Anti cd40 pe

For each assay, cell viability was tested with efluor 506 staining. Expression of activation and maturation markers in DCs was determined by staining with anti-HLA-DR FITC, anti-CD11c efluor 450, anti-CD80 PE-cyanine5, anti-CD86 PE-cyanine7, anti-CD40 PE, and anti-CD83 APC (all from eBioscience). Also, the expression of DC-SIGN and SIGLEC-1 in DCs was determined using anti-CD209 PerCP-cyanine5.5 and anti-CD169 super bright 600 (eBioscience). The frequency of infected CD4+ T cells was evaluated by detecting intracellular p24 (anti-p24 PE, Beckman-coulter, Brea, CA) (S2 Fig). Fluorescence minus one (FMO) or isotype controls were performed for all markers used. The acquisition was performed on an LSR Fortessa (BD) flow cytometer, and analysis was performed using the FlowJo v.7.6 software.

For immunophenotyping, activated DCs or tolerogenic DCs were incubated with monoclonal antibody (mAb)- fluorochrome or biotin conjugates: anti-CCR7-PerCP, anti-CD11c-FITC, anti-CD11b-PE, anti-CD40-PE, anti-CD80-PE, anti-CD86-PE, anti-MHC-II-biotin, and anti-PD-L1-biotin (eBioscience Inc.; San Jose, CA) or with the corresponding isotype controls for 20 min at 4°C in the dark and washed twice with saline solution with 1% FBS. PE-avidin was added to the cell suspensions previously incubated with biotin-mAb conjugates, for 20 min at 4°C in the dark, followed by washing once with 1% FBS saline solution. For each sample, data from 100,000 cells was acquired by three-color flow cytometry, using a BD LSRFortessa SORP cytometer and a BD FacsDiva v.6.2 software (Becton Dickinson; Heidelberg, Germany). Cell-free supernatants of mDCs or tDCs were collected 24 h after stimulation and stocked at −20°C until used for cytokine measurements. The concentrations of IL-6, IL-10 and IL-12 cytokines were measured by ELISA, using specific antibody kits (R&D Systems, Minneapolis, MN), according to manufacturer's instructions.

On day 10, LPS-stimulated cDCs and unstimulated cells were harvested by centrifugation and resuspended in PBS. An average of 5 × 10⁵ cells per stain was subjected to subsequent analyses. Prior to staining, Fc receptors on cDCs were blocked by preincubation with anti-CD16/CD32 antibodies (Biolegend) for 5-10 min on ice. Surface staining was performed by incubating the cells with fluorochrome-conjugated specific antibodies, or the corresponding isotype controls, for at least 20 min in dark on ice. The following antibodies and isotype controls (unless stated otherwise, all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany) were employed: anti-CD11c-FITC/-APC (#130-102-466/-493), anti-MHC-II-APC (#130-102-139), anti-CD40-PE (#130-102-599), anti-CD80-PE (#130-102-613), anti-CD83-PE (#130-104-474), anti-CD86-APC (#130-102-558), anti-hamster IgG-FITC/-PE/-APC (eBiosciences, San Diego, CA, USA), anti-rat IgG2a-PE (eBiosciences), anti-rat IgG2b-APC (Immunotools), and REA Control. Flow cytometric analyses were performed on a FACSCalibur (BD Biosciences). A total of 20,000 events per sample were acquired, and data were evaluated using the CellQuestPro software (BD Biosciences).