Chronic engagement abrogates NKG2D function. (A) RMA cells stably transfected with the NKG2D ligand H60 bind soluble NKG2D. (B) The lytic capacity of NK cells exposed for 3 days to RMA-H60 or control RMA cells was determined. To exclude cold-target competition, unlabeled RMA-H60 cells were added to control NK cells during the lysis assay (at a percentage equivalent to that remaining in the RMA-H60 cultures [ie, < 5%]) (▪). These results are from one representative experiment of 14 performed. The lysis curves were shifted relative to the content of NK cells in the effector cell populations. To demonstrate the Ly49D dependence of CHO lysis, Ly49D was blocked with mAb 4E5 (▦ for controls and for RMA-H60-exposed NK cells). (C) NK cells were cultured in the presence of IL-2 plus RMA-H60 (▦) or RMA cells (□). The lytic activity of NK cells against RMA-H60 was determined after 24, 48, and 72 hours of coculture. NK cell activity was tested in triplicate at effector-target (E/T) ratios of 50:1 and 10:1. The data are representative of 2 independent experiments. (D) NK cells were cultured (for 72 hours in IL-2) separate from RMA-H60 (▪) or control RMA cells (□) using transwell plates. The lytic activity of these NK cells against RMA-H60 was determined. (B-D) Graphs depict the mean (±SD) of triplicate determinations at various E/T ratios.