Anti cd83

Freshly isolated or 24-h cultured PBMCs were cell surface stained and analyzed by flow cytometry [FACS Canto, Becton Dickinson (BD), Heidelberg, Germany]. The following mAbs were used at optimal dilutions: anti-slan (M-DC8 hybridoma supernatant raised in our laboratory) followed by anti-μ-PE or -FITC [goat F(ab′)2 anti-mouse IgM, Beckmann Coulter], anti-CD83, anti-CD86, and anti-HLA-DR (all from BD Pharmingen) and appropriate isotype-matched control mAbs.

The expressions of HLA, costimulatory molecules and GFP to DCs were analyzed by flow cytometry (FACS Caliber, BD Biosciences). The cells were washed once in PBS and stained for 30min with PE-conjugated anti-HLA-A, -B, -C, anti-4-1BBL, anti-CD80, anti-CD83, and anti-CD86, FITC-conjugated anti-HLA-DR, -DP, -DQ (BD Biosciences, USA). And the cells were washed followed fixed using 1% paraformaldehyde solution. Approximately more than 10,000 cells were acquired and the data were analyzed by using FLOWJO software (Tree Star, USA).

Monocytes were isolated from PBMC of healthy donors using CD14 microbeads (Miltenyi Biotec, Germany). The cells were resuspended in VLE-RPMI 1640 (Biochrom AG, Germany) with 1.5% human serum and seeded at 5 × 10⁶ cells in 25 ml Nunclon™flasks (Nunc, Germany). On day 0, 100 ng/ml GM-CSF and 20 ng/ml IL-4 were added to the cultures. At day 1, 1 ng/ml TGF-β1 and 20 ng/ml IL-10 were added only to tolDC. On the following day, maturation cocktails^{30 (link)} (Table 2) were added to tolDC and C5-DC to induce maturation. For immunophenotyping of tolDC and C5-DC, the following antibodies were used: anti-CD14 (clone: M5E2), anti-CD80 (clone: L307.4), anti-CD83 (clone: HB15e), anti-B7-H1 (clone: MIH1), anti-B7-DC (clone: MIH18) (all from BD Biosciences, USA) and anti-CD86 (clone: 2331, Pharmingen). Stained cells were analyzed by using the LSRII (BD Biosciences). Data were processed by using FlowJo software (Tree Star, Ashland OR, USA).Table 2

Maturation cocktail for generation of tolDC and C5-DC.

	TolDC	C5-DC
GM-CSF (Leukine sargramostim, USA)	100 ng/ml	100 ng/ml
IL-4 (R&D, USA)	20 ng/ml	20 ng/ml
IL-1β (R&D, USA)	10 ng/ml	10 ng/ml
TNF-α (PEPRO-TECH, USA)	20 ng/ml	20 ng/ml
PGE2 (Sigma, USA)	250 ng/ml	1 µg/ml
R848 (In vivo Gen)	—	1 µg/ml
IFN-γ (Boehringer Ingelheim, Germany)	—	5000 U/ml
IL-6 (R&D, USA)	15 ng/ml	—
TGF-β1 (PEPRO-TECH, USA)	1 ng/ml	—
IL-10 (PEPRO-TECH, USA)	20 ng/ml	—

Cells were harvested and counterstained immunophenotypically using anti‐CD83, anti‐CD80, anti‐CD86, anti‐CD40 and anti‐CD11c (BD Pharmingen).

BMDCs were washed and resuspended in ice-cold PBS containing 5% fetal bovine serum to prevent nonspecific binding and incubated with anti-CD83, anti-CD80, anti-CD86, and anti-CD40 (BD Pharmingen, San Diego, CA, USA) for 30 minutes at 4°C. After extensive washing, the stained cells were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) and Cell Quest software.
Intracellular levels of ROS were measured with DCFH-DA molecular probes (Molecular Probes-Invitrogen, Carlsbad, CA, USA). Cells were incubated with 10 μM DCFH-DA for 30 min at 37°C and then washed and resuspended in PBS at 1 × 10⁶ cells/mL. The DCs were analyzed using flow cytometry. The fluorescence was determined at 503/529 nm and expressed as a percentage of the control.

Analysis of surface marker expression was carried out by flow cytometry as described previously [28 (link)] using FITC or PerCP/Cy5.5-conjugated antibodies. FITC-conjugated antibodies used were anti-CD3 (BD Biosciences, cat # 349201), anti-CD28 (BD Biosciences, cat # 561790), anti-CD83 (BD Biosciences, cat# 556910), anti-CD86 (Abcam, Cambridge, MA, cat # Ab77276), anti-CD54 (Santa Cruz, Dallas, TX, cat # SC-107), and IgG2a isotype control (Sigma, cat # F6522). PerCP-Cy5.5-conjugated antibodies anti-CD86 (cat # 374215), anti-CD54 (cat # 353119), anti-CD80 (cat # 305231), and IgG1κ isotype control (cat # 400150) were purchased from Biolegend, San Diego, CA.