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The role of antigen in the development of B-cell chronic lymphocytic leukemia

Hoogeboom, R.

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Citation for published version (APA):Hoogeboom, R. (2013). The role of antigen in the development of B-cell chronic lymphocyticleukemia.

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Robbert HoogeboomThe role of antigen in the developm

ent of B-cell chronic lym

phocytic leukemia

Robbert H

oogeboom

Uitnodiging

voor het bijwonen van de openbare verdediging van het proefschrift van

Robbert Hoogeboom

Getiteld:

The role of antigen in the development of B-cell chronic lymphocy-

tic leukemia

Op 8 mei 2013 om 14.00 in de Agnietenkapel van de Universiteit van Amsterdam,

Oudezijds Voorburgwal 231 te Amsterdam

Paranimfen:

Jeroen [email protected]

Sander [email protected]

Hoogeboom cover.indd 1 13-03-13 09:34


Robbert Hoogeboom

Hoogeboom.indd 1 19-03-13 08:43

Dissertation by Robbert Hoogeboom, University of Amsterdam, The Netherlands

The Research for this dissertation was conducted at the department of Pathology of the Academic

Medical Center (AMC) in Amsterdam, The Netherlands and supported by the Dutch Cancer Society,

grant UVA2006-3644.

Printing of this thesis was financially supported by the Dutch Cancer Society, the University of

Amsterdam and by the Department of Pathology of the AMC.

Cover illustration: Marjolijn Hoogeboom

Lay-out: Eelco Roos

Printed by Digital Printing Partners (DPP) in Houten, The Netherlands



Academisch proefschrift

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties ingestelde

commissie, in het openbaar te verdedigen in de Agnietenkapel

op woensdag 8 mei 2013, te 14:00 uur

door

Robbert Hoogeboom

geboren te Leiderdorp


Promotiecommissie

Promotor: Prof. dr. C.J.M. van Noesel

Copromotor: Dr. R.J. Bende

Overige leden: Prof. dr. R.A.W. van Lier

Prof. dr. C.E. van der Schoot

Prof. dr. E.F. Eldering

Dr. A.P. Kater

Dr. A.W. Langerak

Faculteit der Geneeskunde


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Contents

Chapter 1General introduction

Chapter 2MALT lymphoma derived rheumatoid factors are high-affinity non-polyreactive antibodies Blood, 2010; 116 (10): 1818 - 1819

Chapter 3A novel chronic lymphocytic leukemia subset expressing mutated IGHV3-7-encoded rheumatoid factor B-cell receptors that are functionally proficientLeukemia (2013); 27 (3): 738 - 740

Chapter 4A mutated B-cell chronic lymphocytic leukemia subset that recognizes and responds to fungiJ. Exp. Med, 2013; 210 (1): 59 - 70

Chapter 5B cell chronic lymphocytic leukemias expressing stereotypic IGHV4-34-encoded immunoglobulins are selected for a distinct N-acetyllactosamine epitopeManuscript in preparation

Chapter 6An in vitro culture system that selectively induces plasmacytoid differentiation and antibody secretion of B-cell chronic lymphocytic leukemiaManuscript submitted

Chapter 7General Discussion

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Chapter 8 – AppendixPrecursor B lymphoblastic lymphomas originating from follicular lymphomas express hypermutated immunoglobulin gamma heavy chains and surrogate light chainsManuscript in preparation

Chapter 9 SummaryNederlandse samenvatting

CV

Dankwoord

117

141


Chapter 1General introduction


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General introduction

Immunity in a “nutshell”

The B-cell receptor Diversity of the BCR repertoireB-cell receptor diversification and the germinal centre reactionDiversity and B-cell receptor self-reactivityB-cell receptor signaling

Malignant transformation of B-cells LymphomagenesisBCR repertoire in lymphomas

B-cell chronic lymphocytic leukemia CLL leukemogenesisThe B-cell receptor repertoire of CLLBiological and clinical features related to BCR stereotypy

Scope of this thesis

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Immunity in a “nutshell”The human body is constantly threatened by pathogens and the immune system continuously tries to clear these threats. The immune system consists of layers of defense that differ in specificity. Physical barriers, such as the skin and internal epithelial layers, provide a first line of defense. When pathogens breach these barriers, pattern recognition receptors sense general microbial motifs, such as polysaccharides and CpG-DNA, and initiate the so-called innate immune response. Phagocytic cells will bind, internalize and exterminate the invading pathogens and subsequently transport the antigens to draining lymph nodes, where they are presented to B- and T-cells that belong to the adaptive immune system.B- and T-cells express randomly generated antigen receptor genes, encoding the B-cell receptor (BCR) on B-cells and the T-cell receptor (TCR) on T-cells. B and T-cells are activated when these antigen receptors sense their cognate antigen, finally resulting in expansion and differentiation into effector and memory cells. Memory B- and T-cells are long-lived cells and quickly propagate to large numbers of effector cells upon re-encounter with their cognate antigen. Effector T-cells include T-helper cells, which direct the cellular and humoral immune response, and cytotoxic T-cells, which are capable of lysing infected cells. Effector B-cells are plasma cells, specialized in secreting large amounts of soluble BCRs, called antibodies (Ab) or immunoglobulins (Ig), which specifically bind and opsonize antigens. Abs are recognized by the complement system or by receptors on phagocytic cells and are essential for true elimination of harmful agents.

The B-cell receptorDiversity of the BCR repertoireTo be able to eliminate all potential threats, a near-unlimited Ab repertoire is required. To generate this diversity, every precursor B-cell will generate a unique BCR by somatic recombination of Ig heavy and light chain variable region gene segments. First, the variable domain of the Ig heavy chain (IGHV) is generated by rearranging an Ig heavy chain variable gene segment (IGHV) next to an Ig heavy chain diversity gene segment (IGHD) and an Ig heavy chain joining (IGHJ) gene segment (Figure 1). In total, the human genome encodes 39-46 IGHV, 23 IGHD and 6 IGHJ-genes, resulting in >6000 possible combinations1. Subsequently, the Ig light chain genes are rearranged in a similar fashion. The variable domain of the Ig light chain (IGLV) consists of gene segments that encode either the kappa or lambda isotype. The kappa Ig light chain gene repertoire contains 34-37 Ig kappa light chain variable genes (IGKV) and 5 Ig kappa light chain joining genes (IGKJ)2. The lambda repertoire


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consists of 30-33 Ig lambda light chain variable genes (IGLV) and 4 Ig lambda light chain joining genes (IGLJ)3. The total BCR diversity that potentially can be generated by the combined repertoire of IGHV and IGLV genes is estimated to be about 2-4 x 106. Diversity of the BCR is further increased by utilization of the IGHD-gene in any of the three reading frames and by exonucleases that may remove nucleotides from the junctions. Moreover, the enzyme Terminal deoxynucleotide Transferase (TdT) randomly inserts non-templated nucleotides, so-called N-nucleotides, which can potentially encode any amino acid, during the rearrangement process4. As a result the diversity that can be generated is estimated to be more than 1012 (Figure 1).The IGHV and IGLV both consist of four framework regions (FR) and three complementary determining regions (CDR). The FRs are important in maintaining the overall structure and the CDRs form the antigen binding site. The junction of the IGHV, IGHD and IGHJ gene segments, including the N-nucleotides, is the most diverse region of the BCR and forms the IGHV-CDR3. The IGHV-CDR3 is considered unique for every B-cell and is the most important region for determining the antigen specificity5.

