Polyfunctional T-cell Immune Response Analysis

Flow cytometry based ICS assay was done as previously described [35 ,49 (link),50 (link)]. In brief, 10⁶ PBMCs were pulsed with peptide at 10uM in the presence of co-stimulatory antibodies (anti-CD28 and anti-CD49D) and anti-CD107a-FITC (all from BD Biosciences) for 2 hrs at 37°C. Monensin and brefeldin A (both from BD Biosciences) were then added and the cultures incubated for an additional 12 hrs. Next day, the cells were labeled with LIVE/DEAD cell dye (Invitrogen) and surface stained with anti-CD3-Pac Blue, anti-CD8-V500, and anti-CD4-Alexa 780 (both from BD Biosciences). The cells were permeabilized and labeled with anti-IFN-γ-Alexa 700, anti-IL-2-APC, anti-TNFα-PECy7, and anti-Granzyme A-PE (all from BD Biosciences). CD3 events greater than 100,000 were acquired on an LSR II (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar). Polyfunctionality analysis was performed using Boolean gating and SPICE and Pestle software (version 5.1; NIAID).