Reagents and equipment
Human recombinant cytokines (GM-CSF, IL-4, IL-1β, TNF-α, and IL-6) were purchased from R&D Systems (Minneapolis, MN). Prostaglandin E2 (PGE2) was purchased from Sigma-Aldrich (St. Louis, MO). Dendritic cell culture medium (DC-CM) consisted of Iscove's Modified Dulbecco's Medium (IMDM) containing Glutamax, 20 μg/ml gentamycin, 50 μM 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA), and 2% normal human AB serum (Valley Biomedical, Winchester, VA). Roller bottles (850 cm2 or 490 cm2) with vented caps were obtained from Fisher-Costar (Houston, TX). A Stovall Low Profile Roller apparatus (Stovall Life Science Inc., Greensboro, NC) was used for the roller bottle cultures. Flat-bottom static T-175 culture flasks (175 cm2 area) with vented caps were obtained from Nunc (Rochester, NY). All flow cytometry antibodies and 7-aminoactinomycin D (7-AAD) were purchased from BD Biosciences (La Jolla, CA).
Sources of PBMC for DC generation
PBMC were obtained from peripheral blood leukopheresis products obtained from non-mobilized normal donors (LifeBlood, Memphis, TN), or G-CSF-mobilized normal donors (AllCells, Berkeley, CA). Products were collected in the presence of Anticoagulant Citrate Dextrose Formula A (Gambro). In addition, peripheral blood buffy coats (Gulf Coast Regional Blood Bank, Houston, TX) were also used for some experiments. In some experiments HLA-A*0201+ positive non-mobilized leukopheresis products were used to generate DC (LifeBlood, Memphis, TN). The HLA-A*0201 status was further confirmed by flow cytometry after receipt of the sample in the laboratory. All leukopheresis products and buffy coats were used within 24 hours post-collection. The PBMC were isolated by diluting with HBSS, centrifuged at 400 × g for 20 min over Histopaque-1077 (Sigma-Aldrich). The interface cells were collected, pooled, and washed with HBSS until the contaminating platelets were removed. PBMC not used immediately were frozen in human AB serum with 10% DMSO (33.3 × 106) cells/ml and stored in the vapor phase of liquid nitrogen.
Dendritic cell culture in roller bottles
Washed PBMC from leukopheresis products or buffy coats were diluted to 30 × 106 cells/ml in DC-CM and 30 ml (900 × 106 cells) were seeded into 850 cm2 roller bottles with vented caps (Fisher-Costar, Houston, TX). The bottles were placed on the roller bottle apparatus in a 37°C, 5% CO2 incubator and rolled at low speed (1 rpm) for 2 to 3 h. The bottles were then taken out and agitated to loosen any non-adherent cells and the floating cells removed. The bottles were then washed 2 times with 80–100 ml warm DC-CM by rolling the bottle inside a laminar flow hood. After removal of the second wash, 150–180 ml of DC-CM containing 1,000 U/ml GM-CSF and 1,000 U/ml IL-4 was added to each bottle. The bottles were placed back on the roller bottle apparatus in the incubator and rolled at 2 rpm for 4–5 days. A dendritic cell maturation cocktail consisting of a final concentration of 10 ng/ml IL-1β, 10 ng/ml TNF-α, 15 ng/ml IL-6, and 1 μg/ml PGE2 (ITIP) [13, 16]. After 20–24 h the floating cells were harvested in all bottles and analyzed for mature DC content. In some experiments, an alternative maturation cocktail called the "Pittsburgh Protocol" (25 ng/ml IL-1β, 50 ng/ml TNF-α, 1,000 U/ml IFN-γ, 20 μg/ml poly I:C, and 3,000 U/ml IFN-α) was used to generate so-called α Type-1DC (α DC1) was added on day 4 or 5 . In some experiments, 450 cm2 roller bottles (Fisher-Costar) were used with PBMC seeded at 250 to 450 × 106 cells per bottle.
Dendritic cell generation in flat-bottom static T-175 flasks
Washed PBMC from leukopheresis products or buffy coats were seeded into T-175 culture flasks in 15 ml of DC-CM (175 × 106 cells per flask). The flasks were incubated as above for 2 to 3 h and non-adherent cells were removed. The flasks were then washed with 50 ml of warm DC-CM and 60 ml of DC-CM containing 800 U/ml GM-CSF and 1,000 U/ml IL-4 was added. The cells were incubated for 4–5 days and matured for 20–24 h and analyzed for mature DC content and function as above.
Determination of mDC yield and phenotype
Isolated cells were washed in DC-CM and viable cell recovery determined with Trypan Blue staining and counting live cells on a hemocytometer using a light microscope. The total floating cells isolated were divided by the number of culture vessels to determine the yield per flask or per bottle. For cell surface staining, the cells were washed 2 times in cold FACS Wash Buffer (FWB) consisting of D-PBS, 1% BSA and re-suspended at 10 × 106/ml in cold FACS Stain Buffer (FSB) consisting of D-PBS, 1% BSA, and 5% normal goat serum. The cells were stained using anti-CD83-PE, anti-CD80-FITC, anti-CD86-APC, CD11c-FITC and CD14-PE (all from BD Biosciences, La Jolla, CA) on ice for 20 min and washed with cold FWB and re-suspended in 0.35 ml cold FWB. 7-AAD (2 μg/ml) was added 5–10 min before FACS analysis to exclude dead cells and enumerate mDC viability. The samples were run on a FACScalibur or FACScanto flow cytometer and analyzed using FlowJo 7.2.2 software (Tree Star Inc., Ashland, OR).
Functional analysis of isolated mDC
DC isolated from roller bottles and static flask cultures were assayed for their ability to induce allo-antigen T-cell responses and CD8+ T-cell recall responses against HLA-A2-binding epitopes from flu, CMV, and EBV . For allo-antigen responses, 50,000 monocyte-depleted PBMC (2-hour plastic-non-adherent PBMC) from a normal donor other than that used to generate the DC were incubated in U-bottom 96-well plates with different numbers of DC or PBMC stimulators (50,000, 25,000, 10,000, 5,000, 1,000, 500, 200, or 100 cells). On day 6, 1 μCi/well of 3H-thymidine was added to each well and the cells harvested the next day and total cpm/well determined. Recall antigen CD8+ T-cell responses were done in ELISPOT plates (Millipore) using 5 × 105 monocyte-depleted autologous PBMC incubated with peptide-pulsed mDC harvested from roller bottles or static flask cultures. The mDC were pulsed with 5 μg/ml of the HLA-A2-binding epitopes from influenza A matrix (GILGFVFTL), CMV pp65 (NLVPMVATV), and EBV BMLF1 (GLCTLVAML) for 90 min, washed and added to the responder cells in the ELISPOT plates . The plates were incubated overnight and processed as described before .