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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jul 1, 2012.
Published in final edited form as:
PMCID: PMC3119737
NIHMSID: NIHMS294653

A role for IL-27 in limiting T regulatory cell populations1

Abstract

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that T regulatory cells (Treg) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Treg in vivo. The studies presented here show that mice that over-express IL-27 had decreased Treg frequencies and developed spontaneous inflammation. While IL-27 did not cause mature Treg to downregulate forkhead box P3, transgenic over-expression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. Together, these data identify an indirect role for IL-27 in shaping the Treg pool.

Keywords: T cells, Cytokines, Inflammation

Introduction

IL-27 is a member of the IL-6/IL-12 family of cytokines, a group of factors remarkable for their pleiotropic effects on Th function, differentiation, and development (13). IL-27 is a heterodimer composed of the IL-27p28 and Epstein-Barr virus-induced 3 (EBI3) subunits, that signals through a receptor that consists of WSX-1 and glycoprotein 130 (4, 5). Ligation of the receptor complex activates STAT proteins that promote immune-regulatory gene expression programs (48). Initially, IL-27 was described as a pro-inflammatory molecule produced by APC that supports Th1 responses (4); naive T cells exposed to IL-27 activated STAT1 and upregulated T-box expressed in T cells and IL-12Rβ2 expression, rendering these cells sensitive to IL-12 signals (68). Subsequent reports revealed that IL-27 can also limit inflammation. Thus, when mice deficient in WSX-1 were infected with Toxoplasma gondii or Trypanasoma cruzi, they mounted a protective Th1 response and controlled parasite replication, yet succumbed acutely to a lethal T cell-mediated inflammatory disease, suggesting that IL-27 plays a suppressive role during these infections (9, 10). Other studies in various models of inflammation have expanded our understanding of the anti-inflammatory properties of IL-27 in Th1 (1113), Th2 (1416), and Th17 responses(1719).

Though we now appreciate that IL-27 can have both pro- and anti-inflammatory effects in vivo, the biological function of IL-27 during immune homeostasis remains unclear. WSX-1−/− and IL-27p28−/− animals do not display any overt immunological defects, suggesting that IL-27 signaling is not required for the development of a normal immune system. However, there is evidence that IL-27 may have a role in modulating the T regulatory cell (Treg) population. Treg are vital in preventing autoimmunity and uncontrolled inflammation in the steady state, and suppress inappropriate immune responses through a variety of mechanisms (2022). Accordingly, there is significant interest in identifying the factors that can influence Treg homeostasis and function (23). Naive CD4+ T cells cultured with TGF-β (24) and IL-2 (25) in vitro upregulate the transcription factor forkhead box P3 (Foxp3), indicating a differentiation to the Treg fate (26, 27). When these inducible Treg are exposed to IL-27 during differentiation, Foxp3 upregulation is inhibited (2830). Furthermore, natural T regulatory cells (nTreg) express high levels of WSX-1 (31). Additionally, IL-27 can inhibit the production of IL-2 (32, 33), a vital factor that supports the generation and maintenance of the Treg pool(3439).

In order to investigate the role that IL-27 plays in Treg homeostasis, we utilized a transgenic mouse model in which the IL-27p28 and EBI3 subunits are over-expressed (IL-27 tg mice). IL-27 tg mice succumbed at 8–11 wk of age to a systemic inflammatory condition, characterized by immune-pathology in multiple tissues, increased percentages of activated T cells, and elevated cytokine levels. In accordance with this uncontrolled inflammation, IL-27 tg mice lacked Treg in lymphoid organs. In vitro and in vivo studies showed that IL-27 did not cause Foxp3 downregulation in mature nTreg; however, in a bone marrow (BM) chimera model, when IL-27 was present during the differentiation or generation of the Treg pool, reconstitution of the Treg population was inhibited. Consistent with these findings, IL-27 tg mice had a marked defect in their capacity to produce IL-2, an important cytokine for Treg homeostasis. Together, these data suggest that IL-27 can limit the generation or maintenance of the Treg population by inhibiting IL-2 production and thus promote inappropriate inflammation.

Materials and Methods

Mice

C57BL/6 mice were obtained from the Jackson Laboratory, and CD45.1+ congenic mice were obtained from Taconic Farms. Foxp3GFP mice have been described previously (40). Mice that over-express either IL-27p28 or EBI3 were generated by Zymogenetics, Inc. The murine IL-27p28 or EBI3 open reading frames were PCR amplified, and the resulting cDNA was cloned into an Eμ lck transgene expression vector (41), driving expression in T and B cells. Expression cassettes were microinjected into B6C3f1 oocytes fertilized by C57BL/6 males, using procedures described (42). Stable lines were generated by breeding founders to C57BL/6 mice. Hemizygous female EBI3 tg mice were bred with hemizygous male IL-27p28 tg mice to produce IL-27 tg mice that carry over-expressed alleles of IL-27p28 and EBI3. Mice were maintained in specific pathogen-free conditions at the University of Pennsylvania animal facility, according to institutional and federal regulations.

Pathology

Tissues were fixed in buffered paraformaldehyde and embedded in paraffin. Sections were cut and stained with H&E, trichrome, or periodic acid schiff/Alcian Blue by the University of Pennsylvania School of Veterinary Medicine Pathology Laboratory. Images were captured using a Nikon Eclipse E600 microscope, a Photometrics Cool Snap EZ CCD camera, and Nikon NIS Elements software.

