Lymphoprep

PBMCs were isolated by Ficoll-Paque density gradient (Lymphoprep, Proteogenix) from the blood of patients and healthy donors. Fresh or cryopreserved PBMCs were used for the assays. Control PBMCs were obtained from the Etablissement Français du Sang blood bank. PBMCs were cultured at 37°C in 5% CO₂ in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (GIBCO). PBMCs were treated with ruxolitinib 1 µM or BX795 2 µM. HEK 293T and 293FT cells (ATCC) were grown in 6-, 12-, or 96-well plates at 37°C in 5% CO₂ in DMEM (GIBCO) supplemented with 10% (vol/vol) fetal bovine serum (GIBCO). Control and STING KO THP-1 monocytic cell lines were previously generated in the Manel laboratory (Cerboni et al., 2017 (link)). WT and cGAS KO THP-1 cells were from InvivoGen (THP1 Dual). MAVS KO THP-1 cell lines were recently generated in the Rehwinkel laboratory (Hertzog et al., 2020 (link)
Preprint). All THP-1 cell lines were cultured at 37°C in 5% CO₂ in RPMI (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (GIBCO). THP-1 cells were stimulated for 24 h with 1 µg/ml poly(I:C) (High Molecular Weight; InvivoGen), 1 µg/ml 2′3′cGAMP (InvivoGen), or 0.25 µg/ml HT-DNA (Sigma-Aldrich) combined with Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions.

Whole blood samples were collected from the patient and control donors. PBMCs were isolated by Ficoll-Paque density gradient (Lymphoprep; Proteogenix) from blood samples using standard procedures. Expansion of T cell blasts were obtained by incubating PBMCs for 72 h with 2.5 µg ml⁻¹ PHA (Sigma-Aldrich) in Panserin 401 (Pan Biotech) supplemented with 5% human male AB serum (BioWest), 100 U ml⁻¹ penicillin, and 100 µg ml⁻¹ streptomycin. After 3 d, dead cells were removed by Ficoll-Paque density gradient, and blasts were maintained in culture with 100 or 1,000 UI ml⁻¹ IL-2.