70 expression was analyzed in 10 cases, with

seven cases showing ZAP-70 positivity (>20%), despite 5 of these cases

45

having mutated IGHV genes. Similarly, analysis of CD38 expression levels

in 43 patients illustrated that while 20 cases were CD38 positive (>20%),

only 8 of these had unmutated genes. Thus mutated IGHV3-21 CLLs appear

to be atypical in terms of ZAP-70 and CD38 positivity. Moreover, FISH

analysis (on 55 cases) showed that a slightly higher proportion of 11q dele-

tions (27%) were observed in both mutated and unmutated IGHV3-21 cases

than is typically observed in CLL in general (15-20%)126,130,213.

As previously mentioned, the frequency of IGHV3-21 cases appears to vary

greatly between materials from different geographical locations, yet the rea-

son for this discrepancy remains unclear. Studies from countries in northern

Europe (including Ireland and the UK) appear to have higher frequencies of

IGHV3-21 cases than southern European countries131,171-174. Differences in

relative frequency of genes, such as IGHV3-21, are possibly due to biases in

material selection, where cases collected via referral centres tend to follow a

more aggressive clinical course which may cause increased relative frequen-

cies of, for example, IGHV1-69 and IGHV3-21 expressing cases. Converse-

ly, it is possible that the differences in IGHV gene frequencies simply reflect

different levels of antigen exposure in the environment or biased gene usage

due to shared genetic background.

In this study, we reconfirmed the molecular and clinical features of the

IGHV3-21 subset and demonstrated that the frequency of restricted

IGHV3-21 BCRs is more widespread than previously anticipated. This was

supported by a recent IGHV3-21 CLL study conducted in the Mediterranean

region, where IGHV3-21 cases with similarly restricted BCRs were ob-

served205. They identified 7 of 16 cases carrying almost identical HCDR3

motifs to those observed in our study, while the remaining 9 cases showed

heterogeneous HCDR3s. They also found that those cases exhibiting stereo-

typed HCDR3s had progressive disease and displayed CD38 positivity yet,

in contrast to our findings, the non-stereotyped subgroup exhibited a more

variable clinical course with only 44% (4 of 9 cases) showing disease pro-

gression. This observation was subsequently confirmed in a study from the

same group where they analysed 32 IGHV3-21 cases206. Fifty percent of

those cases carried a stereotyped HCDR3 and 44% were associated with

homologous LC rearrangements. They reconfirmed their previous finding

that stereotyped IGHV3-21 cases had a more progressive disease than hete-

rogeneous IGHV3-21 even though both groups were comparable with re-

gards to patient age and clinical stage. However, the overall survival did not

differ between the two groups206. Additionally, a more recent study by Bom-

ben et al. of 37 Italian CLL cases reported that IGHV3-21 cases with homo-

logous HCRD3s more frequently expressed positivity for CD38 and ZAP-

70, markers associated with poorer prognosis, than non-stereotyped IGHV3-

21214.

46

More recently, Ghia EM et al. examined 63 IGHV3-21 cases from the US

(comprising 2.3% of their whole cohort) and observed that 40% of their pa-

tients carried the ARDANGMDV motif in the HCDR3 and were paired with

an IGLV3-21 gene rearrangement215. Of the forty cases that expressed an

IGLV gene, 31 of these carried an IGLV3-21 rearrangement. Furthermore, 5

of 7 examined cases showed a functionally rearranged IGKV allele. With

regards to clinical correlation, there was no difference in the time to treat-

ment observed between the group expressing the ARDANGMDV motif and

those cases with a heterogeneous HCDR3215.

IGHV3-21 gene usage in CLL tumours has obvious clinical relevance, since

these patients show a short median survival regardless of the mutation status

of their IGHV genes or their geographical origin. According to some studies,

it also appears that stereotyped IGHV3-21 cases have a more progressive

disease than heterogeneous IGHV3-21 cases205,206. Finally, the very high

degree of BCR homology between patients once again corroborates the con-

cept of antigen selection in CLL, one that most likely influences disease

course.

Stereotyped subsets and clinical correlations

Subsequent to the publication of paper II, Belessi et al. reported on a fre-

quent somatically introduced deletion of a serine codon in 16/63 (25%) mu-

tated IGHV3-21 cases displaying stereotyped HCDR3s. On comparison with

non-CLL sequences and non-homologous CLL cases, this deletion in the

HCDR2 was evidently CLL specific207. This finding offered the first evi-

dence that cases with stereotypical HCDR3s could also be affected in a simi-

lar fashion by the SHM process, suggesting a “stereotypical” response to

antigen. Stamatopoulos et al. went on to perform a comprehensive study

from a large cohort of 916 CLL patients and analyzed a total of 927 CLL

sequences for HCDR3 similarity206. In total, 48 IG subsets with >60% se-

quence homology at the amino acid level were defined, with 26 subsets

comprising of 3 to 20 sequences while the remaining subsets included pairs

of sequences. Unlike our previous studies, sequences did not necessarily

have to use the same IGHV gene to be assigned to the same subset. For ex-

ample, one subset was characterised by homologous HCDR3s with common

usage of the IGHD6-19 and IGHJ4 genes, yet multiple IGHV genes includ-

ing IGHV1-2, IGHV1-3, IGH1-18, IGH1-8 and IGHV5-a were utilised. It is

interesting to note that all of these IGHV genes are members of the same

IGHV clan. All these homologous subsets were assigned names, simply

termed subset#1 to subset#48 This study demonstrated that a remarkable

22% of CLL patients carried stereotyped HCDRs. Interestingly, a notably

larger proportion of unmutated sequences (35%) belonged to a subset com-

47

pared to mutated sequences (11%). Comparison to non-CLL IG sequences

revealed that the phenomenon of restricted HCDR3s was evident only in

CLL sequences. Moreover, particular subsets had peculiar biological and

clinical characteristics. Cases using IGHV4-34/IGKV2-30 were typically

young with indolent disease and, unusually for CLL, were IgG switched.

