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

This information is current as Bertrand Dubois, Catherine Massacrier, Béatrice Vanbervliet, of September 29, 2021. Jérome Fayette, Francine Brière, Jacques Banchereau and Christophe Caux J Immunol 1998; 161:2223-2231; ; http://www.jimmunol.org/content/161/5/2223 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Critical Role of IL-12 in Dendritic Cell-Induced Differentiation of Naive B Lymphocytes1,2

Bertrand Dubois, Catherine Massacrier, Be´atrice Vanbervliet, Je´rome Fayette, Francine Brie`re, Jacques Banchereau,3 and Christophe Caux4

Dendritic cells (DC) are potent APCs initiating immune responses. In a previous report, we demonstrated that DC directly enhance both proliferation and differentiation of CD40-activated naive and memory B cells. The present study deciphers the molecular mechanisms involved in DC-dependent regulation of B cell responses. Herein, we have identified IL-12 as the mandatory molecule secreted by CD40-activated DC that promote the differentiation of naive B cells into plasma cells secreting high levels of IgM. In fact, IL-12 synergizes with soluble IL-6R ␣-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 Downloaded from 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. The Journal of Immunology, 1998, 161: 2223–2231. http://www.jimmunol.org/ endritic cells (DC)5 are bone marrow-derived leukocytes critical importance as illustrated in the hyper IgM syndrome (15) with potent immunostimulatory properties (1). In the pe- or mice deficient for CD40 or CD40L (16, 17). D riphery, DC such as epidermal Langerhans cells, capture Although studies have underlined the specific role of DC in invading Ag/pathogen and migrate via the lymph stream into humoral responses (4–7), the extent to which DC can directly reg- draining lymph nodes. In the paracortical areas, these DC, called ulate B cell activation requires further analysis. Recent progress in interdigitating cells present processed peptides to naive T cells and the propagation of DC in vitro from precursors (18) and in their initiate the immune response. This T cell activation is followed by purification from blood (19) or tonsils (20, 21) has enabled such cognate interaction between Ag-specific T cells and B cells (2), studies. Recently, several findings have suggested that DC may by guest on September 29, 2021 leading to primary Ab production. The essential role of DC in directly regulate B cell activation during the course of the humoral humoral response has been documented in vitro (3) and in vivo response (22–25). In particular, we showed that DC, generated by (4–7) and is thought to be the consequence of the requirement for culturing cord blood CD34ϩ progenitors for 12 days in presence of DC for naive T cell priming. Once activated, T cells express CD40 GM-CSF ϩ TNF-␣ (26, 27), strongly enhance growth and differ- ligand (CD40L), which in return signal DC to up-regulate costimu- entiation of CD40-activated B cells (22). In the present study, latory molecules (CD54, CD80, CD86) and to secrete cytokines aimed at characterizing the molecules involved in the effects of DC (IL-1, IL-6, IL-8, IL-12, TNF-␣) (8, 9). In parallel, CD40L ex- on B cells, we demonstrate that 1) DC-derived IL-12 is mandatory pressing T cells together with cytokines promotes B cell survival in inducing naive, but not memory, B cell differentiation, and that (10), proliferation (11), as well as B cell differentiation and isotype 2) DC strongly enhance Ig production by naive and memory B switching (12, 13). Thus, among the signals involved in DC/T cells by potentiating the IL-6-dependent pathway of B cell differ- cell/B cell cooperation, CD40/CD40L interactions (14) appear of entiation. 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. Laboratory for Immunological Research, Schering Plough, Dardilly, France Received for publication January 14, 1998. Accepted for publication May 4, 1998. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Hemopoietic factors, cytokines, Abs, and cell lines with 18 U.S.C. Section 1734 solely to indicate this fact. ϫ 6 1 rhGM-CSF (specific activity: 2 10 U/mg, Schering-Plough Research Preliminary results were presented at the 4th International Symposium on Dendritic Institute, Kenilworth, NJ) was used at a saturating concentration of 100 Cells in Fundamental and Clinical Immunology, held in Venice, Italy, October 1996. ng/ml (200 U/ml). rhTNF-␣ (specific activity: 2 ϫ 107 U/mg, Genzyme, 2 B.D. was supported by Foundation Marcel Me´rieux, Lyon, France, and J.F. by Ecole Boston, MA) was used at an optimal concentration of 2.5 ng/ml (50 U/ml). Normale Supe´rieure de Lyon, France. Recombinant human stem cell factor (specific activity 4 ϫ 105 U/mg, R&D 3 Current address: Baylor Institute for Immunology Research, 3500 Gaston Avenue, Systems, Abington, U.K.) was used at an optimal concentration of 25 ng/ P.O. Box 710699, Dallas, Texas 75246. ml. rhIL-2 (specific activity: 3 ϫ 106 U/mg, Amgen, Thousand Oaks, CA) ϫ 7 ϫ 5 4 Address correspondence and reprints requests to Dr. Christophe Caux, Laboratory was used at 20 U/ml. rhIL-12 (1 10 U/mg) and rhsgp80 (1 10 U/mg) for Immunological Research, Schering-Plough, 27 chemin des Peupliers, BP 11, were purchased from R&D Systems and used at 1 ng/ml and 100 ng/ml, 69571 Dardilly, France. E-mail address: [email protected] respectively. rhIL-6, purified from Escherichia coli by ion-exchange chro- Ͼ 5 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; GM- matography ( 95% pure), was obtained from DNAX (Palo Alto, CA) and CSF, granulocyte-macrophage CSF; h, human; SAC, Staphylococcus aureus strain used at 10 ng/ml. Formalinized particles of Staphylococcus aureus strain Cowan I; PE, phycoerythrin; SEA, staphylococcal enterotoxin A; SEB, staphylococ- Cowan I (SAC) were purchased as Pansorbin from Calbiochem-Behring cal enterotoxin B; SAg, superantigen; GCDC, germinal center DC. (La Jolla, CA) and were used at a final concentration of 0.01%.

