Ifn γ pe clone b27

Following CD4⁺ T-cell isolation, cells were stimulated with EBOV at the indicated MOI for 48 h and surface stained for CD25-Alexa.Fluoro.700 clone M-A251 (BioLegend) and CD69-PE/Dazzle clone fn50 (BioLegend). Cells were fixed and permeabilized using the True Nuclear transcription factor kit (BioLegend) and stained for intracellular Ki-67-BVLT.421 clone B56 (BD Biosciences), IL-2–APC clone MQ1-17H12 (BD Biosciences), TNF-α–FITC clone Mab11 (BD Biosciences), and IFN-γ–PE clone B27 (BD Biosciences). Samples were then analyzed using a FACS Fortessa instrument (BD Biosciences). Cytokines and chemokines were detected and quantitated from supernatants by Eve Technologies (Calgary, Alberta, CA). Heat maps were generated using GENE-E (Broad Institute; http://www.broadinstitute.org/cancer/software/GENE-E/index.html).

For both the in vitro and ex vivo studies PBMC were activated for four hours with CD2/CD3/CD28 T cell activation beads (Miltenyi Biotec, Cologne, Germany) in the presence of GolgiStop and GolgiPlug protein transport inhibitors (BD Bioscience, Franklin Lakes, NJ), and anti-human CD107a APC conjugated antibody (clone H4A3, BD Bioscience). Cell surface marker staining was performed for the in vitro studies using LIVE/DEAD Aqua fixable dead cell stain (Thermo Fisher Scientific), CD4 APC Cy7 (clone RPA-T4, BD Bioscience), CD3 BV785 (clone OKT3, BioLegend, San Diego, CA) CD8α BV650 (clone RPA-T8, BioLegend), and IFNγ PE (clone B27, BD Biosciences). Cell surface marker staining was performed for the ex vivo study using LIVE/DEAD Aqua fixable dead cell stain, CD4 BUV395 (clone SK3, BD Biosciences), CD3 BUV496 (clone UCHT1, BD Biosciences) and CD8α BUV805 (clone SK1, BD Biosciences). Surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions.