PBMC Isolation and Stimulation for COVID-19 Vaccine Response

Peripheral blood mononuclear cells (PBMCs) from a small subset of the vaccinated individuals (8 patients with MS and 7 HCWs) were isolated on density gradient centrifugation (SepMate-50 cat#85460 or SepMate-15 cat#85420, StemCell Technologies) according to manufacturer's procedure. The 7 HCWs, used as control group, were employed as controls in another publication.^{27 (link)} All samples were frozen in heat-inactivated fetal bovine serum (FBS; Euroclone SpA) with 10% DMSO and stored in liquid nitrogen. PBMCs were thawed, counted, assessed for viability, and rested for 2–4 hours at 37°C in RPMI+10% FBS prior to further use. Complete medium was freshly prepared as follows: RPMI-1640, 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin (Euroclone SpA). Cells were seeded at a concentration of 2.5 × 10⁶ cells/mL in a 96-multiwell flat-bottom plate (COSTAR; Sigma Aldrich) and stimulated with spike peptide pool at 1 µg/mL or staphylococcal enterotoxin B (SEB) at 200 ng/mL, as a positive control. Anti-CD28 and anti-CD49d monoclonal antibodies (BD Biosciences) were added at 2 µg/mL to costimulate cells. After 1 hour of incubation at 37°C (5% CO₂), 1 µL/mL of Golgi Plug (BD Biosciences) was added to cell cultures to inhibit cytokine secretion. Following an incubation of 16–24 hours, cells were stained as described below.