Suppression of Murine Allergic Airway Disease by IL-2: Anti-IL-2 ...

The Journal of Immunology

Suppression of Murine Allergic Airway Disease by IL-2:Anti-IL-2 Monoclonal Antibody-Induced Regulatory T Cells1 Mark S. Wilson,* John T. Pesce,* Thirumalai R. Ramalingam,* Robert W. Thompson,* Allen Cheever,† and Thomas A. Wynn2* Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10ⴙCD4ⴙCD25ⴙ and Foxp3ⴙCD4ⴙCD25ⴙ) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10ⴙ/ⴙ, but not IL-10ⴚ/ⴚ Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease. The Journal of Immunology, 2008, 181: 6942– 6954.

A

breakdown in immunological tolerance can give rise to T cell-mediated syndromes including autoimmune (1–3) and allergic diseases (4 – 8). Endogenous regulatory T cells (Tregs)3 are a crucial T cell compartment, maintaining peripheral tolerance by suppressing autoreactive T cell responses (reviewed in Ref. 9) and orchestrating a balanced immune response to foreign Ags (reviewed in Refs. 4, 7, 8, 10, and 11). Dysfunctional Tregs have been identified in allergic individuals (5, 12) and glucocorticoid-resistant patients (13), implying that this defect contributes to the development of atopy and subsequent allergic disorders. Successful immunotherapy and treatment of allergic individuals often correlate with an increase in Tregs (14 – 16), supporting the notion that Tregs are central regulators of allergic reactivity. Furthermore, several murine studies illustrate a significant contribution by Tregs in restraining pulmonary inflam-

*Immunopathogensis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and †Biomedical Research Institute, Rockville, MD 20852 Received for publication June 11, 2008. Accepted for publication September 12, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.

2

Address correspondence and reprint requests to Dr. Thomas A. Wynn, Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, 50 South Drive, Room 6154, Mail Stop Code 8003, Bethesda, MD 20892. E-mail address: [email protected]

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Abbreviations used in this paper: Treg, regulatory T cell; ABPAS, Alcian blueperiodic acid Schiff; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; rh, recombinant human; rm, recombinant murine; SEA, soluble egg antigen from S. mansoni; S:S, SEA sensitization; S:S:S, SEA sensitization, challenge, and further challenge; tiger, IL-ten ires gfp-enhanced reporter; WT, wild type.

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mation and preventing immune-mediated pathology following exposure to aeroallergens. For example, depleting CD25⫹ Tregs by using PC61 Ab (17) converted a usually unresponsive strain, C3H/ HeJ, to a responsive phenotype following airway allergen challenge. Adoptive transfer of Tregs (18 –23) into allergen-sensitized animals also reduced airway inflammation and pathology, revealing a similar function for Tregs. Recent studies demonstrated that although IL-2 is not required for thymic Treg development, it is essential for optimal extrathymic Treg homeostasis (24 –29). These studies tie together observations made in IL-2⫺/⫺ mice (30) and endogenous Treg-deficient (Foxp3⫺/⫺) mice (31), both of which succumb to hyperproliferative autoimmune disorders. Thus, although IL-2 was previously considered a pan-T cell growth factor, contrasting functions are emerging, with IL-2 possibly playing a more critical role in tolerance via the maintenance of Treg populations (29, 32–34). In the present study, we coupled two observations, Treg dependence on IL-2 and Treg-mediated control of airway allergy, and asked whether supplementing exogenous IL-2 could be used to preferentially expand endogenous Treg cells and inhibit allergic inflammation and airway hyperreactivity. Using several airway allergy systems, we also examined whether IL-2 in complex with anti-IL-2 mAb could boost CD4⫹ Treg frequencies (35), with the aim of suppressing allergen-induced airway inflammation through Treg expansion. We demonstrate that rIL-2 exacerbates airway inflammation; however, IL-2 administered as a complex with anti-IL-2 mAb considerably reduced airway inflammation and hyperreactivity. Whether IL-2:anti-IL-2 complexes were administered before airway challenge or therapeutically after airway inflammation, a significant reduction in airway pathologies was observed. Both natural (Foxp3⫹) and


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FIGURE 1. rhIL-2 treatment before allergen sensitization has little affect on airway inflammation, whereas rhIL-2 treatment after allergen sensitization exacerbates airway inflammation. Mice were sensitized and challenged. Groups of mice were treated with low (2,500 U) or high (25,000 U) doses of IL-2 either pre-sensitization (A–D) or post-sensitization (E–H). Animals were sacrificed 24 h after the final airway challenge. Data shown are from one of three experiments. d, Day; i.t., intratracheal; S1, sensitized 1; S2, sensitized 2; C1, challenge 1; C2, challenge 2. A, Total airway infiltrates and airway eosinophilia recovered in BAL. B, BAL fluid IL-5 and eotaxin-1 measured by ELISA. C, Five-micrometer sections were cut from paraffin-embedded

6944 inducible (IL-10gfp⫹) Treg populations increased following IL2:anti-IL-2 treatment, and through the use of reconstituted RAG2⫺/⫺ mice we demonstrate that IL-10-producing Tregs are a critical population regulating airway allergy following IL-2: anti-IL-2 treatment.

Materials and Methods Animals Female BALB/c, BALB/c RAG 2⫺/⫺, BALB/c IL-10⫺/⫺, C57BL/6, and C57BL/6 IL-10⫺/⫺ mice 6- to 8-wk old were obtained from National Institute of Allergy and Infectious Diseases (NIAID) facilities at Taconic. IL-10gfp reporter mice designated as tiger (IL-ten ires gfp-enhanced reporter; where ires is internal ribosomal entry site) were generated by Kamanaka and colleagues (36) and bred as homozygotes for the transgene. IL-10gfp reporter mice (tiger) and Foxp3rfpIL-10gfp were kindly provided by Dr. Richard Flavell (Yale University, New Haven, CT). CD4Cre/⫺ STAT5flox mice were kindly provided by Dr. Arian Laurence (NIAID, National Institutes of Health (NIH), Bethesda, MD) and Foxp3gfp reporter mice were provided by Dr. N. Peters (NIAID, NIH), originally generated by Bettelli and colleagues (37). All animals were housed under specific pathogen-free conditions at the NIH in an American Association for the Accreditation of Laboratory Animal Care-approved facility. The NIAID animal care and use committee approved all experimental procedures. A minimum of five mice per group was used in each experiment, unless indicated in the figure legends.

Reagents Soluble Schistosoma mansoni egg Ag (SEA) was prepared from sterile LPS-free eggs isolated from the livers of infected mice. Recombinant human (rh)IL-2 was obtained from National Cancer Institute (NCI) Preclinical Repository. Recombinant murine (rm)IL-2 and anti-mouse IL-2 (clone JES6-1A12) were purchased from eBioscience with isotype control (rat IgG2a) purchased from BD Pharmingen. IL-2:anti-IL-2 complexes were prepared at room temperature with a 1:10 ratio of IL-2:anti-IL-2 (2.5 ␮g of rmIL-2 and 25 ␮g of anti-IL-2) and diluted in sterile PBS for i.p. delivery.

Allergen-induced airway inflammation Mice were immunized by i.p. inoculation with 10 ␮g of SEA from S. mansoni in PBS or with 10 ␮g of chicken egg OVA (Sigma-Aldrich) emulsified in alum (Imject alum; Pierce) and boosted again with 10 ␮g of Ag on day 14. On days 28 and 31 mice were anesthetized with a mixture of xylazine and ketamine and given an intratracheal airway challenge with 10 ␮g of SEA in PBS or 10 ␮g of OVA in PBS, respectively. Mice were killed 24 h after the final airway challenge (day 32) to assess airway inflammation and airway hyperresponsiveness (AHR), with OVA and SEA sensitization and challenge designated as O:O and S:S, respectively. For established airway inflammation model, mice were sensitized and challenged as above, with additional airway challenges given on days 39 and 42, with mice analyzed on day 43. Established allergy model is depicted as sensitization, challenge, and further challenge (S:S:S).

