Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells
Toshiro Futagawa1,2,
Hisaya Akiba1,3,
Tomohiro Kodama1,4,
Kazuyoshi Takeda1,3,
Yasuyuki Hosoda2,
Hideo Yagita1,3 and
Ko Okumura1,3
1 Departments of 1Immunology, and
2 Thoracic and Cardiovascular Surgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corp., 2-3 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
4 First Department of Surgery, Tohoku University School of Medicine, 1-1 Seiryo-cho, Aoba-ku, Sendai 980-8574, Japan
Correspondence to:
H. Yagita
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Abstract
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4-1BB (CDw137) and its ligand (4-1BBL) have been implicated in cellular immune responses. To further characterize the expression and function of 4-1BBL, we newly generated an anti-mouse 4-1BBL mAb (TKS-1), which can inhibit the interaction of 4-1BBL with 4-1BB. Flow cytometric analyses using TKS-1 and an anti-mouse 4-1BB mAb indicated that 4-1BB was inducible on both CD4+ and CD8+ splenic T cells by stimulation with immobilized anti-CD3 mAb, but 4-1BBL was not expressed on resting or activated T cells. 4-1BBL expression was inducible on splenic B cells by stimulation with anti-IgM antibody plus anti-CD40 mAb, on peritoneal macrophages by stimulation with lipopolysaccharide (LPS) and on splenic dendritic cells (DC) by stimulation with anti-CD40 mAb or LPS. Interestingly, splenic DC expressed 4-1BB constitutively, which was down-regulated by anti-CD40 stimulation. Co-culture of splenic DC with 4-1BBL-transfected cells or 4-1BBL-expressing tumor cell lines led to cytokine (IL-6 and IL-12) production and co-stimulatory molecule up-regulation by splenic DC, indicating that 4-1BBL can directly activate DC. Moreover, IL-12 production by anti-CD40-stimulated DC was partially inhibited by TKS-1. These results suggest that 4-1BB expressed on DC may be involved in DC activation through DCtumor interaction and DCDC interaction.
Keywords: co-stimulatory molecules, dendritic cells, tumor immunity
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Introduction
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4-1BB (CDw137) is a member of the tumor necrosis factor (TNF) receptor superfamily of type I membrane proteins and was originally identified as an inducible gene in activated T cells (1,2). A ligand for 4-1BB (4-1BBL) has been cloned in mice and humans, and it was shown to be a type II membrane protein of the TNF superfamily (3,4). Members of the TNFTNF receptor superfamily have been shown to play critical roles in regulating cellular activation, differentiation and apoptosis (5). It has been shown that 4-1BB is expressed on activated CD4+ and CD8+ T cells as well as on activated NK cells (6,7). The expression of 4-1BBL has been found on activated B cells, macrophages and dendritic cells (DC) as estimated by binding of soluble 4-1BB proteins (3,4,8,9). Several in vitro studies have shown that the 4-1BBL4-1BB interaction provided a co-stimulatory signal to T cells, resulting in increased proliferation and cytokine production (912). In addition, it has been reported that the administration of agonistic anti-4-1BB mAb could eradicate established s.c. tumors in mice possibly by directly co-stimulating T cells and NK cells (7,13). The stimulation with agonistic anti-4-1BB mAb has also been shown to preferentially induce the proliferation of CD8+ T cells, and to enhance the generation of cytotoxic T lymphocytes (CTL) in graft-versus-host response and allograft rejection (14). Furthermore, recent studies using 4-1BBL-deficient mice revealed a role for 4-1BBL in allograft rejection and anti-viral CD8+ T cell responses (15,16). These results suggest that the 4-1BBL4-1BB interaction plays an important role in cellular immune responses.
To further characterize the expression and function of 4-1BBL in physiological and pathological conditions, we established a functional blocking mAb against mouse 4-1BBL. Our present study formally defined the expression of 4-1BBL and 4-1BB on T cells, B cells, macrophages and DC. Unexpectedly, we found that murine splenic DC constitutively expressed 4-1BB and were activated by 4-1BBL to produce cytokines and to up-regulate co-stimulatory molecules. Possible relevances of this finding to the DC activation by tumor cells and to the immuno-stimulatory effect of anti-4-1BB mAb in vivo are discussed.
