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Apr 1, 2010 - Taipei, Taiwan, ROC; 4Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC and.

Gene Therapy (2010) 17, 1011–1021 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 www.nature.com/gt

ORIGINAL ARTICLE

Cytokine gene-modulated dendritic cells protect against allergic airway inflammation by inducing IL-10+IFN-g+CD4+ T cells C-Y Hsu1, S-J Leu2, B-L Chiang3, HE Liu4, H-C Su5 and Y-L Lee2 1 Department of Internal Medicine, Cathay General Hospital, Taipei, Taiwan, ROC; 2Department of Microbiology and Immunology, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC; 3Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, ROC; 4Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC and 5 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC

Asthma is characterized by allergen-induced airway inflammation orchestrated by Th2 cells. Dendritic cells (DCs) were found to efficiently prime naive T-helper cells. Thus, modification of DC function may be used as an ideal tool to treat allergic asthma by changing CD4+ T-cell differentiation or suppressing Th2 development. In this study, we examined whether a DC-based vaccine can be applied to DCs modified with interleukin (IL)-10- and IL-12-expressing adenoviruses to prevent ovalbumin (OVA)-induced asthma in mice. Herein, we show that these modified DCs efficiently moderated the characteristics of asthma, including expressions of OVAspecific antibodies, airway hyperresponsiveness, eosinophilic airway inflammation, and Th2 cytokines production. Additionally, IL-10 and IL-12 gene-modified DCs enhanced the

development of both T-helper type 1 (Th1) and IL-10+IFN-g+ (interferon-g) double-positive T cells in vivo. In vitro-generated OVA-specific IL-10+IFN-g+CD4+ T cells inhibited the proliferation of naive CD4+ T cells, and this suppressive effect was a cell contact-dependent mechanism. Furthermore, we showed that combined cytokine-modulated DCs could alleviate established allergic airway inflammation. Taken together, these results suggest that IL-10 and IL-12 gene-modulated DCs are effective in suppressing asthmatic airway inflammation through both immune deviation and immune suppression and are a potential therapeutic approach for asthma. Gene Therapy (2010) 17, 1011–1021; doi:10.1038/gt.2010.39; published online 1 April 2010

Keywords: asthma; dendritic cells; adenoviruses; IL-10; IL-12; T cells

Introduction Allergic asthma is a disease characterized by elevated serum immunoglobulin E (IgE), airway hyperresponsiveness (AHR), and eosinophilic inflammation. It is recognized that CD4+ T-helper type 2 (Th2) cells and their cytokines (interleukin (IL)-4, IL-5, and IL-13) are responsible for initiating and maintaining allergic disorders.1,2 Numerous studies indicated that regulating asthma and allergic diseases is complex, involving several different mechanisms and cell types, and might not be reflected in a simple, dichotomous balance of polarized cells, as suggested by the T-helper type 1 (Th1)/Th2 paradigm.3,4 Dendritic cells (DCs), as professional antigen-presenting cells, have an important function in antigen presentation in the airways, and the cytokine profile secreted by DCs can influence differentiation of T cells.5,6 IL-10 was originally described as a Th2-associated Correspondence: Dr Y-L Lee, Department of Microbiology and Immunology, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan, Republic of China. E-mail: [email protected] Received 9 April 2009; revised 14 August 2009; accepted 16 August 2009; published online 1 April 2010

cytokine that antagonizes Th1 responses.7 Subsequent studies showed that IL-10 can downregulate Th2 clones and their production of IL-4 and IL-5.8 In addition, IL-10 inhibits the synthesis of proinflammatory cytokines and chemokines by monocytes/macrophages, neutrophils, and eosinophils.9–12 IL-10 was also shown to be produced by specialized subsets of regulatory T cells (Tregs), such as CD25+FoxP3+ Tregs and T-regulatory 1 cells (Tr1), and the regulatory activities of T cells were found to be closely related to their IL-10-producing ability.13–16 These findings led to the hypothesis that the expression of IL-10 in the airway environment may inhibit the development of airway inflammation. Another key cytokine produced by DCs involved in Th polarization is IL-12. In general, IL-12 upregulates interferon (IFN)-g production by T cells and natural killer cells, promotes Th1 responses, and suppresses the expression of Th2 cytokines.17,18 A previous study showed that production of IFN-g in combination with IL-10 was induced in T cells by IL-12.19,20 Furthermore, this combined production of IFN-g and IL-10 was postulated to occur in immunoregulatory T cells that protect against severe inflammatory pathologies.21 Recently, it was shown that sublingual immunotherapy with a Dermatophagoides monomeric allergoid downregulated allergen-specific IgE and was associated

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with a significant increase in T cells producing both IL-10 and IFN-g.22 In addition, an inducible Th1-like Treg cell, which produces both IL-10 and IFN-g, was reported to block the development of allergen-induced AHR in a murine model of asthma.23 From this evidence, we hypothesized that induction of the combined production of IFN-g and IL-10 in T cells may be a synergistic combination that can inhibit Th2 immune responses. In this study, we investigated the ability of IL-10 and IL-12 gene-modulated DCs to treat antigen-induced asthma and explored the possible mechanism of this regulation.

