Ficoll density gradient centrifugation

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinised blood samples using a Ficoll density gradient centrifugation (U-04; Eurobio, Les Ulis, France). Then, 10⁵ cells/well were incubated for 24 h in supplemented culture medium (RPMI 1640; Eurobio) in a 96-well cell culture plate at 37 °C under 5% CO₂. PBMCs were then stimulated in duplicate with phytohaemagglutinin (PHA) at 4 µg/mL (R30852801; Remel™, Dartford, Kent, UK) and incubated for 72  h. Then, cellular pellets were analysed for T-cell proliferation using the Click-It^® EdU AF488 flow kit (C10420; Life Technologies, Carlsbad, CA, USA) to measure incorporation of 5-ethynyl-2′-deoxyuridine (EdU) according to the previously published protocol [21 (link)]. The proportion (%) of EdU+ proliferating cells (among CD3⁺ T-cell) was obtained by flow cytometry analyses performed on a BD LSR Fortessa™ flow cytometer (BD Biosciences, San Jose, CA, USA). For each experiment, a minimum of 2.5 × 10³ CD3⁺ T-cells was recorded. Data were analysed using BD FACSDiva Software (version 8.0.3; BD Biosciences).

PBMCs were obtained by Ficoll density gradient centrifugation (Eurobio, FR, EU). They were rapidly thawed at 37°C, extensively washed, and kept at room temperature or overnight at 37°C before assessing their viability. PBMCs (0.15 × 10⁶) per well were cultured in 96-well plates with AIM V medium (Gibco, FR, EU) enriched with R-848 (5 μg/ml; resquimod), high–molecular weight poly-IC (polyinosine-polycytidylic acid) (10 μg/ml; both Invivogen, FR, EU), interleukin-2 (20 IU/ml; IL-2; PROLEUKIN aldesleukin, Novartis Pharma, CH, EU), and the peptide of interest (10 μg/ml) at day 0. After 3, 6, and 10 days, 100 μl of medium was replaced by enriched fresh medium (IL-2 and peptide only at day 6 and IL-2 only at day 10) and splitted if necessary. On day 12, cells were collected and counted for analysis.

Thymuses were obtained from MG patients (12–41 year-old) undergoing thymectomy at the “Hôpital Marie Lannelongue” or “Centre Hospitalier Universitaire de Strasbourg.” The clinical details of the 44 patients included in the study are described in Table 1. Normal thymuses were obtained from infants (4 days to 11 years) and adults (13–35 years) undergoing cardiac surgery at the “Hôpital Marie Lannelongue.”
Thymocytes were isolated from thymuses by mechanical dissociation of fresh thymic tissue, as previously described (26 (link)). The cells were filtered through cell strainer device to remove thymic tissues and washed once with HBSS.
Blood was obtained from MG patients (19–45 years old) just before thymectomy or during follow–up consultation, and from sex-matched control donors (20–64 years old) from the “Etablissement Français du Sang” (EFS). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Eurobio, Les Ulis, France).
These investigations were approved by the local Ethics Committee (“Comité Consultatif de Protection des Personnes”), Ile de France VII (Kremlin Bicêtre, France). The relevant authorization numbers are ID RCB 2006-A00164-47 and 2010-A00250-39.