We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1β (MIP-1β) and MIP-1α, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1α/β in response to BCR triggering. In contrast to MIP-1α/β, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1α/β production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1β and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1α Ab suggesting that MIP-1β plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1α/β, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.

Chemokines ensure the continuous recirculation of immune cells among the various anatomical microenvironments, which is essential for maintaining immunological homeostasis in vivo. They also control the selective recruitment of specific subsets of leukocytes at sites of immune responses and inflammation (1, 2, 3). Chemokines rapidly stimulate integrin-dependent lymphocyte adhesion and are important regulators of other biological functions, including lymphocyte activation/differentiation, and cytotoxicity (4, 5). They are immobilized by components of the extracellular matrix or are sequestered on the cell surface by membrane-bound glycosaminoglycans and create the local gradient of attractant required for the selective compartmentalization of immunocompetent cells and for direct cell-to-cell interactions (6, 7). Much is known about T cell trafficking in lymphoid tissue, but few reports have focused on the production of chemokines by B cells or the regulation of B cell migration.

One recently identified chemokine, B cell-attracting chemokine 1, is involved in the B cell positioning in the primary follicles of the spleen and Peyer’s patches (8, 9). Another CXC chemokine, stromal cell-derived factor-1α (SDF-1α),3 expressed around germinal centers (GC), may attract lymphocytes to this site (10). Two functionally related CC chemokines, C6kine/Exodus-2/secondary lymphoid tissue chemokine and MIP-3β/Exodus-3/CKβ11/EBV-induced molecule 1 ligand, are probably major mediators of lymphocyte trafficking into and through the secondary lymphoid organs (11, 12). Both chemokines preferentially attract naive T cells, so they are probably not involved in cognate T/B cell interactions.

Ag receptor engagement profoundly affects the migration of mature B cells, blocking their homing to primary follicles (13, 14). Indeed, B cells responding to Ag undergo arrest in the outer T cell zone and proliferate in response to ligation of a critical number of B cell Ag receptors (BCR) (15). Thereafter, maturation of the B cell response depends on the availability of primed T cells and the ability of Ag-specific B cells to selectively recruit them. Ag-binding B cells are programmed to die unless they are rescued by signals delivered by primed T cells (15, 16). Ag-specific B cells must develop efficient mechanisms enabling them to establish cognate T/B interactions to stop the BCR-induced cell death program. This first step of cognate T/B interactions takes place rapidly after Ag stimulation in vivo and is followed by the migration of selected lymphocytes into the follicles (14, 15, 17). The subsequent differentiation of B cells within the follicles requires further interactions not only with Ag-specific T cells but also with follicular dendritic cells (FDC). These interactions generate a secondary repertoire of high affinity Abs and memory B cells (18). Data obtained in vivo suggest the existence of highly sophisticated control mechanisms ensuring the rapid colocalization of Ag-specific T and B cells at precise anatomical sites during an ongoing immune response, but the factors regulating this process are unknown.

Here we show that Ag receptor engagement, but not stimulation by CD40 mAb and/or IL-4, induced the coordinated production of two T cell chemoattractants, MIP-1α and MIP-1β, by B cells. In chemotaxis assays conditioned medium from BCR-stimulated B cells had chemotactic activity and selectively recruited T cells of the helper/effector phenotype. Thus, BCR-activated B cells may create chemoattractive gradients favoring cognate interactions with T cells.

Cell surface Ags were detected using the following mAbs: anti-CD19-PE, anti-CD14-FITC, anti-CD62L-FITC, and anti-CD44-FITC (all from Diaclone, Besançon, France); anti-IgM-FITC (Southern Biotechnology Associates, Birmingham, AL); anti-IgD-PE (PharMingen, San Diego, CA); anti-CD38-PE and anti-CD95-PE (from Becton Dickinson, Mountain View, CA); and anti-CD20-FITC (Immunotech, Marseille, France). The CD77 Ag was visualized by indirect immunofluorescence with uncoupled rat anti-CD77 mAb, provided by J. Wiels (19), and FITC-conjugated goat anti-rat IgM Ab (SBA). Anti-CD4, -CD3, and -CD8 mAbs (Becton Dickinson); anti-CD45RO and -CD45RA mAbs (Diaclone); and anti-CCR5 (2D7) mAb (National Institute of Biological Standards and Controls, Potters Bar, U.K.) and then by dichlorotriazinylaminofluorescein (DTAF)-conjugated goat anti-mouse IgG (H+L) F(ab′)2 (Immunotech) were used for indirect immunofluorescence. For intracellular labeling, cells were permeabilized with saponin before staining with uncoupled anti-Ki67 (Dako, Glostrup, Denmark), and anti-bcl2 (Becton Dickinson) mAbs and then by DTAF-conjugated goat anti-mouse IgG (H+L) F(ab′)2 (Immunotech) as previously described (20). Mouse isotype-matched FITC- and PE-conjugated control Igs were purchased from Diaclone and Becton Dickinson, respectively. Uncoupled control mouse Igs were purchased from ICN (Costa Mesa, CA). A FACScan flow cytometer (Becton Dickinson) with a logarithmic scale was used for immunofluorescence analysis. After gating on viable cells, 5000 cells/sample were analyzed.

Human mononuclear cells were obtained from palatine tonsils removed from children with chronic tonsillitis by gentle dissociation with forceps. B cell-enriched populations were obtained by one cycle of rosette formation and depletion of residual T cells with CD2 magnetic beads (Dynabeads M-450, Dynal, Oslo, Norway). The resulting B cell populations consistently contained ≥95% CD19+, ≤1% CD14+, and ≤1% CD3+ and DRC1+ cells. For some experiments tonsillar B cells were separated into IgD+ and IgD populations by incubating them for 30 min with anti-IgD mAb (IADB6, SBA) and removing IgD+ cells with goat anti-mouse IgG magnetic beads (Dynal). IgD B cells were further separated into CD44+ and CD44 B cells using a similar protocol with CD44 mAb (BF24, Diaclone). All purification procedures were conducted at 4°C to prevent apoptosis. The B cell phenotypes of the various subsets are illustrated in Fig. 1.

FIGURE 1.