Bronchoalveolar lavage (BAL) fluid and differential cell counts Twenty-four hours after the final challenge, mice were anesthetized with sodium pentobarbital. The trachea was cannulated and airspaces were lavaged with an initial 500 ␮l of sterile PBS, followed by two 500-␮l PBS washes. Fluids were centrifuged at 1200 ⫻ g, and pellets recovered from all three lavage washes were pooled into 1 ml of PBS for “total BAL cell” counts and cellular analysis. The supernatants of the initial 500 ␮l of BALF were stored at ⫺80°C for biochemical analyses. Cytospins were prepared by spinning 5 ⫻ 105 cells onto poly-L-lysine-coated slides and fixing them with methanol followed by Diff-Quik (Boehringer) staining. Differential cell counts were performed at ⫻100 magnification; a minimum of 200 cells were counted for each slide.

Airway hyperresponsiveness AHR was measured 24 h after the final SEA or OVA challenge by whole body plethysmography (Buxco Electronics) in response to inhaled metha-

IL-2:ANTI-IL-2-DRIVEN Tregs choline (Sigma-Aldrich). Mice were exposed to increasing doses of methacholine (3–50 mg/ml for 2 min) and measured for 5 min following dosing, as previously described (38, 39).

ELISA Cytokines were measured by ELISA using suppliers’ guidelines. Capture (2 ␮g/ml) and biotinylated detection Abs (2 ␮g/ml) for IL-5 were from BD Pharmingen. Capture Abs (0.4 ␮g/ml) and biotinylated detection Abs (0.4 ␮g/ml) for eotaxin-1 were from R&D Systems. In vivo serum IL-5 was measured according to Ref. 40. Immunosorbent plates were washed with 0.05% Tween 20 in PBS (PBST) and blocked with 5% milk in PBST. Assay standard diluted in PBST plus 1% BSA were added for 2 h at 37°C. The biotinylated detection Ab was added for 2 h at 37°C. Peroxidaselabeled streptavidin (1/1000 for 1 h at 37°C) and ABTS peroxidase substrate (both from Kirkegaard & Perry Laboratories) were used to detect biotinylated Ab. OD was read at 405 nm in an ELISA reader (Molecular Devices).

Flow cytometry Cells were stained with Abs diluted in PBS with 0.5% BSA (Sigma-Aldrich) and 0.05% sodium azide (Sigma-Aldrich) for 20 min at 4°C. For detection of CD4⫹CD25⫹IL-10gfp⫹ cells, monoclonal rat anti-mouse CD4 (L3T4, clone RM4 –5; isotype Rat IgG2a) and rat anti-mouse-CD25 (PC61; isotype Rat IgG1) were used. For detection of intracellular IL-13, cells were stimulated with PMA and ionomycin in the presence of brefeldin A for 3 h, after which cells were surface stained for CD4 and/or CD25, permeabilized in Cytofix/Cytoperm (BD Pharmingen), washed in Perm/ Wash buffer (BD Pharmingen) according to the manufacturer’s recommendations, and stained with PE-labeled rat anti-mouse IL-13 (unlabeled Ab provided by Dr. L. Li, Centocor). Intracellular Foxp3 was detected following the manufacturer’s recommendations using PE anti-mouse/rat Foxp3 staining kit (eBioscience). IL-10gfp reporter mice designated as tiger allowed us to identify IL-10-producing cells ex vivo without subsequent manipulation. For cell division assays, CFSE (2.5 ␮M/1 ⫻ 106 cells in 1 ml) was used to label parent cells at room temperature for 15 min before culture. CFSE-labeled cells were cultured with SEA (10 ␮g/ml) or Con A (1 ␮g/ml) for 5 days at 37°C. The expression of surface markers and intracellular cytokines were analyzed on a FACSCalibur flow cytometer using FlowJo software (Tree Star).

Histopathology Formalin (4% paraformaldehyde in PBS)-fixed lungs were processed and embedded in paraffin for sectioning (Histopath of America). H&E stains or Giemsa stains were used for analysis of airway inflammation and pathological changes. Perivascular and peribronchial inflammation evaluations were scored by a blinded observer on an arbitrary 1– 4⫹ basis. Goblet cells were stained with Alcian blue-periodic acid Schiff (ABPAS) with positive bronchial epithelial cells also scored on an arbitrary 1– 4⫹ basis.

RNA isolation and purification and real-time PCR Total RNA was extracted from lung tissue in 1 ml of TRIzol reagent (Invitrogen). The samples were homogenized using a tissue Polytron device (Omni International), and total RNA was extracted according to the recommendations of the manufacturer and further purified using RNeasy Mini kit from Qiagen. Individual sample RNA (1 ␮g) was reverse transcribed using Superscript II reverse transcriptase (Invitrogen Corp.) and a mixture of oligo(dT) and random primers. Real-time RT-PCR was performed on an ABI Prism 7900HT sequence detection system (Applied Biosystems). Relative quantities of mRNA for several genes were determined using SYBR Green PCR Master Mix (Applied Biosystems) and the comparative threshold cycle method as described by Applied Biosystems for the ABI Prism 7700/7900HT sequence detection systems. In this method, mRNA levels for each sample were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) levels and then expressed as a relative increase or decrease compared with levels in naive controls. All primers have previously been described (38).

lung tissue and stained with H&E to show cellular infiltration. five lungs from each group were scored in a blinded fashion and are shown at ⫻10 magnification. D, Lung tissue mRNA for Gob5. E, Total airway infiltrates and airway eosinophilia recovered in BAL. F, BAL fluid IL-5 and eotaxin-1 measured by ELISA. G, Five-micrometer sections were cut from paraffin-embedded lung tissue and stained with H&E to show cellular infiltration. five lungs from each group were scored in a blinded fashion and are shown at ⫻10 magnification. H, Lung tissue mRNA for Gob5. ⴱ, p ⱕ 0.05.


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FIGURE 2. rmIL-2 in complex with anti-mouse IL-2 mAb (JES6-1) significantly reduces airway inflammation and AHR. Mice were sensitized and challenged as in Fig. 1. Groups of mice were treated with either rmIL-2 (2.5 ␮g), anti-IL-2 mAb (25 ␮g), isotype control (25 ␮g), or a complex of rmIL-2 (2.5 ␮g):␣IL-2m Ab (25 ␮g) 7 days before the first airway challenge. Mice were sacrificed 24 h after the final airway challenge. Data shown is one of four experiments. A, Total airway infiltrates recovered in BAL B, BAL eosinophilia. C, BAL fluid IL-5 measured by ELISA. D, BAL fluid eotaxin-1 measured by ELISA. E, Five-micrometer sections were cut from paraffin-embedded lung tissue and stained with Giemsa (left panels, ⫻10 magnification) to show cellular infiltration with parallel sections or stained with ABPAS (right panels, ⫻40 magnification) to show mucin⫹ goblet cells lining the airways. Five lungs from each group were scored in a blinded fashion. F, Lung tissue mRNA for IL-13 and BAL cell intracellular cytokine staining, showing CD4⫹ lymphocytes stained with FITC-labeled anti-IL-13 and allophycocyanin-labeled anti-IL-5 following a 3-h stimulation with PMA and ionomycin in the presence of brefeldin A. G, AHR measured in C57BL/6 (left) and BALB/c (right) mice following S:S and airway challenge with and without IL-2:anti-IL-2 treatment. Enhanced pause (Penh) measurements were made 24 h after the final airway challenge using a Buxco Electronics system with mice exposed to increasing doses of methacholine. One of three representative experiments is shown. ⴱ, p ⱕ 0.05.

