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Originally published online as doi:10.1189/jlb.0706481 on June 15, 2007

Published online before print June 15, 2007
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(Journal of Leukocyte Biology. 2007;82:567-575.)
© 2007 by Society for Leukocyte Biology

Critical role of monocytes to support normal B cell and diffuse large B cell lymphoma survival and proliferation

Chris G. Mueller*,{dagger},1, Charlotte Boix*, Wing-Hong Kwan*, Cécile Daussy*, Emilie Fournier*, Wolf H. Fridman* and Thierry J. Molina{ddagger}

* INSERM, U872, Centre de Recherches Biomédicales des Cordeliers Université Pierre et Marie Curie (Paris VI) et René Descartes (Paris V), UMR S 872, Paris, France;
{dagger} CNRS, Laboratory of Therapeutic Immunology and Chemistry, IBMC, Strasbourg, France; and
{ddagger} Université Paris-Descartes, Faculté de Médecine, AP-HP, Hôtel-Dieu, Service d’Anatomie et de Cytologie Pathologiques, Paris, France

1 Correspondence: CNRS, Laboratory of Therapeutic Immunology and Chemistry, IBMC, Strasbourg, France. E-mail: c.mueller{at}ibmc.u-strasbg.fr


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ABSTRACT
 
Large B cell lymphomas can comprise numerous CD14+ cells in the tumor stroma, which raises the question of whether monocytes can support B cell survival and proliferation. We show that the coculture of monocytes with B cells from peripheral blood or from diffuse large B cell lymphoma enabled prolonged B cell survival. Under these conditions, diffuse large lymphoma B cells proliferated, and addition of B cell-activating factor of the TNF family (BAFF) and IL-2 enhanced cell division. Monocytes and dendritic cells (DC) had similar antiapoptotic activity on healthy B cells but displayed differences with respect to B cell proliferation. Monocytes and cord blood-derived CD14+ cells promoted B cell proliferation in the presence of an anti-CD40 stimulus, whereas DC supported B cell proliferation when activated through the BCR. DC and CD14+ cells were able to induce plasmocyte differentiation. When B cells were activated via the BCR or CD40, they released the leukocyte attractant CCL5, and this chemokine is one of the main chemokines expressed in diffuse large B cell lymphoma. The data support the notion that large B cell lymphoma recruit monocytes via CCL5 to support B cell survival and proliferation.

Key Words: macrophages • chemokines • tumor immunity


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INTRODUCTION
 
Dendritic cells (DC) contribute to the shaping of the B cell immune response, classically exemplified by their ability to prime antigen-specific T cells, which then provide help in the form of the CD40 ligand (CD40L) to B cells. Activated by CD40L, DC then also provide direct help to B cells by the release of cytokines such as IL-12 or IL-6 [1 ]. Moreover, DC can present unprocessed antigen via the IgG receptor Type II to facilitate access of peripherally sampled antigen to lymph node-restricted, naive B cells [2 ]. However, it has emerged that myeloid cells other than DC can support B cell function. For instance, macrophages support BCR-stimulated B cell proliferation [3 ], and activated monocytes can induce CD40-independent Ig class switching [4 ]. B cell-activating factor of the TNF family (BAFF), related to CD40L, has been implicated in these processes, and BAFF is expressed by DC, monocytes, and macrophages [5 ]. BAFF rescues mouse transitional Type II (T2) B cells from apoptosis [6 ] as well as a human T1-like cell line after BCR engagement [7 ].

This has spurred investigation into the role of BAFF in the survival and proliferation of neoplastic B cells. Most neoplastic B cells (Hodgkin and non-Hodgkin lymphoma) express one of three receptors, which bind BAFF or the related a proliferation-inducing ligand (APRIL), namely BAFF-R, B cell maturation antigen (BCMA), and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) [8 9 11 ]. BAFF or APRIL has been detected in B cell lymphoma, and the source may be the B cells themselves [8 ,10 ,11 ] or accessory cells such as infiltrating macrophages [12 ]. BAFF has antiapoptotic effects on neoplastic B cells [8 ,10 11 12 13 14 15 ]; however, B cell proliferation could not be induced [8 ,13 ,14 ], unless costimulation was provided in the form of BCR engagement or cytokines [13 ,14 ]. We have observed recently abundant CD14+ cells in large diffuse B cell lymphoma (DLBCL) and in splenic marginal zone lymphoma (SMZL), but CD1a+ DC or CD169+ sinusal macrophages were absent.