Cell surface phenotype of human tonsillar B cell subsets. A, Tonsillar B cell subsets were isolated according to the expression of surface IgD and CD44 antigen as described in Materials and Methods. B, Phenotypic analysis by flow cytometry showed that the total B cell population was 93 ± 4% CD19+, 56 ± 5% IgM+, 59 ± 6% IgDhigh, 81 ± 16% CD44+, 21 ± 6% CD38high, 15 ± 7% CD77high, 42 ± 12% CD95+, 21 ± 6% Ki67+, 71 ± 12% Bcl2+, and 42 ± 6% CD62L+; naive B cells were 96 ± 4% CD19+, 88 ± 13% IgM+, 83 ± 4 IgDhigh, 97 ± 2 CD44+, 7 ± 3% CD38high, 3 ± 3% CD77high, 7 ± 3% CD95+, 2 ± 2% Ki67+, 90 ± 8% Bcl2+, and 74 ± 10% CD62L+; memory B cells were 92 ± 6% CD19+, 33 ± 12% IgM+, 20 ± 6 IgDhigh, 73 ± 17% CD44+, 10 ± 10% CD38high, 3 ± 3% CD77high, 54 ± 8% CD95+, 4 ± 2% Ki67+, 86 ± 4% Bcl2+, and 73 ± 13% CD62L+; and GC B cells were 94 ± 3% CD19+, 13 ± 2% IgM+, 11 ± 4% IgDhigh, 11 ± 10% CD44+, 90 ± 2% CD38high, 44 ± 12% CD77high, 84 ± 4% CD95+, 75 ± 2% Ki67+, 7 ± 4% Bcl2+, and 13 ± 5% CD62L+. Data are representative of five independent experiments.

FIGURE 1.

Cell surface phenotype of human tonsillar B cell subsets. A, Tonsillar B cell subsets were isolated according to the expression of surface IgD and CD44 antigen as described in Materials and Methods. B, Phenotypic analysis by flow cytometry showed that the total B cell population was 93 ± 4% CD19+, 56 ± 5% IgM+, 59 ± 6% IgDhigh, 81 ± 16% CD44+, 21 ± 6% CD38high, 15 ± 7% CD77high, 42 ± 12% CD95+, 21 ± 6% Ki67+, 71 ± 12% Bcl2+, and 42 ± 6% CD62L+; naive B cells were 96 ± 4% CD19+, 88 ± 13% IgM+, 83 ± 4 IgDhigh, 97 ± 2 CD44+, 7 ± 3% CD38high, 3 ± 3% CD77high, 7 ± 3% CD95+, 2 ± 2% Ki67+, 90 ± 8% Bcl2+, and 74 ± 10% CD62L+; memory B cells were 92 ± 6% CD19+, 33 ± 12% IgM+, 20 ± 6 IgDhigh, 73 ± 17% CD44+, 10 ± 10% CD38high, 3 ± 3% CD77high, 54 ± 8% CD95+, 4 ± 2% Ki67+, 86 ± 4% Bcl2+, and 73 ± 13% CD62L+; and GC B cells were 94 ± 3% CD19+, 13 ± 2% IgM+, 11 ± 4% IgDhigh, 11 ± 10% CD44+, 90 ± 2% CD38high, 44 ± 12% CD77high, 84 ± 4% CD95+, 75 ± 2% Ki67+, 7 ± 4% Bcl2+, and 13 ± 5% CD62L+. Data are representative of five independent experiments.

Close modal

T cells were recovered from the first rosetting cell fraction and were depleted of residual B cells using CD19 magnetic beads (Dynal). T cells were further depleted of CD8+ and CD45RA+ cells by incubation for 30 min with CD8 magnetic beads (Dynal) and CD45RA (ALB11, Immunotech)-coated goat anti-mouse IgG magnetic beads (Dynal). The resulting populations contained ≥95% CD4+CD45RO+ T cells.

All cells were cultured in RPMI 1640 medium (Life Technologies, Paisley, Scotland) containing 10 mM HEPES, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 10% heat-inactivated FCS (complete medium (CM)). B cells (1 × 106 cells/ml) were activated by incubation in CM for 2 days, unless otherwise indicated, with polyclonal anti-IgM Ab coupled to beads (Irvine Scientific, Santa Anna, CA; 5 μg/ml), CD40 mAb (G28.5; 1 μg/ml), IL-4 (Schering Plough, Kenilworth, NJ; 20 ng/ml), or a combination of these. In some experiments B cells were also stimulated in CM by Staphylococcus aureus Cowan I (SAC; 1/104, v/v). The concentration of endotoxin in the culture medium and the concentrations of the reagents used were consistently <1 ng/ml.

Purified CD4+CD45RO+ tonsillar T cells (1 × 106 cells/ml) were cultured in 12-well plates in CM for 12 days with 400 IU/ml IL-2 (Chiron, Amsterdam, The Netherlands). These IL-2-conditioned T cells were >95% CD45RO+, >80% CD4+, <20% CD8low, >75% CCR5+, <2% CD14+, and <1% CD20+ as assessed by flow cytometry.

The HK cell line was obtained from Y. S. Choi (Alton Ochsner Medical Foundation, New Orleans, LA). It has been shown to preferentially adhere to and cooperate with GC B cells (21). The HK cell line was treated with 1× trypsin/EDTA (Life Technologies) and was cultured at a density of 105 cells/ml in CM for 3 days. On the third day, the supernatant was discarded, and HK cells were cultured in fresh CM for 4 more days. For the coculture assay, HK cells were gamma irradiated at 30 grays (137Cs source) 1 day before coculture and were seeded at 2 × 103 cells/well in 96-well microtiter plates (Costar, Cambridge, MA). B cells (105/well) were added and cultured with HK cells either alone or with various stimuli in 200 μl of CM, for 2 (chemokine production) or 3 (proliferation assay) days. In some experiments HK cells were seeded at 2 × 103 cells/well in 96-well microtiter plates for 4 h, treated by incubation with 1% paraformaldehyde in 1× PBS for 15 min, and extensively washed before coculture with B cells.

Proliferation was measured by supplying the cultures with a pulse of 0.5 μCi/well of [methyl-3H]thymidine (Amersham, Les Ulis, France) for the last 12 h of the third day of incubation. Cells were collected by filtration through a glass-fiber filter, and [3H]thymidine incorporation was measured in a beta scintillation counter (Betaplate 1205, EGG Wallac, Turku, Finland). Results are expressed as counts per minute (mean of triplicates ± SD). In some experiments mouse anti-human IL-8, goat anti-human MIP-1α, and anti-human MIP-1β neutralizing Ab (all from R&D Systems, Abingdon U.K.) were added to a final concentration of 5 μg/ml.

