Cd80 apc

Dendritic cells were stimulated with inactivated A. fumigatus conidia and germ tubes (MOI = 1), 100 µg/ml zymosan depleted [a yeast cell wall preparation, which was treated with hot alkali to remove all TLR-stimulating properties to selectively activate Dectin-1 (dZym, InvivoGen)] or 1 mg/ml lipopolysaccharid (LPS, Sigma) for 24 h. Subsequently, cells were harvested, washed, and resuspended in cold Hank’s balanced salt solution (HBSS, Sigma) containing 2 mM EDTA (Sigma). The following antibodies were used for extracellular staining: HLA-ABC-PE (BD Biosciences), HLA-DR-PE (BD Biosciences), CD1a-APC (BD Biosciences), CD14-FITC (BD Biosciences), CD80-APC (Miltenyi Biotec), CD86-PE (BD Biosciences), Dectin-1-PE (R&D) (human cells) and HLA-2K^b-FITC (BD Biosciences), HLA-Ia/I-E-PE (BioLegend), CD11c-APC (BioLegend), CD80-FITC (BioLegend), CD86-PE (BD Biosciences), and Dectin-1-PE (R&D) [murine cells]. 5 × 10⁵ cells were stained for 15 min at 4°C. Subsequently, cells were washed twice and 10⁴ viable cells according to FSC-SSC properties were acquired using a FACS Calibur (BD Biosciences) flow cytometer and CellQuest Pro software (version 5.2). Data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

Lymphocyte surface phenotypes were evaluated by flow cytometry on cryopreserved PBMCs: CD4-APC-H7, CD8-PErCP-Cy5.5, CD38-PCY-7, HLA-DR-BV421, CD45RA-APC-H7, CCR7- PECy5, LIVE/DEAD-V500, Granzyme B-PE, Perforin-FITC, CD16-APC, CD14-BV421, CD56-PE (BD Biosciences, San Jose, CA, USA) and CD19-PercPVio700, CD80-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). Combinations used were: CD4/CD8/CD38/HLA-DR (T-cell activation), CD14/CD16 (monocyte), CD16/CD56/CD3 (NK cells), CD11c/CD3 (DC), CD3/CD19/CD80 (B-cell activation), CD4/CD8/CD45RA/CCR7 (T-cell maturation). T-cell subsets were defined as naïve CCR (C-C chemokine receptor)7+CD45RA+, central memory (CM) CCR7+CD45RA−, effector memory (EM) CCR7−CD45RA−, terminally differentiated (TD) CCR7−CD45RA+. T follicular helper (Tfh) CD4+CxCR5+CD45RA+, T helper 17-like (Th17) CD4+CCR6+CD161+, T regulatory-like (Treg) CD4+CD127-CD25+. Briefly, 1 × 10⁶ PBMCs were stained with the appropriate antibodies for 20 min at 4°C in the dark and then washed and acquired using FACSVerse™ cytometer (BD Biosciences).

M1-like macrophages were harvested, washed with 1X PBS and then stained with the following anti-human monoclonal antibodies: CD209-FITC, CD64-PE-Vio770, CD80-APC, CD86-PE (all from Miltenyi Biotec, Auburn, CA, USA). For each antibody, isotype controls were used according to fluorescence and antibody specificity (REA293 Isotype control antibody, human IgG1—APC, FITC and PE-Vio770, Miltenyi Biotec, Auburn, CA, USA). Cells were incubated for 20 min at RT in the dark. Cells acquisition has been performed with a 16-colors BD FACS Celesta SORP Cell Analyzer (BD Biosciences, San Jose, CA, USA) with the same instrument setting. At least 10⁴ cells were analyzed using Kaluza Version 2.1.1 software (Beckman Coulter, Carlsbad, CA, USA).