Critical Role of IL-12 in Dendritic Cell-Induced Differentiation of Naive B Lymphocytes^1 2

Dendritic cells (DC)⁵ are bone marrow-derived leukocytes with potent immunostimulatory properties (1). In the periphery, DC such as epidermal Langerhans cells, capture invading Ag/pathogen and migrate via the lymph stream into draining lymph nodes. In the paracortical areas, these DC, called interdigitating cells present processed peptides to naive T cells and initiate the immune response. This T cell activation is followed by cognate interaction between Ag-specific T cells and B cells (2), leading to primary Ab production. The essential role of DC in humoral response has been documented in vitro (3) and in vivo (4, 5, 6, 7) and is thought to be the consequence of the requirement for DC for naive T cell priming. Once activated, T cells express CD40 ligand (CD40L), which in return signal DC to up-regulate costimulatory molecules (CD54, CD80, CD86) and to secrete cytokines (IL-1, IL-6, IL-8, IL-12, TNF-α) (8, 9). In parallel, CD40L expressing T cells together with cytokines promotes B cell survival (10), proliferation (11), as well as B cell differentiation and isotype switching (12, 13). Thus, among the signals involved in DC/T cell/B cell cooperation, CD40/CD40L interactions (14) appear of critical importance as illustrated in the hyper IgM syndrome (15) or mice deficient for CD40 or CD40L (16, 17).

Although studies have underlined the specific role of DC in humoral responses (4, 5, 6, 7), the extent to which DC can directly regulate B cell activation requires further analysis. Recent progress in the propagation of DC in vitro from precursors (18) and in their purification from blood (19) or tonsils (20, 21) has enabled such studies. Recently, several findings have suggested that DC may directly regulate B cell activation during the course of the humoral response (22, 23, 24, 25). In particular, we showed that DC, generated by culturing cord blood CD34⁺ progenitors for 12 days in presence of GM-CSF + TNF-α (26, 27), strongly enhance growth and differentiation of CD40-activated B cells (22). In the present study, aimed at characterizing the molecules involved in the effects of DC on B cells, we demonstrate that 1) DC-derived IL-12 is mandatory in inducing naive, but not memory, B cell differentiation, and that 2) DC strongly enhance Ig production by naive and memory B cells by potentiating the IL-6-dependent pathway of B cell differentiation. These findings demonstrate that DC-derived IL-12 can directly signal B cells and thus influence the initiation and subsequent development of the humoral response.

rhGM-CSF (specific activity: 2 × 10⁶ U/mg, Schering-Plough Research Institute, Kenilworth, NJ) was used at a saturating concentration of 100 ng/ml (200 U/ml). rhTNF-α (specific activity: 2 × 10⁷ U/mg, Genzyme, Boston, MA) was used at an optimal concentration of 2.5 ng/ml (50 U/ml). Recombinant human stem cell factor (specific activity 4 × 10⁵ U/mg, R&D Systems, Abington, U.K.) was used at an optimal concentration of 25 ng/ml. rhIL-2 (specific activity: 3 × 10⁶ U/mg, Amgen, Thousand Oaks, CA) was used at 20 U/ml. rhIL-12 (1 × 10⁷ U/mg) and rhsgp80 (1 × 10⁵ U/mg) were purchased from R&D Systems and used at 1 ng/ml and 100 ng/ml, respectively. rhIL-6, purified from Escherichia coli by ion-exchange chromatography (>95% pure), was obtained from DNAX (Palo Alto, CA) and used at 10 ng/ml. Formalinized particles of Staphylococcus aureus strain Cowan I (SAC) were purchased as Pansorbin from Calbiochem-Behring (La Jolla, CA) and were used at a final concentration of 0.01%.

The following blocking Abs were used at 10 μg/ml: mouse anti-IL-2 mAb (R&D Systems) and mouse anti-gp80 mAb (Diaclone, Besançon, France, and R&D Systems), goat anti-IL-12 and goat anti-IFN-γ Abs (R&D Systems), mouse anti-IL-12 mAb (IgG1, clone C8.6), kindly provided by A. O’Garra, DNAX, Palo Alto, CA), rat anti-hIL-10R mAb (clone 3F9) and anti-hIL-10 mAb (clone 12G8, kindly provided by K. Moore, DNAX), mouse IgG1, goat IgG, and rabbit IgG isotype control Abs (R&D Systems), mouse anti-CD40 (mAb89) and anti-CD40L (LL48) mAbs (produced in our laboratory).

