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Modulation of Dendritic Cell Differentiation and Maturation by Decoy Receptor 3 Tsui-Ling Hsu, Yung-Chi Chang, Siu-Ju Chen, Yong-Jun Liu, Allen W. Chiu, Chung-Ching Chio, Lieping Chen and This information is current as Shie-Liang Hsieh of September 25, 2021. J Immunol 2002; 168:4846-4853; ; doi: 10.4049/jimmunol.168.10.4846 http://www.jimmunol.org/content/168/10/4846 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Modulation of Dendritic Cell Differentiation and Maturation by Decoy Receptor 31

Tsui-Ling Hsu,* Yung-Chi Chang,* Siu-Ju Chen,* Yong-Jun Liu,§ Allen W. Chiu,† Chung-Ching Chio,† Lieping Chen,‡ and Shie-Liang Hsieh2*

Decoy receptor 3 (DcR3), a soluble receptor belonging to the TNFR superfamily, is a receptor for both Fas ligand (FasL) and LIGHT. It has been demonstrated that DcR3 is up-regulated in lung and colon cancers, thus promoting tumor growth by neutralizing the cytotoxic effects of FasL and LIGHT. In this study, we found that DcR3.Fc profoundly modulated dendritic cell differentiation and maturation from CD14؉ monocytes, including the up-regulation of CD86/B7.2, and the down-regulation of -CD40, CD54/ICAM-1, CD80/B7.1, CD1a, and HLA-DR. Moreover, DcR3-treated dendritic cells suppressed CD4؉ T cell prolif ؉ ؉

eration in an allogeneic MLR and up-regulated IL-4 secretion of CD4 CD45RA T cells. This suggests that DcR3.Fc may act not Downloaded from only as a decoy receptor to FasL and LIGHT, but also as an effector molecule to skew T cell response to the Th2 phenotype. The Journal of Immunology, 2002, 168: 4846–4853.

ecoy receptor 3 (DcR3),3 also known as TR6 or M68, is surveillance during lymphomagenesis, or that virus-infected lym- a member of the TNFR superfamily and is a decoy re- phoma cells with DcR3 expression might be selected during mul- ceptor for Fas ligand (FasL) and LIGHT (homologous to tistep tumorigenesis (6). In addition, expression of DcR3 can be D http://www.jimmunol.org/ lymphotoxins, shows inducible expression, and competes with detected in malignant glioma cells as well as in human glioblas- HSV glycoprotein D for herpesvirus entry mediator, a receptor tomas, and its expression correlates with the grade of malignancy expressed by T lymphocytes) (1–3). Like osteoprotegerin (4), a (7). Besides tumor cell patients, the DcR3 gene is also overexpressed member of the TNFR superfamily, DcR3 lacks a transmembrane in silicosis or systemic lupus erythematosus patients (8). Because domain and is regarded as a secreted, rather than a membrane- LIGHT is expressed in dendritic cells (DCs) and acts as a costimu- associated, molecule. Moreover, DcR3 can apparently neutralize latory factor essential for priming T cell responses (9–11), we ques- the biological effects of FasL and LIGHT by inhibiting the FasL- tioned whether DcR3 could suppress immunity by interfering with the Fas interaction (1) or by inhibiting LIGHT binding to both the maturation and differentiation of DCs. lymphotoxin (LT)-␤ receptor (LT␤R) and the herpes virus entry by guest on September 25, 2021 Growing evidence has demonstrated that members of the TNF mediator (2, 5). DcR3 gene expression is increased in malignant superfamily transduce signals after engagement with their recep- tissue (1) and DcR3 protein is overexpressed in human adenocar- tors (12–21). In our recent study, we further demonstrated that cinomas of the esophagus, stomach, colon, and rectum (3). More- cross-linking of TNF-related activation-induced cytokine by im- over, DcR3 protein was overexpressed in a substantial number of mobilized soluble receptor activator of NF-␬B.Fc fusion protein tumors in which gene amplification could not be detected (3). A recent study further demonstrated that DcR3 is amplified and over- activated p38 mitogen-activated protein kinase and enhanced ␥ expressed in virus (EBV or human T cell leukemia virus-I)-asso- IFN- secretion via reverse signaling through TNF-related activa- ciated lymphomas (6). These results suggest that EBV and human tion-induced cytokine (22), and cross-linking of TNF-related ap- ␥ T cell leukemia virus-I may use DcR3 to escape from immune optosis-inducing ligand, enhanced proliferation, and IFN- secretion of T cells (23). However, even though “reverse signaling” could be triggered by *Department of Microbiology and Immunology, and Immunology Research Center, National Yang-Ming University, Taipei, Taiwan; †Department of Surgery, Chi-Mei immobilized receptor.Fc or agonistic mAb, there is no evidence to Foundational Hospital, Tainan, Taiwan; ‡Department of Immunology, Mayo Gradu- demonstrate that the soluble receptor.Fc fusion protein can trigger § ate and Medical Schools, Mayo Clinic, Rochester, MN 55905; and Department of signaling and modulate cell function. In this study, we report that Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, Palo ϩ Alto, CA 94304 soluble DcR3.Fc binds to CD14 monocytes and interferes with Received for publication October 22, 2001. Accepted for publication March 5, 2002. their differentiation and maturation into DCs. The expression of The costs of publication of this article were defrayed in part by the payment of page HLA-DR and other costimulatory molecules, such as CD40 and charges. This article must therefore be hereby marked advertisement in accordance CD80/B7.1, was suppressed. In contrast, the costimulatory mole- with 18 U.S.C. Section 1734 solely to indicate this fact. cule, CD86/B7.2, was up-regulated under the same conditions. 1 This work was mainly supported by Grant NHRI-CN-BP-8902S from the National Health Research Institute, Taiwan; National Sciences Council Grants NSC90-2320- Moreover, DcR3.Fc-treated DCs biased T cell differentiation to the B-010-109 and NSC90-2318-B-010-009-M51; and Grant GMYM 8902 from the Chi- Th2 phenotype in allogeneic MLR. Similar results were not ob- Mei Foundational Hospital (Tainan, Taiwan). served when Fas.Fc or LT␤R.Fc was used in place of DcR3.Fc. 2 Address correspondence and reprint requests to Dr. Shie-Liang Hsieh, Department Because DcR3.Fc fusion protein has been shown to have the sim- and Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan. E-mail address: [email protected] ilar binding affinity and specificity as that of DcR3 (24, 25), this 3 Abbreviations used in this paper: DcR3, decoy receptor 3; FasL, Fas ligand; LT, raises the argument that DcR3 produced by many human tumor lymphotoxin; LIGHT, homologous to LTs, shows inducible expression, and competes cells might have similar function to DcR3.Fc and could directly with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes; LT␤R, LT␤ receptor; DC, dendritic cell; sLIGHT, soluble LIGHT; suppress host anti-tumor immunity by altering DCs function and CD40L, CD40 ligand. skewing the immune response from Th1 to Th2.

