Anti cd44 v450

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.

Draining lymph nodes and spleen cells were harvested and stained for surface markers with anti-CD4-FITC or, separately, anti-CD8-FITC, anti-CD44-V450, and anti-CD62L-APC antibodies (BD Biosciences). To determine intracellular Foxp3 expression, cells were fixed and permeated by Foxp3/Transcription Factor Fixation/Permeabilization kits (eBioscience), and then stained for intracellular FoxP3 with anti-Foxp3-APC monoclonal antibody (eBioscience). The frequencies of CD4⁺Foxp3⁺ Tregs and effector CD8⁺ T cells were finally analyzed through FACSCalibur (BD Biosciences).