Cd86 pe

PBMCs from healthy donors were isolated by using Ficoll-Paque Plus (GE Healthcare, UK) density gradient centrifugation from heparinized blood. Monocytes were isolated using anti-human CD14 microbeads by positive magnetic selection in autoMACS (Miltenyi Biotec, Germany) according to the manufacturer's recommendations. Purified monocytes were cultured in RPMI containing 10% FBS and recombinant human GM-CSF and IL-4 (100 ng/mL) as previously described (42 (link)). After 6 days of culture, immature HmoDCs were harvested (2 × 10⁵ cells/well) and stimulated with LPS (100 ng/mL, Sigma-Aldrich, USA), rAl-CPI-only (100 ng/mL), or incubated with rAl-CPI or PBS as a control 30 min prior to the addition of LPS (43 (link)). The expression of surface markers was assessed using anti-human HLA-DR–FITC, CD86-PE, and CD83-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell viability was routinely checked by using trypan blue cell exclusion in the light microscope and propidium iodide staining in flow cytometric analysis (Supplementary Figure 3). CD4⁺CD45RA⁺CD45RO⁻ naïve T cells were purified from PBMCs by negative selection using CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). T cell subset purity was routinely above 92%. HmoDCs were used to stimulate allogeneic CD4⁺ T cells by co-culture (1:5) and cytokines produced after 6 days of coculture were measured by ELISA.

Flow cytometric analyses were conducted using an LSR II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Version 7.6.5; Flowjo). All antibodies against human or mouse cells were used at appropriate dilutions, as determined by previous titration. Doublet discrimination was carried out and non-viable cells were excluded by 4,6 diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich). Mouse cells were characterized using the antibodies as follows: CD3-V500, CD4-V450, CD11c-APC Cy7, SiglecF-AF647, Ly6G-FITC, CD11b-V500, B7-1-V450, B7-2-PE Cy7, CD62L-FITC (BD Biosciences); mPDCA1-APC, B220-PE (Miltenyi Biotec); CD8-PE Cy7, F4/80-PerCP, SiglecH-PE, TLR4-AF488, TLR9-FITC, MHC-I-FITC, MHC-II-V450, PD-L1-PerCP, ICOS-L-PE, OX40L-APC (eBioscience); TLR7-PE (Abcam); p75NTR-AF488 (Advanced Targeting Systems), CD45-Pacific Blue (Biolegend), and panTrk-FITC (Cell Signaling Technology). Human cells were characterized using the antibodies as follows: TrkA-PE (R&D Systems); BDCA-2-FITC, BDCA-4-PE, p75NTR-APC, p75NTR-FITC, p75NTR-PE (Miltenyi Biotec); CD45-V500, CD3-PE, CD4-FITC, CD8-PerCP, FcεRIα-FITC, IL-3R-PE Cy7, CD184-PE Cy7, MHC-I-PE Cy7, MHC-II-PE, CD80-V450, CD86-PE, CD83-V500, OX40L-V500, PD-L1-PE Cy7, CCR7-V450, CCR9-APC (BD Biosciences), CD3-APC eFluor780, CD4-APC, CD8-PE Cy7, CD25-PE (eBioscience), and CD69-PerCP Cy5.5 (BioLegend).