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Figure 1: Diversity of the B-cell receptor repertoireThe variable domain of the BCR is generated by rearranging IGHV, IGHD and IGHJ gene segments and IGKV/IGLV or IGKJ/IGLJ gene segments for the Ig heavy and light chains, respectively. The total BCR repertoire that can be generated is estimated at >1012 unique BCRs.


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B-cell receptor diversification and the germinal centre reactionAfter successful rearrangement of the IGHV and IGLV genes, immature B-cells migrate to the spleen to complete maturation. Subsequently, mature naïve B-cells enter the circulation and secondary lymphoid tissues. BCRs of naïve B-cells generally bind antigens with low-affinity. When naïve B-cells encounter their cognate antigens, their IGHV and IGLV genes may be subjected to the process of somatic hypermutation (SHM). During SHM, the enzyme Activation-induced Cytidine Deaminase (AID) randomly introduces DNA alterations that give rise to further diversification of the BCR repertoire and may result in a higher affinity for the antigen6-8. SHM occurs in both germinal centre (GC) B-cells and in marginal zone (MZ) B-cells. MZ B-cells (also known as IgM memory B-cells) surround primary and secondary follicles in the spleen and mucosa-associated lymphoid-tissues. MZ B-cells generally respond to polyvalent T-cell independent antigens, such as bacterial polysaccharides9;10. Where and when MZ B-cells mutate their IGHV and IGLV genes and whether this is antigen-dependent is a longstanding matter of debate11-14. B-cells that recognize T-cell dependent antigens receive T-cell help through CD40-CD40L interactions, which initiates a GC reaction15. During a GC reaction, B-cells proliferate and mutate their IGHV and IGLV genes, while competing for survival signals. The B-cell clones that have obtained the highest affinity for the antigen, receive stronger activation signals, internalize and present more antigen to T-cells and subsequently receive more T-cell help. B-cells with lower-affinity for the antigen lose the competition for antigen and are prone to apoptosis16-18. The key principle of this process is that only the B-cell clones with the highest affinities are selected for terminal differentiation into memory B-cells and plasmablasts.During a GC reaction, GC B-cells may switch the gene that encodes for the constant domain of the Ig heavy-chain in a process called class switch recombination (CSR), which is also mediated by AID8. Before affinity maturation, naïve B-cells express the Cμ constant region simultaneously with Cδ, resulting in membrane expression of IgM and IgD, respectively. After affinity maturation, the isotype may switch into IgG, IgA and IgE (encoded by Cγ, Cα or Cε, respectively). All isotypes have distinct characteristics. For instance, IgM is secreted as a pentamer, resulting in high-avidity and IgA is the only isotype that can endure the harsh conditions of the gastro-intestinal tract. Moreover, each isotype is recognized by specific Fc-receptors, resulting in activation of distinct effector cell types, e.g. IgG activates Fcγ-expressing phagocytic cells and IgE specifically activates basophiles and mast cells via the Fce-receptor.

Diversity and B-cell receptor self-reactivityAlthough a broad diversity is required for efficient antigen recognition and elimination, the processes associated with the generation of this diversity also have a downside.


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It is inevitable that a large proportion of the IGHV-rearrangements results in self-reactive and poly-reactive BCRs19. Three mechanisms exist to silence self-reactive BCRs in pre-B-cells: receptor editing, anergy and deletion of the clone. During receptor editing, the IGLV of a self-reactive BCR is replaced by a secondary rearranged IGLV20;21. Alternatively, low-affinity self-reactive B-cells may be reprogrammed to a less-responsive state termed anergy. Anergic B-cells have higher levels of basal BCR signaling, lower membrane BCR expression and/or disturbed subcellular localization of co-stimulatory receptors, such as Toll-like receptors (TLR), resulting in a higher activation threshold for antigen-dependent BCR-signaling22-25. When receptor editing is ineffective and anergy is inappropriate, self-reactive B-cells are deleted by apoptosis26;27. 40% of immature and transitional B-cells express a self-reactive BCR and 7% of transitional B-cells express a poly-reactive BCR (Figure 2)19. However, during development to mature naïve B-cells, the percentage of clones with self-reactive BCRs further decreases to 20%, indicating a second checkpoint for deletion of self-reactivity at the transitional B-cell stage19. In MZ B-cells the percentage of self- and poly-reactive BCRs drops to 1-2%, suggesting that self- and poly-reactive B-cells are largely excluded from T-cell independent antigen responses9. In contrast, self- and poly-reactivity is increased in IgG-memory and IgD-only memory B-cell fractions28;29. Remarkably, the increase in self- and poly-reactivity in IgG-memory B-cells is often caused by SHM28. However, in terminally differentiated bone marrow plasma cells, which harbor higher loads of SHM than IgG-memory B-cells, the frequency of self- and poly-reactivity decreases again to 2-27% (Figure 2), suggesting selection against secreted self- and poly-reactive Abs30. Interestingly, self- and poly-reactivity may contribute to more efficient antigen binding due to heteroligation. According to this model, scarce antigens, such as HIV gp140, may be recognized by one antigen binding site of a BCR, whereas the other antigen binding site binds to a self-antigen, which may result in a higher affinity binding of the antigen31.

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Figure 2: Poly- and self-reactivity of the BCR repertoire during B-cell developmentPoly- and self-reactivity of the BCR repertoire is counterselected at the pre-B-cell stage, the transitional B-cell stage, the marginal zone B-cell stage and during terminal differentiation into plasma cells.


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B-cell receptor signalingThe BCR has two important functions. It regulates B-cell activation and differentiation through BCR signaling and it mediates antigen internalization and processing for presentation in MHC-II to CD4+ T-cells. The BCR forms a protein complex that consists of a membrane bound Ig and the Igα (also known as CD79a and mb-1) and Igβ (also known as CD79b and B29) heterodimer. Igα and Igβ are required for trafficking of the Ig to the membrane, for transmembrane signaling and for BCR endocytosis32-34. When BCRs encounter their cognate antigen, they cluster and form an immunological synapse35. BCR cross-linking by an antigen results in phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAM) of Igα and Igβ by the protein tyrosine kinase Lyn (Figure 3)36. Two mechanisms may regulate ITAM phosphorylation upon BCR cross-linking, i.e. BCR cross-linking either results in a conformational change, allowing recruitment of Lyn, or alternatively BCR cross-linking excludes negative regulators of BCR signaling, such as the phosphatases CD45 and CD14837. Subsequent to Lyn phosphorylation, Spleen tyrosine kinase (Syk) is recruited and


1Figure 3: BCR cross-linking by antigen induces four signaling pathwaysCross-linking of BCRs by antigen induces phosphorylation of Igα and Igβ by Lyn and recruitment of Syk. Syk phosphorylation activates a signaling complex containing Btk, Blnk, PI3K and PLCγ2, which triggers four downstream signaling routes (indicated in orange, yellow, dark blue and light blue). BCR signaling induces proliferation, differentiation, survival or apoptosis, depending on the differentiation state of the B-cell and the availability of co-stimulatory signals. See text for details.


