IL-12 Up-Regulates CD40 Ligand (CD154) Expression on Human T Cells Xiaohui Peng, Jacques E

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IL-12 Up-Regulates CD40 Ligand (CD154) Expression on Human T Cells Xiaohui Peng, Jacques E. Remacle, Ahmad Kasran, Danny Huylebroeck and Jan L. Ceuppens This information is current as of September 27, 2021. J Immunol 1998; 160:1166-1172; ; http://www.jimmunol.org/content/160/3/1166

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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. IL-12 Up-Regulates CD40 Ligand (CD154) Expression on Human T Cells1

Xiaohui Peng,* Jacques E. Remacle,† Ahmad Kasran,* Danny Huylebroeck,† and Jan L. Ceuppens2*

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4؉ Th1 cells and their IFN-␥ production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA Downloaded from level. T cells costimulated by IL-12 provided more efﬁcient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction. The Journal of Immunology, 1998, 160: 1166–1172.

he CD40 ligand (CD40L3 or CD154), also called gp39, is ated through IFN-␥. However, antimicrobial activity of IL-12 in a type II transmembrane protein with sequence homology experimental visceral leishmaniasis has also been demonstrated in http://www.jimmunol.org/ T to TNF-␣ and is transiently expressed on activated T cells IFN-␥ gene knockout mice (25). Moreover, IL-12 has been found (1, 2). Its receptor, CD40, is a 45- to 50-kDa glycoprotein, member to inﬂuence IgG production, especially IgG1 and IgG2b (26–31). of the TNF receptor superfamily (1), and is expressed on B cells, These data indicate that IL-12 has effects on macrophages and B monocytes, dendritic cells (DC), and thymic epithelial cells (3–5). cells that are not mediated through IFN-␥. In view of the impor- The CD40L-CD40 interaction was ﬁrst shown to be critical for Th tance of the CD40L-CD40 interaction for humoral immunity, and cell-dependent Ab responses (6–9), as also evidenced by the fail- for antimicrobial activity of macrophages, we considered CD40L ure of isotype switching in humans and mice with a mutated expression as a potential alternative target of IL-12 activity. We

CD40L (10, 11). The CD40L-CD40 interaction induces production here report that IL-12 indeed enhances functional CD40L expres- by guest on September 27, 2021 of proinflammatory cytokines (TNF-␣ and IL-1␤) as well as ge- sion on T cells. latinase production by monocytes and macrophages (12, 13) and is required for macrophage tumoricidal activity and microbicidal ac- Materials and Methods tivity (14). Furthermore, CD40L-CD40 interaction is important for Isolation of peripheral blood T cells up-regulation of APC functions, including up-regulation of B7 expression and induction of IL-12 production (15–19). IL-12 is a All subjects donating blood for this study were healthy volunteers of both heterodimer cytokine comprised of a 40-kDa (p40) and a 35-kDa sexes, aged 20 to 50 yr. PBMC were isolated from heparinized venous blood on Ficoll-Hypaque density gradients. After washing, the cells were (p35) subunit that are linked by a disulfide bond. IL-12 is produced resuspended at a concentration of 5 ϫ 106 cells/ml in complete culture by macrophages, B cells, and DC and exerts multiple effects on medium consisting of RPMI 1640 (Boehringer Ingelheim, Heidelberg, NK cells and T cells (20). As its main activity, IL-12 promotes the Germany) supplemented with 2 mM L-glutamine, penicillin (100 U/ml), differentiation of naive CD4ϩ T cells into Th1 effector cells (21– streptomycin (100 ␮g/ml), and 10% iron-supplemented bovine calf serum 23) and induces IFN-␥ production by T cells and NK cells in (HyClone, Logan, UT). Monocytes were removed by cold agglutination by rotating tubes for 30 min at 4°C. The agglutinated monocytes were sedi- synergism with IL-2, mitogens, phorbol diester, anti-CD3 mAb, or mented out, and the lymphocytes were further purified using Lympho- alloantigens (24). Most in vivo effects of IL-12 seem to be medi- kwik-T (One Lambda, Inc., Los Angeles, CA) supplemented with complement-fixing anti-NK (anti-Leu 11b (anti-CD16), anti-Leu 7 (anti- CD57), and anti-NKH-1A (anti-CD56)) mAb as previously reported (32). The resulting T cell preparations contained Ͼ99% CD3ϩ and Ͻ1% CD16ϩ *Laboratory of Experimental Immunology, Department of Pathophysiology, Faculty ϩ ϩ of Medicine, Catholic University of Leuven; and †Laboratory of Cell Growth, Dif- cells; CD14 monocytes and HLA-DR cells were not detected. These ferentiation, and Development, Flanders Interuniversity Institute for Biotechnology, cells could not be activated by PHA or rIL-2 alone (10 U/ml). Catholic University of Leuven, Belgium Received for publication June 16, 1997. Accepted for publication October 17, 1997. Cell lines The costs of publication of this article were defrayed in part by the payment of page The P815 cell line is an NK-resistant DBA/2 derived murine mastocytoma charges. This article must therefore be hereby marked advertisement in accordance cell line that expresses mouse Fc␥RII and Fc␥RIII. The P815/CD80 cell with 18 U.S.C. Section 1734 solely to indicate this fact. line, transfected with human CD80 cDNA, was a gift from L. L. Lanier 1 This work was supported by Grant G0169.96 from the Fonds voor Wetenschappelijk (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, Onderzoek (Brussels, Belgium) and Grant OT93/32 from the Research Council of the CA) (33). The 3T6 mouse fibroblast cell line, transfected with CD40L, was Catholic University of Leuven. a gift from K. Thielemans (Free University of Brussels, Brussels, Bel- 2 Address correspondence and reprint requests to Dr. Jan L. Ceuppens, Laboratory of gium). The P815 cells and the 3T6 cells were cultured in complete culture Experimental Immunology, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Bel- medium supplemented with gentamicin (50 ␮g/ml), sodium pyruvate (1 gium. E-mail address: [email protected] mM/ml), nonessential amino acids (1/100), 50 ␮M 2-ME, and 10% FCS 3 Abbreviations used in this paper: CD40L, CD40 ligand; DC, dendritic cells. (Gibco, Paisley, Scotland). Geniticin (1 mg/ml) was added every 1 to 2 wk

