Skip to main content
NCBI home page
Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2010 May 21;88(3):537–546. doi: 10.1189/jlb.0110044

Natural Foxp3+ regulatory T cells inhibit Th2 polarization but are biased toward suppression of Th17-driven lung inflammation

Teri Girtsman 1, Zeina Jaffar 1, Maria Ferrini 1, Pamela Shaw 1, Kevan Roberts 1,1
PMCID: PMC2924601  PMID: 20495073

Abstract

nTregs prevent autoimmunity and modulate immune and inflammatory responses to foreign antigens. CD4+Foxp3+ nTregs from DO11.10 mice were expanded ex vivo, and their effectiveness in suppressing the development of lung inflammatory responses, elicited by differentiated CD4+ T cells following antigen inhalation, was examined. Effector DO11.10 CD4+ Th2 cells, when adoptively transferred into BALB/c mice that subsequently inhaled OVA, elicited a pronounced pulmonary, eosinophilic inflammation. Surprisingly, the cotransfer of expanded nTregs failed to suppress the Th2-mediated airway inflammation. Nevertheless, expanded OVA-specific CD4+Foxp3+ nTregs were highly effective at inhibiting the polarization of naïve CD4+ T cells into a Th2 phenotype. This suppression was reversed by an antibody to GITR but was not affected by the presence of the soluble OX40L. Further analysis revealed that although nTregs also failed to inhibit the lung neutrophilic inflammation induced by effector CD4+ Th1 cells, they markedly suppressed pulmonary inflammation elicited by CD4+ Th17 cells but not AHR. The suppression of the Th17-mediated response was evident from a striking reduction in the proportion of OVA-specific T cells expressing IL-17 and the numbers of neutrophils present in the airways of Th17 recipient mice. Collectively, these results demonstrate that expanded nTregs clearly limit the Th2 polarization process and that Th17-mediated inflammatory responses are particularly prone to the immunoregulatory properties of nTregs. These findings thus indicate that expanded nTregs are restrictive in their ability to suppress airway inflammatory processes and AHR.

Keywords: regulation, nTregs, CD4+, airway inflammation

Introduction

Effector CD4+ T cells are typically characterized as Th1, Th2, and Th17 on the basis of their cytokine profiles. Th1 cells secrete IFN-γ and are essential for controlling intracellular pathogens, whereas Th2 cells, which produce IL-4, IL-5, and IL-13, clear helminth infections and drive the inflammatory response in allergic asthma. Human asthma is characterized by chronic mucosal inflammation, associated with pulmonary eosinophilia, mucous hypersecretion, airway remodeling, and AHR to a variety of environmental stimuli that include allergen exposure [1]. Conversely, CD4+ Th17 cells, which are characterized by their secretion of a distinctive set of effector cytokines, which include IL-17, IL-17F, IL-21, and IL-22, mediate autoimmunity, inflammation, and mucosal host defense against pathogens [2]. More recently, it has been suggested that Th17 cells and IL-17 may also contribute to the allergic inflammation in asthma, particularly in cases of severe disease [3,4,5]. It has been proposed that chronic airway inflammation in asthma results from a failure in regulatory processes that are normally operative in the absence of disease. However, work delineating the nature and relative importance of specific regulatory pathways in limiting CD4+ T cell-mediated inflammation is in its infancy.

Tregs play an important role in preventing mucosal inflammatory processes, particularly in the intestine [6], and have been reported to elicit immune suppression to inhaled antigens [7]. In recent years, multiple Treg phenotypes have been described [8]. However, the two major types of Tregs described are the nTregs, which develop in the thymus [9, 10], and iTregs, which form in the periphery [11]. nTregs and iTregs express a key regulatory transcription factor, Foxp3 [12, 13]. The nTregs are CD4+CD25+ T cells that are pivotal for the maintenance of peripheral tolerance and preventing the onset of autoimmune disease [10, 14, 15]. Moreover, nTregs have been shown to inhibit the development of allergic lung inflammation [7, 16, 17]. CD4+Foxp3+ iTregs are induced from CD4+CD25− precursors when encountering antigen in the presence of IL-2 and TGF-β [11, 18]. Although similar in function, iTregs and nTregs differ in principal antigen specificities and costimulatory molecules required for their generation [11]. Moreover, their response to IL-6 is markedly different, as this cytokine can convert nTregs to IL-17-producing Th17 cells, and iTregs are resistant to such conversion [11]. Conversely, the induction of iTregs is inhibited by IL-4, which promotes the differentiation of a Foxp3-negative IL-9- and IL-10-expressing effector phenotype [19]. nTregs and iTregs mediate the suppression of T cell effector function through several mechanisms that require direct cell contact [20] or the production of immunosuppressive cytokines, such as IL-10 [21] and TGF-β [22]. A thorough analysis demonstrating the sensitivity of naïve CD4+ T cells and effector CD4+ T cell responses to suppression by nTregs has not been reported. A major limitation in using nTregs to limit inflammatory responses is the low frequency of Tregs present in lymphoid tissues. To circumvent this problem, several investigators have expanded nTregs in vitro prior to transfer into animals or patients [23]. Human CD4+CD25+ Tregs from peripheral blood specific for human leukocyte antigen A2 have been purified and expanded successfully by TCR stimulation in the presence of high doses of IL-2 [24]. In addition, antigen-specific murine nTregs have been expanded in culture in the presence of IL-2, anti-CD3, and anti-CD28, remaining phenotypically and functionally pure [25].

In this study, we used an adoptive transfer model to investigate the effectiveness of antigen-specific Foxp3+ nTregs in limiting the inflammatory responses elicited by effector CD4+ T cells to inhaled antigen. CD4+Foxp3+ nTregs from DO11.10 mice could be expanded in culture, with retention of Foxp3 expression and regulatory function, when cultured in the presence of OVA323–339 peptide, IL-2, and IL-4. Effector CD4+ Th2-polarized cells, when transferred into mice that subsequently inhaled OVA, developed a pronounced airway eosinophilic inflammation. Surprisingly, the cotransfer of expanded nTregs into mice failed to limit the pulmonary inflammation elicited by effector CD4+ Th2 cells. Nevertheless, by monitoring reduction of GFP expression by CD4+ T cells from IL-4 reporter mice, we found that expanded nTregs were highly effective at inhibiting the polarization of naïve CD4+ T cells into a Th2 phenotype in vitro. Interestingly, the transfer of nTregs markedly suppressed Th17-driven, pulmonary, inflammatory responses but not Th1-mediated inflammation. Collectively, these results demonstrate that antigen-specific nTregs can be expanded ex vivo and were highly effective at inhibiting Th2 polarization but restrictive in their ability to suppress lung inflammatory processes to inhaled antigens.

MATERIALS AND METHODS

Animals

DO11.10 transgenic, C.129-Il4tm1Lky/J (IL-4 reporter or 4get), and BALB/c mice (all from The Jackson Laboratory, Bar Harbor, ME, USA) were used throughout (6–8 weeks old) and housed in specific pathogen-free facilities at the University of Montana (Missoula, MT, USA). All experiments were approved by the Institutional Animal Care and Use Committee and performed according to National Institutes of Health guidelines.

