Reprints Available Directly from the Publisher Photocopying Permitted by License Only Pathogenesis of Low-grade B-cell Lymphomas of Mucosa-associated Lymphoid Tissue

Low-grade MALT-type lymphomas are malignancies of mucosal marginal-zone B cells and preceded by reactive inflammatory lymphoid tissue. Experimental observations suggest that antigen and CD40 Ligand act during cognate T/B cell interaction and are crucial for germinal center B-cell maturation generating marginal-zone B cells. To investigate the mechanisms underlying the development of extranodal MALT-type lymphomas, the immunoglobulin receptor was sequenced and analyzed for antigen specificity using heterohybridoma technology. Furthermore, CD40 ligand expression was evaluated by immunohistochemistry and by semiquantitative RT-PCR, and ligand binding to the CD40 of tumor B cells was studied using the CD40 system. Hypermutations were found in low-grade lymphomas throughout CDR1-CDR3 suggestive of positive selection through their antigen receptor. Different VH families were used and more than 69% of tumor immunoglobulins bound different mucosal antigens. CD40L expression was found in the tumor marginal zone in substantial amounts. The in vitro proliferation response of all low-grade MALT-type lymphomas was dependent on anti-CD40-mediated signals and cytokines. Our data provide evidence that autoantigen as well as the CD40L expressed by activated nonneoplastic T cells may drive the evolution of low-grade MALT-type lymphomas either directly or by paracrine mechanisms and that antigen may contribute to lymphoma pathogenesis.


Univerisitit
Wtirzburg, Josef-Schneider str.2, 97080 Wtirzburg, Germany tissue (MALT).Highly organized lymphoid tissue of MALT-type, however, is formed as a consequence of chronic inflammation.Since morphological and func- tional features of this lymphoma are similar to primary MALT, it has been designated secondary MALT (Greiner and Mtiller-Hermelink, 1996).The conditions leading to secondary MALT are con- sidered as important preconditions of lymphomagenesis at extranodal sites (Isaacson, 1994).

It has been suggested that marginal-zone B cells are the normaI counterpart to MALT-type lymphoma tumor cells.The former are noncirculating memory B cells and generated during T cell-dependent antigen responses (Dunn Walters et al. 1995).Experimental observations suggest 'that antigen and CD40/CD40 ligand (T-BAM, TRAP) act during cognate T/B cell interaction and are crucial checkpoints for germinalcenter B cell maturation generating marginal-zone B cells (Arpin et al., 1995; Han et al., 1995).The maturation of B cells into efficient producers of high- affinity and high-specificity antibodies is regulated by various T cell subsets through cell-surface molecules and soluble cytokines (Banchereau et al., 1994).In particular, IL-2, IL-4, IL-6, and IL-10 have been demonstrated to be crucial for B cell maturation (Fluckiger et al., 1993; Briere et al., 1994; Burdin et  al., 1995).Therefore, T cells may determine not only the type of B cell immune response but may also provide key molecules in B cell lymphoma initiation and progres- sion as well.Both, the histopathological and clinical features of MALT-type lymphomas suggest the possibility that the lymphomagenesis is, at least in part, antigen-dependent.


RESULTS

Tumor Antigen Receptor Is Hypermutated and


Recognizes Autoantigen

Analysis of the VH regions of MALT lymphomas showed usage of different VH families (Table I) (Qin  et al., 1995).Additionally, compared with the germ- line, the VH gene of each tumor contained at least 14 substitutions.They were distributed in a pattern characteristic f

somatic mutations were
ighly concentrated in the CDRs and FRs with a clustering of replacement [R] mutations in the CDRs, but only few in the FRs.Three of the four genes detected here, hv1263, hv3005, and VH4-21, are frequently used in a variety of autoantibodies, such as cold agglutinins, rheumatoid factors and anti-DNA antibodies (Dersimonian et al., 1987; Sanz et al., 1989; Olee et al.,  1991).


Analysis of the Tumor Immunoglobulin


Specificity

The antibody specificity of monoclonal antibodies derived from 13 low-and high-grade MALT-type lymphomas of the stomach, thyroid, salivary gland, and the lung were analyzed by using heterohybridoma technology.Tumor immunoglobulin from 9 of 13 patients (69%) had autoantibody activity (Table I

munoglobulin
have been found to be autoantigens mainly expressed in mucosal tissues, for example, of thyroid, parotid, lung, or stomach tissue.The human Abs bound to different self-determinants and fullfilled the definition ANA, salivary gland, thyroid epithelium, glandular epithelium in the stomach Thyreoglobulin of an autoantibody since they are self-reactive, but not self-specific.These monoclonal antibodies (mAbs)

were highly specific for the identified antigen and bound none of the other tested antigens that are commonly recognized by polyreactive natural auto- antibodies (e.g., rheumatoid factor, myosin, and actin).

