Il 10 apc

For cell surface molecule staining, cells were blocked with anti-CD16/CD32 Abs (eBioscience, San Diego, CA, USA) for 20 min followed by surface molecule staining using anti-CD45-PerCp (BD Pharmingen), anti-Siglec-F-PE (eBioscience), anti-CD11c-FITC (eBioscience), and Fixable Viability Dye-eFlour 506 mAbs (eBioscience). After incubating on ice in the dark for 40 min, the cells were washed and resuspended with a fix buffer (eBioscience) for flow cytometry analysis. For intracellular cytokine staining, lung and spleen single-cell suspensions were cultured at concentrations of 5 × 10⁶ cells/mL and 7.5 × 10⁶ cells/mL, respectively, in the complete RPMI 1640 medium with Brefeldin A Solution (eBioscience), which includes PMA, ionomycin, and BFA, for 5 hrs. After incubation, cells were collected and blocked with anti-CD16/32 Abs for 20 min and stained with anti-B220-FITC (BD Pharmingen), anti-CD3e-PE-Cy7 (eBioscience), and Fixable Viability Dye-eFlour 506 mAbs (eBioscience) for 40 min. Cells were then fixed and washed in permeabilization buffer and stained with anti-IFNγ-APC, IL-10-APC, or IL-4-APC (eBioscience), or with isotype control Abs, for 40 min. Cells were washed twice with permeabilization buffer and analyzed by flow cytometry, which was based on the gating strategy shown in Figure S3.

Cell surface antigens were stained first. The dendritic cell antibody panel quantified the following: CD11c-APC-eFluor780, CD80-PE-Cy7, CD86-FITC, and CD40-eFluor450 (all mAbs are from eBioscience now Thermo Fisher, Waltham, MA). The CD4+ T_Reg cell panel included the following: CD3ε-PE, CD4-PE-Cy7, and CD25-PerCP-Cy5.5s (all mAbs are from eBioscience). For T_Reg intracellular staining, cells were fixed and permeabilized with the one step eBioscience Fix-Perm Foxp3 Buffer Staining Kit (eBioscience). The panel included Foxp3-eFluor450 and CTLA4-APC (mAbs are from eBioscience). Intracellular staining was also used to detect TGFβ-PE, IL12 p40-PE, IL10-APC, and IFNγ-APC production (mAbs are from eBioscience) after ex vivo nonspecific antigen stimulation with a Leukocyte Activation Cocktail with GolgiPlug (BD Biosciences) for flow cytometry. Mouse INFγ Single-Color ELISPOT to determine antigen-specific response of T cells was from Cellular Technology Limited (CTL), Cleveland, OH. For attempting to assess SIRT1 expression by flow cytometry, we used antibodies from Santa Cruz and Abcam.