Cd44 v450

Following the centrifugation of BAL fluid to isolate leukocytes, a total cell count was performed using a Neubauer Haemocytometer and trypan blue to exclude dead cells. Samples were then treated with anti-CD16/32 (BD Pharmingen) and stained at 4 °C for 25 min with the following pre conjugated mAbs against the indicated antigens: MHCII-FITC, NK1.1-FITC, CD4-PerCP Cy 5.5, CD11c-PECy7, B220-PE Texas Red, Ly6G-V450, CD44-V450, TCRβ-APC, CD11b-APCCy7, CD3-APC (all BD Biosciences); 7/4-PE (AbD Serotec), CD8-PECy7 (Invitrogen), F4/80-APC (Invitrogen). Samples were then washed before analysis on a CyAn™ ADP Flow cytometer using Summit software (Beckman Coulter). Cell subsets were defined as the following: eosinophils (F4/80^intCD11b^intSSC^hi), 7/4⁻ monocytes (F4/80^lowCD11b^intSSC^low7/4⁻), 7/4⁺ monocytes (F4/80^lowCD11b^intSSC^low7/4⁺), myeloid dendritic cells (DCs) (CD11b⁺CD11c⁺MHCII⁺), CD4⁺ T cells (CD3⁺αβTCR⁺CD4⁺), CD8⁺ T cells (CD3⁺αβTCR⁺CD8⁺), NK (NK1.1⁺αβTCR⁻CD3⁻) and NKT (NK1.1⁺αβTCR⁺CD3⁺) cells.

Draining lymph node (dLN) and spleen cells were harvested and stained with anti-CD4-FITC, CD8-FITC, CD44-V450, CD62L-PE-Cy7, FoxP3-APC, EOMES-PE-Cy7, Bcl-6-PE, and anti-Thy1.1 (CD90.1)-PE Abs (eBioscience or BD Biosciences). To determine intracellular expression of FoxP3, EOMES, and Bcl-6, cells were fixed and permeated according to the protocol of Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBioscience). Then, cells were stained with anti-FoxP3, EOMES, or Bcl-6 Abs and finally analyzed using FACSAria III (BD Biosciences). To purify CD8+ T_CM cells, cells were stained with anti-CD8-FITC, CD44-V450, and anti-CD62L-PE-Cy7 Abs and CD8+CD44^highCD62L^high T cells were sorted out via FACSAria III (BD Biosciences). To purify CD3+ T cells, splenocytes were stained with anti-CD3-PE Ab and CD3+ cells then were sorted out.

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.