The Tumor Antigen Receptor Is Functionally Active By using an anti-idiotypic antibody highly specific for a gastric IgA-expressing low-grade MALT lymphoma immunoglobulin receptor (idiotype), induction of the proliferation response after cross-linking was induced by additional stimulation with a mitogen.A specific growth promotion effect of the

ti-idiotype antibody in combination with SA
was noted in the lymphoma cells but not in controls.The effect of the anti- idiotype antibody induced proliferation was more pronounced than the effect of cytokine or mitogen stimulation alone (Figure 1A).This may indicate a dependency on additional signals.Furthermore, stim- ulation with the anti-idiotype antibody induced a differentiation of tumor cells, as demonstrated by an enhanced secretion of tumor IgA ( 104 purified B cells were cultured in triplicate over 5 days.[3H]TdR uptake was measured after a 16-hr pulse.Note the enhanced proliferation of MALT tumor B cells but not of controls using the anti-ld antibody.(B) Differentiation of MALT tumor B cells using the anti-ld antibody as demonstrated by IgA-secretion.This effect of the anti-idiotype antibody was found only in the tumor, but not in tonsilar or nodal umor B cells.Within the latter, only SAC was able to induce differentiation as demonstrated by IgM-secretion (C).CD40 Receptor Is Expressed by Tumor B Cells and CD40 Ligand Is Present in Tumor Tissue All investigated and freshly isolated MALT lym- phoma B cells expressed high levels of CD40 on their cell surfaces (Fig. 2).Nontumor T cells expressing CD40L were found within the tumor marginal zone by in situ immunohistochemistry.By using a semi- quantitative RT-PCR,

40L was found to be increased in tumor tissues of low-grade MALT-type lymphomas in cont
ast to controls (reactive, nonneo- plastic tissues) (Fig. 3).


Functional Consequences of Antigen Receptor and CD40 Engagement in MALT Lymphoma

To determine whether the tumor cell response in low- grade lymphoma is due to a stimulatory effect of antigen or due to effects solely mediated by tumor- isotype control CD40-Ugond GAPDH Fig. 3 Semiquantitative RT-PCR using GADPH as a standard showing CD40L message in cas

of low-grade MALT-type B cell lymphomas.

infiltrating T cells the B cells were
ultured in the so- called "CD40-system," which allowed to study the properties of tumor B cells and nontumor T cells independently (Fig. 4).Herein the proliferative B cell response of all low-grade MALT-type lymphomas 0,25
[3H]TdR cpm/lymphocyte MALT- lymphoma i CD40 expression
Fig. 2 CD40 histogram of a multicolor FACS analysis of MALT lymphoma B cell suspension freshly isolated after surgery and gated for viable lymphocytes, CD19+, CD40+, and 7-AAD, as described (Schmid et al., 1992).

Eltonsil [] MALT #4   Fig. 4 Anti-CD40-activat d normal and lymphoma B cells show different growth kinetics in culture period. 5X104 purified B cells were cultured in triplicate over 5 days.[3H]T dR upta are representative of three independent experiments.investigated was stro

ly dependen
on anti-CD40- mediated signals, complemented by cytokines pro- duced by T helper cells of the Th2 type (IL-4 and/or IL-10).Thl cytokines (IL-2 and/or INFy) had little effect.No difference to B cells isolated from normal tonsils was detected.Furthermore, low-grade MALTtype lymphoma B cells were induced to secrete large amounts of tumor immunoglobulin in response to IL-10 (Table III).This antibody secretion by tumor B cells was significantly more pronounced than in normal B cells and could not be enhanced by adding IL-2 or IL-4 alone or in combination.

In contrast to CD40 ligand signaling, B-cell- receptor (BCR) triggering induced spontaneous cell death in normal tonsil but not in MALT lymphoma B cells in vitro (Fig. 5) reminiscent of apoptosis of germinal-center B cells in vivo.


DISCUSSION

To get an insight into the pathogenesis of MALT-type lymphomas, it was the aim of the present study to investigate the eventual correlation between two observations made independently with B cell lympho- mas in general and MALT lymphomas in particular: The first observation concerns the high frequency of "ant -self" or autoimmune reactivity of immunoglobulins secreted both by nodal lymphomas like CLL and follicular B cell lymphomas as well as by lymphomas of MALT (Hussell et al., 1993; Greiner et  al., 1994a).The second observation made only in MALT lymphoma is that they are preceded by reactive inflammatory tissue.So far, however, it has not been defined whether antigen and paracrine factors contribute to lymphoma development.alone or recombinant human CD40 ligand (CD40L) or anti-BCR (a-lg) as an antigen trigger, as described (Galibert et al., 1996).Viable cells were visualized using Trypan blue staining.Note a marked decrease of tonsil but not tumor B cells using a-Ig.