Taken together, the data suggest that the presence of myeloid cells and BAFF may be optimal for B cell survival and proliferation. Different groups have previously shown the beneficial effect of monocytes/CD14+ cells or DC for B cell differentiation [4 ,16 ], B cell proliferation [3 ,16 ], or malignant B cell survival [8 ,12 ]. However, monocytes/CD14+ cells and DC have not been analyzed side-by-side. Here, we compare these cells in the presence or absence of BAFF for the ability to support survival and proliferation of healthy and DLBCL B cells as well as normal B cell differentiation to plasmablasts. In addition, we analyzed the chemokine expression of B cells. Our results show that under most conditions, monocytes/CD14+ cells are as efficient as DC for normal B cell survival, proliferation, and differentiation. Addition of BAFF and IL-2 to the coculture enhances cell division. Furthermore, activated, normal B cells and DLBCL B cells can produce CCL5. Therefore, production of CCL5 by DLBCL would attract blood monocytes to support lymphoma survival and growth efficiently.


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MATERIALS AND METHODS
 
Growth factors, cytokines, and antibodies
Complete medium was RPMI-1640 medium supplemented with 10% endotoxin-free FCS (Australian origin from Invitrogen, Cergy-Pointoise, France), 2 mM L-glutamine (Invitrogen), and antibiotics (Invitrogen). B cells were activated with 5 µg/ml rabbit F(ab')2-Ig anti-human IgG and IgM (Jackson Immunoresearch, West Grove, PA, USA), henceforth referred to as anti-BCR antibodies. Recombinant BAFF, BAFF-R-Fc, or BCMA-Fc, anti-BAFF mAb for ELISA, were kindly provided by Biogen-Idec, Inc. (Boston, MA, USA). M-CSF, IL-4, and TNF-{alpha} were from R&D Systems (Abbington, UK), IL-2 from Chrion, Inc. (Berkeley, CA, USA), GM-CSF from Schering-Plough, Inc. (Kenilworth, NJ, USA), and stem cell factor from Amgen, Inc. (Thousand Oaks, CA, USA). CD40L-expressing mouse fibroblasts were used at a ratio of five B cells:one fibroblast. Activating anti-CD40 mAb (Clone G28.5) was purified by protein A binding from hybridoma supernatant, originally provided by Ed Clark (University of Washington, Seattle, WA, USA).

Cell purification and culture
Peripheral blood buffy coats from healthy donors were obtained from the Établissement Français du Sang, Hôpital Hôtel-Dieu (France), and the Bourg-La-Reine Maternity Ward (France) provided cord blood with informed consent. The material was processed according to institutional guidelines. Mononuclear cells were obtained by centrifugation through a Ficoll cushion (PAA Laboratories, Pashing, Germany). B cells were purified by CD19-coated magnetic beads (Miltenyi, Paris, France), and the negative fraction was enriched for monocytes by centrifugation through a 52% Percoll gradient (Sigma-Aldrich, Saint Quentin Fallavier, France). Floating cells were collected and negatively selected using a monocyte isolation kit (Dynal, Compiègne, France) to a purity of commonly 95%. CD14+ cells and DC were grown directly from cord blood CD34+ cells, as described previously [17 ]. After the culture period with M-CSF (CD14+ cells) or GM-CSF (DC), the cell populations comprised 70–80% CD14+ or DC, respectively, and were used without further cell sorting. Earlier experiments had shown that magnetic bead or flow cytometry-purified CD14+ cells or CD1a+ DC resulted in the same B cell survival. B cell lymphoma cell suspensions were made from splenic or nodal tumors obtained from the Department of Pathology of the Hôpital Hôtel-Dieu or the European Hospital George Pompidou (Paris, France). The cell suspensions were frozen in FCS containing 10% DMSO. After thawing, the cell suspension was cleared though a Ficoll gradient, and B cells were isolated using anti-CD19-coated magnetic beads (Miltenyi).

