Natural Killer Cell-Mediated Shedding of ULBP2
Figure 4
The spontaneous and NK cell-mediated shedding of ULBP2 from tumor cells.
(A, B) Apoptosis-induced shedding of ULBP2 is more intense than spontaneous shedding of it. (A) 2.5×106 Jurkat cells were treated with 2 µg/ml ActD, 4 µM CPT or 20 µM ETO for the indicated time in 0.5 ml serum-free RPMI 1640 medium. (B) 5×105 cells H9 were treated with 2 µg/ml Act D, 4 µM CPT or 50 µM ETO for the indicated time in 0.5 ml serum-free RPMI 1640 medium. The supernatant was collected for ELISA assay. DMSO was used as a negative control. (C, D) NK cell-induced shedding of ULBP2. 1×105 IL-2 expanded human NK cells were co-cultured with the indicated number of Jurkat (C) or H9 cells (D) for 2 hours, and then culture media were collected for ULBP2 ELISA. (E) Absence of ULBP2 in exosomes. 2×107 H9 cells were resuspended in 10 ml serum-free RPMI 1640 medium and treated with ActD or CPT overnight. Exosome preparations from the resulting culture supernatants were used to coat 4-µm-diameter aldehyde/sulfate latex beads by passive adsorption. The coated beads were used for detection of CD63 and ULBP2 by flow cytometric analysis. Beads with exosomes coating are showed in solid lines, and beads without exosomes coating are showed in gray-shaded histograms as controls.