Cell-free supernatants of 2 × 105 B cells/well were collected on day 2 unless otherwise indicated and were stored at −20°C until tested. Chemokine production was measured with specific ELISA kits purchased from R&D Systems (MIP-1α and MIP-1β) and Diaclone (IL-8) according to the manufacturer’s recommendations. Results are expressed as the mean concentration (picograms per milliliter; ±SD) of triplicate determinations.

RNA was extracted from 10 × 106 B cells using RNAzol (Bioprobe, Systems, Montreuil, France) and was treated with 10 U of RNase-free DNase (Boehringer Mannheim, Meylan, France) for 30 min at 37°C. The mixture was subjected to phenol/chloroform extraction, and RNA was precipitated in ethanol, recovered by centrifugation, and suspended in 10 μl of water. The cDNA was prepared by reverse transcription using Superscript (Life Technologies). Samples were treated at 42°C for 60 min, and the cDNA concentration was measured by spectrophotometry.

Competitive PCR for β-actin was performed for each sample as previously described (22). In brief, 50 ng of each cDNA was denatured for 5 min at 94°C, and β-actin was amplified in the presence of graded concentrations of pQB2 plasmid as competitor. Competitive PCR for MIP-1α was performed in the same conditions as those used for β-actin except that the amount of cDNA input was equivalent to 106 molecules of β-actin cDNA. Competitive PCR for MIP-1β was performed with the same amount of cDNA as that used for MIP-1α, but 35 cycles of PCR were performed, each cycle consisting of 1 min at 94°C, 1 min at 55°C, and 1.5 min at 72°C. The MIP-1α and MIP-1β competitor was pQB2 plasmid as previously described (23). The primers used for β-actin, MIP-1α, and MIP-1β are shown in Table I. A PTC-100TM Programmable Thermal Controler (MJ Research, Watertown, MA) was used for all PCR reactions.

Table I.

QC-RT-PCR amplification primersa

Sequence of Primers for PCRSequence of Biotin-Labeled Primers
SenseAntisenseRecognizing the cDNARecognizing the competitor
MIP-1α 5′-GTCATCTTCC TAACCAAGCG-3′ 5′-TGTGGCTGTT TGGCAACAAC-3′ 5′-biotin GCCGGCAGG TCTGTGCTGACCCC-3′ 5′-biotin GGGATCCTT TGCATCGAACT-3′ 
MIP-1β 5′-AGGAAGCTTC CTCGCAACTT-3′ 5′-AGTCCTGAGT ATGGAGGAGA-3′ 5′-biotin CCAGCAGCC TCTGCTCCCAGCC-3′ 5′-biotin AGACCCCA GCAGAGAATGGAA-3′ 
β-actin 5′-GGGTCAGAAG GATTCCTATG-3′ 5′-GGTCTCAAAC ATGATCTGGG-3′ 5′-biotin CGACGAGGC CCAGAGCAAGAGA-3′ 5′-biotin GTGAGGGAC ATGCTCACGCAGC-3′ 
Sequence of Primers for PCRSequence of Biotin-Labeled Primers
SenseAntisenseRecognizing the cDNARecognizing the competitor
MIP-1α 5′-GTCATCTTCC TAACCAAGCG-3′ 5′-TGTGGCTGTT TGGCAACAAC-3′ 5′-biotin GCCGGCAGG TCTGTGCTGACCCC-3′ 5′-biotin GGGATCCTT TGCATCGAACT-3′ 
MIP-1β 5′-AGGAAGCTTC CTCGCAACTT-3′ 5′-AGTCCTGAGT ATGGAGGAGA-3′ 5′-biotin CCAGCAGCC TCTGCTCCCAGCC-3′ 5′-biotin AGACCCCA GCAGAGAATGGAA-3′ 
β-actin 5′-GGGTCAGAAG GATTCCTATG-3′ 5′-GGTCTCAAAC ATGATCTGGG-3′ 5′-biotin CGACGAGGC CCAGAGCAAGAGA-3′ 5′-biotin GTGAGGGAC ATGCTCACGCAGC-3′ 
a

The software program BISANCE (Université Paris V, CITI 2) was used for designing the primer pairs and selecting annealing temperatures using sequences obtained directly form GenBank, with the exception of the β-actin sequences.

Aliquots of the amplified products of competitive PCR were subjected to an additional elongation cycle in the presence of biotinylated oligonucleotides recognizing either the cDNA or the competitor (Table I) and digoxigenin-labeled dUTP. Labeled products were quantified by ELISA in streptavidin-coated microtiter plates using peroxidase-conjugated Fab fragments of sheep anti-digoxigenin Ab. All reagents were obtained from Boehringer Mannheim. Results are expressed as the number of chemokine mRNA copies per 106 copies of β-actin mRNA in the sample.

IL-2-stimulated CD4+CD45RO+ T cells were extensively washed in RPMI for the chemotaxis assay. Cell migration was assessed in a 48-well microchemotaxis chamber (NeuroProbe, Gaithersburg, MD) as previously described (24). The lower wells of the chamber were filled with 27.5 μl of conditioned medium from SAC-activated B cells or control chemokines diluted in HEPES-buffered RPMI 1640 medium, pH 7.4, containing 1% heat-inactivated human plasma (assay buffer). The upper wells were filled with 50 μl of cells (2 × 106 cells/ml) in assay buffer. The lower and upper wells were separated by polyvinylpyrolidone-free polycarbonate membranes with 3-μm pores (Osmonics, Livermore, CA). The surface facing the lower wells was coated with 5 μg/ml collagen type IV (Sigma, St. Louis, MO) by incubation for 1 h at 37°C before the assay. Assays were conducted at 37°C for 3 h in a humidified atmosphere of 5% CO2 in air. Filters were collected, and the surface facing the upper well was washed carefully with 1× PBS, fixed, and stained with Diff-Quick dye. The number of cells that had migrated to the underside of the membrane was counted in five randomly selected high power fields (×400 magnification). All assays were performed in triplicate. Results are expressed as the mean ± SD of the number of cells in five high power fields for each well.

