Cd209

Cells were washed with ice cold PBS. After FcR blockage (Miltenyi, Germany), cells were stained with the respective antibodies in PBS supplemented with 0.5% FCS, 2.5 mM EDTA for 20min at 4°C. Following antibodies were purchased from Biolegend (USA): CD19 (HIB19), CD11b (CBRM1/5), CD23 (EBVCS-5), CD14 (M5E2); R&D: VSIG4 (polyclonal), MARCO (polyclonal), CCR7 (150503); Becton Dickinson (BD, USA): CD56 (B159), HLA-DR (L243), CD209 (DCN46), CD226 (DX11); Data acquisition was performed with a LSR II (BD). Analyses were performed with FlowJo software (Tree Star).

All experiments were performed after the approval of the institutional Committee for Ethics in Research (Fundação Pró-Sangue, CEP#03, FMUSP). Healthy donors’ peripheral blood was obtained from leukoreduction chambers (41 (link)) after signed written informed consents. PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare, Uppsala, Sweden). For the generation of monocyte-derived DCs, PBMCs were either plated for 2 h for adherence of monocytes to the plastic and subsequent removal of non-adherent cells (42 (link)) or by positive magnetic selection (Milteny Biotec, Bergisch Gladbach, Germany) for the isolation of CD14⁺ monocytes. The monocytes were cultured at 37°C and 5% CO₂ in RPMI-1640 medium supplemented with 10% FBS, antibiotic-antimycotic (Thermo Fisher Scientific) and 50 ng/ml of IL-4 plus 50 ng/ml of GM-CSF (both from PeproTech, Rocky Hill, NJ, USA) for 5 days to obtain immature monocyte-derived dendritic cells (iDCs) (43 (link)). At day 5, iDCs cells were harvested, washed, counted, and activated for 24 h with previously transduced SK-MEL-147 cells at a 1:1 and 1:10 ratios. Cells were harvested and analyzed by flow cytometry using CD209 (DCN46, #551545, BD), HLA-DR (G46-6, #556643, BD), CD80 (L307.4, #340294, BD), CD83 (HB15e, #561132, BD), CD86 (2331, #561124, BD) or used for the priming of autologous T cells.