One IGHV1-69 subset (IGHV1-69/IGHD3-10/IGHJ6, subset#5), though

comprised of unmutated IGHV genes, was also associated with more indo-

lent disease compared to other IGHV1-69 unmutated cases (e.g. the IGHV1-

69/IGHD2-2/IGHJ6 subset or subset#3). In contrast, the IGHV1,5/IGKV1-

39/1D-39 ‘mixed’ subset displayed a notably progressive disease206. These

findings, along with the IGHV3-21 phenomenon again underscored the po-

tential biological significance of the existence of highly similar BCRs in

CLL patients.

Light chain gene usage in CLL

Since most previous studies on LC gene frequencies in CLL were performed

in the context of analysis of CLL subsets with specific IGHV usage and

HCDR3 features, Stamatopoulos et al. aimed to perform a completely un-

biased analysis of IGK/L gene usage in CLL216. Analysis of 276 unselected

CLL patients revealed that half of 179 IGK sequences carried somatic

hypermutations (above the 2% cut-off) and the most commonly rearranged

genes were; IGKV3-20 (A27), IGKV1-39/ID-39 (O2/O12), IGKV1-5 (L12),

IGKV4-1 (B3) and IGKV2-30 (A17), in order of frequency. Of the lambda-

expressing cases, IGLV3-21 (VL2-14), IGLV2-8 (VL1-2) and IGLV2-14

(IGL1-4) were the most commonly represented genes. They also identified a

number of LC subsets which displayed highly similar K/LCDR3s and were

frequently paired with a homologous IGHV gene; for example, the IGKV2-

30 gene was most often recombined with the IGKJ2 gene and paired with an

IGHV4-34 rearrangement216. Thus, akin to IGHV gene usage in CLL, there

appeared to also be restricted LC gene usage also, particularly within stereo-

typed HCDR3 subsets. This implied that certain LC IG genes are selected

for, most likely because they are beneficial in antigen binding and confer an

advantage to the clone. Following this, the Chiorazzi group performed a

similar analysis of light chain gene usage in CLL on 206 patients217. They

also observed an over-representation of certain IGK/L genes and preferential

pairing of specific IGK/LV genes with IGK/LJ genes. On comparison with

the repertoire of LC gene usage in normal IgM CD5+ and CD5- B cells, they

reported that the IGK/LV gene usage did not differ significantly to that of

normal B cells217.

48

Stereotyped patterns of somatic hypermutation in CLL (Paper III)

Up to this point there had been much focus on IGHV gene usage in CLL, yet

relatively little was known about SHM targeting of the IGHV genes. We

therefore set out to examine SHM patterns in the clonotypic rearranged

IGHV genes and determine if they could be specific to CLL, especially in

subset of cases with stereotyped BCRs. Thus, the patient cohorts from paper

I and II were merged with sample collections from 5 other collaborating

institutions in France, Spain, Italy and Greece (including the material from

the Stamatopoulos et al. study mentioned above206). A total of 1967 IGH

sequences from CLL patients were analyzed for their IGHV gene usage,

HCDR3 features and SHM patterns. This collection of sequences was di-

vided into four major identity groups; ‘truly unmutated’ (100% germline

identity), ‘borderline mutated’ (98-98.9% germline identity), ‘minimally

mutated’ (98-99.9% germline identity) and ‘mutated’ (<98% germline iden-

tity). The IGHV gene repertoire of these four identity groups differed consi-

derably. The IGHV1-69 and IGHV1-2 genes predominated among the ‘truly

unmutated’ and ‘minimally mutated’ groups, respectively, while the IGHV3-

21 gene predominated in the ‘borderline mutated’ group. Among 1233 “mu-

tated sequences”, it was once again evident that certain IGHV gene such as

IGHV4-34, IGHV3-7 and IGHV3-23, were more frequently mutated, as is

typically observed in CLL (Figure 8).

0%

20%

40%

60%

80%

100%

IGH

V1-69

IGH

V4-34

IGH

V3-23

IGH

V3-7

IGH

V3-21

IGH

V4-39

IGH

V3-30

IGH

V1-2

IGH

V3-33

IGH

V3-33

<98% 98-98.9% 99-99.9% 100%

Figure 8. Distribution of rearrangements of the 10 most frequent IGHV genes of the series according to mutational status