Human recombinant SDF-1α (Diaclone), MIP-1α, (Diaclone), and MIP-1β (R&D) were used at a concentration of 100 ng/ml, and IL-8 (72 amino acid form) (Diaclone) was used at a concentration of 50 ng/ml. Neutralizing polyclonal anti-MIP-1α Ab, anti-MIP-1β Ab, and monoclonal anti-IL-8 Ab (R&D) were used at a concentration of 5 μg/ml. The anti-CCR5 (2D7) mAb (National Institute of Biological Standards and Controls) was used at a concentration of 10 μg/ml for blocking experiments. In some experiments T cells were preincubated at 37°C for 2 h with 200 ng/ml of Bordetella pertussis toxin (Calbiochem, La Jolla, CA) before chemotaxis assay.

Student’s t test was used to detect significant differences. A p value of <0.05 was regarded as significant.

Human tonsillar B cells cultured for 2 days in CM spontaneously released 352 ± 243 pg/ml MIP-1α, 361 ± 401 pg/ml MIP-1β, and 1867 ± 1152 pg/ml IL-8 into culture supernatants (n = 8). In the same culture supernatants, the concentrations of the other two CC chemokines tested, MCP-1 and RANTES, were much lower: 64 ± 24 and 93 ± 24 pg/ml, respectively. Activation of tonsillar B lymphocytes by anti-IgM Ab significantly increased MIP-1α and MIP-1β production (Fig. 2 A; 3.6 ± 2.3 times more for MIP-1α and 7.2 ± 3.2 times more for MIP-1β; n = 5), whereas the addition of CD40 mAb with or without IL-4 had no effect on their secretion. In contrast, the addition of IL-4 and CD40 mAb increased the anti-IgM Ab-induced secretion of MIP-1α and MIP-1β by 1.5- to 2.3-fold. In these five separate experiments using five different B cell donors, the increased production of MIP-1α/β observed after stimulation with anti-IgM Ab alone (p < 0.05) or with anti-IgM Ab and CD40 or IL-4 (p < 0.005) was significant.

FIGURE 2.

MIP-1α and MIP-1β production by tonsillar B cells. A, Tonsillar B cells (2 × 105/well, ≥95% purity) were cultured in medium alone or with the activators indicated. Cell-free culture supernatants were harvested on day 2, and MIP-1α and MIP-1β production was assessed by ELISA. Results (picograms per milliliter) are the means of triplicate determinations from five donors. The SD was <10% of the mean. B, B cells were cultured in medium alone or with the activators indicated for various times. MIP-1β production was assessed by ELISA in cell-free culture supernatants harvested at the time points indicated. Results (picograms per milliliter) are the means of triplicate determinations. The SD was <10% of the mean. Representative data from four independent experiments are shown. Student’s t test was used to compare the effect of various stimuli on chemokine production (p values).

FIGURE 2.

MIP-1α and MIP-1β production by tonsillar B cells. A, Tonsillar B cells (2 × 105/well, ≥95% purity) were cultured in medium alone or with the activators indicated. Cell-free culture supernatants were harvested on day 2, and MIP-1α and MIP-1β production was assessed by ELISA. Results (picograms per milliliter) are the means of triplicate determinations from five donors. The SD was <10% of the mean. B, B cells were cultured in medium alone or with the activators indicated for various times. MIP-1β production was assessed by ELISA in cell-free culture supernatants harvested at the time points indicated. Results (picograms per milliliter) are the means of triplicate determinations. The SD was <10% of the mean. Representative data from four independent experiments are shown. Student’s t test was used to compare the effect of various stimuli on chemokine production (p values).

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MIP-1β production involved rapid protein release. After 12 h, significantly more immunoreactive MIP-1β was detected in culture supernatants from anti-IgM Ab-stimulated B cells than in those from medium- or CD40 mAb-treated cells. After BCR triggering, the concentration of MIP-1β increased until 48 h and then plateaued. The plateau value for MIP-1β at 48 h was 30 times higher after activation with SAC than after activation with anti-IgM Ab (Fig. 2 B). Similar results were observed for MIP-1α production (data not shown). The addition of 10 μg/ml cycloheximide during BCR triggering totally abolished the increase in MIP-1α/β production, suggesting that it was dependent on de novo protein synthesis (data not shown).

In contrast, the production of IL-8 was not significantly affected by any of the stimuli used, and that of RANTES and MCP-1 remained <120 pg/ml. The addition of the cytokines, IL-2, IL-12, IL-13, or IFN-γ, did not significantly affect spontaneous or BCR-induced MIP-1α, MIP-1β, and IL-8 production (data not shown). Thus, B cells selectively released two potent T cell chemoattractants, MIP-1β and MIP-1α, after BCR triggering, whereas IL-8 production was insensitive to in vitro B cell activation.

MIP-1β and MIP-1α transcript levels were determined by competitive RT-PCR before and after B cell activation. Unstimulated B cells contained low levels of both mRNA species before activation (46 × 104 copies of MIP-1α mRNA and 35 × 104 copies of MIP-1β mRNA/106 copies of β-actin mRNA; Fig. 3,A). The amounts of both mRNA species rapidly increased after BCR cross-linking, peaking at 8 h and decreasing thereafter. At 8 h, the number of MIP-1α mRNA copies reached a maximum of 735 × 104/106 copies of β-actin mRNA, much higher than the 58 × 104 copies in medium-treated cells (12.7 times higher). The activation of B cells by anti-IgM Ab resulted in 9.3 times more copies of MIP-1β mRNA (1490 × 104 copies/106 copies of β-actin in BCR-stimulated cells vs 159 × 104 in medium-treated cells; Fig. 3 B). There were time-dependent changes in MIP-1α/β mRNA levels after BCR cross-linking, in contrast to the steady state levels of both species in medium- and CD40-treated B cells.

FIGURE 3.

Kinetics of MIP-1α and MIP-1β mRNA expression. Tonsillar B lymphocytes (≥95% purity) were cultured for the times indicated in medium alone or with anti-IgM Ab or CD40 mAb. A, RT-PCR analysis using a primer pair designed to amplify the mRNA species encoded by the human MIP-1α, MIP-1β, and β-actin genes was performed as described in Materials and Methods. B, Stimulation indexes for MIP-1α and MIP-1β expression in B cells after stimulation with anti-IgM or CD40 mAb vs medium, 8 h postactivation. Representative data from three independent experiments are shown.

FIGURE 3.