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a signaling complex is formed that in addition to Syk and Lyn includes B-cell linker protein (BLNK, also known as SLP-65), Phospholipase C gamma 2 (PLCγ2), Bruton’s tyrosine kinase (Btk) and phosphatidylinositol-3-kinase (PI-3K)38. This signaling complex activates several different pathways. A first pathway involves activation of the guanine exchange factor Vav, p38 and JNK-kinase. A second pathway includes activation of Ras, Raf1 and subsequently extracellular regulated kinase (Erk). In parallel, protein kinase B (PKB, also known as Akt) becomes activated by PI3K, resulting in translocation of nuclear factor of activated T-cells (NF-AT) to the nucleus. Additionally, a pathway initiated by PLCγ2 induces intracellular Ca2+-fluxes and downstream translocation of the transcription factor nuclear factor kappa B (NFkB) to the nucleus38. Activation of all these pathways is required for a complete antigen response. Suboptimal cross-linking, for instance by monovalent soluble antigens, only partially activates the downstream signaling pathways39. Recently, it was shown that BCR signaling does not cease during endocytosis. Instead, the cellular location of the BCR may regulate the balance between the distinct signaling routes and thus the downstream outcome of signaling40.

Depending on the differentiation status of the B-cell and the availability of co-stimulatory signals, such as CD40- and TLR-mediated signaling, BCR signaling may induce survival, activation, proliferation, apoptosis or further differentiation. In pre-B-cells, BCR signaling is required to test that the BCR is functional and not reacting with self-antigens, resulting in survival and differentiation or resulting in receptor editing, anergy or apoptosis as discussed. In mature B-cells, a tonic signal from the BCR is required for survival, whereas antigen-specific signaling induces activation and proliferation41.

Malignant transformation of B-cellsLymphomagenesisDuring development, B-cells are repeatedly exposed to processes that bear an intrinsic risk of genomic instability and subsequent lymphoma development42. In pre-B-cells, the generation of a functional BCR by gene rearrangement requires double-stranded DNA breaks. In mature B-cells, the genome is subjected to modifications during SHM and CSR, which are also associated with double-stranded breaks. Lymphomas can be classified based on histological and cytological features, immunophenotype, location and genomic aberrations (Table 1). Many of these characteristics reflect the differentiation state of the B-cell from which the lymphoma has originated. For instance, precursor B-cell acute lymphoblastic leukemias may

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express the pre-BCR and TdT, whereas typical GC-derived lymphomas, such as follicular lymphomas (FL) and germinal center B-cell-like (GCB) diffuse large B-cell lymphomas (DLBCL), generally express the GC-markers CD10 and BCL6. Mantle cell lymphomas (MCL) mainly reside in the mantle zone of germinal centers and usually harbor a t(11;14) translocation involving BCL1, resulting in overexpression of the cell-cycle regulator cyclin D1. Extranodal mucosa-associated lymphoid-tissue (MALT) lymphomas arise at sites of chronic inflammation caused by bacterial infection or autoimmune disease. Approximately 25% of MALT-lymphomas carries a t(11;18) translocation, resulting in expression of a API2-MALT1 fusion protein that drives NFkB-activation. The NFkB inhibiting enzyme A20 (TNFAIP3) is frequently inactivated in t(11;18)-negative MALT-lymphomas43-45. FLs, Burkitt’s lymphomas (BL) and DLBCLs all originate from (post-)GC-cells. FLs retain the follicular architecture of the GC and virtually always carry a t(14;18) translocation, causing constitutive expression of the anti-apoptotic protein BCL2. Burkitt’s lymphomas are characterized by a t(8;14) translocation, resulting in over expression of the cell-cycle regulator C-Myc. Based on gene expression profiling, DLBCLs can be subdivided into activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subtypes46. GCB-type DLBCLs frequently harbor t(14;18) translocations and/or aberrations involving BCL6, a cell-cycle regulator and repressor of terminal differentiation47. ABC-type DLBCLs display constitutive NFkB signaling due to activating mutations in MYD88, CARD11, Igα and Igβ48-50 or due to inactivating mutations in NFkB inhibitors, such as A2051. In addition, Blimp-1, a transcription factor driving terminal differentiation,


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MZ: Marginal zone ; GC: Germinal center

Table 1: Common genomic aberrations in B-cell lymphomas

Lymphoma subtype Origin Most common genetic aberrations

Mantle cell lymphoma (MCL) Naive B-cell CyclinD1, t(11;14) (>95%)

MALT-lymphoma MZ B-cell MALT1, t(11;18) (~25%) A20 mutation (~20%)

Follicular lymphoma (FL) GC B-cell BCL2, t(14;18) (~25%)

Burkitt’s lymphoma (BL) GC B-cell Myc, t(8;14) (~100%)

Germinal center B cell-like diffuse large B cell lymphoma (GCB DLBCL) GC B-cell

BCL2, t(14;18) (~20%) BCL6 mutation/translocation (~30%)

Activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) Post GC B-cell

CARD11 mutation (~10%) CD79A/B mutation (~25%) MYD88 mutation (~30%) A20 mutation (~25%) Blimp-1 mutation (~25%)


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is frequently inactivated52;53. In summary, a large proportion of B-cell lymphomas carries genomic aberrations involving genes that regulate the cell-cycle (CyclinD1, C-Myc), inhibit apoptosis (BCL2), repress differentiation (BCL-6 and Blimp-1) or drive NFkB activation (MALT1, MYD88, CARD11, Igα /β and TNFAIP3). The genomic aberrations in high-grade lymphomas, including BLs and DLBCLs, often render the cells independent of the microenvironment for survival and proliferation. In contrast, the aberrations in low-grade lymphomas, such as FLs and MALT-lymphomas, are not sufficient for autonomous survival and proliferation, suggesting that additional signals from the microenvironment are required for maintenance and expansion of these tumors. In support, BCL2-translocations have been identified in non-transformed B-cells in healthy donors, indicating that this aberration is insufficient to drive transformation into a FL without additional aberrations or stimuli54.It has been hypothesized that in lymphomas without NFkB-activating genomic aberrations, chronic antigen stimulation may lead to chronic NFkB-signaling and a variety of circumstantial evidence indeed supports a role for antigen-dependent BCR signaling in the development of lymphomas42;55. All low-grade lymphomas and most high-grade lymphomas express a functional BCR and translocations involving the Ig locus are virtually always found at the non-productively rearranged Ig loci, leaving BCR expression intact42. Furthermore, somatic mutations in Ig genes that disturb BCR functioning or expression have not been identified in lymphomas, despite evidence for ongoing SHM42. Moreover, biased usage of IGHV has been reported for the majority of low-grade lymphomas, including MCL, MALT-lymphoma, splenic marginal zone B-cell lymphomas (SMZL) and B-cell chronic lymphocytic leukemia (CLL). Intriguingly, stereotypic IGHV-CDR3 amino acid motifs have been described in groups of MCL, MALT-lymphoma, SMZL and in particular in CLL (as discussed in detail below).A biased BCR repertoire not necessarily indicates a role for antigen in lymphomagenesis. For instance, healthy GC B-cells also have a restricted BCR repertoire when compared to naïve B-cells56, suggesting that a restricted BCR repertoire may also be a reflection of antigenic selection prior to and unrelated to transformation. However, the occurrence of groups of lymphomas with nearly identical BCRs, strongly suggests that antigen-dependent BCR signaling plays a pivotal role in the development of these groups of lymphomas. The challenge is now to identify what antigens these stereotypic BCRs bind. In this thesis, we investigated the antigen specificity of stereotypic BCRs derived from MALT-lymphomas and CLL.

