). It is noted that the B-CLLs that displayed in vitro RF reactivity (POR, AIG, and SMI) as well as the two B-CLLs (CLL-011 and ID-74) that displayed CDR3 homology to two unmutated RFs, all belong to the IgV-unmutated B-CLL subset. This clearly contrasts with the MALT lymphomas with V1-69-RF, V3-7-RF or WOL-RF homology as these RFs as well as the MALT lymphoma Igs are encoded by heavily mutated IgVH genes. Finally, none of the 308 B-CLLs showed CDR3 homology to V1-69-, V3-7-, or WOL-RFs.
RF activity of recombinant lymphoma-derived IgM antibodies
To prove that MALT lymphomas with RF-CDR3 homology possess IgG-binding activity, lymphoma-idiotype-derived Abs (LIDA) of IgM class were generated of M5, M6, M11, and M41-A, C, and D. Since, due to shortage of DNA, we could not resolve the IgV
of M41 we combined each of the IgVH variants of the M41-A, C, and D subclones with the RF-canonical IgV
chain of M22. Recombinant IgM LIDAs were also produced of 6 MALT lymphomas (M8, M9, M14, M21, M22, and M23), which are all devoid of RF-CDR3 homology. As additional controls, IgM LIDAs were generated of four follicular lymphomas (FL1, FL6'94, FL8'92, and FL13), one B-CLL, B-CLL26, and two anti-Rhesus(D) Ab producing B cell lines (8D8 and LOS3; reference 57). The IgVH and IgV
sequences of these FLs and B-CLL have been previously reported by us (18, 58, 59). The lymphomas originally expressed IgM, with exception of M14, M22 (both IgA+), and M23 FL6'94, FL8'92 (both IgG+) (Tables I and II). RF-ELISA studies pointed out that the LIDA of M5, M6, and M11 were indeed strongly reactive with human IgG (Fig. 3). The LIDA of M9 and M22, both with a IgVH-CDR3 region not completely fulfilling our homology criteria but both with the RF-canonical V1-69/JH4-A27(kv325) combination of IgVH and IgV
chains, also displayed strong RF activity (Tables IIIV and Fig. 3). Of note, also sera of patient M22, which contained high concentrations (22 mg/ml and 6.5 mg/ml) of lymphoma-related IgA paraprotein, displayed strong IgA-RF activity in ELISA (Fig. 3). This thus independently confirmed our finding with the M22-LIDA and underscored the validity of our approach of recombinant lymphoma Ab production in the eukaryotic expression system used. Moreover, the LIDA of M21, not even harboring an RF-canonical IgVH rearrangement but with an A27(kv325)-encoded IgV
chain, also possessed strong RF activity. In contrast, none of the LIDAs of MALT lymphomas M8, M14, M23, the four FLs, the B-CLL nor the anti-Rhesus(D) Abs, all, except M23, lacking canonical IgVH RF rearrangements, bound to IgG (Fig. 3). Finally, LIDA M41-A/M22 (the dominant subclone with 19 mutations) and M41-C/M22 with 20 mutations bound IgG in ELISA whereas the LIDA of M41-D/M22 (a subclone with only two mutations) did not (Figs. 13).

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Figure 3. RF activity of lymphoma-idiotype-derived antibodies. (a) Titration of IgM LIDAs M5, M6, M9, M11, M21, M22, M41-A, M41-B, FL6'94, B-CLL26, 8D8, and RF control serum in the IgM-RF ELISA. The LIDAs M8, M14, M23, M41-D, FL1, FL8'92, FL13, and LOS3 did not react with human IgG and showed a similar binding curve as that of FL6'94. (b) Binding activity of IgM LIDAs, RF control serum, and anti-Rh(D) control IgM Abs (LOS3 and 8D8) in the IgM-RF ELISA. All samples were tested at a stratified concentration of 500 ng/ml IgM. The ABS 450 nm is plotted without subtraction of the background (BG) ABS 450 nm. (c) IgA-RF ELISA of two serum samples of M22 and as controls an IgA anti-Rh(D) Ab (VT-7G3), an IgA-RF-containing serum, the RF control serum that was also used in the IgM-RF ELISA, and two negative-control serum samples, respectively. All samples were tested at a stratified concentration of 20 µg/ml IgA. The net ABS 450 nm is plotted with subtraction of the background ABS 450 nm.