Materials and Methods incubated at 4°C for 20 min with 50 ␮l UltraAvidin-PE (Leinco Technol- Production of receptor.Fc fusion proteins ogies, Ballwin, MO) or streptavidin-FITC (BD PharMingen) diluted (1/200) in FACS staining/washing buffer. The sources of mAbs are as LT␤R.Fc protein was produced as previously described (10). To generate follows: anti-CD1a-FITC (clone HI149; BD PharMingen), anti-CD11c-PE the DcR3.Fc, the open reading frame of the human DcR3 gene was isolated (B-ly6; BD PharMingen), anti-CD54 (clone 8.4A6; Ancell, Bayport, MN), by RT-PCR using the forward primer: 5Ј-GGAATTCAAGGACCAT anti-CD80-PE (clone L307.4; BD PharMingen), anti-CD83-FITC (clone GAGGGCGCTG-3Ј and the reverse primer: 5Ј-GGAATTCGTGCACA HB15e; BD PharMingen), anti-CD86-PE (clone 2331 FUN-1; BD PharM- GGGAGGAAGCGC-3Ј. The amplified product was ligated in-frame into ingen), anti-CD40-FITC (clone LOB716; Serotec, Oxford, U.K.), anti- the EcoRI-cut pUC19-IgG1-Fc vector containing the cDNA of the human HLA-DR-FITC (clone B-F1; Serotec), anti-CD14-PE mAb (clone IgG1 Fc. The fusion gene was then subcloned into the pBacPAK9 vector UCHM1; Serotec), biotin-conjugated anti-FasL (clone NOK-1; BD Phar- (Clontech Laboratories, Palo Alto, CA) and cotransfected with linearized Mingen), anti-TNF-␤/LT-␣ mAb (clone 9B9; Boehringer Mannheim, BacPAK6 DNA (Clontech Laboratories) into Sf21 cells. The supernatant Mannheim, Germany), and anti-DC-lysosome-associated membrane gly- from recombinant virus-infected Sf21 cells was filtered and purified on coprotein mAb (clone 104.G4; Immunotech, Marseille, France). protein A-Sepharose beads. The bound DcR3.Fc protein was then eluted with 0.1 M glycine buffer (pH 3.0) followed by dialysis against PBS. Immunoprecipitation of FasL and LIGHT Culture of CD14ϩ monocyte-derived DCs and preparation of COS7 cells were transfected with the pFLAG-FasL, pFLAG-LIGHT, or ϩ pFLAG-CMV2 vector by the calcium phosphate method. Three days after CD4 T cells transfection, cells were harvested and resuspended by lysis buffer (1% PBMCs were isolated by standard density gradient centrifugation with Ficoll- Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8), 1 mM PMSF, 2 ␮ ␮ Paque (Amersham Pharmacia Biotech, Piscataway, NJ) from the heparinized g/ml aprotinin, and 2 g/ml leupeptin), followed by incubation with anti- ϩ ␤ whole blood of normal individuals. Subsequently, CD14 cells were purified LIGHT polyclonal Ab (11), DcR3.Fc, or LT R.Fc. Immunoprecipitates