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IL-2:ANTI-IL-2-DRIVEN Tregs

FIGURE 3. rmIL-2:anti-mouse IL2 mAb treatment significantly reduces established airway inflammation and AHR. Animals were sensitized with 10 ␮g of SEA on day 1 and day 14 followed by two intratracheal airway challenges with 10 ␮g of SEA on days 28 and 31, designated S:S. Additional groups of mice received either PBS or a complex of rmIL-2 (2.5 ␮g):anti-IL-2m Ab (25 ␮g) 3 days before the final airway challenges (days 39 and 42), designated S:S:S. Animals were sacrificed 24 h after the final airway challenge (day 43). A, Total airway infiltrates recovered in BAL. B, BAL eosinophilia. C, BAL fluid IL-5 levels measured by ELISA. D, BAL fluid eotaxin-1 levels measured by ELISA. E, Five-micrometer sections were cut from paraffin-embedded lung tissue and stained with H&E (left panels, ⫻10 magnification) to show cellular infiltration with parallel sections stained with ABPAS (right panels, ⫻40 magnification) showing mucin⫹ goblet cells lining the airways. Five lungs from each group were scored in a blinded fashion. F, AHR). Enhanced pause (Penh) measurements were made 24 h after the final airway challenge using a Buxco Electronics system with mice exposed to increasing doses of methacholine. ⴱ, p ⱕ 0.05.

Statistical analysis Data sets were compared by Mann-Whitney U test or one-way ANOVA as specified in the figure legends. Differences were considered significant at p ⱕ 0.05.

Results

Unlike pre-treatment, which had little impact on airway parameters (Fig. 1, A–D), IL-2 treatment of allergen-sensitized mice increased airway eosinophilia (Fig. 1E), tissue inflammation (Fig. 1G), and Gob 5 gene expression (Fig. 1H). Thus, the initial hypothesis proved incorrect, with rIL-2 exacerbating rather than reducing airway inflammation.

SEA-induced airway disease is exacerbated with rIL-2 treatment A robust and reliable model of airway inflammation and hyperresponsiveness, using SEA as an experimental allergen, was established in C57BL/6 mice (supplemental figure 1).4 To test whether rIL-2 could expand Tregs and modify airway inflammation, we pre-treated mice (Fig. 1; treatment regimen A, top left) or treated allergen-sensitized mice (Fig. 1; treatment regimen B, top right) with rIL-2 at either a high (25,000 IU) or low (2,500 IU) dose. 4

The online version of this article contains supplemental material.

IL-2 complexed with anti-IL-2 mAb suppresses allergic airway disease Boyman and colleagues (35) recently described the expansion of Foxp3⫹ Treg cells with rmIL-2 complexed with a specific anti-mouse IL-2 mAb (clone JES6-1). Building upon their observations, we cultured splenocytes from naive Foxp3gfp reporter mice with IL-2, anti-IL-2, or a complex of IL-2 and antiIL-2 in the presence or absence of stimulatory conditions (antiCD3 and anti-CD28). We confirmed the findings of Boyman


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FIGURE 4. IL-2:anti-mouse IL-2 mAb treatment increases CD4⫹CD25⫹IL-10⫹ and CD4⫹CD25⫹Foxp3⫹ T cells in the airways and lung following airway challenge. BAL and Lung cells were recovered from C57BL/6 tiger IL-10gfp reporter mice 24 h after the final airway challenge of SEA-sensitized mice with or without IL-2:anti-IL-2 treatment, as in Fig. 3. Cells were stained with anti-CD4, anti-CD25, and intracellular Foxp3 as designated before acquisition and analysis by FACS. A, BAL and lung cells stained with anti-mouse CD4 and shown as CD4 vs IL-10gfp. B, BAL and lung cells stained with anti-mouse CD4 and anti-mouse CD25; data shown is gated on CD4⫹ lymphocytes. C, BAL and lung cells stained with anti-mouse CD4, anti-mouse CD25, and anti-mouse/rat Fox p3. Data shown is gated on CD4⫹ lymphocytes. D, Lung cells recovered from Foxp3rfpIL-10gfp dual reporter mice showing either Foxp3rfp⫹ cells stained with anti-CD103⫹ showing IL-10gfp or Foxp3⫺ cells stained with anti-CD103⫹ showing IL-10gfp. Data shown are gated on CD4⫹ lymphocytes. E, Lung tissue mRNA for Foxp3. F, Lung tissue mRNA for IL-10. ⴱ, p ⱕ 0.05.

(35) and also observed optimal expansion of CD4⫹CD25⫹ Foxp3⫹ lymphocytes after 48 (data not shown) or 72 h of culture with 300 ng/ml IL-2 and 3 ␮g/ml anti-IL-2 (supplemental figure 2A) or simply by adding 3 ␮g/ml anti-IL-2 when cells were stimulated with anti-CD3 and anti-CD28. To test whether the expansion of Tregs was dependent upon IL-2 signaling via

STAT5, splenocytes were taken from mice with a specific deletion of STAT5 in CD4⫹ cells (CD4Cre/⫺ STAT5flox). IL-2 in complex with anti-IL-2 increased CD4⫹Foxp3⫹ cells from 7.02 to 12.3% in wild-type (WT) splenocytes, whereas Tregs from CD4Cre/⫺STAT5flox mice failed to expand under similar conditions (supplemental figure 2B).

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We therefore adopted a regimen using an optimal ratio of IL-2 (2.5 ␮g) to anti-mouse IL-2 (25 ␮g) and administered this complex to allergen-sensitized mice 7 days before airway challenge (day 21). Airway infiltration was reduced in IL-2:anti-IL-2-treated mice (Fig. 2A), with significant reductions in BAL fluid eosinophilia (Fig. 2B) and in eosinophil growth and chemotactic factors, IL5 (Fig. 2C) and eotaxin-1 (Fig. 2D), respectively. Treatment with PBS, rmIL-2 alone, anti-mouse IL-2 alone, or an isotype control had no significant impact on airway infiltration. Broncho-vascular inflammation as well as mucus-producing goblet cells were significantly reduced in IL-2:anti-IL-2-treated mice (Fig. 2E). One explanation for reduced mucus production was the suppressed IL13⫹IL-5⫹ CD4 lymphocytes and IL-13mRNA (Fig. 2F) in lung tissue of mice following IL-2:anti-IL-2 treatment compared with untreated mice. Because IL-13 also contributes to the development of AHR, we tested whether IL-2:anti-IL-2 treatment influenced airway responsiveness to a cholinergic stimulant. SEA-sensitized and -challenged mice, with or without IL-2:anti-IL-2 treatment, were exposed to increasing concentrations of methacholine with AHR measurements recorded. SEA-challenged C57BL/6 and BALB/c mice developed significant AHR; however both strains treated with IL-2:anti-IL-2 had significantly lower AHR (Fig. 2G). Reduced airway eosinophilia, goblet cell hyperplasia, and AHR following IL-2:anti-IL-2 treatment were observed using a second experimental allergen (chicken egg OVA) with BALB/c mice (supplemental figure 3), corroborating the data obtained using SEA in C57BL/6 and BALB/c mice. Thus, IL-2:anti-IL-2 treatment proved to be a successful strategy to reduce airway inflammation and AHR in two mouse strains and with two different allergens. IL-2:anti-IL-2 treatment is effective in established airway disease To test the therapeutic potential of IL-2:anti-IL-2 treatment, we adopted a model of established airway inflammation. Briefly, following two primary airway challenges (days 28 and 31) of allergen-sensitized animals, IL-2:anti-IL-2 was administered (day 36) before re-exposure to two further challenges (days 39 and 42). Mice were analyzed for airway inflammation and lung function on day 43, 24 h after the final challenge. Analysis of mice 12 days after the primary airway challenges without subsequent manipulation (S:S) revealed measurable airway eosinophils, IL-5, and eotaxin-1 in BAL recoveries, compared with PBS-challenged mice (Fig. 3, A–D). A second challenge (S:S:S; days 39 and 42) invoked an influx of airway eosinophils accompanied by significant BAL fluid IL-5 and eotaxin-1 secretions (Fig. 3, A–D). Therapeutic administration of IL-2:anti-IL-2 before the second airway challenge (day 36) significantly abrogated subsequent airway and tissue inflammation (Fig. 3E). Inflammatory foci as well as goblet cell mucus and airway mucus plugging observed in tissue sections were also reduced in IL-2:anti-IL-2-treated mice (Fig. 3E). The prevention of subsequent airway infiltration and excess mucus production also corresponded with reduced AHR (Fig. 3F), indicating that IL-2:anti-IL-2 has significant therapeutic potential. IL-2:anti-IL-2 complexes increase the frequency of CD4⫹Foxp3⫹IL-10⫹ T cells Many markers of Tregs have been used to identify, purify, and characterize populations of T cells with regulatory and suppressive functions. In particular, naturally occurring Foxp3⫹ Tregs and inducible IL-10-producing Tregs (Tr1 cells) have received significant attention as regulators of airway allergy (4, 19). To identify a testable mechanism, we monitored the effects of IL-2:anti-IL-2 treatment on both naturally occurring (Foxp3⫹) and inducible (IL10-producing) Treg responses, with intracellular staining for