Preparation of polarized CD4+ Th1, Th2, and Th17 cells

To drive T cell differentiation into a Th2 or Th1 effector phenotype, PLNs were obtained from DO11.10 mice, and CD8+ T cells were depleted using MACS beads (Miltenyi Biotec, Auburn, CA, USA). Cells (5×105/ml) were then incubated in complete RPMI for 4 days in the presence of OVA323–339 peptide (1 μg/ml; Mimotopes, San Diego, CA, USA) and IL-4 (2 ng/ml; R&D Systems, Minneapolis, MN, USA) plus anti-IFN-γ antibody (5 μg/ml R4-6A2; ATCC, Manassas, VA, USA) for preparing Th2 cells or IL-12 (1 ng/ml; R&D Systems) plus anti-IL-4 antibody (5 μg/ml 11B11; ATTC) for preparing Th1 cells. After 4 days of incubation, cells were restimulated as before but this time, also in the presence of IL-2 (10 ng/ml; R&D Systems) for 4 days further. On Day 8, class II+ cells were removed using anti-class II antibody (M5/114) and MACS beads. To generate Th17 effector cells, CD4+ T cells (5×105/ml), purified from DO11.10 PLNs, were incubated with OVA323–339 peptide (1 μg/ml) and mitomycin C-treated APCs (1×106/ml) in the presence of TGF-β (2 ng/ml; eBioscience, San Diego, CA, USA), IL-6 (10 ng/ml; R&D Systems), anti-IFN-γ antibody (5 μg/ml HB-170), and anti-IL-4 antibody (5 μg/ml 11B11). After 4 days of culture, the cells were restimulated as before in the presence of IL-23 (10 ng/ml; R&D Systems). Eight day-polarized effector Th1, Th2, or Th17 cells were collected for adoptive transfer experiments or functional analyses.

Preparation and expansion of CD4+ CD25+ Foxp3+ nTregs in culture

CD4+CD25+ nTregs were purified from DO11.10 PLNs using MACS magnetic cell sorting beads (Miltenyi Biotec). Purified nTregs (5×105/ml) were cultured in complete RPMI media in the presence of OVA323–339 peptide (1 μg/ml), IL-2 (10 ng/ml; R&D Systems), IL-4 (2 ng/ml; R&D Systems), anti-IFN-γ antibody (5 μg/ml; ATCC), and mitomycin C-treated APCs (2×106/ml BALB/c spleen cells). After 4 days of culture, the cells were restimulated as before without adding further APCs but with the addition of exogenous IL-2 (10 ng/ml) and IL-4 (2 ng/ml) for 4 additional days. The expansion of CD4+CD25+ T cells in the presence of exogenous IL-2 alone was limited and was typically, approximately a threefold increase in cell numbers over 8 days. Typically, 3 × 106 CD4+CD25+ T cells yielded 30–50 × 106 cells after an 8-day expansion (in the presence of IL-2 plus IL-4), and the viability was >95%. Eight day-expanded CD4+ nTregs were harvested, and a sample was taken for Foxp3 staining using a mouse Treg flow kit (BioLegend, San Diego, CA, USA) to verify expression of Foxp3 (>90%).

Adoptive transfer of CD4+ T cells into mice and OVA aerosol challenge

Eight day-polarized DO11.10 CD4+ Th1, Th2, or Th17 cells (which were devoid of γδ T cells) were adoptively transferred alone into BALB/c recipients (8×106 cells/mouse injected into the tail veins) or cotransferred with nTregs (3×106 cells/mouse). Control mice were injected with nTregs alone or received no cells. All mice (four/group) were then intranasally challenged by exposure to aerosolized solutions of OVA (0.5%, Grade V; Sigma-Aldrich, St. Louis, MO, USA) for 20 min/day over 7 consecutive days using a Wright’s nebulizer.

Level of pulmonary inflammation and AHR

On Day 8, BAL was performed (3×0.5 ml PBS) to collect BALF for analysis. BALF of four animals was pooled, and EPO levels present in the BAL cells were determined by colorimetric analysis as described before [26]. Cell-differential percentages were determined by light microscopic evaluation of Hema-3-stained cytospin preparations and expressed as absolute cell numbers. AHR, in anesthetized and tracheotomized mice, which were ventilated mechanically, was assessed in response to methacholine inhalation by measurement of airway resistance and dynamic compliance (Buxco Research Systems, Wilmington, NC, USA). Lung tissue was dispersed by collagenase (Type IV; Sigma-Aldrich) to prepare LMCs for functional analysis.

Monitoring the regulation of Th2 differentiation of CD4+ T cells

To monitor the effect of nTregs on Th2 polarization in vitro, IL-4 reporter mice, designated 4get mice (IL-4 GFP-enhanced transcript), were used. These mice provide a means to monitor IL-4 production, as any cells that express IL-4 also coexpress GFP. This allows reliable monitoring of CD4+ Th2 differentiation in vitro. To evaluate Th2 polarization of CD4+ T cells, PLN cells from 4get mice (106/ml) were cocultured with expanded DO11.10 nTregs (2.5×105/ml). Cells were cultured for 4 days in the presence of plate-bound anti-CD3 (2 μg/ml) using Th2-polarizing conditions, IL-4 (2 ng/ml), IL-2 (10 ng/ml), and anti-IFN-γ antibody (5 μg/ml). To monitor the level of Th2 polarization, cells were harvested on Day 4, and the cellular expression of GFP was determined in conjunction with staining using CD4 allophycocyanin-Cy7 and KJ1-26-PE (PharMingen, San Diego, CA, USA). Flow cytometric analysis of GFP expression by CD4+ T cells was completed using a FACSAria (BD Biosciences, San Jose, CA, USA; using FACS DiVa software). DO11.10 Tregs that stained with PE-labeled anticlonotype KJ1-26 were removed from the analysis by electronic gating to ensure the IL-4 production of the CD4+ IL-4 reporter cells was analyzed in isolation. In some experiments, the effect of addition of anti-GITR antibodies (20 μg/ml clone DTA-1; kindly provided by Dr. Shimon Sakaguchi, Kyoto, Japan) and OX40L (20 μg/ml; Xenova, Cambridge, UK) on nTreg-mediated suppression of Th2 polarization was determined.

FACS analysis

The number of KJ1-26+ T cells or CD11b+GR1+ neutrophils present in the BALF was examined by FACS analysis. Cells were stained and analyzed on a FACSAria (BD Biosciences) using FACSDiVa software to enumerate CD4+ T cells (using anti-CD4 GK1.5-allophycocyanin-Cy7 mAb, BD PharMingen) and OVA-specific T cells (KJ1-26 PE, Caltag Laboratories, S. San Francisco, CA, USA), GR1+ (anti-Ly-6G mAb, BD PharMingen), and CD11b+ cells (anti-CD11b FITC mAb, Miltenyi Biotec).