by high-gradient magnetic sorting using the VARIOMACS technique with were collected on protein A beads (Amersham Pharmacia Biotech). Sam- Downloaded from anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Im- ples were fractionated by SDS-PAGE and then probed with anti-FLAG M2 mature DCs were generated from adherent PBMCs by culture in RPMI 1640 Ab (Sigma-Aldrich) by Western blot analysis. medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS Cytotoxicity assay (Life Technologies), 800 U/ml human GM-CSF (Leucomax; Schering- Plough, Kenilworth, NJ), and 500 U/ml human IL-4 (R&D Systems, Minne- HT-29 cells were seeded in ﬂat-bottom 96-well microtitration plates apolis, MN) in the presence or absence of human IgG1 (3 ␮g/ml; Sigma- (Costar) at a density of 5 ϫ 103/well overnight at 37°C, followed by in- Aldrich, St. Louis, MO), LT␤R.Fc (3 ␮g/ml), or DcR3.Fc (3 ␮g/ml) for 6 days

cubation with recombinant soluble LIGHT (sLIGHT) (25 ng/ml) and http://www.jimmunol.org/ (immature DCs). To prepare mature activated DCs, immature DCs were fur- IFN-␥ (10 U/ml; Boehringer Mannheim), in conjunction with IgG1 (1 ␮g/ ther incubated with gamma-irradiated (5500 rad) CD40 ligand (CD40L)-ex- ml), TNFRI.Fc (1 ␮g/ml), LT␤R.Fc (1 ␮g/ml), or DcR3.Fc (1 ␮g/ml) for pressing L cells (DNAX Research Institute, Palo Alto, CA) at a ratio of 3:1 for 4 days. To quantitate FasL-induced apoptosis, cells were cultured in flat- ϩ ϩ 36 h. To purify naive CD4 CD45RA T cells, PBMCs were first isolated by bottom 96-well microtitration plates (Costar) at a density of 105/well in the ϩ ϩ Ficoll-Paque centrifugation, and CD4 cells were then enriched by a CD4 T presence of FasL (Upstate Biotechnology, Lake Placid, NY) and IgG1, ϩ cell isolation kit (Miltenyi Biotec). After depletion of non-CD4 T cells, CD4 Fas.Fc, DcR3.Fc, or LT␤R.Fc in the concentration of 1 ␮g/ml for 16 h. The T cells were then positively selected by CD45RA microbeads (Miltenyi Bio- survival rate was determined by MTT assay as previously described (26). tec) using the VARIOMACS technique. The purity of naive CD4ϩCD45RAϩ Survival rate was determined by OD570 of cells treated with FasL vs OD570 T cells was over 95% by flow cytometry analysis. of cells cultured in medium only.

