Suppression of murine allergic airway disease by IL-2:anti-IL-2 monoclonal antibody-induced regulatory T cells

Abstract

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-10(+)CD4(+)CD25(+) and Foxp3(+)CD4(+)CD25(+)) 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-10(+/+), but not IL-10(-/-) 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.

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 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. *, p ≤ 0.05.

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 μg), anti-IL-2 mAb (25 μg), isotype control (25 μg), or a complex of rmIL-2 (2.5 μg):αIL-2m Ab (25 μg) 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. *, p ≤ 0.05.

FIGURE 3

rmIL-2:anti-mouse IL-2 mAb treatment significantly reduces established airway inflammation and AHR. Animals were sensitized with 10 μg of SEA on day 1 and day 14 followed by two intratracheal airway challenges with 10 μg 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 μg):anti-IL-2m Ab (25 μg) 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. *, p ≤ 0.05.

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 Foxp3^rfpIL-10^gfp dual reporter mice showing either Foxp3^rfp+ cells stained with anti-CD103⁺ showing IL-10^gfp or Foxp3^- cells stained with anti-CD103⁺ showing IL-10^gfp. Data shown are gated on CD4⁺ lymphocytes. E, Lung tissue mRNA for Foxp3. F, Lung tissue mRNA for IL-10. *, p ≤ 0.05.

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 μg):anti-IL-2m Ab (25 μg) 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 μm sections of paraffin embedded lung tissue stained with ABPAS. Five lungs from each group were scored in a blinded fashion. *, p ≤ 0.05.

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 μg):anti-IL-2m Ab (25 μg) 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 μg/ml SEA (B, middle row), or 1 μg/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.

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 and challenged as in Fig. 1. Groups of mice were treated with a complex of rmIL-2(2.5 μg):anti-IL-2m Ab (25 μg) 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. *, p ≤ 0.05.