Temporal Kinetics of iNKT Activation

The iNKT kinetics assay was set up with 2 million PBMCs in 1 ml R10 per condition in a 24-well plate. PBMCs from each donor (n = 4) were stimulated with 1 μg/ml α-Galactosyl Ceramide glycolipid (α-GalCer, also known as KRN7000, Cayman Chemicals, USA). PBMC activation was assessed at 2 h, 4 h, 6 h, 8 h, and 10 h post-stimulation. Further, PBMCs were stimulated for 10 h, centrifuged for 5 min (1500 rpm) and resuspended in R10 for a rest period of 2 h, 4 h and 6 h, respectively. The negative control was an unstimulated ex vivo control and positive control was a 6 h stimulation using Phorbol 12-myristate (PMA—25 ng/ml) and 13-acetate plus ionomycin (Io—500 ng/ml) (Sigma Aldrich, USA). Prior to stimulation, all conditions and controls were pre-stained with the anti-Vα24-Jα18 TCR antibody (6B11, Biolegend, USA), as short-term iNKT stimulation causes iNKT TCR internalization^{21 (link)}. Addition of Golgi stop and Golgi plug (BD Biosciences) which allowed intracellular IFN-γ accumulation, were added 2 h, 1 h, and 30 min post-stimulation in the 8 h and 10 h timepoints, 4 h and 6 h timepoints, and 2 h timepoint, respectively. iNKT cell IFN-γ production was the primary outcome. At the end of each timepoint, cells were stained for flow cytometry.