Murine cholangiocyte 603B line 70 was kindly provided by Yoshiyuki Ueno (Tohoku University, Sendai, Japan) and G. Gores (Mayo Clinic, Rochester, MN). The murine iNKT hybridoma cells (DN32) was provided by Dr Albert Bendelac (University of Chicago, Chicago, IL) and human hepatic stellate cell line (LX2) was obtained from Dr. S.L. Friedman (Mount Sinai School of Medicine, NY) 71.
Primary mouse hepatic NKT cell isolation and culture
Primary hepatic leukocytes were isolated from C57BL/6 mice following a series of enzymatic digestion (collagenase 0.05%, DNAse I 0.002%), mechanical digestion (Seward Stomacher 80; Biomarker Lab systems) and 30% Percoll density gradient. For each experiment, leukocytes were isolated from eight healthy adult mice and then pooled. All experiments were replicated at least one time. Hence, over 100 mice were used for to assess expression of Hh signaling components and analyze effects of Hh pathway activation on primary hepatic iNKT cells (see below).
Immediately after isolation, hepatic leukocytes were washed, re-suspended in flow cytometry buffer (eBiosciences) (1×106 cells/mL) and incubated with anti-mouse CD16/32 (Mouse Fc Block, 1 μg/106 cells; BD) for 30 min. Leukocytes were then stained with FITC-conjugated CD3 (Santa Cruz; sc18843) and PE-conjugated PBS57loaded-CD1d-tetramers (provided by NIH, Atlanta, GA) and sorted using FACS. FACS was performed at the flow cytometry core facility at the Human Vaccine Institute, Duke University Medical Center, using the FACS VantageSE (Beckton Dickinson). Cells were kept at 4°C throughout the purification protocol. Primary iNKT cells were sorted as CD3+ CD1d-tetramer+ double-positive cells. Purity of sorted NKT fractions was checked by FACS re-analysis (>90% purity).
Primary hepatic iNKT cells were cultured in complete NKT media (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL streptomycin and 100 units/mL penicillin, 10 mM HEPES, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate and 5.5 uM 2-mercaptoethanol) 72. For growth and viability assays, cells were cultured in CD3-coated, sterile, 96-well, black, tissue culture plates (Costar 3603, Corning, NY), at a concentration of 1×105 cells per well, with recombinant mouse IL-2 (10 ng/mL; Biolegend), recombinant mouse IL-12, (1 ng/mL; R&D) and anti-CD28 (1μg/mL; eBiosciences).
In additional experiments, primary hepatic leukocytes were incubated in RPMI with the NKT cell ligand, αGalCer (100 ng/mL), in the absence of anti-CD3, anti-CD28, IL-2 and IL-12, for 24 h. 100 ng/mL of αGalCer was used in experiments, as this dose has been shown to elicit the maximum iNKT responses 54, 73. The co-expression of CD3 and CD1d-tetramers was used to identify iNKT cells within each culture.
In experiments where recombinant Shh protein (0–1000 ng/mL) (StemCell Tech, Canada), 5E1-neutralizing antibody (10 μg/mL) (Iowa Hybridoma bank, University of Iowa) or isotype control antibodies were utilized, these were added at the initiation of cell-culture. As the range of Shh concentration in disease states is currently unknown, we have utilized a spectrum of Shh dosing (0–1000 ng/mL) as previously described 36, 74. In all experiments, cultures were harvested for analysis 24 or 72 h later, as specified. In each experiment, all assesses were performed in triplicate. Every experiment was replicated at least one time.