The murine fibroblastic cell line stably transfected with human CD40L (CD40L L cells) was produced in the laboratory as described earlier (28). Mouse fibroblastic L cells transfected with the human CD32/FcγRII (29) were used as control L cells.

Umbilical cord blood samples were obtained according to institutional guidelines. Cells bearing CD34 Ag were isolated from mononuclear fractions through positive selection, using anti-CD34 mAb (10 μg/ml, Immu-133.3, Immunotech, Marseille, France) and goat anti-mouse IgG coated microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). Isolation of CD34⁺ progenitors was achieved using Minimacs separation columns (Miltenyi Biotec) as described (27). In all experiments the isolated cells were 80% to 99% CD34⁺ as judged by FACs staining with anti-CD34 mAb. After purification, CD34⁺ cells were cryopreserved in 10% DMSO.

Cultures were established in the presence of stem cell factor, GM-CSF, and TNF-α, as described (26, 30), in endotoxin-free medium consisting of RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated FBS (Life Technologies), 10 mM HEPES, 2 mM l-glutamine, 5 × 10⁻⁵ M 2-ME, penicillin (100 U/ml), and streptomycin (100 μg/ml). After thawing, CD34⁺ cells were seeded for expansion in 25- to 75-cm² culture vessels (Limbro, Flow Laboratories, McLean, VA) at 2 × 10⁴ cells/ml. Optimal conditions were maintained by splitting these cultures at day 5 with medium containing fresh GM-CSF and TNF-α (cell concentration: 1–3 × 10⁵ cells/ml). For most experiments, cells were routinely collected after 12 days of culture, labeled with FITC-conjugated anti-CD1a mAb (Ortho Diagnostic Systems, Raritan, NJ), and CD1a⁺ DC were isolated using a FACstarplus Cytometer (Becton Dickinson, Mountain View, CA). The procedure of staining and sorting was performed in the presence of 5 mM EDTA to avoid cell aggregation. Reanalysis of the sorted population showed a purity higher than 98%.

Mononuclear cells from tonsils were isolated by a standard Ficoll-Hypaque (density = 1077 g/ml) gradient method. Tonsillar B cells were first enriched in the E⁻ fraction and then submitted to anti-CD2, -CD4, -CD8, -CD14, -CD16 mAb negative selection with magnetic beads coated with anti-mouse IgG (Dynabeads; Dynal, Oslo, Norway). In the isolated population (total B cells), >99% expressed CD19 and CD20 and <1% expressed CD2 or CD14. Isolation of sIgD⁺ B cells naive B cells was performed using a preparative magnetic cell sorter (MACS, Miltenyi Biotec GmBH) as described elsewhere (31). IgD was expressed on >99% of the sIgD⁺ B cell subpopulation as assessed by fluorescence analysis using a FACScan (Becton Dickinson). For certain experiments, sIgD⁻ B cells were further separated into CD38⁻CD39⁺ memory B cells using anti-CD38 mAbs (Immunotech) and bead depletion as described earlier (32).

Mononuclear cells were first isolated from adult peripheral blood. CD4⁺ T cells were then purified by immunomagnetic depletion using a mixture of mAbs: OKT8 (CD8), IOM2 (CD14), ION16 (CD16), ION2 (HLA-DR) (immunotech), NKH1 (CD56) (Ortho Diagnostic Systems, Raritan, NJ), 4G7 (CD19) and mAb89 (CD40). After two rounds of bead depletion, the purity of CD4⁺ T cells was routinely higher than 97%.

Cultures were conducted in modified Iscove’s medium, supplemented with 5% inactivated FBS, 2 mM l-glutamine, and 0.08 μg/ml gentamicine (Schering-Plough, Levallois Perret, France). Irradiated CD40L L cells (2.5 × 10³) (7500 rad) were seeded together with 10⁴ B lymphocytes (either memory or sIgD⁺ B cells) in the presence or the absence of 10⁴ in vitro-generated DC (irradiated 3,000 rad) in 96-well culture plates. B cell proliferation was monitored by tritiated thymidine ([³H]TdR) incorporation after 6 days of coculture. Cells were incubated for the last 16 h with 1 μCi of [³H]TdR. Experiments were conducted in triplicate, and results were expressed as cpm ± SD. For determination of Ig production, supernatants were recovered after 13 days and used for indirect ELISA (33). Phenotype of the cultured cells was routinely performed using FITC-labeled anti-CD3, anti-CD19 (Immunotech), and FITC-labeled IgG1 (Kallestad, Austin, TX) and showed the absence of detectable contaminating T cells throughout the culture.

