Chronic NKG2D stimulation impairs multiple distinct NK cell activation pathways. (A) B- and T-cell–depleted splenocytes were exposed to irradiated RMA-H60 (•) or control RMA cells (○). After 3 days of coculture, residual tumor cells were removed and the cytolytic activity of NK cells toward RMA, RMA H60, anti-Thy1 mAb (αThy1)–coated RMA (ADCC), RMA/S, and RMA m157 target cells was determined. To ensure Ly49H dependence of RMA m157 lysis, the Ly49H receptor was blocked with mAb 3H10 (□, controls; and ■ for RMA H60 exposed NK cells). The lysis curves were shifted relative to the content of NK cells in the effector cell populations as determined by flow cytometry. Data shown are representative of 3 to 4 experiments performed. (B) After 3 days of coculture with RMA H60) or control RMA cells (□), NK cells were restimulated with the indicated cell line or plastic coated mAb. Bars represent the mean percentages (± SD) of intracellular IFNγ plus NK cells in 4 to 8 independent experiments. Statistical significance of differences relative to the corresponding controls was determined using the 2-tailed Student t test: *P < .02; **P < .10; or not significantly different, P > .05. (C,D) After 3 days of coculture with either RMA (○) or RMA H60 cells (•), residual tumor cells were removed and NK cells were further cultured for 18 hours (C) or 42 hours (D) in the presence of IL-2. The cytolytic activity of these NK cells toward RMA H60 (left) anti-Thy1–coated RMA (middle) and RMA/S cells (right) was determined. The data are representative of 2 or more independent experiments. Error bars are SD.