Cd86 pe it2.2

PBMCs were isolated from whole blood according to a previous report [23 (link)]. PBMCs were stained with fluorescent dye conjugated to antibodies. For pDC, anti-Human CD123-FITC (AC145) (Miltenyi Biotec., Bergisch Gladbach, Germany), BDCA4-APC (AD-17F6) (Miltenyi Biotec.), CD86-PE (IT2.2) (eBioscience, San Diego, CA, USA), and HLA-DR-PerCP (L243) (BD Bioscience, NJ, USA) were used. For mDC, anti-human Lineage Cocktail 1-FITC (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) (MφP9, NCAM16.2, 3G8, L27, SJ25C1, SK7) (BD Bioscience), CD11c-APC (MJ4-27G12) (Miltenyi Biotec.), CD1c-PE-Cy7 (L161) (BioLegend, MS, USA), CD86-PE (IT2.2) (eBioscience), and HLA-DR-PerCP (L243) (BD Bioscience) were used. CD123⁺BDCA4⁺ cells were defined as pDC and Lin1⁻CD1c⁺CD11c⁺ cells were identified as mDC. The expression levels of HLA-DR and CD86 were used as maturation markers of pDC and mDC. After staining, cells were analyzed by flow cytometry using FACS Cant II (BD Biosciences). Data were analyzed using the FlowJo software (Treestar, ON, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood [17 (link)]. To evaluate pDC activity, the PBMCs were incubated with anti-human CD123-FITC (AC145) (Miltenyi Biotec., Bergisch Gladbach, Germany), BDCA4-APC (AD-17F6) (Miltenyi Biotec.), CD86-PE (IT2.2) (eBioscience, San Diego, CA, USA), and HLA-DR-PerCP (L243) (BD Bioscience, Franklin Lakes, NJ, USA) and with anti-human Lineage Cocktail1-FITC (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) (MφP9, NCAM16.2, 3G8, L27, SJ25C1, SK7) (BD Bioscience), CD11c-APC (MJ4-27G12) (Miltenyi Biotec.), CD1c-PE-Cy7 (L161) (BioLegend, San Diego, CA, USA), CD86-PE (IT2.2) (eBioscience), and HLA-DR-PerCP (L243) (BD Bioscience) to evaluate mDC activity. CD123 positive + BDCA4 positive cells were defined as pDCs, and Lin1 negative + CD11c positive + CD1c positive cells were defined as mDCs. The expression intensities of the cell surface molecules CD86 and HLA-DR were used as a marker of pDC and mDC activation. Data were collected by flow cytometer (FACS Cant II, BD Biosciences), and FlowJo software (Treestar, Ashland, OR, USA) was used for data analysis.

DC phenotypes were determined by analysis of median fluorescence intensities of surface marker expression measured on a FACS Canto II flow cytometer (BD Biosciences). Cells were harvested and resuspended in FACS buffer (PBS, 1% BSA, 2 mM EDTA) before staining (30 min, 4 °C, dark) using the following antibodies: CD1a-BV421 (HI149), CD40-FITC (SC3) and CD86-PE (IT2.2) (eBioscience), CD80-APC-H7 (L307.4) and PD-L1-PE-Cy7 (MIH1) (BD Biosciences), HLA-DR-APC (LN3, Invitrogen), PD-L2-APC-Vio770 (Miltenyi) as well as LD (eBioscience Fixable Viability Dye eFluor 780 and eFluor 506). Analysis of the median fluorescence intensity of the mentioned markers within the living CD1a⁺ moDC population was performed with FlowJo Software. Cytokine ELISA in supernatants of infected cells was performed according to the manufacturer’s instructions: IL-1β (R&D) and IL-6, IL-8, IL-12, TNF-α (all Peprotech).