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We next tested the binding capacities of the RF+ LIDAs of M5, M6, M9, and M11 with recombinant IgG1 and IgG3 preparations. The LIDA of M5, M6, and M9 reacted with IgG1 only. M11 reacted with both IgG1 and IgG3, and may thus be a pan-IgG reacting RF (unpublished data). In chronic gastritis, RF-expressing B cells may theoretically be stimulated by IgG coated on H. pylori or due to existence of cross-reacting epitopes between H. pylori and IgG-Fc. Upon comparison, one H. pylori (strain 26695, GenBank/EMBL/DDBJ accession no. AE000511) peptide, of the gene product "virulence-associated protein homologue VacB" (GenBank/EMBL/DDBJ accession no. AAD08293), was found to share 68% homology with aa 336354 of the IgG1 Fc at the CH2-CH3 junction. However, none of M5, M6, M8, M9, M11, or M14 LIDA, with or without RF activity, reacted with a synthetic "336-354" peptide, nor did this peptide block the binding of the RF-LIDAs to IgG. In addition, no binding of any of these LIDAs to H. pyloriinfected HM02 epithelial cells (strains 26695 and 1061) was observed. In addition, all LIDAs lacking RF-activity (M8, M14, M23, FL1, FL6'94, FL8'92, FL13, and B-CLL26) showed no antinuclear antibody (ANA) activity on Hep2 cells either (unpublished data).
To explore whether other BCR ligands are present within the tissue of MALT lymphomas or FLs, we also produced LIDAs of the IgG class of 3 (non-RF-CDR3-homologous) MALT lymphomas (M8, M14, and M15) and of 4 FLs (FL1, FL3'93, FL6'94, and FL63; reference 18). These LIDAs were FITC-labeled and tested immunohistochemically for reactivity on the corresponding lymphoma tissues. In none of these experiments however we detected reactivity with any tissue components (unpublished data).
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Discussion
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We systematically analyzed the immunoglobulin repertoires of a comprehensive panel of mature B-NHLs. Unbiased comparison of IgVH-CDR3encoded aa sequences of individual B-NHLs with all IgVH-CDR3 presently available in GenBank revealed distinct patterns of the various B-NHL entities. This provided interesting clues concerning their potential ligands, which was functionally confirmed for the group of MALT lymphomas.
FLs, DLBCLs, BLs, and myelomas all exhibited a low degree of overall IgVH-CDR3 sequence homology (Table V). In none of these 147 B-NHLs, recurrent IgVH-CDR3 motifs were found. The majority of the homologous lymphomas expressed CDR3 that resembled those present in normal B cells. A few, however, shared homology with B-CLL and MALT lymphomas. One DLBCL displayed homology with gastric- and salivary gland-MALT lymphomas as well as with V3-7-RFs (Table V). Thus, in general FL, DLBCL, BL, and myelomas, all carrying significantly hypermutated IgV genes, seem to recognize unique epitopes, suggesting that they arise randomly out of the pool of B cells selected for nonself-antigens, most likely during the germinal center reaction. This is in accordance with previous observations that the germline IgVH gene usage of these B-NHLs is similar to that of normal peripheral B cells (18, 60). In contrast, B-CLL and MCL cases showed a high degree of overall CDR3 homology (31 and 44%, respectively) (Table V). Focusing on B-CLL that shared CDR3 homology with at least two other B-CLL (which held for 37 out of the 308 B-CLL analyzed), we distinguished eight CDR3-homology groups (Table VI). These homology groups in part overlap with B-CLL subgroups as reported by others (4952). Inter-B-CLL homology was largely confined to the IgVH-unmutated subset, which shows a strong bias toward V1-69 usage (53): 62 out of 165 (38%) IgVH unmutated B-CLL expressed V1-69, most often (34/62, 55%) combined with JH6. In addition, the previously described poly-autoreactivity of a significant fraction of B-CLL was also clearly reflected in our study: Eighteen B-CLLs shared CDR3 homology with either of five B-CLL for which reactivity with auto-Ags such as IgG (RF), cardiolipin or myoglobulin has been reported (29, 30). Although the number of available MCL IgVH-CDR3 sequences was limited, we observed overall homology for almost half of the cases with CDR3 of unmutated IgVH genes of normal B cells. Still, MCLs are different from B-CLLs with respect to the IgVH repertoire bias, i.e., with preferential usage of V3-21 and V4-34 IgVH genes by MCLs (61, 62).