FIGURE 5. IL-2: Anti-IL-2 mAb mediated suppression is dependent upon IL-10. WT and IL-10⫺/⫺ mice were sensitized and challenged as in Fig. 1. Groups of mice were treated with a complex of rmIL-2(2.5 ␮g): anti-IL-2m Ab (25 ␮g) 7 days before the first airway challenge. Animals were sacrificed 24 h after the final airway challenge. A, Total airway infiltrates recovered in BAL of WT and IL-10⫺/⫺ mice following airway challenge. B, BAL eosinophilia. C, Peribronchial/vascular inflammation scored from H&E-stained lung sections. Five lungs from each group were scored in a blinded fashion. D, ABPAS⫹ cell score taken from 5 ␮m sections of paraffin embedded lung tissue stained with ABPAS. Five lungs from each group were scored in a blinded fashion. ⴱ, p ⱕ 0.05.

Foxp3 and gfp expression using C57BL/6-IL-10gfp tiger reporter mice (36). Following SEA challenge, IL-10-producing cells were predominantly found within CD4⫹ lymphocyte populations in the


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FIGURE 6. In vivo treatment of allergic mice with IL-2:anti-IL-2 mAb promotes ex vivo polyclonal CD4⫹Foxp3⫹ cell expansion and reduces CD4⫹Foxp3⫺ cell proliferation in an IL-10-dependent manner. WT and IL-10⫺/⫺ mice were sensitized and challenged as in Fig. 5. Groups of mice were treated with a complex of rmIL-2 (2.5 ␮g):anti-IL-2m Ab (25 ␮g) 7 days before the first airway challenge. Mice were sacrificed 24 h after the final airway challenge with thoracic lymph nodes aseptically excised. Thoracic lymph node cells were labeled with CFSE and stimulated for 5 days with medium alone (A, top row), 10 ␮g/ml SEA (B, middle row), or 1 ␮g/ml Con A (ConA) (C, bottom row). After 5 days, cells were stained with anti-mouse CD4 and anti-mouse Foxp3. Data shown are gated on CD4⫹ lymphocytes.

BAL (Fig. 4A, top panels) and lung (Fig. 4A, bottom panels) cell homogenate (Fig. 4A). Following IL-2:anti-IL-2 treatment, IL-10producing CD4⫹ cells in the BAL increased from 5.91 to 13.5% of total lymphocyte populations, with a similar fold increase in the lung tissue (0.36% in untreated mice compared with 0.88% in IL-2:anti-IL-2-treated mice). Costaining with CD25 revealed an increase in IL-10gfp⫹CD4⫹CD25⫹ cells in the BAL (7.60 to 13.1%) and lung (0.54 to 2.38%) following IL-2:anti-IL-2 treatment (Fig. 4B) with IL-10gfp⫹ cells largely found in a CD25high compartment. Similar increases in CD4⫹CD25highIL-10gfp⫹ cells were observed in the thoracic lymph nodes and spleen (data not shown). Furthermore, intracellular staining of BAL and lung cells with anti-mouse Foxp3 revealed a 2-fold increase in the percentage of CD4⫹CD25⫹Foxp3⫹ lymphocytes following IL-2:anti-IL-2 treatment (Fig. 4C), with slight reductions in the percentage of CD8⫹ and B220⫹ lymphocytes in the lung (data not shown). Finally, using dual Foxp3rfp IL-10gfp reporter mice we observed the greatest increase in the frequency of IL-10-producing cells within the CD4⫹Foxp3⫹CD103⫹ compartment within the lung of IL-2:anti-IL-2-treated mice. Whole lung tissue levels of mRNA for both Foxp3 (Fig. 4E) and

IL-10 (Fig. 4F) were elevated in whole lung tissue following IL-2:anti-IL-2 treatment. These observations support our hypothesis that IL-2:anti-IL-2 treatment preferentially expanded both inducible (CD4⫹CD25⫹IL-10gfp⫹) and natural (CD4⫹CD25⫹Foxp3⫹) Tregs, correlating with suppressed allergen-induced airway inflammation and hyperreactivity. Suppression of airway inflammation by IL-2:anti-IL-2 is dependent on IL-10 To test the requirement of IL-10 following IL-2:anti-IL-2 treatment, WT or IL-10⫺/⫺ mice were sensitized and challenged, as described above, with groups of mice treated with IL-2:antiIL-2 7 days before airway challenge. The absence of IL-10 resulted in an increase in total airway infiltrates and airway eosinophils (Fig. 5, A and B) as previously reported (38, 41, 42). However, unlike WT mice, which had reduced airway infiltrates following IL-2:anti-IL-2 treatment, IL-10⫺/⫺ mice had no change in airway infiltrates following treatment, indicating that IL-10 is required for IL-2:anti-IL-2-mediated suppression of airway inflammation (Fig. 5C). Lung histology confirmed that

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FIGURE 7. IL-2:anti-IL-2 mAb treatment requires Treg-derived IL-10 to control allergic airway inflammation. RAG2⫺/⫺ mice were reconstituted with Wt CD4⫺ cells, WT effector (CD4⫹CD25⫺), and either WT or IL-10⫺/⫺ Treg (CD4⫹CD25⫹) cells for 6 wk. Animals were then sensitized