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BCR repertoire in lymphomas

Mantle cell lymphomaNearly half of the BCRs expressed by MCLs are encoded by only four IGHV genes, i.e. IGHV3-21, IGHV4-34, IGHV1-8 and IGHV3-2357. Moreover, approximately 10% of MCLs express stereotypic IGHV-CDR3, suggesting that stimulation by distinctive antigens plays a role in the development of at least subsets of MCLs. In support, in MCL harboring somatically mutated IGHV, several shared SHM have been identified57.

MALT-lymphomaMALT-lymphomas display a restricted, somatically mutated BCR repertoire that varies among MALT-lymphomas depending on their localization. Up to 40% of gastric and salivary gland MALT-lymphomas express stereotypic BCRs with proven specificity for the Fc-tail of IgG, so called rheumatoid factors (RF)58. MALT-lymphomas of the ocular adnexae also express a biased IGHV-repertoire (~20% expresses IGHV4-34)59-61, whereas the IGHV repertoire of cutaneous extranodal MALT-lymphomas is not obviously restricted62. A proportion of gastric, cutaneous and ocular adnexae MALT-lymphomas is associated with H. pylori, B. burgdorferi and C. psitacii infection, respectively. Intriguingly, these lymphomas may respond to bacterial eradication63-65. Yet, these lymphomas do not express BCRs with high-affinity for these pathogens66. Most likely, these tumors are sustained by a chronically inflamed microenvironment, which is resolved by the treatment with antibiotics.

Splenic marginal zone B-cell lymphomasHepatitits C virus (HCV)-associated SMZL also frequently express membrane bound rheumatoid factors67. Among non-HCV-associated SMZL, 30% express the IGHV1-2*04 allele, suggesting that an, as yet unidentified, superantigen may drive development of this subset of SMZL68. Interestingly, five recombinant Ig from IGHV1-2*04-expressing SMZL displayed poly- and self-reactivity69.

Follicular lymphomaIn contrast to other low-grade lymphomas, no IGHV-CDR3 stereotypy has been reported for FLs. Intriguingly, FL IGHV frequently harbor SHM-induced glycosylation motifs, which may result in BCR cross-linking by environmental lectins70;71. In addition, it was recently reported that 26% of FLs express self-reactive BCRs and that stimulation with cognate self-antigens induces BCR signaling in vitro72.


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High-grade lymphomasHigh-grade lymphomas appear less dependent on the BCR for their survival. Nevertheless, physiological BCR signaling may contribute to disease expansion. This notion is experimentally supported by data from the Eμ-Myc mice crossed with transgenic mice that express BCRs specific for hen egg lysozyme (HEL). These mice develop aggressive mature B-cell lymphomas and when malignant B-cells from these mice are exposed to HEL, even more aggressive tumors arise73.

B-cell chronic lymphocytic leukemiaCLL leukemogenesisB-cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal accumulation of CD5+ B-cells74. Two types of CLL are being distinguished, carrying either unmutated IGHV (U-CLL) or somatically mutated IGHV (M-CLL), which are associated with unfavorable and favorable prognosis, respectively75;76. In contrast to M-CLL, U-CLL generally express CD38 and ZAP-70 and expression of these markers correlates with disease aggressiveness75;77. However, despite their diverse clinical behavior, both U-CLL and M-CLL generally share similar gene expression profiles78;79, resembling that of CD5+ B-cells80. Over 80% of CLL harbor chromosomal abnormalities, often already present in monoclonal B-cell lymphocytosis (MBL), a precursor state of CLL81. Moreover, evidence suggests that the MBL/CLL precursor cell might even acquire its first genomic “hit” in the bone marrow at the pre-B-cell stage82. Deletion of 13q is present in ~55% of CLL and is the most common abnormality, followed by deletion of 11q (18%), trisomy 12 (16%) and deletion of 17p (7%)83. The minimally deleted region of 13q contains the miR15a/16-1 cluster that controls cell cycle regulation84. The deletion of 11q usually involves the ATM gene, which plays a role in the detection of DNA damage and the activation of p53, a central regulator of the DNA damage response pathway85. p53 is also frequently inactivated by mutations and by deletion of 17p. The molecular mechanism by which trisomy 12 contributes to CLL progression is currently not understood. Recently, whole genome sequencing revealed recurrent point mutations in NOTCH1, SF3B1 and MYD8886-89. Activating mutations of the transcription factor NOTCH1 occur in ~10% of CLL (preferentially U-CLL) and are associated with trisomy 1286;87. Disruption of the spliceosome component SF3B1 is found in 5-10% of CLL and deregulates alternative splicing88;89. MYD88 activating mutations were identified in

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In the peripheral blood compartment, CLL cells circulate as resting cells that are stuck in the G0 phase79. CLL cells proliferate in so-called proliferation centers in lymph nodes, where they may encounter their cognate antigen. In accordance, CLL express immunophenotypic markers associated with antigen activation90 and lymph node CLL cells have gene expression profiles indicative of ongoing BCR signaling91, suggesting that chronic antigen stimulation may be involved in the pathogenesis of CLL. This hypothesis is supported by the remarkable therapeutic effects of agents that interfere with the BCR signaling pathway, i.e. Btk and Syk inhibitors92;93.

The B-cell receptor repertoire of CLLCompelling evidence for antigen-dependent BCR signaling in CLL is provided by studies on the BCR repertoire. It has long been recognized that CLL express a


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Figure 4: Stereotypic IGHV and IGLV rearrangements in CLL(A) List of common stereotypic CLL subsets. U: unmutated IGHV, M: mutated IGHV. (B) IGHV-CDR3 amino acid sequences of four subset #8 CLL. (C) IGHV-CDR3 amino acid sequences of four subset #4 CLL. Adapted from Stamatopoulos et al97.


