PBMC Activation and Immunization

Peripheral blood mononuclear cells (PBMCs) were separated from EDTA-anticoagulated blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Freshly isolated PBMCs from four donor MCMs heterozygous for the M1 and M2 MHC haplotypes were pooled and resuspended in RPMI-1640 containing 10% fetal calf serum (FCS; R10) and 5 μg/ml phytohemagglutinin-P (Sigma-Aldrich). The culture was incubated overnight at 37°C in a 5% CO₂ atmosphere. Activated PBMCs were harvested the following morning, washed twice with phosphate-buffered saline (PBS), resuspended in PBS, and loaded into tuberculin syringes. Each macaque was then immunized with up to 4 × 10⁷ activated PBMCs as described previously (29 (link)).