Antibodies and Reagents
Biotinylated goat anti-human AR and biotinylated goat normal IgG (isotype control), and APC conjugated anti-human CCR4 (205410) were obtained from R&D Systems (Minneapolis, MN). LEAF™ purified anti-human CD3ε (OKT3), APC-Cy7 conjugated anti-human CD4 (RPA-T4), Pacific Blue or APC-Cy7 conjugated anti-human CD69 (FN50), PE-Cy5 conjugated anti-human CD154 (24–31), PerCP-Cy5.5 conjugated anti-human CD27 (O323), Alexa Fluor 700 conjugated anti-human CD62L (DREG-56), Pacific Blue conjugated anti-human CXCR3 (TG1/CXCR3), PE-Cy7 or PE-Cy5 conjugated anti-human CD123 (6H6), Alexa Fluor 700 conjugated anti-human IL-2 (MQ1-17H12), FITC conjugated anti-human IL-4 (MP4-25D2), and PerCP-Cy5.5 conjugated anti-human IL-17A (BL168) were purchased from BioLegend (San Diego, CA). Functional grade purified anti-human CD28 (CD28.2), PE conjugated anti-human CD45RA (HI100), FITC conjugated anti-human CD45RO (UCHL1), PE-Cy5 conjugated anti-human CD19 (HIB19), PE-Cy7 conjugated anti-human IFNγ (4S.B3), and APC-conjugated streptavidin were obtained from eBioscience (San Diego, CA). Alexa Fluor 488 conjugated anti-human CXCR5 (RF8B2) and PE-Cy7 conjugated anti-human CCR7 (3D12) were purchased from BD Bioscience (San Jose, CA). Qdot® 605 conjugated anti-human CD3 (UCHT1), PE-Texas Red and Qdot® 705 conjugated anti-human CD8α (3B5), PE-Texas Red conjugated anti-human CD4 (S3.5), TRI-COLOR and Qdot® 800 conjugated anti-human CD14 (TüK4), Qdot® 655 conjugated anti-human CD45RA (MEM-56), Pacific Blue conjugated anti-human TNFα (MP9-20A4), and LIVE?DEAD Fixable Yellow Dead Cell Stain Kit were obtained from Invitrogen (Carlsbad, CA).
7-Aminoactinomycin D (7-AAD) and TAPI-1 was obtained from Calbiochem (Gibbstown, NJ). cAMP agonist (8-CPT-cAMP) and cAMP antagonist (Rp-8-Br-cAMP) were purchased from BioLog (Bremen, Germany). Phorbol 12-myristate 13-acetate (PMA), ionomycin, monensin, PGE2, forskolin and 3-Isobutyl-1-methylxanthine (IBMX), adenosine were obtained from Sigma (St.Louis, MO).
Human Peripheral Blood T cell Isolation and Activation
Heparinized blood was obtained from healthy donors under a protocol approved by the University of Rochester Medical Center Research Subjects Review Board. Written, informed consent was obtained from all subjects. PBMC were isolated by Ficoll-Hypaque (Cellgro, Herndon, VA) density gradient centrifugation. Cells were suspended in complete RPMI-8 (RPMI-1640 medium containing 100U penicillin/streptomycin (Invitrogen) supplemented with 8% heat-inactivated fetal calf serum (FCS, HyClone, Logan, UT)). In the experiment treating cells with adenosine, serum-free medium X-VIVO™ 20 (Lonza, Walkersville, MD) was used.
To purify human naïve and memory CD4 T cells from PBMC, fresh PBMC were stained with antibodies specific for cell surface markers and CD4+CD8-CD14-CD123-CD45RA+CD45RO- (naïve CD4 T cells) and CD4+CD8-CD14-CD123-CD45RA-CD45RO+ (memory CD4 T cells) were sorted on a FACSAria (BD Bioscience, San Jose, CA).