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 1167 for selecting transfected cells. The cells were given fresh medium every 2 Table I. rIL-12 up-regulates CD40L expression on anti-CD3-activated or 3 days. T cells

Isolation of tonsillar B and T lymphocytes Culture Additiona Mean Fluorescence Intensityb Fresh tonsils were obtained from children undergoing tonsillectomies. The Resting T Ͻ10 tissue was dispersed by cutting with scalpel blades. Fragments were al- Anti-CD3 97 Ϯ 34 * lowed to pass through a filter, and cell suspensions were collected. B cells Anti-CD3 ϩ rIL-12 193 Ϯ 39 ] were prepared by two cycles of rosetting with AET-treated SRBC and a Purified T cells (5 ϫ 105/ml) were stimulated with immobilized anti-CD3 mAb removal of E-rosetting cells on Ficoll-Hypaque density gradients. T cells OKT3 in the presence or absence of rIL-12 (1 ng/ml). were prepared by two cycles of Lymphokwik-T (One Lambda Inc., Canoga b ϩ After 40 h, cells were incubated with either anti-CD40L-FITC or isotype Park, CA). The resultant B cell preparations contained 95% CD20 cells, matched mouse IgG and analyzed on a FACSort with the Lysis II software (Becton ϩ and the tonsillar T cell preparations contained 97% CD3 cells as deter- Dickinson). The results are expressed as mean of mean fluorescence intensity (MFI) mined by FACS analysis. Ϯ SEM from six independent experiments. * p Ͻ 0.05.

Cytokines and mAbs Analysis of Th cell activity for B cell proliferation and IgG production Recombinant IL-12 was a gift from Genetics Institute (Cambridge, MA). Recombinant IL-4 was obtained from Innogenetics (Gent, Belgium). Anti- Tonsillar B cells (1 ϫ 105/well) and tonsillar T cells (irradiated, 3000 rad, CD3 mAb UCHT-1 (IgG1) was a gift from Dr. P. Beverley, Imperial Re- 105/well) were cultured with soluble anti-CD3 mAb UCHT-1 (1 ␮g/ml) search Cancer Fund (London, U.K.). The clones producing anti-CD3 mAb and mitomycin C-treated P815 mouse mastocytoma cells (1 ϫ 106/ml),