For certain experiments, naive B cells (10⁴) were cultured in presence of irradiated (4000 rad) allogeneic CD4⁺ T cells (10⁵) and increasing numbers of irradiated DC (3,000 rad) with or without superantigens (SEA + SEB, 10 ng/ml each). In certain culture conditions, DC were incubated for 1 h with SEA + SEB, washed 4 times, irradiated (4000 rad), and used to stimulate cocultures of T and B cells.

In other experiments, B cells and DC were cultured in separate compartments using transwells (Costar, Wilmington, MA). DC (10⁵) cultured in the presence or the absence of CD40 triggering (2.5 × 10⁴ irradiated CD40L L cells or CD32 L cells used as control) in the lower compartment (in a total volume of 0.8 ml) were assayed for their ability to stimulate growth and differentiation of 1.5 × 10⁴ B cells activated by 3.75 × 10³ irradiated CD40L L cells in the upper compartment of the transwells (in a total volume of 0.2 ml). DNA synthesis of B cells was performed by transferring, at day 6, the cells present in the top of the transwells into flat-bottom 96-well plates and pulsing them with [³H]TdR for the last 16 h of the culture period.

For phenotypic studies, 10⁵ naive B cells were cultured over 2.5 × 10⁴ irradiated CD40L L cells and IL-2, with or without 10⁵ DC. Cultures were performed in 24-well culture plate in a total volume of 1 ml. For plasma cell formation study, cells were recovered after 8 days and processed for FACS stainings using anti-CD20-FITC, anti-CD38-PE (Becton Dickinson), and anti-CD19-PEcy5 (Immunotech). The percentage of CD20⁻CD38^high cells was analyzed on a FACsCalibur (Becton Dickinson) by gating on CD19⁺ B cells. In some experiments, CD19⁺ B cells from 8 days coculture with irradiated CD40L L cells + IL-2 + DC were sorted into CD20⁺CD38⁺ and CD20⁻CD38²⁺ cells using a FACStar^Plus cytometer (Becton Dickinson).

Irradiated L cells (10⁵; CD40L L cells or control L cells) were seeded together with 5 × 10⁵ naive B cells or 5 × 10⁵ DC per well (24-well culture plates) with or without IL-2 (total volume = 0.5 ml). Cytokine concentrations in 48-h cell-free supernatants were measured by two-site sandwich ELISA. Kits of dosage were purchased from Medgenix Diagnostics (Brussels, Belgium) for IL-6 and from R&D Sytems for sgp80 and IL-12/p70 (high sensitivity). The sensitivities of these kits were: 31 pg/ml for sgp80, 16 pg/ml for IL-6, and 1.2 pg/ml for IL-12/p70.

Sorted CD19⁺ B cells were cytocentrifuged for 5 min at 400 rpm on microscope slides and used for May-Gründwald-Giemsa staining or fixed in cold acetone. Anti-Ig staining was performed using mouse anti-κ and anti-λ mAbs conjugated to peroxidase (Tago, Burlingame, CA) and developed by 3-amino-9-ethylcarbazole. Staining with mouse anti-IgM (Immunotech) was performed using the APAAP technique (Dako, Trappes, France) revealed by the fast blue substrate.

Naive B cells were cultured over irradiated CD40L-transfected L cells with or without irradiated DC derived from cord blood CD34⁺ progenitors (DC). As previously described (22), DC enhanced CD40-induced naive B cell proliferation (threefold enhancement, Fig. 1,A) and sustained subsequent differentiation, provided that exogenous IL-2 was added (Fig. 1,B). Indeed, while IL-2 by itself has no significant effect on CD40-dependent B cell activation (Refs. 13, 34, and 35, and Fig. 1, A and B), in the presence of DC, IL-2 significantly enhanced B cell proliferation (Fig. 1,A) and induced a strong differentiation into IgM-secreting cells (Fig. 1 B). In such cocultures, in the absence of CD40 signaling, DC do not induce B cell proliferation and differentiation (data not shown). The use of CD32 L cells and anti-CD40 Ab (data not shown) or a soluble fusion protein of mouse CD8α and human CD40L (22) led to similar results, although lower in magnitude.