DN32 hybridoma cells were cyto-spun onto VWR superfrost® plus micro slides (VWR) using the Shandon Cytospin 4 (Thermo Scientific, UK) at 300 rpm for 3 min. Slides were air-dried and then fixed with cold (−20°C) methanol for 5 min. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide and non-specific binding of antibodies blocked using Dakocytomation serum-free protein block (Dako). Slides were then incubated with primary antibodies over night in 4°C. After washing with TBS-Tween20 0.1%, HRP-conjugated secondary antibodies were added for 30 min. Antigens were detected by the addition of liquid diaminobenzidine substrate (Dako), with haematoxylin (Sigma, MHS16) counterstain. Isotype-matched antibodies were used as negative controls. Primary antibodies used were as follows: Shh H-160 (200 μg/mL, 1:100 dilution; Santa Cruz), ptc G19 (200 μg/mL, 1:50 dilution; Santa Cruz) and Gli2 (1 mg/mL, 1:50 dilution; abCam). Secondary antibodies used were as follows: ECL™ donkey anti-rabbit IgG HRP-linked whole antibody (1:1000 dilution, Amersham, GE Healthcare, UK) and donkey anti-goat IgG HRP-linked antibody (200 μg/mL, 1:1000 dilution; Santa Cruz).
DN32 hybridoma cells were homogenized using standard RIPA buffer (TBS, 1% NP-40, 0.1% SDS) containing Protease Inhibitor Cocktail Tablets from Roche (Indianapolis, IN). Protein concentration was measured using BCA Protein Assay Kit from Pierce Biotechnology (Rockford, IL). Approximately 15–20 μg of protein was loaded per lane on Tris-Glycine 4–20% gels (Invitrogen, Carlsbad, CA). Separated proteins were then transferred to nitrocellulose membranes (0.45 μm, Invitrogen). After blocking with 5% non-fat milk (Carnation, Swampscott, MA) in TBS (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCL) containing 0.1% Tween-20 (TBS-T), nitrocellulose membranes were incubated with primary antibodies (Shh: 1:100 dilution) overnight at 4°C. ECL™ donkey anti-rabbit IgG HRP-conjugated secondary antibody (Amersham, UK) was added after washing, at a dilution of 1:2000 in 5% non-fat milk for 1 h. SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) was used to detect specific antibody-HRP complexes.
Intrahepatic leukocytes were labeled with anti-mouse CD3-FITC (Santa Cruz), PBS-loaded CD1d-tetramers-allophycocyanin or PE (NIH tetramer core facility, Atlanta), Shh-PE (R&D systems), CD4-Pacific blue (BioLegend), CD8-allophycocyanin (AbCam) or matched isotype controls and analyzed using the FACS VantageSE (Beckton Dickinson).
iNKT cell viability assays
iNKT cells (primary murine liver iNKT cells or DN32 iNKT cells) were cultured in 96-well tissue culture plates, as above. 1×105 cells were seeded in each well and either vehicle (no Shh) or a various doses of Shh (StemCell Tech, Canada, 10–1000 ng/mL) were added. Cultures were harvested 72 h later and iNKT cell numbers were determined by the commercially available Cell Counting Kit-8 (CCK-8, Dojindo, Maryland). Briefly, 10 μL of the CCK8 substrate was added to cell cultures 72 h after plating (end of incubation period) and absorbance in each well measured. A calibration curve was prepared using wells containing a fixed numbers of viable cells. A FLUOstar OPTIMA micro-plate reader (BMG Labtech, Durham, NC) was used for absorbance measurements.
iNKT cell apoptosis assays
Apoptotic activity was assayed using the Apo-ONE Homogeneous Caspase 3/7 Apoptosis Assay (Promega, Madison, WI), according to the manufacturer's instructions. Results were expressed as relative fluorescent units (RFU). A FLUOstar OPTIMA micro-plate reader (BMG Labtech, Durham, NC) was used for fluorescence measurements. Apoptotic activity of primary hepatic leukocyte in culture was assessed by FACS analysis of Annexin-V-FITC (BioVision, CA) staining. Identification of iNKT cell fraction was determined by CD3-CD1d-tetramer double positive staining.