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Methods
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Animals and cell lines
Six-week-old male C57BL/6, DBA/2 and BALB/c mice, and six-week-old female SD rats were purchased from Charles River (Atsugi, Japan). Mice transgenic for DO11.10 TCR
ß (DO-Tg) on a BALB/c background were kindly provided by Dr D. Y. Loh (Nippon Roche, Kamakura, Japan) and were maintained by interbreeding. Murine mastcytoma P815, murine T lymphomas L5178Y, EL-4, WR19L and BW5147, murine B lymphomas A20.2J, 2PK-3 and BAL17, murine macrophage cell lines J774.1, P388D1 and RAW264.7, and murine myeloma P3U1 (P3X63Ag8U.1) were purchased from ATCC (Manassas, VA). The hybridomas producing mAb against MHC class II (M5/114), heat-stable antigen (J11d), B220 (RA3-3A1), CD8 (3.155) and Thy1.2 (J1j) were also obtained from ATCC. The hybridoma producing mAb against CD4 (RL172) was kindly provided by Dr T. Tanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Murine B lymphoma BCL1-B20 was provided by Dr K. Takatsu (Institute of Medical Science, University of Tokyo, Japan). Granulocyte macrophage colony stimulating factor (GM-CSF)-producing B16 melanoma (GM-CSF/B16) was provided by Drs K. Hanada and H. Hamada (Cancer Institute, Tokyo, Japan). Mouse CD40 ligand-transfected P815 (CD40L/P815) was prepared as previously described (17). These cells were cultured in RPMI 1640 medium containing 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 0.1 mg/ml penicillin and streptomycin, and 50 µM 2-mercaptoethanol. A normal rat kidney cell line NRK-52E was kindly provided by Dr T. Otsuka (Institute of Cytosignal Research, Tokyo, Japan), and cultured in DMEM containing 10% FCS, 10 mM HEPES, 4 mM L-glutamine, 0.1 mg/ml penicillin and streptomycin, and 50 µM 2-mercatoethanol.
Antibodies and reagents
Phycoerythrin (PE)-conjugated goat anti-human and anti-rat IgG antibodies were purchased from Caltag (South San Francisco, CA). Purified anti-CD16/32 (2.4G2), CD3 (145-2C11) and CD40L (MR1) mAb, FITC-conjugated anti-CD4 (RM4-4), CD8 (53-6.7), B220 (RA3-6B2), CD11c (HL3), CD80 (16-10A1) CD86 (GL1) mAb, biotinylated anti-4-1BB (1AH2) mAb, rat IgG isotype control, and PE-labeled streptavidin were purchased from BD-PharMingen (San Diego, CA). Anti-CD40 mAb (HM40-3) and biotinylated anti-CD70 mAb (FR70) were prepared as described previously (18,19). Anti-CD28 mAb (PV-1) (20) was kindly provided by Dr R. Abe (Science University of Tokyo, Noda, Japan). Goat anti-mouse IgM F(ab')2 antibody was purchased from Jackson ImmunoResearch (West Grove, PA). Lipopolysaccharide (LPS) and geneticin were purchased from Sigma (St Louis, MO).
Construction of mouse 4-1BBIg fusion protein
To make a fusion protein consisting of the extracellular domain of murine 4-1BB (amino acids 1528) and Fc portion of human IgG1 (4-1BBIg), RT-PCR was performed using total RNA from anti-CD3-activated splenic T cells. 5'-TCACTCGAGATGGGAAACAACTGTTACAAC-3' and 5'-GGTAAAGACACTGAGGTCTCCTAGGTAGT-3' were used as 5' and 3' primer respectively. The PCR product was cloned into the XhoI and BamHI sites of pBluescript SK(+) which contained the gene encoding Fc portion of human IgG1 (provided by Dr B. Seed, Harvard Medical School, Boston, MA). The insert encoding 4-1BBIg was subcloned into the XhoI and NotI sites of a mammalian expression vector pMKITneo (provided by Dr K. Maruyama, Tokyo Medical Dental University, Tokyo). Stable transfectants producing 4-1BBIg was generated by electroporation into COS7 cells and selected with 1 mg/ml of geneticin. 4-1BBIg fusion protein was purified using Protein GSepharose column (Pharmacia Biotech, Uppsala, Sweden).
Preparation of mouse 4-1BBL transfectants
A cDNA fragment encoding the entire open reading frame of mouse 4-1BBL was prepared by RT-PCR from 2PK-3 mRNA. 5'-TCACTCGAGATGGACCAGCACACACTTGATG-3' corresponding to nucleotides 123142 of mouse 4-1BBL cDNA tagged with an XhoI site and 5'-GGCTGTTGGGTACCCTTACTCGCCGGCGAAT-3' corresponding to nucleotides 721739 tagged with a NotI site were used as 5' and 3' primers respectively, according to the published sequence (3). The PCR product was cloned into pMKITneo vector, and transfected into NRK-52E, L5178Y and P815 cells by electroporation. Mock-transfected cells were also produced by electroporation of the pMKITneo vector without an insert. After selection by 1 mg/ml geneticin, transfectants stably expressing 4-1BBL were identified by staining with 4-1BBIg.
Generation of anti-mouse 4-1BBL mAb
A SD rat was immunized with 4-1BBL-transfected NRK cells 5 times at 7-day intervals. Three days after final immunization, the splenocytes were fused with P3U1 cells as described (19). After hypoxanthineaminopterinthymidine selection, one hybridoma producing mAb TKS-1 (rat IgG2a,
) was identified by its strong reactivity with 4-1BBL-transfected L5178Y cells, but not with mock-transfected L5178Y cells, and cloned by limiting dilution. TKS-1 was purified from ascites by standard procedures with caprylic acid and purity was verified by SDSPAGE analysis.