The following blocking Abs were used at 10 ␮g/ml: mouse anti-IL-2 and showed the absence of detectable contaminating T cells throughout the mAb (R&D Systems) and mouse anti-gp80 mAb (Diaclone, Besançon, culture. France, and R&D Systems), goat anti-IL-12 and goat anti-IFN-␥ Abs For certain experiments, naive B cells (104) were cultured in presence (R&D Systems), mouse anti-IL-12 mAb (IgG1, clone C8.6), kindly pro- of irradiated (4000 rad) allogeneic CD4ϩ T cells (105) and increasing num- vided by A. O’Garra, DNAX, Palo Alto, CA), rat anti-hIL-10R mAb (clone bers of irradiated DC (3,000 rad) with or without superantigens (SEA ϩ 3F9) and anti-hIL-10 mAb (clone 12G8, kindly provided by K. Moore, SEB, 10 ng/ml each). In certain culture conditions, DC were incubated for DNAX), mouse IgG1, goat IgG, and rabbit IgG isotype control Abs (R&D 1 h with SEA ϩ SEB, washed 4 times, irradiated (4000 rad), and used to Systems), mouse anti-CD40 (mAb89) and anti-CD40L (LL48) mAbs (pro- stimulate cocultures of T and B cells. duced in our laboratory). In other experiments, B cells and DC were cultured in separate com- The murine fibroblastic cell line stably transfected with human CD40L partments using transwells (Costar, Wilmington, MA). DC (105) cultured (CD40L L cells) was produced in the laboratory as described earlier (28). in the presence or the absence of CD40 triggering (2.5 ϫ 104 irradiated Mouse fibroblastic L cells transfected with the human CD32/Fc␥RII (29) CD40L L cells or CD32 L cells used as control) in the lower compartment were used as control L cells. (in a total volume of 0.8 ml) were assayed for their ability to stimulate growth and differentiation of 1.5 ϫ 104 B cells activated by 3.75 ϫ 103 Generation of DC 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 trans- Umbilical cord blood samples were obtained according to institutional ferring, at day 6, the cells present in the top of the transwells into flat- guidelines. Cells bearing CD34 Ag were isolated from mononuclear frac- bottom 96-well plates and pulsing them with [3H]TdR for the last 16 h of ␮ tions through positive selection, using anti-CD34 mAb (10 g/ml, Immu- the culture period. 133.3, Immunotech, Marseille, France) and goat anti-mouse IgG coated For phenotypic studies, 105 naive B cells were cultured over 2.5 ϫ 104 microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). Isolation of 5 ϩ irradiated CD40L L cells and IL-2, with or without 10 DC. Cultures were CD34 progenitors was achieved using Minimacs separation columns performed in 24-well culture plate in a total volume of 1 ml. For plasma (Miltenyi Biotec) as described (27). In all experiments the isolated cells cell formation study, cells were recovered after 8 days and processed for Downloaded from were 80% to 99% CD34ϩ as judged by FACs staining with anti-CD34 ϩ FACS stainings using anti-CD20-FITC, anti-CD38-PE (Becton Dickin- mAb. After purification, CD34 cells were cryopreserved in 10% DMSO. son), and anti-CD19-PEcy5 (Immunotech). The percentage of Cultures were established in the presence of stem cell factor, GM-CSF, CD20ϪCD38high cells was analyzed on a FACsCalibur (Becton Dickinson) ␣ ϩ ϩ and TNF- , as described (26, 30), in endotoxin-free medium consisting of by gating on CD19 B cells. In some experiments, CD19 B cells from 8 RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with days coculture with irradiated CD40L L cells ϩ IL-2 ϩ DC were sorted 10% (v/v) heat-inactivated FBS (Life Technologies), 10 mM HEPES, 2 ϩ ϩ Ϫ 2ϩ Plus Ϫ5 into CD20 CD38 and CD20 CD38 cells using a FACStar cytom- mM L-glutamine, 5 ϫ 10 M 2-ME, penicillin (100 U/ml), and strepto-

