Induction of IL-10 producing CD4⁺ T cells with regulatory activities by stimulation with IL-10 gene-modified bone marrow derived dendritic cells

Abstract

Dendritic cells (DCs) can induce both tolergenic as well as effective immune responses in the lung. Pulmonary DCs producing interleukin (IL)-10 mediated tolerance induced by respiratory exposure to antigen. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DC. To assess whether IL-10 producing DCs can exert the tolergenic effect through the differentiation of regulatory T cells, bone marrow derived DCs were genetically modified by IL-10 expressing adenovirus. IL-10 gene modified DCs (Ad-IL-10-DC) displayed a characteristic phenotype of immature DCs. Here we showed that in vitro repetitive stimulation of naïve DO11·10 CD4⁺ T cells with Ad-IL-10-DCs resulted in a development of IL-10 producing T-cell regulatory cells. These T cells could not proliferate well but also lost their ability to produce interferon-γ upon restimulation with irradiated splenocytes and ovalbumin peptide. Furthermore, in co-culture experiments these T cells inhibited the antigen-driven proliferation of naïve CD4+ T cells in a dose-dependent manner. Our findings demonstrated that IL-10 producing DCs had the potential to induce the differentiation of Tr1-like cells and suggested their therapeutic use.

Introduction

CD4⁺ T cell subtypes have been distinguished on the basis of their cytokine profile, function, and more tentatively by surface antigen expression. Th1 and Th2 clones have been generated against a range of infectious pathogens and also distinct subtypes of T cells with regulatory function, termed Tr1 [1–3] or Th3 cells [3–5], that secrete high levels of interleukin (IL)-10 or TGF-β respectively. However, little is known about the induction of T regulatory cells, the antigens they recognized or the factors that control their differentiation from naïve T cells.

Dendritic cells (DCs) are considered to be the key antigen-presenting cell (APC) for activation of naïve T cells [5,6]. Such antigenic presentation to T cells could lead to either potent activation (immunogenicity) or inhibition (tolerance) of effector immune functions [7,8]. Under experimental conditions, a number of agents have been reported to modulate the immunostimulatory potential of DCs [9,10]. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DCs. Modulation by IL-10 results in DCs that are no longer capable of presenting Ag for immunity, but induce Ag-specific anergy in both CD4⁺ and CD8⁺ T cells [9,11–13]. Furthermore, pulmonary DCs from tolerized mice expressing IL-10 drive the generation of a population of IL-10 producing regulatory T cells (T reg) in draining lymph nodes capable of suppressing subsequent responses to antigenic challenge [14,15]. In addition, T cells with regulatory activity have been found to be closely related to their IL-10 producing ability.

The goal of the present study was to evaluate how adenoviral gene transfer of IL-10 modulates DC maturation and the capacity to stimulate specific T-cell responses. Additionally, this study aims to explore whether IL-10 producing DCs can induce tolerance through the development of Treg cells.

Materials and methods

Mice

Female BALB/c mice and BALB/c mice expressing a transgene for the DO11·10 TCR specific for amino acids 323–339 of ovalbumin (OVA) and I-A^d (DO.11·10 TCR transgenic [Tg] mice) [16] were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were bred and maintained in the Animal Center of the College of Medicine of National Taiwan University. All mice were used between 6 and 10 weeks of age and were age-matched within each experiment. The animal study protocol was approved by the Animal Research Committee of College of Medicine, National Taiwan University.

Reagents and Abs

The OVA _323–339 peptide was synthesized and purified by high-performance liquid chromatography (GeneMed, South San Francisco, CA). Recombinant mouse IL-2, IL-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) were purchased from Pepro Tech Inc. (Rocky Hill, NJ). The mAb KJ1·26, which recognizes the transgenic TCR complex specific for OVA _323–339 peptide, and its isotype-matched control IgG were purchased from Caltag Laboratories (Burlingame, CA). Other antibodies were obtained from BD PharMingen (San Diego, CA).