Preparation and culture of T cells, B cells and macrophages
Splenic T cells and B cells were purified as previously described (19). Briefly, splenic T cells were purified by passage through a nylon wool column (Wako, Osaka, Japan), and treatment with a mixture of hybridoma supernatants (anti-MHC class II, anti-heat-stable antigen and anti-B220) and Low-Tox rabbit complement (Cedarlane, Hornby, Ontario, Canada). Purified T cells (>95% CD3+) were stimulated with immobilized anti-CD3 mAb (5 µg/ml) for 2472 h. For preparing small resting B cells, splenocytes were treated with a mixture of hybridoma supernatants (anti-Thy-1.2, anti-CD4 and anti-CD8) and Low-Tox rabbit complement. After Percoll (Pharmacia Biotech) gradient centrifugation, small B cells were collected from the 60/70% interface. Purified B cells (>95% B220+) were stimulated with anti-IgM antibody (10 µg/ml) and/or anti-CD40 mAb (5 µg/ml) for 2472 h. Peritoneal macrophages were obtained from BALB/c mice which i.p. received 2 ml of 4% thioglycolate (Sigma) 4 days before. Peritoneal exudate cells were harvested by peritoneal lavage with ice-cold PBS and depleted of non-adherent cells after 1 h culture on plastic dishes, and then stimulated with LPS (10 µg/ml) or anti-CD40 mAb (5 µg/ml) for 2472 h.
Generation of Th1 and Th2 cells
For generation of Th1 and Th2 cells, CD4+ CD62L+ naive T cells were purified from the spleen of DO-Tg mice by using an autoMACS column with FITC-conjugated anti-CD4, anti-FITC multisort kit and CD62L-coupled microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Purified naive CD4 T cells (5x105/well; >95% CD4+ CD62L+) were stimulated with irradiated BALB/c splenocytes (5x106/well) and 1 µM of ovalbumin 323339 peptide in 24-well plates in the presence of 20 U/ml of IL-2, 50 U/ml of IL-12 and 10 µg/ml of anti-IL-4 mAb (11B11) for Th1 development or in the presence of 20 ng/ml of IL-4 for Th2 development respectively. T cells were harvested at day 7 and re-stimulated in the same conditions. After 4 days of secondary stimulation, T cells were split with a medium containing IL-2 for 48 h and live cells were isolated using M-SMF (Japan Immunoresearch, Takasaki, Japan). Thereafter, Th1 and Th2 cells were stimulated with immobilized anti-CD3 mAb (5 µg/ml) and soluble anti-CD28 (5 µg/ml) for 2472 h.
Flow cytometric analysis
Cells (0.51x106) were first preincubated with unlabeled anti-CD16/32 mAb to avoid non-specific binding of antibodies to Fc
R and then incubated with a saturating amount of 4-1BBIg, FITC- or PE-labeled mAb, or biotinylated mAb. After washing with PBS twice, the cells were incubated with PE-labeled goat anti-human IgG antibody for 4-1BBIg or PE-labeled streptavidin for biotinylated mAb. After washing with PBS twice, the stained cells (live gated on the basis of forward and side scatter profiles and propidium iodide exclusion) were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) and data were processed using the CellQuest program (Becton Dickinson).
Preparation and stimulation of splenic DC
DC were prepared from the spleen of GM-CSF/B16-bearing C57BL/6 mice as previously described (21). Briefly, GM-CSF/B16 (2x106 cells) cells were inoculated s.c. in the right flank of C57BL/6 mice. After 2 weeks, when the size of the s.c. tumor reached 18x18 mm, the markedly enlarged spleen was removed, cut into small pieces and filtered through a steel mesh. After hemolysis, cells (1.6x107/well) were placed in six-well culture plates. After incubation for 2 h at 37°C, non-adherent cells were removed by gentle pipetting and washed with RPMI 1640 medium prewarmed to 37°C. The remaining adherent cells were cultured for 16 h and DC that lost adherence to the plates were harvested by gentle pipetting. The purified splenic DC (2x105/well; >90% CD11c+) were cultured with anti-CD40 mAb (5 µg/ml), LPS (10 µg/ml) or irradiated (100 Gy) transfectants or tumor cell lines (5x104/well) in 96-well round-bottomed plates in the presence of TKS-1 or anti-CD40L mAb (10 µg/ml) at 37°C for 2448 h. To determine cytokine production, cell-free supernatants were collected and subjected to ELISA using the OptEIA mouse IL-6, IL-12 p40 and IL-12 p70 set (BD-PharMingen) according to the manufacturer's instruction.
For preparing normal splenic DC, spleens from normal C57BL/6 mice were digested with 400 U/ml of collagenase (Wako) in the presence of 5 mM EDTA, separated into a low-density fraction on Optiprep gradient (Axis-Shield, Oslo, Norway) and cultured overnight with 10 ng/ml of GM-CSF, as described previously (22,23). Non-adherent cells collected after overnight culture were incubated with anti-CD11c-coupled magnetic beads and the bound cells were isolated by the autoMACS column. The purified normal splenic DC (2x106/ml; >90% CD11c+) were cultured with anti-CD40 mAb (5 µg/ml) at 37°C for 2472 h.