To determine the nature of the molecules involved in the DC stimulatory effects (membrane bound and/or soluble) as well as the importance of CD40 triggering on DC, we performed cultures in transwells. Naive B cells were seeded on the top of the transwells together with CD40L L cells, and DC were cultured with CD40L L cells or control CD32 L cells in the bottom. As previously reported (22), B cells needed to be activated through their CD40 to respond to DC stimulation (data not shown). DC, activated or not through CD40, enhanced proliferation of CD40-activated B cells (Fig. 1,C, fivefold enhancement of [³H]TdR uptake). In contrast, the induction of IgM production, which requires the presence of IL-2 (Fig. 1,B), was strictly dependent on CD40 engagement on DC (Fig. 1 C). Thus, DC enhance CD40-induced naive B cell proliferation through the production of soluble molecules independently of CD40 triggering. In contrast, the induction of IL-2-dependent naive B cell differentiation, which is also borne by soluble entities, requires CD40 engagement on the DC.

Since DC-dependent naive B cell differentiation is mediated by soluble molecules, Ab against different cytokines or their receptors were tested for their capacity to affect this response. Among known soluble mediators, IL-10 can be produced by both DC (36) and B cells (37), and has been demonstrated to synergize with IL-2 to induce strong B cell differentiation (38). However, the use of mAbs directed against IL-10R (or IL-10, not shown) ruled out the possible contribution of IL-10 in all the effects described in this study (Table I). Anti-gp80 mAb (IL-6Rα) was found to strongly inhibit the IL-2-dependent IgM production in the presence of DC (68% inhibition, r = 60–87, n = 6). Furthermore, Abs to IL-12 completely blocked DC-induced IgM secretion by naive B cell in presence of IL-2 (96% inhibition, r = 94–98, n = 6). Of note, none of the two Abs had significant effect on CD40-dependent naive B cell proliferation in presence or absence of DC (Table I). The inhibition of IgM production by both Abs was dose dependent (Fig. 2, A and B) and reached a plateau at relatively low doses of Abs (1 μg/ml for anti-gp80 and 5 μg/ml for anti-IL-12 Ab). Comparable results were obtained with anti-IL-6 or anti-gp130 mAb and other anti-IL-12 Ab, including both monoclonal and polyclonal Ab (data not shown). As shown in Figure 2,C, the Ab-dependent inhibition of IgM secretion was specific since it could be reversed by addition of 10 ng/ml and 200 ng/ml of IL-12 and IL-6, respectively. In addition, the growth stimulation in response to IL-2 observed in the presence of DC (Fig. 1 A) was significantly affected by neutralization of IL-6R or IL-12 (data not shown), suggesting that proliferation and differentiation of naive B cells induced by IL-2 + DC are regulated by similar mechanisms. An indirect effect of DC-derived IL-12 on eventually contaminating T cells and NK cells was ruled out by the lack of CD3 and CD56 mRNA, as measured by RT-PCR and by the lack of effect of neutralizing anti-IFN-γ Abs (data not shown).

To determine whether the contribution of IL-6 and IL-12 in naive B cell differentiation was restricted to the effects of DC, naive B cells were cultured in the presence of CD40L L cells, SAC particles, and IL-2 (a DC-independent system allowing significant IgM production). Blocking endogenous IL-6R signaling (Table I) led to a 68% inhibition of IgM secretion (r = 41–80, n = 3), a finding in agreement with the autocrine B cell differentiation activity of IL-6 (37, 39). In contrast, anti-IL-12 Abs had no significant effect on B cell differentiation in the absence of DC (Table I).

Taken together, these results demonstrate that DC promote a naive B cell differentiation pathway that is dependent on IL-6 and IL-12.