FACS analysis of intracellular Foxp3 and cytokine expression

For intracellular Foxp3 analysis, PLNs or expanded Tregs were first stained with CD4/CD25 mAb and then fixed and permeabilized using the Foxp3 staining kit (BioLegend). After permeabilization, the cells were intracellularly stained using Foxp3 mAb or isotype control and analyzed by FACSAria.

For analysis of intracellular IL-17, IL-4, or IFN-γ, cells (1×106 cells) were first stimulated with 50 ng/ml PMA + 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of 1 μl of the protein transport inhibitor BD GolgiPlug containing brefeldin A (BD Biosciences) for 4 h at 37°C. Cells (0.5×106) were then blocked using 2.4G2 supernatant (ATCC) and stained using PE- or allophycocyanin-conjugated KJ1.26 mAb (eBioscience) or FITC- or allophycocyanin-Cy-7-conjugated CD4 (clone GK.1, BD Biosciences). Following treatment with fixation and permeabilization buffers (BioLegend), cells were intracellularly stained using the following antibodies (all from BioLegend): allophycocyanin- or PE-conjugated anti-IL-17 (clone TC11-18H10.1), allophycocyanin-conjugated anti-IL-4 (clone 11B11), FITC-conjugated anti-IFN-γ (clone XMG1.2), allphycocyanin-conjugated anti-γδ TCR (clone GL3), or isotype control (BD Biosciences) and analyzed by FACSAria.

Cytokine measurement

To examine cytokine production, 8 day-polarized Th1, Th2, or Th17 cells (5×105/ml) or LMCs were stimulated with immobilized anti-CD3 (2 or 5 μg/ml, 2C11; ATCC) or OVA323–339 peptide (1 μg/ml) for 24 h, and the supernatants harvested for measurement of IL-4, IL-5, and IFN-γ by ELISA (eBioscience). The level of cytokines IL-21, IL-22, and IL-23 in the BALF was also measured by ELISA (R&D Systems).

Statistical analysis

Data are summarized as means ± sem. Data obtained from adoptive transfer experiments were analyzed using the Mann-Whitney U test, and differences were considered statistically significant, with P < 0.05.

Online Supplemental material

Intracellular IL-4, IFN-γ, and IL-17 expression by polarized CD4+ Th1 and Th2 cells by FACS analysis is shown in Supplemental Figure 1. Data are representative of two independent experiments.

RESULTS

Characterization CD4+CD25+Foxp3+ nTregs in naïve DO11.10 mice

We used the OVA-specific TCR transgenic mouse DO11.10 as a source of antigen-specific CD4+CD25+Foxp3+ T cells and determined their ability to suppress Th1-, Th2-, and Th17-driven pulmonary inflammation. nTregs were identified in DO11.10 mice by staining with the antibodies to CD25 and Foxp3 (Fig. 1A). Typically, 4–6% of naive PLN DO11.10 CD4+ T cells constitutively express CD25 (data not shown). Memory CD4+ T cells [27] and nTregs [28] have been identified previously in TCR transgenic mice. Using intracellular staining, we found a high proportion of Foxp3+ cells in DO11.10 mice expressed the OVA-specific, transgenic TCR, as evidenced by staining with the anticlonotypic antibody KJ1-26 (72.4%; Fig. 1A) and thus, would be expected to respond to the OVA323–339 peptide. In addition, Foxp3 was expressed by CD4+ cells (5.25%), and a high proportion of CD25+ cells was Foxp3+ (52.1%; Fig. 1A). To verify that CD4+CD25+ cells were Tregs, we used three-color staining of the PLNs using anti-CD4, -CD25, and -Foxp3 antibodies. This approach revealed that 85–95% of the CD4+CD25+ cells and 2% of CD4+CD25− cells expressed Foxp3 (data not shown). Magnetic bead sorting of CD4+CD25+ Tregs from the lymph nodes of DO11.10 mice yielded an average of 2 × 105 viable cells/mouse.

Figure 1.

Figure 1.

Open in a new tab

Identification and expansion of OVA-specific CD4+ Tregs in DO11.10 mice. PLN cells were obtained from DO11.10 mice and stained using anti-CD4, -CD25, and -Foxp3, isotype control antibodies, and the anticlonotypic antibody KJ1-26. (A) The frequency of PLN CD4+, CD25+, and KJ1-26+ cells that expressed Foxp3 was determined by FACS analysis. The proportion of Foxp3+ Tregs was assessed by intracellular staining using Alexa Fluor 488 anti-Foxp3 antibody. (B) Expression of Foxp3 by expanded antigen-specific CD4+ nTregs. CD4+CD25+ T cells purified from DO11.10 were expanded in culture for 8 days as described in Materials and Methods. The expanded Tregs were stained with KJ1-26, anti-CD4, or isotype control antibodies and the proportion of Foxp3+ Tregs assessed by FACS analysis following intracellular staining using Alexa Fluor 488 anti-Foxp3 antibody. Data are representative of three separate experiments.

The limited numbers of nTregs in DO11.10 mice made adoptive transfer experiments of these cells difficult. To circumvent this problem, nTregs purified from DO11.10 mice were expanded in culture in the presence of the OVA323–339 peptide and exogenous IL-2 and IL-4. We have reported previously that CD4+CD25+Foxp3+ T cells proliferate in the presence of IL-2 and IL-4 with retention of Foxp3 expression and suppressive function [17]. Importantly, IL-4 has been shown to inhibit the generation of iTregs [19]. Intracellular Foxp3 staining demonstrated that the majority of expanded CD4+CD25+ Tregs continued to express Foxp3 (92.5%) and was OVA-specific (76.1% KJ1-26+Foxp3+; Fig. 1B). In contrast, activated CD4+CD25− cells cultured under identical conditions expressed minimal levels (0.1%) of Foxp3 protein (data not shown). The expanded CD4+CD25+ cells provided a source of nTregs for use in in vitro and in vivo experiments.

Role of antigen-specific Foxp3+ nTregs in regulating effector Th2- or Th1-mediated lung inflammation

