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The Journal of Immunology, 2005, 175: 5135-5145.
Copyright © 2005 by The American Association of Immunologists

Attenuation of Th1 Response in Decoy Receptor 3 Transgenic Mice1

Tsui-Ling Hsu*, Ying-Yu Wu*,{dagger}, Yung-Chi Chang*, Chih-Ya Yang*, Ming-Zong Lai{dagger}, Wenlynn B. Su* and Shie-Liang Hsieh2,*,{ddagger},§

* Institute of Microbiology and Immunology, National Yang-Ming University, {dagger} Institute of Molecular Biology, Academia Sinica, {ddagger} Genomics Research Center, Academia Sinica, and § Immunology Research Center, Taipei Veterans General Hospital, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The soluble decoy receptor 3 (DcR3) is a member of the TNFR superfamily. Because DcR3 is up-regulated in tumor tissues and is detectable in the sera of cancer patients, it is regarded as an immunosuppressor to down-regulate immune responses. To understand the function of DcR3 in vivo, we generated transgenic mice overexpressing DcR3 systemically. In comparison with HNT-TCR (HNT) transgenic mice, up-regulation of IL-4 and IL-10 and down-regulation of IFN-{gamma}, IL-12, and TNF-{alpha} were observed in the influenza hemagglutinin126–138 peptide-stimulated splenocytes of HNT-DcR3 double-transgenic mice. When infected with Listeria monocytogenes, DcR3 transgenic mice show attenuated expression of IFN-{gamma} as well as increased susceptibility to infection. The Th2 cell-biased phenotype in DcR3 transgenic mice is attributed to decreased IL-2 secretion by T cells, resulting in the suppression of IL-2 dependent CD4+ T cell proliferation. This suggests that DcR3 might help tumor growth by attenuating the Th1 response and suppressing cell-mediated immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Decoy receptor 3 (DcR3)3/TR6/M68 was discovered in a search of the human genome database for sequences with homology to the TNFR superfamily (1, 2) as well as in a screening to identify novel secreted proteins (3). It competes for the binding of the following ligands to their respective receptors: Fas ligand (FasL) with Fas, LIGHT (receptor homologous to lymphotoxins that exhibits inducible expression, competes with HSV glycoprotein D for the HVEM, and is expressed by T lymphocytes) with herpesvirus entry mediator (HVEM) and lymphotoxin-{beta} receptor (LT{beta}R), and TNF-like molecule 1A (TL1A) with death receptor 3 (DR3) (1, 2, 4). There is strong evidence that DcR3 is overexpressed in various tumors, including lung and colon cancers (1), gastrointestinal tract tumors (3), virus-associated lymphomas (5), malignant gliomas (6), and pancreatic cancers (7). Wu et al. (8) reported that 56% of tumor patients are serum DcR3 positive, and >70% of patients with gastric, liver, and gallbladder carcinomas have elevated serum DcR3 levels (>20 pg/ml). In addition, overexpression of DcR3 has been reported in cases of silicosis or systemic lupus erythematosus (9) as well as in bacterial Ag-stimulated monocytes and myeloid DCs (10). DcR3 can block the effects of its known ligands (FasL, LIGHT, and TL1A) and contributes to tumor growth by impeding the immune response as well as inducing angiogenesis (11). In contrast, DcR3 can also act as a regulator for the differentiation and maturation of myeloid cells, possibly through interaction with a novel ligand(s) (12, 13, 14, 15, 16).

In a previous study we demonstrated that Fc-tagged human DcR3 (DcR3.Fc) has strong modulatory effects on DC differentiation and activation and gives rise to induction of a Th2-phenotype in naive CD4+ T cells stimulated by DcR3.Fc-treated human monocyte-derived DCs (12). In line with observations in the human system, Wu et al. (13) reported that DcR3.Fc also modulates the differentiation and activation of mouse bone marrow-derived DCs (BMDCs), and that DcR3.Fc-treated mouse BMDCs induce an increased IL-4/IFN-{gamma} ratio when cocultivated with T cells. Accordingly, DcR3.Fc-treated BMDCs inhibit the onset of diabetes, a Th1-related autoimmune disease, when adoptively transferred into NOD mice (13). To further investigate the therapeutic potential of DcR3 in autoimmune diabetes, Sung et al. (17) generated transgenic NOD mice that specifically express DcR3 in pancreatic {beta} cells. It is clear that DcR3 prevents the onset, disease progression, and severity of spontaneous and cyclophosphamide-induced diabetes. Interestingly, a decline in the percentage of Th1 cells in the pancreatic lymph nodes (LNs) of DcR3 transgenic NOD mice was observed (17). However, this phenotype appears to be very localized, because the number of Th1 cells is unchanged in the spleens of these mice. DcR3 protein is undetectable in the sera of DcR3-overexpressing NOD mice, confirming the localized effect of pancreatic DcR3. Given that serum levels of DcR3 increase in tumor patients as well as in patients with Th2-associated allergic diseases, such as atopic dermatitis, atopic rhinitis, and asthma (18), DcR3 might act as a systemic regulator. To investigate the systemic effects of DcR3 in vivo, transgenic mice that express DcR3 under the control of ubiquitously expressed phosphoglycerate kinase (PGK) promoter were generated.

