CCL19 and CXCL12 Trigger in Vitro Chemotaxis of Human Mantle Cell Lymphoma B Cells

964 Vol. 10, 964–971, February 1, 2004 Clinical Cancer Research

Anna Corcione,1 Nicoletta Arduino,2 INTRODUCTION Elisa Ferretti,1 Lizzia Raffaghello,1 Mantle cell lymphoma (MCL) is a B cell tumor that, in the 3 4 3 majority of cases, originates from the clonal expansion of a Silvio Roncella, Davide Rossi, Franco Fedeli, ϩ Luciano Ottonello,2 Livio Trentin,5 naive CD5 B cell localized in the mantle zone of secondary 2 5 lymphoid follicles (1, 2). MCL B cells express monoclonal Franco Dallegri, Gianpietro Semenzato, and surface immunoglobulin usually belonging to IgM and IgD 1 Vito Pistoia isotypes (3, 4), as well as CD5, CD20, CD79a, and CD79b 1Laboratory of Oncology, G. Gaslini Institute, Genova; 2Laboratory (1–4), but not CD10 or CD23 (5). MCL is associated with the of Phagocyte Physiopathology and Inflammation, Department of 3 t(11;14)(q13;q32) which results in the positioning of the bcl-1 Internal Medicine, University of Genova, Genova; Laboratory of gene (11q13) near the immunoglobulin heavy-chain gene locus Pathology, S. Andrea Hospital, La Spezia; 4Division of Internal Medicine, Department of Medical Sciences, Amedeo Avogadro, (14q32). This translocation causes bcl-1 up-regulation and cy- University of Eastern Piedmont, Novara; and 5Academic Department clin D1 overexpression (6, 7). On clinical grounds, generalized of Clinical and Experimental Medicine, Clinical Immunology Branch, lymphadenopathy and bone marrow infiltration are the most Padua University School of Medicine, Padova, Italy common manifestations (1, 2, 8–10). Splenomegaly is detected in 30–60% of the patients and peripheral blood lymphocytosis ABSTRACT in 10–69% of cases (1, 2, 8–10); gastrointestinal (1, 2, 8, 9, 11) and central nervous system (1, 7–10, 12) involvement have been Purpose: Few data are available in the literature on also reported. chemokine receptor expression and migratory capability of Chemokines are low molecular weight cytokines, special- mantle cell lymphoma (MCL) B cells. Information on these ized in the mobilization of leukocytes and other cell types issues may allow us to identify novel mechanisms of chemo- (13–16). Recent studies have shown that neoplastic cells of kine-driven tumor cell migration. hematopoietic and nonhematopoietic origin express various che- Experimental Design: The research was designed to mokine receptors, and overexpression of some of these has been investigate: (a) expression of CCR1 to CCR7 and CXCR1 to related to tumor progression and metastasis (17–19). As for CXCR5 chemokine receptors; and (b) chemotaxis to the B-cell derived lymphoproliferative disorders, CXC chemokine respective ligands in MCL B cells and in their normal -؉ receptor (CXCR) 4 and CXCR5 have been detected in neoplas counterparts, i.e., CD5 B cells. tic B cells from B-cell acute lymphoblastic leukemia (B-ALL; Results: Malignant B cells from MCL patients and Ref. 20–23), B-cell chronic lymphocytic leukemia (B-CLL; normal counterparts displayed similar chemokine receptor Ref. 20, 24, 25), follicular lymphoma (FL; Ref. 26), and hairy profiles. MCL B cells were induced to migrate by CXCL12 ؉ cell leukemia (HCL; Ref. 21). CXCR3 has been found to be and CCL19, whereas normal CD5 B cells migrated to the former, but not the latter chemokine. Overnight culture of expressed in B cells from B-CLL (20, 27), marginal zone MCL B cells and their normal counterparts with CXCL12 lymphoma (20), and a fraction of HCL and B-cell acute lym- cross-sensitized other chemokine receptors to their ligands phoblastic leukemia cases (20). CC chemokine receptor (CCR) in some tumor samples but not in CD5؉ B cells. 