With the aim of performing a more extensive analysis of BCR stereotypy, all

Swedish cases were re-assigned to subsets according to their HCDR compo-

49

sition. In doing so, many of our subsets defined in paper I became part of a

large IGHV1/5/7 ‘mixed’ subset. Similarly, our IGHV3-21 stereotyped sub-

set in paper II became part of the much larger IGHV3-21 subset in this pa-

per. From this combined analysis, it emerged that almost a third (27%) of

sequences belonged to one of 110 different subsets with stereotyped

HCDR3. Notably 43% of “truly unmutated” sequences belonged to one of

these subsets, compared to only 16% of the ‘mutated’ group (p<0.001) (Fig-

ure 9). The fact such a large proportion of unmutated CLL cases displayed

stereotyped HCDR3s supports the argument first raised by the Chiorazzi

group that even though B cells bearing unmutated IGHV genes do not ap-

pear to have undergone the typical GC and SHM process, they may never-

theless be antigen experienced 177. The largest defined subsets were as fol-

lows; subset #1 was comprised of 53 minimally mutated/truly unmutated

sequences which utilised IGHV genes of the same clan (IGHV1-2/IGHV1-

3/IGHV1-1/IGHV5-a/IGHV7-4-1), subset #2 contained 56 IGHV3-21 cases,

followed by a number of subsets predominantly using the IGHV1-69 gene

(subset#7, n=28; subset#3, n=25; subset#6, n=18) and 27 IGHV4-34 se-

quences which belonged to two different subsets (subset#4, n=20; subset#16,

n=7).

Figure 9. The proportion of sequences belonging to a subset in the four germline

identity groups.

The largest mutated subgroups were then examined in terms of SHM charac-

teristics at a number of levels: (i) the distribution of mutations across the

HFRs/HCDRs, (ii) the targeting of the well defined DGYW/WRCH (4-NTP)

hotspot motifs, (iii) the spectra of nucleotide substitutions, and (iv) mutation

targeting of certain superantigenic-binding motifs. At cohort level, SHM

patterns were found to be typical of a canonical SHM process. However,

there were a number of exceptions, in particular in the IGHV3-21 and IGH4-

34 subgroups. IGHV3-21 sequences had the highest R mutation targeting of

the HCDR2 relative to all other genes. Conversely, IGHV4-34 sequences

had the lowest R mutation frequency within the HCDR2, even when com-

0

20

40

60

80

100

%

trulyunmutated

minimallymutated

borderlinemutated

mutated

Subset Non-Subset

50

pared to IGHV4-34 sequences from autoreactive and normal B cells. This

scarcity of R mutations in the HCDR2 of IGHV4-34 sequences was there-

fore considered a CLL-biased finding. In addition, it emerged that IGHV3-

21 displayed an under-representation of G-to-A changes compared to other

IGHV3 groups and an over-representation of T-to-A substitutions, which

was ‘CLL biased’.

A further 5303 non-CLL IGH sequences were collected from public data-

bases in order to identify the most important/relevant amino acid changes

occurring in the CLL cohort. We observed that shared or ‘stereotyped’ ami-

no acid changes (i.e. the same amino acid replacement at the same position)

did indeed exist in a number of CLL subgroups and that these changes were

CLL-biased in that they did not appear, or occurred at a much lower fre-

quency, in the non-CLL cohort. Most of these changes occurred significantly

more frequently in cases with stereotyped rather than heterogeneous HCDR3

sequences and thus could also be considered subset-biased. The IGHV3-21

group (subset #2) had subset biased-changes at two positions. One of these

changes was a serine deletion within the HCDR2 previously reported by

Belessi et al207. Recurrent changes were also observed in one IGHV4-34

subset (subset #16) at 3 positions, another IGHV4-34 subset (subset#4) and

IGHV4-4 (subset #14) at 4 positions. Likewise, the IGHV1-2*02 had a sin-

gle amino acid change which was subset-biased and caused the IG sequence

to become more like the germline configuration of another IGHV1-2 allele,

once again illustrating selection even for individual amino acid changes.

Table 2. Frequency of stereotyped amino acid changes in the IGH genes in CLL subsets compared to non-subset and non CLL IG sequences.

IGHV3-21 sequences Change CLL-Subset#2 CLL-heterogeneous Non-CLLIMGT-HCDR1/34 S-to-N 9/56 39507 7/95IMGT-HCDR2/61 S deletion 18/56 0/29 1/95IMGT-HFR3/66 Y-to-H 7/56 2/29 3/95

IGHV4-34 sequences Change CLL-Subset#4 CLL-heterogeneous Non-CLLIMGT-HCDR1/28 G-to-D 5/20 0/108 6/320IMGT-HCDR1/28 G-to-E 8/20 6/108 18/320IMGT-HCDR1/32 G-to-D 7/20 20/108 49/320IMGT-HFR2/40 S-to-T 10/20 29/108 45/320IMGT-HFR2/45 P-to-S 10/20 17/108 33/320

IGHV4-34 sequences Change CLL-Subset#16 CLL-heterogeneous Non-CLLIMGT-HCDR1/28 G-to-E 6/7 6/108 18/320IMGT-HFR2/40 S-to-T 4/7 29/108 45/320IMGT-HFR2/45 P-to-S 3/7 17/108 33/320

IGHV4-4 sequences Change CLL-Subset#14 CLL-heterogeneous Non-CLLIMGT-HCDR1/33 S-to-N 3/4 0/17 8/90IMGT-HCDR2/57 Y-to-H 4/4 3/17 7/90IMGT-HCDR2/58 H-to-P 3/4 0/17 0/90IMGT-HFR3/78 I-to-M 4/4 6/17 8/90

51

The IGHV4-34 gene is known to encode BCRs that bind to self-antigens and

has been observed to be inherently auto-reactive in its germline state195,196,198.