Transduction of bone marrow derived DCs with recombinant adenovirus

For construction of adenovirus containing mouse IL-10 cDNA, a shuttle vector containing human phosphoglycerate kinase gene promoter (hPGK) was used. The pAdhPGK adenovirus serotype 5(Ad) vector, which contains the entire coding sequence of IL-10 (Ad-IL-10), was provided by Dr L-Y Chau (Academia Sinica, Taipei, Taiwan) [17]. As a control, Ad-Mock was also made by the pAdhPGK vector without carrying cytokine transgene. Recombinant adenovirus was generated by homologous recombination and amplified in 293 cells as previously described [17]. After propagation on 293 cells, the recombinant viruses were purified from infected cells 42–48 h after infection by three freeze-thaw cycles followed by successive banding on cesium chloride density-gradient centrifugation. The purified viruses were dialysed and stored at −70°C until the experiment. Viral titres were assayed by standard endpoint dilution assay using the 293 cells [18]. Furthermore, IL-10 expression was confirmed by measuring the culture supernatants of infected 293 cells (data not shown) [19].

Bone marrow derived DCs (BMDCs) were prepared as described previously [20,21]. Briefly, bone marrow cells from femurs and tibias were depleted of red cells by using an ACK lysis buffer. Approximately one million cells were placed in 24-well plates in 1 ml of medium that was supplemented with recombinant murine GM-CSF (500 U/ml) and IL-4 (1000 U/ml) (Pepro Tech Inc., Rocky Hill, NJ). The culture medium was RPMI-1640 medium supplemented with 5% heat-inactivated foetal calf serum, 4 mm L-glutamine, 25 mm HEPES (pH 7·2), 50 µm 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin and 0·25 µg/ml amphotericin. Every other day, the medium was removed by aspiration to remove the lymphocytes and fresh medium containing GM-CSF and IL-4 was added. On day 6 of the culture, non-adherent cells (BMDCs) were collected and infected with Ad-IL-10 [multiplicities of infection (MOI) = 5000] or Ad-mock (MOI = 5000) for 48 h. On day 8, Ad-IL-10 infected DCs (DC-IL-10) and Ad-mock infected DCs (DC-mock) were harvested and the surface markers expression was analysed by flow cytometry.

The optimal MOI of Ad-IL-10 infection was chosen by the evaluations of IL-10 production in vitro of infected DC. Day 6 DCs were transfected with different MOI of Ad-IL-10 for 48 h. Level of IL-10 in the culture supernatants was analysed by ELISA assay.

Flow cytometry

A FACSCalibur (Becton Dickson) was used for analytical flow cytometry and data were processed with CellQuest Pro (Becton Dickson) software. BMDCs transduced with recombinant adenovirus were stained with rat antimouse monoclonal antibodies to IA^d (MHC classII), CD80 (B7-1), CD86 (B7-2), ICOSL, OX40L, ICAM-1, and CD11c (eBioscience, San Diego, CA).

Cell purification and culture

CD4⁺T cells were purified from spleens of DO11·10 mice after incubation with L3T4 (anti-CD4) magnetic beads with AutoMACS (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.

For generation of T-cell lines, naïve purified DO11·10 CD4⁺T cells were stimulated at 2 × 10⁶ cells/ml with adenovirus-infected DCs at DC/T cell ratios of 1:10 and 1 µg/ml OVA _323–339 peptide in 24-well plates. The T cells were re-stimulated with the same adenovirus-infected DCs and OVA _323–339 peptide under the same conditions used for the initial stimulation once a week for four cycles. T-cell lines were collected for further experiment.

In vitro stimulatory ability of adenovirus-infected DCs

To examine the stimulating ability of adenovirus-infected DCs, OVA-specific T-cell proliferation and cytokine secreting levels were determined. For the proliferation assay, freshly isolated DO11·10 CD4⁺ T cells (2 × 10⁵ cells/ml) were co-cultured with adenovirus-infected DC and 1 µg/ml OVA peptide at an indicated DC/T cell ratio. Cells were cultured in a total volume of 200 µl in 96-well round-bottom tissue culture plates for 3 days. Cultures were then pulsed with 1 µCi [³H] thymidine for the last 18 h of culture and the [³H] thymidine deoxyribose incorporation was measured by scintillation counter.

For in vitro cytokine analysis, adenovirus-infected DCs and naïve DO11·10 CD4⁺ T cells were co-cultured under the same conditions used for the proliferation assay. After 48 h, supernatants were collected and cytokine production analysed by ELISA.

