C57BL/6 and B6.PLThy1a (Thy1.1) mice were from The Jackson Laboratory. OT-II TCR transgenic mice, bred in house on the BL/6 background, were used as a source of Vβ5/Vα2/Thy1.2 CD4+ T cells responsive to the peptide OVA-323–339. Foxp3/GFP reporter mice, kindly provided by Dr. Alexander Rudensky (Sloan-Kettering Institute, NY) were bred in house with OT-II TCR transgenic mice and also used as a source of Vβ5/Vα2/Thy1.2 CD4+ T cells. All experiments were conducted following the guidelines of the La Jolla Institute for Allergy and Immunology’s Institutional Animal Care and Use Committee.
Airway tolerance and lung Inflammation
Airway tolerance was induced similar to previously described protocols (7
). Briefly, on day 0, mice were exposed to 100 μg soluble OVA (Worthington Biochemical Corporation) in PBS, or to PBS alone, given i.n. on 3 consecutive days. To assess the extent of tolerance, 9 days later, mice were sensitized by i.p. injection of 20 μg OVA protein (chicken egg albumin; Sigma-Aldrich), adsorbed to 4 mg aluminum hydroxide (Alum; Pierce Chemical Co.). On day 24 or later, mice were then challenged via the airways with OVA aerosol in a whole body Plexiglas box (10 mg/ml in 15 ml of PBS) for 20 min, once a day for four consecutive days, by ultrasonic nebulization. Tolerance was shown by a lack of lung inflammation, assessed as described below.
To prevent airway tolerance, soluble OVA was mixed with HDM (100 μg), the Nod2 ligand MDP (50 μg), or LPS (1 μg), given i.n. For TGFβ and IL-10R blockade experiments, one single dose of 200 μg anti-IL-10R (1B1.3a), 200 μg anti-TGF-β (1D11), or control IgG was given i.p. at the time of initial exposure to OVA.
Lung Inflammation and airway hyperresponsiveness (AHR)
Bronchoalveolar lavage (BAL) was performed 24 h after the last OVA aerosol challenge. BAL fluid was examined for cytokine content by ELISA (BD Biosciences). For cytological examination, BAL cells were spun on a slide using a Cytospin (Thermo Shandon, Pittsburgh, PA), fixed, and stained with Protocol HEMA3 (Fisher Scientific Company, L.L.C.). Differential cell count was then performed on at least 500 cells in each cytospin slide. For lung histology analysis, 5-μm sections were cut and stained with H&E (hematoxylin and eosin) for examining cell infiltration.
Airway hyperresponsiveness was assessed 24 h after the final OVA challenge in intubated and ventilated mice (FlexiVent ventilator; Scireq) anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally. The dynamic airway resistance was determined using Scireq software in mice exposed to nebulized PBS or Methacholine (3, 24, 48 mg/ml).
T cell preparation and adoptive transfer
Naïve OVA-specific CD25−Foxp3− CD4+ cells were isolated from spleen and lymph nodes of OT-II/Foxp3/GFP reporter mice with naïve CD4+CD62L+ T Cell Isolation Kits (Miltenyi Biotec, CA). Cell suspensions were incubated with the biotinylated antibody cocktail, supplemented with biotinylated anti-mouse CD25, followed by magnetic anti-biotin microbeads and negative selection on LS MACS columns according to the manufacture’s instructions. The CD4+ cell purity was >90%, with >95% of resulting cells expressing the Vα2Vβ5 transgene and less than 0.1% CD4+CD25+Foxp3+ cells. These cells were either used in vitro, or were adoptively transferred (5 × 106 cells) i.v. into B6.PL Thy1.1 congenic mice to allow T cell responses to be analyzed under tolerogenic or inflammatory conditions.
To track the induction of Foxp3+ and LAP+ CD4+ T cells after adoptive transfer and induction of tolerance, lymph nodes and spleen from individual OVA-challenged mice were harvested, homogenized, and treated with red blood cell-lysing buffer (Sigma, MO) to prepare single cell suspensions. Lung tissues were minced into fragments of less than 1mm in size and digested with Collagenase D (3 mg/ml) and DNase (10 μg/ml) before homogenizing to prepare single cell suspensions. After Fc block with the 2.4G2 mAb, cells were stained with anti-Thy1.2 (53–2.1), anti-CD4 (RM4–5) (BD Biosciences) and anti-LAP (27232) (R&D systems) antibodies. Foxp3 expression was assessed by analyzing GFP expression. All samples were run on a FACS LSRII (BD Biosciences) with FlowJo (Tree Star) software.
