Cryopreserved PBMCs were thawed, washed and rested in RPMI 1640 media containing 10% heat-inactivated fetal bovine serum (FBS) overnight prior to antigen stimulation with a concentration of 2 x 10 6 cells /mL. PBMCs were counted with Guava easyCyte (Millipore) using Guava ViaCount reagent (Luminex) and GuavaSoft v.2.6 software. Samples with less than 66% viability were discarded. Cells were stimulated with a pool of early secretory Mtb antigen target-6 (ESAT-6) and culture filtrate protein (CFP-10) (BEI Resources). PMA (25ng/mL)/ionomycin (1ug/mL) was used as a positive control and DMSO (0.5%) was used as a negative control. In addition, costimulatory antibody anti-CD28/49d, cytokine secretion inhibitor Brefeldin A and Monensin were added to each stimulation cocktail. Cells were lysed and permeabilized with FACS Lyse and FACS Perm-II buffer and stained on a BD LSRFortessa with an antibody panel developed for analyzing CD4 and CD8 T-cell responses (ICS) including IFN-, TNF, IL-2, and IL-17a [1] (link) (Table S1). [24] Flow cytometry data were analyzed in FlowJo™ v10.7.
Each sample was compensated and gated manually.
Each sample was compensated and gated manually.