Allogeneic MLR by guest on September 25, 2021 CD14ϩ monocyte-derived DCs were harvested and gamma-irradiated (3000 Results ϩ ϩ rads) followed by incubation with 5 ϫ 104 allogeneic CD4 CD45RA naive Modulatory effect of DcR3 on DC differentiation and maturation T cells/well in U-bottom 96-well microtitration plates (Costar, Cambridge, MA) at ratios of 1:10 to 1:300. After 4 days, [3H]thymidine (Amersham Phar- It has been reported that DcR3 is up-regulated in certain tumors macia Biotech) was added (0.5 ␮Ci/well) and the cells were incubated for (1–3, 6, 7) and tumor-associated DCs usually have a low T cell another 16 h. The cells were harvested on a cell harvester (Skatron, Lier, stimulatory capacity (reviewed by Banchereau et al.; Ref. 27). Norway), and the incorporated radioactivity was measured using a beta Therefore, we asked if DcR3 could modulate the functions of DCs. counter (model LS3801; Beckman Coulter, Brea, CA). We first tested the binding specificity of the DcR3.Fc to FasL and ϩ ϩ In vitro stimulation of CD4 CD45RA T cells LIGHT. DcR3.Fc precipitated both FasL and LIGHT (Fig. 1A), CD4ϩCD45RAϩ naive T cells (2 ϫ 105/well) were plated into U-bottom and both DcR3.Fc and Fas.Fc inhibited activation-induced apopto- ϩ 96-well microtitration plates (Costar) and cultured with gamma-irradiated sis of Jurkat cells and CD4 T cells (Fig. 1, B and C). Furthermore, DCs (at a DC-T ratio of from 1:10 to 1:300). After 3 days, half of the DcR3.Fc inhibited LIGHT and IFN-␥-mediated apoptosis of culture medium was replaced by fresh RPMI 1640 medium (Life Tech- HT-29 cells to a similar degree as LT␤R.Fc did (Fig. 1D). To nologies). On day 6, cells were washed with PBS and incubated with PMA (10 ng/ml) and A23187 (1 ␮g/ml) for 24 h. The supernatants were har- further confirm the binding specificity between DcR3.Fc and FasL, vested and stored at Ϫ20°C. The concentrations of IL-4 and IFN-␥ were Jurkat cells were treated with rFasL in the presence of receptor.Fc measured by OptEIA ELISA (BD PharMingen, San Diego, CA). fusion proteins. As shown in Fig. 1E, DcR3.Fc has a similar effect Analysis of marker expression and cytokine secretion by DC as that of Fas.Fc to inhibit FasL-induced Jurkat cell apoptosis. These results indicate that DcR3.Fc interacted with FasL and ϩ Before staining, CD14 monocyte-derived DCs were harvested and LIGHT, and that it possessed the same binding specificity as pre- washed twice with FACS staining/washing buffer (1% FCS and 0.1% NaN3 in PBS), followed by incubation with various mAbs or anti-LIGHT poly- viously described (1, 2, 24, 25). clonal Ab (11) in staining buffer at 4°C for 20 min. For the samples that We then tested the functions of DcR3 in modulating DC mat- ϩ were incubated with nonfluorochrome-conjugated Abs, cells were then in- uration and differentiation. CD14 monocytes were differentiated cubated with appropriate FITC-conjugated secondary Abs at 4°C for 20 into immature DCs by culture with GM-CSF and IL-4 in conjunc- min after being washed three times with FACS staining/washing buffer. tion with DcR3.Fc, LT␤R.Fc, Fas.Fc, or human IgG1 for 6 days. Cells were fixed with 1% paraformaldehyde in PBS for 30 min at 4°C before the fluorescence was analyzed with a FACSort (BD Biosciences, The cells were then stimulated with CD40L to induce DC matu- Mountain View, CA). Alternatively, cells were stained with biotinylated ration. We found that DcR3.Fc, but not LT␤R.Fc, Fas.Fc (data not DcR3.Fc or LT␤R.Fc as follows: CD14ϩ monocytes and DCs were first shown) or human IgG1 modulated DC differentiation and matura- 6 incubated with 100 ␮l human IgG (100 ␮g/10 cells; Calbiochem, San tion (Fig. 2). Before CD40L stimulation, it was apparent that the Diego, CA) in FACS staining/washing buffer at 4°C for 10 min to prevent nonspecific binding, followed by the addition of 2 ␮g of biotinylated expression of CD1a, a hallmark of the DC phenotype, was sup- DcR3.Fc, LT␤R.Fc, or IgG1 in 50 ␮l FACS staining/washing buffer. After pressed by DcR3.Fc in immature DCs in a dose-dependent manner washing with FACS staining/washing buffer three times, cells were further (Fig. 2A, upper panel), while CD86/B7.2 was up-regulated in a 4848 DcR3 MODULATES DENDRITIC CELL DIFFERENTIATION

FIGURE 1. Speciﬁc interaction between DcR3. Fc and its ligands. A, Im- munoprecipitation of FasL and LIGHT by DcR3.Fc. COS7 cells were transfected with pFLAG-FasL, pFLAG- LIGHT, or pFLAG-CMV2 by the calcium phosphate method. After 3 days, cell lysates were incubated with anti- LIGHT polyclonal Ab, DcR3.Fc or LT␤R.Fc, followed by incubation with protein A-conjugated Sepharose beads. Finally, the immunoprecipitates were fractionated on SDS-PAGE and probed with anti-FLAG M2 mAb. B and C, In- hibition of activation-induced apoptosis in Jurkat cells and CD3ϩ T cells by DcR3.Fc. Jurkat cells (106/well) were stimulated with PMA (20 ng/ml) and Downloaded from ionomycin (2 ␮g/ml) for 18 h (B), while CD4ϩ T cells (5 ϫ 105/well) were activated with immobilized anti- CD3 (20 ␮g/ml) for 5 days (C), in 96- well microtitration plates, followed by restimulation with OKT-3 (20 ␮g/ml) for 24 h in the presence of Fas.Fc (10 http://www.jimmunol.org/ ␮g/ml), DcR3.Fc (10 ␮g/ml), or human IgG1 (10 ␮g/ml). Percentages of cell death were determined by PI staining. Statistical analysis by two-tailed Stu- dent’s t test revealed signiﬁcant differ- ences between IgG1- and DcR3.Fc- p Ͻ 0.05). The data ,ء) treated samples represent the mean Ϯ SD for three ex-

