Peptide synthesis, reconstitution and storage
The HLA-A*0201-restricted Influenza A virus matrix protein M158–66 peptide (FluM158–66, GILGFVFTL), the HLA-A*0201-restricted CMV phosphorylated matrix protein 65495–503 peptide (CMVpp65495–503, NLVPMVATV), and the HLA-Cw*0702-restricted irrelevant peptide Mage-12 (Mage12170–178, VRIGHLYIL), were synthesized with a purity from 90 to 100% as determined by High Pressure Liquid Chromatography (HPLC) analysis (Princeton Biomolecules, Langhorne, PA, USA). Each peptide was dissolved in 25% dimethyl sulfoxide (DMSO) at the final concentration of 1 mg/mL. Up to 50% DMSO was added to those mixtures of peptide that did not dissolve immediately. The peptide solutions were stored at 4°C and replaced every month.
After obtaining informed consent, a lymphapheresis was performed on four healthy subjects at the Department of Transfusion Medicine (DTM), Warren G. Magnuson Clinical Center (CC), NIH, Bethesda, Maryland using a CS3000 Plus blood cell separator (Fenwal, Baxter Healthcare Corporation, Deerfield, IL). All donors were HLA-A*0201 homozygous. High resolution HLA class I genotyping was performed by sequence-specific PCR using genomic DNA (HLA Laboratory, DTM, CC, NIH, Bethesda, MD). The presence of IgG and IgM CMV antibodies in each donor was analyzed by passive latex agglutination (CMVSCAN kit, Becton Dickinson Microbiology System, Cockeysville, MD). Two of the three donors were seropositive for CMV. The donors were not tested for antibodies to Influenza A since almost all individuals are seropositive.
Preparation of PBMCs for qRT-PCR kinetic by ex vivo sensitization (EVS)
PBMCs from apheresis products were separated from red blood cells and platelets by Ficoll (Pharmacia Biotech, Wilkstrom, Sweden) density gradient centrifugation. To eliminate residual erythrocytes, an ACK Lysis Buffer (Bio Whittaker, Walkersville, MD) at 1:10 dilution was used while platelets were removed by washing cells twice in HBSS (Biofluid, Rockville, MD) at 800 RPM for 10 min. For the short-term qRT-PCR assays, immediately after PBMCs were isolated, cells were plated in a 96 U-bottom well plate with RPMI (Biofluid, Rockville, MD) complete medium (10% human serum, Gemini Bio-Products, Woodland, CA; supplemented with 1% HEPES, Biofluid) at concentrations of 1 × 105
, 2 × 105
, 1 × 106
, 2 × 106
cells/200μl (starting concentrations of 5 × 105
, 1 × 106
, 5 × 106
, 1 × 107
cell/ml). 2 × 105
cells/200μl has been considered the standard concentration of cells enable to produce a significant amount of IFN-γ transcripts, as previously reported [11
] The cells were incubated overnight (18 hours) at 37°C (humidity 90%, CO2
5%) to minimize background expression of cytokine due to lymphocyte manipulation. The next day, the PBMCs were directly exposed to individual peptides at a final concentration of 1 μl/mL and incubated at 37°C from 3 to 48 hours according to experimental conditions. After each incubation period the cells were harvested and used for the measurement of cytokine transcription by qRT-PCR following total RNA extraction and cDNA synthesis.
Total RNA extraction
After incubation, cells where harvested, washed, and total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. To optimize lysis and homogenization when greater than or equal to 1 × 107 cells were tested, 700 μl of lysis buffer was used. Total RNA was eluded from columns into a final volume of 30 μl RNase free water and stored at -80°C or immediately used for cDNA synthesis.
Complementary DNA synthesis
One to 5 μg total RNA stored at -80°C or rested at 4°C (on ice) immediately after extraction were transcribed into cDNA using the SuperScript pre-amplification system (Invitrogen, Carlsbad, CA). Briefly, total RNA was heated at 70°C for 10 minutes with 1 μl oligo (dT) primers (0.5 μg/μl) in a final volume of 12 μl. After cooling samples at 4°C for 1 min, 7 μl of RT-PCR mixture (2 μl 10 × RT Buffer, 2 μl 25 mmol of MgCl2, 1 μl dNTP 10 mmol, and 2 μl 1 M DTT) were added to samples. They were held at 42°C for 5 min. Then, 1 μl Superscript RT II (50 U/μl) was added to each sample (final volume 20 μl) and cDNA synthesis was performed using these PCR parameters: 42°C for 50 min and 70°C for 15 min. The samples were thus held at 37°C for 20 min with 1 μl RNase to avoid RNA contamination. Synthesized cDNA was stored at -20°C until use.
Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
Measurement of cytokine mRNA expression by qRT-PCR was performed utilizing an ABI prism 7900 Sequence Detection System (Applied Biosystem, Foster City, CA). IFN-
IL-2, IL-4, IL-10, and
-actin genes primers and TaqMan probes (Applied Biosystem, Foster City, CA) (Table ) [9
] were designed to span exon-intron junctions in order to prevent amplification of genomic DNA and to produce amplicons <150 base pairs (bp) enhancing the efficiency of PCR amplification. TaqMan probes were labeled at the 5'-end with the reporter dye molecule FAM (6-carboxyfluorescein; emission λmax
= 518 nm) and at the 3'-end with the quencher dye molecule TAMRA (6 carboxytetramethylrhodamine; emission λmax
= 582 nm). In order to create a standard curve, the cDNA was generated by reverse transcription using a technique identical with the one used for the preparation of test cDNA. Thereafter, cDNA of IFN-
IL-2, IL-4, IL-10, and
-actin genes was amplified by means of regular PCR using the same primers designed for the qRT-PCR and following these parameters: 10 min at 95°C (1 cycle), 30 sec at 95°C, 30 sec at 60°C and 2.5 min at 72°C (40 cycles) and 5 min at 72°C (1 cycle); final volume 50 μl. Amplified cDNA was then purified and quantitated by spectrophotometry (OD260). The number of cDNA copies was calculated using the molecular weight of each gene amplicon. Serial dilutions of the amplified gene at known concentrations were tested by qRT-PCR. Quantitative RT-PCR reactions of cDNA specimens and cDNA standards were conducted in a total volume of 50 μl with 1× TaqMan Master Mix (Applied Biosystem, Foster City, CA) and primers and probes at optimized concentrations (primer 400 nmol and probe 200 nmol) in a 96-well optical reaction plate (Applied Biosystem, Foster City, CA). Thermal cycler parameters were 2 min at 50°C, 10 min at 95°C, and 40 cycles involving denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min; final volume 50 μl. Real time monitoring of fluorescent emission from the cleavage of sequence specific probes by the nuclease activity of Taq polymerase allowed definition of the threshold cycle during the exponential phase of amplification. Standard curves generated for IFN-
IL-2, IL-4, IL-10, and
-actin genes were found to have excellent PCR amplification efficiency (90–100%; 100% indicates that after each cycle the amount of template is doubled) as determined by the slope of the standard curves. Linear regression analysis of all standard curves was > 0.99. Standard curve extrapolation of gene copy number was performed for all genes studied. Normalization of values was performed by dividing the copies of the genes of interest (IFN-γ, IL-2, IL-4 and IL-10) by the copies of the reference gene (β-actin). All standard and sample PCR assays were performed in triplicate and reported as the average.
Quantitative real time PCR primers and probes for evaluating the four cytokines of interest and the reference genes.
