Effect of miR-155 overexpression on NK-cell cytotoxic effector functions. (A) FACS-sorted NK1.1⁺CD3⁻ NK cells from wt and miR-155 tg mice were used as effector cells in a 4-hour ⁵¹Cr release assay using YAC-1 and RMA-Rae1β tumor cells as targets. (B) Eight days after expansion in IL-2, sorted wt and miR-155 tg NK cells were assayed for ADCC against ⁵¹Cr-labeled P815 Ab-coated targets cells. (C) The wt and miR-155 tg NK cells were mixed with RMA-Rae1β tumor cells at a ratio of 2:1 and injected subcutaneously into the flank of Rag2^−/−xII2rg^−/− recipient mice (left). RMA-Rae1β tumor cells alone were injected as control. Tumor volumes were calculated every 2 days. The graph summarizes mean ± SEM data from 2 experiments with a total of 7 mice for the wt and the miR-155 tg groups. The percent survival of mice that had been inoculated with either wt or miR-155 tg NK cells in combination with RMA-Rae1β tumor cells at a ratio of 2:1 or RMA-Rae1β tumor cells alone as control is shown (right). Data from 3 independent experiments using 11 mice for each group are summarized in the Kaplan-Meier survival plots. (D) Resting (left) and IL-2–activated (8 days; right) NK1.1⁺CD3⁻ wt and miR-155 tg NK cells were analyzed for granzyme B, granzyme M, perforin, and actin protein levels by immunoblot. (E) The wt and miR-155 tg NK cells labeled with PE-conjugated anti-NK1.1 Ab were incubated with GFP⁺ YAC-1 tumor cells (left). Conjugate formation was analyzed at time 0 and after 10 minutes of incubation by flow cytometry. NK-cell target cell conjugates were gated and identified as NK1.1⁺GFP⁺ cells. The percentages of conjugates are shown on top of the representative dot plots. The graph summarizes the data of conjugate formation obtained from 3 wt and 3 miR-155 tg NK cell samples coincubated with YAC-1 tumor cells (right). *Statistically significant; see text for details.