Reagents and animals
C57BL/6J and C57BL/10ScnJ mice were from Jackson Laboratories (Bar Harbor, ME). MyD88−/− mice were from Shizuo Akira. All mice were housed under specific pathogen-free conditions. CpG-A oligodeoxynucleotide (ODN) 2336 (5’-ggG GAC GAC GTC GTG ggg ggG-3’) was from Sigma-Aldrich (St. Louis, MO). Synthetic triacylated lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine (Pam3CSK4) and ultrapure LPS from E. coli 0111:B4-were from Invivogen (San Diego, CA). Recombinant human IFN-β was from Peprotech (Rocky Hill, NJ). Recombinant human IFN-α2a and murine IFN-α4 and IFN-β were from PBL InterferonSource (Piscataway, NJ).
Cells and media
Incubations were carried out at 37°C with 5% CO2. DCs were grown in complete RPMI medium consisting of RPMI with L-glutamine and glucose, supplemented with 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1% penicillin/streptomycin (all from Hyclone, Logan, UT). Ag processing assays were done in complete DMEM medium consisting of DMEM with L-glutamine and glucose (Hyclone), supplemented with 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES (Hyclone) and 1% penicillin/streptomycin.
To make murine DCs, bone marrow cells were isolated from mouse femurs and tibias. Red blood cells were lysed with ACK lysis buffer (Lonza, Walkersville, MD). To make DCs derived from Fms-like tyrosine kinase ligand (Flt3L)-stimulated murine bone marrow (“Flt3L DCs”), marrow cells were cultured with Flt3L-Ig fusion protein (1 µg/ml) (Bioexpress, West Lebanon, NH) to produce a DC culture containing myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). On day 8, non-adherent cells were collected. Alternatively, marrow cells were cultured with J558L cell-conditioned medium (containing GM-CSF) diluted in complete RPMI medium to produce “GM-CSF DCs”, which are mDCs (49
). On day 7, non-adherent cells were collected.
Studies with cells from human donors were approved by the University Hospitals Case Medical Center Institutional Review Board. Blood was harvested in heparinized syringes. PBMCs were collected as reported (50
). Human mDCs were purified by positive selection for CD1c (BDCA-1) with a kit (Miltenyi, Auburn, CA) as reported (51
). DCs were cultured in IMDM (Lonza) supplemented with 5% pooled human serum (Gemini Bioproducts, West Sacramento, CA) and GM-CSF (Berlex, Montville, NJ, 200 U/ml).
The CD4OVA.1 and CD4OVA.2 T hybridoma cell lines were generated in these studies by incubating splenocytes from OT-II TCR transgenic mice in vitro
with OVA(323–339) peptide (100 nM) for 4 d and immortalizing proliferating T cells by fusion with BW1100 cells. CD4OVA.1 and CD4OVA.2 T hybridoma cells were used to detect OVA(323–339):I-Ab
complexes in Ag processing assays with murine DCs. F9A6 T hybridoma cells (52
) were used to detect Ag85B(97–112):HLA-DR1 complexes presented by human DCs.
Murine DCs (2 – 4 × 105/well) were incubated with medium, Pam3CSK4, LPS or IFN-β for 48 h. Cells were washed with PBS with 0.1% BSA (Sigma-Aldrich). DCs were incubated for 15 min on ice in Fc block (anti-CD16, anti-CD32) (BD Biosciences, San Jose, CA) and stained for an additional 30 min on ice with the following antibodies: phycoerythrin-anti-I-A/I-E (BD Biosciences), fluorescein-anti-CD80 (eBioscience), phycoerythrin-anti-CD86 (eBioscience), phycoerythrin-cyanin7-anti-CD11b (eBioscience), and allophycocyanin-anti-CD11c (eBioscience). Human DCs (2–5 × 104) were incubated in polypropylene tubes for 16 – 18 h in the presence of GM-CSF (200 U/ml) with medium, IFN-α, IFN-β, or Pam3CSK4, washed, and stained with phycoerythrin-anti-CD80 (Biolegend, San Diego, CA), phycoerythrin-Texas Red-anti-HLA-DR (Invitrogen), phycoerythrin-cyanin7-anti-CCR7 (BD Biosciences), and Pacific Blue-anti-CD86 (Biolegend). Cells were washed with PBS with 0.1% BSA, fixed in 1% paraformaldehyde (Polysciences, Warrington, PA), and analyzed on a BD LSR-II flow cytometer (BD Biosciences). Data were analyzed using Winlist (Topsham, ME) or Flowjo (Tree Star, Ashland, OR). Mean fluorescence intensity (MFI) with isotype control Ab was subtracted from MFI with specific Ab to determine “specific MFI”.