Results In vitro stimulatory ability of naive CD4+ T cells with adenovirus (Ad)-infected DCs To evaluate the infection efficiency of adenovirus vectors, bone marrow-derived DCs were transduced with different multiplicity of infections (MOIs) of Ad-mock, Ad-IL10, or Ad-IL-12 for 48 h. Levels of IL-10 and IL-12 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). The data showed that IL-10 production of Ad-IL-10-infected DCs was dose dependent (Figure 1a). Similarly, Ad-IL-12-infected DCs secreted IL-12 in the same pattern. Co-infection with a low dose of Ad-IL-10 (at an MOI of 500) and a high dose of Ad-IL-12 (at an MOI of 5000) did not influence the expression levels of IL-10 or IL-12. In contrast, a combined low dose of Ad-IL-12 (at an MOI of 500) infection with a high dose of Ad-IL-10 (at an MOI of 5000) suppressed the production of IL-12. Neither IL-10 nor IL-12 was detectable in the supernatant of Ad-mockinfected DCs. We then studied the ability of DCs co-infected with Ad-IL-10 and Ad-IL-12 to activate the naive CD4+ T-cell response. Purified ovalbumin (OVA)specific, naive DO11.10 CD4+ T cells were cultured with the OVA peptide and irradiated uninfected DCs (DC), Ad-mock-infected DCs (DC-mock, at an MOI of 5000), Ad-IL-10-infected DCs (DC-IL-10, at an MOI of 500), Ad-IL-12-infected DCs (DC-IL-12, at an MOI of 5000), or DCs co-infected with Ad-IL-10 and Ad-IL-12 (DC-IL-10/ 12) at various ratios of DC and T cells. After 3 days of culture, OVA-specific T-cell proliferation and the cytokine profile were assayed. Although CD4+ T cells incubated with DC-IL-10 showed a lower proliferative response to the OVA peptide at the high ratio of DC/T cells (1:10), there were no significant differences among these groups (Figure 1b). Furthermore, DCs that were infected with Ad-IL-12 induced a high level of IFN-g production and a low level of IL-4 secretion (Figures 1c and d). DC-IL-10/12 reduced serum anti-OVA IgE and enhanced anti-OVA IgG2a levels in an animal model of asthma To investigate the application of DCs co-infected with Ad-IL-10 and Ad-IL-12 to modulate the immune response of allergic diseases, in vitro-modulated DCs were injected intratracheally into the lungs of mice, and an animal model of asthma was subsequently induced by OVA sensitization and challenge (Figure 2a). Groups of mice were immunized with 5  105 OVA-pulsed Admock-infected DCs (DC-mock), Ad-IL-10-infected DCs Gene Therapy

(DC-IL-10), Ad-IL-12-infected DCs (DC-IL-12), combined cytokine-modulated DCs (DC-IL-10/12), or uninfected DCs (DC). Control mice received phosphate-buffered saline (PBS) instead of DCs. Serum samples were collected on the indicated days, and OVA-specific IgE, IgG1, and IgG2a serum antibody titers were determined by ELISA. Although both DC-IL-12 and DC-IL-10/12 efficiently inhibited OVA-specific IgE production (Figure 2b) and enhanced OVA-specific IgG2a secretion (Figure 2d), they did not suppress the secretion of IgG1 (Figure 2c). In addition, mice that received DC-IL-10 did not show reduced IgE production or enhanced IgG2a expression.

DC-IL-10/12 suppressed development of AHR and airway inflammation To assess the preventive effects of DC-IL-10/12 on allergic asthma, both AHR and the accumulation of inflammatory cells in bronchoalveolar lavage (BAL) fluid were measured. One day after the last OVA challenge, the airway responsiveness to aerosolized methacholine of each group of mice was measured (Figure 3a). Mice pretreated with PBS, DC-mock, or uninfected DCs and then sensitized with OVA developed markedly increased airway responsiveness to methacholine stimulation. On the contrary, DC-IL-10, -12, and -10/12 alleviated the development of AHR compared with the other groups. In control mice, exposure to aerosolized OVA induced a marked increase in the number of eosinophils and neutrophils in the BAL fluid (Figure 3b). The delivery of DC or DC-mock did not decrease the airway inflammation in this murine model of asthma. In contrast, mice treated with DC-IL-10/12 showed significantly reduced increases in eosinophils and neutrophils. Administration of DC-IL-12 decreased eosinophil infiltration, but induced mild mononuclear cell infiltration in the lungs. A pathological examination of lung sections showed that the control, and DC- and DCmock-treated mice had greater degrees of inflammatory cell infiltration around the airways (Figure 3c). The extent of damaged and infiltrated cells was less severe in the DC-IL-12- and DC-IL-10-treated groups. Of note, lung tissues from DC-IL-10/12-treated mice showed efficient inhibitory effects on peribronchial inflammation. DC-IL-10/12 decreased the levels of inflammatory mediators in BAL fluid Eotaxin is the most potent chemokine for recruiting eosinophils. Evaluation of the eotaxin level in BAL fluid showed that DC-IL-10/12 slightly reduced the secretion of eotaxin (Figure 4a). Furthermore, we assessed the levels of the important Th2 cytokines, IL-4, and IL-5, in BAL fluid. The data showed that DC-IL-10/12 treatment had synergistic effects on the suppression of Th2 cytokine production compared with the DC-IL-10 or DC-IL-12 groups (Figures 4b and c). Levels of IFN-g and IL-10 increased in mice receiving DC-IL-10/12 By studying IFN-g and IL-10 concentrations in BAL fluid and cell culture supernatant from the spleen, we investigated whether it is possible to affect the immune response through inducing IL-10- and/or IFN-g-producing T cells. It was shown that DC-IL-10/12 upregulated

