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The Journal of Immunology, 2006, 177: 1679-1688.
Copyright © 2006 by The American Association of Immunologists

Dendritic Cells Transduced with SOCS-3 Exhibit a Tolerogenic/DC2 Phenotype That Directs Type 2 Th Cell Differentiation In Vitro and In Vivo1

Yonghai Li, Niansheng Chu, Abdolmohamad Rostami and Guang-Xian Zhang2

Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) have been suggested to direct a type of Th differentiation through their cytokine profile, e.g., high IL-12/IL-23 for Th1 (named DC1/immunogenic DCs) and IL-10 for Th2 (DC2/tolerogenic DCs). Suppressor of cytokine signaling (SOCS)-3 is a potent inhibitor of Stat3 and Stat4 transduction pathways for IL-23 and IL-12, respectively. We thus hypothesize that an enhanced SOCS-3 expression in DCs may block the autocrine response of IL-12/IL-23 in these cells, causing them to become a DC2-type phenotype that will subsequently promote Th2 polarization of naive T cells. Indeed, in the present study we found that bone marrow-derived DCs transduced with SOCS-3 significantly inhibited IL-12-induced activation of Stat4 and IL-23-induced activation of Stat3. These SOCS-3-transduced DCs expressed a low level of MHC class II and CD86 on their surface, produced a high level of IL-10 but low levels of IL-12 and IFN-{gamma}, and expressed a low level of IL-23 p19 mRNA. Functionally, SOCS-3-transduced DCs drove naive myelin oligodendrocyte glycoprotein-specific T cells to a strong Th2 differentiation in vitro and in vivo. Injection of SOCS-3-transduced DCs significantly suppressed experimental autoimmune encephalomyelitis, a Th1 cell-mediated autoimmune disorder of the CNS and an animal model of multiple sclerosis. These results indicate that transduction of SOCS-3 in DCs is an effective approach to generating tolerogenic/DC2 cells that then skew immune response toward Th2, thus possessing therapeutic potential in Th1-dominant autoimmune disorders such as multiple sclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs)3 are major professional APCs that initiate and modulate immune response and may instruct Th0 to differentiate into Th1 or Th2 cells (1, 2). Various DC-derived factors that induce Th cell polarization have been identified, including the Th1 cell-polarizing cytokines IL-12 and IL-23 and the Th2/regulatory T cell-polarizing cytokines IL-10 and TGF-beta (2). It has been shown that DCs not only produce IL-12 and IL-23 but also express high levels of their receptors, indicating an autocrine regulatory loop (3). The positive loop by IL-12 on DCs has been proposed to cause a strong Th1 cell polarization (4, 5). IL-23 can bind to its receptors in DCs and induce IL-12 and IFN-{gamma} production by these cells (6). In mice, DCs producing large amounts of IL-12 and IFN- {gamma} (named DC1) induce Th1 responses, whereas DCs producing lower amounts of IL-12 (DC2) preferentially induce Th2 responses (7, 8, 9, 10, 11). In addition, our laboratory found that APCs of IL-12Rbeta1–/– mice, which lack both IL-12 and IL-23 signaling, possess the capability of inducing Ag-specific Th2 immune response (12).

Suppressor of cytokine signaling (SOCS) proteins, a family of cytokine-induced intracellular proteins, can function as feedback inhibitors to regulate the duration or intensity of the cytokine-induced signal. SOCS-3, one member of the SOCS family, can be induced by various cytokines, such as IL-6, IL-12, and IFN-{gamma}, and negatively regulate the actions of those cytokines as well as functions of Stat transcription factors (13, 14, 15). Overexpression of SOCS-3 down-regulates IFN-{alpha} and IFN-{gamma} responses (16, 17, 18). SOCS-3-transgenic mice exhibit enhanced Th2 differentiation, whereas expression of dominant-negative SOCS-3 interferes with Th2 differentiation (13, 14, 15). Recently, Jo et al. (19) found that intracellular delivery of SOCS-3 significantly reduces production of inflammatory cytokines and attenuates liver apoptosis and hemorrhagic necrosis, thus effectively suppressing the devastating effects of acute inflammation. Furthermore, the SOCS signal may be important for the regulation of DC maturation (20).

Based on these observations, we hypothesized that enhanced SOCS-3 expression in DCs will block IL-12/IL-23 signaling in these cells and drive them to a tolerogenic/DC2 phenotype. In the present study we tested this hypothesis by transducing DCs with a bicistronic lentiviral vector encoding SOCS-3 and by determining the functional phenotype of these DCs. The capacity of SOCS-3-transduced DCs to drive naive CD4+ T cells toward Th2 polarization was determined in vitro and in experimental autoimmune encephalomyelitis (EAE) mice in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and reagents

Female C57BL/6 mice at 7–8 wk of age were purchased from The Jackson Laboratory. Myelin oligodendrocyte glycoprotein (MOG) TCR transgenic mice were a gift from Dr. V. Kuchroo (Harvard Medical School, Boston, MA). The MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide was synthesized at the Protein Chemistry Laboratory of the University of Pennsylvania (Philadelphia, PA) and purified by HPLC to >98% purity. A 293T cell line was obtained from American Type Culture Collection. Recombinant mouse IL-12 and IL-23 were purchased from R&D Systems. LPS was obtained from Sigma-Aldrich.

