Anti cd14 conjugated magnetic beads

Blood samples (leucopacks) were obtained from the local French Blood Establishment (Etablissement français du sang, EFS), which carries out donor inclusions, informed consent, and sample collection. Through a convention established between our laboratory and the EFS (N°7828), buffy coats were obtained and peripheral blood mononuclear cells (PBMCs) were isolated as previously described (33 (link)). Monocytes were purified from PBMCs using anti-CD14-conjugated magnetic beads (Miltenyi Biotec, Bergisch Glabach, Germany) and cultured in Roswell Park Memorial Institute-1640 medium (RPMI, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, Life technologies), 2 mM L-glutamine, 100 U/mL penicillin and 50 µg/mL streptomycin (Life Technologies).
Vγ9Vδ2 T cells were expanded from fresh PBMCs as previously described (34 (link), 35 (link)). Briefly, PBMCs were cultured in RPMI-1640 medium supplemented with 10% FBS, interleukin-2 (IL-2, 200 UI/ml) and Zoledronic acid monohydrate (to a final concentration of 1 µM). IL-2 was added every 2 days beginning on day 5 for 12 days and the purity of the Vγ9Vδ2 T cells was assessed by flow cytometry analysis (>85%) and then frozen at -80°C in 10% dimethyl sulfoxide (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and 90% FBS.

293T and HeLa cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and penicillin and streptomycin. Primary monocytes, monocyte-derived macrophages (MDM), monocyte-derived dendritic cells (MDDC) and T cells were cultured in RPMI 1640 supplemented with 10% FBS and penicillin and streptomycin. Monocytes were purified from leukocyte enriched blood samples obtained from anonymous donors by the New York Blood Center using anti-CD14-conjugated magnetic beads (Miltenyi Biotec) and differentiated into MDM by culturing for 7 days with 50 ng/mL GM-CSF or into MDDC by culturing with 50 ng/mL GM-CSF and 100 ng/mL IL-4 (Invitrogen). MDM and MDDCs were plated in 12 well plates at 1.0×10⁶ cells per well and cultured for 20 h with or without 2000 units of Universal Type I IFN (PBL Biomedical Laboratories) or 2.0 µg/ml γ-IFN after which they were lysed in protease-supplemented lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA and 1% NP40).

The generation of mDDC (monocyte-derived dendritic cells) was performed using established protocols. CD14+ cells were isolated by positive selection using anti-CD14 conjugated magnetic beads (Miltenyi, Germany). The CD14+ cells were cultured for 6 days in RPMI (Sigma, UK) 10% foetal calf serum (sigma, UK) and 5% streptomycin/penicillin (Sigma, UK) of 10 ng ml⁻¹ IL-4 and 50 ng ml⁻¹ GM-CSF (Miltenyi, Germany). These mDDC were subsequently co-cultured with the T-cells enriched from the CD14- fraction of PBMC using anti-CD3 magnetic beads (Miltenyi, Germany) in the presence of SARS-CoV-2 peptides individual and in groups, and control peptides including CEF (Cytomegalovirus/Epstein Barr virus and influenza), CEFT (Cytomegalovirus/Epstein Barr virus/influenza/tetanus) SARS-CoV-2 reference group (including overlapping peptides from the spike, nucleocapsid and membrane) (1 µg ml⁻¹ for each peptide) for antigenicity assays.