To determine the source of cytokines (e.g., B cells and/or DC) involved in DC-dependent IgM production, we measured by ELISA the production of IL-6, sgp80, and IL-12/p70 in supernatants of naive B cells and DC. Whereas naive B cells and DC produce comparable levels of IL-6 upon CD40 triggering, the production of soluble IL-6Rα (sgp80) and IL-12 appears more restricted to DC (Fig. 3,A). Although, the production of sgp80 by DC did not require any activation, CD40 engagement led to enhanced levels (Fig. 3 A). Bioactive IL-12 (p70) was produced in low but significant amounts by DC upon CD40 activation, whereas this cytokine was under the detection limit in B cell supernatants. The secretion of these three molecules was not affected by the presence of IL-2 (data not shown). Because naive B cells and DC produce comparable levels of IL-6, it is likely that IL-12 and sgp80, which are mainly produced by DC, are the key molecules contributing to the effects of DC.

Recombinant IL-6, sgp80, and IL-12/p70, were then tested, at optimal concentrations, for their ability to induce CD40-activated naive B cells to proliferate and secrete IgM. In the absence of IL-2, none of the cytokines, used alone or in combinations, led to significant induction of IgM production by naive B cells (Fig. 3,B). In combination with IL-2, sgp80 was totally inefficient in driving B cell differentiation, whereas IL-6 or IL-12 induced low but reproducible IgM production (0.31 ± 0.04 and 0.77 ± 0.06 μg/ml, respectively, n = 5, background 0.08 μg/ml). The effect of IL-2 + IL-12 was strongly potentiated by either addition of sgp80 or IL-6 (5 μg/ml of IgM compared with 15.1 μg/ml obtained with DC). The most potent effect was achieved by combining the three molecules (sgp80, IL6, and IL-12) together with IL-2, thereby reconstituting 30 to 150% of the IgM production (n = 7) obtained in presence of DC (Fig. 3,B). The same combination of cytokines modestly enhanced CD40-activated B cell proliferation (Fig. 3 B), suggesting that another DC product, as yet unidentified, could be involved in DC-dependent B cell proliferation.

Altogether, these data suggest that DC-derived IL-12 synergizes with IL-6R signaling to induce naive B cell differentiation in presence of IL-2. Furthermore, these results imply that IL-2 directly acts on naive B cells and renders them responsive to DC factors.

The differentiation of B cells into plasma cells is characterized by the down-regulation of CD20 and up-regulation of CD38 (40, 41). The analysis of plasma cell formation in culture of CD40-activated naive B cells with IL-2 in presence or absence of DC was performed after 8 days using triple immunofluorescence staining. Whereas naive B cells alone (gate on CD19⁺ cells) did not differentiate into CD20⁻CD38^high cells (0.2%, Fig. 4,A), addition of DC induced significant plasma cell formation (9.3%, Fig. 4,B). Sorted CD20⁻CD38²⁺ cells present characteristics of plasma cells, e.g., oval cells, compact, dense, eccentric nuclei, with basophilic cytoplasm and strong intracytoplasmic anti-Ig staining (Fig. 4, C-E). Their spontaneous production of IgM upon reculture (data not shown) further confirm that these cells are IgM plasma cells. In contrast, sorted CD20⁺CD38⁺ cells are blastic cells with weak intracytoplasmic anti-Ig staining (Fig. 4, F-H).

We next wondered whether IL-6/sgp80 and IL-12 were involved in the formation of plasma cells induced by IL-2+DC. Fig. 4, I-L, shows the effects of anti-gp80 and anti-IL-12 Abs on DC-induced plasma cell formation. Blocking IL-12 strongly impaired the development of plasma cells obtained after 8 days of coculture (85% inhibition). In contrast, Ab to gp80 only slightly affect the generation of plasma cells (22% inhibition). A near complete inhibition was observed when both Abs were added together. In addition, DC potentiate spontaneous IgM production by sorted IgM plasma cells (data not shown). This effect on IgM secretion was blocked by anti-gp80 mAb by 83%, while anti-IL-12 Ab had no effect.

Taken together these results suggest that IL-12 is mandatory for DC-induced differentiation of naive B cells into IgM plasma cells, whereas IL-6/IL-6R signaling may intervene at a later stage of B cell differentiation.