eter (Becton Dickinson). http://www.jimmunol.org/ mycin (100 ␮g/ml). After thawing, CD34ϩ cells were seeded for expansion in 25- to 75-cm2 culture vessels (Limbro, Flow Laboratories, McLean, VA) Determination of sgp80, IL-6, and IL-12/p70 production at 2 ϫ 104 cells/ml. Optimal conditions were maintained by splitting these 5 cultures at day 5 with medium containing fresh GM-CSF and TNF-␣ (cell Irradiated L cells (10 ; CD40L L cells or control L cells) were seeded ϫ 5 ϫ 5 concentration: 1–3 ϫ 105 cells/ml). For most experiments, cells were rou- together with 5 10 naive B cells or 5 10 DC per well (24-well ϭ tinely collected after 12 days of culture, labeled with FITC-conjugated culture plates) with or without IL-2 (total volume 0.5 ml). Cytokine anti-CD1a mAb (Ortho Diagnostic Systems, Raritan, NJ), and CD1aϩ DC concentrations in 48-h cell-free supernatants were measured by two-site were isolated using a FACstarplus Cytometer (Becton Dickinson, Moun- sandwich ELISA. Kits of dosage were purchased from Medgenix Diag- tain View, CA). The procedure of staining and sorting was performed in nostics (Brussels, Belgium) for IL-6 and from R&D Sytems for sgp80 and the presence of 5 mM EDTA to avoid cell aggregation. Reanalysis of the 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 population showed a purity higher than 98%. by guest on September 29, 2021 Giemsa and immunostainings Isolation of T and B cells Sorted CD19ϩ B cells were cytocentrifuged for 5 min at 400 rpm on mi- B cells. Mononuclear cells from tonsils were isolated by a standard Ficoll- croscope slides and used for May-Gru¨ndwald-Giemsa staining or fixed in Hypaque (density ϭ 1077 g/ml) gradient method. Tonsillar B cells were Ϫ cold acetone. Anti-Ig staining was performed using mouse anti-␬ and anti- first enriched in the E fraction and then submitted to anti-CD2, -CD4, ␭ mAbs conjugated to peroxidase (Tago, Burlingame, CA) and developed -CD8, -CD14, -CD16 mAb negative selection with magnetic beads coated by 3-amino-9-ethylcarbazole. Staining with mouse anti-IgM (Immunotech) with anti-mouse IgG (Dynabeads; Dynal, Oslo, Norway). In the isolated Ͼ Ͻ was performed using the APAAP technique (Dako, Trappes, France) re- population (total B cells), 99% expressed CD19 and CD20 and 1% vealed by the fast blue substrate. expressed CD2 or CD14. Isolation of sIgDϩ B cells naive B cells was performed using a preparative magnetic cell sorter (MACS, Miltenyi Bio- tec GmBH) as described elsewhere (31). IgD was expressed on Ͼ99% of Results the sIgDϩ B cell subpopulation as assessed by fluorescence analysis using CD40-activated DC produce soluble factor(s) that promote IL-2- a FACScan (Becton Dickinson). For certain experiments, sIgDϪ B cells dependent differentiation of naive B cells were further separated into CD38ϪCD39ϩ memory B cells using anti- CD38 mAbs (Immunotech) and bead depletion as described earlier (32). Naive B cells were cultured over irradiated CD40L-transfected L T cells. Mononuclear cells were first isolated from adult peripheral blood. cells with or without irradiated DC derived from cord blood CD4ϩ T cells were then purified by immunomagnetic depletion using a CD34ϩ progenitors (DC). As previously described (22), DC en- mixture of mAbs: OKT8 (CD8), IOM2 (CD14), ION16 (CD16), ION2 hanced CD40-induced naive B cell proliferation (threefold en- (HLA-DR) (immunotech), NKH1 (CD56) (Ortho Diagnostic Systems, Raritan, NJ), 4G7 (CD19) and mAb89 (CD40). After two rounds of bead hancement, Fig. 1A) and sustained subsequent differentiation, pro- depletion, the purity of CD4ϩ T cells was routinely higher than 97%. vided that exogenous IL-2 was added (Fig. 1B). Indeed, while IL-2 by itself has no significant effect on CD40-dependent B cell acti- Cocultures of B cells and DC vation (Refs. 13, 34, and 35, and Fig. 1, A and B), in the presence Cultures were conducted in modified Iscove’s medium, supplemented with of DC, IL-2 significantly enhanced B cell proliferation (Fig. 1A) 5% inactivated FBS, 2 mM L-glutamine, and 0.08 ␮g/ml gentamicine and induced a strong differentiation into IgM-secreting cells (Fig. (Schering-Plough, Levallois Perret, France). Irradiated CD40L L cells 1B). In such cocultures, in the absence of CD40 signaling, DC do (2.5 ϫ 103) (7500 rad) were seeded together with 104 B lymphocytes ϩ not induce B cell proliferation and differentiation (data not shown). (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 The use of CD32 L cells and anti-CD40 Ab (data not shown) or a proliferation was monitored by tritiated thymidine ([3H]TdR) incorporation soluble fusion protein of mouse CD8␣ and human CD40L (22) led after 6 days of coculture. Cells were incubated for the last 16 h with 1 ␮Ci to similar results, although lower in magnitude. 3 of [ H]TdR. Experiments were conducted in triplicate, and results were To determine the nature of the molecules involved in the DC expressed as cpm Ϯ SD. For determination of Ig production, supernatants were recovered after 13 days and used for indirect ELISA (33). Phenotype stimulatory effects (membrane bound and/or soluble) as well as the of the cultured cells was routinely performed using FITC-labeled anti-CD3, importance of CD40 triggering on DC, we performed cultures in anti-CD19 (Immunotech), and FITC-labeled IgG1 (Kallestad, Austin, TX) transwells. Naive B cells were seeded on the top of the transwells The Journal of Immunology 2225