To examine pulmonary inflammation elicited by effector CD4+ Th1, Th2, and Th17 phenotypes, differentiated DO11.10 T cells with known antigen specificity were transferred into mice that inhaled OVA. We subsequently investigated whether pulmonary inflammation induced by the polarized CD4+ T cells would be regulated by expanded OVA-specific CD4+Foxp3+ nTregs. CD4+ T cells from DO11.10 mice were cultured for 8 days under Th2-, Th1-, or Th17-polarizing conditions, and the effector T cells (which were devoid of γδ T cells) were then adoptively transferred alone or cotranferred with expanded nTregs into BALB/c recipient mice that were exposed to OVA aerosols for 7 days. The polarized Th1 cells produced IFN-γ but no IL-4 or IL-5, and Th2 cells secreted high levels of IL-4 and IL-5 but negligible IFN-γ, as reported previously [29]. Consistently, intracellular staining showed that CD4+ Th1 cells expressed IFN-γ but not IL-4 or IL-17, and CD4+ Th2 cells expressed IL-4 but not IFN-γ or IL-17 (Supplemental Fig. 1). In the first instance, recipients of CD4+ Th1 or Th2 cells that inhaled OVA developed a marked mucosal inflammatory response characterized by the infiltration of inflammatory cells into the airways. Specifically, Th2 recipient mice developed a pronounced pulmonary eosinophilia, evidenced by cytochemical staining and increased EPO activity (Fig. 2, A and C), and showed an increase in the level of lymphocytes in the airways (Fig. 2A). In sharp contrast, OVA-challenged control mice, which received nTregs alone or did not receive any CD4+ T cells, showed no evidence of lung inflammation. The cotransfer of Tregs with effector Th2 cells failed to suppress the inflammatory response and eosinophilic infiltration into the BALF (Fig. 2, A and C). Conversely, Th1-induced inflammation resulted in the recruitment of lymphocytes, macrophages, and GR1+CD11b+ neutrophils into the airway (Fig. 2, B and D). Cotransfer of nTregs failed to reduce the level of neutrophils present in the BALF of Th1 recipient mice (Fig. 2, B and D). There was no reduction in the level of OVA-specific Th2 (Fig. 2E) or Th1 cells (Fig. 2F) in the lungs, as the number of CD4+KJ1-26+ present in the lungs was unaffected by cotransfer of nTregs. Recipients of nTregs alone or control mice contained negligible CD4+KJ1-26+ T cells. It is clear from these results that under the described conditions, expanded Foxp3+ nTregs, when cotransferred with fully polarized T effector cells, were unable to regulate Th1- or Th2-mediated airway inflammation.

Figure 2.

Figure 2.

Open in a new tab

Transfer of expanded antigen-specific nTregs did not inhibit Th1- or Th2-mediated airway inflammation. DO11.10 CD4+ Th1 or Th2 cells were injected alone or cotransferred with expanded CD4+Foxp3+ nTregs into BALB/c recipient mice. Control mice received Tregs alone or no cells (None). All animals were exposed to OVA aerosols for 7 days, and BALF was collected for analysis. Cell differential counts in the BALF were determined by light microscopic evaluation of stained cytospin preparations. Results are expressed as absolute numbers of lymphocytes (Lym), macrophages (Mac), eosinophils (Eos), and neutrophils (Neu). (A) Cell differential counts for recipients of Th2 cells alone, Th2 cells plus Tregs, Tregs alone, or control mice were determined and expressed as absolute cell number. (B) Cell differential counts for recipients of Th1 cells alone, Th1 cells plus Tregs, Tregs alone, or control mice were determined and expressed as absolute cell number. (C) EPO levels in the BALF from the Th2 recipient group were assessed by colorimetric analysis. (D) The number of GR1+CD11b+ neutrophils in the BALF from the Th1 recipient group was determined by FACS analysis and expressed as total cell number/mouse. Number of CD4+KJ1-26+ T cells present in BALF from recipients of Th2 (E) or Th1 cells (F) was examined by FACS and expressed as total cell number/mouse. Data are expressed as mean ± sem (n=6).

Expanded Foxp3+ nTregs suppress Th2 polarization and IL-4 expression by CD4+ T cells

We next evaluated whether expanded nTregs could inhibit the initial Th2 polarization of CD4+ cells in vitro. To monitor the effect of nTregs on CD4+ Th2 polarization in vitro, PLN cells from IL-4 reporter mice [30] were cocultured with expanded CD4+Foxp3+ Tregs (ratio of 4:1) in the presence of plate-bound anti-CD3 under Th2-polarizing conditions. After 4 days in culture, the number of GFP-expressing CD4+ T cells was determined by flow cytometry. Importantly, KJ1-26 clonotypic antibody was used, which recognized DO11.10 nTregs specifically but not CD4+ cells from 4get mice. [This allowed us to “gate out” nTregs and exclude them from the analysis, although it is important to note that 12% of these expanded nTregs are KJ1-26− (cf., Fig. 1B), a level that would not be expected to have a significant impact on the analysis.] Thus, using GFP production as a measure of IL-4 expression (in comparison with control cells from 4get mice), our data demonstrated that nTregs from DO11.10 mice markedly inhibited Th2 polarization from naïve T cells. After 4 days in culture, the nTregs inhibited expression of GFP by the 4get cells by 77.5% (34.3%-positive GFP staining in the absence of nTregs compared with 7.7%-positive for the cultures grown in the presence of nTregs; Fig. 3A). Addition of exogenous IL-2 to the polarizing culture resulted in a significant increase in the level of GFP expression (Fig. 3B). The IL-2-induced GFP expression was also suppressed by the addition of nTregs (Fig. 3B). Interestingly, the suppression by nTregs was reversed by inclusion of an antibody to GITR (Fig. 3C), which has been shown to prevent the action of Tregs [31]. In contrast, the addition of a soluble form of OX40L failed to reverse the Treg-mediated suppression of Th2 polarization (Fig. 3C).

Figure 3.

Figure 3.

Open in a new tab

Expanded nTregs suppress the differentiation of CD4+ Th2 cells in vitro. PLN cells from 4get mice were cocultured with or without expanded DO11.10 nTregs (ratio of 4:1, respectively) for 4 days on anti-CD3-coated plates (2 μg/ml) under Th2-polarizing conditions. (A) FACS analysis of GFP expression was performed after excluding CD4+KJ1-26+ Tregs from the analysis by gating out cells staining with allophycocyanin-Cy7-conjugated KJ1-26 antibody. (B) Expanded nTregs mediated suppression of GFP-expressing T cells from 4get mice, even in the presence of exogenous IL-2. PLN cells from 4get mice were cultured on anti-CD3-coated plates (2 μg/ml) under Th2-polarizing conditions in the presence or absence of IL-2 (10 ng/ml) and expanded nTregs. GFP expression by CD4+ T cells was determined by FACS (Tregs were excluded as described before). (C) Requirement for GITR, but not OX40L, in Treg-mediated suppression of GFP-expressing T cells. PLN cells from 4get mice were cocultured with DO11.10 nTregs on anti-CD3-coated plates under Th2-polarizing conditions in the presence or absence of anti-GITR antibody (20 μg/ml) or solubilized OX40L (20 μg/ml). GFP expression by CD4+ T cells was determined by FACS analysis (Tregs were excluded as before). Results are expressed as mean ± sem (n=3) and are representative of three independent experiments.

Expanded Foxp3+ nTregs suppress Th17-mediated airway inflammation and IL-17 expression but not AHR

We next examined the ability of expanded CD4+Foxp3+ nTregs to suppress effector Th17-mediated lung inflammatory responses. The adoptive transfer of polarized CD4+ Th17 cells, which expressed high levels of intracellular IL-17 (Fig. 4A) but not IL-4 or IFN-γ (data not shown) into BALB/c mice and subsequent exposure to aerosolized OVA, resulted in a pronounced increase in airway inflammation, associated with elevated neutrophils, macrophages, and lymphocytes in the BALF (Fig. 4B). Control mice (no transfer) and animals that received only nTregs showed no evidence of lung inflammation following OVA challenge. The cotransfer of nTregs with CD4+ Th17 resulted in a significant suppression in Th17-mediated airway inflammation to inhaled OVA (Fig. 4B), as exemplified by a marked reduction in the level of GR1+CD11b+ neutrophils (from 31.1% in Th17 recipients to 16.5% in Th17 plus the Treg group) present in the BALF (Fig. 4C). The reduction in Th17-mediated inflammatory processes in the airways following cotransfer of Tregs was also associated with a striking decrease in KJ1-26+ T cells (from 9.6% in Th17 recipients to 4.5% in Th17 plus the Treg group) in the BALF (Fig. 4D).