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Results
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Establishment and characterization of anti-mouse 4-1BBL mAb
To characterize the expression and function of mouse 4-1BBL, we generated stable mouse 4-1BBL transfectants and anti-mouse 4-1BBL mAb. The mouse 4-1BBL cDNA was isolated from mRNA of 2PK-3 cells by RT-PCR, cloned into a mammalian expression vector, and transfected into NRK-52E, L5178Y and P815 cells. Stable expression of 4-1BBL on the cDNA transfectants (4-1BBL/NRK, 4-1BBL/L5178Y and 4-1BBL/P815), but not on mock-transfected cells (mock/NRK, mock/L5178Y and mock/P815), was verified by binding of 4-1BBIg (data not shown). We immunized a SD rat with 4-1BBL/NRK cells and fused the splenocytes with P3U1 myeloma cells, and then screened the hybridomas producing mAb that specifically reacted with the 4-1BBL transfectants. As represented in Fig. 1(A)
, one mAb designated TKS-1, which bound to 4-1BBL/NRK and 4-1BBL/P815 cells but not to mock/NRK or mock/P815 cells, was obtained. Moreover, as shown in Fig. 1(B)
, preincubation with TKS-1 blocked 4-1BBIg binding to 4-1BBL/L5178Y cells. These results indicated that TKS-1 is specific for mouse 4-1BBL, and can interrupt the interaction between 4-1BBL and 4-1BB.

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Fig. 1. Characterization of TKS-1 mAb. (A) Reactivity of TKS-1 to mouse 4-1BBL transfectants. NRK-52E- and P815-derived transfectants (4-1BBL/NRK, 4-1BBL/P815, mock/NRK and mock/P815) were stained with biotinylated TKS-1 followed by PE-labeled streptavidin. The bold line indicates staining with TKS-1 and the broken line indicates background staining with control IgG. (B) TKS-1 inhibits 4-1BBIg binding to 4-1BBL transfectants. 4-1BBL /L5178Y cells were pretreated with or without TKS-1 and then stained with 4-1BBIg followed by PE-labeled goat anti-human IgG antibody. The control histogram represents background staining with control IgG and PE-labeled anti-human IgG without 4-1BBIg. (C) TKS-1 inhibits T cell co-stimulatory activity of mouse 4-1BBL. Purified splenic T cells (1x105/well) were co-cultured with irradiated 4-1BBL/P815 or mock/P815 cells (2x104/well) in the presence of anti-CD3 mAb (1 µg/ml) and the indicated concentrations of TKS-1 or control IgG. Proliferative response was assessed at 48 h by pulsing the cultures with 0.5 µCi/well of [3H]thymidine for the last 6 h. Data are expressed as mean ± SD of triplicate wells.
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It has been shown that 4-1BBL provided a co-stimulatory signal for T cell proliferation in mice and humans (4,10). To examine whether TKS-1 can block the co-stimulatory activity of mouse 4-1BBL, splenic T cells were co-cultured with 4-1BBL/P815 or mock/P815 cells in the presence of anti-CD3 mAb and TKS-1, and the proliferative responses were assessed. As shown in Fig. 1(C)
, 4-1BBL/P815 cells co-stimulated the proliferation of anti-CD3-stimulated T cells far more efficiently than mock/P815 cells. This proliferative response co-stimulated by 4-1BBL /P815 cells was blocked by TKS-1, but not by control IgG, in a dose-dependent manner. These results indicated that mouse 4-1BBL exhibits a potent co-stimulatory activity for anti-CD3-stimulated T cell proliferation, which can be blocked by TKS-1.
Expression of 4-1BBL on mouse tumor cell lines
A number of mouse tumor cell lines were tested for 4-1BBL expression by staining with TKS-1 and RT-PCR. Most T lymphoma cell lines (EL4, WR19L and BW5147), B lymphoma cell lines (A20.2J, BCL1-B20 and 2PK-3) and macrophage cell lines (J774.1, P388D1 and RAW264.7) expressed 4-1BBL mRNA as estimated by RT-PCR (not shown) and were reactive with TKS-1 (Fig. 2
), but some T lymphoma (L5178Y) and B lymphoma (BAL17) cell lines did not express 4-1BBL mRNA (not shown) and were not reactive with TKS-1 (Fig. 2
). These results indicated a strict coincidence of 4-1BBL mRNA expression and TKS-1 reactivity.

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Fig. 2. Reactivity of TKS-1 with mouse leukemia cell lines. T lymphoma cell lines (EL-4, WR19L, BW5147 and L5178Y), B lymphoma cell lines (A20.2J, BCL1-B20, 2PK-3 and BAL17) and macrophage cell lines (J774.1, P388D1 and RAW264.7) were preincubated with unlabeled anti-CD16/32 mAb and then stained with biotinylated TKS-1 followed by PE-labeled streptavidin. The bold line indicates staining with TKS-1 and the broken line indicates background staining with control IgG.