Flow cytometry, intracellular staining, and cell sorting

Single cell suspensions were prepared from spleens, lymph nodes (LN), peritoneal exudates cells (PEC), and thymi, and cells were surface stained with the following Ab: FITC anti-CD3e (145-2C11) and anti-CD69 (H1.2F3), PerCP-Cy5.5 anti-CD45.1 (A20), Pacific Blue® anti-CD3e (17A2), allophycocyanin anti-CD25 (PC61.5), anti-CD45.2 (104), and anti-CD62L (MEL-14), allophycocyanin-eFluor 780 anti-CD25 (PC61.5), anti-CD45.2 (104), and anti-CD62L (MEL-14), PE anti-CD25 (PC61.5) and anti-CD69 (H1.2F3), and PE-Cy7 anti-CD44 (IM7) supplied by eBioscience; Pacific Orange® anti-CD8α (53–6.7) and PE-Texas Red anti-CD62L (MEL-14) supplied by Invitrogen; PerCP-Cy5.5 anti-CD4 (RM4–5) and anti-CD8α (53–6.7), PE anti-CD45.1 (A20), PE-Cy5 anti-CD44 (IM7), and PE-Cy7 CD69 (H1.2F3) supplied by BD Biosciences; Alexa Fluor 700 anti-CD3 (17A2) supplied by Biolegend. To stain for Foxp3 or GFP, the eBioscience Foxp3 staining set was used according to manufacturer instructions, and cells were stained with the following Ab: eFluor 450 anti-Foxp3 (FJK-16s) and purified rabbit polyclonal anti-GFP supplied by eBioscience; Alexa Fluor 488 goat anti-rabbit IgG supplied by Invitrogen. To stain for intracellular cytokines, cells were stimulated for 4 h with 50 ng/mL PMA (Sigma), 500 ng/mL ionomycin (Sigma), 10 μg/mL brefeldin A (Sigma), and 1:1,500 GolgiStop (BD Biosciences). Cells were surface stained, fixed in 2% paraformaldehyde, permeabilized in BD Biosciences permeabilization buffer according to manufacturer instructions, and stained intracellularly with the following Abs: FITC anti-TNF-α (MP6-XT22) and PE-Cy7 anti-IFN-γ (XMG1.2) supplied by eBioscience. For cell sorting, cells were surface stained with anti-CD4 and anti-CD8α, and CD4+CD8GFP+ cells were sorted by the University of Pennsylvania Flow Cytometry Core using a 3-laser 6-color FACSAria (BD Biosciences). For flow cytometry, a 4-laser 18-color LSR II (BD Biosciences) or a 3-laser 8-color FACSCanto (BD Biosciences) were used to acquire data. FlowJo 8.7.1 (Tree Star, Inc.) was used to analyze data.

nTreg cultures in vitro

Sorted nTreg (CD4+CD8GFP+) from Foxp3GFP mice were cultured with plate-bound 1 μg/mL anti-CD3, soluble 1 μg/mL anti-CD28, 10 μg/mL anti-IFN-γ (XMG1.2), 10 μg/mL anti-IL-4 (S4B6) (all produced in house), and 50, 100, or 200 U/mL IL-2 for 72 h, with or without 10 ng/mL rIL-6, supplied by eBioscience, or 50 ng/mL rIL-27, supplied by Amgen.

Isolation of CD4+CD25+ cells and cell transfer

CD4+CD25+ cells were isolated from single-cell suspensions from spleen and peripheral LN of CD45.1+ female mice using the CD4+CD25+ cell isolation kit (Miltenyi Biotech, Inc.) according to manufacturer instructions. Recipient mice were anaesthetized using isofluorene gas, and 1.5 × 106 cells were transferred i.v.

BM chimeras

For donor cell isolation, single cell suspensions from BM of 8 wk-old IL-27 tg mice or wild-type (WT) littermates were prepared. BM was isolated by flushing the femur and tibia, lysing RBC, and depleting T cells using the CD90.2 negative selection kit (Miltenyi Biotech, Inc.) according to manufacturer instructions. CD45.1 recipient mice were lethally irradiated with 1000 CyG using a Nordion Gammacell irradiator, anaesthetized with ketamine administered i.p., and given 5 × 106 donor cells i.v. Chimeric mice were treated with sulfamexosone/trimethoprim for 2 wk post-reconstitution, and were analyzed at 5 wk post-reconstitution.

Rules Based Medicine and ELISA

Serum was collected from 7 wk-old IL-27 tg mice and WT littermates and sent to Rules Based Medicine for 60 parameter Ag analysis. Serum samples were mixed with capture microsphere multiplexes and incubated for 1 h at 25°. Biotinylated reporter Ab for each multiplex were added and incubated for 1 h at 25°, and multiplexes were developed for 1 h at 25° with an excess of streptavidin-PE. Prepared samples were analyzed using a Luminex 100 (Luminex Corporation) and proprietary analysis software (Rules Based Medicine). For each multiplex, calibrators and controls were tested, and values for each Ag were determined with 4 and 5 parameters and with weighted and non-weighted curve-fitting algorithms. For supernatant samples, 1 × 106 cells were stimulated with soluble 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28 for 24 or 48 h, and supernatants were collected. Supernatants from in vitro cultures were analyzed by ELISA, using the following Ab pair: anti-IL-2 (JES6-1A12) and biotinylated anti-IL-2 (JES6-5H4) supplied by eBioscience. ELISA plates were developed using ABTS substrate solution (KPL Protein Research Products) and visualized using a micro plate reader (Molecular Devices).