Figure 4.

Figure 4.

Open in a new tab

Transfer of Foxp3+ nTregs suppresses Th17-mediated airway inflammation. DO11.10 CD4+ Th17 cells were adoptively transferred alone or cotransferred with expanded CD4+Foxp3+ nTregs into the BALB/c recipient. Control mice received Tregs alone or did not receive any cells (None). All animals were exposed to OVA aerosols for 7 days, and BALF was collected for analysis. (A) FACS analysis of intracellular IL-17 expression by polarized CD4+ Th17 cells. (B) BALF cell differential counts were determined by light microscopic evaluation of stained cytospin preparations. Results are expressed as absolute numbers of lymphocytes, neutrophils, and macrophages. The effect of Tregs on the proportion of GR1+ neutrophils (C) and CD4+KJ1-26 T cells (D) in the BALF of Th17 recipients was determined by FACS analysis. Data represent means ± sem (n=6). *, P < 0.05, compared with the OVA-challenged Th17 recipient group. FACS results are representative of three independent experiments.

In challenged mice that received CD4+ Th17 and Tregs, it was important to discriminate between KJ1-26+ effector Th17 and Tregs present in the lung. Intracellular staining of IL-17 revealed that the cotransfer of Tregs markedly reduced the proportion of IL-17-expressing KJ1-26+ cells (5.5–2.0%) present in the airway (Fig. 5A). Moreover, cotransfer of nTregs markedly inhibited the production of IL-17 by LMCs from Th17 recipients following restimulation with OVA323–339 peptide or immobilized anti-CD3 (Fig. 5B). In addition to IL-17, the level of IL-22 in the BALF of Th17 recipients was reduced slightly by cotransfer of Tregs, however the levels of IL-21 and IL-23 were not affected significantly by Tregs (Fig. 5C). Mice receiving Th1 cells, Th2 cells, and Tregs alone had negligible levels on Th17-associated cytokines in the BALF following OVA inhalation (Fig. 5C). Interestingly, there is a striking increase in IL-17-producing KJ-126− T cells during Th17-mediated pulmonary inflammation (8.4%; Fig. 5A), which was not inhibited by nTregs. Subsequent experiments revealed that a high proportion of CD4−KJ1-26− IL-17-expressing cells in the airways of Th17 recipients was γδ T cells, which were not present in unchallenged, control animals (Fig. 5D). Concomitant with airway inflammation, Th17 recipient mice displayed augmented AHR, as shown by increased levels of airway resistance and reduced dynamic compliance compared with control mice, which was surprisingly more pronounced than that observed in the Th2 group. However, the AHR was not reversed by the cotransfer of nTregs (Fig. 5E). In summary, these findings demonstrate that although the Th17-mediated lung inflammatory response was markedly suppressed by antigen-specific Foxp3+ nTregs, this was not accompanied by an improvement in airway function.

Figure 5.

Figure 5.

Open in a new tab

Transfer of Foxp3+ nTregs inhibits Th17-mediated airway IL-17 expression but not AHR. DO11.10 CD4+ Th17, Th2, or Th1 cells were injected alone or cotransferred with expanded CD4+Foxp3+ nTregs into BALB/c mice, which were then exposed to OVA aerosols for 7 days. Control mice received Tregs alone or did not receive any cells (no transfer). (A) The proportion of IL-17-expressing, OVA-specific Th17 cells (KJ1-26 cells) present in the BALF was determined by FACS using intracellular staining, as described in Materials and Methods. (B) IL-17 production by LMCs from recipients of Th17 cells, Th17 cells plus Tregs, Tregs alone, or control mice was measured by ELISA after restimulation with the OVA323–339 peptide (1 μg/ml) or immobilized anti-CD3 (5 μg/ml) for 24 h. (C) The level of IL-21, IL-22, and IL-23 in the BALF was determined by ELISA. (D) The proportion of IL-17-expressing γδ T cells present in the BALF of Th17 recipients compared with control mice (no transfer) was determined by FACS analysis using intracellular staining. (E) AHR was determined in response to methacholine inhalation by measurement of lung resistance (RL) and dynamic compliance (Cdyn). Data represent means ± sem (n=4–6). FACS results are representative of three independent experiments.

DISCUSSION

Th2 cytokines, such as IL-4, IL-5, IL-9, and IL-13, play a key role allergic asthma, and consequently, it has been suggested that the underlying cause of the disease arises from the failure to regulate lung mucosal CD4+ Th2 responses. Several Th2 cytokines are known to contribute to the inflammatory process. IL-4 promotes CD4+ T cell commitment to a Th2 phenotype, IL-9 and IL-13 promote airway mucus production and AHR, whereas IL-5 regulates eosinophil development, activation, and tissue recruitment (see review, ref. [32]). More recently it has been proposed that CD4+ Th17 cells may also contribute to the asthmatic response in humans [33, 34] and in mouse models of this disease, where Th17 cells appear to underlie the steroid-resistant component of the inflammatory response [5]. In addition, IL-17 has been shown to play an important role in other airway inflammatory responses that include ozone-induced exacerbation of airway inflammation [35] and endotoxin-induced recruitment of neutrophils to the airways [36].