Intracellular cytokine staining

Intracellular cytokine were detected by flow cytometry using the method of Andersson et al. [20], with modifications [21,22]. T cells were cultured for 6 h at 37°C and 5% CO₂ and stimulated with 50 ng/ml PMA (Sigma-Aldrich, St Louis, MO) and 1 µg/ml ionomycin (Calbiochem, San Diego, CA) in the presence of 1 µM monensin (eBioscience, San Diego, CA). Monensin was added for the final 4 h. Then, 10⁶cells were harvested and stained with fluorescently labelled monoclonal antibodies to CD3 and CD4 (eBioscience). Subsequently, cells were washed, fixed and permeabilized using Cytofix/Cytoperm kit (BD Bioscience – PharMingen, San Diego, CA) and stained with fluorescently labelled monoclonal antibodies to IL-10 (eBioscience). Staining with isotype control antibodies (eBioscience) was performed in all experiments. Ten thousand cells were analysed by flow cytometry. FACS analyses are shown after gating on the CD3⁺ lymphocyte population.

Proliferation and cytokine secreting profiles of T-cell lines

In order to determine the proliferation ability of T-cell lines, T-cell lines (1 × 10⁵ cells) were collected and stimulated with APCs (1 × 10⁵ cells) and OVA peptide (0·6 µM). Un-fractionated BALB/c splenocytes were irradiated at 3600 rads and used as APCs. Proliferation response or cytokine secretion was analysed after stimulation at 72 or 48 h respectively.

In vitro suppression assays

Graded numbers of T-cell lines were added to 1 × 10⁵ freshly isolated naïve DO11·10 CD4⁺ T cells stimulated with 1 × 10⁵ irradiated splenic APCs (3600 rads) and OVA peptide (1 µg/ml) in a total volume of 200 µl in 96-well round-bottom tissue culture plates. DO11·10 CD4⁺ T cell cultures without T-cell lines were stimulated in the same manner as positive controls. Proliferation of T cells was determined by [³H]- thymidine incorporation after 3 days of culture.

Measurement of cytokine production

Levels of interferon (IFN)-γ, IL-2, IL-4, IL-10 and IL-13 in the culture supernatants were evaluated by commercially available ELISA (Duoset, R&D, Minneapolis, MN), according to the manufacturer's instructions.

Statistical analysis

All of the data are expressed as the mean ± standard deviation for each group. Differences between surface markers of different adenovirus treated DC were tested for significance by Mann–Whitney U-test. P values < 0·05 were considered to be significant.

The statistical significance of the differences between different DCs treated T cells was assessed with the Wilcoxon signed-rank test for nonparametric data. P values < 0·05 were considered to be significant.

Results

Determination of the optimal ratio of infectious Ad-IL-10 vectors to DCs

To define the optimal viral load for transduction, day 6 DCs were transduced with different MOI of Ad-IL-10 or Ad-mock for 48 h. Level of IL-10 in the culture supernatant was determined by ELISA. As shown in Fig. 1, the data demonstrated that the IL-10 production of Ad-IL-10-infected DCs (DC-IL-10) was dose dependent. The highest IL-10 production level was achieved at an MOI of 5000 and the concentration was approximately 28 ng/ml. Increasing the MOI (e.g. to 10 000) did not enhance the IL-10 concentration.

Fig. 1.

Level of IL-10 in the culture supernatant of Ad-IL-10-infected DCs showed dose dependent pattern. Day 6 DCs were transfected with different MOI of Ad-IL-10 for 48 h. Level of IL-10 in the culture supernatants was analysed by ELISA assay. Results from triplicate experiments are shown, and presented as mean ± SD (n = 3). IL, interleukin; DC, dendritic cell; MOI, multiplicities of infection; SD, stdnard deviation.

Phenotype of Ad-infected DCs

To determine the potential phenotypic changes in DCs following adenovirus infection, DCs were collected and stained with fluorescent labelled mAbs specific for surface markers related to DC function and classification. As shown in Table 1 and Fig. 1b, each group of adenovirus-infected DCs (DC-IL-10 and DC-mock) expressed similar moderate levels of CD11c, ICAM-1 and OX40L. However, the expression of ICOSL on DC-IL-10 was significantly higher compared with DC-mock (5·9 ± 1·2% versus 3·4 ± 1·5%, P value = 0·005). In addition, the expression of MHC class II (I-A) (74·7 ± 2·8% versus 67·7 ± 6·2%), co-stimulatory molecules CD80 (66·8 ± 13·3% versus 61·1 ± 10·0%) and CD86 (48·6 ± 17·5% versus 38·8 ± 8%) on DC-IL-10 appeared to be lower compared with that of DC-mock. Although, the difference is not so significant, the data demonstrate that Ad-IL-10 affected DCs in the expression of cell surface markers, which is considered to be related to DC function.