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Figure 1 Stimulation of naive CD4+ T cells with adenovirus-infected DCs in vitro. (a) The cytokine expression efficiency of adenovirusinfected DCs. On day 6 of culture, bone marrow-derived DCs were collected and infected with different MOIs of Ad-interleukin (IL)-10 or Ad-IL-12, or Ad-IL-10 combined with Ad-IL-12 for 48 h. Levels of IL-10 and IL-12 in the culture supernatants were analyzed by ELISA. Results from triplicate experiments are shown and expressed as mean±s.e.m. (b) The immunostimulatory capacity of adenovirus-infected DCs. On day 6 of culture, bone marrow-derived DCs were stimulated with the OVA peptide and infected with either Ad-mock (at an MOI of 5000), Ad-IL-10 (at an MOI of 500), or Ad-IL-12 (at an MOI of 5000), or co-infected with Ad-IL-10 and Ad-IL-12 for 48 h. On day 8, Ad-IL-10infected DCs (DC-IL-10), Ad-IL-12-infected DCs (DC-IL-12), co-infected DCs (DC-IL-10/12), Ad-mock-infected DCs (DC-mock), and noninfected DCs (DC) were collected. For the proliferation assay, freshly isolated DO11.10 CD4+ T cells were co-cultured with or without irradiated adenovirus-infected DCs at the indicated DC/T cell ratios for 72 h. Proliferation of T cells was determined by [3H]-thymidine incorporation after 3 days of culture. Results from triplicate experiments are shown and are expressed as mean±s.e.m. (c, d) Cytokine secreting pattern of naive DO11.10 CD4+ T cells after stimulation with or without adenovirus-infected DCs. Irradiated adenovirus-infected DCs pulsed with the OVA peptide were collected and co-cultured at various ratios with naive DO11.10 CD4+ T cells. After 72 h, supernatants were collected, and levels of IFN-g and IL-4 production were analyzed by ELISA. Results from triplicate experiments are shown and expressed as mean±s.e.m. *Po0.05, **Po0.01, and ***Po0.001 vs the DC group. #Po0.05, ##Po0.01, and###Po0.001 vs the DC-mock group. w Po0.05, wwPo0.01, and wwwPo0.001 vs the DC-IL-10 group.

both IFN-g and IL-10 productions in DC-IL-10/12treated mice (Figure 5). Taken together, these results indicate that DC-IL-10/12 are potent suppressors, which inhibit the development of pulmonary inflammation in asthma, and one of these regulatory functions might be due to the induction of IL-10 and/or IFN-g production by OVA-specific T cells.

DC-IL-10/12 enhanced development of IL-10+IFN-g+CD4+ T cells in vivo To investigate the T-cell-polarizing capacity of DCs coinfected with Ad-IL-10 and Ad-IL-12 in vivo, splenocytes from groups of mice were collected after AHR measurement and then analyzed for the intracellular expressions of IL-10 and IFN-g. We determined the numbers of IL-10-

and IFN-g-producing CD4+ T cells, which had significantly increased in DC-IL-10/12-treated mice compared with the other groups (Figures 6a and b). We also found that DC-IL-10/12 slightly enhanced the number of Th1 cells. Consequently, our results showed that DC-IL-12 can induce the development of the Th1-cell population.

OVA-specific IL-10- and IFN-g-producing T cells inhibited naive T-cell proliferation in vitro To analyze the influence of DC-IL-10/12 on differentiation of naive OVA-specific T cells in vitro, three rounds of stimulations of T cells were established. We found that DC-IL-10/12 enhanced development of IL-10+IFN-g+ T cells, and this DC-IL-10/12-driven T-cell line secreted greater amounts of IL-10 and IFN-g (Supplementary Gene Therapy

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Figure 2 Anti-OVA antibody expression levels of cytokinemodulated DC-treated mice. (a) Brief protocol of animal sensitization and challenge. Groups of mice received either uninfected DCs (DC), Ad-mock-infected DCs (DC-mock), Ad-interleukin (IL)-10infected DCs (DC-IL-10), Ad-IL-12-infected DCs (DC-IL-12), or combined cytokine-modulated DCs (DC-IL-10/12) pulsed with OVA on day 1. Control mice (control) were administered PBS instead of DCs. On days 8 and 18, all groups of mice were sensitized by an intraperitoneal (i.p.) injection of 50 mg OVA emulsified in 4 mg alum. Then mice were challenged intranasally with 100 mg OVA on days 20 and 21. Subsequently, mice were exposed to OVA aerosols for 3 consecutive days, and AHR was measured 1 day after the last challenge. BAL fluid was obtained, and lung fixation was performed after measuring the AHR. (b) Immunoglobulin E (IgE), (c) IgG1, and (d) IgG2a anti-OVA antibody expressions from OVAimmunized mice that received uninfected or vector-infected DCs pulsed with OVA were measured by ELISA. Control mice were administered PBS instead of DC treatment. Results are expressed as mean±s.e.m. of five to eight mice in each group. Experiments were repeated three times with similar results. *Po0.05, **Po0.01, and ***Po0.001 vs the control group. #Po0.05 and ###Po0.001 vs the DC group. wPo0.05, wwPo0.01 and wwwPo0.001 vs the DC-mock group.