Bone marrow-derived dendritic cell preparation

Bone marrow cells were flushed from the femurs and tibiae of normal, female, 7- to 8-wk-old C57BL/6J mice. These cells were cultured in 100-mm petri dishes with 10 ml of RPMI 1640 medium containing 10% FBS and 200 U/ml mouse GM-CSF (PeproTech) at 37°C in 5% humidified CO2. Nonadherent granulocytes were removed after 48 h of culture, and fresh medium was added. After 7 days of culture, CD11c+ DCs were purified by immunomagnetic sorting using anti-CD11c-coated magnetic beads and the autoMACS system according to the manufacturer’s instructions (Miltenyi Biotec). The purity of the sorted cells was determined by FACS analysis (>96% for CD11c+ cells). Mature DCs were generated in the presence of 100 ng/ml LPS.

SOCS-3 expression on DCs upon IL-12/IL-23 stimulation

DCs were plated in 12-well plates at 1 x 106 cells/ml and stimulated with IL-12 (100 ng/ml), IL-23 (100 ng/ml), and LPS (100 ng/ml). One microgram of RNA from treated DCs was reverse transcribed using Superscript2 (Invitrogen Life Technologies) according to the manufacturer’s instructions. The SOCS-3 gene was cloned with the sense primer (5'-ATGGTCACCCACAGCAAGTT-3') and the antisense primer (5'-AAGTGGAGCATCATACTGATCC-3'); SOCS-3 expression in response to the above stimuli in DCs was detected by real-time PCR. Briefly, a TaqMan probe for SOCS-3 was purchased from Applied Biosystems. Real-time PCR was conducted at a final volume of 25 µl containing cDNA amplified for 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C.

Generation of SOCS-3-lentiviral vector and transduction into DCs

The SOCS-3 gene was inserted into a bicistronic lentiviral vector. The lentiviral vector particles were produced by using the three-plasmid transient transfection system. Vectors were pseudotyped with the vesicular stomatitis virus G glycoprotein. For the transduction of a 10-cm-diameter dish of 293T cells, the mixture of three plasmids was added into 2x HBSS and added drop by drop onto the 293T cells. The medium was replaced after 5 h of incubation. Supernatants were collected on days 2, 3, and 4 posttransduction, and viral particles were concentrated by ultracentrifugation at 26,000 rpm for 2 h at 4°C. Pellets were dissolved in PBS, resulting in 200-fold concentration. Multiplicity of infection was determined by infecting the NIH 3T3 cell line. For transduction to DCs, enriched viral particles with a multiplicity of infection of 10 were added into DCs at day 6 of culture in the presence of 4 µg/ml Polybrene. The same steps were repeated the next day. In some experiments, the transduced DCs were purified by sorting CD11c+ GFP+ cells with FACSAria (BD Bioscience). The viability of DC cultures was monitored periodically before and after transduction using trypan blue (Sigma-Aldrich) staining. Cells with >85% viability were continued for additional experiments.

Immunohistology staining

Transduced DCs were cytospun onto slides. After being fixed with acetone for 2 min and blocked with 1% BSA in PBS, slides were incubated with biotin-conjugated anti-CD11c mAb (BD Pharmingen) for 1 h. Afterward, rhodamine red-conjugated streptavidin (Jackson ImmunoResearch Laboratories) was added and incubated for 30 min. Slides were covered with mounting medium (Vector Laboratories), and coexpression of CD11c and GFP on the same cells was examined by confocal microscopy.

Immunoblot analysis

Transduced DCs were stimulated by LPS for 4 h and then washed twice. The next morning cells were incubated with IL-12 and IL-23 for 2 h, and then cells were centrifuged and lysed in radioimmune precipitation assay buffer (Sigma-Aldrich) with the addition of protease inhibitors (Roche). Lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blotted with primary Abs, including anti-SOCS-3 (Santa Cruz Biotechnology), phosphorylated Stat3 and Stat3 (Santa Cruz Biotechnology), and phosphorylated Stat4 and Stat4 (Zymed Laboratories). Intensity of bands from the Western blots was quantified by densitometry using ImageJ software (NIH images scan). Signal was normalized for total STAT expression for each loading lane.

In vitro assay of coculture system

The coculture system was set up with MOG-specific CD4+ T cells and FACS-sorted transduced DCs by gate of CD11c+GFP+ cells. CD4+ T cells were purified from the spleen of naive MOG TCR transgenic mice using anti-CD4 coupled magnetic beads (Miltenyi Biotec). The purity of both CD4+ T cells and DCs was ≥95% as determined by FACS. A total of 1.5 x 104 purified MOG-reactive CD4+ T cells and 0.5 x 104 transduced DCs were cocultured in the presence of 25 µg/ml MOG35–55 peptide. This concentration of peptide was chosen because it was optimal for T cell stimulation in our preliminary study (data not shown). After coculture for 5 days, the supernatant was collected for cytokine analysis. The cytokines produced by CD4+ T cells were identified by intracellular staining. For proliferation, purified MOG-reactive CD4+ T cells and transduced DCs were placed in a 96-well plate. Cells were cultured in triplicate with 25 µg/ml MOG35–55, 25 µg/ml OVA, or without Ag. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi of 3H. Cells were harvested and counts read using a beta counter.

