Anti cd86

To analyze DC surface markers, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF for seven days. DCs were treated with 100 ng/mL LPS for 24 h in the absence or presence of 10 mM D-2-HG or L-2-HG. Cells were harvested and stained with anti-CD1a & anti-HLA-DR (Beckman Coulter, Krefeld, Germany), anti-CD80 (Biolegend, San Diego, CA, USA), anti-CD83 (eBioscience, San Diego, CA, USA), and anti-CD86 (BD Bioscience, Franklin Lakes, NY, USA) at 4 °C for 30 min. After washing, DCs were resuspended in FACS wash buffer. Flow cytometric measurement was performed using a BD FACS Calibur instrument (BD Bioscience).
For intracellular detection of IL-12 p35 and IL-12 p40, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF. After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStop^TM, BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/Cytoperm^TM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls. Cells were analyzed using a BD FACS Calibur instrument (BD Bioscience).

Antibody staining for flow cytometry was done in FACS buffer (PBS, 0.1% BSA) at room temperature in the dark for 30 min, followed by washing in FACS buffer and fixation with 2% paraformaldehyde in FACS buffer. Cells were stained with the following antibodies: anti-CD14 (Biolegend, 301806), anti-CD16 (Biolegend, 302018), anti-CD38 (Biolegend, 301806), anti-CD86 (BD biosciences), anti-HLA-DR (Biolegend, 307642), anti-CD163 (Biolegend, 333608) and anti-CD206 (Biolegend, 321109). In addition, cells were stained with live/dead dye (Invitrogen, P30253) to identify live cells.
Flow cytometry data was acquired on a Celesta flow cytometer (BD biosciences) and flow cytometry data was analyzed using Flowing software (Turku Bioscience). Analysis showed that 63.9% of macrophage culture were CD14⁺ macrophages (S1F Fig). CD14⁺ macrophages were positive for macrophage markers CD38 (99%), CD86 (98.5%), HLA-DR (74.9%), CD206 (91.6%), CD163 (61.6%) and CD16 (88%) (S1F Fig).

First, we collected the tumor tissues of the two groups and lysed them with a prewarmed dissociation buffer (1 mg/mL collagenase I and 20 µg/mL DNase I). After 30 min, the lysate was filtered through a 70-µm cell strainer after the termination of digestion with a DMEM high glucose medium (containing 5% FBS). Cells were then blocked with Fc Block CD16/CD32 (1:50 dilution for a concentration of 0.5 µg per well) and stained with the antibodies in a staining buffer (1% BSA): anti-F4/80, anti-CD86 and anti-CD206 (BD Biosciences, CA, USA, 1:5000). Finally, the Canto II flow cytometry system and FlowJo_V10 software were used for flow cytometry analysis.

Cultured cells were collected, washed and resuspended in complete growth medium at 1x 10⁶ cells/ml and stained for 20min at 4°C using the following anti-human antibodies (1:20): APC-labeled CCR7 and the matched isotype control antibody (R&D Systems; Bio-Techne, Zug, Switzerland), CD123 (clone 6H6, Abeomics; LucernaChem, Luzern, Switzerland), CD11c (clone 3.9), HLA-DR (BioLegend through LucernaChem), and FITC-labeled anti-CD40 (Serotec; Bio-Rad, Cressier, Switzerland) or anti-CD86 (BD Biosciences, Allschwil, Switzerland). Unbound antibodies were removed by washing with 1x PBS and cell pellets were resuspended in FACS buffer (1x PBS supplemented with 0.5% FCS). Samples were filtered (50μM Cup Filcons from BD), measured with a BD LSRII or a LSRFortessa flow cytometer using the BD FACSDiva™ 6 software and analyzed with the FlowJo software (BD). SYTOX^® Blue or TO-PRO™-3 iodide (Invitrogen; Thermo Fisher Scientific, Allschwil, Switzerland) was added as a dead cell indicator.

mDCs were resuspended in ice cold PBS supplemented with 2 mM EDTA, 0.1% BSA, and 0.01% sodium azide, blocked with 10% mouse serum (Sigma) and stained with anti‐CD83 (BD), anti‐CD86 (BD), and anti‐HLA‐DR (Biolegend). Surface expression was evaluated relative to unstimulated stained controls on a BD CANTO II and analyzed using FlowJo software (Treestar).