Hence, sequence alterations by SHM may be required to abolish self-

reactivity. Introduction of negatively charged acidic residues into the IGHV

sequence is one way of editing anti-DNA antibodies in mice, since it bal-

ances out the charge of the HCDR3, which are often enriched in positively

charged aromatic amino acids218-220. Thus, it is significant that subset#4 and

subset#16 IGHV4-34 cases, which characteristically carry a pair of basic

residues (lysine/arginine or arginine/arginine) within the HCDR3, also carry

stereotyped mutations which create glutamic and aspartic acid residues in the

HCDR1. The introduction of these negatively-charged residues by SHM is

illustrative of an attempt to eliminate the potential DNA binding properties

conferred by the positively charged HCDR3, thereby making it a more ac-

ceptable specificity in the IG repertoire. Simultaneously, the HFR1 motif

that confers the anti-I/i reactivity is particularly conserved within subset#4

and #16 cases, compared to non-subset IGHV4-34 cases, thus retaining the

possibility that subset#4 or subset#16 B cells could be bound by I/i or the B

cell isoform of CD45 which contains a linear poly-NAL196,199. Supporting

this, Catera et al. recently described recombinant CLL antibodies with BCRs

highly similar to our subset#4 cases bound viable B cells via the NAL epi-

tope221. Similarly, examination of the Staphylococcal protein A (SpA) bind-

ing motif revealed that the IGHV3-21 gene was significantly less targeted at

motif positions than other IGHV3 genes (p<0.01). This remarkable preserva-

tion of the germline configuration observed in superantigenic binding motifs

of IGHV3-21 and IGHV4-34 cases preserves the possibility that these sub-

sets of CLL cells could also receive stimulation signals by superantigenic-

like interactions, possibly in conjunction with stimulation by exogenous/self-

antigens.

In summary, the observation that recurrent mutations predominantly oc-

curred in subsets with stereotyped HCDR3s implicates an antigenic drive

that is acting not only on the HCDR3 but also on the HFRs and other

HCDRs of the BCR. Also, the apparent selection for specific individual mu-

tations, as also seen in the minimally mutated subsets, implies that even very

slight amino acid alterations of germline IG sequences may have a signifi-

cant effect on antigen recognition and thus clonal selection. Finally, the find-

ing that many stereotyped mutations were not only subset-biased but also

CLL-biased further substantiates the role of selection by specific antigen(s)

in CLL leukemogenesis.

52

Examination of the role of light chains in antigen recognition in CLL (Paper IV)

Our previous study revealed that the CLL IGH gene repertoire demonstrates

biases in the usage of certain IGHV genes, remarkable HCDR3 stereotypy and

stereotyped patterns of SHM in subgroups of patients. Thus, our aim in paper IV

was to similarly investigate the LC IG genes in terms of mutation frequency and

targeting and CDR3 stereotypy to elucidate if the LC also plays a significant role

in antigen recognition in CLL. In this study, we examined SHM patterns in a

total of 612 IGKV-J and 279 IGLV-J rearrangements from 725 patients with

CLL. The occurrence of secondary rearrangements of the IG LC gene loci (also

known as receptor editing) is an important diversification process, whereby the

undesirable or harmful BCR specificities can be altered, allowing the cell a

second chance at survival. We therefore also investigated the characteristics of

secondary LC rearrangements within this cohort. A further 2,346 IGKV-J

and 2,363 IGLV-J non-CLL IG sequences were collected from public databases,

to use as a reference data set with the aim of identifying disease-biased features

of SHM in CLL IGK/IGL gene rearrangements.

Restricted LC gene usage was observed in most cases belonging to subsets with

stereotyped HCDR3s. For example, IGKV1-39/1D-39 was used in 30/31 cases

of subset#1 (IGHV1/5/7-IGKV1(D)-39); IGLV3-21 was used in 36/37 cases of

subset#2 and all 15 cases of subset#4 employed the IGKV2-30 gene (See ap-

pendix I). In addition, subset-biased K/LCDR3 motifs were apparent in certain

groups of sequences utilising the same IGKV or IGLV gene. Specifically, all 30

IGKV1-39/1D-39 gene rearrangements of subset#1 carried notably long

KCDR3s (10-11 amino acids) generated by significant N region addition and

were characterised by the frequent creation of a proline at the IGKV-J joint

(26/30 cases). In contrast, all nine IGKV1-39/1D-39 rearrangements of subset#8

had 9 amino-acid-long KCDR3s with a junctional arginine present in 5/9 cases.

Hence, even though the potential for LCDR diversity is relatively limited com-

pared to the HCDR3, there nevertheless appears to be selection for specific resi-

dues within the CDR3.

While the SHM patterns of IGK/IGL gene rearrangements were typical of a

canonical SHM process at cohort level, distinctive patterns of mutational

targeting were clearly evident in certain subgroups of sequences, both in

terms of mutational load and at the level of specific amino acid changes.

Firstly, a clustering of R mutations in KCDR1 was observed for all IGKV

subgroups with the notable exception of the IGKV2 subgroup, which exhi-

bited preferential targeting to the KCDR2, especially in IGKV2-30 rear-

rangements of cases with stereotyped IGHV4-34/IGKV2-30 BCRs (sub-

set#4) (Figure 10). Interestingly, the over-targeting in this region could not

53

be accounted for by a preponderance of inherent hotspot targeting motifs in

the IGKV2 germline compared to other IGKV groups.

0

1

2

3

4

5

6

7

8

9

10

IGKV1 IGKV 2 IGKV3 IGKV4

CDR1 R/S

CDR2 R/S

Figure 10. R/S normalised mutation ratios in the KCDR1 and KCDR2.

