Subjects were recruited from the Cleveland, VA, Case Western Reserve University and University Hospitals of Cleveland according to IRB approved protocols.
Unless otherwise stated, cells were cultured in 96-well round-bottom plates at 37°C in 5% CO2 in X-Vivo 15 serum-free media (Lonza-BioWhittaker, Walkersville, MD, USA) supplemented with Penicillin and Streptomycin (Lonza-BioWhittaker).
Blood was obtained in heparin containing green top Vacutainer tubes (BD Biosciences) and processed within 4 h. PBMCs were isolated from blood samples (geriatric and control) by Ficoll Plaque Plus (GE Healthcare/Amersham Biosciences) density gradient centrifugation according to the manufacturer’s instructions. PBMCs were resuspended in X-Vivo-15 serum-free media and counted. In some experiments, pDCs were depleted from PBMCs using a CD304 (BDCA-4/Neuropilin-1) Microbead Kit from Miltenyi Biotec (Germany) according to the manufacturer’s instructions. Multiple cell types were also purified from PBMCs using positive selection Microbead Kits from Miltenyi Biotec. The cell types isolated included: monocytes (CD14 beads), pDCs (BDCA-4/Neuropilin-1 beads), B cells (CD19 beads), myeloid DCs (mDCs, BDCA-1 beads), and T/NK cells (CD2 beads).
To assess pDC frequency and effectiveness of pDC depletion, 5×105 PBMCs were stained in X-Vivo-15 media using the following antibodies: anti-HLA-DR-Pacific Blue (Biolegend, San Diego, CA, USA), Lineage Cocktail 2 (Lin2)-fluorescein isothiocyanate (FITC, Becton Dickinson, San Jose, CA, USA), and anti-CD123-PE (eBioscience, San Diego, CA, USA). Cells were incubated in the dark on ice for 30 min, washed with PBS, and resuspended in 2% paraformaldehyde (Electron Microscopy Sciences). pDC frequency was determined by gating on HLA-DR+, Lin−, and CD123+ cells on a clinically certified LSRII flow cytometer (Becton Dickinson).
Influenza A/PR/8/34 (H1N1, Charles River, Wilmington, MA, USA), influenza A/Hong Kong/8/68 (H3N2, BEI Resources, Manassas, VA, USA), and influenza A/Denver/1/57 (H1N1, BEI Resources) in allontoic fluid were stored as aliquots at −80°C.
A/PR/8/34 was UV-inactivated with 18,000 J using a Stratagene UV Crosslinker 1800. UV inactivation of virus was assessed by infecting Vero E6 cells (ATCC, Manassas, VA, USA). Vero E6 cells (3×104/well) were plated overnight in 24-well plates in DMEM (Hyclone, Thermo Fisher Scientific, Waltham, VA, USA) containing 10% fetal calf serum (Hyclone). Media was aspirated and replaced with fresh media containing trypsin (20 µg/ml, Thermo Fisher Scientific). Cells were infected with either live or UV-inactivated A/PR/8/34 at 1.6×105 EID50/well and 0.32×105 EID50/ml for 48 h at 37°C in 5% CO2. Cells were washed with warm PBS, fixed with freshly made 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min at room temperature, washed again, and permeabilized with ice-cold methanol for 20 min at −20°C. Cells were incubated for 1 h at room temperature with blocking buffer (PBS containing 10% goat serum (Invitrogen, Carlsbad, CA, USA) and 5% BSA (Sigma-Aldrich, St. Louis, MO, USA)) and then stained with anti-influenza A monoclonal-FITC antibody (Chemicon-Millipore, Billerica, MA, USA) or isotype control mouse IgG1 (BD Pharmingen, San Diego, CA, USA). Cells were incubated overnight at 4°C, washed three times with PBS, and observed with a Leica DMI 6000 B inverted microscope.
To generate FITC-labeled virus, purified A/PR/8/34 (200 µl, Charles River) was dialyzed with PBS pH 8.2 for 2 h, incubated with 10 µl FITC (FLUOS 20 mg/ml stock in DMSO, Roche, Mannheim, Germany) for 25 min at room temperature and dialyzed twice in 200 ml PBS pH 7.3 for 1 h for each exchange. FITC-A/PR/8/34 was frozen at −80°C in aliquots.