Table 1.

Surface markers of adenovirus-infected DCs.

	CD11c	MHC II	B7-1	B7-2	ICAM-1	ICOSL	OX40L
No infection	22·1 ± 2·7	73·8 ± 9·7	53·9 ± 12·8	29·9 ± 6·4	58·4 ± 5·1	3·2 ± 1·5	33·2 ± 11·0
DC-mock	24·2 ± 2·5	74·3 ± 2·8	66·8 ± 13·3	48·6 ± 17·5	57·4 ± 12·3	3·4 ± 1·5	39·6 ± 19·6
DC-IL-10	28·0 ± 3·4	67·7 ± 6·2	61·1 ± 10·0	38·8 ± 8·0	56·9 ± 3·5	5·9 ± 1·2*	41·5 ± 5·7

Day 6 DCs were infected with Ad-IL-10 or Ad-mock (MOI = 5000) for 48 h. Adenovirus-infected DCs were harvested and the surface markers expression on these DCs was analysed. Expression of different surface markers was assessed by flow cytometry on adenovirus-infected DCs. Data are represented as the mean ± SD. Experiments were repeated in variation three times.

^*P < 0·05, as compared with the DC-mock group. DC, dendritic cell; MOI, multiplicites of infection; SD, stdnard deviation.

In vitro stimulatory ability of naïve CD4⁺ T cells with adenovirus-infected DCs

We then studied the effects of adenovirus transduction and IL-10 expression on the ability of DCs to activate naïve CD4⁺T cells responses. OVA-specific, naïve CD4⁺ T cells were cultured with OVA peptide and DC-IL-10 or DC-mock at various ratios of DC and T cells. After 3 days, the proliferation of OVA-specific T cells was assayed. Supernatants were assayed by ELISA after 48 h of culture for the levels of cytokines such as IFN-γ, IL-2, IL-4 and IL-13.

An important function of DC is to present specific antigens to T cells and initiate adaptive immune response. Because DC-IL-10 had the surface phenotype of immature DCs, we next assessed induction of T-cell proliferation by adenovirus-infected DCs in vitro. In our results, we found that CD4⁺ T cells incubated with the irradiated DCs, which were infected with high MOI of Ad-IL-10 (MOI = 5000), had lower proliferative response to OVA peptide compared with the T cells incubated with DC-mock (DCs infected with mock virus) (P = 0·0417) (Fig. 2a).

Fig. 2.

Stimulation of naïve T cells with adenovirus-infected DCs. (a) The immunostimulatory capacity of Ad-IL-10-infected DCs decreased in vitro. Day 6 DCs were infected with Ad-IL-10 (hi, MOI = 5000; lo, MOI = 500) or Ad-mock (MOI = 500) for 48 h. Adenovirus-infected DCs were harvested and co-cultured at various ratios with naïve DO11·10 CD4⁺ T cells 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 presented as mean ± SD (n = 3). (b) Cytokine secreting pattern of naïve T cells after stimulation with adenovirus-infected DCs. Adenovirus-infected DCs were harvested and co-cultured at various ratios with naïve DO11·10 CD4⁺ T cells. After 48 h, supernatants were collected and levels of IL-2, IL-4, IL-10 and IL-13 production analysed by ELISA. The level of IFN-γ was significantly lower in the T cells co-culture with DC-IL-10-hi compared with that of DC-mock (P value = 0·0313). However, IL-2, IL-4 and IL-13 were not different in the co-culture supernatants between DC-IL-10 or DC-mock groups. Results from triplicate experiments are shown, and presented as mean ± SD (n = 3). The statistical significance was assessed with the Wilcoxon signed-rank test for nonparametric data. P values < 0·05 were considered to be significant. ND, not determined; DC, dendritic cell; MOI, multiplicities of infection; IFN, interferon; IL, interleukin; SD, standard deviation.