In vitro generation and sorting of Foxp3+ and LAP+ Treg cells
Naïve CD4+ T cells from spleen and peripheral lymph nodes of OT-II/Foxp3/GFP reporter mice were isolated as above. APCs from BL/6 spleen cells were made by depleting T cells using complement fixation with Abs to Thy-1.2 (F7D5 and HO.13.4), CD4 (RL172.4), and CD8 (3.155) and were irradiated with 3,000 rad X-ray before use. Cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies) with penicillin, streptomycin, glutamine, HEPES, 2-ME, and 10% FCS (Omega Scientific). For generation of Foxp3+ and LAP+ Treg cells, naïve OT-II cells were plated at 5 × 105 cells per ml with 2 × 106 cells per ml APCs, 1 μM of OVA-323-339, and 10 ng/ml of recombinant human TGF-β1 (PeproTech). At day 4, expanded OT-II cells were separated into CD4+LAP+ or CD4+Foxp3+ (GFP+) populations by sorting using a FACSAria (BD Biosciences). Expression of Treg associated markers, such as CD25, GITR, CD103, intracellular Granzyme B, intracellular CTLA-4, CD44, CD69 and TGFβRII were determined by flow cytometry by using an LSRII (BD Biosciences) with FlowJo software (Tree Star). Antibodies used were anti-LAP (BAF246) and anti-TGFβRII (R&D systems), anti-CD4 (RM4–5), anti-CD25 (PC61), anti-GITR (DTA-1), anti-CD103 (2E7), anti-Granzyme B (16G6), anti-CTLA-4 (UC10-4B9) and anti-CD44 (IM7) (BD Biosciences), anti-CD69 (H1.2F3) (eBioscience).
In vitro suppression assay
Naïve CD4+ T cells were purified from CD45.1+ OT-II TCR-Tg B6 mice and were labeled with PKH26-GL (Sigma) according to the manufacturer’s instructions. To evaluate suppressive function of Foxp3+ and LAP+ T cells, 5 × 104 PKH26-labeled CD45.1+ naïve OT-II cells were cultured with 4 × 105 irradiated T-depleted APCs and 0.5 μM OVA-323-339 for 3 days in the presence of varying numbers of CD45.2+ Treg cells. Cell division of responder T cells was assessed by dilution of PKH26 dye in the gated CD4+CD45.1+ populations. In some experiments, naïve OT-II CD4+ T cell proliferation was assessed by incorporation of [3H]thymidine (1 μCi/well), which was added for the last 8 h of culture.
In vivo studies with in vitro generated LAP+ Treg cells
In vitro generated LAP+ Treg cells (Thy1.2+) were adoptively transferred into Thy1.1 recipient mice. The recipient mice were then immunized and challenged with OVA as described. Mice were either assessed for lung inflammation, or sacrificed on day 2 or day 6 after transfer and pooled LN and spleen cells were harvested to track the transferred cells by flow cytometry as before.
Multiplex cytokine assays
Sorted T cell populations were stimulated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) for 24 h. Cytokine secretion was measured using BioPlex Pro mouse cytokine assay kits (Bio-Rad Inc., Hercules, CA). All assays were carried out directly in a 96-well filtration plate (Millipore, Billerica, MA) at room temperature and protected from light. Briefly, wells were pre-wet with 100 μl assay buffer (PBS containing 1% BSA), then magnetic beads together with a standard, sample, and blank, were added in a final volume of 100 μl, and incubated together at room temperature for 30 min with continuous shaking at 500 rpm. Beads were washed three times with 100 μl wash buffer (PBS containing 1% BSA and 0.05% Tween 20). A cocktail of biotinylated detection antibodies (25 μl/well) was added to the beads for a further 30 min incubation with continuous shaking at 500 rpm. Beads were washed three times, then streptavidin-PE was added for 10 min. Beads were again washed three times and resuspended in 125 μl of assay buffer (PBS containing 1% BSA and 0.05% Tween 20). The fluorescence intensity of the beads was measured using a BioPlex array reader. BioPlex Manager 4.0 software with five-parametric-curve fitting was used for data analysis.
RT and real-time PCR
FACS sorted cells were lysed using TRIzol reagent (Invitrogen). An aliquot of total RNA (5 μg) was reverse-transcribed to cDNA using the SuperScript III (Invitrogen). The oligonucleotide primer sequences of Foxp3 were: forward primer, 5′-GGC CCT TCT CCA GGA CAG A-3′ and reverse primer, 5′-GCT GAT CAT GGC TGG GTT GT. Real-time PCR assay was carried out with LightCycler (Roche Diagnostics, Germany) using LightCycler 480 SYBR Green I master (Roche Diagnostics, Germany). Data are presented as normalized to ribosomal protein housekeeping gene L32.
Where appropriate, data were analyzed using Student’s t test. Unless otherwise indicated, data represent the mean ± SEM. P < 0.05 was considered significant and indicated by *.