All the experiments described above were performed using a fibroblastic cell line stably expressing CD40L, to mimic activated T cells. To extend the relevance of the direct DC/B cell interaction, we analyzed the contribution of DC-derived IL-12 in coculture of B cells, T cells, and DC. Using irradiated allogeneic peripheral blood CD4⁺ T cells, in place of CD40L L cells, did not allow significant IgM production even in the presence of DC (Fig. 5,A). This low rate of B cell help was likely due to a limited recruitment of alloreactive T cells. To increase T cell activation, a combination of superantigens (SAg: SEA + SEB) was added to cocultures. In this system, DC increased in a dose-dependent manner naive B cell proliferation (data not shown) and subsequent differentiation (Fig. 5,A). Comparable results were obtained using SAg-pulsed DC, showing that SAg primarily favors DC-dependent T cell activation. This system is IL-2- and CD40L-dependent, as demonstrated by the near total inhibition of IgM production observed with blocking anti-IL-2 or anti-CD40L Abs (Fig. 5 A).

We next assessed the contribution of IL-6/sgp80 and IL-12 in this system. As shown in Figure 5 B, in presence of T cells and SAg, DC-dependent IgM production by naive B cells was strongly impaired by blocking IL-6R signaling (45% inhibition) or IL-12 (73% inhibition). In addition, anti-IFN-γ Ab had no effect, suggesting that the effect of IL-12 is independent of endogenous production of IFN-γ. Furthermore, blocking IL-12 has no detectable effect on the level of T cell activation as measured by T cell proliferation (data not shown). Thus, in addition to activate T cells, DC directly contribute to naive B cell proliferation and differentiation, in particular through the production of IL-12.

In addition to inducing naive B cell differentiation in presence of IL-2, DC strongly potentiate memory B cell differentiation in absence of exogenous cytokines (22). We thus wondered whether the molecules that contribute to IL-2-dependent naive B cell differentiation would also drive the differentiation of memory B cells. Thus we have assessed the contribution of IL-6 and IL-12 in DC-induced differentiation of naive/memory B cells. As previously shown (Table I), anti-gp80 or anti-IL-12 Abs strongly suppressed naive B cell differentiation induced by DC + IL-2 (Fig. 6). In contrast, only anti-gp80 mAb significantly inhibited IgG production by memory B cells in the presence of DC (Fig. 6). Thus, while IL-6/sgp80 is involved in DC-induced differentiation of both naive and memory B cells, IL-12 is uniquely involved in the DC-induced differentiation of naive B cells toward IgM-secreting plasma cells.

We previously reported that DC, generated by culturing CD34⁺ progenitors with GM-CSF + TNF-α, strongly enhance the proliferation and differentiation of CD40-activated B cells (22, 23). The present study demonstrates that IL-12, produced by DC upon CD40 activation, is the key molecule involved in the differentiation of naive B cells into IgM-secreting plasma cells. The critical role of IL-12 in naive B cell differentiation has been confirmed in coculture of T cells, B cells, and DC, adding further support to the relevance of the direct DC/B cell interaction. Our study further shows that DC amplify the differentiation of both naive and memory B cells through the release of soluble IL-6R α-chain.

The inhibition by Abs against gp80 of the DC and IL-2-dependent IgM secretion of cultured naive B cells indicates that DC contribute to B cell differentiation by modulating the IL-6/IL-6R signaling pathway. Since CD40-activated naive B cells produce IL-6 (Ref. 42 and Fig. 3), which plays an important role in their differentiation (39, 43, 44), it is unlikely that IL-6 produced by DC is the critical molecule involved in the observed enhancement of B cell differentiation. More likely, DC contribute to the IL-6-dependent B cell differentiation by secreting the soluble form of the gp80 IL-6R α-chain. This molecule has previously been shown to potentiate the biologic activity of IL-6 (45, 46) by forming an IL-6/sgp80 complex, which binds with high affinity to the ubiquitous and constitutive gp130-transducing chain (47, 48). Accordingly, a combination of IL-12 and sgp80 can substitute for DC in the IL-2-dependent naive B cell differentiation, thereby demonstrating the critical role for DC-derived sgp80 in this process. Recently, Kaposi’s sarcoma-associated herpesvirus-infected DC have been implicated in multiple myeloma patients (49). Viral IL-6 was found to be transcribed in the myeloma bone marrow DC and has been proposed to play a role in the propagation of malignant plasma cells. The critical role of IL-6/IL-6R signaling in DC-dependent naive and memory B cell differentiation described herein adds further support to this hypothesis.

