Effect of miR-155 overexpression on terminal differentiation of NK cells. (A) Splenocytes from wt and miR-155 tg mice were stained with anti-NK1.1, anti-CD3, anti-CD27, and anti-CD11b Abs. Contour maps represent surface density expression of CD27 and CD11b on gated NK1.1⁺CD3⁻ NK cells. (B) The graphs summarize mean percentage (left) and absolute number (right) of ^+/− SEM of CD11b^lowCD27^high, CD11b^highCD27^high, and CD11b^highCD27^low NK-cell subsets from 10 miR-155 tg and 4 wt mice. (C) 1 × 10⁵ to 2 × 10⁵ CD11b^lowCD27^high NK cells collected from 2 CD45.2⁺ wt and 3 CD45.2⁺ miR-155 tg mice were intravenously injected into sublethally irradiated CD45.1⁺ recipient mice. After 16 days, splenic NK cells were harvested and the progeny of adoptively transferred CD11b^lowCD27^high NK cells from wt and miR-155 tg mice were analyzed via FACS with a gate on CD45.2 expression and assessment for coexpression of CD27 and CD11b. With this, the frequency of CD11b^lowCD27^high, CD11b^highCD27^high, and CD11b^highCD27^low NK-cell subsets was quantified and graphed. (D) Total wt and miR-155 tg NK1.1⁺CD3⁻ NK cells were each costimulated in vitro for 18 hours using IL-12 (20 ng/mL) and IL-18 (10 ng/mL) and analyzed for IFN-γ secretion by ELISA. (E) FACS-sorted CD11b^lowCD27^high, CD11b^highCD27^high, and CD11b^highCD27^low NK-cell subsets from wt and tg mice were stimulated for 18 hours in vitro with IL-12 (20 ng/mL) and IL-18 (10 ng/mL). Supernatants were then collected and analyzed for IFN-γ by ELISA. *Statistically significant; see text for details.