The Journal of Immunology IL-10 is required for IL-2:anti-IL-2-mediated control of tissue inflammation (Fig. 5C) and goblet cell mucus production (Fig. 5D). Suppression of effector cell proliferation by IL-2:anti-IL-2 is IL-10 dependent To address the Ag specificity of IL-2:anti-IL-2-expanded Treg cells, lung-draining lymph nodes were excised 1 day after the final airway challenge, labeled with CFSE, and re-stimulated in vitro with either SEA or Con A or left unstimulated. After 5 days of culture, cells were stained with anti-mouse CD4 and Foxp3. In the absence of additional in vitro stimulation (Fig. 6A, top row), lymph node cells from IL-2:anti-IL-2-treated WT mice had reduced effector cell (CD4⫹Foxp3⫺) proliferation compared with untreated mice (14.3% in untreated mice (S:S), compared with 6.51% in IL-2:anti-IL-2-treated mice (S:S IL-2:anti-IL-2). Similarly, WT cells from IL-2:anti-IL-2-treated mice restimulated in vitro with SEA (Fig. 6B, middle row) or Con A (Fig. 6C, bottom row) consistently had less effector cell (CD4⫹Foxp3⫺) proliferation compared with untreated mice. These data indicate that effector cell proliferation was inhibited in IL-2:anti-IL-2-treated mice following both Ag-driven and polyclonal stimulation. Division of CD4⫹Foxp3⫹ lymph node cells from IL-2:anti-IL-2-treated mice was largely unaffected whether stimulated with SEA or left unstimulated, suggesting that the increased percentage of CD4⫹Foxp3⫹ Treg cells in IL-2:anti-IL-2-treated mice may not be specific to SEA. In contrast to WT mice, lymph node cells taken from IL10⫺/⫺ mice following airway challenge had a sizable increase in effector cell (CD4⫹Foxp3⫺) division following IL-2:antiIL-2 treatment. For example, SEA restimulation of cells from untreated IL-10⫺/⫺ mice had 20.5% division of CD4⫹ cells; however, this number increased to 49% when similar cells were taken from IL-2:anti-IL-2-treated mice. Similar increases in effector cell proliferation were observed following Con A stimulation (untreated, 41.3%; IL-2:anti-IL-2-treated, 65.7%) (Fig. 6). These data clearly indicate that IL-10 is required for IL-2: anti-IL-2-mediated control of effector T cell expansion following allergen challenge. Treg-derived IL-10 is essential for resolution of airway allergy by IL-2:anti-IL-2 To formally test the role of Treg-derived IL-10, RAG2⫺/⫺ mice were reconstituted with WT CD4-depleted splenocytes (reconstituting the B cell and CD8⫹ cell compartments), WT CD4⫹CD25⫺ (effector T cells), and FACS-purified CD4⫹CD25⫹ Treg cells taken from either WT or IL-10⫺/⫺ naive donors, restricting IL-10 deficiency to Tregs. After 6 wk, mice were sensitized with SEA, treated with either IL-2:antiIL-2 or PBS, and challenged with SEA (Fig. 7, Experimental Model, top left). Animals receiving WT Treg cells had reduced airway infiltrates and eosinophilia following IL-2:anti-IL-2 treatment and airway challenge (Fig. 7, A and B), corroborating earlier observations with WT mice and confirming sufficient

6951 reconstitution. A deficiency of IL-10 in the Treg compartment eliminated IL-2:anti-IL-2-mediated suppression of airway inflammation (Fig. 7, A and B). Furthermore, mRNA for IL-10, Foxp3, and Ebi3, one of two subunits of IL-35 (43, 44), were elevated in lung tissue of IL-2:anti-IL-2-treated mice receiving WT but not IL-10-deficient Tregs (supplemental figure 4). A deficiency of Treg-derived IL-10 also left mice unable to control AHR (Fig. 7C), tissue inflammation, and goblet cell hyperplasia (Fig. 7D). The frequency of IL-13⫹IL-5⫹-competent Th2 cells in the lungs of SEA-challenged mice were also reduced following IL-2:anti-IL-2 treatment (Fig. 7E). This reduction in Th2 cells was not observed in similarly treated mice lacking IL-10 in the Treg compartment. Finally, mice receiving WT Tregs, but not IL-10⫺/⫺ Tregs, had reduced vascular lesions and pneumonia following IL-2:anti-IL-2 treatment (data not shown). Thus, despite increased Foxp3⫹ cells in the lung and BAL of mice receiving IL-2:anti-IL-2 treatment, the ability of IL-2:anti-IL-2-induced Tregs to produce IL-10 appears to be critical for the control of Th2 cell-orchestrated airway inflammation, lung pathology, and AHR using this novel cytokine:Ab complex (35).

Discussion Successful allergen-specific immunotherapy remains the only cure for allergic patients, alleviating many symptoms through, at least in part, the expansion of endogenous allergen-specific Tregs (14 –16, 45). However, anaphylaxis is a significant risk (46). Synthetic steroids, such as glucocorticoids, are often the primary treatment for atopic patients that develop allergic reactions; however, broad-spectrum steroid use can compromise mucosal immunity. Interestingly, steroid use can also amplify Tregs (47). Thus, Tregs may be a central factor limiting allergen reactivity. Animal models have also demonstrated that Tregs from various sources can control airway inflammation, hyperresponsiveness, and airway pathology (18 –23). Therefore, even though Treg cells exhibit a clear role in controlling allergen reactivity in both human and preclinical animal studies, building upon and translating this information into novel treatments have been limited (48). We present a novel approach using cytokine:Ab complexes to expand endogenous Treg cells capable of suppressing airway allergy, whether administered before allergen exposure or after airway challenge. Recombinant IL-2 therapy has been used in cancer clinics with variable success (49 –51). Antony and Restifo (52) argue that IL-2 therapy for cancer patients may be detrimental rather than beneficial, based upon the induction of Treg cells by rIL-2. Taking heed of these concerns, IL-2 and diphtheria toxin conjugates to eliminate CD25high Tregs (53), have been tested. However, variable results were also obtained (54). These observations led to the initial hypothesis of the current study that IL-2 treatment could expand Tregs with the ability to suppress inflammatory responses in the lung. Supporting this hypothesis is a body of literature demonstrating that IL-2, either at a high daily dose (25,000 IU of rhIL-2 administered for 5 days; Ref. 55) or in combination with

and challenged as in Fig. 1. Groups of mice were treated with a complex of rmIL-2(2.5 ␮g):anti-IL-2m Ab (25 ␮g) 7 days before the first airway challenge. Mice were sacrificed 24 h after the final airway challenge. One of two representative experiments is shown. A, Total airway infiltrates recovered in BAL following airway challenge. B, BAL Eosinophilia. C, AHR. Enhanced pause (Penh) measurements were mad 24 h after the final airway challenge using a Buxco Electronics system with mice exposed to increasing doses of methacholine. D, Five-micrometer sections were cut from paraffin-embedded lung tissue and stained with H&E (left panels, ⫻10 magnification) to show cellular infiltration with parallel sections stained with ABPAS (right panels, ⫻10 magnification). Five lungs from each group were scored in a blinded fashion. E, BAL cell intracellular cytokine staining showing CD4⫹ lymphocytes stained with FITC-labeled anti-IL-13 and allophycocyanin-labeled anti-IL-5 following a 3-h stimulation with PMA and ionomycin in the presence of brefeldin A. Data shown are gated upon CD4⫹ lymphocytes. ⴱ, p ⱕ 0.05.

