Anti cd28 and anti cd49d

Blood was collected in sodium heparin tubes and processed within 3 h of collection. The whole blood assay was adapted from the protocol described by Hanekom et al.^{46 (link)} Briefly, 0.5 ml of whole blood was stimulated with a pool of 300 Mtb-derived peptides (Mtb300, 2 µg/ml)^{47 (link)} at 37 °C for 5 h in the presence of the costimulatory antibodies, anti-CD28 and anti-CD49d (1 μg/ml each; BD Biosciences) and Brefeldin-A (10 µg/ml; Sigma-Aldrich). Unstimulated cells were incubated with costimulatory antibodies and Brefeldin-A only. Red blood cells were then lysed in a 150 mM NH₄Cl, 10 mM KHCO₃, and 1 mM Na₄EDTA solution. Cells were stained with a Live/Dead Near-InfraRed dye (Invitrogen), and then fixed using a Transcription Factor Fixation buffer (eBioscience), cryopreserved in freezing media (50% fetal bovine serum, 40% RPMI, and 10% dimethyl sulfoxide) and stored in liquid nitrogen until use.

Splenocytes from immunized mice were isolated 2 weeks, 1 month or 4 months after the last immunization, plated at 1–2 × 10⁶ cells/well in 96-well plates, and stimulated with anti-CD28 and anti-CD49d (2 μg/ml each, BD Biosciences), EsxAB, Hla_H35L, FhuD2 or Csa1A (30 μg/ml), or with a combination of antigens (4C-Staph) containing EsxAB, Hla_H35L, FhuD2, and Csa1A (10 μg/ml each) at 37°C for 16–18 h, in the presence of Brefeldin A (5 μg/ml) for the last 4 h. The cells were then stained with Live/Dead Yellow (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, stained with fluorochrome-conjugated mAbs: anti-CD3-PerCP Cy5.5, anti-CD4-V500, anti-IFN-γ-PE, anti-IL-2-APC, anti-TNF-Alexa700, anti-CD44-V450 (BD Pharmingen), anti-CD8-PE Texas Red (Invitrogen), anti-IL-17 PE-Cy7, anti-IL-4-A488, and anti-IL-13-A488 (eBioscience), in Perm/Wash buffer 1× (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 Figure S1 in Supplementary Material.