Statistical analyses

Non-parametric Mann-Whitney, unpaired, two-tailed student’s t-test, and one-way, nonparametric Kruskal-Wallis ANOVA with Dunn’s Comparison post-testing were used to determine statistical significance. A p value of <0.05 was considered significant.

Results

Generation of IL-27 tg mice

To investigate how IL-27 influences the immune system and shapes the Treg pool during the steady state, mice that over-express the IL-27p28 and EBI3 subunits of IL-27 were developed. First, individual transgenic mice that express either IL-27p28 or EBI3 alone were generated using a construct that targets over-expression to T and B cells (Supplemental Fig. 1A). Splenocytes from IL-27p28 tg mice expressed high levels of IL-27p28 mRNA and normal levels of EBI3 mRNA (compared to WT splenocytes), and splenocytes from EBI3 tg mice expressed low levels of IL-27p28 mRNA and high levels of EBI3 mRNA (Supplemental Fig. 1B). Over-expression of IL-27p28 was restricted to T and B cells (data not shown). IL-27p28 and EBI3 tg mice appeared grossly normal, and lived a normal life span. Compared to WT littermates, IL-27p28 tg mice had slightly elevated numbers of T cells in the spleen, but normal Treg numbers, and EBI3 tg mice had T cell counts comparable to WT mice (Supplemental Fig. 1C). IL-27p28 tg mice were bred to EBI3 tg mice to create mice that over-express both subunits (IL-27 tg mice). Splenocytes from IL-27 tg mice expressed high levels of both IL-27p28 mRNA and EBI3 mRNA (Supplemental Fig. 1B).

IL-27 tg mice develop and succumb to an inflammatory disease

IL-27 tg neonates appeared normal; however, starting at 5 wk of age, IL-27 tg mice began to show signs of disease, characterized by weight loss, dry skin, and alopecia, and by 8–11 wk of age, these mice succumbed to this condition (Fig. 1A). Histological analysis of 10 wk-old IL-27 tg mice revealed marked pathological changes and inflammation in multiple tissues. Immune infiltrates were observed in the liver, lungs, pancreas, kidney, and seminal vesicles; hyperkeratosis, acanthosis, and follicular atrophy were observed in the skin; fibrosis was evident in the lung; and the colon showed a loss of goblet cell mucin production and edema in the submucosa and lamina propria (Fig. 1B and Supplemental Fig. 2A). Analysis of IL-27 tg mice at 7–8 wk of age showed lymphopenia in the spleen and LN, but these mice had normal PEC counts (Fig. 2A). IL-27 tg mice had high percentages of total CD8+ T cells in the spleen and PEC (Supplemental Fig. 2B). Additionally, compared with WT littermates, IL-27 tg mice had a higher percentage of CD4+ and CD8+ T cells in the spleen, LN, and PEC that expressed the early activation marker CD69 (Fig. 2B), and a higher percentage of CD8+ and CD4+ T cells in the spleen that produced IFN-γ following stimulation with PMA and ionomycin (Fig. 2C and Supplemental Fig. 2C). CD4+ T cells from IL-27 tg mice did not demonstrate increased expression of IL-17 or RORγt (Supplemental Fig. 2D). Finally, multiple parameter analysis for a panel of serum Ag showed that IL-27 tg mice had elevated levels of numerous cytokines, including IFN-γ, IL-10, IL-5, IL-6, and TNF-α (Fig. 2D and Supplemental Fig. 3).

Fig. 1
IL-27 tg mice succumbed to an inflammatory disease. A. IL-27 tg mice died at 8–12 wk of age. Data represent 8 WT or IL-27 tg mice; p<0.0001 by Kaplan and Meier logrank test. B. Ten wk-old IL-27 tg mice had inflammatory infiltrates (→) ...
Fig. 2
IL-27 tg mice developed systemic inflammation by 7 wk of age. A. Compared to WT mice, IL-27 tg mice were lymphopenic in the spleen and LN, but had similar PEC counts. Data represent SEM for 10 WT and 9 IL-27 tg mice from 5 independent experiments; spleen ...

IL-27 tg mice are deficient in Treg

Previous studies have indicated that IL-27 has pro-inflammatory activities, and this property could potentially account for the inflammatory disease that affects IL-27 tg mice. However, this uncontrolled inflammation is also consistent with a defect in the Treg compartment, as murine models of Treg deficiency, including scurfy mice(4345), Foxp3−/− mice (46), and IL-2−/− and IL-2Rβ−/− mice (3437, 39), have a similar inflammatory phenotype. When expression of the Treg transcription factor Foxp3 was analyzed, 7 wk-old IL-27 tg mice almost completely lacked CD4+Foxp3+ Treg in the spleen, LN, PEC, and thymus, by frequency (Fig. 3A) and total number (Fig. 3B). Treg deficiency was also present at 1.5 and 10 wk of age (data not shown). As Treg have a critical role in limiting inflammation, the lack of Treg in IL-27 tg mice is likely a major factor in the development of inflammatory disease in these animals.

Fig. 3
IL-27 tg mice were deficient in Treg. IL-27 tg mice had very low A. percentages and B. total numbers of Foxp3+ Treg in the spleen, LN, PEC, and thymus at 7–8 wk of age. Plots were gated on CD3+CD4+ cells. Data represent SEM for 10 WT and 9 IL-27 ...