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restricted IGHV repertoire, resulting in an overrepresentation of IGHV1-69, IGHV3-7 and IGHV4-3494. More importantly, over 30% of CLL can be grouped into subsets based on similarities of the amino acid sequences in the highly variable IGHV-CDR3 (Figure 4A)58;94-99. In a recent study containing IGHV sequences of 7596 CLL, 2308 CLL were subdivided over 952 subsets based on a stereotypic IGHV-CDR3 similar to that of at least one other CLL100. In some subsets, CDR3 stereotypy is germline-encoded and primarily the result of non-stochastic combination of IGHV, IGHD, and IGHJ gene segments (combinatorial stereotypy), such as subset #8 which comprises CLL that carry an unmutated IGHV4-39 (IGHV4-39-U) rearrangement with heterogeneous N-regions (Figure 4B)100. In other subsets, the CDR3 amino acid homology is mainly encoded by the non-templated N-nucleotides (junctional stereotypy). For instance, subset #4 contains CLL that express mutated IGHV4-34 (IGHV4-34-M) with a glycine and tryptophane as N1 region and two basic aminoacids (two arginines or an arginine and a lysine) in the N2 region, whereas IGHD gene segment usage is diverse (Figure 4C)99;100. The use of IGLV genes is also restricted in CLL. Moreover, stereotypic IGHV are usually paired with stereotypic IGLV (Figure 4A), resulting in the expression of near-identical BCRs by the leukemic clones of subsets of patients101;102. The occurrence of subsets of CLL expressing nearly identical BCRs strongly suggests that distinctive antigens are involved in the development of CLL. This hypothesis is supported by studies on SHM patterns in CLL IGHV. Stereotypic CLL harbor subset-biased replacement mutations in both the IGHV and IGLV103;104. This feature is most clear in M-CLL, but also minimally mutated U-CLL carry recurrent amino acid changes104. Moreover, within stereotypic subsets composed of CLL using different IGHV, SHM are found that eliminate germline-encoded variation between subset members100. Besides SHM also other mechanisms of diversification are subset-biased. For instance, in subset #2 CLL (IGHV3-21-M) one amino acid in the CDR2 is frequently deleted105 and subset #4 CLL (IGHV4-34-M) and subset #8 CLL (IGHV4-39-U) usually express BCRs of the IgG isotype96. Altogether, studies on the BCR repertoire in CLL univocally point towards an antigen-driven pathogenesis of subsets of CLL.

Biological and clinical features related to BCR stereotypyAntigen-dependent BCR-signaling may also remain important after transformation. This hypothesis is supported by several studies investigating the relation between IGHV structure and disease aggressiveness. CLL expressing a BCR classified in subset #1 (IGHV1-U) have a more progressive disease as compared to CLL expressing the same IGHV genes without stereotypic CDR3 motifs95 and subset

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#2 CLL (IGHV3-21-M) have an unfavorable prognosis independent of the IGHV mutation status106-108. In contrast, M-CLL using other IGHV3-family genes usually give rise to an indolent disease109;110. Altogether, these findings demonstrate that expression of stereotypic BCRs may influence the clinical behavior of CLL.Biologically, stereotypic CLL may also share unique features. Subset #2 CLL (IGHV3-21-M) and subset #4 CLL (IGHV4-34-M) display gene expression profiles subtly different from non-subset M-CLL expressing the same genes111;112. Moreover, subset-biased genomic aberrations have been identified in CLL of subset #2 (IGHV3-21-M), subset #4 (IGHV4-34-M) and subset #8 (IGHV4-39-U), suggesting that antigen-specificity might somehow influence genomic instability111;113;114. This notion is supported by the higher risk for Richter’s transformations of subset #8 CLL (IGHV4-39-U)115;116. Remarkably, patients with a subset #4 CLL (IGHV4-34-M) frequently share increased Epstein-Barr virus (EBV) and cytomegalovirus (CMV) viral loads, indicating a shared history of viral infections117. Collectively, these data point to an association between B-cell receptor specificity and disease biology.In the last few years, it has become clear that the majority of U-CLL express low-affinity poly-reactive BCRs, recognizing a variety of self- and exo-antigens, such as DNA, LPS, insulin, oxidized LDL, and the cytoskeletal antigens vimentin and myosin118-122. In addition, most U-CLL BCRs bind antigens exposed on apoptotic cells, which correlates with disease agressiveness119;122;123. In contrast, M-CLL BCRs do not bind apoptotic cells and are generally not poly-reactive119;121-123. The specificity of M-CLL with stereotypic BCRs has remained unknown.

Scope of this thesisThe studies in this thesis aim to elucidate the role of antigen and BCR stimulation in the development of lymphomas and CLL in particular. In chapter 2, we study the specificity of recombinant MALT lymphoma-derived soluble BCRs. In chapters 3, 4 and 5, we analyze the specificity of stereotypic M-CLL BCRs and the effect of cognate antigen stimulation on primary CLL cells in vitro. In chapter 6, we explore new methods to produce CLL-derived immunoglobulins by in vitro cell culture.

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123. Chu CC, Catera R, Zhang L et al. Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin. Blood 2010;115:3907-3915.

1


MALT lymphoma-derived rheumatoid factors are nonpolyreactive high-affinity antibodies

Robbert Hoogeboom, Richard J. Bende and Carel J.M. van Noesel

Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Blood (2012); 116 (10): 1818-1819

Chapter 2


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2


33

MALT-lymphoma-derived rheumatoid factors are non-polyreactive

To the editor of Blood We have read with particular interest the paper of Craig et al1, describing that 6 of 7 human mucosa-associated lymphoid tissue (MALT) lymphoma-derived immunoglobulins (Igs) bound various antigens with intermediate affinity, thus implying that MALT lymphomas express polyreactive antigen receptors. We previously reported that MALT lymphomas frequently express Igs, which are homologous to canonical rheumatoid factors (RFs), encoded by IGHV1-69-IGHJ4/IGKV3-20 and IGHV3-7-IGHJ3/IGKV3-15 IGHV/IGKV rearrangements and that recombinant Igs derived of these MALT lymphomas indeed bound human IgG2. Interestingly, we noticed that the panel of MALT lymphoma Igs produced by Craig et al also contained 3 cases with homology to IGHV1-69 RFs (hu-1 and hu-7) and IGHV3-7 RFs (hu-2), although it is noted that 2 of these MALT lymphoma RFs (i.e. hu-2 and hu-7) were not co-expressed with the canonical IGKV3-20 and IGKV3-15 genes as described in literature of 14 of 14 IGHV1-69 RF and of 10 of 10 IGHV3-7 RF cases, respectively2-5.Prompted by the results by Craig et al, we tested the binding characteristics of 7 of our MALT lymphoma Igs, 4 of which were homologous to canonical RFs (i.e., M5, M6, M11, and M222) in ELISAs. This study was conducted in accordance with the Declaration of Helsinki and the ethical standards of the research code committee on human experimentation of the Academic Medical Center of Amsterdam. As controls, we included 2 IGHV-unmutated and 2 IGHV-mutated B-cell chronic lymphocytic leukemia (CLL)-derived Igs, which are reported to be polyreactive and nonpolyreactive, respectively6. In contrast to the data presented by Craig et al, 6 of 7 MALT lymphoma Igs, including the 4 RFs, reacted only with IgG or were nonreactive (Figure 1A), as may be expected from somatically mutated Igs. Only MALT lymphoma Ig M23 did bind several antigens. As expected, the 2 mutated CLLs were nonreactive whereas the 2 unmutated CLLs bound to essentially all antigens tested. In general, the M23 Ig showed a lower degree of polyreactivity compared with the unmutated CLL-derived Igs.To further explore the reactivity of the MALT lymphoma Igs, we used the recombinant Igs in immunohistochemical stainings of tissue microarrays (TMAs) containing 21 paraffin-embedded normal human tissues. None of the MALT lymphoma Igs reacted with any of the tissues on the TMA, except for M23, which showed broad reactivity (Figure 1B). Similarly, the unmutated CLL Igs stained all the tissues tested, even at low concentrations. It is noted that the polyreactive MALT lymphoma M23 differs from the other MALT lymphomas in that it harbors a t(11;18)(API-MALT). This chromosomal translocation results in the constitutive activation of the nuclear factor κB (NF-κB) pathway, potentially rendering the cells independent of antigen receptor signals and thus disturbing the process of antigenic selection.