Immunophenotype by FACS and cytokine assay by intracellular staining and ELISA

Cells, including MOG-reactive CD4+ T cells and DCs, were stained with a combination of mAbs against the following Ags: CD4, CD11c, CD40, CD80, CD86, and MHC class II. The appropriate isotype was used as control mAb. Cells were incubated at 4°C for 20 min, washed, and analyzed by FACS. Intracellular staining of cytokines was conducted using Cytofix/Cytoperm kits (BD Pharmingen) following the manufacturer’s recommendations. Briefly, cells were cultured in the presence of MOG35–55 (25 µg/ml) for 5 days. GolgiStop, a protein transport inhibitor containing brefeldin A, was added during the last 4 h of culture. After surface staining of CD4, cells were thoroughly resuspended and fixed in Cytofix/Cytoperm solution for 20 min. Cells were permeabilized with 1x Perm/Wash solution (BD Pharmingen) and stained for intracellular cytokines using PE-conjugated anti-mouse cytokine mAbs (IFN-{gamma} clone XMG1.2, TNF-{alpha} clone MP6-XT22, and IL-4 clone 11B11; BD Pharmingen). Flow Jo software was used to analyze data. The cytokines in supernatants were analyzed by ELISA in accordance with the manufacturer’s instructions (BD Pharmingen).

Th differentiation driven by transduced DCs in vivo

The CD11c+ GFP+ cells were purified by FACS sorting 48 h posttransduction. The purified DCs were pulsed with MOG35–55 (25 µg/ml) overnight, washed twice, and injected s.c. into each hind footpad of recipient mice at 0.5 x 106 cells in 50 µl of sterile PBS, following a protocol described previously (21, 22). The viability of injected cells was >85%. Seven days after injection the recipient mice were sacrificed, and mononuclear cells from draining popliteal and inguinal lymph nodes were harvested. Cells were cultured for lymphocyte proliferative responses to MOG35–55. Supernatants were collected after 72 h of culture for cytokine analysis.

Induction of EAE and SOCS-3-DC treatment

To confirm the immunoregulatory effect of SOCS-3-transduced DCs in vivo, we administered these cells in EAE mice. Female 8- to 10-wk-old C57BL/6 mice were each injected s.c. with 200 µg MOG35–55 in CFA containing 4 mg/ml Mycobacterium tuberculosis over two sites at the back. Two hundred nanograms of pertussis toxin (List Biological Laboratories) was given i.p. on days 0 and 2 postimmunization (p.i.). SOCS-3-transduced DCs were pulsed overnight with MOG35–55 peptide (25 µg/ml), washed, and injected i.v. (2 x 106/0.2 ml PBS each mouse). DCs transduced with GFP only served as control. The viability of these cells was >85%. A clinical scoring system with a scale of 0 to 5, with 0.5 points for intermediate signs, was used as follows (23): 0, normal; 1, flaccid tail, abnormal gait; 2, hind leg weakness or severe ataxia; 3, minimal hind leg movement; 4, hind leg and forelimb paralysis; and 5, moribund or dead. Mice were examined daily by two blinded observers for signs of EAE. To determine the suppressive effects of SOCS3-transduced DCs on different phases of EAE, these DCs were injected at EAE induction (–3 and +7 days p.i.) or disease onset (11, 14, and 17 days p.i.), and mice were sacrificed at days 21 or 24 p.i., respectively. Splenocytes were isolated for analysis of MOG-induced proliferative responses and cytokine production. All work was performed in accordance with the guidelines for animal use and care at Thomas Jefferson University, Philadelphia, PA.

Statistics

Mann-Whitney U test was used for comparison of average clinical scores, and ANOVA was used for other parameters among different groups. All tests were two sided.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Inducible expression of SOCS-3 in DCs by LPS, IL-12, and IL-23

To determine whether SOCS-3 is constitutively or inducibly expressed in DCs, we first examined SOCS-3 expression in DCs stimulated with LPS, IL-12, IL-23, or without stimulation. As shown in Fig. 1, the SOCS-3 gene was expressed at relatively low levels in nonstimulated DCs. IL-23 induced a high level of SOCS-3 expression after 2 h that persisted even after 6–12 h, whereas IL-12 induced a noticeably lower level of SOCS-3 than IL-23. LPS induced SOCS-3 expression that was 22 times higher than in the first hour, but by 6 h SOCS-3 expression returned to the base level. These data indicate that SOCS-3 is inducible upon inflammatory stimulation and may imply an important regulatory role of SOCS-3 in DC function.