MALT lymphomas were found to express a highly distinctive IgVH repertoire, confirming and extending earlier reports by the groups of Miklos et al. (38) and De Re et al. (63) on salivary gland MALT lymphomas and HCV-associated B cell lymphomas, respectively. Out of a total of 100 MALT lymphomas that we analyzed, 33 cases shared CDR3 aa homology with other, previously published, CDR3. Twenty-one of these 33 MALT lymphomas harbored, according to the criteria chosen, significant homology to RF-related CDR3 and, except for one case, could be classified into either of 3 canonical RF groups; V1-69-RFs, V3-7-RFs and WOL-RFs (Table V). In addition, 5 salivary gland MALT lymphomas were included, reported by Bahler et al. (43, 64) and Miklos et al. (38), which did not completely fulfill our stringent criteria for V1-69-RFs, but did express the typical V1-69/JH4 RF gene rearrangement. The RF-homology group solely involved gastric- and salivary gland-MALT lymphomas. The in vitro binding studies with the recombinant LIDA formally proved that MALT lymphomas with canonical RF IgVH- and IgV
-chain rearrangements and RF-CDR3 homology indeed posses strong RF activity (Fig. 3). Moreover, MALT lymphomas M9, M21, and M22, which did not match our criteria for RF homology but of which M9 and M22 expressed the canonical RF V1-69/JH4 rearrangement, also exhibited strong IgG-binding capacity in vitro (Fig. 3). Thus, the actual proportion of MALT lymphomas with specificity for human IgG is likely to be higher than calculated on basis of our arbitrary homology criteria.
The degree of RF-CDR3 homology found in the 21 MALT lymphomas is strikingly high, taking into account that it concerns heavily mutated IgVH genes and that homology included N-region encoded aa residues (Fig. 2). This suggests a distinct pathogenesis and indicates that these lymphomas originate from precursors strongly selected for auto-IgG specificity. The latter notion is well supported by the finding that the LIDA of the major subclone of M41 (M41-A), with 19 somatic mutations, and M41-C, with 20 mutations, exhibited significant intrinsic IgG-binding activity of the expressed IgVH chains, whereas this could not be measured of a presumed ancestral subclone, M41-D, with 2 mutations (Figs. 1 and 3).
MALT lymphomas typically evolve in a background of chronic inflammation due to infection or autoimmunity. Evidence exists that the tumor B cells in gastric MALT lymphoma are not H. pylorispecific but largely depend on CD40 stimulation by H. pylorispecific T helper cells (23). It has recently been reported, in a murine model, that RF-expressing B cells can be selectively activated in a T cell independent manner by IgG-chromatin complexes through the synergistic engagement of the BCR and toll-like receptor 9 (TLR9; reference 65). TLR9 is expressed in the endoplasmic compartment and serves as pathogen sensor that binds unmethylated CpG DNA motifs which are more common in bacterial than in mammalian DNA. In the human system, CpG-DNA was shown to trigger T cell independent proliferation of memory B cells, but not of naive B cells, which correlated with the levels of TLR9 expression of memory and naive B cells, respectively (66, 67). Stimulation of TLR9 may thus, parallel to the CD40/CD40L pathway, operate in lymphoproliferations of MALT. In gastric MALT, RF B cells may receive synergistic signals of the RF-BCR by IgGH. pylori complexes and of TLR9 by H. pylori DNA. Also in inflamed salivary gland tissue in Sjögren's syndrome, as well as in other autoimmune diseases, RF B cells may receive these signals of the BCR and TLR9 by complexes of IgG and DNA released during normal or pathological cell death. This scenario clearly lends support from the fact that virtually all Sjögren's syndrome patients produce antinuclear antibodies (ANA), including anti-SS-A and SS-B antibodies.
The most frequent genetic alteration found in MALT lymphoma is the t(11;18)(q21;q21) encoding an API2-MALT1 fusion product that constitutively activates the NF-
B pathway (68). The t(11;18) is present in
40 and
25% of pulmonary- and gastric-MALT lymphomas respectively whereas it is virtually absent in MALT lymphomas of the salivary gland (
2%; references 6971). We found that
40% of the salivary gland- and
20% of the gastric-MALT lymphomas express RF-like BCRs whereas we did not identify RF-like BCRs in any of the 19 pulmonary MALT lymphomas. In addition, none of the MALT lymphomas with a t(11;18) possessed RF-CDR3 homology (Table VII). Accordingly, the LIDA of the t(11;18)+ lung lymphoma M23 did not bind IgG in vitro (Fig. 3). This tentative inverse relation between RF-specificity and the t(11;18) suggests that MALT lymphomas containing t(11;18) do not depend for their expansion on BCR-, CD40-, or TLR9-mediated NF-
B activation (Table VII). The fact that t(11;18)+ gastric lymphomas are resistant to H. pylori eradication therapy is in support of this hypothesis (14, 15). By contrast, the t(11;18) gastric and salivary gland MALT lymphomas with RF BCR may need chronic stimulation by IgG in Ag-Ab complexes in gastric- and salivary gland-MALT lymphomas e.g. as IgGH. pylori and IgG-ANA complexes, respectively. Finally, the different Ig repertoire of t(11;18)+ MALT lymphomas, as compared with MALT lymphomas devoid of this translocation, indicates that this genetic alteration as such provides growth advantage and occurs before the selection process favoring RF-expressing clones.