To examine pulmonary inflammation elicited by effector CD4+ Th1, Th2, and Th17 phenotypes, differentiated DO11.10 T cells with known antigen specificity were transferred into mice that subsequently inhaled OVA. This approach provided an opportunity to examine the ability of nTregs to suppress functions mediated by the polarized effector CD4+ T cells and the associated inflammation. The transferred CD4+ T cells retained their cytokine profiles evident at the time of injection for up to 10 days in vivo [17]. The airway inflammatory response elicited by each type of effector CD4+ T cell was different with CD4+ Th2 cells promoting a pronounced eosinphilia and CD4+ Th1 and Th17 cells eliciting neutrophil infiltration into the airways. Coincident with the onset of the inflammatory response, a marked increase in AHR was evident in mice that had received Th2 or Th17 cells and had inhaled OVA. CD4+CD25+Foxp3+ nTregs are considered important regulators of immune and inflammatory responses [8]. The therapeutic potential of nTregs in Th1-mediated disease processes was demonstrated first in a murine model of colitis, showing that the disease progression could be reversed by Tregs [37, 38]. Suppression of intestinal inflammation in colitis by the Tregs was dependent on IL-10 [39] and TGF-β production, although these cells appeared not to be the source of the latter cytokine [22]. Few studies have examined the ability of antigen-specific nTregs to suppress lung inflammation mediated by differentiated CD4+ T cells. It is unlikely that CD4+ Th1-, Th2-, and Th17-mediated inflammation will all be equally susceptible to suppression, given their markedly different properties. With respect to the suppressive function of Tregs, it has been proposed that iTregs may be more effective than nTregs at limiting Th17 responses, as the nTreg phenotype is unstable in the presence of IL-6 [11]. Additionally, the role of nTregs in suppressing Th2-mediated inflammation remains controversial, and their action on airway Th17-mediated inflammation and associated AHR has not been fully elucidated. Consequently, it was important to examine the ability of nTregs to mediate immune suppression in a range of inflammatory settings. Typically, nTregs isolated from TCR transgenic mice express the transgenic TCR in combination with an endogenous TCR α-chain. The principal difficulties encountered when investigating the anti-inflammatory properties of Foxp3+ nTregs arise from the low numbers of these cells present in normal animals and the problems associated with preparing nTregs with a known specificity. In this study, we used the DO11.10 TCR transgenic mouse as a source of nTregs. Consistent with previous reports, 4–6% of CD4+ T cells in DO11.10 mice constitutively express CD25 [17]. In addition, these CD4+CD25+ T cells expressed the Foxp3 protein and the transgenic TCR, as determined by staining with the anticlonotypic antibody, KJ1-26. Foxp3-expressing nTregs from DO11.10 mice were expanded in vitro, and their capacity to limit the onset of allergic inflammation mediated by fully differentiated CD4+ Th2 was compared with the suppression evident in Th1- and Th17-mediated inflammation. Our data show that DO11.10 nTregs, expanded in IL-2 and IL-4, retain Foxp3, clonotypic TCR expression (KJ1-26+), and their immune-suppressive function. These nTregs were devoid of iTregs, as they were derived from CD25+ cells and expanded in the presence of IL-4, which has been shown to inhibit the generation of TGF-β-induced Foxp3+ T cells [19].

We found that the cotransfer of expanded Foxp3+ nTregs with polarized Th2 cells into animals that subsequently inhaled OVA was ineffective at inhibiting the airway eosinophilia elicited by the effector Th2 cells. In this context, it has been demonstrated previously that nTregs were poor regulators of Th2 responses and that nTreg-mediated suppression of Th2-mediated inflammation cells was enhanced considerably by the phosphodiesterase 4 inhibitor rolipram [40]. In the present study, the lack of suppression of airway inflammation evoked by Th2 cells was surprising, given our previous observation that depletion of CD4+CD25+ T cells prior to Th2 differentiation resulted in augmented pulmonary eosinophilic inflammation [17]. It is conceivable that the IL-6 produced by CD4+ Th2 cells resulted in destabilization of the nTreg phenotype, resulting in their conversion to CD4+ Th17 cells [41]. Nevertheless, by monitoring reduction of GFP expression by CD4+ T cells from IL-4 reporter mice, we observed that expanded nTregs were highly effective at inhibiting the differentiation of naïve CD4+ T cells into the Th2 phenotype, even in the presence of exogenous IL-2, by a mechanism that could be reversed by an antibody to GITR. IL-2 and progression of the CD4+ cells through several cell cycles have been shown to be essential for effective Th2 polarization. This strongly implies that in the previous study [17], depletion of nTregs enhanced the Th2 polarization process and the stability of the differentiated cells (thus, augmenting the inflammatory response). In marked contrast, transfer of nTregs markedly suppressed airway neutrophilic inflammation elicited by CD4+ Th17 cells and the number of KJ1-26+ T cells expressing IL-17 in the BALF. However, nTregs did not inhibit the AHR, evident in Th17 recipients, or the lung neutrophilic inflammation, evident in animals that received CD4+ Th1 cells and inhaled OVA. The latter finding was unexpected, given that Tregs mediate suppression in models of Th1 disease [37, 38]. Although inflammatory diseases of the intestine have been traditionally associated with an exaggerated Th1 or Th2 response, more recent studies show that Th17 cells and IL-23 play key roles in intestinal inflammation [42,43,44]. These findings raise the possibility that nTregs regulate intestinal inflammation by suppressing the Th17 response.

There are several explanations for why lung inflammation, elicited by effector CD4+ Th17 cells, but not Th1 or Th2 cells, is susceptible to regulation by nTregs. Possibly, resistance to suppression reflects the lack of nTreg stability in the inflammatory site. Alternatively, the high level of susceptibility of Th17 cells to this form of regulation may stem from the functional plasticity that these cells exhibit [45]. Consequently, Th17 cells are relatively unstable with regard to their cytokine-producing phenotype compared with Th1 and Th2 cells and thus, likely to be more susceptible to immune regulation. The mutual requirement for TGF-β in the development of Tregs and Th17 cells implied a shared, developmental pathway for these two T cell subsets. Other cytokines, such as IL-6 [41, 46] and IL-21 [47, 48], induce Th17 differentiation and inhibit Foxp3 expression. Moreover, IL-2 [49] and the vitamin A metabolite retinoic acid [50, 51] have been shown to promote Treg development and inhibit Th17 differentiation. This reciprocal relationship between Treg and Th17 cells would suggest that Th17 cells are more likely to be permissive to Treg suppression. One plausible manifestation of such a form of regulation could arise as a consequence of nTregs, specifically preventing expansion of CD4+ Th17 cells in lymphoid tissues following transfer.

Interestingly, Th17-mediated airway inflammation was associated with a moderate increase in the levels of IL-21, IL-22, and IL-23, as well as IL-17. Transfer of nTregs resulted in a significant reduction in the number of KJ1-26+ T cells in the BALF, and the proportion of KJ1-26+ cells expressing IL-17 was markedly reduced. However, nTregs did not affect the number of host γδ T cells expressing IL-17 in the airways. Similar reductions in IL-22 levels were also evident by nTreg transfer. However, IL-21 and IL-23 were not inhibited by nTregs, suggesting that these cytokines are produced by cells resistant to this form of suppression. In this context, γδ T cells might be the source of IL-21, and the source of IL-23 is likely to be dendritic cells or macrophages present in the airway rather than CD4+ T cells.

Although the Th17-mediated lung inflammation was markedly suppressed by antigen-specific Foxp3+ nTregs, this was not accompanied by an improvement in airway function. Interestingly, nTregs resulted in a significant decrease in the number of KJ1-26+ T cells producing IL-17 in the airways but did not affect host IL-17-expressing γδ T cells. It is conceivable that these IL-17-producing γδ T cells, which appear to be resistant to suppression by nTregs, may promote AHR, as IL-17 has been shown to elevate AHR [5].

In summary, Foxp3+ nTregs play an important role in regulating T cell polarization and lung inflammatory processes to inhaled antigens. Our results demonstrate that the transfer of nTregs suppresses pulmonary inflammation mediated by CD4+ Th17 cells but not AHR. In addition, they were highly effective at suppressing the differentiation of naïve CD4+ T cells into the Th2 phenotype, although nTregs did not inhibit the airway inflammation mediated by fully polarized Th2 cells. A better understanding of the mechanism underlying the immunomodulatory properties exerted by nTregs could provide important information leading to novel approaches to control the airway inflammatory response manifest in bronchial asthma and other diseases of the respiratory tract. Further elucidation of the role of nTregs is at this time necessary and critical to broaden our understanding of mechanisms underlying respiratory diseases, such as asthma, and how they could be conceivably regulated.