Figure 1
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  		FIGURE 1. Inducible expression of SOCS-3 in DCs by LPS, IL-12, and IL-23. Purified CD11c+ DCs at day 8 of culture were incubated with LPS (100 ng/ml), IL-12 (100 ng/ml), and IL-23 (100 ng/ml) at the indicated time points. mRNA expression of SOCS-3 was assayed by real-time PCR. Data presented as the mean values of triplicate cultures ± SD. Dashed line represents the base level of SOCS-3 in resting DCs. One representative experiment of two is shown.

 
High efficiency of SOCS-3 transduction in DCs

To transduce SOCS-3 in DCs, we inserted SOCS-3 into a lentiviral vector (Fig. 2A). We used a bicistronic lentiviral vector characterized by an internal ribosome entry site and a central polypurine tract sequence element. The central polypurine tract element can enhance the ability of nuclear import and increase the efficiency of gene transfer. In the presence of a single internal ribosome entry site, two genes, in this case SOCS-3 and GFP, can be transduced by the same promoter and then translated into two proteins. Transduction efficiency can be monitored by GFP expression in transduced cells.


Figure 2
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  		FIGURE 2. High efficiency of SOCS-3 transduction in DCs. A, Structure of bicistronic lentiviral vector. SOCS-3 gene was inserted downstream of the elongation factor 1{alpha} (EF-1{alpha}) promoter. In the presence of an internal ribosome entry site (IRES), one RNA containing SOCS-3 and GFP can be translated into two proteins in one cell. Abbreviations: cPPT, central polypurine tract; 5'LTR, 5' long terminal repeat; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; SIN, self inactivating. B, Expression of SOCS-3 protein detected by Western blot. Lane 1, nontransduced DCs; lane 2, GFP-transduced DCs; lane 3, SOCS-3-transduced DCs. GAPDH served as loading control. C, Confocal microscopy. DCs transduced with SOCS-3-LV viral particles were stained with biotin-conjugated primary Ab against CD11c, followed by streptavidin-rhodamine red. Overlay of GFP (a) and CD11c (b) confirmed colocalization (c). D, Transduction efficiency analysis by FACS. CD11c+ cells were gated to determine the percentage of GFP+ cells. One representative experiment of two is shown.

 
To verify that SOCS-3 and GFP proteins can be expressed by our constructed vectors, we prepared viral particles and transduced them into DCs. The expressed SOCS-3 protein was determined by Western blotting. As shown in Fig. 2B, a single band was detected by anti-SOCS-3 mAb in SOCS-3-transduced DCs (lane 3), but not in nontransduced or GFP-transduced DCs (lanes 1 and 2).

We next determined the transduction efficiency in SOCS-3-transduced DCs. Transduced DCs were stained with biotin-conjugated anti-CD11c mAb, and the colocalization of GFP and CD11c was confirmed by confocal microscopy (Fig. 2C). Transduction efficiency was ~63.7% as determined by FACS using GFP as readout (Fig. 2D). Taken together, these data demonstrate that SOCS-3 was constitutively expressed in transduced DCs with our bicistronic lentiviral vector.

Impaired IL-12/Stat4 and IL-23/Stat3 signaling in SOCS-3-transduced DCs

To study the influence of overexpressed SOCS-3 on IL-12/Stat4 and IL-23/Stat3 signaling, DCs transduced with SOCS-3 plus GFP and control DCs transduced with GFP only were stimulated with IL-12, IL-23, or medium only. Expression of total and phosphorylated fractions of Stat3 and Stat4 was analyzed by Western blotting. As shown in Fig. 3, IL-12 and IL-23 markedly increased a fraction of phosphorylated Stat4 and Stat3, respectively. Compared with DCs transduced with GFP-lentiviral vector, SOCS-3-transduced DCs contained noticeably lower levels of phosphorylated Stat4 (~22% activity) induced by IL-12 and phosphorylated Stat3 (~31% activity) induced by IL-23. In contrast, no difference was found in total Stat3 or Stat4 expression between SOCS-3-transduced and GFP-transduced DCs (Fig. 3).


Figure 3
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  		FIGURE 3. Impaired IL-12/IL-23 signaling in SOCS-3-transduced DCs. Transduced DCs were incubated with IL-12 or IL-23 (100 ng/ml) for 2 h. Stat3 and Stat4 and their tyrosine phosphorylation (p-Stat3 and pY-STAT4, respectively) were detected by Western blot. One representative experiment of three is shown.

 
Low expression of MHC class II and CD86 in SOCS-3-transduced DCs

We next evaluated the influence of SOCS-3 on DC maturation status by determining the expression of costimulatory molecules (CD40, CD80, and CD86) and MHC class II on transduced DCs. CD86 and MHC class II were down-regulated in SOCS-3-transduced DCs, but not in GFP-transduced DCs. As shown in Fig. 4, 71.2% (mean fluorescence intensity (MFI) = 73 ± 18) of nontransduced DCs and 63.1% (MFI = 54 ± 15) of GFP-transduced DCs expressed CD86, whereas only 49.6% of SOCS-3-transduced DCs expressed this molecule (MFI = 25 ± 6). For MHC class II, 31.4% of SOCS-3-transduced DCs (MFI = 31 ± 10) were positive, whereas 45.5% (MFI = 70 ± 21) in GFP-transduced DCs and 53.7% (MFI = 92 ± 18) in nontransduced DCs were positive (Fig. 4A). Statistical analysis of the percentages and MFI values of CD86 and MHC class II in SOCS-3-transdued DCs showed a significant difference compared with GFP-transduced and nontransduced DCs (Fig. 4B). No differences in CD40 and CD80 were observed among these three groups (data not shown). There was no deleterious effect on cell viability before or after DCs manipulation as determined by trypan blue staining.