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Table VII Relation between the presence of t(11;18) and RF-CDR3 homology and/or RF activity of MALT lymphoma immunoglobulins
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Materials and Methods
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Patient material and immunohistochemistry
Frozen or paraffin-embedded tissue of 23 low-grade and one large cell (M22) MALT lymphomas was obtained from the Westeinde Hospital, The Hague; the Free University Medical Center, Amsterdam; The Netherlands Cancer Institute, Amsterdam and the Academic Medical Center, Amsterdam, The Netherlands.
Tumor cell immunophenotypes were determined by immunohistochemical stainings on acetone-fixed cryostat sections and on formalin-fixed paraffin embedded sections using the highly sensitive Powervision+ detection system (ImmunoVision Technologies). Monoclonal antibodies used: IgM,
- and
-light chains (Becton Dickinson), IgG, IgA, CD20 (L26; DakoCytomation), CXCR3 (1C6; BD Biosciences), and
4ß7 (Act-1).
M22 was a large cell lymphoma consisting of immunoblasts which had developed in a patient suffering from Sjögren's syndrome. The original diagnosis MALT lymphoma was not made in our hospital and unfortunately we were not able to recollect material from previous biopsies. This lymphoma most likely developed from a MALT-associated clone given the expression of IgA, CXCR3, the mucosa homing receptor
4ß7 as well as the obvious plasmacytoid differentiation with the concurrent lymphoma-related paraproteinemia. These are characteristics highly compatible with extranodal marginal zone lymphomas but extraordinary for diffuse large B cell lymphomas.
This study was conducted in accordance with the ethical standards in our institutional medical ethical committee on human experimentation, as well as in agreement with the Helsinki Declaration of 1975, as revised in 1983.
DNA and RNA isolation; cDNA synthesis; and IgVH, IgV
, and t(11;18) RT-PCR
DNA was isolated from paraffin sections by over night proteinase K digestion. RNA was isolated from frozen sections using the TRIzol reagent (Invitrogen) and cDNA was synthesized with Pd(N)6 random primers. The IgVH and IgV
genes were amplified using IgVH and IgV
family-specific leader primers combined with the appropriate reverse primer being either JH, Cµ, C
, C
, J
, or C
. To determine the clonally expressed IgVH gene of the tumor B cells, the CDR3 region was also amplified, directly on cDNA and in nested PCRs on the IgVH family-specific PCR products, using a forward primer specific for the framework region 3 (FR3) in combination with one of the different nested downstream primers specific for JH, Cµ, C
, C
or C
. The PCR programs and primers sequences were described previously (18, 58). Translocation t(11;18) was determined using the primers and the PCR program as described by Liu et al. (15).
Cloning and sequencing
IgV RT-PCR products of MALT lymphomas were either directly sequenced or cloned into pTOPO-TA-vectors and transformed into TOP10 bacteria (Invitrogen), to generate molecular IgV clones. Sequencing on both strands was performed by an ABI sequencer (Applied Biosystems) using the big dye-terminator cycle-sequencing kit. To identify the IgV germline gene used and the somatic mutations therein, the consensus sequence of each MALT lymphoma was compared with published germline sequences, using the Vbase database (72) and DNAplot on internet (http://www.mrc-cpe.cam.ac.uk). The IgV sequences of the MALT lymphomas were deposited on GenBank/EMBL/DDBJ (accession nos. AY281324, AY281325, AY281326, AY281327, AY281328, AY281329, AY281330, AY281331, AY281332, AY281333, AY281334, AY281335, AY281336, AY281337, AY281338, AY281339, AY281340, AY281341, AY281342, AY281343, AY466502, AY466503, AY561708 and AY927657, AY927658, AY927659, AY927660, AY927661, AY927662, AY927663, AY927664, AY927665, AY927666, AY927667, AY927668). The degree of intraclonal variation (ICV) of IgVH genes and IgV
genes was calculated as the mean number of nucleotide differences of each molecular clone as compared with the consensus IgVH or IgV
sequences (18). ICV was considered significant if exceeding 0.4 mutations/clone.
