Release of soluble NKG2D ligands by SEB-activated PBMCs. Total (●) or CD4-depleted (○) PBMCs were activated with SEB and cell-culture supernatants were harvested after 3, 5, and 7 days and tested in ELISA for the presence of the different ligands. Each circle represents the mean of triplicates of one donor tested on both cell populations. The numbers of donors analyzed were 10 (MICA), 17 (MICB), 11 (ULBP1), and 15 (ULBP2). All donors were monitored also for cell-surface expression of the different ligands by flow cytometry, and those negative in both types of assays were excluded from the analysis. For all soluble ligands, absorbance was measured by subtracting readings at 540 nm from readings at 450 nm. Dotted lines show the detection limit of each ELISA, and they were as follows: sMICA = 62.5 pg/mL, sMICB = 156 pg/mL, sULBP1 = 400 pg/mL, and sULBP2 = 50 pg/mL. Solid lines indicate the average of all measurements in the respective group. Statistical analysis on all donors with paired Student t test.