In this study we report that DcR3 transgenic mice show a tendency for Th2-biased immune responses, which is dominated by an increased IL-4/IFN-{gamma} ratio and reduced expression of IFN-{gamma} in Listeria monocytogenes-infected mice; DcR3 transgenic APCs are a major factor in eliciting this response. Therefore, the DcR3 transgenic mice generated in this study provide a useful model system to mimic the overexpression of DcR3 in human pathological conditions and will facilitate the study of its modulatory effect on the host immune response in patients suffering from cancers and autoimmune diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of DcR3 transgenic mice

The PGK promoter was used to drive the expression of the DcR3 transgene. The human DcR3-coding sequence was amplified by PCR (forward primer, 5'-GGAATTCAAGGACCATGAGGGCGCTG-3'; reverse primer: 5'-GGAATTCGTGCACAGGGAGGAAGCGC-3') and subcloned into PstI- and XbaI-cleaved pPGK-NeoR-bpA (19) by blunt end ligation after removal of the NeoR fragment. The transgene was excised and microinjected into FVB-fertilized eggs. DcR3 transgenic mice were screened by PCR using the primers pGKP (sense; 5'-GCCAATAGCAGCTTTGCTC-3') and DcR3–207 (antisense; 5'-TAGGTGGGTGTTTCTGCCAC-3'). HNT transgenic mice on a BALB/c background were obtained from Dr. H. McDevitt (Stanford University, Stanford, CA). Mice carrying the pPGK-DcR3 transgene were backcrossed to BALB/c or HNT transgenic mice (BALB/c background) for at least six generations. Screening of HNT transgenic mice was performed by two-color flow cytometry, using mAbs against murine (m) TCRv{beta}8.3 and CD4. Samples in which >50% of CD4+ cells were TCRv{beta}8.3+ were considered to be HNT positive. MRL/MpJ-lpr/lpr mice were obtained from The Jackson Laboratory and were bred with HNT transgenic mice on a BALB/c background for at least five generations. All mice were bred and housed at the laboratory animal center of National Yang-Ming University. The animal study was approved by the institutional animal care and use committee of National Yang-Ming University. DcR3 transcript was detected by RT-PCR using PBMC cDNA as a template to amplify a 230-bp product: forward primer, 5'-CTCAATGTGCCAGGCTCT-3'; and reverse primer, 5'-AGCTTCAGCTG CAAGGCC3'. Control amplifications were performed using {beta}-actin-specific primers (forward, 5'-GACTACCTCATGAAGATCCT-3'; reverse, 5'-CCACATCTGCTGGAAGGTGG-3').

Fusion proteins and Abs

Recombinant LT{beta}R.Fc, Fas.Fc, and DcR3.Fc proteins were produced as previously described (12, 20, 21). To prevent any response due to receptor.Fc fusion proteins binding to FcRs on murine splenocytes (SPCs), the extracellular domains of human LT{beta}R, Fas, DcR3, and DR3 were amplified by PCR and subcloned into the pcDNA3.1+mIgG2b.Fcmut vector (a gift from Dr. H.-H. Lin, Sir William Dunn School of Pathology, University of Oxford, Oxford, U.K.). The resulting receptor.Fc constructs produce recombinant proteins that are fused with a mutated mIgG2b Fc portion, which does not bind to mFcRs. These receptor.Fc proteins were overexpressed using the FreeStyle 293 Expression System (Invitrogen Life Technologies) and purified on protein A columns. Recombinant human LIGHT was prepared as previously described (20). The cDNAs encoding the human and murine TL1A extracellular domains were PCR amplified and cloned into pFLAG-CMV-1. Proteins were overexpressed and purified using the FreeStyle 293 Expression System and anti-FLAG M2 affinity gels (Sigma-Aldrich), respectively. To generate anti-DcR3 mAb, mice were immunized with DcR3.Fc, and the positive hybridoma clones were selected by ELISA. Recombinant mFasL (aa 132–279) and mLIGHT (aa 72–239) proteins were purchased from R&D Systems. Anti-mCD4-CyChrome (L3T4; clone RM4-5), anti-mTCRv{beta}8 (clone F23.1), anti-mIFN-{gamma}-FITC (clone XMG1.2), anti-mIL-4-PE (clone 11B11), and anti-mCD16/32 (clone 2.4G2) mAbs were purchased from BD Biosciences. Anti-mCD4-allophycocyanin (L3T4; clone RM4-5) was purchased from eBioscience. Anti-mTCR{beta} (clone H57.597) was obtained from American Type Culture Collection.

Detection of DcR3 protein by Western blot and ELISA

To detect the expression of DcR3 protein in transgenic mice, SPCs were harvested and lysed in cell lysis buffer (1% (v/v) Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8), 1 mM PMSF, 2 µg/ml aprotinin, and 2 µg/ml leupeptin). Twenty-five micrograms of total lysate was fractionated by SDS-PAGE and subjected to Western blot analysis using the anti-DcR3 mAb (clone 3H5). The human DcR3 ELISA kit (Anawrahta Biotech) was used to determine the serum levels of DcR3 in transgenic mice.

Isolation of CD4+ T cells and induction of activation-induced cell death (AICD)

To isolate CD4+ T cells, total LN cells or SPCs were collected and stained sequentially with anti-mCD4-FITC mAb (0.5 µg/107 cells) and goat anti-FITC MicroBeads (Miltenyi Biotec). Cells were then purified using the VarioMACS (Miltenyi Biotec) technique and eluted from LS+ MACS columns according to the supplier’s protocol. To induce AICD, CD4+ cells, prepared from murine LNs, were seeded onto 24-well plates (106 cells/ml/well) precoated with anti-mTCR{beta} mAb (clone H57.597; 2.5 µg/0.5 ml PBS/well at 37°C overnight) and cultivated in the presence of IL-2 (500 U/ml; R&D Systems) for 5 days at 37°C in complete medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 mM HEPES, and 50 µM {beta}2-ME). Cells were replenished with a half volume of IL-2-supplemented medium (500 U/ml) on day 3. Dead cells were removed by density gradient centrifugation (Histopaque 1083; Sigma-Aldrich) at 500 x g for 20 min at 25°C, whereas live cells (density of 106/ml) were restimulated with plate-bound anti-mTCR{beta} mAb for 24 h. The percentages of apoptotic cells were determined by annexin V-FITC/propidium iodide (PI) double staining. To block AICD, receptor.Fc fusion proteins (5 µg/ml) or control hIgG1 (5 µg/ml; Sigma-Aldrich) were added to the cell culture at the time of restimulation.