7 has been detected in B-CLL (28) and in tumor cells from Conclusions: CCR7 and CXCR4 ligands may play a key classical Hodgkin’s disease with lymphocyte predominance role in tumor cell migration and spreading in vivo. CXCL12 (29). Finally, CCR5 has been detected in hairy cell leukemia may additionally contribute by sensitizing MCL B cells to cells and on the surface of B-CLL cells (21). In terms of respond to the ligands of other chemokine receptors. functional in vitro activity, only a few studies have addressed chemokine-driven locomotion of neoplastic B cells. CXCL12, by interacting with its specific receptor (i.e., CXCR4), has been shown to enhance chemotaxis of B-ALL (22, 23), B-CLL (24, 25), and FL (26). The CCR7 ligands, CCL19 and CCL21, Received 8/8/03; revised 11/3/03; accepted 11/3/03. enhanced the chemotaxis of B-CLL and Hodgkin’s disease cells Grant support: Grants from Associazione Italiana Ricerca sul Cancro, to CCL19 and CCL21 (28), whereas CXCR3 mediated the ` Milan, Italy, Ministero Istruzione, Universita Ericera, Progetii Cofinan- chemotactic response of B-CLL cells to CXCL10 (27). Scanty ziati, Rome, Italy, Ministero della Salute, Rome, Italy, and Associazione Italiana Leucemie, La Spezia, Italy. data are presently available on chemokine receptor expression in The costs of publication of this article were defrayed in part by the MCL B cells, mostly viewed in the context of other B-cell payment of page charges. This article must therefore be hereby marked malignancies (20, 21, 27). It has been reported that the latter advertisement in accordance with 18 U.S.C. Section 1734 solely to cells are CXCR4ϩ, CXCR5ϩ, and CCR6ϩ (20, 21, 27). How- indicate this fact. Requests for reprints: Anna Corcione, Laboratory of Oncology, G. ever, no study has thus far addressed chemokine-driven loco- Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy; Phone and motion of malignant B cells from this lymphoma entity. Fax: 39-010-3779820; E-mail: [email protected]. To identify chemokine(s)/chemokine receptor(s) involved

Table 1 Main clinical and laboratory features of mantle cell lymphoma patients Age Case no. (years) Gender Sample WBC/mm3 Histological variant Diagnostic assay 1 90 F Lymph node 4.5 ϫ 103 Typical PCRa 2 75 F Lymph node 4.0 ϫ 103 Typical PCR 3 58 M Blood 88 ϫ 103 Blastoid Immunohistochemistryb 4 73 M Blood 270 ϫ 103 Typical Immunohistochemistry 5 49 M Blood 96 ϫ 103 Typical Immunohistochemistry 6 63 F Blood 28 ϫ 103 Typical Immunohistochemistry 7 49 F Blood 10.2 ϫ 103 Typical Immunohistochemistry 8 73 M Blood 15.7 ϫ 103 Typical FISHc 9 59 F Blood 45.5 ϫ 103 Typical FISH 10 66 M Pleural effusion 3.6 ϫ 103 Typical FISH a For bcl-1 rearrangement in lymph node cells. b For cyclin D1 overexpression in bone marrow cells. c FISH, fluorescence in situ hybridization, for t(11;14) detection in blood and pleural effusion cells.

in the chemotaxis of MCL B cells, we have investigated here the 7 patients with peripheral monoclonal lymphocytosis showed expression of a panel of CXC (CXCR1 to CXCR5) and CC lymphadenopathy. (CCR1 to CCR7) chemokine receptors in MCL B cells and in Control B Cells. Seven reactive lymph nodes with fol- ϩ CD5 B lymphocytes, and tested their in vitro migration to the licular hyperplasia from age-matched individuals were tested as respective ligands. sources of normal CD5ϩ B cells. In parallel experiments, cir- culating CD5ϩ B cells from the peripheral blood of four normal PATIENTS AND METHODS donors were tested. MCL Samples. The study was approved by an Institu- MNC were isolated on Ficoll-Hypaque density gradients, tional Review Board. Diagnosis of MCL was