After 7 d in culture, murine DCs were sorted with CD11c microbeads (Miltenyi MACS) and incubated with agonists for 24 h. DCs were washed twice in PBS with 0.1% BSA, incubated on ice for 15 min in Fc block, incubated 30 min with unconjugated anti-I-Ab or isotype control (BD Biosciences), washed 4 times in ice-cold PBS with 0.1% BSA, and resuspended in complete medium. Duplicate samples were cultured at 37°C for 0, 45 or 90 min, chilled on ice, and stained with AlexaFluor488 rabbit anti-mouse Ab (Invitrogen). Samples were washed, fixed, and analyzed by flow cytometry as above. Specific MFI was normalized to the level at time 0.
Ag presentation assay
Murine DCs (5–10 × 104
/well) were incubated for 20–24 h in complete DMEM medium with IFN-β, Pam3
, LPS, or CpG-A ODN with or without OVA. Cells were washed and fixed with 1% paraformaldehyde or washed and incubated 4 h with OVA prior to fixation. DCs were washed, and CD4OVA T hybridoma cells (105
/well) were added and incubated for 24 h. Alternatively, human mDCs (104
per well) were incubated for 16 – 18 h with Pam3
or IFN-α in the continued presence of GM-CSF (200 U/ml), washed and incubated with Mycobacterium tuberculosis
Ag85B (purified as described (53
)) and F9A6 T hybridoma cells (50
/well) for 24 h. Supernatants (100 µl) were harvested, frozen, thawed and used in a CTLL-2 bioassay for IL-2 (54
). Alamar blue (15 µl, Invitrogen, Carlsbad, CA) was added for the last 24 h. Alamar blue reduction was analyzed by reading the difference between OD at 570 nm and OD at 595 nm on a Biorad Model 680 plate reader.
Quantitative RT-PCR (qRT-PCR)
DCs (3 – 5 × 106
/well) were incubated for 18–24 h with medium, IFN-α, IFN-β, Pam3
, LPS, or CpG-A ODN. RNA was purified using the Qiashredder kit and RNEasy Plus kit (Qiagen). RNA yield was quantified by OD and 1 µg of each sample was used to synthesize cDNA using the Quantitect Reverse Transcription Kit (Qiagen); 4% of the total cDNA from each reaction was used in a quantitative PCR reaction with 500 nM of 5’ and 3’ primer for each gene and SYBR green detection (Bio-Rad, Hercules, CA) using the Bio-Rad CFX96 Real Time fluorescence detection system. All conditions were tested in triplicate. Primer sequences are as follows. GAPDH: sense 5’-AACGACCCCTTCATTGAC-3’, antisense 5’-TCCACGACATACTCAGCAC-3’. Total CIITA mRNA: sense 5’-ACGCTTTCTGGCTGGATTAGT-3’, antisense 5’-TCAACGCCAGTCTGACGAAGG-3’. The β chain of MHC-II I-Ab
): sense 5’-AAGATGTTGAGCGGCATCGG-3’, antisense 5’-GTCAGGAATTCGGAGCAGAG-3’. MARCH1: sense 5’-ATGCACGGACAAAGCAATGG-3’, antisense 5’-GTGTGAAGTCACGGGCAATC-3'. Primers were previously described (31
) or designed using Clone Manager Suite v7.11 and Primers Designers v5.11 (Scientific & Educational Software, Cary, NC). A BLAST search was performed to verify specificity.