IL-27 does not cause nTreg to downregulate Foxp3 in vitro or in vivo

One possible explanation for the loss of Treg observed in IL-27 tg mice was that IL-27, like its cousin IL-6 (47), could cause mature nTreg to downregulate Foxp3 expression. To test this in vitro, nTreg were isolated by cell sorting from Foxp3GFP reporter mice and cultured in vitro in the presence of plate-bound anti-CD3, soluble anti-CD28, anti-IFN-γ, anti-IL-4, and IL-2, with or without murine rIL-27 or rIL-6. After 3 d of culture, Foxp3GFP expression was assessed. Culture with rIL-6 did result in Foxp3 downregulation, as previously reported(47), but rIL-27 did not (Fig. 4A).

Fig. 4
nTreg exposed to IL-27 did not downregulate Foxp3. A. Foxp3GFP+ nTreg did not downregulate Foxp3 following 72 h of culture with rIL-27, but did downregulate Foxp3 following culture with rIL-6. Plots were gated on CD3+CD4+ cells. Data represent SEM for ...

To test the effect of IL-27 on nTreg in vivo, a transfer system was developed in which CD4+CD25+ cells (~85% Foxp3+ (data not shown)) were isolated from CD45.1 congenic mice and transferred to 7 wk-old WT or IL-27 tg recipients. Eleven d later, Foxp3 expression on the congenic cells was examined. In WT recipients, approximately 40% of CD45.1+CD4+ cells in the spleen and LN expressed Foxp3 (Fig. 4B), indicating that a proportion of the transferred Foxp3+ cells either downregulated Foxp3 or died, or that a population of Foxp3 cells expanded. However, WT and IL-27 tg recipients had a similar percentage (Fig. 4B) and number (Fig. 4C) of CD45.1+CD4+Foxp3+ cells in the spleen and LN, suggesting that while the transferred congenic cells displayed decreased Foxp3 in a WT recipient compared to the input, this decrease was similar in an IL-27 tg recipient. These findings, in agreement with in vitro data, indicate that the loss of Treg in IL-27 tg mice is not due to Foxp3 downregulation. Of note, transfer of a small number of CD4+CD25+ cells to IL-27 tg mice did not ameliorate liver and pancreas inflammation (Supplemental Fig. 4A), or decrease T cell activation (Supplemental Fig. 4B), and the transferred mice succumbed to inflammatory disease, similar to untransferred controls.

IL-27 limits Treg expansion in a BM chimera model

Previous studies have reported that IL-27 blocks the generation of inducible Treg in vitro by inhibiting Foxp3 upregulation (2830). To determine whether this type of inhibition correlates with the loss of Treg in IL-27 tg mice, a BM chimera model was utilized in which lethally irradiated WT congenic mice were reconstituted with WT or IL-27 tg BM. At 5 wk post-irradiation and reconstitution, the T cell compartment is composed of approximately 50% donor-derived and 50% host-derived cells, and the host-derived population contains a high proportion of radio-resistant Foxp3+ cells that can reconstitute the Treg compartment in irradiated mice given Treg deficient BM (48, 49). Thus, this model provides a system with which to assess whether IL-27 modulates Foxp3 expression in a cell-extrinsic manner during reconstitution of the Treg compartment.

In irradiated WT mice reconstituted with WT BM, ~8% of donor-derived CD3+CD4+ cells in the spleen were Foxp3+, while ~25% of host-derived CD3+CD4+ cells were Foxp3+ (Fig. 5A). The LN and thymus of these mice showed similar patterns of Foxp3+ expression (Fig. 5A). In contrast, irradiated WT mice reconstituted with IL-27 tg BM had a very low percentage of donor-derived CD3+CD4+ T cells that were Foxp3+ cells in all organs examined, and a marked reduction in the percentage of host-derived, CD3+CD4+ cells that were Foxp3+ (Fig. 5A). The defect in the percentage of Foxp3+ cells in both the donor- and host-derived T cell compartments in irradiated WT mice given IL-27 tg BM was also apparent when total numbers of Foxp3+ cells were calculated (Fig. 5B). Thus, irradiated WT mice reconstituted with IL-27 tg BM could not generate a normal Treg pool from donor-derived T cells, and, in a cell-extrinsic manner, IL-27 prevented host-derived cells from reconstituting the Treg compartment, leading to Treg deficiency similar to that observed in whole IL-27 tg mice. Accordingly, irradiated WT mice reconstituted with IL-27 tg BM developed systemic inflammation that mirrored the disease in IL-27 tg mice in the spleen and pancreas (Fig. 5C), and these mice succumbed to this condition (Fig. 5D).

Fig. 5
Irradiated WT mice reconstituted with IL-27 tg BM were Treg deficient. Irradiated WT mice reconstituted with IL-27 tg BM had low A. percentages and B. total numbers of both donor (CD45.2) and host (CD45.1) Foxp3+ Treg in the spleen, LN, and thymus at ...