OKT3 and anti-CD11b OKM1 were obtained from American Type Culture either parental or CD80 transfected, in the presence or the absence of Downloaded from Collection (Rockville, MD). Anti-CD28 mAb 9.3 was a gift from C. June rIL-12 (1 ng/ml) in 96-well flat-bottom microculture plates. Recombinant (Naval Medical Research Institute, Bethesda, MD). Anti-CD2 mAb 9.6 IL-4 (100 ng/ml) was added to all cell cultures. Anti-IL-2 (2 ␮g/ml) and (IgG2a) was obtained from Oncogen (Seattle, WA), and mAb 9–1 was anti-IL-2R mAbs (1 ␮g/ml for each) were also added to block the effects purchased from XOMA Corp. (Berkeley, CA). mAb 9.6 blocks E rosette of endogenous IL-2 on CD40L expression and B cell proliferation. The formation and is directed against the CD58-binding epitope. The mAb 9-1 final volume of each cell culture was 200 ␮l. After 5 days, cells were detects a CD2 neo-epitope on activated T cells. Humanized anti-Tac mAb pulsed with 1 ␮Ci of [3H]thymidine. Eight hours later, cells were har- (directed at the p55 chain of the human lymphocyte IL-2R (CD25)) and vested, and thymidine incorporation was measured using a beta counter. humanized Mik␤1 (a mAb to the p75 chain of the IL-2R (CD122)) were Unseparated tonsillar mononuclear cells (containing B and T cells) (1 ϫ http://www.jimmunol.org/ gifts from Dr. J. Hakimi (Hoffmann-La Roche, Nutley, NJ). A neutralizing 106/ml) were also cultured with soluble anti-CD3 mAb UCHT-1 (1 ␮g/ml) anti-IL-2 mAb (B-G5) was purchased from Innotest (Besançon, France). and mitomycin C-treated P815 mouse mastocytoma cells (1ϫ106/ml), ei- Anti-IL-10R mAb (clone 37607.11) was purchased from R&D Systems ther parental or CD80 transfected, in the presence or the absence of rIL-12 (Minneapolis, MN). A neutralizing anti-IFN-␥ mAb (D9D10) was a gift (1 ng/ml) in 5-ml snap-cap Falcon tubes (Becton Dickinson, Lincoln Park, from Dr. M. de Ley (Department of Biochemistry, Catholic University of NJ) at 37°C in 5% CO2. The final volume of each cell culture was 1 ml. Leuven, Leuven, Belgium). Anti-CD40L mAb M90 (IgG1) (34) was a gift After 7 days, cells were spun down, and cell-free supernatant was har- from W. Fanslow (Immunex Corp., Seattle, CA). Anti-CD40L-FITC (clone vested. The concentrations of human IgG were estimated by ELISA as 24-31) was obtained from Ancell Corp. (Bayport, MN). previously described (9). Statistical analysis Cell culture conditions and FACS analysis for CD40L by guest on September 27, 2021 Statistical analysis was performed with the Wilcoxon test for paired sam- expression ples. p Ͻ 0.05 was considered significant. For stimulation with immobilized anti-CD3, wells were first coated with OKT3 (final concentration, 5 ␮g/ml) in 300 ␮lofPBSfor4hat37°C and Results then washed three times with PBS. Purified peripheral blood T cells (5 ϫ IL-12 up-regulates CD40L expression on anti-CD3-activated T 5 10 ) were cultured either on OKT3-coated plates or with anti-CD3 mAb cells UCHT-1 (1 ␮g) bound to Fc␥R on mitomycin C-treated P815 cells (1 ϫ 106). All cultures were set up in a 1-ml volume in the presence or the Different concentrations of rIL-12 were added to cultures of puri- absence of rIL-12 (1 ng unless otherwise indicated) in 24-well culture fied T cells stimulated with immobilized anti-CD3 mAb OKT3, ϫ 6 plates. CD80 (transfected into P815 cells; 1 10 /ml), soluble anti-CD28 and cells were then stained for CD40L expression. CD40L was not mAb 9.3 (0.5 ␮g/ml), or a pair of anti-CD2 mAbs (9-1 (2 ␮g/ml) and 9.6 (2 ␮g/ml)) were included in the above cultures to provide a second signal. detected on resting T cells or T cells incubated with rIL-12 alone The culture plates were incubated in a 37°C, humidified atmosphere of 5% (data not shown). The expression of CD40L was weakly induced