Figure 4
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  		FIGURE 4. FACS analysis of surface marker of SOCS-3-transduced DCs. A, Surface molecules in transduced DCs were detected by FACS with the gate of CD11c+GFP+. Numbers represent the percentage of positive cells. B, The MFI values and percentages of CD86 and MHC class II are plotted as histograms. Data presented as the mean values of triplicate cultures ± SD. The p values refer to comparison between mice receiving SOCS-3-transduced DCs and mice receiving GFP-transduced or nontransduced DCs. *, p < 0.05; **, p < 0.01. One representative experiment of three is shown.

 
Altered cytokine production in SOCS-3-transduced DCs

We then investigated the effects of overexpressing SOCS-3 on the cytokine profile of LPS-stimulated DCs by assaying the supernatants collected from transduced DCs. As shown in Fig. 5A, SOCS-3-transduced DCs produced reduced amounts of IFN-{gamma} (p < 0.01) and IL-12 p70 (p < 0.05) as compared with GFP-transduced or nontransduced DCs. In contrast, a significant increase of IL-10 was found in SOCS-3-transduced DCs. No significant difference for TNF-{alpha} was observed among the three groups. Further, mRNA expression of the IL-12/IL-23 subunits p40, p35, and p19 was significantly decreased in SOCS-3-transduced DCs as compared with GFP-transduced DCs (all p < 0.05; Fig. 5B).


Figure 5
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  		FIGURE 5. Cytokine profile of DCs transduced with SOCS-3. DCs at day 6 were transduced with GFP-lentiviral vector or SOCS-3-lentiviral vector. After 48 h of first transduction, DCs were stimulated by LPS to mature for 36 h. A, The supernatant was assayed for the levels of IFN-{gamma}, TNF-{alpha}, IL-10, and IL-12 p70 by ELISA. B, The cells were harvested, and their mRNA expression of IL-12 p40, p35, and IL-23 p19 was determined by real-time PCR. Data are presented as the mean values of triplicate cultures ± SD. The p values refer to comparison between mice receiving SOCS-3-transduced DCs and mice receiving GFP-transduced DCs. *, p < 0.05; **, p < 0.01. One representative experiment of three is shown.

 
Effect of transduced DCs on Th phenotype in vitro: a coculture system

To define Th differentiation of CD4+ T cells primed by transduced DCs, MOG-specific CD4+ T cells purified from MOG Tg mice and CD11c+GFP+-transduced DCs were mixed at a ratio of 3:1. As shown in Fig. 6A, low proliferative responses were observed in cocultured cells without Ag stimulation and with control Ag OVA. MOG-reactive CD4+ T cells primed by SOCS-3-transduced DCs exhibited significantly lower MOG-induced proliferative response as compared with those primed by GFP-transduced DCs (p < 0.05).


Figure 6
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  		FIGURE 6. Effects of transduced DCs on Th phenotype in vitro. A, Less proliferation of MOG-specific CD4+ T cells cocultured with SOCS-3-transduced DCs. MOG-specific CD4+ T cells were purified from MOG-TCR transgenic mice and cocultured with DCs transduced with GFP-lentiviral vector or SOCS-3-lentiviral vector in the presence of MOG, OVA, or medium alone. Proliferation was determined with 3H incorporation. Data presented as the mean values of triplicate cultures ± SD. B, Cytokine production of supernatant harvested after 5 days of coculture was determined by ELISA. Bars represent mean values ± S.D. The p values refer to comparison between mice receiving SOCS-3-transduced DCs and mice receiving GFP-transduced DCs. *, p < 0.05; **, p < 0.01. C, Intracellular staining of IL-4 and IFN-{gamma} produced by CD4+ T cells after 5 days of coculture in the presence of MOG. Numbers refer to the percentage of cytokine-producing cells among CD4+ T cells. Shown is one of three independent experiments. D, Decreased intracellular level of TNF-{alpha} in MOG-specific CD4+ T cells. One representative experiment of three is shown.

 
We then assayed the secreted cytokines in the supernatant of the above coculture. Significantly reduced production of IFN-{gamma}, TNF-{alpha}, and IL-17, as well as higher levels of IL-4 and IL-10, were found in cocultures of T cells and SOCS-3-transduced DCs (Fig. 6B). To more accurately study cytokine production by CD4+ T cells cocultured with SOCS-3-transduced DCs, we performed intracellular cytokine staining for IFN-{gamma} and IL-4, two important cytokines in Th1 and Th2. Within the CD4+ subset, a significantly decreased frequency of IFN-{gamma} but an increased frequency of IL-4 were found in the coculture with SOCS-3-transduced DCs. The percentages of IFN-g+ CD4+ cells (mean ± SEM) were as follows: 25.0 ± 3% in the coculture with GFP-transduced DCs and 13.5 ± 4% in the coculture with SOCS-3-transduced DCs (p < 0.05), whereas the percentages of IL-4+ CD4+ cells were 24.2% ± 2% vs 31% ± 1.4% (p < 0.05) (Fig. 6C). The percentage of TNF-a+ CD4+ T cells is 9.2% ± 2.1% in the coculture with GFP-transduced DCs vs 2.5% ± 1.5% in the coculture with SOCS-3-transduced DCs (p < 0.05) (Fig. 6D). Taken together, these data indicate a Th2 shift induced by SOCS-3-transduced DCs.

