4.2.1. Cell Activation

PBMCs were isolated using the Ficoll–Hypaque density gradient method (GE Healthcare, Chicago, IL, USA) and stored in liquid nitrogen before shipping to the South African Tuberculosis Vaccine Initiative (SATVI) for processing. Cryopreserved cells were thawed and rested for two hours in Roswell Park Memorial Institute RPMI 1640 media containing 10% heat-inactivated fecal bovine serum (FBS) prior to antigen stimulation. We stimulated cells with a pool of early secretory antigen (ESAT-6) and culture filtrate protein (CFP-10, peptide pool referred to as ESCF) consisting of 17 and 16 peptides, respectively, overlapping by 10 amino acid sequences (1 μg/mL, GenScript, Piscataway, NJ, USA); *Mtb* cell lysate (MtbLy) (H37Rv; 10 μg/mL, BEI Resources, Manassas, VA, USA), and HIV-1C Gag 15 mers overlapping by 10 amino acids (2 μg/mL, BEI Resources, Manassas, VA, USA). Phytohemagglutinin antigen (PHA; 2 μg/mL) was used as a positive control. A negative control without stimulation (NS) was also included. Stimulations were performed for two hours in a 37 ◦C CO2 incubator, after which Brefeldin A (5 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added and cells were further stimulated for four hours. All stimulations were conducted in the presence of the co-stimulatory antibodies, anti-CD28 and anti-CD49d (1 μg/mL, BD Biosciences, San Jose, CA, USA) for a total of six hours.
