Lymphoprep density gradient centrifugation

Human peripheral blood mononuclear cells (PBMC) were isolated from Buffy coats by Lymphoprep density gradient centrifugation (Axis‐shield, Dundee, United Kingdom and Nycomed, Zurich, Switzerland). PBMC were cultured in RPMI 1640 supplemented with Glutamax and sodium pyruvate (Gibco), 2.5% FCS and 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco). CD4+ T cells were isolated from PBMC using CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's instructions (average purity of 95%) and were used directly in experiments or fractionated into CD45RA+ and CD45RO+ subsets using anti‐CD45RA‐PE (UCHL‐1, Dako) and anti‐PE magnetic beads (Miltenyi Biotec) resulting in > 97% pure T cell subpopulations.

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy volunteer HIV-1 seronegative donors (Red Cross Luxembourg) by Lymphoprep density gradient centrifugation (Axis-Shield, Oslo, Norway). CD4+ T-cells were isolated from PBMCs by negative selection using antibody coated-beads (Miltenyi Biotec, Leiden, Netherlands) and cultured in RPMI 1640 medium supplemented with 10% FCS, 2mM L-Glutamine, 50 μg/mL Penicillin and 50 μg/mL Streptomycin (all from Invitrogen, Merelbeke, Belgium) (CD4 medium). PBMCs were stimulated with 5 μg/ml PHA-P (Sigma, Borneim, Belgium) for 48 hours, then with 10 U/ml interleukin 2 (IL-2, Roche, Mannheim, Germany) for 24 hours before infection.
Monocytes were isolated from fresh PBMCs by adherence to plastic for 1 hour at 37°C in MDM medium (RPMI 1640, 2mM L-Glutamine, 50 μg/mL Penicillin and 50 μg/mL Streptomycin, 10 mM HEPES, 1% MEM vitamins, non-essential amino acids, 50 μM β-mercaptoethanol, 1 mM sodium pyruvate) (all from Invitrogen, Merelbeke, Belgium) supplemented with 2% human AB serum (Sigma, Bornem, Belgium) as described in [98 (link)]. Adherent monocytes were seeded in 96-well plates (3x10⁵ cells/well) and allowed to differentiate into macrophages for 7 days in MDM medium supplemented with 15% human AB serum.

Blood samples (30–35 mL) from an additional 12 patients with SLE (10 with inactive disease and 2 with active disease) and from 7 healthy donors from Croix Rouge Luxembourg, were collected on EDTA. No clinical data was available for these patients and healthy controls. PBMCs were isolated immediately by Lymphoprep density gradient centrifugation (Axis-Shield, Oslo, Norway), washed and seeded in 24-well plates (10⁶ cells/well) in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-Glutamine, 50 µg/mL Penicillin and 50 µg/mL Streptomycin and 10 U/mL interleukin 2 (IL-2, Roche). Cells were treated with 750 U/mL IFN-α (PBL biomedical laboratories) either immediately for 4, 16 or 24 h (5 SLE patients and 4 healthy controls) or put to rest overnight and treated with IFN-α the following morning for 4, 8 or 24 h (7 SLE patients and 3 healthy controls). At the indicated time points, cells were harvested, washed and dry pellets were frozen at − 80 °C. RNA extraction and relative A3A and A3B expression levels (mRNA) were measured as for the other samples and normalized to the geometric mean of RPL13A and GAPDH according to the Pfaffl method^{65 (link)} for each sample (IFN-α-treated compared to the corresponding non treated control).