Flow cytometric analysis

For cell surface staining, 5 x 105 cells were preincubated with rat anti-mCD16/32 mAb (1 µg/ml) at 4°C for 10 min in 50 µl of FACS staining/washing buffer (1% (v/v) FCS and 0.1% NaN3 in PBS) to prevent nonspecific Ab binding through the FcR. Cells were also incubated with fluorochrome-conjugated Ag-specific mAbs at 4°C for 20 min, followed by washing twice with 1 ml of FACS staining/washing buffer. Those cells stained with unlabeled or biotin-labeled mAbs were additionally incubated with fluorochrome-conjugated secondary Ab at 4°C for 20 min. After washing twice with 1 ml of FACS staining/washing buffer, cells were fixed with 1% (v/v) paraformaldehyde/PBS for 30 min at 4°C, then subjected to flow cytometric analysis. Intracellular cytokine staining was performed as vendor’s suggestion (BD Biosciences). All samples were analyzed with FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences).

Cytokine RT-PCR and ELISA

To detect cytokine transcripts, total RNA was extracted from stimulated cells using RNAzol B (Iso-Tex Diagnostics) and reverse transcribed using a ProStar first-strand RT-PCR kit (Stratagene). Specific primer pairs provided by Dr. C.-P. Hu (Veterans General Hospital, Taipei, Taiwan) and Dr. S.-L. Chang (Tzu Chi University, Hualien, Taiwan) were used to amplify target cytokines. Supernatants from stimulated cells were collected and stored at –20°C before cytokine detection by ELISA. Mouse IL-4, IL-10, and IFN-{gamma} ELISA kits were purchased from Pierce Endogen; mouse IL-12 and TNF-{alpha} ELISA kits were purchased from R&D Systems; the mouse IL-2 ELISA kit was purchased from BioSource. Procedures were conducted as suggested by the manufacturers.

Stimulation of HNT CD4+ T cells

The CD4+ T cells from HNT mice were activated by incubation with the antigenic peptide hemagglutinin (HA)126–138 (HNTNGVTAACSHE; synthesized by Sigma-Aldrich and dissolved in PBS) or the control peptide OVA323–339 (ISQAVHAAHAEINEAGR) in vitro. Briefly, 2 x 106 SPCs/ml were suspended in complete medium and stimulated with 10 µg/ml peptide for 6 h (for cytokine RT-PCR) or 3 days (for cytokine ELISA) in the presence or the absence of human IL-2 (500 U/ml; R&D Systems). Alternatively, purified CD4+ T cells (106 cells/ml/well in 24-well plates) were stimulated with immobilized anti-mTCR{beta} mAb (2.5 µg/0.5 ml PBS/well at 37°C overnight) and cultivated for 3 days at 37°C in complete medium. Supernatants were collected to determine the levels of IFN-{gamma} and IL-4 by ELISA.

Listeria infection and restimulation of mesenteric LN cells

Eight- to 12-wk-old wild-type or DcR3 transgenic mice were infected with sublethal doses of L. monocytogenes (250,000 CFU/mouse) by i.p. injection. After 5 days, 2 x 106/ml mesenteric lymphocytes were restimulated with 107 CFU/ml heat-killed L. monocytogenes (HKLM) in 24-well plates. Cells were harvested after 15 or 24 h for surface CD4 and intracellular IFN-{gamma}/IL-4 staining, respectively; culture supernatants were harvested after 24 h for IFN-{gamma}/IL-4 ELISA. HKLM was prepared by incubating the titrated bacteria at 70°C for 60 min. L. monocytogenes was quantified by determining its CFU in overnight-grown cultures. Ten-fold serial dilutions of bacterial culture were spread out on trypticase soy broth agar (Difco Laboratories) plates. The plates were incubated at 37°C for 16 h to determine the number of colonies. To determine the survival rate of mice after infection, mice were infected with lethal doses (106 CFU/mouse) of L. monocytogenes by i.p. injection. The survival rates of wild-type and DcR3 mice were checked daily for 20 days.

Cell proliferation assay

To measure cell proliferation by [3H]thymidine incorporation assay, 2 x 105/200 µl of SPCs were stimulated with HA126–138 or OVA323–339 peptide (10 µg/ml) in U-bottom, 96-well microtiter plates for 3 days. [3H]Thymidine (0.5 µCi/well; PerkinElmer) was added to each well, and the stimulated SPCs were incubated for an additional 16 h. Cells were collected using a cell harvester (Skatron), and the incorporated radioactivity was measured using a beta counter (model LS3801; Beckman Coulter). To detect the level of cell division in stimulated CD4+ T cells, 5 x 107/ml SPCs were incubated with 5 µM CFSE (Molecular Probes) for 5 min at room temperature in PBS, followed by three washes with 5% (v/v) FCS/PBS. CFSE-labeled SPCs were stimulated with peptide for 3 days. CD4+ T cells were distinguished from SPCs by staining with anti-mCD4-allophycocyanin, and the CFSE profiles of proliferating CD4+ T cells were analyzed with FACSCalibur and CellQuest software.