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Expression of 4-1BBL and 4-1BB on T cells
We next examined the expression of 4-1BBL and 4-1BB on splenic T cells by flow cytometric analysis utilizing TKS-1 and an anti-mouse 4-1BB mAb (1AH2). Purified splenic T cells were stimulated for 2472 h with immobilized anti-CD3 mAb. As shown in Fig. 3(A and B)
, 4-1BB expression was detected on both CD4+ and CD8+ T cells after stimulation with anti-CD3 mAb, which appeared at 24 h and reached a peak at 48 h. The addition of anti-CD28 mAb did not change the expression of 4-1BB on anti-CD3-stimulated T cells at any time point (data not shown). In contrast, 4-1BBL was not found on anti-CD3-stimulated T cells (Fig. 3A and B
) even when co-stimulated with anti-CD28 mAb for 6 days (data not shown).

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Fig. 3. Expression of 4-1BBL and 4-1BB on T cells. (A and B) Expression of 4-1BBL and 4-1BB on activated T cells. Splenic T cells (3x106/ml) from C57BL/6 mice were stimulated with immobilized anti-CD3 mAb and harvested at the indicated periods. Cells were double-stained with FITC-labeled anti-CD4 or -CD8 mAb and biotinylated TKS-1, anti-4-1BB mAb or control IgG followed by PE-labeled streptavidin. Histograms shown are staining of electronically gated CD4+ (A) or CD8+ (B) cells. (C and D) Expression of 4-1BBL and 4-1BB on Th1 and Th2 cells. Th1 (C) and Th2 (D) cells (3x106/ml) were stimulated with immobilized anti-CD3 mAb and soluble anti-CD28 mAb, and harvested at the indicated periods. Cells were stained with biotinylated TKS-1, anti-4-1BB mAb or control IgG followed by PE-labeled streptavidin. The bold line indicates staining with the indicated mAb and the broken line indicates background staining with control IgG.
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To further examine the expression of 4-1BBL and 4-1BB on differentiated T cells, we generated Th1 and Th2 cells by stimulating DO-Tg CD4+ T cells in the presence of IL-12 + anti-IL-4 mAb or IL-4 respectively. Both Th1 and Th2 cells were stimulated for 2472 h with immobilized anti-CD3 mAb and soluble anti-CD28 mAb. The Th1 cells produced 10.3 ng/ml IFN-
and <7.8 pg/ml IL-4, and Th2 cells produced 0.2 ng/ml IFN-
and 13.3 ng/ml IL-4 respectively when assessed by ELISA at 24 h after stimulation. As shown in Fig. 3(C and D)
, 4-1BB was highly expressed on both Th1 and Th2 cells, which was slightly down-modulated by anti-CD3 and anti-CD28 stimulation. In contrast, 4-1BBL was not found on either Th1 or Th2 cells even when stimulated with anti-CD3 and anti-CD28 mAb.
These results indicated that mouse 4-1BB is expressed on TCRCD3-stimulated CD4+ and CD8+ T cells and Th1 and Th2 cells, but 4-1BBL appears not to be expressed on activated or differentiated T cells.
Expression of 4-1BBL and 4-1BB on activated B cells and macrophages
We next examined the expression of 4-1BBL and 4-1BB on splenic B cells. Purified splenic B cells were stimulated with anti-IgM antibody, anti-CD40 mAb or both for 2472 h and stained with TKS-1 or anti-4-1BB mAb. As shown in Fig. 4(C)
, 4-1BBL expression was detected on B cells after stimulation with the combination of anti-IgM antibody and anti-CD40 mAb, which appeared at 24 h and reached a peak at 48 h. In contrast, stimulation with either anti-IgM antibody or anti-CD40 mAb alone did not induce 4-1BBL expression (Fig. 4A and B
). On the other hand, 4-1BB was not found on B cells when stimulated with anti-IgM antibody and/or anti-CD40 mAb (Fig. 4AC
). These results indicated that 4-1BBL is specifically expressed on surface Ig- and CD40-stimulated B cells, while 4-1BB appears not to be expressed on B cells.

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Fig. 4. Expression of 4-1BBL and 4-1BB on activated B cells. Purified splenic B cells (3x106/ml) from C57BL/6 mice were stimulated with anti-IgM antibody (A), anti-CD40 mAb (B) or anti-IgM antibody plus anti-CD40 mAb (C). Cells were harvested at the indicated periods and stained with biotinylated TKS-1, anti-4-1BB mAb or control IgG followed by PE-labeled streptavidin. The bold line indicates staining with the indicated mAb and the broken line indicates background staining with control IgG.
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We also examined the expression of 4-1BBL and 4-1BB on peritoneal macrophages. As shown in Fig. 5
, a marginal expression of 4-1BBL was detected on LPS-stimulated macrophages at 24 h, but not on anti-CD40-stimulated macrophages. In contrast, no significant expression of 4-1BB was observed on murine macrophages.

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Fig. 5. Expression of 4-1BBL and 4-1BB on activated macrophages. Peritoneal macrophages (2x106/ml) from BALB/c mice were stimulated with anti-CD40 mAb (A) or LPS (B). Cells were harvested at the indicated periods and stained with biotinylated TKS-1, anti-4-1BB mAb or control IgG followed by PE-labeled streptavidin. The bold line indicates staining with the indicated mAb and the broken line indicates background staining with control IgG.