CO2. After 16, 40, and 64 h of incubation, the cell pellets were stained with by anti-CD3 stimulation and significantly enhanced by rIL-12 (Ta- either anti-CD40L-FITC mAb or isotype-matched negative control Ab for ble I). Figure 1 shows that increasing doses of rIL-12 progressively 30 min at 4°C. Following two washes with PBS, the cells were fixed in 1% enhanced the expression of CD40L on anti-CD3-activated T cells. paraformaldehyde in saline and analyzed on a FACSort flow cytometer (Becton Dickinson, Mountain View, CA). The optimal concentration of rIL-12 was 1 ng/ml. The highest CD40L expression on anti-CD3-activated T cells in the presence RNA isolation and reverse transcription-PCR of rIL-12 occurred after a 40-h cell culture, with a decline of CD40L expression thereafter (Fig. 2). T cell cultures were harvested 4 or 16 h after the initial stimulation as To study whether IL-12 interacts with other helper signals, pu- described above. Approximately 5 ϫ 106 stimulated T cells were pelleted and directly used for RNA isolation. Total RNA was isolated by the gua- rified T cells were cultured with immobilized anti-CD3 mAb nidium isothiocyanate method (35) and quantified spectrophotometrically. OKT3 as the primary signal and with soluble anti-CD28 mAb or a cDNA was prepared from 2.5 ␮g of total RNA using the SuperScript re- pair of anti-CD2 mAbs (9-1 and 9.6) as helper signals. As shown verse transcriptase preamplification system (Life Technologies, Grand Is- in Figure 2, addition of anti-CD28 mAb or anti-CD2 mAbs en- land, NY) with oligo(dT) primer. The cDNA samples were then subjected to PCR analysis using the following primers: 5Ј-ACATACAACCAAACT hanced CD40L expression, but rIL-12 still further augmented and TCTCCC-3Ј and 5Ј-AGATGTTGTTTTACTGCTGGC-3Ј for detection of prolonged the expression of CD40L, again with maximal CD40L CD40L, and 5Ј-GCTCACCATGGATGATGATATCGCC-3Ј and 5Ј- expression after 40 h of culture. GGATGCCTCTCTTGCTCTGGGCCTC-3Ј for detectin of ␤-actin as a cDNA quality control. Samples were amplified by Goldstar Taq polymer- IL-12 enhances CD40L expression on human T cells when ase (Eurogentec, Seraing, Belgium) by 24 cycles of denaturation at 94°C costimulated with CD80 for 30 s, annealing at 60°C (CD40L) or at 56°C (␤-actin) for 30 s, and extension at 72°C for 1 min. PCR products were analyzed by electrophore- In previous studies we showed that the binding of CD28 on T cells sis on 1% agarose gels and visualized by ethidium bromide staining. to its natural ligand CD80 on APC in the presence of anti-CD3 1168 CD40L IS INDUCED BY IL-12

strongly enhanced CD40L expression, thus pointing to a synergy between CD80 and IL-12 for induction of CD40L.

CD40L expression is regulated through IL-2-dependent and IL-2-independent mechanisms Since IL-12 induces IFN-␥ and IL-10 production by T cells (24, 36), we asked whether CD40L induction by IL-12 might be mediated indirectly through these other cytokines. To this end, a blocking mAb against the IL-10R or a neutralizing anti-IFN-␥ mAb was added to the cultures. Neither anti-IL-10R nor anti- IFN-␥ mAb affected the induction of CD40L expression by IL-12 (data not shown). Since endogenous IL-2 plays an important role in the induction of CD40L on T cells (37), we also evaluated FIGURE 1. Recombinant IL-12 up-regulates CD40L expression on anti- whether IL-12 requires the presence of IL-2 to enhance CD40L. T 5 CD3-activated T cells. Puriﬁed T cells (5 ϫ 10 /ml) from two different cells were stimulated with soluble anti-CD3 mAb cross-linked by donors were stimulated with immobilized anti-CD3 mAb OKT3 and dif- either parental P815 cell FcR or CD80-transfected P815 cells. To ferent concentrations of rIL-12 (0.12–2 ng/ml). After 40 h, cells were parallel cultures, a neutralizing anti-IL-2 mAb and two different stained with either anti-CD40L-FITC or isotype-matched mouse IgG and