6952 dexamethasone (300,000 IU IL-2/mouse/day plus dexamethasone) could expand Treg cells and in some cases dampen autoimmune disease severity following MOG35–55-induced experimental autoimmune encephalomyelitis (47). In this and other (56) studies, however, IL-2 alone was insufficient to suppress airway allergy and even had the opposite effect with increased cellular bronchial influx (57). The failure to expand Tregs and control airway inflammation suggests mechanisms beyond simply IL-2 availability governing Treg expansion. IL-2 availability, activation status and location of T cells, and IL-2 receptor expression both on the T cell and in soluble form may all contribute to IL-2 associated Treg expansion. Following observations made by Boyman and colleagues (35) identifying IL-2 in complex with specific mAbs amplifying Tregs, we used this new finding in our hypothesis. Administration of IL-2:anti-IL-2 mAb complexes before airway challenge reduced the allergic cascade and pulmonary pathology. The application of IL-2:anti-IL-2 complexes before initial airway provocation is an unlikely scenario in the clinic. We therefore tested the ability of IL-2:anti-IL-2 complexes to modulate established airway inflammation. Progression of airway inflammation, mucus secretion, and AHR following a second round of airway challenges was prevented after IL-2:antiIL-2 treatment, suggesting that IL-2 complexes may hold therapeutic potential. Previous studies have demonstrated that IL-10 from adoptively transferred Tregs is not essential (18, 58), but rather IL-10 from host CD4⫹ Tregs is required to control airway allergy (58). Whether from adoptively transferred Tregs or host Tregs, IL-10 appears to be a significant mediator controlling airway inflammation (6, 41). Following IL-2:anti-IL-2 treatment we identified a significant increase in IL-10⫹ cells coexpressing CD4 and CD25. The induction of IL-10 appears to be a major functional molecule following IL-2:anti-IL-2 treatment, as the protective effect of IL2:anti-IL-2 treatment was not observed in IL-10-deficient mice. Furthermore, a significant expansion of CD4⫹CD25⫹Foxp3⫹ cells was also observed, with both IL-10 and Foxp3 mRNA elevated in whole lung tissue. Further clarification, using dual reporter mice (Foxp3rfpIL-10gfp) allowed us to identify that IL-10 was produced by both Foxp3⫹ and Foxp3⫺ CD4⫹CD25⫹ cells, with the greatest increase in IL-10 observed within the Foxp3⫹CD103⫹ Treg subset. Taking together the observations that CD4⫹CD25⫹ cells were the dominant population producing IL-10 and that IL-10-deficient mice were not protected from airway inflammation following IL2:anti-IL-2 treatment, we addressed the role of Treg-derived IL-10 using RAG2⫺/⫺ mice reconstituted with IL-10⫹/⫹ or IL-10⫺/⫺ CD4⫹CD25⫹ cells. These experiments demonstrated that CD4⫹CD25⫹ cell-derived IL-10 appears to be a critical source of IL-10 for IL-2:anti-IL-2 mediated-regulation. Although increases in Foxp3⫹ cells were observed in mice with IL-10-defcient Tregs following IL-2:anti-IL-2 treatment, the IL-10 competence of these cells appears paramount and is reflected in unchanged airway infiltrates, tissue inflammation, mucus secretions, and AHR following treatment. Recent elegant studies, using mice with a conditional deletion of IL-10 specifically in Foxp3-expressing cells, corroborate the findings presented in this study (59). Briefly, mice with IL-10-deficient Foxp3⫹ cells develop greater airway inflammation and AHR compared with mice with IL-10-competent Treg cells. Following IL-2:anti-IL-2 treatment of mice with IL-10 competent Tregs, but not IL-10-deficient Tregs, we observed an increase in Ebi3, one subunit of IL-35 (and IL-27), a newly described Tregassociated cytokine (43, 44). Furthermore, Niedbala and colleagues (43, 44) demonstrated that CD4⫹CD25⫹ cells cultured in

IL-2:ANTI-IL-2-DRIVEN Tregs the presence of IL-35 increased IL-10 production. Thus, IL-2:antiIL-2 may promote IL-35 secretion and collaborate in driving IL-10 production by CD4⫹CD25⫹ cells. The precise mechanism of how IL-2:anti-IL-2 complexes, but neither IL-2 or anti-IL-2 alone, promotes Treg expansion is not thoroughly understood. Dissecting the molecular mechanisms is of particular interest before such therapies head toward the clinic. Simplistically, Treg responses often mirror effector T cell responses, offering a balanced T cell response with IL-2 providing a cue for Treg expansion (32). Conceivably, IL-2 in complex with anti-IL-2 may simply provide an IL-2 signal without the effector T cell response, tricking the immune system into expanding Tregs when effector cells are not present. Boyman and colleagues (35, 60) suggest that partial occlusion of IL-2 receptor subunits may explain the diverse effects of different anti-IL-2 mAbs used to form IL-2 complexes. However, the biophysical interactions of IL-2, when in complex with anti-IL-2, with different receptor subunits have not been clearly dissected. Alternatively, cytokines in complex with anti-cytokine Abs can prolong the half-life and activity of cytokines (61– 66). Supporting this, hypersensitivity can be suppressed by IL-2 in complex with IgG2b with increased stability and half-life (67). This may be an important facet of IL-2 Ab complexes, given the short half-life of native IL-2 (68). If the same is true for IL-2 in complex with anti-IL-2, this may explain why Ab complexes with enhanced IL-2 activity, and not IL-2 alone, can expand Tregs and in this case suppress airway allergy. Thus, determining the strength of the IL-2 signal when in a complex with anti-IL-2 and how this alters downstream pathways may uncover properties of this complex. For example, PTEN (phosphatase and tensin homolog deleted on chromosome 10), a lipid phosphatase that negatively regulates the activation of downstream signaling pathways in T cells, can inhibit IL-2-mediated expansion of Tregs (69). PTEN expression following IL-2:anti-IL-2 complex stimulation remains to be addressed. However, simply stabilizing IL-2 is an insufficient explanation, as IL-2 in complex with a different clone of anti-IL-2, S4B61 (which may also stabilize IL-2), dramatically expands memory CD122⫹CD8⫹ cells (35, 70), protecting mice from tumor metastasis (71) and lethal bacterial infection (72) and enhancing CD8 responses (73). Multiple factors likely govern the properties of IL-2:anti-IL-2 complexes. Understanding how these complexes regulate the activity of IL-2 is of particular interest in preclinical studies. Tregs can clearly control undesirable hyperinflammatory conditions such as allergic and autoimmune diseases. However, the challenge of designing safe and effective Treg based immunotherapy has wisely put the brakes on further clinical developments. This study builds upon advances in Treg immunobiology and the regulation of allergic airway inflammation and presents a novel strategy of IL-2:anti-IL-2 complexes to expand endogenous IL10-secreting Tregs that can control Th2-mediated airway pathologies such as allergic asthma.

Acknowledgments We acknowledge the meticulous care of animals used in this study by Alison Bright, Joy McFarlane, Sandy White, and colleagues (SoBran Inc.). M.S.W. thanks Dr. Daniel L. Barber and Dr. Carl G. Feng for helpful and thought-provoking discussions. We thank Dr. Fred Lewis and colleagues (Biomedical Research Institute, Rockville, MD) for SEA.

Disclosures The authors have no financial conflict of interest.