Kinetics of MIP-1α and MIP-1β mRNA expression. Tonsillar B lymphocytes (≥95% purity) were cultured for the times indicated in medium alone or with anti-IgM Ab or CD40 mAb. A, RT-PCR analysis using a primer pair designed to amplify the mRNA species encoded by the human MIP-1α, MIP-1β, and β-actin genes was performed as described in Materials and Methods. B, Stimulation indexes for MIP-1α and MIP-1β expression in B cells after stimulation with anti-IgM or CD40 mAb vs medium, 8 h postactivation. Representative data from three independent experiments are shown.

Close modal

BCR triggering induces both B cell proliferation and MIP-1α/β production, so we tested whether these two processes were regulated by similar signals. BCR triggering was itself sufficient to markedly up-regulate MIP-1β secretion and the proliferation of unfractionated tonsillar B cells (sevenfold increase in both cases; Fig. 4). The proliferation induced by anti-IgM Ab B cell stimulation was increased by the addition of IL-4 or CD40 mAb (10 and 4 times higher, respectively), but BCR-induced MIP-1β production was only 1.5–2 times higher after these additions. MIP-1β production was also unaffected by the potent mitogenic stimulation of the combination of CD40 mAb and IL-4. A similar pattern was observed for MIP-1α production. Therefore, the production of MIP-1α and MIP-1β does not depend on the proliferative state of B cells. This conclusion is further supported by the observation that anti-MIP-1α/β neutralizing Ab did not inhibit B cell proliferation (data not shown). Thus, B cell proliferation and MIP-1α/β production seem to be controlled by independent mechanisms.

FIGURE 4.

MIP-1α/β production and proliferation of tonsillar B cells. Tonsillar B cells (2 × 105 cells/well, ≥95% purity) were cultured alone or in the presence of the activators indicated. B cell proliferation was measured on day 3 by [3H]TdR incorporation. Cell-free culture supernatants were harvested on day 2, and MIP-1α/β production was quantified by ELISA. Results are expressed as counts per minute (±SD) and means (nanograms per milliliter; ±SD) for triplicate cultures. Representative data from four independent experiments are shown.

FIGURE 4.

MIP-1α/β production and proliferation of tonsillar B cells. Tonsillar B cells (2 × 105 cells/well, ≥95% purity) were cultured alone or in the presence of the activators indicated. B cell proliferation was measured on day 3 by [3H]TdR incorporation. Cell-free culture supernatants were harvested on day 2, and MIP-1α/β production was quantified by ELISA. Results are expressed as counts per minute (±SD) and means (nanograms per milliliter; ±SD) for triplicate cultures. Representative data from four independent experiments are shown.

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Tonsillar B cells consist of several phenotypically and functionally different subsets, so we investigated whether particular B cell subsets were involved in BCR-induced chemokine production. MIP-1α/β production was measured in culture supernatants of IgDhigh naive, IgDCD44+ memory, and IgDCD44 GC B cells. Anti-IgM Ab-treated naive B cells produced 11276 pg/ml MIP-1β, whereas anti-IgM Ab-treated memory B cells and GC B cells produced 357 and 266 pg/ml, respectively (Fig. 5). As surface IgM+ (sIgM+) cells make up a small proportion of memory B cells, we compared the production of MIP-1α and MIP-1β in memory and naive B cells after stimulation by SAC, a potent sIg cross-linker acting on the BCR of various isotypes. SAC-activated naive and memory B cells produced large amounts of MIP-1β (51,101 and 62,026 pg/ml, respectively) and MIP-1α (26,046 and 66,932 pg/ml, respectively). Stimulation by anti-IgM Ab did not increase MIP-1α/β production by GC B cells, but 22 times more MIP-1β (3,086 vs 137 pg/ml) and 10 times more MIP-1α (1,776 vs 175 pg/ml) were produced in the presence of SAC. This suggests that production of these two closely related CC chemokines is a constitutive phenomenon accompanying BCR triggering in all subsets of mature B cells.

FIGURE 5.

MIP-1α/β production by various tonsillar B cell subsets in response to BCR triggering. GC (IgDCD44), memory (IgDCD44+), and naive (IgDhigh) B cells (2 × 105/well) were cultured in medium alone or in the presence of anti-IgM Ab or SAC. Cell-free culture supernatants were harvested on day 2, and the production of MIP-1α (□) and MIP-1β (▪) was assessed by ELISA. Results (nanograms per milliliter) are the means of triplicate determinations (±SD). Representative data from four independent experiments are shown.

FIGURE 5.

MIP-1α/β production by various tonsillar B cell subsets in response to BCR triggering. GC (IgDCD44), memory (IgDCD44+), and naive (IgDhigh) B cells (2 × 105/well) were cultured in medium alone or in the presence of anti-IgM Ab or SAC. Cell-free culture supernatants were harvested on day 2, and the production of MIP-1α (□) and MIP-1β (▪) was assessed by ELISA. Results (nanograms per milliliter) are the means of triplicate determinations (±SD). Representative data from four independent experiments are shown.

Close modal

FDC retain Ag in an unprocessed form in vivo and strongly increase B cell survival and proliferation within GC. Therefore, we assessed whether they regulated MIP-1α and MIP-1β production in various B cell subsets. We used the human FDC-like HK cell line to mimic the effect of FDC. Coculture with irradiated HK cells did not significantly affect spontaneous overall B cell proliferation, whereas it strongly increased B cell proliferation in the presence of CD40 mAb with or without IL-4 or anti-IgM Ab (Fig. 6,A). In striking contrast, the production of MIP-1α/β by B cells was not affected by coculture with HK cells alone. However, in the presence of anti-IgM and CD40 Ab or IL4, coculture with HK cells led to a decreased production of both chemokines (Fig. 6 B). Moreover, coculture with HK cells did not affect the production of MIP-1α/β by naive, memory, and GC B cells, whereas it increased their proliferation in the presence of various stimuli (data not shown).

FIGURE 6.

Effect of HK cells on B cell proliferation and MIP-1β release in tonsillar B cells. A, Tonsillar B cells (2 × 105 cells/well) were cultured alone (□) or with irradiated FDC-like HK cell monolayers (2 × 103 cells/well; ▪) in the presence of the activators indicated. B cell proliferation was measured on day 3 by determining [3H]TdR incorporation. Results for proliferation are expressed as counts per minute (mean of triplicate determinations ± SD) for each culture condition. Radioactivity (270 ± 80 cpm) was incorporated into irradiated HK cells only. B, Cell-free culture supernatants were harvested on day 2, and MIP-1α/β production was quantified by ELISA. MIP-1α/β production is expressed as picograms per milliliter (mean of triplicate determinations ± SD) for each culture condition. Representative data from four independent experiments are shown.