IL-27 tg mice are IL-2 deficient

IL-2 is a vital factor for the proper development of the Treg compartment. In the absence of IL-2, or subunits of its receptor, mice are Treg deficient, and die of systemic inflammation (3439). Thus, the ability of IL-27 to inhibit IL-2 production(32, 33) may be one mechanism by which IL-27 limits the size of the Treg population. To determine whether IL-27 over-expression affects IL-2 production, cells from the spleen, LN, and thymus from WT or IL-27 tg mice were isolated, stimulated for 4 h with PMA and ionomycin, and intracellular cytokine staining was used to assess IL-2 production. While WT CD4+ T cells from all tissues examined produced IL-2, a significantly lower percentage of IL-27 tg CD4+ T cells stained positively for IL-2 (Fig. 6A). The total number of CD4+ T cells in the spleen and LN that produced IL-2 was also greater in WT compared to IL-27 tg mice (Fig. 6B). There was an equivalent number of CD4+IL-2+ cells in the thymus of WT and IL-27 tg mice (Fig. 6B), reflecting an increased percentage of single positive CD4+ T cells in the IL-27 tg thymus. When splenocytes from WT irradiated mice reconstituted with WT or IL-27 tg BM were examined for IL-2 production by intracellular cytokine staining, a greater percentage of host-derived and total CD4+ T cells from WT mice reconstituted with WT BM produced IL-2 compared to those from mice reconstituted with IL-27 tg BM (Fig. 6C). When numbers of donor-derived, host-derived, and total CD4+IL-2+ cells in the spleen were calculated, WT mice reconstituted with WT BM had higher numbers of IL-2 producers in all three categories compared to WT mice reconstituted with IL-27 tg BM (Fig. 6D). Finally, when WT and IL-27 tg splenocytes, or splenocytes from irradiated WT mice reconstituted with WT or IL-27 tg BM, were stimulated with soluble anti-CD3 and anti-CD28, there was a major decrease in the amount of IL-2 in culture supernatants from whole IL-27 tg mice and in WT mice that received IL-27 tg BM compared to that in supernatants from whole WT or WT mice that received WT BM (Fig. 6E). These data suggest that the loss of Treg and the inflammatory disease observed in IL-27 tg mice is associated with an IL-2 deficiency, consistent with the major role of IL-2 in Treg generation and maintenance.

Fig. 6
IL-27 tg mice were IL-2 deficient. Following stimulation, there was a lower A. percentage and B. total number of CD4+ T cells that produced IL-2 from IL-27 tg mice compared to WT mice in the spleen, LN, and thymus (except for total numbers in the thymus). ...

Discussion

The data presented here provide new evidence of a role for IL-27 in modulating the size of the Treg pool in vivo. The mechanisms that control Treg homeostasis have been studied intensively, but many questions still remain regarding how this critical cell population is regulated. Various murine models that lack Treg, including the scurfy Foxp3 mutants(4345), Foxp3−/− mice(46), mice in which Foxp3 can be ablated(50), and IL-2−/− and IL-2Rβ−/− mice(3437, 39) have underscored the strict requirement for Foxp3+ Treg in controlling inflammation and autoimmunity. Comparing the course of disease in different Treg deficient models serves to define how each of these factors influences the Treg pool. For instance, scurfy mutants that lack functional Foxp3 and Foxp3−/− mice have the most severe disease and succumb to multi-organ inflammation prior to weaning, emphasizing that Foxp3 is indispensable for Treg establishment (4346). In contrast, IL-2−/− and IL-2Rβ−/− do possess a Treg population, exhibit unique patterns of organ-specific inflammation, and succumb to disease well after weaning (3437, 39), suggesting that though IL-2 signaling may not be required for Foxp3 upregulation, it is vital for normal Treg homeostasis. The data presented here show that compromised IL-2 production in response to IL-27 signaling is associated with Treg deficiency, yet the phenotype of IL-27 tg mice is different than that of IL-2−/− and IL-2Rβ−/− mice. IL-27 tg mice live slightly longer (Fig. 1A) than IL-2−/− and IL-2Rβ−/− mice, and IL-27 tg mice are lymphopenic (Supplemental Fig. 2B), whereas IL-2−/− and IL-2Rβ−/− mice exhibit lymphoproliferation (35). These differences highlight the complex factors that underlie regulation of the Treg pool. Further examination and comparisons of murine models of Treg deficiency will provide insight into how these cells are regulated during homeostasis.

Previous work provides evidence that IL-27 influences Treg in vivo. IL-27 tg mice have been generated that expressed a fused version of IL-27p28 and EBI3 in the liver, and the phenotype in these mice displayed some similarity to that described in our studies, with pathological changes in the liver and shortened survival. That report concluded that IL-27 over-expression caused potentially lethal BM dysfunction; however, the authors of this study did not examine Foxp3 expression, and the inflammatory phenotype observed in these mice suggests that autoimmune or inappropriate inflammation may be present (53). Additionally, recent work from Cox et al. established a role for IL-27 in limiting Treg populations in vivo in a murine transfer model of colitis. The authors reported that when transferred T cells lacked WSX-1, a larger percentage of transferred cells upregulated Foxp3, thus ameliorating colonic inflammation. The authors hypothesized that increased IL-2 production in the absence of WSX-1 was not responsible for increased Treg frequencies. However, this does not preclude an IL-2-dependent mechanism in our study, as the authors did not assess Treg conversion in the absence of IL-2 (54).

