Arnold S. Freedman, MD, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA. E-mail: firstname.lastname@example.org
Summary. Follicular lymphomas (FLs) localize in lymphoid tissues and recapitulate the structure of normal secondary follicles. The chemokine/chemokine receptor pair CXCL13/CXCR5 is required for the architectural organization of B cells within lymphoid follicles. In this study, we showed that CXCL13 was secreted by FL cells. FL cells expressed CXCR5 and migrated in response to CXCL13. Furthermore, we observed a synergistic effect between CXCL13 and CXCL12 (SDF-1), a chemokine produced by stromal cells in lymphoid tissues. The production of CXCL13 by FL cells and CXCL12 by stromal cells probably directs and participates in the accumulation of FL cells within specific anatomic sites.
Follicular lymphomas (FLs) are neoplastic counterparts of normal germinal centre (GC) B cells, and recapitulate the architecture and cytological features of the normal secondary lymphoid follicle. Generally, FLs are widely disseminated diseases, involving lymphoid secondary tissues and the bone marrow (BM). However, the mechanisms driving tissue localization of FL remains unclear.
Chemokines play an important role in the localization of normal B lymphocytes within tissues. The chemokine CXCL13 (BLC-1, BCA-1) is strongly expressed in the follicles of Peyer's patches, spleen and lymph nodes by a subset of follicular dendritic cells (FDCs) and adjacent follicular stromal cells (Ansel et al, 2000). CXCL13 specifically binds to the receptor CXCR5/BLR expressed on mature B cells and on a subpopulation of T-helper memory cells (Dobner et al, 1992). Murine studies have shown that CXCL13/CXCR5 is required for the organization of B cells in lymphoid follicles and for the development of most lymph nodes and Peyer's patches (Forster et al, 1996; Ansel et al, 2000).
We hypothesize that CXCL13 production by FL cells may participate in the accumulation and architectural organization of FL cells. In the present study, we report that FL cells secrete CXCL13, express functional CXCR5 receptors and migrate in response to CXCL13.
Materials and methods
Isolation and purification of GC and FL B cells. FL cells and normal GC B cells were isolated and purified from histologically involved lymph nodes or normal tonsils, respectively, as previously described (Husson et al, 2001). All samples were obtained according to Human Protection Committee validation and informed consent. Following depletion, the purity of FL cells was greater than 98% (Ghia et al, 1998). Similarly, GC B-cell preparations showed 98% CD20+ CD38+ cells with no detectable CD4+, CD8+, CD56+, CD14+, CD44+ and IgD+ cells.
Cell culture and enzyme-linked immunosorbent assay (ELISA). Purified FL and GC B cells were resuspended in Iscove's-modified Dulbecco's medium (IMDM; Mediatech, Herndon, VA, USA) with 2% fetal calf serum (FCS; PAA Laboratories, Newport Beach, CA, USA), 2 mmol/l l-glutamine and 15 µg/ml gentamicin (Life Technologies, Gaithersburg, MD, USA). Cells were stimulated for 72 h with 2 µg/ml human soluble trimeric CD40 ligand (sCD40L) (Immunex, Seattle, WA, USA) and 2 ng/ml recombinant human interleukin 4 (rhIL-4; R & D systems, Minneapolis, MN, USA). ELISA was performed for CXCL13 and CXCL12 (R & D Systems) on culture supernatants.
Immunohistochemistry. Immunohistochemistry on follicular lymphoma cases for primary antibodies CD20 (L26), κ and λ light chain (Dako, Carpinteria, CA, USA) were performed using 5 µm thick frozen sections and detected with standard avidin–biotin methods as previously described (Kutok et al, 2001). Immunohistochemistry for BCA-1/CXCL13 was performed using 5 µm thick formalin-fixed, paraffin-embedded tissue sections. Briefly, slides were deparaffinized and pretreated with 10 mmol/l citrate, pH 6·0 (Zymed, South San Francisco, CA, USA) in a steam pressure cooker (Decloaking Chamber; BioCare Medical, Walnut Creek, CA, USA) as per the manufacturer's instructions, followed by washing in distilled water. All further steps were performed at room temperature in a hydrated chamber. Slides were pretreated with peroxidase block (Dako) for 5 min to quench endogenous peroxidase activity, followed by a 1:5 dilution of goat serum in 50 mmol/l Tris-HCl, pH 7·4, for 20 min to block non-specific binding sites. Primary murine anti-human BCA-1/CXCL13 antibody (R & D Systems) was applied at a 1:10 dilution in 50 mmol/l Tris-HCl, pH 7·4 with 3% goat serum for 1 h. Slides were washed in 50 mmol/l Tris-HCl, pH 7·4, and goat anti-mouse horseradish peroxidase-conjugated antibody (Envision detection kit; Dako) was applied for 30 min. After further washing, immunoperoxidase staining was developed using a diaminobenzidine chromogen kit (Dako), as per the manufacturer's instructions, and counterstained with haematoxylin.
