Berfeldin a

PFCs or PBMCs were incubated at a concentration of 2×10⁶/mL with different doses of rhIL-12 (eBioscience San Diego, CA, USA) or rhIL-15 (Peprotech, Rocky Hill, NJ, USA), and cell-free supernatants were collected at different time points and assessed by ELISA for IFN-γ (BD Bioscience Pharmingen) and IL-22 (R&D System) production according to the manufacturer’s protocols, respectively. For the detection of intracellular cytokines, the cells were cultured for 8 h in the presence of berfeldin A (BFA, 10μg/mL; Sigma-Aldrich, St Louis, MO, USA) and then subjected to flow cytometry analysis. PFCs or PBMCs were incubated at a concentration of 2×10⁶/mL with BCG (20 μg/mL, Chengdu Institute of Biological Products, Chengdu, China) or M. tb-Ag for 8 h in the presence of BFA for the detection of IFN-γ, IL-22 and IL-17 by flow cytometry. Sorted CD45RO⁺ or CD45RO^- NK cells were treated with or without BCG for 48 h. Sorted CD45RO⁺ or CD45RO^- NK cells were co-cultured with purified CD14⁺ cells (at ratio of 4: 1) in the presence or absence of BCG, BCG plus 2B10 (anti-IL-12Rβ1 mAbs, 10μg/mL, Hoffmann-La Roche Inc., USA), LPS (100ng/mL, Sigma-Aldrich, St Louis, MO, USA) and LPS plus 2B10 for 48 h. Cell-free supernatants were harvested and assessed by ELISA for the production of IFN-γ and IL-22 according to the manufacturer’s protocols, respectively.

To analyze trafficking of dye-labeled lymphocytes, tissues were digested and stained with the following antibodies: CD45 BUV395 (BD), CD45 Pacblue (Biolegend), CD11c FITC (Biolegend), CD4 BV711 (Biolegend), CD8 PE (eBioscience), CD8 PE-Cy7 (eBioscience), and CD19 BV510 (Biolegend). For intracellular chemokine staining of CXCL16 (R&D Systems) was performed using the FoxP3 intracellular staining kit (eBioscience); mice were treated i.v. with Berfeldin A (Sigma) for 4 h prior to harvest. For vascular adhesion molecule and chemokine expression a combination of the previous antibodies were used as well as CD31 PE (eBioscience), CD54 FITC (eBioscience), and CD106 PE (eBioscience). All antibody staining was done for 30 min on ice. Samples were collected on either a BD LSRII or LSR Fortessa and analyzed by FlowJo.