B cell survival assay
In 2 ml complete medium in a 24-well plate were cocultured 5 x 105 normal or DLBCL B cells and 105 CD14+ cells, DC, or monocytes. Cultures containing CD14+ cells received 10 ng/ml M-CSF, and those containing DC, 200 U/ml GM-CSF at the beginning of the culture. To some wells, the following reagents were added at Day 0 and refreshed at Day 3: 10 µg/ml anti-BCR, 100 ng/ml BAFF, 10 µg/ml BAFF-R-Fc, or BCMA-Fc; 10 µg/ml human IgG1 (Sigma-Aldrich) or 100 IU/ml IL-2. After 6 days (normal B cells) or 5–7 days (lymphoma), cells were harvested and labeled for CD19 (APC, Beckman-Coulter, Villepinte, France) and analyzed by flow cytometry after adding a known number of nonfluorescent FACS calibration beads (Becton Dickinson, LePont-de-Claix, France). With the beads, the number of viable CD19+ B cells was determined using the following formula: (number of CD19 events/number of bead events) x (bead numberxtotal volume/volume tested).

To test for the release of a soluble survival factor, 2 x 106 CD14+ cells were kept for 3 days alone or with 107 B cells in 6 ml complete medium in the absence or presence of anti-BCR. Cell-free supernatants of these cultures (50% v/v) were added to 5 x 105 B cells, and their survival was tested as described above. For the lymphoma B cell survival experiment containing a graded number of monocytes, 2.5 x 105 B cells were added to monocytes in 1 ml complete medium/well of a 48-well plate.

B cell proliferation measurements
B cells (2.5x105) were labeled with 1 µM CFSE (Invitrogen) and cocultured with 5 x 104 CD14+ cells, DC, or monocytes. DLBCL B cells (5x105) were labeled with CFSE and cocultured with 105 monocytes to combine lymphoma B cell proliferation with viability measure. After 3 days (normal B cells) or 5 or 10 days (DLBCL), cells were labeled for CD19 (APC, Beckman-Coulter), and dilution of the green fluorescent CFSE label in the CD19+ low side-scatter B cell gate was measured by flow cytometry.

Flow cytometry analysis
Forward- and side-scatter plots were used to gate for viable cells. In the B cell-myeloid cell cocultures, B cells could be distinguished from myeloid cells by their smaller size and lower granulation index. Within this gate, B cell-specific markers CD19 or CD20 were found but no CD14+ cells or CD1a+ DC. Lymphoma B cells were larger but still distinct from monocytes by their lower granulation index (see Fig. 4C ). B cells were tested for their purity by cell surface expression of CD19 (APC, Beckman-Coulter) and CD20 (PE, BD PharMingen, San Diego, CA, USA), and contaminating T cells were detected by anti-CD3 labeling (FITC, BD PharMingen). T cell contamination rarely exceeded 5%. To assess plasmocyte differentiation, cells were stained for CD38 (PE, BD-PharMingen) and CD20 (FITC, BD-PharMingen). CD69 expression was measured using anti-CD69-APC (BD-PharMingen). Purity of monocytes, CD14+ cells, or DC was verified by anti-CD14 mAb (APC or FITC, BD-PharMingen) and anti-CD1a mAb (PE, BD-PharMingen), respectively.


Figure 4
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  		Figure 4. Monocytes support DLBCL survival and proliferation. (A) DLBCL B cells (500x103) were cultured with or without increasing numbers of monocytes. After 5 days, viable B cells were counted. (B) DLBCL B cells (500x103) were cultured in the absence or presence of monocytes (ratio five B cells:one monocyte), with or without 100 ng/ml BAFF and/or 100 IU/ml IL-2. Viable B cells were enumerated after 5 days. The data are the mean of triplicates ± SD and representative of three experiments. Significance is indicated by an asterisk, calculated by the two-sided Student’s t-test. (C) CFSE-labeled DLBCL B cells were kept for 5 or 10 days in the presence of monocytes, with or without BAFF and/or IL-2. CFSE dye dilution was assessed by flow cytometry on CD19+ B cells gated for viable cells with low side-scatter (left side). The percentage of cells with CFSE loss is indicated. (D) DLBCL B cells were tested for CD69 expression 5 days after culture with monocytes and the indicated cytokines.

