Cytokine release in response to anti-CD3 and anti-CD28 mAbs with fresh and precultured PBMCs. Supernatants from assays described in the legend for Figure 1 were taken at 48 hours for the determination of cytokine concentrations using the V-plex Proinflammatory Panel 1 (human) Kit (Meso Scale Discovery, Rockville, MD); n = 5 for fresh and n = 7 for HD precultured PBMCs.