anti-IL-2R (anti-Tac and Mik␤1) mAbs were added to block all Downloaded from analyzed on a FACSort (Becton Dickinson) with LYSIS II software (Bec- ton Dickinson). CD40L expression is expressed as the mean fluorescence IL-2 activity (38). As shown in Figure 3, anti-IL-2 mAb and anti- intensity (MFI). IL-2R mAbs strongly, but not completely, reduced CD40L expression on T cells cultured with either anti-CD3 mAb plus CD80- transfected cells. Importantly, rIL-12 still enhanced CD40L mAb enhances and prolongs CD40L expression (9). We therefore expression when IL-2 activity was blocked, indicating that CD40L tested whether CD80 and IL-12 would synergize in inducing induction by rIL-12 is IL-2 independent. Together, the data indi- http://www.jimmunol.org/ CD40L. To this end, we used mouse mastocytoma P815 cells cate that both IL-2-dependent and IL-2-independent signals (CD80 transfected with CD80 and coated with anti-CD3 (via FcR␥ bind- and IL-12) are involved in CD40L regulation. ing) to stimulate T cells. As shown in Figure 3 and Table II, up- regulation of CD40L induction by CD80 was confirmed. IL-12 did Analysis of CD40L mRNA expression in purified human T cells not influence CD40L expression when T cells were stimulated costimulated by IL-12 with anti-CD3 on P815 cells. When, however, T cells were cul- Next, we studied the effect of IL-12 on the expression of CD40L tured with CD80-transfected P815 cells and anti-CD3 mAb, rIL-12 mRNA in activated T cells. T cells were incubated for 4 or 16 h by guest on September 27, 2021

FIGURE 2. Recombinant IL-12 enhances CD40L expression on anti-CD3-activated T cells costimulated with anti-CD28 mAb or anti-CD2 mAbs. T cells (5 ϫ 105/ml) were stimulated with immobilized anti-CD3 mAb OKT3 in the presence or the absence of rIL-12 (1 ng/ml) for different time periods as indicated. Soluble anti-CD28 mAb 9.3 (0.5 ␮g/ml) or anti-CD2 mAbs (9-1 and 9.6; 2 ␮g/ml of each) were added to provide an accessory signal. After culture, these cells were stained with either anti-CD40L-FITC or isotype-matched mouse IgG and analyzed on a FACSort. Cell number is on the y-axis, and ﬂuorescence intensity in arbitrary units on a log scale is on the x-axis. The number above each histogram represents the mean ﬂuorescence intensity (MFI) of the anti-CD40L-stained cells. The MFI of isotype control in each histogram is Ͻ20. Histograms are from a single experiment and are representative of two experiments. The Journal of Immunology 1169

FIGURE 3. Recombinant IL-12 up-regulates CD40L expression through an IL-2-independent mechanism. T cells (5 ϫ 105/ml) were stimulated with anti-CD3 mAb UCHT-1 (1 ␮g/ml) presented on mitomycin C-treated P815 cells or CD80-transfected P815 cells (1 ϫ 106/ml) in the presence or the absence

of rIL-12 (1 ng/ml). A neutralizing anti-IL-2 mAb (2 ␮g/ml) in combination with anti-IL-2R mAbs (anti-Tac and Mik␤1; 1 ␮g/ml for each) were added Downloaded from from the beginning to some cultures as indicated. After 40 h, cells were stained with either anti-CD40L-FITC or isotype-matched mouse IgG and analyzed on a FACScan. Cell number is on the y-axis, and ﬂuorescence intensity in arbitrary units on a log scale is on the x-axis. The number above each histogram represents the mean ﬂuorescence intensity (MFI) of the anti-CD40L-stained cells. The MFI of the isotype control in each histogram is Ͻ25. Histograms are from a single experiment and are representative of three experiments with similar results.