In vitro Induction of Allogeneic Th1 and Th2 cell Lines
Purified human naïve CD4 T cells were stimulated with irradiated (100Gy) allogeneic Epstein-Barr virus (EBV) – transformed B cells (1
1 ratio) in complete RPMI-8 medium at 105
cells/mL in round-bottom 96-well plate. Th1-biased cultures contained recombinant human IL-2 (5 ng/mL, PeproTech), recombinant human IL-12 (20 ng/mL, PeproTech) and anti-IL-4 (5 µg/ml, R&D Systems). Th2-biased cultures contained recombinant human IL-2 (5 ng/mL), recombinant human IL-4 (20 ng/mL, R&D Systems), anti-IL-12 (5 µg/ml, ebioscience) and anti-IFNγ (5 µg/ml, R&D Systems). Fresh medium containing 5 ng/mL IL-2 was added if necessary to cultures showing strong proliferation. The cultures were restimulated and expanded every seven days.
To enrich for cells with the Th1 or Th2 phenotypes, after 14 days priming, Th1 and Th2 cells were stimulated with plate-bound anti-CD3+ anti-CD28 for 8 hours. IFNγ+ Th1 cells and IL-5+ Th2 cells were stained and sorted by the MACS cytokine secretion assay (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The enriched IFNγ+ Th1 cells and IL-5+ Th2 cells were expanded as previously for 14 days.
Intracellular Staining and Cell Surface Staining
For intracellular staining (ICS) of AR, PBMC (106 per well) were stimulated with medium alone, anti-CD3 (5 µg/ml) + anti-CD28 (1 µg/ml), Staphylococcal enterotoxin B (SEB, 1 µg/ml), PMA (10 ng/mL) + ionomycin (500 ng/mL), influenza H1N1 peptides (H1N1 [New Caledonia/New York], 20 ng/mL/peptide), Fel d1 (50 µg/mL, INDOOR, Charlottesville, VA), Der p1 (50 µg/mL, INDOOR) or Tetanus peptides (3 µg/mL/peptide) in round-bottom 96-well plate (Costar, Corning Inc., Corning, NY). Th1 or Th2 cultures were treated with medium alone or PMA + ionomycin. After 10 hours stimulation (with 2 µM monensin present for the last 8 hours), the cells were first stained with LIVE?DEAD Fixable Yellow Dead Cell Stain Kit, and then stained for cell surface markers CD4, CD8, CD14, CD123, and CD45RA. After cells were fixed and permeabilized using Fix-Perm (BD Bioscience), the cells were stained with anti-AR, anti-IFNγ, anti-IL-2, anti-IL-4, anti-IL-17A, anti-TNFα, anti-CD3 and anti-CD69 (or anti-CD154) intracellularly.
For cell surface staining of AR, PBMC were stimulated with medium alone or 1 µg/ml SEB in the presence or absence of 50 µM TAPI-1, an ADAM17 protease inhibitor 
, for 6, 12, and 24 hours. After stimulation, the cells were stained with antibodies against AR, CD3, CD4, CD8, CD14, CD123, CD69 and 7-AAD. Data were acquired using an LSR II flow cytometer (BD Bioscience), and analyzed with FlowJo software (Tree Star Inc., Ashland, OR).
Quantitative Real Time PCR for Gene Expression
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared by reverse transcription from total RNA using MultiScribe™ Reverse Transcriptase (Applied Biosystems, Foster City, CA) with random hexamer primers (Applied Biosystems). Quantitative real-time PCR (RT-PCR) was performed using the Applied Biosystems 7900HT Sequence Detection System. Primers and probes specific for AR, Heparin-binding EGF-like Growth Factor (HB-EGF), IL-2, IFNγ, IL-3, IL-4, IL-5, IL-10, IL-13, CD3d, EGF, Neuregulin (NRG) 1–4, epiregulin (EREG), betacellulin (BTC) and TGFα were all obtained from TaqMan Gene Expression Assays (Applied Biosystems). CD3d gene expression was used as an endogenous control for normalizing mRNA amounts. All samples were run in duplicate and data were analyzed using SDS software (Applied Biosystems).
Measurement of AR Release
Purified CD4 T cells were treated with medium alone or CD3/CD28 beads (cells:beads 2
1) in the presence or absence of 50 µM TAPI-1. After 24 hours, the supernatants were collected and AR was measured using the human Amphiregulin DuoSet ELISA Development kit (R&D Systems). The detection limit of the assay was 7.8 pg/mL. Because the antiserum for the ELISA was produced by immunization with bacterial recombinant human AR, and the standard is also non-glycosylated AR, this ELISA probably underestimates the concentration of normal human glycosylated AR.