In particular, recent work about CD5 chronic lymphatic leukemia (B-CLL) (Schroeder, Jr. and  Dighiero, 1994) or CD5-follicular lymphoma (Kobayashi et al., 1993) has provided strong evidence that a high percentage of B cells that express immunoglobulin with reactivity against self-determi- nants are frequently committed to malignancy.In contrast to MALT lymphoma, a restricted repertoire of immunodominant epitopes on the target antigen(s) of the lymphoma immunoglobulin (idiotype) is dis- played in CLL, follicular and mantle cell lymphoma, as exemplified by a high frequency of shared idiotypes within these tumors (Dighiero, 1992; Rud- ders et al. 1992; Ohno et al., 1995).Consequently, the immunoglobulins of MALT-type lymphomas exhibit a highly, restricted specificity for a variety of distinctive (auto)antigens confined to MALT (e.g., mucosal plasma cells, thyroid, salivary gland, lung and stomach epithelia) that does not share idiotop within extranodal or nod l lymphomas (Greiner et al.  1994a).

This may be explained at the molecular level by the finding that MALT-lymphoma immunoglobulin gene sequences were found frequently in a variety of autoantibodies.They displayed somatic hypermuta- tions, indicating affinity maturation during a specific antigen response that may alter the binding to the initiating antigen thus leading to a variety of different and highly specific autoantibodies (Schroder et al.,  1996).The putative antigen binding to MALT-tumor immunoglobulin is still unknown and.could be an exogenous antigen, a regulatory ce-idiotype antibody or an autoantigen.In this regard, experimental data favor the latter possibility.

Auto eactivity in MALT-type lymphoma may have consequences in at least two ways.First, it emphasizes the recent hypothesis that proliferation in some MALT-type lymphomas may be antigen-driven, and autoimmunity may play a role in their pathogenesis (Mtiller-Hermelink et al., 1995).This hypothesis is strengthened by the observation that the antigen receptor of MALT lymphomas is functionally active and can induce tumor-cell survival (Fig. 5).Second, according to the concept of the recently proposed REAL classification (Harris et al., 1994), the immunological and molecular data have helped to define MALT-type lymphoma with respect to their extraordinary pathophysiological features.

Whether antigen-driven affinity maturation is asso- ciated with secondary events that cause tumor trans- formation in MALT Lymphoma is uncertain.As in most nodal lymphomas, the etiology and pathogenesis of lymphomas derived from MALT are not yet elucidated.In particular, unifying chromosomal abe- rration (Roblick et al., 1993; Wotherspoon et al.,  1995), rearrangements of oncogenes (Marin et al.  1995), or Epstein-Barr virus infections (Ott et al.,  1993; Greiner et al., in press), thought to be first steps in the development of nodal lymphomas, have not been detected in lymphomas of MALT type.It is possible that antigen is necessary for survival of the initiating malignant tumor clone in combination with paracrine factors supported by reactive tumor infiltrat- ing T cells that express CD40 ligand.In this regard, it was important that large amounts of CD40L were detected in MALT lymphoma tissues in vivo and that t

d to the CD40 ligand signal in vitro.The activa
ion of the malignant B cells parallels the activation of normal mature B cells resulting in B-cell proliferation and differentiation depended on the added cytokine.In contrast, CD40 cross-linking was found to inhibit cell proliferation in other lymphoma types, like Burkitt lymphoma, dif- fuse large-cell lymphoma, or lymphoblastoid cell lines (Funakoshi et al., 1994).

Therefore, our data provide certain evidence that autoantigen as well as the CD40L expressed by activated non-neoplastic T cells may trigger evolution of low-grade MALT-type lymphomas either directly or by paracrine mechanisms and that antigen may contribute to lymphoma pathogenesis.


MATERIAL AND METHODS

Tissues, Cell Culture, and B-Cell Purification Non-neoplastic tonsillar tissues and malignant lymphomas were obtained from biopsies after surgical removal for the preparation of cell suspensions, snapfreezing in liquid nitrogen, and routine fixation fo histological examination.In all cases, the diagnosis was confirmed by morphological and immunopheno- typical analysis of fresh-frozen and paraffin-embedded sections.Mononuclear single-cell suspensions were isolated by density-gradient centrifugation and negatively depleted with magnetic beads coupled either with anti-CD2,-CD14 mAbs (Dynal, Germany).Thereafter, lymphoma cells were further purified by negative depletion of non-neoplastic bystander B cells using beads coated with antibodies t heavy and light chains not expressed by the lymphoma, including IgD.The purity of lymphocyte cell suspen ions obtained after magnetic immuno- beads depletion was between 97.5 and 99.7% B cells, as calculated by FACScan analysis.