established washed in PBS containing 1% fetal bovine serum, and cryopre- according to the criteria of the Revised European-American served until tested. Immunophenotypic analyses showed that Classification of Lymphoid Neoplasms classification (30). CD5ϩ B cells in reactive lymph nodes ranged from 5 to 10% Mononuclear cells (MNCs) were isolated on Ficoll-Hypaque MNC. (Sigma Chemical Co., St. Louis, MO) density gradients from Chemokines and Antibodies. All of the chemokines lymph node fragments (2 cases), peripheral blood samples (7 (CCL2, CCL3, CCL19, CCL20, CCL22, CXCL8, CXCL10, cases), and one pleural effusion (1 case). Lymph node MNC CXCL12, and CXCL13) were purchased from Pepro Tech contained on average 70–80% CD19ϩ B cells and 20–30% (Rocky Hill, NJ) and tested at the final concentration of 100 CD3ϩ T cells. In peripheral blood and pleural effusion samples, ng/ml. This concentration was chosen on the grounds of pre- B cells were 80–90% and T cells ranged from 10 to 20%. liminary dose-response experiments carried out with normal Staining for immunoglobulin light chains showed that mono- tonsil B cells (33). In selected experiments, chemokine concen- clonal B cells expressing either ␬ (6 of 10 cases) or ␭ (4 of 10 trations up to 1000 ng/ml were tested. cases) light chain represented at least 85% of CD19ϩ B cells in For B-cell immunophenotyping, the following mAbs were all of the samples, irrespective of their source. Tumor cells used: CD19-phycoerythrin (PE)-cyanin (Cy)5, CD5-FITC expressed surface immunoglobulin, CD19, CD20, and CD5. (Caltag Laboratories, Burlingame, CA); CD20-PE, CD23-PE, Malignant B cells from all of the cases were consistently CD23 CD3-FITC, CD68-PE, and CD56-PE (Becton Dickinson Sys- negative. MNC were cryopreserved in a freezing solution com- tems, San Jose, CA); and anti-immunoglobulin ␬ light chain-PE posed of 50% RPMI 1640 (Sigma), 40% fetal bovine serum and anti-immunoglobulin ␭ light chain-FITC (PharMingen, San (Sigma), and 10% DMSO (Sigma). Cells were kept in liquid Diego, CA). Anti-CCR1 to CCR7-PE mAbs were purchased nitrogen until tested. from R&D Systems Inc. (Minneapolis, MN). Controls were Table 1 summarizes the main clinical and laboratory fea- isotype-matched mAbs of irrelevant specificity conjugated with tures of the patients. MCL diagnosis for cases #1 and #2 (Table the same fluorochromes as test mAbs (PharMingen). Unconju- 1) was established by PCR analysis of DNA extracted from gated anti-CXCR1 to CXCR5 mAbs were purchased from Se- invaded lymph nodes (31). In cases #3 to #7 (Table 1), cyclin rotec Inc. (Raleigh, NC). Controls were isotype-matched uncon- D1 overexpression was detected by immunohistochemical stain- jugated mAbs of irrelevant specificity (Serotec). Cells treated ing of bone marrow biopsies (32). In cases #8 and #9 (Table 1), with anti-CXCR or control unconjugated mAbs were washed the t(11;14) translocation was detected by fluorescence in situ and incubated with PE-conjugated antimouse IgG subclass goat hybridization analysis of peripheral blood MNC (Ref. 2; Table antisera (Serotec). 1). The same technique was used for MCL diagnosis using For fluorescence-activated cell sorter analysis, 10,000 MNC isolated from the pleural effusion (case #10; Table 1). events were acquired after double or triple gating for MCL or Histological analysis of lymph node biopsies showed that control B cells, respectively. The former cells were double cases 1, 2, and 4–10 had typical MCL. Case 3 showed the stained with mAbs against the monoclonal immunoglobulin histological features of MCL, blastoid variant (Table 1). Six of light chains and with each of the mAbs to chemokine receptors.