B cell plasmocyte differentiation
B cell plasmocyte differentiation was induced as described previously [18 ]. Briefly, 3 x 106 B cells were cultured with 1.5 x 106 mitomycin C-treated CD40L+ fibroblasts for 4 days. Then, nonadherent B cells were restimulated with 106 CD14+ cells or DC. After 4 days, the cells were harvested and analyzed for CD38/CD20 expression by FACS, after gating for viable B cells. Supernatant was kept for measure of Ig concentration.

ELISA
BAFF sandwich ELISA was performed using standard procedures with biotinylated detection mAb (Biogen-Idec, Inc.), revealed by streptavidin-coupled HRP and the appropriate color substrate. CD14+ cells were stimulated with 10 ng/ml IL-10 and/or 10 ng/ml IFN-{gamma} for 48 h in complete medium supplemented with 10 ng/ml M-CSF, and DC were stimulated likewise in the presence of 200 U/ml GM-CSF. To induce CCL5 production, 5 x 105 B cells were cultured in 2 ml complete medium and stimulated with combinations of 100 ng/ml BAFF, anti-BCR, and CD40L+ fibroblasts. After 2 days, supernatants were collected, and CCL5 concentration was measured using an ELISA kit (R&D Systems, Abbington, UK). For Ig, plates were coated with 5 µg/ml goat anti-human IgA, IgG, or IgM (Southern Biotech Associates, Inc., Birmingham, AL, USA). Cell-free supernatants or standards were added, and bound Ig was revealed with alkaline phosphatase-coupled goat anti-human IgA, IgM (both at 5 µg/ml), or IgG (2 µg/ml, all from Southern Biotech Associates, Inc.). As a standard, purified, human IgA (Jackson Immunoresearch), IgG, and IgM (Laboratoire de Fractionnement Biologique, Les Ulis, France) were used.

RT-PCR
RNA was prepared from normal, peripheral B cells or malignant, splenic or nodal B cells (RNeasy-Mini, Qiagen, Courtaboeuf, France) and reverse-transcribed using a RT first-strand synthesis kit primed with polyT (Boehringer-Roche, Indianapolis, IN, USA). The quality of the cDNA was tested by PCR for human ß-actin. The sequence of the primers specific for the different chemokines as well as the expected PCR product size have been published [19 ].


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RESULTS
 
Myeloid cells support B cell survival efficiently
When human peripheral blood B cells were kept for 6 days in complete medium, virtually all cells died (Fig. 1A ). However, when cocultured with CD14+ cells, DC, or monocytes, B cell death was clearly reduced. All three cell types proved to be equally efficient in maintaining viable B cells, as differences were marginal and not significant. We performed the same study with addition of exogenous BAFF, which increased viable B cell recovery significantly in the absence of myeloid cells. However, it could not match the B cell survival signals provided by the myeloid cells. When added to the B cell myeloid cell coculture, BAFF showed an additive effect with CD14+ cells and monocytes, but it was less clear with DC. We confirmed that BAFF costimulated BCR-activated B cell proliferation [20 ]; however, BAFF alone or together with the myeloid cells did not trigger B cell division (data not shown). Figure 1B shows that BCR stimulation likewise increased viable B cell recovery, but again, this stimulus was less efficient than a coculture with the myeloid cells. The increase in viable B cells was in part a result of proliferation (see below).


Figure 1
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  		Figure 1. Myeloid cells efficiently support B cell survival. (A) Peripheral blood B cells (500x103) were left in culture, with or without CD34+ cord blood progenitor-derived CD14+ cells, DC, or peripheral blood syngeneic monocytes (Mono; ratio five B cells:one myeloid cell) in the presence or absence of 100 ng/ml recombinant human BAFF. After 6 days, the number of viable B cells was determined. Shown is the mean of at least three independent experiments (±SD). Significance is indicated by an asterisk and is calculated by the two-sided Student’s t-test. (B) B cells (500x103) were cultured, with or without CD14+ cells or DC (ratio five B cells:one myeloid cell) in the presence or absence of 10 µg/ml anti-BCR (a-BCR) antibodies. After 6 days, the number of viable B cells was determined. Shown is the mean of at least three independent experiments (±SD) and significance indicated by an asterisk. (C) B cells were cultured for 6 days in the following conditions: complete medium (–), CD14+ cells (cells), CD14+ cell-conditioned supernatant (supernatant), CD14+ cell, and B cell coculture-conditioned supernatant (CD14 and B cell supernatant) and finally, CD14+ cell and BCR-stimulated B cell coculture-conditioned supernatant (CD14 and B+a-BCR supernatant). The number of viable B cells was determined. Shown is the result of one of two independent experiments.

To investigate if myeloid cells support B cell survival by virtue of soluble factors, we recovered supernatants from CD14+ cells, cultured alone, with resting B cells, or with BCR-activated B cells (Fig. 1C ). Only addition of myeloid cells directly to B cells supported B cell survival efficiently, strongly suggesting that cell contact was required. The supernatant of BCR-activated B cells increased viable B cell recovery marginally, but this may be a result of transferred, anti-BCR antibodies. Therefore, CD14+ cells support peripheral blood B cell survival more efficiently than BAFF or BCR stimulation, and this occurs through cell-bound factor(s).

Endogenous BAFF participates in B cell survival
Next, we tested if CD14+ cells or DC expressed BAFF at their cell surface (Fig. 2A ) or released it into the culture medium (Fig. 2B ). CD14+ cells produced low levels of membrane-bound or soluble BAFF, but DC expressed no detectable BAFF. As IL-10 or IFN-{gamma} stimulate BAFF expression [3 ,5 ], we cultured the CD14+ cells with IL-10 and/or IFN-{gamma}. IL-10 resulted in the strongest BAFF cell-surface expression, and the combination of IL-10 and IFN-{gamma} led to the highest levels of soluble BAFF. As for DC, stimulation with IL-10 and IFN-{gamma} likewise triggered BAFF expression, but the levels were largely inferior to those obtained with CD14+ cells. Then, we addressed the role of endogenous BAFF or the related APRIL in B cell survival by the CD14+ cells or DC. We cultured B cells with the myeloid cells in the presence of human BAFF-R-IgFc fusion protein, human BCMA-IgFc, or control human IgG1 (Fig. 2C ). BAFF-R binds BAFF exclusively, and BCMA binds BAFF and APRIL [6 ]. BAFF-R-Fc and BCMA-Fc decoy proteins reduced viable B cell recovery significantly when cultured with CD14+ cells, but the effect of the fusion proteins was not significant with DC. The data show that BAFF is produced by CD14+ cells and contributes to B cell survival.


Figure 2
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  		Figure 2. BAFF is implicated in B cell survival by CD14+ cells. CD34+ cord blood progenitor-derived CD14+ or DC were cultured for 48 h, with or without IL-10 and/or IFN-{gamma}, and cell surface expression (A) or release (B) of BAFF was measured by flow cytometry or ELISA. Shown are the geometric (Geo) mean expression and the mean BAFF release of four different donors ± SD. (C) B cells were cocultured with CD14+ or DC (ratio 10 B cells:one myeloid cell) in the presence of 10 µg/ml human IgG1, BAFF-R-Fc, or BCMA-Fc. After 6 days, viable B cells were enumerated. The data are the mean ± SD of three independent experiments. Significance is indicated by an asterisk; n.s., Not significant.

CD14+ cells and DC promote B cell proliferation and differentiation
We compared the different myeloid cells with respect to B cell proliferation. This was measured by the CFSE dilution assay (Fig. 3A ). CD14+ cells, DC, or monocytes induced little B cell proliferation in the absence of B cell activation. When B cells were BCR-stimulated, DC clearly enhanced B cell division. When B cells were activated with anti-BCR/anti-CD40/IL-2 treatment, CD14+ and monocytes were more efficient than DC in promoting B cell proliferation. Myeloid cell division was not detected under any of these conditions (data not shown). Then, CD14+ cells and DC were compared for their ability to induce plasmocyte differentiation. For this, B cells were first activated by CD40L-transfected fibroblasts and then, in a second step, recultured with CD14+ cells or DC in the absence of the fibroblasts [18 ]. As shown in Figure 3B , the Ig concentration increased between the first and the second step without marked isotype change. CD14+ cells and DC were equally capable of promoting IgM and IgG production, and IgA remained low. We labeled B cells for CD20 and CD38 to assess the proportion of CD20–38+ plasmocytes (Fig. 3C ). CD14+ cells and DC were able to induce plasmocyte differentiation, although in each experiment, DC were superior to CD14+ cells.