Surface plasmon resonance

Association and dissociation rates of the interaction between human DcR3.Fc and mouse FasL, LIGHT, and TL1A were determined by surface plasmon resonance using a BIAcore X biomolecular interaction analysis system. Human IgG1 (50 µg/ml) was first immobilized on flow channel 1 of a CM5 sensor chip as the blank to determine the bulk effect of injection itself, whereas DcR3.Fc (50 µg/ml) was immobilized on flow channel 2 by amine coupling at pH 5.0. The sensor surface was equilibrated by PBS, and sensorgrams were collected at 25°C at a flow rate of 30 µl/min. A 180-µl injection of mouse FasL, LIGHT, or TL1A was passed over the sensor surface. After the association phase, 60 s of dissociation data were collected. The sensor surface was regenerated after each cycle with a 15-µl pulse of 10 mM glycine (pH 3.0), twice at a 30-s interval. Sets of five analyte concentrations (250, 500, 1000, 1500, and 2000 nM) were collected and analyzed. The final readout of the specific response difference (response units) is determined by subtracting the signal of channel 1 (bulk effect) from that of channel 2.

Statistical evaluation

Values are expressed as the mean ± SEM of at least three experiments. One-way ANOVA and t tests were used to assess the statistical significance of the differences, with a value of p < 0.05 considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation and characterization of DcR3 transgenic mice

To produce a transgene construct, a cDNA encoding full-length human DcR3 was amplified by PCR and subcloned into the pPGK-NeoR-bpA vector (19) in place of the neomycin-resistant gene (NeoR). A transgene encoding DcR3 under the control of the PGK promoter (PGK-DcR3) was excised from the vector and microinjected into FVB-fertilized eggs. To screen for insertion of the transgene into the mouse genome, we performed PCR using primers that anneal to the PGK promoter and the DcR3 coding region. As shown in Fig. 1A, a product of the expected size (320 bp) was amplified from the genomic DNA of transgenic mice. To assay for the DcR3 transcript, total RNA extracted from peripheral blood cells of both DcR3 transgene-positive and -negative mice was subjected to RT-PCR. A DNA fragment with predicted size was amplified from the cDNA template in four of five founder mice that were positive for the PGK-DcR3 transgene (Fig. 1B). We also examined the expression of DcR3 protein in cell lysates prepared from SPCs of both DcR3 transgenic and wild-type littermates by immunoblotting. As shown in Fig. 1C, a 33-kDa protein was observed in cell lysates from DcR3 transgenic, but not wild-type, mice. Because DcR3 is detectable in the sera of certain cancer patients (8), we next performed a sandwich ELISA to determine serum DcR3 levels. As shown in Fig. 1D, the average concentration of DcR3 in all DcR3 transgenic mice tested was 4.7 ng/ml; no protein was detected in the sera of nontransgenic littermates. Therefore, both intra- and extracellular DcR3 could be detected in DcR3 transgenic mice. Taking these results together, DcR3 transgenic mice, as defined by genomic PCR screening, are able to express both DcR3 RNA transcript and DcR3 protein.



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  		FIGURE 1. Characterization and screening of DcR3 transgenic mice. A, PCR analysis for DcR3 transgene integration. Genomic DNA prepared from the tails of mice was amplified by PCR to detect the DcR3 transgene in the mouse genome. A 320-bp DNA fragment corresponding to the DcR3 transgene was amplified from DcR3 transgenic mice. B, RT-PCR analysis for transgene expression. RNA prepared from the peripheral blood of DcR3 transgenic (DcR3 Tg) and wild-type (WT) mice was analyzed by RT-PCR using DcR3-specific primers as described in Materials and Methods. {beta}-Actin primers were used as a control. C, Expression of DcR3 protein in transgenic mice. The expression of DcR3 in SPCs from WT or DcR3 transgenic mice was determined by immunoblotting using an anti-DcR3 mAb (3H5) as probe, followed by incubation with HRP-conjugated anti-mouse IgG. Ab binding was visualized by incubation with ECL detection reagents (Amersham Biosciences), followed by exposure to x-ray film. D, Detection of DcR3 in mouse sera by ELISA. Serum samples were collected by tail bleeding, and the level of DcR3 was determined using an ELISA kit. Each symbol represents the result for one mouse; the horizontal line represents the mean of each group. p = 0.0002, as analyzed by unpaired t test. WT, wild-type mice; DcR3 Tg, DcR3 transgenic mice; ND, nondetectable. E, Suppression of AICD in CD4+ LN cells in DcR3 transgenic mice. AICD was induced by plate-bound anti-mTCR{beta} mAb and IL-2 (500 U/ml). The percentage of apoptotic cells was determined by annexin V-FITC/PI double staining. Values in each quadrant plot were analyzed by CellQuest software. One representative experiment of three is shown.

 
To study Ag-specific T cell responses in the subsequent experiments, DcR3 transgenic mice on an FVB background were backcrossed with HNT-TCR (HNT) transgenic mice on a BALB/c background. The latter carry rearranged TCR{alpha} and TCR{beta} transgenes that recognize influenza HA peptide (HA126–138) in the context of I-Ad (22). Screening of HNT transgenic mice was performed by flow cytometry, as described in Materials and Methods.

DcR3 transgenic mice show inhibition of AICD

To understand whether DcR3 expressed in transgenic mice is functional, we examined the extent of cell apoptosis in AICD, which is mediated by FasL, one of the ligands of DcR3. To address this question, CD4+ LN cells prepared from wild-type or DcR3 transgenic mice were stimulated with anti-mouse TCR{beta} mAb to induce AICD, followed by annexin V-FITC/PI double staining to detect the percentages of apoptotic cells. In the nontransgenic littermates, the percentages of annexin V-FITC+/PI+ and annexin V-FITC+/PI– CD4+ LN cells were 46 and 15%, respectively, 24 h after restimulation in control mice (Fig. 1E, lower panel). In contrast, the percentages of annexin V-FITC+/PI+ and annexin V-FITC+/PI– cells were only 12 and 5%, respectively, for DcR3 transgenic mice treated under the same conditions (Fig. 1E, upper panel). Exogenous DcR3.Fc and Fas.Fc recombinant proteins have similar inhibitory effects on cell apoptosis, whereas LT{beta}R.Fc and IgG1 do not inhibit AICD. In this study we have shown that endogenous DcR3 expressed in transgenic mice, like recombinant DcR3.Fc and Fas.Fc (12), can inhibit cell death. Therefore, we conclude that DcR3 is an effective inhibitor of AICD in vivo.