Reactive lymph node mononuclear cells were subjected to tri- color staining with CD19 and CD5 mAbs, in combination with the different mAbs to chemokine receptors. Cells were scored using a FACScan analyzer (Becton-Dickinson), and data were processed using CellQuest software (Becton-Dickinson). The threshold line was based on the maximum staining obtained with irrelevant isotype-matched mAb, used at the same concentration as test mAb. Negative cells were defined such that Ͻ1% of cells stained positive with control mAbs. Cells labeled with test antibody that were brighter than those stained with isotypic control antibody were defined as positive. Mean fluorescence intensity values of the isotype control and of test mAbs were used to evaluate whether the differences between the peaks of cells were statistically significant with respect to control. The Kolmogorov-Smirnov test for the analysis of histograms was used, according to the CellQuest software user’s guide. Migration Assay. Chemotaxis of malignant and normal B-cell populations was tested using 24 transwell plates (5 ␮m pore-size, polycarbonate membrane; Costar, Cambridge, MA; Ref. 34). Five ϫ 105 mononuclear cells were dispensed in the upper chamber, and different chemokines (100 ng/ml concentration) or medium alone were added to the lower chamber. Unfractionated mononuclear cells, rather than purified cell frac- tions, from neoplastic samples and reactive lymph nodes were tested, to minimize the risk that chemotactic activity could be affected by the separation procedures. Plates were incubated for 2hat37°C. After removal of the transwell inserts, cells from the lower compartments were collected. Furthermore, 0.5 ml of 5 mM EDTA was added to the lower chamber for 15 min at 37°C to detach adherent cells from the bottom of the wells. Detached cells were pooled with the previously collected cell suspensions and counted by trypan blue staining. Transmigrated normal B cells were identified by double staining with CD19 and CD5 mAbs, whereas B lymphocytes from MCL samples were detected by single staining for the monoclonal immunoglobulin light chain expressed by the individual clones. Each experiment was performed in duplicate, and results were means from du- Fig. 1 Expression of CC chemokine receptors on mantle cell lym- plicate wells. phoma (MCL) B cells and CD5ϩ B cells from reactive lymph nodes. Statistical Analysis. Data were expressed as means Ϯ1 Surface expression of CCR1 to CCR7 on MCL B cells from two lymph SD. Analyses were performed with GraphPad Instat (GraphPad nodes (cases 1 and 2), 7 peripheral blood samples (cases 3–9), and 1 ϩ Software, San Diego, CA). Differences were determined by pleural effusion (case 10; left), as well on normal CD5 B cells from 7 one-way ANOVA with Bonferroni multiple comparison tests. reactive lymph nodes (right), was investigated by flow cytometry. MCL Ͻ B cells were double-stained with a monoclonal antibody (mAb) to the Differences were accepted as significant when P 0.05. monoclonal immunoglobulin light chain expressed by the individual clones and with different mAbs to chemokine receptors. Normal CD5ϩ B cells were triple stained with CD19, CD5, and anti-CCR mAbs. RESULTS Results are percentage of positive cells. Expression of Chemokine Receptors on Malignant B Cells from MCL Patients and Their Postulated Normal Counterparts. MCL B cells from 2 lymph nodes (cases #1 (cases #1, 3, 4, and 6–8), CCR3 in 3 of 10 (cases #4, 6, and 7), and 2), 7 peripheral blood samples (cases #3–9), and 1 pleural CCR4 in 9 of 10 (cases #1–9), CCR5 in 2 of 10 (cases #6 and effusion (case #10), as well as CD5ϩ B cells from 7 reactive 8), CCR6 in 7 of 10 (cases #1–4, 6, 7, and 9), and CCR7 in 10 lymph nodes with follicular hyperplasia and from 4 normal of 10. CCR1 was expressed in 2 of 7 CD5ϩ B cell samples from peripheral blood samples, were tested for CCR and CXCR reactive lymph nodes, CCR2 in 1 of 7 (sample #6), CCR3 in 6 expression by flow cytometry. of 7 (samples #1–6), CCR4 in 4 of 7 (samples #1, and 4–6), Fig. 1 shows the percentage of CCR expression in MCL CCR5 in 3 of 7 (samples #1, 3, and 4), and CCR6 and CCR7 in samples (left columns) and in reactive lymph nodes (right col- 7 of 7. Similar results were obtained with CD5ϩ B cells from umns). On the basis of mean fluorescence intensity analysis normal peripheral blood; CCR3, CCR6, and CCR7 were de- using the Kolmogorov-Smirnov test, MCL B cells were found to tected in 4 of 4 samples tested, CCR4 and CCR5 in 2 of 4, express CCR1 in 8 of 10 cases (#1–5 and 7–9), CCR2 in 7 of 10 CCR1 in 1 of 4, and CCR2 in 0 of 4 (data not shown).