Furthermore, we wanted to estimate the cytokine profile of CD4⁺ T cells incubated with the adenovirus-infected DC in response to OVA peptide. In our results, DCs, which were infected with high MOI of Ad-IL-10, could produce a low level of IFN-γ (P value = 0·0313) and decrease the immunostimulatory capacity in vitro(Fig. 2). However, the cytokine levels of IL-2, IL-4 and IL-13 were not different between the co-culture surpernatants of DC-IL-10 or DC-mock.

Genetically engineered DCs expressing high levels of IL-10 polarize naïve CD4⁺ T cells towards Tr1-like T cells in vitro

Following transduction of DCs with Ad-IL-10, we detected significantly higher levels of IL-10 in the supernatant at day 8 (Fig. 1) compared with DCs transduced with the control constructs (DC-mock). IL-10 is considered the driving force for Tr1 cell generation, as shown by experiments in which antigen-specific murine Tr1 cells can be induced ex vivo by repeated TCR stimulation in the presence of high doses of IL-10 [1]. Thus, we first investigated the T-cell polarizing capacity of DC-IL-10 in vitro.

To determine whether the IL-10 secreting levels in DCs are associated with the induction potential of IL-10 secreting T cells. OVA-specific, CD4⁺ T cells were cultured with OVA-pulsed DC-IL-10-hi (MOI = 5000) or DC-IL-10-lo (MOI = 500). After 7 days, the OVA-specific T cells were harvested and re-stimulated with PMA and ionomycin for 6 h. Then, IL-10 production level was quantified on the single-cell level by intracellular cytokine staining. We obtained the numbers of IL-10 secreting T cells, which were significantly higher in the T-cell population primed with DC-IL-10-hi, which can secret high levels of IL-10, compared with the population priming with DC-IL-10-lo (Fig. 3a). In addition, we also found that most IL-10-secreting T cells could produce IL-10 even without re-stimulation of PMA and Ionomycin (Fig. 3b).

Fig. 3.

Ad-IL-10-infected DCs enhance development of IL-10 producing cells. Day 6 DCs were infected with Ad-IL-10 (hi, MOI = 5000; lo, MOI = 500) or Ad-mock (MOI = 500) for 48 h. Adenovirus-infected DCs were harvested and co-cultured with naïve DO11·10 CD4⁺ T cells in the presence of OVA peptide. After 7 days, T cells were collected and re-stimulated with (a) or without (b) PMA and ionomycin for 6 h. IL-10 expression was detected by intracellular cytokine staining and analysed by flow cytometry. Percentage of positive cells is indicated in the upper right of each plot. IL, interleukin; DC, dendritic cell; MOI, multiplicites of infection.

To analyse the influence of adenovirus-infected DCs on the priming and differentiation of naïve OVA-specific T cells, three rounds of stimulations of T cells were established with DC-IL-10 or DC-mock (Fig. 4). Following three rounds of stimulation, the two T-cell populations were then re-stimulated under identical conditions using irradiated BALB/c splenocytes and OVA _323–339 peptide to determine their proliferation ability and their cytokine secretion profiles. Our results showed that mock-T cells (stimulated with DC-mock) produced high levels of IL-2 and IFN-γ. In contrast, the T cells (IL-10-T) generated in the presence of DC-IL-10 secreted lower amounts of IL-2 and IFN-γ (Fig. 4b) but significantly greater amounts of IL-10 (Fig. 5). In addition, the IL-10-T cells could not proliferate as well as mock-T cells upon re-stimulation with irradiated BALB/c splenocytes (Fig. 4a).

Fig. 4.