periments. D, Inhibition of LIGHT-me- by guest on September 25, 2021 diated apoptosis in HT-29 cells. HT-29 cells were incubated with sLIGHT (25 ng/ml) and IFN-␥ (10 U/ml) in the presence of IgG1 (1 ␮g/ml) or Fc fusion proteins (1 ␮g/ml) for 4 days, and the survival rate was determined as described in Materials and Methods. One representative experiment of three is p Ͻ 0.05 when compared ,ء .shown p Ͻ ,ءء ;with IFN-␥ treated samples 0.05 when compared with IFN-␥ and sLIGHT treated samples. E, Neutral- ization of FasL-induced apoptosis in Jurkat cells. Jurkat cells were treated with different concentrations of FasL in the presence of IgG1 (1 ␮g/ml), or Fc fusion proteins (1 ␮g/ml) for 16 h, and the cell survival rate was determined by MTT assay.

dose-dependent manner (Fig. 2A, lower panel). The modulatory the expression of HLA-DR was down-regulated by DcR3.Fc in effect of DcR3.Fc on the expression of CD1a and CD86/B7.2 immature DCs (mean fluorescence intensity ϭ 55 Ϯ 18) compared reaches a plateau at 3 ␮g/ml (Fig. 2A). with those treated with IgG1 (mean fluorescence intensity ϭ We further tested the modulatory effect of DcR3.Fc on the ex- 132 Ϯ 36) and LT␤R.Fc (mean fluorescence intensity ϭ 127 Ϯ pression of other activation and differentiation markers of DCs 18) (Fig. 2B and Table I). Compared with IgG1, it is interesting to (Fig. 2, B and C and Table I). It is interesting to note that the note that DcR3.Fc increased the expression of CD86/B7.2 on im- expression of DC-lysosome-associated membrane glycoprotein (a mature DCs up to 2-fold when compared with control proteins. By marker of mature DCs) was up-regulated by DcR3.Fc, but not by contrast, the expression of CD11c, a lineage marker of myeloid LT␤R.Fc or IgG1 before CD40L stimulation (Fig. 2B). In contrast, cells, was unaffected under the same condition (data not shown). The Journal of Immunology 4849 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Modulation of surface marker expression and cytokine secretion of DCs by DcR3.Fc. A, CD14ϩ monocytes were cultured with GM-CSF (800 U/ml) and IL-4 (500 U/ml) for 6 days to differentiate them into immature DCs in the presence of various concentrations of IgG1 and DcR3.Fc. Surface marker expression was analyzed by flow cytometry analysis and the mean fluorescence intensity was calculated by CellQuest software (BD Biosciences). p Ͻ 0.05 when compared with IgG1-treated samples. The data represent the mean Ϯ SD for three experiments. B and C, Human IgG1 (3 ␮g/ml), DcR3.Fc ,ء (3 ␮g/ml), or LT␤R.Fc (3 ␮g/ml) were added to the culture of DCs in the presence of GM-CSF (800 U/ml) and IL-4 (500 U/ml) (denoted as GMIL4). Surface markers expressed by DCs before CD40L stimulation (B) or after CD40L stimulation (C) were analyzed by flow cytometry analysis (shaded histograms, isotype control Abs; open histograms, specific Abs). Dead cells were gated out by propidium iodide staining. One representative experiment of three is shown.