Chemotaxis of MCL B Cells and CD5؉ B Cells in Response to CCR and CXCR Ligands. MCL B cells were tested for chemotaxis to the ligands of the receptors investigated, with the exception of the CCR3 and CCR5 binding chemokines CCL11 and CCL4, respectively, because neither receptor had been detected on tumor cells. MNC isolated from 2 lymph nodes (cases #1 and 2), 5 peripheral blood samples (cases #4, 5, 6, 8, and 9), and 1 pleural effusion (case #10) were tested in a transwell assay. Neoplastic B cells that migrated in response to chemokines or medium (control) were identified by staining for the monoclonal immunoglobulin light chain expressed by each clonal population. The chemotactic responses of the eight MCL cell suspensions are shown in Fig. 3. By assuming that a chemokine was active in the individual cases when it at least doubled the number of migrated cells in comparison with control cells, it was found that CXCL12 (the ligand of CXCR4) stimulated the chemotaxis of all of the MCL B cell suspensions. CCL19 (a ligand of CCR7) enhanced the locomotion of B cells in 6 of 8 cases (#1, 4, 5, 8, 9, and 10; Fig. 3). Other chemokines, i.e., CCL3 (a ligand of CCR1), CCL2 (that binds to CCR2), CCL22 (a ligand of CCR4), CCL20 (that binds to CCR6), CXCL8 (a ligand of CXCR1 and CXCR2), CXCL10 (a ligand of CXCR3), and CXCL13 (that binds to CXCR5) displayed heterogeneous effects. In particular, CCL3 was found to enhance tumor cell locomotion in 2 of 8 cases (#4 and 8), CCL2 in 1 of 8 cases (#4), CCL22 in 2 of 8 cases (#4 and 5), CCL20 in 1 of 8 cases (#4), CXCL8 in 1 of 8 cases (#4), CCL10 in 3 of 8 cases (#1, 4, and 8), and CXCL13 in 1 of 8 cases (#4; Fig. 3). Notably, case #4 displayed special behavior, because malignant B cells responded to every chemokine tested (Fig. 3). When the results obtained in the individual cases were pooled, only CXCL12 and CCL19 were found to enhance significantly the chemotaxis of MCL B Fig. 2 Expression of CXC chemokine receptors on mantle cell lym- ϩ Ͻ Ͻ phoma (MCL) B cells and CD5 B cells from reactive lymph nodes. cells (P 0.001 and 0.05, respectively). Surface expression of CXCR1 to CXCR5 on MCL B cells from two Because B cells from cases #9 and 10 were unresponsive to lymph nodes (cases 1 and 2), 7 peripheral blood samples (cases 3–9), all of the chemokines other than CXCL12 and CCL19, tumor ϩ and 1 pleural effusion (case 10; left), as well on normal CD5 B cells cells from these cases were tested for chemotaxis to CCL3, from 7 reactive lymph nodes (right), was investigated by flow cytometry. MCL B cells were double stained with a monoclonal antibody CCL2, CCL22, CCL20, CXCL8, CXCL10, and CXCL13 in (mAb) to the monoclonal immunoglobulin light chain expressed by the concentrations ranging from 10 to 1000 ng/ml. No migration individual clones and with different mAbs to chemokine receptors. was ever detected even under these conditions (data not shown). ϩ Normal CD5 B cells were triple stained with CD19, CD5, and anti- In subsequent experiments, chemotaxis of CD5ϩ B cells from CXCR mAbs. Results are percentage of positive cells. 7 reactive lymph nodes with follicular hyperplasia to the following chemokines, CXCL12, CCL19, CCL11, CCL22, CCL4, CCL20, CXCL8, CXCL10, and CXCL13 was investigated. These chemokines were selected on the ground of the consistent expression of Fig. 2 shows the percentage of CXCR expression in the the respective receptors on CD5ϩ B cells; for the same reason, MCL samples (left columns) and in the reactive lymph nodes CCL2 and CCL3 were not tested, because cognate receptors CCR1 (right columns). CXCR1 was expressed by MCL B cells in all and CCR2 had not been detected. In these experiments, MNC were of the cases but 1 (case #10), and CXCR2 to CXCR5 in 10 of tested, and CD5ϩ B cells migrating in the assay were identified by 10. CXCR1 to CXCR5 were detected on CD5ϩ positive B cells double staining for CD19 and CD5. As shown in Fig. 3, (bottom), from all of the reactive lymph nodes (Fig. 2) and all of the only CXCL12 stimulated significantly the chemotaxis of normal normal peripheral blood samples (data not shown). CD5ϩ B cells (P Ͻ 0.001). By comparing the results shown in Figs. 1 and 2, it was Taken together, these results demonstrate that CCR7 was found that the percentages of CCR3ϩ, CCR6ϩ, and CXCR1ϩ functional in tumor cells but not in their normal counterparts, cells were significantly lower in MCL B cells than in control whereas CXCR4 mediated chemotaxis of both cell types. CD5ϩ B cells (P Ͻ 0.001 for the three receptors). No significant Effects of CXCL12 Pretreatment of MCL and CD5؉ B differences in the expression of the remaining chemokine re- Cells on Their Subsequent Responsiveness to Chemokines. ceptors were detected, although a trend to up-regulation of Cross-sensitization or desensitization of chemokine receptors CCR1, CCR2, and CCR4 in tumor cells was observed. may occur in differentiating normal B cells (35) and in follicular