Production of IgM antibodies derived of B-NHL
Recombinant IgM
antibodies of the lymphomas (lymphoma-idiotype-derived Ab [LIDA]) of patients M5, M6, M8, M9, M11, M14, M21, M22, M23, M41, FL1, FL6'94, FL8'92, and B-CLL26 were produced using the pIgH(µ) and pIgL(
) expression vectors as described previously (59). In brief, the IgVH and IgV
sequences of each of these lymphomas, including one EBV B cell clone (8D8), which produces a human monoclonal antibody specific for the erythrocyte Rhesus(D) blood group antigen (57), were each cloned into the pIgH(µ) and pIgL(
) vectors respectively. For production of recombinant antibody, 10 µg pIgH(µ) and 10 µg pIgL(
) were linearized with PvuI and cotransfected into SP2/0 myeloma cells by electroporation. Subsequently, the transfected cells were selected in geneticin-containing medium. The IgVH of M41-A, C, and D were expressed with the IgV
of M22. An Ig-secreting heterohybridoma of an IgM
expressing FL FL13 (18), was produced by electrofusion with F3B6 (73) as described previously (74). LOS3 and VT-7G3 are an IgM
and an IgA
anti-Rhesus(D)-secreting EBV B cell clone, respectively (57). Supernatants were screened for IgM
or IgM
, using ELISAs as described previously (57). The pIgH(µ) and pIgL(
) expression vectors were provided by J. van Es and T. Logtenberg (Utrecht Medical Center, Utrecht, The Netherlands).
LIDA reactivity in rheumatoid factor ELISA and on H. pyloriinfected HM02 cells
LIDA reactivity with hIgG was determined using the IgM Rheumatoid Factor ELISA kit (Sanquin) according to the manufacturer's instructions. The plates were developed using TMB as substrate, as described previously (57). For IgA RF activity, a HRP-labeled rabbit anti-IgA Ab (DakoCytomation) was used. The "336-354" IgG1 Fc peptide was coated at 4 µg/ml, incubated with LIDA, followed by mAb anti-IgM-HRP (MH15/1-HRP; Sanquin), and developed as described previously (57). For blocking RF activity, LIDAs were tested in RF-ELISA in the presence of a 50500 molar excess of the "336-354" IgG1 Fc peptide. To detect LIDA reactivity with H. pylori,
80% confluent cultures of HM02 cells were incubated with 2*106 CFU H. pylori (strains 1061 and 26695) and cultured for 1 wk. The cells were then fixed with methanol-aceton (1:1), immunocytochemically stained with LIDA, HRP labeled rabbit anti-IgM Ab (DakoCytomation) and developed with AEC as substrate.
Online supplemental material
Table S1 summarizes the IgVH-CDR3 amino acid sequence homology of MALT lymphomas M5, M6, M11, and M41 with other normal and malignant B cells as well as with RF-producing B cells. Table S2 gives an overview of the IgV
-CDR3 amino acid sequences of the MALT lymphomas of Table III. The supplemental legend to Table V summarizes all lymphomas used in the IgVH-CDR3 homology analysis. The supplemental legend to Table VI depicts the B-CLL cases belonging to the eight IgVH-CDR3 homology groups and their resemblance to homology groups as described previously (4952). Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20050068/DC1.
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Acknowledgments
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We thank M.E.C.M. Oud, E.J.M. Schilder-Tol, and Dr. M. Spaargaren for performing t(11;18) analyses; J.B.G. Mulder for technical advice and immunohistochemical stainings; Drs. T.A. Out and R.J. van de Stadt for providing sera; Dr. M.H. Delfau-Larue for sharing pathological information on pulmonary MALT lymphomas; Dr. D. Hamann for help with the RF ELISAs; Drs. K. van Amsterdam and A. van der Ende for providing H. pyloriinfected HM02 cells; and C.C.H. Vellema, M.J.H. Berends, L.J.M.J. Delahaye, Dr. E.C.M. Ooms, and Dr. E.H. Jaspars for providing tissue material.
The authors have no conflicting financial interests.
Submitted: 7 January 2005
Accepted: 3 March 2005
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