2


34

2

Figure 1: Reactivity of MALT lymphoma-derived immunoglobulins.(A) Seven recombinant MALT lymphoma-derived IgMs (M5, M6, M8, M11, M14, M22, and M23), 2 unmutated CLL-derived IgMs (CLL46 and CLL55), 2 mutated CLL-derived IgMs (CLL9 and CLL19), and 1 IgM control anti-erythrocyte Rhesus D were tested for reactivity in ELISAs to IgG, ssDNA, insulin, lipopolysaccharide, actin, and vimentin, as described previously2. The OD450 nm is plotted without subtraction of background OD450 nm. MALT lymphoma–derived Igs are represented by blue lines. Red lines represent control Igs. The 4 MALT lymphoma–derived RFs are represented by dashed blue lines. (B) Immunohistochemical stainings of human TMAs, containing 21 normal human tissues, with 7 recombinant MALT lymphoma- derived IgMs, one mutated CLL–derived IgM and one unmutated CLL–derived IgM. Displayed are kidney, duodenum, liver and muscle stained with 5 μg/mL of recombinant IgM, except for CLL55, which was used at 1 μg/mL. Staining was visualized using mouse anti-human IgM (clone MH15, Sanquin), followed by the Powervision+ detection system (ImmunoVision Technologies). Images were acquired with a Leica DM5000B microscope coupled to a Leica DFC500 camera (Leica Microsystems) at the original magnification of 200x.


35

MALT-lymphoma-derived rheumatoid factors are non-polyreactive

Our finding that MALT lymphoma RFs are monoreactive, is concordant with several papers in which IGHV1-69/IGKV3-20 RFs are compared with unmutated IGHV1-69-encoded CLL Igs, demonstrating that mutated IGHV1-69/IGKV3-20 RFs are nonpolyreactive, whereas unmutated IGHV1-69 CLL Igs show low-affinity binding to multiple antigens, including IgG7,8.We conclude that IGHV1-69/IGKV3-20 and IGHV3-7/IGKV3-15 MALT lymphoma-derived Igs are ligand-selected, monospecific high-affinity antibodies.

Acknowledgment: The authors thank A. R. Musler for generating the tissue microarrays.

References1. Craig VJ, Arnold I, Gerke C, et al. Gastric MALT lymphoma B cells express polyreactive, somatically

mutated immunoglobulins. Blood. 2010;115(3):581-591.2. Bende RJ, Aarts WM, Riedl RG, de Jong D, Pals ST, van Noesel CJM. Among B cell non-Hodgkin’s

lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity. J Exp Med. 2005;201(8):1229-1241.

3. Borretzen M, Randen I, Natvig J, Thompson K. Structural restriction in the heavy chain CDR3 of human rheumatoid factors. J Immunol. 1995;155(7):3630-3637.

4. De Re V, De Vita S, Marzotto A, et al. Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood. 2000;96(10):3578-3584.

5. Charles ED, Green RM, Marukian S, et al. Clonal expansion of immunoglobulin M+CD27+B cells in HCV-associated mixed cryoglobulinemia. Blood. 2008;111(3):1344-1356.

6. Hervé M, Xu K, Ng Y-S, et al. Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity. J Clin Invest. 2005;115(6):1636-1643.

7. Martin T, Crouzier R, Weber J, Kipps T, Pasquali J. Structure-function studies on a polyreactive (natural) autoantibody. Polyreactivity is dependent on somatically generated sequences in the third complementarity-determining region of the antibody heavy chain. J Immunol. 1994;152(12):5988-5996.

8. Martin T, Duffy SF, Carson DA, Kipps TJ. Evidence for somatic selection of natural autoantibodies. J Exp Med. 1992;175(4):983-991.

2


A novel chronic lymphocytic leukemia subset expressing mutated IGHV3-7-encoded rheumatoid

factor B-cell receptors that are functionally proficient

Robbert Hoogeboom1, Thera A. Wormhoudt1, Martin R. Schipperus2, Anton W. Langerak3, Deborah K. Dunn-Walters4, Jeroen E.J. Guikema1, Richard J. Bende1

and Carel J.M. van Noesel MD1.

1 Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

2 Department of Hematology, HagaZiekenhuis, The Hague, The Netherlands3 Department of Immunology, Erasmus MC, Erasmus University,

Rotterdam, The Netherlands4 Peter Gorer Department of Immunobiology, King’s College London School of Medicine,

London, United Kingdom

Leukemia (2013); 27 (3): 738 - 740

Chapter 3


38

3


39

A subset of CLL expresses IGHV3-7-encoded rheumatoid factors

Letter to the editor of LeukemiaRheumatoid factors (RFs) are auto-antibodies, mostly of the IgM isotype, that specifically bind to the Fc portion of IgG. RFs are transiently produced in healthy individuals in response to infections and after immunization with mismatched red blood cells (RBCs). RFs are chronically produced in patients suffering from rheumatoid arthritis (RA), Sjögren syndrome (SS) and in hepatitis C virus (HCV)-induced type-II mixed cryoglobulinemia (MC-II) (reviewed in 1). Analyses of RFs in MC-II and in donors immunized with mismatched RBCs revealed four groups of stereotyped immunoglobulin (Ig) VH-DH-JH rearrangements, each with distinct heavy chain complementary determining region 3 (HCDR3) amino acid sequences: two with IGHV1-69/JH4 rearrangements, designated V1-69-RF and RF-WOL, and two with IGHV3-7/JH3 and IGHV4-59/JH2 rearrangements, designated V3-7-RF and V4-59-RF, respectively2-4.Remarkably, similar stereotyped RF-rearrangements are frequently expressed in malignant B-cell lymphomas2;5;6. Previously, we have shown that recombinantly produced V1-69-RF, V3-7-RF and RF-WOL, derived from gastric and salivary gland marginal zone B-cell lymphomas (MZBCL), bind with high-affinity to IgG in vitro2;7. V1-69-RF, V3-7-RF, RF-WOL and V4-59-RF rearrangements are also frequently expressed in HCV-associated lymphomas, splenic marginal zone lymphomas and in individual cases of ocular MZBCLs and diffuse-large B-cell lymphomas (DLBCL)5;6;8-10. Recently, Kostareli et al reported 12 cases of B-cell chronic lymphocytic leukemia (CLL) expressing V4-59-RF10 and Fazi et al reported two cases of V3-7-RF in monoclonal B-cell lymphocytosis11. In this study, we analyzed HCDR3 amino acid sequences of 81 IGHV3-7-expressing CLL and identified 3 cases with strong homology to 25 previously reported V3-7-RFs from both healthy and malignant B-cells (Table 1 and Table S1). These 25 V3-7-RF cases include 9 with proven RF-activity in vitro. The three newly found CLL cases, CLL81, CLL311 and CLL416, utilized the IGHD3-22 gene segment and the IGHJ3 segment shared by 15/24 and 22/24 of V3-7-RFs, respectively. The CLL V3-7-RFs displayed >70% of homology with a V3-7-RF HCDR3 consensus sequence, defined by the amino acids used by the majority of V3-7-RF cases (Table 1). Of note, the V3-7-RFs also shared amino acids encoded by non-templated N-region nucleotides (Figure 1A). Two of the newly identified CLL V3-7-RF cases (CLL81 and CLL416) expressed the stereotyped IGKV3-15 Ig light chain (IGLV) reported in 10/10 V3-7-RF cases (Table 1). In CLL311, the V3-7-RF Ig heavy chain (IGHV) was paired with an IGKV3-20-encoded IGLV. Remarkably, similar IGKV3-20 rearrangements were found in the other groups of stereotyped RFs.The CLL V3-7-RFs were heavily mutated (94.5 ± 1.9% average homology to the

3


40

3

Nam

e (IG

HV3

-7)

IgH

-rea

rran

gem

ent

HC

DR

3H

omol

ogy

(%)

IgL-

rear

rang

emen

tLC

DR

3So

urce

RF

activ

ity

Con

sens

us

CARGDYYDSSGS FIDAFDIW

100%

(18/

18)

CQH YNNWPPWT F

CLL

81 (9

4.4%

)aIG

HV

3-7/

IGH

D3-

22/IG

HJ3

C--------ATY -N-----W

78%

(14/

18)

IGK

V3-

15/IG

KJ1

CQH YNNWPPWT F

CLL

yes

CLL

311

(96.