Effect of SOCS-transduced DCs on directing Th differentiation in vivo

We further determined the effect of SOCS-3-transduced DC on Th differentiation in vivo. Purified transduced DCs were incubated overnight with MOG35–55 (25 µg/ml), washed, and adoptively transferred into the footpads of naive mice by s.c. injection. Draining lymph nodes were harvested after 7 days, and Ag-specific cytokine production was determined. As shown in Fig. 7, lymph node cells from mice receiving GFP-transduced DCs produced low levels of IL-4 and IL-5 and high levels of IFN-{gamma}. In contrast, SOCS-3-transduced DCs primed a Th2-dominant response, i.e., increased IL-4 and IL-5 and decreased IFN- {gamma} production. This finding was confirmed by analyzing the intracellular cytokine production in CD4+ T cells. Compared with GFP-transduced DCs, SOCS-3-transduced DCs were able to prevent Th1 polarization and induce Th2 by increasing the percentage of IL-4+ T cells (1.8 ± 0.7% vs 3.5 ± 1.0%; n = 3; p < 0.05) and reducing the percentage of IFN- {gamma}-producing cells (33.1 ± 4.1% vs 19.0 ± 3.0%; n = 3; p < 0.05) (Fig. 7).


Figure 7
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  		FIGURE 7. SOCS-3-transduced DCs induce a Th2 response in vivo. Transduced DCs were pulsed with MOG35–55 (25 µg/ml) overnight, washed twice, and injected s.c. into each hind footpad of naive B6 mice at 0.5 x 106 cells in 50 µl of sterile PBS. After 7 days lymph nodes were removed, and cells were stimulated with MOG35–55 (25 µg/ml) for 72 h. A, Intracellular staining of IL-4 and IFN-{gamma} produced by CD4+ T cells. Numbers refer to the percentage of cytokine-producing cells among CD4+ T cells. Shown is one of two experiments. B, Supernatants were tested for IL-4, IL-5, and IFN-{gamma} by ELISA. The p values refer to comparison between mice receiving SOCS-3-transduced DCs and mice receiving GFP-transduced DCs. *, p < 0.05; **, p < 0.01. One representative experiment of two is shown.

 
SOCS-3-transduced DCs inhibited development of clinical EAE

Finally, we investigated the in vivo effect of SOCS-3-transduced DCs on clinical EAE. Control mice receiving GFP-transduced DCs exhibited characteristic signs of EAE starting on day 12 p.i. In contrast, mice receiving SOCS-3-transduced DCs at the induction phase of EAE (–3 and 7 days p.i.) developed significantly lower clinical scores compared with control mice injected with GFP-transduced DCs (p < 0.05) (Fig. 8A). Similar suppressive effects were observed in mice receiving SOCS-3-transducing DCs at disease onset (11, 14, and 17 days p.i.) (Fig. 8B). No significant difference was found in the mice receiving transduced DCs at peak EAE (18, 19, and 20 days p.i.) (data not shown). MOG-induced lymphocyte proliferation and production of the Th1 cytokines IFN-{gamma} and IL-17 were suppressed in splenocytes of mice receiving SOCS-3-transduced DCs (p < 0.01 and 0.05, respectively), whereas the level of MOG-induced Th2 cytokine IL-4 was significantly up-regulated in the splenocytes of these mice (p < 0.05; Fig. 8, C and D).


Figure 8
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  		FIGURE 8. SOCS-transduced DCs inhibit development of clinical EAE and induce Th2 response. EAE was induced in female B6 mice with MOG35–55 and CFA. DCs were isolated from the bone marrow of congenic B6 mice and transduced with SOCS-3 or GFP as described in Fig. 2. Transduced DCs were pulsed overnight with MOG35–55 peptide (25 µg/ml), washed, and injected i.v. to each immunized mouse at 2 x 106 in 0.2 ml of PBS. Transduced DCs were injected at EAE induction (–3 and + 7 days p.i.) (A) or disease onset (11, 14, and 17 days p.i.) (B), and mice were sacrificed at days 21 p.i. and 24 p.i., respectively. Clinical disease was evaluated daily by two blinded observers following a 0–5 scale system as described in Materials and Methods. Symbols refer to mean clinical scores (n = 8 in each group). Splenocytes of mice in A were isolated for proliferative response (C) and cytokine production (D). The p values refer to comparison between GFP-DC-injected and SOCS-3-DC-injected mice. *, p < 0.05 and **, p < 0.01. Similar results were obtained for splenocytes of mice in B (data not shown). One representative experiment of two is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study provides evidence that DCs overexpressing SOCS-3, an important suppressor of cytokine signaling, exhibit a tolerogenic/DC2 phenotype that promotes Th2 differentiation and have therapeutic potential for Th1 cell-mediated autoimmune diseases such as EAE/multiple sclerosis.