When DcR3.Fc treated-immature DCs were stimulated with the T cells were incubated with DcR3.Fc-treated mature DCs at CD40L for 36 h, the expression of CD54/ICAM-1, CD80/B7.1 and DC-T ratios of 1:10 and 1:30 (Fig. 3A, upper panel), or with CD86/B7.2 were all up-regulated, while the expression of CD1a DcR3.Fc-treated immature DCs at DC-T ratios of 1:10 (Fig. 3A, was not affected by CD40L stimulation. The expression of CD40 lower panel). is only slightly up-regulated before and after CD40L stimulation In addition to its suppressive effect on T cell proliferation, (mean ﬂuorescence intensity ϭ 17 Ϯ 11 vs 31 Ϯ 23). Compared DcR3.Fc also modulated the secretion of IFN-␥ and IL-4 of with IgG1- and LT␤R.Fc-treated mature DCs, the expression of CD4ϩCD45RAϩ naive T cells. We found that DcR3.Fc-treated CD1a, CD54/ICAM-1, HLA-DR, and CD80/B7.1 is still sup- immature DCs enhanced IL-4 secretion (1.5-fold) compared with pressed, but the expression of CD86/B7.2 is further up-regulated IgG1- or LT␤R.Fc-treated immature DCs (Fig. 3B, lower panel). by DcR3.Fc (Fig. 2C and Table I). Therefore, DcR3.Fc, but not In contrast, DcR3.Fc-treated immature DCs did not signiﬁcantly LT␤R.Fc, profoundly affected the differentiation and maturation affect IFN-␥ secretion (Fig. 3B, upper panel). Therefore, DcR3.Fc- of DCs. treated immature DCs increased the IL-4-IFN-␥ ratio via the en- hancement of IL-4 production in CD4ϩCD45RAϩ naive T cells. Th2 polarization by DcR3-primed DCs We then tested the effect of DcR3-treated mature DCs on IFN-␥ Because DcR3.Fc modulated the expression of surface molecules and IL-4 secretion from CD4ϩCD45RAϩ naive T cells. To ad- important for Ag presentation, we asked the effect of DcR3.Fc- dress this aspect, immature DCs were stimulated with CD40L for treated DC to modulate T cell proliferation and differentiation. As 36 h to induce DC maturation, followed by incubation with allo- shown in Fig. 3A, DcR3.Fc suppressed T cell proliferation when geneic CD4ϩCD45RAϩ naive T cells at various DC-T ratios. We 4850 DcR3 MODULATES DENDRITIC CELL DIFFERENTIATION

Table I. Mean ﬂuorescence intensity of surface markers on receptor.Fc-treated DCsa

Mean Fluorescence Intensity CD1a CD40 CD54 HLA-DR CD80 CD86

GMIL4 73 Ϯ 16 31 Ϯ 975Ϯ 16 105 Ϯ 23 20 Ϯ 576Ϯ 15 GMIL4/IgG1 80 Ϯ 24 29 Ϯ 10 83 Ϯ 18 132 Ϯ 36 21 Ϯ 372Ϯ 14 GMIL4/DcR3.Fc 10 Ϯ 417Ϯ 11 67 Ϯ 15 55 Ϯ 18 10 Ϯ 4 162 Ϯ 43 GMIL4/LT␤R.Fc 57 Ϯ 18 27 Ϯ 11 76 Ϯ 19 127 Ϯ 18 21 Ϯ 384Ϯ 17 GMIL4/CD40L 40 Ϯ 14 47 Ϯ 17 210 Ϯ 62 343 Ϯ 31 54 Ϯ 15 113 Ϯ 52 GMIL4/IgG1/CD40L 38 Ϯ 18 46 Ϯ 14 238 Ϯ 66 287 Ϯ 27 43 Ϯ 16 117 Ϯ 48 GMIL4/DcR3.Fc/CD40L 9 Ϯ 331Ϯ 23 118 Ϯ 36 75 Ϯ 822Ϯ 9 237 Ϯ 52 GMIL4/LT␤R.Fc/CD40L 31 Ϯ 13 40 Ϯ 14 219 Ϯ 61 298 Ϯ 39 46 Ϯ 4 112 Ϯ 42

a CD14ϩ monocytes were cultured in the presence of GM-CSF and IL-4 (GMIL4), or in conjunction with 3 ␮g/ml of DcR3.Fc, or LT␤R.Fc. After 6 days, the developed immature DCs were further stimulated with CD40L-expressing L cells in the presence of Fc fusion proteins for 36 h. The expression of surface markers was analyzed by ﬂow cytometry and the mean ﬂuorescence intensity was calculated by CellQuest software (BD Biosciences). The data represents the mean Ϯ SD from six independent experiments.

found that DcR3.Fc-treated mature DCs did not affect the secretion DcR3 binds to a novel ligand distinct from FasL and LIGHT Downloaded from of IFN-␥ at a DC-T ratio of 1:30 (Fig. 3C, upper panel). Consis- tently, DcR3.Fc-treated mature DCs enhanced IL-4 secretion at a In previous experiments, we demonstrated that DcR3.Fc, but not ␤ DC-T ratio of 1:30 (Fig. 3C, lower panel) to 2-fold compared with LT R.Fc, could modulate the expression of several surface mol- ϩ ϩ IgG1- or LT␤R.Fc-treated mature DCs. Therefore, DcR3-treated ecules and enhance IL-4 secretion of CD4 CD45RA naive T mature DCs also increased the IL-4-IFN-␥ ratio by enhancing IL-4 cells. This suggested that DcR3.Fc and LT␤R.Fc might bind to secretion. According to this data, it is suggested that DcR3.Fc distinct molecules to execute their respective functions. To address http://www.jimmunol.org/ might cause both immature and mature DCs to skew the immune this aspect, we used anti-LIGHT, anti-LT␣, and anti-FasL Abs, as response toward Th2 development. well as DcR3.Fc and LT␤R.Fc fusion proteins, to stain CD14ϩ by guest on September 25, 2021