3 with mitomycin C-treated wild-type P815 cells or CD80-trans- liferation was measured by [ H]thymidine incorporation. As http://www.jimmunol.org/ fected P815 cells in the presence or the absence of rIL-12. Soluble shown in Figure 5, no significant B cell proliferation was found in anti-CD3 mAb was added as a primary signal. Total RNA was the cultures where B cells were cocultured with T cells stimulated isolated, and reverse transcription-PCR reactions using specific with anti-CD3 mAb and P815 cells. However, B cell proliferation primers for CD40L and ␤-actin were performed. As shown in Fig- was induced in the presence of CD80-costimulated T cells, and this ure 4, no CD40L mRNA was detected in T cells cultured with was further enhanced by rIL-12. When anti-CD40L mAb was P815 cells alone (lane 5) or in P815 cells and anti-CD3 mAb in the added, B cell proliferations were completely blocked. A control presence (lane 7) or the absence (lanes 2 and 6) of rIL-12 in either mAb did not show any effect on B cell proliferation. In the exper- 4- or 16-h cell cultures. However, CD80-transfected P815 cells iments shown in Figure 5, IL-2 activity was blocked with mAb to induced early expression (at 4 h) of CD40L mRNA (lane 3)in IL-2 and to IL-2R ␣- and ␤-chains, because IL-2 also strongly by guest on September 27, 2021 anti-CD3 activated T cells. Interestingly, the addition of rIL-12 did influences CD40L expression. However, similar data were ob- not enhance CD40L mRNA at4hofstimulation (lane 4), but tained when IL-2 activity was not blocked (data not shown). To clearly enhanced CD40L mRNA expression at 16 h of stimulation exclude a direct effect of IL-12 on B cells, B cells were cultured (lane 9). Thus, IL-12 seems to have a late effect on with 3T6 mouse fibroblasts transfected with CD40L and exoge- CD40L mRNA. nous rIL-4 in the presence or the absence of rIL-12. Recombinant IL-12 did not have any effect on B cell proliferation in this system T cells costimulated by IL-12 provide more efficient help for (data not shown). Thus, the effect of IL-12 on B cell proliferation B cell proliferation and IgG production via an effect on CD40L-CD40 interaction We studied the functional relevance of IL-12-induced CD40L expression by analyzing the effect of IL-12 on T cell-dependent B cell proliferation and IgG production. This Th cell activity has been shown to be CD40L dependent (7, 8). Tonsillar B cells were cultured with rIL-4 and irradiated autologous T cells. B cell pro-

Table II. rIL-12 enhances CD40L expression on T cells costimulated by CD80 FIGURE 4. Recombinant IL-12 enhances CD40L mRNA expression in T cells stimulated by anti-CD3 and CD80. Purified human T cells (1 ϫ 6 Culture Addition a Mean Fluorescence Intensity b 10 /ml) were cultured for 4 (lanes 2–4)or16h(lanes 5–9) before mRNA extraction. Cultures were performed with mitomycin C-treated P815 cells Ͻ Resting T 10 and anti-CD3 mAb in the absence (lanes 2 and 6) or the presence (lane 7) P815 ϩ anti-CD3 30 Ϯ 3 P815 ϩ anti-CD3 ϩ rIL-12 35 Ϯ 7 of rIL-12 (1 ng/ml) or with CD80-transfected P815 cells and anti-CD3 P815/CD80 ϩ anti-CD3 187 Ϯ 35 mAb in the absence (lanes 3 and 8) or presence (lanes 4 and 9) of rIL-12 P815/CD80 ϩ anti-CD3 ϩ rIL-12 370 Ϯ 61 ]* (1 ng/ml). Lane 5 represents the T cells (1 ϫ 106/ml) that were cultured with mitomycin C-treated P815 cell alone (without anti-CD3 or rIL-12) for a T cells (5 ϫ 105/ml) were cultured with mitomycin C-treated P815 cells or CD80-transfected P815 cells in the presence or absence of rIL-12 (1 ng/ml) in flat- 16 h. Total RNA was isolated by the guanidium isothiocyanate method and bottom 24-well culture plates. Anti-CD3 mAb UCHT-1 was added at a concentration converted to cDNA. The cDNA was used as template for PCR using of 1 ␮g/ml. CD40L primers and as a control ␤-actin primers. The CD40L and ␤-actin b After 40 h, cells were incubated with either anti-CD40L-FITC or isotype- PCR products were verified after gel electrophoresis and ethidium bromide matched mouse IgG and analyzed on a FACSort. The results are expressed as mean of mean fluorescence intensity (MFI) Ϯ SEM from six independent experiments. staining. The top panel represents the CD40L PCR products, and the bot- * p Ͻ 0.05. tom panel represents the ␤-actin PCR products. 1170 CD40L IS INDUCED BY IL-12

FIGURE 6. Costimulation of T cells with rIL-12 results in more efﬁ- cient T cell help for IgG production. Tonsillar mononuclear cells (1 ϫ 106/ml) were cultured with mitomycin C-treated P815 cells (1 ϫ 106/ml) alone or with anti-CD3 mAb in the presence or the absence of rIL-12 (1 ng/ml). The anti-CD40L mAb M90 or the control mAb OKM1 (both at 10 ␮

g/ml) was added as indicated. After 7 days, cell-free supernatant was Downloaded from harvested, and the concentrations of human IgG were measured by ELISA. The results are expressed as the mean Ϯ SEM from ﬁve independent experiments. * indicates p Ͻ 0.05.

