Anti pdl2

Enzymatically digested cell suspensions were incubated for 15 min at
4°C in PBS with 0.5% FCS with the following antibodies: anti-CD16/32,
anti-CD8α, anti-CD11b, anti-CD11c, anti-CD45.1, anti-CD45.2,
anti-CD103, anti-CD169, anti-CD301b, anti-Thy1.1 (all from Biolegend),
anti-PDL2 (BD Biosciences), or anti-SIGN-R1 (R&D Systems). Viability was
determined using Fixable Viability Dye eFluor 780 (Invitrogen). Experiments
examining CCR7 were done by first incubating cells with anti-CCR7 (BD
Biosciences) for 30 min at 37°C prior to additional surface staining
as above. Intracellular cytokine staining was done by first incubating cells
in PMA/Ionomycin (Sigma) in the presence of GolgiPlug for 4 hours prior to
surface staining followed by intracellular staining using BD
Cytofix/Cytoperm kit (BD Biosciences). hCCL1-AF647 (Almac) binding was
accomplished by first incubating 2×10⁶ cells in 50
μl of 0.25 μg/ml hCCL1-AF647 in PBS/0.5% FCS/20mM HEPES for 1
hr at 37°C. Cells then underwent additional antibody staining as
above. All samples were run on BD Fortessa X-20 or BD LSRII and analyzed
using FACS Diva 8 (BD Biosciences) and FlowJo (Version 10) (TreeStar).

DCs (1×10⁵) were stimulated with selected vaccines for 18h, and labeled with fluorochrome-conjugated anti-CD83, anti-CD80, anti-CD86, anti-HLA DR, anti-PD-L1, anti-PD-L2, anti-CCR7, anti-CXCR4 and anti-CD40 antibodies (BD Biosciences). Cell death was assessed by Annexin V and PI staining (BD Biosciences). Labeled cells were analyzed using a FACS Canto (BD Biosciences). IP-10 (BD Biosciences) and IFN-β (R&D Systems) levels were measured by ELISA, and other cytokines were assessed using bead-based cytokine multiplex analysis (Luminex, Bio-Rad).