AUTHORSHIP

Teri Girtsman performed most of the experiments, Zeina Jaffar assisted in designing and performing in vivo experiments and writing the manuscript, Maria Ferrini assisted in performing Th17 experiments, Pamela Shaw assisted with FACS analysis, and Kevan Roberts is the Principal Investigator who is the main contributor in designing and writing the manuscript.

ACKNOWLEDGMENTS

This work was supported by grants from National Institutes of Health (R01-HL079189 to K.R.) and COBRE (P20RR017670). The authors acknowledge the Fluorescence Cytometry Core and thank Mary Buford (Buxco Research Systems) for her technical assistance.

Supplementary Material

[Supplemental Figure]
jlb.0110044_index.html (902B, html)

Footnotes

Abbreviations: AHR=airway hyper-reactivity, ATTC=American Type Tissue Collection, BALF=bronchoalveolar lavage fluid, EPO=eosinophil peroxidase, Foxp3=forkhead box p3, GITR=glucocorticoid-induced TNFR family-related gene, iTreg=induced regulatory T cell, LMC=lung mononuclear cell, nTreg=natural regulatory T cell, OX40L=OX40 ligand, PLN=peripheral lymph node cell, Treg=regulatory T cell

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

References

  1. Holgate S T. The airway epithelium is central to the pathogenesis of asthma. Allergol Int. 2008;57:1–10. doi: 10.2332/allergolint.R-07-154. [DOI] [PubMed] [Google Scholar]
  2. Zhu J, Paul W E. CD4 T cells: fates, functions, and faults. Blood. 2008;112:1557–1569. doi: 10.1182/blood-2008-05-078154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Song C, Luo L, Lei Z, Li B, Liang Z, Liu G, Li D, Zhang G, Huang B, Feng Z H. IL-17-producing alveolar macrophages mediate allergic lung inflammation related to asthma. J Immunol. 2008;181:6117–6124. doi: 10.4049/jimmunol.181.9.6117. [DOI] [PubMed] [Google Scholar]
  4. Chakir J, Shannon J, Molet S, Fukakusa M, Elias J, Laviolette M, Boulet L P, Hamid Q. Airway remodeling-associated mediators in moderate to severe asthma: effect of steroids on TGF-β, IL-11, IL-17, and type I and type III collagen expression. J Allergy Clin Immunol. 2003;111:1293–1298. doi: 10.1067/mai.2003.1557. [DOI] [PubMed] [Google Scholar]
  5. McKinley L, Alcorn J F, Peterson A, Dupont R B, Kapadia S, Logar A, Henry A, Irvin C G, Piganelli J D, Ray A, Kolls J K. TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice. J Immunol. 2008;181:4089–4097. doi: 10.4049/jimmunol.181.6.4089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Groux H, Powrie F. Regulatory T cells and inflammatory bowel disease. Immunol Today. 1999;20:442–445. doi: 10.1016/s0167-5699(99)01510-8. [DOI] [PubMed] [Google Scholar]
  7. Akbari O, Freeman G J, Meyer E H, Greenfield E A, Chang T T, Sharpe A H, Berry G, DeKruyff R H, Umetsu D T. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med. 2002;8:1024–1032. doi: 10.1038/nm745. [DOI] [PubMed] [Google Scholar]
  8. Shevach E M, DiPaolo R A, Andersson J, Zhao D M, Stephens G L, Thornton A M. The lifestyle of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells. Immunol Rev. 2006;212:60–73. doi: 10.1111/j.0105-2896.2006.00415.x. [DOI] [PubMed] [Google Scholar]
  9. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol. 2005;6:345–352. doi: 10.1038/ni1178. [DOI] [PubMed] [Google Scholar]
  10. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–1164. [PubMed] [Google Scholar]
  11. Horwitz D A, Zheng S G, Gray J D. Natural and TGF-β-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other. Trends Immunol. 2008;29:429–435. doi: 10.1016/j.it.2008.06.005. [DOI] [PubMed] [Google Scholar]
  12. Brunkow M E, Jeffery E W, Hjerrild K A, Paeper B, Clark L B, Yasayko S A, Wilkinson J E, Galas D, Ziegler S F, Ramsdell F. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001;27:68–73. doi: 10.1038/83784. [DOI] [PubMed] [Google Scholar]
  13. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057–1061. doi: 10.1126/science.1079490. [DOI] [PubMed] [Google Scholar]
  14. Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, Sakaguchi S. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol. 1999;162:5317–5326. [PubMed] [Google Scholar]
  15. Asano M, Toda M, Sakaguchi N, Sakaguchi S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med. 1996;184:387–396. doi: 10.1084/jem.184.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hadeiba H, Locksley R M. Lung CD25 CD4 regulatory T cells suppress type 2 immune responses but not bronchial hyperreactivity. J Immunol. 2003;170:5502–5510. doi: 10.4049/jimmunol.170.11.5502. [DOI] [PubMed] [Google Scholar]
  17. Jaffar Z, Sivakuru T, Roberts K. CD4+CD25+ T cells regulate airway eosinophilic inflammation by modulating the Th2 cell phenotype. J Immunol. 2004;172:3842–3849. doi: 10.4049/jimmunol.172.6.3842. [DOI] [PubMed] [Google Scholar]
  18. Bluestone J A, Abbas A K. Natural versus adaptive regulatory T cells. Nat Rev Immunol. 2003;3:253–257. doi: 10.1038/nri1032. [DOI] [PubMed] [Google Scholar]
  19. Dardalhon V, Awasthi A, Kwon H, Galileos G, Gao W, Sobel R A, Mitsdoerffer M, Strom T B, Elyaman W, Ho I C, Khoury S, Oukka M, Kuchroo V K. IL-4 inhibits TGF-β-induced Foxp3+ T cells and, together with TGF-β, generates IL-9+ IL-10+ Foxp3(–) effector T cells. Nat Immunol. 2008;9:1347–1355. doi: 10.1038/ni.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shevach E M. Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity. 2009;30:636–645. doi: 10.1016/j.immuni.2009.04.010. [DOI] [PubMed] [Google Scholar]
  21. Asseman C, Mauze S, Leach M W, Coffman R L, Powrie F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J Exp Med. 1999;190:995–1004. doi: 10.1084/jem.190.7.995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Powrie F, Carlino J, Leach M W, Mauze S, Coffman R L. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells. J Exp Med. 1996;183:2669–2674. doi: 10.1084/jem.183.6.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Allan S E, Broady R, Gregori S, Himmel M E, Locke N, Roncarolo M G, Bacchetta R, Levings M K. CD4+ T-regulatory cells: toward therapy for human diseases. Immunol Rev. 2008;223:391–421. doi: 10.1111/j.1600-065X.2008.00634.x. [DOI] [PubMed] [Google Scholar]
