Cd1c pe

The following immunostaining Ab reagents were used for flow cytometry analysis: Mouse-anti-human CD83-PE, CD86-PE, OX40L-PE, CD40L-PE, CD3-FITC, CD4-APC, CD8-APC, CD19-PE, CD3-PE, HLA-DR-FITC (all from BD Biosciences), CD40-PE, CD56-PE (Beckman Coulter, Indianapolis IN U.S.A.), CCR7-FITC (R&D Systems), CD14-PE, CD1c-PE (Miltenyi Biotech, San Diego, CA) and the respective matched isotype controls (BD Biosciences). Prior to analysis for expression of CD40L, isolated CD4⁺ and CD8⁺ T cells were stimulated for 24 h with anti-CD3/CD28 activating Dynabeads (Gibco, Life Technologies). Purity was determined by the exclusive expression of either CD4 or CD8 on the CD3⁺ gated lymphocytes. Purity of blood-isolated DC was delineated using the following gating strategy: lineage (CD3, CD14, CD19, CD56)⁻ and CD1c⁺ HLA-DR⁺. Analysis was performed using the BD Biosciences LSR Fortessa Cell Analyzer and FlowJo version 7.6 software.

Cryopreserved CBMC and PBMC were thawed in the presence of DNAse, assessed for viability using trypan blue exclusion, and rested overnight in serum-free complete medium (XVNS-15; Lonza) plus low dose IL-2 (30 U/ml; Prometheus) in 6 well ULA tissue culture plates (Fisher Scientific). Following overnight rest, CBMC/PBMC were subjected to CD4+ enrichment using negative isolation columns (Miltenyi), and resulting CD4+ T cells labeled with anti-CD4-PECy7 (BD), anti-CD45RA-allophycocyanin (Biolegend), and anti-CD14/CD19/CD123/CD56 (Biolegend)/CD1c -PE (Miltenyi), and sorted to obtain CD4+CD45RA+(CD14/CD19/CD123/CD1c/CD56)^negative T cells using a FACS InFlux sorter (BD). To assess the impact of contaminating CD4+CD45RA+RO+ and CD4+CD45RO+RA− T cells on the effector cytokine response among adult donors, PBMC from a subset of adult donors underwent CD4+ enrichment using negative isolation columns followed by parallel FACS purification for CD4+/CD45RA+/CD14, CD19, CD123, CD1c, CD56^negative T cells and CD4+/CD45RA+/CD45RO− (BV 421; BD)/CD14, CD19, CD123, CD1c, CD56^negative T cells (Supplemental Figure 1 A/B).