Negative regulation of signal transduction pathways is necessary for an appropriate cellular and physiological response to cytokine stimulation. In DCs, proinflammatory cytokines such as IL-12 and IL-23 trigger their extracellular signals to the nucleus through activated STAT proteins, and the duration and the intensity of the cytokine-induced signal are under feedback regulation by a newly described family of intracellular proteins called SOCS. Among them, SOCS-3 has been suggested to be a potent inhibitor of IL-12- and IL-6-induced signaling (24, 25) and to regulate DC maturation (20). SOCS-3 can be induced by infectious agents and proinflammatory cytokines, including IL-12 (24, 26), suggesting that SOCS-3 may act in a classical negative feedback loop to attenuate proinflammatory signaling. However, the role of SOCS-3 in regulating the signaling pathway of IL-23, another important proinflammatory cytokine in the IL-12 family (27), is not known. Our current study shows that, in DCs, SOCS-3 is not only induced by IL-12 and a bacterial product LPS, consistent with previous studies, but also upon stimulation with IL-23, suggesting a negative feedback for IL-23 response.

Transmembrane signaling for IL-12 and IL-23 is mediated by proteins of the Janus kinase and Stat pathway. IL-12 and IL-23 activate a similar spectrum of Jak/Stat molecules: Jak2, Tyk2, Stat3, Stat4, Stat5, and Stat1 (3, 28). However, IL-12 mainly signals through activation of a Stat4/Stat4 homodimer, whereas IL-23 mainly activates a Stat3/Stat4 heterodimer (27, 29). Secretion of IL-12 by DCs leads to induction of T-bet and IFN-{gamma} and, in turn, participates in a positive feedback loop via IL-12 receptor beta1 and beta2 (4, 5). IL-23 can bind to splenic DCs and drive IL-12 and IFN-{gamma} production of these cells (6). We and others have found that APCs deficient in IL-12/IL-23 or their receptors display an immunosuppressive property (12, 13, 30, 31). As the most potent APCs, DCs can either stimulate or regulate immune response depending on their ontogeny, state of differentiation, and maturation, thus expressing variable membrane-bound and soluble molecules (32). The dual function of DCs has made them suitable targets for manipulation, such as genetic modification and drugs to modulate immune response. Due to the potent ability of SOCS-3 to block Stat3 and Stat4 signaling (13, 33), we believe that up-regulation of this molecule could effectively target IL-12/IL-23 downstream signaling and induce a tolerogenic phenotype of target cells. Indeed, data in our present study demonstrate that overexpression of SOCS-3 inhibits the IL-12-induced activation of Stat4 and IL-23-induced activation of Stat3 in DCs. Significantly lower CD86 and MHC class II expression on SOCS-3-transduced DCs suggests a blockade of NF-{kappa}B activation, another pathway for IL-12 signaling (34, 35), The low level of IL-23 in SOCS-3-transduced DCs implies that SOCS-3 influences the autocrine and paracrine effect of IL-23 in DCs, probably by physical interaction of SOCS-3 with IL-23R (13, 33).

The mechanism underlying SOCS-3-induced high IL-10 production is not clear. Two factors may contribute to this phenomenon. The first factor is that SOCS-3 may regulate the proposed IL-12/IL-10 balance. It has been suggested that there is an IL-12/IL-10 immunoregulatory circuit controlling susceptibility to autoimmune diseases (36). In this circuit the disease-promoting effects of IL-12 are antagonized by IL-10, which, in turn, is regulated by the exogenous production of IL-12. Therefore, decreased IL-12 production or its responsiveness could result in an increased IL-10 level, thus exerting a profound effect on the incidence of autoimmune diseases (36, 37). Results derived from our present study support this IL-12/IL-10 circuit model. The second factor is that endogenous SOCS-3 may be a selective inhibitor for IL-12/IL-23 but not for IL-10, similar to a previously proposed role of SOCS-3 in IL-4 signaling (15). In that report, Seki et al. (15) found that forced expression of SOCS-3 inhibited the phosphorylation of IL-12-mediated Stat4 and IL-2-mediated Stat5, whereas IL-4-induced Stat6 phosphorylation was not affected. As a result, the inhibition of IL-12 signaling by SOCS-3 reduces Th1 differentiation, with substantial enhancement of Th2 development (15). It is also possible that whereas SOCS-3 efficiently inhibits IL-12-induced Stat4 and IL-23-induced Stat3 phosphorylation, this approach does not influence Stat1 and Stat3 phosphorylation induced by IL-10. Thus, predominant IL-10 expression in DCs subsequently accelerates the skewing toward Th2 differentiation. IL-10 has been identified as the major cytokine that prevents both differentiation of DCs from monocytes and maturation of DCs by blocking release of IL-12 (38, 39). IL-10 also inhibits expression of costimulatory molecules and, consequently, Th1 response (40, 41). In addition, the ability of IL-10 to inhibit gene expression in monocytes might be associated with its ability to rapidly induce synthesis of SOCS-3 (42), which can act as a mediator for IL-10-induced anti-inflammatory function (18, 25, 43, 44, 45). These results, together with our current observation that SOCS-3 induces IL-10 production in DCs, indicate a syngeneic effect of SOCS-3 and IL-10 in regulating immune response.