DcR3 transgenic mice show an attenuated Th1 and polarized Th2 character upon stimulation

We have shown previously, in both human and murine systems, that DcR3.Fc-treated DCs induce CD4+ T cells to differentiate into a Th2 phenotype (12, 13). To examine the possibility that DcR3 skews the immune system toward a Th2-predominant response when it is expressed systemically in vivo, SPCs prepared from HNT-DcR3 and HNT mice were treated with HA126–138 peptide, which specifically stimulates HNT-bearing CD4+ T cells when presented by I-Ad on APCs (22). Subsequent analysis of cytokine profiles by RT-PCR revealed that the expressions of IL-2 (77%), IL-3 (85%), IL-12 p40 (64%), IFN-{gamma} (49%), and TNF-{alpha} (50%) were down-regulated in HNT-DcR3 SPCs, whereas the expressions of IL-4 (163%), IL-10 (152%), and IL-13 (139%) were up-regulated (Fig. 2A). Because IL-4, IL-10, and IL-13 are characteristic of a Th2-polarized immune response, whereas IL-12 p40, IFN-{gamma}, and TNF-{alpha} are representative cytokines of a Th1 response, this observation suggests that HNT-DcR3 SPCs become biased toward a Th2 response upon HA126–138 peptide stimulation. To test this hypothesis, supernatants from cultured SPCs stimulated with HA126–138 peptide for 3 days were subjected to cytokine ELISA. In accord with the results of RT-PCR, the secretion of IL-4 (p = 0.003) and that of IL-10 (p = 0.015) were significantly up-regulated, whereas production of IL-2 (p = 0.045), IFN-{gamma} (p = 0.048), TNF-{alpha} (p = 0.036), and IL-12 p40 (p = 0.042) was down-regulated, in HNT-DcR3 SPCs (Fig. 2B). Similar results were observed when HA126–138 peptide-primed SPCs were restimulated with immobilized anti-mouse TCR{beta} mAb (Fig. 2C).



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  		FIGURE 2. Cytokine profiles of stimulated-HNT-DcR3 or HNT SPCs. A, Cytokine RT-PCR. cDNAs were reverse transcribed from the total RNA of HA126–138 peptide-stimulated SPCs, isolated from HNT and HNT-DcR3 mice. PCR products were fractionated on 1.5% agarose gels. The intensity of each PCR product was quantified by ImageQuant software (Amersham Biosciences) and normalized against {beta}2-microglobulin. The relative intensity of each PCR product was calculated by dividing the volume of the PCR product amplified from HNT or HNT-DcR3 SPCs by that amplified from HNT SPCs. One set of representative data from three experiments is shown. B, Cytokine ELISA. Supernatants collected from 3-day cultures of HA126–138 peptide-stimulated SPCs were assayed by ELISA kits. The data presented in this study were obtained by pairing the results for HNT and HNT-DcR3 mice from six independent experiments. The p values, calculated by unpaired t tests, are indicated in each panel. C, IFN-{gamma} and IL-4 ELISA of restimulated SPCs. SPCs were stimulated with HA126–138 peptide for 3 days, followed by restimulation with plate-bound anti-mTCR{beta} mAb (H57.597) for 20 h. Supernatants were collected for ELISA. The data presented in this study were obtained by pairing the results for HNT and HNT-DcR3 mice from four independent experiments. The p values, calculated by unpaired t tests, are indicated in each panel. D, Secretion of IFN-{gamma} and IL-4 by anti-mTCR{beta} mAb-stimulated CD4+ T cells. Purified CD4+ T cells were stimulated with immobilized anti-mTCR{beta} mAb as described in Materials and Methods. The amounts of IFN-{gamma} and IL-4 were measured by ELISA.

 
To determine whether the down-regulation of IFN-{gamma} and the up-regulation of IL-4 were due to the APCs of HNT-DcR3 transgenic SPCs, we next investigated IFN-{gamma} and IL-4 secretion by purified CD4+ T cells stimulated with anti-mTCR{beta} mAb. As shown in Fig. 2D, CD4+ T cells purified from HNT and HNT-DcR3 SPCs showed equal amounts of IFN-{gamma} and IL-4 secretion. These data suggested that APCs of HNT-DcR3 transgenic mice were crucial for the polarized Th2 response.