Ad-IL-10-infected DCs skew naïve CD4⁺ T cells towards a distinct Th cell differentiation in vitro.Day 6 DCs were infected with Ad-IL-10 (hi, MOI = 5000; lo, MOI = 500) or Ad-mock (MOI = 5000) for 48 h. Adenovirus-infected DCs were harvested and co-cultured with naïve DO11·10 CD4⁺ T cells in the presence of OVA peptide. After three rounds of stimulation, T cells were collected and stimulated with irradiated BALB/c splenocytes and OVA _323–339 peptide. (a) Proliferative response or (b) cytokine secretion was analysed after stimulation at 72 or 48 h respectively. The data showed that IL-10-T cells could not proliferate as well as mock-T cells. Furthermore, mock-T cells produced high levels of IL-2, IFN-γ and IL-13. In contrast, the IL-10-T secreted lower amounts of IL-2 and IFN-γ (P = 0·0313). Results from triplicate experiments are shown, and presented as mean ± SD (n = 3). The statistical significance was assessed with the Wilcoxon signed-rank test for nonparametric data. P values < 0·05 were considered to be significant. IL, interleukin; DC, dendritic cell; MOI, multiplicities of infection; IFN, interferon; SD, standard deviation.

Fig. 5.

Ad-IL-10-infected DCs skew naïve CD4⁺ T cells towards IL-10 secreting T cells. Cycle-4 T cells were collected and stimulated with irradiated BALB/c splenocytes and OVA _323–339 peptide (1 µg/ml). The IL-10-T-4 secreted large amounts of IL-10 compared with mock-T-4 cells. Results from triplicate experiments are shown, and presented as mean ± SD (n = 3). IL, interleukin; DC, dendritic cell; SD, standard deviation.

In the absence of OVA peptide, no cytokine secretion can be detected from either T-cell population. As shown in Fig. 4, titration of OVA peptide in the final stimulation cultures demonstrated that the secretion of IL-10 by IL-10-T cells was directly linked with antigen dose while the capacity to secrete IL-2 (P = 0·0625) and IFN-γ (P = 0·0313) remained profoundly reduced even in the presence of a high level of antigen (4·0 µg/ml). In addition, IL-10-hi-T cells also secreted less level of IL-13 upon stimulating with a lower level of antigen (2–0·5 µg/ml) (P value = 0·0313). Thus, we concluded that the presence of DC-IL-10 during repeated stimulations of DC-mediated T-cell stimulation results in the generation of a population of T cells that predominantly responds to subsequent antigen exposure by secretion of IL-10.

To analyse the functional properties of these T-cell lines, suppressive ability assay was performed. IL-10-hi-T cells obtained after repeated stimulation with DC-IL-10 strongly suppressed the proliferation of responder naïve CD4⁺ T cells stimulated with irradiated splenocytes and OVA peptide (Fig. 6). Furthermore, the suppressive ability of these T cells is highly related to the IL-10-secreting level of DCs. As a control, mock-T cells had no inhibitory effect. Altogether, these results showed that DC-IL-10 induced the differentiation of Tr1-like cells in vitro.

Fig. 6.

Inhibition of antigen-specific proliferation of navie DO11·10 CD4+ T cells after co-culture with Tr-like cells. Tr-like cells were induced by repetitive stimulation of naïve CD4+ T cells with adenovirus-infected DCs at DC/T cell ratios of 1:10. Seven days after the third re-stimulation, naïve DO11·10 CD4⁺ T cells were stimulated with irradiated splenocytes and OVA peptide in the presence of different numbers of Tr-like cells. [³H] thymidine incorporation was measured after 3 days of culture. Results are expressed as the mean ± standard deviation of triplicate cultures.

Discussion

Regulatory T cells have been suggested to play an important role in mediating peripheral tolerance. It appears that Treg cells are generated and maintained in the periphery, dependent upon the presence of antigen and of co-stimulatory signals [9,23,24]. These insights suggest the important roles for APC phenotype and micro-environmental factors in Treg cell development.

The results of Akbari et al. clearly demonstrate that the development of respiratory tolerance is initiated by uptake of antigen in the lungs by pulmonary DCs, which can produce IL-10 [14]. In addition, pulmonary DC exposed to respiratory allergen could induce the development of IL-10-producing Treg cells. This process is dependent on T-cell co-stimulation via the inducible co-stimulator (ICOS)-ICOSL pathway [15]. These Treg cells, production of which is IL-10-dependent, block the development of airway hypersensitivity. Thus, IL-10 producing DCs appear to contribute an immunoregulatory function in the allergic disease and could be future targets of therapeutic modalities. Moreover, many groups have investigated the capacity of IL-10 gene modified DC to induce the immune tolerance in different animal models of diseases. The experiments described in this study demonstrate that IL-10 gene modified DC drive the development of IL-10-producing Treg cells in vitro. On a more practical level, the development of in vitro culture systems that result in the generation of substantial numbers of Treg cells offers the possibility of substituting an antigen-specific adoptive immunotherapy for the systemic administration of immunosuppressive or immunomodulatory drugs.