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Expression of 4-1BBL and 4-1BB on DC
We also examined the expression of 4-1BBL and 4-1BB on splenic DC. Splenic DC were prepared from the spleen of GM-CSF/B16 tumor-bearing C57BL/6 mice, which generate a large number of splenic DC, and were stimulated with anti-CD40 mAb or LPS for 2448 h. As shown in Fig. 6(A)
, a low level of 4-1BBL was expressed on splenic DC, which was up-regulated by stimulation with anti-CD40 mAb or LPS at 24 h. Unexpectedly, splenic DC constitutively expressed 4-1BB, which was slightly down-regulated by anti-CD40 stimulation (Fig. 6A
). In contrast, the expression of CD70, which is another co-stimulatory molecule belonging to the TNF family, was enhanced by anti-CD40 stimulation (Fig. 6A
). Similar results were obtained when splenic DC prepared from normal C57BL/6 mice were stimulated with anti-CD40 mAb (Fig. 6B
).

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Fig. 6. Expression of 4-1BBL and 4-1BB on splenic DC. Splenic DC (5x106/ml) from GM-CSF/B16-bearing C57BL/6 mice (A) or normal C57BL/6 mice (B) were stimulated with anti-CD40 mAb or LPS. Cells were harvested at the indicated periods and double-stained with FITC-labeled CD11c mAb and biotinylated TKS-1, anti-4-1BB mAb, anti-CD70 mAb or control IgG followed by PE-labeled streptavidin. Histograms shown are staining of electronically gated CD11c+ cells. The bold line indicates staining with the indicated mAb and the broken line indicates background staining with control IgG.
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Activation of splenic DC by 4-1BBL
The above observation implied that a signaling through 4-1BB expressed on splenic DC might activate DC as did CD40, another member of the TNF receptor superfamily (24). To address this possibility, splenic DC prepared from GM-CSF/B16 tumor-bearing C57BL/6 mice were co-cultured with 4-1BBL/P815, CD40L/P815 or mock/P815 cells in the presence of TKS-1 or anti-CD40L mAb for 48 h and then cytokine (IL-6, L-12 p40 and IL-12 p70) production was assessed by ELISA. Up-regulation of CD80 and CD86 on splenic DC was also assessed as an indication of DC activation by flow cytometric analysis. As shown in Fig. 7(A)
, when cultured in medium alone or with mock/P815 cells, splenic DC released only low levels of IL-6, IL-12 p40 and IL-12 p70. Upon stimulation with CD40L/P815, splenic DC released high levels of these cytokines so that IL-6 production was increased 4.4-fold, IL-12 p40 11.3-fold and IL-12 p70 10.9-fold as compared to mock/P815 cells. 4-1BBL/P815 cells also markedly induced the cytokine production by splenic DC so that IL-6 production was increased 4.3-fold, IL-12 p40 7.4-fold and IL-12 p70 6.0-fold as compared to mock/P815 cells. This cytokine production induced by 4-1BBL /P815 or CD40L/P815 cells was inhibited by addition of TKS-1 or anti-CD40L mAb respectively. Although CD80 and CD86 were already expressed at high levels before stimulation, 4-1BBL/P815 enhanced the expression of CD80 and CD86 on splenic DC to a comparable level to that did CD40L/P815 (Fig. 7B
). These results indicated that the 4-1BB molecules expressed on splenic DC were functional and 4-1BBL can activate DC as efficiently as CD40L.

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Fig. 7. Activation of splenic DC by 4-1BBL. (A) Cytokine production. Splenic DC (2x105/ml) were co-cultured with irradiated 4-1BBL/P815, CD40L/P815 or mock/P815 cells (5x104/ml) with or without 10 µg/ml of TKS-1 or anti-CD40L mAb (MR1). Cell-free supernatants were collected at 48 h, and IL-6, IL-12 p40 and IL-12 p70 were measured by ELISA. Data are expressed as mean ± SD of triplicate wells. (B) Up-regulation of co-stimulatory molecules. Splenic DC were harvested at 48 h from the co-cultures as described in (A) and stained with FITC-labeled anti-CD80 mAb, anti-CD86 mAb or control IgG. The bold line indicates staining with the indicated mAb and the broken line indicates background staining with control IgG. Similar results were obtained from three independent experiments.
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4-1BBL-expressing tumor cell lines induce IL-12 production by splenic DC
As represented in Fig. 2
, some tumor cell lines constitutively express 4-1BBL on their cell surface. We then examined whether 4-1BBL expressed on these tumor cells can directly activate DC. Splenic DC prepared from GM-CSF/B16 tumor-bearing C57BL/6 mice were co-cultured with a 4-1BBL+ B lymphoma cell line 2PK-3, a 4-1BBL- B lymphoma cell line BAL17 or 4-1BBL+ macrophage cell line P388D1 in the presence or absence of TKS-1. Cell-free culture supernatants were collected at 48 h, and the production of IL-12 p40 and IL-12 p70 was measured by ELISA. As shown in Fig. 8
, 2PK-3 and P338D1 cells induced production of both IL-12 p40 and IL-12 p70 by splenic DC at comparable levels to that induced by 4-1BBL/P815 cells, which was significantly inhibited by the addition of TKS-1. In contrast, 4-1BBL- BAL17 cells did not induce IL-12 production significantly. These results indicated that 4-1BBL expressed on some tumor cell lines can directly activate DC to produce IL-12.