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Figure 3 Preventive effects of combined cytokine-modulated DCs on AHR and airway inflammation. (a) One day after the last OVA challenge, airway resistance was measured by invasive body plethysmography. Results are expressed as mean±s.e.m. of the pulmonary resistance (RL) in the ratio of RL after PBS nebulization of three independent experiments. Each group contained five to eight mice. (b) Cell compositions in BAL fluid of different groups of mice are expressed as mean±s.e.m. of five to eight mice per group. Cells were counted and classified as monocytes (Mon), eosinophils (Eos), neutrophils (Neu), and lymphocytes (Lym). Experiments were repeated three times with similar results. (c) Lung sections were obtained from PBS-treated control mice (control), DC-treated mice (DC), and those treated with Ad-mock-infected DCs (DCmock), Ad-interleukin (IL)-10-infected DCs (DC-IL-10), Ad-IL-12infected DCs (DC-IL-12), or DCs co-infected with Ad-IL-10 and AdIL-12 (DC-IL-10/12). Sections were stained with hematoxylin and eosin for the morphological analysis. Tissues were examined by light microscopy (original magnification  400). *Po0.05, **Po0.01, and ***Po0.001 vs the control group. #Po0.05, ##Po0.01, and ### Po0.001 vs the DC group. wPo0.05; wwPo0.01 and wwwPo0.001 vs the DC-mock group.

Figure S1). Additionally, this T-cell line expressed minimal levels of IL-4 (18 pg ml 1) and IL-2 (35 pg ml 1) and secreted no IL-5 or TGF-b (data not shown). To

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addition, blocking IL-10-mediated signaling partially reversed this suppressive mechanism (Figure 7b). Furthermore, to address the issue of whether cell–cell contact can mediate the suppression of naive T-cell proliferation by the DC-IL-10/12-driven T-cell line, transwell culture chambers were used. Separation of naive T cells from the T-cell line resulted in abrogation of the suppression of T-cell proliferation, as opposed to cultures in which both naive cells and the T-cell line were present in the lower chamber of the culture (Figure 7b). Taken together, these results showed that DC-IL-10/12 induced the differentiation of IL-10+IFN-g+ Tregs in vitro, and this suppressive effect of Tregs was a cell contactdependent mechanism.

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DC-IL-10/12 alleviated Th2-dependent lung inflammation in sensitized mice We next investigated whether DC-IL-10/12 can suppress established Th2 responses in OVA-sensitized mice. Mice were sensitized to three OVA injections and subsequently treated intratracheally with OVA-pulsed DC, DC-mock, DC-IL-10, DC-IL-12, or DC-IL-10/12 (Figure 8a). Ten days after DC treatment, mice were challenged with OVA for 5 consecutive days. One day after the last OVA challenge, AHR and airway inflammation were assessed. As shown in Figure 8b, administration of DC-IL-12 alone notably reduced the severity of AHR. Similarly, DC-IL-10/12 treatment resulted in a significant decrease in the development of AHR compared with the control group. The reduction in AHR by DC-IL-10/12 was accompanied by a notable inhibition of airway inflammation. Thus, DC-IL-10/12 greatly reduced the numbers of eosinophils (Figure 8c). A similar result was also found in mice treated with DC-IL-12 alone. Additionally, we observed slight enhancement of IFN-g expression and reduced levels of eotaxin and IL-4 production in mice treated with DC-IL12 alone or DC-IL-10/12 (Supplementary Figure S2). In contrast, DC-IL-10 failed to suppress the development of asthma in already sensitized mice.

Discussion

Figure 4 Levels of inflammatory mediators decreased in BAL fluid of mice receiving combined cytokine-modulated DCs. (a) Eotaxin, (b) interleukin (IL)-4, and (c) IL-5 levels in BAL fluids of different groups of mice were measured by ELISA. Results are expressed as mean±s.e.m. of five to eight mice per group. Experiments were repeated three times with similar results. *Po0.05, **Po0.01, and ***Po0.001 vs the control group. #Po0.05, ##Po0.01, and ### Po0.001 vs the DC group. wPo0.05 and wwwPo0.001 vs the DC-mock group.

analyze the functional properties of this co-infected DC-driven T-cell line, a suppressive ability assay was performed. T-cell lines obtained after repeated stimulation with DC-IL-10 or DC-IL-10/12 showed a low proliferative capacity and strongly suppressed the proliferation of responder naive DO11.10 CD4+ T cells at 1:1–1/2:1 ratios of regulator to responder (Figure 7a). In

Although the etiology of asthma is not completely understood, it has become increasingly clear that DCs have an important role in both the sensitization phase and maintenance of the disease. As atopic asthma is associated with Th2-dependent disease, deviation toward other pathways of CD4+ T-cell development might be beneficial. Therefore, genetically modulated DCs, such as IL-12, were tested to see whether they could counterbalance Th2 activity.24,25 These strategies proved to be efficient in inhibiting the induction of antigeninduced Th2 responses, but they had a major drawback of causing Th1-driven inflammatory pulmonary pathology. However, although Th1 cells might not function in the lung mucosa itself to inhibit allergic asthma, it is possible that Th1 cells might act in peripheral lymphoid organs to inhibit the early development of Th2 effector cells. The recent discovery of Tregs is an important advance in knowledge concerning the regulation of immune responses. It was reported that pulmonary DCs from tolerized mice expressing IL-10 drive the generation of a population of IL-10-producing Tregs that Gene Therapy