Secondly, differences in mutational load were observed across the entire LC

gene in groups of sequences utilising the same IGKV or IGLV gene and/or

belonging to stereotyped subsets. In fact, significant differences were even

observed with regard to mutation status among groups of sequences utilising

different alleles of certain IGK/LV genes (specifically, the IGKV1-5,

IGLV1-51 and IGLV3-21 genes). Thirdly, recurrent amino acid changes

were observed at a high frequency in subset#2 (IGHV3-21/IGLV3-21), and

subset#4 (IGHV4-34/IGKV2-30). The subset #2 sequences carried an S-G

change at codon 110 in 46% of subset cases, compared to just 26% of non-

subset CLL cases and 5% of non-CLL cases. The recurrent changes in sub-

set#4 IGHV4-34 cases at codons 31,43 and 66 were present in subset CLL

cases at a frequency of 27-53%, compared to frequencies of only 6-18% of

hetergenous CLL sequences and 1.6-10% of non-CLL IGHV4-34 sequences.

Thus, these distinct amino acid changes were greatly under-represented in

these subgroups and could be considered as “CLL-biased”. In order to verify

that the recurrent change at codon 110 of subset#2 IGLV3-21 cases was a

bona fide mutation, the germline sequences of two cases were sequenced. It

became apparent that the S-to-G change was indeed a true mutation, since

the germline encoded a serine at that position. This analysis also revealed

that a C-to-T silent mutation at codon IMGT/LCDR3-108, observed in 60/92

IGLV3-21 rearrangements, is a previously unidentified allelic variant of the

IGLV3-21 gene.

Table 3. Frequency of stereotyped amino acid changes in the IGK/LV genes in CLL

subsets compared to non-subset and non CLL IG sequences.

IGLV3-21 sequences Change CLL-Subset#2 CLL-heterogeneous Non-CLLIMGT-LCDR3, codon 110 S-to-G 17/37 14/55 9/197IGKV2-30 sequences Change CLL-Subset#4 CLL-heterogeneous Non-CLLIMGT-KCDR1, codon 31 Y-to-H 10/15 1/17 6/62IMGT-KFR2, codon 43 Q-to-H 4/15 2/17 2/62IMGT-KFR3, codon 66 N-to-D 8/15 3/17 1/62

54

It is relevant to note that the IGKV2-30 gene sequences of subset#4 cases

carry several germline-encoded glutamic/aspartic acid residues throughout

its sequence (at codons 33, 68, 74, 86, 95, 97 and 98), along with one stereo-

typed somatically introduced glutamic acid residue in the KFR3. The pairing

of IGHV gene with an IGKV gene so enriched in negatively charged resi-

dues is possibly a mechanism to negate the positively charged HCDR3 of the

IGHV4-34 chain. Similar to the stereotyped changes evident in the HCDR1

and HCDR2 in the IGHV4-34 gene, introduction of the acidic, residue at

codon 66 is a further attempt to counter-balance the overall charge of the

BCR.

Multiple rearrangements were evident in 35% of lambda-expressing cases

and 11% of kappa-expressing cases (p<0.001). Moreover, a significant pro-

portion of CLL cases (63 cases; 26 kappa- and 37 lambda-expressing) with

monotypic LC expression were found to carry at least two potentially func-

tional rearrangements. Notably, 30% of such cases belonged to subsets with

stereotyped BCRs. This finding infers the occurrence of secondary rear-

rangements most likely created in the context of (auto) antigen-driven recep-

tor editing, particularly in the case of stereotyped subsets.

It has been reported that some LC germline-encoded specificities are inherent-

ly “dangerous” as exemplified by the IGKV1-17 gene, which is associated

with a more severe form of lupus nephritis due to the positive charge it bes-

tows on anti-DNA antibodies222. In this study, four of twelve potentially func-

tional IGKV1-17 rearrangements were detected among lambda-expressing

cases and of the remaining eight IGKV1-17 rearrangements in kappa-

expressing cases, two were co-amplified along with a second potentially fuc-

tional IGKV-J rearrangement. These findings are illustrative of an active re-

ceptor editing mechanism in CLL, whereby a primary undesirable/harmful

(yet functional) rearrangement with autoreactive potential in clonal CLL cells

is replaced by a considerably ‘safer’ secondary rearrangement.

In conclusion, the pairing of IGH and IGK/IGL gene rearrangements in CLL

malignant B cells is non-stochastic and the LC gene biases evident in CLL

possibly reflect selection by similar antigens in the CLL cell micro-

environment. Moreover, SHM targeting in CLL LCs appears to be just as

precise and, most likely, functionally driven as in heavy chains since LC

genes also display stereotyped mutations that are CLL- and subset-biased.

Analysis of secondary rearrangements provided evidence to support an ac-

tive process of receptor editing whereby some initial functional IGK rear-

55

rangements are selected against, in favour of a more “acceptable” specificity.

Thus, CLL is characterised not only by stereotyped HCDR3s but, rather by

stereotyped BCRs involving both chains, which create distinctive antigen

binding grooves.

What are the culprit antigens in CLL?

There is of course much interest as to what exactly the stimulating antigens

may be in CLL. In 2008, Lanemo-Myhinder et al. analysed the specificities

of monoclonal IG from 28 CLL cell lines and primary cell cultures223. They

observed that CLL cells expressing different IGHV genes bound a number of

cytoskeletal and cell surface self-antigens. Several of the IGs examined were

subset members; a recombinant subset#2 (IGHV3-21) antibody recognised

cofilin-1, the subset#5 antibody (IGHV1-69) recognised PRAP-1 and a sub-

set#1 antibody bound oxidised LDL. Notably a subset#32 (IGHV3-30.3

UM) antibody displayed cross-reactivity with phosphorylcholine motifs in

Streptococcus pneumoniae polysaccharides, vimentin and oxidised LDL.

