Cd14 positive selection

Monocytes were isolated from Buffy coats obtained from healthy blood donors out of the German Red Cross Blood Service Baden-Württemberg – Hessen after informed consent, as described previously (21 (link)). The isolation was carried out using CD14 positive selection (Miltenyi Biotec), resulting in 90–98% monocyte purity, controlled by flow cytometry. The cells were seeded into cell culture dishes in customized serum-free medium (SFM from Gibco) supplemented with 5 mM glucose at a concentration of 1x10⁶ cells/mL. Macrophage were differentiated in the presence of M-CSF at 5 ng/mL (Peprotech; #A300-25B) and Dexamethasone 10^-8 M (Sigma, #D2915). For M1 polarization IFN-γ was used at the concentration of 100 ng/mL (Peprotech; # 300-02), for M2 polarization, IL-4 was used at the concentration of 10 ng/mL (Peprotech; #200-04). No cytokines were added for M0 differentiation.

PBMCs from 8 healthy HLA-A*0201⁺ donors and 15 CMV D+/R– LT patients were isolated by Ficoll-Paque PLUS gradient centrifugation (GE Healthcare). In some experiments, CD14⁺ and CD8⁺ T cells were isolated from PBMCs using the CD14-positive selection and CD8-negative selection kits, respectively, according to the manufacturer’s instructions (Miltenyi Biotec). For some experiments, naive and memory CD8⁺ T cells were purified by cell sorter (BD FACSAria III Cell Sorter) based on the surface markers CD8, CD45RA, CCR7 and CD62L.

Peripheral blood from the bronchoscopy study participants recruited in Boston was obtained at least 7 days before the bronchoscopy and PBMCs were processed and cryopreserved. Ficoll gradients were used to isolate peripheral blood mononuclear cells (PBMCs). Monocytes and CD4+ T cells were then isolated by CD14+ positive selection (Miltenyi) and CD4+ T cell negative selection (StemCell EasySep), respectively. MDMs were obtained by maturing monocytes in RPMI with 10% GemCell US Origin Human Serum AB (GemBio) for 7 days.

Human DCs were prepared from purified monocytes (MO) by CD14 positive selection (Miltenyi Biotec, Paris, France), cultured in RPMI-1640 (Lonza, Verviers, Belgium), 10% FCS (Life Technologies, Saint-Aubin, France), supplemented with GM-CSF (1000 IU/10⁶ cells) and IL-4 (500 IU/10⁶ cells) (Miltenyi Biotec) (13 (link)). Buffy bags from anonymous healthy donors who gave informed consent were obtained from the Etablissement Français du Sang (EFS, Rungis, France), in accordance with EFS guidelines. After 5 days, MO-DC differentiation was validated by flow cytometry using CD1a and CD14 staining (BD Pharmingen, San Jose, CA, United States). Murine DCs were generated by isolation of bone marrow cells from SureL1 mice cultured for 10 days in RPMI-1640, containing 10% FCS, 50 mM 2-mercaptoethanol and 200 U/ml murine GM-CSF (Cellgenix Technology Transfer, Freiburg, Germany) (28 (link)). The purity of bone marrow derived-DCs (BM-DCs) was assessed by flow cytometry using CD11c staining. FVIII endocytosis by DCs was detected using the monoclonal anti-FVIII IgG, mAb 77IP52H7, conjugated to FITC after permeabilization of cells with 0.1% saponin (14 (link)).

Buffy coats obtained from anonymous healthy blood donors were purchased from the German Red Cross Blood Donor Service Baden-Württemberg & Hessen. Human peripheral mononuclear cells were isolated from the buffy coats by passage over a Leukocyte Separation Medium (PAA Laboratories GmbH) gradient. Monocytes were isolated by exploiting their ability to adhere to plastic, or by CD14 positive selection (Miltenyi Biotec GmbH). Monocytes were then differentiated into hMDM1 and hMDM2 macrophages by the addition of 10 ng/mL recombinant human GM-CSF (R&D Systems) or recombinant human M-CSF for 5 to 7 days, respectively^{25 (link)}.

CD14⁺ cells were purified from frozen PBMCs and CD14-positive selection (Miltenyi Biotec) according to the manufacturer’s protocols. MDMs were generated by seeding three million CD14⁺ cells into one six-well plate (Nunc™, Thermo Fisher Scientific) in IMDM (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific) and M-CSF (50 ng/mL; Miltenyi Biotec) and cultured for 7 to 9 days. Cells were detached from culture plates with Accutase® (Sigma-Aldrich). DLD-1 cells were labeled with the CFSE Cell Division Tracker Kit (BioLegend) according to manufacturer’s instructions. A total of 100,000 DLD-1 cells and 50,000 MDMs were incubated in U-bottom 96-well plates (Corning) with hSIRPα Nbs (1 µM) or KWAR23 (100 nM) and cetuximab (0.66 nM) (MedChemExpress) for 2 h at 37° C, followed by detachment of adherent cells from culture plates with Accutase® (Sigma-Aldrich). For flow cytometry, cells were incubated with CD206 Ab AF647 (clone 15–2, BioLegend) and dead cell marker Zombie Violet (BioLegend). Percent of phagocytosis indicates the percentage of viable CD206⁺CFSE⁺ macrophages.