Fig. 3 Chemotaxis of mantle cell lymphoma (MCL) and normal CD5ϩ B cells to CCR and CXCR ligands. Cell locomotion was investigated using a transwell assay. MNC isolated from two lymph nodes (cases 1 and 2), 5 peripheral blood samples (cases 4–6, 8, and 9), and 1 pleural effusion (case 10) from MCL patients were added in the chemotaxis chamber in the presence (f)or absence (Ⅺ) of 100 ng/ml of the following chemokines, CXCL12, CCL19, CCL3, CCL2, CCL22, CCL20, CXCL8, CXCL10, and CXCL13. In parallel experiments, MNC from 7 reactive lymph nodes were added in the chemotaxis chamber in the presence (f) or absence (Nil, Ⅺ) of 100 ng/ml of the following chemokines, CXCL12, CCL19, CCL11, CCL22, CCL4, CCL20, CXCL8, CXCL10, and CXCL13. Mi- grated cells were recovered from the lower chamber after2hat37°C and counted. Malignant B cells were detected by staining for the monoclonal immunoglobulin light chain expressed by the individual clones, whereas normal CD5ϩ B cells were identified by double staining for CD19 and CD5. For MCL experiments, results for each case are expressed as absolute number of migrated cells. For CD5ϩ normal B-cell experiments, results are mean absolute number of migrated cells from seven independent experiments; bars, ϮSD.

lymphoma B cells (36). To investigate this issue, MCL B cells cell chemotaxis, as it was observed using freshly isolated cells and their normal counterparts were preincubated overnight with (Fig. 4 and, for comparison, Fig. 3). CXCL12 before being tested for chemotaxis to the ligands CD5ϩ normal B cells from the seven reactive lymph nodes indicated above. CXCL12 was selected for these experiments did not migrate to any chemokine with the only exception of because it was the only chemokine that attracted both MCL B CXCL12, after overnight incubation with CXCL12 itself (data ϩ cells and CD5 normal B cells not shown) As shown in Fig. 4, a significant response of malignant B Although the number of MCL samples tested in these cells to CXCL12 and CCL19 as compared with control cells experiments was limited, the above results indicate that tumor (nil; P Ͻ 0.01 and Ͻ0.05, respectively) was consistently ob- cells underwent cross-modulation of different chemokine recep- served with five MCL cell suspensions. Notably, tumor cells tors after exposure to CXCL12, whereas this was never ob- from case #1 acquired migratory competence to CXCL13 under served with their normal counterparts. these conditions (Fig. 4 and, for comparison, Fig. 3). Malignant B cells from case #2, which were unresponsive to CCL19 in the experiments shown in Fig. 3, were found to migrate to the same DISCUSSION chemokine after preincubation with CXCL12 (Fig. 4). Finally, In this study we have demonstrated that malignant B cells tumor cells from case #5 did not migrate to CCL19 (Fig. 4), from MCL patients express different receptors for chemokines whereas freshly isolated cells from the same patient did (see Fig. of the CC and CXC families, with a pattern common to all of the 3). All of the remaining chemokines did not stimulate MCL B samples irrespective of their source (i.e., peripheral blood,

(39, 40). They expand in fetal life, but are strongly down- regulated in postnatal life (40). In mice, CD5ϩ B cells represent a lineage separate from conventional, i.e., CD5Ϫ, B cells (41), whereas this has not yet been clearly established for their human counterparts (40). CD5ϩ B cell expansion takes place in auto- immune diseases, such as rheumatoid arthritis and some hemo- lytic anemias (42). This study shows for the first time that human CD5ϩ B cells express various chemokine receptors, with an overall profile similar to that of MCL B cells, but migrate only to CXCL12. This latter observation is consistent with a previous report showing that mouse peritoneal CD5ϩ B cells are attracted by CXCL12 (43). Failure of chemokine