4%)a

IGH

V3-

7/IG

HD

3-22

/IGH

J3C----F--T--Y -N-----W

78%

(14/

18)

IGK

V3-

20/IG

KJ4

CQQ YGSSPLT F

CLL

ndC

LL41

6 (9

2.7%

)aIG

HV

3-7/

IGH

D3-

22/IG

HJ3

C----F-E---- YN----VW

72%

(13/

18)

IGK

V3-

15/IG

KJ1

CQQ YNNWPPWT F

CLL

ndH

CV

64 (9

6.9%

)IG

HV

3-7/

IGH

D3-

22/IG

HJ3

C-----S----Y Y-E---VW

72%

(13/

18)

MB

L (A

typi

cal-C

LL)

ndH

CV

41IG

HV

3-7/

IGH

D3-

10/IG

HJ3

C-----H-GGN- -------W

78%

(14/

18)

MB

L (C

LL-li

ke)

ndM

6 (9

4.8%

)IG

HV

3-7/

IGH

D3-

22/IG

HJ3

C-----F----- -------W

94%

(17/

18)

IGK

V3-

15/IG

KJ1

CQH YNNWPPWT F

Gas

tric

MZB

CL

yes

Pat

3 (9

9.3%

)IG

HV

3-7/

IGH

D4-

17/IG

HJ3

C----- -- -D YN-----W

72%

(13/

18)

IGK

V3-

15/IG

KJ1

CQQ YNNWPPWT F

Gas

tric

MZB

CL

yes

Cas

e 18

(94.

3%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C----F--T-A -----NVW

67%

(12/

18)

Gas

tric

MZB

CL

ndM

L25

C-----DYD--- -V-----W

83%

(15/

18)

Gas

tric

MZB

CL

ndM

L27

C----------H -S-----W

89%

(16/

18)

Gas

tric

MZB

CL

ndM

L39a

CT----S--DS- -N----VW

67%

(12/

18)

Gas

tric

MZB

CL

ndJN

1823

84 (9

8.8%

)IG

HV

3-7/

IGH

D3-

22/IG

HJ3

C----------Y YH-----W

83%

(15/

18)

Ocu

lar M

ZBC

Lnd

M5

(94.

4%)

IGH

V3-

7/IG

HD

3-3/

IGH

J3C----F W--D -------W

72%

(13/

18)

IGK

V3-

15/IG

KJ1

CQH YNNWPPWT F

Sal

ivar

y gl

and

MZB

CL

yes

SH

/SC

(97.

2%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C------E---N -------W

89%

(16/

18)

IGK

V3-

15/IG

KJ1

CQQ YNNWPPWT F

Sal

ivar

y gl

and

MZB

CL

ndIs

olat

e 10

(98.

3%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C----------Y -S----TW

83%

(15/

18)

Sal

ivar

y gl

and

MZB

CL

ndM

A-1

(99.

7%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C---------- -H-----W

89%

(16/

18)

Sia

lade

nitis

ndM

A-2

(99.

3%)

IGH

V3-

7/IG

HD

3-10

/IGH

J3C-------- -- -V-----W

89%

(16/

18)

Sia

lade

nitis

ndS

S-1

0 (9

5.0%

)IG

HV

3-7/

IGH

D3-

22/IG

HJ3

C---------DY Y------W

83%

(15/

18)

Sia

lade

nitis

ndE

J-7

(93.

7%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C-----S---D- Y- ---VW

72%

(13/

18)

DLB

CL

ndC

ase

5IG

HV

3-7/

IGH

D3-

22/IG

HJ3

C-------T--Y -S-----W

83%

(15/

18)

IGK

V3-

15/IG

KJ1

CQH YNNWPPWT F

HC

V-as

soci

ated

lym

phom

and

Cas

e 6

IGH

V3-

7/IG

HD

3-16

/IGH

J3C-----DN--D -V-----W

72%

(13/

18)

IGK

V3-

15/IG

KJ1

CQQ YNNWPPWT F

HC

V-as

soci

ated

lym

phom

and

Cas

e 10

IGH

V3-

7/IG

HD

5-24

/IGH

J3C-----F-DD-P ---V-NVW

61%

(11/

18)

HC

V-as

soci

ated

lym

phom

and

RF-

M7

(98.

6%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C--------G-D Y------W

83%

(15/

18)

IGK

V3-

15/IG

KJ1

R

heum

atoi

d fa

ctor

yes

RF-

TT9

(96.

2%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C----------N --D---VW

83%

(15/

18)

IGK

V3-

15/IG

KJ1

H

D im

mun

ized

with

RB

Cs

yes

RF-

MR

5 (9

1.6%

)IG

HV

3-7/

IGH

D3-

22/IG

HJ3

CV----N-G-D-G-H----LW

67%

(12/

18)

IGK

V3-

15/IG

KJ1

H

D im

mun

ized

with

RB

Cs

yes

RF-

MR

41 (9

1.0%

)IG

HV

3-7/

IGH

D3-

22/IG

HJ3

C-----V-GNY-G–H-----W

67%

(12/

18)

IGK

V3-

15/IG

KJ1

H

D im

mun

ized

with

RB

Cs

yes

TB-1

-P5

(97.

2%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C-- -----R- -H-----W

78%

(14/

18)

His

tory

of T

Bye

sL1

4453

(91.

3%)

IGH

V3-

7/IG

HD

3-22

/IGH

J3C-------Y--N Y-----AW

78%

(14/

18)

IGK

V3-

15/IG

KJ1

CQH YNNWPPWT F

Rhe

umat

oid

artri

tisye

sU

N_1

8102

(95.

5%)a

IGH

V3-

7/IG

HD

1-26

/IGH

J3C-----SG-YSRR-N----VW

61%

(11/

18)

HD

IgM

-mem

ory

B-c

ell

ndU

N_3

5230

(93.