FIGURE 3. Modulatory effects of DcR3.Fc on T cell proliferation in MLR. A, Allogeneic CD4ϩ T cells were cultured with gamma-irradiated DCs in 96-well microtitration plates for 4 days at different DC-T ratios of 1:10 (Ⅺ) and 1:30 (f), and the T cell proliferation was measured as described in Materials and Methods. B and C, The concentrations of IFN-␥ and IL-4 secreted by CD4ϩCD45RAϩ naive T cells which cocultured with immature DCs (B) or with p Ͻ ,ء ;(mature DCs (C) were determined by ELISA. One representative experiment of three is shown. GMIL4: GM-CSF (800 U/ml) and IL-4 (500 U/ml 0.05 when compared with untreated or IgG1-treated DCs. The Journal of Immunology 4851

The information obtained in this study suggests that DcR3.Fc, when cultured with CD14ϩ monocyte-derived DCs in vitro, can modulate the expression of surface molecules, reduce T cell proliferation, and enhance IL-4 production of CD4ϩCD45RAϩ naive T cells. In contrast, neither LT␤R.Fc nor Fas.Fc had a similar effect, so DcR3.Fc might modulate DC function by interacting with a non-FasL non-LIGHT molecule expressed, at least, on monocytes and DCs. Because DcR3 is up-regulated in cancer patients, DcR3 might be one of the factors responsible for immuno- suppression found in cancer patients.

Discussion Tumor cells can produce factors that promote blood vessel growth (neovascularization) to meet their increasing demand for oxygen and nutrients. In addition, tumor cells express many immunosup- pressive factors, such as TGF-␤, IL-10, DF3/MUC1 (28), and RCAS1 (29), to suppress the host immune response and facilitate tumor growth. DcR3, a decoy receptor capable of neutralizing FasL and LIGHT, has been reported to be overexpressed in tumor Downloaded from cells originating from the gastrointestinal tract and the pulmonary system (1, 3). Thus, it has been speculated that DcR3 might inhibit host immune responses by neutralizing the cytotoxic effects of FasL and LIGHT. A recent study demonstrated that both DcR3.Fc and rDcR3 have