Table I. IL-6 and IL-12 are involved in the DC-dependent IgM production by naive B cellsa

% of Inhibition

[3H]TdR uptake IgM production

Medium DC IL-2 ϩ DC SAC ϩ IL-2 Ab (n ϭ 6) (n ϭ 6) (n ϭ 6) (n ϭ 3)

4 Ϯ 413Ϯ 868Ϯ 868Ϯ 18 Anti-gp80 (0–14) (1–28) (60–87) (41–80) 5 Ϯ 54Ϯ 41Ϯ 1ND Anti-IL-10R (0–15) (0–11) (0–2) 5 Ϯ 54Ϯ 496Ϯ 25Ϯ 8 Anti-IL-12 (0–15) (0–16) (94–98) (0–18)

a Antibody to gp80 or IL-12 strongly inhibit DC-dependent naive B cell differentiation. Highly puriﬁed naive B cells (104) were cultured over 2.5 ϫ 103 irradiated CD40L L cells in various conditions: medium alone, DC (104), IL-2 ϩ DC, or SAC ϩ IL-2. Cultures were performed in presence of either isotype match control Abs or blocking Abs directed against gp80, IL-10R, or IL-12 as detailed in Materials and Methods. Effects of Abs on B cell proliferation and differentiation were analyzed in

six independent experiments (except for CD40L ϩ SAC ϩ IL-2, n ϭ 3). The effect Downloaded from of blocking Ab is expressed as percent inhibition of B cell proliferation (day 6) and IgM secretion (day 13) in presence of the corresponding control Ab.

Anti-IL-12 and anti-IL-6R␣ Abs inhibit the IL-2 and DC- dependent production of IgM http://www.jimmunol.org/ 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 FIGURE 1. CD40-activated DC enhance naive B cell proliferation and mAbs directed against IL-10R (or IL-10, not shown) ruled out the differentiation through the production of soluble factors. A, B, Highly pu- possible contribution of IL-10 in all the effects described in this by guest on September 29, 2021 4 ϩ rified naive B cells (10 ) (sIgD ) were cultured in medium alone or in study (Table I). Anti-gp80 mAb (IL-6R␣) was found to strongly presence of 2.5 ϫ 103 irradiated CD40L L cells with or without exogenous ϩ 4 ϩ inhibit the IL-2-dependent IgM production in the presence of DC IL-2. Irradiated CD1a DC (10 ), generated from cord blood CD34 pro- ϭ ϭ genitors, were used as stimulator cells, as detailed in Materials and Meth- (68% inhibition, r 60–87, n 6). Furthermore, Abs to IL-12 ods.[3H]TdR uptake was determined after 6 days of coculture. Cell-free completely blocked DC-induced IgM secretion by naive B cell in supernatants were recovered after 13 days, and IgM levels were determined presence of IL-2 (96% inhibition, r ϭ 94–98, n ϭ 6). Of note, by indirect ELISA. Results are expressed as mean Ϯ SD of triplicates (one none of the two Abs had significant effect on CD40-dependent experiment representative of four). C, sIgDϩ B cells (1.5 ϫ 104) were naive B cell proliferation in presence or absence of DC (Table I). cultured in the presence of 3.75 ϫ 103 irradiated CD40L L cells with (IgM The inhibition of IgM production by both Abs was dose dependent production) or without IL-2 (proliferation) in the top of the transwell. Ir- (Fig. 2, A and B) and reached a plateau at relatively low doses of 5 ϫ 4 radiated DC (10 ) were cultured in the lower compartment with 2.5 10 Abs (1 ␮g/ml for anti-gp80 and 5 ␮g/ml for anti-IL-12 Ab). Com- irradiated CD40L cells or CD32 L cells used as control. DNA synthesis of parable results were obtained with anti-IL-6 or anti-gp130 mAb B cells of the upper compartment was determined after 6 days of coculture. IgM production was analyzed by ELISA after 13 days and are expressed as and other anti-IL-12 Ab, including both monoclonal and poly- ␮g/ml for 104 seeded B cells. Cultures were conducted in triplicates (one clonal Ab (data not shown). As shown in Figure 2C, the Ab-de- experiment representative of four). pendent 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 together with CD40L L cells, and DC were cultured with CD40L affected by neutralization of IL-6R or IL-12 (data not shown), L cells or control CD32 L cells in the bottom. As previously re- suggesting that proliferation and differentiation of naive B cells ported (22), B cells needed to be activated through their CD40 to induced by IL-2 ϩ DC are regulated by similar mechanisms. An respond to DC stimulation (data not shown). DC, activated or not indirect effect of DC-derived IL-12 on eventually contaminating T through CD40, enhanced proliferation of CD40-activated B cells cells and NK cells was ruled out by the lack of CD3 and CD56 (Fig. 1C, fivefold enhancement of [3H]TdR uptake). In contrast, mRNA, as measured by RT-PCR and by the lack of effect of neu- the induction of IgM production, which requires the presence of tralizing anti-IFN-␥ Abs (data not shown). IL-2 (Fig. 1B), was strictly dependent on CD40 engagement on DC To determine whether the contribution of IL-6 and IL-12 in (Fig. 1C). Thus, DC enhance CD40-induced naive B cell prolif- naive B cell differentiation was restricted to the effects of DC, eration through the production of soluble molecules independently naive B cells were cultured in the presence of CD40L L cells, SAC of CD40 triggering. In contrast, the induction of IL-2-dependent particles, and IL-2 (a DC-independent system allowing significant naive B cell differentiation, which is also borne by soluble entities, IgM production). Blocking endogenous IL-6R signaling (Table I) requires CD40 engagement on the DC. led to a 68% inhibition of IgM secretion (r ϭ 41–80, n ϭ 3), a 2226 IL-12-DEPENDENT NAIVE B CELL DIFFERENTIATION