Incubation of Cells with Influenza and Ligands
cells/well) in 200 µl of X-Vivo media were incubated with or without A/PR/8/34 (low = 0.32×105
/ml, high = 1.6×105
/ml), A/Hong Kong/8/68 (1.8×105
/ml), A/Denver/1/57 (1.1×106
/ml), type “A” CpG ODN 2216 (3 µg/ml, InvivoGen, San Diego, CA, USA), ODN 2216 control that lacks CpG motif (3 µg/ml, InvivoGen), Guardiquimod (1 µg/ml, InvivoGen), or Poly(I:C)-LMW/LyoVec (4 µg/ml, InvivoGen) for 20 h at 37°C. Concentration of reagents was determined in initial experiments by incubating control PBMCs with various dilutions of reagents (virus, TLR ligand) for 20 h, analyzing IFN-alpha in supernatants and selecting concentrations for the subsequent studies that were on the linear part of the IFN-alpha response (data not shown). For inhibitor studies using chloroquine (10 µM, Sigma-Aldrich) or control and IRS 661 ODNs (1.4 µM, manufactured as phosphorothioate ODNs by Invitrogen [19
]), inhibitors were added 15 min prior to the addition of A/PR/8/34 or ligands and remained in the culture throughout. Supernatants were stored in microfuge tubes or 96-well plates at −80°C for further analyses.
For experiments that compared A/PR/8/34-induced IFN-alpha production by various cell types, the following number of cells were added per well to reflect their anticipated proportion in 5×105 PBMCs: pDCs (2×104 cells/well), B cells (5×104 cells/well), mDCs (2×104 cells/well), monocytes (1×105 cells/well), and T cell/NK cells (2×105 cells/well).
IFN-alpha and IFN-beta levels in supernatants were assessed with the Verikine™ Human IFN-Alpha Multi-subtype ELISA kit (detection limit 12.5 pg/ml), and Verikine™ Human IFN-Beta ELISA kit (detection limit 25 pg/ml, PBL Interferon Source, Piscataway, NJ, USA), respectively. The human IFN-alpha kit from eBioscience was used for the pDC depletion, cell type specific, and inhibitor experiments. Kits were used according to the manufacturer’s instructions. All samples and standards were inactivated with a final concentration of 0.5% Triton X-100 (Bio-Rad, Richmond, CA, USA).
Analysis of Uptake of Influenza by pDCs
PBMCs (1×106) were pulsed with or without FITC-A/PR/8/34 for 1 h at 37°C. Cells were washed once with media and chased for 30 min at 37°C. Cells were then washed twice with media, resuspended in MACS buffer (Miltenyi Biotec), and stained with the following antibodies: anti-HLA-DR-Pacific Blue (Biolegend), BDCA-4-APC (Miltenyi Biotec), and CD123-PE-Cy7 (eBioscience). Cells were incubated in the dark on ice for 30 min, washed with PBS, resuspended in 2% paraformaldehyde (Electron Microscopy Sciences), and analyzed by flow cytometry.
PBMC (2×106) were placed in 400 µl X-Vivo medium±1.6×105 EID50/ml A/PR/8/34 at 37°C for 8 h in microfuge tubes. Cells were stained with LIVE/DEAD Violet Fixable Dead Cell Stain Kit (Molecular Probes, Invitrogen), Lin-FITC, CD123-PE, HLA-DR-PECY5 (BD), and Annexin V-APC (eBioscience) in high calcium concentration annexin buffer according to the manufacturer’s instructions. Cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences) in annexin buffer and pDCs analyzed for Annexin V staining by flow cytometry.
The magnitude of IFN-alpha production and pDC frequency was compared between geriatric and control patients. The distribution of these data were evaluated using scatter plots and were shown to be non-normally distributed. Therefore, Mann–Whitney, nonparametric two-tailed t
tests were used for statistical comparisons and graphics. Results in the text present both median and mean. All analyses were performed using GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA, USA, (www.graphpad.com