The study of various models of Treg deficiency will be useful in defining mechanisms that contribute to the inflammation that develops in response to a loss of Treg. In particular, study of the IL-2−/− and IL-2Rβ−/− mouse models has led to a better understanding of how IL-2 signaling regulates T cells. Formerly, IL-2 was considered to be an essential T cell growth factor required for the expansion of effector Th populations in vivo (51, 52). However, the fact that IL-2−/− and IL-2Rβ−/− mice exhibit a lymphoproliferative disease showed that IL-2 signaling is not absolutely required to support Th effector expansion or autoimmune and dysregulated inflammation (3439). Likewise, in IL-27 tg mice, while the ability of IL-27 to suppress IL-2 is associated with a Treg deficit, consistent with the development of systemic inflammation, the pro-inflammatory activities of IL-27 may also influence the inflammation that develops. In agreement with initial studies on IL-27, which described the cytokine as a pro-inflammatory factor that enhanced Th1 responses (4, 68), IL-27 tg mice have increased IFN-γ levels (Fig. 2C and Supplemental Fig. 2C), and IL-27 tg T cells were more likely to express the transcription factor T box expressed in T cells (data not shown). Additionally, though IL-2 is not required for T cell expansion, competition for IL-2 in IL-27 tg mice might influence the generation of the T cell response in the inflamed environment. Thus, in this model, IL-27 may not only limit Treg populations, but may also contribute to the Th1-type inflammation and the effector T cell response that is associated with the Treg deficiency.

While our studies of IL-27 tg mice identify a role for over-expression of this cytokine in Treg homeostasis, there is no evidence to date that IL-27 signaling occurs in the thymus and shapes the Treg pool of normal, WT mice. Mice deficient in IL-27 signaling do not exhibit obvious changes in Treg numbers in the steady state, or during inflammation, raising the question of why these cells express the IL-27R (31). Regardless, our data do not elucidate Treg differentiation pathways in the intact thymus; rather, they highlight a mechanism by which the Treg compartment could be manipulated, particularly when the Treg pool is expanding, as in the BM chimera model described here. There are numerous situations in which Treg are deleterious to the initiation or establishment of a desirable immune response, particularly during vaccination or cancer immuno-therapy (5560). In these scenarios, IL-27 therapy delivered systemically or to the local site of the immune response could help to limit Treg and support the generation of the desired immune response. Indeed, IL-27 therapy in a murine model of neuroblastoma limited IL-2-induced Treg expansion in the tumor microenvironment and correlated with enhanced tumor killing (61). While the balance between regulated inflammation and autoimmunity is always a consideration, the studies presented here describe a new mechanism by which the Treg population can be modulated and point to a pathway that could be manipulated therapeutically.

Supplementary Material

Acknowledgments

The authors would like to thank Yasmine Belkaid and Alexander Rudensky for helpful discussion. Foxp3GFP mice were provided by L. A. Turka (University of Pennsylvania), and IL-27p28 and EBI3 tg mice were provided by N. Hosken and S. D. Levin (Zymogenetics, Inc.).

Abbreviations used in this paper

B.D.
below detection
BM
bone marrow
EBI3
Epstein-Barr virus-induced 3
forkhead box P3
Foxp3
LN
lymph node
MDC
macrophage-derived chemokine
nTreg
natural T regulatory cell
N.D
not detected
PEC
peritoneal exudate cell
Treg
T regulatory cell
WT
wild-type

Footnotes

1This work supported by National Institutes of Health grants T32 AI007532, T32 AI055428, RO1 AI071302, and the State of Pennsylvania.

Disclosures

The authors have no competing financial interests.

