1.1 Primary Human CD8+ Cell Preparation
Human buffy coats were obtained from the Blood Centers of the Pacific, San Francisco, CA. Peripheral blood mononuclear cells (PBMC) were prepared by Histopaque-1077 density gradient centrifugation (Sigma-Aldrich, St. Louis, MO). The resultant PBMC layer was collected and cells were washed twice with Hank’s Balanced Salt Solution (HBSS) (Mediatech Inc., Manassas, VA). PBMC were resuspended in standard growth medium consisting of RPMI 1640 (Mediatech Inc., Manassas, VA), 10% heat-inactivated (56°C, 30min) fetal bovine serum (GIBCOBRL, Carlsbad, CA), 2mM L-glutamine, and 100 μg/ml of penicillin and streptomycin (Tissue Culture Facility, University of California, San Francisco, CA).
Primary CD8+ cells were obtained by positive selection immunomagenetic (IM) bead isolation using Dynal beads (Cat. No. 113.33D, Invitrogen, Oklo, Norway) or Miltenyi beads (Mat. No. 120-000-244, Miltenyi Biotec, Auburn, CA) bearing an anti-CD8 monoclonal antibody. The Dynal IM beads were removed using Detach-a-beads (Invitrogen, Oklo, Norway).
1.2 Primary CD8+ Cell Transfection
The primary CD8+ cells were washed with 1xDulbecco’s Phosphate Buffered saline (DPBS) (without calcium and magnesium, Mediatech Inc., Manassas, VA), then resuspended in Buffer T (Lot No. ALDV0809, Invitrogen, Carlsbad, CA) at the concentration of 20×106/ml. Maxi-prep purified plasmids (1mg/ml in ddH2O) (Cat. No. K2100-17, Invitrogen, Carlsbad, CA) were then added to the cell mixture. The primary cells were transfected under varying conditions using the Invitrogen Neon transfection kit. The cells were resuspended in 500 μl standard growth medium without penicillin and streptomycin, and incubated for 24 hours at 37°C and 5% CO2.
1.3 Removal of Dead and Dying Cells
Transfected primary CD8+ cells were collected and centrifuged at 300g for 5 mins. Supernatants were removed and the cells were resuspended in 100 μl of dead cell-removal beads (Lot No. 5090922114, Miltenyi Biotec, Auburn, CA) per 107 cells as described by the manufacturer. The mixture was incubated at room temperature for 15 min and added to an MS column (Miltenyi Biotec, Auburn, CA). The columns were then washed four times with binding buffer. Live cells were collected from the flow-through.
1.4 Human IFN-α ELISA
The human IFN-α Muti-Subtype ELISA Kit (Pestka Biomedical Laboratories Inc., Piscataway, NJ) was employed in this study. The procedures followed the manufacturer’s instruction.
1.5 IFN Suppression by Transfected primary CD8 + Cells
Two million freshly isolated primary human CD8+ cells were transfected with 100 μl tips containing the IFN-α plasmid or plasmid control. The transfected CD8+ cells were then treated with the dead cell removal kit. After 24 hours, the supernatant of the transfected CD8+ cells was collected and added to HIV-1SF2
(Subtype B, CXCR4 tropism)-acutely infected CD4+ cells plated in triplicate in a 96 well plate (Mackewicz et al., 1994
). At 2-day intervals, 100 μl aliquots of culture supernatants were collected from each well and assayed for reverse transcriptase (RT) activity as described (Hoffman et al., 1985
). Wells were refed with 50 μl of fresh standard growth medium containing 100 units Hu rIL-2 (Invitrogen, Camarillo, CA) and 50 μl of cell supernatant. The percent of HIV suppression was calculated relative to virus replication in untreated cultured HIV-1SF2-infected CD4+ cells.
1.6 Flow Cytometry Assay
The purified CD8+ cells were stained with fluorochrome-conjugated antibodies using a standard protocol. Flow cytometry was performed on the BD FACSort using the FCS Express V3 for data analysis. For measuring dead cells, the Annexin V-APC Apoptosis Detection Kit and Propidium Iodide were obtained from eBioscience. (Cat. No. 88-8007, 00-6990-50) Mouse IgG2a, Mouse IgG1, Human HLA-DR, CD3, CD4, CD8, CD16,56, CD38, CD25 were purchased from BD Biosciences, San Diego, CA.
1.7 Confocal imaging of GFP-positive CD8+ cells
Two million un-stimulated primary CD8+ cells were transfected with the GFP plasmid or buffer alone under 20ms/Pulse1/2200V. After removal of dead cells, the nuclei of CD8+ cells were stained with Hoechst33342 (Invitrogen, Camarillo, CA) (2 μg/ml) at room temperature for 15 min. Cells were washed with 1xDPBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. Confocal microscopy imaging was taken using a Leica CTR 6500 (Leica Microsystems, Heidelberg, Germany) with 20x and 40xZoom4x objectives.