mRNA quantification by real-time PCR
Total RNA was extracted from DN32 hybridoma cells using Trizol (Invitrogen). 1.5 μg of RNA was reverse-transcribed using random primers and Superscript RNase H-reverse transcriptase (Invitrogen). Samples were incubated at 25°C for 15 min, 42°C for 55 min; reverse transcriptase was inactivated by heating at 70°C for 15 min followed by cooling at 4°C for 10 min. mRNA were quantified by real-time reverse-transcriptase-PCR per the manufacturer's specifications (Eppendorf, Mastercycler Real-Time PCR). Amplification was performed using SYBR Green PCR Master Mix (Applied Biosystems). Five microliters of diluted cDNA samples (1:5 dilution) were used for quantitative two-step PCR (a 10-min step at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 65°C) in the presence of 400 nM specific forward and reverse primers, 5 mM MgCl2, 50 mM KCl, 10 mM Tris buffer (pH 8.3), 200 μM dATP, dCTP, dGTP, and 400 μM dUTP and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin Elmer Applied Biosystems). Each sample was analyzed in triplicate. S9 (mouse) or 18S (human) rRNA was used as housekeeping control. Threshold cycles (Ct) were automatically calculated by the iCycler iQ Real-Time Detection System. Ct values were normalized to the housekeeping control to give a relative mRNA level. Sequences of primers used were as follows: MOUSE: 9S: sense: GGGAGCTGTTGACGCTAGAC, anti-sense: CGGGCATGGTGAATAGATTT; shh: sense: CTGGCCAGATGTTTTCTGGT, anti-sense: TAAAGGGGTCAGCTTTTTGG; Gli1: sense: AACTCCACAGGCACACAGG, anti-sense: GCTCAGGCTTCTCCTCTCTC; ptc: sense: ATGCTCCTTTCCTCCTGAAACC, anti-sense: TGAACTGGGCAGCTATGAAGTC; IL-4: sense: TCCTGCTCTTCTTTCTCG, anti-sense: CTTCTCCTGTGACCTCGTT; IL-10: sense: TGTGAAAATAAGAGCAAGGCAGTG, anti-sense: CATTCATGGCCTTGTAGACACC; IL-13: sense: CAGCATGGTATGGAGTGTGG, anti-sense: TGGGCTACTTCGATTTTGGT; IFN-γ: sense: CATCAGCAACAACATAAGCGTCA, anti-sense: CTCCTTTTCCGCTTCCTGA; TNF-α: sense: TCGTAGCAAACCACCAAGTG, anti-sense: AGATAGCAAATCGGCTGACG; CD69: sense: GTACAATTGCCCAGGCTTGT, anti-sense: TCCAATGTTCCAGTTCACCA; CD154: sense: CAGTGGGCCAAGAAAGGATA, anti-sense: GGTATTTGCCGCCTTGAGTA; CD178: sense: CATCACAACCACTCCCACTG, anti-sense: GTTCTGCCAGTTCCTTCTGC; SOCS2: sense: TCAGCTGGACCGACTAACCT, anti-sense: TGTCCGTTTATCCTTGCACA; SOCS3: sense: AGCTCCAAAAGCGAGTACCA, anti-sense: TGACGCTCAACGTGAAGAAG; HUMAN: Gli1: sense: GTGCAAGTCAAGCCAGAACA, anti-sense: ATAGGGGCCTGACTGGAGAT; ptc: sense: ACAAACTCCTGGTGCAAACC, anti-sense: CTTTGTCGTGGACCCATTCT; 18S: sense: TGCATGTCTAAGTACGCACG, anti-sense: TTGATAGGGCAGACGTTCGA.
Human peripheral blood iNKT cells
NKT cells were isolated from healthy donor peripheral blood and iNKT expanded with αGalCer (Axxora, San Diego). Briefly, PBMC were isolated from Buffy Coats by Ficoll-Hypaque density centrifugation. NKT cells were selected by Mo-Flo cell sorting CD3+CD56+ cells. For expansion of iNKT, cells were first cultured for a 2-wk period in RPMI-1640 containing L-glutamine and 10% human serum (HD Supplies) in the presence of αGalCer at 100 ng/mL, supplemented with 100 U/mL IL-2 (Peprotec). iNKT cells were then selected by Mo-Flo cell sorting CD3+ cells expressing the Vα24/Jα18 iNKT TCR (6B11; BD Biosciences). Blood samples were obtained with informed consent of donors and in accordance with local ethical approval 04/Q2708/41 and REC 2003/242 from the South Birmingham Research Ethics Committee, UK.