References 1. Kelchtermans, H., B. De Klerck, T. Mitera, M. Van Balen, D. Bullens, A. Billiau, G. Leclercq, and P. Matthys. 2005. Defective CD4⫹CD25⫹ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-␥. Arthritis Res. Ther. 7: R402–R415. 2. Marazuela, M., M. A. Garcia-Lopez, N. Figueroa-Vega, H. de la Fuente, B. Alvarado-Sanchez, A. Monsivais-Urenda, F. Sanchez-Madrid, and R. Gonzalez-Amaro. 2006. Regulatory T cells in human autoimmune thyroid disease. J. Clin. Endocrinol. Metab. 91: 3639 –3646. 3. Suri-Payer, E., A. Z. Amar, A. M. Thornton, and E. M. Shevach. 1998. CD4⫹CD25⫹ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160: 1212–1218. 4. Hawrylowicz, C. M., and A. O’Garra. 2005. Potential role of interleukin-10secreting regulatory T cells in allergy and asthma. Nat. Rev. Immunol. 5: 271–283. 5. Ling, E. M., T. Smith, X. D. Nguyen, C. Pridgeon, M. Dallman, J. Arbery, V. A. Carr, and D. S. Robinson. 2004. Relation of CD4⫹CD25⫹ regulatory T-cell suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease. Lancet 363: 608 – 615. 6. Urry, Z., E. Xystrakis, and C. M. Hawrylowicz. 2006. Interleukin-10-secreting regulatory T cells in allergy and asthma. Curr. Allergy Asthma Rep. 6: 363–371. 7. Akbari, O., P. Stock, R. H. DeKruyff, and D. T. Umetsu. 2003. Role of regulatory T cells in allergy and asthma. Curr. Opin. Immunol. 15: 627– 633. 8. Wu, K., Y. Bi, K. Sun, and C. Wang. 2007. IL-10-producing type 1 regulatory T cells and allergy. Cell. Mol. Immunol. 4: 269 –275. 9. Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, and T. Nomura. 2006. Foxp3⫹ CD25⫹ CD4⫹ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212: 8 –27. 10. Umetsu, D. T., and R. H. DeKruyff. 2006. The regulation of allergy and asthma. Immunol. Rev. 212: 238 –255. 11. Belkaid, Y. 2007. Regulatory T cells and infection: a dangerous necessity. Nat. Rev. Immunol. 7: 875– 888. 12. Grindebacke, H., K. Wing, A. C. Andersson, E. Suri-Payer, S. Rak, and A. Rudin. 2004. Defective suppression of Th2 cytokines by CD4CD25 regulatory T cells in birch allergics during birch pollen season. Clin. Exp. Allergy 34: 1364 –1372. 13. Xystrakis, E., S. Kusumakar, S. Boswell, E. Peek, Z. Urry, D. F. Richards, T. Adikibi, C. Pridgeon, M. Dallman, T. K. Loke, et al. 2006. Reversing the defective induction of IL-10-secreting regulatory T cells in glucocorticoid-resistant asthma patients. J. Clin. Invest. 116: 146 –155. 14. Gardner, L. M., F. C. Thien, J. A. Douglass, J. M. Rolland, and R. E. O’Hehir. 2004. Induction of T “regulatory” cells by standardized house dust mite immunotherapy: an increase in CD4⫹ CD25⫹ interleukin-10⫹ T cells expressing peripheral tissue trafficking markers. Clin. Exp. Allergy 34: 1209 –1219. 15. Francis, J. N., S. J. Till, and S. R. Durham. 2003. Induction of IL10⫹CD4⫹CD25⫹ T cells by grass pollen immunotherapy. J. Allergy Clin. Immunol. 111: 1255–1261. 16. Hall, G., C. G. Houghton, J. U. Rahbek, J. R. Lamb, and E. R. Jarman. 2003. Suppression of allergen reactive Th2 mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production. Vaccine 21: 549 –561. 17. Lewkowich, I. P., N. S. Herman, K. W. Schleifer, M. P. Dance, B. L. Chen, K. M. Dienger, A. A. Sproles, J. S. Shah, J. Kohl, Y. Belkaid, and M. Wills-Karp. 2005. CD4⫹CD25⫹ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function. J. Exp. Med. 202: 1549 –1561. 18. Wilson, M. S., M. D. Taylor, A. Balic, C. A. Finney, J. R. Lamb, and R. M. Maizels. 2005. Suppression of allergic airway inflammation by helminthinduced regulatory T cells. J. Exp. Med. 202: 1199 –1212. 19. Kearley, J., J. E. Barker, D. S. Robinson, and C. M. Lloyd. 2005. Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4⫹CD25⫹ regulatory T cells is interleukin 10 dependent. J. Exp. Med. 202: 1539 –1547. 20. Cottrez, F., S. D. Hurst, R. L. Coffman, and H. S. Groux. 2000. T regulatory cells 1 inhibit a Th2-specific response in vivo. J. Immunol. 165: 4848 – 4853. 21. Hadeiba, H., and R. M. Locksley. 2003. Lung CD25 CD4 regulatory T cells suppress type 2 immune responses but not bronchial hyperreactivity. J. Immunol. 170: 5502–5510. 22. Jaffar, Z., T. Sivakuru, and K. Roberts. 2004. CD4⫹CD25⫹ T cells regulate airway eosinophilic inflammation by modulating the Th2 cell phenotype. J. Immunol. 172: 3842–3849. 23. Strickland, D. H., P. A. Stumbles, G. R. Zosky, L. S. Subrata, J. A. Thomas, D. J. Turner, P. D. Sly, and P. G. Holt. 2006. Reversal of airway hyperresponsiveness by induction of airway mucosal CD4⫹CD25⫹ regulatory T cells. J. Exp. Med. 203: 2649 –2660. 24. Fontenot, J. D., J. P. Rasmussen, M. A. Gavin, and A. Y. Rudensky. 2005. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6: 1142–1151. 25. D’Cruz, L. M., and L. Klein. 2005. Development and function of agonist-induced CD25⫹Foxp3⫹ regulatory T cells in the absence of interleukin 2 signaling. Nat. Immunol. 6: 1152–1159. 26. Furtado, G. C., M. A. Curotto de Lafaille, N. Kutchukhidze, and J. J. Lafaille. 2002. Interleukin 2 signaling is required for CD4⫹ regulatory T cell function. J. Exp. Med. 196: 851– 857. 27. Malek, T. R., A. Yu, V. Vincek, P. Scibelli, and L. Kong. 2002. CD4 regulatory T cells prevent lethal autoimmunity in IL-2R␤-deficient mice: implications for the nonredundant function of IL-2. Immunity 17: 167–178.

6953 28. Almeida, A. R., N. Legrand, M. Papiernik, and A. A. Freitas. 2002. Homeostasis of peripheral CD4⫹ T cells: IL-2R ␣ and IL-2 shape a population of regulatory cells that controls CD4⫹ T cell numbers. J. Immunol. 169: 4850 – 4860. 29. Nelson, B. H. 2004. IL-2, regulatory T cells, and tolerance. J. Immunol. 172: 3983–3988. 30. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, and I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75: 253–261. 31. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, and F. Ramsdell. 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27: 68 –73. 32. Yu, A., and T. R. Malek. 2006. Selective availability of IL-2 is a major determinant controlling the production of CD4⫹CD25⫹Foxp3⫹ T regulatory cells. J. Immunol. 177: 5115–5121. 33. Malek, T. R., and A. L. Bayer. 2004. Tolerance, not immunity, crucially depends on IL-2. Nat. Rev. Immunol. 4: 665– 674. 34. Fehervari, Z., T. Yamaguchi, and S. Sakaguchi. 2006. The dichotomous role of IL-2: tolerance versus immunity. Trends Immunol. 27: 109 –111. 35. Boyman, O., M. Kovar, M. P. Rubinstein, C. D. Surh, and J. Sprent. 2006. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311: 1924 –1927. 36. Kamanaka, M., S. T. Kim, Y. Y. Wan, F. S. Sutterwala, M. Lara-Tejero, J. E. Galan, E. Harhaj, and R. A. Flavell. 2006. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity 25: 941–952. 37. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, and V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238. 38. Wilson, M. S., E. Elnekave, M. M. Mentink-Kane, M. G. Hodges, J. T. Pesce, T. R. Ramalingam, R. W. Thompson, M. Kamanaka, R. A. Flavell, A. Keane-Myers, et al. 2007. IL-13Ralpha2 and IL-10 coordinately suppress airway inflammation, airway-hyperreactivity, and fibrosis in mice. J. Clin. Invest. 117: 2941–2951. 39. Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin, and E. W. Gelfand. 1997. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156: 766 –775. 40. Finkelman, F. D., and S. C. Morris. 1999. Development of an assay to measure in vivo cytokine production in the mouse. Int. Immunol. 11: 1811–1818. 41. Tournoy, K. G., J. C. Kips, and R. A. Pauwels. 2000. Endogenous interleukin-10 suppresses allergen-induced airway inflammation and nonspecific airway responsiveness. Clin. Exp. Allergy 30: 775–783. 42. Zuany-Amorim, C., S. Haile, D. Leduc, C. Dumarey, M. Huerre, B. B. Vargaftig, and M. Pretolani. 1995. Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice. J. Clin. Invest. 95: 2644 –2651. 43. Collison, L. W., C. J. Workman, T. T. Kuo, K. Boyd, Y. Wang, K. M. Vignali, R. Cross, D. Sehy, R. S. Blumberg, and D. A. Vignali. 2007. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 450: 566 –569. 44. Niedbala, W., X. Q. Wei, B. Cai, A. J. Hueber, B. P. Leung, I. B. McInnes, and F. Y. Liew. 2007. IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur. J. Immunol. 37: 3021–3029. 45. Pereira-Santos, M. C., A. P. Baptista, A. Melo, R. R. Alves, R. S. Soares, E. Pedro, M. Pereira-Barbosa, R. M. Victorino, and A. E. Sousa. 2008. Expansion of circulating Foxp3⫹CD25bright CD4⫹ T cells during specific venom immunotherapy. Clin. Exp. Allergy 38: 291–297. 46. Akdis, M., and C. A. Akdis. 2007. Mechanisms of allergen-specific immunotherapy. J. Allergy Clin. Immunol. 119: 780 –791. 47. Chen, X., J. J. Oppenheim, R. T. Winkler-Pickett, J. R. Ortaldo, and O. M. Howard. 2006. Glucocorticoid amplifies IL-2-dependent expansion of functional FoxP3⫹CD4⫹CD25⫹ T regulatory cells in vivo and enhances their capacity to suppress EAE. Eur. J. Immunol. 36: 2139 –2149. 48. Roncarolo, M. G., and M. Battaglia. 2007. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat. Rev. Immunol. 7: 585–598. 49. Ahmadzadeh, M., and S. A. Rosenberg. 2006. IL-2 administration increases CD4⫹ CD25hi Foxp3⫹ regulatory T cells in cancer patients. Blood 107: 2409 –2414. 50. Wei, S., I. Kryczek, R. P. Edwards, L. Zou, W. Szeliga, M. Banerjee, M. Cost, P. Cheng, A. Chang, B. Redman, et al. 2007. Interleukin-2 administration alters the CD4⫹FOXP3⫹ T-cell pool and tumor trafficking in patients with ovarian carcinoma. Cancer Res. 67: 7487–7494. 51. Zorn, E., E. A. Nelson, M. Mohseni, F. Porcheray, H. Kim, D. Litsa, R. Bellucci, E. Raderschall, C. Canning, R. J. Soiffer, et al. 2006. IL-2 regulates FOXP3 expression in human CD4⫹CD25⫹ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 108: 1571–1579. 52. Antony, P. A., and N. P. Restifo. 2005. CD4⫹CD25⫹ T regulatory cells, immunotherapy of cancer, and interleukin-2. J. Immunother. 28: 120 –128. 53. Litzinger, M. T., R. Fernando, T. J. Curiel, D. W. Grosenbach, J. Schlom, and C. Palena. 2007. The IL-2 immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-mediated T-cell immunity. Blood 110: 3192–3201. 54. Attia, P., A. V. Maker, L. R. Haworth, L. Rogers-Freezer, and S. A. Rosenberg. 2005. Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma. J. Immunother. 28: 582–592.

