Anti b220 ra3 6b2

CoPP or DMSO-treated BM-DCs from BALB/c mice were pulsed with OVA (200 μg/ml; grade V; Sigma-Aldrich) and LPS (1 μg/ml) for 24 hours. The harvested BM-DCs were then transferred intratracheally (1 × 10⁶ cells) or intravenously (2 × 10⁵ cells/recipient) into syngeneic naive mice. After 10 days, the recipients were daily challenged with 3% OVA aerosol for 15 minutes for consecutive 4 days. On the day after last challenge, bronchoalveolar lavage fluids (BALFs) and local draining lymph node cells were collected and the cells were stained with PE-Cy7-anti-CD11c (N418; eBioscience), FITC-anti-I-A^d/I-E^d (M5/114.15.2; eBioscience), PE-anti-CCR3 (83101; R&D Systems), APC-anti-CD3 (145-2C11; BD Biosciences) and anti-B220 (RA3-6B2; eBioscience) Abs. The cellular composition of BALF or lymph node cells was determined by flow cytometry (LSR II; BD Biosciences).

Surface staining of cells was performed by suspending them in blocking solution (10% fetal calf serum in PBS) for 30 min at 4°C in the presence of the following fluorescent-labeled mAbs: anti-B220 (RA3-6B2, eBioscience), anti-CD3 (145-2C11, TONBO Bioscience), anti-CD4 (GK1.5, BioLegend), anti-CD8 (53-6.7, TONBO Bioscience), anti-CD25 (PC61.5, eBioscience), anti-CD21 (7G6, eBioscience), anti-CD23 (B3B4, eBioscience) and anti-LAP (TW7-16B4, BioLegend), anti-CXCR5 (SPRCL5, eBioscience), anti-PD1 (J43, eBioscience), anti-CD138 (281-2, BioLegend), anti-CD19 (1D3, TONBO Bioscience), anti-GL-7 (GL7, eBioscience), anti-IgM (eb121-15-F9, eBioscience), anti-IgD (11-26c, eBioscience), anti-CD40L (MR1, eBioscience), and anti-CD69 (H1.2F3, BioLegend). For intracellular cytokine staining, cells were exposed to monensin, fixed and permeabilized using the BD Cytofix/Cytoperm Plus Kit with BD GolgiStop™ (BD Biosciences) following manufacturer’s indications, and then stained with specific fluorescent-labeled mAbs: anti-IFN-γ (FITC-XMG1.2, TONBO Bioscience) and anti-IL-2 (PE-JES6-5H4, BD Pharmingen). For FoxP3 staining, the Anti-Mouse/Rat Foxp3 PE Staining Set (eBioscience) was used following manufacturer’s indications. Flow cytometry analyses were performed on a FACS Canto II equipped with CellQuest (BD Biosciences) and Flowjo 8.7 software.

Bronchoalveolar lavage fluid (BALF) procedures was generated as described previously with a minor modification (17 (link)). Tracheae was cannulated and the lung was lavaged 2 times with 1 mL PBS (contain 1% FCS). Each fluid was centrifuged and the supernatant was rapidly frozen at −80°C. The cells in BALF were stained with CD11c-PE/Cy7 (N418), MHC class II-FITC (M5/114.15.2), anti-B220 (RA3–6B2; eBioscience, Thermo Fisher Scientific), CCR3-PE (83,101; R&D Systems, Minneapolis, USA), and CD3-APC (145–2C11; BD Biosciences, San Diego, USA) and antibodies and analyzed by flow cytometry (LSR II; BD Biosciences) (17 (link)). The gating strategy of immune cells in BALF was showed in Supplementary Figure 2.