DCs were incubated with medium, IFN-β, LPS or Pam3CSK4 for 20 h at 37°C. Subsequent steps for purification of nuclei were performed at 4° C. Cells were washed in PBS with protease inhibitors (protease inhibitor cocktail (P8340, Sigma-Aldrich) plus 1 mM NaF, 1 mM PMSF and 10 nM calyculin A), pelleted and incubated in 1 ml Nuclei EZ Prep Lysis Buffer (Sigma-Aldrich) with protease inhibitors for 10 min. After centrifugation at 500 × g for 5 min, supernatants were removed and pellets were resuspended in 1 ml Nuclei EZ Prep Lysis Buffer with protease inhibitors for 10 min to remove residual cytosolic material. Nuclei were collected by centrifugation at 500 × g for 5 min, solubilized in 10% glycerol, 2% SDS, 63 mM Tris-Cl, then sonicated and boiled. Protein concentrations were determined using the BCA assay (Thermo Scientific, Rockford, IL) to standardize loading for analysis by 12% SDS-PAGE and Western blotting.
per sample) were incubated for 24 h with medium, IFN-β, LPS or Pam3
, washed once with PBS, and then lysed in 1% Triton X-100 with 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, 25 mM NEM and protease inhibitor cocktail. Samples were centrifuged for 15 min at 14,000 × g
. Supernatants were pre-cleared for 1 hr with 50 µL of rec-Protein G Sepharose 4B (Invitrogen). Immunopreciptiations were performed for 90 min at 4° C using Protein G Sepharose and 4 µg of control antibody (mouse IgG) or Y3P, specific for I-Ab
). The beads were washed three times with ice-cold 0.1% Triton X-100 in 150 mM NaCl, 25 mM Tris-Cl, pH 7.4 and then resuspended in non-reducing sample buffer for analysis by 10% SDS-PAGE and Western blotting.
Samples were analyzed by SDS-PAGE and transferred to Immobilon membranes (Millipore, Billerica, MA). Membranes were blocked using PBS-T with 5% dry milk with the exception of anti-ubiquitin blots, which were performed using 3% BSA in PBS-T. Primary Abs were specific for actin (clone I-19, Santa Cruz Biotechnology, Santa Cruz, CA), C/EBPβ (clone C-19, Santa Cruz Biotechnology), ubiquitin (biotinylated-P4D1, Covance, Princeton, NJ), or I-Aβb (KL295, ATCC). Detection was performed using HRP-conjugated neutravidin (Invitrogen) or HRP-conjugated secondary antibodies (eBioscience) followed by ECL Western Blotting substrate (Thermo Scientific).
DCs (3 – 5 × 106/well) were incubated for 24 h with medium, IFN-α, IFN-β, Pam3CSK4, LPS, or CpG-A ODN. Cells were fixed and permeabilized using a “Fix and Perm” kit (Invitrogen). After permeabilization, cells were stained with biotinylated anti-I-A/I-E (eBioscience) for 30–60 min, washed with PBS, 0.1% BSA, and incubated with streptavidin-Alexafluor488 (Invitrogen) for 30 min. Cells were washed with PBS, 0.1% BSA and mixed with Prolong Gold mounting medium with DAPI (Invitrogen). The resulting cell suspension was placed on glass slides and covered with a poly-lysine coated coverslip (BD Biosciences). Images were captured using a Leica TCS-SP microscope. Visual counts were performed to determine the number of cells with cell-surface MHC-II expression, and at least 100 cells were counted per condition.
DCs (3 – 5 × 106/well) were incubated for 24 h with medium, IFN-α, IFN-β, Pam3CSK4, LPS, or CpG-A ODN. Cells were surface-stained for 30 min on ice with a combination of phycoerythrin-cyanin7-anti-CD11b and phycoerythrin-cyanin7-anti-CD11c (eBioscience). Cells were washed, fixed, permeabilized as for confocal microscopy, and stained for 30 min with phycoerythrin-cyanin5-anti-IA/I-E (eBioscience) and a combination of Alexafluor488-anti-LAMP1 and Alexafluor488-anti-LAMP2 (eBioscience). DAPI (10 µg/ml) was added and cells were analyzed by imaging flow cytometry on an Imagestream cytometer (Amnis, Seattle, WA). Data were analyzed using the IDEA software (Amnis).