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Figure 5 Levels of interferon (IFN)-g and interleukin (IL)-10 increased in mice receiving combined cytokine-modulated DCs. IFN-g and IL-10 production in (a, b) BAL fluid, and in (c, d) culture supernatants of OVA-restimulated T cells from the spleen were analyzed by an ELISA. Results are expressed as mean±s.e.m. of five to eight mice per group. Experiments were repeated three times with similar results. *Po0.05, **Po0.01, and ***Po0.001 vs the control group. #Po0.05, ##Po0.01, and ###Po0.001 vs the DC group. wwPo0.01 and wwwPo0.001 vs the DC-mock group.

can inhibit subsequent responses to antigenic challenge.26 Thus, induction of antigen-specific Tregs targeted to allergies and asthma is considered a promising immunomodulatory strategy to suppress the Th2 response. In this study, our results clearly showed that a single injection of DC-IL-10/12 before mice were sensitized and challenged with OVA strongly decreased the production of allergen-specific IgE, alleviated the severity of AHR, reduced allergen-induced eosinophilic airway inflammation, and decreased the accumulation of proinflammatory Th2 cytokines in the airway. Histological studies also showed that it not only decreased OVA-induced peribronchial inflammation but also avoided Th1-driven inflammatory pathology that was observed in DC-IL-12treated mice. Taken together, these data indicate that DC-IL-10/12 exerted better preventive effects in modulating allergic airway inflammation than did treatment with DC-IL-10 or DC-IL-12 alone. How these cytokine gene-modulated DCs carry out their ‘regulatory’ role is not well understood. One possible explanation for the inhibitory activities of DC-IL-10/12 on lung allergic responses might involve their capacity to produce high levels of IL-10 and IL-12, and directly inhibit allergic airway inflammation. The immunoregulatory properties of IL-10 and IL-12 have led to their therapeutic uses in allergic asthma. Several studies showed that the systemic administration of the Gene Therapy

IL-10 or IL-12 protein can decrease eosinophil infiltration and airway inflammation in murine models of asthma.27,28 Although IL-12 is capable of downregulating allergic inflammation, the increase in TNF-a modulates the inflammatory reaction as was observed after IL-12 therapy. It is noteworthy that IL-10 can reduce TNF-a release, and macrophages are a widely described celltype sensitive to this cytokine. Accordingly, our previous study showed that combined Ad-IL-10 and Ad-IL-12 treatment might suppress the production of TNF-a in lungs and inhibit cellular recruitment into airways of OVA-sensitized mice.29 Another explanation for the inhibitory activities of DC-IL-10/12 might involve their ability to induce the expression and functional activation of T cells. Indeed, both local (BAL fluid) and systemic (spleen) levels of IL-10 and IFN-g in mice that received DC-IL-10/12 were significantly higher than those of mice that received DCs or DC-mock. Moreover, our studies showed that the percentage of IL-10+IFN-g+CD4+ T cells increased in mice receiving DC-IL-10/12 treatment. Furthermore, we showed that DC-IL-10/12 could drive the development of IL-10+IFN-g+CD4+ Tregs in vitro. These antigenspecific Tregs expressed high levels of IL-10 and IFN-g, and inhibited T-cell proliferation in vitro in a cell contactdependent manner. In this study, we also showed that DC-IL-10/12 has inductive effects on Th1 responses, including anti-OVA IgG2a and IFN-g production in mice

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Figure 6 DCs co-infected with Ad-interleukin (IL)-10 and Ad-IL-12 showed enhanced generation of IL-10+interferon (IFN)-g+ CD4+ T cells in vivo. Splenocytes from groups of mice were collected after AHR measurement and restimulated with phorbol 12-myrisytate 13-acetate (PMA) and ionomycin for intracellular expression of cytokines by flow cytometry. Representative fluorescence activated cell sorting (FACS) plots show IL-10 and IFN-g expressions in the CD4+ gate (a), and statistical evaluations across all animals (b) are shown as mean±s.e.m. of five mice in each group.

treated with DC-IL-10/12. Thus, these data support the concept that their preventive activity against the development of allergic reactions is mediated by a combined mechanism of immune suppression and immune deviation. In general, adaptive antigen-specific Tregs do not express inhibitory molecules, such as cytotoxic T-lymphocyte-associated molecule-4 or programmed death-1, and therefore these inhibitory pathways are not likely to be involved in their suppression mechanisms. However, using allogeneic immature DCs, Jonuleit et al.30 showed that the suppressive effect of human Tr1-like cells required cell–cell contact. They showed that neither neutralizing mAbs directed against IL-10 nor blocking cytotoxic T-lymphocyte-associated molecule-4 could reverse the suppressive capacity of Tr1-like cells. Umetsu and co-workers observed that the inhibitory effects of some adaptive antigen-specific Tregs can be abrogated by blocking the inducible costimulator (ICOS)–ICOS-ligand pathway, suggesting that ICOS–ICOS-ligand interactions may be important in driving the development of antigen-specific Tregs.23,31 Therefore, further study of IL-10+IFN-g+CD4+ T cells is required to clearly understand the mechanisms of suppression. Taken together, it is possible that IL-10-producing T cells simultaneously synthesize IFN-g, a combination that is particularly effective in downmodulating allergic asthma. Similarly, the same results were observed with oral probiotics, which seem to be able to alleviate