FIGURE 6.

Effect of HK cells on B cell proliferation and MIP-1β release in tonsillar B cells. A, Tonsillar B cells (2 × 105 cells/well) were cultured alone (□) or with irradiated FDC-like HK cell monolayers (2 × 103 cells/well; ▪) in the presence of the activators indicated. B cell proliferation was measured on day 3 by determining [3H]TdR incorporation. Results for proliferation are expressed as counts per minute (mean of triplicate determinations ± SD) for each culture condition. Radioactivity (270 ± 80 cpm) was incorporated into irradiated HK cells only. B, Cell-free culture supernatants were harvested on day 2, and MIP-1α/β production was quantified by ELISA. MIP-1α/β production is expressed as picograms per milliliter (mean of triplicate determinations ± SD) for each culture condition. Representative data from four independent experiments are shown.

Close modal

In striking contrast to naive and memory B cells, GC B cells spontaneously secreted large amounts of IL-8 (<10, 68, and 1547 pg/ml, respectively; Table II). None of the B cell activators tested induced significant levels of IL-8 production by naive and memory B cells or regulated its production by GC B cells. The IL-8 concentration in the supernatants of GC, but not of naive or memory B cells, was strongly increased in the presence of irradiated HK cells. If HK cells were treated with paraformaldehyde rather than being irradiated before coculture, no increase in IL-8 production was observed, suggesting that HK cells were indeed the main source of IL-8 during HK-B cell coculture (data not shown). IL-8 production during GC B cell/HK cell cocultures was not further increased by B cell activation (Table II). In contrast, stimulation of naive B cells by anti-IgM Ab with or without IL-4 and/or CD40 mAb up-regulated IL-8 production by HK cells. Stimulation by CD40 mAb and IL-4 of naive B cells also increased IL-8 production by HK cells. A similar pattern of IL-8 production was observed for memory B cells (data not shown). These results suggest that strong induction of IL-8 production by HK cells during HK-B cell coculture depends on B cell activation. GC B cells activated in vivo spontaneously induce IL-8 production, whereas naive and memory B cells require further activation in vitro.

Table II.

In vivo and in vitro activated B cells induce IL-8 production by FDC-like HK cellsa

ActivatorsIgDhigh B cellsIgDCD44 B Cells
− HK cells+ HK cellsb− HK cells+ HK cells
None 0c 431 1,547 36,499 
Anti-IgM Ab 4,319 1,496 39,909 
CD40 mAb 40 471 1,876 35,390 
IL-4 442 1,500 35,287 
CD40 mAb+ IL-4 4,948 1,202 49,255 
Anti-IgM Ab+ CD40 mAb 77 6,330 1,354 47,143 
Anti-IgM Ab+ IL-4 17,115 1,485 47,414 
Anti-IgM Ab+ CD40 mAb+ IL-4 52 25,907 1,299 47,765 
ActivatorsIgDhigh B cellsIgDCD44 B Cells
− HK cells+ HK cellsb− HK cells+ HK cells
None 0c 431 1,547 36,499 
Anti-IgM Ab 4,319 1,496 39,909 
CD40 mAb 40 471 1,876 35,390 
IL-4 442 1,500 35,287 
CD40 mAb+ IL-4 4,948 1,202 49,255 
Anti-IgM Ab+ CD40 mAb 77 6,330 1,354 47,143 
Anti-IgM Ab+ IL-4 17,115 1,485 47,414 
Anti-IgM Ab+ CD40 mAb+ IL-4 52 25,907 1,299 47,765 
a

A total of 2 × 105 IgDhigh naive and IgDCD44 GC B cells (>90% purity) were cultured alone or with 2 × 103 HK cells in the presence of the activators indicated. IL-8 was measured on day 2 in cell-free culture supernatants by ELISA. Values (pg/ml) are the means of triplicate determinations. SD were consistently <15% of the mean. Representative data of four independent experiments are shown.

b

The levels of IL-8 in culture supernatants of HK cells cultured alone or in the presence of the activators indicated were <400 pg/ml.

c

Below the detection limit of the ELISA kit (<10 pg/ml).

MIP-1α/β selectively attract activated T lymphocytes, so we assessed the extent to which conditioned medium from activated B cells (sn B SAC) induced the migration of IL-2-activated CD4+CD45RO+ T cells. sn B SAC produced significantly higher levels of T cell migration (2.2 times higher than medium alone). Significant T cell migration with six different sn B SAC were obtained in eight independent experiments (2.0 ± 0.4 times higher migration than control medium; p < 0.005) using T cells from different donors. Dilutions (1/1 to 1/10) of the sn B SAC were tested, and the undiluted supernatant and a 1/1 (50%) dilution of sn B SAC yielded comparable and maximal responses (data not shown). The chemoattractant activity of sn B SAC was stronger than that of 50 ng/ml IL-8, comparable to that of 100 ng/ml MIP-1α and 1.6 and 3 times lower than that of 100 ng/ml MIP-1β and SDF-1α, respectively. SDF-1α, a highly efficient chemoattractant for T cells, was used as a positive control (Fig. 7,A). Pretreatment of CD4+CD45RO+ T cells with 200 ng/ml of Bordetella pertussis toxin inhibited migration toward sn B SAC to background levels, confirming the involvement of Giα protein-coupled receptor(s) (Fig. 7,B). A checkerboard-type analysis showed that the migration of T cells toward sn B SAC was mostly chemotactic and not chemokinetic because the cells did not migrate above the background level if incubated in the absence of a chemotactic concentration gradient (Fig. 7,C). The addition of neutralizing anti-MIP-1β Ab reduced T cell migration by only 27 ± 7%, but this effect was significant (p < 0.005; n = 7). Interestingly, the addition of neutralizing anti-MIP-1α Ab with neutralizing anti-MIP-1β Ab did not increase the blocking effect over that observed with anti-MIP-1β Ab alone. The addition of anti-CCR5 blocking mAb inhibited the chemoattractant activity of sn B SAC by 41 ± 5% (p < 0.05; n = 4). In contrast, neutralizing anti-IL-8 mAb and isotype-matched control Igs did not cause any significant inhibition (Fig. 7 D). Thus, chemokines or chemotactic agents other than MIP-1α/β, produced by BCR-stimulated B cells, also attract T cells of the helper/effector phenotype.

FIGURE 7.