Intracellular staining (ICS) assay
Freshly isolated PBMCs from seropositive donors at a concentration of 1.5 × 106 cells/ml of RPMI complete medium (without HEPES, Biofluid, Rockville, MD) were rested overnight in a 14 mL polypropylene tube (Becton Dickinson, Franklin Lakes, NJ) and then stimulated with peptide at a final concentration of 10 μg/mL each tube. One hour after cell activation, Brefeldin A (Sigma, Saint Louis, MI) at a final concentration of 10 μg/mL was added to each tube culture. After 5 more hours (6 hours total) cells were transferred to 5 mL polsterene round bottom tubes (Becton Dickinson, Franklin Lakes, NJ) and cell incubation was stopped washing cells in 2 mL cold PBS for 5 min (1500, 4°C). Pellets were re-suspended in 1 mL PBS containing 1 mmol EDTA and tubes were incubated at 37°C for 10 min to detach adherent cells. After washing cells with 2 mL cold FACS buffer (PBS plus 0.5%BSA) by centrifugation (5 min, 1500, 4°C), cells were maintained in a 100 mL suspension for monoclonal antibodies (MoAb) extra-cellular staining. Cells were stained with 10 μl of CD3-PE and 10 μl CD8-PerCP (BD, Bioscience, San Jose, CA) for 15 min at 4°C (on ice) in the dark. After surface staining, 2 mL of BD FACS Lysing Solution (BD, Bioscience, San Jose, CA) at 1:10 dilution in PBS was added to each tube and the cells were washed by centrifugation (5 min, 1500, 4°C). Permeabilization of cells was performed by re-suspending cells in 500 μl of FACS Permeabilizing Solution 2 (BD, Bioscience, San Jose, CA) at 1:10 dilution in DEPC water, vortexing them for 30 sec and incubating them at room temperature (RT) for 10 min. The cells were thus washed twice by centrifugation (2 mL cold FACS Buffer, 5 min, 1500, 4°C). For intracellular staining, the cells were stained with either 20 μl of human anti IFN-γ-FITC or mouse IgG1 isotype-FITC (BD, Bioscience, San Jose, CA) and incubated for 30 minutes at 4°C (on ice) in the dark. After staining, the cells were washed once (2 mL cold FACS Buffer, 10 min, 1500, 4°C) and immediately analyzed on a flow cytometer or fixed in 200 μl 4% paraformaldehyde in PBS solution, kept at 4°C in the dark and analyzed later.
Peptide-specific ex vivo (EVS) and in vitro sensitization (IVS) for protein release kinetics
Cell supernatants from ex vivo stimulated PBMCs sensitized at different concentrations and times of exposure were used for the measurement of cytokine protein release with an enzyme-linked immunoabsorbent assay (ELISA) kit (Endogen, Woburn, MA). Briefly, after each time point harvested cells were used for mRNA cytokine transcription while supernatant were used for protein release detection.
In parallel, a 2-week in vitro sensitization was performed using PBMCs from CMV-seropositive donors. Cells were plated at a concentration of 3 × 106/2 ml of medium per well in a anti-human CD3 T-cell activation 24-well plate (Becton Dickinson, Bedford, MD) and directly stimulated with 3 μl/mL of peptide (day 0). One day later (day 1), recombinant human interleukin-2 (rhIL-2, 100 U/ml) and recombinant human interleukin-7 (rhIL-7, 10 ng/ml) (PeproTech, Rocky Hill, NJ) were added to the cell culture. rhIL-2 only (100 U/ml) was continuously administered every other day. At day 15, each group of cells was washed and directly re-stimulated in 2 mL of fresh medium with 3 μl/mL of peptide or was not re-stimulated. Eighteen hours after peptide boosting, the in vitro sensitized PBMCs were harvested. Supernatants were collected to measure IFN-γ protein release using the ELISA assay.
Quantitative RT-PCR results were reported as the number of IFN-
, IL-2, IL-4, and IL-10 gene copies normalized by 105
β-actin gene copies and plotted against the different times of exposure. The cytokine production, as shown, represents the mRNA copy number of the gene of interest from stimulated cells relative to the copy number of the gene of interest from unstimulated cells, in both cases after normalization by the gene of reference (β-actin). Figure shows the results as mRNA copy numbers corrected by β-actin. Student's t test was used to compare cytokine mRNA release as well as cytokine protein expression by PBMCs stimulated under different conditions. ELISA results were extrapolated from a standard curve generated by linear regression. Three-color flow cytometry was performed using a FACS-Calibur flow cytometer and data were analyzed using CellQuest software. For each analysis, 250,000 events were acquired. A light scatter region was designed to include only viable lymphocytes. FACS analysis was performed on gated CD3bright
expression allowing the exclusion of residual contaminating cells.
Figure 3 Effect of cell concentration on the specific kinetics of IFN-γ transcript production during CMVpp65495–503 peptide stimulation. Different concentrations of PBMCs from two CMV seropositive donors and one CMV seronegative donor were ex vivo (more ...)