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Fig. 8. 4-1BBL-expressing leukemia cell lines induce IL-12 production by splenic DC. Splenic DC (2x105/ml) were co-cultured with irradiated mock/P815, 4-1BBL/P815, 2PK-3, P388D1 or BAL17 cells (5x104/ml) in the presence of 10 µg/ml of TKS-1 or control IgG. Cell-free supernatants were collected at 48 h and IL-12 p40 and IL-12 p70 were measured by ELISA. Data are expressed as mean ± SD of triplicate wells. Similar results were obtained from three independent experiments. *P < 0.05.
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Involvement of 4-1BB4-1BBL interaction in CD40-mediated IL-12 production by DC
The above experiments indicated that 4-1BB on DC could induce IL-12 production as potently as CD40. Given that the CD40 stimulation induced 4-1BBL expression on DC (Fig. 6
), 4-1BB4-1BBL interaction on DC may be involved in the CD40-mediated IL-12 production by DC. To address this possibility, splenic DC prepared from GM-CSF/B16 tumor-bearing C57BL/6 mice were stimulated with various concentrations of anti-CD40 mAb in the presence or absence of TKS-1 and IL-12 p40 production was measured at 48 h by ELISA. As shown in Fig. 9
, anti-CD40 mAb could induce IL-12 production by splenic DC in a dose-dependent manner, which was significantly inhibited by TKS-1. These results indicated that the 4-1BB4-1BBL interaction on DC is at least partly involved in the CD40-mediated IL-12 production by DC.

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Fig. 9. Involvement of 4-1BB4-1BBL interaction in IL-12 production by anti-CD40-stimulated DC. Splenic DC (2x105/ml) were cultured with the indicated concentrations of anti-CD40 mAb in the presence of 10 µg/ml of TKS-1 or control IgG. Cell-free supernatants were collected at 48 h and IL-12 p40 was measured by ELISA. Data are expressed as mean ± SD of triplicate wells. Similar results were obtained form three independent experiments. *P < 0.05.
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Discussion
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In this study, we generated an anti-mouse 4-1BBL mAb and formally verified the expression of 4-1BBL and 4-1BB on murine T cells, B cells and macrophages. We also characterized the expression and function of these molecules on DC, and found their novel contribution to DC activation.
Our present results (Fig. 3
) showed that 4-1BB is expressed on TCRCD3-stimulated T cells and Th1 and Th2 cells, but 4-1BBL is not expressed on normal splenic T cells and Th1 and Th2 cells even after activation with anti-CD3 and anti-CD28 mAb. It has been reported that 4-1BBL was expressed in an activated murine T cell clone as estimated by Northern blot analysis (3) and on an activated human T cell clone as estimated by 4-1BBIg binding (4). This discrepancy may represent a difference between primary T cells and long-term cultured T cell clones. Consistent with this notion, most of the T lymphoma cell lines we examined constitutively expressed 4-1BBL (Fig. 2
). Therefore, we presently cannot exclude the possibility that 4-1BBL may be expressed on primary T cells under certain conditions. Further studies are needed to address this possibility.
Our present results (Figs 2 and 4
) showed that 4-1BBL is constitutively expressed on most B lymphoma cell lines and that the 4-1BBL expression was inducible on primary resting B cells upon stimulation via surface IgM and CD40. These results are consistent with previous studies using a 4-1BBalkaline phosphatase (4-1BBAP) fusion protein to detect 4-1BBL (8). The 4-1BBL molecules expressed on B cells, which are activated by surface Ig engagement by antigen and CD40 engagement by CD40L on T cells, may act co-stimulatory for secondary T cell activation in the germinal center. Alternatively, the 4-1BBL molecules on B cells may transmit a co-stimulatory signal into B cells, since it has been reported that 4-1BB-expressing transfectants co-stimulated the proliferation of anti-IgM-primed B cells (8).
Our present results (Figs 2 and 5
) showed that 4-1BBL is constitutively expressed on macrophage cell lines and is induced on peritoneal macrophages by LPS. These results are consistent with previous studies showing constitutive expression of 4-1BBL on murine macrophage cell lines as estimated by 4-1BBAP binding (8) and inducible expression of 4-1BBL in bone marrow-derived macrophages by LPS as estimated by Northern blot analysis (3). In humans, it has been recently reported that peripheral blood monocytes constitutively expressed 4-1BB and cross-linking with immobilized anti-4-1BB mAb induced monocyte activation to produce IL-8 and TNF-
(25). It has been also reported that immobilized 4-1BBIg induced monocyte activation to produce IL-6, IL-8 and TNF-
, suggesting constitutive expression and a signaling of 4-1BBL on human monocytes (26). In contrast, we could not find either 4-1BB or 4-1BBL on unstimulated murine peritoneal macrophages (Fig. 5
). This may represent a species difference. Further studies are needed to explore the macrophage-activating function of 4-1BBL on LPS-stimulated macrophages and its involvement in T cell-mediated activation of macrophages.