Suppression of IL-2-dependent CD4+ T cell proliferation in DcR3 transgenic mice

The decreased IFN-{gamma} secretion described above might due to the lower percentage of IFN-{gamma}-producing CD4+ T cells or to a reduction in the amount of IFN-{gamma} secreted by CD4+ T cells. Intracellular cytokine (IFN-{gamma} and IL-4) staining was performed to address this question. Compared with cells from HNT mice, decreased percentages of IFN-{gamma}+ (24 vs 35% at 6 and 15 h; 35 vs 51% at 24 h) and an increased percentage of IL-4+ (8 vs 4% at 6 and 15 h) CD4+ T cells were observed for HNT-DcR3 mice (Fig. 3A). Moreover, compared with HNT littermates, the proliferation of HA126–138 peptide-stimulated CD4+ T cells in HNT-DcR3 transgenic mice was suppressed, as shown by [3H]thymidine incorporation (Fig. 3B) and flow cytometric analysis of CFSE-labeled CD4+ T cells (80 vs 69%; Fig. 3C). Because the secretion of IL-2 decreased under the same conditions (Fig. 2), we tested whether exogenous IL-2 could restore IFN-{gamma} expression in CD4+ T cells. In the untreated group, the proportions of IFN-{gamma}+ cells in HNT-DcR3 and HNT mice were 24 vs 35% at 15 h and 35 vs 51% at 24 (Fig. 3A), whereas treatment with IL-2 increased the percentages of IFN-{gamma}-expressing CD4+ T cells (50 vs 54% at 15 h; 58 vs 61% at 24 h) in HNT-DcR3 and HNT mice and significantly increased the ratio of IFN-{gamma}+ cells in the former compared with the latter (Fig. 3D). It is interesting to note that IL-2 had no obvious effect on the ratio of IL-4-secreting CD4+ T cells between HNT-DcR3 and HNT mice (from 8 vs 4% to 9 vs 5% at 15 h in IL-2-treated samples). In accordance with the measurements of intracellular IFN-{gamma} and IL-4, the levels of IFN-{gamma} secreted by HA126–138 peptide-stimulated HNT and HNT-DcR3 SPCs were similar after the addition of exogenous IL-2, whereas the differences in IL-4 secretion between stimulated HNT and HNT-DcR3 SPCs remained significant in the presence of exogenous IL-2 (Fig. 3E).



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  		FIGURE 3. Stimulation of HNT-DcR3 and HNT mice in the presence or the absence of IL-2. A and D, Intracellular cytokine staining. SPCs were restimulated with plate-bound anti-mTCR{beta} mAb for 6, 15, or 24 h after 3 days of HA126–138 peptide stimulation in the absence (A) or the presence (D) of human IL-2 (500 U/ml). Cells were triple stained with anti-mIFN-{gamma}-FITC/anti-mIL-4-PE/anti-mCD4-allophycocyanin and subjected to flow cytometric analysis. The cell populations presented in this study are CD4+ T cells; the percentage of positively stained cells is indicated in each quadrant plot. One set of representative data from three experiments is shown. B, [3H]thymidine incorporation assay. SPCs prepared from HNT and HNT-DcR3 mice were stimulated with HA126–138 peptide or OVA323–329 peptide or were left untreated (CTRL) for 3 days, followed by the addition of [3H]thymidine (0.5 µCi/well) for 16 h before harvesting. The data presented in this figure were obtained from two HNT (HNT-1 and -2) and two HNT-DcR3 (HNT-DcR3-1 and -2) mice. *, p < 0.05 compared with both HNT-1 and HNT-2 controls, by paired t test. C, CFSE labeling assay. CFSE-labeled (Figure legend continues)

SPCs were stimulated with HA126–138 peptide for 3 days before flow cytometric analysis. CD4+ T cells were gated, and the fluorescent intensity of CFSE was analyzed. Percentages of dividing cells are indicated in each histogram ({square}, unstimulated CD4+ T cells; {blacksquare}, stimulated CD4+ T cells). One representative set of data from three experiments is shown. E, IFN-{gamma} and IL-4 ELISA of stimulated SPCs incubated with exogenous IL-2. SPCs were stimulated with HA126–138 peptide in the presence or the absence of IL-2 for 3 days. Supernatants were collected for ELISA. One representative set of data from three experiments is shown. *, p < 0.05 compared with the groups of HNT mice treated under the same conditions, by paired t test.

 
To investigate whether a Th2-biased phenotype could be observed in HNT-DcR3 transgenic mice and whether this might affect the outcome of a host immune reaction to pathogen invasion, we examined the level of cytokine expression in DcR3 transgenic mice after infection with L. monocytogenes, an intracellular bacteria that typically induces a strong Th1 response in vivo (23, 24). DcR3-BALB/c mice and nontransgenic littermates were infected with sublethal doses of L. monocytogenes i.p.; mesenteric LN cells from infected mice were then restimulated with HKLM to assess the L. monocytogenes-induced immune response. The amounts of IFN-{gamma} and IL-4 in the culture supernatant were measured by ELISA. As shown in Fig. 4A, IFN-{gamma} secretion was significantly suppressed in the HKLM-restimulated mesenteric LN cells of DcR3-BALB/c mice (0.9 ng/ml), compared with that in wild-type littermates (12 ng/ml). However, the level of IL-4 was too low to be detected under the same conditions. From this observation, it is obvious that IFN-{gamma} secretion is suppressed not only in HNT-DcR3 CD4+ T cells after HA126–138 peptide stimulation, but also in DcR3-BALB/c T cells stimulated with L. monocytogenes.



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  		FIGURE 4. DcR3 transgenic mice infected with L. monocytogenes show attenuated induction of IFN-{gamma}. A, ELISA of IFN-{gamma}. Mesenteric LN cells of infected wild-type (WT) or DcR3 transgenic (DcR3) mice were restimulated with HKLM for 24 h. Culture supernatants were collected and subjected to ELISA. *, p < 0.05 compared with WT control, by paired t test. One representative experiment of three is shown. B, Detection of intracellular IFN-{gamma}. Mesenteric LN cells were restimulated with HKLM for 15 or 24 h before cell harvest and subsequent staining. CD4+ lymphocytes were gated for analysis, and the percentage of IFN-{gamma}-expressing CD4+ T cells is indicated in each quadrant plot. One representative experiment of three is shown. C, DcR3 transgenic mice are more susceptible to L. monocytogenes infection. WT (n = 12) and DcR3 transgenic (n = 12) mice were injected with lethal doses of L. monocytogenes (106 CFU/mouse) i.p. The number of survivors was checked daily for 20 days.