receptors to trigger chemotaxis upon interaction with their specific ligands may be related to low or absent expression or, alternatively, to functional impairment. In this study, many chemokine receptors, although expressed in malignant B cells or in their normal counterparts, were nonfunc- tional. Potential mechanisms underlying this latter phenomenon may be receptor desensitization by homologous or heterologous ligands (35, 44), uncoupling of signal transduction pathways, or defective internalization of the receptor-ligand complex. For example, members of the RGS protein family, when expressed in normal or malignant B cells, turn off signal transduction initiated by the binding of chemokines to their receptors (45, 46). Defective internalization of the CXCL12-CXCR4 complex has been described in freshly isolated germinal center B cells, and related to their inability to migrate to CXCL12, despite high CXCR4 expression (37). A final explanation for the failure of some chemokine receptors to trigger chemotaxis upon interaction with their ligands may be that the model used in this study Fig. 4 Effects of CXCL12 pretreatment of mantle cell lymphoma is not sensitive enough or just not representative, for example, (MCL) B cells on their subsequent responsiveness to chemokines. MNC because of the lack of important coactivators. ϩ cells from 5 MCL samples (cases 1, 2, 5, 8, and 9) were preincubated In this study, normal CD5 B cells did not migrate in overnight with 100 ng/ml CXCL12, washed, and subsequently tested in response to CCL19, indicating that functional CCR7 incompe- f Ⅺ a transwell assay in the presence ( ) or absence (nil, ) of the following tence is characteristic of this cell type and that malignant trans- chemokines, CXCL12, CCL19, CCL3, CCL2, CCL22, CCL20, CXCL8, CXCL10, and CXCL13, tested at the concentration of 100 ng/ml. formation is associated with gain of CCR7 function. Migrated cells were collected and identified by staining for the mono- In an attempt to overcome the refractoriness of various clonal immunoglobulin light chain expressed by the individual clones. chemokine receptors in MCL B cells and their normal counter- Results are expressed as absolute number of migrated cells. parts, cells were incubated overnight with CXCL12 and then subjected to chemotactic assays in the presence of a chemokine panel. lymph node, or pleura). The chemotactic in vitro activity of CXCL12 itself enhanced the in vitro migration of MCL B tumor cells was enhanced significantly only by CXCL12 and cells and their normal counterparts, indicating that CXCR4 was CCL19. Nonetheless, scattered in vitro migration to other che- rapidly recycled to the cell surface after internalization of the mokines was observed in the individual MCL cases, demon- CXCR4-CXCL12 complex. Tumor cells preincubated with strating the heterogeneous behavior of each B cell clone. CXCL12 showed a significant chemotaxis to CCL19, whereas In secondary lymphoid tissues, CXCL12 is expressed by normal CD5ϩ B cells did not. Finally, pretreatment of MCL B stromal cells (37), whereas CCL19 and CCL21, the ligands of cells with CXCL12 endowed them with de novo responsiveness CCR7, are expressed on the surface of high endothelial venules to CXCL13 in one case and to CCL19 in another, and, con- and in the perivascular stroma (38). Similar patterns have been versely, abrogated CCR7 function in a third case. In contrast, detected in infiltrated lymph nodes from B-CLL patients (28). CD5ϩ B cells cultured with CXCL12 did not migrate to other Therefore, it is conceivable that interaction of CXCR4 with chemokines. Thus, CXCL12, besides attracting directly MCL B CXCL12 and of CCR7 with CCL19 and CCL21 in the lymph cells, may also influence their in vivo migration by modulating node microenvironment promotes migration of MCL tumor other chemokine receptors. cells, thus contributing to their local invasive potential. A recent study has suggested the existence of two MCL CD5ϩ B cells represent a lymphocyte subset specialized in subsets, one showing a gene expression profile comparable with the production of low affinity, polyreactive IgM autoantibodies that of normal pregerminal center and memory B cells, and

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Anna Corcione, Nicoletta Arduino, Elisa Ferretti, et al.

Clin Cancer Res 2004;10:964-971.

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