Chemotactic response of CD4+CD45RO+ T cells to conditioned medium from SAC-activated B cells. IL-2-conditioned T cells used in migration assays were >95% CD45RO+, >80% CD4+, and >75% CCR5+. A, Migration of CD4+CD45RO+ T cells toward sn B SAC or toward recombinant MIP-1α, MIP-1β, and SDF-1α (each at a concentration of 100 ng/ml) or IL-8 (50 ng/ml) was assessed in a 48-well microchemotaxis chamber. The assay was performed in triplicate, and migrating cells in five randomly selected high power fields (HPF; ×400) were counted for each well (mean ± SD). Representative results from eight independent experiments performed with T cells from different donors and six different sn B SAC are shown. The significance of differences in T cell migration toward sn B SAC and control medium (background migration) was determined by Student’s t test. B, Chemotaxis of CD4+CD45RO+ T cells toward sn B SAC is mediated by a Giα protein-coupled receptor(s). Lymphocytes were preincubated with B. pertussis toxin (PTX) before the chemotaxis experiment. Representative results (mean of triplicate determinations ± SD) from three independent experiments are shown. C, Checkerboard-type assay of sn B SAC on CD4+CD45RO+ T cells. For the microchemotaxis assay, sn B SAC was added to the upper and/or lower wells of the chamber, as indicated. The assay was performed in triplicate, and migrating cells were counted as described in A (mean ± SD). Representative data from three independent experiments are shown. D, Migration of CD4+CD45RO+ T cells toward sn B SAC alone or in the presence of neutralizing anti-MIP-1α and/or anti-MIP-1β Ab, anti-IL-8 Ab, anti-CCR5 blocking mAb, or control Ig. Representative results (mean ± SD) from four to seven independent experiments performed with cells from various donors are shown. Significant differences in migration toward sn B SAC were determined by Student’s t test.

FIGURE 7.

Chemotactic response of CD4+CD45RO+ T cells to conditioned medium from SAC-activated B cells. IL-2-conditioned T cells used in migration assays were >95% CD45RO+, >80% CD4+, and >75% CCR5+. A, Migration of CD4+CD45RO+ T cells toward sn B SAC or toward recombinant MIP-1α, MIP-1β, and SDF-1α (each at a concentration of 100 ng/ml) or IL-8 (50 ng/ml) was assessed in a 48-well microchemotaxis chamber. The assay was performed in triplicate, and migrating cells in five randomly selected high power fields (HPF; ×400) were counted for each well (mean ± SD). Representative results from eight independent experiments performed with T cells from different donors and six different sn B SAC are shown. The significance of differences in T cell migration toward sn B SAC and control medium (background migration) was determined by Student’s t test. B, Chemotaxis of CD4+CD45RO+ T cells toward sn B SAC is mediated by a Giα protein-coupled receptor(s). Lymphocytes were preincubated with B. pertussis toxin (PTX) before the chemotaxis experiment. Representative results (mean of triplicate determinations ± SD) from three independent experiments are shown. C, Checkerboard-type assay of sn B SAC on CD4+CD45RO+ T cells. For the microchemotaxis assay, sn B SAC was added to the upper and/or lower wells of the chamber, as indicated. The assay was performed in triplicate, and migrating cells were counted as described in A (mean ± SD). Representative data from three independent experiments are shown. D, Migration of CD4+CD45RO+ T cells toward sn B SAC alone or in the presence of neutralizing anti-MIP-1α and/or anti-MIP-1β Ab, anti-IL-8 Ab, anti-CCR5 blocking mAb, or control Ig. Representative results (mean ± SD) from four to seven independent experiments performed with cells from various donors are shown. Significant differences in migration toward sn B SAC were determined by Student’s t test.

Close modal

Maturation of the B cell response to a peptide Ag depends mostly on the limiting amount of T cell help (25). This process depends on the availability of Ag-specific T cells and on the ability of Ag-binding B cells to efficiently recruit them. As B and T cells specific for a given Ag are triggered independently, T/B cell interactions require the redistribution of Ag-primed cells within precise anatomical sites at which cognate T/B interactions take place (26). In contrast to the limited trafficking capacity of Ag-binding B cells, which typically colonize only the adjacent follicle, Ag-primed and expanded T cells actively migrate to multiple, adjacent, and distant follicular sites (27). The selective influx of Ag-specific T cells from T cell zones into the follicles during the primary Ab response in vivo reflects the Ag-driven selection of GC CD4+ T cells reported in several studies (28, 29, 30). It appears that specific chemoattractive gradient(s) selectively produced by Ag-binding B cells enable rare Ag-specific T cells to be quickly selected and recruited to the specialized niches of lymphoid organs.

In this study we have shown that two closely related T cell chemoattractants, MIP-1α and MIP-1β, were rapidly produced by B cells after engagement of their BCR. This triggering, but not stimulation, by CD40 mAb or IL-4 rapidly induced the coordinated expression of their transcripts and proteins. This effect was selective because the production of two other CC chemokines, MCP-1 and RANTES, was not affected by B cell stimulation. BCR triggering by anti-IgM Ab induced a strong, but transient, increase in MIP-1α/β mRNA levels, peaking 8 h after stimulation. This was associated with an early and sustained release of MIP-1α/β proteins. Similar kinetics of protein release were observed after B cell activation by SAC, a potent sIg cross-linker, which resulted in 20–30 times more MIP-1α/β production than that induced by anti-IgM Ab. Rapid induction of MIP-1α/β production by BCR-activated B cells is consistent with previous data showing that the genes encoding MIP-1α/β are immediate early genes (31). The restricted set of chemokines produced by B cells and its strict dependence on BCR triggering suggest that MIP-1α/β plays an important role in Ag-driven B cell differentiation.

BCR engagement is central to three crucial steps in Ag-driven B cell differentiation: 1) during the priming of IgDhigh naive B cells in the T cell-rich extrafollicular area of secondary lymphoid tissue, 2) during Ag-driven positive selection of high affinity clones during GC reaction, and 3) at the postselection stage of IgDlow/− memory B cell terminal differentiation (18, 32). BCR cross-linking by anti-IgM Ab preferentially up-regulated MIP-1α/β expression in naive B cells, but the extensive cross-linking of sIg with SAC led to the production of large amounts of both chemokines in naive and memory B cells. The weaker induction of MIP-1α/β production by anti-IgM Ab presumably reflects the lower frequency of sIgM+ cells in the memory B cell pool. After stimulation with SAC, GC B cells secreted about 20 times less MIP-1α/β than did naive and memory B cells. This low level of production is consistent with the presence of a limited pool of centrocytes expressing low levels of sIg in the GC B cell population. Thus, all functional B cell subsets produce MIP-1α/β in response to BCR engagement.