 
To test whether the decreased secretion of IFN-{gamma} was due to lower responsiveness of CD4+ T cells to L. monocytogenes infection in DcR3-BALB/c mice, the intracellular expression levels of IFN-{gamma} and IL-4 were measured in mesenteric LN cells isolated from L. monocytogenes-infected mice (Fig. 4B). Compared with nontransgenic littermates, the percentage of IFN-{gamma}-expressing CD4+ T cells in DcR3-BALB/c mice was decreased (13 vs 9% at 15 h; 35 vs 10% at 24 h). However, the expression of IL-4 was not detected by intracellular cytokine staining. This also supports the argument that DcR3 skews the immune response toward a Th2 phenotype by suppressing IFN-{gamma}-secreting CD4+ T cell proliferation.

We asked whether decreased IFN-{gamma} expression correlated with the susceptibility to L. monocytogenes infection. To answer this question, mice were injected with lethal doses (106 CFU/mouse) of L. monocytogenes i.p. to study survival rates for 20 days. As shown in Fig. 4C, 83% of L. monocytogenes-infected DcR3-BALB/c mice died within 7 days. In contrast, 50% of nontransgenic littermates were still alive on day 7. The survival rates of L. monocytogenes-infected mice did not change up to 20 days after infection. Thus, the increased susceptibility of DcR3 transgenic mice to L. monocytogenes infection correlated with the reduced percentage of IFN-{gamma}-expressing CD4+ T cells in L. monocytogenes-infected DcR3 mice.

Exogenous TL1A restores DcR3-mediated cytokine secretion

DcR3 has been shown to neutralize the biological effects of three ligands: LIGHT, TL1A, and FasL. Both LIGHT and TL1A have been shown act as costimulators of T cell proliferation and to enhance IFN-{gamma} secretion (4, 20, 25); therefore, we tested whether the Th2-biasing effects of DcR3 occur via the neutralization of endogenous FasL, LIGHT, and TL1A. To address this question, we studied the effects of recombinant Fas.Fc, DcR3.Fc, and DR3.Fc on cytokine secretion from T cells. As shown in Fig. 5A, DcR3.Fc and DR3.Fc, but not Fas.Fc, had similar effects, causing down-regulation of IFN-{gamma} secretion (300 vs 125 ng/ml) and up-regulation of IL-4 secretion (145 vs 170 ng/ml) compared with untreated controls (Fig. 5A) in HNT SPCs. Because DcR3 binds to both LIGHT and TL1A, the observed effects might be attributed to the neutralization of endogenous TL1A and/or LIGHT. To address this question, recombinant LIGHT and TL1A were added to HA126–138 peptide-stimulated SPCs of HNT-DcR3 transgenic mice. As shown in Fig. 5B, TL1A, but not LIGHT, was able to restore IFN-{gamma} secretion. This suggests that decreased secretion of IFN-{gamma} is mediated by the inhibition of endogenous TL1A in HNT-DcR3 transgenic mice. This is in accordance with the previous observation that TL1A, but not LIGHT, can enhance T cell responsiveness to IL-2, so the neutralization of TL1A in HNT-DcR3 transgenic mice impairs IL-2-dependent proliferation of Th1 cells, thus reducing the total amount of IFN-{gamma}. This argument is also supported by the effect of DR3, which binds only TL1A, to suppress IFN-{gamma} secretion and up-regulate IL-4 secretion.



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  		FIGURE 5. Expression IFN-{gamma} and IL-4 in receptor.Fc- or ligand-treated SPCs. A, Cytokine secretion of recombinant receptor.Fc-treated HNT SPCs. SPCs prepared from HNT mice were stimulated with HA126–138 peptide for 3 days in the presence of 3 µg/ml Fas.Fc, DcR3.Fc, or DR3.Fc. Supernatants were harvested and subjected to ELISA. *, p < 0.05 compared with the control group (none), by paired t test. One representative set of data from three experiments is shown. B, Cytokine secretion by recombinant ligand-treated HNT-DcR3 SPCs. SPCs prepared from HNT-DcR3 mice were stimulated with HA126–138 peptide for 3 days in the presence of 1 µg/ml LIGHT or TL1A. Supernatants were collected and subjected to ELISA. One representative set of data from three experiments is shown.

 
Binding affinity of human DcR3 for murine FasL, LIGHT, and TL1A

Because the human DcR3 ortholog is not found in mouse genome, understanding of the interaction between human DcR3 and murine ligands (mFasL, mLIGHT, and mTL1A) is crucial to reveal the mechanism of DcR3-mediated attenuation of the Th1 response in DcR3 transgenic mice. To answer this question, a surface plasmon resonance technique was applied to determine the affinity between human DcR3.Fc and recombinant mFasL, mLIGHT, and mTL1A, respectively. As shown in Fig. 6 and Table I, DcR3 has a higher affinity to mTL1A (54 ± 26 nM) and mFasL (179 ± 46 nM), whereas its affinity to mLIGHT (375 ± 158 nM) is much lower. From the results shown above, we conclude that the attenuated Th1 response is due to the neutralization of mTL1A, thus inhibiting IL-2 secretion and reducing Th1 cell proliferation in DcR3 transgenic mice.



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  		FIGURE 6. Kinetic analysis for the interactions of mFasL, mLIGHT, and mTL1A with DcR3.Fc. Human IgG1 was first immobilized on flow channel 1 of a CM5 sensor chip as the blank to determine the bulk effect of injection itself, whereas DcR3.Fc was immobilized on flow channel 2 for analysis of its kinetic interaction with murine ligands. Murine ligand, as the analyte, was injected at doses of 250–2000 nM. The interactions of DcR3.Fc with mFasL (A), mLIGHT (B), and mTL1A (C) were analyzed by surface plasmon resonance using a BIAcore X.