FIGURE 2. Anti-gp80 and anti-IL-12 Abs in- hibits naive B cell differentiation induced by DC ϩ IL-2 in a dose-dependent manner. Highly puri- ﬁed naive B lymphocytes were cultured over irradiated CD40L-transfected L cells in the presence of IL-2 and DC. Cell-free supernatants were recovered after 13 days, and IgM production was determined by ELISA. A, B, Increasing dose of anti-gp80 or anti-IL-12-blocking Abs, or their corresponding control Abs, were added at the beginning of the coculture. C, Exogenous IL-6 (200 ng/ ml) or IL-12 (10 ng/ml) was added with the highest concentration of blocking Abs (10 ␮g/ml) to reverse cytokine neutralization (one experiment representative of three). Downloaded from

finding in agreement with the autocrine B cell differentiation ac- modestly enhanced CD40-activated B cell proliferation (Fig. 3B), http://www.jimmunol.org/ tivity of IL-6 (37, 39). In contrast, anti-IL-12 Abs had no signif- suggesting that another DC product, as yet unidentified, could be icant effect on B cell differentiation in the absence of DC (Table I). involved in DC-dependent B cell proliferation. Taken together, these results demonstrate that DC promote a Altogether, these data suggest that DC-derived IL-12 synergizes naive B cell differentiation pathway that is dependent on IL-6 and with IL-6R signaling to induce naive B cell differentiation in pres- IL-12. ence of IL-2. Furthermore, these results imply that IL-2 directly acts on naive B cells and renders them responsive to DC factors. DC-derived IL-12 and sgp80 synergize with IL-2 to induce CD40-activated naive B cell to secrete IgM DC induce differentiation of naive B cells toward IgM plasma To determine the source of cytokines (e.g., B cells and/or DC) cells through the production of IL-12 by guest on September 29, 2021 involved in DC-dependent IgM production, we measured by The differentiation of B cells into plasma cells is characterized by ELISA the production of IL-6, sgp80, and IL-12/p70 in superna- the down-regulation of CD20 and up-regulation of CD38 (40, 41). tants of naive B cells and DC. Whereas naive B cells and DC The analysis of plasma cell formation in culture of CD40-activated produce comparable levels of IL-6 upon CD40 triggering, the pro- naive B cells with IL-2 in presence or absence of DC was per- duction of soluble IL-6R␣ (sgp80) and IL-12 appears more re- formed after 8 days using triple immunofluorescence staining. stricted to DC (Fig. 3A). Although, the production of sgp80 by DC Whereas naive B cells alone (gate on CD19ϩ cells) did not dif- did not require any activation, CD40 engagement led to enhanced ferentiate into CD20ϪCD38high cells (0.2%, Fig. 4A), addition of levels (Fig. 3A). Bioactive IL-12 (p70) was produced in low but DC induced significant plasma cell formation (9.3%, Fig. 4B). significant amounts by DC upon CD40 activation, whereas this Sorted CD20ϪCD382ϩ cells present characteristics of plasma cytokine was under the detection limit in B cell supernatants. The cells, e.g., oval cells, compact, dense, eccentric nuclei, with baso- secretion of these three molecules was not affected by the presence philic cytoplasm and strong intracytoplasmic anti-Ig staining (Fig. of IL-2 (data not shown). Because naive B cells and DC produce 4, C-E). Their spontaneous production of IgM upon reculture (data comparable levels of IL-6, it is likely that IL-12 and sgp80, which not shown) further confirm that these cells are IgM plasma cells. In are mainly produced by DC, are the key molecules contributing to contrast, sorted CD20ϩCD38ϩ cells are blastic cells with weak the effects of DC. intracytoplasmic anti-Ig staining (Fig. 4, F-H). Recombinant IL-6, sgp80, and IL-12/p70, were then tested, at We next wondered whether IL-6/sgp80 and IL-12 were involved optimal concentrations, for their ability to induce CD40-activated in the formation of plasma cells induced by IL-2ϩDC. Fig. 4, I-L, naive B cells to proliferate and secrete IgM. In the absence of IL-2, shows the effects of anti-gp80 and anti-IL-12 Abs on DC-induced none of the cytokines, used alone or in combinations, led to sig- plasma cell formation. Blocking IL-12 strongly impaired the de- nificant induction of IgM production by naive B cells (Fig. 3B). In velopment of plasma cells obtained after 8 days of coculture (85% combination with IL-2, sgp80 was totally inefficient in driving B inhibition). In contrast, Ab to gp80 only slightly affect the gener- cell differentiation, whereas IL-6 or IL-12 induced low but repro- ation of plasma cells (22% inhibition). A near complete inhibition ducible IgM production (0.31 Ϯ 0.04 and 0.77 Ϯ 0.06 ␮g/ml, was observed when both Abs were added together. In addition, DC respectively, n ϭ 5, background 0.08 ␮g/ml). The effect of IL-2 ϩ potentiate spontaneous IgM production by sorted IgM plasma cells IL-12 was strongly potentiated by either addition of sgp80 or IL-6 (data not shown). This effect on IgM secretion was blocked