Our present results (Fig. 6
) showed that 4-1BBL is expressed on splenic DC at a low level and up-regulated by CD40-mediated or LPS stimulation. The low-level 4-1BBL expression on splenic DC is consistent with a previous observation using 4-1BBAP (9). The 4-1BBL molecules up-regulated on CD40-stimulated DC may contribute to the cytotoxic T lymphocyte-inducing ability of CD40-stimulated DC (24), since 4-1BB-mediated co-stimulation preferentially activates CD8+ T cells (14). Alternatively, the 4-1BBL molecules on DC may transmit a DC-activating signal as those on B cells and monocytes. Further studies are needed to address these possibilities.
The most interesting finding in this study was the expression of 4-1BB on DC. Unexpectedly, splenic DC constitutively expressed 4-1BB at a high level, which was down-regulated upon CD40 stimulation (Fig. 6
). In contrast, the expression of CD70 was enhanced by anti-CD40 stimulation. These data indicated that the anti-CD40 stimulation specifically reduced the expression of 4-1BB on DC. On the other hand, anti-CD40 stimulation induced the expression of 4-1BBL on DC. Therefore, the reduction of 4-1BB expression seems to be caused by interaction with 4-1BBL expressed on DC themselves. The 4-1BB molecules expressed on DC were functional to activate DC, leading to IL-6/IL-12 production and CD80/CD86 up-regulation (Fig. 7
). The stimulatory effect of 4-1BBL was almost comparable to that of CD40L, which is well known as a potent DC activator (24,27). Since 4-1BBL was inducible on DC by CD40 stimulation (Fig. 6
), we found the 4-1BB4-1BBL interaction at least partly contributed to enhance the IL-12 production by CD40-stimulated DC (Fig. 9
). Therefore, the 4-1BB4-1BBL interaction represents a novel pathway of DC activation by DCDC interaction. We also demonstrated that tumor cell lines that were transfected with 4-1BBL cDNA or spontaneously expressed 4-1BBL could induce IL-12 production by DC in a 4-1BBL-dependent manner (Fig. 8
). Recent studies have shown that DC play a pivotal role in eliciting anti-tumor immunity (27,28). However, mechanisms for the DCtumor interaction remain largely unknown. Our present results suggest a possibility that 4-1BB molecules expressed on DC may be involved in tumor surveillance by DC. Although most murine leukemia cell lines we examined expressed 4-1BBL (Fig. 2
), it is unclear whether these cell lines did express 4-1BBL during tumor development or they gained the 4-1BBL expression during a long-term culture. The expression of 4-1BBL during primary leukemogenesis and the possible involvement of 4-1BBL in tumor surveillance remain to be determined by further studies.
It should be also noted that the 4-1BB-mediated activation of DC might be involved in the anti-tumor effect of 4-1BBL gene transfer and agonistic anti-4-1BB mAb (13,29). These modalities have been thought to exert the immunopotentiating effect by directly co-stimulating T cells and NK cells. However, our present results suggest that DC activation by these modalities might play a rather critical role. This is reminiscent of the anti-tumor effect of CD40L gene transduction and agonistic anti-CD40 mAb. We previously demonstrated that the CD40L gene transduction exerted an anti-tumor effect by inducing IL-12 production and CD80/CD86 expression of antigen-presenting cells, possibly DC (17). A similar critical contribution of DC activation has been suggested to the anti-tumor effect of agonistic anti-CD40 mAb (30,31). Therefore, the 4-1BBL gene transfer and the agonistic anti-4-1BB mAb may exert anti-tumor effects in a similar way. It has been reported that the CD40L gene transfer and the administration of agonistic anti-CD40 mAb in vivo could cause some complications such as pneumonia and lymphoproliferation (32,33). Since the expression of 4-1BB is rather limited to activated T cells and DC, 4-1BB may be a better target than CD40 for the immunopotentiation.
Recent studies using 4-1BBL-deficient mice have revealed a role for 4-1BBL in anti-viral CD8+ T cell responses and allograft rejection (15,16). The anti-mouse 4-1BBL mAb we established in the present study will be useful for further investigating the expression and function of 4-1BBL in physiological and pathological conditions by using various disease models in the murine system.
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Acknowledgments
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We thank Drs O. Shimosato and H. Matsuda for helpful suggestions. This work was partly supported by grants from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Japan.
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Abbreviations
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AP alkaline phosphatase |
CTL cytotoxic T lymphocyte |
DC dendritic cell |
GM-CSF granulocyte macrophage colony stimulating factor |
L ligand |
LPS lipopolysaccharide |
PE phycoerythrin |
TNF tumor necrosis factor |
Received 14 May 2001,
accepted 27 November 2001.
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