 

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  		Table I. Kinetics of ligand binding to DcR3.Fc determined by surface plasmon resonancea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DcR3 has been reported to be a potent modulator of host immunity (7, 26, 27), as well as being able to enhance the differentiation of osteoclasts (16) and angiogenesis (11). DcR3-treated dendritic cells skew host immunity toward a Th2-predominant response in both human (12) and murine (13) model systems, and overexpression of DcR3 in the pancreas inhibits the onset of type I diabetes (17), a Th1-mediated autoimmune disease. However, the systemic effects of DcR3 and the mechanism by which it promotes a Th2-predominant response have not been addressed.

The Th2-prone response, observed in DcR3 transgenic mice, apparently occurs through the suppression of IL-2 secretion, thereby impairing the proliferation of Th1 cells, which rely on IL-2 as a growth factor. This is supported by the observation that addition of exogenous IL-2 abolishes DcR3-mediated Th1 attenuation.

The neutralization of TL1A and LIGHT by DcR3 might also be important in biasing the T cell response. Both LIGHT (20) and TL1A (4, 25) have been reported to be costimulators of T cell proliferation and IFN-{gamma} secretion in studies with human cell culture system. In nontransgenic mice, the addition of DcR3.Fc and DR3.Fc proteins can inhibit IFN-{gamma} secretion. Moreover, the addition of TL1A increases IFN-{gamma} secretion of HNT-DcR3 SPCs significantly, whereas LIGHT has little effect on IFN-{gamma} secretion under the same conditions. Furthermore, the kinetic analysis of the interactions between DcR3.Fc and murine ligands (mFasL, mLIGHT, and mTL1A) revealed that DcR3 had the highest binding affinity to mTL1A and had the lowest affinity to mLIGHT. This indicates that TL1A is the major molecule affected by DcR3 in transgenic mice. It has also been reported that TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo (4). Therefore, it is reasonable to speculate that the DcR3-mediated effect occurs via the neutralization of endogenous TL1A during T cell activation. The lack of TL1A-mediated costimulation impairs responsiveness to IL-2, thus suppressing the proliferation of Th1 (IFN-{gamma}-positive) cells, which depend on IL-2 as their growth factor.

The fact that DcR3 is such a potent immunomodulator makes it a good candidate for immunotherapy. Recently, we have successfully used DcR3.Fc to treat type I diabetes in NOD mice (13). Because the onset of this condition is attributed to the activity of autoreactive Th1 cells (28), the effectiveness of this treatment supports the idea that DcR3.Fc might ameliorate diabetes by inducing Th2 polarization. Moreover, we have found that human DcR3.Fc is able to modulate the surface marker expression as well as the cytokine secretion profile of murine BMDCs; the effects are similar to those seen for human CD14+ monocyte-derived dendritic cells, and DcR3.Fc-treated murine BMDCs also induce an increased IL-4/IFN-{gamma} ratio when cocultivated with T cells. To directly investigate the therapeutic potential of DcR3 in preventing diabetes, transgenic mice that overexpress DcR3 in their pancreatic {beta} cells were generated (17), and transgenic DcR3 was shown to protect mice from autoimmune and cyclophosphamide-induced diabetes in a dose-dependent manner. The observation that transgenic islets have a higher transplantation success rate and a longer survival period than controls clearly demonstrates the possibility of using DcR3 as a therapeutic agent.

Because DcR3 is overexpressed in various cancers, with expression levels being linked to tumor status, it is possible that DcR3 may enable tumor cells to evade immune surveillance by neutralizing FasL- and LIGHT-induced cytotoxicity and blockade of LIGHT- and TL1A-induced T cell costimulation. Moreover, DcR3 has been implicated in the enhancement of tumor growth through neutralizing TL1A, thereby enhancing angiogenesis in HUVECs in vitro and inducing angiogenesis in vivo (11). In combination with its ability to suppress Th1 activities, resulting in a Th2-predominant response, this may allow DcR3 to promote tumor progression through both local and systemic actions. However, there is no evidence that DcR3 is directly involved in tumor genesis, because the incidence rates of tumor formation in DcR3 transgenic mice and nontransgenic littermates are similar up to 12 mo of age (data not shown). Therefore, understanding the pathological actions and the regulation of DcR3 is important in developing new strategies for tumor therapy. It would be very interesting to test whether the neutralization of DcR3 secretion by tumors might enhance the efficacy of chemotherapy or immunotherapy in the future.


   Acknowledgments
 
We thank Dr. Caroline Milner for critical comments on this work. Special thanks go to Drs. Nien-Jung Chen and Shu-Fen Wu for their technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was mainly supported by Grants NSC92-2320-B-010-052, NSC94-2320-B-010-015, and NSC93-2320-B-010-011 from the National Sciences Council, Taiwan. Other support includes Grant 94M002-1 from the Academia Sinica (Taiwan) and Grant VGH37113 from the Taipei Veterans General Hospital. Back

2 Address correspondence and reprint requests to Dr. Shie-Liang Hsieh, Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan. E-mail address: slhsieh{at}ym.edu.tw Back

3 Abbreviations used in this paper: DcR3, decoy receptor 3; AICD, activation-induced cell death; BMDC, bone marrow-derived DC; DR3, death receptor 3; FasL, Fas ligand; HA, hemagglutinin; HKLM, heat-killed L. monocytogenes; HVEM, Herpesvirus entry mediator; LIGHT, receptor homologous to lymphotoxins that exhibits inducible expression, competes with HSV glycoprotein D for the HVEM, and is expressed by T lymphocytes; LN, lymph node; LT{beta}R, lymphotoxin-{beta} receptor; m, murine; PGK, phosphoglycerate kinase; PI, propidium iodide; SPC, splenocyte; TL1A, TNF-like molecule 1A. Back

Received for publication February 2, 2005. Accepted for publication August 11, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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