Recombinant human cd40 ligand

LCs were treated with HPV16 VLPs before stimulation, and surface markers were detected by flow cytometry as previously described (Woodham and others 2015 (link)). Briefly, 10⁶ LCs were treated with 10 μg HPV16 L1L2 VLPs in 1 mL phosphate-buffered saline for 1 h at 37°C or left untreated. LCs were then transferred to 6-well plates (5 × 10⁵ LC/well) in 3 mL of complete medium. After 6 h, the cells were left untreated or treated with IRX-2 at the indicated dilutions. Recombinant human CD40 ligand (R&D Systems) was used as a positive control to confirm LC responsiveness. After 72 h, the cells were collected and stained for surface Langerin, E-cadherin, major histocompatibility complex (MHC) I, MHC II, CD80, CD83, CD86, CD1a, CD40, CCR7, or isotype controls, and fluorescence intensity was measured using flow cytometry.

An affiniPure F (ab’) Fragment Goat Anti-Human IgA/ IgG/IgM (H + L) (anti-B-cell receptor [BCR], used at 10 μg/ml) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Recombinant human CD40 ligand (used at 100 ng/ml), recombinant human cytokines IFN-γ (used at 20 ng/ml), IL-4 (used at 20 ng/ml), IL-17 (used at 100 ng/ml) and IL-21 (used at 50 ng/ml)) were all from R&D Systems (Minneapolis, MN, USA).