by (5 ␮g/ml of IgM compared with 15.1 ␮g/ml obtained with DC). anti-gp80 mAb by 83%, while anti-IL-12 Ab had no effect. The most potent effect was achieved by combining the three mol- Taken together these results suggest that IL-12 is mandatory for ecules (sgp80, IL6, and IL-12) together with IL-2, thereby recon- DC-induced differentiation of naive B cells into IgM plasma cells, stituting 30 to 150% of the IgM production (n ϭ 7) obtained in whereas IL-6/IL-6R signaling may intervene at a later stage of B presence of DC (Fig. 3B). The same combination of cytokines cell differentiation. The Journal of Immunology 2227 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. DC-derived IL-12 synergize with IL-2 and IL-6R signaling to drive naive B cell differentiation. A, Naive B cells or DC were cultured in presence of irradiated CD40L L cells or control L cells for 48 h as described in Materials and Methods, and supernatants were assessed for the presence of sgp80, IL-6, and IL-12 (one experiment representative of three). B, Highly puriﬁed naive B cells (104) were cultured over irradiated CD40L L cells in presence or absence of IL-2. rhsgp80, rhIL-6, rhIL-12 were tested alone or in combinations for their capacity to enhance CD40-induced B cell proliferation and differentiation. Levels obtained by addition of 104 DC are indicated for comparison. Thymidine uptake after 6 days and IgM production after 13 days are shown. Results are expressed as mean Ϯ SD of triplicates (one experiment representative of four).

Contribution of IL-12 and sgp80 in T cell-dependent naive B We next assessed the contribution of IL-6/sgp80 and IL-12 in cell differentiation this system. As shown in Figure 5B, in presence of T cells and All the experiments described above were performed using a ﬁ- SAg, DC-dependent IgM production by naive B cells was strongly broblastic cell line stably expressing CD40L, to mimic activated T impaired by blocking IL-6R signaling (45% inhibition) or IL-12 ␥ cells. To extend the relevance of the direct DC/B cell interaction, (73% inhibition). In addition, anti-IFN- Ab had no effect, sug- we analyzed the contribution of DC-derived IL-12 in coculture of gesting that the effect of IL-12 is independent of endogenous pro- ␥ B cells, T cells, and DC. Using irradiated allogeneic peripheral duction of IFN- . Furthermore, blocking IL-12 has no detectable blood CD4ϩ T cells, in place of CD40L L cells, did not allow effect on the level of T cell activation as measured by T cell pro- signiﬁcant IgM production even in the presence of DC (Fig. 5A). liferation (data not shown). Thus, in addition to activate T cells, This low rate of B cell help was likely due to a limited recruitment DC directly contribute to naive B cell proliferation and differen- of alloreactive T cells. To increase T cell activation, a combination tiation, in particular through the production of IL-12. of superantigens (SAg: SEA ϩ SEB) was added to cocultures. In this system, DC increased in a dose-dependent manner naive B cell Naive and memory B cell differentiation are differentially proliferation (data not shown) and subsequent differentiation (Fig. regulated by DC 5A). Comparable results were obtained using SAg-pulsed DC, In addition to inducing naive B cell differentiation in presence of showing that SAg primarily favors DC-dependent T cell activa- IL-2, DC strongly potentiate memory B cell differentiation in ab- tion. This system is IL-2- and CD40L-dependent, as demonstrated sence of exogenous cytokines (22). We thus wondered whether the by the near total inhibition of IgM production observed with block- molecules that contribute to IL-2-dependent naive B cell differen- ing anti-IL-2 or anti-CD40L Abs (Fig. 5A). tiation would also drive the differentiation of memory B cells. 2228 IL-12-DEPENDENT NAIVE B CELL DIFFERENTIATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Neutralization of IL-12 supress DC-induced differentiation of naive B cells toward IgM plasma cells. Naive B cells (105) were cultured over irradiated 2.5 ϫ 104 CD40L L cells in presence of IL-2, with or without 105 DC. After 8 days of culture, cells were harvested and labeled with anti-CD19-PEcy5, anti-CD20-FITC, and anti-CD38-PE. Analysis of CD20ϪCD38high B cells was achieved by gating on CD19ϩ cells. A, B, The effect of DC on naive B cell differentiation into IgM plasma cell is expressed as percentage of CD19ϩCD20ϪCD382ϩ cells obtained after 8 days of coculture. C-H, Sorted CD19ϩCD20ϩCD38ϩ (F, G, H) and CD19ϩCD20ϪCD382ϩ cells (C, D, E) were stained by MGG (C, F), anti-␬ ϩ anti-␭ (D, G) or anti-IgM Abs (E, H). I-L, Abs against gp80 (J), IL-12 (K), or both (L), or control Abs (I) were added at 10 ␮g/ml from the beginning of the culture of naive B cells and DC in presence of IL-2. CD20 and CD38 expression on B cells was analyzed as described above. (one experiment representative of three). Original magniﬁcations: MGG (ϫ400), anti-Ig stainings (ϫ1000).

