Il 4 af488

Splenocytes were isolated 12 days after vaccination, plated at 1-2x10⁶ cells/well in 96-well plates, and stimulated with vaccine proteins (Hla_H35L, EsxAB, FhuD2 and Csa1A, 10 μg/ml each) together with anti-CD28 and anti-CD49d mAb (2 μg/ml each, BD Biosciences) at 37°C for 16–18 h. Brefeldin A (5 μg/ml) was added for the last 4 h. The cells were then stained with Live/DeadYellow (Invitrogen), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), washed in Perm/Wash buffer (BD Biosciences), incubated with anti-CD16/CD32 Fc block (BD Biosciences) for 20 min at RT, and stained with the following fluorochrome-conjugated mAbs anti: CD3-PerCP Cy5.5, CD4-V500, IFN-γ-PE, IL-2-APC, TNF-AF700, CD44-V450 (BD Pharmingen), CD8-PE Texas Red (Invitrogen), IL-17A-PE Cy7, IL-4-AF488 and IL-13-AF488 (eBioscience) in Perm/Wash buffer (BD Biosciences) for 20 min at RT, washed twice in Perm/Wash buffer, suspended in PBS. Samples were acquired on a LSRII special order flow cytometer (BD Biosciences) and T-cell responses were analyzed using FlowJo software (TreeStar) applying the gating strategy described in S1 Fig.