Figure 3
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  		Figure 3. Myeloid cells promote B cell proliferation and differentiation. (A) CFSE-labeled B cells were cultured with or without CD34+ cord blood progenitor-derived CD14+ cells, DC, or monocytes in the presence or absence of the indicated stimuli (anti-BCR antibodies, anti-CD40 mAb, IL-2). After 3 days, the cells were labeled for CD19, and the CFSE label was measured by flow cytometry, after gating for viable CD19+ B cells. The percentages of CD19+ B cells having lost the CFSE dye are indicated. The data are representative of three experiments. (B) B cells were first kept for 4 days on CD40L-transfected fibroblasts (1st step, ratio two B cells:one fibroblast), and then, nonadherent B cells were recultured for 4 days with CD14+ cells or DC in the absence of fibroblasts (2nd step). Supernatants IgM, IgG, and IgA, after the first and second step, were measured by ELISA. (C) After the second step, cells were labeled with anti-CD20 and anti-CD38, and the expression of these markers was measured by flow cytometry after gating on live B cells. The graph on the right depicts the percentage of CD38+ CD20– plasma B cells in three independent experiments.

Monocytes support B cell lymphoma survival and proliferation
Having shown that monocytes are as efficient as CD34+ progenitor-derived CD14+ cells and DC in supporting healthy B cell survival, we tested the ability of monocytes to support DLBCL B cell survival and proliferation. Figure 4A shows that culturing DLBCL B cells in complete medium for 5 days resulted in complete cell death. However, when cultured with monocytes, live B cells were recovered, and the recovery was proportional to the number of monocytes added. Then, we studied the effects of BAFF and IL-2 (Fig. 4B ). B cell death could not be prevented with exogenous BAFF and IL-2 in the absence of monocytes. However, in the presence of monocytes, BAFF had a marginal effect, and IL-2 increased the number of viable DLBCL B cells significantly. We tested three other DLBCL and one SMZL, where CD14+ cells are also frequently found [21 ]. The data are summarized in Table 1 . Consistently, lymphoma B cells survived only when monocytes were present, and addition of BAFF and IL-2 resulted in the highest B cell recovery.


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  		Table 1. Lymphoma B Cell Survival by Monocytes

We monitored B cell proliferation using the CFSE dye dilution assay. In Figure 4C , side- and forward-scatters for the DLBCL #1 are shown, pointing out cell counting beads, monocytes, and the gated B cell population. Already at Day 5, DLBCL B cells displayed loss of the CFSE dye under all conditions, reflecting cell division. At Day 10, it was apparent that IL-2 accelerated by twofold DLBCL B cell division, and BAFF alone had only a small effect. Additional action of IL-2 and BAFF was noticeable. DLBCL B cells displayed expression of the early activation marker CD69 (Fig. 4D ), and the highest CD69 levels were found with IL-2 and BAFF.

Morphologically, without monocytes, the DLBCL B cells showed cell death (autolysis and pycnosis) (Fig. 5A ), whereas with monocytes, the B cells appeared healthy (Fig. 5B and 5C ). Occasional, excentric nuclei and light basophilic cytoplasm were observed, suggestive of plasma cell differentiation (see arrows in Fig. 5C ). Many cells were CD20+ (Fig. 5D ), and some cells showed intense IgM staining (Fig. 5E ). It appeared that some B cells were in direct contact with monocytes (see arrow in Fig. 5E ). Taken together, these data show that monocytes provide essential survival signals for DLBCL B cells, which cannot be matched by BAFF or IL-2. However, when cultured with monocytes, the lymphomas then respond to BAFF and IL-2.