The strong inhibitory effect of anti-IL-12 Abs on DC and IL-2-dependent IgM secretion suggests that IL-12 plays a critical role in this biologic function. Accordingly, a combination of IL-12 with IL-6 and sgp80 can substitute for DC in the IL-2-dependent differentiation of naive B cells. The bioactive form of IL-12 is exclusively produced by CD40-activated DC, while activated normal B cells do not secrete this cytokine (Ref. 50 and Fig. 4). The effects of IL-12 on B cells described herein are in accordance with two previous reports showing that IL-12 enhanced proliferation and Ig secretion of SAC + IL-2-activated peripheral blood B cells (51, 52). We further demonstrate that DC-derived IL-12 plays a critical role in IgM plasma cell formation. The mechanism by which IL-12 induced naive B cell differentiation remains unknown. Of importance, the present study indicates that the IL-12-dependent B cell differentiation requires IL-2 signaling on B cells, in line with other reports (51, 52). The ability of IL-12 to synergize with IL-2 has also been reported during T cell and NK cell activation (53, 54). While the signals regulating IL-12 receptors expression on B cells (55, 56) are not well defined, the present study demonstrates the expression of a functional IL-12 receptor on B cells following CD40 and IL-6R signaling.

It has been well established by many groups that IL-12 plays a pivotal role in controlling cell-mediated immunity (57, 58, 59). Our findings suggest that B cells may represent an important cellular target of IL-12, in addition to T cells and NK cells. Indeed, recent studies performed in vivo in mouse have suggested it (60, 61, 62), though it could not be demonstrated whether the effects were directly on B cells or indirect through the activation of other cell types subsequently affecting B cells. In particular, IL-12 was found to enhance the secretion of Ag-specific IgG1, IgG2a, and IgG2b when given to mice vaccinated with Shistosoma mansoni cercariae (62). The direct effect of IL-12 on humoral response observed both in vitro and in vivo, together with its critical role in cell-mediated immunity, suggests that this cytokine could constitute a potent vaccine adjuvant in situation that may require both arms of the immune system, such as HIV (63), malaria (64, 65), or certain cancers.

While this study succeeded in deciphering the molecules involved in the DC-dependent naive B cell differentiation, the soluble mediators controlling B cell proliferation remain to be characterized. Not only are B cell proliferation and naive B cell differentiation differentially regulated, but also memory B cell differentiation appears to be independently controlled, since it is unaffected by the presence of IL-12. Furthermore, DC appear to regulate naive B cell differentiation toward IgM-secreting cells and sIgA-expressing cells through different mechanisms, the former being strictly dependent on IL-12 and the latter being partially TGF-β dependent (23).

Figure 7 summarizes the effects of DC on naive B cells in the context of DC/T cell/B cell interactions. Once activated by DC, Ag-specific T cells express CD40L and secrete IL-2. Following cognate interaction with T cells, naive B cells engaged through their CD40, proliferate in response to an unidentified molecule (IL-X), whose production by DC does not require CD40 triggering. Furthermore, in presence of T cell-derived IL-2, IL-12 then stimulates CD40-activated naive B cells to differentiate into IgM-secreting plasma cells, whereas sgp80 may regulate a later stage of differentiation (e.g., Ig secretion). This regulation of naive B cell differentiation by DC might occur during the initiation of primary B cell responses in the extrafollicular areas of secondary lymphoid organs (22, 23, 24, 25). However, DC within germinal centers (GCDC) have recently been identified (20), and preliminary results show that GCDC can support naive B cell differentiation in presence of IL-2. Thus the effect of DC on naive B cell differentiation might also occur within B cell follicles and involve specialized GCDC.

In conclusion, our present study shows the critical role of IL-12, produced by dendritic cells, in the launching of primary B cell responses. It has also highlighted the complex molecular interplay that occurs during the Ag-driven interaction of T cell, B cell, and dendritic cells, the full deciphering of which will necessitate further studies.

We thank I. Durand for FACS analysis, S. Bourdarel for editorial assistance, and doctors from clinics and hospitals in Lyon who provided us with umbilical cord blood samples and tonsils. We thank D. Blanchard, P. Garrone, and Y. J. Liu for helpful discussion and careful reading of the manuscript.