5%)a

IGH

V3-

7/IG

HD

4-23

/IGH

J3C-----GGD-AI -V---A-W

61%

(11/

18)

HD

IgM

-mem

ory

B-c

ell

nd

Per

cent

ages

nex

t to

nam

e re

flect

iden

tity

to g

erm

line

IGH

V3-

7. H

omol

ogy

is c

alcu

late

d as

the

per

cent

age

of a

min

o ac

ids

shar

ed w

ith t

he c

onse

nsus

seq

uenc

e. H

CD

R3:

Im

mun

oglo

bulin

hea

vy c

hain

com

plem

enta

rity-

dete

rmin

ing

regi

on 3

. LC

DR

3: I

mm

unog

lobu

lin li

ght

chai

n co

mpl

emen

tarit

y-de

term

inin

g re

gion

3.

CLL

: ch

roni

c ly

mph

ocyt

ic

leuk

emia

. MB

L: m

onoc

lona

l B-c

ell l

ymph

ocyt

osis

. MZB

CL:

mar

gina

l zon

e B

-cel

l lym

phom

a. D

LBC

L: d

iffus

e-la

rge

B-c

ell l

ymph

oma.

TB

: Myc

obac

teriu

m tu

berc

ulos

is in

fect

ion.

H

D: h

ealth

y do

nor.

RB

Cs:

Red

blo

od c

ells

. a N

ewly

iden

tified

in th

is s

tudy

. nd:

not

det

erm

ined

.

Tabl

e 1:

Ste

reot

yped

V3-

7-R

F re

arra

ngem

ents

and

CD

R3

amin

o ac

id s

eque

nces

from

CLL

, B-c

ell l

ymph

omas

and

non

-neo

plas

tic B

-cel

ls


41


IGHV3-7 germline) and shared replacement mutations Q58P, Y66F and Q90E (Figure 1B and Table S2). Identical somatic mutations are also frequent among the 25 previously reported V3-7-RFs, but are rare among other IGHV3-7-expressing CLL (Table S2). In addition, all CLL V3-7-RFs shared silent mutations at codon 40, which encodes a serine (S40). None of the 23 non-CLL V3-7-RFs carried replacement mutations at S40 either, whereas replacement mutations at S40 were common in the entire cohort of IGHV3-7-expressing CLL (19% of cases) (Table S2), suggesting that the serine at this position is important for IgG reactivity.To prove RF-activity, we recombinantly produced the BCR of CLL81 as soluble IgM (sIgM) as described in the Supplementary Methods. CLL81 sIgM indeed clearly bound to IgG in ELISA, apparently with higher affinity than V3-7-RFs of two MZBCLs (M5 and M6). Soluble IgM of two unrelated IGHV3-7-expressing CLL did not bind to IgG in ELISA (Figure 1C). Binding to IgG was abolished when either the IGHV was replaced with an unrelated IGHV3-7 or when the endogenous IGLV was replaced with that of an unrelated CLL (Figure 1D), showing that the combination of Ig heavy and light chains in V3-7-RF is selected for IgG-binding capability.Several lines of evidence suggest a role for antigen-dependent growth of leukemic cells in CLL. Direct proof, however, is lacking, as the natural ligands of CLL BCRs are generally not known. Now able to address this issue, we found that stimulation of primary cells of patient CLL81 with IgG-aggregates (a model for immune-complexes) induces proliferation in a proportion of CD19+CD5+ tumor cells (Figure 1E), whereas IgG-aggregates did not induce proliferation of IGHV-mutated control CLL cells (Figure 1F). This indicates that at least a proportion of cells express functionally proficient BCRs, implying that interactions with IgG-containing immune-complexes may drive proliferation of leukemic cells in vivo as well.RF-expressing B-cell lymphomas are associated with Sjögren’s syndrome or infections (i.e. HCV, Helicobacter pylori and Chlamydophila psittaci infection)12, suggesting that RF-specificity might provide a growth advantage within the context of chronic inflammation. Serological analysis of patient CLL81 did not reveal RFs or antibodies for HCV, citrullinated protein or nuclear components (data not shown), suggesting that this case is not associated with HCV infection or auto-immune diseases. In general, CLL-like stereotyped BCR sequences are rare in B-cells of healthy donors13-15. Interestingly, we identified two V3-7-RFs (UN_18102 and UN_35230) among 1080 IGHV3-7 BCR sequences obtained from healthy donors (Table 1 and Table S1). These V3-7-RFs may have arisen as a by-product of a vaccination with pneumovax II (Sanofi Pasteur MSD, Maidenhead, UK) and influvac (Solvay, Southampton, UK), received 28 days earlier. V3-7-RFs have also been reported in

3


42

3


43


healthy donors with a history of tuberculosis and after immunization with mismatched RBCs. It is conceivable that conditions that induce inflammation and high levels of immune-complexes drive expansion of RF B-cell clones with a subsequent risk of genetic derailment.In conclusion, we have identified a new subset of CLL expressing stereotyped IGHV3-7-encoded BCRs. These BCRs display CDR3 homology with previously reported rheumatoid factors and specifically bind IgG in ELISA. Binding of the IgG-epitope requires both the stereotyped IGHV3-7 heavy chain as well as the stereotyped IGKV3-15 light chain. Most importantly, we show that interaction of the V3-7-RF-expressing leukemic cells with their cognate antigen induces proliferation, providing proof of concept for antigen-sustained tumor cell expansion.

Conflict of interestThe authors declare no conflict of interest

AcknowledgementsThis work was supported by the Dutch Cancer Society, grant UVA 2006-3644.

3

Figure 1: A subset of CLL expresses mutated IGHV3-7-encoded rheumatoid factors (V3-7-RFs) that are functionally proficient. (A) Immunoglobulin heavy chain complementary determining region 3 (HCDR3) amino acid sequences of three CLL V3-7-RF cases. (B) Schematic representation of the immunoglobulin heavy chains of CLL V3-7-RFs. Lollipop-shaped symbols indicate somatic mutations as compared to the IGHV3-7 germline. Closed and open circles represent replacement and silent mutations, respectively. Boxes highlight shared mutations. (C) Anti-IgG ELISA of CLL81 (V3-7-RF), two non-RF IGHV3-7-expressing CLL (CLL82 and CLL85) and two marginal zone B-cell lymphoma-derived V3-7-RFs (M5 and M6). (D) Anti-IgG ELISA of CLL81 IGHV coupled to the IGLV of CLL82 and CLL81 IGLV coupled to the IGHV of CLL82. Representative CFSE stainings of CD19+CD5+-cells of CLL81 (E) or control IGHV-mutated CLL (F) after 8 days of culture on CD40L-expressing fibroblasts either unstimulated (left panel) or stimulated with IgG (right panel).


44

References

1. Newkirk MM. Rheumatoid Factors: Host Resistance or Autoimmunity? Clinical Immunology 2002;104:1-13.

2. Bende RJ, Aarts WM, Riedl RG et al. Among B cell non-Hodgkin’s lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity. J.Exp.Med. 2005;201:1229-1241.

3. Borretzen M, Randen I, Natvig JB, Thompson KM. Structural restriction in the heavy chain CDR3 of human rheumatoid factors. The Journal of Immunology 1995;155:3630-3637.

4. De Re V, De Vita S, Gasparotto D et al. Salivary gland B cell lymphoproliferative disorders in Sjögren’s syndrome present a restricted use of antigen receptor gene segments similar to those used by hepatitis C virus-associated non-Hodgkins’s lymphomas. Eur.J.Immunol. 2002;32:903-910.

5. De Re V, De Vita S, Marzotto A et al. Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 2000;96:3578-3584.

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