Figure 5
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  		Figure 5. DLBCL B cells display viable cell morphology with monocytes. B cells were kept without (A) or with monocytes in the presence of IL-2 and BAFF (B–E), cytospun, and stained by May-Grünwald-Giemsa (A–C) for CD20 (D) or IgM (E). (C) Arrows point to cells with light, basophilic cytoplasm, suggestive of plasma cell differentiation. (E) Arrow indicates a cell, which is probably a monocyte in contact with IgM+ B cells. Original magnification objective: x10 (A, B); x20 (D, E); after x40 (C).

Activated B cells release the chemoattractant CCL5
To explore if B cells can attract myeloid cells, we analyzed mRNA expression of different chemokines by B cells from two DLBCL: a healthy spleen and blood. Among the large number of chemokines tested, CCL5 (RANTES) was always amplified strongly (data not shown). Figure 6A shows the results of the PCR reaction for CCL1–5 and CCL17–22 of B cells from peripheral blood and a DLBCL case. We then measured CCL5 release by resting or activated peripheral blood B cells. As shown in Figure 6B , resting B cells expressed low but detectable amounts of CCL5, but it is notable that activated B cells increased CCL5 levels, and the highest levels were found in the condition of anti-BCR/CD40L stimulation. We measured CCL5 release in DLBCL B cell cultures (Table 2 ) and found that two of three lymphomas produced CCL5 when cocultured with monocytes and BAFF/IL-2. This shows that some DLBCL B cells can release CCL5, which would increase recruitment of monocytes and other leukocytes.


Figure 6
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  		Figure 6. B cells produce CCL5. (A) B cells purified from healthy peripheral blood and a DLBCL were tested for mRNA expression of CCL1–CCL5 and CCL17–CCL22 by RT-PCR. (B) Measure of CCL5 release by ELISA of peripheral blood B cells, resting or stimulated with the indicated activators. Shown is the mean ± SD CCL5 release in 48 h for one experiment of two, performed in triplicate.


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  		Table 2. CCL5 Production by DLBCL B Cells


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DISCUSSION
 
Among the different non-Hodgkin B cell lymphomas, DLBCL stands out as a lymphoma frequently displaying a host response gene signature [22 23 24 ]. Within this gene group are found genes encoding CD14, M-CSF, CD3, and CCL5. The presence of CD14+ monocytes and CD3+ T cells has been confirmed by immunohistochemical and morphologic analyses [21 ,23 ]. We had further characterized the myeloid cell population, and in spite of prominent CD14 staining, no CD1a+ or CD209+ DC or CD169+ sinusal macrophages were found [21 ].

In this report, we investigated the functional implications of CD14+ cell recruitment, first, in comparison with DC for healthy B cell survival, proliferation, and differentiation and then with respect to DLBCL survival and growth. The DC were generated with GM-CSF from CD34+ progenitors and display an immature phenotype phenotypically and functionally [17 ]. To measure B cell survival, we determined the absolute, viable B cell numbers rather than performing annexin V/propidium iodide staining to avoid a bias introduced by phagocytosis of apoptotic/dead B cells. We found a clear support of B cell survival by monocytes, cord blood progenitor-derived CD14+ cells, and DC, which could not be matched by exogenous B cell survival factor BAFF or BCR stimulation. Endogenous BAFF contributed to B cell survival by CD14+ cells, evident in the partial blocking by soluble decoy receptors. It was observed recently that individuals lacking functional TACI have normal B cell numbers [25 ,26 ] and that monkeys treated with BAFF-blocking antibodies display only a mild reduction in B cell numbers [27 ]. Therefore, antiapoptotic signals other than BAFF or APRIL are likely to be necessary for human B cell survival. The monocyte-derived, nurse-like cells provide support to lymphocytic leukemia B cells via the chemokine CXCL12 (stromal cell-derived factor-1) [15 ], and a follicular DC line maintains lymphocytic leukemia B cell survival via CD44 [28 ]. Recently, it was shown that mouse B cells require CD45 to survive in conditions mimicking a germinal center reaction [29 ].