the same binding affinity to LIGHT and have the same inhibitory http://www.jimmunol.org/ effect on the development of CTL in mice (24) indicating that the biological effect of DcR3.Fc is equivalent to rDcR3 protein. There- fore, the modulatory effect of DcR3.Fc observed in this study should be able to reflect the function of DcR3. In this study, we FIGURE 4. Characterization of the interaction of DcR3.Fc with cell sur- demonstrated that DcR3.Fc could modulate DC differentiation and face molecules. Monocytes (A) and CD14ϩ monocyte-derived immature and activation via a ligand distinct from FasL and LIGHT. Because ϩ mature DCs (B) were stained with the following Abs: anti-LIGHT polyclonal FasL is not expressed on CD14 monocyte-derived DCs cultured Ab, anti-LT␣ mAb, and biotinylated anti-FasL mAb, followed by FITC-con- with GM-CSF and IL-4, and LT␤R.Fc did not affect CD14ϩ jugated secondary Abs or PE-conjugated UltraAvidin. Alternatively, cells monocyte-derived DCs in a similar fashion as DcR3.Fc, the mod- by guest on September 25, 2021 were stained with biotinylated receptor. Fc fusion proteins (LT␤R.Fc and ulatory effects of DcR3 cannot be attributed to neutralization of DcR3.Fc) or biotinylated IgG1, followed by incubation with either PE-conju- LIGHT or FasL. Therefore, our observations suggest that DcR3 gated UltraAvidin or FITC-conjugated streptavidin. The mean fluorescence may act as an effector molecule to modulate DC functions via its intensity of cells was analyzed by FACSort and by CellQuest software (BD binding to surface molecules to trigger “reverse signaling” as Biosciences). Shaded histograms, control staining; open histograms, specific staining. One representative experiment of three is shown. found for other members of the TNF superfamily (12–23). How- ever, we cannot rule out the possibility that DcR3 might interact with an unidentified molecule capable of triggering signals to induce DC differentiation, thus the addition of DcR3 blocks this signaling path- monocytes and immature and mature DCs to define the molecule way and results in the failure to follow the normal process of DC that interacts with DcR3.Fc. As shown in Fig. 4A, DcR3.Fc, but differentiation and maturation in the in vitro culture system. not LT␤R.Fc, bound freshly isolated CD14ϩ monocytes. In con- One of the most striking effects of DcR3.Fc on DC differenti- trast, anti-LIGHT, anti-LT␣, and anti-FasL Abs did not bind ation was the up-regulation of CD86/B7.2 and down-regulation of CD14ϩ monocytes. This suggested that DcR3.Fc could bind to a CD80/B7.1. In addition to activation through TCR by the Ag- surface molecule distinct from LIGHT, the membrane form of LT, MHC complex, the costimulatory signal induced by interaction of and FasL on CD14ϩ monocytes. CD28 or CTLA-4 on the T cell surface with either CD80/B7.1 or DcR3.Fc, LT␤R.Fc, and anti-LIGHT polyclonal Ab all bound to CD86/B7.2 on APC is required for T cell proliferation and cyto- immature DCs. In contrast, only DcR3.Fc bound to mature DCs kine secretion. It has been reported that IFN-␥ could up-regulate, (Fig. 4B). In a previous study, we showed that LIGHT is expressed while IL-10 could down-regulate, both CD80/B7.1 and CD86/ on the surface of immature DCs, but is shed from mature DCs and B7.2 expression (30, 31). Thus, the unique feature of DcR3.Fc is barely detectable by LT␤R.Fc (9). DcR3.Fc detected strong flu- up-regulation of CD86/B7.2 with simultaneous down-regulation of orescence signals in both immature and mature DCs, while CD80/B7.1 might be an invaluable tool to dissect the underlying LT␤R.Fc did not detect any signal in mature DCs, and only a weak mechanism for the regulation of CD80/B7.1 and CD86/B7.2 ex- signal could be detected in immature DCs. From this, we conclude pression. Furthermore, it has been demonstrated that even though that the molecule detected by DcR3.Fc on mature DCs is distinct CD80/B7.1 and CD86/B7.2 equivalently costimulate IL-2 and from LIGHT, indicating that DcR3 binds to both LIGHT and an IFN-␥ production, CD86/B7.2 preferentially induced more IL-4 unidentified novel ligand in immature DCs. Because FasL was not production than CD80/B7.1 (32, 33). In this study, we clearly dem- expressed on freshly isolated monocytes and CD14ϩ monocyte- onstrated that DcR3 up-regulated CD86/B7.2 and down-regulated derived DCs cultured with GM-CSF and IL-4, we conclude that CD80/B7.1 expression on DCs. In addition, DcR3.Fc-treated DCs the specific signal detected by DcR3.Fc on monocytes and mature dramatically enhanced IL-4 secretion by CD4ϩCD45RAϩ naive T DCs must differ from those of LIGHT and FasL. cells. Thus, our observation is in accordance with previous reports 4852 DcR3 MODULATES DENDRITIC CELL DIFFERENTIATION that CD86/B7.2 preferentially activates IL-4 expression and Th2 pression of a decoy receptor for Fas ligand (DcR3) in virus (EBV or HTLV-I) development (32–34). In addition to CD80/B7.1and CD86/B7.2, it associated lymphomas death and decoy receptors and p53-mediated apoptosis. Cancer Lett. 160:89. has been reported that blocking or absence of LFA-1 (CD11a/ 7. Roth, W., S. Isenmann, M. Nakamura, M. Platten, W. Wick, P. Kleihues, CD18)/ICAM-1 (CD54) interaction promotes IL-4 secretion (35– M. Bahr, H. Ohgaki, A. Ashkenazi, and M. Weller. 2001. Soluble decoy receptor 37). In DcR3.Fc-treated mature DC, we found that the expression 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apo- level of CD54/ICAM-1 is suppressed (mean fluorescence inten- ptosis and chemotaxis. Cancer Res. 61:2759. sity ϭ 118 Ϯ 36), compared with IgG1- (mean fluorescence in- 8. Otsuki, T., A. Tomokuni, H. Sakaguchi, T. Aikoh, T. Matsuki, Y. Isozaki, F. Hyodoh, H. Ueki, M. Kusaka, S. Kita, and A. Ueki. 2000. Over-expression of tensity ϭ 238 Ϯ 66) or LT␤R.Fc- (mean fluorescence intensity ϭ the decoy receptor 3 (DcR3) gene in peripheral blood mononuclear cells (PBMC) 219 Ϯ 61) treated mature DCs (Table I). Therefore, another mech- derived from silicosis patients. Clin. Exp. Immunol. 119:323. anism of DcR3.Fc-treated DC to induce Th2 differentiation might 9. Tamada, K., K. Shimozaki, A. I. Chapoval, Y. Zhai, J. Su, S. F. Chen, be through the down-regulation of CD54/ICAM-1, resulting in the S. L. Hsieh, S. Nagata, J. Ni, and L. Chen. 2000. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allo- enhanced secretion of Th2 cytokine. However, we cannot rule out geneic T cell response. J. Immunol. 164:4105. the possibility that other molecules are also responsible for the Th2 10. Zhai, Y., R. Guo, T. L. Hsu, G. 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