clinical symptoms of atopic dermatitis.32 The probiotic not only induces IL-10 production, but also increases the production of IFN-g. Recently, another member of the IL-12 family of cytokines, IL-27, was reported to induce IL-10+IFN-g+ T cells, and this phenotype characterizes CD4+ T cells that were described as key negative regulators of the immune response to Toxoplasma gondii and Leishmania major infection.33–35 Actually, IL-10+CD4+ regulatory cells with a Th1 cell phenotype were described in several infections, including murine Mycobacterium avium and Brucella abortus, as well as in shortterm T-cell clones derived from patients with M. tuberculosis.36–38 However, whether such IL-10+IFN-g+ double-positive T cells as described in these infection models can be defined as Treg or Th1 cells remains to be determined. Finally, the therapeutic effects of DC-IL-10/12 in OVA-sensitized mice were evaluated, and data showed that combined cytokine treatment did not show synergistic efficacy in this murine model of asthma. We propose that Ad-IL-12 but not Ad-IL-10 expressed a direct inhibitory effect on established Th2 immune responses. Thus, the relative efficacies of varying combined doses of Ad-IL-10 and Ad-IL-12 should be further examined to obtain the optimal therapeutic effects before using adenoviral vectors for gene therapy in the future. Furthermore, multiple treatments of OVA-sensitized mice with DC-IL-10/12 might be a better strategy to induce Tregs than mice treated with DC-IL10/12 only a single time. Gene Therapy

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Figure 7 DCs co-infected with Ad-interleukin (IL)-10 and Ad-IL12 induced the development of IL-10+interferon (IFN)-g+ regulatory T cells in vitro. (a) Inhibition of antigen-specific proliferation of naive DO11.10 CD4+ T cells after co-culture with T-cell lines. T-cell lines were induced by repetitive stimulation of naive DO11.10 CD4+ T cells with Ad-mock-infected DCs (DC-mock), Ad-IL-10-infected DCs (DC-IL-10), Ad-IL-12-infected DCs (DC-IL-12), co-infected DCs (DC-IL-10/12), or non-infected DCs (DC) in the presence of the OVA323 339 peptide (1 mg ml 1). Seven days after the third stimulation, different numbers of T-cell lines were stimulated with irradiated splenocytes and the OVA peptide in the presence of purified naive DO11.10 CD4+ T cells (Tn, 2.5  105 cells per well) at suppressor/responder (T-cell line/Tn) ratios of 1:1, 1/2:1, 1/4:1, 0:1, and 1:0. [3H]-thymidine incorporation was measured after 3 days of culture. Results are expressed as mean±s.e.m. #Po0.05, ##Po0.01, and ###Po0.001 vs the DC group. wwPo0.01 and wwwPo0.001 vs the DC-mock group. (b) IL-10 and cell–cell contact mediated the suppression of naive DO11.10 CD4+ T-cell proliferation by the DC-IL-10/12-induced T-cell line. The purified naive DO11.10 CD4+ T cells (Tn, 2.5  105 cells per well) were cultured in 24-well plates in the presence of irradiated splenocytes and the OVA peptide with the DC-IL-10/12-induced T-cell line (2.5  105 cells per well), which was added directly to the culture or to the transwell. Under the same culture conditions, an anti-IL-10 monoclonal antibody (mAb) or isotype control mAb was added to the cultures. Control cultures consisted of polarized T-cell lines in the absence of naive DO11.10 CD4+ T cells, and naive DO11.10 CD4+ T cells in the absence of polarized T-cell lines. Results are expressed as mean±s.e.m. of triplicate cultures.

In conclusion, together with our increased understanding of DC biology, in particular in the area of CD4+ Treg-cell polarization, this knowledge may ultimately result in DC-based vaccines to prevent atopic Th2 sensitization in high-risk individuals. However, further challenges include optimization of the conditions for the in vivo generation of such allergen-specific Tregs with minimal side effects. Achieving this will require a more complete understanding of the inhibitory roles of IL-10- and IFN-g-secreting Tregs and knowledge of how to sustain their longevity in vivo. Certainly, their safety will also need to be established. Gene Therapy

Figure 8 Therapeutic effects of combined cytokine-modulated DCs on AHR and airway inflammation. (a) Brief protocol of animal sensitization and challenge. (b) One day after the last OVA challenge, airway resistance was measured by invasive body plethysmography. Results are expressed as mean±s.e.m. of the pulmonary resistance (RL) in the ratio of RL after PBS nebulization. Each group contained five to eight mice. (c) Cell compositions in BAL fluid of different groups of mice are expressed as mean±s.e.m. of five to eight mice per group. Cells were counted and classified as monocytes (Mon), eosinophils (Eos), neutrophils (Neu), and lymphocytes (Lym). *Po0.05, **Po0.01, and ***Po0.001 vs the control group. #Po0.05, ##Po0.01, and ###Po0.001 vs the DC group. w Po0.05, wwPo0.01, and wwwPo0.001 vs the DC-mock group.

Materials and methods Mice Female BALB/c mice and DO11.10 mice expressing a transgenic T-cell receptor that is specific for amino acids 323–339 of OVA were purchased from the Animal Center of National Taiwan University and maintained in the Animal Center of Taipei Medical University. All mice were used between 6 and 10 weeks of age and were age matched within each experiment. Animal care and handling protocols were approved by the Animal Committee of the College of Medicine, Taipei Medical University.