References

1. Boulay JL, O’Shea JJ, Paul WE. Molecular phylogeny within type I cytokines and their cognate receptors. Immunity. 2003;19:159–163. [PubMed]
2. Hunter CA. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nat Rev Immunol. 2005;7:521–531. [PubMed]
3. Kastelein RA, Hunter CA, Cua DJ. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu Rev Immunol. 2007;25:221–242. [PubMed]
4. Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, Hibbert L, Churakova T, Travis M, Vaisberg E, Blumenschein WM, Mattson JD, Wagner JL, To W, Zurawski S, McClanahan TK, Gorman DM, Bazan JF, de Waal Malefyt R, Rennick D, Kastelein RA. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+ T cells. Immunity. 2002;16:779–790. [PubMed]
5. Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, Phillips JH, McClanahan TK, de Waal Malefyt R, Kastelein RA. WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27. J Immunol. 2004;172:2225–2231. [PubMed]
6. Hibbert L, Pflanz S, de Waal Malefyt R, Kastelein RA. IL-27 and IFN-α signal via Stat1 and Stat3 and induce T-Bet and IL-12Rβ2 in naive T cells. J Interferon Cytokine Res. 2003;23:513–522. [PubMed]
7. Lucas S, Ghilardi N, Li J, de Sauvage FJ. IL-27 regulates IL-12 responsiveness of naive CD4+ T cells through Stat1-dependent and -independent mechanisms. Proc Natl Acad Sci U S A. 2003;100:15047–15052. [PMC free article] [PubMed]
8. Takeda A, Hamano S, Yamanaka A, Hanada T, Ishibashi T, Mak TW, Yoshimura A, Yoshida H. Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment. J Immunol. 2003;170:4886–4890. [PubMed]
9. Villarino A, Hibbert L, Lieberman L, Wilson E, Mak T, Yoshida H, Kastelien RA, Hunter CA. The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity. 2003;19:645–655. [PubMed]
10. Hamano S, Himeno K, Miyazaki Y, Ishii K, Yamanaka A, Takeda A, Zhang M, Hisaeda H, Mak TW, Yoshimura A, Yoshida H. WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production. Immunity. 2003;19:657–667. [PubMed]
11. Rosas LE, Satoskar AA, Roth KM, Keiser TL, Barbi J, Hunter C, de Sauvage FJ, Satoskar AR. Interleukin-27R (WSX-1/T-cell cytokine receptor) gene-deficient mice display enhanced resistance to Leishmania donovani infection but develop severe liver immunopathology. Am J Pathol. 2006;168:158–169. [PMC free article] [PubMed]
12. Wirtz S, Tubbe I, Galle PR, Schild HJ, Birkenbach M, Blumberg RS, Neurath MF. Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27. J Exp Med. 2006;203:1875–1881. [PMC free article] [PubMed]
13. Sonoda KH, Yoshimura T, Takeda A, Ishibashi T, Hamano S, Yoshida H. WSX-1 plays a significant role for the initiation of experimental autoimmune uveitis. Int Immunol. 2007;19:93–98. [PubMed]
14. Artis D, Villarino A, Silverman M, He W, Thornton EM, Mu S, Summer S, Covey TM, Huang E, Yoshida H, Koretzky G, Goldschmidt M, Wu GD, de Sauvage F, Miller HR, Saris CJ, Scott P, Hunter CA. The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity. J Immunol. 2004;173:5626–5634. [PubMed]
15. Miyazaki Y, Inoue H, Matsumura M, Matsumoto K, Nakano T, Tsuda M, Hamano S, Yoshimura A, Yoshida H. Exacerbation of experimental allergic asthma by augmented Th2 responses in WSX-1-deficient mice. J Immunol. 2005;175:2401–2407. [PubMed]
16. Shimizu S, Sugiyama N, Masutani K, Sadanaga A, Miyazaki Y, Inoue Y, Akahoshi M, Katafuchi R, Hirakata H, Harada M, Hamano S, Nakashima H, Yoshida H. Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1) J Immunol. 2005;175:7185–7192. [PubMed]
17. Batten M, Li J, Yi S, Kljavin NM, Danilenko DM, Lucas S, Lee J, de Sauvage FJ, Ghilardi N. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat Immunol. 2006;7:929–936. [PubMed]
18. Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson LM, Villarino AV, Huang Q, Yoshimura A, Sehy D, Saris CJ, O’Shea JJ, Hennighausen L, Ernst M, Hunter CA. Interleukin 27 regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol. 2006;7:937–945. [PubMed]
19. Fitzgerald DC, Ciric B, Touil T, Harle H, Grammatikopolou J, Das Sarma J, Gran B, Zhang GX, Rostami A. Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells. Nat Immunol. 2007;8:1372–1379. [PubMed]
20. Bluestone JA, Tang Q. How do CD4+CD25+ regulatory T cells control autoimmunity? Curr Opin Immunol. 2005;17:638–642. [PubMed]
21. Miyara M, Sakaguchi S. Natural regulatory T cells: mechanisms of suppression. Trends Mol Med. 2007;13:108–16. [PubMed]
22. Wing K, Sakaguchi S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol. 2010;11:7–13. [PubMed]
23. Belkaid Y, Oldenhove G. Tuning microenvironments: induction of regulatory T cells by dendritic cells. Immunity. 2008;29:362–371. [PMC free article] [PubMed]
24. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25 naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med. 2003;198:1875–1886. [PMC free article] [PubMed]
25. Davidson TS, Dipaolo RJ, Andersson J, Shevach EM. Cutting Edge: IL-2 is essential for TGF-β-mediated induction of Foxp3+ T regulatory cells. J Immunol. 2007;178:4022–4026. [PubMed]
26. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity. 2005;22:329–341. [PubMed]
27. Kim JM, Rudensky A. The role of the transcription factor Foxp3 in the development of regulatory T cells. Immunol Rev. 2006;212:86–98. [PubMed]
28. Neufert C, Becker C, Wirtz S, Fantini MC, Weigmann B, Galle PR, Neurath MF. IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1. Eur J Immunol. 2007;37:1809–1816. [PubMed]
29. Stumhofer JS, Silver JS, Laurence A, Porrett PM, Harris TH, Turka LA, Ernst M, Saris CJ, O’Shea JJ, Hunter CA. Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. Nat Immunol. 2007;8:1363–1371. [PubMed]
