Cd14 pe hcd14

In some experiments, myeloid antigen presenting cells (APCs) were removed from the tumor digest cultures. Briefly, tumor was dissociated as above. A small aliquot of dissociated tumor material was used to phenotype APCs by FACS as described above. Following the staining, myeloid cells were removed by positive magnetic selection with CD11b PE (D12), CD11c PE (S-HCL-3, both from BD Biosciences), CD14 PE (HCD14) and CD68 PE (Y1/B2A, both from BioLegend), followed by anti-PE magnetic beads (Miltenyi). Residual cells were stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE, ThermoFischer Scientific) to later assess TIL proliferation (see below). Cultures were then either resupplied with the same amount of APCs or left APC-free. Cultures were treated with PD-1, CTLA-4, control Ab as above, or predicted CD28 antagonist peptide p2TA (1 μg/ml) or p2TA scramble, synthetized at the Protein and Peptide Chemistry Facility of the University of Lausanne. After 3–4 days in culture, CD8 cells were analyzed by FACS to assess CFSE dilution, as described above.

PBMCs were isolated from buffy coat preparations of healthy blood donors (Blood Transfusion Center Solna, Stockholm, Sweden) through Ficoll-Paque Plus separation (GE Healthcare), as previously described (25 (link)). Exosomes (10 µg) were stained with a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), as previously described (25 (link)). Prefiltered (0.22-µm filter) PKH67-stained exosomes were added to PBMCs (2.5 × 10⁵) for 1, 2, or 4 h at 37°C, 5% CO₂. A PKH67 dye pellet centrifuged in parallel with labeled exosomes served as negative background control. PBMCs were stained with the following Abs to distinguish B cells (CD3⁻CD19⁺HLA-DR⁺), monocytes (CD3⁻CD14⁺HLA-DR⁺), and T cells (CD3⁺CD19⁻): CD19-ECD (HD237; B4 lytic; Beckman Coulter); HLA-DR–PE-Cy5 (TU36; BD Biosciences), CD14-PE (HCD14; BioLegend), and CD3 Pacific Blue (SP34-2; BD Biosciences). Exosome binding to live PBMCs (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit; Invitrogen) was measured (~150,000 events) using an LSR Fortessa (BD) or FACSAria (BD) and analyzed using FlowJo software.