Requirements for optimal CD40L induction on T cells are in- completely understood. CD40L is not expressed on resting T cells. http://www.jimmunol.org/ CD3-TCR triggering or ionomycin alone is sufficient to induce some CD40L expression (6, 39, 40). CD40L can be up-regulated by PMA (39, 40), and endogenous IL-2 production was found to be important for the expression of CD40L (37). Moreover, we FIGURE 5. Costimulation of T cells with rIL-12 results in more efficient T cell help for IL-4-dependent B cell proliferation. Purified tonsillar have previously reported that ligation of B7 with CD28 strongly B cells and T cells (irradiated) from two different donors were cultured up-regulates and prolongs CD40L expression on anti- with mitomycin C-treated P815 cells alone or with anti-CD3 mAb in the CD3-activated T cells (9). Roy et al. reported that Ag-induced

presence or the absence of rIL-12 (1 ng/ml). Recombinant IL-4 (100 ng/ml) expression of CD40L on TCR transgenic T cells was inhibited by by guest on September 27, 2021 and anti-IL-2 mAb together with anti-IL-2R mAbs (to block IL-2 activity) Abs to class II MHC, CD4, and LFA-1, but not by CTLA-4 Ig, were added to all cell cultures. The anti-CD40L mAb M90 or the control anti-B7-1, or anti-B7-2 (41). Ding et al. reported that induction of mAb OKM1 (both at 10 ␮g/ml) was added as indicated. After 5 days, cells CD40L is only partially inhibited by CTLA-4 Ig, and that CD40L ␮ 3 ϩ were pulsed with 1 Ci of [ H]thymidine. Eight hours later, cells were could be induced on CD4 T cells from CD28-deficient mice (42). harvested, and thymidine incorporation in B lymphocytes was measured Thus, B7-independent costimulatory mechanisms are also in- using a beta counter. volved in enhancing the expression of CD40L. IL-12, as shown in this study, is such a helper signal that up- regulates CD40L expression. Although IL-12 greatly induces most likely proceeds via modulation of the interaction of CD40L IFN-␥ and IL-10 production (24, 36), it is unlikely that induction on anti-CD3 activated T cells and of CD40 on B cells. of CD40L by IL-12 is an indirect effect of IFN-␥ or IL-10, since To study effects of IL-12 on Ig production by B cells, we cul- a neutralizing anti-IFN-␥ mAb or anti-IL-10R mAb did not block tured tonsillar mononuclear cells with soluble anti-CD3 mAb and the effect of IL-12 on CD40L expression. Moreover, exogenous mitomycin C-treated P815 cells, either parental or CD80 trans- rIL-10 inhibited CD40L expression (our unpublished observa- fected, in the presence or the absence of rIL-12. As shown in tions), and IFN-␥ has been shown by others to inhibit the expres- Figure 6, large amounts of IgG were produced when T cells were sion of CD40L in mice (40). The inhibition of IFN-␥ on CD40L stimulated with anti-CD3 mAb presented on P815/CD80 cells. In- expression was not confirmed in the human system (our unpub- terestingly, the addition of rIL-12 further enhanced IgG produc- lished observations). Although endogenous IL-2 plays an impor- tion. Blocking experiments with anti-CD40L mAb resulted in par- tant role in the induction of CD40L (37), it is not likely that in- tial inhibition of IgG production by B cells. With relevance to the duction of CD40L by IL-12 is mediated through IL-2. First, our effects of IL-12, anti-CD40L inhibited IgG production in the pres- data (43) as well as other reports (44) showed that IL-12 does not ence of rIL-12 to a similar extent as in the absence of IL-12. affect IL-2 production. Second, IL-12 still enhanced CD40L expression in the presence of a neutralizing anti-IL-2 mAb and anti- Discussion IL-2R mAbs that have previously been shown to block all IL-2 In the present study we have shown that IL-12 up-regulates the activity (38). On the other hand, it is clear from these experiments expression of CD40L on anti-CD3-stimulated T cells at both the that IL-12 synergizes with CD80 and IL-2 in inducing CD40L. protein and the mRNA level. This effect is most clear when T cells Therefore, optimal induction of CD40L occurs when IL-12, CD80, are costimulated with CD80. Functional studies confirmed that co- and IL-2 are all present to signal the T cells. stimulation