Numerous studies have demonstrated that DCs are critical in developing a specific immune response in the lungs [25–28], where they may prime naïve CD4⁺ T cells to differentiate into Th1, Th2, or Treg cells. Thus, although the mechanisms governing the development of T cell tolerance or sensitization are not entirely clear, it seems likely that it results in a distinct pattern of modulation involving multiple surface and secreted proteins that are linked with DC maturation and immunostimulatory capacity [29,30]. In this study, the phenotypic characterization of adeno-IL-10 and adeno-mock-infected DCs appeared in different patterns of surface markers (Table 1). DCs infected with Ad-IL-10 for 2 days exhibit surface phenotypes characteristic of immature DCs, decreased surface expression of MHC class II and co-stimulatory molecules. Interaction between CD28 and its ligands CD80 and CD86 has been found to be a dominant co-stimulatory pathway in allergic diseases [31,32]. The OX40 (CD134)-OX40L co-stimulatory pathway also has been shown to have an important role in allergic inflammation [33]. OX40 is present on activated T cells and binds to OX40L on activated B cells, DC and other APC. OX40 is expressed on memory CD4 T cells, controlling the pivotal memory Th2 cells that regulate lung inflammation [34]. Our results indicate that both B7 (CD80/CD86) co-stimulatory molecules are down-regulated after being infected with Ad-IL-10 compared with Ad-mock. Other important co-stimulatory sets of molecules are ICOS and ICOSL. Their importance in the immune response is highlighted by the fact that blocking ICOS-ICOSL interactions inhibits respiratory tolerance and suppresses Treg cells development [15]. In addition, murine models deficient in ICOS have decreased IgE production, Th2 cytokines, and (AHR). Thus, ICOS-ICOSL interaction plays an important role in both Th2 immune response and immune tolerance. In our study, Ad-IL-10-infected DCs showed a significantly higher amount of ICOSL compared with Ad-mock-infected DCs. Thus, our data could demonstrate that Ad-IL-10 affected DCs in the expression of cell surface markers that are considered to be related to DC function.

In Fig. 2, we can see that naïve T cells could secrete a smaller amount of IFN-γ after stimulation of DC-IL-10-hi. In addition, after repetitive stimulation with DC-IL-10, the T cells lost their capacity to synthesize IFN-γ, as shown in Fig. 4. Thus, our DC-IL-10 did have the ability to modulate the T cell priming and function.

Then, we generated T-cell lines following multiple rounds of stimulation by adenovirus-infected DCs and analysed the properties and regulatory capacity of these T-cell lines. Indeed, the features of the T-cell lines generated in our culture system retain lower proliferative capacity in vitro and are likely to be the Tr1 cells that generated in the experimental culture system of Barrat et al.[22]. Production of IL-10 by these cells, while being important for bystander suppression, also has the potential for immune stimulation. However, further experiments using an in vitro transwell culture system and in vivomodels of immune-mediated disease will be necessary to address the suppressive mechanism of these Treg cells. Nevertheless, the strategy of harnessing T-cell regulatory mechanisms for human immunotherapy has distinct advantages over traditional immunosuppressive therapies and there is good evidence to suggest that it can be achieved either through delivery of modulated T cells, or inoculation with immature antigen-bearing DCs.

The importance of antigen dose and relative frequency of regulatory and responder T cells has not been closely examined for therapy involving IL-10 secreting T cells. In our in vitro assay of suppression, it was demonstrated that inhibition of a primary T-cell response to OVA peptide was best achieved at lower antigen dose and with a larger number of DC-IL-10-drived T cells (IL-10-T).

In conclusion, our findings demonstrated that IL-10 producing DCs have the potential to induce the differentiation and development of Tr1-like cells. In the future, we will test both in vitroand in vivo function of these T cells to determine their ability to suppress antigen-specific immune response. We also like to apply these T cells with regulatory activity for the alleviation of airway inflammation in a murine model of asthma. The approach here might shed light on further therapeutic approaches for allergic diseases.