Interestingly, most of the epitopes, such as vimentin and oxidised LDL, rec-

ognised by CLL antibodies in this study are expressed on apoptotic blebs as

neo-antigens in the process of cell breakdown. This kind of cross-reactivity

between bacteria and neo-antigens presented on apoptotic cells was evident

in a number of the other antibodies examined. These observations lead to the

hypothesis that CLL cells possibly originate from a population of B cells

which produce ‘natural IgM antibody’ which is involved in the clearance of

apoptotic cells, yet also bind and eliminate pathogenic bacteria.

Chu et al. also recently investigated the reactivity of monoclonal antibodies

(mAbs) derived from CLL patient cells and encoded by the IGHV1-69,

IGHD3-16, and IGHJ3 genes and carrying a stereotyped HCDR3 (sub-

set#6)224. This stereotyped heavy chain was also paired with an unmutated

LC gene most often encoded by IGKV3-20 which also carried a restricted

KCDR3 sequence. These antibodies were found to strongly bind cytoplasmic

structures present in HEp-2 cells which were subsequently identified to be

non-muscle myosin heavy chain IIA (MYHIIA)224. Like many of the antibo-

dy recognised by Rosèn’s group, MYHIIA appears to be presented on the

surface of cells which are undergoing stress or apoptosis225,226. Catera et al. subsequently reported that 60% of CLL monoclonal antibodies, particularly

those encoded by unmutated IGHV genes, bound structures on the surfaces

of apoptotic cells221. In fact, these antibodies recognised both neo-antigens

which are generated by oxidation, and antigens which are normally ex-

pressed within the cell and are relocated to the cell surface, during the apop-

totic process. Some of the epitopes generated by oxidation are similar to

those on bacteria and other microbes221. This re-confirms the idea that CLL

56

cells that display reactivity to apoptotic cells could be stimulated by self-

antigens displayed on cells in the normal process of cell death and cell

‘clear-up’ and thereby allow the CLL clone to receive transient yet chronic

antigen stimulation. Moreover, it is possible that cross-reactivity with other

infectious agents could allow the clone to propagate further thereby aiding

development and expansion of the leukaemia.

There are, however, many other stereotyped subsets where the stimulating

antigens have not been identified and it is unclear how broad the stimulating

antigen pool may be in CLL, even in the case of stereotyped subsets. Never-

theless, it would perhaps not be unreasonable to speculate that the character

and ubiquity of such antigens may play a very significant role in the clinical

presentation and prognosis in particular CLL groups. Further studies aiming

to correlate BCR structure with clinical parameters and outcome would be

required to fully clarify any relevant correlations.

57

CONCLUDING REMARKS

The bias in IGHV gene usage in CLL and the restricted HCDR3 composition

evident in almost a third of all patients is highly indicative of clonal selection

by specific antigens. Moreover, analysis of LC gene usage in CLL revealed

that there is also a predilection for utilisation of certain LC genes in CLL,

particularly among cases belonging to subsets defined according to HCDR3

homology. This pairing of certain IGH gene rearrangements with specific

IGK/IGL gene rearrangements, which also carry stereotyped K/LCDR3

structures, further substantiates the proposal of antigen selection as a signifi-

cant process in the pathogenesis of CLL. Furthermore, the finding that the

clinically distinct mutated and unmutated groups carry homologous HCDRs

and K/LCDR3s, implies that not just somatically mutated but also unmutated

cases appear to be antigen selected. It is also relevant that certain subsets,

such as the IGHV3-21/IGLV3-21 subset display a poor prognosis regardless

of IGHV mutation status, highlighting the biological influence of BCR ste-

reotypy on disease course.

Recurrent stereotyped mutations were evident within the IGHV genes and on

comparison to heterogeneous CLL and non-CLL sequences were found to be

specific, both to the respective CLL subset and CLL in general. Like IGHV

genes, IGK/LV genes also carried stereotyped mutations which were more

frequent in subset sequences compared to non-subset and/or non-CLL IG

genes. This evidence of stereotyped biased mutations occurring throughout

the IG sequence in both IGH and LC genes is strong evidence that not only

the HCDR3 and K/LCDR3 partake in antigen recognition, but other regions

of the molecule could be also actively involved in antigen binding. In addi-

tion, the stereotyped mutations evident in minimally mutated subsets imply

that there is a functional purpose even for individual amino acid changes.

Furthermore, the IGHV4-34/IGKV2-30 subset demonstrated that somatically

introduced alterations within both the IGHV and IGKV genes can potentially

counter-balance the autoreactive tendencies of positively charged HCDR3s.

This exemplifies the relevance and specificity of such SHM changes, since it

can mean the difference between a cell being allowed to enter the function-

ing B cell repertoire, or being induced to undergo apoptosis. The SHM cha-

racteristics in this and the IGHV3-21/IGLV3-21 also support the notion that

58

self-antigens could be responsible for ongoing stimulation of certain CLL

subsets, where intrinsic germline-encoded motifs are motifs conserved.

