Dcn46

For generation of mo-DCs, monocytes were purified by plastic adherence of PBMCs. For this purpose, cells were seeded (1 × 10⁸/10 ml) into 75 cm² cell culture flasks (Corning, Cambridge, MA, USA) in serum-free X-VIVO 20 medium (Cambrex Bio Science, Verviers, Belgium). After 2 h of incubation at 37 °C/5% CO₂, non-adherent cells were removed. The monocytes were cultured in 10 ml RP10 medium (RPMI-1640 with glutamax-I), supplemented with 10% inactivated foetal calf serum and antibiotics (Invitrogen, Karlsruhe, Germany). GM-CSF (100 ng ml⁻¹; Leukine Liquid Sargramostim; Sanofi, Bridgewater, NJ, USA) and IL-4 (20 ng ml⁻¹; R&D Systems, Wiesbaden, Germany) were added every second day for 7 days. After culture, cells were trypsinized and the reaction was blocked with culture medium. After washing two times with FACS buffer, cells were stained with allophycocyanin-conjugated anti-CD209 Ab (DCN46; BD Biosciences, Heidelberg, Germany) and the indicated workshop Abs using the protocol described above. CD209 was used as the key marker to define mo-DCs.^{24 (link)}

Paraffin sections (4 µm) of the re-excision skin samples of 22 patients were collected, mounted on Superfrost Plus glass slides, and dried overnight at 37°C. After deparaffination, the tissue sections were hydrated through decreasing (v/v) percentages of ethanol and endogenous peroxidase was blocked with 0.1% hydrogen peroxide in methanol. Antibodies against CD1a (MTB1), CD14 (EPR3653), CD83 (1H4B/MONX10851, Monosan, Uden, The Netherlands), Langerin (12D6, Novocastra, Newcastle, UK), CD123 (7G3), DC-SIGN (DCN46, BD Pharmingen, San Jose, USA), CD3 (F7.2.38), and CD68 (KP1, Dako, Heverlee, Belgium) were used. An IgG1 mouse-anti-human isotype control was used (MOPC 21, Organon Teknika-Cappel, Boxtel, The Netherlands). The slides were pretreated with 10 mM TRIS, 1 mM EDTA pH 9 (CD3, CD14, CD123, DC-SIGN, CD68) or 10 mM Na-citrate pH 6 (CD1a, CD83, Langerin). Primary antibodies were applied and visualization was performed with Bondmax (Menarini Group, Malmö, Sweden) for CD3, the Power Vision plusTM system (Immunologic, Duiven, The Netherlands) for CD14, and the EnvisionTM horseradish peroxidase system (DakoCytomation, Glostrup, Denmark) for the other markers, all according to the manufacturer’s instructions.

DCs were harvested on day 5 to 6 of culture, and their immature phenotype CD14⁻CD1a⁺CD209⁺ (M5E2, Biolegend; HI149 and DCN46, both BD Bioscience) was confirmed by flow cytometry analysis. Autologous CD14⁻ PBLs were thawed and added to DCs at a 5:1 ratio to achieve the DC:PBL coculture, which was then stimulated with the immunomodulators at the indicated concentrations (SI Appendix, Table S1).