30. Huber M, Steinwald V, Guralnik A, Brustle A, Kleemann P, Rosenplanter C, Decker T, Lohoff M. IL-27 inhibits the development of regulatory T cells via STAT3. Int Immunol. 2008;20:223–234. [PubMed]
31. Villarino AV, Larkin J, 3rd, Saris CJ, Caton AJ, Lucas S, Wong T, de Sauvage FJ, Hunter CA. Positive and negative regulation of the IL-27 receptor during lymphoid cell activation. J Immunol. 2005;174:7684–7691. [PubMed]
32. Owaki T, Asakawa M, Kamiya S, Takeda K, Fukai F, Mizuguchi J, Yoshimoto T. IL-27 suppresses CD28-mediated IL-2 production through suppressor of cytokine signaling 3. J Immunol. 2006;176:2773–2780. [PubMed]
33. Villarino AV, Stumhofer JS, Saris CJ, Kastelein RA, de Sauvage FJ, Hunter CA. IL-27 limits IL-2 production during Th1 differentiation. J Immunol. 2006;176:237–247. [PubMed]
34. Almeida AR, Legrand N, Papiernik M, Freitas AA. Homeostasis of peripheral CD4+ T cells: IL-2Rα and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J Immunol. 2002;169:4850–4860. [PubMed]
35. Malek TR, Yu A, Vincek V, Scibelli P, Kong L. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rβ-deficient mice. Implications for the nonredundant function of IL-2. Immunity. 2002;17:167–178. [PubMed]
36. de la Rosa M, Rutz S, Dorninger H, Scheffold A. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur J Immunol. 2004;34:2480–2488. [PubMed]
37. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol. 2005;6:1142–1151. [PubMed]
38. Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med. 2005;201:723–735. [PMC free article] [PubMed]
39. Yu A, Malek TR. Selective availability of IL-2 is a major determinant controlling the production of CD4+CD25+Foxp3+ T regulatory cells. J Immunol. 2006;177:5115–5121. [PubMed]
40. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. [PubMed]
41. Iritani BM, Forbush KA, Farrar MA, Perlmutter RM. Control of B cell development by Ras-mediated activation of Raf. EMBO J. 1997;16:7019–7031. [PMC free article] [PubMed]
42. Hogan B, Beddington R, Constantini F, Lacy E. Manipulating the Mouse Embryo. A Laboratory Manual. 2. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 1994.
43. Godfrey VL, Wilkinson JE, Russell LB. X-linked lymphoreticular disease in the scurfy (sf) mutant mouse. Am J Pathol. 1991;138:1379–1387. [PMC free article] [PubMed]
44. Clark LB, Appleby MW, Brunkow ME, Wilkinson JE, Ziegler SF, Ramsdell F. Cellular and molecular characterization of the scurfy mouse mutant. J Immunol. 1999;162:2546–2554. [PubMed]
45. Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, Wilkinson JE, Galas D, Ziegler SF, 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. [PubMed]
46. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4:330–336. [PubMed]
47. Yang XO, Nurieva R, Martinez GJ, Kang HS, Chung Y, Pappu BP, Shah B, Chang SH, Schluns KS, Watowich SS, Feng XH, Jetten AM, Dong C. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity. 2008;29:44–56. [PMC free article] [PubMed]
48. Rivera A, Chen CC, Dougherty JP, Ben-Nun A, Ron Y. Host stem cells can selectively reconstitute missing lymphoid lineages in irradiation BM chimeras. Blood. 2003;101:4347–4354. [PubMed]
49. Komatsu N, Hori S. Full restoration of peripheral Foxp3+ regulatory T cell pool by radioresistant host cells in scurfy BM chimeras. Proc Natl Acad Sci U S A. 2007;104:8959–8964. [PMC free article] [PubMed]
50. Kim JM, Ramsussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol. 2007;8:191–197. [PubMed]
51. Hoyer KK, Dooms H, Barron L, Abbas AK. Interleukin-2 in the development and control of inflammatory disease. Immunol Rev. 2008;226:19–28. [PubMed]
52. Malek TR. The biology of interleukin-2. Annu Rev Immunol. 2008;26:453–479. [PubMed]
53. Seita J, Asakawa M, Ooehara J, Takayanagi S, Morita Y, Watanabe N, Fujita K, Kudo M, Mizuguchi J, Ema H, Nakauchi H, Yoshimoto T. Interleukin-27 directly induces differentiation in hematopoietic stem cells. Blood. 2008;111:1903–1912. [PubMed]
54. Cox JH, Kljavin NM, Ramamoorthi N, Diehl L, Batten M, Ghilardi N. IL-27 promotes T cell-dependent colitis through multiples mechanisms. J Exp Med. 2010;208:115–123. [PMC free article] [PubMed]
55. Shimizu J, Yamazaki S, Sakaguchi S. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J Immunol. 1999;163:5211–5218. [PubMed]
56. Dannull J, Su Z, Rizzieri D, Yang BK, Coleman D, Yancey D, Zhang A, Dahm P, Chao N, Gilboa E, Vieweg J. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest. 2005;115:3623–3633. [PMC free article] [PubMed]
57. Sakaguchi S, Powrie F. Emerging challenges in regulatory T cell function and biology. Science. 2007;317:627–629. [PubMed]
58. Yamaguchi T, Hirota K, Nagahama K, Ohkawa K, Takahashi T, Nomura T, Sakaguchi S. Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity. 2007;27:145–159. [PubMed]
59. Jaron B, Maranghi E, Leclerc C, Majlessi L. Effect of attenuation of Treg during BCG immunization on anti-mycobacterial Th1 responses and protection against Mycobacterium tuberculosis. PLoS One. 2008;3:e2833. [PMC free article] [PubMed]
60. Ruter J, Barnett BG, Kryczek I, Brumlik MJ, Daniel BJ, Coukos G, Zou W, Curiel TJ. Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines. Front Biosci. 2009;14:1761–1770. [PubMed]
61. Salcedo R, Hixon JA, Stauffer JK, Jalah R, Brooks AD, Khan T, Dai RM, Scheetz L, Lincoln E, Back TC, Powell D, Hurwitz AA, Sayers TJ, Kastelein R, Pavlakis GN, Felber BK, Trinchieri G, Wigginton JM. Immunologic and therapeutic synergy of IL-27 and IL-2: enhancement of T cell sensitization, tumor-specific CTL reactivity and complete regression of disseminated neuroblastoma metastases in the liver and BM. J Immunol. 2009;182:4328–4338. [PMC free article] [PubMed]
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