In summary, the finding of almost identical BCRs in distinct patient tumours

is, statistically speaking, highly unlikely to occur by chance when one con-

siders the mechanisms involved in creating antibody diversity. The fact that

similarity in IG sequence extends to the HCDR3, the most hypervariable

region of the molecule, is remarkable and strongly implicates the role of

antigenic selection, at least for certain subgroups of CLL cases. Furthermore,

the observation that such stereotyped BCRs were observed in CLL cases

from different geographical locations worldwide confirmed that the unique

features of stereotyped subsets are not solely accounted for by shared genetic

background within populations. Numerous stereotyped amino acid changes

were identified both in IGHV and IGK/LV genes, indicating that mutations

within the V region of IG genes may confer a functional advantage to clones.

Finally, the evidence of subset-biased mutations in CLL LC genes, along

with the drive for certain secondary LC gene rearrangements strongly imply

that LCs contribute significantly to the specificity of leukemic BCRs, in

association with defined heavy chains.

59

APPENDIX I

% Germline Predominant identity (average) IGKV/IGLV gene

1 53 IGHV1-2 IGHD6-19 IGHJ4 99.9 13-14 amino acids IGKV1-39/IGKV1D-39IGHV1-3IGHV1-18IGHV1-8IGHV5-aIGHV7-4-1

2 58 IGHV3-21 NA IGHJ6 97.2 9 amino acids IGLV3-21Rare IGHV3-48 IGHV3-30, IGHV3-11

3 25 IGHV1-69 IGHD2-2 IGHJ6 100 20-22 amino acids IGKV1-39/IGKV1D-39Rare IGHV4-34, IGHV1-8 IGKV3-11

4 20 IGHV4-34 IGHD5-5 IGHJ6 93.4 20 amino acids IGKV2-30IGHD4-17IGHD3-10

5 17 IGHV1-69 IGHD3-10 IGHJ6 99.9 20-21 amino acids IGKV1-33/IGKV1D-33IGLV3-21

6 18 IGHV1-69 IGHD3-16 IGHJ3 100 20-21 amino acids IGKV3-207 28 IGHV1-69 IGHD3-3 IGHJ6 100 20-25 amino acids IGLV3-9

Rare IGHV1-2, IGHV3-49, IGHV4-34, IGHV4-59

8 18 IGHV4-39 IGHD6-13 IGHJ5 99.8 18-19 amino acids IGKV1-39/IGKV1D-399 13 IGHV1-69 IGHD3-3 IGHJ6 100 20-25 amino acids Mixed

Rare IGHV4-34, IGHV3-23 IGHV3-21, IGHV3-30

12 9 IGHV1-2 IGHD3-22 IGHJ4 100 18-22 amino acids IGKV3-15IGHV1-46

13 4 IGHV4-59 IGHD2-15 IGHJ2 93.7 18 amino acids IGKV3-2016 7 IGHV4-34 IGHD2-15 IGHJ6 94.6 24 amino acids IGKV3-2019 8 IGHV1-69 IGHD3-9 IGHJ4/5 100 19-23 amino acids ND

IGHV3-48Rare IGHV3-74, IGHV4-31

28 5 IGHV1-2 IGHD1-26 IGHJ4 99.7 17-18 amino acids IGKV4-1

59 10 IGHV1-69 IGHD3-3 IGHJ4/5 99.9 12 amino acids IGKV2-28/IGKV2D-28IGHV1-58

HCDR3 lenghtSubset IGHV genes IGHD gene IGHJ gene n

61

ACKNOWLEDGEMENTS

This work was carried out at the Dept. of Genetics and Pathology and the

Dept. of Oncology, Radiology and Clinical Immunology, Rudbeck Laborato-

ry, Uppsala University. Financial support was provided by the Swedish Can-

cer Society and the Swedish Medical Council.

I would first and foremost like to sincerely thank my supervisor Richard Rosenquist Brandell for your knowledge, guidance and your encourage-

ment, especially when moral was low. For having time to discuss, no matter

what the time of day or night, and always being available to help, especially

in times of crisis! I have been very fortunate to have a supervisor who is so

genuinely involved and supportive of my work.

Kostas Stamatopoulos my co-supervisor, thank you for your endless know-

ledge not only on immunoglobulins but on almost everything from the By-

zantine empire to the best wines. I learned a huge amount from you. Thanks

also to you and Nikki for keeping me so well fed (both quality and quanti-

ty!)….I often returned from Thessaloniki with an extra kilo or two; very

useful for the Swedish winters. Chrysoula Belessi, thank you for your great

education on light chains, for your kind smile, and for always, always spot-

ting the mistakes before it’s too late!!

My co-supervisors; Gerard Tobin, for being the source of all knowledge in

the early days; and Gunilla Enblad, for advice and words of encouragement

along the way.

To the rest of my Greek family; Anastasia for being so cool, calm and col-

lected . . . at all times!, for being my personal chauffeur, and taking care of

me so well in Thessaloniki. Nikos, for always saying “there is a faster and

easier way to do this” you made what could have been very painful jobs

much, much easier (Also, thanks for always laughing at my ’jokes’. ...even if

it was mostly out of sympathy). Most of all thank you both for the tremend-

ous amount of work you put into our papers. Effie, for insisting we try some

tsipouro now and again and all those delicious snacks you brought on long

62

days in the office. You all showed me the reason for the reputation of the

warm Mediterranean hospitality, ��

To our European collaborators, Fred Davi and Paolo Ghia for your contri-

bution of data to these studies, your advice, opinions, encouragement and

never-ending enthusiasm. To our Swedish & Finnish collaborators; Anna Laurell, Anna Åleskog, Karin Karlsson, Göran Roos, Mats Merup, Ju-hani Vilpo, for provision of clinical samples and data. Special thanks to

Christer Sundström for clarification on the enigmatic lymphoid tissues.