Thus we have assessed the contribution of IL-6 and IL-12 in DC- shows that DC amplify the differentiation of both naive and mem- induced differentiation of naive/memory B cells. As previously ory B cells through the release of soluble IL-6R ␣-chain. shown (Table I), anti-gp80 or anti-IL-12 Abs strongly suppressed naive B cell differentiation induced by DC ϩ IL-2 (Fig. 6). In contrast, only anti-gp80 mAb significantly inhibited IgG produc- Contribution of IL-6/sgp80 to naive B cell differentiation tion by memory B cells in the presence of DC (Fig. 6). Thus, while The inhibition by Abs against gp80 of the DC and IL-2-dependent IL-6/sgp80 is involved in DC-induced differentiation of both naive IgM secretion of cultured naive B cells indicates that DC contrib- and memory B cells, IL-12 is uniquely involved in the DC-induced ute to B cell differentiation by modulating the IL-6/IL-6R signaling differentiation of naive B cells toward IgM-secreting plasma cells. pathway. Since CD40-activated naive B cells produce IL-6 (Ref. 42 and Fig. 3), which plays an important role in their differentia- Discussion tion (39, 43, 44), it is unlikely that IL-6 produced by DC is the We previously reported that DC, generated by culturing CD34ϩ critical molecule involved in the observed enhancement of B cell progenitors with GM-CSF ϩ TNF-␣, strongly enhance the prolif- differentiation. More likely, DC contribute to the IL-6-dependent B eration and differentiation of CD40-activated B cells (22, 23). The cell differentiation by secreting the soluble form of the gp80 IL-6R present study demonstrates that IL-12, produced by DC upon ␣-chain. This molecule has previously been shown to potentiate CD40 activation, is the key molecule involved in the differentia- the biologic activity of IL-6 (45, 46) by forming an IL-6/sgp80 tion of naive B cells into IgM-secreting plasma cells. The critical complex, which binds with high affinity to the ubiquitous and con- role of IL-12 in naive B cell differentiation has been confirmed in stitutive gp130-transducing chain (47, 48). Accordingly, a combi- coculture of T cells, B cells, and DC, adding further support to the nation of IL-12 and sgp80 can substitute for DC in the IL-2-de- relevance of the direct DC/B cell interaction. Our study further pendent naive B cell differentiation, thereby demonstrating the The Journal of Immunology 2229

FIGURE 6. Differential contribution of IL-6 and IL-12 in DC-dependent naive and memory B cell responses. The effects of anti-gp80 and anti-IL-12 blocking Abs were analyzed in 10 independent experiments in which 104 DC were cultured in presence of either 104 memory B cells and irradiated CD40L L cells (IgG production) or 104 naive B cells ϩ IL-2 ϩ CD40L L cells (IgM production). Blocking Abs or their corresponding control Abs were added at the beginning of the culture. Ig productions were determined after 13 days. The effect of blocking Ab is expressed as percent

inhibition of Ig secretion in presence of the corresponding control Ab. Downloaded from Results are presented as mean inhibition Ϯ SD observed in 10 independent experiments.