of T cells with IL-12 results in more efficient help for The signal transduction mechanism by which IL-12 induces B cell growth and IgG production, and that this helper activity is CD40L requires further investigation. It is also not clear yet dependent on the interaction of CD40L on T cells and of CD40 on whether IL-12 has an effect on the transcription of the CD40L gene B cells. or on CD40L mRNA stability in activated T cells. IL-12 is the only The Journal of Immunology 1171 cytokine identified to date that can induce phosphorylation of formed to study whether the IFN-␥-independent effect is partly STAT4 (45, 46). In STAT4-deficient mice, all IL-12 functions mediated by CD40L. Although the biologic significance of our tested were disrupted, including the induction of IFN-␥, mitogen- findings for APC physiology requires further investigation in esis, enhancement of NK cytolytic function, and Th1 differentia- IFN-␥ knockout mice or IFN-␥R knockout mice, the induction of tion (47, 48). These results demonstrate that STAT4 is essential for CD40L by IL-12 is probably part of an important regulatory circuit mediating responses to IL-12. It will therefore be of major interest between APC and T cells. B7 and IL-12 both enhance CD40L to study binding of phosphorylated STAT4 to the promotor region expression on activated T cells and their IFN-␥ production in a of the CD40L gene. synergistic way (49). CD40L and IFN-␥, in turn, directly stimulate Another interesting point in our experimental results is the syn- IL-12 production by macrophages and DC (17–19), and they en- ergy between CD80 and IL-12 in inducing CD40L. Synergistic hance the expression of costimulatory molecules such as B7-1, effects between both helper signals have also been found for IFN-␥ B7-2, and intercellular adhesion molecule-1 on the APC (5, 15, and IL-10 production (43, 49). IL-12R chains are expressed by 16). Finally, a signal delivered to T cells via CD40L triggering can activated T and NK cells. Two IL-12R chains, termed ␤1 and ␤2, up-regulate T cell activation and synergize with IL-12 to further have been identified to date. The IL-12R ␤1 subunit is expressed induce IFN-␥ production (55). in both Th1 and Th2 cells, while the IL-12R ␤2 subunit is selec- tively lacking in Th2 cells (50, 51). Recent reports showed that Acknowledgments anti-CD28 mAb and IL-2 augment the expression of IL-12R ␤1 We thank P. Beverley (Imperial Research Cancer Found, London, U.K.), and induce high affinity IL-12 binding (which correlates with ␤2- W. Fanslow (Immunex Corp., Seattle, CA), J. Hakimi (Hoffmann-La Downloaded from chain expression) to anti-CD3-activated T cells (52). Lack of high Roche, Nutley, NJ), C. June (Naval Medical Research Institute, Bethesda, affinity IL-12 binding can explain why IL-12 had no effect on MD), L. L. Lanier (DNAX Research Institute of Molecular and Cellular CD40L expression when T cells were stimulated with P815 plus Biology, Palo Alto, CA), M. de Ley (Department of Biochemistry, Catholic anti-CD3 mAb alone. When costimulated with CD80, the strong University of Leuven, Leuven, Belgium), M. O’Donnell (Genetics Insti- effects of IL-12 on T cell CD40L expression most likely result tute, Cambridge, MA), and K. Thielemans (Free University of Brussels, from induction of high affinity IL-12R expression. Brussels, Belgium) for kindly providing the reagents and cells used in this To confirm that the effect of IL-12 on CD40L expression was study. http://www.jimmunol.org/ functionally relevant, we have studied cognate T cell interactions with B cells. We choose not to perform studies with macrophages, References because IFN-␥ induction by IL-12 would interfere strongly with 1. Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlmann, and R. J. Noelle. 1996. the effect of CD40L on these cells. Optimal activation of B cells by Immune regulation by CD40 and its ligand gp39. Annu. Rev. Immunol. 14:591. 2. Stout, R., and J. Suttles. 1996. The many roles of CD40:CD40L interactions in Th cells requires direct contact between the two cell types through cell-mediated inflammatory responses. 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