To all my lecturers at the School of Biological Science, Dublin Institute of Technology for introducing me to the fascinating and often confusing world

of biology. You sowed the seeds of interest that led me to this point.

To all the admin people at Rudbeck; Ulla, Frida, Lena, Birgitta, Elisabeth, Pirkko, Britt-Marie, Gunilla Å, for keeping everything running like

clockwork behind the scenes and patiently answering my infinite questions.

To Kenneth Nilsson, Helena Jernberg Wirklund and Ulf Gyllensten for

providing such a stimulating research environment. To all the people at the

Genome centre past and present for being so fast and efficient with se-

quences. Viktor & Per Ivan for the good news that my hard-drive was ok

after that time I decided to give my laptop a bath.

Microsoft Excel, although you have caused me much heartache, we did

have some good times together too. All is forgiven.

All the kids at molhem; Arifin and Ja for being such genuine and generous

people (and knowing useful things such as which fruit really is the smelliest

in the world). Dijana, for greeting me with ‘hi fifi how is it going for you’,

that is just nice to hear every day. Ingrid T for being a long term companion

on the PhD journey and always asking how things are going, it feels like we

undertook some of the same battles together. Larry, for always having a

solution, and if not at hand, buying one! For being an exceptional travelling

companion and for trying to educate me on the intricacies and wonders of

ALL sports! (I-liked-it-a-lot). . . Lesley, lesley, the energy bunny, how is it

that your batteries never run out?, thank you for your interesting (ok, I mean

weird) views on life, love and white food (not to mention the ones that make

other food turn pink...) and for always knowing where my

keys/wallet/mobile are, or at least where I last had them. If the science thing

doesn’t work out maybe you would be interested in a PA position?? Maria N, the world feels like a much calmer, happier place when you are around,

(especially in this particular group of hyperactives!) Marie S, for being my

most persistent and dedicated language guru; proofreading my dodgy Swe-

dish must have been very disheartening at times (stort, stort tack) and for

63

being such good-humoured and sincere person. . .(and to your just as lovely

husband too!). Mattias, for solving all those niggling computer problems

that just won’t go away and figuring out mysterious data anomalies, it’s

thanks to you my computer never left the building via a window. Mi (sötis),

for being so sweet and thoughtful, and keeping the lab shipshape. You are so

cuuuute! Meena, for very tasty samplings of your home cooking and for

magically getting everything to work in the lab. Nicola for bringing a bit of

Dublin attitude to the office and for keeping me in stitches most of the time. .

you are deadly!!

I’d also like to thank the ‘Old gang’ down on the oncology corridor. Ulf for

your limitless repertoire of rude jokes and for keeping the spirit of rock alive

and well, Mattias B for knowing where to find long lost DNA samples!,

Mia thank you so much for all you help in the early days of the IGHV3-21,

its only now I fully appreciate how much work it (I) was, Åsa for being such

a cheerful office buddy and for inspiring me go to stallet every so often,

Ingrid G, Daniel M, Nongnit, Majlis B and Marie F, for really nice com-

pany and chats over coffee.

To all of the people on the third floor (and some floaters from the second !);

too many great people to mention you all by name, thank you for making

Rudbeck such a entertaining and positive place to work. Thanks especially to

“Rudbeck: The next generation” who try to keep me young in spirit (and

entice me with radioactive-coloured drinks at parties!). Special thanks to

Marina for all the advice on how to get through the thesis writing process in

one semi-sane piece.

To Anna L, Eva H, Lotta B and Rebeqa G for being really fun company on

the many courses and conferences we CLL-addicts seem to find ourselves at.

I’ve been fortunate make many gracious and supportive friends while here in

Sweden; Chris for being the one to moan to when I had the PhD blues, the

mumsiga fikas and all those great evenings involving beer and dancing. Jo-han for being the coolest mathematician I have ever met . . .and keeping the

passion for the irish language alive (Ros na Run should very grateful). Sa-rah, THE girl to talk about girls things with, thank you for taking care of me

as a new immigrant to Uppsala and being such a good friend, Paddy for

being full of so much useful information, everything you say is interesting or

funny and often both. . . Martin, Anne and Fanny for evenings of poker

games, bbqs, smelly fish and many, many laughs. Margaret for ‘you can do

it’ pep talks and making me believe that maybe one day I can manage to fit

in the 503 things you do on an average weekend. Daniel Ö for opening my

eyes to the wonders of eating frog legs in Johnny Foxes; it would probably

not have occurred without your vision.

64

To all my sweet friends in Dublin who I miss very, very much; Babs, Bren-dan, Natasha, Róisín Mc, Róisín and Orla T.

To my Swedish family; the Qvarnströms/Bladfors/Erikssons/Olssons,

thank you for your incredible kindness and warmth and treating me as a

member of your family from day one. I am very lucky to have been

‘adopted’ by such a truly lovely group of people.

Eoin and Niall the finest and funniest brothers a gal could wish for, love ya

both xxx

Mom and Dad, for all the love and support (financial, mental and emotional)

you have given me my whole life, saying thanks is not enough.

Fredrik, for believing in me when I didn’t myself, for your love, most of all

thank you for you.

65

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