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Laboratory for Immunological Research, Schering Plough, Dardilly, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (sgp80), produced by DC, to drive naive B cell
differentiation. IL-12 is critical for the differentiation of naive B
cells into IgM plasma cells, whereas IL-6R signaling mainly promotes Ig
secretion by already differentiated B cells. The differentiation of
naive B cells in cocultures of B cells, T cells, and DC is IL-12
dependent, definitely demonstrating that the role of DC in humoral
responses is not confined to the activation of T cells and further
extending the physiologic relevance of DC/B cell interaction. Finally,
this study also identifies differential requirements for DC-dependent
naive and memory B cell differentiation, the latter being IL-12
independent. Altogether these results indicate that, in addition to
prime T cells toward Th1 development, DC, through the production of
IL-12, may also directly signal naive B cell during the initiation of
the immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
(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- (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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rhGM-CSF (specific activity: 2 x 106 U/mg,
Schering-Plough Research Institute, Kenilworth, NJ) was used at a
saturating concentration of 100 ng/ml (200 U/ml). rhTNF- (specific
activity: 2 x 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 x 105 U/mg, R&D
Systems, Abington, U.K.) was used at an optimal concentration of 25
ng/ml. rhIL-2 (specific activity: 3 x 106 U/mg,
Amgen, Thousand Oaks, CA) was used at 20 U/ml. rhIL-12 (1 x
107 U/mg) and rhsgp80 (1 x 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-
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/FcRII (29) were used as control L cells.
Generation of DC
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-, 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 x 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 x 104 cells/ml. Optimal conditions were
maintained by splitting these cultures at day 5 with medium containing
fresh GM-CSF and TNF- (cell concentration: 1–3 x
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%.
Isolation of T and B cells
B cells. 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).
T cells. 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%.
Cocultures of B cells and DC
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 x 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 x 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 x 104 B cells activated by 3.75 x 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 x 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-CD38high 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 FACStarPlus cytometer (Becton Dickinson).
Determination of sgp80, IL-6, and IL-12/p70 production
Irradiated L cells (105; CD40L L cells or control L cells) were seeded together with 5 x 105 naive B cells or 5 x 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.
Giemsa and immunostainings
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-
and anti-
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1B). 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. 1A) and induced a strong differentiation into IgM-secreting
cells (Fig. 1B). 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 CD8 and human CD40L
(22) led to similar results, although lower in magnitude.
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C, fivefold enhancement of
[3H]TdR uptake). In contrast, the induction of IgM
production, which requires the presence of IL-2 (Fig. 1B),
was strictly dependent on CD40 engagement on DC (Fig. 1C).
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.
Anti-IL-12 and anti-IL-6R Abs inhibit the IL-2 and
DC-dependent production of IgM
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-6R) 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 2C, 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. 1A) 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- Abs (data not shown).
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) 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.
DC-derived IL-12 and sgp80 synergize with IL-2 to induce CD40-activated naive B cell to secrete IgM
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-6R (sgp80)
and IL-12 appears more restricted to DC (Fig. 3A). Although, the production
of sgp80 by DC did not require any activation, CD40 engagement led to
enhanced levels (Fig. 3A). 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.
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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. 3B). The same
combination of cytokines modestly enhanced CD40-activated B cell
proliferation (Fig. 3B), 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.
DC induce differentiation of naive B cells toward IgM plasma cells through the production of IL-12
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-CD38high cells (0.2%, Fig. 4A), addition of DC induced
significant plasma cell formation (9.3%, Fig. 4B). 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).
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, 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.
Contribution of IL-12 and sgp80 in T cell-dependent naive 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. 5A). 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. 5A). 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. 5A).
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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- Ab had no effect, suggesting
that the effect of IL-12 is independent of endogenous production of
IFN-. 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. Naive and memory B cell differentiation are differentially regulated by DC
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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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 -chain. Contribution of IL-6/sgp80 to naive B cell differentiation
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 -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.
Contribution of IL-12 to naive B cell differentiation
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.
Differential regulation by DC of B cell proliferation, naive and memory B cell differentiation
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-ß 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.
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Acknowledgments |
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Footnotes |
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2 B.D. was supported by Foundation Marcel Mérieux, Lyon, France, and J.F. by Ecole Normale Supérieure de Lyon, France.
3 Current address: Baylor Institute for Immunology Research, 3500 Gaston Avenue, P.O. Box 710699, Dallas, Texas 75246.
4 Address correspondence and reprints requests to Dr. Christophe Caux, Laboratory for Immunological Research, Schering-Plough, 27 chemin des Peupliers, BP 11, 69571 Dardilly, France. E-mail address:
5 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; GM-CSF, granulocyte-macrophage CSF; h, human; SAC, Staphylococcus aureus strain Cowan I; PE, phycoerythrin; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; SAg, superantigen; GCDC, germinal center DC.
Received for publication January 14, 1998. Accepted for publication May 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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