MIP-1α/β chemokines have been shown to be locally produced in the lymph nodes of healthy subjects. However, cells producing MIP-1α/β are rare in secondary lymphoid tissue and are detected in extrafollicular T cell zones and within the GC (33, 34). This expression may reflect the low proportion of cells triggered by Ag in vivo. Moreover, both chemokines have been shown to regulate T lymphocyte trafficking into lymph nodes during an immune response in vivo (35). MIP-1α and MIP-1β have a preferential chemotactic effect on CD45RO+ T cells, with MIP-1β acting mostly on CD4+ T cells, so B cell-derived MIP-1α/β probably have a chemoattractant activity affecting T cells of the helper/effector phenotype (36, 37). Consistent with this, IL-2-conditioned CD4+CD45RO+ T cells actively migrated toward a chemotactic gradient(s) present in conditioned medium from BCR-activated B cells. The inhibition of this activity by anti-MIP-1β neutralizing Ab and anti-CCR5 blocking Ab shows that MIP-1β is an important component of the T cell chemoattractant activity of these media. It also shows that chemotactic agents other than MIP-1β exert their effects on CD4+ T cells and are secreted by BCR-activated B cells. One candidate is a CCR4 agonist, macrophage-derived chemokine produced by B cells (38) that preferentially attracts Th2-polarized T cells (39).

Within lymphoid organs, CCR5, the main high affinity receptor for MIP-1β, is present on a small proportion (≤10%) of T lymphocytes in the vicinity of B cell follicles and within the GC (40, 41). This suggests that during the Ag-driven immune response in vivo, CCR5+ T cells may be actively recruited to the anatomical sites of cognate T/B cell interactions. Our results and the presence of CCR5 mostly on the highly migratory CD4+CD45RO+CD26high subset of T cells (42) suggest that these helper/effector CD4+ T cells may be the main target of B cell-derived MIP-1α/β (41). CCR5 is a marker of recently activated T cells, and its expression is strongly IL-2 dependent (43). This suggests that during IL-2-mediated clonal expansion of Ag-specific T cells in secondary lymphoid organs these cells become highly responsive to locally induced MIP-1α/β gradients formed around Ag-primed B cells. These transient Ag-induced gradients of MIP-1α/β may be superimposed on the constitutive gradients of homing chemokines (secondary lymphoid tissue chemokine, EBV-induced molecule-1 ligand, or SDF-1α) within lymphoid tissue. Our data argue that MIP-1α/β gradients play an important role in lymphoid tissue during the adaptive immune response by bringing and keeping together a functional cellular unit, Ag-specific B and T cells, that is required for maturation of the B cell response.

Dendritic cells (DC), which express mRNA for CCR5 and CCR1 and migrate in the presence of MIP-1α/β, are also potential targets for B cell-derived chemokines (44). This possibility is of particular interest given recent data concerning the role of DC in stimulating the primary B cell response (45). It is unknown whether MIP-1α/β recruits the recently identified subset of CD4+CD11c+ DC present within the GC (46). Finally, MIP-1α/β secreted by Ag-binding B cells may act on the B cells themselves, because MIP-1α has been shown to act as a chemoattractant for B cells (47). The principal MIP-1β receptor, CCR5, is not present on B cells, but other recently identified MIP-1β receptors, such as CCR8, are presumably present (48, 49). The last major finding of our study is the lack of effect of HK (FDC)-B cells interactions on MIP-1α/β production by B cells, although these interactions strongly increased the proliferation of activated B cells and Ig secretion (50) (our unpublished observations). FDC-like HK cells do not express MIP-1α/β mRNA or protein (data not shown). This is intriguing because the FDC network in the GC retains Ag in an unprocessed form for the selection of high affinity B cell clones and is thus essential in promoting B cell survival and differentiation (51, 52, 53, 54). IL-8 was spontaneously released by GC B cells, but not by naive or memory B cells. Moreover, IL-8 production by B cells was not significantly regulated by cell activation or coculture with HK cells. Interactions between HK cells and the GC or B cells activated in vitro led to the production of large amounts of IL-8 by HK cells. It is unknown whether activated B cells induce IL-8 production by FDC in vivo. The role of IL-8 in T cell traffic is still controversial, and our in vitro experiments showed that T lymphocyte attraction in response to IL-8 was indeed marginal. However, IL-8 might contribute to regulate the B cell response in secondary lymphoid organs as previously shown (55, 56, 57).

Thus, we provide herein the first evidence that Ag-binding B cells are an important source of two CC chemokines with T cell-specific properties: MIP-1α and MIP-1β. The engagement of the Ag receptor on B cells determined the capacity to recruit T cells of the effector/helper phenotype. Cognate T/B cell interactions are required at all stages of Ag-driven B cell differentiation, so B cell-derived MIP-1α/β may directly affect maturation of the B cell response and, thus, the overall outcome of the adaptive immune response.

We thank Y. S. Choi for providing the HK cell line, D. Treton for excellent technical assistance, and Drs. A. Lange and J. Silber (K. Dluski Hospital, Wroclaw, Poland) for their encouragements and constant support. Anti-CCR5 mAb (2D7) was obtained through the AIDS Reagent Project, National Institute of Biological Standards and Control (Potters Bar, U.K.) from Leukosite, Inc. We also thank J. Wiels (UMR 1598, Villejuif, France) for providing anti-CD77 mAb.

1

This work was supported by grants and a fellowship (to E.A.L.) from the Agence Nationale de Recherche sur le SIDA, Institut National de la Santé et de la Recherche Médicale, and the Association Claude Bernard, the Université Paris-Sud.

3

Abbreviations used in this paper: SDF, stromal cell-derived factor; GC, germinal center; BCR, B cell Ag receptor; FDC, follicular dendritic cells; MIP, macrophage inflammatory protein; PE, phycoerythrin; CCR, CC chemokine receptor; CM, complete medium; SAC, Staphylococcus aureus Cowan I; MCP, monocyte chemotactic protein; sIg, surface immunoglobulin; DC, dendritic cells; sn B SAC, conditioned medium from SAC-activated B cells.

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