It has been suggested that APCs, among which DCs are the most potent, provide T cells not only with an Ag-specific stimulatory signal (signal 1, ligation of TCR) and a series of costimulatory signals (signal 2), but also with signals (proposed as signal 3) that polarize Th cell cytokine production (10, 46). There exists a paradigm of type 1 and type 2 DCs, i.e., DC1 cells produce high levels of IL-12 and promote Th1 cell development, and DC2 cells produce a low level of IL-12 and promote Th2 (10, 46). In the present study, low expression of MHC class II and CD86 in SOCS-3-transduced DCs may induce decreased T cell proliferation due to a suboptimal level of stimulatory signals. The strongly exhibited DC2 phenotype in SOCS-3-transduced DCs potently polarizes the immune system to Th2 responses, demonstrated by decreased production of the inflammatory cytokines TNF-{alpha} and IFN-{gamma} and increased IL-4 production by CD4+ T cells. A significant decrease in IL-17 production in cocultured T cells indicates a low production of biologically active IL-23 (12, 47, 48) of SOCS-3-transduced DCs. Such a decrease can also be due to high IL-4 production, which inhibits differentiation of IL-17-producing CD4+ T cells (49). The shift from Th1 to Th2 was confirmed by adoptive transfer of SOCS-3-tranduced DCs in vivo. The combination of Th2-promoting properties and poor proliferation of Ag-reactive T cells suggests that SOCS-3-transduced DCs possess a "tolerogenic"/DC2 phenotype and would thus be useful in the treatment of Th1-mediated autoimmune diseases.

Although systemic administration of anti-inflammatory cytokines, such as IL-4 and IL-10, could serve as a useful immunotherapy for autoimmune diseases, there are several reasons why treatment by adoptively transferred cells delivering these cytokines might be a more effective and promising approach. First, because of the chronic nature of autoimmune diseases and the short half-life of cytokines, a frequent administration of exogenous cytokine is required with limited effect. In contrast, gene transduction offers a unique way of providing long-term delivery of immunomodulatory molecules in vivo that can antagonize the chronic inflammatory processes (50, 51). Second, the systemic distribution of exogenous cytokines will affect multiple target systems/organs, including the cardiovascular system, liver, spleen, and bone marrow, and can lead to deleterious side effects. For example, administration of IL-4 may cause severe side effects including cardiac inflammation and necrosis, hepatitis, hepatic necrosis, and even death (52, 53). In contrast, local delivery of DC2-type cytokines (low IL-12 and high IL-10) by DCs during APC-T cell interaction would specifically affect the targeted cells (46), thus avoiding the side effects of systemic administration. Because Ag-specific T cells are not easily available for in vitro manipulation, especially in humans, gene-modified DCs provide a practical means to guide the differentiation of these T cells in vivo. Finally, a highly expressed upstream regulatory factor for Th1/Th2 differentiation, such as SOCS-3 in the present study, results in a combination of therapeutic products, including high levels of endogenous, autoantigen-induced IL-4 and IL-10, but low levels of the proinflammatory cytokines IFN-{gamma}, TNF-{alpha}, and IL-17 as shown in Fig. 6. Adoptive cellular gene transduction used in our present study as a treatment for chronic inflammatory diseases is thus clearly advantageous over the injection of exogenous IL-4, IL-10, or any other single cytokine or a mixture of multiple cytokines/cytokine antagonists.

In conclusion, our study demonstrates the suppressive effect of SOCS-3-transduced DCs on EAE and provides evidence that these DCs exhibit a tolerogenic/DC2 phenotype that selectively suppresses Th1 and induces Th2 differentiation in vitro and in vivo. Manipulation of autogenous DCs with SOCS-3 could, therefore, be a potential therapy for Th1 cell-mediated autoimmune disorders such as EAE and multiple sclerosis.


   Acknowledgments
 
We thank Dr. Bogoljub Ciric for critical discussions and Katherine Regan for editorial 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 supported by grants from the National Institutes of Health, the National Multiple Sclerosis Society, and the Groff Foundation. Back

2 Address correspondence and reprint requests to Dr. Guang-Xian Zhang, Department of Neurology, Thomas Jefferson University, 300 Jefferson Hospital for Neuroscience Building, 900 Walnut Street, Philadelphia, PA 19107. E-mail address: guang-xian.zhang{at}jefferson.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; MFI, mean fluorescence intensity; MOG, myelin oligodendrocyte glycoprotein; p.i., postimmunization; SOCS, suppressor of cytokine signaling. Back

Received for publication November 15, 2005. Accepted for publication May 16, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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