Cd123

For neutrophil and monocyte isolation purity analysis: (from BioLegend unless otherwise stated) CD3 (FITC, HIT3a); CD19 (FITC, HIB19); CD20 (FITC, 2H7); CD56 (FITC, MEM-188); CD66b (AF700, G10F5); HLA-DR (V500, G46-6, BD Biosciences); CD14 (PE, M5E2); CD16 (APC-Cy7, 3G8).
For isolation of mononuclear subsets by FACS: (from BioLegend unless otherwise stated) CD1c (PE-Cy7, L161); CD3 (FITC, HIT3a); CD11c (V450, B-ly6, BD Biosciences); CD14 (PE, M5E2); CD16 (APC-Cy7, 3G8); CD19 (FITC, HIB19); CD20 (FITC, 2H7); CD56 (FITC, MEM-188); CD66b (AF700, G10F5); CD123 (PerCP-Cy5.5, 7G3); HLA-DR (V500, G46-6 BD Biosciences).
For MoDC differentiation analysis: CD1a (BV510, BD Biosciences); CD1c (BV421, BD Biosciences); CD11c (PE-Cy7, BioLegend); CD14 (BV711, BioLegend); CD16 (PE, BD Biosciences); CD64 (FITC, BD Biosciences); CD141 (APC, Miltenyi Biotec).
For macrophage differentiation analysis: CD80 (APC, clone 2D10); APC isotype control; CD200 receptor (PE, clone OX-108); PE isotype control; CD1a (FITC) FITC isotype control (All from BioLegend).

To highlight HBsAg uptake/binding on DC subsets, PBMCs were seeded at 1 × 10⁶ cells mL⁻¹ in 48‐well plates (Corning) and exposed for 2 h at 4 or 37°C to 25 µg mL⁻¹ of either native or deglycosylated FL‐HBsAg or FL‐HBcAg in presence or not of a mixture of TLR ligands comprising polyI:C (30 µg mL⁻¹), Imiquimod (R848, 1 µg mL⁻¹), and Class‐A CpG oligonucleotide ODN‐2336 (CpG_A, 1 µm; Invivogen). In some experiments, PBMCs were exposed to increased amounts of FL‐HBsAg from 10 to 75 µg mL⁻¹. After exposure, PBMCs were thoroughly washed with cold PBS2% FBS to remove the unbound particles and cells were then stained for CD11c, HLA‐DR, Lineage cocktail, CLEC9A, CD123 (BD); CD45 (Biolegend), and BDCA1 (Beckman Coulter) antibodies and further fixed with FACS lysing solution (BD). FL‐HBsAg‐positive and FL‐HBcAg‐positive cDC1s, cDC2s and pDCs were then analysed using LSRII Flow Cytometer and FACSDiva software (BD).

Immunophenoptyping of patient and donor PBMNCs was performed using antibodies against CD3, CD4, CD8, CD56, CD19, lineage markers (CD3, CD14, CD16, CD19, CD20 and CD56), CD27, HLA-DR, CD123, and CD11c (BD Biosciences, San Jose, California, USA). CD45RA (Beckman Coulter Inc. Brea CA, USA) was used to detect naïve T-cells and CD127 (BD Biosciences, San Jose, California, USA) was used to detect memory B cells. CD4, CD25, CD127 and Foxp3 (BD Biosciences, San Jose, California, USA) were used to identify regulatory T-cells (Treg) cells.

Endobronchial biopsies were processed and embedded into glycol methacrylate resin (Polyscience), as previously described [80 (link)]. Sections from biopsies (2 μm) were stained in duplicates with anti-CD8, HLA-DR, CD11c, and CD123 (all BD Biosciences) followed by the rabbit anti-mouse (Dako) biotinylated secondary antibody. The immunostaining was performed as previously described [13 (link)]. All sections were visualized with 3-amino-9-ethylcarbazole (AEC), and cell nuclei were counterstained with Mayer hematoxylin (Histo Lab). Finally, all sections were analyzed using a high-resolution digital scanner, NanoZoomer-XR (HAMAMATSU) to convert them into digital images. A blinded analysis was performed using the scanned sections and NanoZoomer Digitial Pathway View2 software (NDP View; HAMAMATSU). The number of positive cells was expressed as cells/mm and cells/mm² of epithelium and lamina propria, respectively. Quantification of HLA-DR molecules was carried out with a Leica DMR-X microscope (Leica Microsystems GmbH) coupled to computerized image analysis (Leica Qwin 5501W; Leica Imaging Systems) as described previously [81 (link)].

Dendritic cell surface markers were assessed by flow cytometry on Day 7 using surface markers CD14, CD11c, CD123, HLA-DR, HLA-ABC, CD83, AND CD86 (BD Pharmingen). Statistical significance was determined using two-tailed t-tests (p < 0.05). For imaging flow cytometry, prior to coculture with DCs, melanoma-derived EVs were isolated and fluorescently stained with general lipid membrane marker PKH67 (Sigma) according to the manufacturer’s instructions. Briefly, 2 µL of PKH was diluted in 0.5 mL Diluent C and then mixed with EV preparation for 4 min. Following the incubation, 1 mL complete culture media was added, and EVs were washed and concentrated over 100-K ultrafilters (Millipore). After 7-day coculture, DCs were analyzed using ImageStream MKII imaging flow cytometer (Amnis) and analyzed using Ideas 6.2 software (Amnis).

All antibodies used for flow cytometry were mouse anti-human mAbs. Anti-human CD45 (Cat No: 340910), CD56 (Cat No: 345811), CD123 (Cat No: 564195), HLA-DR (Cat No:756414) were purchased from BD Biosciences. Anti-human CD3 (Cat No: 317321), CD19 (Cat No: 302239), CD88 (Cat No: 344304), CD89 (Cat No: 354120), CD14 (Cat No: 325618), CD45RA (Cat No: 304142), CD1c (Cat No: 331520), CD141 (Cat No: 344112) were purchased from BioLegend.
Samples were run on LSRFortessa (BD Biosciences) flow cytometer. Initially, all DCs, including pDCs, cDC1s and cDC2s, were gated based on CD45⁺CD3-CD56^-CD19^-CD88^-CD89^-. Subsequently, pDCs were specifically identified by gating on CD123⁺CD45RA⁺. Following the exclusion of pDCs, cDC1s were identified based on the expression of CD141, while cDC2s were identified based on the expression of CD1c. Data were analyzed with FlowJo v.10.8.1.