Monocytes, CD14+ cells, and DC were all capable of promoting B cell proliferation, yet some differences were observed. DC enhanced proliferation of BCR-stimulated B cells, but monocytes or CD14+ cells did not; however, when a CD40 stimulus was provided, CD14+ cells and monocytes were superior to DC. These findings may be related to B cell differentiation. In response to CD40 ligation, DC produce IL-12, which promotes naive B cells to undergo plasma cell differentiation [30 ,31 ]. However, CD40L-activated CD14+ cells release IL-10 (data not shown), which accelerates cell division in the presence of IL-2 by up-regulating IL-2R [32 ]. Consistent with this idea, in the presence of CD40L, DC were slightly superior to CD14+ cells in promoting B cell differentiation to plasmocytes.

The results obtained using normal B cells provided grounds to postulate that monocytes would support lymphoma B cell survival and proliferation. This would be particularly relevant in lymphomas with prominent monocyte infiltration in the tumor stroma, such as DLBCL and SMZL [21 ]. Therefore, B cells from four different DLBCL and one SMZL were cultured with or without monocytes and exogenous BAFF/IL-2. IL-2 was chosen, as this cytokine accelerates B cell proliferation, particularly in the presence of IL-10 [32 ]. After a 6-day culture, viable B cells were only found when cocultured with monocytes. Addition of BAFF and IL-2 led to the highest viable B cell recovery. The presence of BAFF and IL-2 in the absence of monocytes failed to preserve B cell viability. In the tumor environment, IL-2 may be produced by T cells [23 ], and CCL5 can trigger IL-2 release by T cells [33 ]. When cocultured with monocytes, occasional lymphoma B cells were found displaying morphology and intense IgM staining resembling plasma cells. The significance of this finding is currently unclear. It may concur with the ability of CD14+ cells to promote plasmocyte formation when cultured with normal B cells in the presence of CD40L. The experiments were carried out with frozen lymphoma cell suspensions, and the freeze-thaw cycle may have reduced resistance to cell death. The ability to maintain B cell lymphoma in culture with monocytes should facilitate their study and could be used to test the effects of different survival and growth factors. Alternatively, the in vitro effects of antilymphoma treatments such as anti-CD20 antibodies may be explored in the monocyte-B cell culture system.

The supportive role of myeloid cells in B cell survival and proliferation poses the question of why monocytes are abundant in DLBCL and in SMZL but not in other lymphoma types [21 ]. When screening for chemokine expression by different B cells, we found mRNA expression of CCL5 in normal, resting B cells, indicating that B cells have an intrinsic capacity to recruit monocytes via CCL5. However, when testing for CCL5 chemokine release, we found that only activated B cells released important amounts of CCL5. In this respect, B cells may behave analogously to CD8+ T cells, which contain an intracellular CCL5 mRNA pool that is not translated until cell activation [34 ]. When testing DLBCL B cells for CCL5 release, we found that the two nodal but not the splenic lymphoma B cells produced CCL5 and only when cultured in the presence of monocytes and BAFF/IL-2. Activation of B cells under these conditions was also apparent by CD69 expression (data not shown). As B cells died in the absence of monocytes, it is unclear if lymphoma B cells would produce CCL5 when stimulated by BAFF/IL-2 only. It is interesting that CCL-5 transcripts belong to the gene expression signature of the host immune response in DLBCL [23 ], where the lymph node signature is enriched significantly [35 ], thus potentially explaining our findings in lymph nodes and not in the spleen case. The signature of the cell origin is less defined in such cases [23 ], which may explain our findings of numerous CD14+ cells in DLBCL cases, independently of the germinal center origin [21 ]. Therefore, DLBCL may attract leukocytes including monocytes and T cells through CCL5.

Taken together, these findings suggest that means of blocking CCL5 production or function could curtail monocyte recruitment by these lymphomas and thus, reduce lymphoma growth.


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ACKNOWLEDGEMENTS
 
INSERM and the Association pour la Recherche contre le Cancer (ARC) supported the authors. We are most thankful to Biogen-Idec, Inc., for providing BAFF, anti-BAFF mAb, and BAFF-blocking agents and to Amgen, Inc., and Schering-Plough, Inc., for stem cell factor and GM-CSF, respectively. We give many thanks to S. Fisson and D. Damotte for providing tumor samples. We acknowledge B. Marmey for technical help and S. Kaveri for critical reading of the manuscript.

Received July 29, 2006; revised April 3, 2007; accepted May 11, 2007.


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