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Cytokine-expressing adenoviral vectors Construction of a single-chain IL-12 gene was performed as described earlier.39 The p40 and p35 subunits of the murine IL-12 gene were connected by a linker and were used to generate a single-chain IL-12 gene. The mouse single-chain IL-12 or IL-10 gene was inserted into a recombinant adenoviral shuttle vector (pAdhPGK) containing the human phosphoglycerate kinase (hPGK) gene promoter. Then Ad-IL-12 and Ad-IL-10 recombinant adenoviruses were generated by homologous recombination with an adenoviral backbone plasmid (pAdEasy-1) and amplified in the AD293 adenoviruspackaged cell line. As a control, Ad-mock was made from the pAdhPGK vector, which did not carry the cytokine transgene. After propagation in AD 293 cells, the recombinant viruses were purified from infected cells 24–36 h after infection by three freeze-thaw cycles followed by successive banding by cesium chloride density-gradient centrifugation. The purified viruses were dialyzed and stored at 70 1C until the experiment. Viral titers were measured by a standard endpoint dilution assay using AD 293 cells. Preparation of bone marrow-derived DCs Bone marrow-derived DCs were prepared as described earlier.40 Briefly, tibial and femoral bone marrow from 5–6-week-old BALB/c mice was isolated. Bone marrow cells were cultured in RPMI-1640 and 5% fetal bovine serum complete medium with IL-4 (1000 U ml 1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (500 U ml 1) on day 0. Every other day, the medium was removed, and fresh medium containing granulocyte-macrophage colony-stimulating factor and IL-4 was added. The optimal MOI for Ad-IL-10 and Ad-IL-12 infection was chosen by evaluating IL-10 or IL-12 production in vitro by infected DCs. On day 6 of culture, bone marrow-derived cells were analyzed by flow cytometry, gated on CD11c+ (eBioscience, San Diego, CA, USA) cells. The flow cytometry analyses showed that there were 70–80% of DCs in this cell population. In vitro stimulatory ability of adenovirus-infected DCs Day 6 bone marrow-derived DCs were stimulated with 1 mg ml 1 of the OVA peptide and infected with either Ad-mock (DC-mock, at an MOI of 5000), or Ad-IL-10 (DC-IL-10, at an MOI of 500), or Ad-IL-12 (DC-IL-12, at an MOI of 5000), or co-infected (DC-IL-10/12) with Ad-IL-10 (at an MOI of 500) and Ad-IL-12 (at an MOI of 5000) for 48 h. Levels of IL-10 and IL-12 in the culture supernatants were analyzed by ELISA. On day 8, irradiated DC-mock, DC-IL-10, DC-IL-12, DC-IL-10/12, and non-infected DCs were collected for the following proliferation assay. Freshly isolated DO11.10 CD4+ T cells (4  105 cells per ml) were co-cultured with or without adenovirus-infected DCs at the indicated DC/T cell ratios. Cultures were pulsed with 1 mCi [3H] thymidine for the last 18 h of a 72-h culture, and [3H] thymidine deoxyribose incorporation was measured by a scintillation counter. For the in vitro cytokine analysis, irradiated adenovirus-infected DCs and naive DO11.10 CD4+ T cells were co-cultured under the same conditions used for the proliferation assay. After 72 h, supernatants were collected, and cytokine production was analyzed by ELISA.

Sensitization, treatment, and challenge of mice On day 6 of the culture, bone marrow-derived DCs were stimulated with 100 mg ml 1 OVA protein and infected with either Ad-mock (at an MOI of 5000), Ad-IL-10 (at an MOI of 500), or Ad-IL-12 (at an MOI of 5000), or co-infected with Ad-IL-10 (at an MOI of 500) and Ad-IL12 (at an MOI of 5000) for 48 h. On day 8, DC-IL-10, DC-IL-12, DC-IL-10/12, DC-mock, and non-infected DCs (DC) were collected for the animal experiments. Briefly, groups of mice were immunized intratracheally on day 1 with 5  105 OVA-pulsed DC, OVA-pulsed DC-mock, OVA-pulsed DC-IL-10, OVA-pulsed DC-IL-12, and OVApulsed DC-IL-10/12, respectively. Control mice were administered PBS instead of DC treatment. On days 8 and 18, all groups of mice were immunized intraperitoneally with 50 mg of OVA (grade V; Sigma, St Louis, MO, USA) emulsified in 4 mg aluminum hydroxide (Pierce, Rockford, IL, USA). Then mice were challenged intranasally with 100 mg OVA on days 20 and 21. Subsequently, mice were exposed to OVA aerosols (5% OVA in normal saline solution) for 3 consecutive days (days 22, 23, and 24) for 30 min daily, and AHR was measured 1 day after the last challenge (on day 25). Immediately, after measuring AHR, BAL fluid was collected to determine cytokine and chemokine production by ELISA. In a second experiment, the therapeutic effects of cytokine-modulated DCs on the development of asthma in already sensitized mice were studied. Groups of mice were sensitized intraperitoneally with 50 mg OVA in aluminum hydroxide on days 1, 14, 28, and 35. The mice received uninfected DCs, DC-mock, DC-IL-10, DC-IL-12, or DC-IL-10/12 pulsed with OVA on day 28. Control mice were administered PBS instead of DC treatment. Then mice were challenged intranasally with 100 mg OVA on days 38 and 39 and exposed to OVA aerosols for 3 consecutive days (days 40, 41, and 42). After 24 h, the degrees of AHR and airway inflammation were analyzed. Serum antibody assay Serum samples were collected from the retro-orbital venous plexus after intraperitoneal immunization with the OVA antigen. The OVA-specific IgE, IgG1, and IgG2a serum antibody titers were determined by ELISA. Levels of antibodies were compared with IgE, IgG1, and IgG2a standards at predetermined concentrations (immunoglobulin concentrations: 1 mg ml 1 IgE, 15.5 mg ml 1 IgG2a, and 25.6 mg ml 1 IgG1). The concentration of standard serum was arbitrarily assigned 1 ELISA unit. Determination of airway responsiveness Twenty-four hours after the last aerosol exposure, the airway function of mice was assessed, as described earlier, by measuring changes in lung resistance (RL) in response to increasing doses of inhaled methacholine.41 Data are expressed as RL in the ratio of RL after PBS nebulization. BAL fluid and lung histology study The lungs were lavaged through the trachea three times with 1 ml of Hanks’ balanced salt solution, which was free of ionized calcium and magnesium. Total cells in the BAL fluid were resuspended in 1 ml of Hanks’ balanced Gene Therapy