Fluorescence-activated cell sorting (FACS) analysis. Immunophenotyping was performed as previously described (Ghia et al, 1998) using monoclonal antibodies directed against: CD4, CD8, CD14, CD20, CD38, CD44, CD56, IgD, κ or λ light chains (Southern Biotechnology Associates, Birmingham, AL, USA), CXCR4 and CXCR5 (R & D Systems).
Chemotaxis assay. Recombinant CXCL12 and CXCL13 were obtained from R & D Systems. Chemotaxis assays were performed as previously described (Husson et al, 2001). Chemokines were added in the bottom wells and 100 µl (1 × 105) of cell suspension cells, with or without chemokines, were loaded onto the inserts. Cells were collected after 4 h and counted by flow cytometry.
Results and discussion
Normal and malignant B cells were cultured for 3 d and culture supernatants were analysed by ELISA for CXCL13. CXCL13 could not be detected in GC B-cell supernatants whereas low levels (± 50 ng/ml) were detected in FL cell supernatants from five patients. As GC B cells and FL cells undergo apoptosis when left unstimulated in vitro, we postulated that the low levels of chemokine produced at basal conditions could be due to extensive death of the cells (Ghia et al, 1998). Alternatively, this could be due to low levels of constitutive expression. Survival of FL cells (and normal B cells) can be extended in vitro by stimulation through CD40 and IL-4R (Ghia et al, 1998). GC B cells and FL cells were cultured with soluble CD40 ligand (sCD40L) and rhIL-4 for 72 h. Stimulated FL cells showed significantly increased production of CXCL13 (2–15-fold increase), while GC B cells did not. The basal value of CXCL13 secretion in FL was 47 ± 7 ng/ml, whereas following CD40/IL4 stimulation the level of CXCL13 was 259 ± 163 ng/ml. This is representative of five independent determinations (non-parametric t-test P-value = 0·0082). This was in contrast to murine dendritic cells stimulated with CD40L, which downregulated CXCL13 production, suggesting a different mechanism of regulation depending on the cell type (Vissers et al, 2001). To demonstrate that FL cells were producing CXCL13, we performed immunostaining of FL tissue with anti-CD20, anti-κ, anti-λ and anti-CXCL13 antibodies. The lymphoma cells were strongly CD20 positive (Fig 1A) and demonstrated monotypic surface κ light chain expression (Fig 1C), but not for λ light chain. Strong cytoplasmic staining for CXCL13 was seen in the neoplastic cells (Fig 1B, insert) within the follicles and was absent in the interfollicular areas (Fig 1B, large panel). CXCL13 expression by malignant B cells has also been reported in gastric extranodal marginal zone lymphomas. In these cases, the neoplastic B cells were the only apparent source of CXCL13 (Mazzucchelli et al, 1999). As these tumour cells strongly expressed CXCR5, lymphoma cell production of CXCL13 might contribute to their localization and retention within the gastric mucosa.
In FL, there is evidence that other chemokine/receptor pairs are involved in the progressive accumulation of malignant B cells within lymphoid tissues. FL are characterized by the presence of FDCs and lymph node stromal cells that produce the chemokine CXCL12 (Arai et al, 2000). It has been reported that FL cells may be a source of CXCL12 based on polymerase chain reaction studies (Corcione et al, 2000). We could not detect CXCL12 by ELISA in FL cell supernatants (data not shown).
FLs express both CXCR5 and CXCR4 (CXCL12 specific receptor) (Fig 2A) (Jones et al, 2000). We performed a migration assay, using recombinant CXCL13, recombinant CXCL12 as positive control, or the combination of both (Corcione et al, 2000; Jones et al, 2000). FL cells from patients migrated in response to CXCL13 alone albeit to a lesser extent than with CXCL12. The combination of both chemokines had an additive effect on the migration of FL cells as compared with the individual chemokines (Fig 2B). When CXCL13 was present in the upper compartment, and CXCL12 and CXCL13 together in the lower compartment, the migration was the same as when CXCL12 was present alone in the lower compartment. FL cell localization in lymphoid tissues may be supported by CXCL13 and CXCL12 produced by FL cells and stromal cells respectively (checkerboard).
CXCL13 production by FL cells suggests a specific chemokine/receptor interaction, leading not only to neoplastic B-cell accumulation into secondary lymphoid tissues, but also to the typical architectural organization of the disease. It has been suggested that T cells may be involved in the growth and survival of FL (Ghia et al, 1998). CXCR5 is also expressed on a T-cell subset, termed follicular B-helper T (TFH) cells (Breitfeld et al, 2000). These T cells are mainly localized in the mantle and light zone of GCs and are capable of providing help for B-cell activation. CXCL13 production by FL cells may also act as a chemoattractant for TFH cells, thereby contributing to the capacity of malignant cells to create a microenvironment necessary for their survival.
This work was supported in part by NIH grants CA66996 (to A.S.F.), The Leukaemia and Lymphoma Foundation of America, the Norman Hirschfield Foundation, Associazione Italiana per la Ricerca sul Cancro (AIRC), MURST 40%. H.H. was supported by the ‘Cure for Lymphoma Foundation’.