6954 55. Zhang, H., K. S. Chua, M. Guimond, V. Kapoor, M. V. Brown, T. A. Fleisher, L. M. Long, D. Bernstein, B. J. Hill, D. C. Douek, et al. 2005. Lymphopenia and interleukin-2 therapy alter homeostasis of CD4⫹CD25⫹ regulatory T cells. Nat. Med. 11: 1238 –1243. 56. Matsubara, S., K. Takeda, T. Kodama, A. Joetham, N. Miyahara, T. Koya, C. H. Swasey, M. Okamoto, A. Dakhama, and E. W. Gelfand. 2007. IL-2 and IL-18 attenuation of airway hyperresponsiveness requires STAT4, IFN-␥, and natural killer cells. Am. J. Respir. Cell Mol. Biol. 36: 324 –332. 57. Renzi, P. M., L. Xu, X. X. Yang, W. S. Powell, and J. G. Martin. 1999. IL-2 enhances allergic airway responses in rats by increased inflammation but not through increased synthesis of cysteinyl leukotrienes. J. Allergy Clin. Immunol. 104: 145–152. 58. Kearley, J., S. J. McMillan, and C. M. Lloyd. 2007. Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo. J. Exp. Med. 204: 1289 –1294. 59. Rubtsov, Y. P., J. P. Rasmussen, E. Y. Chi, J. Fontenot, L. Castelli, X. Ye, P. Treuting, L. Siewe, A. Roers, W. R. Henderson, Jr., et al. 2008. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28: 546 –558. 60. Boyman, O., C. D. Surh, and J. Sprent. 2006. Potential use of IL-2/anti-IL-2 antibody immune complexes for the treatment of cancer and autoimmune disease. Expert Opin. Biol. Ther. 6: 1323–1331. 61. Finkelman, F. D., K. B. Madden, S. C. Morris, J. M. Holmes, N. Boiani, I. M. Katona, and C. R. Maliszewski. 1993. Anti-cytokine antibodies as carrier proteins: prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes. J. Immunol. 151: 1235–1244. 62. Phelan, J. D., T. Orekov, and F. D. Finkelman. 2008. Cutting edge: mechanism of enhancement of in vivo cytokine effects by anti-cytokine monoclonal antibodies. J. Immunol. 180: 44 – 48. 63. Jones, A. T., and H. J. Ziltener. 1993. Enhancement of the biologic effects of interleukin-3 in vivo by anti-interleukin-3 antibodies. Blood 82: 1133–1141. 64. Montero-Julian, F. A., B. Klein, E. Gautherot, and H. Brailly. 1995. Pharmacokinetic study of anti-interleukin-6 (IL-6) therapy with monoclonal antibodies: enhancement of IL-6 clearance by cocktails of anti-IL-6 antibodies. Blood 85: 917–924.

IL-2:ANTI-IL-2-DRIVEN Tregs 65. Sato, J., N. Hamaguchi, K. Doken, K. Gotoh, K. Ootsu, S. Iwasa, Y. Ogawa, and H. Toguchi. 1994. Enhancement of the anti-tumor activity of recombinant interleukin-2 (rIL-2) by immunocomplexing with an F(ab⬘)2 fragment of murine monoclonal antibody against rIL-2. Biol. Pharm. Bull. 17: 1101–1105. 66. Sato, J., N. Hamaguchi, K. Doken, S. Iwasa, Y. Ogawa, and H. Toguchi. 1994. Pharmacokinetic alteration in rats of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2. Biol. Pharm. Bull. 17: 535–538. 67. Ruckert, R., U. Herz, R. Paus, D. Ungureanu, T. Pohl, H. Renz, and S. Bulfone-Paus. 1998. IL-15-IgG2b fusion protein accelerates and enhances a Th2 but not a Th1 immune response in vivo, while IL-2-IgG2b fusion protein inhibits both. Eur. J. Immunol. 28: 3312–3320. 68. Lotze, M. T., Y. L. Matory, S. E. Ettinghausen, A. A. Rayner, S. O. Sharrow, C. A. Seipp, M. C. Custer, and S. A. Rosenberg. 1985. In vivo administration of purified human interleukin 2: II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL 2. J. Immunol. 135: 2865–2875. 69. Walsh, P. T., J. L. Buckler, J. Zhang, A. E. Gelman, N. M. Dalton, D. K. Taylor, S. J. Bensinger, W. W. Hancock, and L. A. Turka. 2006. PTEN inhibits IL-2 receptor-mediated expansion of CD4⫹ CD25⫹ Tregs. J. Clin. Invest. 116: 2521–2531. 70. Cho, J. H., O. Boyman, H. O. Kim, B. Hahm, M. P. Rubinstein, C. Ramsey, D. M. Kim, C. D. Surh, and J. Sprent. 2007. An intense form of homeostatic proliferation of naive CD8⫹ cells driven by IL-2. J. Exp. Med. 204: 1787–1801. 71. Kamimura, D., Y. Sawa, M. Sato, E. Agung, T. Hirano, and M. Murakami. 2006. IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb. J. Immunol. 177: 306 –314. 72. Kamimura, D., and M. J. Bevan. 2007. Naive CD8⫹ T cells differentiate into protective memory-like cells after IL-2 anti IL-2 complex treatment in vivo. J. Exp. Med. 204: 1803–1812. 73. Mostbock, S., M. E. Lutsiak, D. E. Milenic, K. Baidoo, J. Schlom, and H. Sabzevari. 2008. IL-2/Anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8⫹ T cell responses and increases the ratio of effector/ memory CD8⫹ T cells to regulatory T cells. J. Immunol. 180: 5118 –5129.