Computational protocol: Effect of Maraviroc Intensification on HIV-1-Specific T Cell Immunity in Recently HIV-1-Infected Individuals

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Protocol publication

[…] PBMC were thawed and rested overnight at 37°C in RPMI1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100μg/ml streptomycin, and 2 mM glutamine (R10). The following day, the cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen), washed and stained with the following antibodies: anti-CD3-APC-Cy7, anti-CD4-V450, anti-CD8-PE-Cy7, anti-CD45RA-APC (BD Biosience), and anti-CCR7-PE (e-BioScience). The cells were washed and fixed with 1% Formaldehyde in PBS. All data were collected on a BD LSR II flow cytometer (BD Biosience) and analyzed using FlowJo 8.7.7 (TreeStar). [...] Thawed PBMC were rested overnight at 37°C in R10. The following day, costimulatory antibodies (anti-CD28 and anti-CD49d at 1μg/ml; BD Biosciences) and monensin (GolgiStop; BD Bioscience) were added, and cells were stimulated with the different peptide pools (5μg/ml per peptide) as indicated. A negative (no peptide) and a positive control (phorbol-12-myristate-13-acetate (PMA at 10 ng/ml and ionomycin, 1μM) were included in each assay. Following incubation for 6 hrs, the cells were washed with PBS containing 1% FCS and the fluorescent reactive dye (Invitrogen) for dead cells was added. Cells were washed again, and stained with anti-CD3-V450, anti-CD8-PerCP, and anti-CD107a-PE (BD BioScience). Following washing, the cells were fixed and permeabilized using Fix & Perm cell permeabilization reagents (Invitrogen). The cells were then stained with anti-MIP-1β-FITC, anti-IL-2-PE-Cy7, anti-IFN-γ-APC (BD Bioscience). Data were collected on a BD LSR II flow cytometer (BD Biosience) and analyzed using FlowJo 8.7.7 (TreeStar). After gating for each effector function, a Boolean gate platform was used to create the full array of possible combinations and SPICE software (version 5.22) was used to analyze the polychromatic flow cytometry data. We applied a threshold for positive responses using negative values distribution after background subtraction (i.e. unstimulated cultures), as previously described . […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Human immunodeficiency virus 1, Human immunodeficiency virus 2