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 http://www.jimmunol.org/ FIGURE 5. Contribution of IL-6/sgp80, IFN-␥, and IL-12 in the differentiation of naive B cell induced by DC and T cells. A, Naive B cells (104) CD40 and IL-6R signaling. were cultured in presence of irradiated allogeneic CD4ϩ T cells (105) with It has been well established by many groups that IL-12 plays a increasing numbers of DC. Cultures were performed with or without SEA pivotal role in controlling cell-mediated immunity (57–59). Our ϩ SEB (10 ng/ml each) in presence of the indicated blocking Abs or ﬁndings suggest that B cells may represent an important cellular control Abs (10 ␮g/ml). In one culture condition, DC were pulsed with 10 target of IL-12, in addition to T cells and NK cells. Indeed, recent ng/ml of SEA ϩ SEB for 1 h before coculture with T and B cells. IgM studies performed in vivo in mouse have suggested it (60–62), production was determined after 13 days. B, The effects of blocking Abs though it could not be demonstrated whether the effects were di- ϩ were compared in the two systems of naive B cell differentiation: B rectly on B cells or indirect through the activation of other cell

ϩ ϩ ϩ ϩ ϩ by guest on September 29, 2021 CD40L L cells IL-2 DC (left part) and B T SAg DC (right types subsequently affecting B cells. In particular, IL-12 was found part). (one experiment representative of three). 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 critical role for DC-derived sgp80 in this process. Recently, Ka- in vitro and in vivo, together with its critical role in cell-mediated posi’s sarcoma-associated herpesvirus-infected DC have been im- immunity, suggests that this cytokine could constitute a potent plicated in multiple myeloma patients (49). Viral IL-6 was found vaccine adjuvant in situation that may require both arms of the to be transcribed in the myeloma bone marrow DC and has been immune system, such as HIV (63), malaria (64, 65), or certain proposed to play a role in the propagation of malignant plasma cancers. cells. The critical role of IL-6/IL-6R signaling in DC-dependent naive and memory B cell differentiation described herein adds fur- Differential regulation by DC of B cell proliferation, naive and ther support to this hypothesis. memory B cell differentiation While this study succeeded in deciphering the molecules involved Contribution of IL-12 to naive B cell differentiation in the DC-dependent naive B cell differentiation, the soluble me- The strong inhibitory effect of anti-IL-12 Abs on DC and IL-2- diators controlling B cell proliferation remain to be characterized. dependent IgM secretion suggests that IL-12 plays a critical role in Not only are B cell proliferation and naive B cell differentiation this biologic function. Accordingly, a combination of IL-12 with differentially regulated, but also memory B cell differentiation ap- IL-6 and sgp80 can substitute for DC in the IL-2-dependent dif- pears to be independently controlled, since it is unaffected by the ferentiation of naive B cells. The bioactive form of IL-12 is ex- presence of IL-12. Furthermore, DC appear to regulate naive B cell clusively produced by CD40-activated DC, while activated normal differentiation toward IgM-secreting cells and sIgA-expressing B cells do not secrete this cytokine (Ref. 50 and Fig. 4). The effects cells through different mechanisms, the former being strictly depen- of IL-12 on B cells described herein are in accordance with two dent on IL-12 and the latter being partially TGF-␤ dependent (23). previous reports showing that IL-12 enhanced proliferation and Ig Figure 7 summarizes the effects of DC on naive B cells in the secretion of SAC ϩ IL-2-activated peripheral blood B cells (51, context of DC/T cell/B cell interactions. Once activated by DC, 52). We further demonstrate that DC-derived IL-12 plays a critical Ag-specific T cells express CD40L and secrete IL-2. Following role in IgM plasma cell formation. The mechanism by which IL-12 cognate interaction with T cells, naive B cells engaged through induced naive B cell differentiation remains unknown. Of impor- their CD40, proliferate in response to an unidentified molecule tance, the present study indicates that the IL-12-dependent B cell (IL-X), whose production by DC does not require CD40 trigger- differentiation requires IL-2 signaling on B cells, in line with other ing. Furthermore, in presence of T cell-derived IL-2, IL-12 then reports (51, 52). The ability of IL-12 to synergize with IL-2 has stimulates CD40-activated naive B cells to differentiate into IgM- also been reported during T cell and NK cell activation (53, 54). secreting plasma cells, whereas sgp80 may regulate a later stage of 2230 IL-12-DEPENDENT NAIVE B CELL DIFFERENTIATION

FIGURE 7. A model for DC-induced differentiation of naive B cells into IgM plasma cells: role of IL-12 and IL-6/gp80. Results obtained in vitro using Downloaded from irradiated CD40L transfected L cells as surrogate activated T cells or purified CD4ϩ T cells are replaced in the context of the triad of DC, Ag-specific T cell and B cell. Ag-bearing DC activate T cells through their TCR and CD28, leading to expression of CD40L and secretion of IL-2. In return, activated T cells stimulate both B cells and DC. CD40 engagement on B cells induce their proliferation, which is strongly enhanced by IL-X, a DC-derived molecule, as yet not identified, which does not require CD40 stimulation. In addition, activated T cells induce DC to produce IL-12/p70 and up-regulate sgp80 secretion by engaging CD40. In presence of IL-2, IL-12 and sgp80 induce naive B cell differentiation into IgM plasma cells. IL-12 plays a critical role in DC-induced IgM plasma cell formation whereas sgp80/IL-6 may act at a later stage of B cell differentiation. http://www.jimmunol.org/

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