Anti human pe tnf α

PBMCs were infected with rscVSVs encoding full length or fragments of LASV proteins and EGFP at multiplicity of infection (MOI) of 15. To ensure T cell responsiveness in PBMC cultures, anti-human CD3 (OKT-3) (60 μg/ml) and CD28 (9.3) (20 μg/ml) antibodies were used as a positive control. After 4 hours, brefeldin A was added (4 μg/ml), and infected PBMCs were incubated overnight (total of approximately 16 hours) at 37°C in 5% CO_2. PBMCs were washed in PBS and stained with anti-human brilliant violet 421 CD4 (RPA-T4) (BioLegend) and FITC CD8a (HIT8a) (Biolegend) for 1 h at 4°C in FACS buffer (PBS containing 2% FCS and 0.2% Azide). Cells were washed in FACS buffer, fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. Intracellular staining with anti-human PE TNF-α (BD Biosciences), PE/Cy7 IFN-γ (4S.B3) (BD Biosciences), and APC IL-2 (MQ1-17H12) (BD Biosciences) antibodies followed for 1 h at 4°C. Cells were washed and resuspended in FACS buffer for analysis using a LSR II (Becton Dickinson), and analyzed with FlowJo software (TreeStar, Inc). T cells incubated with peptides instead of rscVSVs were treated similarly except brefeldin A was added after one hour of peptide incubation (10 μg/ml; unpurified, Anaspec) and cells were stained four hours later.

Plasmids used to rescue rscVSVs expressing LASV antigens have been described (8 (link), 42 (link), 43 (link)). LASV sequences were based on the Josiah strain from lineage IV. rscVSVs were rescued and characterized as described (8 (link)). PBMCs from LF survivors were incubated with rscVSVs at a multiplicity of infection (MOI) of 15 in a U-bottom 96-well plate. After 4 h, brefeldin A (4 μg/ml) was added, and cells were incubated overnight (∼16 h) at 37°C in 5% CO₂ before staining with the following antibodies: anti-human brilliant violet 421 CD4 (clone RPA-T4; BioLegend), fluorescein isothiocyanate (FITC) CD8a (clone HIT8a; BioLegend), anti-human PE TNF-α (BD Biosciences), PE/Cy7 IFN-γ (clone 4S.B3; BD Biosciences), and APC IL-2 (clone MQ1-17H12; BD Biosciences). BD fixation/permeabilization kit was used for intracellular stains. Positive controls were treated similarly except for incubation with unlabeled anti-human CD3 (clone OKT3; BioXCell; 60 μg/ml) and CD28 (clone 9.3; BioXCell; 20 μg/ml) instead of rscVSVs.