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salt solution, and total cells were determined using a hemocytometer. Cytocentrifuged preparations were stained with Liu’s stain (Chi I Pao, Taipei, Taiwan) for the differential cell counts. At least 200 cells were counted and differentiated by standard morphological criteria. After the lavage, the lungs were immediately removed and fixed in 10% v/v buffered formalin (in 0.01 M PBS; pH 7.2). Pulmonary tissues were subsequently sliced and embedded in paraffin, and cut into 5-mm-thick sections. These frozen sections were stained with hematoxylin–eosin and examined by light microscopy for histopathological changes.

Measurement of culture supernatant and BAL fluid cytokines Culture and BAL supernatant cytokine levels were determined using commercially available ELISAs according to the manufacturer’s instructions. ELISA kits for detecting IL-4, IL-5, IL-10, IL-12, IFN-g, and eotaxin were obtained from R&D Systems (Duoset, R&D Systems, Minneapolis, MN, USA). Intracellular cytokine staining Intracellular cytokines were detected by flow cytometry using the method of Batten et al.30 with modifications. Splenocytes from groups of mice were collected after AHR measurement and restimulated for 6 h with 20 ng ml 1 phorbol 12-myrisytate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) and 1 mg ml 1 ionomycin (Calbiochem, San Diego, CA, USA). Brefeldin A (5 mg ml 1; Sigma-Aldrich) was added for the final 4 h of stimulation. Then, 106 cells were collected and stained with phycoerythrin-Cy5-conjugated anti-mouse CD4. After fixation and permeabilization, cells were stained with a combination of phycoerythrin-conjugated antimIL-10 and fluorescein isothiocyanate-conjugated antimIFN-g. Staining with isotype control antibodies was performed in all experiments. All mAbs were obtained from eBioscience. After washing, staining was analyzed by means of flow cytometry on an fluorescence activated cell sorting Calibur with CellQuest software (BD Biosciences, Mountain View, CA, USA). In vitro purification and generation of T-cell lines CD4+ T cells were purified from spleens of DO11.10 mice after incubation with L3T4 (anti-CD4) magnetic beads with MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Positively selected cells and the non-selected fractions were collected for further analysis. The resulting T-cell preparations containing 95–99% CD4+ cells were used as DO11.10 T cells without further purification. To generate T-cell lines, 106 cells per ml of naive purified DO11.10 CD4+ T cells were stimulated with or without adenovirus-infected DCs at a DC/T cell ratio of 1:2.5 and with 1 mg ml 1 of the OVA323 339 peptide in the presence of IL-2 (10 units per ml). T cells were re-stimulated with the same adenovirus-infected or uninfected DCs and the OVA323 339 peptide plus IL-2 under the same conditions used for the initial stimulation every 7-day interval for three cycles. T-cell lines were collected for further experiments. Gene Therapy

In vitro suppression assays Polarized T-cell subsets were tested for their ability to suppress the proliferation of naive T cells in response to OVA-specific activation. Thus, graded numbers of threecycle T-cell lines were added to 2.5  105 freshly isolated naive DO11.10 CD4+ T cells stimulated with 2.5  105 irradiated splenic antigen-presenting cells and the OVA peptide (1 mg ml 1) in 24-well plates. Control cultures consisted of polarized T-cell lines in the absence of naive DO11.10 CD4+ T cells, and naive DO11.10 CD4+ T cells in the absence of polarized T-cell lines. Additionally, either anti-IL-10 mAb (10 mg ml 1) (R&D) or an isotypematched control mAb was added to cultures of 2.5  105 cells of T-cell line and 2.5  105 naive DO11.10 CD4+ T cells stimulated with irradiated splenocytes and the OVA peptide. In transmembrane culture, the 2.5  105 cells of the T-cell line with irradiated splenic antigenpresenting cells and the OVA peptide in the outer well and 2.5  105 naive DO11.10 CD4+ T cells with irradiated splenic antigen-presenting cells in the inner well were stimulated with the OVA peptide in 24-well transwell culture plates (Corning Costar, Corning, NY, USA). Proliferation of T cells was determined by [3H]-thymidine incorporation after 3 days of culture. Statistical analysis Results are expressed as mean±s.e.m. Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s post hoc test. A P-value of o0.05 was considered statistically significant.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This study was supported by grants from Cathay General Hospital (95 CGH-TMU-06) and from the National Science Council of the Republic of China (NSC95-2314-B-038-052).

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