Purified normal and tumor B cells were cultured in RPMI1640 supplemented with 1% gentamycin, 10% FCS, and 50/zg/ml transferrin (Sigma, St. Louis).For activation through CD40 antigen, B cells were cultured in the presence of 0.1/g/ml anti-CD40 mAb 89 (J.Banchereau, Dardilly, France) presented by a mouse Ltk-cell line stably expressing CDw32 according to the experimental procedure described previously (Banchereau and Rousset, 1991).Recom- binant cytokines were added at the onset of culture.


DNA sy

hesis was deter
ined as measured by [3H]

TdR incorporation as described (Greiner et al.,  1994b).

Tumor Immunoglobulin and c-Idiotypic Antibodies MALT-type lymphoma immunoglobulins were pro- duced by fusing lymphoma cells with the hetero- myeloma NSO and selected by corresponding to the lymphoma immunoglobulin as described recently (Greiner et al., 1994b).Monoclonal anti-idiotypic antibodies w

e prod
ced by immunizing mice with purified tumor immunoglobulin.Mouse immuno- globulin-secreting hybridomas were cloned by limit- ing dilution and selected by exhibiting a restricted specificity in immunohistochemistry, Western-blot, and competitive ELISA for the tumor antigen receptor and not for controls (Greiner et al., 1991).


Flow Cytometry

Flow cytometric analysis was performed on a FACS- can (Becton-Dickinson) with an Argon ion laser tuned at 488 nm using LYSIS II for data acquisition and analysis using tr

le immunostaini
g with directly conjugated antibodies: CD19 (HD 37, sigma); kappa (Dako, Hamburg); lambda (Dako); CD3 (UCHT-1, Sigma); CD14 (Leu-M3; Becton-Dickinson); CD40 (mAb89; Banchereau and Rousset, 1991).ELISA Supernatants were assayed in ELISA as described recently (Greiner et al., 1994a) using microtiter plates (Falcon, FRG) coated with goat anti-human heavy- chain (Dako).After blocking with 0.5% BSA, undi- luted samples were added for 2 h at 37C.Finally, the plates were developed with rabbit anti-human heavychain HRP conjugate (Da o) and orthophenyl-dia- mine with hydrogen peroxide in citrate phosphate buffer and read at 490 nm in an automatic ELISA reader (Merlin, FRG).The sensitivity of the assay was < 10 ng/ml.


RNA Extraction

All tumor tissue blocks were snap-frozen in liquid nitrogen and stored at -70C until extraction of RNA.Total RNA was prepared (TRIzol reagent; Life Technology, Paisley, UK) from 20 sections of about 10 #m from the frozen tumor tissue blocks.Integrity of RNA was controlled by electrophoresis through a 2% formaldehyde-agarose gel and the yield of RNA was quantitated by measuring the optical density.To check for carryover of material during the isolation step, extraction buffer without tissue was used as a negative control.cDNA Synt esis First-strand synthesis was performed with 1 /zg of total cellular RNA.RNA, 2/g of dT-15 primer, and DEPC-treated water to give a final volume of 8 #1 were incubated for 10 min at

C.After chilling on ice, a master mix consi
ting of dNTPs and dithiother- eitol (final concentrations of mmol/1 each and 10 mmol/1, respectively), 25 U of recombinant RNAse inhibitor (Promega, Heidelberg), RT-buffer and 200 U of moloney-murine leukemia virus reverse transcriptase (GIBCO BRL) were added to a final reaction volume of 25/zl.After 70 min of incubation at 37C, the samples were heated to 98C for 4 min.The efficiency of cDNA synthesis was estimated by PCR with glyceraldehyde-3-phosphate dehydro- genase (GAPDH)-specific primers.



Fig.Stimulation of purified tumor B cells of IgA-expressing MALT-type lymphoma and follicular nodal lymphoma as neoplastic and tonsilar as non-neoplastic B cells as control.(A)5  104 purified B cells were cultured in triplicate over 5 days.[3H]TdR uptake was measured after a 16-hr pulse.Note the enhanced proliferation of MALT tumor B cells but not of controls using the anti-ld antibody.(B) Differentiation of MALT tumor B cells using the anti-ld antibody as demonstrated by IgA-secretion.This effect of the anti-idiotype antibody was found only in the tumo