  24. Jiang S, Tsang J, Game D S, Stevenson S, Lombardi G, Lechler R I. Generation and expansion of human CD4+ CD25+ regulatory T cells with indirect allospecificity: potential reagents to promote donor-specific transplantation tolerance. Transplantation. 2006;82:1738–1743. doi: 10.1097/01.tp.0000244932.29542.9e. [DOI] [PubMed] [Google Scholar]
  25. Tang Q, Henriksen K J, Bi M, Finger E B, Szot G, Ye J, Masteller E L, McDevitt H, Bonyhadi M, Bluestone J A. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 2004;199:1455–1465. doi: 10.1084/jem.20040139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee S C, Jaffar Z H, Wan K S, Holgate S T, Roberts K. Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation. J Immunol. 1999;162:6867–6879. [PubMed] [Google Scholar]
  27. Lee W T, Cole-Calkins J, Street N E. Memory T cell development in the absence of specific antigen priming. J Immunol. 1996;157:5300–5307. [PubMed] [Google Scholar]
  28. Piccirillo C A, Letterio J J, Thornton A M, McHugh R S, Mamura M, Mizuhara H, Shevach E M. CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor β1 production and responsiveness. J Exp Med. 2002;196:237–246. doi: 10.1084/jem.20020590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jaffar Z, Ferrini M E, Buford M C, Fitzgerald G A, Roberts K. Prostaglandin I2-IP signaling blocks allergic pulmonary inflammation by preventing recruitment of CD4+ Th2 cells into the airways in a mouse model of asthma. J Immunol. 2007;179:6193–6203. doi: 10.4049/jimmunol.179.9.6193. [DOI] [PubMed] [Google Scholar]
  30. Mohrs M, Shinkai K, Mohrs K, Locksley R M. Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity. 2001;15:303–311. doi: 10.1016/s1074-7613(01)00186-8. [DOI] [PubMed] [Google Scholar]
  31. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 2002;3:135–142. doi: 10.1038/ni759. [DOI] [PubMed] [Google Scholar]
  32. Wills-Karp M. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu Rev Immunol. 1999;17:255–281. doi: 10.1146/annurev.immunol.17.1.255. [DOI] [PubMed] [Google Scholar]
  33. Zhao Y, Yang J, Gao Y D, Guo W. Th17 immunity in patients with allergic asthma. Int Arch Allergy Immunol. 2009;151:297–307. doi: 10.1159/000250438. [DOI] [PubMed] [Google Scholar]
  34. Wong C K, Lun S W, Ko F W, Wong P T, Hu S Q, Chan I H, Hui D S, Lam C W. Activation of peripheral Th17 lymphocytes in patients with asthma. Immunol Invest. 2009;38:652–664. doi: 10.1080/08820130903062756. [DOI] [PubMed] [Google Scholar]
  35. Pichavant M, Goya S, Meyer E H, Johnston R A, Kim H Y, Matangkasombut P, Zhu M, Iwakura Y, Savage P B, DeKruyff R H, Shore S A, Umetsu D T. Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17. J Exp Med. 2008;205:385–393. doi: 10.1084/jem.20071507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ferretti S, Bonneau O, Dubois G R, Jones C E, Trifilieff A. IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. J Immunol. 2003;170:2106–2112. doi: 10.4049/jimmunol.170.4.2106. [DOI] [PubMed] [Google Scholar]
  37. Mottet C, Uhlig H H, Powrie F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol. 2003;170:3939–3943. doi: 10.4049/jimmunol.170.8.3939. [DOI] [PubMed] [Google Scholar]
  38. Maloy K J, Powrie F. Regulatory T cells in the control of immune pathology. Nat Immunol. 2001;2:816–822. doi: 10.1038/ni0901-816. [DOI] [PubMed] [Google Scholar]
  39. Uhlig H H, Coombes J, Mottet C, Izcue A, Thompson C, Fanger A, Tannapfel A, Fontenot J D, Ramsdell F, Powrie F. Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis. J Immunol. 2006;177:5852–5860. doi: 10.4049/jimmunol.177.9.5852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Bopp T, Dehzad N, Reuter S, Klein M, Ullrich N, Stassen M, Schild H, Buhl R, Schmitt E, Taube C. Inhibition of cAMP degradation improves regulatory T cell-mediated suppression. J Immunol. 2009;182:4017–4024. doi: 10.4049/jimmunol.0803310. [DOI] [PubMed] [Google Scholar]
  41. Bettelli E, Carrier Y, Gao W, Korn T, Strom T B, Oukka M, Weiner H L, Kuchroo V K. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. [DOI] [PubMed] [Google Scholar]
  42. Ahern P P, Izcue A, Maloy K J, Powrie F. The interleukin-23 axis in intestinal inflammation. Immunol Rev. 2008;226:147–159. doi: 10.1111/j.1600-065X.2008.00705.x. [DOI] [PubMed] [Google Scholar]
  43. Brand S. Crohn’s disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn’s disease. Gut. 2009;58:1152–1167. doi: 10.1136/gut.2008.163667. [DOI] [PubMed] [Google Scholar]
  44. Fujino S, Andoh A, Bamba S, Ogawa A, Hata K, Araki Y, Bamba T, Fujiyama Y. Increased expression of interleukin 17 in inflammatory bowel disease. Gut. 2003;52:65–70. doi: 10.1136/gut.52.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhu J, Paul W E. Heterogeneity and plasticity of T helper cells. Cell Res. 2010;20:4–12. doi: 10.1038/cr.2009.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mangan P R, Harrington L E, O'Quinn D B, Helms W S, Bullard D C, Elson C O, Hatton R D, Wahl S M, Schoeb T R, Weaver C T. Transforming growth factor-β induces development of the T(H)17 lineage. Nature. 2006;441:231–234. doi: 10.1038/nature04754. [DOI] [PubMed] [Google Scholar]
  47. Korn T, Bettelli E, Gao W, Awasthi A, Jager A, Strom T B, Oukka M, Kuchroo V K. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature. 2007;448:484–487. doi: 10.1038/nature05970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhou L, Ivanov I I, Spolski R, Min R, Shenderov K, Egawa T, Levy D E, Leonard W J, Littman D R. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol. 2007;8:967–974. doi: 10.1038/ni1488. [DOI] [PubMed] [Google Scholar]
  49. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133:775–787. doi: 10.1016/j.cell.2008.05.009. [DOI] [PubMed] [Google Scholar]
  50. Elias K M, Laurence A, Davidson T S, Stephens G, Kanno Y, Shevach E M, O'Shea J J. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Blood. 2008;111:1013–1020. doi: 10.1182/blood-2007-06-096438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, Cheroutre H. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007;317:256–260. doi: 10.1126/science.1145697. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Figure]
jlb.0110044_index.html (902B, html)
jlb.0110044_1.pdf (456.9KB, pdf)

Articles from Journal of Leukocyte Biology are provided here courtesy of The Society for Leukocyte Biology

RESOURCES