Cd83 pe

To detect the effect of DAC on the DC surface markers, DAC-treated and -untreated DCs were stained by anti-human CD86-APC, CD80-PE, HLA-DR-PE/Cy5, CD83-PE and CD40-PerCP-Cy5.5 (all antibodies were from BioLegend, San Diego, USA) at 4°C for 30 minutes. The expression of cell surface markers was detected by flow cytometry (FACSAira, BD Biosciences, Franklin Lakes, USA) and analyzed as the median fluorescence intensity (MFI). The histograms with overlays were made by FlowJo software (Treestar, Inc., San Carlos, USA).
To investigate whether DAC affects the function of DCs in relation to its effect on differentiating T cells into Th1/Th17 subsets, CD4⁺ cells were co-cultured with DAC-treated or -untreated DCs for 5 days as previously described [11 (link)]. Then 100 ng/ml PMA (Sigma-Aldrich, St. Louis, USA) and 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, USA) were added to the cells at 37°C for 1 hour and subsequently incubated for an additional 4 hours with 10 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, USA). Anti- IFN-γ-FITC and IL-17A-PE antibodies (both from eBioscience, San Diego, USA) were used to perform the intracellular staining for 30 minutes at 4°C. The frequencies of CD4⁺ IFN-γ⁺ and CD4⁺ IL-17A⁺ cells were detected with the FACSAira flow cytometer (BD Biosciences, Franklin Lakes, USA).

Immature DCs and mature DCs were stained with a LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies, Grand Island, NY, USA), then fixed with 2% PFA. After fixation, the cells and appropriate controls were prepared. All samples were stained with HLA-DR-PE/Cy7 (Biolegend, San Diego, CA, USA), CD83-AF647 (Biolegend, San Diego, CA, USA), and CD83-PE (Biolegend, San Diego, CA, USA). Cells were analyzed on a BD LSR II (BD Biosciences, San Jose, CA, USA). Flow data were analyzed using FlowJo v9.6.1 software.

Fluorescence-conjugated antibodies were used to stain DCs: ACE2-APC, CD1a-Alexa Fluor 488, CD40-APC, CD80-PerCP/Cy5.5, CD83-PE, CD86-Alexa Fluor 488, C-X-C chemokine receptor type 4 (CxCR4)-PE, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)/CD209-PerCP/Cy5.5, human leukocyte antigen (HLA)-DR-PE, HLA-ABC-APC, and programmed death-ligand 1 (PD-L1)-FITC (all from BioLegend). Isotype-matched antibodies were used as controls. Shortly, DCs were washed and resuspended in phosphate-buffered saline (PBS) + 1% FBS. Then, 3 µL of fluorescence-conjugated antibodies were added to cells and incubated for 30 min, at 4 °C, in the dark. Cells were subsequently washed, resuspended in PBS + 1% FBS, and analysed in an Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analysed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA), and the results are presented as mean fluorescence intensity (MFI), obtained by the subtraction of isotype control values.